okadaic-acid and phosphatidylethanol

Ca(2+)-dependent and protein kinase C-dependent mechanisms of phospholipase D (PLD) activation were studied in rat hepatocytes by measuring phosphatidylethanol (Peth) formation in the presence of ethanol. Stimulation of Peth formation by 12-O-tetradecanoyl-phorbol 13-acetate (TPA), vasopressin, or A23187 was inhibited by multiple protein kinase C inhibitors or by protein kinase C down-regulation, indicating that this enzyme is involved in the action of all these agents. A controlled elevation of the cytosolic Ca2+ concentration ([Ca2+]cyt) over the range of 0.1-2.0 microM activated Peth formation in the absence of other agonists. Staurosporin potentiated Ca(2+)-induced Peth formation by shifting the [Ca2+]cyt dose-response curve to the left. Other protein kinase C inhibitors (calphostin C, bisindolylmaleimide) inhibited Ca(2+)-mediated Peth formation, but this inhibition was reduced in staurosporin-treated cells. Okadaic acid potentiated PLD activation by TPA, but suppressed PLD activation by elevated [Ca2+]cyt. Desensitization of TPA-induced PLD activity did not affect PLD activation by Ca2+. These data indicate that [Ca2+]cyt and protein kinase C control distinct pathways of PLD activation, but the Ca(2+)-mediated pathway is suppressed by a staurosporin-sensitive protein kinase. Both mechanisms contribute to vasopressin-induced Peth formation in intact hepatocytes. Activation of protein kinase A enhanced vasopressin-induced Peth formation, but not TPA-stimulated or Ca(2+)-stimulated stimulated Peth formation. Protein kinase A acted by enhancing hormonal Ca2+ mobilization, rather than by directly activating PLD, and thereby shifted the balance of Ca(2+)-dependent and protein kinase C-dependent activation mechanisms of PLD in intact cells.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Animals; Calcium; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Ethers, Cyclic; Glycerophospholipids; Isoquinolines; Liver; Male; Okadaic Acid; Phosphatidic Acids; Phospholipase D; Piperazines; Protein Kinase C; Rats; Rats, Sprague-Dawley; Staurosporine; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin; Vasopressins