Abstract
The effect of ethanol on receptor-mediated phospholipase C-linked signal transduction processes was investigated in isolated rat hepatocytes. Pretreatment of the cells with ethanol (6-300 mM) markedly inhibited a subsequent stimulation of phospholipase C by vasopressin, angiotensin II, or epidermal growth factor. By contrast, the effects of the alpha 1-adrenergic agonist phenylephrine and of glucagon were not affected by ethanol pretreatment. Ethanol inhibited the agonist-induced decrease in polyphosphoinositides, the formation of inositol phosphates, and the increase in cytosolic free Ca2+ levels, as detected with the intracellular Ca2+ indicator indo-1. The effects of ethanol were concentration dependent and were pronounced at low concentrations of agonists but were not significant at saturating levels. Pretreatment of the cells with the protein kinase C inhibitor H7 partly prevented the inhibition by ethanol of vasopressin-induced phospholipase C activation. By contrast, pretreatment of the cells with (Rp)-adenosine cyclic 3':5'-phosphorothioate [Rp)-cAMP-S), a competitive inhibitor of protein kinase A, potentiated the inhibitory effect of ethanol on the Ca2+ mobilization by vasopressin. (Rp)-cAMP-S similarly potentiated the inhibition of phospholipase C by the protein kinase C-activating phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). The kinase A inhibitor also made the Ca2+ mobilization by phenylephrine sensitive to ethanol, indicating that the formation of cAMP in the cells played a role in suppressing the sensitivity to ethanol. Pretreatment of the cells with ethanol enhanced the inhibitory effects of TPA on the vasopressin-induced phospholipase C activation at all concentrations of the hormone; however, these synergistic effects were prevented when TPA was added prior to ethanol, a condition that prevents the activation of phospholipase C by ethanol. The data indicate that ethanol causes desensitization of the receptor-mediated phospholipase C secondary to the ethanol-induced activation of phospholipase C and activation of protein kinase C. Ethanol treatment also affects the sensitivity of the phospholipase C system to control by protein kinases A and C. The data indicate that ethanol can affect the control of intracellular signal transduction processes in liver cells under physiologically relevant conditions.
Highlights
The effect of ethanol on receptor-mediated phospho- In a recent series of studies in isolated hepatocytes and lipase C-linked signal transduction processes was in- other cells (1-6), we have demonstratedthatethanol,in vestigated in isolatedrat hepatocytes
Effects of Ethanol on Agonist-induced Ca'+Mobilization-The vasopressin-induced activation of polyphosphoinositidespecific phospholipase C in intact hepatocytes causes an increase in cytosolic free Ca2+levels by releasing Ca'+ from intracellular stores and activating Cain2f+lux from the extracellular medium
Pretreatment of the cells withethanol (100 mM) mimicked theessentialfeatures of thisinhibition (Fig. 1, middle truces).Ethanol itself causes a transient Ca2+increase, which we had found earlier to be due to the activation of phospholipase C (1).This response decays over a period of 12 min, and thceytosolic Ca2+ concentration returns to resting levels (1,3).the subsequentresponse to submaximal concentrations of vasopressin (0.1 or 0.4 nM) was inhibited, with respect to both the initial rateof Ca2+ increaseand the steady-state Ca2+concentration attained in those cells (Fig. 1,A and B )
Summary
Cells were loaded with the indicator by a 40-min incubation with indo-1 AM (5 PM) in the presence of0.0012% Pluronic F-127 followed by a 20-min incubation in the standard incubation buffer containing 0.2% bovine serum albumin. Ence by NAD(P)H-dependent fluorescence is minimized, and the effects of ethanol can be studied more readily than with quin-2- or fura-2-loaded cells. All inositol phosphate and phosphoinositide assays were carried out in duplicate or triplicate, and resulotsf three or more individual experiments were used to calculate the mean & S.E.Experiments usingindo-1-loadedcells thatarepresentedas Ca2+-dependent indo-1fluorescence traces were from individual experimentsrepresentative of a t least three similar ones. Reagents-(R,,)-CAMP-S was kindly donated by Sandoz Research Institute.Indo-1 (K+ salt)andindo-1 AM were purchased from Molecular Probes; ionomycin was obtained from Calbiochem; radiochemicals were purchased from Amersham Corp.; myo-[2-3H]inositol (15 Ci/mmol in 95% ethanol) was purified as described previously (1). Other chemicals and biochemicals were obtained from Sigma or Fisher
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