Calcium-Dependent Phospholipase A<sub>2</sub> Mediates the Production of Endothelium-Derived Hyperpolarizing Factor in Perfused Rat Mesenteric Prearteriolar Bed

Abstract

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 A<sub>2</sub> (PLA<sub>2</sub>) and diacylglycerol (DAG) lipase, to EDHF-mediated dilation of the rat mesenteric bed. We tested the hypothesis that PLA<sub>2</sub>-mediated release of AA is essential for the production of EDHF. Mesenteric beds were perfused with physiological salt solution (PSS) containing indomethacin and nitro-<i>L</i>-arginine methyl ester to block cyclooxygenase and nitric oxide synthase, respectively, and constricted with cirazoline (an α<sub>1</sub>-adrenoceptor agonist). Bolus applications of acetylcholine and histamine caused dose-dependent dilation of the constricted beds. The 85-kDa PLA<sub>2</sub> inhibitor, arachidonyl trifluoromethyl ketone (AACOCF<sub>3</sub>), at 3 µ<i>M</i>, 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<sub>3</sub> also blocked cirazoline-stimulated release of 6-keto-PG<sub>1α</sub>, but did not alter the vasodilation initiated by sodium nitroprusside (a nitric oxide donor), cromakalim (a K<sup>+</sup> channel activator), or by Na<sup>+</sup>/K<sup>+</sup>-ATPase activation, as measured by KCl vasodilation in preconstricted beds perfused with K<sup>+</sup>-free PSS. The 14-kDa PLA<sub>2</sub> inhibitor, oleyloxyethyl phosphorylcholine, also blocked EDHF vasodilation and also significantly inhibited K<sup>+</sup> channel activity. Neither the Ca<sup>2+</sup>-independent PLA<sub>2</sub> 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<sub>1α</sub>. We conclude that Ca<sup>2+</sup>-dependent PLA<sub>2</sub>, rather than DAG lipase, generates the AA for the production of EDHF in the perfused rat mesenteric bed.