20-Hydroxyeicosatetraenoic Acid Mediates Angiotensin Ii-Induced Phospholipase D Activation in Vascular Smooth Muscle Cells
Angiotensin II (Ang II) promotes arachidonic acid (AA) release via activation of cytosolic phospholipase A2 (cPLA2) and D (PLD) in rabbit vascular smooth muscle cells (VSMC). Ang II also stimulates the Ras/MAP kinase pathway, and MAP kinase activates cPLA2 and PLD in VSMC. The increase in Ras/MAP kinase activity elicited by Ang II in VSMC is mediated by 20-hydroxyeicosatetraenoic acid (HETE) and to a lesser extent by 12(S)-HETE, metabolites of AA generated by cytochrome P450 (CYP) and lipoxygenase (LO), respectively, upon activation of cPLA2 by calcium calmodulin-dependent kinase II. The purpose of this study was to determine if Ang II-induced PLD activation stimulation in VSMC from rabbit aorta is mediated by the Ras/MAP kinase pathway via AA metabolites generated by cPLA2. Ang II (100 nM) increased PLD activity measured as the production of 3H-phosphatidylethanol from phospholipids in VSMC prelabeled with 3H-oleic. Inhibitors of PLD (C2-ceramide, 10 μM), phosphatidate phosphohydrolase (propranolol, 10 μM) and diacylglycerol lipase (RHC 80267, 10 μM) attenuated Ang II-induced AA release. PLD activity was not altered by propranol and RHC 80267. Therefore, Ang II-induced AA release is partly mediated through PLD activation. Ang II-induced PLD activation was decreased by MAFP (50 μM), a cPLA2 inhibitor. Inhibitors of lipoxygenase (baicalein, 5 μM) and CYP (ODYA, 5 μM) but not cycloxygenase (indomethacin, 10 μM) attenuated Ang II-induced PLD activation. AA metabolites of CYP, 20-HETE (0.25-1 μM) and 12(S)-HETE increased PLD activity. Inhibitors of ras farnesyltransferase (FPT III, 25 μM, 18 hr) and MAP kinase kinase (UO126, 10 μM) significantly attenuated the increase in PLD activity elicited by 20-HETE. These data suggest that the CYP metabolite, 20-HETE, generated from AA following cPLA2 activation by Ang II, stimulates Ras/MAP kinase pathway, which in turn activates PLD and release additional AA.