Phospholipase D in rat myocardium: formation of lipid messengers and synergistic activation by G-protein and protein kinase C.
Activation of phospholipase D (PLD) and phosphoinositide-specific phospholipase C (PI-PLC) by fluoride, to stimulate heterotrimeric G-proteins, and by phorbol esters, to stimulate protein kinase C (PKC), was studied in rat atria. Fluoride and 4beta-phorbol-12beta,13alpha-dibutyrate (PDB), in contrast to 4beta-phorbol-13alpha-acetate (PAc), activated PLD, catalyzing the formation of [3H]-phosphatidylethanol ([3H]-PETH), [3H]-phosphatidic acid ([3H]-PA), choline and sn-1,2-diacylglycerol (DAG). Basal PLD activity was resistant to drastic changes in Ca2+ and to Ro 31-8220, a PKC inhibitor, but was decreased by genistein, an inhibitor of tyrosine kinase, and increased by vanadate, a tyrosine phosphatase inhibitor; both effects were, however, very small. Fluoride-evoked PLD activity was resistant to Ro 31-8220 and to genistein, but was Ca2+-dependent. The rate of fluoride-induced PLD activation was maintained for at least 60 min. In contrast, PDB-mediated PLD activity was blocked by Ro 31-8220 and was resistant to extracellular Ca2+-depletion and desensitized within ca. 15 min. PDB markedly potentiated the fluoride-evoked generation of [3H]-phosphatidylethanol and of choline, but inhibited the formation of [3H]-inositol phosphates ([3H]-IP(1-3)). Ethanol (2%) blocked the PDB-evoked generation of both [3H]-phosphatidic acid and of sn-1,2-diacylglycerol, whereas fluoride-evoked responses were reduced only to approximately 50%. In conclusion, the trimeric G-protein-PLD pathway in heart tissue did not enclose PKC activation and was long-lasting and Ca2+-dependent; there was no evidence for an involvement of tyrosine phosphorylation. However, PKC activation modulated G-protein-coupled PLD and PI-PLC activities in opposite directions. PLD activity significantly contributed to the mass production of sn-1,2-diacylglycerol in the heart. The evidence for a pathophysiological role of PLD activation in cardiac hypertrophy and in ischemic preconditioning is discussed.
Department of Pharmacology, University of Mainz, Germany.
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Lindmar
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Löffelholz
D000818:Animals; D004357:Drug Synergism; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D050356:Lipid Metabolism; D008055:Lipids / Q000502:physiology; D008297:Male; D009206:Myocardium / Q000201:enzymology* / Q000378:metabolism; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010703:Phorbol Esters / Q000494:pharmacology; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010739:Phospholipase D / Q000187:drug effects / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D015290:Second Messenger Systems / Q000502:physiology*; D010738:Type C Phospholipases / Q000378:metabolism
D008055:Lipids; D010703:Phorbol Esters; C050164:phorbol 13-monoacetate; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D043265:Phosphatidylinositol Diacylglycerol-Lyase