Prostacyclin formation elicited by endothelin-1 in rat aorta is mediated via phospholipase D activation and not phospholipase C or A(2)

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that also stimulates production of prostacyclin (PGI(2)) from arachidonic acid. The purpose of this study was to determine the contribution of phospholipases (PLs) A(2), C, and/or D in ET-1-induced PGI(2) formation in the rat aorta, measured as immunoreactive 6-ketoprostaglandin (PG) F-1 alpha. ET-1 increased 6-keto-PGF(1 alpha) formation, which was not affected by a PLA(2) inhibitor, 7,7-dimethyl eicosadienoic acid (DEDA). Furthermore, ET-1 failed to stimulate PLA(2) activity measured in the cytosol (cPLA(2)), using phosphatidylcholine, L-a-1-palmitoyl-2-arachidonyl[C-14] as a substrate. However, the adrenergic agonist norepinephrine increased 6-keto-PGF(1 alpha) formation, which was attenuated by DEDA, and enhanced PLA(2) activity. ET-1 enhanced PLC activity, as indicated by increased inositol phosphate production, which was prevented by a PLC inhibitor, U-73122. However, ET-1-induced 6-keto-PGF(1 alpha) production was not altered by U-73122. An inhibitor of PLD activation, C-2-ceramide, attenuated ET-1-induced PLD activity, as indicated by the production of phosphatidylethanol. Furthermore, ET-1-induced 6-keto-PGF(1 alpha), formation was inhibited by C-2-ceramide as well as by ethanol treatment. Moreover, inhibitors of phosphatidate phosphohydrolase (propranolol) and diacylglycerol lipase (RHC-80267), attenuated ET-1-induced 6-keto-PGF(1 alpha) formation. Finally, ET-1-induced activation of PLD was not attenuated by a selective PKC inhibitor, bisindolylmaleimide I. These data suggest a novel pathway for ET-1-induced PGI(2) formation in the rat aorta involving activation of PLD but not cPLA(2) and independent of PLC or PKC activation.