Jooniul o/ ~ i ~ ~ i r o i ~ / r c ~ ~ n i s r r ~ ~ Raven Press, Ltd., New York 0 1989 International Society for Neurochemistry

Effect of Acute Ethanol on Release of Endogenous Adenosine from Rat Cerebellar Synaptosomes

Mike Clark and M. Saeed Dar

Department of Pharmacology, School of Medicine, East Carolina University, Greenville, North Carolina, U.3.A

Abstract: The effects of pharmacologically relevant concen- trations of ethanol on the release of endogenous adenosine from rat cerebellar synaptosomes were investigated. Release was conducted for 5 , 10, 30, or 60 s after which time the incubation medium (containing the released adenosine) was rapidly separated from the synaptosomal membranes by vac- uum filtration. The adenosine content of the filtrate was measured by HPLC-fluorescence detection. Both basal and KCI-stimulated adenosine release consisted of an initial rapid phase, for the first 10 s, that was followed by a relatively slower phase. Basal endogenous adenosine release was esti- mated as 199 -+ 14 pmol/mg protein/5 s. Potassium (chloride)

increased adenosine release from the basal level to 433 2 83 pmol/mg protein/5 s. Ethanol caused a dose-dependent in- crease of adenosine release. The interaction between dilazep and ethanol indicates that ethanol-stimulated release does not involve the dilazep-sensitive transport system. The results support previous findings that indicate that cerebellar aden- osine is involved in the mediation of ethanol-induced motor disturbances in the rat. Key Words: Adenosine-Cerebel- lum-Release-Ethanol-Synaptosomes-Rat. Clark M. and Dar M. S. Effect of acute ethanol on release of endoge- nous adenosine from rat cerebellar synaptosomes. J. Neu- rochem. 52, 1859-1865 (1989).

Adenosine displays several membrane-bound re- ceptor-mediated physiological responses such as suppression of neurotransmitter release (Hollins and Stone, 1980a; Corradetti et al., 1984), inhibition of spontaneous neuronal activity (Kostopoulos et al., 1975; Phillis and Wu, 1981), and modulation of cyclic AMP levels (Fredholm et al., 1983). There is substantial evidence that adenine compounds are released in the CNS as a result of both electrical (Daval and Barberis, 1981; Lee et al., 1982; Jonzon and Fredholm, 1985) and chemical (Bender et al., 198 1; Daval and Barberis, 198 1 ; Barberis et al., 1983) stimulation. Adenosine comprises the bulk of the released adenine compounds (Bender et al., 1981; Daval and Barberis, 1981). The adenosine receptors and uptake sites are important systems to investigate for the development of poten- tially useful psychotherapeutic agents (Marangos and Boulenger, 1985).

Experiments in our laboratory (Dar et al., 1983; Dar and Wooles, 1986; Dar, 1988) and those of others (Proctor and Dunwiddie, 1984; Fredholm et al., 1985; Gordon et al., 1986) have provided evidence for aden- osine involvement in the CNS effects of ethanol. We recently observed a functional correlation between

acute ethanol-induced motor disturbances and in- creased density of adenosine A, binding sites in the cerebellar cortex of the rat (Clark and Dar, 1988~). Dar et al. ( 1983) observed a functional correlation be- tween chronic ethanol-induced tolerance to ethanol and an adenosine agonist, and an antagonist and a decrease in adenosine binding in whole brain in mice during ethanol withdrawal. The adenosine uptake mechanism serves a critical role in the termination of adenosine action in the CNS (Phillis et al., 1979). The release of adenosine into the synaptic cleft obviously will be a critical determinant of the magnitude and duration of the purine’s action. Therefore, to investigate further the modulatory role of adenosine in ethanol- induced motor dysfunctions, the present study was camed out to determine whether pharmacologically relevant concentrations of ethanol alter the release of endogenous adenosine from rat cerebellar synapto- somes.

MATERIALS AND METHODS Preparation of synaptosomes

Synaptosomes were prepared according to the method of Gray and Whittaker (1962) as modified by White (1975).

Received June 24, 1988; revised manuscript received November 9, 1988; accepted November 2 I , 1988.

Address correspondence and reprint requests to Dr. M. S. Dar at Department of Pharmacology, School of Medicine, East Carolina University, Greenville, NC 27858, U.S.A.

The present address of Dr. M. Clark is Biological Psychiatry Branch, National Institute ofMental Health, Bldg 10, Rm 3N212, Bethesda, MD 20892, U.S.A.

Abbreviation used: ANOVA, analysis of variance.

1859

1860 M. CLARK AND M. S. DAR

Groups of four male Sprague-Dawley rats (150.-200 g; Charles River, Raleigh, NC, U.S.A.) were decapitated. The cerebellae were pooled and homogenized in 10 ml of0.32 M sucrose. All sucrose solutions were buffered to pH 7.5 with 5 mM Tris-HCI and maintained at 4°C. The homlogenate was centrifuged at 1,000gfor 5 min. All centrifugations were at 4°C. The pellet was resuspended and recentrifuged. The supernatants were combined and centrifuged at 12,000 g for 20 min. The resulting pellet was resuspended in 12.2 ml of 0.32 M sucrose and layered over a discontinuous :sucrose density gradient (0.8 M and 1.2 M ) and subjected to cen- trifugation at 150,000 g for 60 min. The synaptosomal frac- tion (0.8 M/1.2 M interface) was collected, diluted to 40 ml with 0.32 Msucrose, and centrifuged at 20,000 g for 20 min. The resulting synaptosomal pellet was resuspended in I . 1 ml of incubation solution (pH 7.5-7.6), which contained in final concentration: 120 mM NaCI, 4.75 mM KCI, 1.18 mM MgS04, 26 m M NaHC03, 1.2 mM KH2P04, 1.7'7 m M CaCl2, 5.5 mM glucose, and 58.5 mM sucrose (no oxygen- ation). The synaptosomal suspension was kept on ice throughout the experiment except when the suspension was quickly mixed (2-3 s). An aliquot of this suspension was taken for protein analysis (Lowry et al., 195 I) .

Release of endogenous adenosine by cerebellar s ynaptosomes

The method used to study the release of adenosine by cer- ebellar synaptosomes was different from the traditionail pre- loading technique in which synaptosomes are incubated with [3H]adenosine or [3H]adenine, washed, and then incubated for release of [3H]adenosine. Therefore, synaptosomes were not preincubated with [3H]adenosine. We selected the HPLC- fluorescence detection technique for the study of endogenous adenosine release because of the apparent advantages of not using labeled compounds, not dealing with the extent of me- tabolism of the labeled drug, and the relative ease of the pro- cedure.

Release of endogenous adenosine was initiated by adding 40 pl (200-280 p g of protein) of synaptosomal preparation (maintained at 0°C) to tubes containing 960 pl of incubation solution maintained at either 37°C or 0°C. The release was allowed to continue for 5, 10, 30, or 60 s at either 37'8C (to obtain total values) or 0°C (to obtain blank values). After the incubation, the incubation solution (which now contained the released adenosine) was rapidly separated from the syn- aptosomes by vacuum filtration through Whatman GF/C glass microfiber filters. For the various release experiments conducted, the incubation medium in some tubes contained (in final concentration) 50 mMKC1,0.5 mMEGTA (Si,gma, St. Louis, MO, U.S.A.), 50 ptMdilazep (courtesy of Dr. S. J. Mustafa, who received it as a gift from Degussa Pharma Gruppe, Frankfurt, F.R.G.), and/or 25, 50, 75, or 100 mM ethanol. Therefore, the synaptosomes were exposed to ethanol, KCI, EGTA, and/or dilazep only for the duration of the incubation for release (i.e., 5, 10, 30, or 60 s).

Measurement of released adenosine Adenosine content of the filtrate (i.e., released adenosine)

was measured by HPLC-fluorescence detection by a slight modification of the method previously described by Clark and Dar (1988b). In brief, 100-p1 aliquots of the filtrate were placed into 300-pl plastic conical vials. Two microlitei*s of chloroacetaldehyde (Fluka, Buchs, Switzerland) were added to produce a final concentration of 135 mM. The vials were capped, well mixed, and submerged in boiling water for 30

min to complete the conversion of adenosine to the fluores- cent compound 1 ,N6-ethenoadenosine. Twenty microliters of the derivatized adenosine were injected into the HPLC system. The HPLC system and conditions were identical to those used previously (Clark and Dar, 19886). In brief, a 5- pm reverse-phase column (CIS pBondapak, 4 mm X 15 cm, Waters Associates, Milford, MA, U.S.A.) and an automated injector (WISP/7 IOB, Waters Associates) were used. The col- umn was connected to a fluorescence detector (McPherson Model FL-749, Acton, MA, U.S.A.) with High Sensitivity Attachment, excitation monochromator set at 270 nm (un- corrected), a secondary filter with a cut-off below 360 nm, and gain set at 950 V. The mobile phase consisted of 0.05 M sodium acetate buffer (pH 5.0), to which sodium octyl- sulfonate (1 mM, Pfaltz and Bauer, Waterbury, CT, U.S.A.) and 3% acetonitrile were added. Isocratic separation of aden- osine was accomplished at ambient temperature (22-24°C) and at a flow rate of 1.5 ml/min.

Statistics The time course of adenosine release was statistically tested

by a two-way analysis of variance (ANOVA) with Newman- Keuls post hoc analysis for a significant interaction between incubation time and treatment (presence or absence of KCI). This was followed by one-way ANOVA at appropriate in- cubation times to test for statistically significant increases in basal release of adenosine by KCl. The rest of the adenosine release data were analyzed with one-way ANOVA and New- man-Keuls post hoc comparisons. Data were considered sta- tistically significant at p < 0.05.

RESULTS

Release was measured at either 37°C (for total val- ues) or 0°C (for blank values). Net release was calcu- lated as the difference between total and ice-cold blank values. Figure 1 shows the relationship between the concentration of synaptosomal membrane protein and the amount of endogenous adenosine released during a 30-s incubation period. The protein concentration ranged from 70 to 484 pg in a single assay, and a linear relationship between protein concentration and the amount of adenosine released (total, blank, and net) was observed at all protein concentrations tested (Fig. 1). Based on this information a synaptosomal protein concentration range of 200-280 pg was used routinely in all the release experiments. Figure 2 shows the time course of endogenous adenosine release. There was a relatively large variability between experiments for the total and ice-cold blank values generated (Fig. 2). However, the variability within a single experiment was small. On the other hand, as noted by the small SEM in the net release curve (Fig. 2), the actual amount of adenosine released remained quite constant between experiments. Figure 3 clearly shows that adenosine re- lease was composed of an initial rapid phase that was followed by a slower phase. The initial rate of release was quite rapid and peaked within 10 s. The rate of adenosine release was nearly constant after 10 s.

KCl markedly stimulated adenosine release during the initial rapid phase (Fig. 3). Basal release in the pres- ence of KCl was increased 120, 31, 28, and 17% at 5 ,

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ETHANOL AND RAT CEREBELLAR ADENOSINE RELEASE 1861

1200 1400 T

" (il 1 - in 1000

200 + O L- 0 . 0

A Total

Ice-cold Blank

Net

FIG. 1. Relationship between release of endogenous adenosine and amount of synaptosomal membrane protein. The results are from a single assay in which each measurement (point) was done in triplicate. Total and ice-cold blank values were obtained at 37°C and O'C, respectively. Net release was calculated as the difference between values from total and ice-cold blanks.

I 0 . 1 0 . 2 0 . 3 0 . 4 0 . 5

P r o t e i n (mgl

10, 30, and 60 s, respectively. Because release of aden- osine in the presence or absence of KCI was most rapid during the early time period, all subsequent release ex- periments were conducted for a 5-s incubation period.

Figure 4 shows the dose-response relationships among various pharmacologically relevant concentra- tions (25-100 mM) of ethanol and the amount of basal release of endogenous adenosine. Basal release of adenosine was estimated as 199 t- 14 pmol/mg protein/ 5 s. Release was significantly increased, 50, 7 1,68, and 103% by 25,50,75, and 100 mMethano1, respectively. Figure 4 indicates a dose-dependent stimulation of adenosine release by increasing concentrations of ethanol.

Figure 5 shows the effects of KCl and EGTA on the release of endogenous adenosine in the presence or ab- sence of 25 mM ethanol. Release was significantly in- creased by ethanol (25 M), KCl(50 mM), and EGTA

(0.5 mM): 50, 118, and 214%, respectively. The stim- ulatory effect of KCl was neither additive nor poten- tiated by ethanol-induced stimulation of adenosine re- lease (Fig. 5). On the other hand, EGTA-stimulated release was significantly (35%) inhibited by 25 mM ethanol. Basal release of endogenous adenosine was inhibited 56% by 50 pM dilazep (Fig. 6). However, release in the presence of both 50 pM dilazep and 25 mM ethanol was not different from control (basal re- lease) (Fig. 6).

DISCUSSION

Based on studies conducted with brain tissues, adenosine release in the CNS is apparently of physio- logic importance. Release of [3H]adenosine from pre- loaded synaptosomes prepared from whole brains of mice (Gonzales and Leslie, 1985) and from the cerebral

FIG. 2. Time course of endogenous adenosine re- lease from rat cerebellar synaptosomes at either

obtained for the two incubation temDeratures. The c ena

37°C (total) or 0°C (ice-cold blank). Net release was calculated as the difference between release values

I

a,

results are the means & SEM of four separate ex- periments done in triplicate.

u 400

f 0 I

0 10 20 30 40 50 60 70

T i m e (secl

J. Neurochem., Vol. 52, No. 6. 1989

1862 M. CLARK AND M. S. DAR

400

300

2 0 0

100

' O o 0 T I

--

--

--

--

FIG. 3. Time course of basal (control) release of en- dogenous adenosine from rat cerebellar synaptosomes and the effects of 50 mM KCI. The results are the means & SEM of four separate experiments done in triplicate. Net release values are shown. *p < 0.05 and **p < 0.01, compared with control. 200 1 I . Control

4 50 W K C I

0 10 20 30 40 5 0 60 70

cortices of rats (Bender et al., 1981) and guinea pigs (Kuroda and McIlwain, 1974) has been demonstrated. According to these investigations, the release of aden- osine appeared to be at least partially Ca2+ dependent. Kuroda and McIlwain ( 1974) found that electrncally stimulated increases of [3H]adenosine efflux were di- minished about one-half by tetrodotoxin. These find- ings support the notion that adenosine release IS in- volved in neurotransmission. Data from the present investigation (Fig. 3) clearly showed that adenosine re- lease was composed of an initial rapid phase during the first 10 s. KCI increased basal adenosine release by 120% during the first 5 s and by <35% during the sub- sequent 10, 30, or 60 s of release (Fig. 3), findings that may indicate the importance of the rapid compa'nent of endogenous adenosine release.

The value obtained in the present study for basal release of endogenous adenosine during a 30-s incu- bation period (5 19 k 136 pmol/mg protein/30 s; Fig. 2) was much higher than that obtained by Bender et al. (198 1) for basal release of [3H]adenosine from pre- loaded cerebral cortical synaptosomes (2 pmol/mg protein/30 s). The marked increase (260-fold) in the value of basal adenosine release that we observed com- pared with that reported by Bender et al. (198 1) ob- viously was due to the different experimental methods used in these two studies. According to the procedure of Bender et al. (198 l), the synaptosomes were pre- loaded with [3H]adenosine by incubation at 37°C for 30 s, then washed and used in the purine release ex- periments. We incubated the synaptosomes for 5-60 s soon after they were prepared. Prior to the incubation,

5 0 0 T "I

161

151

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t" - u a, m 500

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X X LD \ ( u c

e 111 400

C L a 300

\

0 200 a

100

I.

' I 0 25 50 75 100

C o n t r o l E t h a n o l (mM1

FIG. 5. Effects of 50 mM KCI and 0.5 mM EGTA on basal release of endogenous adenosine from rat cerebellar synaptosomes in the presence (hatched bars) or absence (open bars) of 25 mM ethanol. Each bar represents the mean -t SEM. Numbers in parentheses represent the number of separate experiments (each done in trip- licate). "p < 0.01 compared with basal release. ' p < 0.05 compared with EGTA in the absence of ethanol.

FIG. 4. Effects of various concentrations of ethanol on basal re- lease of endogenous adenosine from rat cerebellar synaptossmes. The number of separate experiments (each done in triplicate) per- formed at each concentration is shown in parentheses. Ethanol significantly ('p < 0.05; " p < 0.01) enhanced adenosine release at each concentration.

J Neuiochem, b'ol 32. No 6, 1489

ETHANOL AND RAT CEREBELLAR ADENOSINE RELEASE 1863

0 c o n t r o l "'[ D i l a z e p (50 uM1

i z 1 5 0 1

D i l a z e p (50 uM1 + Ethanol (25 mM1

2 c g."

FIG. 6. Effects of dilazep on basal and ethanol-stimulated release of endogenous adenosine from rat cerebellar synaptosomes. Each bar represents the mean f SEM of four separate experiments done in triplicate. **p < 0.01 compared with control and with ethanol groups.

the synaptosomes were always kept at 0-4°C in an ice bucket, as stated in Materials and Methods. During this incubation (e.g., 30 s; Figs. 2 and 3), release of significant amounts of endogenous adenosine took place. Similar significant release of endogenous aden- osine would have occurred during [3H]adenosine pre- loading by Bender et al. (198 1) when synaptosomes were incubated under conditions very similar to ours. The procedure used by Bender et al. (198 1) obviously was not designed to detect this release of endogenous adenosine because the synaptosomes were washed, re- moving the endogenously released adenosine, at the end of the [ 3H]adenosine preloading incubation.

A less significant factor also contributing to the smaller release value in the Bender et al. study (1 98 1) might have been isotopic dilution of [3H]adenosine that was caused by marked release of endogenous adenosine during the preloading incubation. The consequent re- duction of the specific activity of [3H]adenosine would result in an artifactually low adenosine release rate in the case of the preloading method. Moreover, we have observed that adenosine release is composed of both rapid and slow phases and that the rate of release be- comes slower after 30-60 s of incubation, so preloading the synaptosomes in a longer incubation period (i.e., resulting in a slower release rate) may also partially account for the differential values of basal adenosine release obtained with the two experimental techniques. Therefore, a substantial amount of adenosine release must already have occurred (during preloading) before the actual incubation to study the release of [3H]adenosine as carried out by Bender et al. (1981). In addition, the cerebellum, a key brain area involved in the coordination of motor activity (Sage, 1984), was used in the present investigation to prepare the syn- aptosomes and it appears to have a relatively higher basal adenosinergic activity than the cerebral cortex, the brain area used by Bender et al. (1 98 1) to prepare

the synaptosomes. Some of the evidence suggesting higher adenosinergic activity in the cerebellum than in other brain areas includes relatively higher basal aden- osine levels (Clark and Dar, 1988b), higher density of adenosine Al binding sites (Goodman and Snyder, 1982), and a significant increase in the maximum number of Al binding sites caused by acute ethanol (Clark and Dar, 1988a), compared with other areas of the brain. However, both methods appear to be effective for measuring drug-induced alterations of adenosine release because similar results were obtained by Bender et al. (1981) and in the present investigation (Fig. 5) for the effects of compounds such as KCl and EGTA.

We observed that 50 mM KCl stimulated release of endogenous adenosine from cerebellar synaptosomes. Likewise, Bender et al. (1 98 1) and Gonzales and Les- lie (1985) observed that 50 mM KCl increased [3H]adenosine efflux from cerebral cortical synapto- somes and a P2 crude synaptosomal preparation, re- spectively. It is noteworthy that Hollins and Stone (1 980b) found that KC1-induced [3H]adenosine efflux from cerebral cortical slices did not begin until the de- polarizing agent was removed. These temporal differ- ences in the effects of KCl may reflect differences in synaptosomal and slice preparations. However, using cerebral cortical slices, Hollins et al. (1980) reported that completely omitting K+ from the incubation me- dium slightly decreased purine efflux but restoring K+ to a normal concentration (6.24 mM) resulted in a large increase of purine efflux.

As the synaptosomes prepared by the procedure used by Bender et al. (198 1) and in the present study are not considered pure, nonneuronal contaminants such as glial cells may provide an additional source of the released endogenous adenosine measured in the present investigation.

It was suggested that adenosine uptake is mediated by a nucleoside carrier in the membrane (Bender et al., 1981). The uptake system reported by Bender et al. ( 198 1) was temperature dependent and was inhib- ited by various adenosine analogues and pyrimidine nucleosides. It is likely that adenosine release and up- take is mediated via the same carrier mechanism. Sup- port for this notion was available from two observations in the present study. First, release of endogenous aden- osine was temperature dependent. Second, 50 pM dil- azep inhibited release of endogenous adenosine by 56%. Dilazep is known to inhibit the uptake of adenosine (Mustafa, 1979; Phillis and Wu, 1982). Indeed, 50 pM dilazep inhibited [3H]adenosine uptake into rat cere- bellar synaptosomes by 83% (Clark and Dar, 1989). Because dilazep inhibits both the release and uptake processes for adenosine in rat cerebellar synaptosomes, the same site of action (the nucleoside carrier) appears most likely to be involved; this suggests that adenosine release and uptake occur via the participation of the same transport molecule. However, as dilazep inhibited the release by 56% whereas the same concentration of dilazep inhibited uptake by 83%, two different mech-

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1864 M. CLARK AND M. S. DAR

anisms of release may be occurring simultaneously (i.e., release involving the dilazep-sensitive transport site and release occurring independently of the dilazep-sensitive carrier mechanism). Jonzon and Fredholm ( 1 985) sug- gested that adenosine release occurs mainly via a car- rier-mediated process rather than by exocytosis. They observed that electrically evoked labeled-purine release from rat hippocampal slices was inhibited by the nu- cleoside uptake inhibitor dipyridamole, where<as the release of noradrenaline and y-aminobutyric acid was enhanced by known inhibitors of their uptake. On the other hand, by using a combined in vivo/in vitro method, Lee et al. (1982) were able to selectively stim- ulate (electrically) pre- and postsynaptic neurons and to measure release of labeled adenosine derivatives. They reported evidence for presynaptic release of pu- rines by an activation-coupled mechanism. Results from the present study suggest the importance lof the nucleoside camer mechanism for adenosine release under basal conditions, as noted by its sensitivity to dilazep treatment. However, another release mecha- nism is apparently important. The apparent absence of dilazep-mediated inhibition of basal adenosiine re- lease in the presence of ethanol (Fig. 6) suggested at least two interesting possibilities. First, it simply may mean that ethanol completely blocked the effect of dil- azep by acting directly at the nucleoside carrier mech- anism, where dilazep also acts. On the other hand, the data (Fig. 6) may suggest that ethanol acts on adenosine release by a mechanism independent of the dilazep- sensitive carrier mechanism. Hence, ethanol diQ not directly interfere with the inhibitory effect of dilazep, and the net value of adenosine release obtained in the presence of both dilazep and ethanol (Fig. 6) actually represents the arithmetic difference between the ethanol-induced increase (Fig. 5 ) and the dilazep-in- duced inhibition (Fig. 6) of endogenous adenosine re- lease. When the inhibitory effect of dilazep (56%, Fig. 6) is compared with the release of adenosine in the presence of both dilazep and 25 mM ethanol (98%, Fig. 6), the full stimulatory action of ethanol is still present (i.e., adenosine release in the presence of 25 mM ethanol is 15 1% of control, Fig. 5; and from Fig. 6, 56% + 98% = 154%).

In agreement with the findings of Bender et al. (1981), who measured release of [3H]adenosine from preloaded cerebral synaptosomes, we found that basal release of endogenous adenosine from cerebellar syn- aptosomes was markedly increased when EGTA was included in the incubation medium that contained Ca2+ (Fig. 5 ) . Also, when Ca2+ was not included in the incubation medium (data not shown), basal adenosine release was increased to the same extent as in incu- bation medium containing Ca2+ and EGTA. The in- creased release of adenosine in the absence of extra- cellular Ca2+ (whether Ca2+ was omitted from thle in- cubation medium or EGTA was added) suggests that extracellular Ca2+ inhibits the release of adenosine:. Be- cause 25 mMethanol paradoxically inhibited the stim- ulatory effects of EGTA on adenosine release (Fig. 5) ,

J Neurochem , Vol 52, N o 6, 1989

an interaction between ethanol and Ca2+ flux was ap- parently involved. Ethanol, in pharmacologically rel- evant concentrations, does alter Ca2+ mobilization (Hoffman and Tabakoff, 1985).

Ethanol (25- 100 mM) significantly increased basal release of adenosine in a dose-dependent manner. This suggests that degrees of ethanol intoxication (which are related to blood ethanol levels) may be functionally correlated with progressive increases in adenosine re- lease and consequently higher activity of the adenosine system with increasing brain ethanol concentrations. We have recently provided evidence that acute ethanol increases the density of adenosine A, binding sites in the cerebellar cortex of the rat and that this increase in binding is functionally correlated with potentiation of ethanol-induced motor dysfunctions by adenosine agonists (Clark and Dar, 1988~). The finding that ethanol substantially increases release of endogenous adenosine from cerebellar synaptosomes gives further support to the working hypothesis that cerebellar adenosine, at least partially, modulates the motor dis- turbances elicited by acute ethanol. It remains to be established whether chronic ethanol causes compen- satory effects to adenosine binding, uptake, and/or re- lease as a means of tolerance to ethanol-induced motor impairment.

Acknowledgment: This work was supported in part by the North Carolina Alcoholism Research Authority Grant 8605.

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