Calcium and Sedative-Hypnotic Drug Actions

CALCIUM AND S EDATIVE-HYPNOTIC DRUG ACTIONS By Peter L. Carlen Neurology Program Alcoholism and Drug Addiction Research Foundation Playfalr Neuroscience Unit Toronto Western Hospital, and Departments of Mediclne (Neurology) and Physiology and Institute of Medical Science University of Toronto Toronto, Ontarlo, Canada M5S 2S1

and Peter H. Wu Neurology Program Alcoholism and Drug Addiction Research Foundation Department of Pharmacology Unlversity of Toronto Toronto, Ontario, Canada M5S 2S1 I.

Introduction

11. Behavioral Effects 111. Electrophysiology

A.

Calcium Currents Calcium-Dependent Ionic Currents Calcium Currents and Second Messenger Systems Acute Sedative-Hypnotic Drug Actions in Relationship to Calcium and Calcium-Dependent Currents E. Tolerance, Dependence, and Chronic Neuronal Effects of SedativeHypnotic Drugs in Relationship to Calcium and Calcium-Dependent Currents F. Aging Interactions IV. Biochemistry A. Effects of Sedative-Hypnotic Drugs on Influx of Calcium B. Effects of Sedative-Hypnotic Drugs on Calcium Receptors C. Effects of Sedative-Hypnotic Drugs on Intracellular Regulation of Calcium Ion Concentration D. Calcium Second Messenger Systems and Drug Action V. Conclusions References

B. C. D.

1. introduction

Calcium participates in a large number of neuronal biological processes and its physiological role has been very extensively reviewed in recent years 161 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 29

Copyright 0 1988 by Academic Press, Inc. AU rights of reproductionin any form reserved.

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(Hagiwara and Byerly, 1981; Tsien, 1983, 1987; Eckert and Chad, 1984; Rasmussen and Barrett, 1984; Rubin et al., 1985; Augustine et al., 1987; Miller, 1987; Ewald and Levitan, 1987). Our laboratory has conducted cellular electrophysiological experiments on the effects of commonly used and abused sedative-hypnotic drugs on central mammalian neurons and has reached the conclusion that many of their effects are calcium mediated. This review will focus on neurophysiological and biochemical data related to the interactions of sedative-hypnotic drug neuronal actions with calcium-mediated metabolic processes and calcium-dependent currents. For the sake of brevity, we have restricted the scope of the work cited herein. The second section will be a brief discussion of the behavioral effects of the three types of sedative-hypnotic drugs to be discussed in this article, i.e., alcohol, barbiturates, and benzodiazepines. These behavioral effects include acute sedative-hypnotic actions, tolerance, dependence phenomena (e.g., drug withdrawal hyperexcitability) and, at least in the case of chronic ethanol administration, brain damage. A description of the acute and chronic cellular neuronal electrophysiologicaleffects of these drugs will comprise the third section. We are stressing electrophysiological data initially because we feel that the expression of brain activity (and hence behavior) is via a summation of cellular and local circuit neuronal events, which, however, are ultimately based on biochemical and biophysical processes. The fourth section will entail a description of neuronal biochemical processes affected by these sedativehypnotic drugs. II. Behavioral Effects

Many animal and human behavioral experiments have demonstrated the sedative actions of ethanol, barbiturates, and benzodiazepines (Boisse and Okamato, 1980; Harvey, 1985; Ritchie, 1985), although some barbiturates are better anticonvulsants than sedatives, others are convulsants, and some benzodiazepines have nonsedative or antagonistic properties (Henkeler et al. , 1981). Ethanol in low doses is stimulatory in certain species (Masur and Boerngen, 1980; Erickson and Kochbar, 1985) and it is a well known clinical observation that the elderly can show enhanced sensitivity or sometimes even a paradoxical excitatory response to sedative drugs such as benzodiazepines and barbiturates. This review will focus on those drugs that are considered to have primarily sedative properties. These sedative drugs all demonstrate a certain degree of cross-tolerance to each other (Kalant et ol., 1971; Okamoto, 1978; Boisse and Okamoto, 1980; Commissaris and Rech, 1983). Also, all three classes of drugs show the

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development of tolerance (Kalant et al., 1971; Boisse and Okamoto, 1980) to their sedative actions with repeated administration or even following a single dose (acute tolerance). After more prolonged administration, drug “dependence” can develop, which means that following drug withdrawal a central nervous system (CNS) hyperexcitable state results, often manifested by behavioral hyperactivity, shakes, tremors, and even seizures gaffe, 1985). Chronic administration of alcohol is well known to cause brain damage (Carlen et al., 1981). It is unclear whether chronic intake of benzodiazepines or barbiturates can cause any long-term brain dysfunction. Finally, extracellular brain calcium has been implicated in mediating the acute behavioral responses to ethanol (Erickson et al., 1980; Little et al., 1986; Morrow and Erwin, 1986). 111. Electrophyslology

A. CALCIUM CURRENTS Calcium currents and their electrophysiological actions are being extensively studied in many different types of neurons, and several recent reviews have been published (Hagiwara and Byerly, 1981; Tsien, 1983, 1987; Rubin et al., 1985; Augustine et al., 1987; Ewald and Levitan, 1987; Miller, 1987). Ca ions play an essential role in presynaptic transmitter release (Katz and Miledi, 1967), although presynaptic depolarization also plays a role (Dude1 et al., 1983; Parnas et al., 1986). When an action potential invades the nerve terminal, voltage-dependent Ca channels open, allowing Ca ions to flow from the extracellular fluid with a Ca concentration of approximately M to the intracellular compartment with a much lower Ca concentration of lo-’ M or less. The rise in intracellular Ca in the presynaptic terminal somehow triggers the exocytotic release of quanta of neurotransmitter. Hence alterations in presynaptic Ca function can have profound effects on neurotransmitter release. T o study Ca currents directly by electrophysiological techniques, it is much easier to impale with a microelectrode the larger postsynaptic element (usually the soma) than the presynaptic terminal (the squid giant synapse being an exception, e.g., Augustine and Charlton, 1986). The advent of patch-clamp and single-electrode voltage-clamp techniques has permitted more accurate delineation of the various Ca currents of mammalian neurons. At present, it seems that there are probably three different types of neuronal Ca channels (“T,” “N,” and “L”) as measured by somatic recordings (Nowycky et al., 1985; Tsien, 1987). The T current is activated at relatively

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hyperpolarized potentials and is rapidly inactivating. The N current is activated at more depolarized voltages and is also rapidly inactivating. The L current is activated at relatively depolarized voltages and is slowly inactivating. All currents have different sensitivities to various Ca current-blocking agents. Yaari et al. (1987) showed, using whole-cell patch voltage-clamp recordings of acutely dissociated embryonic rat hippocampal neurons, low voltage-activated fully inactivating somatic Ca channels and high voltage-activated slowly inactivating Ca channels, predominantly located in the developing dendrites. Our laboratory, using the single-electrode voltage clamp in rat dentate granule neurons in in vitro slices, has demonstrated three types of Ca currents (Carlen et al., 1986; Nielsen et al. 1987), roughly corresponding to the descriptions of Nowycky et al. (1985) in chick dorsal root ganglion neurons. Brown and Griffith (1983) demonstrated a persistent slow inward Ca current in hippocampal CAI and CA3 neurons probably corresponding to the L current.

B. CALCIUM-DEPENDENT IONICCURRENTS Once intracellular Ca is raised, ion channel function can be affected. In 1972, Krnjevic and Lisiewicz showed in cat spinal motoneurons that intracellular Ca ion injection activated a potassium conductance kK). This powerful intrinsic inhibitory mechanism, i.e., Ca-dependent gK7 was also demonstrated in Aplysiu neurons by Meech (1972). During a train of Nadependent spikes in central neutrons, some inward Ca current is also generated, causing a postspike train afterhyperpolarization (AHP) which is due to Ca-dependent gK (Krnjevic el al., 1978; Hotson and Prince, 1980; Gustafsson and Wigstrom, 1981). Recently it has been shown in hippocampal neurons that the AHP is composed of two Ca-dependent gKs,an early (I,) and a late component (IAHP)(Lancaster and Adams, 1986). In addition, a Ca-activated gc, (Owen et ul., 1986) and a Ca-activated cation (Na and K) current (Kramer and Zucker, 1985) have been described. Several neurotransmitters activate Ca-dependent currents in neurons. For example, our laboratory has shown that the CAI neuronal dendritic hyperpolarizing response to focally applied y-aminobutyric acid (GABA) is a Cadependent gK (Blaxter et u l . , 1986). Finally, increased intracellular Ca concentration per se inactivates Ca currents in many neurons (Eckert and Chad, 1984), and this mechanism is key to the development of our working hypothesis as to how ethanol and barbiturates affect neurons and interact with Ca currents (discussed below). Recent observations suggest that Cadependent inactivation of the Ca current results from the activation of a Cadependent phosphatase during cellular entry and accumulation of Ca, and this inactivation is subsequently removed as the phosphatase activity

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declines and dephosphorylated sites are rephosphorylated through the action of an endogenous kinase (Chad and Eckert, 1986).

C. CALCIUM CURRENTS AND SECOND MESSENGER SYSTEMS There is now a growing literature concerning the interaction of cellular second messenger systems, neurotransmitters and neurohormones, and Ca currents (Kaczmarek and Levitan, 1987;Worley et al., 1987). There are two presently well studied receptor-regulated second messenger systems active in the CNS: (1) cyclic adenosine monophosphate (AMP), which activates specific phosphorylating enzymes called protein kinases, and (2) the phosphoinositide cycle whereby receptor activation causes the release of inositol-l,4,5-triphosphate(InsPs) and diacylglycerol (Rasmussen and Barrett, 1984). Guanosine triphophate-binding proteins (G proteins) operate in both of these systems. Tsien (1987) reviewed the modulation of voltagegated Ca channels in the surface membrane of excitable cells. These are complex interrelationships between the several ways of regulating intracellular Ca ion concentration and the effects of Ca ions on second messenger systems (Rasmussen and Barrett, 1984). Ca interacts with different types of protein kinases (Nestler et a f . , 1984; Nishizuka, 1986), with cyclic AMP, with guanine nucleotide-binding proteins (Dolphin, 1987), and with inositol phospholipids (Downes, 1983). Recent electrophysiological experiments have demonstrated interactions between second messenger systems, Ca currents, and Ca-dependent currents. Phorbol esters, which activate protein kinase C , blocked the ZAHp in hippocampal CAI neurons (Baraban et a l . , 1985; Malenka et al., 1986). Phorbol ester and protein kinase C enhanced a voltage-sensitive Ca current in Alplysia neurons (DeRiemer et al., 1985), whereas protein kinase C activation in embryonic chicken dorsal root ganglion neurons attenuated a voltage-dependent Ca current (Rane and Dunlap, 1986). Ca action potentials in Alplysia bag cell neurons were enhanced by intracellular microinjection of the catalytic subunit of cyclic AMP-dependent protein kinase C (Kaczmarek et al., 1980). Higashida and Brown (1986) gave evidence in voltage-clamped cultured neuroblastoma-glioma hybrid cells that extracellular application of bradykinin, which forms intracellularly InsPs, or intracellular injection of Ca or InsPs activated a Ca-dependent g ,. Paupardin-Tritsch et al. (1986)showed that intracellular injection of a cGMP-dependent protein kinase enhanced a Ca current and potentiated the serotonin-induced Ca current increase in snail neurons.

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D. ACUTESEDATIVE-HYPNOTIC DRUGACTIONS IN RELATIONSHIP TO CALCIUM AND CALCIUM-DEPENDENT CURRENTS Over the past several years, this laboratory has been examining the central actions of sedative-hypnotic drugs using intracellular electrophysiologicd recording techniques from mammalian neurons in in uitro brain slices. We have studied clinically relevant sedative doses of ethanol (Q20 mM), benzodiazepines (QlO-aM), and pentobarbital (QlO-*M). Most of this work has been presented elsewhere (Carlen et a l . , 1982a,b, 1983a,b; 1985a-c; Carlen, 1987; Durand and Carlen, 1984; O’Beirne et al., 1987). In hippocampal CAI neurons, all sedative drugs, when applied via the bath perfusate or by drop application, caused, within 1 to 2 min, a neuronal hyperpolarization of the neuron, usually, but not always, associated with decreased input resistance (or increased conductance). Spontaneously active neurons diminished or ceased firing. Intracellular injection of chloride had no effect, suggesting that this hyperpolarization was most likely due to an increased gK. Intracellular injection of cesium, which decreases several gKs, blocked the ethanol-mediated hyperpolarization (Carlen et al. , 1982b). Nicoll and Madison (1982) also showed that several general anesthetics hyperpolarized vertebrate neurons, probably by increasing gK. Extracellular perfusion of tetrodotoxin (TTX), which blocks Na channels and secondarily spike-evoked synaptic transmission, had no effect on these drugs’ actions. Zero Ca perfusate with added MnC12 also did not block the hyperpolarizing action of ethanol and pentobarbital, but did block the hyperpolarization caused by midazolam. We shall discuss this point later. To further examine sedative drug activation of the inhibitory gKs, other voltage responses dependent upon gK activation were examined. All three drug types enhanced the long-lasting orthodromic inhibitory postsynaptic potential (IPSP), which is mediated by a gK which is probably not C a dependent, although there is some debate on this issue. The postspike train long-lasting AHP is a Ca-activated gK (Ic and IAHP). In zero Ca perfusate, this AHP potential disappears. This conductance is ubiquitous throughout the cental nervous system and is a powerful intrinsic neuronal inhibitory mechanism. Enhancement of the inhibitory long-lasting AHP (IAHP) was the single most consistent response to ethanol (Carlen d al., 1982), pentobarbital (O’Beirne et al., 1987), midazolam (Carlen d al., 1983b), and clonazepam (Gurevich et al., 1984) in CAI neurons. The enhanced AHP was also seen with ethanol in dentate granule neurons (Niesen et al., 1986) and with pentobarbital in CA3 neurons (O’Beirne d al., 1987). These findings fed us to hypothesize that these drugs could act in large part by affecting C a currents or intracellular Ca metabolism and that the sedative drug-induced hyperpolarization and enhanced AHPs were due to increased Ca-dependent K currents.

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In TTX perfusate, higher threshold Ca spikes can be elicited in CAI neurons. Ethanol had no effect or slightly decreased the Ca spikes (Carlen et al., 1982a). Pentobarbital decreased the Ca spikes (O’Beirne d al., 1987). Since both these drugs tended to increase the membrane conductance, the decrease of the Ca spike could be in part due to a conductance increase to which these mainly dendritic Ca spikes could be very sensitive (MacDonald and Schneiderman, 1986). However, diminished Ca spikes were noted in cells with no apparent decrease in input resistance. This effect could be due to the inactivating effect of raised intracellular Ca on some inward Ca currents (Eckert and Chad, 1984). On the other hand, midazolam enhanced the Ca spikes, even though it also caused decreased input resistance (Carlen et al., 1983b). Zero Ca perfusate, with MnCh added, blocked the hyperpolarizing effect of midazolam, but not that of ethanol or pentobarbital. A specific benzodiazepine antagonist, Ro14-7437, caused a depolarization of hippocampal CAI neurons with increased input resistance, decreased AHPs, and, in the presence of TTX, diminished Ca spikes (Carlen et al., 1983a). Like midazolam, its effects were blocked by zero Ca perfusate. These data led us to the conclusion that the hyperpolarization caused by ethanol (Carlen et al., 1982a) and pentobarbital (O’Beirne et al., 1987) could be caused, at least in part, by an increased Ca-mediated gK which was triggered by raised [Ca], from intracellular stores, whereas the actions of the benzodiazepines were dependent upon extracellular Ca (Carlen et al. , 1983b). It is easy to explain the enhanced AHPs seen in the presence of midazolam as due to increased inward Ca current during the preceding train of spikes. However, based on the above data showing that ethanol and pentobarbital do not enhance Ca spikes, it seems that the enhanced AHPs in the presence of these two drugs are not due to enhanced inward Ca currents. There is more than one type of voltage-dependent Ca current in these neurons (Brown and Griffith, 1983): a spikelike current, which could be related to the N current, and a much more slowly inactivating current, which could be the L current as described by Tsien (1987). The interaction of sedative drugs with voltage-clamped Ca currents in central mammalian neurons has not been investigated to date except for one report by Llinas and Yarom (1986) showing that the low-threshold Ca current in inferior olivary neurons was blocked by low concentrations of monohydroxyl alcohols including ethanol (0.1 to 1 mM). If ethanol and pentobarbital do not enhance inward Ca currents, then their AHP-enhancing action could be related to some mechanism which enhances the gK sensitivity to the transiently increased [Ca],, resulting from a train of spikes. This could be related to second messenger activation.

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As mentioned above, ethanol and pentobarbital caused a tonic hyperpolarization, often with increased conductance, both in normal and in Cafree perfusate (Carlen d al., 1982a, 1985a; O’Beirne et ul., 1987). Also both drugs had no effect or decreased Ca spikes. Based on these data, we hypothesized that ethanol could act by raising intracellular free ionic Ca concentration from intracellular stores (Carlen et al., 1982a), which would tend to diminish inward Ca currents (Eckert and Chad, 1984). However, raised intracellular Ca concentration per se could hyperpolarize the neuron by augmenting g, (Krnjevic and Lisiewicz, 1972), even in the presence of zero Ca perfusate. Mention should be made here of probably the most favored explanation to date of sedative-hypnotic drug action, i.e., activation of GABA-mediated C1 conductance. In our hands, at pharmacologically relevant doses for sedation, no augmentation of GABA-mediated gc, was seen (Carlen et al., 1985b), although higher doses of pentobarbital (lom4M) (O’Beirne et al., 1987) and midazolam M) (Carlen et al., 1983b) did augment GABA actions. Nestros ( 1 980) reported that ethanol augmented GABA-mediated neurotransmission in cat cortex in uiuo using extracellular recordings. Shefner et al. (1982) showed that 30 mM ethanol enhanced the GABA action on locus coeruleus neurons using in uitro intracellular recordings. However, she demonstrated that the main mechanism of inhibition of spontaneous firing of these neurons was augmentation of the late postspike AHP (a Ca-mediated g,) and by decrease of the rate of depolarization preceding each spike (Shefner and Tabakoff, 1984). These data were interpreted as being due to ethanol enhancement of a long-lasting gK. Biochemical experiments, however, give much evidence for C1 channel and GABA involvement in ethanol actions ( M a n and Harris, 1987; Ticku and Burch, 1980). We shall now report the electrophysical work of other laboratories regarding the acute effects of sedative-hypnotic drugs with relation to Ca and Cadependent currents, starting with relevant experiments using alcohol. At the vertebrate neuromuscular junction in a perfusate without Ca and with added EGTA (a Ca chelator), ethanol increased spontaneous nonspike-induced neurotransmitter release (miniature endplate potentials), a finding compatible with increased presynaptic Ca concentration from intracellular sources (Quastel et al., 1971). Carlen and Corrigall (1980) also demonstrated raised spontaneous neurotransmitter release from rat neuromuscular junctions in the presence of ethanol. Bergman d al. (1974), using high doses of ethanol (>lo0 mM) in three different types of Aplysia neurons, showed blockade of inward Na and Ca currents. Schwartz (1983) showed in voltage-clamped Aplysiu neurons that the leakage and slow inward Ca currents were decreased, and outward K currents were increased by 4% ethanol. More recent experiments (Schwartz, 1985) demonstrated that the Ca-activated K current was increased and the

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Na- and Ca-dependent slow inward currents were reduced by ethanol. The reduction of the Ca-dependent slow inward current was not prevented by intracellular injection of EGTA, suggesting that ethanol-induced increased intracellular Ca concentration was not solely responsible for the reduced Ca current, although this author suggested that ethanol could act by increasing intracellular Ca concentration. Also in Aplysia neurons, Camacho-Nisi and Treistman (1985) showed that a relatively low dose of ethanol (50 mM) reversibly decreased the amplitude of the Ca current with no effects on Na currents. In single bullfrog atrial cells, ethanol, in anesthetic doses of 100 to 500 mM, significantly depressed the slow inward Ca current and the repolarizing K current (Takeda et al., 1984). In rat cultured dorsal root ganglion neurons, ethanol in pharmacologically relevant concentrations (0.05 to 0.30 g % ) decreased gK as a result of decreased inward Ca current (Oakes and Pozos, 1982a). C a spikes were also diminished (Oakes and Pozos, 1982b), although very low concentrations of ethanol, leaking from a nearby microejection pipette containing low concentrations of ethanol (0.05 to 0.10 g%), increased the Ca spike. This low-dose effect may be related to the excitatory behavioral effects of ethanol seen at lower doses in some species. Pozos and Oakes (1987) reported that other alcohols, containing one to five carbons, all produced a decrease in the Ca spike duration. They presented preliminary results in cultured neuroblastoma cells loaded with the intracellular fluorescent Ca dye indicator, fura 2, that ethanol doses of 0.15 g% and higher can increase intracellular free C a concentration. Vassort et al. (1986) demonstrated in squid axons injected with Arsenazo I11 that alcohols with a chain of 5 to 10 carbons increased intracellular free [Cali. Further biochemical evidence and discussion supporting the idea of ethanol and barbiturates raising intracellular free [Cali is presented in Section IV. In our experiments, pentobarbital acted similarly to ethanol postsynaptically on hippocampal pyramidal cells (Carlen et al., 1985a; O’Beirne et al., 1987). Ca spikes were diminished by M pentobarbital. Ca spikes were also decreased in cultured mouse spinal cord neurons by pentobarbital (25 to 600 p M ) , by phenobarbital (100 to 500 luM) (Heyer and MacDonald, 1982), and by convulsant barbiturates (Skerritt and MacDonald, 1984). In the leech Retzius neuron, several different barbiturates prolonged the action potential, compatible with the interpretation that barbiturates block a voltagedependent inward Ca current which activates a gK necessary for normal repolarization (Kleinhaus and Prichard, 1977). More recently it was demonstrated that Ca action potentials in leech nociceptive neurons were blocked by phenobarbital, pentobarbital, and methohexital (Johansen and Kleinhaus, 1986). Calcium spikes in Aplysia neurons were also blocked by pentobarbital (Goldring and Blaustein, 1982). Pentobarbital, on Helix

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neurons, increased the rate of voltage-dependent inactivation and decreased the maximal peak amplitude of the voltage-clamped Ca current (Nishi and Oyama, 1983a,b). Biochemical data also show that barbiturates and other sedative hynotics inhibit depolarization-induced Ca uptake into synaptosomes (Leslie, 1987). Benzodiazepines block Ca uptake in synaptosomes in micromolar concentrations (Leslie, 1987). Electrophysiological experiments in our laboratory on CAI neurons showed neuronal inhibition (i.e., membrane hyperpolarization and increased AHPs) by clonazepam (Gurevich et al. , 1984) and by midazolam (Carlen et al., 1983b) at low nanomolar concentrations. Midazolam, at 5 x M, consistently enhanced Ca spikes elicited in TTX. Skerritt et a!. (1984) showed that diazepam and its p-chloro derivative, Ro5-4864, inhibited intracellularly injected current-induced high-frequency repetitive firing in cultured mouse spinal cord neurons, with 50% inhibition by diazepam at 87.5 n M and by Ro5-4864 at and by diazepam (10 p M ) . C a action potentials were inhibited by 10 # diazepam and 10 pi4 Ro5-4864. The Kd of the high-affinity benzodiazepine sites in mammalian brain are in the lower nanomolar range. Cherubini and North (1985) observed that benzodiazepines, midazolam and diazepam in 100-300 pM concentrations, were able to decrease calcium action potentials in guinea pig myenteric neurons and the effects were reversibly blocked by Ro15-1788, a benzodiazepine antagonist.

E. TOLERANCE, DEPENDENCE, AND CHRONIC NEURONAL EFFECTS OF SEDATIVE-HYPNOTIC DRUGS IN RELATIONSHIP TO CALCIUM AND CALCIUM-DEPENDENT CURRENTS Unlike the biochemical literature, there is relatively little work done on the electrophysiologically measured effects of prolonged administration of sedative-hypnotic drugs in relationship to Ca or Ca-dependent currents. In the mammalian neuromuscular junction, ethanol increases spontaneous neurotransmitter release as measured by increased frequency of miniature endplate potentials (Quastel et al., 1971; Curran and Seeman, 1977; Carlen and Corrigall, 1980), which is a Ca-dependent process. Curran and Seeman (1977) showed tolerance to this action of ethanol in neuromuscular preparations removed from rats chronically exposed to ethanol, whereas Carlen and Corriglall (1980) did not. However, Carlen and Corrigall (1980) did show tolerqce to the in uitro ethanol-induced depression of the orthodromically evoked hippocampal CAI field potential at doses greater than 100 mM (supraanesthetic) and Durand el al. (1981) showed what appeared to be acute

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tolerance to perfused ethanol (100 mM) which initially depressed the CAI field potential, but within 30 min of perfusion, the field potential began to increase in size. This could be an in vitro correlate of acute tolerance. Rougier-Naquet et al. (1986) demonstrated hyperexcitability of CAI neurons in slices from rats undergoing ethanol withdrawal after 4 days of ethanol intubation (8- 12 g/kg/day) and in slices from ethanol-naive animals following 2 hr of slice perfusion with 100 mM ethanol. In both cases, the neuronal hyperexcitability associated with alcohol withdrawal showed, in intracellular recordings, diminished AHPs and decreased spike frequency adaptation to a 600-msec depolarizing current pulse. These results support the hypothesis that neuronal inhibition is depressed after ethanol withdrawal by means of decreased Ca-mediated gK. Whether this is related to alterations in Ca currents is not known. Hyperexcitability was also noted in in vitro hippocampal CAI neurons from drug-naive rats exposed to 20 n M clonazepam-containing perfusate for 2 or more hours and then withdrawn from drug (Davies et al., 1985, 1987) or in neurons from the rats fed clonazepam (10 to 50 mg/day in the rat chow) for 1 month following abrupt drug withdrawal (Davies and Carlen, 1984). No work to our knowledge has been done on the electrophysiology of neurons after subacute or chronic administration of barbiturates. Long-term administration of ethanol in the diet for 5 months is an adequate period for the development of behavioral and neuronal morphological deficits in rats. In hippocampal slices removed from such animals chronically exposed to ethanol via a liquid diet and then allowed to withdraw for 3 weeks, Durand and Carlen (1984) showed decreased AHPs and IPSPs in both dentate granule and CAI neurons. Certainly decreased &(s) helps to explain these findings. In the case of the decreased AHP, this ethanolinduced diminution implies a decreased Ca-mediated gK, which was hypothesized to be related to a state of chronically raised [Ca],, which is also toxic to cells and could be a cause of alcohol-induced brain damage (Durand and Carlen, 1984).

F . AGINGINTERACTIONS There are almost no data, to our knowledge, concerning the electrophysiology of sedative drug-neuronal interactions in aged compared to younger mature animals. Recent work from our laboratory (Niesen et al., 1986) in rat hippocampal dentate granule neurons showed that 20 m M ethanol inhibited neurons from young mature rats (6-8 months) by hyperpolarizing the membrane and augmenting the AHP. Presumably, these effects are similar to those noted in CAI neurons (Carlen et al., 1982a).

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However, in neurons from old rats (26-28 months), ethanol caused a slight depolarization, diminished spike-frequency adaptation to a 600 msec depolarizing current pulse, and diminished AHPs and IPSPs. The longlasting AHP is mediated totally, and the spike-frequency adaptation in part, by Ca-dependent g,. Hence, in old neurons, a moderately intoxicating dose of ethanol caused a neuronal disinhibition related in part to an altered effect on Ca-mediated g,.

IV. Biochemistry

In the preceding section, we presented evidence that the sedative-hypnotic drugs, i.e., ethanol, barbiturates, and benzodiazepines, may exert their pharmacological actions via Ca-dependent mechanisms. In this section we will present biochemical evidence that these drugs affect calcium homeostasis in neurons and might even alter second messenger function. The chemical gradient for C a across the neuronal membrane is approximately lO'-foId in favor of the extraneuronal calcium concentration. The influx of Ca into neurons is regulated by calcium channels. The intraneural Ca concentration is tightly regulated by the interplay of several factors, i.e., Ca channel influx, Na-Ca exchange, an ATP-dependent Ca pump, Ca-binding proteins, and intracellular C a uptake and release by the smooth endoplasmic reticulum and mitochondria (Rasmussen and Barrett, 1984). Sedativehypnotic drugs can affect intraneural C a by altering any or all of these factors. An alteration in intracellular C a concentration not only affects ionic conductances such as g,, but can also affect intraneuronal metabolic processes, since C a per se is a second messenger and affects other second messenger systems (Rasmussen and Barrett, 1984).

A. EFFECTS OF SEDATIVE-HYPNOTIC DRUGS ON INFLUX OF CALCIUM 1. Ethanol-Acute Eflects For the past 10 years, many groups have shown that ethanol, pentobarbital, and, to a lesser extent, benzodiazepines inhibit depolarization-evoked Ca influx into synaptosomes. It has been accepted that depolarizationinduced C a influx represents a mechanism for the mediation of presynaptic neurotransmitter release (Katz and Miledi, 1967). However, reviews of work done in this area indicate methodological problems (see Leslie, 1987). Earlier studies looking at the effects of ethanol on potassium-depolarized Ca uptake over a time course of minutes (slow-phase), have yielded conflicting

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results. Harris and Hood (1980) observed the inhibition of potassiumevoked Ca uptake by a concentration as low as 45 m M ethanol, whereas Blaustein and Ector (1975) did not observe any inhibition at an ethanol concentration of 100 mM. The reasons for these types of conflicting results could be that: 1. Ca influx through Ca channels and Ca channel opening time are known to occur in the millisecond range, whereas these studies observed Ca influx over a time course of minutes. These influx studies could therefore be looking at the summation of various cellular processes controlling [Cali. Ethanol may not affect all these processes equally. 2. Raised intracellular Ca plays a large role in the inactivation of many Ca channels (Eckert and Chad, 1984), and the inactivation time for the T and N types of C a channels is in the range of tens to hundreds of milliseconds. When Ca influx was examined over the time course of minutes, most Ca channels could have been largely inactivated. 3. The Ca uptake activity varies with different brain regions (Stokes and Harris, 1982). A synaptosomal preparation containing different amounts of tissue from the various brain regions may contribute to the differences seen in several laboratories.

Fast-phase Ca influx has been described by Nachshen and Blaustein (1980) and by Leslie et al. (1983a). This process probably correlates with the time course and pharmacology of L-type Ca channels (slowly inactivating Ca channels) (Leslie, 1987), possibly representing the neuronal mechanism of transmitter release (phasic release). Using a cortical synaptosomal preparation, Leslie et al. (1983a) and Daniel1 and Leslie (1986) showed that the fast-phase Ca influx was inhibited by low doses of ethanol, whereas the slow-phase Ca influx was insensitive to low concentrations of ethanol. In clonal neural cells, Messing et al. (1986) showed that acute exposure to ethanol produced a concentration-dependent decrease in depolarizationevoked 45Cauptake.

2 . Ethanol- Chronic Eflects Chronic administration of ethanol to rats results in a shift in the dose-response curve toward less ethanol inhibition of Ca influx. For example, Leslie et al. (1983b) demonstrated the development of tolerance in rats to the ability of ethanol in uitro to inhibit fast-phase Ca uptake in cerebral cortex. Wu et al. (1986) showed that the depolarization-induced Ca uptake measured at 20 sec following K depolarization was markedly inhibited in hippocampus and cerebral cortex but not in hypothalamus from rats made dependent on

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ethanol. In cultured clonal neural cells exposed to ethanol for 2-10 days, Messing et al. (1986) showed an increase in 45Cauptake to depolarization and an increase in the number of Ca channel-binding sites. The 45Cauptake was restored to normal following withdrawal of ethanol from the cultures. Very little has been done on human brain C a metabolism in alcoholism, Kapur et al. (1985) showed that cerebrospinal fluid (CSF) ionic Ca was lower in recently abstinent chronic alcoholic patients compared to neurological controls, whereas the total CSF Ca, serum ionic Ca, and total serum Ca were unchanged. How do biochemical studies explain electrophysiological data? Bear in mind that in most biochemical studies, the effects of ethanol on Ca entry are carried out in synaptosomal preparations (containing intact resealed presynaptic terminals), whereas the electrophysiological studies usually measure the postsynaptic neuronal somatic responses. T o date, we are not aware of any detailed studies comparing the properties of Ca channels in presynaptic and postsynaptic neuronal membranes and their sensitivities to the effects of sedative-hypnotic drugs.

3 . Barbiturates-Acute

Effects

Barbiturates inhibit synaptosomal calcium influx (Hood and Harris, 1980; Leslie et al., 1980b; Elrod and Leslie, 1980). It is interesting to note that pentobarbital reduced depolarization-induced Ca influx into synaptosomes and the Ca action potential in neurons in similar concentrations (Ondrusek et al., 1979; Heyer and MacDonald, 1982; Leslie et al., 1980b; Elrod and Leslie, 1980; Werz and MacDonald, 1985). Chandler et al. (1986) reported that 5-(2-cydohexylideneethyl)-5-ethylbarbituric acid (CHEB), a convulsant type of barbiturate, also inhibited the voltage-dependent Ca channels in brain synaptosomes. Since both convulsant and anticonvulsant barbiturates exert their effects through inhibition of voltage-dependent C a channels, it is difficult to explain the difference in these drugs’ actions based on Ca interactions alone.

4 . Barbiturates-Chronic

Effects

Chronic pentobarbital treatment results in an adaptive change of brain calcium channels similar to that observed with chronic ethanol treatment. The K-evoked Ca uptake was less sensitive to barbiturates in the chronically pentobarbital-treated animals (Leslie et a l . , 1980b).

5 . Benzodiazepines-Acute

Effects

There are high-affinity (Braestrups and Squires, 1977) and low-affinity (Shoemaker et a l . , 1981) benzodiazepine binding sites in brain. There is

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evidence that the peripheral type of benzodiazepines receptors is also present in brain (Shoemaker et al. , 1979). Electrophysiological and pharmacological studies show that peripheral-type benzodiazepine receptors are coupled to calcium channels in the heart (Mestre et al., 1985). Using the guinea pig papillary muscle preparation, Mestre et al. (1985) showed that Ro5-4864, an agonist of the peripheral type of benzodiazepine receptor, decreased the tension of the papillary muscle. This effect was reversed by increasing extracellular Ca concentration and also by PK 11195, an antagonist of the micromolar affhity peripheral-type benzodiazepine receptor. Biochemical studies on the effects of benzodiazepines on voltage-dependent Ca uptake have shown that benzodiazepines significantly inhibit "fast-phase' ' voltagedependent Ca uptake into mouse brain synaptosomes at concentrations which correlate with the KdS of low-affinity (micromolar) benzodiazepine receptors (Taft and Delorenzo, 1984). The ability of these benzodiazepines to inhibit Ca uptake is directly correlated to their hypnotic potency, suggesting that inhibition of presynaptic calcium entry may be linked with the hypnotic action of benzodiazepines (Leslie et al., 1986). However, Carlen et al. (1983b) demonstrated central mammalian neuronal inhibition by increased g, with concomitantly increased Ca spikes at nanomolar concentrations of midazolam. The Kdsof high-affinity benzodiazepine receptors are in the lower nanomolar range, as is the CSF concentration of these highly protein-bound drugs (Kanto et d.,1975).

6 . Benzodiazepines-Chronic Efects Chronic administration of chlordiazepoxide to animals resulted in decreased inhibitory effects of benzodiazepines on the K-evoked synaptosomal Ca uptake (Leslie et al.. 1980b), suggesting that voltage-dependent Ca channels underwent adaptive changes. It is important to note that, once again, the observation was made in presynaptic terminals. There are no data indicating whether the voltage-dependent Ca channels, which show adaptive changes to the effects of benzodiazepines, will also show adaptive changes to the effects of ethanol or pentobarbital, or vice versa, although it is assumed that all three drug classes show a certain degree of cross-tolerance following chronic treatment (Boisse and Okamoto, 1980). Finally, it should be mentioned that the data concerning sedative-hypnotic drug action on biochemically measured neurotransmitter release are quite confusing. This presumably reflects many methodological issues, in addition to the conceptual problem of possible sedative drug-induced increased presynaptic Ca concentration which would promote spontaneous transmitter release, but could have variable effects on action potential-evoked neurotransmitter release, depending on the degree of Camediated inactivation of the action potential-evoked nerve-terminal Ca current.

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B. EFFECTS OF SEDATIVE-HYPNOTIC DRUGS ON CALCIUM RECEPTORS 1. Ca Blockers Ca enters neurons via voltage-sensitive calcium channels. One way to investigate the functional roles of the Ca channel is to use drugs which are selective for blocking C a channels called Ca-channel blockers or antagonists. This area has been recently reviewed by Miller (1987) and Greenberg (1987). Cachannel antagonists include dihydropyridines (DHP), phenylalkamines, diltiazem, and bepridil. Although there is some controversy, reports indicate that there is single class of DHP-binding sites, since in either synaptosomal preparations or crude brain homogenates, binding studies show one class of binding site (Miller, 1987; Snyder, 1984). A recent study (Miller, 1987) using a brain synaptosomal preparation showed that Ca influx through voltagesensitive Ca channels was not blocked by DHPs when K depolarization was used as a stimulus, but the C a influx induced by Bay K 8644, a calciumchannel agonist, was completely blocked by DHPs in nanomolar concentrations, indicating that brain calcium channels appear to vary in sensitivity to the types of stimuli applied.

2. Ethanol-Acute

Effects

Hoffmeister et al. (1982) and Itil et al. (1984) observed that nimodipine enhanced sedative-hypnotic drug action. Isaacson et al. (1985) showed that nimodipine potentiated the sleep time and hypothermia induced by ethanol. These findings suggest that the sedative-hypnotic drugs, including ethanol, interact with DHP cerebral binding sites. In in vitro DHP binding studies, Greenberg and Cooper (1984) and Harris et al. (1985) observed that ethanol displaced [3H]DHP binding in brain. However, Harris et al. (1985) reported that I A4 ethanol was required to displace this binding by 30-40%. Greenberg and Cooper (1984) observed that ethanol inhibited specific [3H]nitrendipine binding with a K, value of 460 mM by decreasing binding affinity without altering the maximal number of binding sites (calcium channels), suggesting a competitive type of inhibition by ethanol. Furthermore, ethanol at a lower concentration did not modify the inhibitory actions of verapamil or diltiazepam on [3H]nitrendipine binding. These observations suggest that ethanol may not directly interfere with the operation of the calcium channel but may exert actions on the surrounding microenvironment of calcium channels which causes a change in the binding affinity of calcium-channel antagonists.

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We also observed that ethanol inhibited [SH]nitrendipinebinding to brain cortical synaptosomal membranes in vitro (P. H. Wu et al., unpublished data). The I C ~ O values for inhibition of [3H]nitrendipine binding by methanol, ethanol, propanol, butanol, and n-amylalcohol were 5400, 1075, 450, 170, and 75 mM, respectively. There is a linear relationship between these IC50 values and the carbon chain length, suggesting that the inhibitory effects on DHP binding may be related to the ability of alkanols to perturb brain synaptosomal membranes. Leslie (1987) concluded that, since very high ethanol concentrations are required to inhibit DHP binding, it is unlikely that ethanol exerts its pharmacological action by specifically and directly inhibiting voltage-dependent calcium entry into presynaptic nerve terminals.

3 . Ethanol- Chronic E’ects The effects of chronic ethanol administration on DHP binding have been shown by Lucchi et al. (1985). Ethanol was administered to rats through the drinking water. The very high-affinity and Ca-independent DHP binding (Kd = 0.05 “M) was slightly increased, whereas the Cadependent DHP binding was reduced to one-fifth of the controls in brain synaptosomal membrane preparations. Consistent with this binding study, Lucchi et al. (1985) also found that striatal slices from chronically ethanolfed rats showed greatly reduced K-stimulated Ca uptake. These results suggest that the effects of ethanol on calcium entry regulation may be a mechanism for the development of ethanol-induced tolerance, dependence, or even neurotoxicity. Wu et al. (1987) administered ethanol (10 g/kg/day) to groups of Wistar rats by intubation. Animals were sacrificed during the treatment period ranging from 1.5 to 9.5 days. The DHP-binding sites were labeled by [3H]nitrendipine. The results indicated that chronic ethanol treatment for 5.5 days resulted in an increase (approximately 40%) in the estimated B,, and Kd of the DHP-binding sites. It is interesting to note that the maximal increase in DHP binding occurred after 3.5 days of ethanol treatment. Wu et al. (1987a) also noted that the increase in DHP binding seemed to correlate with the time course for the development of behavioral tolerance to ethanol, suggesting that calcium channels may participate in the mechanism of ethanol tolerance. If “plasticity” of calcium channels is involved in the development of tolerance to ethanol, blockage of voltage-dependent calcium channels should be able to modify the development of ethanol tolerance. Wu et al. (1987b) showed that chronic nifedipine treatment of rats delayed the development of behavioral tolerance to ethanol in a moving-belt impairment test. The experiments showed that the time course for the development of tolerance to chronic ethanol

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administration (3 g/kg/day) was 21 days and 35 days, respectively, for the sham-treated and nifedipine-treated animals. In a recent report, Little et al. (1986) showed that calcium-channel blockers were able to abolish, prevent, or reduce seizures induced by audiogenic stimuli following ethanol withdrawal.

4. Barbiturates Pentobarbital anesthetic potency was significantly increased by verapamil (10 mg/kg), flunarizine (40 mg/kg), and nitrendipine (100 mg/kg) ( D o h and Little, 1986). Harris et al. (1985) showed that [3H]nitrendipine binding to rat cortical membranes was reduced by phenobarbital and pentobarbital at supraanesthetic ICSO values of 0.40 and 0.76 mM, respectively. These drugs reduced the Kd with little effect on the Bms.However, Harris et ~ l(1985) . found that the DHP-binding sites in their preparation did not seem to involve the voltage-dependent calcium channels since DHPs were not able to inhibit Ca influx. There are little data regarding the chronic effects of barbiturates.

5. Benzodiazepines Draski et al. (1985) showed that nimodopine (5 mg/kg) significantly potentiated the hypothermia and diminished motor activity induced by 2.5 or 5.O mg/kg diazepam, suggestingthat nimodipine may interact with diazepam receptor sites. On the other hand, nifedipine blocked the hypnotic effect of flurazepam. Also nifedipine (1 CUM) and nitrendipine (1 CUM) antagonized the effects of diazepam to increase Ca uptake (Mendelson et ul., 1984). These data suggest that calcium channels may be the sites where diazepam exerts its pharmacological action. This notion was further supported by Taft and Delorenzo (1984), who showed that micromolar concentrations of diazepam inhibited Ca uptake through voltagesensitive Ca channels and demonstrated that benzodiazepines function as Cachannel antagonists using [3H]nitrendipine as a Ca receptor probe. There is evidence suggesting a dissociation of calcium channels from peripheral-type benzodiazepines receptor sites (Bolger et al., 1986; Doble et al., 1985). However, the fact that calcium-channel blockers are able to block the behavioral effects of diazepam as well as the Ca-uptake mechanism regulated by benzodiazepines suggests a complex relationship between these two sites. We are not aware of data concerning the effects of chronic benzodiazepine treatment on DHP binding sites or benzodiazepine receptors.

C . EFFECTS OF SEDATIVE-HYPNOTIC DRUGS ON INTRACELLULAR REGULATION OF CALCIUM ION CONCENTRATION

Intracellular calcium ions are subjected to many biochemical processes which serve to regulate their intracellular concentrations (Rasmussen and

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Barrett, 1984). These biochemical processes include calcium binding proteins, uptake by subcellularorganelles, extrusion of cytosolic Ca by an ATP-dependent Ca pump (Ca-ATPase), and the Na-Ca membrane exchange mechanism.

1. A TP-Dependent Calcium Pump The ATP-dependent Ca uptake into the smooth endoplasmic reticulum has been localized on the inner surface of synaptic plasma membrane fragments (McGraw et al., 1980) and has similar functions to the Ca pump of red cell membranes. Following acute administration of ethanol (4 glkg, i.p.) to mice, Garrett and Ross (1983) showed that, with the loss of the righting reflex [blood alcohol level (BAL) = 600 mg/%], Ca-ATPase and ATP-dependent C a uptake were inhibited. However, at the time of recovery of the righting reflex, the ATP-dependent Ca uptake remained inhibited while Ca-ATPase had returned to control levels, suggesting that ethanol may act to uncouple the enzyme and uptake process. T o elucidate the mechanism of the effects of ethanol on Ca-ATPase, Garrett and Ross (1983) showed that ethanol disrupted intracellular membranes such as smooth endoplasmic reticular membrane, resulting in fewer calcium ions being bound and hence increased free [Cali. Chronic ethanol administration results in the inhibition of ATPdependent Ca uptake in synaptic membranes (Ross, 1986). Membranes from ethanol-treated mice exhibited reduced capacity to take up Ca, and the addition of calmodulin to this preparation stimulated the chronic ethanolinhibited ATP-dependent Ca-uptake system. This suggests that chronic ethanol may cause a decrease in the calmodulin-regulated ATP-dependent Ca-uptake, thereby resulting in the diminution of the cytosolic buffering of intracellular Ca, causing a rise in cytosolic [Ca], in central neurons (Ross, 1986). Rudeen and Guerri (1985) observed that prenatal (in utero) and postnatal ethanol exposure decreased Ca-dependent ATPase activity in different brain regions. The longer the period of time that ethanol was consumed by the mother and prenatally and by the newborn postnatally, the greater the inhibitory effects of alcohol on this enzyme activity.

2 . Ca-Binding Protein The effects of ethanol treatment on calcium-binding activity in synaptosomal membranes prepared from hippocampus, cortex, and cerebellum were reported by Virmani et al. (1985). Using a chelator fluorescence probe (chlortetracycline) and 45Ca binding methods, Virmani et al. (1985) observed that, in acutely intoxicated and later dependent rats in withdrawal, the synaptosomal membranes from the hippocampus showed a more

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drastic increase in calcium-binding activity than did the synaptosomal membranes from the cortex and the cerebellum. However, the increase in Ca binding and Ca-binding sites seen by Virmani et al. (1985) cannot differentiate whether the binding occurs mainly on outer or inner synaptosomal membranes. Other lines of evidence indicate that ethanol does not inhibit calcium binding to Ca-binding protein in uztro. Ishii and Ohnishi (1985) employed a chelex resin method which is sensitive enough to measure free Ca concentration as low as low9M . They demonstrated that an ethanol concentration as high as 25% in nitro did not influence the Ca binding of troponin. Behavioral studies investigating the ethanol-induced sleeping time in mice correlated Ca prolongation of ethanol-induced sleeping time to activation of tyrosine and tryptophan hydroxylase through a calmodulin and calmodulin-dependent protein kinase mechanism (Sutoo ef a/., 1985). The inconsistent results in this area suggest that the effects of ethanol on intracellular protein C a binding probably do not account for the major effects of ethanol intoxication. We are unaware of data relating sedative-hypnotic drug action to intracellular Ca ion regulation by the smooth endoplasmic reticulum or mitochondria.

3. Nu-Ca Exchange Activating the Na-Ca exchange process reduces intracellular Ca which exchanges for extracellular Na (Michaelis and Michaelis, 1981). This has been shown to be a high-capacity intracellular C a regulating system with a K, of 40 ,uM (Gill et al., 1981). Michaelis et al. (1985) observed that the Na-Ca exchange activity in synaptic plasma membranes was inhibited even by low concentrations of ethanol (less than 25mM), while this concentration of ethanol did not have any effects on the ATPase-dependent Ca pump. Ethanol increases membrane fluidity (Chin and Goldstein, 1981). However, increasing membrane fluidity by inserting cis-vaccenic acid increased Na-Ca exchange activity, increasing the extrusion of intracellular C a (Michaelis et al., 1985). Whether the inhibitory effects of ethanol on the Na-Ca exchange antiporter system which thereby increases [Ca], can be extended to other sedative-hypnotic drugs is not yet known.

D. CALCIUM SECOND MESSENGER SYSTEMS AND DRUG ACTION Ca serves as an intracellular messenger (Williamson et a l . , 1981) for signal transduction when cell-surface receptors are activated. There are also other second messenger systems (Nairn et al., 1985; Worley et al., 1987) including cyclic AMP and cyclic GMP. It has been estimated that the cytosolic free C a is

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maintained in the range of 0.1-0.2 f l o r less (Rasmussen and Barrett, 1984). However, when neurons are stimulated by a-adrenergic or cholinergic agonists, the intracellular Ca concentration can rise up to 0.8 pA4 by mobilizing intracellular calcium stores (Reinhart et al., 1983; Berridge, 1984). The second messenger system involving Ca mobilization requires the breakdown of cellular membrane phosphatidylinositol by a phosphoinositide-specific phospholipase C in the plasma membrane, resulting in the intracellular release of inositol 1,4,5-triphosphate (Ins-l,4,5-P3) and diacylgylcerol (Nishizuka, 1984). Ins-1,4,5-P~causes the release of calcium from the endoplasmic reticulum (Streb et al., 1983; Gandhi and Ross, 1987). Diacylglycerol, in the presence of normal cytosolic levels of calcium, can increase the affinity of protein kinase C to calcium and can activate the enzyme to phosphorylate a variety of proteins and ion channels (see Section II1,C). Thus, receptor-mediated activation of phospholipase C leads to a complex network of intracellular signals resulting in increased phosphorylation of various soluble and membrane-bound proteins, thereby changing the state of neuronal function. Modification of the Ca second messenger system can therefore produce profound effects on neuronal activity. The precursor for the formation of inositol phosphates is the phosphatidylinositides in the cell membrane. The membrane phosphatidylinositides are in turn synthesized from myoinositol, which is formed de nouo from glucose-6-phosphate undergoing cyclization and dephosphorylation (Eisenberg, 1967), and can be obtained from the blood circulation by an active uptake system (Caspary and Crane, 1970; Hauser, 1969). Drugs that have effects on the synthesis or active uptake of myoinositol could therefore affect inositol phosphates and the Ca second messenger system. Allison and Cicero (1980) showed that acute ethanol administration (3 gkg) significantly depressed myoinositol-l-phosphate levels in the cortex by 60% within 30 min. In fact, a significant depression of myoinositol-l-phosphate was found 5 min after injection. The decreased myoinositol-1 -phosphate levels returned to normal 24 hr after the initial injection. The decreased myoinositol-lphosphate levels were correlated with blood alcohol levels and the time course of the change was correlated with the behavioral changes. It was suggested that myoinositol-1 -phosphate might participate in the acute pharmacological effects of ethanol. Allison et al. (1976) observed that lithium markedly increases myoinositoll-phosphate levels in brain cortex. Lithium has been shown to attenuate alcohol self-administration in both humans and animals, and to reverse some of the acute pharmacological effects of ethanol (Kline et al., 1974; Judd et al., 1977), suggesting that increased myoinositol-1 -phosphate can antagonize ethanol effects.

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i t has been shown (John et al., 1985) that synaptosomal phospholipase A2 and the phospholipid base-exchange enzymes are highly dependent on extracellular C a concentration and that these enzymes were significantly inhibited by the presence of 50 mM ethanol in vitro. When ethanol was administered to rats chronically, the activities of phospholiphase A2 and phospholipid base-exchange enzymes were increased according to the time course of treatment. The increase in the enzyme activities persisted through the period when the animals were undergoing a physical withdrawal from ethanol. The synaptic membranes obtained from chronically ethanol-treated animals showed less sensitivity to ethanol effects in uitro, suggesting that ethanol tolerance and dependence are associated with changes in membrane phospholipid metabolism and enzyme activities. Hudspith et al. (1985) showed that phospholipase C activity was inhibited in the presence of 50 m M ethanol in uitro but was slightly increased in the brains of animals treated chronically with ethanol. Also, the phosphatidylinositol turnover as the result of membrane depolarization was increased in the brains of the chronically ethanol-treated animals. These results suggest compensatory alterations in the activity of Ca-activated enzymes of phospholipid metabolism in brain tissue during the continued presence of ethanol in uiuo. Since polyphosphoinositides may occur in two forms in rat brain-metabolically inert and metabolically labile (Eichberg et al., 1971; Hauser et al., 1971; Urna and Ramakrishnan, 1983)-the increase or decrease in the metabolically active forms could therefore account for the observations of Hudspith et af. (1985). However, Shah et al. (1984) found that when female rats were allowed to consume ethanol during gestation and lactation, the turnover of the metabolically labile pools of phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate were impaired in the ethanol-exposed pups compared to the nonexposed pups. This result suggests that either the metabolically labile polyphosphoinositides easily accessible to hydrolyzing enzymes were absent in the brain tissue of ethanol-fed pups or the responsible degradative enzymes had become inactivated as the result of the chronic ethanol intake. If chronic ethanol administration indeed decreases the metabolically active polyphosphoinositides, the depolarization of brain synaptosomal membranes should not bring about an increase in the turnover of phosphatidylinositols, unlike what was shown by Hudspith et al. (1985). This apparent disagreement was resolved recently by a study reported by Hoek et al. (1987), who demonstrated the short-term effects of ethanol on C a homeostasis in isolated hepatocytes. Ethanol caused a rapid transient activation of the Ca-dependent phosphorylase a, not associated with changes in CAMPlevels. The activation peaked after 20-30 sec and declined slowly over a period of 5-10 min. Maximal activation was found with 200 mM ethanol,

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but a significant effect was observed with 25 m M ethanol. The effects of ethanol on Ca mobilization were investigated in the hepatocytes loaded with the intracellular calcium indicator, quin-2. The addition of ethanol caused a transient increase in cytosolic free Ca, with the same time course as noted for the effects of ethanol on the activation of the phosphorylase a activity. Once the hormone-sensitive Ca pools in the cell were depleted, the effects of ethanol on Ca mobilization were diminished, suggesting that the Ca mobilization affected by ethanol was activated by a second messenger system. To further elucidate the mechanism, Hoek et al. (1987) labeled hepatocytes with 32P,and the metabolism of inositol phosphates was investigated. Addition of ethanol to 32P-labeledhepatocytes caused a 5-7 % decrease in the level of [32P]phosphatidylinositol 4,5-bisphosphate and a 10-15 % increase in [32P]phosphatidylinositol-4-phosphateand [32P]phosphatidicacid. In the my~-[~H]-inositol-labeled hepatocytes, ethanol induced a 50-100% increase in the levels of inositol 1,4,5-triphosphate, inositol 1,3,4-triphosphate, and inositol biphosphate. More importantly, the changes in inositol 1,4,5-triphosphate levels due to ethanol (200 mM) paralleled the time course of the elevation of cytosolic free calcium levels and the activation of phosphorylase a. These results indicate that ethanol (acutely in vitro) increased the membrane phosphatidylinositide turnover via activation of hormone-sensitive phosphoinositide-specificphospholipase C activity. T h e activation of phospholipase C enhanced Ca mobilization through the inositol second messenger system. Whether ethanol affects CNS second messenger systems in a similai- manner is a matter for further research. V. Conclusions

The relationship between calcium and sedative-hypnotic drug action is an emerging and fruitful area of research. We feel that the most productive approach is to combine, where possible, cellular electrophysiological studies with biochemical investigations. Electrophysiology, in part, suffers from being too specific (i.e., sampling of one neuronal type at a time), whereas biochemistry can be too nonspecific, since usually brain fractions involving many different types of neurons and other tissues are sampled. In the future, electrophysiological data should be obtained from several brain regions and more effort should be spent on examining the electrophysiological correlates of tolerance, dependence, drug-induced brain damage, and aging. Rather than examining C a spikes, more specific analyses of drug effects on whole-cell and membrane-patch voltage-clamped Ca currents should be undertaken. Future biochemical research could include more specific regional brain or the purer

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cell culture tissue measurements, better measures of acute drug actions, and further investigations into drug interactions with the [Ca], regulating mechanisms, including cytoplasmic organelles, and with the Ca second messenger system. More attention must be paid to lower and pharmacologically relevant drug concentrations, since many studies report drug effects using supra-anesthetic o r neurotoxicological concentrations. Technological advances will permit clearer answers to more precise questions. These include in vivo positron emission tomographic scanning, in vivo magnetic resonance imaging with spectroscopy, ion-selective microelectrodes, enzyme-specific microelectrodes, Ca-specific indicator dyes with neural imaging, and whole-cell and patch-clamp recording techniques. For example, patch-clamp recordings will permit direct measures of drug interaction with single ionic channel events. With regard to the data reviewed herein, all three drug types seem to block inward C a currents in neurons and depolarization-induced synaptosomal Ca influx, although micromolar concentrations of a benzodiazepine were required (Skerritt ef al., 1984; Taft and DeLorenzo, 1984). On the other hand, our laboratory showed enhanced Ca action potentials by nanomolar concentrations of midazolam, which may be pharmacologically more relevant. However, it must be mentioned that in guinea pig myenteric neurons, Cherubini and North (1985) showed decreased C a action potentials with picomolar concentrations. Our working hypothesis for acute neuronal sedative-hypnotic drug action is that ethanol and barbiturates, in pharmacological doses, increase intracellular Ca, which secondarily tends to inactivate inward Ca currents (Eckert and Chad, 1984), which would explain their inhibitory effects on depolarization-induced synaptosomal C a influx. Based on the hippocampal neuronal data wherein ethanol- and pentobarbital-induced hyperpolarizations were present even in zero C a perfusate and based on the synaptosomal Ca influx data, the source of the drug induced raised [Ca], was deduced to be intracellular. Published work to date on ethanol (none found for barbiturates or benzodiazepines) suggests that the Na-Ca pump or the CaATPase-dependent pump are inhibited, thereby raising [Ca],. The role of other [Cali homeostatic mechanisms remains to be explored for all three drug types. The data dealing with Ca-channel blockers and drug actions are incomplete and sometimes controversial. However, the study of Mendelson et al. (1984), showing that Ca-channel blockers antagonized the hypnotic effects of flurazepam and diminished the diazepam-induced increased synaptosomal Ca uptake, supports our hypothesis that phamacologicaal doses of benzodiazepines act by increasing [Ca], by increasing inward C a currents (Carlen et al., 1983b).

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This review has stressed the concept that acute sedative-hypnotic drug action is related to changes in intracellular Ca concentration. A transient rise in [Cali not only triggers ionic conductance changes (in this case increased gK which hyperpolarizes the neuron), but also interacts with the Ca second messenger system, thereby possibly activating several longer-term neuronal processes, depending on the cell type being examined. We hypothesize that the longer-term effects of sedative-hypnotic drugs such as tolerance and dependence phenomena will significantly involve Ca-second messenger interactions. Acknowledgments This work was supported by the MRC, OMH, and ABMRF. Secretarial assistance was provided by Yvonne Bedford.

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