title: The role of cytosolic Ca2+, protein kinase C, and protein kinase A in hormonal stimulation of phospholipase D in rat hepatocytes.
abstract: 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.
authors.affiliation: Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
authors.forename: L
authors.initials: L
authors.lastname: Gustavsson
authors.forename: G
authors.initials: G
authors.lastname: Moehren
authors.forename: M E
authors.initials: ME
authors.lastname: Torres-Marquez
authors.forename: C
authors.initials: C
authors.lastname: Benistant
authors.forename: R
authors.initials: R
authors.lastname: Rubin
authors.forename: J B
authors.initials: JB
authors.lastname: Hoek
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004988:Ethers, Cyclic / Q000494:pharmacology; D020404:Glycerophospholipids; D007546:Isoquinolines / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D008297:Male; D019319:Okadaic Acid; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D019311:Staurosporine; D013729:Terpenes / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin; D014667:Vasopressins / Q000494:pharmacology*
chemical_list: D000470:Alkaloids; D004988:Ethers, Cyclic; D020404:Glycerophospholipids; D007546:Isoquinolines; D010712:Phosphatidic Acids; D010879:Piperazines; D013729:Terpenes; C051521:phosphatidylethanol; D014667:Vasopressins; D019319:Okadaic Acid; D019284:Thapsigargin; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D010739:Phospholipase D; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium