THE ROLE OF CYTOSOLIC CA2-KINASE-C, AND PROTEIN KINASE-A IN HORMONAL-STIMULATION OF PHOSPHOLIPASE-D IN RAT HEPATOCYTES(, PROTEIN)

Ca2+-dependent and protein kinase C-dependent mechanisms of phospholip ase D (PLD) activation were studied in rat hepatocytes by measuring ph osphatidylethanol (Peth) formation in the presence of ethanol. Stimula tion of Peth formation by 12-O-tetradecanoylphorbol 13-acetate (TPA), vasopressin, or A23187 was inhibited by multiple protein kinase C inhi bitors or by protein kinase C down-regulation, indicating that this en zyme is involved in the action of all these agents. A controlled eleva tion of the cytosolic Ca2+ concentration ([Ca2+]cyt) over the range of 0.1-2.0 muM activated Peth formation in the absence of other agonists . Staurosporin potentiated Ca2+-induced Peth formation by shifting the [Ca2+]cyt dose-response curve to the left. Other protein kinase C inh ibitors (calphostin C, bisindolylmaleimide) inhibited Ca2+-mediated Pe th 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 P LD 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 Ca2+-mediated pathway is suppressed by a staurospo rin-sensitive protein kinase. Both mechanisms contribute to vasopressi n-induced Peth formation in intact hepatocytes. Activation of protein kinase A enhanced vasopressin-induced Peth formation, but not TPA-stim ulated or Ca2+-stimulated Peth formation. Protein kinase A acted by en hancing hormonal Ca2+ mobilization, rather than by directly activating PLD, and thereby shifted the balance of Ca2+-dependent and protein ki nase C-dependent activation mechanisms of PLD in intact cells.