FURTHER EVIDENCE AGAINST INHIBITION OF SYNTHESIS OF RAT LIVER TRYPTOPHAN PYRROLASE BY CHRONIC ETHANOL ADMINISTRATION

The decrease in rat liver tryptophan pyrrolase activity causedby chronic ethanol administration has previously been shownto be reversed by addition in vitro of NAD⁺ or NADP⁺ This reversalis now shown to be unaffected by pyrazole, thus suggesting non-involvementof alcohol dehydrogenase in the reversal. By contrast, whenpyrrolase synthesis is inhibited, the decreased activity cannotbe reversed by NAD⁺ or NADP⁺ in vitro. These results providefurther evidence for involvement of NADH and NADPH in the pyrrolaseinhibition by chronic ethanol administration and against thesuggested inhibition of synthesis of the apoenzyme.     

EFFECTS OF ETHANOL ON TESTICULAR STEROIDOGENESIS IN THE RAT

The effects of ethanol and NAD⁺ on the pregnenolone-to-testosteronepathway of testicular steroidogenesis in lysed Leydig cell preparationswere investigated. Testosterone and four precursor steroidswere determined by gas chromatography/mass spectrometry afterincubation of the cell preparations with 100 µM of pregnenoloneand appropriate concentrations of ethanol (1–100mM). Concentrationsof all 4-ene-steroids measured were significantly decreasedeven at the lowest concentration of ethanol. When NAD⁺ (0.1mM) was added to the incubation medium, the levels of progesteroneand 17-hydroxyprogesterone returned to control values, whereasthose of androstenedione and testosterone remained decreased.These results suggest that ethanol may inhibit testicular steroidogenesisby suppressing at least two steps in the pregnenolone-to-testosteronepathway, the pregnenolone-to-progesterone step catalysed byNAD⁺-dependent 5-ene-3ß-hydroxysteriod dehydrogenase/isomeraseand the 17-hydroxyprogesterone-to-androstenedione step catalysedby the NAD⁺-independent C_17–20 lyase.     

ALDEHYDE DEHYDROGENASE ACTIVITY AND ACETATE PRODUCTION BY AEROBIC BACTERIA REPRESENTING THE NORMAL FLORA OF HUMAN LARGE INTESTINE

We have recently proposed the existence of a bacteriocolonicpathway for ethanol oxidation, i.e ethanol is oxidized by alcoholdehydrogenase of intestinal bacteria resulting in high intracoloniclevels of reactive and toxic acetaldehyde. This study was aimedto examine aldehyde dehydrogenase (ALDH) activity, acetaldehydeconsumption and production of acetate by aerobic bacteria (n=27),representing the normal human colonic flora. Most bacterialstrains did not show any membrane-associated aldehyde dehydrogenase,but possessed marked cytosolic NADP⁺- and NAD⁺-dependent aldehydedehydrogenase activity, ranging from 155 nmol of NAD(P)H produced/min/mgof protein to zero with acetaldehyde as substrate. NADP⁺-linkedALDH activity was significantly higher than NAD⁺-linked activityin most of the tested bacteria. In addition, aerobic bacteriametabolized acetaldehyde effectively in vitro and this couldbe inhibited by cyanamide in nearly half of the tested strains.Production of acetate from acetaldehyde ranged from 2420 nmol/10⁹colony-forming units to almost negligible. In conclusion, manyhuman aerobic colonic bacteria possess significant aldehydedehydrogenase activity and can, consequently, produce acetatefrom acetaldehyde in vitro at least under the partially aerobicconditions proposed to prevail on the colonic mucosal surface.Individual variation in the capability of colonic flora to removetoxic acetaldehyde may be one factor regulating intracolonicacetaldehyde levels, as well as the rate of bacteriocolonicpathway for ethanol oxidation.     

THE EFFECT OF NALOXONE ON THE HEPATOCELLULAR REDOX STATE AND SERUM ETHANOL CONCENTRATIONS FOLLOWING ACUTE ETHANOL ADMINISTRATION

Naloxone hydrochloride (2.0 mg/kg) has been found to reversethe significant decreases in the hepatic cytosolic and mitochondria1[NAD⁺]/[NADH] ratios observed after acute ethanol administrationin rats. This correction of the ethanol-induced changes in thehepatocellular redox state by naloxone was, however, not aswiatedwith any lowering of serum ethanol concentrations or an observablereduction in the extent of intoxicatlon. Thls lack of antagonismof alcohol intoxication by naloxone was not affected by thefeeding status of the animals, the time point after naloxoneadministration at which serum ethanol concentration was determinedor the method used for ethanol analysis. Thus this study hasfailed to confirm that naloxone antagonises acute alcohol intoxication,in spite of 11s potent abllity to reverse the ethanol-inducedchanges in the hepatic redox state.     

CHARACTERISTICS OF ALDEHYDE DEHYDROGENASES OF CERTAIN AEROBIC BACTERIA REPRESENTING HUMAN COLONIC FLORA

We have proposed the existence of a bacteriocolonic pathwayfor ethanol oxidation resulting in high intracolonic levelsof toxic and carcinogenic acetaldehyde. This study was aimedat determining the ability of the aldehyde dehydrogenases (ALDH)of aerobic bacteria representing human colonic flora to metabolizeintracolonically derived acetaldehyde. The apparent Michaelisconstant (K_m) values for acetaldehyde were determined in crudeextracts of five aerobic bacterial strains, alcohol dehydrogenase(ADH) and ALDH activities of these bacteria at conditions prevailingin the human large intestine after moderate drinking were thencompared. The effect of cyanamide, a potent inhibitor of mammalianALDH, on bacterial ALDH activity was also studied. The apparentK_m for acetaldehyde varied from 6.8 (NADP⁺ -linked ALDH of Escherichiacoli IH 13369) to 205 µM (NAD⁺ -linked ALDH of Pseudomonasaeruginosa IH 35342), and maximal velocity varied from 6 nmol/min/mg(NAD⁺ -linked ALDH of Klebsiella pneumoniae IH 35385) to 39nmol/min/mg (NAD⁺ -linked ALDH of Pseudomonas aeruginosa IH35342). At pH 7.4, and at ethanol and acetaldehyde concentrationsthat may be prevalent in the human colon after moderate drinking,ADH activity in four out of five bacterial strains were 10–50times higher than their ALDH activity. Cyanamide inhibited onlyNAD⁺ -linked ALDH activity of Pseudomonas aeruginosa IH 35342at concentrations starting from 0.1 mM. We conclude that ALDHsof the colonic aerobic bacteria are able to metabolize endogenicacetaldehyde. However, the ability of ALDHs to metabolize intracolonicacetaldehyde levels associated with alcohol drinking is ratherlow. Large differences between ADH and ALDH activities of thebacteria found in this study may contribute to the accumulationof acetaldehyde in the large intestine after moderate drinking.ALDH activities of colonic bacteria were poorly inhibited bycyanamide. This study supports the crucial role of intestinalbacteria in the accumulation of intracolonic acetaldehyde afterdrinking alcohol. Individual variations in human colonic floramay contribute to the risk of alcohol-related gastrointestinalmorbidity.     

COMPARISON OF THE ACUTE EFFECTS OF ETHANOL ON LIVER AND SKELETAL MUSCLE PROTEIN SYNTHESIS IN THE RAT

(1) The acute effects of ethanol on protein synthesis by liverand skeletal muscle were investigated in young (95–100g) rats. Rats were injected intraperitoneally with ethanol,75 mmol/kg body wt; controls were injected with isovolumetric0.15 M NaCl. After 140 min rates of protein synthesis were measuredby injection of a large dose of L[4³H]phenylalanine and at 150min rats were killed. (2) Fractional rates of protein synthesisin control animals were approximately four to five times greaterin liver than muscle. Absolute rates were, however, comparablein liver and skeletal muscle. Ethanol reduced the fractionalrate of liver protein synthesis by 5–20%; the responsefor muscle was relatively greater (25–30%). The decreasein the amount of protein synthesised by muscle was also greaterthan that by liver. (3) After 150 min, plasma gamma-glutamyltransferase, alanine aminotransferase, alkaline phosphatase,lactate dehydro-genase and creatine kinase activities were alldecreased by 25–60%. Aspartate aminotransferase activitywas increased by 42%, though this was not statistically significant.(4) Increased plasma glucose and triglycerides in ethanol-dosedrats indicated that limitations in substrate supply were notmediating factors in reducing protein synthesis. Ethanol wasalso able to exert its effects in the presence of elevated insulinlevels. A direct effect of ethanol, or its metabolites, on proteinsynthesis, is therefore implied.     

CHARACTERIZATION OF RAT TESTICULAR ALCOHOL DEHYDROGENASE

A protein from rat testes that catalyzes the oxidation of ethanolin the presence of NAD⁺, but not NADP⁺, has been characterizedenzymatically and compared to that of hepatic alcohol dehydrogenaseobtained from the same animals. The testicular enzyme, likethe hepatic enzyme, has a K_m value for ethanol in the 0.5–1.0-mMrange and can utilize other alcohols such as n-propanol, n-butanol,and isobutanol, although the K_m values for these other alcoholsare considerably lower (0.03–0.08 mM) that that for ethanol.The testicular enzyme is more heat-labile than is the hepaticenzyme. Finally, the testicular enzyme catalyzes the oxidationof retinol and its retinol dehydrogenase activity is inhibitedby ethanol.     

THE ROLE OF GLUCOSE AND INSULIN IN THE EFFECT OF ETHANOL ON PROTEIN SYNTHESIS IN ISOLATED RAT HEPATOCYTES

The incorporation of ¹⁴C-valine into liver protein was studiedin isolated rat liver parenchymal cells. Various glucose levelsin the incubation media did not affect the rate of ¹⁴C-valineincorporation into proteins. The insulin stimulated incorporationof ¹⁴C-valine into proteins was also unaffected by the variousglucose levels. Ethanol decreased the incorporation of ¹⁴C-valineinto liver proteins, affecting stationary and export proteinsto the same extent. This inhibitory action of ethanol on valineincorporation was reversed by increasing exogenous glucose concentrations.The combination of insulin and high glucose level totally preventedthe ethanol inhibition.     

INCREASED MALATE DEHYDROGENASE ACTIVITY IN BLOOD FROM NON-DRINKING ALCOHOLICS

Since cytosolic malate dehydrogenase has been shown to playa role in the regulation of liver cytosolic [NAD⁺]/[NADH] redoxstate during ethanol metabolism, it is possible that differencesin this enzyme could cause differences in response to ethanol.The present study demonstrates that the isozyme pattern of thiscytosolic enzyme in whole blood samples is the same as thatin liver and that the pattern does not differ in alcoholic andcontrol subjects. A marginally significant elevation of activityof malate dehydrogenase in blood from alcoholic subjects isreported. Further studies are needed to confirm this latterfinding and to assess fully its possible significance.     

ALTERATION OF ACETALDEHYDE METABOLISM IN CARBON TETRACHLORIDE-INTOXICATED RAT LIVER: ANALYSIS USING LIVER PERFUSION SYSTEM

The hepatic metabolism of acetaldehyde in carbon tetrachloride(CCl₄)-intoxicated rats was studied using a non-recirculatinghaemoglobin-free liver-perfusion system. Acetaldehyde uptakeby the liver from acutely CCl₄-treated animals (4.16 mmol/kg,i.p.) at 24 hr after the treatment was not significantly altered,whereas that by the liver from chronically CCl₄-treated animals(2.08 mmol/kg,i.p., twice a week, for 8–12 weeks) wasdecreased by approximately 50% when it was determined in thepresence of 0.01–5 mM acetaldehyde. In liver from ratschronically intoxicated with CCl₄, the following important biochemicalchanges were observed: (1) The activity of low K_m aldehyde dehydrogenase(ALDH) in hepatic mitochondria was decreased by approximately75%. (2) The basal levels of the lactate/pyruvate (cytosolic[NADH]/[NAD⁺]) ratio as well as the ß-hydroxybutyrate/acetoacetate(mitochondrial [NADH]/[NAD⁺]) ratio were elevated by more than2-fold. (3) Mitochondrial NADH oxidation was also reduced byapproximately 35% of the control level. (4) The basal levelof hepatic oxygen uptake was attenuated by approximately 50%,and the infusion of acetaldehyde (0.01–5.0 mM) causeda further decrease in the uptake. (5) The rate of ethanol productionfrom acetaldehyde by the catalytic action of alcohol dehydrogenasewas found to be unaltered when low concentrations of acetaldehyde(0.01–0.2 mM) were used, whereas a significant suppressionof the rate of ethanol production was detected in the presenceof high concentrations of acetaldehyde (0.6–5 mM). Thesedata suggest that the changes in activity of the lowK_m mitochondrialacetaldehyde dehydrogenase and those in mitochondrial NADH oxidationcoupled with mitochondrial respiration may, at least in part,play important roles in the decreased hepatic acetaldehyde metabolismobserved in chronically CCl₄-treated rats.     

ETHANOL-INDUCED INHIBITION OF TESTOSTERONE BIOSYNTHESIS IN VITRO: LACK OF ACETALDEHYDE EFFECT

Acute ethanol intake has been shown to decrease plasma testosteronelevels in rodents and man. This is mainly due to inhibitionof testosterone synthesis, but there is still some controversyabout the mechanisms responsible for the inhibition. These mechanismswere studied in the present experiments with isolated rat Leydigcells. Leydig cells were incubated for two hours in sealed plasticvials with various concentrations of ethanol (0.3–40 mM)or acetaldehyde (5–40 µM). Very low ethanol concentrations(2.5 mM) reduced testosterone production significantly, whereaseven 40 µM acetaldehyde added repeatedly to the incubationmedium had no effect on testosterone levels. An inhibitor ofalcohol dehydrogenase, 4-methyl-pyrazole, abolished the ethanol-inducedinhibition of testosterone synthesis. A suggested mechanismfor the inhibition is an elevated free [NADH]/[NAD⁺] ratio inLeydig cells caused by the metabolism of ethanol.     

Pre-natal and post-natal effects of alcohol in the rat--II. Changes in gamma-aminobutyric acid concentration and adenosine triphosphatase activity in the brain

The effects of chronic alcohol administration during the pre-nataland/or post-natal period on concentration of aminobutyric acid(GABA) in the amygdaloid cortex and striatum and on activitiesof Na⁺, K⁺- and Mg²⁺ -activated adenosine triphosphatases (ATPases)in the mid-brain and hippocampus were studied. There was a smalldecrease in the GABA content of both brain regions in thoseoffspring that had received alcohol immediately before, butnot in those that were free of the drug at the time of death.This suggests that the changes in GABA concentration are dueto a direct pharmacological effect of ethanol. A decrease alsooccurred in the activity of the Mg²⁺ -activated ATPase in thoseoffspring exposed to alcohol during the period of weaning. Thiseffect could not be attributed to a direct action of the drug,but may be indicative of a longer-term influence of ethanolon membrane transport processes. The increase in the activityof the Na⁺, K⁺ -activated ATPase in the mid-brain of rats thatwere exposed to the drug pre-natally and post-natally and followingweaning is probably due to a direct effect of the drug.     

ACUTE EFFECTS OF ETHANOL ON PROTEIN SYNTHESIS IN THE RAT

The effect of ethanol, administered acutely (75 mmol kg^–1),on protein synthesis in the whole body and in the tissues ofthe rat was investigated. Ethanol decreased whole-body proteinsynthesis by 41% and also that in individual tissues (liver:60%; muscle: 75%; heart: 45%; kidney: 59%; spleen: 73%; plasmaprotein: 44%; lung: 64%). These data indicate a generalisedeffect upon protein synthesis and are discussed in relationto the possible mode of action of ethanol.     

EFFECTS OF BRAIN MEMBRANES ON 1H NUCLEAR MAGNETIC RESONANCE SIGNAL INTENSITY OF ETHANOL IN VITRO

In vivo proton nuclear magnetic resonance (¹H NMR) studies ofethanol in animal and human brains have shown that only a fractionof ethanol in brain is visible by NMR. The goals of these invitro ¹H NMR experiments were to determine: (I) whether theinteraction of ethanol with brain membranes in vitro diminishesethanol visibility; and (2) if a magnetization transfer (MT)effect can be observed for the interaction of ethanol with brainmembranes in vitro. Furthermore, pilot studies were performedto determine if the brain membranes from rats chronically exposedto ethanol had a different effect on ethanol NMR visibilityand spin-spin relaxation time (T2) than brain membranes obtainedfrom control rats. Results show that the NMR visibility of ethanolis lower in rat brain membrane suspensions in vitro as comparedto ethanol in saline solutions. The factors decreasing ethanolNMR visibility are T2 relaxation, water presaturation time,and off-resonance saturation by a frequency-dependent MT pulse.One-pulse NMR measurements without water presaruration showedthat ethanol visibility was significantly increased by 15% inbrain membrane suspensions of ethanol-fed rats, suggestive ofdecreased ethanol partitioning compared to controls. Furthermoreethanol in brain membrane suspensions from ethanol-fed ratsshowed smaller MT effects than from control rats. These resultsprovide a mechanism for decreased NMR visibility of ethanolin brain, and suggest that chronic exposure to ethanol producesmembrane changes which result in increased NMR visibility.     

THE EFFECTS OF CHRONIC ETHANOL ADMINISTRATION ON STIMULATED PAROTID SECRETION IN THE RAT

Young adult male rats were maintained on a regimen of twicedaily intragastric administration of 4 ml of 35% (v/v) ethanolin water (daily dose approximately 7.0 g/kg) or 4 ml of calorificallyequivalent sucrose solution over approximately 100 days. A secondcontrol group received no intragastric solutions. Under Valium-Hypnormanaesthesia, parotid saliva was collected by intra-oral ductcannulation following stimulation by pilocarpine (10 mg/kg,subcutaneously) or by isoprenaline (30 mg/kg s.c). In the ethanol-dosedrats the initial parotid salivary flow rate was raised in comparisonto control animals by 39% after pilocarpine stimulation andby 37% after isoprenaline stimulation. The protein concentrationwas significantly reduced in saliva from ethanol-dosed ratscompared to control groups but the amylase activity per ml ofsaliva was not significantly different from control groups.After pilocarpine stimulation, the [K⁺] was significantly higherin ethanol-dosed rats than in controls, but the [Na⁺] was effectivelylower (allowing for flow rate differences). The blood-ethanollevel was zero at the time of collection of saliva, suggestingthat the salivary differences are due to functional adaptationinduced in acinar and ductal epithelia by prolonged repeatedexposure to high doses of ethanol.     

THE EFFECT OF CHRONIC ETHANOL CONSUMPTION ON IRON ABSORPTION IN RATS

Alcoholics often have an increased amount of iron in the liverwhich may contribute to the development of alcoholic liver disease,although the mechanism is unknown. It has been shown that chronicethanol intake decreases the enterocyte turnover and enhancesgalactose absorption. Whether it affects iron absorption isstill controversial. The aim of this study was to investigatethe effect of chronic ethanol ingestion on whole body iron absorptionin rats. Twenty-eight adult male Sprague-Dawley rats were pair-fed aliquid diet containing either ethanol as 36% of total caloriesor an isocaloric diet where fat was substituted, for ethanol.On the 28th day, four-hour fasted rats were given an oral doseof ⁵⁹Fe (0.5µCi) and were immediately counted by a wholebody counter. ⁵⁹Fe levels were then monitored over the followingnine days. Although ethanol- and control-fed rats had a similarhepatic iron content (59.5±5.8 vs 60.2±7.4 µg/100mg dry liver weight) (mean±S.E.M.), the ⁵⁹Fe total bodycontent was greater in the ethanol group (75%±3%) comparedwith the control group (45%±4%). These results show thatchronic ethanol ingestion increased iron absorption in rats.A reduction of enterocyte turnover may play a role in determiningthis effect.     

Oral Administration of Nicotinamide Mononucleotide Increases Nicotinamide Adenine Dinucleotide Level in an Animal Brain

As a redox-sensitive coenzyme, nicotinamide adenine dinucleotide (NAD⁺) plays a central role in cellular energy metabolism and homeostasis. Low NAD⁺ levels are linked to multiple disease states, including age-related diseases, such as metabolic and neurodegenerative diseases. Consequently, restoring/increasing NAD⁺ levels in vivo has emerged as an important intervention targeting age-related neurodegenerative diseases. One of the widely studied approaches to increase NAD⁺ levels in vivo is accomplished by using NAD⁺ precursors, such as nicotinamide mononucleotide (NMN). Oral administration of NMN has been shown to successfully increase NAD⁺ levels in a variety of tissues; however, it remains unclear whether NMN can cross the blood–brain barrier to increase brain NAD⁺ levels. This study evaluated the effects of oral NMN administration on NAD⁺ levels in C57/B6J mice brain tissues. Our results demonstrate that oral gavage of 400 mg/kg NMN successfully increases brain NAD⁺ levels in mice after 45 min. These findings provide evidence that NMN may be used as an intervention to increase NAD⁺ levels in the brain.     

EFFECT OF ETHANOL ON NEUROMUSCULAR FUNCTION IN THE RAT DIAPHRAGM PREPARATION

The effect of ethanol on neuromuscular transmission in the isolatedphrenic nervediaphragm preparation of the rat was studied byanalysing its effect on directly and indirectly-elicited twitchtension, tetanic and post-tetanic twitch tensions, and on thephenomenon of post-tetanic twitch potentiation (PTP). The resultsshow that ethanol (1.0 x 10^–4-1.0 M) depresses neuromusculartransmission, by reducing the amplitudes of both directly andindirectly elicited twitch and tetanic contractions. Ethanolproduces a greater reduction in the amplitude of indirectlythan directly-elicited twitch tension and the mean concentrationof ethanol which produced a 50% reduction was 38 mM. High concentrationsof ethanol (> 10^–3M) completely blocked the indirectly-elicitedtwitch tension in 4 min exposure. Ethanol also reduced the tetanictension and increased the PTP value. There was no tetanic fadein the presence of ethanol, but the peak tetanic tension wasreduced by 20–50%. It is suggested that ethanol inhibits neuromuscular transmissionin the rat phrenic nerve-diaphragm preparation, possibly viaa mixture of pre- and postjunctional mechanisms and producesa greater reduction in the twitch than tetanic tension.     

THE EFFECTS OF THE NITRIC OXIDE SYNTHASE INHIBITOR 7-NITROINDAZOLE ON ETHANOL PHARMACOKINETICS IN RATS AFTER ACUTE AND CHRONIC ETHANOL ADMINISTRATION

The aim of this work was to study the effects of the nitricoxide synthase (NOS) inhibitors 7-n,troindazole (7-NI) and N^G-nitro-L-arginine(L-NOARG) on the effects and pharmacokinetics of ethanol inrats. Ethanol at a dose of 4 g/kg, i.p. induced sleep in rats(sleep time: 117.2 ± 30.7 min). Administration of theNOS inhibitors 7-NI (20 mg/kg, i.p.) and L-NOARG (20 mg/kg,i.p.) 30 min before ethanol significantly increased the durationof ethanol-induced sleep. L-NOARG also significantly increasedthe toxicity of ethanol as evidenced by increased post-experimentallethality Ethanol at a dose of 2 g/kg (i.p.) did not inducesleep in vehicle-treated rats; however, the combined administrationof ethanol (2 g/kg) and 7-NI at doses of 40, 80, and 120 mg/kgcaused sleep, for 49.4 ± 3.7, 204.0 ± 13.3, and447.5 ± 62.8 min, respectively. L-NOARG (20 mg/kg) hadno effect on ethanol concentrations in blood after acute ethanoladministration (4 g/kg). 7-NI in lower doses (20 and 40 mg/kg)had no effect and in higher doses (80 and 120 mg/kg) significantlyslowed ethanol clearance during the 12 h after ethanol administration.The effect of 7-NI (20 mg/kg) on ethanol pharmacokinetics afterchronic ethanol administration (inhalation for 18 days) wasalso studied. The administration of 7-NI immediately after theend of ethanol exposure had a pronounced effect on ethanol pharmacokinetics;in 7-NI-treated rats the fall in ethanol concentrations wassignificantly slower as compared with vehicle-treated rats.In 7-NI- treated rats, blood-ethanol levels were higher at 3,6, 9, and 12 h after the end of ethanol exposure.     

AN ANAESTHETIC DOSE OF ETHANOL INHIBITS CYCLIC AMP FORMATION BY ALTERING THE FUNCTION OF GS PROTEIN

The effects of ethanol on cerebral -aminobutyric acid (GABA)_Breceptor and its related signal transduction systems were studied.[³H]GABA binding to cerebral GABA_B receptors in crude synapticmembranes from rat brain was unaffected by ethanol at concentrationsup to 100 mM, although 100 mM ethanol suppressed not only thebasal but also forskolin-stimulated cAMP formation in cerebralcortical membrane vesicles. The suppressive effect of baclofenand the stimulatory effect of forskolin on cAMP formation wasalso found to be unaltered by ethanol. Pretreatment of the membranevesicles with islet-activating protein (IAP), which eliminatesthe function of G₁ protein by its ribosylation, had no effecton the suppressive effects of ethanol on basal and forskolin-stimulatedcAMP formation. In contrast, pretreatment of membrane vesicleswith ‘low pH’, which is known to suppress the functionof G_s protein, eliminated the suppressive effect of ethanolon cAMP formation. These results suggest that an anaestheticdose of ethanol, which is below that necessary to cause changesin receptor binding, may not affect the functions of GABA_B/G₁,G₀ protein/adenylate cyclase system, but suppresses cAMP formationsystems which are coupled with G_s protein.