20-hydroxyeicosatetraenoic acid mediates angiotensin II-induced phospholipase D activation in vascular smooth muscle cells

Angiotensin II (Ang II) activates cytosolic phospholipase A(2) (cPLA(2)) and phospholipase D (PLD) in rabbit vascular smooth muscle cells (VSMCs). Ang II also activates ras/mitogen-activated protein (MAP) kinase in VSMCs; this activation is mediated by 20-hydroxyeicosatetraenoic acid (HETE) and 12(S)-HETE, which are metabolites of arachidonic acid generated by cytochrome P450 4A and lipoxygenase, respectively, produced on activation of cPLA(2). The purpose of this study was to determine if Ang II-induced PLD activation in VSMCs is mediated through the ras/extracellular signal-regulating kinase (ERK) pathway by arachidonic acid metabolites that are generated consequent to cPLA(2) stimulation. Inhibitors of PLD (C-2 ceramide), phosphatidate phosphohydrolase (propranolol), and diacylglycerol lipase (RHC 80267) attenuated Ang II-induced arachidonic acid release. Ang II-induced PLD activation, as measured by [H-3]phosphatidylethanol production, was inhibited by C-2 ceramide but not by propranolol or RHC 80267. Ang II-induced PLD activation was decreased by the inhibitor methyl arachidonylfluorophosphate (MAFP) and the antisense oligonucleotide of cPLA(2). Inhibitors of lipoxygenases (baicalein) and cytochrome P450 4A (ODYA) attenuated Ang II-induced PLD activation. 20-HETE and 12(S)-HETE increased PLD activity. Inhibitors of ras farnesyltransferase (FPT III and BMS-191563) and MAP kinase kinase (UO126) attenuated the increase in PLD activity elicited by 20-HETE and Ang II. PLD2 was the main isoform activated by Ang II in VSMCs. These data suggest that the CYP4A metabolite 20-HETE, which is generated from arachidonic acid after cPLA(2) activation by Ang II, stimulates the ras/MAP kinase pathway, which in turn activates PLD2 and releases further arachidonic acid for prostaglandin synthesis through the phosphatidate phosphohydrolase/diacylglycerol lipase pathway.