INTERNATIONAL SOCIETY FOR BIOMEDICAL RESEARCH ON ALCOHOLISM

Sixth Congress-June 21-27, 1992 Bristol, England POSTER ABSTRACTS (pp. 600-641) PLENARY ABSTRACTS (pg. 642) SYMPOSIUM ABSTRACTS (pp. 643-656) 1

HAS THE CONTRIBUTION OF ALCOHOL DEHYDROGENASE TO ALCOHOL METABOLISM BEEN PREVIOUSLY OVERESTIMATED? R.G. Thurmana. B.U. Bradforda and D.T. Forma#, Dept. of Pharmawlog and Biochemistry and Pathologyb, U.of N. Carof$a at Chapel Hill, NC, USA After the alcohol dehydrogenase-deficient deer mouse (ADH- mutant was dosed with SM) m /k of 4methylpyrazok (4MP) Lp., rates of ethano? (€7 and methanol (M) metabolism were decreased significantly b 41% and 35%, respectively. In perfused liver. rates of metabolism were also decreased up to 77% by 4MP. When livers were perfused with M, a selective substrate for catalase. 4MP (1.5 mM) decreased rates of M metabolism by 58%. 4MP caused negligible changes in total hepatic catalase activity or in rates of oxidation of E by microsomes but inhibited catalase-dependent alcohol metabolism by limiting the supply of H t 0 , ~ ’ . In liver homogenates, 4MP competitively in ibited fatty acyl CoA synthetase, a key enzyme involved in the supply of substrate (e.g., palmitate) for catalase-Hfl2. When the reaction product palmitoyl CoA was used, the addition of 4MP did not alter enzyme activity. These data indicate that fatty acyl CoA synthetase is inhibited by 4MP, thus reducing the availabilii of for catalasedependent alcohol met8 oltsm m a r results were obtained with methanol in deer mice containing alcohol dehydrogenass (ADH’). Taken together, these data support the hypothesis that the contribution of alcohol dehydrogenase to alcohol metabolism, based largely on selective inhibition of ADH by 4MP, may have been previously overestimgted (AA-03624).

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CHARACTERISTICS OF HELICOLiACnR PYLORI ASSOCIATED ALCOHOL DEHYDROGENASE ACTlVlTY R. Roine, K.S. S h e l a , I. H i d k - N h , T.U. Koruaen ud M. Warplm. R w m h Unit of Alcohol D~eases,ud Deptmsnt of B.cteriology ud Immunology, University of Helsinki. Finlad. It is g c n c d y accepted G x I infection with Hdicc&crrr pylon is largely responsible for the O C C Y ~ D C Cof gastritis md duodenal ulcer disease. Furthcmrom, H. pylon’ infection hc IUely been Iinked also aith h e development of gastric cancer. We have recently demonstrated ttd H. pylon’ psresscs significant dcohol dchydmgems (ADW activity ad can produce luge ammm of ncsuldehydc from dhaml in h a Acetaldehyde, a highly ructivc d toxic mhallncc may bc u t important explanatory factor in Lbc pthogcawis of belicohacter M S O C U I morbidity. ~ Tbe lim of Ibc p e n t study wm to furlher chancterizc some basic aspacts of ADH derived from two H. pylon st&s (NCTC 11637 Md NCTC 11638). ADH activity was determind rpactrophotornetncally from the cylosol of the bacteria. Both strainr showed higher ADH a c t n ~ t i eat~ high (0.5-2.5 M) than at low (12.5-SO mM) ethanol concentrations. The K . for a h o l for rtraia 11637 was ulculated to bc 103 mV mI thal for strain 11638 69 mM. When t k e d in a pH rage from 7.2 to 10.5, marimal ADH activity wm observed U pH 9.6. Addition of 4m*hylpymzole lo the mction mixture clearly inhibited H. pylon‘ ~ ~ s ~ ~ iADH a t e activity d both U low ud high ethanol coxcnlralions. At 0.5 M ethawl coacmlntion, c h i d i n e inhibited significantly ADH concentmtions likely to cccur in tbc gastric lumm after ~c 0th drug (ADH activity U I mM, S mM. d 10 mM ciwtidine BOX, 46%. md 36% of b-eline, reapactivcly; p < 0.05). In conclusion, H. pylon usocided AD11 may, It least in prt, Lm respnslble for the ethanol oxidetion sboun to =cur in the stomach, M wmc chamcto~ticsof helicobactsr ADH bur m c m b h e to hose repodd for gastric rnucose.1 ADH activity.

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ROLE OF EXTRAHEPATIC ALCOHOL DEHYDROGENASE (ADH) IN T H E METABOLISM OF E W O L AND BUTANOL IN DEER MICE T. Cronholm’. C. Norsien-HWgl. G. EkstrOm’. J.A Handler’. R.G. Thurman:and M. Ingelman-Sundberg’, ‘Department of Physiological Chemistry, Karolinska Institutet, Stockholm, Sweden and ZDepartment of PharmawloD, School of Medicine, University of North Carolina, Chapel Hill, North Carolina, U S A A strain of deer mice (Pcromyscus manuulanrs) that lacks detectable ADH-activity in the liver has been used enensively for siudies on non-ADH systems for ethanol elimination. With the aid of deuteriated ethanols we have previously observed dehydrogenasedependent ethanol elimination in these deer mice ( A D H ) (Norsten et al.. J. Biol. Chem. 261.5593-5597, 1989).Attempts have now been made 10 localize and charaaerize the responsible activity. I n VIVO administration of [l.l-‘H2]ethanol and unlabeled butanol resulted in the appearance of [2H]butanol in blood. Liver perfusions showed significant, but slow, intermolecular 2H lransfer beween labeled butanols. possibly explained by low A D H activity detected in the inner mitochondria1 fraction of liver homogenates. Tbis could not explain the elimination and hydrogen exchange in vivo. Thus it was concluded that the ADH activity was mainly extrahepatic. Significant NAD-dependeniethanol-o~dizingactivitywas d e t m e d in cyIosol oblained from gastric mumsa, and revenibility was confirmed with *H-substrates. On a protein basis the activity was similar in gastric mucosa of ADH- deer mice and liver of ADHpossessing deer mice, indicating that the gaslric acti\ity might explain ethanol elimination in the ADH- deer mice. This was funher supported by similar kinetic isotope effects UI nvo and in qWsol from gastric mucosa. and by the insensitivity of the gastric activiry in vino to inhibition with 4-methylpyazole as has previously been observed lor ethanol elimination in ADH’ deer mice.

SLY DIFFEREWES OF HTPITIC ALCUBOL D~YDRffiEW3E(ADHI ACTIVITY M D

€WRESSIOY OF .U)H m R M T. Ida. S.Xunitoh. T.Tmaka. T. Ya.ashita,

T. Yonna” , S.Uishipuchi’: snd S.otania’. “ D f p a r k n t o f Public Health. “Third Department of Internal kdicme. “ D e D a r k n t of Bioche.istry.Osaka City University W i d Sclaol, Osaha. Japan.

HeDat.lc ADlt IS t h e .osT LsDOTtJnt enl.ne for the R t a b o l l s Of ethanol and. i t s a c t i v i t y . in part. r e c n l a t e s t h e ethanol elimination r a t e IEERI. Several hormones have effert on the Mil activ i t y ad testosterone i s b o r n to drcrelue t h i s enrm activity. In t h i s study. n have muestigated the differences of hepatic MH a c t i v i t y and i t s rRNd expression in male. female and castrated Vistar rats. The castrated r a t s KTV r e a d testis 2 weeks before t h i s experiment. The h e p a t i c AD8 a c t i v i t i e s of female and castrated r a t s .ere a i g n i f i c z a t l y higher than t h a t of male r a t s (151=4.8. 166.855.2 V.S. 98.1k3.2 nIU/w protein. respectivel y ] . Furtherare. t h e expression of hepatic MH WAS of fnale and castrated r a t s have m c r t s d compare t o t h a t nf . a l e rats. I n c o n t r a s t . t h e r e .ere D O signrf~caot differences in t h e EER after ?g/k of cthmol & m i s t r a t i o n i . 0 . in 3 WOODS.From these r e s n l t s . I t IS suggested t h a t t h e hepatic MH a c t i v i t y is reg”l a t e d by t e s t o s t e r o n e a t t h e level of transcription and that the incease of hepatic ADR a c t i v i t y hps so effect on the FIR.

* Poster Abstracts pp. 600-64 1 600

Plenary Abstracts pg. 642 Symposium Abstracts pp. 643-656

5 TN1'IIANIICI.CAI: CLASS I I 1 AI.COIIOL I)I:IIYDIIOCENASI: IN

8 xllllYTATIU4 UP THE LlVm E T W O L - U T A B D -

ASTKOCYTES AND IIEPATOCYTES. IF.J . Iborra, 'J.Renau-Piqueras, M.PortolCs, 'EI.D.Boleda, 2X .Pares, 'C.Guerr i. 'Ctr Invest. Hosp. "La Fe", Valencia; 'Dept. Bioquimica, Univ. Autbnoma, Barcelona;'Inst. Invest. Citol6gicas, Valencia, Spain. Alcohol dehydrogenase (ADH) includes, at least, four isoenzyme classes with very different capacities for ethanol oxidation. Class 111 ADH (which is present in all tissues and is highly conserved throughout evolution) has a low activity with ethanol, and has been identified as a gl utaLhione-dei)eiidend formdldel~yrledrhydi ogenase. Immunocytochemical studies reveal the presence of class I11 ADH in both cytoplasm and nucleus of astrocytes and hepatocytes. In the riucleus the labeling was found mainly over condensed chromatin. The gold pdi-cicle density over the nucleus was greater i n differentiaLed than i n proliferating astrocytes. In contrast, class T Allll wds foilrid o n l y i n tile cyto~~idslil. Similar results were fouiid when the activity of class I T T ADll was medsured using both octanol

LlZING SYSTEM TO A C D H U L lU SHUHT- xN1, LGBG-SLLEP MTS. L.H.Bardina and V.I.Satanovskaya. I n s t i t u t e o f Biochemistry, Acad. S c l . o f Belarus. Grodno, Belarus. On the b a s i s o f ethanol-induced s l e e p time (ethanol 3.5g/kg,i.y.)rnale r a t s wer e divided i n t o short-sleep (SS-up t o 20 min) and long-sleep (LS>lZO m i n ) groups. During 7 days ethanol was administered i. p . a t a dose o f 3 . 5 d k g once a day.Contro1 r u t s were given iootonlc BaCl solution under the same cond1tions.hfter 24h f o l lowing the l a s t ethanol aduinistration, the r a t s were decapitated. It was shown that the SS and LS r a t s d i d n o t d i f f e r in a c t i v i t i e s o f l i v e r alcohol dehydrogen a a e s and t h e microsomal ethanol oxidlzing system (MEUS), whereas the c a t a l a s e a c t i v i t y was higher ( p 8 . 0 2 ) i n the control SS r a t s . The development of alCOhOl tolerance was accompanied by induction of b&Us by two-fold i n LS r a t s and by three-fold i n SS rats as w e l l as by a decrease o f the yeroxidatic cataluse a c t i v i t y ( ~ 0 . 0 2 ) . No d i f f e r e n c e s were found i n the l i v e r trldehgrde dehydrogenases act i v i t i e s (high arld low forms).Thus, the metabolic a h y t a t i o n o h h h e l i v e r t o ethanol i s probably r e a l i z e d mainly t h o ugh the systems of i t s u t i l i z a t i o n .

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l'hese results demonsLrate the presence of class I11 ADH in the nucleus and suggest that one of the functions of this enzyme could be to protecl DNA front Cnri~~aIileliyileLoxicity.

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ALTERATIONS INDUCED BY PRENATAL EXPOSURE TO ETHANOL ON P62 AND FODRIN, TWO CALMODULIN-BINDING PRYTEINS, IN ASTRCGLIAL CELLS. 41. Faura, M. Portol&s, IF.J.Iborra, IJ. Renau-Piqueras, R. SQez, 3J.Serratosa, 40.Bachs, 2C. Guerri. 'Ctr.Invest .Hosp. "La Fe", 2Inst .Invest. Fitol., Valencia, 3Dept.Farmacol.Toxicol. ,CSIC, Dept.Biol.Cell.,Fac.Med.,Univ.Barcelona.Spain. Calmodulin (CaM), an important regulator of the calcium signal, has been implicated in DNA replication, DNA repair, the expression of some genes, and phosphorylation of nuclear proteins. CaM function requires some targets, i.e. CaM binding-proteins (CBP). We have analyzed the effect of prenatal exposure to alcohol (PEA) on the distribution of two CBP (p62 and fodrin), in astrocytes in primary culture. p62, is related with the condensation of chromatin, and fodrin is a spectrin-like protein. In controls, p62 was present in the nucleus of astrocytes, as well as in the nucleus of precursor glial cells. PEA treatment decreased the amount of p62 in the nucleus of proliferating astrocytes. Fodrin appeared associated to peripheral condensed chromatin. Ethanol treatment does not alters the amount of fodrin but changes the pattern distribution. Since these proteins and CaM are involved in the regulation of important nuclear functions, the alterations found after PEA could explain some of ethanol-induced injury on nervous cells during development.

?ZlHANOL PRODUCTION BY HELICOBACIER PYLON

K.S.Sdmcl., R. Roinc, I. H-k-Nihnnc, T.U. Kosunen and M.S d q u r o , Revlrth Unit of Alcohol Di-. md k p r n m c n t of Buleriolop. and Immunology, Univcnity of Helsinki. Finland. We havc recently dcmonstnted thal Hdic-cr pylon' m a e d iignifiunt dcohol dshydrogcnase (ADH) activity which un produec l +ifm, and therefore contribute lo H. pyton' acetaldehyde from e t h ~ o in misted gutric morbidiy. In hcterid fcrmenution. acetddehydc is d u d to ethanol with simulunmvi oxidation d NADH lo NAD. I h c p u r p c of the preen1 rmdy WM to determine whether R pyton' it upablc of producing e t h ~ o via l uctddchydr d u c t i o n during in ~ m culturn. o To that aim we incubated two Hclicobactn strains (NCTC 11637 and NCKC 11638) in a liquid bmh medium under micrmercbic unditiona for two days. E t h ~ o lmd acellldchydc were determined from the CulNrC wlution by h u d rplce g~ chromalognphy at 37 'C. For s o m p u i m , three oUIcr bacteria dso cmt.ininpADH (fichmicb cdi, Mebn'rl1opnmtmrn;~c. and cmnpYl&clrrjejm~ were cultured in a similu manner. Ethanol was found in the culture medii of both H.pylon' lvnins (65 pM and lBO)lM,NCTC 11637md NCTC 11638). Aa m u n cthurd production, this q u d s to 6500 Md 18033 pmol per 10' bacteria. By conVLlt. rlhlnol productionof E. cdi waa only 0.4 pmol pr 10' hcteria. K.pncwnatiar and C. jrjvn. did not pmducc c t h ~ o l .Acetaldehyde WM below the dctstion limit in the c v l h l m of dl bacteria atudied. Our mulu ivogcst that H. pylon' ia capable of producing e t h ~ oinl who. Accordingly, the biological role of hclicabsctcr ADH m l d be to ferment glueme 01 some &her rubstnte n4 icetddehydc lo ethanol. M d by that m e m i provide energy in rnicmembis conditionn,.lf this m u m in rim, the hc1eri.l production of &MOI may uhlmtc, at I& in put, gMkC ADH and mstqucntly be one dclerminlnl in the lint p m mctlbli8m of ethanol. According LO the pruent data. fcrmcntation mediated by helicobseter ADH apnd lo be msaiated with musunble acetaldehyde conccnlmtions. Potmtidly toxic leveli of acetaldehyde un be produced via heliubcler ADH from added ethanol M prcMnted in oyr earlier rtudiu.

10 GLUTATHIONE-DEPENDENT FORMALDEHYDE DEHYDROGENASE/

OXIDATIVE CHANGES INDUCED BY ETHANOL IN BRAIN AND ADAFTATIVE RESPONSES AFTER CHRONIC ETHANOL

CLASS I11 ALCOHOL DEHYDROGENASE: COFACTOR SPECIFICITY AND COMPETITION BETWEEN ALCOHOLS AND HEMITHIOACETALS AS SUBSTRATES M.Koivusalo and L.Uotila, Department of Medical Chemistry,University of Helsinki,Helsinki,Finland. We have previously shown that the cytoplasmic formaldehyde dehydrogenase and Class I11 alcohol dehydrogenase are identical enzymes. The purified enzymes from rat liver and pig kidney cortex have been further characterized. The thiol specificity of the formaldehyde dehydrogenase reaction has been studied with several thiols. hnega-thiohexanoate and omega-thiooctanoate could be used instead of GSH but with lower efficiency. Cysteinylglycine, N-acetylcysteine,mercaptoethanol, dithiothreitol and dithioerythritol were not active as cofactors. Several NAD(P1 analogs were active as cofactors. Acetylpyridine-adenine dinucleotide IAPAD) gave the highest maximal activity in both reactions but the K -value was much higher than with NAD. The oxidakons Of S-hydroxymethylglutathione and the alcohol substrates were mutually competitive. 12-Hydroxydodecanoate inhibited competitively the production of S-formylglutathione from formaldehyde and CSH when assayed at 240 nm. Similarly when the NADH production was measured with varying 1 2-hydroxydodecanoate concentrations and constant concentrations of formaldehyde and GSH, the kinetics were COmpetitfVe.

CONSUMPTION. 'C.Guerri, 'C.Montoliu, 'S.VallCs, 2E.Fornas, 'J. Renau-Piqueras, 'Inst. Invest. Citol6gicas, 'Ctr. Invest., Hosp. "La Fe", Valencia, Spain. Lipid peroxidation has been implicated in the toxicity of alcohol on liver. However, the importance of this mechanism in brain is not clear. In the present work we have investigated the effects ethanol "in vitro" and "in vivo" in chronic alcohol-fed rats, on the levels of malonyldialdehyde(MDA) and on the activities of superoxide dismutase(S0D). catalase and glutathione peroxidase(GSH-Px) which are implicated in the elimination of free radicals. Our results demonstrate that when brain homogenates were incubated with ethanol(25.50, 100 and 200mM), a dose-dependent increase in MDA formation was found. However, chronic alcohol consumption does not increase the basal levels of brain MDA, but induces an increase in the activities of brain SOD and catalase and reduces the glutathione(GSH1 levels. No variations in GSH-Px activity was observed. These results suggest that chronic alcohol consumption induces an adaptative response to lipid peroxidation in brain and that this treatment produces an oxidative stress which is reflected in the decrease of GSH levels.

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EXPRESSION OF TUMOR-ASSOCIATED ALOEHYDE DEHYOROGENASE (ALDH3) IN THREE TUMOR CELL LINES A . Eckey. C. Wolff. 0. tleier-Tackmann, D.P. Aganrsl and H.W. Goedde. Instiute of Human Genetics, University of Hamburg, FRG.

Aldophosphemide. the cytotoxic metabolite of cyclophosphemide can be detoxified by aldehyde dehydrogenese (ALDH). Thus resistance of certain tumor cells t o cyclophosphamide i s sttrlbuted t o the celluler ALOH activity. I n experimental r a t l i v e r tumors a unique ALOH (ALDHB) has been observed which is u s u a l l y not expressed i n normal aemmslian liver but i n l u n g and gastric mucosa. A similar atypical ALDH isozyme, detected previously by us i n a human primary l i v e r tumor, was found t o be immunologically identicel t o the gastric ALOH3 (Aganral e t e l . , Prog. C l i n . Blol. Res., V o l . 290, AR. Liss. New Vork, pp 119-132, 13891. The aim of the present study was t o examine the expression of ALOHS activity i n human c e l l lines derived from 8 liver t u m o r (HepGZ), a l u n g edenacarcinome (A549) and from a pharynx carcinoma (UMSCCZ) . I n HePGZ c e l l s , the enzyme activity and antigen levels were found t o be relatively low but were enhanced several times when the c e l l s were grown i n the presence of the carcinogen 4-methylcholanthrene. I n A543 end UMSCCP c e l l s , the expression of ALDH3 was manyfold higher t h a n i n the corresponding normel tissues. However, no further induction was observed w i t h 4methylcholanthrene. These results indicate t h a t ALDH3 is inducible i n different c e l l s during carcinogenesis, and may account f o r the acquired cyclophosphemide resistance cornonly observed i n tumor calls.

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ADAPTATION OF THE LIVER ETHANOL-METABOLIZING SYSTEMS TO ALCOHOL I N SHORT- AND LONG-SLEEP RATS L.R. B a r d i n a and V . I . S a t a n o v s k a y a , I n s t i t u t e of B i o c h e m i s t r y , Acad. S c i . of B e l a r u s , Grodno, Belarus On t h e b a s i s of e t h a n o l - i n d u c e d s l e e p t i m e ( e t h a n o l 3.5g/kg, 1 . p . ) m a l e r a t s were d i v i d e d i n t o s h o r t - s l e e p (SS-up t o 20 min) and l o n g s l e e p (LS->120 min) g r o u p s . D u r i n g 7 d a y s e t h a n o l v a s a d m i n i s t e r e d 1.p. a t a d o s e o f 3.5gIkg once a day. C o n t r o l r a t s v e r e g i v e n i s o t o n i c N a C l s o l u t i o n u n d e r t h e same c o n d i t i o n s . A f t e r 24h f o l l o w i n g t h e l a s t e t h a n o l admini s t r a t i o n , t h e e t h a n o l t r e a t e d r a t s as w e l l as c o n t r o l s were d e c a p i t a t e d . I t was shovn t h a t t h e SS and LS r a t s d i d n o t d i f f e r i n a c t i v i t i e s of l i v e r a l c o h o l d e h y d r o g e n a s e s and t h e microsomal e t h a n o l o x i d i z i n g s y s t e m (HEOS), w h e r e a s t h e c a t a l a s e a c t i v i t y was h i g h e r (pZ&YD LIVEROF THE RAT.

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PLATELET ADENYJATE CYCLASE ACl'IVITY I N LONG-TERM ABSTIKENT ALCOEOLICS. 8 . Ozawo. Y . Katamura. T. Ashizawa. M . Watanabe. S . Hatta.. N . Takahata and ;.Saito. Dept. of Neuropsychiatry and Pharmacolow. Sapporo Medical College, Sapporo. Japan. P l a t e l e t adenylate cyclase(AC) a c t i v i t y was determined i n long-term ahstinent a l c o h o l i c s who have f i r s t degree r e l a t i v e s with alcoholism. Control s u b j e c t s were matched with s e x and age. The basal a c t i v i t y of AC was the same i n p l a t e l e t s from the a l c o h o l i c s and c o n t r o l . hut the p l a t e l e t AC a c t i v i t y a f t e r stimulation with guanine nucleotide (GppNEp) was s i g n i f i c a n t l y lower i n a l c o h o l i c s . However. there was no s i g n i f i c a n t d i f f e r e n c e i n the EC value for Gpp(NE)p stimulation of AC hz?ween a l c o h o l i c s and c o n t r o l . AC a c t i v i t y i n the presence o f saturated l e v e l of GppNEp (Vmax) w a s s i g n i f i c a n t l y lower i n a l c o h o l i c s . Binding of photos e n s i t i v e GTP analog (AAGTP) to G s was decreased i n the p l a t e l e t membrane from a l c o h o l i c s . Amount o f G s but not Gi was s i g n i f i c a n t l y decreased i n p l a t e l e t from a l c o h o l i c s as a s s e s s e d by i m u n o b l o t t i n g . I t i s suggested that amount of G s p r o t e i n Is decreased i n a l c o h o l i c s and p l a t e l e t AC is a b i o l o g i c a l marker f o r a l c o h o l i c s .

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INVESTIGATION OF HUMAN CYTOCHROME P4502Et EXPRESSION IN THE MAMMALIAN CELL LINE, PC12 J Mapoles'. J F. MenezY,A. Alexander* and F. Benhou' * Hepalobilary Research Center, Univ. Colorado Health Sci. Cen:er, Denver, USA. ' L a b . Biochimie. Fac. M a . , Brest, France. We have constructed a mammalian cell line which expresses cyiochrome P4502E1 (P450alcohol), in order to study lhe effects of intracellularty generated acetaldehyde on rnicrosomal function and to study Ihe regulatton of P4502E1 at the post-translational level. A mammalian expression piasmid was constructed using the human cDNA for P4502Ei and driven by rhe Rous Sarcoma virus promoter. This plasmid was colransfecled with the plasmid RSVneo into lhe mammalian cell line PC12. These cells express a high level of NADPH c)lochrome reductase (65 nmd/min/mg rnicrosomal protein In comparison to about 80 in the human itver) which is required for cflochrome activity. and they do nor express P4502E1. Clones resistant lo the neomycin analog G418 were screened for chlonoxazone hydroxylation as an indicator of P4502Et expression (Peter el al. Chem Res Toxicol, 1990. 3, 566) Expression of the enzyme was confirmed in a clone, DB7, by Northern and Western blot analysis and by detection of enzyme activily in microsomes Kinetics of chlorzoxazone were determined 10 be Km = 300 pM; Vm = 60 pmol/rnin/mg microsornal protein This acttvity was inhibited by IOpM dielhytdilhiocarbamate (DETC) a specific inhibitor of the enzyme P4502Ei. The production of acetaldehyde from ethanol was found 10 be 1.8 nmol/rnin/mg microsomal protein, using DB-7 rnicrosomes. When tested for induction afier 3 days of exposure lo 50 rnM ethanol. 20 mM acetone or 10 rnM pyrazote. no evidence oi induction was found. However these compounds decrease the inhibition of chlorzoxazone metabolism produced by DETC. Supported by a Collaborative Research Grant forn NATO

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EFFEClS OF €TUANOLM D AC€TALUW?)E ON TBE MTURATION OF BEPATIC SECXEIORY GLYCOPROTEMS A. Takada. H. Karahara. S. Takase. f l . Tsuchlshlma. X-E. Sang. Dlv. of Gastroenterology. Dept. of I n t e r n a l . fled.. Kanazawa fled. Univ.. Ucklnada, Ishlkawa, 920-02. Japan, The e f f e c t s of e t h a n o l and acetaldehyde on t h e s a t u r a t i o n of h e p a t i c s e c r e t o r y g l y c o p r o t e l n s In c u l t u r e d hepatocytes from r a t s were analyzed u s l n g a pulse-chase l a b e l i n g method. Hepatocytes were obtained from male r a t s and c u l t u r e d f o r 48 hour Hepatic secretow p r o t e i n s were p u l s e l a b e l e d w i t h 55-S methlonlne f o r 1 hour. The l a b e l e d p r o t e i n s were s e p a r a t e d by twodimensional e l e c t r o p h o r e s i s of l s o e l e c t r i c focusing and SDS-PAGE a f t e r inmunoprecipitation u s i n g a n t i - r a t b e r m antibody. D i r e c t a u t o r a d i o g + m u s i n g X-ray f l l n on t h e slab g e l s was performed t o d e t e c t Isotope-labeled p r o t e i n s . A s p o t f o r t r a n s f e r r l n was d e t e c t e d at 77.000 in molecular weight and 5.2-5.4 i n PI In t h e c o n t r o l c u l t u r e s . Followlng treatment w i t h nonensln f o r 3 hour6, t h e s p o t f o r t r a n s f e r r i n was d e t e c t e d a t 5.5-5.8 i n PI and at 5.4-5.8 f o l l o r l n g t r e a t m e n t with tunicaqvcln f o r 3 hours. The s p o t s f o r t r a n s f e r r i n were s h i f t e d t o t h e more h a s i c s i d e by both t r e a t m e n t s . which i n h i b i t g l y c o s y l a t i o n of g l y c o p r o t e l n . The s p o t f o r t r a n s f e r r i n was d e t e c t e d at 5.4-5.8 f o l l o w i n g treatment with acetaldehyde f o r 3 hours and at 5.3-5.7 a f t e r additional treatment f o r 3 hours f o l l o w i n g pre-treatment f o r 6 hours with e t h a n o l , l n d i c a t l n g t h a t t r a n s f e r r i n was s h i f t e d to t h e more b a s l c s i d e . However. t h e s h i f t of t r a n s f e r r i n was not observed following t r e a t m e n t with e t h a n o l for 3 hours. These r e s u l t s s u g g e s t t h a t g l y c o s y l a t i o n of s e c r e t o r y glycoprotelns a t t h e Golgl a p p a r a t u s mw be i n h i b i t e d by acetaldehyde. b u t n o t by e t h a n o l i t s e l f . and t h a t t h i s I n h l b l t i o n may b e r e l a t e d t o t h e development of alcoholic l l v e r Injury.

EFFECT OF MANGANESE Oh' THE DEVELOPMENT OF N E R V E CELLS CULTURED FROM IN UTERO ALCOHOL

142

ETHANOL INCRWSES THE HORMONE-STIMULATED CAMP RESPONSE {N CULTURED HUMAN PLACENTAL TROPHOBLASTS PI Karl A Divald SE Fisher Department of Pediatna North Shore Unlvernny HospilaLComell University Medical College Manhasset New York 11030 The placenta n the pnncipal maternaWelal interface in human pregna-cy It facilitates the transfer 01 essential nulnenls and produces hormones which help maintain pregnancy Impairmen: of placenlal cell function by ethanol (EtOH) or d s me'abolites could adverrely affect fetal developmenl thereby contributing to the palhophysiology d the felal alwhol ryndrcme One mechanism by which ElOH might impair plac.$nlal cell function is through a'teratlons in inIracellular signal pathways Therefore we exposed cultured normal human placental Irophobiasls to EtOH a i d evaiuated res?in; and nononed-st$mulatedCAKP prduct8on METHODS Culfured trophoblasts Were exposed lo EIOV (280300 mg/dl) for up to 72 h r s Control and EtOH exposed cells were also treated with epinephrine (1*M) or adenosine (50pN) Levels of CAMP were measured by rad~oimmunoassay RESULTS Acute EtOH treatment (10 rnir) had no effect on eliher restlng or stimulated CAMP levels Long term ElOH exposure (18 lo 72 hrs) di0 no! affecl resting CAMP prodiction howeve' there was a signticant increase In the CAMP response to bol? hormones Epinephrine Control = 483 2 98 EtOH (72 hrs) = 776 2 167 pmol cAMP/mg pro1 ( m e a n t SE p 4 05 N=7 paired t-'est) Adenosine Cont = i E 4 i 4 6 E ~ O H= 30 2 t 8 3 (pc0 05 N=B] This pattern occurred with or withod the phosphodiesterase inhibitor Roiipram When the catalflic subunit of adenylate cyclase was activated by fonkolin (ZOmM) EtOH treatment resulted in 53 9 2 6 4% higher cAMP levels CONCLUSION In cultured human troplrohlasts exposure to EtOH for 16 hours or more increases the CAMP response to hormone stirnulatior The eihanced cAMP response mny be due in part to increased adenyl cyciase activity

143

ETHANOL ALTERS HORMONE PRODUCTION BY CULTURED HUMAN PLACENTAL TROPHOBLASTS. P I Karl, K Alpy S E Fisher. Depaflmenl of Pediatrics. North Shore Universnv HosDilal Cornell universny Mediwl college. Manhasset. New York iic13d Ethanol (ElOH) abuse during pregnancy can lead to abnormal fetal g r M h and developmenl. hallmarks of lhe fetal alwhol syndrome (FAS) One factor in lhe palhophyriology d the FAS may be impaired placenlal function. The plawnla transfers nutnents and provides endocnne maintenance of pregnancy We studled the effect of ElOH on hormone production by cunured human irophoblasts. METHODS: Normai human placenlal trophoblasls were maintained in culfure and exposed lo EtOH (280-300 mgldl) for up to 96 hrs Conlrol cells were similarly maintained without ElOH. Production of human placental laclogen (hPL). human chorionic gonadotropin (hCG) and progesterone (Prcg), a s well a s the cAMP response lo adenosine lreafment. were assessed by radioirnmunnassay RESULTS: EtOH exposure increased trophoblasl production of hCG and Prog. but not hPL (mean 2 SE: 'p-2 01. palred 1-test):

EXPOSEDRATS M. Ledig, J.C. Copin and G.m o l e y , Laboratoire de Neurob i o l o g i e Ontogtnique, C e n t r e de Neurochimie d u CNRS, S t r a s b o u r g , France. Using cultured n e r v e c e l l s as a m o d e l s y s t e m of brain d e v e l o p m e n t , an alcohol antagonizing e f f e c t of manganese (Mn:.) was shown. M n z - w a s a d d e d , e i t h e r in vitro (2 p M ) to t h e cell cultures, or in vivo to the alcohol diet (25 mg CI2 Mn/l of 20 O/c v/v ethanol) of p r e g n a n t rats. Neurons w e r e cultured for one week f r o m c o n i c a l brain c e l l s of 14 d a y - o l d e m b r y o s . G l i a l c e l l s were cultured up to 4 w e e k s f r o m c o r t i c a l brain c e l l s o f neur b o r n rara. T h e b i o c h e m i c a l p a r a m e t e r s we e x a m i n e d were : proleins, e n z y m a t i c m a r k e r s of n e r v e cell maturation (enolase isoenzymes. g l u t a m i n e s y n t h e t a s e ) a n d superoxide d i s m u t a s e a scanvenger o f free radicals produced d u r i n g alcohol d e g r a d a t i o n . T h e results w e r e c o m p a r e d to appropriate controls. T h e alcohol antagonizing effect was f o u n d 10 be restricted essentially to glial cells. T h e effect on t h e m a r k e r s o f cell m a t u r a t i o n w a s o b s e r v e d i n t h e proliferative period until 2 w e e k s of culture w h e n hln?' w a s a d d e d e i t h e r in v i w together w i t h t h e e t h a n o l d i e t or in vitro 10 glial cells c u l t u r e d f r o m only alcohol e x p o s e d embryos. T h e r e was no e f f e c t in vitro w h e n Mn?. has a l r e a d y been added in vivo. For s u p e r o x i d e d i s m u t a s e o n l y t h e in vilro treatment w a s effective. Our d a t a s u e r e s t that Mnl- may act as a g r o w t h p r o m o t i n g factor o v e r c o m i n g at least panially t h e a l c o h o l i n d u c e d r e t a r d a t i o n o f nerve c e l l d e v e l o p m e n t observed in fetal alcohol syndrome.

140

THE EFFECT OF U~I-BUTANOLON RAT SERUM AMTNO ACIDS M. Hagman, T. Eriksson and K E . Kitson*, Depament of Pharmacology. Gothenburg University, Gothenburg. Sweden and Depament of Chemisoy and Bmhemisoy. Massey University, Palmerston North, New Zealand. Acute adminisoation of ethanol induces a rapid decrease in the wncenwuons of most plasma amino acids. Boh metabolic, i.e. depcndent on the oxidation of erhanol, and non-meiakolic mechanisms have been proposed. In this sNdy we used rerr-butanol as a 1001 Lo elucidate the mechanism behind the amino acid decreasing effect of ethanol ferr-Bulanol is considered 10 have nonspecific physio-chemical effects on cell membranes similar to thore of ethanol. but unlike ethanol it is only minimally rnetahlized. Our results show that t-butanol exens an amino acid decreasing effezl similar 10 Iha! of ethanol. mdicahng lhat lhe mechanism of h e amino acid decrwing effect of ethanol is primanly of a physicechemical nature rafher than a result of the oxidation of ethanol. However, u'e also found thal the effect of ethanol on amino acids was smaller in this panicular group of rats compared wih the results from several other SNdieS. Preliminary results indicate h a t the rats we used in the present study have low alcohol dehydrogenase activity. ?his raises a question as 10 whether here is a correlation between ADH activity and amino acid metabolism during ethanol oxidation.

SELECTIVE SUPPRESSION BY ETHANOL OF GLYCOGEN SYNTHESlS I N OXlDATIVE SKELETAL WSCLES T. N. P a l m e r . R. T h a m b i r a J a h a n d D. Xu, D e p a r t m e n t of B i o c h e m i s t r y , The U n i v e r s i t y o f W e s t e r n Australia. Perth. Australia S k e l e t a l muscle is heterogeneous i n s t r u c t u r e a n d f u n c t i o n . One f a c e t of t h i s h e t e r o g e n e i t y r e l a t e s t o d i f f e r e n c e s between o x i d a t i v e and non-oxidative muscles i n t h e i r capacicy f o r g l u c o s e u t i l i z a t i o n . O x i d a t i v e muscles have f a r h i g h e r r a t e s of g l u c o s e u t i l i z a t i o n i n t h e p o s t a b s o r p t i v e s t a t e . E t h a n o l is r e c o g n i s e d t o c a u s e a c u t e i n s u l i n r e s i s t a n c e and a b n o r m a l i t i e s i n p e r i p h e r a l g l u c o s e u t i l i z a t i o n . The s t u d y a d d r e s s e d t h e q u e s t i o n of w h e t h e r t h i s i m p a i r m e n t 15 m e d i a t e d by d i f f e r e n t i a l e f f e c t s of e t h a n o l o n o x i d a t i v e and non-oxidative muscles. E t h a n o l a d m i n i s t r a t i o n i n t h e r a t was a s s o c i a t e d w i t h ( a ) a r e d u c e d g l y c a e m i c r e s p o n s e t o o r a l or i n t r a p e r i t o n e a l g l u c o s e . and ( b ) t h e a l m o s t c o t a l a b o l i t i o n of glycogen d e p o s i t i o n i n o x i d a t i v e muscles ( v i z . s o l e u s , diaphragm). T h i s e f f e c t was s e l e c t i v e i n t h a t e t h a n o l p r o d u c e d n o e q u l v a l e n t impairment i n glycogen d e p o s i t i o n i n n o n - o x i d a t i v e m u s c l e s . The i n h i b i t i o n of g l y c o g e n s y n t h e s i s i n o x i d a t i v e muscles was ( a ) dosed e p e n d e n t , ( b ) independent o f plasma i n s u l i n l e v e l s , ( c ) and r e l a t e d (p u.u I o c x ~ ~ i i i tlic i ~ cellect ol Ro 154513 o n t h r behaviour and Iwlarsiuin (jOiiihl)-evohd “Cd’ u p t a k by syiiaploneuroromrs i d d i d fruiii the rat braiii cudex Wlowiiig diroiiic ethanol administratiw. hl.ile WiiLlr rats were adiiiinisleral ct1ia1iol in dtinhing water i i i iiicre‘bidig cuiicentrdtiws l r u n 2% to I O Q during 2 inonths (the liinl dil, iiiwlc 0letti;ud was l j g). Witlidnival for 10-24 hrfm Lliruiiic ~ t l i a i i adiiiinirrratioii ~l rrsulted in the devcluyinciit of withdraaal bigis: iii)wloiiic jerhs r i d trctncx riid elcrated polasriumevohed “Ca” uptahe by 3 2 6 In contrast. in ethanol-intoxicated miiiruls iI .5 lu d t u r witlidrawal) no c h ~ i i p in s 4’Ca1’ uplake were olijclicd >I. cornpared to coiil~ulaniiiials. Administration ofRo 15 4513 (4 ni&) to ethanol-intoxicated rats resulted iii the apparance of nillidrawal aiyiis aiid increased polasriutit-ebuked ‘Ca:‘ uptake by 356% a, coiiiptred to the corresponding cotitrd. Administration ul H o l i 4 j l J to 24lir-witlidrawn rats did not f u r t k r increase the scvciity ol’signs a i d “Ca:. uplahe. 1 l i e s data suggest that Ro 154513 i n q prcLipilde nillidmwl dyiis ill ethanol-iiituricaled rdb and ;hi> effect is diic to its actioii uii p o t a s i i u i i - w o h d ‘Ca:. uptake.

reinforcement.

I n a s e p a r a t e set o f s t u d i e s i n anesthetized rats, t h e d i r e c t a p p l i c a t i o n o f e t h a n o l t o t h e n. accumbens was e x a m i n e d u s i n g i n v i v o electroc h e m i s t r y . A t d o s e s of ethanol r a n g x f r o m 5mM t o IOOmM, no release o f d o p a m i n e was o b s e r v e d . The i m p l i c a t i o n of t h e s e f i n d i n g s t o t h e manner i n w h i c h e t h a n o l accesses t h e mesoaccumbens dopamine p a t h w a y will b e d i s c u s s e d .

Symposium 7 Behavioural Assessment of Alcohol Withdrawal Organizer: SE File (UK) s7-1

BEHAVIOR G E N E T I C ANALYSES O F D R U G WITHDRAWAL John Crabhe. Ph.D.. Research Career Scientist. Dept. of Veterans Affairs Medical Center and Professor of Medical Psychology and Pli~rmac~ilo&y, Oregiin Health Sciences Univeriily, Piirlland. Oregon, USA Many studies with rodent models have illustrated the role of genetic factors in determining the severity of ethanol withdrawal. This paper reviews typical findings using inbred mouse strains and mice selectively hred to be ethanol Withdrawal SeizureProne (WSP) or -Resistant (WSR). A consistent finding has been that genotypes susceptible to alcohol withdrawal are also susceplihle to withdrawal from Other drugs with CNS depressant effects. A new method for rough genetic mapping of genes associated with withdrawal, Quantitative Trait Loci (QTL) gene mapping, has been employed in the BXD RI recombinant inbred mouse series. Several QlU are associated with alcohol withdrawdl, and one area of Chromosome 2 contains a QTL marker lor a gene affecting withdrawal from ethanol, nilriius oxide. and high-pressure neurological syndrome-induced convulsions.

S7-2

S7-5

E.H. J o y c e ’ a n d T.W. Robbins..

.

Academic ‘ D e p a r t m e n t of P s y c h i a t r y , C h a r i n g C r o s s a n d W e s t m i n s t e r M e d i c a l S c h o o l , UK, a n d * * D e p a r t m e n t of E x p e r i m e n t a l P s y c h o l o g y , U n i v e r s i t y of C a m b r i d g e , UK.

It h a s b e e n s u g g e s t e d t h a t b o t h K o r s a k o f f a n d non-Korsakoff a l c o h o l i c s s h a r e certain neuropsychological d e f i c i t s following alcohol withdrawal. S i n c e b o t h g r o u p s h a v e i n common l o n g term a l c o h o l a b u s e , t h i s h a s been p o s i t e d as e v i d e n c e t h a t a l c o h o l p e r se can c a u s e i r r e v e r s i b l e c o g n i t i v e impairment. T h i s hypot h e s i s h a s b e e n e x a m i n e d by c o m p a r i n g age a n d I9 m a t c h e d g r o u p s of d e t o x i f i e d K o r s a k o f f a n d n o n K o r s a k o f f a l c o h o l i c s a n d normal c o n t r o l s on p e n c i l a n d p a p e r a n d c o m p u t e r i s e d (CANTAB) n e u r o p s y c h o l o g i c a l tests. The r e s u l t s i n d i c a t e t h a t Korsakoff, b u t n o t non-Korsakoff, a l c o h o l i c s have neuropsychological d e f i c i t s d i r e c t l y a t t r i b u t a b l e t o f r o n t a l - l o b e and medial temporallobeldiencephalic dysfunction. A l t h o u g h nonK o r s a k o f f a l c o h o l i c s showed c e r t a i n n e u r o psychological abnormalities t h e s e could not be r e l a t e d t o d a m a g e of s p e c i f i c n e u r o n a l s y s t e m s . T h e r e l a t i o n s h i p t o these f i n d i n g s a n d l e n g t h O f abstinence w i l l be discussed.

ULTRASONIC VOCALIZATION DURING ETHANOL WITHDRAWAL IN RATS AS AN INDEX OF WITHDRAWALINDUCED ANXIETY Darin Knapp. Jane Saien and LA Pohormky The feasibilily of using ultrasonic vocalization as an index 01 anxiety during withdrawal from ethanol is k i n g assessed in rau. Tactile stimulation elicits ultrasonic vocalization in 100% of rats during withdrawal. None of the dextrin-maltose control emit ultrasonic vocalization. The latency and duration of ultrasonic vocalizations during the withdrawal period, and comparison to such other withdrawal signs as tremon will be compared. In addition the effect 01 diazepam, an anxiolytic on the ultrasonic vocalization during withdrawal will be described.

s7-3

COGNITIVE DEFICITS I N KORSAKOFF AND NON-KORSAKOFF ALCOHOLICS FOLLOWING ALCOHOL WITHDRAWAL AND THEIR RELATIONSHIP TO LENGTH OF ABSTINENCE

A COMPARISON OF ETHANOL & BENZODIAZEPINE WITHDRAWAL A C U T E ANXIETY RESPONSES A N D LONG-TERM COGNITIVE DEFICITS Sandra E. File, Psychopharmacology Resrarch Unit. UMDS Guy‘s Hosp:tal. London S E I 9 R T These studies cornpared the behavioural effects o f withdrawal from a 3 - 4 week period of treatment with ethanol or benrodiazepines on acute anxrety responses and aggressive behaviour and cognitive performance 2-6 months later The anxiogentc withdrawal responses were assessed in the elevated plus-marc and the social interaction lest. 7 Sh after wirhdrauil of 10% ethanol from a liquid diet a n d 24h afrer the last dose of chlurdtazepoxide ( I 0 mgfkglday) or diazepam ( 2 mgfkgtday). In both cases the i n c r e v e d anxiety could be reversed by benzudtarepines. a treatment which rnrmraincd the dependence process. In both cases the GABA agonist. baclofen, reversed the anxiety and i t is suggested th:t this was due to an action i t heteroreceptors to decrease 5 - H T release in the hippocampus The S-HT a onist. buspirone has similar effects. again probably due t o l ~ p ~ s ) n a p rai xc i o n . u hereas the reversals of anxiety by 5 - H T receptor antagonists are posrsynaptic. It is suggested that d e s e drugs could be used to treat the withdrawal anxiety, while permitting normal recovery from the dependence process. Finally. it is suggested that the short and long-term reversals of withdrawal anxiety by the benzodiatepine antagonist. flumatenil. indicate that this drug not only treats the increased anxiety. but reverses the dependence Process by reselling the GABA-bentodiazepine receptor complex to i t s drug-naive stare. O n l j the rats preriously exposed to theethanol diet showed long-term behavioural changes. There rats were more submissive to un1:eated r a u intruding into :heir home-cage terrirory a n d showed impairments in between-day habituation. passive avoidance a n d working and reference memory in positively rewarded tasks

Symposium 8 Relevance of Nutrition in Studies of Alcohol-Related Diseases Organizers: B Watzl (Germany) RR Watson (USA)

s8-1

NUTRITIONAL AND ALCOHOLINDUCED MODERATION OF KUPFFER CELL F U N C T I O N David L E a r n e s t , M.D., Professor of Meilicine, Gastroenterology S e c t i o h University of Arizona College of Medicine, Tucson, Arizona 85724, U S A H e p a t i c Kupffer cells a r e t h e largest g r o u p o f fwed tissue macrophages i n t h e body. They play a central role in i m m u n e function by phagocytosis. antigen presentation and secretion o f a wide variety of cyfokines having b o t h direct a n d imrnunodulatory functions. Alcohol excess a n d certain nutritional deficiencies can affect Kupffer cell function. TXis discussion will present data which show separate, progressive a n d addictive effects of Kupffer cell function of feeding nutritionally defined liquid diets and alcohol. The results justify including s e p a r a t e assessment of t h e effects of diet in future studies regarding alwhol-induced alterations i n i m m u n e function.

sg-2 s7-4 648

DIETAFCYFATIY ACIDS AND wonok EFFECTS ON CELLULAR MEMBRANES R. C. Reitz, Dept. of Biochemistry, University of Nevada, Reno. NV 89557, U.S.A. The consumption of e t h a n o l h a s been shown t o exert profound effects on cellular membranes which resul:

s8-5 NUTRITION IN THE PATHOGENESIS OF

in damage and/or adaptation. Both membrane lipids and proteins are effected, but because of the physicochemical properties of ethanol, many of the membrane effects are directly related to the interaction of ethanol with the lipid component of the membrane. In addition t o the direct Lipid-ethanol interaction, ethanol has been shown to dramatically alter lipid metabolism. Triaqlglycerol accumulates dramatically in the liver, and hiosynrhesis of the polyunsaturaled fatty acids seems to be altered via effects upon the acyl-CoA desaturases. Because precursors of both families of unsaturated fatty acids, i.e. 0 3 and 0 6 families, cannot be synthesized de novo, they must be supplied from dietary sources. Thus, the unsaturated membrane fatty acid composition depends upon these dietary fats and their metabolism via the desaturases. Further, the level of dietary fat seems t o play a very important role in ethanol induced damage to various cellular membranes. Diets with high levels of fat greatly enhance liver steatosis as well as liver membrane damage and liver fibrosis. By altering the composition of dietary fat to include either more saturated fatty acids, higher levels of a specific 0 6 fatty acid, alinolenic acid, o r higher levels of the 0 3 fatty acids, biochemical, physiological and neurobehavioral effects of ethanol have been shown t o be modulated. Therefore, it appears that dietary fatty acids may play an important role in altering some of the deleterious effects of ethanol.

s8-3

ALCOHOLIC LIVER DISEASE

S.W. French, Harbor-UCLA Medical Center, Torrance, California USA. Human population studies indicate that the incidence of cirrhosis varies in proportion t o the per capita alcohol and unsaturated fat consumed. Experimental studies on rats fed ethanol chronically by indwelling gastric cannula indicated that the type of fat fed with alcohol alters the pathologic changes found in the liver. This report compares these changes f o r the following sources of dietary fat: tallow, lard, olive oil, fish oil, safflower oil, corn oil and sunflower oil. The severity of the liver pathology correlated with the unsaturated fatty acid content of the fat., When studies were done using diets deficient in Vitamin A or deficient in vitamins, minerals, choline and protein the pathologic changes were accentuated.

Symposium 9 Ethanol and Excitatory Neurotransmitter Amino Acids Organizer: B Shanley (Australia)

ANTIOXIDANT STATUS AND ALCOHOL-RELATEDDISEASES A. Bjornehoe, National Institute of Forensic Turtcology, Oblo. Norway. Chronic cthanol comumption is assuciated with incrcaul insidcnca of a varicty of illnesses, including cancer. SluU~eshave s h o w that ethanol consumption may result in increased oxidative stress with f o m t i o n of lipid proxiddr and f r e t radials. The svweptibility of a given tissue to peroxidation is, however, a function of the overall balance k t w e n prcnxiddnls and snlioxidrnt defense ryrtsms. The latter involw both intra- and extraccllulrr protective factors whers nutrients play an imporIan1role. lmpairul nutritional status of different vitamins and of tnce tlemdnts have been rapnul in ulcoholics. R d u c d ILVCIS vitamin E have k n found in Y m m of alcoholics with and without liver disuse- and in liver bmprier from alcoholics w l h cirrliubs T h e findings may he due to the incruwd oriditwe strcss as rcpond in experimrnlal animls, and be imponant nincc vitamin E i s the major. tf not the only. lipid roluhlr free radical %avenger in wmc tirrucs. Reduced levclr of serum s l d n i u m havc b e n found in s e v c r . ~ Istudies of alcoholics. Alterations of lscorbic acid and glutathione status have also bcen report&, but these results ars somewhat conflictmg. R d u d hepatic activity of CuIZn-ruproxidc dwnutaM has hccn found m the liver of exprtmcntal rnimalr fdcthilnol. hut ncmnrl i ~ ~ t i v i l i eofs this eitzymc IIAVC k e n rcprtcd i n hlwxl frwn ~ICOJII~IICI. Hcduid n~lior~danr rapxity nuy promuis the gsncrrum of hcc rad~crlsw d lipid peroxides which may damayc cells d i r ~ t l y induct , inflmmmilron

OL AND s9- 1 ETHAh' B. Tabakoff

THE " D A RECEPTOR and P.L. Hoffman, Dept of Pharmacology, Univ of C o l o r a d o Sch of bled, Denver, CO, USA E t h a n o l s e l e c t i v e l y m o d u l a t e s th'e a c t i v i t y Of s e v e r a l r e c e p t o r - g a t e d i o n c h a n n e l s . The NPLDA r e c e p t o r - g a t e d c a t i o n c h a n n e l is p a r t i c u l a r l y s e n s i t i v e t o a l c o h o l ' s a c t i o n s . We have d e m o n s t r a t e d t h a t a l c o h o l ' s e f f e c t s c a n be r e v e r s e d by g l y c i n e and s t r u c t u r e - a c t i v i t y studies indicate that t h i s reversal occurs t h r o u g h i n t e r a c t i o n s of g l y c i n e w i t h t h e s t r y c h n i n e i n s e n s i t i v e g l y c i n e s i t e on t h e " D A r e c e p t o r . C h r o n i c t r e a t m e n t of a n i m a l s , o r c e l l s i n c u l t u r e , w i t h e t h a n o l p r o d u c e s an upr e g u l a t i o n of t h e " D A r e c e p t o r , b u t h a s l i t t l e o r no e f f e c t on k a i n a t e r e c e p t o r - c o u p l e d c h a n n e l s . Our s t u d i e s a l s o d e m o n s t r a t e t h a t mice s e l e c t i v e l y bred f o r a l c o h o l withdrawal s e i z u r e s e n s i t i v i t y (WSP) mice have h i g h e r l e v e l s of NMLlA r e c e p t o r s i n t h e hippocampus t h a n withd r a w a l s e i z u r e r e s i s t a n t (WSR) mice. S t u d i e s using specific antagonists acting a t the " D A r e c e p t o r complex a l s o i n d i c a t e t h a t h W A r e c e p t o r u p - r e g u l a t i o n may b e r e s p o n s i b l e f o r components of CNS h y p e r e x c i t a b i l i t y s e e n i n a l c o h o l w i t h d r a w i n g a n i m a l s and humans.

and ilccelsrdlc ccdlagcn ayiitlicais Thebc cvcnts may p w g r c ~ sto ti>,ue cI.~n~dyc cud dirciru. The ~nqx,n;mced rrd$cals111 uaiiier U U I I J ~ Nand ~ proniotim i b prc>cntly given n d i attciitiim TIICrule a11 I ~ p d peroxides and free rrdiial. in alcthol relrtd dlwrss and CIIICCT is, hwcvcr, .IS yet ~ i n r o ~ l v e dMore . rcwarch B S r q u i r d in this ticld to c ~ l d l i l i 4 thc i I ~ d C t l i c ~ cIC.ICIIVC ~ W C I in C ~IIIE pthugcnc\i. 01

i(1'IhII ICIII1L.d ,l,,CJIC, And 1') the a n t m I d m t d d h r c I I I ~ I

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MODULATION OF CANCER GROWTH BY VITAMIN E (E) AND

S9-2 E " 0 L

ALCOHOL C.D. Eskelsont.4. O.E. Odeleye2.4. R.R. Wason2.4. D.L. Ean~est~.~. Depl. of Surgery].Dcpt. of Family and Comkunity Medicinel. Dcpt. of Intcmal Medicinef,and the NlAA Alcohol Research Ceitler'. The University of Arizona H e l l h Sciences Ccnlcr.Tucson. A 2 85724 Seventy-fivepcrccnt of esophagealcancers are alcohol.rclated yet dcohol is considered a tumor promolor and not a carcinogen. Ethanol is oridilrd to acetaldehyde by: alcohol dchydrogcrwse, catalaw ~d the niicrosomal ethanol oridiiing system (MEOS). Free radicals (FR) arc released when ethanol is oxidized by the MEOS. Thcreforc,living systems have a FR burden when hey mewbolize ethanol. FR rapidly react with biological mxcrials. it.. lipids, proteins. nucleic acids. etc., forming toxic FR products. This study fucuscs on ihe effcclsof FR and/or FR pruducls on cancer-promotionduriiig alcohol ntetabltrm. Mice wcrc dividcd intu 8 grwps and fed nulritiorully dcqwte liquid dies. Groups 1-4 were orally givcn the esophngca!-carcinogcnN-nitrosmcthylbmrylamme (NhIBzA). The groups were lhen supplemenledas follows: I - 5% EtOH 2 - 160 IU of E%g of diet; 3.5% EtOH wich E: 4 - XgUlU diet: 5 chanol; 6 - E enriched diets; 7 .E enriched diets with 5% ethmol; 8 .liquid diet only. Following fccding fur 17 weeLs, livers and crophagus were removed and Ihc FR burdcn in ihc liver measured by lipid pcroxidc pruducls ivid Ihe nwntcr of tymo~sin Ihe esophagusdcierniincd. The h w ruggot that FR andlor FR products the cancer proniotcn during ethanol metabolism. since diets supplemenled with high levels of vitmin E. which inhibits FR activity and thus the formation of FR products. suppresses cancer "promotion by cthuiul." Therefore. alcohol is not a good cancer promoter, but the FR produccd during ethanol metabolism. andlor the products of FR activily. are the cancer-promoters writ& to ethulol. ~

Supponcd by N l A M 08237

649

ACTION O N EXCITATORY AMINO ACID ACTNATEDIONCHANNEl3 F.F.Weight. R. Peoples, J. Wright, D. Lovinger and G. White, Lab. Molecular & Cellular Neurobiology, Nad. Inst Alcohol Abuse & Alcoholism. Rockvile, MD 20852, USA The effect of ethanol on excitatory amino acid activated ion channels was investigated using patch-clamp recording methods t o study hippocampal neurones in tissue culture. In whole-cell recording, intoxicating concenuations of ethanol (5-50 mM) inhibit the ion cumnt activated by N-methyl-Daspartate W A ) in a concenuationdcpmdent manner with an IC50 of 30 mM. Kainate- and quisqualate-activated currents. on the other hand, are inhibited by anesthetic concentrations o f ethanol (50-200 mM) in a concenuation dependent manner with an IC50 of 200 mM. The percent inhibition of NMDA-activated c u m n t by ethanol is similar over a range of membrane wtentials from -60 to +60 mV, and efhans docs not alter h e reversal potential of KMDA cumnt. In addition, the pement inhibiuon of h M D A current by ethanol docs not differ with d f f e r e n t concentrations of NMDA (10-1oOOpM), glycine (0.I-lOOpM). Mg2* (0-500 pM),Zn?* (0-20pM), kewmine (0-10 pM),spermine (0& I pM), prorons (pH 6.0-8.0) or &thiothreitol (0 & 2 m.U). In single-channel experiments. the inhibition of NMDA currcnt by ethanol is not associated with a change ofchannel conductance. but is associated with a decrease in both the probability of channel opening and channel open time. The results suggest thar ethanol inhibits NMDA current by altering gating of the channel. rather than by affecring channel conductance, ion permcancc or regulatory sites on

rhe channel.

S9-3 Ethanol inhibits NMDA receptor mediated regulation of immediate early genes. P.A. Wilce, F. Le, D.A.

Symposium 10 Biological Markers of Previous Alcohol Consumption Organizer: M Phillips (USA)

Hume and B.C.Shanley. Alcohol Research Unit. Department of Biochemistry, University of Queensland, St Lucia Qld 4072 Australia. We have investigated Ihe effect of acule and chronic elhanol treatnient on the NMDA receptor using the expression of c-fos as marker. NMDA induced rapid and transient expression of c-fos which was blocked by acute administration of either low doses of ethanol or the NMDA receptor antagonist MK801 but not by the GABA- benzodiazepine receptor agonist clonazepam. The effect of ethanol was not a global non-specific effect since even at higher doses, ethanol failed to inhibit cyos induction by kainic acid or by caffeine. Withdrawal of ethanol after chronic treatment resulted in c-fos expression in the cortex, hippocampus and the cerebellum 10-12 hours later. This could be prevented by prior administration of MKSOI. The results emphasise the importance of Ihe NMDA receptor in the action of ethanol and the potential use of immediate early gene expression in neuropharmacology.

s9-4

S9-5

S 10- I OVERVIEW PROBLEMS, PITFALLS AND OPPORTUNITIES IN MONITORING ALCOHOL CONSUMPTION R. K. Fuller, National Institute on Alcohol Abuse and Alcoholism, Rockville, MD, U.S.A. Otherwise well-designed clinical trials may yield spurious results if methods used to measure treatment response are not valid or reproducible. The most commonly used measure to assess drinking behavior in alcoholism treatment studies is patients' self-reports. Orrego et al. (1979) compared daily urines analyzed for ethanol with self-reports. They found that 50% of the subjects denied drinking at some time when the urine ethanol was positive and 25% consistently lied. Fuller et al. (1988) compared self-reports with collateral reports. The cohabiting collaterals reported twice the frequency of drinking (median values) as the patients. These studies and others indicate that, in addition to self-report, corroborating data are necessary to measure drinking behavior. Advantages and disadvantages of methods that have been used to corroborate self-report such as collateral reports; breath, urine, and blood ethanol tests; and liver tests will be discussed. Methods under development include blood "markers" and devices which measure ethanol in body fluids.

ETHANOL EFFECTS O N NMDA-STIMULATED LEVELS OF EXTRACELLULAR NEUROTRANSMITTERS BY IN VIVO MICRODIALYSIS R.A. Gonzales and L. C. Roper, University Of Texas, Austin, Texas, USA To determine whether t h e inhibition of NMDA receptor function by ethanol observed in vitro may he reflected in vivo, w e measured t h e effects of ethanol administration on levels Of extracellular dopamine (DAI using microdialysi 5 . R a t s were implanted with a guide cannula under anesthesia one week prior to placement o f a microdialysis prohe into t h e striaturn. Basal levels of dialysate DA were around 0.5 pq/Ul. NMDA infusion into t h e striatum of an awake rat caused a concentration-dependent stimulation of extracellular DA levels. A o n e hour infusion of NMDA caused increases in dialysate DA from 3.5-49 fold over basal at concentrations from 0.3-10 mu. MK-001 ( 2 5 uM) and DNPX ( 1 0 uM1 siqnificantly inhibited the stimulation Of dialysate DA levels by 1 mM NHDA for 1 0 minutes. This suggests complex regulation of extracellular DA levels in t h e striatum by NMDA. Ethanol (0.5-4 g/kq,i.p.) did not alter t h e dialysate DA levels in t h e presence or absence of NMDA. Ethanol's actions on extracellular DA may not be predicted from a sinqle effect of ethanol on one transmitter system but depend on t h e inteqration of multiple signals in this brain reqion. (Supported by a grant from NIAAA, L A A ~ U ~ R ~ I

GLUTAMATE RECEPTOR CHANGES IN BRAIN SYNAPTIC MEMBRANES FROM HUMAN ALCOHOUCS. E.K. Mlchaells, Oepartmenl of Phannacology & Toxleology, UnlvwJity of Kansas, Lamence, KS 68045 Ethanol admlnlstered acutely Inhibits glutamate neurotransmlssion, especially glutamate acllvatlon of NMDA receplors. Several years ago we suggested that braln neurons may adapt Lo thls lnhlbitlon by lncreaslng the receplors for L-glutamlc acid. Chronlc alcohol treatment of experimental anlrnals Increased L['Hjglutamate blndlng sites and lrnmunoreactlve sites In neuronal membranes labeled by an antlglutamate blndlng protein antlbody. Brain membranes from human alcohollcs 81.90 exhibited Increases In llgand binding to agonlst recognition sltes. We obtalned evidence also for dlfferentlal regoiatlon of expresslon of NMDA receptor agonlst vs antagonist blndlng sites In bralns from human alcoholics. These observatlons wlll be dlscussed In the context of cunent molecular bloiogical Information about the NMDA receptors.

SIO-3COKPARISON OF NEW NETHODS

OF' CARBOHYDRATEDEFICIENT TRANSFERRIN fCL7Tl KEASUREKENTS : APPLICATION TO A PUBLIC HEALTH APPROACH FOR THE PREVENTION OF ALCOHOLIC CIRRHOSIS. C.S. Lieber, Bronx VAnC L Kt. Sinai School of Medicine, New York, NY (USA). Recently, Xin et al. (Alcoholism: Clin Exp Res 15: 814 1991) developed an isoelectric focusi;g/Western blot (IEF/WB) procedure for CDT measurement which compares favorably with the micro anionexchange chromatography/radioimmunoassay of Stibler et al. (Alcoholism: Clin Exp ReS 10:535, 1986). The latter is now marketed in a modified version (Stibler et al. Alc. Alc. Suppl 1:451, 1991). which correlates with IEF/WB but yields nearly 5-fold lower values and sianificantlv lesser sensitivity for detec-&g recen't drinkers who are also less well discriminated from non-drinking patients with liver diseases. using IEF/WB, we now screen far heavy drinking. Complications of liver disease are then detected with standard liver function tests. Next w e screen for precirrhotic lesions by serum collagen propeptides (Fab PIIIP) or, when possible, liver biopsy. Patients with perivenular and perisrnusoidal fibrosis at high risk to rapidly develop cirrhosis upon continuation of drinking are subjected to intensive treatment, instituted prior to their medical or social disintegration, with the goal to prevent progression to cirrhosis.

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650

Symposium 12 Tissue Culture Methods in Alcohol Research Organizers: JM Littleton (UK) P Hoffman (USA)

SIO-4 TRANSDERMAL DOSIMETRY n. P h i l l i p s , S t . Vincent's

Medical Center of Richmond and New York Medical College. New York. USA

The transdermal dosimeter (TDD) is a device f o r continuously c o l l e c t i n g f l u i d from t h e s u r f a c e of t h e s k i n f o r periods up to e i g h t days. Ethanol is excreted through t h e s k i n and captured i n the TDD i n a q u a n t i t y which v a r i e s with t h e amount consumed during the period the TDD was worn. The c u r r e n t v e r s i o n of t h e TDD provides:

S12-1ETHANOL-INDUCED TERATOGENIC ALTERATIONS I N DEVELOPINGICARDIOMYOCYTES~IN CULTURE E . D . 4 d i c k e s , T . J . M o l l n e r a n d M.C. l l a k o i d , P e p a r t m e n t s of P a S h o l o g y a n d Neurology and Pharmacology , C r e i g h t o n U n i v e r s i t y S c h o o l of M e d i c i n e , Omaha, NE.

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high s p e c i f i c i t y for e t h a n o l improved s e n s i t i v i t y ; e t h a n o l i s bound t o a c t i v a t e d carbon, reducing l o s s e s from back-diffusion across the s k i n improved adhesion with reduced r i s k of sample loss by shedding or leakage . d e t e c t i o n of unauthorized tempering using adhesive tape which d i s p l a y s a v i s u a l s i g n a l of removal an o f f i c e - b a s e d assay employing a modified breach alcohol m o n i t o r

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USA

T o u n d e r s t a n d t h e m e c h a n i s m of e t h a n o l t e r a t o g e n i c i t y , a n i n v i t r o c a r d i a c myoc y t e model is r e p o r t e d . Dose a n d t i m e d e p e n d e n t e f f e c t s of e t h a n o l a r e t e s t e d on g r o w t h p h a s e e v e n t s . The p h e n o t y p i c expression, growth r e t a r d a t i o n and pers i s t e n c e on p o s t - h y p e r p l a s t i c d e v e l o p m e n t a r e e x a m i n e d t o d e t e r m i n e t h e p e r i o d of v u l n e r a b i l i t y a n d t h r e s h o l d of d a m a g e . Ethanol administered to t h e cultures causes a dose dependent decrease i n t h e c e l l p o p u l a t i o n e x p a n s i o n , e a r l y commitm e n t t o t h e GO c o m p a r t m e n t a n d fewer cells i n t h e a c t i v e c y c l i n g compartments (S+GZ/M), a n d d e l a y e d e x p r e s s i o n o f matu r a t i o n r e l a t e d s a r c o m e r i c p r o t e i n . The r e s u l t a n t p o p u l a t i o n r e p r e s e n t s a n illequipped, protein d e f i c i e n t , functionally i n a d e q u a t e c e l l m a s s , i n c a p a b l e of p a r t i c i p a t i n g i n subsequent embryogenic events. These i n vitro s t u d i e s p a r a l l e l & v i v o e v e n t s j u s t i f y i n g t h e model u s e .

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The TDD provides an o b j e c t i v e method f o r d e t e c t i n g and quantifying alcohol consumption i n an o u t - p a t i e n t population.

Symposium 11 The Role of Calcium Charrnels in Alcohol Dependence and Withdrawal Organizer: JH Little (UK)

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S11-1 INCREASESINSYNAPTICEXCITATIONLEADINGTO ACTIVATION OF ENHANCED CALCIUM CURRENTS DURING ETHANOL WITHDRAWAL IN VITRO M.A. Whittingfon. J.D.C. Lambert' and H.J. Little, Pharmacology Department, University of Bristol, U.K. and 'Institute of Physiology, University of Aarhus, Denmark. Ethanol wifhdrawal is characferised by a period of cenfral nervous system hyperexcitability. Increases in the numher of dihydropyridine-sensitive cdcium channels have been implicated in the generation of this hyperexcifabilify. To investigate the basis of the neuronal changes seen during ethanol withdrawal we measured the following: Pharmacologically isolated GABA, receptor-mediated. and NMDA receptor-mediated synaptic transmission; Whole cell calcium currents under voltage clamp. Recordings were taken from CAI pyramiddl cells in hippocampal slices taken from mice that had been drinking 24% ethanol (v/v) as sole fluid fur 18 weeks, and frum cuntrol slices. Results revealed no change in the maximum sue of GABA,-mediated inhibitow postsynaptic polentials (IPSPs) during the period of epileptiform activity. The size of NMDA recepformediated excitatory postsynaptic potentials (EPSPs) was increased and resulted in synaptically evoked calcium spikes in CAI pyramidal cells during this period of withdrawal. Voltage clamp studies showed an increase in high voltageactivated calcium currents at depolarised membrane potentials. These data suggested n synergism between increased excitatory transmirsion and increased calcium channel function leading fo epileptiform acfivity during ethanol withdrawal.

S u p p o r t e d b y N I A A A AA 07358 (EDA)

S 12-2

ETHANOL ACTICUS MI C12* CURRENTS P e t e r L . Carlcn. YD. FRcP(c), AddiCtim Research Fan;latim, PIeyfair Yeuroscicme Unit, University Toronto

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Ethanol a c t i w on n u r o r r m r a i t t c r receptor mediated i m l c charnels (e.9. G a m . Y M A I have received attenrim. nweve ethanol a110 a f f e c t s VOItage d e p n j e n t t). ChaMCIs and ta" activated K+ charnels. S m e of t h ~ + m t i m sof LthaMl can h e x p l a i d by altered i n t r a c e l l u l a r t a regulation. This lab uses sharp i n t r a c c l t u l r r and whole c e l l microelectrode rrcerdingr f r m ~yurms i n b r a i n slices. Both current- a d V O l t ~ O e ~ C 1 m Q recordings a r t mployed. SinPle-eleCtrode voltage clanp recordinps from h i - a p l dcnrstc piarwle ( P O neyrons have d c m m i t r a t d 3 types Of Ca currents; a 10" threshold t anrient 1 - t y p ta2+ current. a hioh threshold transient Y ' t y g caE current, a d a high ~urrent. threshold 110vly i m c t i w t i n g L-type Ca

s 12-3 EVALUATION OF

i n the hiph threshold currents, e r p c i a l l y the L - t y p current h i c h i s a Currmt blwcired by d i h y d r w r i d l n C . l C i m cham1

an agonists. This e f f e c t was biocked by cheliting i n t r a i r l l u l ~ r by including EGTA i n the recording clectrodt.

[a2+ currents were a t s o mawid i n w r m s f

m slices d z r g o i n p acute ethanol uithdraual. These slices ~ t f efrm drup naive r a t s . and e r e eaposed t o 100 nW ethanol for > 2 hwxlrr. Upon ~ l c o h a l uithdrarsi. the neurons exhibited y p r c x c i t a b i l i t y within 10 to 15 minutes. The high threshold Cap' ~ u r r m t swere eohamed. Finally, h o l e c e l l voltage recordings hawe been Dbtlined f l m OG seizure prone (YSP) a r d withdrawal EeizUre resistant ( Y S r o nice. Preliminary data rhou that i n DG - r M S f r m YSP nice. the depalarizing afterpatenrials are en9anced. reflcctinp ~ m r e a s e dI - t y p e currents.

neurons of iithdra.al

Slppartcd by ABMRF a d HRC

ETHANOL EFFECTS ON PLC SIGNAL CELL LINES OF NEURONAL ORIGIN C.Alling, O.Bergman, C . k n n o n and P.Simonsr0n fmm Dept. of Psychiatry and Neurccbemistry,Lund University. Lund. Sweden. Human neuroblastouta cells SH-SYSY and neuroblprtouta-gliomncells 108-15 have been used u models for the elucidation of the effstr of ethanol on receptor mediated phorpholip= C (PLC) activity and c-fos mRNA expression and on protein kin= C (PKC) activity. Cells were exposed lo ethanol (0-200 mM) for v m g periods up to seven days. Agoorst stimulated events were obtained in NO 108 cells Uilh brndyLinin and in SH-SYSY cells with whchol. Chmnic ethanol exponvt reduced the agonist stimulated IP3 fomlion in NO 108-IScells and in SH-SY5Y cells. 100 mM elhanol for seven days incwed ths membme bound and the cytosolic form of PKC activity in SH-SYSY cells. Carbachol (I mM) induced a mpximal c-foa mRNA response aftcr 40 minutes in SH-SYSY cells that also could bc obtained lhrouph PKC stimulationby TPA.

TRANSDUCTION PATHWAYS USING

Acute ethanol (20 nW) prfured m t o DG neurons ceused a decrease

tk

PROTEIN KINASES AND E N H A N C E M ~OF NEURKE OUTGROWTHBY ETHANOL IN pC12 CELLS R.O. Messing and M. Henteleff. E. Gallo Clinic 8 Research Center, Dept. of Neurology. University of California, San Francism, CA. USA. We used the PC12 cell line as a model system to study the formation of neural processes. Ethanol increased neurite outgrowth in PC12 cells cultured with nerve growth factor (NGF) or basic fibroblast growth factor (bFGF). Similar enhancement of dendrilic growth occurs in some brain regions in rodents dependent on ethanol. Phorbol esters that activate protein kinase C (PKC) also enhanced NGFinduced neurite outgrowth in PC12 cells. Cells cultured in efhanol had increased levels of PKCB and PKCs and increased PKC-mediated phosphorylation. Treatment with 1 &M phorbol 12-myristate, 13-acetate for 24-48 hours dramatically reduced levels of PKCa p, 6, and E. Elhanol failed lo enhance NGF- or bFGF-induced neurite outgrowth in these PKCdepleted cells. Activation of cAMP-dependent prolein kinase (PKA) also increased NGF-induced neurite outgrowth. However, ethanol enhanced neurite outgrowth in the mutant PC12 clone A126-1B2, which is deficient in PKA activity and does not respond to CAMPanalogs. These findings suggest that PKC 8, or E , but not PKA. IS necessary for enhancement of neurite outgrowth by ethanol.

65 1

s 12-1

ETHANOL AND GLUTAHATE RECEPTORS IN CULTURED CEREBELLAR GRANULE CELLS P.L. Hoffman, K.R. I o r i o . L.J. R e i n l i b ' and 8 . Tabakoff, Univ o f Colorado Hlth S c i C t r . Denver, CO USA and 'Natl I n s t Alcohol Abuse and Alcoholism, R o c k v i l l c . KD USA Primary c u l t u r e s of c e r e b e l l a r g r a n u l e c e l l s have been used t o s t u d y t h e e f f e c t s o f e t h a n o l on t h e f u n c t i o n o f t h e mDA and k a i n a t e s u b t y p e s of glutamate receptor. Acutely, ethanol i s a potent and s e l e c t i v e i n h i b i t o r o f NHDA-stimulated c a l c i u m i n f l u x and c y c l i c GHP a c c u m u l a t i o n ; h i g h e r c o n c e n t r a t i o n s of e t h a n o l are n e c e s s a r y t o i n h i b i t the response t o k a i n a t e . After chronic exposure of t h e c e l l s t o e t h a n o l t h e r e i s a n upr e g u l a t i o n of NWA r e c e p t o r f u n c t i o n (measured an an i n c r e a s e i n i n t r a c e l l u l a r c a l c i u m ) . However, t o kainate o r depolarizing the response c o n c e n t r a t i o n s of KC1 i s n o t a l t e r e d . s u g g e s t i n g that, i n cerebellar granule C e l l s . neither r a i n a t e r e c e p t o r s n o r L-type (nifedipine9ensitive)voltage-sensitive c a l c i u m c h a n n e l s a r e i p - r e g u l a t e d a f t e r c h r o n i c e t h a n o l e x p o s u r e . The ihsnge i n NMDA r e c e p t o r f u n c t i o n may s e r v e as a nodel t o a s s e s t h e mechanism f o r i n c r e a s e s i n M D A r e c e p t o r s i n o t h e r b r a i n a r e a 8 of e t h a n o l lependent a n i m a l s . which may c o n t r i b u t e t o iymptoms of e t h a n o l w i t h d r a w a l . Supported i n ) a r t by ADAMHA. PKS ( M 9 0 0 S )

s 13-2 1.1 I A H $1 A c'oI.(

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I.Plinius Maiiir Sucicry ibr study and treatment tit' alciiholism in Eiinqw. ?.UnivenitS Lihre de Bnixelles, Clinique d'Alcooltigie. Campus Rlugmann - PI. Van Gehuchtzn 4, - 1020 Bmsrels - RELGILIM. Many phmacological agents have heen pnip'sed tiir b e treatment of drinking pn)hlems. However. there are many pitfdlls in the asbesinlent niruch drug\. In this prewntaticin. we will review and illusmte them thmugh some examples. The spontdneous iiutcome