Endothelin-1 activates phospholipase D and thymidine incorporation in fibroblasts overexpressing protein kinase C beta 1.
Endothelins (ETs) are a family of extremely potent vasoconstrictor peptides. In addition, ET-1 acts as a potent mitogen and activates phospholipase C in smooth muscle cells and fibroblasts. We examined the effects of ET-1 on phosphatidylcholine (PC) metabolism and thymidine incorporation in control Rat-6 fibroblasts and in cells that overexpress protein kinase C beta 1 (PKC). PC pools were labeled with [3H]myristic acid, and formation of phosphatidylethanol (PEt), an unambiguous marker of phospholipase D (PLD) activation, was monitored. ET-1 stimulated much greater PEt formation in the PKC overexpressing cells. ET-1 action was dose-dependent with a half-maximal effect at 1.0 x 10(-9) M. With increasing ethanol concentrations, [3H]PEt formation increased at the expense of [3H]phosphatidic acid (PA). Propranolol, an inhibitor of PA phosphohydrolase, increased [3H]PA accumulation and decreased [3H]diacylglycerol (DAG) formation. These data are consistent with the formation of [3H]DAG from PC by the sequential action of PLD and PA phosphohydrolase. Phorbol esters are known to stimulate thymidine incorporation and PLD activity to a greater extent in PKC overexpressing cells than in control cells. ET-1 also stimulates thymidine incorporation to a greater extent in the PKC overexpressing cells. The effect of ET-1 on thymidine incorporation into DNA in the overexpressing cells was also dose-dependent with a half-maximal effect at 0.3 x 10(-9) M. Enhanced PLD activity induced by ET-1 in the overexpressing cells may contribute to the mitogenic response, especially in light of a possible role of the PLD product, PA, in regulation of cell growth.
Pai
J K
JK
Schering-Plough Research, Bloomfield, New Jersey 07003.
Dobek
E A
EA
Bishop
W R
WR
D000818:Animals; D002453:Cell Cycle / Q000187:drug effects*; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D004075:Diglycerides / Q000378:metabolism; D016232:Endothelins / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D000431:Ethanol / Q000378:metabolism; D005347:Fibroblasts / Q000201:enzymology; D020404:Glycerophospholipids; D066298:In Vitro Techniques; D010711:Phosphatidate Phosphatase / Q000378:metabolism; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D011433:Propranolol / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004075:Diglycerides; D016232:Endothelins; D020404:Glycerophospholipids; D010712:Phosphatidic Acids; C051521:phosphatidylethanol; D000431:Ethanol; D004247:DNA; D011433:Propranolol; D011493:Protein Kinase C; D010711:Phosphatidate Phosphatase; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate