guanylyl-imidodiphosphate and Alcoholism

In the present study, to investigate the possibility that chronic ethanol treatment might alter Ca2+-inhibited type 5 adenylyl cyclase (AC) activity, we examined the effect of chronic ethanol treatment on striatal dopaminergic signal transduction, especially the AC system, in mice. We fed male C57BL/6 mice for 7 days with a 5% ethanol-containing or control liquid diet. Basal and forskolin-stimulated AC activities were reduced in striatal membranes of ethanol-treated mice. 5'-guanylylimidodiphosphate-stimulated AC activity was also decreased in ethanol-treated mice. But no significant differences were observed in the levels of the guanine nucleotide binding protein subunits Gs alpha and Gi1alpha&2alpha, determined by immunoblotting, between ethanol-treated and control mice. These results indicated that the function of the catalytic subunit of AC was decreased in the straitum of chronically ethanol-treated mice. We further examined the inhibitory regulation of AC activity in the context of a change of type 5 AC. Inhibition of forskolin-stimulated AC activity by 10 microM free Ca2+ was smaller in ethanol-treated mice than in control mice. However, the protein level of type 5 AC in the striatum, determined by immunoblotting, was not significantly different between ethanol-treated and control mice. These findings suggest that Ca2+-inhibited, presumably type 5, AC activity is reduced in mouse striatum by chronic ethanol treatment, and that this reduction is not due to a decrease in type 5 AC expression.

Topics: 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; Adenylyl Cyclase Inhibitors; Alcoholism; Animals; Calcium; Corpus Striatum; Cyclic AMP; Dopamine; Dopamine Agonists; Enzyme Inhibitors; Ethanol; Guanylyl Imidodiphosphate; Immunoblotting; Male; Mice; Mice, Inbred C57BL; Signal Transduction

The possibility that long-term ethanol ingestion might alter either vasoactive intestinal peptide (VIP) content, VIP binding to membrane receptors, G-protein levels or adenylate cyclase activity in rat prostate was tested, as ethanol produces serious alterations in the hypothalamic-pituitary-gonadal axis and several modifications on different elements on signal transduction pathways in other systems.. Prostatic membranes from control and ethanol-treated (for 4 weeks) rats were used to study adenylate cyclase stimulation as well as for the immunodetection of stimulatory (alpha(s)) and inhibitory (alpha(i)1-2) G-protein subunits. Studies on VIP binding and cross-linking to receptors were performed using [125I]VIP. Prostatic VIP content was estimated by radioimmunoassay. GTPase activity was quantified by measuring the amount of 32Pi released from [gamma-32P]GTP.. Chronic ethanol ingestion resulted in an increased presence of VIP in the rat prostate without any change on the VIP receptor/effector system in this gland. By contrast, the basal adenylate cyclase activity as well as the dose-dependent stimulation of this enzyme by either the nonhydrolyzable GTP analogue Gpp(NH)p or the beta-adrenergic agonist isoproterenol were enhanced in prostatic membranes after ethanol intake. Moreover, an increase in the content of G-protein subunits (alpha(S) and alpha(i)1-2) was observed without any change in GTPase activity in this condition. These modifications were accompanied by a significant decrease in rat prostate weight and, consequently, the height of the secretory epithelium in this gland.. Considering the role of VIP in the mechanisms of secretion and cell proliferation in the prostate, the observed increases in the prostatic content of VIP and G-protein subunits make conceivable that VIP and cAMP signal transduction could be involved in the atrophy of the rat prostate and in the alterations in the composition of seminal fluid that appear in the alcoholic syndrome.

Topics: Adenylyl Cyclases; Alcoholism; Animals; Atrophy; Colforsin; Cyclic AMP; Enzyme Activation; Ethanol; GTP Phosphohydrolases; GTP-Binding Proteins; Guanylyl Imidodiphosphate; In Vitro Techniques; Male; Prostate; Protein Conformation; Rats; Rats, Wistar; Receptors, Vasoactive Intestinal Peptide; Signal Transduction; Vasoactive Intestinal Peptide

The levels of G alpha i2-protein and the G beta gamma-heterodimer were measured in platelet membranes of non-alcoholics, non-alcoholics after an ethanol load (1 g/kg body weight) and of alcoholics under various conditions. The findings were correlated with the activation of the adenylyl cyclase (AC) by various agents. The activation of AC was facilitated by acute ingestion of ethanol. This could not be explained by changes of the G-proteins determined because the levels of the G alpha i2-protein increased, whereas those of the G beta gamma-proteins remained in the control range. The alcoholics were divided into two groups on the day of admission: those with ethanol still present in the blood (intoxicated alcoholics) and those acutely withdrawn within the last 48 h (ethanol absent from the blood). The intoxicated alcoholics had elevated G alpha i2-protein levels in contrast to the acutely withdrawn patients, who did not. This observation suggests rapid changes of the G-protein levels. By analysing the inhibitory efficacy of the G-proteins on AC, it was found that the concentration of the G beta gamma-heterodimer, but not that of the G alpha i2-proteins, correlated with the inhibitory efficacy. The basal activity of the AC was reduced as well as the activation by some compounds. Eight days later (short-term withdrawal) both the levels of G alpha i2 and G beta gamma were elevated. Again, the inhibitory efficacy of the G-proteins correlated with the G beta gamma-heterodimer levels but not with those of the G alpha i2-protein. Furthermore, the changes of the G beta gamma-protein levels between the first and the eighth day correlated with the changes of the inhibiting efficacy. Only a trend was observed with respect to a lowered basal activity if compared with the intoxicated non-alcoholics. The activation of AC by guanylylimidyldiphosphate [Gpp(NH)p] and Gpp(NH)p + ethanol (200 mM in vitro) was still reduced. Observations after 3 and 6 months of abstinence demonstrated elevated G alpha i2- and G beta gamma-protein levels. This suggests residual marker properties of the G-proteins whereby the activity of AC was normal. Only the reduced stimulation by Gpp(NH)p + ethanol in vitro (200 mM), compared with the respective stimulation of AC of intoxicated non-alcoholics, suggested some residual disturbances of the signal transduction during long-term abstinence.

Topics: Adenylyl Cyclases; Alcohol Drinking; Alcoholism; Blood Platelets; Cell Membrane; Ethanol; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Humans; In Vitro Techniques; Temperance

Low concentrations of ethanol (10-100 mM), added to assays in vitro, altered agonist (isoproterenol) binding to mouse cerebral cortical beta-adrenergic receptors in a reversible manner. Ethanol decreased the affinity of the high affinity form of the receptor for isoproterenol but had no effect on the affinity of the low affinity form of the receptor, the proportion of high and low affinity forms of the receptor, the total number of agonist-binding sites, or antagonist binding. The selective effect of ethanol on the properties of the high affinity agonist-binding site suggested that ethanol alters the characteristics of the complex of the receptor and Gs, the guanine nucleotide-binding protein. In cerebral cortical membranes of mice that had ingested ethanol chronically, isoproterenol binding data were best fit by a one-site model, even in the absence of guanine nucleotides. This change, when considered together with previously reported changes in adenylate cyclase activity, is reminiscent of heterologous desensitization of the beta-adrenergic receptor. Thus, both acute and chronic ethanol administration may produce changes in adrenergic function in brain.

Topics: Adrenergic beta-Agonists; Adrenergic beta-Antagonists; Alcoholism; Alcohols; Animals; Cell Membrane; Cerebral Cortex; Ethanol; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Iodocyanopindolol; Isoproterenol; Male; Mice; Pindolol; Receptors, Adrenergic, beta; Substance-Related Disorders

Chronic ingestion of ethanol, which produced tolerance and physical dependence, resulted in altered function of the cerebral cortical beta-adrenergic receptor-coupled adenylate cyclase system in mice. Although there was no change in basal adenylate cyclase activity, or in the activity of the digitonin-solubilized catalytic unit, stimulation of adenylate cyclase activity by the nonhydrolyzable guanine nucleotide analog guanylylimidodiphosphate [Gpp(NH)p] was reduced in brains of ethanol-fed animals. Ethanol added in vitro increased adenylate cyclase activity, and this enhancement, in the presence of Gpp(NH)p, was also reduced in cortical membranes of ethanol-fed mice. Furthermore, the maximal response to isoproterenol was decreased, and the EC50 for isoproterenol stimulation of adenylate cyclase activity was increased in ethanol-fed animals. The results are consistent with a qualitative or quantitative defect in the function of the stimulatory guanine nucleotide-binding protein (Ns), as well as in the beta-adrenergic receptor, after chronic ethanol exposure. In part, these changes appear to be similar to those that occur during heterologous desensitization of various receptor systems, and may be associated with dependence on or tolerance to ethanol.

Topics: Adenylyl Cyclases; Alcoholism; Animals; Cerebral Cortex; Guanylyl Imidodiphosphate; Isoproterenol; Kinetics; Male; Mice; Mice, Inbred C57BL; Receptors, Adrenergic, beta