Effects of psychoactive drugs on cyclic nucleotides in the central nervous system

Progres.s in Netlrobiology Vol. 21. pp. I to 13311983 Printed m Great Britain. All rights reservcd

113(11-(1082/83$0.00 + .50 Copyright © 1983 Pergamon Press Ltd

E F F E C T S O F P S Y C H O A C T I V E D R U G S ON CYCLIC N U C L E O T I D E S IN T H E C E N T R A L N E R V O U S S Y S T E M G E N E C . PALMER Director ~)f Research, Frist-Massey Neurological Institute, Suite 104, 3.56 24th A venue North, Nashville, Tennessee 37203, U.S.A. (Received 4 Jan 1983)

Contents 1, Introduction 2. Notes on methodology 2,1, Whole cell preparations 2.2. Broken cellular preparations 2.3. lit vivo studies 2.3.1. Rapid fixation of tissues 2.3.2. hmtophoresis 2.3.3. Direct electrical stimulation of adrenergic pathways 2.3.4. Intra-cerebro-ventricular drug administration 2.3.5. Body fluid m e a s u r e m e n t s 2.3.6. Postmortem assays 2.3.7. Histochemical--immunofluorescent techniques 3. Adenylate cyclase 3.1. Distribution and cellular localization 3.2. Development and aging of adenylate cyclase 3.2.1. Cyclic A M P levels 3.2.2. Adenylate cyclase 3.2.3. Catalytic and G T P sites 3.2.4. Adenosine and depolarizing agents 3.2.5. Adrenergic systems 3.2.6. Dopamine 3.2.7. Serotonin 3.2.8. Histamine 3.2.9. Metabolic role of cyclic A M P in development 3.2.10. Conclusions 3.3. Ionic requirements 3.4. Catalytic site 3.5. Transducer-GTP-sensitive site 3.6. Activation of adenylate cyclase 3.6.1. Adrenergic agents 3.6.2. Dopamine 3.6.3. Histamine 3.6.4. Serotonin (5-HT) 3.6.5. Prostaglandins 3.6.6. Adenosine 3.6.7. A m i n o acid transmitters 3.6.8. Depolarizing agents 3.6.9. Electrical stimulation 3.6.10. Steroids 3.6.11. Peptides 4. The phosphodiesterases and calmodulin 4.1. Distribution and localization 4.2. Development and aging 4.3. Properties and enzyme subtypes 4.4. Calmodulin 4.5. Regulation of phosphodiesterases 5. Guanylate cyclase-cyclic G M P 5.1. Distribution 5.2. Development and aging 5.3. Properties and enzyme subtypes 5.4. Activators and inhibitors of cyclic G M P 5.5. Regulation of cyclic G M P

4 5 5 5 6 6 6 6 6 7 7 7 7 7 8 8 8 9 9 10 11 12 13 13 14 14 14 15 15 15 17 17 18 19 19 20 21 21 21 23 23 23 23 25 26 26 26 26 27 27 28 28

(Dedicated to my long term colleague and friend Dr. Albert A. Manian on his retirement from the Psychopharmacology Section of N I M H . ) JPN 2 1 : 1 / 2

A

1

2

G.C. PALMER 6. Cyclic AMP/cyclic GMP interactions 7. Metabolic actions of cyclic nucleotides 7.1. Protein kinases 7.1.1. Cyclic AMP and calcium-dependent forms 7.1.2. Cyclic GMP-dependent forms 7.2. Development and aging profiles of protein kinase 7.2.1. Cyclic GMP-dependent form 7.2.2. Cyclic AMP dependent and independent forms 7.3. Cyclic AMP-mediated induction of enzymes 7.3.1. Activation of tyrosine hydroxylase 7.3.2. Glycogenolysis 7.3.3. Serotonin N-acetyltransferase 8. Neuroleptics 8.1. Introduction 8.2. Clinical studies 8.2.1. Postmortem tissue 8.2.2. CSF 8.2.3. Plasma 8.2.4. Urine 8.2.5. Blood cells 8.3. In vivo studies--neuroleptics 8.3.1. Cyclic AMP 8.3.2. Cyclic GMP 8.4. Guanylate cyclase-cyclic GMP 8.4.1. Guanylate cyclase 8.4.2. Cyclic GMP 8.5. Protein kinases 8.6. Miscellaneous actions of neuroleptics 8.7. Dopamine receptors 8.8. Norepinephrine receptors 8.9. Histamine receptors 8.10. Serotonin receptors 8.11. Phosphodiesterases--calmodulin 8.12. Chronic treatment with neuroleptics 8.12.1. Calmodulin 8.12.2. Dopamine and phosphodiesterase 8.13. Conclusions 9. Antidepressants 9.1. Overview 9.2. Tricyclic antidepressants 9.2.1. Dopamine receptors 9.2.2. Norepinephine receptors 9.2.3. Histamine receptors 9.2.4. Serotonin receptors 9.2.5. Cholinergic receptors 9.2.6. Miscellaneous actions of tricyclic antidepressants 9.2.7. Clinical studies 9.3. Monoamine oxidase inhibitors 9.3.1. Acute effects 9.3.2. Chronic effects 9.4. Electroconvulsive therapy (ECT) 9.4.1. Acute ECT 9.4.2. Chronic ECT 9.4.3. Clinical studies 9.5. Summary 10. Stimulants--amphetamines and cocaine 10.1. Amphetamine 10.1.1. Introduction 10.1.2. Acute effects 10.l.3. Chronic studies 10.1.4. Conclusions 10.2. Cocaine 11. Agents that deplete monoamines 11.1 Alpha-methyl-p-tyrosine 11.2. Reserpine 11.3. 6-Hydroxydopamine 11.4. Lesions 11.5. Kainic acid 11.6. Conclusions 12. Lithium 12.1. Clinical aspects 12.2. Antiadrenergic actions 12.2.1. Human studies 12.2.2. Iontophoretic studies

3(1 31 31 31 33 33 33 33 34 35 35 36 36 36 37 37 37 38 38 38 38 38 39 39 39 40 4O 40 41 41 42 43 43 44 45 45 45 46 46 46 46 47 51 53 53 54 55 55 55 56 57 57 57 57 57 58 58 58 59 59 60 6O 61 61 62 63 65 66 67 67 67 68 68 69

PSYCHOACTIVEDRUGS, CYCLIC NUCLEOTIDESAND THE CNS 12.3. Effects of Li ÷ on cyclic nucleotide systems in brain 12.3.1. Acute effects on m o n o a m i n e systems 12.3.2. Chronic effects on m o n o a m i n e systems 12.3.3. O t h e r cyclic nucleotide systems 12.3.4. Peripheral organs 12.4. Conclusions 13. Anti-Parkinson agents , 13.1. Introduction 13.2. Clinical studies 13.3. Cyclic nucleotides and anti-Parkinson drugs 13.3.1. Agents enhancing D A 13.3.2. Cholinergic agents 13.4. Ergot alkaloids 13.4.1. d ' L S D (d'lyserg]c acid diethylamide) 13.4.2. Other ergot alkaloids 14. Hallucinogens 14.1. Phencyclidine 14.2. Mescaline a n d o t h e r major hallucinogens 14.3. Harmaline 14.4. Tetrahydrocannabinol (THC) 14.5. Yohimbine 15. Opiates 15.1 Introduction 15.2. Role of cyclic nucleotides in the actions of opiates 15.3. Direct effect of opiates on cyclic nucleotide systems 15.3.1. Tissue culture models 15.4. Striatal mechanisms and neurotransmitter related events 15.4.l. Prostaglandins 15.4.2. In vivo levels of cyclic nucleotides 15.4.3. Influence on adenylate cyclase activity 15.5. Neurotransmitter-cyclic nucleotide systems in other brain regions 15.5.1. Cerebellum 15.5.2. Nucleus accumbens 15.5.3. A m y g d a l a 15.5.4. C e r e b r u m 15.5.5. Periaqueductal gray 15.5.6. Brain stem 15.5.7. Diencephalon 15.5.8. Whole brain 15.6. Adrenergic receptors 15.7. Plasma levels of cyclic nucleotides 15.8. Conclusions 16. Convulsants and anticonvulsants 16.1. Introduction and significance of cyclic nucleotides in seizures 16.2. Effects of anticonvulsants on steady-state levels of cyclic nucleotides elicited by seizures 16.3. Effects of anticonvulsants in whole cell preparations 16.4. Effects of seizure conditions on cyclic nucleotide enzymes 16.4.1. Adenylate cyclase-DA receptors 16.4.2. Phosphorylation mechanisms 16.4.3. Anticonvulsant action on cyclic nucleotide enzymes 16.5. Conclusions 17. Drugs with CNS depressant activity 17.1. Benzodiazepines 17.1.1. Background 17.1.2. Benzodiazepines as phosphodiesterase inhibitors and adenosine uptake blockers 17.1.3. In vivo-rapid fixation of tissue 17.1.4. O t h e r studies 17.1.5. E n h a n c e m e n t of adenosine action 17.1.6. Conclusions 17.2. Ethanol 17.2.1. Background 17.2.2. Peripheral systems 17.2.3. CNS actions 17.2.4. Conclusions 17.3. Barbiturates 17.3. l. Introduction 17.3.2. Cyclic nucleotide systems 17.3.3. Conclusions 17.4. General anesthetics Acknowledgements References

3 69 69 70 70 71 72 72 72 72 73 73 73 74 74 74 75 75 75 76 76 76 77 77 77 78 78 80 81 81 82 83 83 83 84 84 84 84 84 85 85 85 85 86 86 88 89 90 90 90 91 91 92 92 92 93 94 94 94 95 95 95 95 96 99 100 100 100 100 101 102 102

2

(1,('. PA]MFI¢ Abbreviations

Adenylatc Cyclasc 5'-AMP ATP CNS CPZ CSF CyclicAi~,IP CyclicGMP DA d'LSD ECT EGTA GABA Gpp(NH)p GTP Guanyiate eyclase 5-HT l -DOPA NE Phosphodiesterase Protein Kinase THC

IE('4.e,. I 1: ATF'l~YlOl;hosphatclyasc (cydizin~)] Adcnosmc-5'-n]c,nol'~hosphate Adenosine 5'-triphosphatc Central nerw)us system (Thlorpromazme Cerebrospinal fluid Adenosine 3'.5' monophosphate Ouanosinc 3' ,5'-monophosphate Dopamine d'Lyscrgic acid diethylamide Elect roconvulsiveIherapy El hyleneglycoI-Bis-(beta-ammoethyl ether) N, N'tetra acetic acid Gamma amino butyric acid 5'-Guanylyl imidodiphosphate Guanosine-5'-Iriphosphatc [EC 4.6. t.2; GTP pyrophosphate-lyase (cyclizing)] 5-O fl-tfyplamine or serotonin l.-Dihydroxyphcnylalanine Norcpinephrinc or noradrenalin (EC 3.1-t. 17: 3' ,5' cyclicnucleotide-5'-nucleotidohydrolasc) (EC 2.7.1.37: ATP protein phosphotransferase) Tctrahydrocannabinol

1. Introduction

The discovery of adenylate cyclase-cyclic A M P (adenosine 3; 5 ' - m o n o p h o s p h a t e ) , as well as the enzyme(s) responsible for cyclic A M P metabolism, the phosphodiesterases, by Sutherland and coworkers (see Robison et al., 1968, for review) has been one of the milestones of biomedical research. As an aftermath of these experiments entire journals including annual review volumes, are now devoted toward reporting the outcome of the vast research efforts directed toward understanding the p h e n o m e n o n of receptor-induced alterations in cellular metabolism and function. The current massive investigations toward understanding the molecular properties of cellular receptor interactions in addition to the role of calmodulin are further offshoots emanating from cyclic nucleotide investigations. With regard to the central nervous system (CNS) an initial research effort implicated altered functional levels of catecholamines as molecular substrates in mental illness (Schildraut and Kety, 1967). Later work considered the role of m o n o a m i n e enzymes and reuptake processes under similar experimental conditions. Research in cyclic nucleotides with regard to brain receptor function lagged until 1968 when Kakiuchi and Rail developed the procedure whereby incubated tissue slices were revealed to accumulate cyclic A M P in response to designated monoamines. R e c e p t o r blocking agents specifically antagonized the action of these monoamines. As an aftermath of these key observations the field for cyclic nucleotide action in the brain rapidly expanded. Since that time cyclic A M P and cyclic G M P (guanosine 3 ' , 5 ' - m o n o p h o s p h a t e ) have been implicated in many neuropathological conditions namely, psychosis, depression, mania, ischemia, epilepsy, trauma, Parkinsonism, anesthesia, opiate action and hypertension. The location of an enzyme, adenylate cyclase, within the cell m e m b r a n e is a handy site in which a receptor may discriminate the action of particular neurotransmitter and couple it to an intracellular metabolic event. Moreover, other species of neurotransmitters have been shown to modulate, augment or inhibit neurotransmitter action on adenylate cyclase. F u r t h e r m o r e , both the receptor and catalytic components of adenylate cyclase display the capability to either "up regulate" or "down regulate" in response to an environmental or pharmacological challenge. Another important p h e n o m e n o n first described by Nelson Goldberg (Goldberg et al., 1975) was the ability of cyclic A M P and of cyclic G M P to influence the intracellular concentrations of one another. This review principally focuses upon the action of neuropharmacological agents on cyclic nucleotide enzymes within the brain. Sections 2-7 discuss in brief the background

PSYCHOACTIVEDRUGS, CYCLICNUCLEOTIDESAND THE CNS

5

information necessary for understanding neuropharmacological drug action. No attempt is made to review exhaustively these topics, many of which are already considered in recent articles. In the sections on neuro-psychopharmacology all aspects of drug action on cyclic nucleotide related systems are stressed which may diverge somewhat from only a consideration of the more glamorous aspects or current trends within a particular field. It is my feeling that all data even that which is currently thought to be insignificant must be weighed toward a complete evaluation of a particular drug in question. Where a topic has been recently reviewed, it is discussed briefly. The majority of the review is directed toward understanding cyclic nucleotides in mammalian brain. Peripheral tissue findings are only presented in order to amplify a particular topic.

2. Notes on Methodology

No one method for measurement of cyclic nucleotide levels or the relative activities of associated enzymes offers definitive answers. Each procedure must be weighed against several factors depending upon the experimental protocols attempted. As one reads the scientific literature he/she will quickly realize that individual groups of investigators usually employ only one or a few of the available techniques. Technical problems, as well as a degree of scientific expertise of the laboratories embarking on a study are important factors to consider.

2.1. WHOLE CELL PREPARATIONS

Preparations containing intact tissue elements are of value because receptor systems are left intact and rather large increases in cyclic nucleotides can be measured following addition of neurohumoral agonists, depolarizing agents or adenosine. This procedure is useful for examining the actions of surface or receptor acting drugs either upon basal levels of cyclic nucleotides or responses evoked by agonists. Limitations to the procedure are the distortion and damage of tissues during cellular isolation or tissue slice preparation. Also the osmotic nature of the incubation medium itself may cause tissue damage. Tissue culture methods have additional problems in that one is usually evaluating hormone responses in metabolically derranged cells plus inherent problems with contamination. Moreover, if pure cellular strains are used, i.e. neuroblastoma, then the normal physiological contributions of supporting cells e.g. gila are lost. Other limitations include cellular extrusion of cyclic nucleotides into the medium and the unique metabolic properties of fetal tissues if used as a tissue source (Robison et al., 1968; Daly, 1977; Nathanson, 1977; Garthwaite et al., 1979). 2.2. BROKEN CELLULAR PREPARATIONS With this method the actions of transmitters and drugs can be examined upon intracellular process, namely, the cyclic nucleotide enzymes themselves. A large sample preparation can be prepared from one brain region and from this same sample a wide variety of dose-response relationships of drugs and hormones may be compared to a respective set of controls. Moreover, some hormonally elicited responses for adenylate cyclase activation, i.e. dopamine (DA) and serotonin (5-HT) become unmasked upon cellular disruption. Preparations for receptor ligand binding investigations can be compared to the ability of specific agonist/antagonists to modify adenylate cyclase. The reaction and kinetic properties of the enzymes can be tightly controlled. In addition, the roles of guanine nucleotides and the catalytic site may be studied. Limitations to broken cellular preparations are a diminished degree, or complete absence of, adenylate and guanylate responses to specific neurohomoral agents. If isolated cells or organelles are used, there is an inherent problem of purity (Robison et al., 1968; Daly, 1977; Nathanson, 1977).

6

(J. (_~. PAI MER

2.3. In Vivo STUDIES 2.3.1. Rapid fixation o f tissues A variety of procedures have been developed as a means to measure the steady-state, in vivo level of compounds that rapidly turn over in the brain. Cyclic AMP does offer a tremendous challenge in this area because levels rapidly rise during periods of trauma, seizures, decapitation, anoxia, ischemia, sample preparation, etc. The levels of cyclic G M P do not turn over with the same rapid pace (less than I sec) as do levels of cyclic AMP, but nevertheless the same traumatic conditions do produce changes in cyclic GMP. Rapid freezing of small animals, mice or gerbils is most likely sufficient for accurate measurement of cyclic nucleotides in brain structures located near the surface i.e. cortex and cerebellum. This technique of decapitation plus rapid freezing in liquid nitrogen is obsolete in larger animals-rats. Commercial microwave ovens do not yield accurate in vivo data unless they are highly modified in that a high intensity beam is tuned and focussed upon the skull of the animal. With these high intensity-focussed instruments fixation of mouse brain occurs in less than 250 msec and thus valuable analytical information is attained. Moreover, this method of fixation allows for the convenience of brain dissection at room temperature. The "freeze-blowing" procedure was developed by Veach (see Lust et al., 1973) and likewise achieves rapid brain fixation. The major problem is that only whole brain assays can be conducted (Jones and Stavinoha, 1979; also Guidotti et al., 1974; Lenox et al., 1977; Dodson et al., 1979; Schneider et al., 1981 ). 2.3.2. lontophoresis In order to correlate relationships between cyclic nucleotide action and neurotransmitter-electrophysiological activities, Siggins et al. (1969, 1971, 1974), Lake et al. (1973), and Stone et al. (1975) have conducted extensive investigations using the technique of microiontophoresis. The method first revealed that cyclic AMP acted similarly to norepinephrine (NE) and cyclic GMP acted similarly to acetylcholine with respect to their actions on the firing patterns of individual neurons. The major drawback with this work is that cyclic AMP is normally formed intracellularly as a consequence of extracellular NE action. Thus application of the cyclic nucleotide to a cell surface is unlikely to be its usual site of action. In addition some investigators have not demonstrated concordant actions between cyclic AMP and NE (Lake et al., 1973). 2.3.3. Direct electrical stimulation o f adrenergic pathways Investigators have applied electrical current to the locus coeruleus or preganglionic fibers and measured cyclic AMP levels in brain regions and cells innervated by these afferent pathways. When combined with proper techniques for rapid fixation, followed by nucleotide measurement by either chemical analyses or visualization by immunofluorescence the procedure is extremely useful for defining with precision the cellular location of catecholamine or afferent pathways functionally linked to cyclic nucleotides. The method is of further benefit for evaluation of injected drug responses on the particular systems under investigation (Siggins et al., 1973; Korf and Sebens, 1979; McAfee et al., 1980). 2.3.4. Intra-cerebro-ventricular drug administration

Injection of drugs, neurotransmitters or cyclic nucleotides into the lateral ventricles has been employed to measure behavioral responses, drug agonist/antagonist interactions and subsequent cyclic nucleotide levels after suitable tissue fixation. The specificity of the responses and the nonphysiological routes of drug transport from cerebro-spinal fluid (CSF) to brain are the major obstacles to this method. In addition intracerebral injection of cyclic nucleotides can produce a variety of effects ranging from seizure behavior to anesthesia (Herman, 1973; Cohn et al., 1975, 1978).

PSYCHOACTIVE DRUGS, CYCLICNUCLEOTIDES AND THE CNS

7

2.3.5. B o d y fluid measurements Cyclic nucleotides have been measured and compared to mental conditions using such diverse fluids as plasma (including studies in platelets, erythrocytes and leukocytes), urine and CSF. The first two sites are obviously susceptible to contaminating factors from peripheral tissues. The CSF measurement which should be a potentially valuable procedure for patients has not yielded concordant findings among the various laboratories. Although somewhat difficult, the methods use the "push-pull" perfusion apparatus in which drugs, transmitters, etc. are "pushed" (infused) into the ventricles or into a specified region and CSF is "pulled out". Nucleotide assays are then performed at designated time points. This latter method has not been extensively used by many laboratories in the cyclic nucleotide field (Abdulla and Hamadah, 1970; Robison et al., 1970; Paul et al., 1971b; Myllyla et al., 1975; Belmaker et al., 1976; Korf et al., 1976; Lykouras et al., 1979; Pandey and Davis, 1979; Schoener et al., 1979). 2.3.6. P o s t m o r t e m assays Postmortem analysis of cyclic nucleotide enzymes, correlated to ligand binding studies, offers limited but useful information for human studies and should probably be reserved for that type of investigation (Nagatsu et al., 1978; Lee et al., 1978b). 2.3.7. Histochemical--immunofluorescent techniques Suitable methodology has been developed to visualize cyclic AMP, cyclic GMP, calmodulin, adenylate cyclase, guanylate cyclase, protein kinases and phosphodiesterase at intracellular loci within the CNS. The future development of monoclonal antibodies to some of these enzymes will be of further aid for the determination and characterization of enzyme subtypes e.g. soluble vs particulate guanylate cyclase (Florendo et al., 1971; Rechardt and Harkonen, 1977; French et al., 1978; Sugimura and Mizutani, 1978; Wood et al., 1980; Ariano and Matus, 1981; Cumming et al., 1977, 1979, 1981, 1982; Zwiller et al., 1981b; Ariano et al., 1982).

3. Adenylate Cyclase The early discoveries of Sutherland (for review see: Robison et al., 1968) led literally to an explosion of scientific data in an attempt to understand the mechanisms whereby the hormonal activation of adenylate cyclase exerts metabolic control over cells. The following section serves only as an introduction to the characteristics of the enzyme and its regulation by neurohumoral substances. The development and aging profiles of adenylate cyclase are explored in greater depth because this topic has not been recently reviewed. 3.1. DISTRIBUTION AND CELLULAR LOCALIZATION

Klainer et al. (1962) first reported a slight stimulation of adenylate cyclase by catecholamines in broken cell preparations of mammalian brain. Since activation was of such a small degree of magnitude, work suffered in the CNS until Weiss and Costa (1967, 1968b) began observing prominent adenylate cyclase activation in the pineal. Work by De Robertis et al. (1967) localized the greatest adenylate cyclase activity to synaptosomal fractions. The work with neurohumorally elicited cyclic AMP in incubated tissue slices by Kakiuchi and Rail (1968) essentially initiated the extensive studies in the brain. By the use of various techniques hormonally-elicited adenylate cyclase has been found in every cell type in the CNS. The enzyme is, however, unequally distributed, displaying higher activity in gray matter than in white matter. Certain brain regions (cerebral cortex, olfactory bulb, hippocampus) contain higher specific activities than others (pons, medulla and spinal cord). Essentially all the enzyme activity in brain is confined to the particulate fraction (Weiss and Costa, 1968a; Williams et al., 1969).

S

(;. ('. PAl MER

3.2. DEVELOPMENT AND AGING OF ADENYI_ATE CYCI.ASE 3.2.1. Cyclic AMP levels

The data for endogenous levels of cyclic AMP during development are summarized in Table 1. The chick retina at the 14th embryonic day contains measurable levels of cyclic AMP. In most regions of the rat brain, cyclic AMP levels have been detected either at or shortly before birth. Usually by young adulthood, levels are maximal or do not change during subsequent maturation. With regard to aging, cyclic AMP in the mature (18 month) rat cerebellum declined to 14% of the maximal levels observed in the young adult (Austin et al., 1978). Zimmerman and Berg (1974, 1975) reported in the rat that the maximal steady-state levels of cerebral cyclic AMP occurred at 2 months postpartum, thereafter (6-24 months) cyclic AMP was less. This finding was likewise confirmed in midbrain regions and the hypothalamus (Purl and Volicer, 1981). Measurements of endogenous cyclic nucleotides do n o t clearly reflect the metabolic activity in the brain because of the multiplicity of factors influencing their synthesis and metabolism. 3.2.2. Adenylate cyclase

The human fetus contains detectable adenylate cyclase activity in the brain at the 12th fetal week. No earlier data are available (Menon et al., 1973). At day 6 of embryonic age the chick retinal enzyme has been observed (DeMello, 1978). In all studies with rodents the enzyme is detectable in all brain regions that have been evaluated either shortly before or after birth. A maximal level of enzyme activity is attained by days 12-20 postpartum (see Table 1, for specifics). There are some instances when basal adenylate cyclase is influenced during development. "Quaking" mice have elevated cerebral enzyme activity at neonatal days 25-35. In contrast adenylate cyclase in the upper brain stem is considerably higher at 20-40 days after TABLE 1. APPEARANCE AND MATURATION OF BASAl. ACTIVITY OF ADENYEATE CYCI ASE AND CYCLIC A M P CON IENI IN BRAIN

Adenylate cyclasc Species

region

Chick retina R a t - - Whole brain Cerebrum Cerebellum Brainstem Colliculus Striatum Olfactory bulb Pineal Human fetus

Cyclic AMP

Appear

Maxiinal

Appear

Maximal

Refs.

embryo-6

embryo-16

embryo-14

embryo-18

11 -1 0 1t 1

17-311 211 25 15 16 9-12

2 5,9 1,4,6.7,10.13 13 13 3

1 1 (I 3rd mo.

5 11 1t l)

30 no change 311 nochange

15

3

21 no change

1 12 8

Fluoride-Sensitive Adenylate Cyclase Rat brain region

Whole brain Cerebrum Olfactory bulb Caudate Cerebellum

Appear

Maximal

Decline

Rcfs

9 11-19 7

13 20-35 21 3 mo. 31 15 25

23 35-180 35 2 yr adult n o change

9 1.4,6,7,9,14 1 11 12 12 12

Brain stem

Pineal

0

0 = day of birth; All figures unless indicated are days of development. References: (1) Cousin and Davrainville, 1980a; (2) De Mello, 1978; (3) Enjalbert et al., 1978; (4) Harden et al., 1977b; (5) Hommes and Beerc, 19711; (6) Kaufman et al., 1972; (7) Kohrman, 1973; (8) Menon et al., 1973; (9) Schmidt et al., 19711; (10) Von Hungen et al., 1974a; (11) Walker and Walker, 1973a; (12) Weiss. 1971 ; (13) Weiss and Strada, 1973; (14) Zimmerman and Berg, 1975.

PSYCHOACTIVE DRUGS, CYCLICNUCLEOTIDES AND THE CNS

9

birth. The ability of fluoride to activate the enzyme was the same in both brain regions of either quaking or control mice (Sapirstein et al., 1980). In rats fed a growth restricted diet, the basal activity of adenylate cyclase was reduced in the developing olfactory bulb, but was unchanged in the cerebrum (Cousin and Davrainville, 1980b). Injections of 6hydroxydopamine to newborn rats did not change the cerebral activity of basal or fluoride-responsive adenylate cyclase throughout development (Harden et al., 1977a). Aged rats (2 yrs.) were initially found to be deficient in basal adenylate cyclase activity in the cerebral cortex (Kauffman et al., 1972). However, there is little agreement between groups of investigators as to the influence of aging on basal adenylate cyclase. Walker and Walker (1973a) reported that enzyme activity in senescent rats was unchanged in the cortex and hippocampus, but increased 2 fold in the caudate and cerebellum. Zimmerman and Berg (1975) did not observe any change in cerebral enzyme activity when measured from 1-24 months after birth. Puri and Volicer (1977) observed only a slight elevation in striatal adenylate cyclase in old (30 month) rats. 3.2.3. Catalytic and G T P sites In only one study has the actual GTP-transducer site been looked at with respect to development and maturation. Limbird and co-workers (1980) showed that during the maturation of rat reticulocytes of erythrocytes there was a parallel loss of adenylate cyclase responsiveness to catecholamines and guanine nucleotides without a loss of basal activity. Whether this loss of GTP-receptor coupling process is significant in the brain remains to be seen.

Adenylate cyclase in fetal human brain is activated by fluoride (Menon et al., 1973) and most studies with rodent brain reveal fluoride-activation at birth or shortly before. In the rat olfactory bulb, fluoride is not effective until postnatal day seven (Cousin and Davrainville, 1980a). There is some argument, especially in the cerebral cortex of rats, as to when the enzyme sensitivity to fluoride becomes evident. The dates range from one day before birth (Kohrman, 1973) to 7-19 days postpartum (Kauffman et al., 1972; Schmidt et al., 1970--whole brain only; see Table 1). In rats fed a growth restricted diet fluoride was less effective in the stimulation of cerebral and olfactory bulb adenylate cyclase (Cousin and Davrainville, 1980b). In contrast when newborn rats whose food intake (undernourished) was limited, the ability of fluoride to activate_cerebral adenylate cyclase between postnatal days 10-19 was greater than controls. The maturation of fluoride-sensitive adenylate cyclase is associated with a decline in enzyme responsiveness. The decline may occur in some brain regions at the young adult stage and at various periods thereafter up to 2 years of age. Not enough detailed work has been performed to draw any definite correlations with regard to development and aging of the catalytic site of adenylate cyclase. With the discovery of forskolin, a specific agonist of the catalytic site, perhaps more complete data will be forthcoming. 3.2.4. Adenosine and depolarizing agents In tissue slices of rat cerebral cortex, adenosine produced an accumulation of cyclic AMP at eight days after birth. Activity rose to a maximum from 12-18 days and then declined in the adult. Adenosine plus NE gave a greater than additive response with a huge rise in cyclic AMP synthesis evident at 14 days followed by a decline thereafter (Perkins and Moore, 1973a). The enhanced adenosine responses at 12-18 days might be associated with the phenomenon of preinnervation supersensitivity as described by Schmidt and co-workers (1970, 1980a). The tremendous rise (at 14 days) in cortical cyclic AMP elicited by NE plus adenosine was not associated with any prominent change in either basal or fluoride-activated adenylate cyclase (Perkins and Moore, 1973a). The ability of the neurotoxin, kainic acid to elicit accumulation of cyclic AMP in rat cerebellar slices progressively declined in tissue taken from 3, 12 and 24 month old animals (Schmidt and Thornberry, 1978).

10

G.C. PALMER 3.2.5. Adrenergic systems

3.2.5.1. D e v e l o p m e n t The first investigation concerning the profiles of adrenergic receptor development in the brain was conducted by Schmidt and colleagues (1970). Tissue slices prepared from whole rat brain displayed a capability to synthesize cyclic AMP in response to added NE beginning at the third day postpartum. The maximal response was reached by day 9 with a decline to the adult pattern of activity at day 22. At this time the elevated responsivenes at day 9 was thought to relate to either some type of "preinnervation supersensitivity" phenomenon in that vital regulatory processes were absent or because of the low levels of endogenous transmitters at this developmental stage, the receptors had "up regulated". Using tissue slices of rat cerebrum, Perkins and Moore (1973a) showed that the appearance of the NE response occurring at day 10 postpartum was independent from adenosinemediated activation of cyclic AMP (day 6). The maximal NE response or NE plus adenosine was, at days 14-16, followed by a decline thereafter. The NE response was pharmacologically characterized as a beta receptor. In an interesting investigation with tissue slices in the rabbit brain, Schmidt and Robison (1971) demonstrated that brain regions which did not contain NE-sensitive cyclic AMP systems in the adult, namely the hippocampus, hypothalamus and cerebrum, did indeed possess a highly active system prior to fetal day 14. The physiological basis for the rapid disappearance of these receptors is unknown. Other, whole cell preparations of central tissue (fetal rat brain cultures, chick embryonic neuronal cultures and chick postnatal tissue slices) readily accumulated cyclic AMP in response to beta adrenergic agonists and prostaglandin El (Gilman and Schrier, 1972; Nahorski et al., 1975; Ciesielski-Treska and Ulrich, 1980). With broken cellular preparations, NE stimulation of pineal adenylate cyclase is absent at birth, appears at day 3 and rapidly reaches the adult level by day 6 (Weiss, 1971). Von Hungen et al. (1974a) and Kohrman (1973) using particulate fractions of rat cerebrum demonstrated the presence of NE-adenylate cyclase at one day before birth. A maximal stimulation was present by 14-25 days with a gradual decline in enzyme activation in the 1 year old adult. This broken cell data did not correlate with tissue slice work mentioned in the preceeding paragraph. Norepinephrine elicited a marked activation of adenylate cyclase in the 6-day old rat striatum and hippocampus. The enzyme from the 1-42 day-old cerebellum and medulla was only marginally responsive to NE (Von Hungen etal., 1974a). In an attempt to correlate beta receptor ligand binding to isoproterenol activated adenylate cyclase in the rat cerebrum, Harden et al. (1977a,h) first detected the presence of these receptors at day 6 with maximal numbers seen between days 16-45. Isoproterenolsensitive adenylate cyclase was prominent at day 7-10 and the maximal response occurred from days 16 to 45. Cantor et al. (1981a) likewise correlated the appearance of beta receptors in the rat pineal to NE activation of adenylate cyclasc. Subtypes of beta adrenergic receptors within the same brain region develop at different periods. In the rat cerebellum the betaj receptors gradually increase from birth to 25 days and then decline to practically undetectable levels in the adult. The beta2 receptors show a steady, rapid rise from postnatal day 8-42 with a gradual elevation seen thereafter. The cerebral beta I receptors appear at day 8, rapidly rise by day 20 and decline in numbers between 8 and 28 days. The cerebral beta 2 receptors reach a peak by day 28 and remain fairly constant up to 3 months of age (Pittman et al., 1980b). The developmental patterns of NE receptor coupled adenylate cyclases are markedly altered by disruptive agents. Postnatal injection of 6-hydroxydopamine into rats reduced the adult cerebral content of NE, elevated beta receptor numbers, enhanced catecholamine stimulation of cyclic AMP, and destroyed the NE-induced turnover of phospholipids (Palmer and Scott, 1974; Harden et al., 1977a; Austin et al., 1978). When X-irradiation was directed to the posterior cortical surface of neonatal rats the adult hippocampus and pia-arachnoid were highly damaged with regard to NE activation of adenylatc cyclase. The posterior cortex and associated capillaries were essentially unaf-

PSYCHOACTIVE DRUGS, CYCLIC NUCLEOTIDES AND THE CNS

11

fected by postnatal X-irradiation (Chronister et al., 1980; Palmer et al., 1982). Removal of the superior cervical ganglion or decentralization of it at birth did not affect the numbers of beta receptors in the developing rat pineal (Cantor et al., 1981a). This latter observation indicated that receptors in the pineal are not influenced by innervation in contrast to what was observed in cerebral tissue. 3.2.5.2. Aging When 3 month old rats were compared to 24 month old animals, NE was less effective in activation of adenylate cyclase from the aged caudate, cerebrum, hippocampus and especially the cerebellum (Walker and Walker, 1973a). With incubated tissue slices Schmidt and Thornberry (1978) reported that of several brain regions only the aged cerebellum revealed an attenuated response to NE. This work was supported by receptor ligand binding techniques in which the aged cerebellum began to lose beta2 receptors at one year of age, however, the reduction on cyclic AMP responses was not evident until 24 mo. (Maggi et al., 1979; Greenberg and Weiss, 1978; Pittman et al., 1980a). Misra et al. (1980) reported a deficiency in beta adrenergic receptors in aged rat cerebral cortex, but this finding was not corroborated by Pittman et al. (1980a). The rat striatum and pineal lost beta receptors during aging and the older pineals were unable to develop beta adrenergic supersensitivity in response to denervation techniques (Greenberg and Weiss, 1978). In one human study cerebellar but not cerebral beta receptors were shown to decline from birth to 80 years of age (Maggi et al., 1979). The decline in beta adrenergic activity with aging in some brain regions may at times be correlated with altered activities of several monoamine systems, namely decreased NE uptake mechanisms and increased metabolic activities of monoamine oxidase and catechol-O-methyltransferase. However, these parameters vary widely among brain regions and animal species (Austin et al., 1978; Pradham, 1980).

3.2.6. D o p a m i n e 3.2.6.1. D e v e l o p m e n t Many of the central mechanisms for DA-neurotransmission processes develop in an independent manner from one another. For example, in the rat brain the specific DA uptake system and the capacities for storage and release are present at birth. The ability of fetal monoamine neurons to store DA and NE is many fold greater than their capability for biosynthesis. Moreover, the storage capacity is highly sensitive to reserpine depletion. Dopamine induced activation of adenylate cyclase precedes the development of presynaptic components in the striatum. The DA-stimulation of inorganic phosphate incorporation into phospholipids at synaptic terminals occurs during the second postnatal week and is correlated with both receptor binding sites and the presence of the presynaptic uptake system (Coyle and Campochiaro, 1976; Coyle and Molliver, 1977; Enjalbert et al., 1978; Schmidt etal., 1980a; Deskin etal., 1981). Dopamine activation of adenylate cyclase was found to be present in several regions of the rat brain by day 1 postpartum. The respective brain regions containing the DAresponsive enzyme were: striatum, cerebrum, subcortex, hippocampus and hypothalamus. Enzyme sensitivity to DA was essentially absent in the medulla and cerebellum. Activity to DA was higher in the newborn cerebrum than in the adult (Von Hungen et al., 1974a). X-irradiation directed to the posterior cortex of neonatal rats produced a diminished capability of DA to activate,adenylate cyclase in the adult hippocampus (Chronister et al., 1980). In the well-developed guinea pig brain the degree of DA-adenylate cyclase in the newborn colliculi, hypothalamus and spinal cord was similar to the adult (Enjalbert et al., 1978). The newborn monkey retina displays a sensitivity to DA-adenylate cyclase that is 63% of the adult (Makman et al., 1975) while in the chick, DA-sensitive adenylate cyclase

II

( ; . ( ' . P M MIR

becomes evident at an early stage. It is absent at embryonic day 6 but appears at day 7 and rapidly reaches the maximum activity by embryonic day 8 (DeMello, 1978). In the rat retina DA-adenylate cyclase does not appear until day 6 postpartum, attains maximal activity (preinnervation supersensitivity?) by day 14 and declines by 5()% by day 2 t) (Makman et al., 1975). Interestingly if newborn rats receive X-irradiation tile sensitivity of retinal adenylate cyclase to DA is greatly enhanced in the adult. This is in contrast to similar treatment in other brain regions where adenylate cyclase responses are usually diminished. In other brain regions (cortex and hippocampus) the DA fibers are extrinsic in origin while in the retina they arise from intrinsic, amacrine cells. Perhaps these cells are highly susceptible to damage and the cells receiving DA synapses are not and thus become supersensitive (up regulate) to DA. Alternatively, in the hippocampus and cortex it appears that the receptors alone are damaged by the influence of X-irradiation (Palmer el al., 1982). 3.2.6.2. A g i n g A host of recent investigations principally in preparations of rat striatum, have revealed in aged animals (12-30 months) a prominent decrease in both the binding of D A ligands and the ability of D A to elicit activation of adenylate cyclase. In some investigations there was noted a slight rise in basal adenylate eyclase which could partially account for the deficits in DA-adenylate cyclase. However, other investigators did not observe such changes in basal activity (Walker and Walker, 1973a; Govoni et al., 1977.1978, 1980; Puff and Volicer, 1977; Schmidt and Thornberry, 1978; Misra et al., 1980; Cubells and Joseph, 1981 ; Levin el al., 1981). Govoni et al. (1980) attributed the loss in receptors to a decreased affinity for ligand binding of spiroperidol while the data of Misra et al. (1980) and Levin et al. (1981) revealed the change to involve a decrease in the maximum number of striatal DA-receptors. In further work Cubells and Joseph (1981) provided behavioral evidence that DA-2 receptors (not coupled to adenylate cyelase) likewise declined in senescent rats. Moreover, D A levels declined in certain rat brain regions with advancing age (Austin et al., 1978). McGeer and McGeer (1977) reported severe losses in tyrosine hydroxylase in the postmortum striatum of aged humans. On the other hand, supersensitivity of stereotyped behaviors was seen in aged rats subjected to testing by apomorphine and d-amphetamine (Flemenbaum, 1979). Taken together the findings assert that DA function involving multiple receptors is attenuated during the molecular processes of aging. Diminished activation of DA-adenylate cyclase, and in some instances decreased DA ligand binding, has been observed in other brain regions of the aged rat i.e. nucleus accumbens, substantia nigra, olfactory tubercle, frontal cortex and hypothalamus, but not in the cerebellum, hippocampus, posterior cerebrum or medulla (Walker and Walker, 1973a; Govoni et al., 1977, 1980; Austin et al., 1978). Likewise, in the aged rabbit frontal cortex, hypothalamus and anterior limbic region, the ability of DA to stimulate adenylate cyclase declined by 50% as measured at either 5½ months or 5½ years of age. The activity of the retinal enzyme was unchanged (Makman et al., 1980). An interesting observation in the rat retina was made by Riccardi and coworkers (1981). In this tissue DA turnover~ receptor numbers and even methionine-enkephalin binding sites were increased in old (24 month) rats. This finding was opposite to that observed for most of the other tissues discussed. When rats were raised on a restricted diet consisting of alternate days of feeding and fasting the age associated loss in striatal DA receptors was retarded. Moreover, survival time of the rats was increased by 4(1% (Levin el al., 1981 ). The data with D A and aging though far from complete provide rather interesting inroads towards an initial understanding of these complex physiological processes in the brain. The striatal data indicate a correlation between loss of DA function to changes in coordination and behavior associated with aging in humans. 3.2.7. S e r o t o n i n Von Hungen et al. (1974a, 1975a) performed the first experiments relative to a discovery

PSYCHOACTIVE DRUGS, CYCLIC NUCLEOTIDES AND THE CNS

13

of 5-HT sensitive cyclases in mammalian brain, as well as, the developmental aspects of this indoleamine. Before this time it was doubted that 5-HT was coupled in any manner to adenylate cyclase. In a 10,000 x g particulate fraction of immature brain, Von Hungen demonstrated the presence of 5-HT-sensitive enzymes in the cerebral cortex, subcortical regions, striatum, hypotbalamus, hippocampus, cerebellum, inferior and superior colliculi. In the latter two structures the enzyme was highly sensitive to 5-HT, tryptamine and 5-methbxytryptamine. The greatest magnitude of activation occurred at birth (80%). Activity dropped by days 6-15 and in most cases was absent in the adult. One problem with the absence of a 5-HT response in the adult may have been the degree of maturation of monoamine oxidase A. Inhibitors for this enzyme were not present in their assays. Alternatively, Enjalbert et al. (1978) and Nelson et al. (1980) described the presence of 5-HT adenylate cyclase in eight brain regions. In the colliculi, for example, in the presence of monoamine oxidase inhibitors, the enzyme was sensitive to 5-HT at birth and rapidly reached a peak from day 6 to adulthood. Moreover, in all brain regions (except cerebellum) from newborn rats, appearance of 5-HT-cyclase correlated with binding of 5-HT to the crude mitochondrial fraction. This correlation was not evident in the adult (except striatum and cerebellum). In both rat and guinea pig cortical tissues there was little change in the degree of 5-HT stimulation of adenylate cyclase between the newborn and adult. Injection of kainic acid into 9 day-old striata decreased within one day the activity of 5-HT cyclase. The cyclase was diminished to a greater extent than corresponding 5-HT binding. Lesions to the raphe nuclei at day 4 did not subsequently alter 5-HT cyclase activation. The studies indicated the possible presence of separate receptors for 5-HT i.e. either coupled or uncoupled to adenylate cyclase. Little investigative work has been performed on 5-HT and aging. In many species 5-HT content and turnover remained unchanged or even increased during maturation. In the aged rat there is a decreased content of 5-HT and tryptophan hydroxylase in hippocampus, pons and the raphe nucleus. The significance of this to receptor action is unknown (Pradhan, 1980). 3.2.8. Histamine In incubated tissue slices of rabbit cerebrum the presence of histamine-sensitive accumulation of cyclic AMP was detected at 1-2 days prior to birth. From 4-8 days postpartum, a huge elevation (preinnervation supersensitivity?) in cyclic AMP production in response to histamine was evident. Thereafter the sensitivity declined to day 20 at which responses similar to the adult were seen (Palmer et al., 1972a). In rabbit frontal cortex, hypothalamus and anterior limbic lobe the maximal response of histamine-adenylate cyclase declined by 50% in animals aged 5½ years (Makman et al., 1980). The presence of H-1 receptors, histamine levels and histidine decarboxylase was detected at birth in synaptosomes from several rat brain regions. The activities of both ligand binding and enzyme activity paralleled one another (except cerebrum) and reached maximal activity by 20 days postpartum. Moreover, areas poor in neuronal histamine had initially high amine levels which subsequently declined as development progressed (Tran et al., 1980; Subramanian et al., 1981). In one study with adenylate cyclase, when rats received a sequence of X-irradiation shortly after birth and were sacrificed at 6 weeks, the hippocampal enzyme was less sensitive to histamine. There was considerable damage (histological observation) to the dentate gyrus, but whether this structure received any histamine innervation was not determined (Chronister et al., 1980). The latter data might reveal a nonspecific receptor damage because D A and NE systems were also less responsive. 3.2.9. Metabolic role o f cyclic A M P in development The well-known action of cyclic AMP to stimulate protein kinase and metabolic events is discussed in Section 7. However, without delving into the extensive literature of tissue culture, cyclic A M P has been shown by many investigators to induce differentiation in

14

(L C'. PAIMI r

various culture preparations that include: (a) alteration of the shape of cultured schwannoma cells (Sheppard et al., 1975); (b) stimulation of microtubule dependent axonal elongation (Roisen et al., 1972; Shapiro, 1973); (c) modification of enzymes, cellular RNA and protein synthesis (Prashad et al., 1977, Lira and Mitsunobu, 1972; Waymire et al., 1978). For the most part tumor cell lines were used in these investigations and as to how these data reflect normal patterns of development remains to be seen. 3.2.10. C o n c l u s i o n s Development of receptor mediated responses does not follow any uniform pattern within individual brain regions of a particular species. However, several different types of developing neurohumoral-sensitive adenylate systems have been described which at times correlate with receptor ligand binding data, as well as, other parameters of monoamine metabolism. These types are typified by the following examples: (1) some brain regions exhibit highly sensitive receptors shortly after birth which disappear in the adult; (2) certain neurotransmitter responses display evidence of preinnervation supersensitivity; (3) specified receptors gradually develop to a maximal activity and then remain unchanged throughout the life of the animal; (4) where two or more hormones activate adenylate cyclase in the same tissue, their pattern of development is independent of one another; (5) receptor subtypes develop and age at independent rates even in the same brain region; and (6) one other receptor type has been described in peripheral organs in which during a critical period of embryonic development the hormonal activation suddenly "turns on" with marked sensitivity for 1-2 days and just as suddenly it becomes quiescent (Waterman et al., 1977). Moreover, receptors are highly sensitive to environmental stimuli. 3.3. IONICREQUIREMENTS The most important ion for adenylate cyclase activation is free Mg2+. The receptorcyclase complex possesses specific sites for free Mg2+. The ion promotes increases in both agonist affinity for the receptor and for the V.naxof the catalytic site of adenylate cyclase, without any change in the K m for the Mg2+-ATP substrate. Manganese or cobalt may substitute for Mg2+. The enzyme is inhibited by lead, mercury or copper presumably because of their binding capacities for cysteine residues on enzymes. Calcium plays an unusual role with regard to adenylate cyclase activation. In most preparations small concentrations stimulate while larger concentrations inhibit hormonal activation of the enzyme. In specific brain regions the cyclase may be activated by Ca2+-calmodulin interactions (for reviews see: Daly, 1977; Brostrom et al., 1978a,b; Cech et al., 1980). 3.4.

CATALYTIC SITE

Adenylate cyclase consists of at least three subcomponents. The receptor is activated by specific hormones, the response is magnified via a GTP-sensitive coupling (transducer) site and the catalytic site in the presence of Mg2+ completes the sequence of the reaction in which ATP is transformed into cyclic AMP. For a considerable time period it was thought that the metabolic poison, fluoride, was a specific activator of the catalytic site. Some of the findings in support of this contention were that high speed (100,000 × g) particulate fractions of brain were insensitive to catecholamines while fluoride evoked a huge stimulation of the enzyme. Moreover, receptor blocking agents did not influence fluoride activation of the enzyme (Robison et al., 1968; Weiss, 1969). However, as work progressed it was found that fluoride activation of adenylate eyclase did to some extent require GTP for full expression of activity (Abramowitz et al., 1979). In recent years the diterpene, forskolin, has been shown to be a highly potent activator of the catalytic site of adenylate cyclase, an event occurring in broken, as well as, whole cell preparations. Moreover, differences exist as to the mode of action of fluoride and forskolin which suggests that they do not act similarly on adenylate cyclase preparations (see: Seamon and Daly, 1981, for review).

PSYCHOACTIVEDRUGS, CYCLIC NUCLEOTIDESAND THE CNS

15

3.5. TRANSDUCER-GTP-SENSITIVESITE Hormone induced receptor activation is a complex process which requires first a chemical attraction of the hormone for the receptor. By a process of phospholipid methylation hormone/receptor interactions increase cell membrane fluidity and may orient the "free floating" receptor to achieve an interaction with the GTP-sensitive transducer (or regulatory site) (Hirata et al., 1979). The transducer site originally described by Rodbell (1980) apparently consists of at least two subunits, one of which promotes receptor binding to hormones and the other, a GTPase which limits hormonal receptor interactions. The GTPase produces a GDP which acts to deactivate the enzyme. Supposedly, a continuous occupancy of the receptor by the hormone and a continuous supply of GTP keeps the enzyme activated, thereby producing a steady supply of cyclic AMP. The active form of the receptor does not arise unless both hormone and nucleotide interact with it simultaneously. Agents resistant to GTPase action such as Gpp(NH)p (5'-guanylyl imidodiphosphate) do not generate GDP and thus the hormone receptor interaction remains in a permanently activated state. The only manner in which to deactivate this system would be competition with excess GTP. Agents like cholera toxin activate adenylate cyclase by inhibiting GTPase. Thus guanyl nucleotides have at least three actions: (1) they increase basal activity by increasing the affinity of Mg2+-ATP interaction at the catalytic site; (2) they accelerate the rate of hormone binding and release from its receptor, they actually decrease the affinity of agonists but not antagonists to receptors, this association with GTP promotes a more rapid hormone/receptor interaction; and (3) they promote coupling of free floating membrane receptors to adenylate cyclase (also see: Levitzki, 1978; Abramowitz et al., 1979; Pike and Lefkowitz, 1981). As will be evident in the following discussions, almost all hormonal/modulator interactions of adenylate cyclase require the presence of guanyl nucleotides for full expression of enzyme activity. The detailed experiments for GTP activation of adenylate cyclase have been principally conducted in peripheral tissues and it is possible that these processes could differ somewhat, albeit to a slight extent, in the brain.

3.6.

ACTIVATION OF ADENYLATE CYCLASE

3.6.1. Adrenergic agents 3.6.1.1. Alpha adrenergic receptors Recent reviews have discussed the localization and possible functions of alpha receptors in both brain and peripheral tissues (Hoffman and Lefkowitz, 1980; Exton, 1982). In simplest terms the alpha~ receptor is thought to be located at postsynaptic sites, stimulated selectively by alpha-methylnorepinephrine, and blocked rather specifically by prazosin. Stimulation of alpha1 receptors alters Ca 2+ flux, increases cell membrane turnover of phosphadityl inositol and perhaps elicits a calmodulin-Ca 2+ dependent activation of a protein kinase. The alpha2 receptor is postulated to be presynaptic, antagonized by the hallucinogen, yohimbine, and is activated by the popular antihypertensive agent, clonidine. Alpha2 stimulation mediates a feedback inhibition of NE release. Alpha2 receptors may likewise be postsynaptic and in both locations are thought to diminish cyclic AMP levels via a GTP dependent process. In central tissue, alpha2 reduction in cyclic AMP is best demonstrated in the neuroblastoma × glioma hybrid cell line where NE inhibits both basal and prostaglandin E 1 activation of adenylate cyclase (Sabol and Nirenberg, 1979; Kahn et al., 1982). In cultured glia cells alpha agonists partially block beta adrenergic, adenosine, prostaglandin E1 or cholera toxin-induced accumulations in cyclic AMP (McCarthy and DeVellis, 1978). Postsynaptic localizations of alpha2 receptors have been demonstrated in rat cortex as revealed by an enhanced binding of respective ligands following destruction of presynaptic NE endings with 6-hydroxydopamine (U'Prichard et al., 1979; Dausse et al., 1982).

1(3

G . C . PALMER

There are, however, many conflicting experiments which do not indicate that alpha receptors are either negatively coupled to adenylate cyclase or independent thereof. In early work Chasin and coworkers (1971), Palmer et al. (1973) and Perkins and Moore (1973b) demonstrated that alpha adrenergic antagonists were effective inhibitors of NE-elicited accumulations of cyclic AMP in tissue slices of rat brain. This work has been substantiated in a wide variety of central preparations and in addition, alpha agonists, e.g. methoxhmine, phenylephrine and clonidine were shown to elevate adenylate cyclase cyclic AMP (Sattin et al., 1975; Skolnick and Daly, 1975; Ahn et al., 1976: Schwabe and Daly, 1977; Jones and McKenna, 1980). On the other hand, independent investigators have been unable to show cyclic AMP stimulation with alpha agonists and in certain instances alpha agonists actually inhibit NE-elicited accumulations of cyclic AMP (Tsang and Lal, 1977; Robinson et al., 1978; Schultz and Kleefeld, 1979). In other work, various research groups provided evidence that alpha stimulation of cyclic AMP was associated with: (1) Ca e~ influx (Schwabe and Daly, 1977); (2) adenosine release (Sattin et al., 1975; Skolnick and Daly, 1975; Schultz and Kleefeld, 1979; Nimit et al., 1981); or (3) the presence of low concentrations of prostaglandin Ex (Partington et al., 1980). Jones and McKenna (1980) showed that in rat spinal cord alpha adrenergic stimulation of cyclic AMP was, however, unrelated to the action of adenosine. Furthermore, Ahn et al. (1976) felt that there were similarities between DA and alpha adrenergic activation of adenylate cyclase (see also review by Nathanson, 1977). The question of the role of the alpha receptor with regard to adenylate cyclase is indeed complex and remains to be resolved. Perhaps several conditions may exist depending upon the species, cell type, and brain region i.e. (1) alpha receptors coupled to adenosine; (2) coupled to Ca2+; (3) negatively coupled to adenylate cyclase; (4) positively coupled to adenylate cyclase; (5) acting independently of adenylate cyclase; and (6) coupled to cyclic GMP. 3.6.1.2. Beta adrenergic receptors Apparently two subtypes of beta receptor occur in nature. The beta~ type typically stimulates the heart, a selective agonist is dobutamine and a specific antagonist is practolol. The betaz subtype is commonly associated with bronchodilation, is activated by fenoterol and rather specifically blocked by butoxamine. Both types of receptors mediate adenylate cyclase activation in the brain, via a GTP dependent process. Brain studies have been hampered to some extent because of the lack of selective agonists capable of eliciting the full degree of activity as observed with NE, epinephrine or isoproterenol. An exception is fenoterol. Beta receptor binding is independent of adenylate cyclase activation. Both processes, however, require GTP (Hegstrand et al., 1979). The beta receptor and adenylate cyclase are products of separate genes (Insel et al., 1976). Activation of the beta receptor involves reactive sulfhydryl groups (Stadel and Lefkowitz, 1979) and is associated with an increased rate of methylation of membrane phospholipids with a concomitant increase in membrane fluidity. In the presence of ions and GTP perhaps this altered physical state of the cell membrane is necessary for coupling to adenylate cyclase (Hirata et al., 1979). A recent review by Minneman et al. (1981) thoroughly discusses the subtypes of beta receptors. Beta receptor activation of adenylate cyclase has been identified in all brain regions and cellular types. Some cells i.e. capillaries and pia-arachnoid contain predominantly beta~ type receptors (Palmer, 1980; Palmer and Palmer, 1983). Moreover, certain brain regions of the cat possess principally beta2 receptors e.g. the cerebellum, while in the cerebrum and the hippocampus the betal receptors are in the majority (Dolphin et al., 1979). In the rat cerebral cortex chronic tricyclic antidepressant treatment or administration of 6-hydroxydopamine leads to exclusive decreases and increases of the betai receptor. Thus the two receptor subtypes are independently regulated (Minneman etal., 1979), besides displaying differential patterns of development and maturation (Pittman et al., 1980b). While this introduction to beta receptors is rather brief the following subsections regarding psychoac-

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17

tive drugs will reveal the importance of these molecules in both normal and abnormal brain function. Moreover, the popular antihypertensive agent, propranolol exerts a significant portion of its therapeutic actions by blocking beta adrenergic receptors in the brain (Amer, 1977). 3.6.2. D o p a m i n e The importance of DA neurotransmitter/modulator systems with regard to control of mood and behavior cannot be overemphasized and will become apparent in many of the following sections. The initial work that led to a vast amount of scientific research as to the nature of and the role of the DA receptor was the discovery of a DA-sensitive adenylate cyclase in homogenates of rat caudate nucleus by Kebabian and co-workers (1972). Before that time investigators had been unsuccessful in their attempts to link DA action to adenylate cyclase. In earlier work incubated tissue slices or high speed particulate fractions from brain had been used. Why the DA-adenylate cyclase does not function unless the cells have undergone disruption remains a major unsolved puzzle. Only a limited number of experiments, chiefly with retina (Bucher and Schorderet, 1974) have consistently displayed a DA-sensitive enzyme in an intact cellular preparation. Dopamine responsive adenylate cyclase systems have been reported in most areas of the rat brain, namely the striatum (Kebabian et al., 1972), median eminence, olfactory tubercle, nucleus accumbens, amygdala (Clement-Cormier and Robison, 1977), substantia nigra (Gale et al., 1977), layers of frontal cortex (Krieger et al., 1979), other cortical areas, thalamus, hypothalamus, medulla (Leysen and Laduron, 1977), hippocampus (Chronister et al., 1980) and spinal cord (Gentleman et al., 1981). Many similar brain regions from other species likewise contain DA-sensitive adenylate cyclase (see Daly, 1977). Two brain regions which do not contain the enzyme are cerebellum (Leysen and Laduron, 1977) and the A-10 region containing the DA neurons projecting to the mesolimbic system (Lorez and Burkard, 1979). Most cell types in the brain (neurons, glia, choroid plexus and capillaries) have a DA-adenylate cyclase. Pia-arachnoid, however, does not appear to contain such DA coupled receptors (Palmer et al., 1976b; 1980c). Under conditions of cellular fractionation the highest activity of adenylate cyclase to DA in the rat striatum was associated with the crude synaptosome fraction. When the MI subsynaptosomal fraction was further separated by sucrose density gradient centrifugation, the fraction at the 1.0 M interface contained the highest activity (Clement-Cormier and George, 1979). One important discovery within the past five years has been the observation that not all DA receptors are coupled to activation of adenylate cyclase. A series of recent reviews have addressed this problem and as a consequence of receptor ligand analyses on brain particulate preparations the existence of multiple DA receptors has been reported. Depending upon the investigator these various receptor subtypes number from 2 (Kebabian and Calne, 1979; Schmidt, 1979) to 3 (Sokoloff et al., 1980) or 4 (Seeman, 1980). One problem with these types of investigations are the differential localizations of DA receptors in the brain, i.e. postsynaptic sites, presynaptic sites, synapses on incoming neuronal endings, receptors on glia, capillaries, neuronal perikarya, dendritic spines etc. All these specialized structures would be expected to differ slightly in their biochemical, as well as, functional properties. Insufficient studies have been performed on purified cell types within particular brain regions. One general agreement is that the receptor sites for DA-sensitive adenylate cyclase are not characteristic of high affinitysites. What postsynaptic functional roles these DA receptors play with regard to cellular physiological events is not clear at this time. 3.6.3. Histamine In their salient work Kakiuchi and Rail (1968) observed that following addition of histamine to tissue slices of rabbit cerebellum or cerebrum there was a marked production of cyclic AMP. The response was selectively blocked by antihistamines. The aftermath of

18

(}. ('. PALMER

this work was slow in developing but within a few years the discovery of HI and H2 receptors plus the fact that histamine was a likely candidate for a central neurotransmitter/ neuromodulator led to a considerable volume of research (for reviews see: Green and Hough, 1981; Schwartz et al., 1981b). Further work demonstrating that tricyclic antidepressants and neuroleptics were potent antihistamines provided a clinical link for these investigations (Sections 8 and 9). Histamine-sensitive adenylate cyclase systems have been described in all cellular elements (except pia) and several regions (cerebrum, cerebellum and nucleus accumbens) of the rabbit brain (Kakiuchi et al., 1968; Shimizu et al., 1970; Forn and Krishna, 1971; Spiker et al., 1976; Chronister et al., 1982). Other species displaying prominent histaminesensitive adenylate cyclases are: guinea pig cerebrum, hippocampus, amygdala, hypothatamus and striatum (Shimizu et al., 1970; Forn and Krishna, 1971 ; Chasin et al., 1973; Dismukes et al., 1976; Ahn and Makman, 1977; Psychoyos, 1978; Kanof and Greengard, 1979; Tuong et al., 1980; Coupet and Szuchs-Myers, 1981 ; Hough and Green, 1981); rat hippocampus, cortex and hypothalamus (Palmer and Palmer, 1978; Portaleone et al., 1978; Chronister et al., 1980; Tuong el al., 1980), chicken brain (Nahorski et al., 1977); but such an action in the mouse cerebrum is questionable (Forn and Krishna, 1971 ; Quach et al., 1980). Species apparently not possessing a histamine-responsive enzyme include the gerbil, hamster, cat and monkey (Forn and Krishna, 1971; Hough and Green, 1981). Considerable investigative efforts have not unraveled the controversy as to whether the histamine-sensitive adenylate cyclase system in the brain is coupled to H~, He or both receptors. Many such characterizations have been made on broken cell preparations, a procedure which may easily destroy receptor specificity. For example the histamine inhibition of high frequency hippocampal inputs to the nucleus accumbens of the rabbit is mediated exclusively via H 2 receptors. The adenylate cyclase in this tissue is equally activated by either H1 or H2 agonists (Chronister etal., 1982). The following is a partial list of receptor subtypes identified: (a) Guinea pig cerebrum and hippocampus--Hl, H2 (Dismukes et al., 1976; Psychoyos, 1978). (b) Guinea pig cerebrum--H1 (Coupet and Szuchs-Myers, 1981). (c) Guinea pig cerebrum and hippocampus--H2 is more prominent than Hi (Kanof and Greengard, 1979; Tuong et al., 1980). (d) Chick brain--H2 (Nahorski et al., 1977). (e) Rat hypothalamus--H~, H2 (Portaleone et al., 1978). (f) Mouse cerebrum--HL (Quach et al., 1980). (g) Rat cerebrum--H2; cerebral neurons--H~, H2 (Palmer and Palmer, 1978). (h) Rabbit microvessels and choroid plexus--H 1, H 2 (Palmer et al., 1980c). The activation of cyclic AMP by histamine in the guinea pig cerebrum does not require calcium. If adenosine levels were diminished by preincubating the tissue slices with adenosine deaminase, histamine was no longer effective. These data indicate that a histamine evoked adenosine release may be responsible for cyclic AMP synthesis (Schwabe et al., 1978). Quach et al. (1980) using the mouse cortex demonstrated that histamine-elicited glycolysis was mediated by an H~ receptor. Whether the glycolysis was firmly linked to cyclic AMP was not illustrated. For more detailed discussions see reviews by Green and Hough (1980) and Schwartz et al. (1981b). 3.6.4. Serotonin ( 5 - H T ) With the early employment of tissue slices 5-HT was generally shown not to be effective in the stimulation of cyclic AMP (Kakiuchi and Rall, 1968; Palmer et al., 1973; Ferrendelli et al., 1975). However, Daly and coworkers (see Daly, 1977, 1979) did report that this monoamine would potentiate or act synergistically with other agents, namely catecholamines, histamine and adenosine to promote cyclic AMP synthesis in tissue slices of guinea pig brain. Furthermore, ganglia from insects were shown to be selectively

PSYCHOACTIVE DRUGS, CYCLIC NUCLEOTIDES AND THE C N S

19

responsive to 5-HT and octopamine. The response to 5-HT was specifically blocked by 5-HT antagonists (see Nathanson, 1977). In other work, Von Hungen and coworkers (1974a, 1975a) demonstrated the presence of 5-HT-sensitive adenylate cyclase in broken cell preparations from newborn rat brain regions. The response declined as maturation occurred. The field remained inactive until recently when serotonin-sensitive adenylate cyclases were observed in cultured neuroblastoma (NCB-20 brain hybrid) (MacDermot et al., 1979), glia membranes from horse striatum (Fillion et al., 1980), homogenates from several immature rat brain regions (Enjalbert et al., 1978; Nelson et al., 1980), and synaptosomes from mature rat brain (Pagel et al., 1976). No such systems were evident in cerebral microvessels (Baca and Palmer, 1978). Binding and kinetic data revealed that the 5-HT receptor linked to adenylate cyclase was different than high affinity binding sites observed in rat brain. The enzyme did require GTP for full activity (Nelson et al., 1980). Moreover, prolonged exposure of the neuroblastoma cells did not develop a densitization of the 5-HT-adenylate cyclase (MacDermot et al., 1979). 3.6.5. Prostaglandins Prostaglandin-sensitive accumulation of cyclic AMP has been demonstrated in tissue slices from most areas of the rat brain (Dismukes and Daly, 1975a). The E series compounds, notably E1 are the most active. Intravenous injections of E prostaglandins elevated cyclic AMP content in rapidly fixed brain (see: Nathanson, 1977). Moreover, E prostaglandins and the I2 compound elevated cyclic AMP in neuroblastoma. Prolonged exposure to any of these agents produced "down regulation" to the remaining active prostaglandins (Howlet, 1982). In addition, prostaglandin El and E2 elicit cyclic AMP synthesis in cultured microvessels (Karnushina et al., 1982), glial cells (Ortmann and Perkins, 1977) and neuroblastoma x glioma hybrid cells (Moylan and Brooker, 1981). Incubation of cultured astrocytes with either dibutyryl cyclic AMP or prostaglandin El promotes both process elongation and induction of enzymes associated with GABA and glutamate metabolism (Tardy et al., 1981). Broken cellular preparations, on the other hand, do not always display a sensitivity to prostaglandins. In some brain regions stimulation occurs, in others cyclic AMP-coupled catecholamine responses are inhibited. Also the short lived intermediates from arachidonic acid i.e. thromboxanes and endoperoxides could contribute to the discrepant findings. Even though high affinity prostaglandin receptors have been identified, whether or not all are associated with adenylate cyclase activation remains to be seen. Prostaglandins have been shown to act presynaptically and depending on the brain region may either evoke or prevent release of monoamines. Iontophoretically applied prostaglandin El to cerebellar Purkinje cells prevents the NE-induced depressant action, but no effect on the cyclic AMP depressant action is observed (Hoffer et al., 1969). However, in the caudate prostaglandin, E~ potentiates the action of DA and cyclic AMP on cell firing. In some instances prostaglandin-cyclic AMP production is blocked by opiates. These diverse actions have indicated that prostaglandins may function as co-transmitters in the brain (for reviews see: Daly, 1977; Nathanson, 1977). 3.6.6. A d e n o s i n e The discovery that an adenosine-induced accumulation of cyclic AMP was present in tissue slices of mammalian brain is attributed to Sattin (1981, for review). At least three types of adenosine receptors have been subsequently identified in central tissue. The A 1 receptor possesses a high affinity for adenosine and acts to inhibit adenylate cyclase. A low affinity receptor for adenosine (A2) likewise acts on the external cell surface but stimulates adenylate cyclase. The methylxanthines inhibit both the A1 and A2 receptors while the third site (P-receptor) is located intracellularly and is not influenced by the methylxanthines. Both the A1 and A2 receptors are unequally distributed throughout the brain. Adenosine receptors are present in all types of brain cells, including microvessels. There are some problems with methodology when evaluating adenosine-sensitive adenylate

2{)

( i . ( ' . P,x.l MER

cyclases in preparations consisting of broken cells. Cellular disruption may further unmask inhibitory P sites. Preincubation with adenosine deaminase is necessary to reduce the endogenous levels of adenosine. Deaminase-resistant analogs of adenosine must be used to evoke enzyme activation. The analog so selected should possess minimal activity toward the Ai receptor. Lastly non-methylxanthine inhibitors of phosphodiesterase should bc used during incubation of the enzyme. In broken cells adenosine-elicited adenylate cyclase is not 6bserved in all brain regions, however, in tissue slice preparations adenosine is usually active. For example in the spinal cord adenosine-activated adenylate cyclase is thought to be weak, however, in whole cell preparations Jones (1981 ) recently showed that adenosine would produce a five-fold accumulation of cyclic AMP. Current evidence suggests that most adenosine receptors are located presynaptically. In this vein, iontophoretic application of adenosine produces a hyperpolarization with no change in membrane resistance. Therefore, it has been shown that adenosine interfers with presynaptic release of many transmitters, but has no effect on excitatory responses produced by acetylcholine or glutamate at post-synaptic loci. In other situations adenosine, added in combination with NE, histamine or 5-HT results in either a synergistic action or a potentiation of cyclic AMP production. In this context NE acts through an alpha receptor and histamine via an H~ response. Adenosine-adenylate cyclase additionally requires Mg 2+ and G T P for maximal activity. Even though the existence of purinergic nerve pathways have not been described, adenosine is released following nerve stimulation and displays many properties with respect to CNS function; (a) Behaviorally, adenosine is a CNS depressant and therefore the sedative properties of benzodiazepines might be due to their capacity to prolong adenosine action by preventing its reuptake. Moreover, the stimulant properties of methylxanthines are thought to be a result of blocking adenosine receptors. Methylxanthines, in addition, by mobilizing Ca 2+ block opiate-induced analgesia. The various receptors responsible for these actions have not been characterized with any degree of precision. (b) Electrophysiologically, adenosine reduces spontaneous neuronal activity and blocks trans-synaptic potentials at specified sites. (c) Adenosine is released during ischemia or anoxia and dilates cerebral blood vessels presumably to protect the region from reduced blood flow and oxygen. At this time cyclic AMP levels are raised. The headache observed during withdrawal from caffeine may be a result of an enhanced dilation of these blood vessels. (d) Adenosine acts as an anticonvulsant and both it and cyclic AMP levels are elevated after convulsions, a process thought to tone down the excitatory state of the brain. High doses of methylxanthines cause convulsions. Several recent reviews thoroughly discuss the vast amount of work performed on this subject by Daly (1979) and coworkers (1981), Phillis and Wu (1981a), W i n n e t al. (1981) and Premont et al. (1979). Also see Cardinali (1980) for a discussion on methylxanthines. 3.6.7. A m i n o acid transmitters Excitatory amino acids such as glutamate and aspartate enhance the accumulation of both cyclic AMP and cyclic G M P in tissue slices from different brain regions of various species. A requirement for calcium appears necessary for these responses to occur. In further work glycine produces a slight elevation in cyclic nucleotide content, G A B A and kainic acid appear to elevate only cyclic GMP. Structure-activity relationships revealed that 1-cysteine sulfinic acid was the most potent excitatory amino acid. The 'T' isomers of the amino acids were more powerful than "d" isomers. Glutamate topically applied to the rat cerebral cortex also augmented cyclic AMP levels as measured in rapidly fixed tissue. Unfortunately this model system with amino acid-induced accumulation of cyclic nucleotides has not yielded much data concerning the actions of psychoactive drugs on the CNS (Ferrendelli et al., 1974; Shimizu et al., 1975; Daly, 1977; Krivanek, 1977; Schmidt and Thornberry, 1978).

PSYCHOACTIVE DRUGS. CYCLIC NUCLEOTIDES AND THE C N S

2|

3.6.8. Depolarizing agents Either high levels of potassium or agents allowing an influx of sodium effectivelY increase the synthesis of cyclic AMP and cyclic GMP in incubated tissue slices of mammalian brain. A requirement for calcium has been demonstrated and these agents may additionally release adenosine. Agents evaluated in these whole cell preparations are Na +, K+-ATPase inhibitors (ouabain and cassaine), agents that increase sodium permeability of excitable membranes (veratridine and batrachotoxin), elevated levels of sodium and NH'j, and the calcium ionophore, A-23187. The latter only elevates cyclic GMP. The action of many of these agents is blocked by nonspecific membrane stabilizers like cocaine. Specific agents that block sodium permeability namely tetrodotoxin prevent the action of ouabain, veratridine and batrachotoxin. Both veratridine and K ÷ when applied topically to the rat cerebrum increase cyclic AMP in rapidly fixed tissue. The use of depolarizing agents has proved of value in studying the effects of antiepileptic drugs on central cyclic nucleotide systems (Shimizu et al., 1970; Shimizu and Daly, 1972; Huang et al., 1972; Zanella and Rall, 1973; Ferrendelli et al., 1976; Krivanek, 1978; Yamamoto et al., 1978; Jones, 1982). 3.6.9. Electrical stimulation Kakiuchi et al. (1969) first noted an ll-fold increase in cyclic AMP levels in tissue slices of guinea pig cerebral cortex in response to a sequence of applied electrical pulses. Cyclic AMP elevation was a function of the duration of electrical stimulation. The effect was prevented by prior incubation with theophylline indicating that electrically-induced release of adenosine was the responsible putative transmitter. Prior treatment of the animals with reserpine in order to reduce NE levels did not decrease the effectiveness of electrical pulses. However, when histamine or NE were included in the incubation medium the action of the electrical pulses was enhanced. In further work the phenomenon was described for cerebellar tissue and the action of electrical pulses was not dependent on Ca 2+ or M f + (Zanella and Rall, 1973). Electroconvulsive shock readily induces the formation of both cyclic AMP and cyclic GMP as determined in rapidly inactivated brain tissue (see Sections 9 and 16). Electrical stimulation of the rat locus coeruleus rapidly produces an increase in cyclic AMP in rat Purkinje cells (Siggins et al., 1973) and in frontal cortex, striatum, hypothalamus and hippocampus (Korf and Sebens, 1979; Korf et al., 1979). Pretreatment of the rats with reserpine prevents the increased cyclic AMP in the cortex suggesting that the action is dependent upon physiological release of monoamines. Furthermore, the action of electrical stimulation in the cortex was mediated by both alpha and beta adrenergic receptors. McAfee and coworkers (1971, 1980) demonstrated an enhanced accumulation of cyclic AMP in rabbit and rat superior cervical ganglia following presynaptic stimulation using physiological pulses. Ganglia not containing nerve cell bodies (nodose ganglion) or postganglionic stimulation of the superior cervical ganglion failed to increase cyclic AMP. The electrical action was blocked by muscarinic antagonists. Subsequent work revealed that muscarinic stimulation released DA from small intensely fluorescent interneurons and DA evoked the cyclic AMP accumulation. Work attempting to relate these effects to changes in post synaptic potentials led to considerable Controversy (Phillis, 1977; Busis et al., 1978; McAffee et al., 1980). Nevertheless these important findings stimulated further work in the field. In the bullfrog sympathetic ganglion, electrical stimulation caused both cyclic AMP and cyclic GMP levels to rise (Busis et al., 1978). Preganglionic stimulation of the guinea pig superior cervical ganglion likewise evoked an increase in cyclic GMP via a muscarinic mediated receptor (Wamsley et al., 1979). 3.6.10. Steroids Brostrom et al. (1974) observed an augmented sensitivity of basal, fluoride and NE-adenylate cyclase in cultured glioma celia following preincubation with glucocor-

22

(i. C, PALMER

ticoids. In later work incubation of human astrocytoma cells with hydrocortisone, corticosterone or dexamethasone led to an elevated rate of conversion of labelled adenine to cyclic AMP under either basal or prostaglandin Et stimulated conditions. This effect was prevented in the presence of progesterone, testosterone or the protein synthesis inhibitor, cycloheximide. The glucocorticoids did not alter the normal astrocytoma response to isoproterenol (Foster and Perkins, 1977). In addition, Foster and Harden (1980) showed that preincubation of human astrocytoma with dexamethasone increased basal, GTP. fluoride and isoproterenol-elicited adenylate cyclase, as well as the density of beta receptors. Since cycloheximide again prevented the response these workers postulated that glucocorticoids evoke a synthesis of a protein that modified the sensitivity of adenylate cyclase. However, when rats were adrenalectomized and NE pathways lesioned, an elevated beta receptor number was observed in the hippocampus (Roberts and Bloom, 1981). Moreover, adrenalectomized rats also displayed an enhanced sensitivity of NEcyclic AMP in tissue slices of forebrain (Mobley and Sulser, 1980). In the latter two situations the observed phenomena were reversed by corticosterone treatment. Thus glucocorticoids play undetermined roles in the modulation of central adenylate cyclasc. Most likely their inhibitory actions on phosphodiesterase can be ruled out because of the huge doses needed to block this enzyme (Weinryb et al., 1972). The sex hormones have also been demonstrated to influence the activity of central catecholamine receptors and related cyclic nucleotide actions. A few such studies will be mentioned here. Two laboratories have shown that incubation of hypothalamic tissue with estrogens promotes an increase in cyclic AMP. Also acute administration of estrogen to intact rats elevates the hypothalmic content of the cyclic nucleotide. These actions are blocked by both alpha and beta blockers, as well as, catechol estrogens (incubated tissue only). Estrogen might act to increase stores of catecholamines or facilitate their action (Gunaga and Menon, 1973; Gunaga et al., 1974; Paul and Skolnick, 1977). If rats were ovariectomized and treated chronically with 17 alpha-ethynylestradiol, beta adrenergic responses were diminished in the cerebral cortex. When cycling female rats were compared t o age-matched males the ability of isoproterenol to elevate cerebral cyclic AMP was reduced along with the number of beta receptors. These attenuated responses were eliminated with ovariectomy (Wagner et al., 1979a; Wagner and Davis, 1980). Long term ovariectomy of rats resulted in attenuated DA adenylate cyclase in the striatum and nucleus accumbens (Kumakura et al., 1979). Estrogen given to male rats (6 days) caused an increase in the number of striatal DA receptors and stereotyped behavior. This action was prevented by hypophysectomy (Hruska et al., 1980). On the other hand, when rats TABLE 2. A ( ' I ION OF PEHIDES ON CYCLIC NUCI.EOTIDES IN FHE BRAIN

Hormone

Action

Vasoactive intestinal peptide (VIP)

Stimulates adenylate cyclase and cyclic AMP

Somatostatin

Decrease cyclic AMP, increase cyclic GMP Increase regional cyclic AMP Increase cyclic AMP Increase cyclic A M P and neurite extension Increase adenylate cyclase

Secretin Substance P Glucagon ACTH MSH release inhibiting factor

Increase cyclic AMP Increase cyclic AMP Increase cyclic GMP

Tissue

References

Synaptosomes Brain regions Cultures Retina Brain slices IV* Cultures, retina Neuroblastoma Particulate fractions Retina IV Synaptosomes

2 7 1() 5,8 1 4 5,10 6 3 5,8 I1 x)

* IV - intraventricular administration. References: (1) Catalan et al., 1979; (2) Deschodt-t,anckman et al.. 1977; (3) Duffy and Powell. 1975; (4) Herchl et al., 1977); (5) Longshore and Makman, 1981; (6) Narumi and Maki, 1978; (7) Quik et al., 1978; (8) Schorderet e t a l . , 1981; (9) Spirtes et al., 1980, (10) Van Calker et al., 1980; (11) Wiegant e t a l . , 1981.

PSYCHOACTIVEDRUGS, CYCLICNUCLEOTIDESANDTHE CNS

23

were withdrawn from chronic haloperidol, administration of estrogen led to a "down" regulation of the usual increase in striatal DA receptors and stereotyped behaviors (Fields and Gordon, 1982). The actions of sex hormones in the brain are a complex process affecting many pathways. Stevens (1979) discussed the central DA suppressive action of prolactin, estrogen and leuteinizing hormone with regard to the temporal relationships often found between adolescence and schizophrenia. She suggested that the surge of hypothalamic peptides and gonadal steroids at puberty must be accompanied by appropriate "feed back" adjustments of DA mechanisms in both the mesolimbic and tuberoinfundibular system. Failure to do so would result in behavioral pathology. 3.6.11. Peptides

The field of brain peptide neurotransmitters/neuromodulators is rapidly expanding. A review by Snyder (1980) discusses many facets of current investigative efforts. The opiates are also presented in this article, as well as, in Section 15. Many of these peptides do influence cyclic nucleotide accumulation in the brain. A partial list of compounds is evident in Table 2.

4. The Phosphodiesterases and Calmodulin

The topics of phosphodiesterases in the brain as well as the roles of calmodulin have been reviewed in depth elsewhere (Daly, 1977; Kebabian, 1977; Strada and Thompson, 1978; Klee et al., 1980; Roufogalis, 1980; Weiss et al., 1980; Brostrom and Wolff, 1981 ; Palmer, 1981a; Vincenzi, 1981; Cheung, 1982). Therefore, this section will only provide a brief overview of the subject. One area that will be examined in greater depth will be the developmental and aging profiles of phosphodiesterase. The levels of cyclic nucleotides are controlled within specific microenvironments inside individual brain cells by several mechanisms: (1) active or passive extrusion of cyclic nucleotide into the CSF; (2) desensitization of receptors; (3) presence of calmodulin inhibitory proteins; (4) the presence of an opposing cyclic nucleotide; and (5) the phosphodiesterases. 4.1.

DISTRIBUTION AND LOCALIZATION

As would be expected, phosphodiesterases have been observed both histochemically and biochemically to occur in all cell types and many associated organelles within the CNS to include: neurons, glia, capillaries, pia, synaptic vesicles, microfilaments and choroid plexus. Greatest enzyme activity is found at pre- and postjunctional synaptic sites. The distribution of phosphodiesterase does not coincide with the density of monoamine innervation within the brain. Gray matter-rich areas of the brain contain higher activities of phosphodiesterase than areas with large amounts of white matter such as spinal cord, medulla-pons and cerebellum. Substantial levels of enzyme actvity are thus found in the cerebral cortex, striatum, hippocampus and thalamus. 4.2.

DEVELOPMENT AND AGING

Three reviews have discussed the role of phosphodiesterase with regard to its appearance, maturation and aging in the brain (Weiss and Strada, 1973; Schmidt et al., 1980a; Palmer, 1981). The usual problems are apparent in that many investigators begin their developmental studies only at birth and then compare newborn data to that of the adult. Usually insufficient data points are collected to determine with any degree of precision the time of peak activity and or influences of aging. Moreover, the various enzyme subtypes are usually not evaluated nor are the different brain regions and cellular types looked at. Postsynaptic localization of phosphodiesterase has been cytochemicallydetected along the developing dendrites of the molecular layer of the rat cerebrum (Adinolfi and Schmidt,

24

(~. ('.PAI MER

1974). Cumming and coworkers (1982) localized the presence of calmodulin in rat cerebellar Purkinje cells beginning at postnatal day 10. We observed the presence of high and low Km cyclic AMP phosphodiesterase in cerebral pia and capillaries between postnatal days 1 and 3. Moreover, the enzymes did not display any prominent change in activity at various time periods up to 8 weeks of age (Palmer and Palmer, 1983). Nevertheless, a body of information describing a variety of preparations and brain regions has indicated in rat and mice brains that cyclic AMP phosphodiesterase makes its appearance shortly after birth with a peak activity around 16 days which so remains throughout the life of the animal. The exceptions are the rat cerebellum in which enzyme activity is higher at birth, and in the striatum where the low K,,, enzyme declines with age (Weiss and Strada, 1973; Strada et at., 1974; Purl and Volicer, 19771. Table 3 is a collection of data from a variety of sources and summarizes these findings. Calmodulin was measured in three rat brain regions. The cerebellum at birth contained considerably lower levels of calmodulin than did the brain stem and cerebral cortex. The activator from all three regions was fully functional and readily stimulated the "Peak II" species of high Km cyclic AMP dependent phosphodiesterase isolated from rat cerebrum (Strada et al., 1974). The low calmodulin content in the newborn cerebellum was substantiated by the work of Cumming et al. (1982). Using immunofluorescence techniques calmodulin was not detected in Purkinje cells until day 10 after birth. The appearance of calmodulin, as well as, the various forms of phosphodiesterase neither correlated with the onset of neurohumoral receptors nor with the receptor activation of adenylate cyclase (see Section 3.2). Kinetic profiles of different regions of the rat brain revealed that the postnatal elevation in cerebral phosphodiesterase activity was due to an elevation in the high K,,,-cyclic AMP dependent form. When the various forms of phosphodiesterase were isolated by poly-

TABLE 3. CYCI,IC NUCLEOT1DE PHOSPHOD1ESTERASES DURING DEVELOPMENTAND AGING

Time (day) of appearance

Species Rat

Preparation--region Whole brain homogenate Whole brain homogenate Nerve ending membranes Cerebral homogenate Cerebrum (low K,,,) Cerebral capillaries and pia

First appear 0 I() 10 3 11 (I

St r i a t u m * ( l o w K,,,)

Mice

Guinea pig Human

Cerebellum (low Kin) Cerebellum (calmodulin) Brain stein (low K,~I) Olfactory bulb Olfactory bulb (low K,,,) Cerebrum Retina inner layer low K m Retina outer segments Hypothalamus Whole brain low K,. low K,n cyclic G M P Cerebrum-soluble Cerebrum particulate Cerebrum soluble cyclic G M P Cerebrum particulate cyclic GMP

2 I[) 2 7 1 1-6 1 6 1~5 - 7 -7 14-2(/ fetal weeks weeks

Peak activity 23 17(1 III to 0 20-170 21 day-2 yrs 16 day-2 yrs 0-adult

Decline

5.1 I Ill

None None

4 to 30 too.

311 mo.

2 10 25~6(1 1('~65 7 35 35 15 2(140 20-40 20 - 7 to 300

30

300 young adult adult adult

Authors

5 2,8.15 13 15 9 1(|

13,14 3 1~. 14 2 2 1.12 12 12 1? 4 4 7,14 7 7 7

11= day of birth, all ages are given in days unless otherwise noted. All activity is high K,, cyclic A M P phosphodiesterase and "'low K,,I'" is also cyclic A M P phosphodiesterase unless otherwise denoted. References: (11 Adinolfi and Schmidt, 1974; (2) Cousin and Davrainville, 19811b: (3) C u m m i n g et al., 1982; (4) Davis and Kuo, 1976; (5) Gaballah and Popoff, 1971; (6) Hommes and Beere, 197(I; (7) Kang, 1977: (8) K a u f f m a n n et al.. 1972: (9) Palmer and Palmer, 1983; (lll) Purl and Volicer, 1977; (11) Schmidt e t al., 1970; (12) Schmidt and Lolley. 1973; (13) Strada etal.. 1974; (141 Weiss and Strada, 1973: (15) Zimmerman and Berg, 1975.

PSYCHOACTIVEDRUGS. CYCLICNUCLEOTIDESAND THE CNS

25

acrylamide gel electrophoresis from newborn rat cerebellum, the five peaks of enzyme activity corresponded to that observed in the adult. In similar studies with the cerebrum "Peak I" of phosphodiesterase was not detected in the newborn and "Peak II" (calmodulin activated enzyme) though present, was considerably less than in the adult (Weiss and Strada, 1973; Strada et al., 1974). In the human brain the mature cortex, as compared to a 14 week fetal enzyme, had 10 and 15-20 times the activity for respective hydrolysis of cyclic AMP and cyclic GMP. In the fetus the enzyme was principally associated with soluble preparations whereas, with maturity a shift to the particulate fraction was noted in the high Km cyclic GMP activity and in the low Km activities for both nucleotides (Kang, 1977). It might be that the fetal membrane systems are labile with regard to their ability to bind tightly the enzymes usually associated with the particulate fractions. Therefore centrifugation procedures would readily disrupt these cell membranes. However, these phenomena could represent a latent development of this species of particulate phosphodiesterase. Phosphodiesterases are subject to certain types of control mechanisms during ontogeny. Mothers of newborn rats were fed for 3 weeks on an isocaloric diet containing 8% casein as compared to the control diet (18.5%) protein. At 21 days the undernourished litters were provided with the control diet. Up to 21 days, but not at 35 days postpartum, high Km cyclic A M P phosphodiesterase was significantly less in the cerebral cortex of the growthrestricted animals (Cousin and Davrainville, 1980b). In contrast, Kauffman and coworkers (1972) did not find any changes in cerebral phosphodiesterase activity when rats were raised under conditions of undernourishment (less milk intake). Genetically abnormal C3H mice as compared to control D B A strain have a similar developmental pattern of retinal phosphodiesterase from birth to about 6 days postpartum. At this time period the high K m enzyme makes its appearance and the normal enzyme activity rises 8-10 fold. The C3H mice, however, do not develop the high Km enzyme. The high Km enzyme coincides with the differentiation and growth of photoreceptor outer segments. In the C3H mice this layer undergoes degeneration. In other brain regions, hypothalamus and cortex there were no differences in C3H and control phosphodiesterases (Schmidt and Lolley, 1973). 4.3.

PROPERTIES AND ENZYME SUBTYPES

In the earlier work it was thought that only one form of cyclic nucleotide phosphodiesterase activity was responsible for controlling the metabolism of cyclic AMP and GMP. The K m for total phosphodiesterase activity was however, considerably greater than the tissue levels of cyclic AMP. With further detailed kinetic investigations using LineweaverBurk plots the profiles of enzyme activity were at times nonlinear. These findings suggested the existence of different enzyme forms or one enzyme displaying cooperative kinetic activity. Some further work in peripheral tissue has indicated a form(s) of phosphodiesterase capable of interconverting between a low cyclic nucleotide affinity state (high Km form capable of metabolizing the nucleotide when intracellular concentrations are excessive) to a high affinity state (low K mform acting to maintain physiological levels of the cyclic nucleotides). Additionally the enzyme may contain distinct sites for both cyclic nucleotides (Strada et al., 1981). The extensive employment of several biochemical isolation techniques has revealed the existence of several forms of phosphodiesterases each subject to different Km rates for either cyclic AMP or cyclic GMP, varying sensitivities to inhibitors and activators, pH, heat, and ionic requirements, as well as, cellular and subcellular Iocalizations within a specific brain region. In certain brain regions as many as four to five separate enzyme forms have been identified (Uzunov et al., 1978). In addition, the steady-state levels of one nucleotide will inhibit or increase the activity of the enzyme subtype responsible for metabolism of the other cyclic nucleotide. Moreover, the stimulation of cyclic A M P dependent enzymes by calcium and calmodulin are not constant events in specific cell types within a particular brain region. In some situations cyclic GMP dependent enzymes are preferentially activated by calmodulin. Usually, however, the

26

G . C . PALMER

calmodulin-activated species of phosphodiesterase is a high Km soluble variant of the enzyme. 4.4. CALMODULIN

The current, popular topic of calmodulin has been exhaustively reviewed. The small mol. wt molecule (16,700), found ubiquitously in eukaryotic cells, is heat stable, acidic in nature, globular in form until it bonds 4 calcium ions and then it assumes a helical state. A host of molecular actions has been associated with calmodulin: (1) activation of high K m cyclic AMP, low Km cyclic AMP and low K,n cyclic GMP dependent phosphodiesterases; (2) control of adenylate cyclase activation by neurohumoral agents; (3) activation of cyclic AMP-independent protein kinase-phosphorylation of specific presynaptic proteins which control release of neurotransmitters and vesicular-presynaptic membrane interactions; (4) it promotes phosphorylation of tubulin, and disassembly of microtubules; (5) it induces fast axoplasmic transport; (6) regulation of Ca 2+, Mg2÷-ATPase consonant with cellular extrusion of calcium; (7) activation of phospholipase A inducing a release of arachidonic acid; (8) it stimulates glycogen metabolism; (9) control of contractile processes; (10) synthesis of neurotransmitters; (11) binding to the postsynaptic protein, calcineurin whose function is undetermined; and (12) activation of NAD + kinase. In addition there are present in the brain certain proteins that bind to and inhibit the action of calmodulin. Calmodulin action has been shown to be influenced by certain psychoactive drugs, namely the phenothiazines, phenytoin, diazepam and possibly opiates (also see DeLorenzo, 1981). 4.5.

REGULATION OF PHOSPHODIESTERASES

Cyclic nucleotide phosphodiesterases are inhibited by a variety of agents. The methylxanthines are the most extensively studied group of compounds, however, large concentrations are required and current thinking indicates that their inhibition of adenosine receptors best describes their primary site of action. Other inhibitors that have been identified are papaverine, benzodiazepines, neuroleptics, glucocorticoids, vinblastine, potassium, and apomorphine. Activators of phosphodiesterase are: imidazole, vitamin E derivatives, persistently elevated levels of cyclic AMP, oleic acid, phosphatidyl-inositol, lysophosphatidyl choline, phospholipase C, Triton X-100, brain gangliosides, and light (retina) (see Palmer, 1981). Fell (1980) has postulated a sequence of events to explain some of the interactions of cyclic AMP phosphodiesterase. He feels that the high K m enzyme is present in large amounts in the cytosol and mainly acts to control the level of cyclic AMP inside the cell. The low Km form is principally localized in the plasma membrane and is readily accessible to exert a more exacting control over cyclic AMP formed at this critical loci as a consequence of neurohumoral activation.

5. Guanylate Cyclase-Cyclic GMP 5 . 1 . DISTRIBUTION

All types of cells within the brain contain guanylate cyclase and cyclic GMP as measured either by biochemical analysis or immunofluorescence techniques. Thus neurons, synaptic complexes, glia, capillaries, pia and choroid plexus possess the components of the guanylate cyclase system, but the function of cyclic GMP in the brain remains an enigma. One obvious reason for the limited information on cyclic GMP has been the mystique associated with DA and NE receptor events coupled to cyclic AMP and their possible correlation to mental disorders. Therefore the major thrust of research has been conducted upon these latter systems. Cyclic GMP is normally found in brain regions at concentrations which are an order of magnitude below that of cyclic AMP. The cerebellum and the retina are two exceptions and contain the highest levels of cyclic GMP in the CNS. Moreover,

PSYCHOACTIVEDRUGS, CYCLICNUCLEOTIDESAND THE CNS

27

levels in the retina are 10 fold greater than the cerebellum. On the other hand, activities of guanylate cyclase do not vary to any great degree among the various regions of rodent brain. The striatum and nucleus accumbens do have the greatest activity. When various cells were isolated from the rat cerebral cortex relatively low guanylate cyclase activities were observed in perikarya, glia and capillaries, while the pial activity was similar to the cerebral cortical homogenates. Procedures for cellular isolation may have damaged critical cellular structures and in addition, endogenous activators, ions, cofactors etc. could have leached out of cells (Gordis et al., 1974; Kebabian et al., 1975b; Rubin and Ferrendelli, 1976; Cumming et al., 1977, 1979; Hofman e t a l . , 1977; Cam etal., 1978; Greenberg etal., 1978; Chan-Palay and Palay, 1979; Karnushina et al., 1980; Ariano et al., 1981, 1982; Palmer, 1981c; Zwiller et al., 1981a; for review see: Ferrendelli, 1978). 5 . 2 . DEVELOPMENT AND AGING

Few investigative efforts have been directed toward understanding the influence of development and aging mechanisms on guanylate cyclase-cyclic GMP systems in the brain. Using homogenates of rat cerebellum Spano and colleagues (1975) reported a sharp elevation in guanylate cyclase activity at postnatal day 6, a peak in activity was reached at day 10 with a decline to adult activity by day 20. Endogenous cerebellar cyclic GMP content could not be detected until day 12. When tissue slices of rat cerebellum were stimulated by kainic acid there was a progressive decline in the ability of the neurotoxin to stimulate cyclic GMP during aging i.e. 3-24 months (Schmidt and Thornberry, 1978). Steady-state levels of cyclic GMP declined with age in the rat cerebral cortex, striatum, cerebellum and hypothalamus (Puri and Volicer, 1981). As to whether this observation reflects a change in guanylate cyclase or a deficiency of neurotransmitter-Ca2+ coupled processes is unknown. In studies with mice who possessed inherited genetic disorders e.g. homozygote "wobbler" and "stagger" mice the cerebellar content of cyclic GMP was drastically reduced when compared to sex and age-matched clinically unaffected controls. In addition guanylate cyclase activity in stagger mice was less at all stages of development (first appearance at postnatal day 5 with a gradual increase to a maximum at day 25) (Brooks et al., 1978; Spinka et al., 1980). 5.3. PROPERTIES AND ENZYME SUBTYPES

Recent reviews have discussed in depth the properties of guanylate cyclase in the brain and other tissues. Current thinking supports the idea that two forms of guanylate cyclase exist in most central and peripheral tissues. Basically soluble enzymes have greater specific activities than particulate enzymes. In addition, different substances are thought to stimulate selectively only one enzyme form. However, the processes of high speed centrifugation and partial purification make many of these suppositions tenuous. For example, loosely bound particulate forms could be shorn free from their membrane sites by the processes of homogenization and centrifugation. In addition many intracellular cofactors, activators, ions or even inhibitors could be removed from particulate sites during solubilization. These reasons may partially explain the inconsistencies among the various laboratories. Recently, however, monoclonal antibodies have been prepared for the soluble enzyme from lung (Lewicki et al., 1980). Hopefully future work along these lines of investigation in the CNS will resolve many of the problems listed. Manganese is the principal ionic cofactor for guanylate cyclase activation, however, calcium may substitute if the content of Mn 2+ is lowered (Olson et al., 1976). In addition, Mg2+ has been identified as an important cofactor, especially for enzyme activation by nitroso compounds (Ignarro et al., 1981). A wide variety of agents stimulate guanylate cyclase the most potent being agents that in the presence of vital cofactors (hematin, catalase, heme compounds, and various nitroso derivatives) generate NO or free radicals. In addition, various long chain unsaturated fatty acids, catecholamines and solubilization procedures activate the enzyme (see Table 4 for partial list; Daly, 1977; Ferrendelli, 1978; Murad et al., 1978).

( i . ( ' . P~[Mll~

2~

TABIF 4. AGFNIS INFI UFN('INGGUAN'~IAll: ()h( I,XSI [N ItH ('NS Agent

Response

Cigarette smoke NH~OH NaN3 Nitroso compounds Superoxide dismutase Chelating agents Ca -'+ with low Mn ~+ Mg :+ Endogenous activators Cyclic AMP protein kinase Somatostatin Catecholamines Lubrol PX Biotin Gila monster venoms Macrophage migration inhibiting factor Arachidonic, Linolenic Linoleic, Oleic, Palmitoleic and Myristoleic acids Arachidonic acid

Preparation

increase mcreasc increase increase increase mcrease increase increase increase inhibit mcrease inhibit increase mcrease increase increase decrease increase

cortex, cerebellum synaptosomes, cortex, neurons synaptosomes, cortex, neurons cerebellum soluble synaptosomcs human caudatc all brain cell types neuroblastoma, brain neuroblastoma cerebellum cortex human caudate synaptosomes cerebellum cerebellum 5 7 day-aged brain 14 day-aged brain cortex or soluble

increase inhibit

enzymes--whole brain soluble enzymes

Rclerc neck, 2 4.0.11.14, 15 4.9,11,14,15 8,20,22 21 ~ 12-10 1.8 I 10

17 5 11 18 19 7

7

3,22 22

References: (1) Amano et ell., 1979; (2) Arnold et al.. 1977; (3) Asakawa et al., 1978; (4) Deguchi, 1977; (51 Frey etal., 1980; (6) Frey e t a l . , 1981; (7) Gerber etal., 1981; (8) Ignarro etul., 1981; (9) Kimura etal., 1975; (10) Kumakura et al., 1978; (11) Nakane and Deguchi, 1978; (12) Nakazawa et al., 1976; (13) Olson et al., 1976; (14) Palmer et al., 1981; (15) Palmer, 1981c; (16) Sulakhe et al., 1976; (17) Vesely, 1980; (18) Vesely, 1982a; (19) Vesely, 1982b; (20) Yoshikawa and Kuriyama, 1980a, (21) Yoshikawa and Kuriyama, 1980b: (22) Zwiller etal., 1981a.

5.4.

ACTIVATORS AND INHIBITORS OF CYCLIC G M P

Tables 5 and 6 contain lists of compounds or neurohumoral agents that influence the accumulation of cyclic GMP in either whole cell preparations or under in vivo conditions. Upon examination of these tables, it becomes readily evident that muscarinic agonists are highly active in eliciting synthesis of cyclic GMP. These agents are selectively inhibited by atropine. In some tissue preparations acetylcholine induces a rather potent response and in other brain regions an elevation of only 50% over control values is attained. Guanylate cyclase is probably not the site of the muscarinic receptor in the brain, but neuronal depolarization evoked by acetylcholine initiates calcium movement into the cell with concomitant activation of the enzyme. Calcium ions are also critical for cyclic GMP elevation by depolarizing agents. Norepinephrine elevates cyclic GMP in pineal and guinea pig brain slices by beta adrenergic mechanisms, but acts through an alpha receptor in neuronal × glioma hybrid cells (O'Dea and Zatz, 1976; Ohga and Daly, 1977aa,b; see Section 15). Under in vivo conditions agents eliciting seizure activity elevate cyclic GMP while conditions in which G A B A levels are enhanced decreases the steady-state levels of the cyclic nucleotide.

5.5.

REGULATION OF CYCIJC G M P

A wide variety of psychopharmacologicai agents produce alterations in cyclic GMP in the brain. These drugs are discussed in detail in the succeeding sections. Moreover, as will be presented, cyclic GMP measurements have been made in extracellular fluids of patients suffering from a wide variety of neurological symptoms. Perhaps the strongest case for a role for cyclic GMP is during seizure activity and conditions of ischemia. In addition, as revealed in Section 6, one of the major roles of cyclic GMP is to oppose or inhibit the action of cyclic AMP. Metabolic studies with cyclic GMP are lacking for the CNS. A few behavioral investigations indicated that steady-state levels of cyclic GMP rise in specified rodent brain regions (notably the Cerebellum) as a consequence of stressful situations,

PSYCHOACTIVEDRUGS,CYCLICNUCLEOTIDESANDTHECNS TABI.E 5. AGENTS THATELEVATECYCLICGMP

Agent Muscarinic Agonists Acetylcholine Carbamylcholine Pilocarpine Bethar~echol Methacholine Eserine Histamine Tetramethylammonium

29

IN WHOLECELLPREPARATIONS

Preparations

References

cortex, ganglia neuroblastoma, cortex, N-G hybrid, ganglia cortex, N-G hybrid, cerebellum cortex, ganglia, hippocampus, nerve cells cortex cortex nerve cell cultures N-G (Neuroblastoma-glioma) hybrid

10,13,17,20 2,8,17,20,21,23 8,13,17 3,10,13,20 13 17,18 20 8

neuroblastoma, cerebellum, cortex, nerve culture ne uroblastoma, cerebellum, cortex cerebellum, cortex ganglia (sympathetic) (superior cervical) cerebellum, primary nerve culture

1,5,19,21 5,19,21 5,15 23,24

N-Me-N'-Nitro-N-

cerebellum, primary nerve culture several brain regions several brain regions cortex, cerebellum several brain regions cortex, cerebellum neuroblastoma, cortex, cerebellum cortex, cerebellum glioma neuroblastoma

1 6,7,11 6,11 11 6,7,22 11 18,19,21 19 12 21

Nitrosoquanidine A23187 Ca 2+ Mn z+ Alpha Agonists Isoproterenol Prostaglandin E~ Adenosine Phosphatidic Acid

neuroblastoma cerebellum, cortex neuroblastoma cortex, cerebellum, glioma, pineal cortex, cerebellum, glioma, pineal ne uroblastoma, cerebellum, cortex cerebellum, cortex neuroblastoma

2,21 15 4 1,9,1 l, 12,14,15 11,12,14,15 15,21 15 16

Depolarizing Agents KCI Ouabain Veratridine Electrical Mast cell degranulating peptide Sea anemone toxin Glutamate Aspartate Glycine Kainic Acid GABA NaN3 NH~OH Nitroprusside

1

References: (1) Ahnert et al., 1979; (2) Bartfai et al., 1978; (3) Black et al., 1979; (4) EbFakahany and Richelson, 1980; (5) Ferrendelli et al., 1973; (6) Foster and Roberts, 1980; (7) Garthwaite et al., 1979; (8) Gullis et al., 1975; (9) Haidamous et al., 1980; (10) Kebabian et al., 1975a,b; (11) Kinscherf et al., 1976; (12) Kon and Breckenridge, 1979; (13) Lee etal., 1972; (14) O'Dea and Zatz, 1976; (15) Ohga and Daly, 1977a,b; (16) Ohsako and Deguchi, 1981 ; (17) Palmer and Duszynski, 1975; (18) Palmer et al., 1980a; (19) Palmer et al., 1981; (20) Richelson, 1978a; (21) Saito and Deguchi, 1979; (22) Schmidt and Thornberry, 1978; (23) Wamsley et al., 1979; (24) Weight et al., 1974.

physical s h a k i n g , cold, s w i m m i n g in ice w a t e r , f o r c e d r u n n i n g , fighting a n d h e a t ( M a o e t 1974; D i n n e n d a h l , 1975; F e r r e n d e l l i , 1978; M e y e r h o f f e t a l . , 1979). Cyclic G M P d i s p l a y s a c i r c a d i a n v a r i a t i o n in rat b r a i n , b e i n g h i g h e r at night ( d u r i n g a c t i v e - w a k e n i n g ) in the c e r e b e l l u m , c o r t e x a n d s t r i a t u m . C o n v e r s e l y h y p o t h a l a m i c v a l u e s w e r e h i g h e r d u r i n g d a y l i g h t ( C h o m a e t a l . , 1979). Cyclic G M P has b e e n a s s o c i a t e d with e x c i t a t o r y - c h o l i n e r g i c t r a n s m i s s i o n p r o c e s s e s in the brain. U s i n g i o n t o p h o r e t i c t e c h n i q u e s b o t h a c e t y l c h o l i n e a n d cyclic G M P d i s p l a y e d m a i n l y e x c i t a t o r y a c t i o n s w h e n a p p l i e d to the surface of rat c o r t i c a l - s p i n a l n e u r o n s . O n the o t h e r h a n d , a c e r t a i n p e r c e n t a g e of n e u r o n s w e r e d e p r e s s e d in r e s p o n s e to cyclic G M P . E l e c t r i c a l m e a s u r e m e n t s of n e u r o n s e x p o s e d to e i t h e r a g e n t have likewise b e e n s h o w n not to be r e l a t e d . V a r i o u s technical factors such as choice o f n e r v e p r e p a r a t i o n , resting rate of n e u r o n a l firing, as well as, the a n e s t h e s i a e m p l o y e d m a y all c o n t r i b u t e to the d i s c r e p a n t findings ( S t o n e e t a l . , 1975; K r n j e v i c e t a l . , 1976; for review see: Phillis, 1977). In o t h e r e l e c t r o p h y s i o l o g i c a l w o r k , s t i m u l a t i o n o f p r e g a n g l i o n i c fibers e l e v a t e d b o t h cyclic G M P a n d cyclic A M P in s y m p a t h e t i c ganglia. T h e a c t i o n was b l o c k e d by the m u s c a r i n i c a n t a g o n i s t , a t r o p i n e . M u s c a r i n i c agonists m i m i c k e d the action of s t i m u l a t i o n of cyclic G M P . E v i d e n c e has b e e n p r e s e n t e d to show t h a t the slow e x c i t a t o r y p o s t s y n a p t i c p o t e n t i a l al.,

30

(}. ( ' . PAl MFR

TABI.IE 6. AGENTS AFFbCTING ill t.'iuo LEVEl S OF C'~ct I( G M P ~N ~nF BRAIN

Agent

Rcsponse

Oxotremorine Arecoline Physostigmine Atropine, Kainic Acid Papaverine Soman Aminooxyacetate Pentylenetetrazol Isonicotinic Acid Electroconvulsive Shock Intracerebro-GABA Muscimol Baclofen Picrotoxin Cold Stress Int racerebro-Glutamate Isopropyl-bicyclic-Phos. 3-Acetylpyridine Apomorphine

incrcasc increase increase decrease decrease decrease increase decrease increase decrease increase decrease decrease decrease increase increase increase increase decrease increase

Preparation scvcral brain regions ccrebellum, cortex cortex (cerebellum~ecrease) mouse cerebellum (no change--rat cortex) cerebellum cerebellum cerebellum cerebellum several brain regions cerebellum, medulla, hypothalamus cerebellum, cortex cerebellum cerebellum cerebellum cerebellum cerebellum, medulla, hypothalamus cerebellum cerebellum cerebellum cerebellum

Rcfcwcncc 5.9.14 -1 4 10.14 2 11 10 In 6,7,14 12 11 S, 13 13 7 7 12 13 13 t3 1.3

References: (1) Biggio et al., 1977b; (2) Biggio et al., 1978a:(3) Breese et al., 1979, (4) Dinnendahl and Stock, 1975; (5) Ferrendelli et al., 19711; (6) Ferrendetli and Kinscherf, 1977a; (7) Gumulka et al., 1979a; (8) Kouyoymdjian et al., 1978; (9) Lenox et al., 1980; (10) Lundy and Magor, 1978; (11) Lust etal., 1976, 1981a (12) Mao etal., 1974; (13) Mansson, 1980; (t4) Rubin and Ferrendelli, 1977. g e n e r a t e d in ganglia by acetylcholine is mediated by cyclic G M P . Inconsistent data from different laboratories using similar, as well as, other techniques and species has m a d e such a simplistic role for cyclic G M P an unlikely mechanism concerning muscarinic transmission in sympathetic ganglia ( G r e e n g a r d , 1976; Phillis, 1977; Busis e t a l . , 1978; Libet, 1979).

6. Cyclic AMP/Cyclic GMP Interactions M a n y situations exist in both central and peripheral tissues in which one cyclic nucleotide appears to exert a controlling influence over the action of the other. In some cases the control is m e d i a t e d at the r e c e p t o r sites and in others the presence of a particular nucleotide m a y inhibit or e n h a n c e the phosphodiesterase-elicited metabolism of the other nucleotide. Yet in other situations modulators of cyclic G M P - p r o t e i n kinase exist which antagonize cyclic A M P - p r o t e i n kinase with ultimate influence on subsequent metabolic events. These conditions w h e r e b y either cyclic A M P and cyclic G M P seem to impose contrasting or even antagonistic influences over one a n o t h e r have been t e r m e d by G o l d b e r g and co-workers (1975) the " Y i n - Y a n g " hypothesis. T h e Y i n - Y a n g symbolizes a dualism b e t w e e n o p p o s i n g natural forces, but also takes into account that such an interaction m a y e v o k e a mutual synthetic process. This latter premise was recently d e m o n s t r a t e d in a culture system of neural retinal cells. In this case both cyclic nucleotides exhibited stimulatory effects on m a c r o m o l e c u l a r synthetic processes (Kalmus et a l . , 1982). A c c o r d i n g to G o l d b e r g , cyclic nucleotide interactions consist of two basic types of events. In the t y p e - A condition the process is facilitated by cyclic A M P and suppressed by cyclic G M P . T h e available data with central tissue indicates that this form m a y be m o r e prevalent in the CNS. In the type-B condition the response is p r o m o t e d by cyclic G M P and inhibited by cyclic A M P . P e r h a p s a type-C condition should be considered where the two nucleotides act in concert and a t y p e - D situation in which a particular nucleotide acts only m o n o directionally and passively reverts to the non-functional state once the facilitory signal is removed. In their excellent review G o l d b e r g et al. (1975) discuss several examples of the Y i n - Y a n g hypothesis with regard to the better studied peripheral tissues. In Table 7 are listed some of the experimental evidence for m o d u l a t i o n of cyclic A M P responses by cyclic G M P in the CNS. T h e experiments, m a n y of which e m p l o y widely

PSYCHOACTIVEDRUGS, CYCLICNUCLEOTIDESANDTHE CNS

31

TABLE7. CYCI.ICAMP/CYcLIC GMP INTERACTIONSIN CNS Agent

Interaction(s)

Muscarinic agonists, cGMP Muscarinic agonists

Inhibit catecholamine-, adenosine- or prostaglandin-elicitedcyclic AMP Inhibit adenylate cyclase Inhibit DA-adenylate cyclase

Oxotremorine

Decrease cAMP in vivo

Protein kinase modulator cGMP

Inhibits cAMP protein kinase Stimulates cGMP protein kinase Stimulates dephosphorylation of tubulin that was phosphorylated by cAMPprotein kinase Inhibits neuronal firing Excites neuronal firing Decreases aggressive behavior

NE, cAMP Ach, cGMP cAMP cGMP cGMP, carbachol

Increases aggressive behavior Stimulates phosphodiesterase hydrolysis of cAMP

Tissues

Reference

Glioma Cerebrum N-G hybrid Caudate SC-ganglion Cerebellum and cerebrum Guinea pig brain Rat brain tubulin

1,4 9 8 12,13 6 2

Pyramidal neurons, rat Rats and mice

11

Cerebrum

7 10

5 3

Abbreviations: cAMP and cGMP mean either dibutyryl cyclic AMP or cyclic AMP and dibutyryl cyclic GMP or cyclic GMP. N-G hybrid = neuroblastoma × glioma hybrid; SC = superior cervical; Ach = acetylcholine. References: (1) Bottenstein and De Vellis, 1978; (2) Ferrendelli etal., 1970; (3) Filburn etal., 1978; (4) Gross and Clark, 1977; (5) Kantak et al., 1981; (6) Kebabian etal., 1975a,b; (7) Kuo etal., 1978; (8) Nathanson etal., 1978; (9) Palmeretal., 1980a; (10) Sandoval and Cutrecasas, 1976; (11) Stone etal., 1975; (12) Tang and Cotzias, 1977a; (13) Walker and Walker, 1973b.

diverse techniques do indicate a type-A mechanism exists in the brain (Goldberg et al., 1975). In several situations, namely ischemia and convulsive episodes, cyclic AMP and cyclic GMP levels rise or fall out of phase with one another. For example following decapitation cyclic AMP levels in the cerebellum rapidly rise while cyclic GMP content drops (Lust and Passonneau, 1979; Lust et al., 1981a,b). In the following sections on drugs and the metabolic aspects of cyclic nucleotides (Section 7) the opposing actions of cyclic GMP/cyclic AMP will be evident. An especially interesting observation is that cyclic GMP appears to mediate analgesia in the CNS while cyclic AMP shortens or antagonizes the action of anesthetic agents (Cohn et al., 1974, 1978; see Section 17). The clinical relevance of many of these phenomena remain to be established.

7. Metabolic Actions of Cyclic Nucleotides 7.1.

PROTEIN KINASES

This topic has been widely reviewed and the following is only a cursory discussion. Much credit for the work in this field goes to the laboratory of P. Greengard and coworkers. In addition, other investigators have made important observations as to the site of action and the metabolic roles for cyclic nucleotides in the brain. The cyclic AMP induced activation of protein kinase(s) most likely represents a common pathway of most or all metabolic processes elicited by either cyclic AMP or cyclic GMP (for reviews see: Daly, 1977; Nathanson, 1977; Phillis, 1977; Greengard, 1978; Kometiani et al., 1978; Ehrlich, 1979; Williams, 1979). 7.1.1. Cyclic A M P and calcium-dependent forms Protein kinases are ubiquitous in the brain and are located at various intracellular loci in both neurons and glia (Cumming et al., 1981). Cyclic AMP-induced phosphorylation takes place at multiple intracellular sites, however, the highest densities of protein kinases are found associated with synaptic processes. Metabolic conditions influenced by protein kinase-elicited phosphorylation include: histone modification, ribosome activation,

32

G . C . PAl m~-r

glycogenolysis, structural protein alterations (myelin basic protein, tubulin, actomyosin. neurofilaments), Mg2+, Ca2+-ATPase activity, enzyme induction, visual processes. permeability of membranes to ions, transmitter synthesis and transmitter release. Protein kinase consists of a regulatory subunit (cyclic AMP binding protein) bound to a catalytic moiety. When cyclic AMP binds to the regulatory site of this inactive dimer the catalytic unit is released in an active state to phosphorylate specified substrate proteins. The latter in turn accept phosphate from ATP via protein kinase and phosphorylation of the substrate protein occurs at principally serine residues. Phosphorylation evokes a change in the charge on the substrate protein promoting either an activation or inhibition of its activity. Inactivation of the substrate protein is achieved by phosphoprotein phosphatases. Two types of protein kinases apparently exist in nature differing in the chemical compositions of their respective regulatory subunits. Cyclic AMP-dependent protein kinasc requires Mg2+ or Mn 2+ for activity while Ca 2+ inhibits the enzyme. Both cyclic AMP dependent and independent forms of protein kinase are known to exist in the CNS. Under proper conditions the dependent form may be activated 2(J-fold in the presence of the cyclic nucleotide. Neurohumoral agents, depolarizing substances or electrical stimulation under a variety of experimental conditions result in enhanced rates of phosphorylation of central tissues. Greengard and coworkers (see: Greengard, 1978) have presented evidence in which adrenergic transmission to superior cervical ganglion cells consonant with cyclic AMP activation of protein kinase is responsible for ionic permeability changes yielding the slow inhibitory action potentials observed. Thus, protein kinase activity may alter the fluid properties of neuronal membranes. Current investigations are attempting to define which central proteins are phosphorylated by either cyclic AMP-dependent or independent protein kinases. Much work has been directed toward separation of synaptic proteins using polyacrylamide gel electrophoresis. However, phosphorylated proteins from other intracellular sites have also been described. From several proteins isolated by separation procedures only a limited number are subject to protein kinase elicited phosphorylation. Independent investigative groups have utilized varying techniques and nomenclature to describe the various proteins phosphorylated by protein kinase. The following is a partial list of some of these proteins: (a) Protein I is unique to synaptic elements in the nervous system. It consists of two subunits Ia and Ib. Protein Ia is probably located postsynaptically, activated by neurohumoral or depolarizing agents, cyclic AMP and maybe calcium. It is thought to be associated with membrane permeability. Protein Ib is presynaptic, activated by Ca 2+caimodulin-protein kinase and may control transmitter release. Phosphorylation of these proteins is extremely rapid (Forn and Greengard, 1978; Greengard, 1978; Williams. 1979; Kennedy and Greengard, 1981). (b) Protein II is found in several tissues and might be a component of the regulatory subunit of protein kinase. Cyclic AMP apparently regulates dephosphorylation of this protein (Nathanson, 1977; Greengard, 1978; Williams, 1979). (c) Proteins DPH-M and DPH-L are thought to be located in synaptic vesicles, are activated by a Ca2+-calmodulin process and have been shown to regulate neurotransmitter release, an event inhibited by diazepam and phenytoin. These proteins are similar to alpha and beta tubulin (DeLorenzo, 1981). (d) Tubulin kinase is activated by a Ca2+-calmodulin kinase and the resultant effect is a modification of the physical properties of tubulin (DeLorenzo, 1981; DeLorenzo et al., 1981). (e) Proteins F and H are synaptic proteins in rat striatal membrane fractions. During chronic exposure to morphine their phosphorylation rate is decreased. Alternatively, one day after a learning experience the rate is increased (Ehrlieh, 1979). (f) Retinal proteins--Rhodopsin is phosphorylated after light absorption by a protein kinase in the outer membrane segments. This cyclic AMP-independent enzyme might control the light sensitivity of the rods as well as calcium permeability in these structures. Cyclic nucleotide dependent protein kinases have likewise been identified in the retina (Farber and Lolley, 1979).

PSYCHOACTIVEDRUGS, CYCLICNUCLEOTIDESAND THF CNS

33

7.1.2. Cyclic GMP-dependent Jbrms Cyclic GMP activation of protein kinase producing phosphorylation of histones has been observed in the cerebellum and cerebrum of rodent brain and the caudate of humans. Moreover, with advancing age the level of cyclic GMP dependent kinase increases. The role of this enzyme system in the brain is presently unknown, but the enzyme differs from the cyclic AMP dependent protein kinase in its requirements for ions, pH sensitivity and the presence of endogenous modulators. The latter stimulate the cyclic GMP-dependent enzyme and inhibit the cyclic AMP dependent enzyme (Kuo et al., 1978; Boehme et al., 1979).

7.2.

DEVELOPMENT AND AGING PROFILES OF PROTEIN KINASE

7.2.1. Cyclic G M P dependent form The developmental and aging aspects of cyclic GMP dependent protein kinases have been for the most part ignored by investigators in the field. In rat and guinea pig cerebellum the enzyme activity is absent at birth, appears before postnatal day 10, the activity peaks at about day 25 (only the rat was studied at this time) and remains unchanged in the adult (Kuo et al., t976; Bandle and Guidotti, 1979). The enzyme development in the rat cerebellum correlates with the appearance of Purkinje call dendrites and the synaptic connections made therein with the climbing and parallel fibers. However, no correlation can be made between ontogenesis of the enzyme between the two species because of the time differential in the gestational period of the two rodents. The rat takes 3 weeks and is born immature, while the guinea pig gestation at about 60 days results in a more developed animal.

7.2.2. Cyclic A M P dependent and independent forms A series of investigations using various enzyme sources--whole brain fractions, homogehates, ribosomal proteins, brain regions, and synaptic membranes have generally agreed on one basic theme. There is little difference in enzyme activity among appearance, maturation or senescence with regard to central activity of cyclic AMP dependent and independent species of protein kinase. The enzyme(s) possess almost full activity before birth and has been looked at in a variety of species to include: rat brain (Gaballah et al., 1971; Takahashi et al., 1975; Kinnier etal., 1979; Shambaugh et al., 1978; Lohmann etal., 1978; Schmidt and Sokoloff, 1973; Miyamoto et al., 1980; Schmidt et al., 1979, 1980a); bovine cerebrum (Reichlmeier, 1976); and human cerebrum (Schmidt et al., 1980b). This general statement, however, is subject to modification because Takahashi etal. (1975) and Miyamoto et al. (1980) reported that cyclic AMP dependent protein kinase gradually declined after birth in rat brain while Ca2+-dependent protein kinase gradually increased to a maximum at 28 days postpartum. On the other hand, using fractions of the rat cerebellum Shambaugh et al. (1978) showed that the majority of the protein kinase in this tissue was the cyclic AMP dependent enzyme and activity in the cytosol was increased 5-fold between 4 and 20 days postpartum. Moreover, the cyclic AMP dependent enzyme activity did not change during development. Administration of thyroid hormone (T3 or T4) to rats produced no change in the developmental profiles of protein kinase (Schmidt, 1973). The protein kinase studies show the presence of a metabolically active protein kinase at an early age of development prior to appearance of functional receptors coupled to adenylate cyclase. Moreover, during aging protein kinase did not change. However, as discussed in Section 3.2 the receptor site of this entire enzyme complex is the most labile to alterations in synaptic input, drug manipulation and development. Therefore, if any part of this entire system is associated with the pathology of the aging process the protein kinases are spared.

3-i

( ; . ('+ P.\I M[R

7.3.

CYCHC AMP-MEDIATED

INDUCTION OF ENZ'.'MI-S

Addition of cyclic AMP analogs and phosphodiesterase inhibitors to cultures of ganglia, neuroblastoma or fetal brain cells results in differentiation. Axons become elongated and elaborate nerve processes. Cyclic A M P also prevents cultured cells from reaching confluency. Similarly, nerve growth factor produces many of these effects. It has been postulated that cyclic AMP and nerve growth factor stimulate the assembly of microtubules as cytochalasin-B and colcemid retard axonal elongation. Increasing the dose of these two metabolic promoters overcomes this drug elicited inhibition (Hier et al., 1973; Roisen and Murphy, 1973; Shapiro, 1973" Bondy et al., 1974; Zwiller et al., 1977). Table 8 shows that various enzymes and metabolic processes are augmented when cyclic A M P levels are raised in the various preparations examined. In some cases of enzyme induction cyclic AMP influences both transcription and translational processes. In other situations either one or the other mechanisms are activated by cyclic AMP. Most likely receptor activation by neurohumonal agents elicits cyclic AMP formation with stimulation of protein kinase

TABI.E 8. AI TERATIONSOF CI-NrRAI MF.'IABOLISMBYCYCLIC A M P I INKEI) SYSl EMS System

Response

Preparations

I. Cyclic A M P phosphodiestcrasc (low K.,) 2. Tryptophan hydroxylasc 3. D A s y n t h e s i s a n d DA-beta-hydroxylase 4. Na + K + ATPase 5. Glutamic acid decarboxylasc 6. N A D ( P ) H

+:'

glioma, neuroblastoma, ral pineal

+ +

Agents

Rclcrcncc

rat brain stem

c A M P , NE PGE~, lsop Pdcl cAMP

20,23,27 3tl,32,33 37 2.35

cAMP Pdcl Protein kinasc cAMP

(~,%21.3S

-+

striatum, CSF, ncuroblastoma,N-gliomahybrid brain plasma m e m b r a n e s chick brain

+

rat cerebrum

II

+ + +

C6-glioma (Nuclear) neuroblastoma neuroblastoma

Lactate dehydrogcnasc Nerve growth factor E-Prostaglandins Plasminogen activator Acetylcholine transferase Glutamate uptake Diphosphoinositide kinase Glyceralphosphate dehydrogcnase 18. D N A synthesis

+ + + + + + + +

C6 glioma C6 glioma ncuroblastoma, glioma neuroblastoma neuroblastoma neuroblastoma, glioma rat, rabbit whole brain rat cerebral culture

c A M P , Pdel lsop Isop cAMP Pdcl, c A M P NE, P G E t , A d e n . NE, c A M P , lsop NE. [sop cAMP P G E t , E 2, c A M P PGE~, Pdel, c A M P cAMP cAMP NE, c A M P , Pdel

19. 20. 21. 22.

+

7. Protein kinasc 8. c A M P b i n d i n g protein 9. Ornithinc dccarboxylase 10. 11. 12. 13. 14. 15. 16. 17.

Protein synthesis Protein synthesis Protein synthesis Polyadenylic potymerasc 23. R N A synthesis 24. Thy-l-cell surface antigen

neuroblastoma, glioma

no change +

+

glioma, neurohlastoma fetal gila, neuroblastoma glioma rat brain (nuclear and cytoplasmic R N A ) glioma, neuroblastoma several lines of cultured nerve cells

c A M P , Pdel, NE, lsop cAMP cAMP cAMP cAMP Pdel cAMP c A M P , Pdel lsop

15 19

32 27,2,'4, 1,7 5,12,17 31 8 13 24 3 36 4 16.22,25 14,26,28 27,34 111 20 3 I8

"'+'" = enhanced response; " - ' " = inhibited response. Abbreviations: c A M P = dibutyryl cyclic A M P : Pdcl = phosphodiesterase inhibitors; PG = prostaglandins; lsop = isoproterenol; Aden = adenosine. References: (1) Bachrach, 1975; (2) Boadle-Biber, 1980; (3) Borg et al., 1979; (4) Breen et al., 1978; (5) De Vellis and Kukes, 1973; (6) Fraeyman et al., 1981 ; (7) Gibbs e t a l . , 1980; (8) H a m p r e c h t et al., 1973; (9) H a m p r e c h t et aL, 1974; (llJ) Horak and Koschel, 1977; (11) Keller and C u m m i n s , 1980; (12) K u m a r e t a l . , 198(I; (13) Laug e t a l . , 1976; (141 Lira and Mitsunobu, 1972; (15) Lingham and Sen, 1982; (161 Mares e t a L , 1981 ; (17) Miles e t a l . , 1981 ; (I 8)Morris et al., 1981 ; (19) Nistico e t a l . , 1980; (20) Oleshansky and Neff, 1975; (21) Patrick and Barchas, 1976; (22) Prasad et al., 1972; (23) Prasad and K u m a r , 1973; (24) Prasad and Mandal, 1973; (25) Prasad et al., 1975; (26) Prashad et al., 1977; (27) Prashad and Rosenberg, 1978; (28) Prashad et al., 1979; (29) Schumm and Richter, 1982; (30) Schwartz et al., 1973; (31) Schwartz and Costa, 1977; (32) Schwartz and Costa, 1980a,b; (33) Schwartz, 1982: (34) Shapiro, 1973; (35) Tagliamonte etal., 1971 : (36) Torda, 1972; (37) U z u n o v etal., 1973: (38) Waymire et al., 1978.

PSYCHOACTIVE DRUGS, CYCIJC NUCLEOTIDES AND THE CNS

35

leading to phosphorylation of histones followed by mRNA synthesis and translation into specific proteins. It is beyond the scope of this review to discuss each process in detail but three events, activation of tyrosine hydroxylase, glycogen metabolism and melatonin synthesis have been studied in more depth. 7.3.1. Activation o f tyrosine hydroxylase

Within cellular elements of the peripheral nervous system--adrenal medulla and superior cervical ganglion--arguments have been presented in support of and against a second messenger role for cyclic AMP mediation of the transynaptic induction of tyrosine hydroxylase, the rate limiting enzyme in catecholamine synthesis (see reviews: Costa et al., 1974; Thoenen, 1974). Within the central nervous system studies have shown that cyclic AMP, phosphodiesterase inhibitors or neurohumoral activation of adenylate cyclase increase the activity of tyrosine hydroxylase. A wide variety of brain regions and cell cultures have yielded positive findings in this regard, while cyclic GMP has been considered to be ineffective (Ebstein et al., 1974; Harris et al., 1975; Patrick and Barchas, 1976; Boarder and Fillenz, 1978; Waymire et al., 1978; Bustos and Roth, 1979; Tank and Weiner, 1981). The activation of tyrosine hydroxylase by cyclic AMP involves a change in the kinetics of the enzyme. A decreased Km for pteridine cofactors and an increased K i for feedback inhibition by DA becomes apparent (Harris et al., 1975). Using a purified enzyme fraction from rat caudate, activation of tyrosine hydroxylase was preceded by cyclic AMP stimulation of protein kinase concomitant with an elevated rate of tissue phosphorylation (Joh et al., 1978). Ebstein et al. (1974) showed a DA-induced inhibition of tyrosine hydroxylase. This inhibition was overcome in the presence of dibutyryl cyclic AMP or the DA receptor blocker, haloperidol. Therefore, a presynaptic DA autoreceptor appeared to be the likely site for initiation of the series of metabolic events responsible for feedback inhibition of tyrosine hydroxylase. 7.3.2. Glycogenolysis The well-known cascade of events in the liver involving hormone actions on receptors, with subsequent production of cyclic AMP, activation of protein kinase, phosphorylation yielding an inhibition of synthetic enzymes and an activation of catabolic enzymes for glycogen metabolism is a well-described process (Larner, 1977). In central tissues, however, such an elegant sequence of events is inconclusive. Breckenridge and Norman (1965) were the first to suggest an action of cyclic AMP on the breakdown of glycogen in the brain. Injections of amphetamine or caffeine enhanced the conversion of inactive forms of phosphorylase to the active forms. Moreover, intraventricular administration of NE, dibutyryl cyclic AMP or isoproterenol produced glycogenolysis in rapidly frozen mouse brain, an event blocked by beta blockers but only partially by alpha blockers (Leonard, 1972). Similar findings were reported for "freezeblown" brains of young chickens. In this study catecholamines or histamine injections induced cyclic AMP formation while glycogen levels declined (Edwards et al., 1974). Stressful events such as electroconvulsive shock or decapitation elicit cyclic AMP formation which is accompanied by conversion of phosphorylase to the active form (Lust et al., 1976). Under in vitro conditions cyclic AMP elicited an enhanced rate of aerobic glycolysis in slices of rabbit forebrain (Dittmann and Herrmann, 1970). In more extensive work with tissue slices catecholamines, histamine, serotonin, dibutyryl cyclic AMP, phosphodiesterase inhibitors and K + depolarization all caused glycogen hydrolysis in rat brain. The action of NE was mediated by beta receptors. In addition, in the striatum the agents were observed to increase lactate formation and decrease oxygen and hexose uptake. Cyclic AMP appeared to convert brain metabolism from an aerobic to an anerobic (glycolytic) utilization of glucose (Quach et al., 1978; Wilkening and Makman, 1979). The localization of cyclic AMP mediated glycogenolysis may have in part a glial origin. Cultures of C6 glioma rapidly degrade glycogen in the presence of catecholamines,

36

( ~ ('. P \ [ M I ~

dibutyryl cyclic AMP, histamine and phosphodicsterase inhibitors. Propranolol (beta blocker) inhibits both adrenergic-induced glycogenolysis, as well as, stimulation of adenylate cyclasc. Histamine did not activate adenylate cyclasc (Newburgh find Rosenberg, 1972; Opler and Makman, 1972). These pertinent findings suggest that the long processes of astrocytes surrounding the neurons contain glycogen stores thai might provide a nutritive function during periods of enhanced activity, stress or anoxia. The amouni of glycogen in the brain, however, is insufficient to meet energy demands for any period of time and these small stores may provide only a modest margin of safcty during emergencies.

7.3.3. Serotonin N-acetyltran,sjorase The pineal gland receiving exclusive innervation of adrenergic nerve endings via the superior cervical ganglion has provided an ideal model system to evaluate the control of cellular responses through a beta-adrenergic receptor-adenylate cyclase system. Cyclic AMP so generated mediates the induction of the enzyme serotonin N-acetyltransferase. The degree to which NE activates adenylate cyclase in the pineal depends upon the light-dark cycle the animal receives, as well as the stage of ovarian function. Estradiol released from the ovary inhibits NE-sensitive adenylate cyclase ultimately reducing the content of melatonin synthesized by the gland. Melatonin possesses antigonadotropic activity exerting a feedback control of the gonads. Thus in the rat, darkness increases the sympathetic release of NE to the pineal elevating cyclic AMP which in turn acts through protein kinase to initiate transcription progressing to translation of serotonin N-acetvltransferase with an increased production of melatonin. Light decreases sympathetic activity to the pineal along with attenuated activities of the melatonin synthesizing enzymes hydroxyindole-O-methyltransferase and serotonin N-acetyltransfcrase. This subject has been reviewed in depth (Axelrod, 1974; Strada and Martin, 1979). The major problem with many metabolic conditions involving cyclic nucleotides is that non-neuronal (pineal) and cancer-like cells have afforded the best model systems for evaluation. Likewise, fetal cell cultures do not display the same metabolic profiles as the adult tissue. Nevertheless, useful information has been obtained in which to serve as guideposts for future work.

8. The Neuroleptics 8.1. INTRODUCTION Recent reviews and texts have addressed the role of catecholamine neurotransmitter levels in the limbic-mesolimbic system with regard to the pathogenesis of schizophrenia and psychosis. Since the brain functions as a finely turned instrument any overactivity or hypoactivity of a number of transmitter systems acting at critical synapses could produce symptoms of schizophrenia. The best evidence indicates a major role for an overactive D A system in this disorder. However, good evidence is available for involvement of NE and serotonin systems as well. Less prominent are the observations that opiate systems, prostaglandins and histamine may be contributing factors. Neurotensin, a neuromodulator, appears to possess endogenous neuroleptic activity and thus an underbalance of this peptide could likewise be associated with the disease process. There are essentially two types of psychosis; (1) organic i.e. drug-induced; and (2) functional i.e. schizophrenia. The latter has links to genetics, as well as environmentalpsychosocial stress factors. In contrast to mania, schizophrenia is more prominent in people from poorer socio-economic environments. Major symptoms of schizophrenia are: lowered level of consciousness, delusions, hallucinations, emotional flattening, motor activity---either retardation or hyperkinesia, loss of initiative, and incoherent train of thought. Likewise, the disorder is manifested by different subtypes, namely paranoid, chronic, schizophreniform, catatonic and hebephrenia. Schizophrenic symptoms fire best

PSYCHOACTIVEDRUGS, CYcIJc NUCLEOTIDESAND THE CNS

37

controlled by neuroleptic drugs which have a major property in reducing DA activity in the brain. However, these agents are highly nonspecific from a pharmacological standpoint as they block release of transmitters--presynaptic receptors, avidly bind to cholinergic, histamine, serotonin and adrenergic receptors, block reuptake sites, interact with calmodulin, possess local anesthetic properties, expand and/or solubilize cellular membranes, release calcium from mitochrondria, induce presynaptic synthesis of monoamines and produce a wide variety of active/inactive/toxic metabolites that influence therapeutic outcome. The diverse classes and examples of neuroleptics are phenothiazines (chlorpromazine, fluphenazine, thioridazine), thioxanthenes (chlorprothixene), butyrophenones (haloperidol, spiroperidol), indoles (molindone), dibenzoxazepines (loxapine), diphenybutylpiperidines (pimozide), dibenzodiazepines (clozapine) and rauwolfia alkaloids (reserpine). The major questions to be addressed in this section are what roles do the cyclic nucleotide-receptor coupled systems play with regard to schizophrenia and the therapeutic agents used to control the associated symptoms? Likewise, what particular DA, adrenergic, histamine and serotonin receptor subtypes are influenced? Immediately after the salient finding of a DA-sensitive adenylate cyclase in the striatum by Kebabian et al. (1972) the publication explosion commenced in an attempt to pinpoint this enzyme system as the molecular site so altered by the schizophrenic disease process. With the advent of more studies on a variety of different receptors inhibited by neuroleptics and iigand binding techniques, many of these earlier, simple hypotheses became subject to question. Moreover, DA receptor-ligand binding investigations have added further to the confusion. Seeman (1982) has described at least 4 DA receptor subtypes and some of these subtypes are further divided into subcategories. Few investigations have taken into account the physiological and pathological correlates of ligand binding patterns to DA uptake or release sites, as well as cellular, regional or species variabilities. The majority of the work has been performed in striatum, but DA receptors are different in the mesolimbic nuclei. The latter because of insufficient quantities of tissue have not been rigorously evaluated. In the following subsections only a summary of the extensive literature will address the effects of neuroleptics on DA mechanisms. Recent reviews have considered this transmitter system in detail (for further reading on hypotheses and neuroleptic actions see: Crow et a/.1976; Van Praag, 1977; Richelson, 1981; for cyclic nucleotides see: Daly, 1977; Nathanson, 1977; Palmer and Manian, 1979; Schultz, 1979; Palmer, 1981b; Ragheb and Ban, 1982). 8.2.

CLINICAL STUDIES

The data in this subsection are insufficient and at time are incompatible for drawing firm conclusions regarding a role of cyclic nucleotide receptor-coupled events in the pathology of schizophrenia. 8.2.1. P o s t m o r t e m tissue Dopamine content and receptor density were significantly increased over controls in the caudate, putamen and nucleus accumbens in tissue removed post-mortem from both neuroleptic treated and drug-free schizophrenics (Bird et al., 1977; Crow et al., 1978; Lee et al., 1978b). Binding of apomorphine to presynaptic receptors was unchanged in these investigations (Lee et al., 1978b). In one study with incubated tissue slices from human cerebellum cyclic AMP accumulation to added NE, isoproterenol, clonidine, serotonin or DA was completely suppressed by chlorpromazine (CPZ) (Tsang and Lal, 1978). Calmodulin content did not differ in schizophrenic brain (Vargas and Guidotti, 1980). 8.2.2. C S F Lumbar levels of cyclic nucleotides from chronic schizophrenics with tardive dyskinesia were not different from controls. However, when valproate (anticonvulsant) or cyproheptadine (antiserotonin) were given the nucleotide levels increased, but this could not be

correlated to clinical improvement of the tardive dyskinesia (Nagao et al., 1979). Gomc3 and coworkers (198(/) demonstrated a rise in CSF levels of NE and cyclic AMP in chronic schizophrenics. Alternatively, Biederman et al. (1977), reported that when neuroleptic treatment elicited a faw)rable clinical response, cyclic AMP levels dropped. On the other hand, no differences in CSF cyclic nucleotidc values were seen in untreated schizophrenics vs controls (also see Smith et al., 1976). Using CSF levels of cyclic GMP Smith et al. (1976) reported elevated amounts in schizophrenics pretrcated with probenecid while Ebstcin et al. (1976a) reported elevated levels of cyclic GMP only after 2 months of neurolcptic treatment. 8.2.3. P l a s m a Plasma levels of cyclic AMP were not altered in schizophrenic patients, however, after neuroleptic treatment the levels were decreased, an event unrelated to clinical improvement (Hansen, 1972; Stefanis et al., 1977; Lykouras et al., 1980). 8.2.4 Urine Patients with psychotic depressions had lower levels of cyclic AMP excreted in the urine than controls (Abdulla and Hamadah, 197(I; Paul et al., 1970a, 1971a; Sinanan et al., 1975 ; Jarrett et al., 1977). Alternatively Somerville (1973) and Brown et al. (1972) were unable to confirm these findings in psychotic depressives, while similar negative data for patients with paranoid psychosis or periodic catatonia were given by Geisler et al. (1976) and Perry et al. (1973). 8.2.5. B l o o d cells 8.2.5.1. Platelets In two investigations of platelets taken from schizophrenic patients, prostaglandin E~ stimulation of cyclic AMP was lower than normals, especially in males (Kafka el al., 1979: Rotrosen et al., 1980). In contrast Pandey and colleagues (1977) demonstrated an enhanced ability of prostaglandin Et to increase cyclic AMP in platelets taken from acute but not chronic schizophrenics. 8.2.5.2. L e u k o c y t e s Both NE and isoproterenol were less effective in eliciting an elevation of cyclic AMP in leukocytcs taken from schizophrenics (Pandey et al., 1979). 8.3. In Vivo STUDIESNEUROLEPTICS 8.3.1. Cyclic A M P 8.3.1.1. Urine Subchronic injections of small doses of CPZ to rats over a period of one week elevated both urine volume and the 24 hr content of cyclic AMP. Upon cessation of treatment control values were attained within 7 days (Palmer and Evan, 1974). Keatinge etal. (1975), however, were unable to confirm these findings. 8.3.1.2. P l a s m a Nakadate and coworkers (1980a,b), showed that following either subcutaneous or intraventricular injection of CPZ to mice, plasma levels of cyclic AMP became elevated. The action was mediated exclusively via beta receptors acting presumably in the CNS to affect adrenal release of epinephrine in the periphery. Epinephrine may have then caused urinary cyclic AMP levels to rise (see also Palmer and Evan, 1974). Pretreatment with reserpine, but neither 6-hydroxydopamine nor alpha-methyl-p-tyrosine prevented the plasma cyclic AMP rise in response to CPZ.

PSYCHOACTIVE DRUGS, CYCLIC NUCLEOT1DES AND THE C N S

39

8.3.1.3. Trauma Decapitation of rats rapidly elevated levels of central cyclic AMP, an event blocked by trifluoperazine and CPZ, but not by trifluoperazine-sulfoxide or promethazine (Uzunov and Weiss, 1971). Somewhat similar findings were reported by Singh et al. (1980). A huge elevation in cyclic AMP was seen in mouse cerebral cortex following a stab wound. The rise was prevented by CPZ, trifluoperazine and theophylline, In this study neither pretreatment by reserpine nor beta blockers were effective. Watanabe et al. (1975) concluded that the cyclic AMP response was probably mediated by adenosine. Any trauma or injury releases a large quantity of neutrotransmitter substances and the resultant rate of depolarization could also account for these data. However, neuroleptics do not appear to inhibit adenosine receptors (Blumberg et al., 1976; Cotter et al., 1978). 8.3.1.4. Electrophysiological Unilateral stimulation of the rat locus coeruleus elevated the cyclic AMP content in the rapidly fixed cerebrum. The action was, however, insensitive to the DA blocker, haloperidol, or to clozapine. Most likely the response was exclusively due to NE pathways (Ader et al., 1980). Iontophoretic application of DA to striatal neurons depresses the firing rates and CPZ antagonizes this action of DA (Siggins et al., 1974). 8.3.1.5. Rapidly fixed brain

Acute injections of CPZ or haloperidol decreased cyclic AMP in mouse cerebellum, cerebrum and diencephalon. Subchronic injections, however, elevated the steady-state levels of the cyclic nucleotide (Palmer et al., 1977a, 1978). 8.3.2. Cyclic G M P The cerebellum of mice and rats readily accumulates cyclic GMP in response to DA stimulation (apomorphine, amphetamine) of the afferent pathways to the Purkinje cells. Enhancement of GABA inhibitory pathways to the cerebellum diminishes this excitatory activity and decreases cyclic GMP, The site of DA action has been shown to originate in the striatum. Thus only intrastriatal injections of DA agonists elicit this response which is inhibited stereochemically by a variety of pharmacologically active neuroleptics namely, CPZ, haloperidol, ( - ) spiroperidol, sulpiride, and (+) butaclamol. Destruction of striatal neurons with kainic acid prevents both the action of DA agonists on the elevation of cyclic GMP and the effect of neuroleptics which when given alone decrease the steady state levels of cerebeilar cyclic GMP. Subchronic injections of haioperidol or CPZ reveal that a tolerance to the reduction in cyclic GMP becomes evident, At this point in time apomorphine evokes an even greater cyclic GMP accumulation. Biggio et al. (1978b), concluded that this cerebellar system could serve as an illustrative model to determine the state of activation of striatal DA-receptors (also see: Ferrendelli et al., 1972; Burkard et al., 1976; Gumulka etal., 1976; Biggio and Guidotti, 1977; Biggio et al., 1978a,b; Corda etal., 1979). Neuroleptics likewise reduce cyclic GMP in the cerebral cortex and tolerance becomes apparent by 24 hr (Palmer et al., 1978). 8.4. GUANYLATECYLASE-CYcLICGMP 8.4.1. Guanylate cyclase Limited data is available in order to resolve any conclusions as to the manner in which neuroleptics influence guanylate cyclase. Of several derivatives of CPZ tested, o,nly the 8-OH-metabolite effectively inhibited basal, calcium or NaN 3 and NH2OH activation of the enzyme, as evaluated in soluble and particulate fractions of mouse brain (Pajer et al., 1981). The action of neuroleptics to inhibit a calmodulin-Ca2+ activation of particulate guanylate cyclase was demonstrated in tetrahymena pyriformis (Nagao et al., 1981). The particulate enzyme from another non-neural tissue was inhibited by phenothiazines and imipramine (Fujimota and Okabayashi, 1975). However, the enzyme in the guinea pig

4(~

(;. (', P-xJ Ml~

cerebellar homogenate was subject to inhibition by only high concentrations ol phenothiazine-likc compounds (Ohga and Daly, 1977a). Fujimota and Okabayashi (1975) felt that the particulate enzyme was especially susceptible to drug inhibition because of the membrane stabilizing actions of the phenothiazines. 8.4.2. Cyclic G M P The neuroblastoma culture system (NIE-115) readily accumulated cyclic GMP in response to carbamylcholine. Phenothiazines with strong anticholinergic actions (clozapine and thioridazine) markedly inhibited this system while agents with lesser degrees of anticholinergic activity (haloperidol, trifluoperazine) were over a 100-fold less potent (Richelson, 1977; Richelson and Divinitz-Romero, 1977). The calcium elicited accumulation of cyclic GMP in guinea pig cerebellar slices was partially inhibited by CPZ or imipramine and potently blocked by promethazine (Ohga and Daly, 1977a). Some phenothiazines, i.e. 8-OH-CPZ, and thioridazine elevated cyclic GMP in rat cerebral slices via a calcium dependent process. Other phenothiazines inhibited cyclic GMP stimulation to carbamylcholine in this tissue (Palmer et al. 1976a). Thus the production of cyclic GMP by acetylcholine and calcium-induced depolarization may be an active site for phcnothiazine action, in keeping with their prominent anticholinergic side effects.

8.5.

PROTEIN KINASES

This area of investigation has been neglected from a standpoint of evaluating the action of psychoactive drugs on cyclic nucleotide systems. Petzold and Greengard in 1973 (unpublished personal communication) reported that high concentrations of 7,8-diOH- or 7,8-dioxo- but not 7,8-diMe-derNatives of CPZ blocked cyclic AMP dependent protein kinase in bovine brain. Maxwell (1975) found that inhibition of this enzyme by CPZ was of a noncompetitive nature. Hullihan el al. (1979) observed that the DA-induced phosphorylation of rat caudate nucleus was inhibited by neuroleptics. The rate of phosphorylation of proteins in guinea pig cerebral slice was enhanced by electrical activity and monoamines. Chlorpromazine was only an effective inhibitor of the electrical pulse (Williams and Rodnight, 1976). In more recent work phosphorylation of calcium or phospholipid, but not cyclic nucleotide dependent protein kinases, was blocked by several pharmacologically active neuroleptics (Schatzman et al., 1981; Wrenn et al., 1981). Two calcium-sensitive protein kinases that phosphorylate protein 1, a specific synaptic protein, were inhibited by trifluoperazine. Drug action was most likely an interaction of the calmodulin system (Kennedy and Greengard, 1981 ). 8.6.

M1SCEI.I,ANEOUS ACTIONS OF NEUROI.EPT1CS

High concentations of dihydroxylated inetabolites of CPZ, promazine, perphenazine and prochlorperazine, as well as, the parent compounds, were required to inhibit fluoride-sensitive adenylate cyclase from rat brain homogenates, particulate fractions, or isolated neuronal and glial fractions (Uzunov and Weiss, 197 l : Palmer and Manian, 1979). Phenothiazines such as CPZ, trifluoperazine, thioridazine and fluphenazine were potent inhibitors of the rapid uptake of adenosine by rat cerebral cortical synaptosomcs. This action was postulated to be a significant factor in the ability of these agents to have sedative properties. Interestingly the less clinically potent agent, clozapine, was weaker than the other agents (Phillis and Wu, 198lb). Earlier Huang and Daly (1972) had reported that CPZ elevated basal levels of cyclic AMP in guinea pig cortical slices. The increased availability of adenosine may have accounted for these findings. Adenosine-activated adenylate cyclase in tissue slices was insensitive to neuroleptic inhibition (Blumberg et al., 1976: Cotter et al., 1978). Local anesthetic action may account for the ability of CPZ to reduce cyclic AMP accumulation in cortical slices evoked by electrical pulses (Kakiuchi et al., 1969). This

PSYCHOACTIVEDRUGS, CYCLICNUCLEOTIDESAND THE CNS

41

property of CPZ was likewise attributed to prevention of veratridine-induced cyclic AMP production in guinea pig cortex (Ohga and Daly, 1977a). For a detailed account of neuroleptic actions on peripheral tissues see the review by Palmer (1979). 8.7.

DOPAMINE RECEPTORS

A tremendous literature has appeared since the initial discoveries by Kebabian et al. (1972), when it was found that D A was capable of stimulation of adenylate cyclase when tissue homogenates were employed. This study led immediately to speculations that this system would be the molecular site of action for neuroleptics and thus the search for the molecular deficit associated with schizophrenia would be ended. A rapid series of publications followed which examined the structure activity and stereospecific relationships of neuroleptic inhibition of DA-adenylate cyclase. A variety of tissues and brain regions were evaluated with the rat striatum receiving the greatest intensity of investigative efforts. Initial work revealed that DA-adenylate cyclase was potently blocked in a stereospecific manner by pharmacologically active neuroleptics and their active metabolites (Clement-Cormier et al., 1974; Horn et al., 1974). Inactive metabolites were ineffective (Palmer and Manian, 1979, for review). The major problem arose in that agents like clozapine or thioridazine, reported to have less D A blocking activity and hence weaker clinical efficacy, were almost as potent as powerful D A blockers and antischizophrenic agents such as fluophenazine and haloperidol. A clinically effective antipsychotic, sulpiride, was shown to be a D A receptor blocker but lacked any inhibition of the DA-adenylate cyclase system. Further problems arose when it was shown that binding of D A ligands occurred with high degrees of affinity to subcellular fractions devoid of DA-adenylate cyclase activity. Such inconsistencies led investigators to postulate the existence of different subtypes of D A receptors in the brain. The problem was further confounded when it was revealed that neuroleptics exerted antagonism of histamine, serotonin, NE and acetylcholine receptors, as well as, calmodulin. However, these parameters were not inhibited as potently as the D A receptors. The existence of different subtypes of DA receptors was further supported with the discovery of presynaptic autoreceptors. However, many of these receptor subtypes identified by ligand binding could easily be identified as uptake sites, storage sites or located on glia. Thus the original idea that DA-sensitive adenylate cyclasc might be the molecular site for schizophrenia or Parkinsonism has been severely modified to include contributions of other D A receptors as well. It would be almost inconceivable to review extensively the vast literature on neuroleptics and D A receptors especially when many excellent recent reviews have been written (Snyder el al., 1974; Iversen, 1975: Kebabian and Calne, 1979; Leysen, 1979; Libet, 1979; Palmer and Manian, 1979; Schultz, 1979; Schmidt, 1979; Stevens, 1979; Calne, 1980; Laduron, 1980; Schachter et al., 1980; Seeman, 1980; Cools, 1981; Kebabian and Cote, 1981). 8 . 8 . NOREPlNEPHRINE RECEPTORS

In the late 1960's and early 1970's prior to the discovery of the DA-sensitive adenylate cyclase, the majority of the work with neuroleptics was concerned with the NE system which was evaluated in incubated tissue slices from many brain regions. Kakiuchi and Rail (1968) were first credited with observing an inhibition of NE-cyclic AMP by CPZ using incubated slices of rabbit cerebellum. Since that time many similar observations have been made with a variety of pharmacologically active neuroleptics and their active metabolites (7-OH-CPZ, beta-OH-CPZ, quaternary-CPZ, 7-OH-quaternary-CPZ, 7-QH-perphenazine, 7-OH-prochlorperazine, 7-OH-fluphenazine, 2- or 3-OH-promazine and 7OH-CPZ-Me-I). In general butyrophenones, 8-OH-derivatives of phenothiazines, diOHderivatives of phcnothiazine and methoxy derivatives of CPZ were considerably less potent. The agent, phenothiazine and sulfoxide derivatives of CPZ or trifluoperazine displayed no potency. All tissues responding to NE (except striatum) were sensitive to inhibition by the neuroleptic agents: rat pineal, brain stem, cerebellum, limbic forebrain,

42

G, C. PALMER

hypothalamus, cerebrum, guinea pig, mouse and human cerebrum and mouse limbic forebrain. The brain region appearing most sensitive to the action of NE was the limbic forebrain where even the DA blocker pimozide was highly potent (Uzunov and Weiss. 1971 ; Huang and Daly, 1972, 1974; Palmer et al., 1972b; Forn et al., 1974; Blumberg et al., 1976; Harris, 1976; Skolnick et al., 1976; Sawaya et al., 1977; Tsang and Lal, 1977: Cotter et al., 1978; Palmer and Manian, 1979). In broken cellular preparations epinephrine or NE-responsive adenylate cyclase was inhibited by trifluoperazine, CPZ, 7-OH-CPZ, 7-OH-CPZ-Me-I, fluphenazine, thioridazine, pimozide and pindolol. In these cell free preparations the following regions and species have been evaluated: cat brain (cortex, cerebellum and hippocampus), rat brain (pineal, cerebral cortex), mouse brain (frontal cortex) and monkey brain (frontal cortex) (Uzunov and Weiss, 1971; Ahn et al., 1976; Bockaert et al., 1977; Cotter et al., 1978; Palmer et al., 1978; Dolphin et al., 1979). Norepinephrine-adenylate cyclase in the homogenate of the rat striatum was insensitive to neuroleptic action (Walker and Walker, 1973b; Harris, 1976). Isoproterenol activation of the enzyme in monkey or rat frontal cortex and cat brain was resistant to neuroleptic inhibition but was blocked by propranolol. The latter agent was not potent with respect to NE-adenylate cyclase. The data indicated that ncuroleptics do not block NE strictly via a beta adrenergic receptor, but instead illustrate that NE interacts at DA-like receptors (Ahn et al., 1976; Bockaert et al., 1977; Dolphin e t a l . , 1979). In microiontophoretic investigations fluphenazine and alpha (but not beta) flupenthixol counteracted the inhibition of Purkinje cell firing produced by NE. The agents also attenuated the Purkinje cell inhibition initiated by electical stimulation of its noradrenergic input, the locus coeruleus (Freedman and Hoffer, 1975; Skolnick et al., 1976). For a more detailed review of NE and neuroleptic actions, see Palmer and Manian (1979). 8.9. HISTAMINE RECEPTORS Rail and Kakiuchi (1968) using tissue slices of rabbit cerebellum first reported that CPZ inhibited cyclic AMP accumulation in response to histamine. Similar data were reported in the guinea pig brain (Huang and Daly, 1972; Free et al., 1974). Using a similar preparation of cerebral cortex it was found that antipsychotic agents possessing stronger anticholinergic actions were the most potent with regard to an antagonism of histamineelicited cyclic AMP. In this manner promethazine, clozapine, butaclamol and thiordiazine were the most potent along with quaternary and 7-OH metabolites of CPZ. Methylated and sulfoxide CPZ derivatives and haloperidol were the weakest agents. In isolated neuronal and glial fractions these active neuroleptics were more potent in blocking histamine-sensitive adenylate cyclase in glia than in neuronal perikarya ( S p i k e r e t a l . , 1976; Palmer and Manian, 1979). In guinea pig cortical homogenates binding of H-1 ligands was potently inhibited by promethazine, CPZ, fluphenazine and thioridazine. The degree of inhibition was a 1,000 fold greater than with haloperidol and loxapine. Adenylate cyclase activation by histamine was inhibited in the same order of potency but the drugs with the greater anticholinergic actions were about 15-fold more potent than haloperidol, loxapine and fluphenazine (Coupet and Szuchs-Myers, 1981). Quach et al. (1979) reported somewhat similar H-1 binding data for displacement of mepyramine in mouse cerebrum, following in vivo administration of neuroleptics. The guinea pig cortical and hippocampal system also possess a powerful H-2-sensitive adenylate cyclase. Kanoff and Greengard (1978) reported a potent inhibition of histaminesensitive adenylate cyclase by "anticholinergic" neuroleptics in a hippocampal preparation (also see Tuong et al., 1980). In conclusion, as with the cholinergic-induced stimulation of cyclic GMP, inhibition of histamine sensitive cyclic AMP systems in brain occurs to a greater extent with agents possessing strong sedative-anticholinergic actions. This antimuscarinic activity is of course

PSYCHOACTIVE DRUGS, CYCLIC NUCLEOTIDES AND THE C N S

43

associated with neuroleptics causing a lesser incidence of extrapyramidal reactions and tardive dyskinesia (Miller and Hiley, 1974). 8.10. SEROTONINRECEPTORS Serotonin activation of adenylate cyclase in neuroblastoma-brain hybrid cells was effectively inhibited by fluphenazine while pimozide was five-fold less active (MacDermot et al., 1979). This enzyme system in glial membranes (horse striatum) was inhibited by pimozide, haloperidol and droperidol at an IC50 of 0.1 kLM (Fillion et al., 1980). In an evaluation of DA and 5-HT responsive adenylate cyclases from regions of the newborn rat brain, clozapine, thioridazine, CPZ, fluphenazine and haloperidol displayed more potent antagonism toward the DA system. However, in the colliculi 5-HT-adenylate cyclase was inhibited according to an identical potency of the previously listed drugs (Enjalbert et al., 1978). Pagel et al. (1976) reported that the 5-HT-sensitive adenylate cyclase in mature rat brain was insensitive to antagonism by fluphenazine. When newborn rat brains were examined, both binding of 5-HT and the responsive enzyme were inhibited by several pharmacologically active neuroleptics (Von Hungen et al., 1975a; Nelson et al., 1980). Chlorpromazine, but not pimozide inhibited 5-HT elicited cyclic AMP in human cortical slices (Tsang and Lal, 1977). 8.11. PHOSPHODIESTERASES--CALMODULIN In the earliest experiments various phenothiazines were added to phosphodiesterase preparations consisting of homogenates or partially purified enzymes. In general, concentrations of about 100 /xM of CPZ, CPZ-free radical, trifluoperazine, chlorprothixene, thioridazine, perphenazine, dihydroxy, and benzo [b]-derivatives of CPZ were required to inhibit enzyme activity by 50% (ICs0). Some brain regions (cerebellum) were more resistant to inhibition than others. Moreover, haioperidol was an extremely weak antagonist of phosphodiesterase (Honda and Imamura, 1968; Roberts and Simonsen, 1970; Uzunov and Weiss, 1971; Beer et al., 1972; Weinryb et al., 1972; Berndt and Schwabe, 1973; Petzold and Greengard, personal communication in 1973; Palmer and Manian, 1979; Palmer, 1981; Sweatt e t a l . , 1982). The biochemistry and pharmacology of calmodulin has been discussed (Section 4). Basically the antipsychotic drugs inhibit calmodulin-calcium interactions with adenylate cyclase or the activatable forms of phosphodiesterase. The action has been called specific but some problems have arisen. The neuroleptics inhibit DA receptors in a stereospecific manner with a greater degree of potency than their ability to antagonize calmodulin, which does not display stereospecificity. Brostrom and coworkers (1978a,b) had described for several years an inhibition by CPZ of an adenylate cyclase system in a cerebral particulate fraction of brain. The inhibition involved both calcium and the so called calcium dependent regulatory protein now known as calmodulin. Other work by Gnegy and coworkers (1976, 1977a,b) described essentially the same type of calmodulin dependent activation of adenylate cyclase in the striatum. The interaction was acutely inhibited by neuroleptics but long term neuroleptic treatment led to an elevated tissue level of calmodulin. The majority of the work performed with neuroleptic influences on calmodulin-calcium activation of phosphodiesterase was accomplished by the group of Weiss (Levin and Weiss, 1975, 1977, 1978, 1979; Weiss and Wallace, 1980; Prozialeck etal., 1981 ; Prozialeck and Weiss, 1982). Brain regions were shown to contain several types of phosphodie~terase, one form (high Kin-cyclic AMP type) was markedly activated by calcium-calmodulin and inhibited by EGTA (chelates calcium) and CPZ. Excess calcium was necessary to overcome inhibition by EGTA while excess calmodulin overcame CPZ inhibition, suggesting an interaction by neuroleptics at a molecular site different from calcium. It was later shown that trifluoperazine and other neuroleptics could bind to a variety of proteins at low affinity-calcium independent sites, but only with calmodulin did the neuroleptics bind with

a high affinity-calciunl dependent reaction. This neurolcptic blockade of calmodulinphosphodiesterase was specific for this protein when tested on cahnodulin isolated from several brain and peripheral rcgions taken from a variety of specie,,, including h u m a n s Pharmacologically active antipsycholics all bound to the same site on calmodulin and were considerably more potent phosphodieslerasc inhibitors or competitive inhibitors of displacement of trifluoperazine binding than werc antidepressants, antianxicty agents, stimulants, hallucinogens, catecholamines, promethazine and CPZ-snlfoxide. Two agents shown to be DA blockers without antipsychotic activity (perlapine and metoclopramide) and the antipsychotic, molindone displayed weak action toward displacement of bound trifluoperazine from calmodulin. Photochemical activation of phenothiazines yielded an irreversible covalent binding of neuroleptics to calmodulin. This irradiated calmodulin-~ neuroleptic complex was unable to activate phosphodicstcrase. In an examination of the IC~ values, for neuroleptic inhibition of calmodulin, and their partition coefficients for hydrophobicity there was found to be a good correlation between these two parameters. Even though hydrophobicity of the phcnothiazine ring was important for binding to calmodulin the nature of the side chain on the molecule provided for an additional electrostatic interaction between the drug and the protcin. In other work, Filburn et al. (1979) showed neuroleptic inhibition of a calcium, calmodulin-sensitive form of cyclic G M P dependent phosphodiesterase from brain. In addition, the calcium, calmodulin stimulation of Mg 2+, Ca2+-ATPase was potently blocked by CPZ derivatives, some of which did not possess antipsychotic activity. All CPZ analogs did, however, possess a nonspecific hydrophobic site for calmodulin attachment (Roufogalis, 19g 1). Sweatt and coworkers (1982) using a series of benzo[bJ-derivatives of CPZ were unable to correlate the drug potency of calmodulin-phosphodiesterase inhibition to that observed with antagonism of DA-adenylate cyclase in enzymes prepared from rat striatum. Norman and Drummond (1979) looked at a variety of different neuroleptic compounds with respect to calmodulin-phosphodiesterase inhibition and reported no correlation between clinical efficacy of the neuroleptics to their ability to inhibit calmodulin. The major problem was a lack of stereospecific interaction. A caffeine or theophylline activation of phosphodiesterase was reported by Dunlop et al. (1981). The enzyme stimulation was blocked by trifluoperazine. This nqethylxanthine reaction was thought to take place because caffeine and theophylline mobilized calcium from the tissue. The data with phosphodiestcrase inhibition by neuroleptics are compatable with some of the in vivo findings (reported in previous sections) in which steady-state levels of cyclic AMP are increased in brain and body fluids following neuroleptic treatment. Moreover, the calcium-calmodulin induced phosphorylation of synaptic vesicle protein was also demonstrated to be sensitive to neuroleptic antagonism (DeLorenzo, 1981). With such a diverse profile of metabolic activity for calmodulin, the clinical significance fl)r interactions with neuroleptics would at best be only speculative. 8.12. CHRONI("TREATMENTWITH NEUROLEP'rlCS A series of behavioral investigations pointed out the fact that chronic treatment of laboratory animals with neuroleptics led to heightened behavioral responses to DA agonists i.e. apomorphine or amphetamine. Moreover, supersensitive responses (locomotor activity and stereotypy) were found in both the striatum and nucleus accumbens (Jackson et al., 1975; Sayers et al., 1975: Christensen et al,, 1979: Smith and Davis, 1976). From these initial experiments many investigators set out to discover a possible molecular site associated with tardive dyskinesia. The findings taken together are intriguing but as usual many discrepancies were found. The essential elements of these findings were that chronic neuroleptic treatment produces alterations in DA receptor activities principally in the striatum. In this vein, DA receptor activity is increased, cahnodulin content is augmented, low Km phosphodiesterase activity is diminished and it is equivocal whether D A receptors coupled to adenylate cyclase are enhanced. In some recent work NE receptors may change.

PSYCHOACTIVE DRUGS, CYCIJC NUCLEOTIDES AND THE CNS

8.12.1.

45

Cahnodulin

Gnegy and coworkers (1976, 1977a,b, 1980; also see Lucchelli, 198(/) have conducted extensive work on the calmodu[in system as a molecular site of DA supersensitivity in the rat striatum, as a consequence of chronic neuroleptic administration. Agents such as (+)-butaclamol, and haloperidol when given on a chronic basis (for 10 or more days) followed by a period of withdrawal, evoked increases in the striatal content of calmodulin. behavioral responses to apomorphine, and DA-sensitive adenylate cyclases (decreased affinity for DA). Inactive isomers such as (-)-butaclamol or agents that do not produce tardive dyskinesia, namely clozapine, did not elicit these responses. Moreover, these phenomena of supersensitivity were unique to the striatum and not to the frontal cortex or nucleus accumbens. The content of calmodulin was even lower in the hypothalamus. 8.12.2. D o p a m i n e and p h o s p h o d i e s t e r a s e Despite the fact that central DA receptors increase in both content and responses to physiological stimuli subsequent to chronic neuroleptic administration (Yarbrough, 1975; Burt el al., 1977; Friedhoff el al., 1977; Clement-Cormier, 1980; Clow et al., 1980; Reeches et al., 1982), the data indicating that these receptors are associated with DA-adenylate cyclase are controversial. This enhanced D A binding has been demonstrated in the rat striatum, substantia nigra and mesolimbic region. In one study both acute and chronic injections of teflutixol actually resulted in a reduced ability for haloperidol to bind to mouse striatal preparations. Similarly, acute injections of neuroleptics usually inhibited DA-adenylate cyclase (Hyttel, 1978; Clow et al., 1980), however, another study reported instead an increased enzyme sensitivity to D A (lwatsubo and Clouet, 1975). The experiments of Gnegy et al. (1976, 1977a,b, 198(I) along with those by Friedhoff el al. (1977), Kaneo et al. (1978) and Clow et al. (1980) revealed an enhanced ability of DA to stimulate adenylate cyclase in the striatum following an appropriate withdrawal interval from chronic neuroleptic treatment. M o r e o v e r , / - D O P A treatment prevented this process. In one study the DA-sensitive enzyme in the nucleus accumbens actually declined (Kaneo et al., 1978). The failure of others (Rotrosen el al., 1975; Palmer and Wagner, 1976; Roufogalis el al., 1976; Hyttel, 1978; Clement-Cormier, 1980; Magistretti and Schorderet, 1980) to demonstrate analogous findings could be related to several factors namely, insufficient drug dosage, insufficient duration of injection, insufficient withdrawal period, and the lack of relationship of D1 receptors associated to the process of supersensitivity. Clow et al. (1980) found that at least 6 months treatment of trifluoperazine was necessary to elicit DA-adenylate cyclase hypersensitivity. Many studies were conducted for shorter periods of time. On the other hand, the elevated binding of D A ligands to the striatal preparations following haloperidol was confined to microsomal fractions, devoid of DA-adenylate cyclase (Clement-Cormier, 1980). In a single investigation Fredholm (1977) injected rats for 18 days with haloperidol and observed a diminished activity of low Km cyclic AMP phosphodiesterase in the striatum. The lowered activity could be a contributing factor to the elevated DA-adenylate cyclase reported above. Some interesting sidelines have arisen from these investigations with chronic neuroleptics. Increased NE-sensitive adenylate cyclase and beta receptor binding have been reported in the rat cerebellum, caudate, cerebrum and olfactory tubercle (ClementCormier, 1980; Weiss and Greenberg, 1980). On the other hand, in the initial work, Shultz (1976) showed that chronic injections of CPZ to rats promoted a time-dependent decrease in the ability of NE to accumulate cyclic AMP in cortical slices. However, no time was allowed for withdrawal and the presence of the parent drug and its metabolites in the incubation procedures could have interfered with the enzyme. 8.13. CONCLUSIONS

A number of molecular events associated with the central action of neuroleptics can be correlated to an influence on cyclic nucleotide systems. In many cases, the major exception being calmodulin, analogies can be drawn to clinical or physiological events. An incom-

plete list of these situations might include: (a) antidopaminc----contvol of schizophrcni;~. induction of Parkinsonism, chronic supersensitivity--tardive dyskinesia; (b) antiadrcncrgic control of schizophrenia, control of mania and orthostatic hypotension: (c) :mticholincrgic or antihistaminic-sedation, antagonism of DA-Parkinsonism in the striatum, and peripheral anticholinergic side effects: and (d) antiscrotonin-rcduction of hallucinations.

9. Antidepressants 9.1. OVERVIEW

Depression or the "blues" is a complex disease and represents the most common psychiatric disorder seen in the United States today. There are genetic links associated with the disorder(s). Some patients experience only depression (unipolar) and others, both depression and cycles of mania (bipolar). In addition many forms of depression have been subclassified into primary and secondary, and even further subcategories are used to characterize the various patterns of symptoms observed in clinical practice. The major symptoms of depression usually include: (1) a persistent, dysphoric mood; (2) appetite-weight loss or gain; (3) sleep disturbance; (4) loss of energy; (5) psychomotor-agitation or retardation; (6) anhedonia--inability to derive pleasure; (7) self-reproach, guilt; (8) slow thinking; (9) suicide-death thoughts, plus many more associated symptoms. Depression may also be episodic or non-recurring in nature. The popular monoamine hypothesis of affective disorders by Schildkraut and Kety (1967) suggests that depression is a result of functional deficiency of either NE or serotonin at critical sites within the brain. Thus the possibility of at least two subtypes of depression can be determined based on central neurotransmitter action. Even though a variety of tests and measurements have been made on NE and serotonin such a simplistic view has been fraught with equivocal data. Nevertheless, it is widely accepted that the major acute effects of antidepressants are to increase the availability of NE and/or serotonin in the brain. Thus: (1) the monoamine oxidase inhibitors prevent the metabolism of NE and serotonin: (2) electroconvulsive therapy apparently releases catecholamines; (3) tryptophan loading elevates serotonin levels; (4) the tricyclic antidepressants selectively inhibit the uptake of serotonin (tertiary amine tricyclics) or NE (secondary amine tricyclics); (5) some atypical antidepressants (e.g. iprindole and mianserin) are thought to block presynaptic receptors which control the release of catecholamines; and (6) amphetamines which elevate mood by releasing NE and DA, blocking their reuptake, inhibiting monoamine oxidase and via metabotites exerting "false" transmitter actions. Amphetamines are discussed in Section 10. The one problem with these compounds is that all but amphetamines require a lag period of at least 2-3 weeks before antidepressant therapy is achieved (electroconvulsive therapy is slightly shorter). Recent findings which report that all these modes of administration which enhance the action of NE, produce after prolonged treatment, a subsensitivitv of either or both beta and alpha2 adrenergic receptors, have generated some lively arguments as to just where and how antidepressants exert their therapeutic molecula, actions (for further reading see: Maas, 1979; Richelson, 1979b; Collis and Shepherd, 1980: Akiskal, 1981; Harrison-Read, 1981; Schaffer et al., 1981; Sugrue, 1981). The following discussion is mainly concerned with the action of antidepressants on cyclic nucleotide systems in the brain. The evidence is further supported by receptor ligand binding techniques and other data, however, these are not extensively reviewed. 9.2. TRICYCLIC ANTIDEPRESSANTS 9.2.1. Dopamine receptors 9.2.1.1. Acute Nomifensine, an atypical tricyclic antidepressant, which may block DA uptake, when injected into rats increased in a dose-dependent manner the steady state levels of striatal

PSYCHOACTIVE DRUGS, CYCLIC NUCLEOTIDES AND THE CNS

47

cyclic AMP (Gerhards et al., 1974). In keeping with these in vivo studies daily administration of amitryptyline to mice for 3 days led to a substantial increase in levels of brain cyclic AMP as measured following rapid fixation with focussed-high intensity microwave irradiation. The drug acted similarly in a peripheral tissue-lung (Palmer et al., 1977a). These findings suggest two possibilities for tricyclic antidepressant action, either an inhibition of phosphodiesterase or potentiation of central catecholamine action. Alternatively, when evaluated under in vitro conditions the tricyclic antidepressants in the following order of potency were effective inhibitors of DA-adenylate cyclase in the striatum: amitriptyline, doxepin > Cl-imipramine, nortriptyline > imipramine, desipramine, protriptyline and melitracene (Karobath, 1975). In further work Cl-imipramine was clearly the most potent tricyclic antidepressant to inhibit DA-adenylate cyclase in rat striatum. The next most potent compounds were hydroxy metabolites of Cl-imipramine i.e. 8-OH- or 2-OHanalogs, and these were more potent than imipramine and 2-OH-imipramine. Desipramine and its 2-OH-metabolite were the weakest agents. The data indicate the importance of considering not only the parent compound during drug action but the active vs inactive metabolites as well (Palmer et al., 1977b; Keller et al., 1980). Additional work with ligand binding techniques, single unit recording, and measurement of DA turnover rates indicated that tricyclic antidepressants, especially Cl-imipramine were effective blockers of DA receptors. The agents were, however, slightly less potent than phenothiazines (Chiodo and Antelmen, 1980; Keller et al., 1980). In contrast tricyclics were not shown to influence spiroperidol binding to rat brain (Hall and Ogren, 1981). 9.2.1.2. Chronic No studies have looked at the sensitivity of DA-adenylate cyclase following chronic tricyclic antidepressant administration. In behavioral, single unit recording and ligand binding work, chronic tricyclic antidepressant treatment led to: supersensitivity of mesolimbic DA receptors (Spyraki and Fibiger, 1981), subsensitivity of DA auto-receptors in the striatum (Serra et al., 1980) or substantia nigra (Chido and Antelman, 1980) and no change in DA turnover (Keller et al., 1980). Prolonged tricyclic antidepressant treatment may in susceptible individuals produce Parkinsonism and tardive dsykinesia and this might explain the DA antagonism of these compounds. 9.2.2. Norepinephrine receptors 9.2.2.1. Introduction There is general agreement that acute treatment with tricyclic antidepressants leads to a blockade of either monoamine reuptake or an inhibition of central adrenergic receptors coupled to adenylate cyclase. Acute inhibition of either beta or alpha~ adrenergic receptors may account for the ability of tricyclic antidepressants to control agitated depressions, produce hypotension and cause sedation. Chronic treatment with tricyclic antidepressants, on the other hand, results in a "down regulation" of beta and alpha2 adrenoreceptors. In order to explain the relevance of receptor desensitization the following possibilities are offered: (1) Chronic blockade of reuptake of NE raises the level of the monoamine in the synaptic junction with concomitant stimulation of the alpha2 presynaptic receptors. When these receptors which control the release of NE become desensitized, the "brake" mechanism controlling transmitter release is lost and even more NE is available in the synaptic junction to effect mood elevation at postsynaptic sites. This supposition would support the monoamine hypothesis of affective disorders; (2) "Down regulation" of postsynaptic beta receptors in the face of elevated NE (due to blockade of reuptake) could be an adaptive or tolerance mechanism in which the organism adjusts itself to the adverse effects of antidepressants, namely, sedation and hypotension; (3) "Down regulation" of postsynaptic beta receptors suggest that during depression receptors are supersensitive and this contention leads to the following alternative conclusion: (a) Norepinephrine is an inhibitory transmitter in the brain and receptor supersensitivity lends itself to slowing

4~

( ; . ( ' . P,\J '~i R

down of "affect", hence depression. Receptor subscnsitivity after antidepressant trc~lment therefore reduces the depressant action of NE and hence mood elewttion; and (h) the depressive state is due to lowered levels of functionally available NE and the receptors attempt to compensate for these deficits by becoming superscnsitive. When suflicicnl N E is now made available by antidepressant treatment the receptors then return to their neutral state and function under normal conditions. The idea that reccpt~,r "'down regulation" is required before antidepressant action is achieved is especially attractive because most mood elevating drugs take 2-3 weeks to yield therapeutic benefit. The argument against this statement is the immediate mood elevation produced by amphetamines. Chronic amphetamine treatment likewise leads to "down regulation'" of adrenergic receptors, an action that may account for its potentiality to produce tolcrancc. The following discussion does provide evidence for and against these diverse and provoking hypotheses which have been recently advanced by independent investigators, each promoting one or more theoretical aspects, to account for their experimental cvidencc (for further discussions see: Richelson, 197gb; Sulser, 1978: U'Prichard el al., 197S: Maas. 1979; Collis and Shepherd, 1980: []arrison-Read, 1981; Maj, 1981; Sugrue, 1()81). 9.2.2.2. A c u t e actions In most investigations tricyclic antidepressants at concentrations from 100 to {). 1 /,LM inhibit NE stimulation of cyclic AMP in tissue slices, cultured astroglia, and broken cellular preparations from a variety of central and even peripheral tissues. Hydroxylated metabolites of imipramine and desipramine have the least potency in this regard. Not enough detailed studies have been conducted to determine the relative potencies of the clinically used compounds (Huang and Daly, 1972; Palmer et al., 1972b; Palmer, 1973, 1976; Frazer et al., 1974: Free et al., 1974; Sawaya et al., 1977; Jones, 1978: 11ertz el al., 1980). Acute injections of imipramine (with or without concurrent tri-iodothyronine), but not desipramine, led to a reduction in NE-elicited cyclic AMP in rat cortical slices. Moreover, under in vitro conditions imipramine did not block the action of isoprotcrenol on cyclic AMP in these preparations, indicating a possible alpha adrenergic blocking action for the tricyclics (Frazer et al.. 1974, 1978). In some reported instances tricyclics clcw~tcd both basal (high doses) and NE (low doses) accumulation of cyclic AMP (Huang and Daly, 1972, 1974; Berndt and Schwabe, 1973; Jones, 1978). Huang and Daly (1974) rcported th at this observed phenomenon resulted from an inhibitory action of tricyclics on adenosinc uptake. In one negative study Schmidt and Thornberry (1977) did not observe any effect of desipramine or fluoxetine on NE-cyclic AMP in rat limbic forcbrain. Tricyclic antidepressants bind (in vitro) to alpha~ adrenergic receptors with a greater degree of potency than to alpha2 receptors. Little or no interference with beta adrcncrgic binding occurs with tricyclic antidepressants (U'Prichard et al., 1978; Brown et al., 198(I; Maggi et al., 1980; Hall and Ogren, 1981). In another acute study direct ionophorctic ejection of desipramine onto rat Purkinje cells depressed cellular discharge. At lower doses desipramine potentiated NE-induced depression of Purkinje cell firing rates. The action of desipramine was abolished by destruction of the NE fiber inputs to the cerebellum using 6-hydroxydopamine (Schultz et al., 1981). 9.2.2.3. C h r o n i c effects" Frazer and coworkers in 1974 reported the initial findings which opened up a tremendous investigative effort as a means to unravel the puzzle concerning the molecular sites of actions in the brain which are influenced by chronic antidepressant therapy. In their salient work, rats were treated for 5 days with imipramine and afterwards they noted a decreased capability of NE to stimulate the production of cyclic AMP in incubated tissue slices of cerebral cortex. Schultz in 1976 extended this observation and found that by day 3 of imipramine administration, the subsensitivity became apparent and by day 10 the most extensive reduction in NE-cyclic AMP occurred. Moreover, the subsensitivity was even greater if at least a 24 hr lag period took place betweeen the last injection and sacrifice. In

PSYCHOACTIVE DRUGS, CYCLIC NUCLEOTIDES AND 'IHE CNS

49

further work with the rat cortical preparation desipramine and iprindole were equally effective (Frazer el al., 1978, Wolfe et al., 1978). Moreover, the latter investigators reported that the sensitivity of rat cortical slices to the specific beta agonist, isoproterenol was decreased after tricyclic antidepressant treatment. If the adrenergic nerve endings were destroyed immediately after birth, desipramine was no longer capable of producing receptor hyposensitivity. This suggested that either a continued presence of NE was requirc~t in the synaptic cleft or that the subsensitivity occurred at presynaptic adrenergic receptors (Wolfe et al., 1978). Little evidence exists that presynaptic beta receptors are present in brain (Dahlof, 1981). Chronic treatment of rats with nisoxetine (specific blocker of NE uptake) produced a decrease in NE-cyclic AMP in the rat cortex while fluoxetine an inhibitor of 5-HT uptake was ineffective (Mishra et al., 1979). Within the cerebral cortex the reduction in cyclic AMP stimulation by NE was greatest at about 5 days after the last drug treatment, suggesting that the presence of the drug was not responsible for the observed hyposensitivity. About 7 days later cyclic AMP stimulation returned to normal values (Wolfe et al., 1978). Moreover, the decreased sensitivity of catecholamine elicited adenylate cyclase was also observed in broken cell preparation, however, no change was seen in enzyme sensitivity to fluoride or GTP analogs. In addition, the level of phosphodiesterase was unchanged (Turck et al., 1980). Cyclic GMP systems have not been evaluated after chronic tricyclic antidepressant treatment. In a novel experiment, electrical stimulation of the rat locus coeruleus elevated cyclic AMP content in the rapidly inactivated cerebral cortex. This stimulation of cyclic AMP was attenuated after 2 weeks administration of the following antidepressants: desipramine > imipramine = nomifensine. Neither iprindole, mianserin nor CI-imipramine were effective (Korf et al., 1979). A great deal of work with the rat limbic forebrain region has been performed by Sulser and coworkers (Vetulani et al., 1976a,b; Mishra et al., 1979, 1980a; Mishra and Sulser, 1978, 1981; Sulser, 1978). In this tissue chronic administration of desipramine, iprindole. Cl-imipramine and amitriptyline reduced the ability of NE to stimulate cyclic AMP in tissue slices. The specific 5-HT reuptake inhibitor, ftuoxetine, was not effective. In some instances drug levels were measured in tissue removed 24 hr after the last injection and detectable levels were not evident. It would have been of interest, however, if any active metabolites could have been determined to rule out whether any endogenous antidepressant-like compounds affected the experimental outcome. In a somewhat similar chronic investigation it was shown that the tetracyclic, atypical antidepressant, mianserin, and the 5-HT uptake blocker, zimelidine, both depressed the cyclic AMP responses to NE but not to isoproterenol. Interestingly, desipramine reduced both NE and isoproterenol parameters of stimulation. In addition, the ability of beta adrenergic ligands to bind to respective receptors was reduced after desipramine, an event not shared by mianserin and zimelidine (Mishra e l a l . , 1980a). Even though female rats have been shown to have a lesser capability to synthesize cyclic AMP in response to NE, the receptor hyposensitivity in the limbic forebrain produced by chronic desipramine was identical to males (50% reduction; Mishra and Sulser, 1981). Schmidt and Thornberry (1977) attempted to observe whether endogenous stores of 5-HT would influence NE-elicited cyclic AMP in the limbic forebrain. Neither reduction in synthesis (p-chloro-phenylalanine) or inhibition of uptake (fluoxetine) were effective. Moreover chronic desipramine produced results similar to those of Sulser's group. When mouse astrocytes were grown in culture in the presence of amitriptyline, the stimulation of cyclic AMP by isoproterenol was less than controls (Hertz el al., 1981). This observation is important because many of the responses reported with both ligand binding techniques and cyclic AMP activation do not take into account any contributions of the glia. More importantly is the fact that chronic desipramine does not reduce beta adrenergic sensitivity in some peripheral tissues (rat heart and diaphragm; Frazer et al., 1978), suggesting that "down regulation" of receptors is unique to brain. Both chronic imipramine and desipramine reduce NE-cyclic AMP in the rat cerebellum (Schultz et al., 1981 ) and pineal (Moyer el al., 1981). The latter actions of desipramine and

5tl

(i. ('. P',l ,,u I¢

iprindole were: blocked by pineal denervation (ganglionectomy); occurred in both incubated cells or homogenatcs; and were uninfluenced by phosphodiestcrasc activity. In receptor ligand binding investigations a host of investigators have determined that chronic injections of desipraminc, amitriptylinc, nortriptylinc, imipramine, Cl-imipraminc, iprindole, trazodonc, doxepin, and mianserin all reduced the density of beta adrenergic receptors in several areas of the rat brain (Banerjec~ e t a l . , 1977; Clements-Jewery, 19"}8, Wolfe et al., 1978: Bergstrom and Kellar, 1979a; Greenberg and Weiss, 1979: Minneman

el al., 1979; K i n n i e r el al., 198(1; M i s h r a el al., 1 9 8 0 a ; S e l l i n g e r - B a r n c t t e

el a / . ,

1980: Moyer el al., 1981: Schultz et al., 1981 ). In two contrasting studies mianserin and zimelidine were ineffective (Mishra et al., 1980a" Sellinger-Barnette et al., 1980). Interestingly, even though cerebral beta receptor hyposensitivity was evident within one week after desipramine treatment, a period of drug administration of 6 weeks was necessary before "down regulation" was seen in subcortical structures (Bergstrom and Kellar, 1979a). In addition, the striatum and cerebellum were resistant to the development of beta receptor subsensitivity in the face of a prolonged sequence of tricyclic antidepressant administration (Bergstrom and Kellar, 1979a: Greenberg and Weiss, 19791. However. the drugs did produce subsensitivity of NE-cyclic AMP in the cerebellum (Schultz eta/., 1981 ). Minneman et al. (1979) showed that chronic desipramine produced a selective "'down regulation" of cortical beta~ receptors while the beta2 receptors were resistant. Neonatal administration of 6-hydroxydopamine or glanglionectomy in the adult obliterated the subsequent actions of the tricyclics on beta receptor subsensitivity in the cerebral cortex (Wolfe et at., 1978) and pineal (Moyer et at., 1981) respectively. The latter findings indicated that the constant presence of NE in the synaptic cleft was a prerequisite for development of "down regulation" of beta receptors. In general the effects of antidepressants on beta receptor ligand binding parallel to some extent the observations on the subsensitivity of adenylate cyclase. In some studies with chronic tricyclic antidepressant treatment, alpha= receptors become subsensitive as accessed by ligand binding techniques. Thus a variety of unrelated investigations do support this contention. Two weeks of amitriptyline treatment to rats reduced the binding of clonidine to the alpha2 receptors in the amygdala, hippocampus, caudate and locus eoeruleus but not in the hypothalamus. Neither acute injections of clonidine were effective nor did amitriptyline added in vitro displace clonidine binding (Smith, C. B. et al., 1981). In peripheral tissue (aorta) chronic desipramine produced an alpha2 receptor subsensitivity to evoked physiological responses (Crews and Smith, 1978). If single cell recordings were made in the locus coeruleus, one week of desipraminc treatment reduced the sensitivity of alpha, receptors in that brain region (McMillen et al., 1980). Preskon et al. (1980a,b) reported that various tricyclic antidepressants enhanced the capillary permeability of water and ethanol into the brain. Chronic drug treatment led to an even greater efficiency of molecular exchange across the blood-brain barrier. Since NE action is involved in this phenomenon, it is likely that tricyclics acted to diminish the action of the alpha2 receptors. Combinations of desipramine and phenoxybenzamine (alpha blocker) led to an accelerated "'down regulation" of beta receptors in the rat cerebrum. Crews et al. (1981) postulated that phenoxybenzamine inhibited the alpha= receptor which in turn allowed for greater release of NE in the synapse. Thus, reuptake of NE was prevented (desipramine) and more NE was available to desensitize the receptors. Assessment of alpha2 receptor sensitivity would have been desirable in this study. However, it would have been difficult as phenoxybenzamine binds tightly to receptors and its presence would affect attachment of a competing ligand. Other alphae blockers should have been used, as it would be important to determine whether simultaneous alpha= and beta receptor subsensitivity to tricyclics took place in the same brain region. In patients, chronic desipramine led to an attenuated ability of injected clonidine to increase blood pressure and plasma levels of the NE metabolite (3-Methoxy-4-OH-phenethylene-glycol, M H P G ) , an event thought to occur via a depressed alpha= receptor (Charney et al., 1981). The binding of the nonspecific alpha receptor ligand, dihydroergocriptine in the rat cerebrum was not influenced by a 12-week administration of desipramine (Bergstrom and Kellar.

PSYCHOACTIVE DRUGS, CYCLIC NUCLEOTII)ES AND THE C N S

51

1979a). Moreover, chronic mianserin appeared not to influence alpha2 receptor sensitivity in rat brain (Sugrue, 1980). This discussion while disjointed and not an exhaustive review of alpha2 receptor actions does point to a new approach toward understanding the molecular loci affected by chronic tricyclic treatment. In other work, chronic desipramine or imipramine reduced the binding sites for imipramine in rat hippocampus and cortex (Kinnier et al., 1980; Raisman et al., 1980). Whether the sites are alpha2, beta or reuptake sites is not known. Chronic desipramine likewise reduced the responses of iontophoretically applied NE to cerebellar Purkinje cells, an event roughly correlated with decreased cyclic AMP responses and beta receptor binding (Schultz et al., 1981). Olpe and coworkers (1980, 1981) demonstrated that chronic treatment of rats with desipramine, Cl-imipramine, maprotiline and mianserin, but not iprindole, led to a decreased sensitivity of cingulate cortical neurons to respond to iontophoretically released NE. 9.2.3. H&tamine receptors 9.2.3.1. Introduction A recent proliferation of scientific literature has failed to establish a link between the therapeutic effects of tricyclic antidepressants and their inhibitory action on central histamine receptors. Some authors have suggested that inhibition of H2 receptors represents a common site of action of antidepressants and this was related to their therapeutic efficacy. The hypothesis fails to take into account several aspects including: the immediate mood elevating actions of agents devoid of histamine blocking action e.g. amphetamine, the action of monoamine oxidase inhibitors, the activation of adenylate cyclase by H~ receptors and the action of the tricyclics as extremely potent H~ blockers. However, the latter site of action is thought to relate principally to the sedative producing properties of H~ antagonists. Another consistent problem has been the lack of cbronic investigations. Most likely the antihistaminic actions represent the well-known anticholinergic-antihistamine side effects encountered during the initial phases of tricyclic antidepressant therapy (see Section 9.2.5). Nevertheless, the findings have stimulated a lively debate and generated some interesting scientific findings (Green and Maayani, 1977; Kanof and Greengard, 1978; Richelson, 1979a,b; Schwartz et al., 1981a,b; Chronister et al., 1982; Maayani et al., 1982). 9.2.3.2. A c u t e Studies In the rabbit cerebral cortex we evaluated the action of imipramine and its analogs toward antagonism of histamine-elicited accumulation of cyclic AMP. In both slices and broken cells imipramine, Cl-imipramine, 8-OH-Cl-imipramine and 2-OH-Cl-imipramine were considerably more potent than desipramine and 2-OH-desipramine. The subtypes of receptors involved were not determined, but the data again indicated the importance of active metabolites when considering the action of parent compounds (Palmer etal., 1977c). Richelson (1978a, 1979a,b) performed an extensive study with the cultured mouse neuroblastoma cell line. These cells generate a marked elevation in cyclic GMP in response to added histamine and the phenomenon appears mediated exclusively by H1 receptors. In fact doxepin and amitriptyline were found to be the most potent of all known H1 blockers. All tricyclics tested were likewise relatively potent blockers on either Hi elicited cyclic GMP synthsis or H~ receptors. The secondary amine tricyclic antidepressants (nortriptyline > protriptyline > desipramine) were considerably less active in both testing procedures than were tertiary amine tricyclics. The data correlated with the H~ induced sedative properties of tertiary amine tricyclics. In the rat and mouse brain little work has been accomplished as a means to link the histamine receptor blocking activities of tricyclic antidepressants to those of cyclic nucleotide generation (Maayani et al., 1982). Doxepin binding to rat brain displayed a regional distribution that was correlated with the presence of H~ receptors. Binding was

52

(i

( . I'x]~I~l~

greatest in the hypothalamus IMIowed by in descending order of receptor density, midbrain, cerebrum, brain stem, striatum, thalamus and cerebellum. Tertiary tricyclics were the most potent antidepressants toward inhibition of doxcpin binding (Taylor and Richelson, 1980, 1982). ttall and ()gren ( 1981 )reportcd the following order of potency for displacement of the t41 ligand, mcpyramine from rat brain: mianserinc and amitriptylinc > imipramine, maprotiline > Cl-imipraminc and nortriptylinc (also sec Tran et al., 1981 h)r analogous data). Injections of radioactive mepyramine into mice labeled the ttl receptors in rive. Pretreatment of animals with antidepressants was shown to inhibit mepyramine labeling. This displacement correlated in some respects to in vitro studies. As usual doxepin was a highly potent agent, interestingly, so were the atypical antidepressants, mianserin and iprindole (l)iffley et at., 1980). The major controversy surrounding antidepressant action at histamine receptors has occurred in the guinea pig brain-cerebrum and hippocampus. Using guinea pig cortical vesicles prelabeled with adenine, Psychoyos (1978, 1981) showed that tricyclic antidepressants in the following order of potency inhibited H t agonist-elicited accumulation of cyclic AMP: amitriptyline, mianserin, triimipramine, maprotiline, imipramine and fluoxetine. In an evaluation of both H t, H~ binding and inhibition of adenylate cvclase, Coupet and Szuchs-Myers (1981) concluded that tricyclic antidepressants were more potent inhibitors of H1 receptor mediated events. Tran et al.'s (1981) data with doxepin binding further supported this contention. In contrast data by Green and Maayani (1977), Kanoff and Greengard (1978), Tiiong et al. (1980), Schwartz et al. (1981), and Maayani et al. (1982) using either broken cell preparations or tissue slices and employing selective H, agonists argue that tricyclic antidepressants primarily act to inhibit t-1~receptors in guinea pig brain. The order of potency for inhibition generally followed the patterns described above for Hi blockade, i.e. the tertiary amines and atypical antidepressants were considerably more potent than secondary amines. From a historical standpoint it is of interest that in the first study performed with guinca pig slices desipramine had no reported action on histamine-cyclic AMP (Kodama et al., t971). 9.2.3.3. C h r o n i c studies In one recent work Pandey and colleagues (1982) injected guinea pigs on a chronic basis with tricyclic antidepressants (amitriptyline and desipramine), a monoamine oxidase inhibitor (phenelzine) or chlorpromazine. When tissue slices of cerebral cortex were evaluated 24 hr after the last injection, histamine-evoked synthesis of cyclic AMP was diminished in the animals receiving the antidepressants, but not the phenothiazinc. Maayani et al. (1982) were unable to observe any change in hippocampal H, receptor sensitivity after injection of amitriptyline to guinea pigs on a chronic basis. These discrepancies could result from regional specificity, the use of different enzyme preparations, or reflect an action unique to the 1t~ receptor.

9.2.3.4. C o n c l u s i o n The data are unequivocal in that tricyclic and the so called atypical antidepressants are extremely potent inhibitors of histamine (H~ and H2) elicited cyclic AMP and cyclic G M P systems in mammalian brain. Histamine receptors are inhibited to a greater extent by tertiary amine tricyclics than by their corresponding secondary amine metabolites or analogs. Antagonism of H~ receptors may be related to the sedative-anticholinergic properties that these agents, especially the tertiary tricyclics, share with muscarinic blockers. In many patients these sedative-anticholinergic effects are transient and in perhaps the one chronic study revealing a "down regulation" of histamine-adenylate, cyclase corresponds to the development of this tolerance mechanism. What role H~ receptors play in the physiology of brain behavior is unknown. It appears unlikely that histamine receptor blockades as a parallel to mood elevation, because other compounds which elevate mood have little antihistamine actions.

PSYCHOACTIVE DRUGS, CYCLIC NUCI.EOTIDES AND THE CNS

53

9.2.4. Serotonin receptors To my knowledge no detailed work has been accomplished with regard to tricyclic antidepressant action on cyclic nucleotide systems in the brain that are activated by serotonin. Huang and Daly (1972) did report a reduction by imipramine of K + plus 5-HT elicited cyclic AMP in guinea pig cerebral cortex. Mianserin is an atypical antidepressant with relatively potent 5-HT receptor antagonist activity. This agent did indeed inhibit 5-HT stimulated adenylate cyclase in vitro. In addition, mianserin only inhibited slightly the isoproterenol stimulation of adenylate cyclase (Enjalbert et al., 1978; MacDermot et al., 1979; Nelson etal., 1980). Under acute in vitro conditions, tricyclic antidepressants (imipramine and CI-imipramine) decrease the affinity of 5-HT for receptors in brain membranes (Fillion and Fillion, 1981) and similarly displace LSD and 5-HT from rat brain binding sites (Hall and Ogren, 1981). When animals were chronically administered tricyclics (approximately 21 days) there was a reduction in the number of 5-HT receptor binding sites in several areas of the rat brain. The action was postsynaptic, because destruction of presynaptic nerve terminals with 5, 6 diOH-tryptamine concomitant with tricyclic administration did not modify 5-HT binding (Segawa et al., 1979; Maggi et al., 1980; Wong and Bymaster, 1981). There was some controversy as to whether chronic administration of the specific 5-HT uptake inhibitor, fluoxetine, would induce 5-HT receptor subsensitivity, Maggi et al. (1980) reported negative data while Wong and Bymaster's (1981) findings were affirmative. Tricyclic antidepressant administration required a rather long period of time (at least 3 weeks) before 5-HT receptor hyposensitivity became evident. Shorter time periods of drug administration were not effective (Bergstrom and Kellar, 1979a; Savage etal., 1979). Chronic electroconvulsive therapy did not alter 5-HT receptor sensitivity in rat brain regions (Kellar et al., 1981). Serotonin-2 binding sites were reduced in the frontal cortex of mice following both acute and chronic injections of the atypical antidepressant, mianserin (Blackshear and Sanders-Bush, 1982). The limited amount of data along with the findings with monoamine oxidase inhibitors (see 9.3) do reveal a "'down regulation" of brain 5-HT receptors after a chronic period of drug administration. Whether these observations represent an adaptation to a side effect of the drug or are of therapeutic benefit is not known. 9.2.5. Cholinergic receptors The tricyclic antidepressant drugs are well-known for their anticholinergic properties which occur in both central and peripheral tissues. Common symptoms of this antimuscarinic action include: dry mouth, urinary retention, blurred vision, exacerbation of narrow angle glaucoma, sedation, constipation, confusion, speech blockage, hot dry skin and delirium. An antidote for overdose is the acetylcholinesterase inhibitor physostigmine (Richelson, 1979b). With such a well-defined property of tricyclic antidepressants it is somewhat surprising that so few different experimental techniques have been utilized to evaluate their effects on cholinergic systems. This anticholinergic action is not without clinical correlations, because for highly susceptible individuals (e.g. an elderly man with benign prostatic hypertrophy) suffering from depression a selection of a less potent anticholinergic tricyclic might prove to be the drug to initiate therapy (Richelson, 1979b). The majority of the work associated with tricyclic antidepressant action on cyclic GMP systems in neural tissue has been performed by Richelson and coworkers (see below). They have used exclusively the mouse neuroblastoma cell culture which potently responds to either cholinergic-muscarinic agonists or histamine to synthesize cyclic GMP. The equilibrium dissociation constant (K~) values for anticholinergic action of tricyclic antidepressant inhibition of cyclic GMP are approximately 2 orders of magnitude weaker than corresponding antihistamine actions. The tertiary amine tricyclics are considerably more powerful than secondary amines in their ability to modify muscarinic induced cyclic GMP. Furthermore, the order of antimuscarinic potency on cyclic GMP correlates with their ability to modify either carbamylcholine-induced contractions in guinea pig ileum or

5-I

( ; . ( ' . l)\l M~k

muscarinic receptor antagonism in human caudate. The order of potency for lricyclics t~, inhibit cyclic GMP generation is: amitriptyline > doxepine, imipramine > trimipramine 3, 3-CI-imipramine, nortriptyline > desipramine, protriptyline > 3-C1-2-OH-imipramine -'::. 2-OH imipramine > didesmethyl-imipramine (Richelson, 1978b; 1979b; Richelson and Divinitz-Romero, 1977; Petersen and Richelson, 1982). In addition, tricyclic antidepressants inhibit quinuclidinyl benzylate (QNB) binding to cholinergic receptors in ral brain (Hall and Ogren, 1981). In one chronic study a 2l day tricyclic administration did not alter the patterns of cholincrgic binding in rat cerebrum, striatum, and hippocampus (Maggi et al., 1980). In a clinical investigation, tricyclic antidepressant treatment resulted in diminished levels of cyclic GMP in the CSF (Smith et at., 1976). Whether this was a result of central cholinergic receptor blockade was not determined. It would be of interest if some brain model system were utilized for chronic type studies. An important fact would be to determine if tolerance to the side effects of tricyclic antidepressant therapy would be manifested as a densitization of muscarinic receptors mediating the synthesis of cyclic GMP.

9.2.6. Miscellaneous actions of trio'clic antidepressants 9.2.6.1. Adenosine In a single investigation Sattin et al. (1978) showed that large concentrations of tricyclic antidepressants when added to incubated slices of guinea pig cerebral cortex evoked a stimulation of cyclic AMP production. The antidepressant action was antagonized by theophylline indicating an action of tricyclics either to release or to prevent the uptake of adenosine. Adenosine when iontophoretically applied to cortical neurons slowed their rate of firing, an action augmented by the coejection of tricyclic antidepressants. 9.2.6.2. lnhibitiott ~f o'clic A M P phosphodiesterases In the early work on cyclic nucleotide systems there seemed to be one answer as to the manner in which the tricyclic antidepressants exerted their influence on cyclic nucleotide systems. Many reviews quoted such work with phosphodiesterase but failed to mention thc relatively large drug doses needed to achieve enzyme inhibition. However, m vivo studies (see Section 9.2.1.1) do indicate that therapeutic levels of the agents raise the steady-state levels of cyclic AMP. This may not be solely attributed to their catecholamine potentiating action because amphetamine generally does not act in this regard (Section 11). Furthermore, the concentrations of tricyclics shown to inhibit phosphodiesterase were equal or smaller than methylxanthines. It is doubtful that inhibition of phosphodiesterase is a direct therapeutic effect because amphetamines, monoamine oxidase inhibitors and electroconvulsive therapy all elevate mood without inhibiting the enzyme. Yet the ability of tricyclic antidepressants to increase the availability of monoamines along with their inhibition of phosphodiesterase would be expected to augment cyclic AMP levels a t synaptic loci. A wtriety of different preparations have been used to evaluate tricyclic antidepressant inhibition of cyclic AMP phosphodiesterase: Human platelets and brain (Pichard et al., 1972); mouse brain (Roberts and Simonsen, 1970); rabbit brain (Honda and lmamura, 1968); heart (Ramsden, 1970; Weinryb et al., 1972); and rat brain including isolated cortical neuronal and glial-enriched fractions (Muschek and McNeill, 1971 ; Weinryb etal., 1972; Berndt and Schwabe, 1973: Levin and Weiss, 1976; Palmer, 1976). Phosphodiesterase in central tissues was more susceptible to drug inhibition than in peripheral tissues. Levin and Weiss (19751 reported that tricyclic antidepressants inhibited Cae+-calmodulin activated phosphodiesterase, albeit to a lesser extent than phenothiazines. When tested on the neuronal and glial fractions 2-OH-desipramine was more potent than many parent compounds (Palmer, 19761.

PSYCHOACTIVEDRUGS, CYCLICNUCLEOT1DESAND THE CNS

55

9.2.6.3. Fluoride-sensitive adenylate cyclase

In one study with homogenates of neuronal and glial fractions from rat cerebral cortex, Cl-imipramine, iprindole and nortriptyline were the most potent of 10 tricyclic antidepressants and hydroxylated metabolites to inhibit fluoride activated adenylate cyclase. The ICs0 value never went below 0.5 mM and this value was higher than similar drug actions on phospbodiesterase (Palmer, 1976). 9.2.6.4. Peripheral tissues " Tricyclic antidepressants inhibited catecholamine-induced release of free fatty acids from incubated fat cells, in keeping with their potential to block adrenergic receptors (Himms-Hagen, 1970; Nakano and Ishii, 1970). 9.2.6.5. Cyclic G M P The work with acetylcholine and histamine is discussed above (9.4 and 9.5). Desipfamine was shown to block the NE-elicited, but not the K +-induced, accumulation of cyclic GMP in rat pineal and posterior pituitary glands ( O ' D e a et al., 1978). 9.2.7. Clinical studies Several groups of investigators have shown a decrease in the 24-hr urinary content of cyclic AMP in severely depressed patients including those suffering from neurotic, endogenous and psychotic depressions. The degree of attenuated cyclic AMP levels was not correlated with lack of physical activity. In some studies urinary cyclic AMP levels became elevated during therapeutic management with antidepressants (Abdulla and Hamadah, 1970; Paul et al., 1970a, 1971 a,b; Sinanan et al., 1975). I n a rat model, reserpine decreased urinary cyclic AMP while tricyclic antidepressant injections led to elevated levels (Palmer and Evan, 1974; Keatinge et al., 1975). On the other hand, Smith et al. (1976) were unable in patients to correlate any changes in CSF levels of cyclic AMP or G M P to any type of mental illness. Prostaglandin E 1-induced formation of cyclic AMP in platelets was not correlated with any degree of depressive illness or other behavioral disorder, except acute schizophrenia when an increase was seen (Wang et al., 1974; Pandey and Davis, 1979). These workers, however, showed a deficit in the ability of isoproterenol to stimulate cyclic AMP in intact leukocytes taken from depressed patients. Thus the possibility exists that depressives may have a peripheral beta receptor deficit (also see Pandey et al., 1979). More work will be required to determine if such a model system will be useful as a clinical test for adrenergic type depressions. Eleven depressed patients were treated with a beta2 adrenergic agonist, salbutamol, and adrenergic sensitivity was evaluated by measuring the plasma cyclic AMP increase after treatment. Salbutamol exhibited both antidepressant efficacy and produced a subsensitivity of the cyclic AMP responses with a time course paralleling the therapeutic effects (Lerer et al., 1981). These data implicate beta2 receptors in depression. On the other hand, Minneman et al. (1979) reported that chronic tricyclic antidepressant injections to rats led to a "down regulation" of only beta~ receptors. However, before any hypothesis can be made with regard to a particular subtype of receptor with respect to depression, more investigations will be required to identify the brain regions and species of neurons responsible for the illness. Betat-beta2 as well as alphas-alpha 2 receptor interactions will also have to be considered. 9.3. MONOAMINE OX1DASE INHIBITORS

9.3.1. A c u t e effects In the initial work Huang and Daly (1972) were unable to observe any action of several monoamine oxidase inbibitors to influence the accumulation of cyclic AMP in incubated

5(~

( ~ . ( ' . PX[MtR

tissue slices of guinea pig cerebral cortex. Whether or not a species difference existed between monoamine oxidase activity in the rat and guinea pig was not known: but studies with all m a j o r regions of the rat brain (cerebrum, brain stem, thalamus, hypothalamus, hippocampus, cerebellum, and limbic forebrain) revealed enhanced basal levels of cyclic A M P in incubated tissue slices (Pahner et al., 1973: Vetulani el al.. 1976a,b). In these studies pargyline did not enhance the action of N E on cyclic AMP, however, when the time course of NE-elicited cyclic A M P accumulation was measured, pargyline did magnify the action of N E (Palmer, 1973). When the monoamine oxidase inhibitors were injected into animals and cyclic A M P was measured following rapid inactivation of the brain by microwave irradiation, pargyline elevated cyclic A M P in the mouse cerebrum (Pahner et al., 1977a) and deprenyl was active in the rat cerebellum (Mao el al., 1974). Paul and coworkers (1970b) were unable to determine such an effect. Even when beta-phenylisopropylhydrazine was administered to mice prior to I,-DOPA, followed by rapid freezing of the brain, whole brain cyclic A M P levels only tended to increase but the levels were not significant (Paul e l al., 1970b). Acute injections of harmaline into rats did augment cyclic G M P levels in the cerebellum, an action associated with excitatory neurotransmission processes and not thought to occur via monoamine oxidase inhibition (Mao el al., 1974, 1975a,b; O p m e e r et al., 1976; Biggio el al., 1977a). 9.3.2. C h r o n i c effects Injections of m o n o a m i n e oxidase inhibitors over a period of time (4-2l days) in general produce two major phenomena: (1) a decrease in beta adrenergic receptor numbers: and (2) a decrease in the ability of beta adrenergic agonists and NE to elicit a synthesis of cyclic AMP. The majority of these investigations were performed on rat cerebral cortex, limbic forebrain, and in one instance in the pineal gland. Monoamine oxidase inhibitors that are of the "A'" type (clorgyline-metabolize preferentially NE and 5-HT) and nonspecitic inhibitors (phenelzine) were therefore more potent than the "B" type inhibitors (deprenyl). At least 9 days after the last injection of the m o n o a m i n e oxidase inhibitor were necessary before both the diminished beta receptor activity and the beta adrenergic stimulation of cyclic A M P returned to normal. Moreover, these actions of chronic monoamine oxidase inhibitors on NE-cyclic A M P occurred in either broken cell or tissue slice preparations. Chronic injections of monoamine oxidase inhibitors in combination with desipramine did not further reduce either beta receptors or NE-cyclic A M P to levels below that seen with tricyclic antidepressants alone. If central N E stores were depleted by reserpine during monoamine oxidase inhibitor administration, no "down regulation" of either beta receptors or NE-adenylate cyclase occurred, indicating that elevated N E levels were required to produce the subsensitivity (Vetulani et al., 1976a,b; Wolfe el al., 1078: Sawtge el al., 1979, 1980: Sellinger-Barnette el al., 1980; Moyer el al., 1981 : Cohen el al.. 1982). Chronic injections (21 days) of the m o n o a m i n c oxidase-A inhibitor, clorgylinc, led to a reduction in numbers of alpha2 (62T,,), alphat (36%) and beta (34%) adrenergic receptors in rat cortex. In the brain stem the alpha2 receptor decrease was approximately equal to the beta receptors (60 vs 74%). Cohen el al. (1982) postulated that depression was due to a supersensitivity of the alpha_~ system which would lead to less NE release for availability at postsynaptic receptors. Therefore, monoamine oxidase inhibitors act to increase NE and this increase resets the activity of presynaptic alpha 2 receptors. They also felt that the use of selective types of m o n o a m i n e oxidase inhibitors might lead to better therapeutic m a n a g e m e n t of depression, because pargyline, a more specific ~'B" type of enzwne inhibitor, was ineffective. Chronic administration of monoamine oxidase " A " inhibitors but not "'B- type inhibitors, led to a reduction in the binding of 5-HT in the rat cerebral cortex. Administration of 5-HT agonists yielded identical results, however, if agents that deplete central levels of 5-HT were also given with the monoamine oxidase inhibitor, no subsensitivity of 5-HT binding was evident. The data indicated that the action of monoamine oxidase inhibitors

PSYCHOACTIVE DRUGS, CYCLIC NUCLEOTIDES AND THE CNS

57

was indirect, and only elevated levels of 5-HT were necessary to desensitize the receptors (Savage et al., 1979, 1980). Chronic injections of phenelzine to guinea pigs led to a reduction in the ability of histamine to evoke synthesis of cyclic AMP in slices of cerebral cortex (Pandey et al., 1982). 9.4. ELECTROCONVULSIVETHERAPY(ECT) 9.4.1. A c u t e E C T The observation that acute administration of electroshock to animals led to a steadystate elevation in cyclic nucleotides and adenylate cyclase is discussed in detail in (Section 16---Convulsants and Anticonvulsants). Moreover, electrical stimulation applied directly to incubated tissue slices augments levels of cyclic AMP (Section 3.6.8). 9.4.2. Chronic E C T Acute E C T to rats raises the basal and hormonal activation of cortical adenylate cyclase (Pandey et al., 1976) while chronic (3 days or more) application of E C T to rats produces, as with other antidepressants, both a reduction in beta receptor numbers and beta adrenergic stimulation of cyclic AMP. Apparently, 5-HT, DA, muscarinic or alpha adrenergic receptor numbers are unchanged in the cerebral cortex of rats subsequent to chronic ECT. However, when single unit recordings were made, the D A autoreceptors in the rat substantia nigra were desensitized after chronic ECT. The effect of E C T was specific because foot shock stress did not influence beta receptor activity in the brain. Moreover, the onset of ECT-induced subsensitivity, in keeping with its clinical profile, is faster than that of the tricyclic antidepressants. The action of chronic E C T was shown to diminish beta receptor numbers in only selected brain regions namely, the cerebrum, hippocampus, and limbic forebrain. No such changes were observed in the striatum, hypothalamus or cerebellum. Electroconvulsive shock prevented the development of the supersensitivity of cyclic AMP generating systems seen after destruction of adrenergic nerve endings with 6-hydroxydopamine or depletion of NE by reserpine. Therefore, before E C T is effective in reducing the sensitivity of the postsynaptic beta adrenergic receptor systems, a critical level of NE must be sustained within the synaptic cleft (Vetulani and Sulser, 1975; Vetulani et al., 1976a,b; Gillespie et al., 1979; Bergstrom and Kellar, 1979b; Chiode and Antelman, 1980; Kellar et al., 1981). More work is definitely needed with this ECT model. Unanswered questions are: how does chronic E C T affect the sensitivity of guanylate cyclase-cyclic GMP systems, the phosphodiesterases, and what role does adenosine play with regard to the experimental outcome?; Would treatment with methylxanthines to block adenosine receptors prevent the formation of the receptor hyposensitivity? Electrical stimulation does release adenosine from central tissue and this may account for the continued activation of adenylate cyclase (Phillis and Wu, 1981). 9.4.3. Clinical studies In a clinical investigation Hamadah and coworkers treated 13 depressed women with ECT. Twelve patients showed elevated levels of urinary cyclic AMP on the day of treatment. These levels dropped within 24 hr. Control patients did not show an increase in urinary cyclic AMP. In contrast, Moyes and Moyes (1976) were unable to detect significant alterations in urinary cyclic AMP in in-patients undergoing ECT. 9.5. SUMMARY The data from the vast work with tricyclic type antidepressants is summarized in Table 9. From the diverse data reported, intriguing possibilities have been presented. A few major areas remain to be explored: The role of the alphal receptor, role of guanylate cyclase, and subsequent influences on the protein kinases. The latter may prove to be especially important because of its unique action in initiating or slowing down the

5~

( ] . ( ' . PALMER

TABLE 9. SUMMARY OF EFFECTS OF TRICYCLIC AND A FYPICAL ANTIDEPRESSANTS ON RECEPIOR [N[IIAI~ D CYC l I( A M P SYSIEMS

Receptor

Acute action

Clinical correlate

Chronic actions

Clinical correlate Parkinsonism Tardivcdyskinesia

DA-

Block

Parkinsonism

?

NE alpha~

Block

Hypotension sedation Agitated depression

?

NE alpha: NE beta

? Block

Serotonin Acetylcholine

Block Block

Histamine

Block

Sedation? Hypotension

Subsensitivity Subsensitivity

Anticholinergic profile Sedation-anticholinergic profile

Subsensitivity ? Subsensitivity

Antidepressant Antidepressantor tolerance Antidepressant Tolerance to antihistamine-anticholinergic side effects

metabolic events initiated by receptor-coupled cyclic nucleotide activation. Furthermore. it seems new approaches (e.g. electrophysiological, in vivo studies) need to be explored, rather than further continuation of more kinetic analysis associated with receptor ligand binding parameters or adenylate cyclase activation. In addition, Kostowski (1982) raised some interesting points in that NE interactions with 5-HT or D A at critical synapses might produce different clinical symptoms. In general, all antidepressants including amphetamine and even methylxanthines (Goldberg et al., 1982) possess the capability of: (1) enhancing the functional activity levels of monoamines; and (2) inducing some type of receptor subsensitivity after continued administration. Most likely both events are necessary to achieve antidepressant therapy.

10. Stimulants--Amphetamines and Cocaine 10.1. AMPHETAMINE 10.1.1. Introduction Amphetamines have major excitatory effects on the brain, attributed to their ability to enhance the action of both D A and NE. Enhancement of catecholamine action is most likely achieved by (in the following order of importance): (1) release of D A and NE; (2) inhibition of reuptake; (3) inhibition of monoamine oxidase (larger doses); (4) rapid formation of active metabolites (p-OH-amphetamine and p-OH-norephedrine) which act like false transmitters evoking a sustained displacement of catecholamines from storage granules; and (5) a degree of receptor action (more prominent in peripheral tissues ). The " d " form of amphetamine is faster acting than the "l" isomer. With this propensity to elicit catecholamine action, in keeping with the monoamine hypothesis of central disorders, amphetamines in large concentrations readily cause a psychosis almost indistinguishable from acute paranoid schizophrenia. The only major differences are the preoccupation with sexual activity and stereotyped motor movements (long term ingestion) seen in amphetamine users. Amphetamine psychosis can be controlled by neuroleptics. There is some debate as to whether amphetamines produce physical dependence (addiction). There is a lack of drug craving during withdrawal, however, behavioral and autonomic symptoms are present and long lasting. With such powerful catecholamine actions of amphetamines, it would be expected that dramatic changes in cyclic nucleotides would be observed during experimental testing. Such has not been the case as will be evident in the following discussion (for further reading see: Snyder et al., 1974).

PSYCHOACTIVE DRUGS, CYCLIC NUCLEOTIDES AND THE CNS

59

10.1.2. A c u t e effects 10.1.2.1. In vivo studies As early as 1972 Ferrendelli and colleagues observed a dose-response increase in cyclic GMP in the rapidly frozen mouse cerebellum following an acute injection of amphetamine. The action was blocked by neuroleptics, but not atropine, indicating an action of amphetamine on catecholamine mechanisms. In further work amphetamine along with other excitatory or stress-induced responses (harmaline, pentylenetetrazol or physical shaking) elevated cyclic GMP principally in the molecular layer of the vermis. Likewise, the molecular layer in the hemispheres and the granular layer showed significantly elevated levels of the nucleotide (Rubin and Ferrendelli, 1977). Additional workers have confirmed these observations and correlated the rise in cyclic GMP to drug induced stereotyped behavior (Gumulka et al., 1976a,b; Haidamous et al., 1980). Two possibilities for this cerebellar action of amphetamine were offered by these investigators: (1) amphetamine elicits indirect excitation of the primary cerebellar afferent pathways-climbing and/or mossy fibers; and (2) the phenomenon occurs as a result of alpha adrenergic receptor activation. Acute injections of amphetamine reportedly produced no effects on steady state levels of cyclic AMP in several brain regions. Since cyclic AMP turns over rapidly in the brain and since larger animals were used, the methods of rapid fixation had not reached the level of sophistication as that presently available (Paul etal., 1970b; Schmidt etal., 1972; Palmer et al., 1973). With the use of better techniques for tissue inactivation, acute injections of amphetamine have been shown to elevate cyclic AMP in rat striatum (Gerhards et al., 1974; Kennedy and Zigmond, 1979), frontal cortex and olfactory tubercle (Kennedy and Zigmond, 1979), and to decrease the nucleotide in the lung (Palmer et al., 1977a). Moreover, p-hydroxyamphetamine injections augmented cyclic AMP in the cerebrum and depleted levels in the lung (Palmer et al., 1977a). The cerebellar content of cyclic AMP was unaffected by amphetamine (Ferrendelli et al., 1972; Gumulka et al., 1976a,b). 10.1.2.2. In vitro studies In incubated slices of mouse cerebellum amphetamine had no effect on cyclic GMP but levels of cyclic AMP were depleted to a small extent (Rubin and Ferrendelli, 1977). In regions of the rat brain, amphetamine was without any influence on basal levels of cyclic AMP (Kakiuchi and Rail, 1968; Weiss and Costa, 1968b; Palmer et al., 1973; Schorderet, 1977; Sulser, 1978), but along with the p-hydroxy metabolite the NE-induced elevation of the nucleotide was inhibited in the hypothalamus (Palmer, 1973). In contrast, amphetamine, p-hydroxyamphetamine and most isomers of p-hydroxy-norephedrine acted to augment the increase in cyclic AMP to low doses of NE in rat limbic forebrain (Mobley et al., 1979). In other in vitro investigations phosphodiester~se activity was not influenced by amphetamine (Beer et al., 1972; Weinryb et al., 1972). Amphetamine antagonized lipolysis in fat cells and blocked catecholamine stimulation of adenylate cyclase in lung and heart (McNeill and Muschek, 1972; Weinryb etal., 1972). 10.1.3. Chronic studies Prolonged injections of amphetamine for 6 days to mice resulted in a highly significant reduction in the usual cyclic AMP response to NE when cerebral slices were examined in vitro. Adenosine-elicited cyclic AMP was likewise reduced, however, to a smaller extent. The decreased responsiveness of the cyclic AMP system was not due to any change in the affinity of NE, but rather a reduction in the maximal activation of the enzyme. Alternatively this action of amphetamine could not be detected in broken cell preparations. After one week of cessation of amphetamines, normal cyclic AMP responses to NE were attained in the tissue slices (Martes et al., 1975; Baudry et al., 1976). Similar data with chronic amphetamine action were reported by Mobley et al. (1979) using the rat limbic forebrain preparation. Moreover, amphetamine given with iprindole reduced the time

(~{I

(i. (.

P \ I MIR

period in which subsensitivity of NE-cyclic AMP occurred. The NE-subscnsitivitv was associated with a decreased concentration of beta receptors. In this study both the decreased NE-cyclic AMP and beta receptors occurred in the limbic forebrain and cerebral cortex. However, the cortical tissue's ability to recover to normal values following cessation of amphetamine was considerably longer in duration than in the limbic forebrain (Manier et al., 1980). Other investigators have likewise shown that chronic amphetamine reduces the density of beta and DA receptors in the brain (Nielsen e t a l . , 1980; Banerjee et al., 1979). Chronic amphetamine injections do not appear to influence urinary levels of cyclic AMP in rats (Palmer and Evan, 1974). I(t. 1.4. ( ' o n c h t s i o n s Amphetamines rejected in vivo most likely elevate cyclic AMP by their well-known actions associated with augmentation of catecholamine levels in the brain. Two observations are apparent under in vitro conditions. The inhibition by amphetamine of neurohumoral elicited cyclic AMP at high agonist levels could represent an attraction (without any intrinsic activity) toward the receptor site which it competes for with NE. However, amphetamine also slightly magnifies the action of low concentrations of NE in tissue slices, presumably by blocking NE reuptake or monoamine oxidase inhibition. The acute stimulation of cerebellar cyclic AMP by anaphetamine may be an indirect activation of afferent fibers to the cerebellum. Chronic amphetamine decreases the sensitivity of beta adrenergic receptors coupled to adenylate cyclase. This may account for the observed tolerance associated with amphetamine administration. Noteworthy was the obserwttion that chronic treatment with large doses of amphetamines elevated both the receptor density and ability of NE to stimulate cyclic AMP. Banerjcc et al. (1979) concluded that these observations were associated with the acute psychosis seen in patients ingesting large amounts of amphetamine. 1(1.2. COCAINE Use of cocaine or "'coke" as it is commonly called has been dated to at least 500 AD in Peru. With the latest fashionable trend for usage, cocaine is considered by many to be a seriously abused drug. The agent is difficult to study in the laboratory because of its added property of local anesthesia. Thus all enzymatic systems associated with excitable membranes would be influenced by the drug. Cocaine does produce sympathomimetic actions presumably by blocking reuptake of catecholamines, however, it action in the brain remains an enigma. Mental feelings are practically indistinguishable from amphetamine, but the onset and duration of cocaine action is shorter. It produces an exaltation of spirit, freedom from fatigue and a sense of well being. Cocaine may cause psychosis, but this action occurs with less frequency than with amphetamine. Chronic use results in paranoia, stereotyped behavior and formication ("coke bugs" or feelings of worms crawling under the skin). Physical dependence is not thought to occur, but upon withdrawal there are feelings of dysphoria, fatigue, anxiety and depression similar to amphetamine (Ray, 1978). In incubated tissue slices prepared from rat brain regions, cocaine did not enhance the action of large concentrations of NE with respect to cyclic AMP accumulation (Palmer, 1973). On the other hand, a slight cocaine-induced enhancement of cyclic AMP in response to low levels of NE was noted in tissue slices of rat cortex (Kalisker et al.,1973) and limbic forebrain (Mobley et al., 1979). In keeping with the latter observation Pandey et al. (1975) reported enhanced basal levels of cyclic AMP in cortical slices prepared from rats that received an acute injection of cocaine. Under in vivo conditions tyrarnine injections into rats resulted in elevated levels of cyclic AMP in the plasma. Pretreatment with cocaine abolished this tyramine action presumably by inhibiting the uptake of tyramine into presynaptic nerve endings and chromaffin cells where it would normally act to displace NE from vesicular storage sites (Kunitada et al., 1978). Post et al. (1979) treated monkeys for either one or ten weeks with cocaine. No significant changes in CSF cyclic AMP were observed overall between controls and drug

PSYCHOACTIVE DRUGS, CYCIJC NUCI.EOT1DES AND TIfE C N S

61

injected animals. However, some cocaine animals developed large fluctuations in their levels of cyclic AMP. These workers concluded that the observation was a result of cocaine-induced activation of central catecholamine pathways. This contention is supported by Banerjee et al. (1979). At one and twelve hours following cocaine injections there was an increase in NE-elicited cyclic AMP in rat brain tissue slices concomitant with elevated binding of beta receptor ligands. The limited data available point out essentially one action of cocaine on cyclic nucleotide systems in the brain, i.e. magnification of catecholamine action. It is unknown whether blockade of monoamine uptake is the mechanism solely responsible for these observations. In some recent metabolic work Hanbauer et al. (1980) evaluated the action of both amphetamine and cocaine on calmodulin function in the striatum. In their view soluble(cytoplasmic) calmodulin activates phosphodiesterase while that derived from membraneparticulate fractions stimulates adenylate cyclase. Amphetamine injections increase the synaptic membrane levels of the activator. The precise inter-relationships of these drugs on striatal DA transmission remains to be determined.

11. Agents that Deplete Monoamines In this section it will be readily apparent that agents interferring with monoamine pathways, synthesis or storage sites within the brain eventually evoke a supersensitivity of the cyclic AMP generating systems to catecholamines. In some cases receptor sensitivity is specific as to either the subtype of receptor influenced or to particular brain regions evaluated. A review by Wagner et al. (1979b) discusses the phenomenon of supersensitivity from another standpoint, with regard to organization and differences among rates of appearance, acute vs chronic supersensitivity, etc. 1 1.1. ALPHA-METHYI,-D-TYROSINE Alpha-methyl-p-tyrosine inhibits tyrosine hydroxylase, the rate limiting step in catecholamine biosynthesis. The agent does have neuroleptic properties and enhances the therapeutic efficacy of drugs such as phenothiazines and reserpine. Use of the drug is limited because of its short duration of action. With regard to effects on cyclic nucleotide systems only limited information is available. When injected over a period of time (daily for 3 days) alpha-methyl-p-tyrosine decreased steady state levels of cyclic AMP in cerebrum and diencephalon of mice whose brains were rapidly inactivated by focused microwave irradiation. In contrast, cyclic AMP was elevated in the lung (Palmer et al., 1977a). The urinary excretion of cyclic AMP was decreased within the first 24 hr period following injected alpha-methyl-p-tyrosine, but upon continued administration cyclic AMP levels became elevated over controls and remained so elevated for 4 additional days (Palmer and Evan, 1974). In another study the noradrenergic pathway of rats when stimulated (locus coeruleus) increased the steadystate content of cerebral cyclic AMP. This response was prevented if rats were pretreated with alpha-methyl-p-tyrosine plus reserpine, indicating that released NE was responsible for the elevated cyclic AMP (Korf and Sebens, 1979). Under in vitro conditions when alpha-methyl-p-tyrosine was given for 13 days yon Voigtlander et al. (1973) were unable to observe any change in adenylate cyclase responsiveness to DA in homogenates of mouse striatum. In a behavioral study in rats, repeated administration of reserpine plus alpha-methyl-p-tyrosine yielded supersensitive stereotyped motor activity in response to amphetamine or apomorphine (Friedman et al., 1975). To account for the discrepancies in these findings, there could be either a species difference between rats and mice, or perhaps the D A receptors responsible for supersensitivity are not coupled to adenylate cyclase. Nevertheless, the data with alpha-methyl-ptyrosine are insufficient to draw any conclusions.

02

( ; . ( ' . PAIMIR

11.2. RESERPINE Reserpine depletes central monoamines, principally NE, D A and 5-[tT (and maybe histamine) by inhibiting the uptake or transport of the monoamine into the intrancuronal storage granule. The free monoamines are then destroyed by monoaminc oxidases. Reserpine was the first successful neuroleptic and had been used in the Eastern countries for centuries until the 1950's when its benefits were appreciated in the West. Reserpine depletion of monoamines has been employed in a number of studies as an animal model for depression. Depression, as well as an overactive cholinergic system ("reserpine syndrome") are the chief side effects of reserpine in humans. Its use as a neuroleptic has been superceded by the phenothiazine and butyrophenone neuroleptics. About the only clinical use for reserpine today is as an adjunct drug for hypertension. When reserpine is injected into animals for as little a duration as 2 days and for periods up to a week, the beta adrenergic receptors coupled to adenylate cyclase bccomc hypersensitive. Most brain regions and all brain cells (except glia) apparently "'up regulate" in response to reserpine-induced monoamine depletion. These data are sun1 marized in Table 10 and it is evident that not enough detailed data are available for many regions. The cerebral cortex has been the most extensively studied using tissue sliccs~ homogenates and isolated cellular preparations. As a general agreement among investigators beta receptors become supersensitive in response to reserpine. Dopaminergic supersensitivity is only observed in disrupted cellular preparations. The striatum is interesting in that NE-hypersensitivity is readily evident but in only one study did DA-cliciI an enhanced activation of adenylate cyclase as a consequence of reserpinization and that occurred at only the highest D A concentrations. Perhaps in this situation excess D A was activating NE receptors. In further work with the rat cerebrum following reserpine, enzyme responses to histamine, serotonin, fluoride or veratridine were unchanged from controls. However, adenosine responses were elevated by 30% (Dismukes and Daly, 1974: Palmer et al., 1976b). In most experiments hypersensitivity of beta-receptor clicilcd responses coupled to adenylate cyclase reach the maximum after 4-6 days following drug injections, however, hypersensitivity can be detected as early as 5-24 hr. After the last injections normal responses to NE do not return until an elapsed time period of 9-1 (~days TABLE 10. RESERPINE-INDUCEDSUPERSENSIHVITYOF CATECEtOLAMINE-EIICITEDADENYLATECYCI ASE Rt'SPONSES IN RAT BRAIN Catecholamine Brain preparation Whole brain--slices Cerebrum--slices Cerebrum--homog. * Cerebral N e u r o n s - - h o m o g . Cerebral-glia.--homog. Cerebral-capillaries--homog. Cerebral-pia--homog. Hypothalamus--slices Hypothalamus--homog. St riatum--homog. Hippocampus--slices Pineal--homog. Pineal---cultured Limbic forebrain--slices Brain stem--slices Cerebellum--slices Thalamus--slices

lsoproterenol

Increase Increase Increase None Increase

NE Increase Increase Increase Increase None Increase Increase Increase Increase Increase Increase Increase

I ncrease Increase

Increase None None None

DA

None Increase Increase None lncreasc

Increase None or Increase : :~

References 16 2,t~ I 1.15 3,10.11 11 11 12 12 9 1 Ill, 13,15 9 4 5 3,7,14 9 9 9

* Homog. = homogenate; **Observed only with large doses. References: (1) Ahn and Makman, 1977: (2) Baudry el al., 1976; (3) Blumberg et al., 1976; (4) Cantor et al., 19glb; (5) Deguchi and Axelrod, 1973: {6) Dismukes and Daly, 1974; (7) Gillespie el al., 1980; (g) Palmer, D. S. e l a l . , 1976; (9) Palmer, et al.. 1973: (1(I) Palmer and Wagner, 1976: ( l l ) Palmer et al., 1976b; (12) Palmer and Palmer, 1983; (13) Rotrosen ¢'t al., 1975: (14) Vetulani e t a l . , 1976b; (15) Wagner el al., 197g: (16) Williams and Pitch, 1974.

PSYCHOACTIVE DRUGS, CYCLIC NUCI.EOTIDES AND THE CNS

63

(Dismukes and Daly, 1974; Baudry et al., 1976). Most investigators reported that beta adrenergic supersensitivity was a result in an increased maximal response of the enzyme to NE (Dismukes and Daly, 1974; Baudry et al., 1976; Blumberg et al., 1976; Palmer et al., 1976b). On the other hand, Palmer, D. S. et al. (1976) showed an increased affinity for NE to activate adenylate cyclase following reserpine. Two studies have correlated the reduction in spontaneous motor activity elicited by reserpifie to the development of the enhanced sensitivity of NE-elicited adenylate cyclase in the rat cerebral cortex. This inverse correlation could not be made with striatal tissue. The data indicated that the catecholamine mediated cyclic AMP generating system in the rat cerebral cortex has an inhibitory influence on spontaneous behavioral activity (Williams and Pirch, 1974; Wagner et al., 1978). These findings are somewhat in agreement with that of Skolnick and Daly (1974) who found that spontaneous motor activity in four distinct rat strains was positively correlated with NE-cyclic AMP in slice preparations of midbrainstriatal tissue and negatively correlated with NE-cyclic AMP in slices of cerebral cortex. If amphetamine was administered in conjunction with reserpine to rats, the beta adrenergic hypersensitivity of the adenylate cyclase system did not develop in either the cerebrum or the limbic forebrain. Since amphetamine enhances the action of NE by increasing its release, blocking reuptake and monoamine oxidase, the results would be expected (Gillespie et al., 1980). Young rats readily demonstrated an ability to increase the density of beta adrenergic receptors following reserpine treatment. These enhanced receptor numbers found in the cerebellum, cortex and pineal were absent in aged animals. Therefore, do the processes of aging strongly influence the capacity of an organism to "up or down" regulate its receptors in response to an environmental challenge (Greenberg and Weiss, 1979)? U'Prichard and Snyder (1978) reported an augmented number of both alpha and beta adrenergic receptors in rat forebrain in respone to reserpine injections. What role the alpha receptors play in the phenomenon of hypersensitivity is unknown. Of historical interest, examination of the data of Kakiuchi and Rall's (1968) original findings did reveal that reserpine treatment of rabbits for 3 days led to an increased ability of cerebellar slices to accumulate cyclic AMP to added NE and histamine. Under in vivo conditions employing rapid fixation of brain tissue, acute reserpine depleted cyclic GMP content in the mouse cerebellar molecular layer (vermis > hemispheres) (Ferrendelli et al., 1972; Rubin and Ferrendelli, 1977). Daily administration of reserpine for 3 days increased cyclic AMP in the diencephalon (1 day) and lung (3rd day), but not the cortex of mice (Palmer et al., 1977a). Acute (1-2 days) reserpine injections diminished urinary excretion of cyclic AMP in rats followed by a rebound-enhanced cyclic AMP excretion for the subsequent duration of treatment (4 additional days) (Palmer and Evan, 1974). Keatinge et al. (1975) reported analogous findings for acute reserpine, but prolonged treatment caused a continued depression of urinary cyclic AMP. In some additional unrelated experiments, reserpine in vitro inhibited a partially purified phosphodiesterase prepared from rabbit cerebrum (Honda and Imamura, 1968). Subchronic administration of reserpine promoted a change in the affinity (decrease in ECs0) of NE to initiate glycogenolysis in slices of mouse brain. The action was mediated exclusively through beta receptors and no change was noted in the ability of dibutyryl cyclic AMP to elicit glycogenolysis (Quach et al., 1978). This sudy establishes a functional correlation between "up regulation" of receptors in response to lowered availability of a neuromodulator and a metabolic event. It could be assumed that in order to meet emergency energy demands the receptors coupled to glycogenolysis are adjusted to maintain homeostasis. 11.3. 6-HYDROXYDOPAMINE 6-Hydroxydopamine has proven to be a useful tool in which to achieve a selective degeneration of afferent catecholamine fibers in both the brain and peripheral tissues. The "so-called" selectivity for catecholamine pathways (especially NE) may result from the

(~4

( ;. ('. PAl MH{

specific uptake mechanisms in these neurons for the neurotoxm, lmracellu[ar damage is thought to occur from an oxidation process generating superoxide and hydroxy radicals which in turn oxidize sulfhydryl groups on enzymes and also lead to pcroxidalion of membrane lipids (Kostrzewa and Jacobwitz, 1974; Sachs and Jonsson, 1975). Recen! questions have. however, been put forth that debate against the selectivity of ¢~-hwlroxxdopamine action. The agent, in addition produces extensive damage to the blood-brain barrier~ causes tissue cavitation and gliosis at the site of injection. Therefore. sclcc{ivc degeneration of catecholamine containing neurons cannot account for all the actions caused by 6-hydroxydopamine (Cooper, P. H. et al., 1982). Behavioral alterations havc also been observed in rats receiving 6-hydroxydopamine. In my first experience, I noted that rats remained quiescent following intraventricular injection but when handled would develop an aggressive biting attack. In some rats loud noises within the laboratory produced seizures (Pahner, 1972). Several observations have becn made with regard to other 6-hydroxydopamine-evoked behavioral alterations: injected newborns develop behavioral suppression in rearing and conditioned avoidance behavior, as well as altcrations in growth (Nyakas el al., 1973; Smith el al., 1973), impaired learning of complex behavioral tasks (Mason and lversen, 1974), enhancement of audiogcnic seizures (Bourn el al., 1972), irritability, vigorous, spontaneous fighting, and an incrcase in shock-induced aggression (Nakamura and Thoenen, 1972: Thoa el al., 1972). Intraventricular, intranigral, intracisternal or intraperitoneal (neonatal animals only) injections of 6-hydroxydopamine caused within a few days a hypersensitivity of central cyclic nucleotide systems to a variety of neurohumoral agonists. This phenomenon was revealed in most regions of the rat brain, except cerebellum, mesencephalon and maybe the medullary region. The resistance of the cerebellum to 6-hydroxydopamine may be due to a drug-promoted hyperinnervation of adrenergic nerve endings to this tissue (Harden et al., 1979). However, this contention is not supported by iontophoretic studies (Siggins el al., 1971). Despite the limited studies for many brain regions, it appears that several neuromodulator systems are capable of eliciting the hypersensitivity of cyclic AMP in response to 6-hydroxydopamine injections. In this vein, alpha and beta adrenergic agonists, prostaglandins, DA and NE + adenosine combinations are effective in increasing the maximal response of the enzyme in the rat brain, while adenosine alone, NaF, veratridine and G T P are without an action. Some rat strains and the guinea pig cortex do not develop any supersensitivity to 6-hydroxydopamine (see Table 11 for details). The non-specific nature of the receptor-mediated responses evoked by 6-hydroxydopamine are in contrast with the reserpine findings in which the heightened receptor sensitivity was confined to beta adrenergic receptors. In two investigations the authors suggested that supersensitivity to 6-hydroxydopamine consisted of two components: (1) a short term destruction of presynaptic nerve endings in which the uptake mechanism was destroyed. thus NE added to tissue slices now retained a higher concentration in the synaptic cleft in which to elevate cyclic AMP. This component was not accompanied by an increase in receptor density; and (2) a longer term postsynaptic component requiring at least 3 days in which the NE sensitivity was associated with an increase in beta~ receptor numbers (Kalisker e t a l . , 1973; Sporn e t a l . , 1977; Minneman e t a l . , 1979). In most cases the degree of elevation in either DA or beta adrenergic stimulation of adenylate cyclase was to a greater extent than the increase in corresponding receptor numbers (Sporn el al., 1976, 1977; Minneman et al., 1979; Mishra et al., 1980; Biswas and Jonsson, 1981). The supersensitivity to 6-hydroxydopamine was long lasting, remained evident for periods up to 100 days following injection (Palmer and Scott, 1974; Krueger el al., 1976; Skolnick and Daly, 1977; Harden el al., 1979), and was not accompanied by any change in phosphodiesterase activity (Skolnick el al., 1978). 6-Hydroxydopamine-induced supersensitivity of rotational behavior elicited by injections of D A agonists has been directly correlated with an increased capacity of DA ligands to bind to rat striatal preparations. A direct correlation between behavior and DA-adenylate cyclase, however, could not be made (Waddington et al., 1979; Mishra et al., 1980). If the nigral-striatal tract was lesioned with 6-hydroxydopamine, caudate neurons showed an

PSYCHOACTIVEDRUGS, CYCLICNUCLEOTIDESAND THE CNS

65

TABLE 11. ENHANCEMENTOFNEUROHUMORAL-INDUCEDACCUMULATIONOFCYCLICAMP IN RAT BRAINFOEI,OWING 6-HYDROXYDOPAMINE Region Cortex Striatum Homog. Mouse homog. Slices Spinal cord Mesencephalon Midbrain Medulla pons Brain s t e m Limbic forebrain Hippocampus Hypothalamus Cerebellum Cortex F344 rat Cortex--Guinea Pig

Increased responses to agonists

References

NE, isoproterenol, methoxamine, PGEt, adenosine + NE No response to NaF, adenosine or veratridine

1,2,4,7,9-11,15

No change* or increase to DA No change to DA DA, NE. None to isoproterenol, NaF, or adenosine NE No change to NE or isoproterenol NE No change to NE NE NE and isoproterenol, none to adenosine NE (low doses) NE None None None

5",6,14 13 5,6 3 9 1 1 1 12 8 7 1,7 9 9

Data are for tissue slices except where noted. Homog. = homogenate. References: (1) Dismukes and Daly, 1975b; (2) Huang et al., 1973; (3) Jones, 1980; (4) Kalisker etal., 1973; (5) Krueger et al., 1976; (6) Mishra etal., 1974, 1980b: (7) Palmer, 1972; (8) Segal etal., 198l; (9) Skolnick and Daly, 1977; (10) Skolnick etal., 1978; (11) Sporn et al., 1976, 1977; (12) Vetulani et al., 1976b; (13) Von Voigtlander et al., 1973; (14) Waddington et al., 1979; (15) Weiss and Strada, 1972.

even greater depression in their firing rate in response to DA agonists (Siggins et al., 1974). In three in vivo investigations, constant intraventricular infusion of NE prevented the subsequent increase in beta adrenergic receptors in the cerebrum of rats undergoing 6-hydroxydopamine treatment (Biswas and Jonsson, 1981). Low doses of clonidine which presumably inhibit the release of NE by interacting with presynaptic receptors, in turn decrease steady-state levels of cyclic GMP in rat cerebellum. Destruction of presynaptic adrenergic nerve endings with 6-hydroxydopamine abolished the action of clonidine (Haidamous et al., 1980). In speculation, maybe clonidine interacted with alpha receptors negatively coupled to cyclic AMP but positively coupled to cyclic GMP. The tyramineinduced elevations in plasma cyclic AMP were abolished when rats were pretreated with 6-hydroxydoparnine (Kunitada et al., 1978), indicating an indirect action by tyramine on a system requiring intact adrenergic nerve endings. The DA-induced increase in incorporation of 32p into homogenates of rat striatum was further enhanced following 6-hydroxydopamine injections into the nigra-striatal pathway. The rate of cyclic AMP stimulation of phosphorylation into caudate proteins was, however, unaffected (Hullihan et al., 1979). ll.4. LESIONS The initial study which actually opened up the investigation in order to determine the molecular substrates of the phenomenon of adrenergic denervation supersensitivity was conducted by Weiss and Costa (1967). When pineal glands of rats were denervated by removal of the superior cervical ganglia, the sensitivity of adenylate cyclase to NE was enhanced after 3 weeks. Furthermore, this NE-adenylate cyclase was accompanied by an increase in beta receptor numbers (Cantor et al., 1981b) and an enhanced induction by catecholamines of the enzyme involved in melatonin synthesis, serotonin-N-acetyltransferase (Deguchi and Axelrod, 1972, 1973). In further work pineal denervation revealed that the ability of NE, K + or ouabain to elevate cyclic GMP was impaired, suggesting a presynaptic calcium-dependent mechanism responsible for this event (O'Dea and Zatz, 1976). Lesions to the rat locus coeruleus or medial forebrain bundle resulted in enhanced cyclic AMP responses when cortical slices were exposed to NE, isoproterenol, and histamine

00

(]. C'. PAl Mt R

plus a phosphodiesterase inhibitor. No changes were evident to adenosine, prostaghmdin E~ or serotonin or the DA-adenylate cyclase in the striatum (Eccleston, 1973; Dismukes el al., 1975; Phillipson et al., 1977). Furthermore the histamine response was of an ft-2 receptor nature and the medial forebrain lesions likewise evoked an enhanced NE-cyclic AMP in the hippocampus (Dismukes et al., 1975). Radiofrequency lesions to the rat substantia nigra or ventral tegmental area caused depletions in DA content and the sensitivity of adenylate cyclase to D A was heightened in the striatum and nucleus accumbens-olfactory tubercle region (Mishra et al., 1974; Rosenfeld et al., 1979). Transection of the spinal cord led to a 5-10 fold elevation in DA-adenylate cyclase in tissue evaluated below the lesion (Gentlemen e t a l . . 1981 ). On the other hand, when the caudate nucleus of the rat was completely denervated (blood supply intact) on one side and examined six weeks later for adenylate cyclase activity, the enzyme was less responsive to D A and NE. Low K m phosphodiesterase was, however, elevated. Histological evaluation revealed viable neurons except that the dendritic spine apparatus was immature. The investigators assumed that the desensitivity was a result of tonic loss of the ability of at least two or more transmitters to maintain the spine apparatus. This loss of tone caused the spines to degenerate (Pajer el al., 1982). After a hemisection of the rat brain, DA-sensitive adenylate cyclase in the substantia nigra completely disappeared, while 6-hydroxydopamine destruction of the substantia nigra did not alter the enzyme. This work provides evidence that the DA-sensitive adenylate cyclase in the substantia nigra is not coupled to D A autoreceptors, but instead the D A fibers may end on incoming terminals associated with G A B A neurons (Premont et al., 1976). Naftchi et al. (1981) showed an elevated content of cyclic AMP and tyrosine hydroxyl asc activity in the rat brain stem following 5 days transection of the spinal cord. At 7 days post-section these levels were returned to normal. The significance is unknown, unless the NE neurons sending fibers into the cord were attempting to adjust to the environmental stress. 11.5.

KAINIC A C I D

A recent review by McGeer and McGeer (1982) describes the relevant data with regard to the action and hypothetical roles of the neurotoxin, kainic acid, in the CNS. The agent possesses a special property in that intrinsic neurons and respective dendrites are selectively destroyed following focal injection of the drug. Therefore, axons "en passant" and incoming fibers are not damaged. Apparently even greater selectivity is achieved with kainic acid, because neurons containing glutamate receptors are particularly susceptible. It has been proposed that destruction of intrinsic neuronal perikarya by kainic acid in the striatum may produce a model of Huntington's disease in animals. Agents such as kainic acid also serve to perform selective lesions in other brain regions and have proven useful in studies of neuroendocrinology, epilepsy, retinal degeneration and thalamic-induction of acute myocardial necrosis. Striatal tissue may contain as many as five different sites for D A action: (1) glial cells; (2) neuronal cell bodies; (3) intrinsic dendrites; (4) presynaptic-autoreceptors; and (5) incoming afferents containing different transmitters from various brain regions (Pajer et al., 1982). Various strategies have been attempted using measurements of endogenous transmitters, cyclic nucleotides, neurotransmitter enzymes and patterns of ligand binding in an attempt to elucidate the function of each of these DA-receptor endings. Sokoloff et al. (1980) evaluated the role of 4 types of D A receptors in the striatum and concluded that DI and D2 were similar and located on postsynaptic dendrites, as evidenced by a reduction in receptors and DA-adenylate cyclase following kainic acid, while respective activity increases were seen after 6-hydroxydopamine. The U 3 receptors were autoreceptors and were uninfluenced by kainic acid, but were decreased subsequent to destruction of incoming nerve endings by 6-hydroxydopamine. Receptors of the D4 type were located on incoming afferents from various structures. Moreover, kainic acid caused only a slight reduction in receptor densities while 6-hydroxydopamine produced a slight elevation in D4

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67

receptor numbers. Another phenomenon associated with kainic aid is a proliferation of glia and this has been attributed to some supersensitive striatal reponses (cyclic AMP) in slices exposed to isoproterenol, NE, D A and prostaglandin E1 (Minneman et al., 1978; Zahniser et al., 1979; Ariano et al., 1980). A similar finding was reported in the rat hippocampus. Normal beta receptor-elicited cyclic AMP synthesis was assumed to be a beta~ response. After kainic acid a 12-15 fold hypersensitivity became evident, but was now predominantly of a beta2 nature (Segal et al., 1981). Other findings associated with intrastriatal administration of kainic acid to rats were decreases in: basal adenylate cyclase, DA-adenylate cyclase, in all types of phosphodiesterase, guanylate cyclases, cholinergic binding, specific binding of spiroperidol, and no change in adenosine-elicited cyclic AMP. In other work there was a decrease in phosphodiesterase and DA-adenylate cyclase in the substantia nigra, while no change in nigral binding of spiperone was noted. Kainic acid evoked an increase in striatal G A B A and a decrease in benzodiazepine receptor content (Di Chiara et al., 1977; Schwarcz and Coyle, 1977; Minneman et al., 1978; Schwarcz et al., 1978; Quick et al., 1979; Zahniser et al., 1979; Ariano et al., 1980; Wallaas, 1981). l 1.6. CONCLUSIONS The agents discussed in the preceding section have proven to be useful in determining both ule location of and the nature of receptors responsible for the phenomenon of adrenergic denervation supersensitivity. In general, depletion of monoamine with reserpine produces an "up regulation" of beta1 receptors associated with neuronal function. On the other hand, 6-hydroxydopamine lesions to afferent catecholamine pathways and physical placement of lesions yields enhanced cyclic AMP responses to a wider variety of agents. These data and that observed with kainic acid suggests a lack of involvement of adenosine receptors in mechanisms responsible for hypersensitivity. Not enough data are available with alpha-methyl-p-tyrosine to draw any definite conclusions. Hopefully future tools like kainic acid will be developed in which to localize other diverse pathways or receptor sites (benzodiazepine, prostaglandin, neuropeptides, etc.) within the CNS.

12. Lithium

12.1. CLINICAL ASPECTS Manic illness is characterized by a variety of behavioral traits associated with hyperactive conditions lasting a week or more which include: elated and/or irritable mood, talkativeness, accelerated speech, accelerated motor activity, flight of ideas, decreased sleep, poor judgement, distractability, and the absence of drug-induced states. There may be also the presence of a depressive state, i.e. so-called bipolar or manic-depressive illness. The mood shifts may cycle with intervening periods of normality and the presence of one symptom may be predominant to a greater degree than the other. The disease like all affective disorders is more prevalent in women than men, but generally has an earlier onset with regard to the patient's age than pure depressive illness. All social-economic classes and martial states seem to be affected by mania to an equal extent in contrast to schizophrenia which has a greater prevalence among single patients and those from lower social-economic classes. A genetic deficit is thought to be an underlying cause of the disease because its incidence is strongly correlated to family history (Shopsin, 1979 for details). With the initial discovery by Cade in 1949, Li + has proven to be the agent of choice in the therapeutic management of manic-depressive (bipolar) illness (Shopsin, 1979, for review). The popular monoamine hypothesis for central disorders, namely, schizophrenia, depression and mania, states that the latter is predominantly mediated by a functional excess of N E activity at undetermined sites in the brain. On the other hand, depression is mediated by functional deficits of central NE or 5-HT (Schildkraut and Kety, 1967; Garver and Davis, 1979). However, the situation is much more complex because the powerful

I"~

(J. ( ' . [',',,I M}R

neuroleptics such as haloperidol which are relatively specific DA receptor blockers arc extremely effective in reducing the symptoms of mania. These agents arc widely used in the initial management of manic illness. The issue is even more complicated and may involve multiple transmitters, because elcwition of central acetylcholine levels in manic patients appears to ameliorate the symptoms of mania (Garvcr and Davis, 1979). If, on the other hand, NE is the predominant transmitter deficit, one might speculate that changes in cyclic nlJcleotide levels would be prevalent during the course of the disorder. And indeed! Several investigators havc reported elevations in either plasma or urinary cyclic AMP levels during mania or on the clay of rapid switch from depression to mania (Paul et al., 1971a,b). These elevated cyclic AMP levels were attenuated after successful therapeutic treatments with either Li* or phenothiazines and were furthermore shown not to be associated with the state of physical activity of the patients (Abdulla and Hamadah, 1970: Paul et al., 1971b; Sinanan el al., 1975; Lykouras et al., 1979). As with most such studies conflicting data have been presented. Neither Robison et al. (1970) nor Smith et al. (1976) were able to observe any change in cyclic AMP levels of CSF in manic patients. In further work, leukocytes isolated from patients with schizophrenia and affective disorders had lower cyclic AMP sensitivities to NE or isoproterenol than normals (Pandey et al., 1979). Unfortunately animal models to evaluate the molecular mechanisms of this disorder arc sorely needed. In two animal experiments when Li + was administered to rats to achieve therapeutic plasma concentrations the urinary and plasma cyclic AMP levels rose in conjunction with the polyuria. At two weeks when the urine volume dropped towards normal levels, cyclic AMP content remained elevated. By 60 days there were no differences in cyclic AMP in the urine (Palmer and Evan, 1974; Christensen et al., 1977). Again these were normal animals and the significance of the findings is unclear. 12.2. ANTIADRENERGICACTIONS The major molecular action of Li ~ is to normalize the excitability of nervous tissue. The ion readily accumulates and displaces Na + from neurons rendering the tissue less responsive to stimuli (Samuel and Gottesfeld, 1973). With regard to antiadrenergic actions of Li ~ it acts to: reduce the release of biogenic amines during electrical stimulation in brain slices: increase the amine reuptake into brain synaptosomes and in platelets; produce an alteration in biogenic amine metabolism consistent with these changes; and block the post-decapitation (NE-induced?) rise in cerebral cyclic AMP in rats (Murphy et al., 1973; Uzunov and Weiss, 1971 ). 12.2.1. t t u r n a n studies In humans injection of epinephrine produces an increase in plasma cyclic AMP via a drug-specific interaction with beta adrenergic receptors. In addition, epinephrine increases plasma cyclic G M P by an action involving alpha adrenergic receptors. Glucagon infusion likewise elevates plasma cyclic AMP. Lithium treatment to either manic (who gave normal pretreatment responses) or control patients abolished the rises in plasma cyclic AMP and cyclic GMP following epinephrine infusions. The action of glucagon was unaffected by Li + administration. Thus lithium's effects were specific and were unrelated to a preexisting pathology or to receptor alterations. Moreover, haloperidol did not block the actions of epinephrine indicating that the site of action of Li + was confined to adrenergic receptors (Belmaker et al., 1976, 1980; Ebstein et al., 1975, 1976b, Oppenheim et al., 1979). Belmaker and coworkers (1976) attempted to explain the dichotomy between the haloperidol-antidopamine and Li÷-antinoradrenergic actions with regard to their treatment profiles for manic-depressive illness. They suggested that neuroleptics principally reduce the related symptoms of motor hyperactivity while Li + normalizes mood. The findings of Paul et al. (1971b) tend to support this view. During clinical manifestations of mania, urinary cyclic AMP was elevated and after Li + treatment the levels dropped. Alternatively, during the depressive cycle of the disease urinary cyclic AMP content was lower and became elevated following institution of Li + therapy.

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12.2.2. lontophoretic studies When N E is applied via iontophoresis to single cortical pyramidal and cerebellar Purkinje cells it produces a depression of cellular firing rates, an action mimicked by cyclic A M P (Siggins et al., 1969; Phillis, 1977). Lithium was shown by Phillis and Limacher (1974) to antagonize the inhibitory actions of NE. Similar Li + data for cerebellar Purkinje cells were reported by Siggins and Schultz (1979) and furthermore Li + blocked the reduction in Purkinje cell firing rates elicited by direct stimulation of the locus ceruleus. When administered alone to control rats Li + directly depressed Purkinje cell discharges. On the other hand, if rats were pretreated with 6-hydroxydopamine to destroy adrenergic nerve terminals, Li + no longer had a direct effect. This finding indicated that Li + antagonized the action of endogenously released NE. The influence of chronically administered Li + is in contrast to actions observed with acute drug treatment. When Li + was chronically given to rats, the antagonism by Li + of the reduction in Purkinje cell firing in response to iontophoretically applied N E was either now absent, or Li + slightly enhanced the action of NE. Moreover, Li + had no effect on the depression of cell firing as a consequence of stimulation of the locus ceruleus. Chronic Li + treatment was not associated with any change in the binding of beta adrenergic ligands in the cerebellum; but the NE-induced increase in cyclic A M P accumulation was enhanced (Schultz et al., 1981).

12.3.

EFFECTS OF LI + ON CYCLIC NUCLEOTIDE SYSTEMS IN BRAIN

12.3.1. Acute effects on m o n o a m i n e systems 12.3.1.1. Norepinephrine and D A As a general p h e n o m e n o n the NE-sensitive adenylate cyclases are more susceptible to inhibition by Li + than are corresponding D A systems. The N E response occurs at more equivalent therapeutic ranges while the anti-DA effects of Li + occur at toxic drug levels. Perhaps the tremors and Parkinson-like symptoms seen in Li + treated patients are an anti-DA action manifested at these toxic concentrations (Reches et al., 1978). In whole pineal glands or in tissue slices of rat hypothalamus and cerebral cortex, but not brain stem, low doses of Li + antagonized the accumulation of cyclic A M P elicited by N E or isoproterenol (Forn and Valdecasas, 1971; Palmer et al., 1972b; Ebstein et al., 1980; Zatz, 1979). Moreover, Li + did not influence the binding of beta receptor ligands in the pineal (Zatz, 1979); indicating that the anti-NE actions were not directly associated with N E receptor recognition. When the receptor antagonist action of Li + was examined with respect to adenylate cyclase activity in broken cellular preparations, N E was more potently inhibited than DA. In the first such study with a crude mitochondrial fraction from rabbit cerebrum, Dousa and Hechter (1970a) used large concentrations of Li + (25-50 mM) and reported an inhibition of both epinephrine and fluoride activated adenylate cyclase. In the second study Walker (1974) employed a frozen-thawed homogenate preparation from several rat brain regions. This procedure suffers in that m o n o a m i n e activation of adenylate cyclase is reduced. Nevertheless small doses of Li + (0.5 mM) effectively blocked NE-adenylate cyclase in all brain regions. This concentration had no effect on DA-adenylate cyclase in the caudate. In further work with the crude synaptosomal enzyme from guinea pig caudate and cortex only NE- and isoproterenol-sensitive adenylate cyclases were antagonized by doses of Li + in the therapeutic range (0.75-1.5 mM). Inhibition ofChe D A system in the caudate required Li + levels well within the toxic range (Reches et al., 1978; Geisler and Klysner, 1978; Ebstein et al., 1980). In either broken cell preparations or tissue slices neither rubidium nor cesium ions affected NE-elicited adenylate cyclase (Ebstein et al., 1980). A n o t h e r possibility, however, for the apparent selective action for Li + on N E - r e c e p t o r coupled adenylate cyclase is the sole use of the caudate nucleus to examine the action of D A . Possibly this brain region is insensitive to Li + actions. One such study needed to correct this problem would be to use a brain region like the frontal cortex which

711

(i. (', PAI,MFr

displays a sensitivity to DA, NE, and 5-HT. Therefore, if Li + possesses an exclusive neurotransmitter specificity, this experiment should demonstrate such an effect. Lithium at low doses does increase the basal level of cyclic A M P in incubated tissue slices of rat cortex (Ebstein et al., 1980). This action might be an activation of adenylate cyclasc by Li + in glial cells because Schimmer (1971, 1973) using cultured glial cells showed that 15 + elevated basal cyclic nucleotide levels and enhanced epinephrine or fluoride actiwJtion of the enzyme. Small concentrations of Li* inhibited the stimulation of cyclic G M P in response to NE in intact rat pineal glands. Under conditions when the pineal was rendered either supersensitive or subsensitive to catecholamines, the action of Li + was unchanged with respect to inhibition of NE-induced accumulation of either cyclic A M P or cyclic G M P (Zatz, 1979). 12.3.1.2. Histamine In two studies Li + was shown to inhibit histamine elicited cyclic A M P accumulation. In the rabbit brain cortex Li + at 2 mM was effective (Forn and Valdecasas, 1971), while Y a m a m o t o et al. (1978) reported that only huge concentrations were active in the guinea pig cortex. In the latter study no dose response relationships to Li + were obtained. 12.3.2. Chronic effects on m o n o a m i n e systems Pert and coworkers (1978) reported that when rats were chronically treated with haloperidol, the animals displayed symptoms of increased locomotor activity, stereotyped m o v e m e n t s to an a p o m o r p h i n e challenge, and an increased binding of the D A ligand, spiroperidol, to the striatum. Lithium-treated animals failed to develop evidence of these super-sensitive responses to D A and a clinical correlate was drawn in that Li + treatment in conjunction with neuroleptics might prevent tardive dyskinesia. In further work of this nature, chronic Li + treatment to rats prevented the development of reserpine-induced supersensitivity of cortical beta adrenergic receptors (Treiser and Kellar, 1979). When tissue slices of the cerebral cortex were incubated in the presence of N E the supersensitivity of the reserpine-induced accumulation of cyclic A M P was likewise prevented after chronic Li +. In this study chronic Li + had no effect on NE-elicited cyclic A M P in non-reserpinized animals. The preincubation procedure for the tissue slices most likely washed out contaminating-endogenous Li + accumulated by this nerve tissue, therefore indicating that the drug altered the properties of the N E receptor site (Hermoni et al., 1980). In another brain region, the rat cerebellum, chronic Li + was reported to enhance (slightly) the NE-induced production of cyclic A M P in tissue slices (Schulz et al., 1981). In contrast to these latter findings, Ebstein et al. (1980) showed that after cessation of chronic Li + (4 days), cyclic A M P accumulation in response to N E was not elevated in rat cortical slices. In fact when the tissues were prepared on the day of the last Li + treatment NE responsiveness was instead, attenuated. Taken together the effects of chronic Li + appear to prevent the development of D A and beta adrenergic receptor supersensitivity, presumably via a stabilization of both pre- and post synaptic receptor sites as evidenced by the work of Verimer et al. (1980). The ion itself appears to promote little change on the synaptic m e m b r a n e systems, however, this supposition is equivocal.

12.3.3. Other cyclic nucleotide systems

12.3.3.1. A d e n o s i n e The adenosine sensitivity of the crude synaptosomal preparation of adenylate cyclase in the guinea pig cortex and caudate was inhibited by Li +. In this case the ion was more potent toward the caudate enzyme (Ebstein etal., 1977). In tissue slices from this tissue Li + at one huge concentration (121 mM) blocked cyclic A M P accumulation elicited by adenosine ( Y a m a m o t o et al., 1978).

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71

12.3.3.2. Depolarizing agents The rise in cyclic G M P in mouse cerebellar slices produced by either sea anemone toxin or mast cell degranulating peptide was abolished if Li + was substituted for Na + in the buffer medium (Ahnert et al., 1979a,b). Lithium, in this case, most likely diminished membrane hyperexcitability and reduced Na + depolarization which in turn elicits Ca 2+ influx into the cells with concomitant guanylate cyclase activation. In other work, large concentrations of Li + inhibited electrical-induced accumulation of cyclic AMP in guinea pig cortical slices (Yamamoto et al., 1978). The K + stimulated increase in both cyclic AMP and GMP in rat pineal was prevented in the presence of 2 mM Li + (Zatz, 1979). 12.3.3.3. Fluoride Sodium fluoride activation of adenylate cyclase was inibited by rather large levels of Li + in rabbit cerebral cortex (Dousa and Hechter, 1970a; Forn and Valdecasas, 1971). The experiments reported in this section reveal a generalized membrane stabilizing action of Li + toward the action of agents which elicit depolarization of nerve cells.

12.3.4. Peripheral organs 12.3.4.1. Kidney The well-known side effect of Li + , namely polyuria is most likely caused by the selective inhibition of vasopressin activated adenylate cyclase in the renal medulla. A resistance to the effects of polyuria develops after several weeks and this might likewise be correlated to vasopressin sensitivity of adenylate cyclase. The effect of Li + in the kidney is rather specific for vasopressin because this ion had no influence on phosphodiesterase, protein kinase, or fluoride and other hormonally induced activations of adenylate cyclase (Dousa and Hechter, 1970b; Dousa, 1974; Geisler et al., 1972; Wraae et al., 1972). Lithium likewise was shown to block vasopressin, or dibutyryl cyclic AMP-induced permeability actions in the toad bladder (Harris and Jenner, 1972). 12.3.4.2. Thyroid The goiterogenic actions of Li + are thought to be caused by its ability to block thyroid stimulating hormone (TSH) action on adenylate cyclase in the thyroid gland. Moreover, both acute and chronic effects of Li + are manifested through an impaired ability of the gland to release iodide. Concordantly, Li + only weakly inhibited the binding of TSH to thyroid membranes (Williams etal., 1971 ; Wolff and Jones, 1971 ; Moore and Wolff, 1974). 12.3.4.3. Platelets The stimulation of cyclic AMP by prostaglandin El or prostacyclin in human platelets is prevented by therapeutic concentrations of Li +. In humans prostaglandins (E~ and prostacyclin) inhibit platelet aggregation and NE counteracts these prostanoid actions. In the rabbit Li + does not inhibit prostacyclin activation of adenylate cyclase. In keeping with its antiadrenergic action, the antagonism by N E and D A of prostaglandin E~ actions on human platelets is counteracted by Li + (Murphy et al., 1973; Wang et al., 1973; Imandt et al., 1981). The clinical significance of these observations of Li + action on platelets awaits further clarification. 12.3.4.4. Other tissues Lithium blocks in a dose dependent manner the stimulation of cyclic AMP by epinephrine in rat skeletal muscle preparations (Frazer et al., 1975). In heart muscle, Li + reduces the N E activation of phosphorylase a (Frazer et al., 1972). Large doses of Li + block glucagon-adenylate cyclase in liver (Dousa and Hechter, 1970a).

72

( ; . ( ' . PAl MIR

12.4. CONCLUSIONS The general molecular site of action for Li + appears to be one of membrane stabilization. Most likely the agent slows down or limits the processes of Na+-induced depolarization in brain tissue. As a more specific site of central action the ion exerts an antiadrenergic action as evidenced by: (1) a more selective inhibition of NE-sensitive cyclic AMP systems; (2) antagonism of the depressant effects of iontophoretically applied NE to central neurons" and (3) prevention of reserpine-induced supersensitivity of beta receptors--coupled to adenylate cyclase. Overall, cyclic nucleotide generating systems are especially vulnerable to inhibition by Li + and some clinical analogies can be drawn. Thus if mania is a result of excessive NE function in the brain the anti-NE actions correlate. Lithium produces polyuria and a goiterogenic effect in patients most likely because of respective inhibition of vasopressin and TSH-sensitive adenylate cyclases in the renal medulla and thyroid. Moreover, only higher doses of Li + inhibit DA-sensitive adenylate cyclasc and a correlation could be drawn with the more toxic actions of Li +, namely tremor, Parkinsonism and even tardive dyskinesia. Muscle fasciculations during Li + therapy might be attributed to its actions in these tissues. It should be remembered that these suppositions are tenuous as no mention is made of the influences of Li + on many ionic exchange mechanisms in both central and peripheral systems as discussed by Shopsin (1979) and Ramsey (1981).

13. Anti-Parkinson Agents 13.1. INTRODUCTION Parkinson's disease results from a decreased functional activity of D A nerve endings within the striatum. New terminology has therefore named the disease "nigra-striatal D A deficiency syndrome". For reasons unknown, the D A neurons in the substantia nigra become destroyed and the appropriate striatal postsynaptic receptor sites "up regulate" to compensate for the loss of functional DA. At this stage of the disease, agents that increase the physiological pools of D A are effective therapies to combat the disease e.g. L-DOPA plus peripheral decarboxylase inhibitors, amantadine, monoamine oxidase inhibitors (largely experimental) or anticholinergic agents (to counteract excessive acetylcholine overactivity which is under normal control by DA). However, many patients after prolonged therapy show either a "burn out" in which L-DOPA is no longer effective or "on-off" reactions. The "burn out" reaction may result from continued destruction of D A neurons and an end point is reached when precursors of D A cannot be taken up into the neurons, enzymatically formed into DA, and released in sufficient quantities to achieve any therapeutic benefit. At these latter stages of Parkinsonism only anticholinergic agents or a drug possessing direct D A receptor agonist activity would prove effective. At present bromocriptine is the best such agonist available to the clinician. The search for more efficacious agents has centered around agents with dopamimetic activity, namely the ergot alkaloids. The major symptoms of Parkinsonism are difficulty in initiating voluntary movement, slowness of movement, muscular rigidity and tremors. The drugs currently available do not necessarily result in complete disappearance of all symptoms. With regard to cyclic nucleotide systems in the striatum, the ergot class of compounds appears to stimulate the enzyme at low doses, but inhibits D A activation at higher concentrations. On the other hand, D 2 receptors not coupled to adenylate cyclase, are more specifically activated by ergot compounds. Therefore current efforts are directed toward development of more specific D A receptor agonists. However, the relative roles of either type of receptor in Parkinsonism have not been clarified (for more detailed discussions se~: Agid et al., 1979; Marx, 1979; Fann and Wheless, 1981; Schmidt, 1981). 13.2. CLINICAL STUDIES When striata were removed from postmortem Parkinson patients and examined for adenylate cyclase activity, it was shown that in drug treated patients (L-DOPA or

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73

amantadine) the activity of both basal and DA-elicited adenylate cyclase was less than controls. There were no differences observed with respect to age or sex of the patients. In one patient given only anticholinergic drugs, the DA-adenylate cyclase was hypersensitive (Shibuya, 1979). The latter observation, DA-supersensitivity, was also reported by Nagatsu et al. (1978). Moreover, Lee and coworkers (1978a) looked at DA receptor ligand binding patterns in the striatum of drug-free, L-DOPA and control patients. In drug-free patients there was an increased binding of haloperidol (postsynaptic sites) and a decreased binding of apomorphine (presynaptic sites) in the putamen and caudate nuclei. Controls were the same as L-DOPA patients. In correlative studies with rats, chronic treatment with L-DOPA produced a decreased affinity for D A with regard to adenylate cyclase activation in the striatum (DA1 receptor activity). Moreover, the ability of spiroperidol to bind to striatal fractions was elevated (D2 receptor activity). Thus the DI receptors "down regulated" while D 2 receptors "up regulated" in response to L-DOPA (Wilner etal., 1980). L-DOPA has also been shown to prevent the supersensitivity of DA-adenylate cyclase in rats receiving chronic neuroleptic treatment (Friedhoff et al., 1977), as well as, produce a decrease in NE-adenylate cyclase in the cerebellum (Wilner et al., 1980). Alternatively, Shibuya (1979) did not observe any change in the DA responses of the enzyme in the caudate of rats receiving 6 months treatment with L-DOPA. Neither acute nor chronic L-DOPA were able to change the CSF level of cyclic AMP in Parkinson patients. Likewise, control levels of cyclic AMP were unchanged between normals and Parkinson patients (Cramer et al., 1973). A similar negative result was seen in rabbit CSF after L-DOPA (Sebens and Korf, 1975). In contrast Belmaker et al. (1978) reported a 40-50% reduction in cyclic AMP and an 80-90% reduction in cyclic GMP in the CSF of Parkinson patients. L-DOPA treatment did not alter the level of either nucleotide. These investigators postulated that the low cyclic GMP value was related to the decreased choline acetyltransferase observed in Parkinsonism (McGeer and McGeer, 1976) and that the attenuated cyclic AMP was consistent with diminished DA activity. In concordant work with rats L-DOPA but not the DA agonist, pribedil, increased cisternal cyclic AMP an action mediated, however, via beta adrenergic receptors (Kiebling et al., 1975). 13.3.

CYCLIC NUCLEOTIDES AND A N T I - P A R K I N S O N D R U G S

13.3.1. Agents enhancing D A Amantadine and L-DOPA when injected in vivo did not alter cyclic AMP content in either the medial forebrain or cerebellum of rapidly inactivated mouse brain tissue. On the other hand, cyclic GMP levels were elevated (Gumulka et al., 1976a,b). Bromocriptine, however, decreased steady-state levels of cyclic GMP in rat cerebellum (Trabucchi et al., 1978). Under in vitro conditions, adenylate cyclase in the mouse caudate was stimulated to a slight extent by amantadine but not by L-DOPA and piribedil (Tang and Cotzias, 1977a). Piribedil and the corresponding metabolite, S-584 did elevate (slightly) an adenylate cyclase prepared from capillary fractions of rat cerebrum (Baca and Palmer, 1978). In tissue slices of limbic forebrain piribedil and S-584 blocked NE-cyclic AMP (Sawaya et al., 1977). Tang and Cotzias (1977b) showed that the intensity of neurological responses evoked by L-DOPA in mice depended upon sex, genetic strains and previous drug treatment. Dopamine-adenylate cyclase was found to be correlated to neurological responses and genetic strains. 13.3.2. Cholinergic agents Walker and Walker (1973b) demonstrated an acetylcholine inhibition of DA-sensitive adenylate cyclase in the rat caudate. These data were confirmed by Tang and Cotzias (1977a), however, in their work an anticholinergic drug was as effective as acetylcholine. In keeping with the idea that acetyicholine exerts opposing actions on DA systems in the striatum, dexetimide blocked both DA and apomorphine elevated adenylate cyclase (Puri

74

O.C. PALMER

et al., 1978). Other instances in which cholinergic agonists oppose cyclic AMP systems are

discussed in Section 6. 13.4. ERGOT ALKALOIDS

13.4.1. d ' L S D (d ' lysergic acid d i e t h y l a m i d e ) d'LSD is the most potent hallucinogen known and possesses a wide variety of actions on central cyclic nucleotide systems which include: (a) an activation at low doses of adenylate cyclase in retina, striatum, limbic cortex and cerebral cortex (Von Hungen et al., 1974b; 1975b; Da Prada et al., 1975; Bockaert et al., 1976; Schmidt and Hill, 1977; Ahn and Makman, 1979; Watling and Dowling, 1981); (b) at high doses d 'LSD inhibited DA-sensitive adenylate cyclase (see authors), as well as NE-adenylate cyclase in brain stem, hypothalamus and cerebrum (Palmer and Burks, 1971 ; Von Hungen et al., 1974b, 1975b), GTP activated adenylate cyclase in monkey limbic cortex (Ahn and Makman, 1979), and the histamine (H2) sensitive enzyme in monkey cerebrum, guinea pig hippocampus and cerebrum (Green et al., 1977; Ahn and Makman, 1979); (c) d'LSD exhibited specific alpha and beta adrenergic blockade of dog artery and rabbit heart contractile responses, respectively; and (d) the inactive metabolite of d'LSD, i.e. 2-bromo LSD exerted effects as potent as the parent compound (Palmer and Burks, 1971 ; Von Hungen, 1974b, 1975b; Green et al., 1977). When injected in vivo d ' L S D did not alter steady-state levels of cyclic AMP in the rapidly fixed cerebrum and diencephalon of mice (Palmer et al., 1977a). 13.4.2. Other ergot alkaloids To summarize, a majority of the considerable data associated with the action of ergot alkaloids on central adenylate cyclase systems reveal that ergots at low concentrations stimulate striatal or retinal adenylate cyclase, while at higher levels the agents block the DA stimulated enzyme. The stimulatory actions of ergots, like d'LSD are blocked by neuroleptic agents. The agents behave as partial agonists occupying the receptor but having little intrinsic activity, compete with DA and thus act as antagonists. The drugs listed below have varying degrees of potency and notably bromocriptine and lergotrile which are used to treat Parkinsonism generally possess weaker activity toward stimulation of adenylate cyclase. The agents evaluated have been (DH = dihydro): DH-ergotamine, ergotamine, bromocriptine, ergocristine, ergonovine, ergometrine, 2-bromo-alpha-ergocriptine, hydergine, DH-ergotoxin, nicergoline, pergolide, lisuride, ergocornine, elymoclavine and agroclavine (Montecucchi, 1976; Schorderet, 1976; Schmidt and Hill, 1977; Fuxe et al., 1978; Markstein et al., 1978; Pagnini et al., 1978; Portaleone, 1978; Trabucchi et al., 1978; Saiani et al., 1979; Rosenfeld et al., 1980; Wong and Reid, 1980; Watling and Dowling, 1981). In addition, many of the ergots inhibited NE and/or isoproterenolinduced cyclic AMP accumulation in rat striatal slices (Markstein et al., 1978), rat hypothalamic slices (Palmer et al., 1973) and mouse limbic forebrain slices (Sawaya et al., 1977). Nicergoline did not, however, influence NE, adenosine or K ÷ activation of cyclic AMP in rat cortical slices (Montecucchi, 1976). Moreover, neither bromocriptine nor hydergine influenced adenylate cyclase in rat hypothalamus, but were active in the striatum (Portaleone, 1978). A variety of ergots were shown to inhibit cyclic AMP phosphodiesterases to a potency level equal to that of the methylxanthines. Dihydro derivatives of ergostine, ergoptine, ergocristine, ergocornine, ergocryptine, ergosine, ergotamine, ergonine and ergovaline were inhibitory in respective order of potency on an enzyme prepared from cat brain (Iwangoff and Enz, 1972). Pagnini et al. (1978) reported that bromocriptine at low doses activated phosphodiesterase and at higher doses inhibited the enzyme. Dihydroergotoxin and ergotamine along with nicergoline inhibited phosphodiesterase in rat cerebrum (Montecucchi, 1976). Under in vivo conditions bromocriptine was shown by Trabucchi et al. (1978) to elevate cyclic AMP in rapidly inactivated rat striatum. Preinjection of haloperidol prevented this action. Pagnini et al. (1978) and Portaleone (1978) found that

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injected bromocriptine or hydergine led to augmented adenylate cyclase activity. Whether these latter findings are related to phosphodiesterase antagonism is unknown. Fuxe et al. (1978) have conducted an extensive evaluation of ergots employing histochemical, biochemical and behavioral techniques. Their conclusion was that the behavioral and anti-Parkinson action of ergots can be explained by their action as partial agonists/antagonists at several DA receptor sites in the brain, an event varying from one DA receptor population to another. In conclusion the role of D1 receptors coupled to adenylate cyclase as a molecular substrate in Parkinsonism seems likely but the data are equivocal and must further take into account the D 2 receptors (Calne, 1978).

14. Hallucinogens With the exception of work done with d'LSD (see previous Section 13.4) little work has been accomplished regarding actions of hallucinatory substances on cyclic nucleotide systems in the brain. It seems somewhat surprising because of the widespread effects many of these compounds have on central monoamine mechanisms. Moreover, the prevalent publicity these compounds have recently enjoyed in the popular press should have resulted in more research directed toward understanding the molecular sites of action of the hallucinogens. 14.1. PHENCYCLIDINE Phencyclidine also known as crystal, angel dust, PCP, and a host of other street names was initially produced as an anesthetic. Because of the widespread adverse reactions associated with its use namely, hallucinations, coma, violent behavior, blank stare, delirium, seizures and psychotic reactions, the agent is now only clinically used as an animal anesthetic. Phencyclidine does possess actions which might suggest a role as a DA agonist; it blocks reuptake of DA, increases tyrosine hydroxylase activity in vitro, decreases tyrosine hydroxylase in vivo, releases DA from "DA-loaded" synaptosomes, produces stereotyped behavior and induces "turning to the side of the lesion" in rats which have received unilateral disruption of the nigro-striatal pathway (Leelavathi et al., 1980). Pandey et al. (1975) reported that phencyclidine injections (i.p.) raised the basal activity of adenylate cyclase in the rat cerebrum. In another study acute and chronic doses (30 days followed by 24 hr withdrawal) of phencyclidine diminished both DA-adenylate cyclase and low K mcyclic AMP phosphodiesterase in the rat striatum. The agent had no effect on DA receptor ligand binding in vitro but did increase binding following an acute injection (Leelavathi et al., 1980). In one other investigation guanylate cyclase in the supernatant fraction from rat tissues including cerebellum and cerebrum was potently stimulated by phencyclidine, cyclohexyl piperidine and their morpholine analogs. Interestingly these compounds do not appear to generate free radicals or the NO ion (Vesely, 1979). 14.2. MESCALINE AND O T H E R M A J O R HALLUCINOGENS

Using tissue slices of rat brain, mescaline, psilocybin, ibotenic acid, dimethyltryptamine, diethyltryptamine and dipropyltryptamine increased the accumulation of cyclic AMP. Inactive congers of LSD, 1-LSD and bromo-LSD were ineffective. Injections of the pharmacologically active agents produced elevations of the nucleotide in the cerebrum and brain stem but not the cerebellum. However, the method of in vivo determination Qf cyclic AMP in this earlier work did not allow for rapid fixation of brain tissue. Interestingly trifluoperazine injections prevented the cyclic AMP increase, suggesting a DA or NE action by the hallucinogens (Uzunov and Weiss, 1972). In contrast, in the rabbit retina or in broken cell preparations of rat striatum, cortex and hippocampus, no influence on adenylate cyclase could be detected by mescaline, dimethyltryptamine, psilocin or bufotenin (Von Hungen et al., 1974b, 1975b; Bucher and Schorderet, 1974).

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14.3. HARMALINE Harmaline has both monoamine oxidase inhibitor, as well as, hallucinatory properties (Longo, 1972). However, its unique effects upon augmenting cerebellar cyclic GMP content is a direct action on the inferior olivary nucleus to enhance the firing rate of climbing fibers which evoke the discharge rate of cerebellar Purkinje cells. The effect of harmaline is counter-acted by agents enhancing G A B A action, diazepam and muscimol. The increase in cerebellar cyclic GMP is greatest in the molecular layer especially that of the vermis. Other monoamine oxidase inhibitors do not share this action of harmaline. In incubated slices of cerebellum, however, harmaline decreases cyclic GMP, but elevates cyclic AMP (Mao et al., 1975a; Opmeer et al., 1976; Biggio et al., 1977b; Rubin and Ferrendelli, 1977). 14.4. TETRAHYDROCANNABINOL(THC) Some recent experiments with cannabinoids indicated that possible sites of central actions involved beta adrenergic receptor activity. In humans propranolol counteracted the "high" and the impaired performance seen following THC. Chronic administration of THC to animals increased the activity of hypothalamic monoamine oxidase. Delta9 and l l-hydroxy-delta9 THC injections resulted in an enhanced affinity for beta adrenergic ligands to bind to receptors in mouse cerebral cortex. Chronic administration of the two compounds, on the other hand, led to reduced numbers of beta adrenergic receptors. The nonpsychogenic compound, cannabidiol, was ineffective. These authors felt that THC enhanced or facilitated adrenergic activity in the brain, an action that eventually led to "down regulation" of receptors after chronic treatment (Hillard and Bloom, 1982; also see Banerjee et al., 1975). With regard to cyclic nucleotide systems deltas-THC but not deltag-THC increased the steady state levels of cyclic AMP in the rat midbrain but lowered the levels in the cerebellum and medulla (Askew and Ho, 1974). In contrast Dolby and Kieinsmith (1974) found that low THC doses increased while high THC doses lowered cyclic AMP content in mouse cerebellum, cortex and medulla. However, in both investigations only low intensity commercial microwave ovens were used to sacrifice the animals. Injection of delta,)-THC caused reduced activities of adenylate cyclase and phosphodiesterase in the rat midbrain while phosphodiesterase activity was augmented in the cerebellum (Askew and Ho, ! 974). Rather high concentrations of delta~-THC blocked the accumulation of cyclic AMP in cultured fibroblasts exposed to epinephrine and prostaglandin El. Lower levels of THC activated cyclic AMP-dependent protein kinase and prevented the release of cyclic AMP from the cells into the culture medium (Kelly and Butcher, 1973; 1979). 14.5. YOHIMBINE Yohimbine has long been known to have hallucinogenic properties (Longo, 1972), but recently one of its major mechanisms of action was ascribed to a blockade of presynaptic alpha2 receptors. Whether this presynaptic-type receptor is coupled to adenylate cyclase activation is not known with any degree of certainty. Like most so called specific antagonists, yohimbine acts to some extent to inhibit p0stsynaptic alpha receptors. Some alpha receptor responses in the brain are coupled to adenylate cyclase activation (see Section 3.6.1.1 .) and it is not surprising that yohimbine displays a potency in blocking the action of NE on cyclic AMP production in tissue slices of mouse limbic forebrain (Sawaya et al., 1977) and rat spinal cord (Jones and McKenna, 1980). In the neuroblastoma × glioma hybrid cells, catecholamines lower cyclic AMP by means of an alpha 2 type receptor activation. Yohimbine potently reversed the epinephrine-induced inhibition of basal adenylate cyclase in these cells (Kahn et al., 1982). No definite conclusions can be drawn which correlate in any manner between the hallucinogenic property of these psychoactive drugs to their ability to influence cyclic nucleotide systems in the brain. However, some detailed work has been accomplished with d ' L S D and this is discussed in Section 13.4.

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15. Opiates 15.1. INTRODUCTION The discovery within the past decade of opiate receptors and associated endogenous peptides that mimic the action of natural and synthetic opiates has been one of the salient findings of bio-medical research. The importance of this research cannot be minimized because of the need for better analgesics without addiction potential. Another thrust of this work has been to develop agents to treat or prevent addiction and associated withdrawal mechanisms. Furthermore, investigations may uncover drugs which might have a more specific action on a particular opiate receptor subtype without producing the host of other effects associated with opiate administration. No attempt will be made here to discuss all the important events associated with the tremendous amount of research conducted within the past few years. The discussion will, however, attempt to unravel the data concerning the influence of opiates on cyclic nucleotide systems. (For reviews of opiate action see Goldstein, 1976; Bunney etal., 1979; deWied, 1980; Simon, 1981 ; Berger et al., 1982; Koob and Bloom, 1982). A recent review by Dr. Maria Wolleman (1981) using a somewhat different format evaluates the role of cyclic AMP and opiate action. A variety of opiate-like peptides related to the endorphins and enkephalins have been shown to exert different actions in the brain. A recent novel hypothesis that opiates cause schizophrenia was supported by findings that certain patients could be successfully treated with the highly specific opiate antagonists, naloxone or naltrexone. Dialysis treatment which presumably removed opiate-like compounds also ameliorated the symptoms of schizophrenia. This work has, however, not gone unchallenged, but it is not inconceivable that opiate alterations in specific brain regions might either directly produce or indirectly (e.g. affect release of DA) cause mental disorders. In fact des-tyrosine-gamma-endorphin possesses neuroleptic action in both man and animals while des-tyrosine-alpha endorphin produces behavioral actions resembling to some extent, amphetamines. A deficiency of one compound or a functional excess of another within a particular brain region could easily result in symptoms of schizophrenia, mania or depression. Other opiate receptors appear to mediate principally pain suppression while different subtypes are associated with tolerance and withdrawal, and yet others stress and related endocrine events. Further actions of opiates include pupillary constriction, suppression of cough, decreased sensitivity to PCO2, sleep, euphoria, vomiting, and diminished respiration. Many of these actions can be traced to specific brain nuclei. The opiate receptors have unequal distributions in the brain with unusually high levels reported in nerve endings of the amygdala, striatum, periaqueductal gray, substantia gelatinosa, intralaminar nucleus of the thalamus and nucleus accumbens. The cerebellum contains low levels of opiate receptors but metabolic actions produced by opiates in this tissue are prominent via indirect pathways originating from the striatum (see discussion below). Opiates normally act in a stereospecific manner (one exception being the brain stem cough center), the '1' isomers being the most potent. The presence of excess Na + (perhaps due to depolarization) is associated with decreased affinity for agonists and enhanced affinity for antagonist binding. Moreover, highly active peptidases exist in brain which limit the action of the enkephalins. Opiates likewise deplete brain Ca 2+ and thus exert indirect, as well as, direct actions on a wide variety of neurotransmitter systems, ultimately affecting the synthesis and degradation of cyclic nucleotides. Thus ample evidence exists that these agents exert highly important neuromodulator actions in the brain (for detailed discussions see: Cardenas and Ross, 1975; Bunney et al., 1979; Ross et al., 1979; deWied, 1980; Simon, 1981; Berger et al., 1982). 15.2.

ROLE OF CYCLIC NUCLEOTIDES IN THE ACTION OF OPIATES

Some of the initial work which suggested that opiates might interact with cyclic nucleotide systems was conducted by Ho and coworkers (1973a,b). Intracerebral injections of cyclic AMP into mice resulted in administering larger doses of morphine in order to achieve analgesia. This and many other studies suggested that opiates possess anti-cyclic

78

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AMP like actions. In further work by Ho et al. (1973a,b, 1979) intraccrcbral injections of cyclic AMP accelerated the time period in mice to develop both tolerance and physical addiction to morphine. This phenomenon was blocked by administration of the protein synthesis inhibitor, cycloheximide. Withdrawal behavior from morphine addiction was found to be suppressed by electroacupuncture. Furthermore, preadministration of phosphodiesterase inhibitors (sc) or dibutyryl cyclic AMP (intracerebrally) completely antagofiized the effect of electroacupuncture. Ho et al. (1979) felt that acupuncture evoked a release of beta-endorphin whose action was counteracted by cyclic AMP. Moreover, Collier and Francis (1975) and Roy and Collier (1975) found that administration of cyclic AMP but not cyclic GMP to addicted rats or mice could intensify naloxone-precipitatcd withdrawal behavior. Naloxone may also increase central cyclic GMP levels (Gumulka et al., 1979b). In many instances (to be discussed below) opiates act differently in mice from behavioral and biochemical standpoints than in rats. In rats intracerebral injections of cyclic AMP analogs produced analgesia, an event reversed by naloxonc (Levy et al., 1981 ). The analgesic action of cyclic G M P was not sensitive to reversal by naloxone (Cohn et al., 1978). In recent experiments Hosford and Haigler (1981) evaluated the site of action of morphine and met-enkephalin in the mesencephalic reticular formation of rats. Spontaneous neural firing to nociceptive stimuli (foot pinch) was inhibited by iontophoretic ejection of the opiates in the vicinity of the neurons. The opiate inhibition of nociceptivc stimulus-induced firing was prevented by iontophoretically applied cyclic AMP analogs or phosphodiesterase inhibitors. Occupation of opiate receptors must therefore block in an unidentified manner, adenylate cyclase because dibutyryl cyclic AMP analogs bypass this site within the cell membrane by acting inside the cell. Karras and North (1979) were unable to observe any relationship between morphine and cyclic AMP with regard to firing of neurons in the guinea pig myenteric plexus. Similarly with anesthesized spinal, cats in which micropipettes were placed in the substantia gelatinosa, cyclic AMP action was not correlated to the ability of morphine to suppress noxious stimuli (Duggan and Griersmith, 1979). Possibly the positive correlations between morphine and cyclic AMP represent either regional or species specificity. 15.3.

DIRECT EFFECT OF OPIATES ON CYCLIC NUCEEOTIDE SYSTEMS

In the following discussion it will become evident that considerable controversy is associated with the number of experiments concerning opiate action in the various areas of the brain. Some of the controversies may be resolved because of limitations in employing various techniques for rapid fixation of brain tissue. Likewise, rats and mice under certain situations respond oppositely to opiates. The best model system that has yielded consistent results is the neuroblastoma x glioma hybrid cell, while the model system perpetuating the most controversy has been associated with D A mechanisms in the rat striatum, It seems with the well developed background of information regarding opiate administration during acute, chronic, and withdrawal situations in mice that much useful data comparing in vivo findings could be generated. Yet many investigators have attempted to decapitate, use nonfocussed microwave irradiation, or quick freeze adult rats and then correlate observed levels of cyclic nucleotides to basal activities of enzymes. Moreover, many different modes of drug injection are utilized in the experiments so comparisons of data are at times almost impossible to make. Out of necessity the data are principally presented according to cellular system or to brain regions.

15.3.1. Tissue culture models 15.3.1.1. A c u t e studies An extensive literature employing tissue cultures of the neuroblastoma x glioma hybrid (NG108-15) has indicated a use for this model system in evaluation of opiate action on

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cyclic nucleotide systems. The cell line possesses some unique characteristics, namely: (a) abundant opiate receptors; (b) capability for enkephalin synthesis; (c) receptors associated with stimulation of adenylate cyclase activity-prostaglandins El and adenosine; (d) receptors linked to adenylate cyclase in an inhibitory fashion---opiates, alpha adrenergic, muscarinic-cholinergic and perhaps beta adrenergic; (e) receptors linked to stimulation of cyclic GMP-opiates; alpha adrenergic and muscarinic-cholinergic; (f) low activities of enkephhlin metabolizing peptidases; (g) the presence of possibly two guanyl nucleotide binding sites associated with adenylate cyclase----one stimulatory the other inhibitory to hormone action; and (h) calcium activation of adenylate cyclase at lower concentrations and inhibition at higher calcium levels (Gullis et al., 1975a; Sharma etal., 1977; Klee, 1978; Bunney et al., 1979; Brandt et al., 1980; Wilkening et al., 1980; Propst and Hamprecht, 1981 ; Gwyn and Costa, 1982; Gilbert, 1982). Several investigators have consistently shown that upon acute addition of opiates either directly to cultures of neuroblastoma × glioma hybrids or to analogous cellular homogenates there was a respective reduction in cyclic AMP levels and inhibition of adenylate cyclase activity. Throughout all the studies the action of opiates was sterospecific in that /-isomers were several times more potent than corresponding d-isomers. Moreover, the inhibitory actions of opiates on either basal and prostaglandin El or adenosine-activated adenylate cyclase were reversed stereospecifically by only ( - ) naxloxone. Naloxone did not reverse the inhibitory actions of NE and carbamylcholine on cyclic AMP stimulation. The opiate and neurohumoral inhibition of adenylate cyclase-cyclic AMP was shown to be readily reversible, indicating that the direct presence of the drug was necessary for the observed action. The potency of opiates to inhibit basal or prostaglandin Erstimulated adenylate cyclase correlated in most instances with their affinity for opiate receptors. Enkephalin peptides (i.e. N-leu-5-enkephalin, D-ala2-D-leu-enkephalin, leu-enkephalin and met-enkephalin) were somewhat more potent than endorphins (beta, alpha, gamma and beta-lipotrophin) and natural or synthetic opiates (morphine, levorphanol, etorphine and methadone). Agents acting as partial agonists in other opiate assays behaved as partial agonists on adenylate cyclase. In addition to classical morphine binding sites, other more specialized enkephalin binding sites were thought to be present in these cells and the presence of Na + and GTP for expression of enkephalin action was likewise demonstrated. Even if adenylate cyclase had been preacti,,ated with cholera toxin (inhibits hydrolysis of GTP), enkephalins potently inhibited either basal or prostaglandin E1 activation of the enzyme. Thus, acute studies with opiates indicate a potent and highly specific receptormediated influence with regard to reducing the activity of adenylate cyclase (Sharma et al., 1975a,b; Traber et al., 1975a,b; Wahlstrom et al., 1977; Klee, 1978; Blume et al., 1979; Bunney et al., 1979; Wilkening et al., 1980; Gilbert et al., 1982). A direct receptor mediated inhibition of adenylate cyclase in neuroblastoma × glioma hybrids may not be the sole molecular mechanism responsible for opiate action. In these cells opiates stereospecifically enhance the synthesis of cyclic GMP. Likewise carbamylcholine and NE are effective (Gullis et al., 1975b; Traber et al., 1975b; Propst and Hamprecht, 1981). In more detailed work with the N4TG1 neuroblastoma cell, Gwynn and Costa (1982) correlated the pharmacological potency of opiates to increase cyclic GMP with their ability to displace binding of etorphine. The short term desensitization of opiate mediated cyclic GMP was not accompanied by any decrement in the number of agonist binding sites. Similarly there was no cross desensitization between azide, carbachol or etorphin to influence cyclic GMP. A possible mechanism to explain the acute action of opiates in simultaneously inhibiting cyclic AMP and stimulating cyclic GMP would be the well known opposing actions between the two cyclic nucleotides (Section 6). Thus the formation of cyclic GMP, an action requiring Ca 2, could effectively limit the synthesis of cyclic AMP (see Wilkening et al., 1980). On the other hand, naturally occurring opiates would activate GTPase and thereby reduce cyclic AMP while the synthesis of cyclic GMP would remain unimpeded. The mechanism occurs only in cells with opiate receptors (Koski and Klee, 1981; Christie-Pope et al., 1982). Even though opiate-induced GTP hydrolysis occurs in hybrid cells, the situation is further complicated because in these cells

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GTP may mediate, via two separate guanyl nucleotide binding sites, either inhibition or stimulation of adenylate cyclase (Koski and Klee, 1981). It also remains to be seen what related roles NE and carbamylcholine have with regard to the overall metabolic events associated with the two cyclic nucleotides. The action of these entirely different ncurotransmitters is identical to the opiates and each are expressed via separate, but highly specific receptor mechanisms. 15.3.1.2. C h r o n i c studies Exposure (4 or more hr) of neuroblastoma x glioma hybrid cultures to opiates or enkephalin-like peptides has been suggested to be a useful model system to evaluate mechanisms of opiate dependence and withdrawal (Sharma et al., 1975a,b, 1977; Klee, 1978; Bunney et al., 1979). After such exposure to opiates, the depressed levels of cyclic AMP observed after acute exposure have now returned to normal values. This acquisition of tolerance does not involve changes in the content of opiate receptors, but rather a compensatory increase in basal activity of adenylate cyclase. Moreover, chronic exposure to opiates followed either by naloxone or by washing the cells yielded a greater activation of adenylate cyclase by prostaglandin E1 or adenosine. The stimulatory "withdrawal-like" action by prostaglandin E~ and adenosine was not related to any increase in receptor affinity for either agonist, but appeared to require, in part, an increased synthesis of adenylate cyclase. In this regard cycloheximide blocked the etorphin-dependent increase in adenylate cyclase after 10 and 20 hr of incubation, but not at 4 hr. Moreover, the percent stimulation of adenylate cyclase to fluoride or GTP analogs was greater in control cells than after chronic exposure to opiates. Interestingly chronic incubation with both carbamylcholine and NE produced results analogous to the opiates. Klee (1978; also see Bunney et al., 1979) has suggested the concept of dual regulation of adenylate cyclase as a biochemical model for both acute and chronic opiate effects. In the acute situation morphine inhibits adenylate cyclase and depresses cyclic AMP levels. The cell compensates for chronic morphine action by increasing the number of molecules of adenylate cyclase thereby restoring cyclic AMP to normal values (dependence and tolerance). Upon rapid removal of morphine or administration of naloxone (withdrawal) the level of cyclic AMP is raised to abnormally high levels. This is followed by a gradual return to the normal level of adenylate cyclase. The only problem with this hypothesis is that the roles and effects of cyclic AMP have not been evaluated. 15.4. STRIATAL MECHANISMS AND NEUROTRANSMITTER RELATED EVENTS Opiates appear to influence a host of neurotransmitter systems, in particular the activities of catecholamine neurons. A brief discussion of DA influences is warranted because of the sensitivity of DA-adenylate cyclase in the corpus striatum. Most likely endogenous opiates regulate the release of neurotransmitters via inhibition or stimulation of this mechanism. If lesions were made to specific NE or DA pathways in the brain, the nucleus accumbens, for example had decreased uptake of NE and DA followed by a drop in the number of opiate receptors (Schwartz et al., 1979). Furthermore, Tang and Cotzias (1978) demonstrated that both morphine and DA inhibited in an additive manner the ability of naloxone to bind to nerve membrane fractions of the rat caudate nucleus. Morphine, L-DOPA and apomorphine all increased motor activity in mice and the two DA agonists prolonged morphine-induced analgesia. Areas rich in DA i,e. caudate and nucleus accumbens have an extremely high density of opiate receptors and enkephalin content as revealed by immunohistochemical localization (Snyder and Innis, 1979; Elde et al., 1976). A review by Lai (1975) summarized many of the actions of narcotics on central DA systems and in addition introduced possible interactions between opiates and the prostaglandins. The latter produce pain and alterations in body temperature which are counteracted by opiates. There are contrasting data, however, because Leysen and Laduron (1977), showed that the regional distribution of DA-adenylate cyclase and neuroleptic binding sites did not correlate with the distribution of opiate receptors. The

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inherent problem is that with respect to the wide variety of opiate receptor responses present in the central nervous system one would only expect a small portion to be associated with DA systems. Within the striatum, however, both neuroleptic and opiate receptors are localized principally to the microsomal fraction while DA-adenylate cyclase was observed in the mitochondrial fraction (Leysen and Laduron, 1977). Unfortunately for central tissue there are no simple cellular models to evaluate opiate-cyclic nucleotide interactions as was discussed for the neuroblastoma x glioma hybrid. The majority of the work discussed in the following paragraphs unless otherwise indicated was performed on the rat striatum. In these studies opiate action was consistently reversed by naloxone. Many conflicting results are presented and the reader should be aware that to draw positive conclusions from these data is at best difficult. Moreover, organization of the data from such divergent investigations does not readily produce a cohesive story of events. 15.4.1. Prostaglandins These systems in the rat striatum are discussed first because the initial work of Collier and Roy (1974) opened up the field of investigation. In their experiments they observed that morphine, methadone and heroin effectively inhibited the activation of adenylate cyclase by prostaglandins E1 and E 2. It was, however, difficult for other investigators using broken cellular fractions to replicate these studies, principally because of the relative insensitivity of the preparations to prostaglandins (Van Inwegen et al., 1975; Havermann and Kuschinsky, 1978b). In addition, administration (i.v.) of prostaglandin E~ to mice elevated the steady-state level of cyclic AMP in the striatum but the action was not affected by either acute or chronic administration of morphine (Von Voigtlander and Losey, 1977). However, tissue slices of striatum readily accumulate cyclic AMP as a consequence of prostaglandin E2 addition. In a naloxone-reversible manner morphine, met-enkephalin, levorphanol (but not dextrorphan) blocked this action of prostaglandin E2. Morphine was not effective when prostaglandin stimulation was enhanced by adding K + or adenosine to the tissue slices, indicating rather specific actions of opiates at prostaglandin-like receptors (Havermann and Kuschinsky 1978b).

15.4.2. In vivo levels of cyclic nucleotides 15.4.2. I. Cyclic AMP--acute investigations When rats or mice were acutely injected with rather high doses of morphine and sacrificed by rapid freezing the striatum contained elevated levels of endogenous cyclic AMP. The effect lasted from 1-30 min postinjection and was prevented by naloxone pretreatment (Bonnet, 1975; Clouet and Iwatsubo, 1975; Clouet et al., 1975; Von Voigtlander and Losey, 1977). In similar experiments injections of naloxone were, however, found to be ineffective (Dias, 1979). On the other hand Slater and Blundell (1979) reported a decrease in striatal cyclic AMP in the rat following morphine while in the mouse the cyclic nucleotide was elevated. They also observed that morphine affected behavioral responses in the rat (blocked some amphetamine behaviors and spontaneous activity) that were opposite from the mouse (increase in running). They concluded that morphine elicited a release of DA in the mouse while it blocked DA release in the rat. However, Arbilla and Langer (1978) showed an opiate-sensitive inhibition of NE release in the rat cerebellum, but no such event was evident with DA release in the striatum. 15.4.2.2. Cyclic AMP--chronic investigations Chronic injections of morphine into mice or rats resulted in a striatal cyclic AMP content that was unchanged (Clouet and Iwatsubo, 1975), elevated (Bonnet, 1975; Clouet et al., 1975; Merali et al., 1975) or decreased (Slater and Blundell, 1979) with respect to controls. Again, any changes were reversed by naloxone.

~2

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15.4.2.3. Cyclic A M P - - w i t h d r a w a l Chronically treated rats in which withdrawal to morphine addiction was induced by naloxone did not have any change in the striatal level of cyclic AMP, despite elevated levels of NE and DA (Mehta and Johnson, 1975). However, Volicer et al. (1977) reported elevated levels of the nucleotide, during withdrawal. 15.4.2.i. Cyclic' G M P In rapidly frozen striatum, acute injection of morphine led to attenuated levels of cyclic GMP (Bonnet, 1975). Alternatively, using microwave inactivation of tissue, Racagni et al. (1976) found an elevated striatal content of cyclic GMP within 5~40 min following morphine. As a further complication O'Callaghan et al. (1979) oserved no change in striatal cyclic GMP after acute injections of morphine. No change in striatal cyclic GMP was observed in rats undergoing withdrawal from morphine dependence (Volicer et al., 1977). In tissue slices of rat striatum opiates including enkephalins (in the presence of peptidase inhibitors) stereospecifically enhanced the accumulation of cyclic GMP. Basal levels of the cyclic nucleotide were not influenced by opiates (Minneman and Iversen, 1976). 15.4.3. Influence of adenylate cyclase activity 15.4.3.1. Basal, G T P and fluoride activity Morphine and naturally occurring opiates have been reported under in vitro situations to decrease basal activity of adenylate cyclase-cyclic AMP in the caudate nucleus of the rat and rabbit, as well as, the rabbit nucleus accumbens. Interestingly, the opiates likewise reduced adenylate cyclase elevation to GTP, but were without an effect on the GTPase resistant analog 5'guanylylimido-diphosphate. Inhibition of GTP-activated adenylate cyclase was amplified in the presence of Na + (Minneman and Iversen, 1976; Tang and Cotzias, 1978; Cooper et al., 1982; Christie-Pope et al., 1982). These data support the interesting finding of Koski and Klee (1981) in which opiates were found to stimulate directly the GTPase in neuroblastoma × glioma cells. Several investigators were either unable to show any influence of opiates on basal adenylate cyclase activity (Van Inwegen et al., 1975) or reported on enhancement of basal activity that was especially evident following acute injections of morphine (Merali et al., 1975). In these latter experiments injected morphine did not alter activity (Clouet and Iwatsubo, 1975; CIouet et al., 1975; Puri etal., 1975, 1976). In a pertinent investigation, incubation of striatal slices in the presence of morphine caused an enhanced calmodulin release into the cytosol. The phenomenon was blocked by the DA receptor antagonist, haioperidol and the opiate antagonist, naltrexone. The release of calmodulin was coincident with a shift of biphasic Kmphosphodiesterase to an enzyme form possessing exclusively low Km kinetics. Depletion of rat striatal monoamines by pretreatment with reserpine did not yield the above results indicating a monoamineopiate interaction at postsynaptic sites coincident with calmodulin-phosphodiesterase alterations. This phenomenon was not present in similarly evaluated cerebellar tissue (Hanbauer et al., 1979). During withdrawal from morphine a 2-fold increase in striatal adenylate cyclase (basal) occurred within 1-72 hr and control levels were attained by 96 hr (Puri et al., 1976). Tolerance to morphine was likewise associated with elevated adenylate cyclase (Merali et al., 1975). Alternatively, Van Inwegen and coworkers (1975) were unable to confirm these observations. 15.4.3.2. DA-adenylate cyclase Under conditions following either acute or chronic injections of morphine into rats there was, in addition to elevated basal levels, an enhanced stimulation of striatal adenylate cyclase by DA (Clouet and Iwatsubo, 1975; Merali et al., 1975; Purl et al., 1975; Tang and

PSYCHOACTIVEDRUGS, CYCLICNUCLEOTIDESAND THE CNS

83

Cotzias, 1978). In more detailed work, implantation of rats with morphine pellets revealed that all components of the striatal adenylate cyclase system were augmented e.g, DA, GTP, 5'-guanylyl-imidodiphosphate, MnZ+-ATP and basal activity (Parenti et al., 1982). In some instances the relative percent stimulation of adenylate cyclase over basal activity was not reported and upon inspection of the data the percent activation of adenylate cyclase to DA in the control was thus equal in magnitude to the morphine-treated animals. Moreox;er, Puri et al. (1976) failed to replicate their earlier work and Van Inwegen et al., 1975) and Havemenn and Kuschinsky (1978a) either saw none or only slight inhibitory effects of morphine on DA-adenylate cyclase. During withdrawal from opiates, the activation of rat striatal adenylate cyclase by DA was lower than controls and normal enzyme sensitivity returned by 96 hr after withdrawal (Purl et al., 1976; Merali et al., 1975). Again the investigation of Van Inwegen and coworkers (1975) failed to support these observations. 15.4.3.3. O t h e r striatal systems Morphine treatment of rats did not change the activities of striatal phosphodiesterase nor did it influence the cyclic AMP dependent incorporation of labeled phosphorous into protein-I-kinase (Merali et aI., 1975; Sieghart et al., 1979). However, Merali et al. (1975) did show enhanced protein kinase activity in the rat brain P2 synaptosomal fraction during morphine dependence. Activity was drastically reduced in morphine withdrawn rats but was again elevated when rats were given methadone.

15.5. NEUROTRANSMITTER--CYcLIC NUCLEOTIDE SYSTEMS IN OTHER BRAIN REGIONS

15.5.1. C e r e b e l l u m Acute injections of morphine into rats or mice decreased the steady-state levels of cyclic AMP in the cerebellum. Chronic treatment and withdrawal led to elevated cyclic AMP levels. The increase observed during withdrawal was blocked by the beta blocker, propranolol. No change in phosphodiesterase activity accompanied these alterations (Clouet et al., 1975; Charalampous and Askew, 1977; Mehta and Johnson, 1975). Lichtenstein (1976), however, found that heroin injections augmented cyclic AMP within 30 rain in the rat. In the majority of investigations acute administration of morphine, other opiates and enkephalin into rats, mice or monkeys promptly resulted in a decrease in the in vivo content of cyclic GMP in the cerebellum. This observation in the rat was evident following intrastriatai injection of the opiates and was not found when the opiate was given directly into the cerebellum or substantia nigra. Based on other work it was thought that opiates decreased cerebellar cyclic GMP via opiate receptor influences in the striatum which act to diminish excitatory input to the "opiate receptor poor" cerebellum by way of the afferent mossy fibers (Katz and Catravas, 1976; Pert et al., 1976; Biggio et al., 1977c, 1978b,c; Katz et al., 1978). In support of these findings Gumulka et al. (1979b) observed a naloxoneinduced elevation in cerebellar cyclic GMP. However, based on further observations they felt that high doses of naloxone exerted antagonism of GABA receptors, thereby increasing excitatory input into the cerebellum concomitant with increased synthesis of cyclic GMP. Askew and Charalampous (1976) found conflicting results. Acute morphine increased cerebellar cyclic GMP in the mouse along with a decrement in cyclic GMP phosphodiesterase activity. Chronic morphine and associated withdrawal therefrom yielded decreased guanylate cyclase activity in conjunction with lower levels of cyclic GMP. 15.5.2. N u c l e u s a c c u m b e n s In homogenates of rabbit nucleus accumbens enkephalins and beta endorphin reduce either the basal or GTP-induced activity of adenylate cyclase (Christie-Pope et al., 1982).

84

(~.('

PAixl~l¢

Iwatsubo (1977) reported that acute morphine injections into rats increase both basal and DA-adenylate cyclasc. Multiple doscs further incrcascd the DA sensitivity of the cnzwnc. 15.5.3. ,4 mygdala

Acute opiate treatment m rats produced a stereospecific increase in basal adenylate cyclase'in nerve ending preparations of amygdala. Using rapid fixation of tissue acute morphine treatment increased cyclic AMP within 15 min and normal levels returned shortly thereafter. Morphine tolerant rats had consistently high amygdaloid content of cyclic AMP. Large doses of opiates non-stereospecifically inhibited adenylate cyclase while lower doses werc without visible effects on the basal activity of the enzyme (Clouet et al., 1975). Dias et al. (1969), however showed that the naloxone-induced elevation in cyclic AMP was prcvented by pretreatment with haloperidol and propranolol. Dias therefore felt that endogenous opiates exerted a tonic influcnce on DA and NE neuromodulator systems in this region of high opiate receptor density (Pert et al., 197(~). Moreover, Wilkening et al. (1976) observed a potent inhibition by opiates of DA-adenylatc cyclase in the monkey amygdala. 15.5.4. C e r e b r u m

Following 30 rain administration of heroin cyclic AMP content rose in the cerebral cortex of the rat (Lichtenstein, 1976). However, similar injections of morphine did not change basal adenylatc cyclase or phosphodiesterasc, but during withdrawal from chronic morphine a slight decrease in adenylate cyclase was reported (Merali et al., 1975; Van lnwegen, et al., 1975). In the initial work, chronic infusion of morphine (1-4 weeks) produced an increase in basal adenylate cyclase without affecting phosphodiesterase (Naito and Kuriyama, 1973). Opiate peptides directly inhibited adenylate cyclase in homogenates of rat cerebral cortex (Wollemann et al., 1079), 15.5.5. Periaqueductal gray

This region is high in opiate receptors (Pert et al., 1976) and is thought to be a major site of analgesia for opiates. Morphine treatment of rats yielded a time dependent and stereospecific depression of steady-state levels of cyclic GMP. Moreover, tolerance developed to these effects of morphine, lew)rphanol, and to the partial agonists pentazocine and cyclazocine (O'Callaghan et al., 1979). 15.5.6. Brain stem Acute morphine decreased in vivo levels of cyclic AMP in rat brain stem, an event not seen during chronic treatment (Clouet et al., 1975). Met-enkephalin stimulated adenylate cyclase in the rat brain stem while beta-endorphin inhibited it (Wollemann et al., 1979). Acutely administered morphine lowered the steady-state levels of both cyclic nucleotides in the rat substantia nigra (Bonnet, 1975). 15.5.7. Diencephalon Injected morphine produced a time-dependent decrease in cyclic GMP in the centromedian nucleus in the rat thalamus without affecting the hypothalamus (O'Callaghan et al., 1979). In earlier work Bonnet (1975) likewise showed a morphine-induced depletion of cyclic GMP in both the rat thalamus and hypothalamus, while cyclic AMP levels were not changed. Alternatively, Lichtenstein (1976) reported an elevation in steady-state levels of cyclic AMP in the hypothalamus within 30 min subsequent to heroin infusion, while Clouet et al. (1975) found the nucleotide to be lowered 2 hr after morphine injections. During dependence to morphine, adenylate cyclase activity did not change in the thalamus-hypothalamus, but the enzyme activity declined following withdrawal (Merali et al., 1975).

PSYCHOACTIVEDRUGS, CYCLICNUCLEOTIDESAND THE CNS

85

15.5.8. W h o l e brain Merali et al. (1975) isolated crude synaptosomes from whole rat brain and observed a naloxone reversible elevation in cyclic AMP content, adenylate cyclase activity and cyclic AMP-protein kinase activity following injections with morphine. The cyclic AMP content would most likely not be a valid measurement because of the stress of animal sacrifice and time required for tissue isolation. Schmidt and Way (1976) were unable to show an action of morphine on either cyclic AMP content in rat brain (sacrificed by decapitation) on any effects of prostaglandins and/or morphine on adenylate cyclase activity. 15.6.

ADRENERGIC RECEPTORS

Following treatment of mice for 2-4 weeks with morphine, synaptosomal fractions from the cerebral cortex Contained higher adenylate cyclase and protein kinase activities while enzyme sensitivity to NE was reduced. If the mice were treated with the narcotic antagonist, levallorphan, normal enzyme responses to NE were now evident (Kuriyama et al., 1978). Continued work with the rat brain preparation indicated that opiates acted to block release of NE from the rat cerebral cortex, and other brain regions (Arbilla and Langer, 1978) via a presynaptic localization of opiate receptors (Llorens et al., 1978). The action of cyclic AMP to accelerate tolerance and physical dependence to opiates was prevented by beta adrenergic blockers (Ho et al., 1975). Furthermore, chronic opiate administration to rats and monkeys resulted in increased numbers of beta adrenergic receptors in the cerebrum, cerebellum and brain stem accompanied by an enhanced stimulation of adenylate cyclase by NE and isoproterenol (Llorens et al., 1978; Nathanson and Redman, 1981; Mattio and Kirby, 1982). These rather provocative studies with NE should be a basis for looking at the role of opiates on another transmitter system. It seems that much repeated and conflicting work was attempted on the: (1) DA system; (2) basal adenylate cyclase; and (3) measurement of steady-state levels of cyclic nucleotides. Other work with opiates hopefully might include the neglected histamine system. 15.7. PLASMA LEVELS OF CYCLIC NUCLEOTIDES

Data have been reported concerning altered levels of plasma cyclic AMP in response to an opiate challenge. In rats and mice acute opiate injections increased plasma cyclic AMP while the levels were normal during tolerance. Naloxone-precipitated abstinence yielded huge plasma levels of cyclic AMP (Ho et al., 1979; Muraki et al., 1979, 1981 ; Nakaki et al., 1981). Moreover, intracerebral administration of morphine or beta-endorphin likewise produced an acute elevation in plasma cyclic AMP. The action of opiates was prevented by beta but not alpha adrenergic blocking agents. Morphine most likely augmented plasma cyclic AMP concentrations by effecting a release of catecholamines from the adrenal medulla (Muraki etal., 1979; 1981; Nakaki etal., 1981). In recent work Muraki etal. (1982) found that plasma cyclic nucleotide responses to morphine varied considerably among different strains of mice. 15.8. CONCLUSIONS Divergent and conflicting results with opiates do not allow for many firm conclusions concerning their action on cyclic nucleotide systems in the brain. The neuronal z glioma hybrid cells offer the most consistent results and indicate that opiates elevate cyclic GMP and inhibit basal and prostaglandin or adenosine accumulation of cyclic AMP presumably via either stimulation of GTPase or by an opposing action by cyclic GMP. Tolerance to opiates is reflected through elevated activity of adenylate cyclase which is manifested upon withdrawal. Additional work with the beta adrenergic receptor indicates NE-sensitivity of adenylate cyclase which develops in rodent brain during tolerance. Acute injections of opiates generally elevate cyclic AMP and basal enzyme activity in some brain regions. Acute opiate administration depletes cerebellar cyclic GMP via an indirect process involving activation of opiate receptors in the striatum. Despite the well-described

~(~

(;. ('. P,xl MI-R

behavioral and chemical actions of opiates on central DA systems, no clear cut data have evolved with regard to cyclic nucleotide mechanisms. For some reason the incubated tissue slice method has been largely ignored in studies associated with opiate action. This technique looked highly promising in one prostaglandin study and could be extended to include other transmitters. Opiate effects on cyclic G M P mechanisms should likewise be expanded. It is indeed unfortunate that with all the expended efforts stated herein that no clear cut picture of opiate action has emerged. Some encouraging findings are the observations that opioid peptides may exert some of their effects on neuronal function by affecting the phosphorylation of specific synaptic proteins (Davis and Ehrlich, 1979).

16. Convulsants and Anticonvuisants

16.1.

INTRODUCTION AND SIGNIFICANCE OF CYCLIC NUCLEOTIDES IN SEIZURES

Epilepsies are a group of diverse central disorders with a common symptom of seizures which tend to be recurrent. Basically two conditions cause seizures i.e. not enough inhibitory action within a specified core of neurons, or too much excitatory action. Design of therapeutic agents has been directed to some extent to correct these abnormalities. A wide variety of substances (drugs, drug-induced withdrawal, hormones, poisons, etc.), as well as, trauma, fevers and alterations in central metabolic states elicit seizures. The International League Against Epilepsy has developed a new classification to aid the physician in establishing a proper format for therapeutic management of seizures. This classification consists of four broad categories each containing several subtypes of symptomology: (1) Partial seizures--these may have complex symptoms, usually are difficult to manage, and may include psychomotor problems; (2) bilaterally symmetrical-generalized seizures--these include grand mal (tonic-clonic) and petit mal (absence seizures); (3) unilateral seizures; and (4) unclassified seizures--status epilepticus, chemically-, hormonally- or metabolically-induced (see White, 1981). The following paragraphs will illustrate that there is a definite link between seizure activity and the production of cyclic nucleotides in the brain. Moreover, data will be presented to indicate a cyclic nucleotide site of action for antiepileptic drugs. Sattin (1971) initially demonstrated an increase in cyclic AMP levels in rapidly fixed brain as a response to seizures produced by either electroconvulsive shock or administration of hexafluorodiethyl ether. A host of subsequent studies using focal epilepsy, electroconvulsive shock, drugs, toxic substances, and freezing lesions have generally shown a direct relationship between convulsant activity and elevations in either or both cyclic AMP and GMP. In these investigations a variety of techniques were employed to measure cyclic nucleotide alterations in rapidly fixed tisssue from various species (Table 12; see reviews by Ferrendelli et al., 1979; Lust et al., 1981a). For the most part all brain regions, except striatum show the usual seizure-induced increase in cyclic nucleotides. However, both the magnitude and the time course of these cyclic nucleotide elevations differ considerably between the different brain regions. Moreover, the rise in either cyclic AMP or cyclic GMP were not in phase with one another with respect to a particular brain region, or to a time of appearance, or with regard to a course of action, and dose of convulsant agent. In a series of experiments various cell layers of the cerebellum, cerebrum and hippocampus were assayed as a means to locate the cellular site of cyclic nucleotide change, elicited by convulsant agents. A few specific differences were seen but generally cyclic nucleotide levels were increased throughout all cell layers. For example, during the first clonic seizure-induced by pentylenetetrazol cyclic AMP rose (2-fold) in the molecular and granular layers of the cerebellar cortex while cyclic GMP rose (4-fold) in the molecular layer and 2.5 fold in the granular layer (Ferrendelli et al., 1979, 1980). After maximal electroshock seizures both cyclic nucleotides increased in all layers of the cerebellum, however, the time of appearance of the maximal increase differed between nucleotides, as well as, between the different cell layers for a particular cyclic nucleotide (McCandless et al., 1979a,b; Lust e t a l . , 1981b).

PSYCHOACTIVE DRUGS, CYCLIC NUCLEOT1DES AND THE CNS

87

TABLE 12. STIMULATIONOF CYCLIC NUCLEOTIDES itl vivo IN BRAIN BY AGENTS PRODUCING CONVULSIVE EPISODES Agent

Species

Brain regions

Pentylenetetrazol

mouse guinea pig

Bicyclic phosphorus esters Electroconvulsive shock

mice

cerebrum, cerebellum, median forebrain, hippocampus, striatum cortex, cerebellum subcortex

Freezing lesion KC1 spreading depression Focal penicillin Bicuculline Caffeine

Harmaline Hexafluorodiethylether Homocysteine Isoniazid 3-Mercaptopropionate Oxotremorine Picrotoxin Soman Veratridine

rabbit mouse mouse rat rat rat cat mouse mouse mouse mouse mouse mouse mouse mouse mouse mouse mouse rat

CSF forebrain cerebellum cerebrum cerebrum cerebrum cerebrum forebrain forebrain cerebrum, subcortex, and cerebellum cerebellum cerebrum cerebrum cerebrum,cerebellum cerebrum cerebellum cerebellum cerebrum cerebrum

cAMP

cGMP

Reference

usually increase during tonic-clonic

I'

2,6,9,10, 16,17

~

1'

1,12

1" 1' '~ " " ]" NC** 1" NC ~,

ND* ND 1" ND ND ND 1' ~" ND "

15 19 9,10,14 20 8 7 18 10,21 18 2

ND 1" 1" 1" 1' ND NC 1' t

1' ND ND t 1" 1" 1' 1' ND

16 19 5 6,10,13 4 16 6,11,16 1 8

* ND = not determined, ** NC = no change. References: (1) Coult et al., 1979; (2) Ferrendelli etal., 1980; (3) Folbergrova, 1977; (4) Folbergrova, 1980; (5) Folbergrova, 1981 ; (6) Gumulka et al., 1979a; (7) Krivanek and Mares, 1977; (8) Krivanek, 1978; (9) Lust etal., 1978; (10) Lust etal., 1981a; (11) Mao etal., 1975b; (12) Mattsson et al., 1977; (13) McCandless etal., 1979a; (14) McCandless etal., 1979b; (15) Myllyla, 1976a; (16) Opmeer etal., 1976; (17) Palmer etal., 1979; (18) Raabe etal., 1978; (19) Sattin, 1971;(20) W a l k e r e t a l . , 1973; (21) Wasterlain and Csiszar, 1980.

Another problem associated with the experiments involving cyclic nucleotide alterations and seizure activity is the general lack of correlated electrical recording studies. In one study with focal cortical epilepsy Raabe et al. (1978) measured an increase in cyclic GMP, but not cyclic AMP in only the region of the epileptogenic focus. Cyclic GMP was highest during electrically recorded ictus, dropped somewhat during post-ictus and was further decreased (yet remained elevated over control) during the interictal interval. In Section 6 there is a discussion of the opposing interactions between cyclic AMP and cyclic GMP. The work by Siggins and coworkers (1969) demonstrated that cyclic AMP mechanisms were associated with a reduction in the firing of cerebellar Purkinje cells. Further work with cortical pyramidal cells indicated inhibition of firing rates by cyclic AMP and an increase in firing patterns by cyclic GMP (Stone et al., 1975, Stone and Taylor, 1977). With regard to seizure activity, cyclic GMP had been shown to be elevated in the preconvulsive stage and the increased cyclic AMP was not always apparent until evidence of convulsions were manifested. These observations ledFerrendelli et al. (1979; 1980) and Lust et al. (1981) to postulate that the early appearance and large increase in cyclic GMP was associaed with, or contributed to, the onset of excitatory transmission. The later rise in cyclic AMP could be a protective inhibitory mechanism designed to dampen the excitatory action of cyclic GMP. However, the elevation in cyclic AMP could also be a result of anoxia-ischemia and the associated release of adenosine and catecholamines (Lavyne et al., 1975; Lust and Passonneau, 1979; Winn et al., 1981). However, the contribution of anoxia to cyclic AMP levels is thought to be minimal during seizures because of regional differences in response to convulsants and because pentylenetetrazol evokes the synthesis of cyclic AMP. This latter type of seizure is essentially devoid of anoxia (Ferrendelli et al., 1979; Lust et al., 1981a).

SS

( i . C . P:,LMEI¢

16.2. EFFECTS OF ANTICONVULSANTS ON STEADY-STAI-E LEVEES OF CYcl.l(" NUCLEOTIDES ELICITED BY SEIZURES It a p p e a r s that the b i o c h e m i c a l m o d e l of seizure elicited synthesis of cyclic n u c l e o t i d e s might be a useful t e c h n i q u e to e v a l u a t e the m o l e c u l a r site o f action of a n t i c o n v u l s a n t drugs. In clinical i n v e s t i g a t i o n s two studies failed to s h o w any c o r r e l a t i o n b e t w e e n levels of cyclic A M P and G M P w h e n e p i l e p t i c p a t i e n t s w e r e c o m p a r e d to c o n t r o l s ( R o b i s o n e t a l . , 1970: T r a b u c c h i e t a l . , 1977). O n the o t h e r h a n d Myllyla e t a l . (1975) did r e p o r t an i n c r e a s e d C S F c o n t e n t of cyclic A M P in p a t i e n t s which h a d a seizure within t h r e e days p r i o r to sampling. T h e r e is s o m e a r g u m e n t as to w h e t h e r C S F has any p h y s i o l o g i c a l r e l a t i o n s h i p to c h a n g e s in i n t r a c e l l u l a r levels o r t u r n o v e r rats of cyclic n u c l e o t i d e s in the brain. In f u r t h e r w o r k , M y l l y l a (1976a) a d m i n i s t e r e d e l e c t r o c o n v u l s i v e shock to r a b b i t s a n d o b s e r v e d a rise in cyclic A M P which c o u l d be p r e v e n t e d by p r i o r t r e a t m e n t with p h e n y t o i n , p h e n o b a r b i t a l o r c a r b a m a z e p i n e . E v i d e n t l y m o r e w o r k is r e q u i r e d with this C S F m o d e l . In a d d i t i o n , cyclic G M P s h o u l d be m e a s u r e d a n d p e r h a p s c o r r e l a t i o n s to seizure activity could be m a d e to levels of r e l e a s e d t r a n s m i t t e r s u b s t a n c e s . A n o t h e r p r o c e d u r e for e v a l u a t i o n o f a n t i c o n v u l s a n t a g e n t s i n v o l v e d a d m i n i s t r a t i o n of large i n t r a h y p o t h a l m i c d o s e s of d i b u t y r y l cyclic A M P which c a u s e d seizures in chickens. T h e action of the cyclic A M P a n a l o g was p r e v e n t e d b y t r e a t m e n t with p h e n y t o i n a n d p h e n o b a r b i t a l while d i a z e p a m was ineffective (Nistico, 1977). T h e m o s t p o p u l a r m e t h o d o f e x a m i n i n g the effects of a n t i s e i z u r e drugs on c o n v u l s a n t - e l i cited cyclic n u c l e o t i d e p r o d u c t i o n is the t e c h n i q u e of r a p i d fixation of tissue. H o w e v e r , unless the b r a i n is fixed by r a p i d f r e e z i n g (with assay o f a r e a s close to the surface) o r f o c u s s e d m i c r o w a v e i r r a d i a t i o n , d a t a for cyclic A M P will v a r y ( J o n e s and S t a v i n o h a , 1979: see Section 2.3.1). In T a b l e 13 are listed a s u m m a r y o f findings with a n t i c o n v u l s a n t agents. F o r the most p a r t the m o u s e b r a i n has b e e n utilized exlusively for this t y p e of w o r k . In g e n e r a l m o s t a n t i c o n v u l s a n t a g e n t s including the G A B A agonist, b a c l o f e n , definitely b l o c k the c o n v u l s a n t - i n d u c e d rise in cyclic G M P . T h e effects with cyclic A M P usually follow this p a t t e r n , b u t d i f f e r e n c e s are a p p a r e n t . A n u n u s u a l o b s e r v a t i o n is that r e d u c i n g the action of central m o n o a m i n e s with p r o p r a n o l o l , or r e s e r p i n e , inhibits the c o n v u l s a n t TABLE 13. INHIBITORY ACTIONS OF ANTICONVUI.SANI S ON SEIZURE-INDUCED EI.EVAFIONS IN CYCLIC N U ( I.EO I'll)l:b IN RAPIDI Y FIXED MOUSE BRAIN

Amiconvulsant Phenytoin

Phenobarbital Pentobarbital Valproate Ethosuximide Diazepam Clonazepam Bacloten Reserpine Propranolol

Convulsant Electroshock

Brain region

Cerebellum cortex Pentylenetetrazol Cerebellum cortex 3-Mercaptopropionate cortex Pemylenetetrazol Cerebellum cortex 3-Mercaptopropionate cortex Picrotoxin Cerebellum and harmaline cortex lsoniazid Cerebellum Pentylenetetrazol whole brain Pentylenetetrazol cortex, cerebellum Picrotoxin Cerebellum lsoniazid Cerebellum Pentylenetetrazol Cerebellum lsoniazid Cerebellum Picrotoxin and Cerebellum isoniazid Cerebellum Pentylcnetetrazol Cerebellum Pentylenetetrazol whole brain Homocystein cortex

Cyclic AMP

CyclicGMP

partial block no effecl block block block block block block noeffect no effect ND block block no effect ND block ND ND ND ND block block

block block block block no effect block block block block block block block block block block block block block block no effect no effect NO

Reference t~,7,8 I, 11 I, 11 3 1,10.1 | I, 111,11 2,3 10 1() 7.9 1 Ill, 11 8,111 7. I 1 7 5 5 _4 I 4

ND = not determined. References: (1) Ferrendelli et al., 1979; (2) Folbergrova, 1977; (3) Folbergro~a, 19811: (4) Folbergrova, 1981 ; (5) Gumulka et al., 1979a; (6) Johnson and Ricker, 1982: (7) Lust et al., 1978: (8) Mao et al., 1975a,b; (9) McCandless et al., 1979a,b; (10) Opmeer et al,, 1976, ( 11) Palmer et al.. 1079.

PSYCHOACTIVEDRUGS, CYCLIC NUCLEOTIDESAND THE CNS

89

induced rise in cyclic AMP alone, suggesting that a release of NE is essential for this phenomenon. Moreover, these agents did not prevent convulsions (Ferrendelli et al., 1979; Folbergrova, 1977). The role of adenosine in cyclic AMP elevations during seizure activity is somewhat equivocal, Sattin (1971)first noted that the adenosine receptor blockers, theophylline and caffeine, when injected prior to electroconvulsive shock partially reduced the increase in cyclic AMP in rapidly frozen mouse brain. Folbergrova (1977) was unable to prevent 3-mercaptopropionate seizure-induced elevations in cyclic AMP by pretreatment with theophylline. The idea that adenosine is released during seizures followed by its stimulation of cyclic AMP--in which both agents act to inhibit neuronal firing (Phillis and Wu, 1981) is an intriguing possibility with regard to the latent protective role of cyclic AMP during convulsive episodes. Unfortunately sufficient experimental evidence is lacking to support this hypothesis. Anticonvulsant drugs injected in vivo influence to some extent the central content of cyclic nucleotides in rapidly fixed control animals. The mouse cerebellum appear to be especially a site for the depressant actions of antiepilepsy drugs on steady-state levels of cyclic GMP. Thus, acetazolamide, baclofen, carbamazepine, clonazepam, diazepam, pentobarbital, phenytoin, procylidine, trimethadione, and valproate all possessed this ability (McCandless et al., 1979a; Lust et al., 1978; Gumulka et al., 1979a; Opmeer et al., 1976). Drug action on cerebral cyclic GMP was considerably less potent. In a few situations phenytoin and carbamazepine acted to decrease cyclic AMP in the mouse cerebrum or cerebellum (Palmer et al., 1979; Lust et al., 1978). Diazepam and clonazepam increased steady-state levels of cyclic AMP (Palmer et al., 1979), presumably due to two actions, prevention of adenosine uptake (Phillis and Wu, 1981) and inhibition of cyclic AMP phosphodiesterase (Palmer, 1979; see Section 17.1). 16.3.

EFFECTS OF ANTICONVULSANTS IN W H O L E C E L L PREPARATIONS

Various experimental protocols have utilized the technique of incubated tissue slices in an attempt to define with a greater degree of precision the transmitter substances that are influenced by antiepileptic agents. A substantial amount of work in this area has been performed by the group of Ferrendelli (Ferrendelli and Kinscherf, 1978b; for review see Ferrendelli et al., 1979). In their experiments tissue slices of mouse cerebellum increased both cyclic AMP and GMP in response to ouabain (inhibits Na + , K+-ATPase), veratridine (prevents closure of ion specific Na + channels), K + depolarization and glutamate (activates specific receptors associated with excitation). Anticonvulsant agents namely, phenytoin, carbamazepine, phenobarbital, primidone, phensuximide, methsuximide, alpha-methylalpha-phenylsuximide and high levels of clonazepam which block maximal electroshock seizures, prevented the veratridine- and ouabain-induced formation of cyclic nucleotides. Agents generally used for absence (petit mal) seizures or to prevent pentylenetetrazol convulsions (low doses of clonazepam, valproate, ethosuximide, and trimethadione were either ineffective or only partially prevented the formation of cyclic GMP alone. These agents had essentially no actions with respect to cyclic nucleotide production elicited by potassium or glutamate. In similar work with mouse cerebellum and cerebral cortex (Palmer et al., 1979; 1981) and rat cerebral cortex (Lewin and Bleck, 1977) it was likewise shown that antiseizure drugs only prevented the ouabainAnduced formation of cyclic AMP and cyclic GMP. The elevation of cyclic AMP by NE was selectively inhibited by carbamazepine, a compound with a tricyclic structure resembling imipramine or phenothiazines (Palmer, 1979; Palmer et al., 1979). At only high anticonvulsant concentrations were cyclic AMP responses to adenosine or K + affected. Likewise, the ability of hydroxylamine to elevate cyclic GMP in the mouse brain was essentially unaffected by antiepilepsy drugs. Similarly the action of sodium azide or cyclic GMP formation was inhibited by only phenytoin and carbamazepine (Palmer et al., 1981). With regard to Ferrendelli's initial work it was further shown that phenytoin blocked the influx of Ca 2+ into brain synaptosomes (Ferrendelli and Kinscherf, 1977; Ferrendelli,

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1980). Moreover, Study (1980) showed that phenytoin inhibited the calcium-dependent increases in cyclic GMP produced by either K + depolarization or by muscarinic activation of neuroblastoma cells. The additional fact that tetrodotoxin, a selective inhibitor of Na + channels, blocked only ouabain or veratridine elevations in cyclic nucleotides led Ferrendelli to postulate that the major site of phenytoin and perhaps other anticonvulsant action was to limit Ca2+-Na + influx during depolarization (Ferrendelli and Kinscherf, 1977b; 1978a,b; Ferrendelli et al., 1979; Ferrendelli, 1980). Dretchen et al. (1977) showed a phenytoin reduction in adenylate cyclase activity after the induction of repetitive after-discharges (cat nerve-muscle preparation). The effects of phenytoin could be reversed by calcium or mimicked by verapamil (calcium current antagonist). These data further supported Ferrendelli's hypothesis that phenytoin blocks a cyclic nucleotide mediated Ca 2+ influx associated with transmitter release. Pentylenetetrazol evoked a small increase in cyclic AMP in incubated tissue slices from rat cerebral cortex. The response was inhibited by the adenosine receptor blocking agent, theophylline. Alternatively, additions of adenosine and 2-Cl-adenosine magnified the action of pentylenetetrazol suggesting that the increase in cyclic AMP was due to a drug-induced release of adenosine. In keeping with the inability of anticonvulsants to affect adenosine-sensitive adenylate cyclases, neither phenytoin, phenobarbital nor ethosuximide prevented the action of pentylenetetrazol (Lewin et al., 1976). Interestingly in mouse cerebral tissue slices, clonazepam and to a lesser extent diazepam, enhanced both basal levels of and adenosine-induced accumulations of cyclic AMP (Palmer et al., 1979). The latter findings could be attributed to either a benzodiazepine potentiation of adenosine actions by blocking adenosine uptake into neurons (Phillis and Wu, 1981) or an inhibition of phosphodiesterases by benzodiazepines (Palmer, 1981a). 16.4. EFFECTS OF SEIZURE CONDITIONS ON CYCLIC NUCLEOTIDE ENZYMES 16.4.1. Adenylate cyclase-DA receptors In only a limited number of investigations have the cyclic nucleotide enzymes been looked at with regard to experimental epileptic conditions. In the "kindling" rat model, subconvulsive application of pentylenetetrazoi or electrical stimulation was administered to the amygdala on a chronic basis until such stimulation elicited a specified series of tonic-clonic seizures. Following chronic electrical stimulation there was observed a decrease in both basal and DA stimulated adenylate cyclase in the amygdala while only a diminished basal activity of the enzyme was seen in the cerebral cortex. In conjunction with these findings the ability of labeled spiroperidol to bind to DA receptors in the amygdala was likewise reduced (Gee et al., 1980). Despite these changes in adenylate cyclase-DA receptor sensitivity, Walker et al. (1981) were unable to demonstrate any change in steady-state levels of cyclic AMP or GMP as assessed by focussed microwave fixation of the brain. In further work Gee et al. (1981) found that whereas acute application of pentylenetetrazol had no effect on adenylate cyclase or DA receptor binding, chronic treatment did, however, reduce specifically the high affinity spiroperidol binding in the amygdala and low affinity binding in the frontal cortex. Thus two differing experimental protocols do not yield the same answers. Electrical stimulation might influence all types of DA receptors while pentylenetetrazol decreases t h e n u m b e r of those DA receptors uncoupled to adenylate cyclase. Moreover, the two agents produce different types of seizure activity in the brain. Dopamine does appear to suppress seizure activity, possibly through its action associated with the generation of slow inhibitory postsynaptic potentials (Gee et al., 1981; Phillis, 1977; McAfee and Greengard, 1972). 16.4.2. Phosphorylation-mechanisms

Ehrlich et al. (1980) showed an increased phosphorylation of specific brain proteins of rodents as a consequence of electroconvulsive shock. Furthermore, Strombom and

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coworkers (1979) using quick frozen tissue from mice brain reported an enhanced rate of phosphorylation of proteins Ia and Ib in response to seizures elicited by picrotoxin or pentylenetetrazol. The type I proteins are specific for neural elements i.e. presynaptic vesicles, membranes and subsynaptic material in the postsynaptic complex. A calcium stimulated phosphorylation of specific presynaptic proteins (DPH-L and DPH-M) of the rat brain was blocked by phenytoin in keeping with experiments by Ferrendelli and Kinscherf (1977b) where, in incubated tissue slices phenytoin antagonized the calcium influx into cells during cellular depolarization. The site of action of antiepileptic drugs at the level of specific protein phosphorylation should be an important consideration for future work. In this aspect some interesting new data have been reported with respect to benzodiazepine action (see Section 17.1).

16.4.3. Anticonvulsant action on cyclic nucleotide e n z y m e s In tissue homogenates of rodent brain, of the drugs tested (phenobarbital, carbamazepine, phenytoin, diazepam and clonazepam), only carbamazepine had any inhibitory action toward DA- or NE-activated adenylate cyclase. The benzodiazepines enhanced the activity of adenylate cyclase especially when phosphodiesterase inhibitors were omitted from the assay (Palmer, 1979; Coult and Howells, 1979). In keeping with their potential to prevent the metabolism of cyclic nucleotides (see Section 17.1) these agents were the only antiepilepsy drugs capable of inhibiting cyclic AMP phosphodiesterase under conditions of low substrate or high sustrate with or without calmodulin and calcium. The latter action on phosphodiesterase appeared not to exhibit any special specificity (Palmer, 1979). Furthermore, these drugs were relatively ineffective toward guanylate cyclase prepared from mouse cerebellar and cortical homogenates. Carbamazepine at high concentrations did block calcium activation of guanylate cyclase (under conditions of low Mn2+). In addition, along with phenytoin, carbamazepine inhibited enzyme activation by sodium azide, but not by hydroxylamine (Palmer et al., 1981). The cycic nucleotide enzymes thus do not appear to be a suitable preparation in which to evaluate the site of action of anticonvulsants.

16.5. CONCLUSIONS The studies quoted herein indicate a direct relationship between the onset and duration of seizure activity to central elevations in cyclic AMP and cyclic GMP. Moreover, in most situations especially with cyclic GMP the seizure-elicited rise in cyclic nucleotides was prevented by pretreatment with clinically useful antiepileptic agents. In incubated tissue slices the stimulation of cyclic nucleotide accumulation by agents evoking sodium depolarization appears to be selectively inhibited by antiseizure drugs. The drugs have little action on cyclic nucleotide enzymes--adenylate and guanylate cyclases, or the phosphodiesterases. However, some recent evidence reveals an anticonvulsant action at the level of the synaptic membrane involving phosphorylation of specific proteins. These studies should be extended to include a greater number of agents. One well known difficulty in attempting work of this nature is that different classes of antiepilepsy drugs have other, perhaps more specific, sites of action on a variety of membrane, transmitter or enzymatic systems. Other problems with drugs are their differences in treating the different categories of epilepsy and preventing the various types of experimental seizures. Furthermore, there is a definite lack of clinical correlates to the cyclic nucleotide fluctuations observed under experimental situations using cellular or animal models. Most likely, cyclic nucleotide studies connected to measuring both transmitter release (especially GABA and glutamate) and electrical recording sequences might be of greater benefit. Nevertheless these molecules especially cyclic GMP are tied into the seizure process, while cyclic AMP is delayed and exerts a dampening or inhibitory effect and helps prevent the spread of the seizure current.

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17. Drugs with CNS Depressant Activity 17.1. BEN ZODIAZEPINES

17.1.1. Background Estimates in the United States alone range up to 100 million prescriptions per year for benzodiazepines whose use, misuse, overuse and abuse potentials have been amply described. Four major clinical uses of benzodiazepines are attributed to: (1) their ability toward controlling symptoms of anxiety; (2) sleep-inducing (hypnotic) effects; (3) centrally-induced muscle relaxant properties; and (4) anticonvulsant actions. The first three actions, especially anti-anxiety, are the most prevalent areas in which misuse and abuse occur. While recent biochemical advances have determined a molecular site (benzodiazepine receptor) for benzodiazepine action, any relationship to the clinically observed effects remains an enigma. In addition, an endogenous substance within the brain that acts on benzodiazepine receptors has not yet been discovered. To state that such a substance does not exist may be erroneous because, for example, the opiate receptor was well described prior to the discovery of enkephalin and endorphins. Anticonvulsant properties are the only definite clinical actions of benzodiazepines that can be correlated to their G A B A facilitatory action at the benzodiazepine receptor. The major problem with the association of benzodiazepine action to antianxiety properties is the disease itself which can also be effectively controlled in some cases by the beta adrenergic antagonist, propranolol. Anxiety is manifested by a variety of symptoms which include: (a) behavioral--nervousness, tension, headache, irritability, alarm, mild depression, worry, insomnia, subjective quality of fear, dread, or apprehension of impending danger; and (b) somatic--tremor, palpitations, sweating, urinary frequency, gastrointestinal complaints, subjective aches and pains, tightness in the throat and chest, dry mouth, dizziness, weakness and difficulty in breathing. The more persisting severe symptoms should be differentiated from normal anxieties that wax and wane in everyday life. Unfortunately many patients cannot cope with even minor upsets. With such a wide variety of symptoms it would be almost impossible to determine a specific site of benzodiazepine action in the brain. Some of the high density areas for benzodiazepine receptors, however, may be related to their pharmacological effects (e.g. amygdala, hippocampus and frontal cortex with anxiolytic activity; cortex, hippocampus and amygdala with anticonvulsant activity; cerebellum and reticular formation with ataxia and muscle relaxant activity). The benzodiazepine receptor is principally observed in the brain in association with nerve ending fractions. Binding of benzodiazepines is saturable and stereospecific, and solubilization indicates a mol. wt of 200,000 daltons. The occupation of the benzodiazepine receptor promotes a change of the G A B A receptor to one of a high affinity state. The receptor for benzodiazepines is a separate entity from that for G A B A . Furthermore, another protein substance, GABA-modulin, which is thought to limit the receptor affinity of G A B A , may be a site for benzodiazepine action. Thus the benzodiazepine receptor enhances the action of G A B A and increases chloride conductance in target neurons. Multiple receptors for benzodiazepines have been reported to exist, but whether these receptors exhibit "up and down" regulation like D A or NE receptors is a subject of considerable controversy. In some interesting animal work it was shown that emotional or fearful strains of mice had lower receptor numbers than more courageous strains (for reviews of this discussion see: Vacik and Palmer, 1980; Lader, 1981 ; Muller, 1981 : Speth etal., 1981; Suzman, 1981). In the following discussion it will be evident that benzodiazepines do influence cyclic nucleotide systems in the brain. The evidence, however, is somewhat limited and corollaries to the action of cyclic nucleotide and either the benzodiazepine receptor or clinical conditions cannot be made at this time. Basically, three major observations will be

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shown to occur concerning benzodiazepine action on cyclic nucleotides: (1) phosphodiesterase inhibition; (2) decreased production in vivo of cyclic GMP in the cerebellum; and (3) inhibition of adenosine uptake. 17.1.2. Ben zodiazepines as phosphodiesterase inhibitors and adenosine uptake blockers 17.1.2.1. Whole cell preparations

The elevation of cyclic nucleotides by benzodiazepines in tissue culture or brain slice preparations may actually be attributed to two common properties, namely, inhibition of cyclic nucleotide phosphodiesterase and potentiation of adenosine action by preventing adenosine uptake thus preventing its inactivation by adenosine deaminase. In cultured neuroblastoma or neuroblastoma x glioma hybrid cells diazepam enhanced the accumulation of cyclic AMP under conditions of basal or prostaglandin El-elicited activity. When a methylxanthine phosphodiesterase inhibitor was administered along with diazepam, the elevation in cyclic AMP was less than with the benzodiazepine alone. The xanthine analog blocked adenosine receptors and therefore the blockade of adenosine reuptake by diazepam was compromised. The remaining elevation in cyclic AMP was attributed to antagonism of phosphodiesterase (Schultz and Hamprecht, 1973; Propst et al., 1979). In incubated tissue slices of mouse cerebellum or cerebrum, guinea pig cerebral cortex and rat cerebrum benzodiazepines elevated basal levels of cyclic AMP and GMP (Schultz, 1974a,b, 1975; Rubin and Ferrendelli, 1977; Palmer et al., 1979; Traversa and Newman, 1979). Nahorski and Rogers (1976) were, however, unable to see any effect of medazepam in similar preparations of chick or rat cerebral cortex. In addition, diazepam magnified the cyclic AMP accumulation induced by adenosine (Schultz, 1974a; Palmer et al., 1979; Traversa and Newman, 1979), histamine, NE, isoproterenol and NE plus histamine (Schultz, 1974a,b, Traversa and Newman, 1979). These latter two groups of investigators also found that other benzodiazepines and metabolites of diazepam were similarly effective, however, diazepam was the most potent agent. Effective agents were d', dl or l' isomers of oxazepam, des-methyl-diazepam, methyloxazepam, and chlordiazepoxide. If the slices were pretreated with theophylline and adenosine deaminase there was a marked reduction in the ability of the benzodiazepines to elevate cyclic AMP, further suggesting a benzodiazepine inhibition of the uptake of adenosine (Traversa and Newman, 1979). In another adenosine experiment, Schultz (1975) using guinea pig cerebral slices found that NE plus histamine readily caused the tissue to become refactory toward further stimulation of cyclic AMP. The following agents, however, restored the response to additional NE plus histamine combinations: methylxanthines, benzodiazepines and adenosine. In tissue slice studies related to the antiseizure capability of benzodiazepines, the agents blocked the rise in cyclic AMP and GMP evoked by sodium-induced depolarizations (Ferrendelli and Kinscherf, 1978b; Palmer et al., 1979, 1981 ; see Section 16). Likewise, in Section 16 is discussed the role of benzodiazepines in vivo in regard to preventing the seizure-induced rise in steady-state levels of cyclic nucleotides. 17.1.2.2. Broken cell-phosphodiesterase studies Weinryb and coworkers (1972; see also Beer et al., 1972) first reported in a rat brain preparation that diazepam was more potent than chlorodiazepoxide in inhibiting cyclic AMP dependent phosphodiesterase. Other non-benzodiazepine antianxiety agents, meprobamate or hydroxyzine were not effective. Diazepam was likewise more potent as an enzyme inhibitor than the methylxanthines. In further work these investigators established a correlation between the potency of dibutyryl cyclic AMP, benzodiazepine and methylxanthines to diminish anxiety (conflict test) to their ability to inhibit phosphodiesterase. Nonrelated psychotropic drugs did not manifest this correlation. When evaluations of either cyclic AMP or cyclic GMP phosphodiesterases from cat and rat brain regions or neuroblastoma were made, medazepam and diazepam were found to be the most potent inhibitors of enzyme action. The drugs were almost as potent as papaverine. For inhibition

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of cyclic AMP phosphodiesterase, medazepam acted most effectively in the spinal cord > mesencephalon, p o n s > cerebrum, hippocampus, thalamus, olfactory bulb > caudate > cerebellum. For the cyclic GMP-dependent enzyme the olfactory bulb was inhibited to the greatest extent and the caudate, cerebellum and hippocampus the least. The caudate and cerebellum do, however, have extremely high specific activities of phosphodiesterase. In several brain regions the following order of potency for benzodiazepine inhibition of phosphbdiesterase was observed diazepam > chlordiazepoxide, N-methyl-oxazepam, N-demethyl-diazepam > oxazepam (Dalton et al., 1974). In the rat and mouse brain. diazepam was more potent than clonazepam in enhancing activity of adenylate cyclase or inhibition of low Kmcyclic AMP phosphodiesterase. The high Km form of the cyclic AMP dependent enzyme was also inhibited by benzodiazepines especially in the presence of Ca > and calmodulin (Levin and Weiss, 1975; Palmer, 1979). 17.1.3. In vivo--rapid fixation o f tissue

Even though benzodiazepines are fairly effective phosphodiesterase inhibitors and additionally enhance adenosine action, only a few studies measuring steady-state levels of cyclic nucleotides demonstrated a positive effect. Injections of diazepam and clonazepam did to some extent elevate cyclic AMP in mouse cerebellum and cerebral cortex (Palmer et al., 1979; Chan and Heubusch, 1982). On the other hand, similar injections of diazepam consistently lowered the steady-state levels of cyclic GMP in the cerebellum; the major changes being observed in the vermis and associated granular and molecular layers. The hemispheres were also affected in this manner. Moreover, agents that induced seizures or antagonized GABA (picrotoxin, harmaline, pentetrazol, oxotremorine and thyrotrophic releasing factor) when given to mice consistently elevated cerebellar concentrations of cyclic GMP. Pretreatment of the animals with diazepam blocked this cyclic GMP increase. Combinations of diazepam and ethanol were even more potent. If diazepam or GABA agonists were applied to the surface of the cerebellum, cyclic GMP content was attenuated. In some rather detailed and involved experimentation Biggio et al. (1977a) hypothesized the following mechanisms responsible for the diazepam reduction in cerebellar cyclic GMP. The activity of Purkinje cells is regulated by two excitatory inputs--the mossy and climbing fibers, plus an inhibitory GABA input via interneurons. Activation of the excitatory inputs (seizures, etc.) elevates cyclic GMP. If cerebellar GABA fibers are activated directly by agonists (muscimol or benzodiazepines) excitatory firing in the Purkinje cells is inhibited and levels of cyclic GMP fall. Apparently the same type of mechanism occurs in the substantia nigra. This work is a summary of that performed by Mao et al. (1975a,b); Opmeer et al. (1976); Biggio et al. (1977a,b); Rubin and Ferrendelli (1977); Waddington and Longden (1977); Lust et al. (1978); Mailman et al. (1978); and Chan and Heubusch (1982). In Section 15 it was shown that morphine and opiates likewise lower cerebellar cyclic GMP but this occurs via an indirect pathway originating from the striatum. 17.1.4. Other studies Injected diazepam was found to decrease the plasma levels of cyclic AMP in mice an event reversed by theophylline (Singh et al., 1980). The fall in cyclic AMP could be an adenosine-induced activation of At receptors that are inhibitory to stimulation of adenylate cyclase. DeLorenzo et al, (1981) reported a benzodiazepine antagonism of phosphorylation of brain synaptic membranes. The agents inhibited a Ca2+-calmodulin-protein kinase system. The author stated that the potency of benzodiazepines to effect enzyme inhibition was related to their efficacy to prevent seizures. 17.1.5. E n h a n c e m e n t o f adenosine action Diazepam was shown to potentiate the depressant actions of adenosine on the firing of cerebral neurons. This depression could be blocked by methylxanthines which inhibited

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the adenosine receptors. The ability of benzodiazepines to inhibit uptake of adenosine and thereby prolong its action was correlated with their clinical, anticonflict and receptor binding potencies. This of course is somewhat in contrast to Beer's et al. (1972) correlations of antianxiety action to inhibition of phosphodiesterase. However, recent work indicates that potent phosphodiesterase inhibitors are also potent adenosine uptake inhibitors. Certainly the sedative actions of benzodiazepines could be attributed to prolonging the depress'ant actions of adenosine in the brain (for Review see Phillis and Wu, 1981 ; Section 3.6.6). 17.1.6. Conclusions Several mechanisms of action may be attributed to benzodiazepines and these may influence the rate of accumulation of cyclic nucleotides in the brain by direct means--inhibition of phosphodiesterase and adenosine uptake, as well as, indirectly via an activation of GABA pathways in the cerebellum. Much work needs to be done, especially with regard to whether cyclic nucleotides play any role in sleep-wakefulness or in anxiety. Moreover, little is known about neurochemical changes during dependence and withdrawal from benzodiazepines. It should also be borne in mind that many of the benzodiazepine actions discussed occur at higher concentrations than their affinity for the benzodiazepine receptor. 17.2. ETHANOL 17.2.1. Background The progressive disease process, alcoholism, is the most severe drug abuse problem affecting mankind. The facts are well-known concerning the ability of ethanol to cause physical addiction manifested by withdrawal upon cessation of ingestion. The withdrawal process is characterized within a few hours by a hyperexcitability including tremors, anxiety, hyper-reflexes, insomnia and at 24 hr hallucinations may become prominent worsening with full-blown delirium tremens and even convulsive episodes at any period up to 96 hr. Continuing investigations have attempted to elucidate either the mechanism of action of ethanol or define the molecular substrates underlying the addiction process. Genetic roles, as well as, several of the monoamine neuromodulators have been implicated in these conditions. The acetaldehyde metabolites of alcohol have been shown to condense with catecholamines forming tetrahydroisoquinolines or other similar alkaloids which can serve as precursors of opiates, indicating a possible molecular site for an ethanol dependence mechanism. As with the other depressant agents, acute ethanol ingestion alters the fluidity of biological membranes changing their molecular conformation. Chronic alcohol, instead, appears to diminish this fluidizing effect. Such alterations in biomembranes may be linked to conditions in which alcohol either causes release of certain neurotransmitters and peripheral hormones, or inhibits the release and action of others (Myers, 1978; Mendelson and Mello, 1979). With these widespread biomembrane and hormonal effects, it is likely that the cyclic nucleotides and their respective enzymes responsible for their formation and degradation would be influenced by ethanol. Moreover, since cyclic GMP may be associated with excitatory transmission processes, it is apparent that central levels of this nucleotide would be especially susceptible to the depressant actions of ethanol. This depressant effect might lead to increased synthesis of excitatory receptors which could be especially active during the withdrawal process. 17.2.2. Peripheral systems Ethanol in low concentrations in vitro has been shown to activate basal and enhance NaF-stimulation of adenylate cyclase, as well as inhibit phosphodiesterases from a variety of tissue sources including human specimens (see Table 14). In one particular tissue, the gastric mucosa, oral ethanol, stimulates the output of gastric acid at low concentrations but is either inactive or inhibitory at higher concentrations. Cyclic AMP may be an intracellular

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mediator of gastric acid secretion and studies have shown that low concentrations t2-5% ) of ethanol stimulate mucosal cyclic AMP, elevate adenylate cyclasc (basal and NaFinduced) and inhibit phosphodiesterase. At ethanol levels over 10% (20% level is the equivalent of a martini on an empty stomach) all these systems are inhibited (Puuruncn et at., 1976). In some instances acute ethanol induces a subsensitivity of hormonal receptors. For example, ethanol administration to rats produces within 2 hr time a 4-fold reduction in plasma elevation of cyclic AMP in response to i.v. glucagon or isoproterenol. This receptor subsensitivity is most likely a result of the immediate release of catecholamincs and glucagon due to ethanol and the receptors then become desensitized in response to a further challenge by the agonists (lhrig et al., 1978; French et al., 1979). A somewhat similar observation was revealed in fasted rats who received intragastric ethanol. However, the isolated perfused liver did not display any of these metabolic changes to ethanol (Jauhonen et al., 1975). On the other hand, when isocalorically pair fed rats (controls were given dextrose to match calories consumed by the ethanol rats) were given chronic ethanol the liver contained an adenylate cyclase system that was less responsive to NE. After three days of alcohol withdrawal the enzyme sensitivity was normal (French et al., 1976). Alcohol blood levels were not correlated with any of these responses. These studies show that the action of ethanol on observed biochemical responses is indeed complex. Release of hormones may produce receptor stimulation or tachyphylaxis depending on the time interval. In the meantime, ethanol may have a direct effect on the enzymes themselves; the response is either stimulatory or inhibitory depending on the level. Furthermore, chronic depressive actions of ethanol may yield data opposite to that obtained during the hyperexcitability associated with the withdrawal process. 17.2.3, C N S actions 17.2.3.1. In vivo studies When ethanol was acutely given to rats (6 gm/kg po) or mice and the brain rapidly inactivated by freezing or focussed microwave irradiation, the levels of cyclic GMP were reduced in the cerebellum. This occurred even under conditions when Ca =+ content was elevated. Ethanol in turn prevented the usual rise in steady state levels of cyclic G M P in the cerebellum as produced by injections of arecoline and nicotine or heat and cold stress. However, injections of thyrotropin releasing hormone counteracted the depressant actions of ethanol on cyclic GMP. In one strain of mice, cerebellar cyclic GMP levels were even smaller than controls in response to an ethanol challenge (Chan and Heubusch, 1972; Mailman et al., 1978; Church and Feller, 1979; Dodson and Johnson, 1979, 1980; Lundberg et al., 1979; Volicer and Klosowicz, 1979; Yanaglsawa et at., 1979; Ferko et al., 1982). When studies of this type were evaluated under more detailed conditions, a single acute dose of ethanol decreased both cyclic AMP and cyclic GMP in most regions of the rapidly inactivated rat brain, namely cerebrum, pons, medulla, and cerebellum. However, while the cyclic GMP levels were depressed for up to 4 hr post-ethanol, the cyclic AMP reductions were transient with a duration of about ½ hr. Combinations of ethanol plus chlordiazepoxide gave an even greater depression of cyclic G M P (Volicer and Hurter, 1977; Chan and Heubusch, 1982). In another study testing the effect of acute ethanol on several brain regions, only the cerebellum and cerebrum displayed a reduction in cyclic GMP. The striatal level of cyclic AMP was lowered, the only region so affected (Fcrko et al., 1982). Under conditions of chronic ethanol administration (3 times/day for 7 days) steady-state levels of cyclic G M P alone were lower in cerebrum, pons, medulla and cerebellum (Volicer and Hurter, 1977). These chronic studies are not without controversy because Shen et al. (1977) reported that central cyclic AMP levels were depressed and Redos et al. (1976) were unable to demonstrate any changes in steady-state cyclic AMP levels following either acute or chronic alcohol administration. Some of the observed effects may be regional as levels

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of cyclic GMP in the vestibular region rise during chronic ethanol ingestion (Eliasson et al., 198l). When animals were given alcohol on a chronic basis rather dramatic changes in the in vivo levels of cyclic nucleotides were observed upon withdrawal. After discontinuance of ethanol rats displayed twitching, tremors and seizures and this evidence of hyperactivity was correlated with transiently elevated levels of cyclic AMP in the cerebral cortex, pons and me~luila. Moreover, cyclic GMP was increased in the cerebellum and medulla (Volicer and Hurter, 1977; Shen et al., 1977). Alternatively, Redos et al. (1976) reported no cyclic AMP alterations in any brain region during withdrawal. In humans who had abstained at least 30 days from ethanol, cyclic AMP content in the CSF was lower following 24 hr post-ingestion of ethanol (Orenber et al., ! 976). Hawley and coworkers (1981) failed to observe any changes in CSF concentrations of GABA, cyclic AMP or cyclic GMP in alcoholic patients undergoing acute withdrawal and during convalescence. The only consistent change was increased CSF levels of NE during withdrawal. In the rat CSF, an acute dose of ethanol depressed the levels of both cyclic nucleotides for a 24 hr period (Weitbrecht and Cramer, 1980). Normally in animals, steady-state levels of cyclic AMP in brain rise tremendously during trauma or postdecapitation, however, this event was prevented by ethanol pretreatment (Volicer and Gold, 1973; Myllyla, 1976b). In some studies in which dibutyryl cyclic AMP was given by i.v. infusion into rats, it was shown to enhance the induction of acute tolerance to ethanol (Wahlstrom, 1976). In another experiment central or peripheral administration of dibutyryl cyclic AMP, catecholamines, prostaglandins E~ and E2, amphetamine, L-DOPA, apomorphine and 5-HT antagonists all lessened the degree of ethanol withdrawal symptoms in mice. The majority of these agents are directly or indirectly associated with enhanced catecholamine activity which would be expected to elevate cyclic AMP (Collier et al., 1976). Since this cyclic nucleotide is associated with slow inhibitory transmission, perhaps a cyclic AMP increase is a safety mechanism to help the organism quiet down the excess hyperactivity of the withdrawal process, provided the catecholamine concentrations are not too high to cause psychosis. The cyclic GMP mechanism associated with cholinergic stimulation worsens the ethanol-induced withdrawal system in mice and this situation would be associated with the increased excitation during withdrawal (Collier et al., 1976). Maybe this is another case in which the two cyclic nucleotides oppose or attempt to regulate the action of one another. Insufficient evidence, however, limits the feasibility of this supposition. The dopamine depolarization- or dibutyryl cyclic AMP-induced enhancement of tyrosine hydroxylase activity as observed in the rat striatum was blocked by 0.2-0.8% ethanol. Ethanol did not have any effect on tyrosine hydroxylase by itself and most likely acted either on the synthesis of the enzyme or its activation (Gysling and Bustos, 1977). 17.2.3.2. In vitro studies In experiments directly associated with cyclic nucleotide enzymes it has been repeatedly shown that ethanol in central and peripheral tissues directly activates basal, as well as, hormonally elicited adenylate cyclase. In general, ethanol inhibits phosphodiesterases (Table 14). The elevation in basal adenylate cyclase activity and hormone responses to ethanol in vitro (whole cellular preparations) is most likely due to a change in osmolarity of the system. When neuroblastoma cultures were incubated with corresponding concentrations of salts or nonpermeable sugars to correct for osmotic imbalances, the ethanol enhancement of prostaglandin E1 stimulation of cyclic AMP was abolished (Stenstrom and Richelson, 1982). In a novel experiment mice were treated chronically with ethanol and by 10-14 days there were both elevated levels of cyclic GMP and enhanced activities of guanylate cyclase in the vestibular nuclei. After 4 weeks ethanol administration cyclic GMP levels rose even JFN 21:i/2-~

( i ('. PALMER TABI,E 14. lit Vitro Af'IIONS OF ~FFIANOI,ON CY(I Ic NLIC!EOIII)F ~YSFFMS Organ system

Aclions

References

Adenylate Cyclase Ratliver Rat liver Rat adipo,se Rat and human intestine

Hamster palate Dog and human gastric mucosa

Increase glucagon and basal Increase glucagon and basal Increase basal Increase basal Increase basal Increase basal and NaF

Oorman and Bitensky, 197(t Greene etal., 1971 Greene etal., 1971 G r e e n e e t a l . , 1971 Palmer etal., 1980b Puuruncn etal., 1976 and Karppanen etal., 1976 Tague and Shanbour, 1974 Jauhonen etal., 1975 G r e e n e e t a l . , 197l Kuriyama and Israel, 1973 Rabin and Mollinoff, 1981 Stenstrom and Richelson, 1982

Mouse brain regions

Blockbasal and NaF No effects Increased basal and NaF lncrease basal, catecholamine and NaF

Neuroblastoma

Increase prostaglandin E~, not basal

Phosphodiesterase Rat gastric mucosa Human gastric mucosa

Inhibitory Inhibits low K m

Tague and Shanbour, 1974

Dog gastric mucosa

Inhibitory

Mouse brain

No change

Puurunen etal., 1976 Kuriyama and lsrael, 1973

Rat gastric mucosa

Perfused rat liver Rat brain

Karppanen el al. , 1976

higher and this was correlated with a latent depression of cyclic GMP phosphodiesterase. In mice strains that rejected ethanol the early rise in cyclic GMP was less pronounced. Whether guanylate cyclase elevation is a result of tolerance to the ataxic effects of ethanol cannot be determined at this time~ but hopefully other brain regions will be evaluated in this regard (Eliasson et al., 1981). Up to 24 hr following alcohol withdrawal in mice the sensitivity of adenylate cyclase to D A and NE was diminished in the striatum but not in the mesolimbic system. Binding of the dopamine ligand spiroperidol was, however, not attenuated in the striatum and the authors concluded that a deficient mechanism must exist for coupling between receptors and the catalytic site of adenylate cyclase. Ethanol added to the experiments in vitro did not influence their outcome (Tabakoff and Hoffman, 1979). The sensitivity of rat cerebellar adenylate cyclase to calcium activation was unchanged following either 3 weeks administration of ethanol or during the withdrawal process (Von Hungen and Baxter, 1982). When maternal rats were fed ethanol after mating the newborn animals had both decreased body and brain weights. At the 21 day fetal age the brains contained elevated levels of NE and adenylate cyclase activities. At postnatal day 4 adenylate cyclase in the cerebellar region was augmented. No changes in brain levels of cyclic AMP or activities of phosphodiesterase were observed during the course of the investigation (Mena et al.,

1982). In work with incubated tissue slices some controversy exists regarding the role of alcohol on the sensitivity of catecholamine-elicited cyclic AMP systems. French el al. (1974) pair fed rats (isocalorieally) either alcohol or dextrose for 16 weeks and noted a subsensitivity of cyclic AMP accumulation to NE and histamine additions to incubated cerebral slices from the ethanol rats. When similarly treated rats were removed from ethanol and allowed to withdraw for 3 days the NE, histamine and 5-HT accumulations of cyclic AMP were greatly increased over controls (French and Palmer, 1973; French el al., 1975). In support of the latter findings, the binding of the beta adrenergic ligand, dihydroalprenolol, was increased in both the heart and brain of rats at 48-72 hr after cessation of chronic ethanol. These time periods for withdrawal correspond to the appearance of hallucinations and delirium tremens (Banerjee and Khanna, 1978). Since the formerly suppressed receptors might now be over-responding to the agonists, the enhanced activity of NE related systems might be associated with the hyperactive behavior and mental problems encountered.

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99

Smith and coworkers (1981) did not note the attenuated NE-sensitivity of cyclic AMP in cortical tissue slices from rats receiving only 96 hr of ethanol. In fact both basal activity and NE actions were augmented. During withdrawal, however, NE elicited cyclic AMP synthesis was even greater and correlated with hyperactivity. In one study, in contrast to Banerjee and Khanna's (1978) findings in the heart, Sabourault andcolleagues (1981) reported that neither alpha nor beta receptors changed following 3 weeks of ethanol inhalatibn. 17.2.3.3. Condensation products of ethanol metabolism The condensation products of ethanol metabolites, i.e. 3,4 dihydroxyphenylacetaldehyde, in combination with DA form tetrahydropapaveroline alkaloids that have a degree of action on cyclic nucleotide systems. Tetrahydroisoquinoline, salsolinol, tetrahydropapaveroline and their N-methylated derivatives displayed antagonism toward DA-adenylate cyclase in the rat caudate. Alternatively, with the beta adrenergic receptor in the rat erythrocyte, tetrahydropapaveroline isomers had weak agonist activity and could compete with isoproterenol to limit stimulation of adenylate cyclase (Sheppard and Burghardt, 1974). With central tissue, Nimitkitpaisan and Skolnick (1978) showed a relatively strong potency of tetrahydroisoquinoline toward blocking isoproterenolinduced cyclic AMP formation in rat brain. Moreover, this alkaloid prevented the binding of the radioligands, dihydroalprenolol (beta receptors) and haioperidol (DA receptors) to brain membrane fractions. Alpha adrenergic ligand binding was little affected by tetrahydroisoquinoline, while salsolinol, salsoline and reticuline were more potent in inhibiting alpha ligand binding. These latter agents were somewhat ineffective on DA and beta receptors. The findings indicate that the ratio of the condensation products may further play a regulatory role with respect to ethanol's action on catecholamine receptors and associated cyclic nucleotide actions in the CNS.

17.2.4. Conclusions Taken together the experimental findings from a variety of laboratories employing diverse techniques and investigative approaches indicate that the depressant actions of acute ethanol readily diminished the cyclic GMP system in the brain to a somewhat greater extent than the cyclic AMP system. One might argue that since ethanol decreases the regional availability of calcium in the brain (Ross et al., 1974) that this is associated with lowered activation of guanylate cyclase. The latter speculation, however, has not been confirmed. In general, withdrawal from chronic alcohol ingestion is manifested by enhanced sensitivities of cyclic AMP systems to catecholamines. However, cyclic GMP may also be elevated, as well. In one case guanylate cyclase activity was increased. The cyclic AMP systems become most prominent within 72 hr of abstinence, a time when hyperactivity and delirium tremens is present in humans. Could the elevated guanylate cyclase-cyclic GMP be associated with excitatory behavior and the augmented cyclic AMP levels be a protective mechanism acting in opposition to dampen the hyperexcitability? Other unexplained observations were the consistent stimulation of basal adenylate cyclase by ethanol in vitro in spite of the decrease in cyclic AMP seen following acute administration. However, inhibition of phosphodiesterase offers one answer to this phenomenon. The condensation products appear to have limited degrees of anti-catecholamine action. These agents have been postulated to be involved in the addition process and may act on a chronic basis to limit NE and DA action. The latter receptors might then "up regulate" and become supersensitive upon withdrawal. Major questions remaining unanswered include the precise interneuronal transmission signals so influenced by ethanol at various stages of its action--acute, chronic, addiction potential and the phenomenon of withdrawal. Furthermore, the relationships between cyclic GMP and cyclic AMP should be analyzed during these time periods.

101)

(} (

P,,I,,lll~

17.3. BARBII'URATES 17.3.1. Introduction With the past problems associated with "sleeping pill" addiction and the present popularity of quaaludes, it is surprising that so little neurochemical work has been done with these compounds on central cyclic nucleotide systems. The lack of data is especially evident regarding chronic cxperirnents and the related processes of physical dependence and withdrawal. No data are available as to the effects of barbiturates on in vitro systems especially tissue culture and incubated tissue slices. The uniform depressant actions of these drugs do impose limitations when studying enzyme components of biomembranes. 17.3.2. @ c l i c nucleotide systems The initial work using methods of rapid freezing did show that acute injections of barbiturates led to decreased steady-state levels of cyclic AMP in the rat cerebral cortex (Breckenridge, 1964; Volicer and Gold, 1973). In contrast Paul and coworkers (1970b) were unable to observe any effect of pentobarbital on in vivo levels of cyclic AMP in the mouse cerebrum, however, the drug prevented the post-decapitation rise in the cyclic nucleotide. In further work acute injections of phenobarbital and pentobarbital were shown to increase cyclic AMP in the rat cerebellum, amygdala, pituitary, and cerebrum (Kimura et al., 1974; Kant et al., 1980). Under in vitro conditions brominated barbiturates were potent inhibitors of basal adenylate cyclase in homogenates of guinea pig lung (Weinryb et al., 1971). Furthermore, the glucagon-sensitive but not the fluoride-sensitive, site of adenylate cyclase was inhibited by phenobarbital in plasma membranes from rat liver. These data indicated a drug-induced receptor uncoupling action in the outer membrane components of the plasma membrane (Houslay et al., 1981). One consistent observation with measurement of steady-state levels of cyclic GMP in the brain was a barbiturate-induced depletion of the nucleotide in almost all brain regions, especially the cerebellum (Lust et al., 1976: Opmeer et al., 1976; Lenox et al., 1979: Lundberg et al., 1979; Dodson and Johnson, 1980; Kant et al., 19g0). During withdrawal from chronic barbiturate administration cyclic GMP content became elevated in the cerebellum (Lenox et al., 1979). Pretreatment of mice with pentobarbital prevented the stimulation of steady-state brain levels of cyclic GMP as an outcome of injections of harmaline, oxotremorine, picrotoxin and pentetrazol (Opmeer et al., 1976). The role of barbiturates as anticonvulsants is discussed in Section 16. A series of studies have revealed that intracerebral administration of dibutyryl cyclic AMP shortens sleep time, reverses the centrally-induced cardiovascular and respiratory depression, and overcomes the analgesic and anesthetic properties of barbiturates. In a similar manner the actions of chloral hydrate, paraldehyde, halothane, diazepam, ketamine and ethanol were affected by dibutyryl cyclic AMP. Intracerebral dibutyryl cyclic AMP does have CNS stimulant properties complete with convulsive episodes. However, dibutyryl cyclic AMP must also have unique antianesthetic properties because CNS stimulants (convulsant-analeptics) like amphetamine, caffeine, pentylenetetrazol, strychnine, ethamivan, doxapram and theophylline do not reverse amobarbital overdose, narcosis or anesthesia. Picrotoxin displayed a limited degree of antianesthetic properties but severe toxicity problems were likewise evident. The steep dose response curve, mode of administration, and low margin of safety, however, negates a clinical use of dibutyryl cyclic AMP as an effective antidote for overdose of depressants. Alternatively, NE which stimulates cyclic AMP in all brain regions under in vitro conditions, prolonged amobarbital narcosis in rats (Cohn e t a l . , 1973, 1974, 1975; Kraynack e t a l . , 1976; Isom e t a l . , 1978). 17.3.3. Conclusions Barbiturates act as central depressants and one site of action appears to be an inhibition of steady-state levels of cyclic GMP that were produced by agents associated with excitation and which likewise produce seizures. These experiments lend further evidence

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101

that cyclic GMP is associated with excitatory transmission processes in the brain. A considerable amount of work is required, especially with guanylate cyclase and the role of cyclic AMP using whole cell preparations to access the sensitivity of adenylate cyclase during barbiturate dependence and withdrawal. Apparently the agents have little or no effects on phosphodiesterase (Weinryb et al., 1972). The idea that cyclic AMP counteracts barbiturate overdose is intriguing but of little practical clinical value at this time. The inhibition of the rate of phosphorylation of the synaptic protein, protein I, by barbiturates indicates another possible site of action of these agents (Schulman et al., 1980). 17.4.

GENERAL ANESTHETICS

The general anesthetics while acting principally as central depressants may influence cyclic nucleotides as a result of their well-known side effects; namely excitement during induction (ether, cyclopropane and halothane) and hallucinatory properties (ketamine). For example diethyl ether produces an elevation in plasma cyclic AMP in rats, an event that is abolished by agents which deplete catecholamines (Kunitada et al., 1978). Like other CNS depressants ether decreases drastically the levels of cyclic GMP in frozen mouse cerebellum (Lust et al., 1976; Dodson and Johnson, 1980). On the other hand, in studies involving less rapid fixation of rat brain tissue ether produced increases in levels of cerebellar cyclic AMP and cyclic GMP. Furthermore, ether elevated cyclic GMP in rapidly frozen heart and lung, an action prevented by atropine. The latter data reveal a cholinergic mechanism responsible for the observed effect because cholinergic agonists did elevate cyclic GMP in these organs under in vitro conditions (Kimura et al., 1974). Halothane anesthesia at concentrations of 0.5-10% enhanced the activity of adenylate cyclase-cyclic AMP in a variety of tissues namely the rat caudate (Woo et al., 1979), human platelets (Walter et al., 1980), rat aorta (Yang et al., 1973; Sprague et al., 1974), rat uterus (Triner et al., 1977), rat liver (Rosenberg and Pohl, 1976), and cultured neuroblastoma (Seager, 1975). Moreover, responses to neurohumoral agents, GTP analogs and fluoride were augmented in the presence of halothane (Triner et al., 1977; Rosenberg and Pohl, 1976). Exposure of related anesthetics (isoflurane, fluroxene and methoxyflurane) likewise elevated cyclic AMP levels in cultured neuroblastoma (Seager, 1975). The halothane activation of adenylate cyclase in human platelets was associated with an inhibition of platelet aggregation. Moreover, halothane did not inhibit either high or low Km cyclic AMP phosphodiesterase (Walter et al., 1980). Under in vivo conditions employing rapid fixation of tissue halothane increased cyclic AMP in rat brain (Biebuyck et al., 1975). Divakaran et al. (1980) have addressed the controversy as to whether halothane elevates or depresses brain steady-state concentrations of cyclic AMP. If oxygen was used as the carrier gas for the anesthetic, cyclic AMP levels increased. If the drug was administered in air cyclic AMP levels dropped, albeit slightly. This was supposed to resolve discrepancies between their findings and those of Nahrwold et al. (1977) who used halothane in air. However, Kant et al, (1980) found that halothane in air elevated in vivo levels of cyclic AMP in cerebellum, brain stem, hypothalamus and pituitary of rats sacrificed using focussed microwave irradiation. Since Divakaran et al. (1980) sacrificed their rats by decapitation and freezing, there may have been additional problems of fixation artifacts in their work. Most investigations report that halothane depressed the steady-state levels of cyclic GMP in all brain regions of rapidly fixed animals (Nahrwold et al., 1977; Lundberg et al., 1979; Divakaran et al, 1980; Kant et al., 1980). Ketamine injections prevented the post decapitation rise in cyclic AMP in several areas of the rat brain (Lachowicz and Wojtkowiak, 1980). Ketamine under in vitro conditions elevated cyclic AMP in cultured neuroblastoma cells (Seager, 1975). In conclusion perhaps the general activation of the adenylate cyclase mechanism by anesthetic agents is reflected by the inhibitory action of cyclic AMP on central neurons. The general depression of cyclic GMP could be a direct depressant effect on the excitatory neurotransmitter systems responsible for its formation. More data is needed on the cyclic

1112

(A. (~;. PALMi:R

G M P s y s t e m . I n o n e s t u d y i n t r a v e n t r i c a l l y a d m i n i s t e r e d d i b u t y r y l , cyclic G M P p r e v e n t c d a d v e r s i v e r e s p o n s e s in r a t s t o n o x i o u s s t i m u l i , a n a c t i o n n o t a n t a g o n i z e d b y n a l o x o n c ( C o h n et al., 1978). M a y b e o n e n u c l e o t i d e m e d i a t e s c e n t r a l d e p r e s s i o n (cyclic A M P ) , t h e • o t h e r a n a l g e s i a (cyclic G M P ) .

Acknowledgements I a m g r a t e f u l t o S. J o P a l m e r , J u d i N a y l o r a n d T o n i T u b e r v i l l e f o r h e l p i n p r e p a r i n g t h i s manuscript. I wish to especially thank the Frist-Massey Neurological Institute for support.

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