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The dose-effect relationship between blood alcohol concentration (BAC) and altered platelet function was examined in whole blood in a rat model of alcohol exposure by inhalation, using the impedance method of ex vivo whole blood platelet aggregation. With rates of alcohol addition to the chamber air inflow from 29 to 56 mg ethanol/l air/min, BAC was dependent on duration of exposure and concentration of alcohol in the air. Next, 3, 6, and 9 h exposures to the highest delivery rate were used, and platelet aggregability was tested. After 9 h, BAC reached 453 +/- 16 mg% and aggregation responses to three doses of collagen were significantly lower than in control blood (p < 0.01). Less consistent inhibition was observed with arachidonic acid and ADP, and also when exposure duration was reduced. However, some significant inhibition of collagen-induced aggregation (p < 0.05) was observed with BAC as low as 127 +/- 15 mg%. These experiments demonstrate that in vivo alcohol exposure inhibits, in a concentration-dependent manner, ex vivo rat whole blood platelet aggregation, at BACs readily attained in humans by ingestion.

Ethanol administration to rats by ethanol vapor inhalation (14 days) results in a 40-50% reduction in the level of gamma-aminobutyric acidA (GABAA) receptor alpha 1 subunit mRNAs [4.4 and 4.8 kilobases (kb)] in the cerebral cortex. The level of alpha 2 subunit mRNA (8.0 kb) was also reduced by 29%, whereas there was no effect of prolonged ethanol exposure on the level of alpha 3 subunit mRNA (3.1 kb). Ethanol exposure did not alter the steady state levels of cerebral cortical glutamic acid decarboxylase or beta-actin mRNAs. Moreover, no alterations in the levels of total RNA, poly(A)+ RNA, or rRNA were observed, suggesting that the ethanol-induced reductions in GABAA receptor alpha 1 and alpha 2 subunit mRNAs were not the result of a generalized effect of ethanol administration on transcription or mRNA turnover. These ethanol-induced reductions in GABAA receptor alpha subunit mRNAs may underlie alterations in GABAA receptor function or number observed following prolonged ethanol exposure in rats.

The spasmogenic properties of a stable thromboxane mimic, U46619, were determined in the isolated rat aorta after 1, 4, 8, or 12 hr of acute ethanol exposure in vitro or after 12 days of chronic exposure by inhalation. Acute ethanol exposure (87-822 mM) increased the baseline tension of aortic rings in a concentration-dependent manner. Moderate concentrations of ethanol (11 and 43 mM) decreased the maximum tensile response of rat aortic rings to U46619 after 8 and 4 hr of exposure, respectively. In contrast, higher ethanol concentrations (87 and 411 mM) did not significantly effect the maximum tensile response to U46619. Ethanol at 822 mM completely inhibited the response to U46619 and this inhibition could be 85% reversed after removal of ethanol. The inhibitory effect of 1.64 M ethanol was irreversible. Sensitivity to U46619 was inversely related to the ethanol concentration (greater than or equal to 43 mM) and incubation time. Similarly, chronic exposure to moderate blood ethanol levels (92-198 mg/100 ml) for 12 days decreased the maximum tensile response whereas high levels did not effect aortic contractility in vitro. Sensitivity of aorta from rats with mean blood ethanol levels greater than or equal to 92 mg/100 ml decreased at least one order of magnitude. The results suggest that the effect of physiologically tolerable ethanol concentrations on the amplitude of the tensile response to U46619 is biphasic both during acute exposure in vitro or following a chronic exposure period in vivo and the inhibitory effect on sensitivity for U46619 is both concentration- and time-dependent in the rat aorta preparation.

In a previous study we reported the presence of specific nonopiate beta-endorphin (BE) binding sites in peripheral tissues of the rat (Dave JR, Rubinstein N, Eskay RL: Evidence that beta-endorphin binds to specific receptors in rat peripheral tissue and stimulates the adenylate cyclase-adenosine 3',5'-monophosphate system. Endocrinology 117:1389-1396, 1985). The objective of this study was to determine the effect of chronic ethanol administration in vivo on specific nonopiate binding of BE in hepatic and kidney membranes. Experimental animals were exposed continuously for 14 days to ethanol vapor in an inhalation chamber at vapor levels sufficient to maintain blood ethanol levels greater than 120 mg/100 ml, whereas control animals were maintained in ethanol-free chambers. Chronic ethanol treatment decreased [125I]BE binding to hepatic and kidney membranes by approximately 35%, which was due to a decrease in the number of BE binding sites. Chronic ethanol treatment decreased immunoreactive BE in plasma by greater than 80%. In vitro exposure of hepatic and kidney membranes from control animals to ethanol resulted in a dose-related enhancement of BE binding with maximal enhancement of 50-65% being observed at 0.2% ethanol concentration. In contrast, the addition of ethanol in vitro to hepatic and kidney membranes derived from rats chronically exposed to ethanol in vivo did not affect BE binding. Our findings demonstrate that chronic alcohol exposure lowers immunoreactive BE in plasma and reduces BE binding in hepatic and kidney membranes. The observed reduction of BE binding may be due to ethanol-induced changes in membrane composition.

Animals exposed continuously for 14 days to ethanol vapor in an inhalation chamber at sufficient ethanol vapor concentration to maintain blood ethanol levels from 100-250 mg/100 ml exhibited approximately 36% lower corticotropin-releasing factor binding and 24% lower adenylate cyclase activity in anterior (AL) and neurointermediate lobe (NIL) membranes of the pituitary gland compared to controls not treated with ethanol. To determine the effect of chronic ethanol exposure on proopiomelanocortin (POMC) biosynthesis, the levels of POMC mRNA in the AL and NIL were quantified by Northern blot and slot blot techniques. Ethanol treatment for 1, 7, or 14 days produced a time-related decrease in POMC mRNA levels, relative to total RNA levels, in both the AL and NIL. Ethanol treatment caused a greater reduction in NIL POMC mRNA than in AL POMC mRNA. Exposure to ethanol vapors for 14 days decreased immunoreactive beta-endorphin in plasma by approximately 82%. The observed reduction of immunoreactive beta-endorphin in plasma after long term exposure of rats to ethanol may be related to the alcohol-mediated decrease in corticotropin-releasing factor binding and adenylate cyclase activity, which, in turn, leads to decreased intracellular POMC levels through reduced production of POMC mRNA in the AL and NIL of the rat pituitary gland.

Rats were exposed to alcohol vapors by an inhalation technique and blood pressure and its reactivity and platelet aggregation were measured. Acute exposure to alcohol levels which produced moderate blood alcohol concentration (BAC) was associated with increased vascular production of prostacyclin (PGI2) and plasma catecholamines, whereas platelet production of thromboxane (TXA2) decreased. Blood pressure is elevated in these animals, however, the platelet aggregating and pressor effects of noradrenaline (NE) were decreased. Chronic exposure to high BAC is associated with a dramatic reduction in vascular and platelet prostaglandin (PG) production and a marked increase in plasma catecholamine levels. Platelet aggregation decreased in these animals, however, the pressor effect of TXA2 and NE was significantly increased. The fatty acid precursors to PG were reduced by 50% in the lipid extracts of these preparations. These findings suggest that alterations in fatty acid metabolism may lead to a functional deficiency in PG production from dependent rats. Qualitative differences may exist between acute and chronic exposure with respect to the cardiovascular state. Locally produced PG and circulating catecholamines may mediate alcohol-induced alterations in vascular smooth muscle tone and platelet aggregation.

The effect of chronic ethanol exposure on the function of the gamma-aminobutyric acid (GABA) receptor-coupled chloride ion channel was studied in synaptoneurosomes from rat cerebral cortex. Ethanol was administered to rats by inhalation resulting in high blood ethanol levels which have previously been shown to produce tolerance and dependence. After 14 days of ethanol exposure, the stimulation (apparent Vmax) of 36Cl- uptake by the GABA agonist muscimol was decreased by approximately 26%. Similarly, stimulation of 36Cl- uptake by the barbiturate pentobarbital was also reduced to the same extent and there was an increase in the apparent EC50 concentration. Direct stimulation of 36Cl- uptake by ethanol was unchanged after chronic ethanol exposure when measured in the same membrane preparations in which a decrease in muscimol and pentobarbital-stimulated 36Cl- uptake were observed. However, ethanol's ability to potentiate muscimol-stimulated 36Cl- uptake was abolished completely in synaptoneurosomes from ethanol-treated rats. The decrease in GABA receptor-coupled chloride ion channel function observed after chronic ethanol administration was observed only in animals where the mean blood ethanol concentration was greater than or equal to 150 mg%. These blood levels have previously been shown to be associated with ethanol withdrawal after ethanol discontinuation in this rat strain. Four days after ethanol administration, after all signs of ethanol withdrawal had subsided, the decrease in muscimol and pentobarbital-stimulated 36Cl- uptake was reversed. These data suggest that subsensitivity of the GABA receptor-coupled chloride ion channel after chronic ethanol administration may contribute to ethanol tolerance and the ethanol withdrawal syndrome.

Ethanol exposure leads to a loss in membrane polyunsaturated fatty acids (PUFA). It is proposed that polyunsaturated species of phospholipids are not randomly distributed, but are concentrated in the cytosolic leaflets of the plasma membrane and are preferentially associated with membrane proteins. These lipids affect the physical state of environments surrounding membrane proteins and thereby serve to regulate many cellular functions. Disruption of these environments may occur even when a small percentage of total polyunsaturates is lost due to ethanol exposure. One possible mechanism of ethanol-induced polyunsaturate loss may be activation of a phospholipase A2 enzyme which is selective for these species of phospholipids. Fatty acids released would stimulate the production of prostaglandins and/or leukotrienes. Similarly, the released docosahexaenoate can be metabolized by rat brain to leukotriene-like compounds which are biologically active in smooth muscle systems. This metabolism is stimulated by ethanol in human platelets, in vitro.

An inhalation method of ethanol administration was used to study the effects of 14 days of ethanol administration on the immune and hematopoietic systems of the rat. A decrease in cellularity was found in the spleen, thymus, and bone marrow of ethanol-treated rats. Although the red blood cell count, white blood cell count, and hemoglobin concentration were not significantly different between treatment and control groups, treatment with ethanol altered the relative proportion of lymphocytes and polymorphonuclear leukocytes in the peripheral blood. The granulocyte-macrophage progenitor cells in the bone marrow were unaffected by ethanol treatment, but a significant decline in the number of erythroid progenitor cells was noted in ethanol-treated rats. Splenic lymphocytes, although fewer in number in the ethanol-treated rats, showed no significant difference in ability to proliferate when stimulated by nonspecific mitogens.

RO 15-4513, an imidazodiazepine that has been reported to reverse some of the behavioral effects of ethanol, was given to DBA/2 mice. Although no animals treated with a 6 mg/kg dose of this drug had seizures, 20% of animals given 20 mg/kg of this drug had tonic seizures. Ethanol withdrawal was induced in DBA/2 mice treated with 4-methyl pyrazole using an inhalation paradigm. Mice were more likely to have a seizure during ethanol withdrawal if treated with RO 15-4513 (6 mg/kg) than if they received the vehicle. These data suggest administering RO 15-4513 as an alcohol antagonist to alcoholic subjects may increase the incidence of seizures.

Spontaneous 6-keto-PGF1 alpha and TXB2 levels in isolated rat aortic rings increased in a concentration dependent manner after a 0.5 hour incubation with moderate or high ethanol concentrations (11 mM to 218 mM). After a 1 hour incubation with moderate concentrations of ethanol less than or equal to 22 mM) spontaneous prostaglandin (PG) production did not increase although high concentrations (87 mM and 218 mM) increased both 6-keto-PGF1 alpha and TXB2 levels. Similarly, in the presence of 40 microM Na-arachidonate, high ethanol concentrations increased PG production after 0.5 and 1 hour incubation. In addition, either a 4 or an 8 hour exposure to high ethanol concentrations increased spontaneous PG production. A moderate concentration of ethanol (22 mM) increased the 6-keto-PGF1 alpha/TXB2 ratio whereas high levels (greater than or equal to 87 mM) depressed the ratio after 0.5 and 1 hour exposure. This effect was short-lived since after 4 or 8 hours incubation with high ethanol concentrations the 6-keto-PGF1 alpha/TXB2 ratio was markedly increased. The alcohol-induced changes in both spontaneous and arachidonate-stimulated PG levels were concentration dependent and related to the incubation time. Furthermore, these data suggest that there may be unbalanced production of PGI2 and thromboxane A2 in vascular tissue exposed to alcohol.

Rats were exposed to alcohol vapor for 6 days and the mean blood ethanol concentration (BEC) was obtained for each subject. Blood pressure and its reactivity to noradrenaline and a thromboxane-mimic U46619 were directly measured on day 6 via a catheter implanted in the tail artery of normal and ethanol-treated animals. The mean BEC for each subject correlated with mean arterial blood pressure (MAP); an increase in BEC was associated with a decrease in MAP (p less than 0.02). The mean MAP of subjects with BEC less than 168 mg% was 8% higher than normal (not significant), whereas, the mean MAP of subjects with BEC greater than 182 mg% decreased 27 +/- 4% (p less than 0.01). Conversely, the pressor response to U46619 was markedly enhanced (p less than 0.005) in rats with mean BEC greater than 182 mg% at all doses investigated (12.5-3200 ng per rat). Increases in the pressor response to noradrenaline in ethanol-treated rats were significant only when maximally stimulated by 400 and 800 ng doses (p less than 0.03). A 3-fold increase in sensitivity for U46619 was seen in subjects with high mean BEC, however, sensitivity for noradrenaline did not significantly change. Vasoreactivity was not effected in rats with mean BEC less than 168 mg%. These data demonstrate that a moderate mean BEC for 6 days induces a tendency towards a mild hypertension, whereas, high mean BEC induces marked hypotension which is associated with hyperreactivity. Long-term exposure to high blood ethanol concentrations may predispose the alcohol-dependent rats to hypertensive disease and vasospastic disorders, at least partially, as a result of enhanced sensitivity to prostaglandins such as thromboxane.