Calcium-dependent phospholipase A2 mediates the production of endothelium-derived hyperpolarizing factor in perfused rat mesenteric prearteriolar bed

The isolated, perfused rat mesenteric bed releases a cytochrome P450-linked metabolite of arachidonic acid (AA) as endothelium-derived hyperpolarizing factor (EDHF) in response to acetylcholine and histamine. This study assessed the relative contribution of two AA-generating pathways, phospholipase Az (PLA(2)) and diacylglycerol (DAG) lipase, to EDHF-mediated dilation of the rat mesenteric bed. We tested the hypothesis that PLA(2)-mediated release of AA is essential for the production of EDHF. Mesenteric beds were perfused with physiological salt solution (PSS) containing indomethacin and nitro-1-arginine methyl ester to block cyclooxygenase and nitric oxide synthase, respectively, and constricted with cirazoline (an alpha(1)-adrenoceptor agonist). Bolus applications of acerylcholine and histamine caused dose-dependent dilation of the constricted beds. The 85-kDa PLA(2) inhibitor, arachidonyl trifluoromethyl ketone (AACOCF(3)), at 3 mu M profoundly blunted decreases in perfusion pressure initiated by 1 nmol acetylcholine (94.3 +/- 1.7%) and by 100 nmol histamine (88.5 +/- 3.3%) to 9.6 = 7.5 and 8.6 +/- 6.0%, respectively. AACOCF(3) also blocked cirazoline-stimulated release of 6-keto-PG(1 alpha), but did not alter the vasodilation initiated by sodium nitroprusside (a nitric oxide donor), cromakalim (a K+ channel activator), or by Na+/K+-ATPase activation, as measured by KCl vasodilation in preconstricted beds perfused with K+-free PSS. The 14-kDa PLA(2) inhibitor, oleyloxyethyl phosphorylcholine, also blocked EDHF vasodilation and also significantly inhibited K+ channel activity. Neither the Ca2+-independent PLA(2) inhibitor, HELSS [E-6-(bromomethylene)-tetrahydro-3-(1,naphthalenyl)-2H-pyran-2-one], nor DAG lipase inhibitor, RHC-80267 [1,6-bis-(cyclohexyloximino-carbonylamino)-hexane] altered EDHF-mediated vasodilation. However, RHC-80267 blocked cirazoline-stimulated release of 6-keto-PGF(1 alpha). We conclude that Ca2+-dependent PLA(2), rather than DAG lipase, generates the AA for the production of EDHF in the perfused rat mesenteric bed.