Endothelin activation of phospholipase D: dual modulation by protein kinase C and Ca2+.
Previous work from this laboratory has identified an endothelin (ET) type A (ETA) receptor on cultured rat renal medullary interstitial cells (RMIC), coupled to phosphatidylinositol-specific phospholipase C (PI-PLC), dihydropyridine-insensitive receptor-operated Ca2+ channels, and phospholipase A2. The current studies explored a role for ET stimulation of phosphatidylcholine-specific phospholipase D (PC-PLD) in intracellular signaling of this cell type. ET stimulated PLD activation, as measured by phosphatidic acid (PA) or phosphatidylethanol (PEt) accumulation, in a time- and concentration-dependent manner. Inhibition of diacylglycerol (DAG) kinase by ethylene glycol dioctanoate or 6-(2)4-[(4-fluorophenyl)-phenylmethylene]-1-piperadinyl]ethy l-7-methyl-5H - thiaxolo-[3,2-alpyrimidin]-5-one (R 59022) failed to blunt PA accumulation, indicating that PLD, and not DAG, was the source of PA. Inhibition of PA phosphohydrolase (PAP) by propranolol increased late accumulation of PA, suggesting that the prevailing metabolic flow was in the direction of PA to DAG. Phorbol 12-myristate 13-acetate (PMA) augmented ET-evoked PEt accumulation, whereas downregulation of protein kinase C (PKC) obviated agonist-induced PEt production. PMA augmentation of PLD activity proceeded independent of cytosolic free Ca2+ concentration. Ca2+ derived from either intracellular or extracellular sources enhanced ET-related PEt accumulation but was without effect in PKC-downregulated cells. Collectively, these observations indicate that ET stimulates PLD production in RMIC. PKC is the major regulator of this process, with Ca2+ playing a secondary, modulatory role. In addition, these data suggest that PC-PLD is coupled to the ETA receptor.
Department of Medicine, School of Medicine, Hadassah University Hospital, Jerusalem, Israel.
M M
MM
Friedlaender
D
D
Jain
Z
Z
Ahmed
D
D
Hart
R L
RL
Barnett
E P
EP
Nord
D000818:Animals; D002118:Calcium / Q000502:physiology*; D016232:Endothelins / Q000494:pharmacology*; D004789:Enzyme Activation; D020404:Glycerophospholipids; D015759:Ionomycin / Q000494:pharmacology; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D016232:Endothelins; D020404:Glycerophospholipids; D010712:Phosphatidic Acids; C051521:phosphatidylethanol; D015759:Ionomycin; D011493:Protein Kinase C; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium