Gustavsson L, Moehren G, Torres-Marquez M E, Benistant C, Rubin R, Hoek J B
Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
J Biol Chem. 1994 Jan 14;269(2):849-59.
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.
通过在乙醇存在的情况下测量磷脂酰乙醇（Peth）的形成，研究了大鼠肝细胞中磷脂酶D（PLD）激活的钙依赖和蛋白激酶C依赖机制。12-O-十四烷酰佛波醇-13-乙酸酯（TPA）、血管加压素或A23187对Peth形成的刺激被多种蛋白激酶C抑制剂或蛋白激酶C下调所抑制，表明该酶参与了所有这些试剂的作用。在没有其他激动剂的情况下，将胞质钙浓度（[Ca2+]cyt）在0.1-2.0微摩尔范围内进行可控升高可激活Peth的形成。星形孢菌素通过将[Ca2+]cyt剂量反应曲线向左移动来增强钙诱导的Peth形成。其他蛋白激酶C抑制剂（钙磷蛋白C、双吲哚马来酰亚胺）抑制钙介导的Peth形成，但在经星形孢菌素处理的细胞中这种抑制作用减弱。冈田酸增强了TPA对PLD的激活，但抑制了[Ca2+]cyt升高对PLD的激活。TPA诱导的PLD活性脱敏并不影响Ca2+对PLD的激活。这些数据表明，[Ca2+]cyt和蛋白激酶C控制PLD激活的不同途径，但钙介导的途径被一种对星形孢菌素敏感的蛋白激酶所抑制。这两种机制都有助于完整肝细胞中血管加压素诱导的Peth形成。蛋白激酶A的激活增强了血管加压素诱导的Peth形成，但不增强TPA刺激或钙刺激的Peth形成。蛋白激酶A通过增强激素钙动员起作用，而不是直接激活PLD，从而在完整细胞中改变了PLD钙依赖和蛋白激酶C依赖激活机制的平衡。
