Enhanced sensitivity of pituitary beta-endorphin to ethanol in subjects at high risk of alcoholism.

BACKGROUND: Previous studies have demonstrated that a moderate dose of ethanol induced a significant increase in the plasma beta-endorphin content of subjects from families with a history of alcoholism (high risk (HR)), but not subjects from families without a history of alcoholism (low risk (LR)). The objective of this study was to examine the response of the pituitary beta-endorphin and adrenal cortisol systems to various concentrations of ethanol in male and female subjects at high and low risk of the future development of alcoholism. METHODS: All subjects participated in four experimental sessions. In each session the subjects were given a drink containing one of the following doses of ethanol: 0, 0.25, 0.50, and 0.75 g of ethanol per kilogram of body weight (for a 60- to 70-kg individual). Blood samples were taken at 0 minutes and at 15, 45, 120, and 180 minutes after the drink for estimation of the blood alcohol, plasma beta-endorphin, and plasma cortisol levels. RESULTS: The concentration of alcohol in the blood at various intervals after the drink was similar among the subjects, regardless of the risk group. Ethanol increased the plasma level of beta-endorphin-related peptides of the HR but not of the LR subjects in a dose-dependent manner. All subjects showed a small decrease in plasma cortisol level with time, but ethanol ingestion did not significantly alter the plasma cortisol levels. CONCLUSION: This study indicates that the pituitary beta-endorphin system, but not the adrenal cortisol system, of the HR subjects shows an enhanced sensitivity to ethanol, which may be an important factor in controlling ethanol consumption.

Effects of ethanol treatment and withdrawal on biosynthesis and processing of proopiomelanocortin by the rat neurointermediate lobe.

Male Sprague Dawley rats were chronically pair-fed with liquid diets containing 6.5% (vol/vol) ethanol, or equicaloric sucrose. After 21 days the ethanol-containing diet was discontinued and both groups were fed the sucrose diet. Groups of animals were killed on day 22 (0 day of ethanol withdrawal) and 1, 3, 8, and 15 days after ethanol withdrawal and the neurointermediate lobes (NILs) were removed and incubated with [3H]phenylalanine for 3 h. Chronic ethanol treatment induced an increase in the biosynthesis and release of beta-endorphin-like peptides by the rat NIL. After ethanol withdrawal the beta-endorphin-like immunoreactivity content in the NIL and the in vitro release of immunoreactive beta-endorphin (beta EP) by the NIL were significantly lower than in the controls on the first day, whereas no significant difference was found on days 3, 8, and 15 after ethanol withdrawal. The in vitro incorporation of [3H]phenylalanine into POMC, beta-lipotropin and beta EP was found to be higher in the ethanol-treated animals than in the controls on days 0, 1, and 3 after ethanol withdrawal, with no significant difference on days 8 and 15 after ethanol withdrawal. Furthermore, in both the ethanol-treated animals and their pair-fed controls the rate of incorporation of [3H]phenylalanine into total proteins, POMC, beta-lipotropin, and beta EP was significantly higher on days 8 and 15 after ethanol withdrawal than on the day of ethanol withdrawal (day 0), suggesting the implication of a nutritional factor. HPLC analysis of the beta EP peptides indicated that the percentage of acetylated forms of beta EP was higher in the NIL of the alcohol-treated animals, especially on days 8 and 15 after ethanol withdrawal. This observation suggests that though the rates of biosynthesis and release of beta EP-related peptides have returned to normal at 15 days after ethanol treatment, the activity of the enzyme responsible for the acetylation of beta EP remained elevated.

Chemical characterization of adrenocorticotropin and a novel peptide biosynthesized from a pituitary adenoma of a patient with Cushing's disease.

ACTH and another unidentified peptide have been biosynthesized in vitro and purified from a pituitary adenoma of a patient with Cushing's disease. The new peptide had an apparent molecular weightof 4000 daltons and was more acidic than ACTH as analyzed by sodium dodecyl sulfate/urea and acidic (pH 4.5) polyacrylamide gel electrophoreses, respectively. Sequence studies revealed the presence of [35S]methionine at position 3 of the new peptide. High performance liquid chromatographic analysis of [35S]methionine-labeled tryptic fragments of the peptide revealed that this peptide contained gamma MSH- (1-7) within its sequence. Our observations raise the possibility that this peptide contains at its N-terminus gamma MSH, a putative peptide proposed by Nakanishi et al.

The effect of electroconvulsive therapy on endorphins in depression.

The time course study of the endorphin response to electroconvulsive therapy (ECT) was carried out in 10 patients (including one control who did not receive active ECT) at the first and sixth ECT. Results showed a significant rise of plasma endorphin levels after ECT. This increase returned to the pre-ECT level within 1 hr after ECT. There was a pre-rise of plasma endorphin level, which probably was stress related and which was also observed in the control case.

Hyperlipoproteinemia induced by a transplantable pituitary tumor in the rat.

The MtT-F4 tumor, a transplantable pituitary tumor of rats, induces significant hyperlipidemia in male Fisher 344 rats. The increasive hypercholesterolemia was accompanied by hypertriglyceridemia only in the first month of tumor implantation. Clofibrate feeding inhibited the development of hypercholesterolemia and maintained normal serum triglyceride levels. In contrast to the changes in lipoprotein cholesterol distribution and profile found in experimental hyperlipidemia induced by high fat and cholesterol feeding, the hypercholesterolemic tumor-bearing rats showed no accumulation of cholesterol in the very low density and intermediate density lipoproteins, and no appearance of a new class of lipoprotein, B-VLDL. An HDLc-like lipoprotein appeared as hypercholesterolemia developed. Increased amounts of cholesterol were deposited in the aorta. The effects are attributed to the lipolytic hormones secreted by the tumor and antagonism to their action by clofibrate.

Effects of voluntary ethanol drinking on [125I]insulin-like growth factor-I, [125I]insulin-like growth factor-II and [125I]insulin receptor binding in the mouse hippocampus and cerebellum.

Chronic exposure to ethanol can induce widespread cell loss in the brain, in some cases even causing dementia. Although the underlying mechanism associated with ethanol toxicity has not yet been established, it is suggested that one of the ways in which ethanol disrupts neuronal functioning/survival is by targeting the actions of mitogenic growth factors. Insulin-like growth factors-I and -II and insulin are structurally related polypeptides with potent mitogenic and metabolic effects on the central and peripheral nervous systems. These growth factors and their respective receptors are widely distributed throughout the brain, including the hippocampus and cerebellum. Evidence indicates that ethanol can decrease plasma levels of insulin-like growth factors and can also inhibit the growth-promoting and cell survival effects of these growth factors under in vitro conditions. The present study was designed to determine if voluntary ethanol consumption over a 21-day period could alter [125I]insulin-like growth factor-I, [125I]insulin-like growth factor-II and [125I]insulin receptor-binding sites in the hippocampus and cerebellum-areas known to be severely affected following chronic exposure to ethanol. C57BL/6 mice were presented with either water only or a choice of water and a 10% v/v ethanol solution. Mice with access to the ethanol solution drank an average of 5.35+/-0.77 g of ethanol/kg body weight per day. [125I]Insulin-like growth factor-I receptor-binding sites were found to be significantly increased in all subfields of the hippocampal formation, but not in the cerebellum, of ethanol-treated mice compared to controls. [125I]Insulin-like growth factor-II and [125I]insulin receptor-binding sites, on the other hand, did not exhibit any alterations either in the hippocampus or cerebellum following chronic exposure to ethanol. These results, in keeping with earlier reports, suggest that hippocampal insulin-like growth factor-I is more sensitive to ethanol treatment than either insulin-like growth factor-II or insulin, and the observed increase in the [125I]insulin-like growth factor-I receptor levels possibly reflects an activity-dependent response to prevent/slow down neuronal degeneration and/or to regulate subtle functional alterations that follow chronic exposure to ethanol.

An in vivo profile of beta-endorphin release in the arcuate nucleus and nucleus accumbens following exposure to stress or alcohol.

The aim of the present study was to determine the effects of distinct categories of stressors on beta-endorphin (beta-EP) release in the arcuate nucleus (ArcN) and nucleus accumbens (NAcb) using in vivo microdialysis. Adult male rats were implanted with a cannula aimed at either the NAcb or the ArcN. On the day of testing, a 2 mm microdialysis probe was inserted into the cannula, and artificial cerebrospinal fluid was infused at 2.0 microl/min. After three baseline collections, animals either had a clothespin applied to the base of their tail for 20 min (a physical/tactile stressor), were exposed to fox urine odour for 20 min (a psychological stressor/species-specific threat), or were administered 2.4 g ethanol/kg body weight, 16.5% w/v, i.p. (a chemical/pharmacological stressor) with control animals receiving an equivalent volume of saline. Both tail-pinch and fox odour significantly increased beta-EP release from the ArcN (P<0.05), whilst only tail-pinch enhanced beta-EP release from the NAcb (P<0.01). On the other hand, alcohol stimulated beta-EP release in the NAcb as compared with saline-treated controls (P<0.01), but not in the ArcN. Although the increase in extracellular beta-EP produced by the other stressors was relatively rapid, there was a 90-min delay before alcohol administration caused beta-EP levels to exceed that of saline-injected controls. In conclusion, the fact that physical and fear-inducing psychological stressors stimulate beta-EP release in the ArcN and only physical stressors stimulate beta-EP release in the NAcb, indicates that stressors with different properties are processed differently in the brain. Also, an injection of alcohol caused a delayed increase of beta-EP in the NAcb but not the ArcN, indicating that alcohol may recruit a mechanism that is, at least partially, distinct from stress-related pathways.

Characterization of beta-endorphin peptides in the spinal cord of the rat.

The objective of the present studies was to estimate the total content of beta-endorphin-like immunoreactivity (beta-EPLIR) and to characterize the beta-endorphin-like peptides in distinct regions of the spinal cord using gel filtration and reverse phase high performance liquid chromatography. The concentration of beta-EPLIR expressed as pg per mg tissue was similar in the four regions of the spinal cord. Sephadex G-75 chromatography demonstrated the presence, in all four regions of the spinal cord, of beta-endorphin (beta-EP) immunoreactive peptides eluting at the positions of standard beta-EP and beta-lipotropin (beta-LPH) peptides as well as a high molecular weight form eluting prior to beta-LPH. High performance liquid chromatography of the beta-EP-sized peptides indicated some differences in the relative proportions of the various beta-EP-sized peptides among the four regions of the spinal cord, which suggest a different origin of the beta-EP fibers terminating in different regions of the spinal cord as well as different physiological importance of the beta-endorphin peptides in the various spinal cord regions.

Endorphins in inbred mice with variable sensitivity to ethanol: effect of acute ethanol treatment.

Resting levels of beta-endorphin like immunoreactivity were determined in the hypothalamus, neurointermediate lobe and anterior lobe of the pituitary gland and the serum of inbred mice strains (C57BL/6, BALB/C and DBA/2). C57BL/6 mice showed the highest content of beta-endorphin like immunoreactivity in the neurointermediate lobe, anterior lobe and serum. Animals were injected i.p. with either an ethanol solution (3g ethanol/Kg b. wt.) or saline. 45 minutes post ethanol treatment the beta-endorphin like immunoreactivity content was increased in the serum of all three strains of mice studied and was decreased in the hypothalamus of the C57BL/6 mice. Studies with reverse phace HPLC indicated some differences in the relative proportions of the various forms of beta-endorphin in some tissues among the three strains of mice. These results suggest that genetically determined differences in endorphin levels may be involved in some of the genetically determined differences in responses to ethanol.

Rats exposed prenatally to alcohol exhibit impairment in spatial navigation test.

Prenatal exposure to ethanol causes learning disabilities and low I.Q. scores. The objective of the present studies was to investigate whether exposure of rats to ethanol in utero, would induce a deficit in spatial memory in adult life. Pregnant rats were fed with an ethanol diet from day 1 of pregnancy till parturition. Control rats were either pair-fed with an isocaloric sucrose diet or were fed with lab-chow ad libitum. On the first day of birth, offspring exposed to ethanol in utero were placed with a control mother fed with lab-chow, while offspring of the lab-chow fed dams were placed with ethanol-treated dams. At 40, 60 and 90 days postnatally, behavioral testing was performed using the Morris swim maze, a test of spatial memory. Results indicated that the offspring exposed to ethanol in utero presented deficits in spatial memory processes. Ethanol did not completely block the learning of the swim maze task but the alcohol-exposed offspring exhibited longer latencies to perform the task, swam longer distances prior to locating and climbing onto the platform, and when the platform was removed, searched for it in all 4 quadrants of the pool. Restricted caloric intake during gestation and maternal behavior in early postnatal life also induced deficits in the performance on the swim maze task. However, these deficits were mild and short-lasting being absent at 60 and 90 days of age. In contrast, the deficits induced by ethanol were more severe and longer-lasting, being present in adult life.

The beta-endorphin response to stress during postnatal development in the rat.

The plasma beta-endorphin and adrenocortical responses to ether and handling stress were examined in animals of various ages. At each age studied there was a significant, stress-induced elevation of plasma beta-endorphin-like immunoreactivity levels. Moreover, basal beta-endorphin-like immunoreactivity levels were higher in animals 3, 7, and 14 days of age than in adults. In contrast, plasma corticosterone levels in the Day 7 pups were not elevated by stress; a finding consistent with several previous reports on the absence of increased release of adrenocorticotropin and corticosterone during stress in the neonate. These data suggest that the release of adrenocorticotropin and beta-endorphin in response to stress differ in the developing animal.

Characterization of the effects of acute ethanol administration on the release of beta-endorphin peptides by the rat hypothalamus.

In the present studies the direct effect of ethanol on the release of beta-endorphin by the rat hypothalamus was investigated. When various concentrations of ethanol (10-120 mM) were added into the incubation medium, it was noticed that though low concentrations of ethanol (10, 20 and 30 mM) induced a pronounced increase in the release of beta-endorphin-like peptides from the hypothalamus, high concentrations of ethanol (40, 60 and 120 mM) induced a less pronounced increase. Exposure of hypothalamus to depolarizing concentrations of potassium chloride (following washing of the ethanol), provoked a significant release of beta-endorphin-like peptides, regardless of the ethanol concentration the tissues were exposed prior to the stimulation with the potassium chloride. Chromatographic analysis of the incubation media with Sephadex-G-75 revealed that the hypothalamus released mainly beta-endorphin-sized peptides. Analysis of the beta-endorphin-sized peptides with reverse-phase high performance liquid chromatography indicated the presence of beta-endorphin-(1-31) as well as non-acetyl and acetyl beta-endorphin-(1-27). Thus ethanol exerts a biphasic effect on the release of beta-endorphin-like peptides by the rat hypothalamus, with low concentrations inducing a dose-dependent increase, reaching maximum at 20 mM ethanol, and with higher concentrations of ethanol inducing a less pronounced increase in the release of beta-endorphin-like peptides, leading to an inverted U-shaped dose response relationship of ethanol and release of beta-endorphin-like peptides from the rat hypothalamus.

Enhanced activity of the brain beta-endorphin system by free-choice ethanol drinking in C57BL/6 but not DBA/2 mice.

The objective of the present studies was to investigate the effect of voluntary ethanol consumption for 21 days on the brain beta-endorphin system of C57BL/6 (alcohol-preferring) and DBA/2 (alcohol-avoiding) strains of mice. As expected, C57BL/6 mice consumed a significantly higher quantity of the 10% ethanol solution than the DBA/2 mice. Under basal conditions the content of beta-endorphin like peptides differed only in the nucleus accumbens, higher levels being found in the DBA/2 mice. Voluntary ethanol consumption induced an increase in the hypothalamic content of mRNA coding for proopiomelanocortin, associated with a significant increase in the tissue content of beta-endorphin-like peptides in the arcuate nucleus and septum of the C57BL/6 mice, but did not alter the activity of the brain beta-endorphin system of the DBA/2 mice. Since voluntary ethanol consumption was not associated with nutritional deficits and stress, the ethanol-induced enhanced activity of the brain beta-endorphin system of the C57BL/6 mice must be a direct effect of ethanol and may be important in controlling the voluntary ethanol consumption by this strain of mice.

Alterations in brain levels of atrial and C-type natriuretic peptides after chronic moderate ethanol consumption in spontaneously hypertensive rats.

Atrial (ANP) and C-type (CNP) natriuretic peptides have been found in brain regions associated with fluid homeostasis and blood pressure. Since chronic moderate ethanol consumption has been shown to prevent the age-dependent increase in blood pressure in experimental animals, the objective of the present studies was to investigate the effect of ethanol (20% (v/v) for 8 months) on the total content and concentration of ANP and CNP in the brain of spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. Ethanol increased the content and concentration of both ANP and CNP in the hypothalamus, pons and medulla of SHR rats. In contrast, in the WKY rats ethanol had no effect on the levels of ANP in any of the brain regions studies, but enhanced the concentration of CNP in the hypothalamus and medulla. Thus, ethanol induced changes in the content of natriuretic peptides in distinct brain regions associated with control of cardiovascular activity. Such changes may be partially responsible for the effect of chronic moderate ethanol consumption on blood pressure.

Effect of chronic moderate ethanol consumption on heart brain natriuretic peptide.

There is experimental evidence indicating that chronic moderate ethanol consumption delays the age-dependent increase in blood pressure. Since the brain natriuretic peptide (BNP) is a potent hypotensive hormone, the effect of chronic ethanol treatment on the heart BNP system was investigated, using spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. Chronic moderate ethanol consumption resulted in significantly lower circulating BNP levels for both SHR (206.9 +/- 18.5 vs. 306.9 +/- 28.1 pg/ml, n = 12, P < or = 0.05) and WKY rats (131.3 +/- 20.7 vs. 220.6 +/- 25.0 pg/ml, n = 12, P < or = 0.05). Left and right atrial BNP content and concentration in WKY rats and left atrial BNP content and concentration in SHR rats were augmented by the ethanol treatment, but not atrial BNP mRNA. In ventricular tissue, alcohol had no effect on total BNP content of either SHR or WKY rats, but it induced a significant elevation in ventricular BNP concentration (microgram/mg protein) and BNP mRNA in SHR, but not WKY rats. Thus, chronic ethanol treatment resulted in specific alterations in the activity of the heart BNP system.

Effect of chronic morphine treatment on beta-endorphin biosynthesis by the rat neurointermediate lobe.

The effect of chronic morphine treatment on the in vitro biosynthesis of beta-endorphin by rat pars intermedia was investigated. Tolerance and physical dependence were induced in 200 g rats by the subcutaneous implantation of 75 mg morphine pellets for either 3 days or 15 days. Immediately following sacrifice of the animals the neurointermediate lobes were removed and incubated with [3H] phenylalanine. The protein extracts of the lobes were analyzed for the incorporation of the labelled amino acid into total protein, pro-opiomelanocortin, beta-lipotropin (beta-LPH) and beta-endorphin. the biosynthesized products were purified by immunoprecipitation with an antiserum to beta-endorphin. The identity and purity of beta-endorphin were verified by polyacrylamide disc gel electrophoresis with sodium dodecyl sulfate, and microsequencing. The identity of pro-opiomelanocortin (POMC) was verified by peptide mapping of its tryptic digestion products. The results showed that morphine treatment induced a decrease in the incorporation of the radioactive amino acid into total protein, pro-opiomelanocortin, beta-LPH and beta-endorphin. The decrease was more pronounced for the incorporation into beta-LPH and beta-endorphin than into pro-opiomelanocortin and total proteins, suggesting an effect of morphine treatment on the processing of the pro-opiomelanocortin to its final maturation products.

Alcohol-seeking behavior: the roles of the hypothalamic-pituitary-adrenal axis and the endogenous opioid system.

Both the hormones of the hypothalamic-pituitary-adrenal (HPA) axis and the endogenous opioid system are activated in response to stress as well as after alcohol consumption, supporting the hypothesis that stress can influence both alcohol consumption and craving for alcohoL Activation of the HPA axis by stress or alcohol results in the production of glucocorticoid hormones, such as cortisol. Those hormones, in turn, are important for the release of the brain chemical dopamine in certain brain areas that are associated with the rewarding and reinforcing effects of alcohol and other drugs. Alcohol-induced release of certain endogenous opioids similarly results in dopamine release in those brain regions. Through this mechanism, both the HPA axis and the endogenous opioid system may influence alcohol consumption. Consequently, genetically determined differences in the activities of the HPA axis and endogenous opioid system may help determine a person's alcohol consumption level and vulnerability to alcoholism.

Long-term ethanol alters the binding of 3H-opiates to brain membranes.

In order to examine whether ethanol treatment has selective or differential effects on brain binding sites for opiates, male Sprague Dawley rats were fed for 15 or 21 days with a complete liquid diet containing 6.5% ethanol (v:v) or an isocaloric amount of sucrose. The binding of 3H-DADL-enkephalin, 3H-dihydromorphine and 3H-naloxone to the brain membranes from rats treated with ethanol was increased. However, addition of ethanol directly in the incubation medium decreased the binding of 3H-DADL enkephalin and increased the binding of 3H-dihydromorphine to brain membranes from both control and ethanol treated rats. Direct exposure of brain membranes to ethanol caused no significant change in the binding of 3H-naloxone. Thus chronic ethanol ingestion alters the binding of opiate ligands to brain membranes. Furthermore, the direct effect of ethanol appears to be different for the different classes of opiate binding sites.

Effect of acute ethanol in vivo and in vitro on the beta-endorphin system in the rat.

Acute ethanol treatment in vivo (i.p. injection of 3.5 g ethanol/Kg B. wt.) stimulated the release of beta-endorphin like peptides by the pituitary gland as was indicated by the increased content of beta-endorphin like immunoreactivity (beta-EPLIR) in the plasma. Furthermore, a significant decrease in the anterior lobe content of beta-EPLIR was observed, while the decrease in the neurointermediate lobe beta-EPLIR content at 45 min after the i.p. ethanol injection was not statistically significant. In vitro incubation of neurointermediate lobes, from animals injected with either ethanol or saline, in the presence of 3H phenylalanine indicated that the content of beta-EPLIR in the incubation medium was increased, the content of the newly biosynthesized 3H-phenylalanine labelled proteins in the neurointermediate lobe extract was decreased, while the content of 3H-phenylalanine labelled pro-opiomelanocortin, beta-lipotropin and beta-endorphin in the neurointermediate lobes extract were not significantly changed by the ethanol treatment, though a small increase was observed. When neurointermediate lobes from untreated control animals were incubated for 3 hrs with 3H-phenylalanine in the presence or absence of 300 mg ethanol per 100 ml incubation medium, there was no significant difference in the beta-EPLIR content in the incubation medium, or in the content of 3H-phenylalanine labelled proteins, pro-opiomelanocortin, beta-lipotropin and beta-endorphin in the neurointermediate lobe extract. These results suggest that ethanol has little or no direct effect on the beta-endorphin peptides in the pars intermedia cells.

Inbred strains of mice with variable sensitivity to ethanol exhibit differences in the content and processing of beta-endorphin.

The content of beta-endorphin-like immunoreactivity (beta-EPLIR) in the anterior and neurointermediate lobe of the pituitary gland, the hypothalamus and the serum of the c57BL/6, BALB/C and DBA/2 inbred strains of mice was estimated at the resting state as well as 45 min after i.p. injection of either ethanol solution (3.0 g/kg.b.wt.) or saline. At the resting state, the neurointermediate lobe and the serum of the c57BL/6 mice showed the highest content of beta-EPLIR, while no statistically significant difference was noticed in the total beta-EPLIR content in the anterior lobe and hypothalamus. At 45 min post-ethanol treatment the beta-EPLIR content was increased in the serum of all three strains of mice studied and was decreased in the hypothalamus of the c57BL/6 mice only. Further analysis of the beta-endorphin peptides using sephadex G-75 chromatography and reverse phase high performance liquid chromatography indicated strain differences in the relative proportions of the various forms of beta-endorphin in the anterior lobe, neurointermediate lobe and the hypothalamus. These strain specific differences in the content and post-translational processing of beta-endorphin may be involved in some of the genetically determined differences in responses to ethanol by these inbred strains of mice.