Calcium-Dependent Phospholipase A₂ Mediates the Production of Endothelium-Derived Hyperpolarizing Factor in Perfused Rat Mesenteric Prearteriolar Bed

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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 A₂ (PLA₂) and diacylglycerol (DAG) lipase, to EDHF-mediated dilation of the rat mesenteric bed. We tested the hypothesis that PLA₂-mediated release of AA is essential for the production of EDHF. Mesenteric beds were perfused with physiological salt solution (PSS) containing indomethacin and nitro-L-arginine methyl ester to block cyclooxygenase and nitric oxide synthase, respectively, and constricted with cirazoline (an α₁-adrenoceptor agonist). Bolus applications of acetylcholine and histamine caused dose-dependent dilation of the constricted beds. The 85-kDa PLA₂ inhibitor, arachidonyl trifluoromethyl ketone (AACOCF₃), at 3 µM, 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. AACOCF₃ also blocked cirazoline-stimulated release of 6-keto-PG1α, 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 PLA₂ inhibitor, oleyloxyethyl phosphorylcholine, also blocked EDHF vasodilation and also significantly inhibited K⁺ channel activity. Neither the Ca²⁺-independent PLA₂ 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-PGF1α. We conclude that Ca²⁺-dependent PLA₂, rather than DAG lipase, generates the AA for the production of EDHF in the perfused rat mesenteric bed.