0145-6OO8/92/ 1606-I 1 lOS3.00/0 ALCOHOLISM: CLINICAL A N D EXPERIMENTAL RESEARCH

Vol. 16, No. 6 November/December 1992

Effect of Ethanol Administration and Withdrawal on Serotonin Receptor Subtypes and Receptor-Mediated Phosphoinositide Hydrolysis in Rat Brain Subhash C. Pandey, Mariann R. Piano, Dorie W. Schwertz, John M. Davis, and Ghanshyam N. Pandey

The effect of short-term (15 days) and long-term (60 days) ethanol treatment and withdrawal on agonist-stimulated phosphoinositide (PI) hydrolysis, serotonin receptor subtypes (~HTIA and 5HT2), and a,-adrenergic receptors were studied in rat cerebral cortex. Shortterm ethanol treatment had no significant effect on serotonin (5HT), norepinephrine (NE), and calcium ionophore (A23187)-stimulated [3H]-inositol-l-phosphate ([3H]-IP,) formation and 5-HT2receptors as measured by ‘251-lysergicacid diethylamide (‘%LSD) binding, in rat cerebral cortex. However, 15 days of ethanol treatment, followed by 24 hr of withdrawal resulted in a decrease in. , 6 of ’251-LSDbinding without significant change in K,, as well as a decrease in 5HTstimulated [3H].IP, formation in rat cerebral cortex. SHTIA and a,adrenergic receptors were determined by using [3H]-8-hydroxy-2(di-N-propylamin0)tetralin and [aH]-prarosin as radioligand, respectively. We also observed that long-term ethanol treatment had no significant effect on B,. and KD of 5HT2, ~ H T ~and A , wadrenergic receptors, as well as NE and A23187-stimulated [3H]-IP, formation, but significantly decreased the 5HT-stimulated [3H]-IPl formation in rat cerebral cortex. It is possible that a decrease in 5HT-induced PI turnover after long-term ethanol exposure may be due to a decrease in coupling of 5HT2 receptors to G protein or PLC enzyme, whereas the decrease in 5HT-induced PI turnover after withdrawal may be due to a decrease in functional 5HT2receptor number. Key Words: Serotonin-2(5-HT2) Receptor, Serotonin-1A(5HTlA) Receptor, a,-Adrenergic Receptor, Ethanol, PhosphoinositideTurnover.

EVERAL INVESTIGATORS have shown that ethanol S produces changes in the lipid components of cellular membranes, which in turn interfere with neuronal function.’-’ The biological functions of membrane-bound proteins such as receptors, are dependent on their lipid surrounding~,~,’ and thus both structural and functional properties of neural membranes could be affected by ethanol exposure. A number of neurotransmitters are known to initiate signal transduction by interaction with their receptors and activation of either adenylate cyclase or phosphoinositide (PI) signalling systems. Activation of neuroFrom the Illinois State Psychiatric Institute and College of Medicine, University of Illinois (S.C.P., J.M.D., G.N.P.), and College of Nursing, University of Illinois (M.R.P., D. W.S.),Chicago, Illinois. Received for publication January 31, 1992; accepted May 19, 1992 These studies were supported in part by a grant from the National Institute of Mental Health to Dr. G.N. Pandey (ROI MH36169-08) and from the Alcoholic Beverage Medical Research Foundation to Dr. Dorie Schwertz. Reprint requests: Dr. Ghanshyam N. Pandey, Research Department, Illinois State Psychiatric Institute, 1153 North Lavergne Avenue, Chicago, I L 60651. Copyright 0 1992 by The Research Society on Alcoholism. 1110

transmitter receptors, such as serotonin-2 (5HT2) and aladrenergic receptors, stimulate phosphoinositide (PI) hydrolysis by phospholipase C (PLC) enzyme and produces inositol trisphosphate (IP3) and diacylglycerol (DG), both of which act as intracellular second messengers.8-12 A significant amount of work has been reported regarding the effect of alcohol on the CAMP-adenylate cyclase signaling system.I3-l5 However, very little is known with regard to how alcohol modifies the receptor-mediated hydrolysis of phosphoinositide in brain. Allison and Cicero16first reported that in vivo acute ethanol decreases inositol- 1-phosphate levels in rat brain. Gonzales and Crews” reported that chronic administration of ethanol had no effect on NE-stimulated PI hydrolysis in rat cortical slices. Ritchie et a1.18 reported that chronic ethanol exposure produced a significant increase in NE-stimulated PI hydrolysis in astrocytes. In contrast, chronic ethanol treatment decreased NE-stimulated PI hydrolysis in liver without changing the number of al-adrenergic receptors.” These results thus suggest that phosphoinositide metabolism may be affected by ethanol exposure. Alterations in serotonergic function have been implicated in alcoholism.20-22This is based on the observation that levels of 5HT and its metabolite, 5-hydroxyindole acetic acid, are decreased in brain from alcohol-preferring rats in comparison to alcohol non-preferring rat^.'^.^^ Furthermore, enhancement of 5HT transmission by the systemic administration of inhibitors of 5HT uptake (fluoxetine and fluvoxamine) reverses the intake of ethanol by alcohol-preferring rats and by other rats as ell.^^,^^ Wong et al.27reported increased 5HT1 receptors (3H-5HT binding sites) but no change in 5HT2 receptors as measured by [3H]-ketanserinbinding in brain of alcohol-prefemng rats. Wong et a1.28also reported that B,,, of [3H]-S-OH-2-(diN-propy1amino)tetralin (8-OH-DPAT) binding to ~ H T ~ A receptors are increased in brains of alcohol prefemng rats as compared to alcohol nonpreferring rats. Whereas effects of acute and chronic exposure of alcohol on NE-stimutheir lated PI hydrolysis in rat brain have been effect on al-adrenergic, serotonin receptor subtypes (~HTIA 5HT2) , and 5HT-stimulated PI hydrolysis in rat brain has not yet been investigated. The present investigation examines the effect of short-term (15 days) and long-term (60 days) ethanol treatment on serotonin receptor subtypes (5HTlA,5HT2), and al-adrenergic receptors, Alcohol CIin Exp Res, Vol 16, No 6, 1992: pp I 1 10-1 I16

ETHANOL EFFECTS ON SEROTONIN RECEPTORS AND BRAIN PI TURNOVER

1111

as well as NE, 5HT, and A23 187-stimulatedPI hydrolysis (100 pl) were placed into different tubes containing 50 PM pargyline and for 10 min at 37T, after which NE M), 5HT in rat cerebral cortex. The effect of short-term ethanol Mpreincubated ), or A23 187 (50 PM) was then added in a total incubation volume of exposure followed by 24 hr of withdrawal was also inves- 500 81. The tubes were further incubated for 45 rnin at 37'C in an tigated. incubator chamber saturated with 02/C02. The incubations were stopped MATERIALS AND METHODS Materials Drugs were obtained from Sigma Chemical Co., St. Louis, MO. Dowex AGI-XI (100-200 Mesh) was obtained from Bio-Rad Laboratories (Richmond, CA), ['H]-Myoinositol, '251-lysergic acid diethylamide (LSD), ['HI-prazosin, and ['HIS-OH-DPAT were purchased from New England Nuclear, Boston. Scintillation cocktail (3a70B, 4a20) was obtained from Research Products International Corporation (Mount Prospect, IL). Solvents and other chemicals were analytical grade from Fisher Scientific Co.

by the addition of 1.88 ml of CHC13/MeOH (1:2 V/V), followed by 0.62 ml CHCI3 and 0.62 ml HzO. The samples were mixed for 10 rnin and then centrifuged at 4000 X g for 15 min. A portion of organic phase (500 pl) was transferred into scintillation vials with 15.0 ml of 4a20 cocktail to determine the radioactivity in phospholipids. An aliquot of the upper phase (1.5 ml) was removed and applied to a 2.0-ml Dowex AGI-X8 column (formate form, 100-200 mesh). The columns were washed with 4.0 ml of 5 mM sodium tetraborate solution to elute the free inositol and glycerophosphoinositol.[3H]-IPIwas then eluted from the column with two 8.0-ml aliquots of 0.2 M ammonium formate and 0.1 M formic acid. The radioactivity in the eluates was then counted in a liquid scintillation counter. PI hydrolysis is expressed as percent of total ['H]-inositol incorporated (DPM from ['H]-IPI fraction/DPM from column + DPM in chloroform and methanol layer x 100).

Ethanol Treatment and Brain Collection Male virus-free Sprague-Dawley rats (Saskco King), weighing 200 to 250 g were used in all experiments. After a brief acclimation period, rats were individually housed and offered 100 ml of the Lieber-DeCarli control diet as their sole source of food or fluid. On the following day rats were divided into two groups. One group continued to receive the control liquid diet, while the other group received the ethanol-containing (9% v/v) Lieber-DeCarli liquid diet ad libitum.29These diets were isocaloric. Animals were pair-fed for 15 days (short-term) or 60 days (longterm). A third group of rats was studied after 15 days of ethanol feeding and 24 hr of withdrawal (ethanol-withdrawn groups). We did not determine the effect of ethanol withdrawal after 60 days of ethanol treatment since this may result in high mortality rate and audiogenic seizures. Hunter et al." observed that after 30 days of ethanol treatment (liquid diet) followed by withdrawal, 40% of the animals experienced severe audiogenic seizures, while 10%of animals died. Animals were weighed twice a week. Fresh diet was provided between 6 and 7 PM every night. On days 15 and 60, 20 pl of tail vein blood was obtained at 8:OO AM for analysis of blood ethanol levels by a gas chromatographic technique." At this time, both the 15 days and 60 days ethanol treated animals exhibited signs of intoxication such as sedation and ataxia. The rats were decapitated, brains were removed, and cerebral cortex and hippocampus were dissected out on an ice-chilled plate. The cerebral cortex was frozen at -80°C until used for measurement of ['HI -8-OH-DPAT, 125[I]-LSD,and ['HI-prazosin binding. Fresh cerebral cortex was used immediately to measure NE, 5HT, and A23 187-stimulated ['H]-IPI formation as an index of PI hydrolysis.

Determination of Phosphoinositide Hydrolysis in Rat Cortical Slices PI hydrolysis in rat cortical slices was determined according to the method of Berridge et a1.:' with slight modifications as recently described by Pandey et a]." The cerebral cortex was washed with Krebs-Ringer (KR) buffer (NaCI, 118 mM; KCI, 4.7 mM; CaCL, 0.75 mM; KH2P04, 1.18 mM; MgSO.,, 1.18 mM; NaHCO,, 24.8 mM; glucose, 10 mM, pH = 7.4), which had been bubbled with 02/C02 mixture (955). Washed cerebral cortex was then cross-chopped (350 p ~with ) the McIlwain tissue chopper. The slices were dispersed gently in 15.0 ml KR buffer and incubated at 37°C for 15 min. This washing process was repeated one more time. Cortical slices were allowed to settle under gravity and excess buffer was removed. Washed cortical slices were suspended in 5.0 ml of oxygenated KR buffer, and 40 pci of ['HI-myoinositol was added. The suspension was incubated for 1 hr at 37°C in an incubator chamber saturated with 02/C02mixture (955). In order to remove the unincorporated [3H]-myoinositol, cortical slices were washed five times with oxygenated KR buffer (30 ml). Final washing was done with oxygenated KR buffer containing 10 mM LiCl. The cortical slices were allowed to settle under gravity, and excess buffer was removed. Packed cortical slices

pH]-Prazosin Binding to al-Adrenergic Receptors in Rat Cortex al-Adrenergic receptor number and KO were determined in cortical membranes using ['HI-prazosin binding as described by Stockmeier et al.34 Cerebral cortex samples were homogenized using a Brinkman polytron (setting 7 for 15 sec) in 10 volumes of 50 mM Tris buffer (pH = 7.7), then centrifuged at 49,000 X g for 15 min. The supernatant was discarded and the pellet was resuspended in 10 volumes of buffer and centrifuged as above. The pellet was washed twice by resuspension in fresh 50 mM Tris buffer (pH = 7.7) followed by centrifugation.The final pellet was resuspended in Tris buffer, and 500 ~1 aliquots were added to triplicate tubes containing ['HI-prazosin (0.2-1.5 nM) in the presence and absence of 10 p~ phentolamine in a total incubation volume of 1.O ml. The tubes were incubated for 30 rnin at 25°C. The reaction was terminated by addition of 5.0 ml cold Tris buffer (pH = 7.7) and rapid filtration through Whatman GF/B filters using Brandel cell harvester and immediate rinsing with 10.0 ml of cold Tris buffer (pH = 7.7). The filters were transferred into vials and scintillation cocktail was added. The radioactivity trapped on the filters was measured by liquid scintillation counting. Specific binding was defined as difference in binding observed in the presence and absence of 10 PM phentolamine and ranged from 90% to 60% depending upon the concentration of radioligand.

Determination ofthe pH]-&OH-DPAT Binding to ~ H T IReceptors A in Rat Cortical and Hippocampal Membranes ~ H T , receptors A were determined according to a method of Pandey et al.35Briefly, cortices and hippocampi were homogenized in 10 volumes of 0.32 M sucrose using a polytron (setting 9,30 sec), and then centrifuged at 1000 x g for 10 rnin to remove nuclei and cell debris. The resulting supernatant was centrifuged at 70,000 x g for 15 rnin and the pellet thus obtained was suspended in 10 volumes of Tris buffer (50 mM, pH = 7.5). After incubation at 37°C for 15 min, the suspension was centrifuged again at 70,000 X g for 15 min. The final pellet was suspended in incubation buffer (50 mM Tris, 4 mM CaCL, 0.1% ascorbic acid, pH = 7.7). ['HI-8-OH-DPAT binding was carried out in triplicateby incubating an aliquot (500 pl) of membrane suspension (37"C, 30 min) with ['HI-SOH-DPAT at five to six concentrations (between 0.5 and 10 nM) with or without 10 p~ 5HT in a total volume of 1.0 ml. ['HI-8-OH-DPAT bound to the membrane was separated by filtration through a Whatman (GF/ B) filter and washed four times with 5.0 ml cold buffer (50 mM Tris, pH = 7.5) using a Brandel cell harvester. The filters were placed in vials with scintillation cocktail and counted. Specific binding was defined as the difference between total binding and the binding observed in the presence of 10 p~ 5HT, and ranged from 90% to 70% depending upon the concentration of ['HI-8-OH-DPAT.

PANDEY ET AL.

1112

Effect of Short- Term Ethanol Treatment and Withdrawal 5HT2receptors were studied by 12'I-LSDbinding to cortex membranes on Phosphoinositide Hydrolysis according to the method described by Elliot and Kent,36 with some The effect of 15 days of ethanol treatment and 24 hr of modification. The tissue was homogenized in 10 volumes of hypotonic ethanol withdrawal on basal and agonist-stimulated [3H]medium ( 5 mM Tris and 0.1 % EDTA, pH = 7.5) using a polytron setting IPI formation in rat cortical slices was examined. No at 7 for 20 sec and centrifuged at 49,000 X g for 15 min at 4°C. The pellet obtained was washed twice with 10 volumes of hypotonic buffer significant differences were found among control, ethanol and centrifuged as above. The final pellet was suspended in incubation fed and withdrawn groups in basal, NE, and A23187buffer (50 mM Tris, 120 mM NaCI, 5 mM KCl, 1 mM MgCl2, 0.05% stimulated [3H]-IP,formation (Table 2). We observed that ascorbic acid, pH = 7.4). The receptor binding assay was camed out in 5HT significantly stimulated [3H]-IPI formation in rat triplicate in plastic tubes containing incubation buffer, '251-LSDranging cortical slices of all three groups (Table 2). The increase from 0.25 to 3.0 nM (six different concentrations), 40 pl cortical membrane suspension with or without 1 p~ ketanserin, in a total incubation in 5HT-stimulated [3H]-IPI formation in the short-term volume of 100 p1 and incubated at 37°C for 90 min. The incubation was ethanol group was somewhat less than in the control terminated by rapid filtration over Whatman GF/B filters, washed 3 group, but this difference was not statistically significant. times with 5 ml ice-cold 50 mM Tris buffer (pH = 7.7) containing 0.01 % bovine serum albumin. Filters were dried and then counted in a Gamma However, the increase in [3H]-IPIformation was signifiCounter. Specific binding was defined as the difference between binding cantly ( p < 0.01) attenuated in the ethanol-withdrawn observed in the presence or absence of 1 p~ ketanserin and ranged from group compared to the control group. '251-LSD Binding to SHT2 Receptors in Rat Cortex

80% to 60% depending upon the concentration of ligand. In all binding assays, B,,, and KO were computed by Scatchard analysis using the EBDA p r ~ g r am,and ~ ' protein content was determined by the method of Lowry et aL3'

Effect of Long- Term Ethanol Treatment on Phosphoinositide Hydrolysis The effect of long-term ethanol treatment on maximal NE (100 PM),5HT (100 PM),and A23 187 (50 pM)-stimuStatistics lated PI hydrolysis in rat cerebral cortical slices was invesStatistical analyses were performed using analysis of variance; if tigated. Basal, A23 187, and NE-stimulated [3H]-IPI forsignificance was found, post hoc comparisons were made using multiple mation was not affected by 60 days of ethanol exposure, comparison procedure (control vs. ethanol treated vs. withdrawn). Student's t test was used to compare the differences between two groups as compared to control rats (Table 3). However, 5HT (100 (control vs. ethanol or control vs. withdrawn group). A value of p < 0.05 pM)-stimulated increase in IPI formation was significantly was considered to be significant. attenuated ( p < 0.05) after 60 days of ethanol treatment (Table 3). RESULTS

Effect of Chronic Ethanol Treatment on al-Adrenergic Receptors in Cerebral Cortex All animals gained weight over 15 and 60 days of Changes in the number and affinity of al-adrenergic ethanol treatment. There were no significant differences in body weights between the short-term, long-term receptors in the cortex of 60-day ethanol-fed rats and ethanol-fed and ethanol-withdrawn groups and their re- Table 2. Effect of Short-Term (15 days) Treatment and Withdrawal on Agonistspective liquid control groups (Table 1). Stimulated r3H1-IP, Formation in Rat Cerebral Cortex We measured the blood ethanol levels at 15 and 60 days Short-term Ethanol Control ethanol-fed withdrawn of ethanol consumption, and found that the blood ethanol Agonist group group group level was not significantly different among the two groups Basal 3.27 f 0.09 3.36 k 0.12 3.26 f 0.12 (Table 1). Blood ethanol levels after 24 hr of withdrawal In the presence of NE 7.03 f 0.61 6.50 f 0.44 6.34 f 0.56 NE-stimulated 3.81 k 0.64 3.14 k 0.39 3.08 f 0.51 in the 15 days ethanol-fed group were 0 mg%. Very subtle In the presence of 5.27 k 0.23 5.58 k 0.29 5.52 f 0.28 signs of withdrawal were observed in the ethanol-withA23187 drawn group, and these included enhanced startle reflex, A23187-stimulated 2.01 k 0.28 2.28 f 0.24 2.01 f 0.20 In the presence of 5HT 4.05 f 0.12 3.96 f 0.11 3.61 k 0.12 chattering teeth, and spontaneous squealing. This with5HT-stimulated 0.81 k 0.12 0.62 f 0.11 0.38 f 0.06' drawal state was characterized and rated as described by Control, 15-day ethanol-fed and withdrawn (24 h) rats were sacrificed and Majchro~icz.~~ Table 1. Body weights (g) of Ethanol-Fed and Control Rats and Blood Ethanol Level Ima%) in Ethanol-Fed Rats at the Various Time Points Studied

Short-term Groups Control Ethanol Ethanol withdrawn

Long-term

4

Body weight Blood ethanol Body weight Blood ethanol 376 f 12 344 f 17 345 f 6

164 f 20 0

349 f 5 367 f 10

198 f 34

The ethanol-withdrawn group received the Lieber-DeCarli control liquid diets instead of the ethanol diet on the 15th night. Values are mean f SEM from eight rats in each group.

cerebral cortices were dissected out. Cortical slices were prepared and labeled with [3H]-myoinositolaccording to the procedure as described in "Methods"section. [3H]-Myoinositolprelabeled cortical slices were stimulated by NE M). 5 HT M), and A23187 (50 PM). Released [3H]-IP, were separated by dowex column chromatography. Results are expressed as percent of total [3H]-inositolincorporated (dpm). Values are mean f SEM from seven to nine rats in each group. * Significantly different from control ( p < 0.01). In a typical experiment in control rat cortical slices total incorporated counts (dpm) in basal, NE, A23187, and 5HT tubes were 174056, 168910, 186451, and 185381, respectively, and radioactivity in [3H]-IP, fraction in basal, NE, A23187. and 5HT tubes were 5792,11828,10432, and 7723, respectively. (In this particular experiment [3H]-IP, formation in terms of YO of total count incorporated in Basal, NE, A23187, and 5HT tubes were 3.32, 7.00, 5.59, and 4.1 7, respectively.)

ETHANOL EFFECTS ON SEROTONIN RECEPTORS AND BRAIN PI TURNOVER Table 3. Effect of Long-Term Ethanol Treatment on Agonist Stimulated [3H]-IP, Formation in Rat Cerebral Cortex Agonist Basal In the presence of NE NE-stimulated In the presence of A23187 A23187-stimulated In the presence of 5HT 5HT-stimulated

Control group

Long-term ethanol-fed group

3.87 f 0.21 8.39 f 0.47 4.45 f 0.46 7.81 0.39 4.09 f 0.45 5.07 f 0.27 1.21 f 0.22

3.81 f 0.14 8.29 f 0.50 4.48 f 0.44 7.50 0.28 3.82 f 0.32 4.55 f 0.17 0.75 f 0.11

*

120

1

*

Control (Sucrose-fed), 60-days ethanol-fed rats were sacrificed and cerebral cortices were dissected out. Cortical slices were prepared and labeled with [3H]myoinositol according to the procedure as described in "Methods" section. [3H]M). 5-HT myoinositol prelabeled cortical slices were stimulated by NE M), and A23187 (50 p ~ ) .[3H]-IP1thus formed were separated by dowex column chromatography. Results are expressed as percent of total [3H]-inositolincorporated (dpm) as described in "Methods" section. Values are mean -c SEM from eight rats in each group. * Significantly different from control group ( p < 0.05). In a typical experiment in control rat cortical slices total incorporated counts (dpm) in basal, NE, A23187, and 5HT tubes were 147977, 172036, 105494, and 157326, respectively, and radioactivity in [3H]-IP, fraction in basal, NE, A23187, and 5HT tubes were 4812, 14538, 7988, and 7092, respectively. (In this particular experiment [3H]-IPl formation in terms of YOof total count incorporated in basal, NE, A23187, and 5HT tubes were 3.25, 8.45, 7.57, and 4.5, respectively). Table 4. Effect of Long-Term (60 days) Ethanol Treatment on el-Adrenergic and 5HT2 Receptors in Rat Cerebral Cortex [3H]-prazos~nbinding

1113

'Z51-LSDbinding

Groups

B , (fmol/ mg protein)

KO (PM)

Bmax(fmol/ mg protein)

KD (nM)

Control Ethanol

138 f 10 150 k 13

88 -c 5.4 73 f 4.8

151 f 15 158 k 18

0.87 2 0.11 0.94? 0.11

Cortical membranes were prepared from rats maintained on a liquid diet containing ethanol or sucrose for 2 months. Cortical membraneswere used to measure the B,, and KO for lz5I-LSDbinding to 5HTz receptors and [3H]-prazosinbinding to wadrenergic receptors. Values are mean f SEM from seven to eight rats in each group.

sucrose-fed rats was assessed. Scatchard analyses of saturation experiments with different concentrations of 13H]prazosin revealed that neither the Bmax nor KO for [3H]prazosin binding to a '-adrenergic receptors in cortical membranes from ethanol-fed rats was significantly different from that of control rats (Table 4).

0.5

1.5

1.0

2.0

3.0

2.5

3.5

Concentration o f 1251-LSD (nM)

Fig. 1. Saturation isotherm of '251-LSDbinding to 5HTz receptors in rat cerebral cortex. Each point is the mean of duplicate determinations. Inset, Scatchard plot of the specific binding of lZ51-LSDto 5HT2receptors in cortical membranes. Bound = lZ51-LSDspecifically bound ( f m o p g protein), B/F = bound over free lz5I-LSD (fmol/mg protein x nM). For this particular experiment, binding indices are KO = 0.96 nM, B,, = 151 f r n o p g protein, CCo = 0.95. Table 5. Effect of Short-Term Ethanol Administration and Withdrawal on 5HT2 Receptors in Cerebral Cortex 1Z51-LSDbinding

Groups

Em, (fmol/ mg protein)

KO (nM)

Control Ethanol Withdrawn

233 f 16 217 f 21 178 f 8'

1.0 f 0.12 1.2 f 0.10 1.2 f 0.12

Control, 15-day ethanol-fed and withdrawn (24 hr, see "Methods") rats were sacrificed and homogenates of the cerebral cortex were assayed to determine 5HT2 receptors using '251-LSDas radioligand. B,, and KD were obtained from Scatchard analyses of the binding data. * Significantly different from control group ( p < 0.01). Values are mean i SEM from seven rats in each group.

fed rats (Tables 4 and 5). This suggests that the decrease in 5HT-stimulated IP, formation in 60-day ethanol-fed rats could be due to ethanol-induced changes in postreceptor events. Ethanol withdrawal after short-term ethanol treatment had a different effect on 5HT2 receptors. The mean B,,, Efect ofEthan01 Treatment on 5HT2 Receptors in Rat of '251-LSDbinding in cortex of ethanol-withdrawn rats Cerebral Cortex was significantly ( p 0.01) lower than B,,, in control We used high aflinity radioligand (I2%LSD) for meas- rats (Table 5). No significant differences were found uring the 5-HT2 receptors and observed that binding of among groups in KDof I23-LSD binding (Table 5). These '251-LSDto cerebral cortex membranes was saturable and data suggest that our findings of a decrease in 5HTreached a plateau between 2.0 and 3.5 nM (Fig. 1). The stimulated IPI formation in ethanol-withdrawn rats, may Scatchard plot from a typical saturation experiment indi- be due to a decrease in the number of functional 5HT2 cated a single class of high affinity binding sites, with KO receptors. of 0.96 nM and B,,, of 151 fmol/mg protein (Fig. 1). In order to examine if changes in 5HT-stimulated PI ~ A in Rat hydrolysis are related to changes in 5HT2 receptors, we Effect ofEthan01 Treatment on ~ H T Receptors Brain examined 5HT2 receptors number and affinity in the cerebral cortex of 15 and 60 days ethanol-fed, ethanolIt has been shown that binding of [3H]-8-OH-DPAT to withdrawn and control rats. There was no significant 5HTlAreceptors in cortex and hippocampus is signifidifference in B,,, or KO values of '251-LSDbinding be- cantly higher in the alcohol-prefemng rats than in nontween control, 15 days ethanol-fed, and 60 days ethanol- preferring rats.28We therefore examined the effect of 60-

-=

Ill4

PANDEY ET AL.

day ethanol treatment on [3H]-8-OH-DPAT binding to 5HTIAreceptors in cortex and hippocampus. Because ~ H T receptors ~ A are located presynaptically and postsynaptically in the cortex and postsynaptically in the hippocampus, both brain regions were assayed.35 Table 6 shows the effects of long-term ethanol treatment on t3H]8-OH-DPAT binding in rat cortical and hippocampal membranes. No significant effect of 60 days ethanol treatment was found on &,,or KD for [3H]-8-OH-DPAT binding in either brain area. DISCUSSION

The major finding of the present investigation is that phosphoinositide signal transduction via the 5HT2 receptor is altered by ethanol treatment and withdrawal. After 15 days of ethanol exposure, 5HT-stimulated IPl formation was reduced to 76% of control production and by 60 days of ethanol exposure, 5HT-elicited IPI was significantly reduced to 62% of control. This finding suggests that the effect of chronic ethanol exposure on 5HT2 receptor-mediated IPI production is gradual and dependent on the duration of exposure. Interestingly, at the same time points of ethanol exposure, no changes were found in the B,,, or apparent KO of 5HT2 receptors as measured by '"I-LSD binding studies. Because no change in the 5HT2 receptors was detected, one could hypothesize that ethanol exposure produces a change in post-receptor events. Our data suggests that the activity of PLC is not altered, leaving us with the speculation that 5HT2 receptor-(; protein or G protein-PLC interactions may be affected by chronic ethanol consumption. Confirmation of this speculation will require further studies. Our observation that chronic ethanol treatment results in decreased 5HT-induced PI turnover is similar to the findings of Simonsson and Allir~g,~'who observed a decrease of 5HT-induced PI turnover in platelets of alcoholic patients. Another possible explanation for the observed ethanolinduced decrease in 5HT-stimulated IP1 production is that the specific radioactivity of the phosphoinositide pool, which was available for hydrolysis, was decreased by chronic ethanol exposure. However, this explanation is unlikely because we did not observe any significant change in the basal levels of inositol phosphate production or NETable 6. Effect of 60 Days of Ethanol Treatment on WT,* Receptors in Different Areas of Rat Brain [3H]-8-OH-DPATbinding Cortex Groups Control Ethanol

B, 79 f 7 81 f 5

Hippocampus KO

Bmm

KO

2.1 & 0.15 2.1 f 0.11

296 f 34 272 f 27

1.7 f 0.18 1.7 f 0.26

Cortical and hippocampal membranes were prepared from rats which were maintainedon a liquid diet containing ethanol or sucrose for 2 months Membranes were incubated with various concentrations of [3H]-80H-DPATand nonspecific binding was determined in the presence of M 5-HT 6,- (fmol/mg protein) and KO (nM) were obtained from Scatchard analyses of the binding data Values are mean SEM from eight rats in each group

*

or A23 187-stimulated [3H]-IPI formation in control or ethanol-treated rat cortical slices. The effect of withdrawal after 15 days of treatment on 5HT2 receptors and 5HT2-receptor mediated IP1 production is very interesting and different from the findings after 15 days of ethanol treatment alone. Ethanol withdrawal produced a 53% decrease in SHT-stimulated IPI production and a concomitant 24% decrease in B,, of '251-LSDbinding. The apparent KO of the 5HT2 receptor was not altered by short-term ethanol treatment and withdrawal. This data suggests that withdrawal after 15 days of ethanol consumption, unlike ethanol consumption alone, results in down regulation of 5HT2receptor number but has no effect on receptor affinity. Our investigation demonstrates that A23 187-stimulated PI hydrolysis in rat cortex is not altered by either shortterm or long-term ethanol treatment or by withdrawal. A23187 is a calcium ionophore that has been shown to increase phospholipase C activity through mobilization of ~ a l c i u m . ~This ' , ~ ~finding suggests that ethanol treatment does not cause an abnormality in PLC enzyme activity or in the ability of the enzyme to be stimulated by a nonreceptor-mediated event such as calcium mobilization. Our observation that A23 187-stimulated PI-turnover in rat cortex does not change following chronic ethanol treatment is similar to the findings of Gonzalez and Crews." It has been shown that B,,, and KOof [3H]-5HTbinding sites, as a measure of 5HTI receptors, are increased in brain from alcohol-preferring rats in comparison to alcohol non-preferring rats.27Wong et a1.28also reported that the number of 5HTla receptors is increased in brains of alcohol-preferring rats in comparison with alcohol nonpreferring rats. This finding directed our examination of effect of long-term ethanol treatment on 5HTIAreceptor subtypes, as measured by [3H]-8-OH-DPATbinding. We observed that chronic ethanol treatment had no significant effect on either B,,, or KD of [3H]-8-OH-DPATbinding in rat cerebral cortex and hippocampus, suggesting that the ~ H T receptor ~ A is not affected in rat brain by chronic ethanol exposure. Previous studies have demonstrated that chronic ethanol treatment had no significant effect on NE-induced PI turnover; however, the effect on al-adrenergic receptors was not measured. '8,43 Using rat liver slices, Gonzales and Crews' showed that chronic ethanol decreases NE-stimulated [3H]-IP~formation but does not change al-adrenergic receptors. We investigated the effect of long-term ethanol treatment on [3H]-prazosinbinding and NE-stimulated PI hydrolysis and found that neither NE-stimulated [3H]-IPI formation nor al-adrenergic receptors were affected by long-term ethanol treatment. We also observed that short-term ethanol treatment alone and after 24 hr of withdrawal had no effect on NE-stimulated PI turnover in rat cerebral cortex. Our finding provides further evidence that the PLC enzyme itself is not altered by ethanol treatment.

ETHANOL EFFECTS ON SEROTONIN RECEPTORS AND BRAIN PI TURNOVER

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In conclusion, our results suggest that: (1) long-term ethanol treatment causes a significant decrease in 5HTinduced PI turnover without changing the 5HT2 receptor number or apparent affinity in rat cerebral cortex, (2) long-term ethanol treatment has no effect on 5HTIAreceptor number or affinity in cortex or hippocampus, (3) short-term ethanol treatment elicits a 24% reduction (not significant) in 5HT-stimulated IPI formation and has no effect on 5HT2 receptors, and (4)ethanol withdrawal after short-term ethanol treatment results in down regulation of 5HTz receptors and a significant decrease in 5HTstimulated PI turnover in rat cerebral cortex. Because 5HT-mediated PI turnover appears to become increasingly reduced with longer duration of ethanol treatment while no concomitant changes in 5HT2 receptor number or apparent KO are detected, we hypothesize that ethanol may be affecting the coupling of 5HT2 receptor to G protein and/or phospholipase C enzyme. The decrease in 5HT-induced PI turnover after ethanol withdrawal may be due to a decrease in the number of 5HT2 receptors. Further experiments are necessary to clarify the mechanism(s) for the relationship between ethanol’s effect on 5HT-induced PI hydrolysis and brain function.

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