Adenosine: The prototypic neuromodulator

Neurochem. Int. Vol. 14, No. 3, pp. 249-264, 1989 Printed in Great Britain.All rights reserved

0197-0186/89$3.00+ 0.00 Copyright © 1989PergamonPress plc

ADENOSINE: THE PROTOTYPIC N E U R O M O D U L A T O R MICHAEL WILLIAMS Research Department, Pharmaceuticals Division, CIBA~eigy, Summit, NJ 07901, U.S.A. A paper in honour o f Henry Mcllwain for his contribution to neurochemlstry

Al~lraet--Purinergic modulation of mammalian tissue function was first demonstrated in 1929. However, the lability of the purine and its ubiquitous distribution and function in mammalian tissues, led to a degree of scepticism as to any discrete role for the compound that resulted in a 40 year hiatus related to a concerted effort to determine the functional role of the nucleoside. Prompted by the discovery of cyclic AMP, biochemical studies in Henry McIlwain's laboratory at the Institute of Psychiatry provided some of the first evidence that adenosine might function as a chemical messenger. The subsequent discovery of cell surface recognition sites, or receptors, for adenosine that exist in two main subclasses termed A~ and A2 together with the availability of selective receptor antagonists generated data implicating adenosine in the pharmacological actions of several classes of centrally active agents including: anxiolytics, antipsychotics, antidepressants, cognitive enhancers and anticonvulsants. In addition, adenosine has potent effects on cardiovascular, pulmonary, renal and immune function. Effects on these systems, especially the latter, have important global implications for CNS function. These data indicate that adenosine, like the many peptides currently being studied, is a homeostatic neuromodulator and as such may represent the prototypic agent of this type. Increased efforts in the area of medicinal chemistry together with the discovery of novel structural classes that antagonize adenosine function will provide the tools for a more precise understanding of the role(s) of the purine. This may lead to new classes of therapeutic agent that act to alter modulatory, as opposed to transmitter-related, responses.

Despite the passage of some 60 years since Drury and Szent-Gyorgi (1929) demonstrated the cardiovascular actions of adenosine nucleotides and the nucleoside, it is only in the past decade or so that the molecular basis for the actions of adenosine have become defined (Daly, 1982; Dunwiddie, 1986; Williams, 1987; Bruns et aL, 1988). While many research centers throughout the world have contributed in the evolution of the concept of adenosine as a neuromodulatory agent, the studies initiated by Henry McIlwain in the late 1960s in collaboration with Ted Rail and Shiro Kakiuchi (Kakiuchi et al., 1968) on adenosine-related alterations in cyclic AMP formation in mammalian brain slices, were a pivotal step in developing the concept of cell surface adenosine receptors and in defining their function in the CNS. In subsequent studies, using the same biological systems developed by McIlwain, Sattin and Rall (1970), observed that the methylxanthines, caffeine and theophylline, previously assumed to function as phosphodiesterase inhibitors, blocked the effects of adenosine in increasing brain slice cAMP levels. This led to the suggestion that xanthines were in fact adenosine receptor antag-

onists and thus provided the first tools to study adenosine function in a variety of mammalian tissues. In the subsequent 20 years, while numerous biochemical, behavioral and electrophysioiogical studies have been used to better understand the role of adenosine in CNS function and the existence of receptor subtypes, the work of McIlwain and Rail must stand as seminal in providing the basis for what is now known about the role of the purine and the continuing escalation in publications in this area. Indeed, few researchers who were at the Institute of Psychiatry in the late 1960s and early 1970s escaped Henry McIlwain's enthusiasm and commitment to the understanding of adenosine-related processes in the CNS (McIlwain, 1972). More importantly, this purine nucleoside provided a conceptual framework, parallel to that emerging for the more popular peptides, for analysing and delineating the processes of neuromodulation and neurohumoral homeostasis. Adenosine as a neuromodulator

In comparison to "traditional" neurotransmitters such as GABA and dopamine, adenosine has poorly definable pathways leading to its synthesis and

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degradation. Adenosine, as both phosphorylated nucleotide and enzyme cofactor, is involved in nearly every aspect of cell function (Arch and Newsholme, 1978). However, using immunohistochemical techniques, Brass et al. (1986), have provided evidence for the possible existence of"adenosinergic pathways" in rat brain whose putative activity appears increased by ischemia. Similarly, Nagy et al. (1984) have identified discrete adenosine deaminase containing pathways in the basal ganglia. In contrast to adenosine, dopamine has a defined synthetic pathway with appropriately selective enzyme/uptake mechanisms responsible for the termination of its actions. Given the ubiquitous distribution and also the concept of cellular adenylate charge (Atkinson, 1978), it appeared that since adenosine was everywhere, it could have no specific function in any tissue. Furthermore, the loss of adenosine from one cell to the extracellular space would alter the adenylate charge and thus conceivably result in a downstream energy flow if the nucleoside were taken up rather than being catabolized in the synaptic cleft. While the transfer of adenylate charge from one cell to another would be an attractive nuance in that the purine could cause postsynaptic changes that would be effected intracellularly by the same molecule initiating the extracellular event, theoretically it would mean a net loss of energy from the cell initiating the response mediated via adenosine. Whether such an event is detrimental in the overall function of a given tissue is debatable. Certainly the loss of amino acid building blocks from a cell in the process of hormone function or peptide neurotransmission appears to have no net negative ramifications that have thus far been considered. It appears highly probable therefore, that adenosine in fact acts in most tissues as a paracrine homeostatic modulatory agent such that any potential "randomness" in its effects occurs, like those in response to conventional hormones, by virtue of their ability "to bind with high affinity to specific receptors presumably arrayed on neuronal surfaces that interface with the ambient intercellular mileu" (Schmitt, 1982). This somewhat unorthodox mode of action which would appear to suggest a lack of specificity may in fact reflect a global role for the purine and would certainly be consistent with the large increases in its extracellular availability following trauma. Both cerebral (Berne et al., 1974; Hagberg et al., 1987) and myocardial (Gidday et al., 1984) ischemia resulting in large increases in adenosine due to the breakdown of ATP.

Adenosine availabili O,

As noted, the initial suggestion that adenosine was a modulator of cyclic AMP formation arose from the studies of Kakiuchi et al. (1968) who identified adenosine as the chemical mediator of the effects of electrical stimulation on brain slice cyclic AMP levels• Continuing studies on the nature of adenosine release (Pull and McIlwain, 1972; Kuroda and McIlwain, 1973, 1974) have shown that depolarizationqnduced, calcium dependent release can occur. Other workers have found, however, that purine release is calcium independent (Fredholm and Vernet, 1979). Furthermore, it is unclear whether neurons or glia represent the source of the purine (Stone et al., 1989). Fredholm et al. (1982) have suggested that adenosine is released by a non-vesicular process. Given that quantal release of the purine is controversial, it is uncertain as to what role conventional release mechanisms are involved in making adenosine available. In heart tissue however, it appears that adenosine availability can be "back-titrated" by increasing the oxygen supply (Bardenheuer and Schrader, 1986). Despite the conflicting results related to the mechanisms regulating adenosine availability, the physiological stimuli regulating this process appear to be directly related to homeostasis. In the heart, this functional role enables adenosine to prevent the neutrophil related tissue related damage occurring during reperfusion (Mullane, 1988) as well as acting as an antiarrhythmic agent (Wainwright and Parratt, 1988). Potentiation of the effects of endogenous adenosine produced as the result of ischemia by AICA riboside, a constituent of purine pathways, may represent a novel approach to the prevention of such damage by modifying the deleterious actions of neutrophils during tissue reperfusion (Engler, 1987). The mechanism for the actions of AICA-riboside arc currently unknown. Adenosine and its stable analogs can also prevent neuronal cell loss following ischemia (Evans et al., 1987; Goldberg et al., 1988) and thus the purine functions as an autacoid to prevent tissue inJury. ADENOSINE R E C E P T O R S

The initial classification for purinergic receptors was pharmacological in nature related to the effects that purine nucleosides and nucleotides produced in classical tissue preparations Burnstock (1978) (Table 1). Those receptors termed P~ were most responsive to adenosine while P2 receptors were ATP selective. While it was clear that alkylxanthines represented effective antagonists for P~ receptors (Daty,

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Adenosine: the prototypic neuromodulator

Receptor P~

Subclasses A~(Ri) A2(Ra)

Table I. Adenosinereceptorclassification Agonist pharmacology CPA/> CHA > R-PIAt> 2-CADO/>NECA > S-PIA> CV 1808 NECA > MECA>/2-CADO> CV 1808= R-P1A> CPA/>S-PIA

1982), all putative antagonists for P: receptors have proven to be non-specific (Williams, 1987). Working independently, two groups studying the biochemical effects of adenosine on cyclic A M P production in various cell preparations, identified two types of PI receptor. Londos and Wolff (1977) termed the P~ receptor, the R receptor, the R standing for "ribose" indicating that an intact ribose group was necessary for ligand activity. This site was further delineated into two subtypes, Ra and Ri, depending on the effects of ligands on cyclic A M P formation. At Ra receptors, adenosine increased cyclic A M P formation via activation of adenylate cyclase while at the Ri receptor, activation led to an inhibition of cyclase activity. A second adenosine recognition site, the P site identified by Haslam et al. (1978) is an intracellular site located on the catalytic subunit of adenylate cyclase. It is not to be confused with either the P~ or P2 receptor. Its physiological role has yet to be determined (Daly, 1985). Studies in neuroblastoma cell lines by Van Calker et al. (1969), led to the identification of similar classes of P~ receptor although the terminology used was different. The Ri receptor was termed A~ while the Ra receptor was termed A 2. This nomenclature is now universally accepted (Hamprecht and Van Calker, 1985). While this classification was initially based on the ability of adenosine analogs to modulate adenylate cyclase activity, these receptors have become more properly classified in terms of their structure-activity requirements (Hamprecht and Van Calker, 1985). This has become necessary since cyclic A M P appears unlikely to be the only second messenger through which the purine elicits its effects. Considerable evidence has accumulated for modulation of calcium and potassium channel related events that regulate transmitter release (Fredholm and Dunwiddie, 1988). The purine has also been reported to modulate phosphatidylinositol (PI) production (Delahunty and Linden, 1988) although data from studies in which the effect of adenosine on histamine-evoked changes in IMP accumulation (Petcoff and Cooper, 1987; Hill and Kendall, 1987) have suggested that the effects of the purine on PI turnover may be secondary to

Antagonist CPX t> BW-A 844U i> XAC > PACPX> CPT > CPQ > PD 113297 PD 115199~>CGS 15943> HTQZ > DMPX > ADQZ

alterations in intracellular calcium concentrations (Cooper and Caldwell, 1989). Adenosine receptor activation occurs at both preand postsynaptic locii. Thus, the effects of adenosine may be indirect, inhibiting the release of a variety of conventional neurotransmitters from presynaptic terminals or direct, by affecting postsynaptic processes. Cyclic A M P is unlikely to be the second messenger for the presynaptic actions of the purine (Fredholm and Lindgren, 1988). Adenosine can alter both pre and postsynaptic calcium fluxes (Schubert et al., 1986). While arachidonate pathway products do not appear to represent a major second messenger system for adenosine at this time (Axelrod et al., 1988), prostacyclin production in rabbit heart can be increased by adenosine via a receptor-linked process (Karwatowska-Prokoczuk et al., 1988). As with other neurotransmitter/neuroeffector systems (Green, 1987) adenosine receptor subtypes may be more rationally delineated on the basis of their pharmacological characteristics rather than effector systems. In considering the structure-activity relationship (SAR) for agonists at A~ and A2 receptors (Table 1), substitutions in positions N 6, 2 and 5'-confer differing degrees of selectivity and activity (Bruns et aL, 1988). The N 6 substituted analogs, R-phenyl-isopropyladenosine (PIA), cyciopentyladenosine (CPA) and cyclohexyladenosine (CHA) are potent A~ selective ligands having 90-1000 selectivity for this receptor over the A2 receptor with IC5o values in the 1-10 nM range. The 2-substituted chloro derivative, 2-CADO, is slightly AI selective. 5'-N-ethylcarbaxamido adenosine (NECA) although used for a considerable period of time as an "A2 selective" ligand, is in fact non-selective, with CV 1 8 0 8 (2-phenylaminoadenosine) and a series of N 6 substituted analogs (Bridges et al., 1988), being the most A2-selective (56-fold) compounds thus far reported. The SAR for A~ and A 2 receptors is, however, seriously confounded by marked species differences in their activities (Murphy and Snyder, 1982; Daly et al., 1983; Hamilton et al., 1985; Ferkany e t al., 1986; Stone et al., 1988).

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The two adenosine receptor subtypes are distributed heterogenously in mammalian tissues with the highest density in the CNS (Goodman and Snyder, 1982; Goodman et al., 1983; Reddington et al., 1987; Snowhill and Williams, 1986; Williams, 1987; Jarvis et al., 1989), further reinforcing the role for the purine as a paracrine neuroeffector (Schmitt, 1982). Evidence is also accumulating for the potential existence of other adenosine receptor subtypes (Bruns et al., 1986, 1988; Riberio and Sebastio, 1986). While such data is preliminary in nature and based on an SAR relationship devoid of selective receptor ligands, A2a and A2b a s well a s A 3 receptors and their respective effector systems are the focus of intensive research efforts and may be anticipated to further delineate the role of adenosine in the CNS. R e c e p t o r structure

As with many other receptors (Koblika et al., 1988; Wheatley et al., 1988), efforts are underway to isolate and sequence those for adenosine. This has important implications in regard to subtyping of such entities since, unlike the /3-adrenoceptor, the spectrum of chemical entities having agonist or antagonist activity at either A~ or A 2 receptors is very limited. The A~ receptor solubilized from various brain regions has a molecular weight of 280,000 Da (Stiles, 1985; Stiles and Jacobson, 1987). However, photoaffinity labeling has resulted in the identification of a protein with M 34,000-38,000 (Choca et al., 1985; Stiles et al., 1985), while irradiation inactivation/target size analysis has estimated this same site to have an M of 63,000-65,000 (Reddington et al., 1987). Chemical modification studies (Klotz, 1988; Klotz and Lohse, 1986) have indicated that the A~ receptor is glycoprotein in nature and has at least two histidine sites that are involved in binding of ligands, one for agonists and one for antagonists, both of which would favor the binding of hydrophobic ligands. Similar studies o n A 2 receptors and on receptors of both subtypes from peripheral tissues are ongoing, but hampered by the lack of selective ligands (Lohse et al., 1988). ADENOSINE ANTAGONISTS

Theophylline and caffeine were the first reported adenosine receptor antagonists. Like nearly all alkylxanthines, these compounds were, however, active as phosphodiesterase inhibitors. The synthesis of a large number of modified xanthines, especially those with phenyl substituents in the 8-position (Daly, 1985; Hamilton et al., 1985; Bruns et al., 1988;

Trivedi et al., 1989; Williams, 1989) led to compounds that were both selective and far more potent as adenosine receptor antagonists. Some, such as 8-cyclopentyltheophylline (CPT) are A~ selective with subnanomolar affinity and have been used as radioligands (Bruns et al., 1987). Others such as 1,3dipropyl-7-propargyl xanthine (DMPX) while A 2 selective (Ukena et al., 1986) are relatively weak (Ki ~ 600 nM). More recently, three classes of non-xanthine adenosine receptor antagonist have been described (Davies et al., 1984; Hamilton et al., 1987a; Trivedi and Bruns, 1988; Francis et al., 1988; Bruns et al., 1988). Of these, the quinazoline, HTQZ and CGS 15953 are of especial interest in that they are A2 selective, 26- and 7-fold respectively with ICs0 corresponding values of 120 and 3 nM. Such compounds represent important advances in probing the function of adenosine in vivo by providing novel structures with defined activity. One drawback, however, is that such compounds have low aqueous solubility (Bruns et al., 1988; Williams, 1989). THERAPEUTIC IMPLICATIONSOF ADENOSINE ACTIONS

There is a wealth of evidence to indicate the existence of a purinergic inhibitory tone (Harms et al., 1978) in many mammalian tissues. Thus adenosine normally regulates the release of a variety of neurotransmitters, its actions being proportional to the degree of activity of the tissue and the amount of adenosine consequently available in the microenvironment of the synapse. Under adverse conditions when adenosine levels are markedly increased, the purine can function ostensibly to prevent tissue damage and provide any traumatized tissue with an environment conducive to recovery. In cardiac tissue, the role of the purine as a cardioprotectant has been postulated (Gerlach et al., 1987; Engler, 1987). C e n t r a l nervous s y s t e m

The release of many diverse classes of transmitter can be inhibited by adenosine including: glutamate, GABA, norepinephrine, dopamine, serotonin and acetylcholine. Based on the observed behavioral effects of both agonist and antagonist administration, it is apparent that the actions of adenosine are rational inasmuch as it does not appear that effects on inhibition of excitatory (glutamate) and inhibitory (GABA) neurotransmitter release cancel each other out. In addition, the role of autoreceptors within the context of adenosine actions may still have to be

Adenosine: the prototypic neuromodulator resolved from a theoretical viewpoint. In addition to such indirect actions occurring at the presynaptic level, adenosine also has direct actions at the postsynaptic membrane which can add an additional layer of complexity to the effects of the purine, again dependent on the state of the tissue at the time of adenosine availability. In considering the potential role(s) of adenosine antagonists, the involvement of adenosine in normal tissue function requires consideration. Inevitably however, the observed effects of the purine and its more stable analogs may reflect an atypical situation such that the actions of an antagonist may not always be construed in terms of an effect opposite to that of an agonist. At the whole animal level, adenosine is a potent CNS depressant (Phillis and Wu, 1981; Snyder et al., 1981; Dunwiddie, 1985) inhibiting spontaneous and evoked cell firing and locomotor activity. The purine may also function as an endogenous anticonvulsant (Dragunow et al., 1985; Dragunow, 1988). Conversely, the xanthine as well as non-xanthine adenosine antagonists are potent central stimulants. The antagonists also increase locomotor activity in animals (Snyder et al., 1981) and have proconvulsant, and at high doses, convulsant activity (Dunwiddie and Worth, 1982). The purine has thus been implicated in the mechanism of action of nearly every major class of psychotherapeutic agent, a fact that may be attributed to the global effects of the purine on neurotransmitter release (Fredholm and Dunwiddie, 1988) as well as the delicate balance existing between the various neurotransmitter/ neuromodulator pathways. (1) Epilepsy. Caffeine and theophylline at high doses elicit convulsions and at lower doses function as proconvulsants acting to increase the efficacy of a range of chemical convulsants (Dunwiddie and Worth, 1982). Endogenous adenosine levels are increased as a result of the ischemia associated with an epileptic attack. There is considerable evidence to indicate that the purine is an endogenous anticonvulsant (Dragunow, 1988). In kindling models of epilepsy, adenosine can prolong the spread of postictal depressions (Rosen and Berman, 1985). The actions of the anticonvulsant, carbamazepine have been linked to effects, albeit weak, on central adenosine receptors (Marangos et al., 1983). Barbiturates which also possess anticonvulsant activity interact with A~ receptors (Lohse et al., 1985). An extrapolation of the proposed anticonvulsant role of the purine relates to the ability, already described, of adenosine to prevent the cell damage associated with cerebral ischemia (Evans et al., 1987;

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Goldberg et al., 1988). Antagonists of the actions of the excitatory amino acid glutamate at N-methylD-aspartate receptors have anticonvulsant activity (Lehmann et al., 1987) and are also effective antiischemic agents (Jarvis et al., 1988). The release of glutamate can be modulated by adenosine (Dolphin and Archer, 1983) presumably via receptors located on excitatory neurons (Goodman et al., 1983). It is highly probable, therefore, that the anticonvulsant and anti-ischemic actions of adenosine are a result of both the inhibition of glutamate release and the attenuation of the central inflammatory response. The effect of exogenous adenosine analogs on chemically-induced convulsions can vary (Dunwiddie and Worth, 1982) indicating possible differences in the adenosine systems involved in the actions of such compounds. (2) Anxiolytic actions. A considerable body of evidence exists implicating the involvement of central purinergic systems in the mechanism of action of the benzodiazepine (BZ) anxiolytics (Williams, 1983; Phillis, 1984; Phillis and O'Regan, 1988). Various adenosine ligands, agonists as well as antagonists, interact with the central BZ receptor (Asano and Spector, 1979; Marangos et al., 1979), while caffeine is anxiogenic (Charney et al., 1985). CGS 8216, a prototypic non-xanthine adenosine receptor antagonist (Williams and Risley, 1982) is also an inverse agonist at the central BZ receptor (Czernik et al., 1982). Xanthines can antagonize BZ effects on cell firing and behavior (Phillis and Wu, 1981) and both the BZs and adenosine are effective muscle relaxants (Bruns et al., 1983; Turski et al., 1984). Chronic treatment with theophylline can uncouple the functional state of the central BZ receptor complex (Roca et al., 1988). Interesting, NMDA receptor antagonists also share this property (Lehmann et aL, 1987). However, alterations in endogenous adenosine levels using the adenosine deaminase inhibitor, EHNA have given conflicting data as to the involvement of the purine in the actions of the BZ (Nostrand et al., 1983; Gundlach and Johnston, 1988). Seizures induced by caffeine are blocked by the BZ receptor antagonist, Ro 15-1788 (Albertson et al., 1982) while electrophysiological studies have indicated that the fl-carbolines, the most active ligands at the central BZ receptor yet identified, can block the actions of adenosine (Phillis and O'Regan, 1988) although such antagonism, given the lack of effect of these compounds in binding assays (Williams et al., 1981) may represent a functional rather than pharmacological effect.

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Stress, an important component of anxiety, can be augmented by caffeine and theophylline (Henry and Stephens, 1980). Central A~ receptors appear to play a role in stressqnduced gastric ulcer formation (Geiger and Glavin, 1985: Ushijima et al., 1985). Despite this comparatively large body of overall convincing evidence, the precise nature of the adenosine/anxiety link remains unresolved but nonetheless intriguing. (3) Analgesia. As with the putative anxiolytic actions of adenosine, those related to its involvement in nocioception are complex. Xanthines have been reported to both induce and prevent pain as well as antagonize the effects of morphine (Ho et al., 1973; Jurna, 1984; DeLander and Hopkins, 1987) while IBMX can induce a "quasi-morphine withdrawal syndrome" (Collier et al., 1981) that involves increases in norepinephrine turnover (Galloway and Roth, 1983). The antinociceptive actions of adenosine appear to be A 2 receptor mediated at the spinal level (DeLander and Hopkins, 1987). NECA can thus potentiate morphine-induced analgesia, as can the adenosine uptake inhibitor, NBI (Nitrobenzylthioinosine; DeLander and Hopkins, 1987). There are, however, other reports (Gourley and Beckner, 1973) that adenosine can antagonize morphineinduced analgesia. Caffeine is an effective analgesic adjuvant (Laska et at., 1984). Morphine-dependent mice show increases in A~ receptor density (Ahlijanian and Takemori, 1986). (4) Alcohol and adenosine. Theophylline reduces the duration of ethanol-induced sleep in rodents (Dar et al., 1983). Conversely, the adenosine uptake inhibitor, dipyridamole can prolong ethanol-induced hypnosis and potentiate the associated motor incoordination (Dar et al., 1983). Ethanol-sensitive "long sleep" mice are more sensitive to the sedative and hypothermic actions of R-PIA than the ethanolinsensitive "short sleep" mouse (Proctor and Dunwiddie, 1984). Assessment of the effects of ethanol treatment on adenosine-stimulated adenylate cyclase activity in the neuroblastoma-glioma cell line, NG 108-15, has provided a model of acute ethanol intoxication, tolerance and dependence (Gordon et al., 1986). Ethanol treatment in rats can upregulate rat cerebellar A~ receptors, an effect that can be antagonized by theophylline (Clark and Dar, 1988). Taken together, these studies indicate that adenosine may be involved in the molecular events associated with ethanol-related motor dysfunction and sedation. (5) Dopamine-related disease entities'. Caffeine can elicit a self-destructive behavior in rats (Minana et at., 1984) similar to that seen in Lesch-Nyhan syndrome,

a genetic disorder associated with a deficit in the purine metabolizing enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGPRTase). This effect of the xanthine is also similar to that observed following apomorphine administration (Muller and Nyhan, 1982). Dopaminergic hyperactivity in the basal ganglia is thought to be the primary lesion in this disease state (Lloyd et al., 1981), an effect that may be due to a reduction in adenosine function which would facilitate dopaminergic neurotransmission by reducing presynaptic inhibitory processes. This, and a supersensitivity of dopamine Dj receptors, has been proposed as a cause of the self-mutilation behavior associated with Lesch-Nyhan syndrome (Criswell et al., 1988). Theophylline, caffeine and the Az-selective xanthine, DMPX (Ukena et al., 1986) increase locomotor activity in rodents (Snyder et al., 1981; Seale et al., 1989), an effect mediated via A_, receptor blockade (Bruns et al., 1988) associated with alterations in dopamine system function (Fuxe and Ungerstedt, 1974). Increased locomotor activity in rats that have been unilaterally lesioned in the nigrostriatal dopamine pathway occurs as contralateral rotational behavior (Fredholm et al., 1983). This test procedure is routinely used to identify compounds with putative antipsychotic potential, typically those that reduce dopaminergic neurotransmission either by direct postsynaptic blockade or by presynaptic dopamine autoreceptor activation. Interestingly, within the context of the involvement of adenosine in dopamine related locomotor behaviors, selective A 2 receptor agonists have been reported to have activity in test procedures predictive of antipsychotic activity (Heffner et al., 1985). A potential mechanism for this effect could be the activation of presynaptic A 2 receptors that inhibit striatal dopamine release. Caffeine has been used as an adjunctive therapy with dopamine agonists in the treatment of Parkinsonism (Fuxe and Ungerstedt, 1974). (6) Depression. Adenosine has been implicated in the actions of tricyclic antidepressants (Sattin et al., 1979; Stone, 1983). Chronic electroconvulsive therapy (ECT), a procedure used in the treatment of chronic depression can increase A~ receptor density in rat brain (Newman et al., 1984). Chronic treatment with tricyclic antidepressants (Williams et al., 1983) or with lithium (Newman et al., 1984), the latter a common treatment for depression, failed to have any effect on A~ receptor density. The recently reported trazoloquinoxalinamine adenosine antagonists (Trivedi and Bruns, 1988) have been patented as novel antidepressants (Sarges, 1985). The link between adenosine and depression is however, far

Adenosine: the prototypic neuromodulator from strong and the observed data may result from non-specific alteration in the CNS activity. Also, it is noteworthy that nearly all preclinical models for depression, biochemical or behavioral, are highly empirical. (7) Cognition and cerebral blood flow. Xanthines are central stimulants in most mammalian species (Snyder, 1985; Williams and Jarvis, 1988). While this is usually ascribed to the antagonism of the sedative actions of endogenous adenosine in the CNS, xanthines can also increase cerebral blood flow (Sollevi, 1986), thus increasing oxygen and glucose availability to the brain. Caffeine can increase cerebral glucose utilization while decreasing cerebral blood flow (Grome and Stefanovich, 1986), an action thought to involve antagonism of adenosine-induced vasodilitation processes. Adenosine appears to be a key regulator of CNS glucose utilization (Magistretti et al., 1986). A role for purines in the genesis of migraine may occur: (Burnstock, 1985). (8) Respiration. Breathing involves actions of endogenous adenosine (Hedner et al., 1982; Eldridge and Millhorn, 1987), caffeine and aminophylline being respiratory stimulants (Richmond, 1949). In preterm infants, hypoxemia can increase adenosine levels, an effect that leads to a fatal depression of central respiratory center activity (Lagercrantz et al., 1984). Apnea, or sudden infant death syndrome (SIDS), can be effectively treated with methylxanthines (Arnada and Turmen, 1979). The respiratory effects of the purine differ between adults and the very young (Ribero et al., 1988). In adults, adenosine stimulates respiratory ventilation, an effect dependent on carotid body chemosensory activity. Respiration is however inhibited in preterm rabbits (Hedner et al., 1984) or neonates (Lagercrantz et al., 1984). These actions appear to be mediated via A2 receptor activation (Monteiro and Ribeiro, 1987). Adenosine thus appears to be a primary mediator of carotid body chemoceptor function (Ribeiro and McQueen, 1981). Peripheral effects o f adenosine

Adenosine has been implicated in a multitude of processes related to the normal function of tissues and tissue systems outside the CNS. While usually of less interest to those involved in CNS research, research related to these systems has shown them to have a major impact on CNS function. (a ) Cardiovascular actions. The hypotensive actions of adenosine occur directly via the ability of the purine to dilate coronary vessels and reduce cardiac output as well as indirectly via its involvement in renin release and renal function, as well as in the

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homeostatic integration of chemoceptor and baroceptor function. In addition to the peripheral actions of adenosine in regulating blood pressure, there is a prominent contribution from the CNS as evidenced by the actions of centrally administered adenosine analogs (Mueller et al., 1984; Barraco, 1988). Adenosine is a potent regulator of coronary blood flow (Drury and Szent-Gyorgyi, 1929; Berne, 1963; Berne et al., 1974; Sollevi, 1986; Sparks and Bardenheuer, 1986) and has negative dromotropic, chronotropic and inotropic actions (Honey et al., 1930; Bellardinelli et al., 1983; Clemo and Bellardineili, 1985). The former actions of the purine are mediated via interactions with A2 receptors (Hamilton et al., 1987a,b; Oei et al., 1988) while the effects on cardiac rhythmicity involve A~ receptors in the atrioventricular node (Evans et al., 1982). The two receptor subtypes appear quite similar pharmacologically to those present in brain tissue (Hamilton et al., 1987a,b; Oei et al., 1988) requiring caution in the dissociation of the peripheral and central effects of systemically administered adenosine (Snyder et al., 1981; Barraco et al., 1986; Phillis and DeLong, 1986; Barraco, 1988). Thus, in cardiac tissue, adenosine effects can be direct involving potassium channel activation (Rardon and Bailey, 1984; DiMarco et al., 1985; Fredholm and Dunwiddie, 1988) as well as indirect, involving antagonism of catecholamine actions on adenylate cyclase activity (Rardon and Bailey, 1984) Adenosine may also be involved in the cardiotonic actions of the xanthines (West et al., 1987). In contrast to caffeine, the thio-xanthine, S-caffeine, has negative inotropic and chronotropic activity (Fassina et al., 1985). Since both S-caffeine and caffeine inhibit cPDE activity, the involvement of increased cAMP levels in the cardiotonic actions of such compounds appears unlikely. Adenosine antagonists also cause atrial or ventricular arrythmias and sinus tachycardia (Ogilvie, 1985). Adenosine is involved centrally in blood pressure regulation at the level of the nucleus tractus solitarius (Barraco, 1988), an area involved in baroceptor integration. It is not, however, established as to whether adenosine, as in the situation in regard to chemoceptor function (Riberio and McQueen, 1984), is the chemical mediator of such effects. Adenosine levels in heart are in a state of constant flux, increases being observed during hypoxia, ischemia or reactive hyperemia (Sollevi, 1986). The purine is therefore physiologically available, not only to activate adenosine receptors in cardiac tissue, but also those on circulating blood cells such as neutrophils. Thus, in addition to acting as a local

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autacoid (Engler, 1987), the purine can theoretically recruit other tissue systems to deal with trauma. (b) Renal effects. Adenosine can reduce renal blood flow by a transient vasoconstrictor action. Thus unlike most vascular beds which dilated by the purine, adenosine constricts those in the kidney (Osswald,1984). Mechanistically, these effects on vascular bed function appear to be mediated by opposing effects on voltage sensitive calcium channel activity (Churchill and Bidani, 1989). By antagonizing the actions of adenosine, caffeine is an effective diuretic, producing its effects by inhibiting solute resorption through increased glomerula filtration rate and renal vasodilation (Osswald, 1984). In addition to affecting renal blood flow, glomerular filtration rate, urine flow, natriuresis and activity in afferent and efferent nerves in the renal vasculature, adenosine is a potent modulator of renin release having biphasic actions. Activation of A~ receptors inhibits renin release while at higher concentrations, the purine activates A 2 receptors to stimulate renin release (Itoh et al., 1985; Churchill and Bidani, 1989). By acting as a signal transducer in response to increased renal sodium loads (Murray and Churchill, 1985) and affecting the amount of renin available in the circulation, adenosine can regulate blood pressure indirectly, another consideration in assessing the actions of systemically administered purine analogs. ( c ) Pulmonary function and asthma. Adenosine has been implicated in the etiology of asthma by virtue of its ability to potentiate, sequentially, in response to an allergic challenge, mast cell degranulation, histamine release and broncho-constriction (Holgate et al., 1987). The usefulness of xanthines as antiasthmatics has however been known for over a century (Salter, 1859) and the demonstrated efficacy of theophylline in the treatment of asthma has made this particular therapeutic application of the xanthines among the most widely studied (Svedmyr, 1985). There is, however, some controversy as to the mechanism of the anti-asthmatic actions of the xanthines. While considerable evidence supports an action as a phosphodiesterase inhibitor, a postulate supported by the demonstrated efficacy of enprofylline (3-propylxanthine), a xanthine that has relatively weak activity as an adenosine antagonist, as an anti-asthmatic (Persson et al., 1986), adenosine antagonism appears also to be a possible mechanism of anti-asthmatic action. In contrast to the reported actions of adenosine in facilitating histamine release in rat and guinea pig, in human basophils, the purine inhibits mediator release (Marone et al., 1979;

Church and Holgate, 1986). As in the kidney, this anomaly has been explained by biphasic actions of adenosine. When given before IgE, adenosine can inhibit histamine release. When added after, it enhances release (Church et al., 1983). Similarly, administration of the purine to asthmatics causes broncho-constriction but has no effect in normal subjects (Holgate et al., 1987). Thus, in continuing the theme of the present overview, the effects of adenosine, in its role as a homeostatic agent, are very much dependent on the state of the tissue in which the studies reported are performed. Adenosine analogs have been patented as novel anti-allergic substances (Schaumann et al., 1987). (d) Inflammation. The process of inflammation is involved in a wide variety of diseases. Antiinflammatory agents such as the steroids and NSAIDs are, however, typically associated with such gross manifestations of the inflammatory response as arthritic joint pain and headache. Inflammation is, however, a far more global phenomenon leading to cell death and tissue damage and has important implications for CNS function. Cytokines and other inflammatory mediators can evoke superoxide release from neutrophils, a process that can be blocked by the activation of A 2 receptors on the neutrophil cell surface (Cronstein et al., 1983: Cronstein and Hirschhorn, 1989). Anecdotal accounts related to the use of the cytokine, interleukin-I in the treatment of cancer, have indicated that this agent can induce psychotic episodes. Furthermore, platelet activating factor (PAF), also an inflammatory mediator has CNS actions (Korenecki and Ehrlich, 1988). A series of adenosine analogs have recently been described as antiinflammatory agents in whole animal procedures, although the mechanism has not yet been elucidated (Krenitsky et al., 1988). Neutrophil activation is also part of the inflammatory response; thus the "adenosine potentiator", AICA-riboside (Engler, 1987) has the potential to reduce the tissue damage associated with inflammation by preventing superoxide production. (e) Immune function. With the advent of molecular biology, it has become increasingly apparent that the immune system and the CNS have a reciprocal relationship. Altered immune competence can affect behavior while the CNS can regulate immune system function, especially under stress related situations (Ader 1981). As in the stress associated with anxiety (vide infra), the CNS can affect immune competence, the accounts relating to "attitude" overcoming such disease states as cancer being legion.

Adenosine: the prototypic neuromodulator Abnormalities of purine metabolizing enzymes underlie certain types of severe combined immunodeficiency diseases (Waldmann, 1988; Cronstein and Hirschhorn, 1989). Adenosine receptors are present on lymphocytes (Marone et al., 1978, 1979; Cronstein et al., 1983; Lappin and Whaley, 1984; Polmar et al., 1988). Adenosine can inhibit lymphocyte proliferation (Hirschhorn et al., 1970; Nishida et aL, 1984), an effect apparently mediated via cell surface receptor activation (Sandberg, 1983). Adenosine can prevent T-cell mediated cell lysis (Wolberg et al., 1978) but has no effect on killer cell function (Nishida et al., 1984). The purine can also modulate the expression of T-lymphocyte "subset specific" surface antigens (Birch et al., 1982, 1986). Such effects are restricted to T4 ÷ and T8 ÷ cell populations, especially those with mixed epitopes, favoring that for T8 (Birch and Polmar, 1986; Samet, 1985), resulting in the loss of "helper" T cell function via apparent activation of a subset of suppressor cells. This latter response impairs B-cell differentiation. Polmar (1984) has suggested that adenosine receptor-related effects on immune cell function relate to an immunohomeostatic system that limits autoimmune responses to "self antigens" in response to ischemic, allergic and inflammatory reactions. This may be a continuation of the general antiinflammatory action of the purine.

ADENOSINE---AGLOBAL HOMEOSTATIC MEDIATOR?

From a rather inauspicious start in the late 1920s, fuelled by the discovery of the putative second messenger, cAMP, in the 1960s, studies defining the role of adenosine in mammalian tissue function have increased exponentially in the past 20 years. Such studies have been directed to nearly every organ system with, in general, a unifying role related to homeostatic function (Newby, 1984; Williams, 1984). The effects of adenosine are complex, depending on the tissue, the type of receptor activated, the state of the tissue being studied and the species. This complexity has been exaggerated by the paucity of bioavailable pharmacological tools available to define A~ and A 2 mediated events. It is only in the last 10 years that any concerted effort has been made in the medicinal chemistry arena to synthesize receptor selective agonists and antagonists. And it is only in the last 4 years that potent, non-xanthine adenosine receptor antagonists have been discovered (Bruns et al., 1988; Williams, 1989).

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As an example of the unifying concept of adenosine action, one may consider the evidence implicating adenosine as an anxiolytic, as a muscle relaxant, as a potentiator of the effects of alcohol, as an anticonvulsant and as an antidepressant. All such proposed activities for the purine are also shared by the centrally active BZs, a coincidence of some note. In addition, differences in the biophysical properties of the central BZ receptor complex have been reported in the alcohol sensitive/insensitive long term and short term sleep mice (Mclntyre et al., 1988). It is of considerable interest that the BZs, along with morphine, were among the first drugs shown to be acting via their own receptors in the mammalian CNS. While the enkephalin's and endorphins have been firmly established as endogenous ligands for the various classes of opiate receptors, this has not been the case for the diazepam receptor. While many, many types of compound, including a number of peptides have been reported as endogenous ligands for the central BZ receptor (Haefley et al., 1985) none have been unequivocally identified as being active both in vitro and in vivo paradigms associated with BZ receptor activation. As already noted, however, adenosine receptor ligands can modulate BZ related processes and vice versa. Furthermore, a wide variety of compounds with a basic BZ pharma,cophore have been shown to have activity at a range of neuropeptide receptors, including those in the periphery for cholecystokinin (Chang et al., 1985; Evans et al., 1987), PAF (Kornecki et al., 1984), x-opiates (Romer et al., 1982), TRH (Sharif and Burt, 1984; Rinehart et al., 1986) and perhaps those for atrial naturietic factor (Webb et al., 1989). At this time it is premature to suggest, based on this tentative relationship, that adenosine and peptides subserve a similar function. It would not be improbable, however, that adenosine, based on its well established homeostatic role, would be the prototypic modulator with the peptides acting as fine tuning entities in more defined situations to regulate cell function. Based on the origin of both compounds, their role is certainly older established within the heirarchy of evolution than those entities" which we normally think of as neurotransmitters. Current approaches in biological research relate to the discrete actions of chemical messengers rather than their more global actions, a realistic approach related to solving tangible problems rather than the more elusive "big picture". The basic goals of both academic and industrial research relate to improving knowledge and in doing so, life. In academia this relates to a better under-

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standing of the function of nature and m the life sciences, an understanding of the disease process, both genesis and etiology. At the industrial level, such knowledge is transformed into medication and represents an improvement in life, a fact attested to by the dramatic reduction in confined psychotics with the advent of chlorpromazine. However, drug therapy is often discrete, with pathophysiology rather than the compound providing specificity of action. Thus, even while " n o r m a l " cell and tissue function may be affected by a drug, it is assumed (and hoped) that a diseased target tissue will be the primary beneficiary. Such an approach to health care has been highly successful and represents an basic tenet of Western drug therapy. In Sino-Japanese medicine, as well as in the resurgence of homeopathic medicine in Europe there is a tradition of prevention rather than cure. It is tempting to speculate (and certainly a review article in honor of Henry Mcllwain is the ideal place for such speculation, a precedent well established by "The P r o f " ) that traumatic insults, probably minor in nature, impact the body to varying degrees, reordering the system. As part of this process, cellular energy levels are altered with a corresponding decrease or increase in the availability of adenosine and its nucleotides and a resultant imbalance in overall tissue homeostasis. Compounds that may affect adenosine (such as the BZs?) may then function as drugs by restoring, probably transiently, the equilibrium in energy. If such speculations were true, then drugs specifically and potently modulating adenosine-related process may prove to be highly beneficial. Order and purpose in apparently random, non-specific circumstances are not without precedent (Gleick, 1987). Given that a good deal of evidence from conditions where trauma is extreme (ischemia, anoxia) is not inconsistent with such a role for the purine, it is conceivable that adenosine is the prototypic neuromodulator and, depending on one's semantics, autacoid and primary homeostatic effector. When reading Henry Mcllwain's more recent work, especially that related to the rheosome concept (Mcllwain, 1978), one may wonder, whether in studying the effects of adenosine on c A M P formation in the late 1960s, there may not have been an inkling, not only of the complexity of the potential role for this nucleoside in tissue function, but also an appreciation of the seminal nature of the purine as a prototypic neuroeffector agent. As molecular techniques continue to improve and become increasingly sophisticated, many of Henry

Mcllwain's ideas will be subject to further experimental evaluation. It is to be hoped that they will prove to be as intriguing, as complex and as thought provoking as his efforts which presaged and were seminal to the current explosion in adenosine related research.

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