ABSTRACT
The vasodilatation of the isolated, perfused mesenteric bed of the rat to acetylcholine is mostly independent of nitric oxide and prostanoids. This nitric oxide-/prostanoidindependent vasodilatation is blocked by inhibitors of Ca 2 + -activated potassium channels and thus presumably is mediated by endothelium-derived hyperpolarizing factor ( E D H F ) . The hypothesis was tested that epoxyeicosatrienoic acid (EET), is the E D H F responsible. Vascular responses to acetylcholine were studied in isolated mesenteric beds perfused with physiological salt solutions containing N^-nitro-L-arginine methyl ester and indomethacin to block the synthesis of nitric oxide and prostanoids, respectively. The following were determined: (a) NADPH-dependent metabolism of radio-labeled arachidonic acid in homogenates of, and microsomes from, mesenteric blood vessels by a reverse-phase high performance liquid chromatography ( H P L C ) ; (b) E E T release from the mesenteric vascular bed by an HPLC-coupled, electrospray ionization tandem mass spectrometry ( H P L C - M S - M S ) . Acetylcholine elicited dilatation of the mesenteric vascular bed accompanied by release of 1 4[C]-arachidonic acid (after labeling) during constrictions caused by an a\ -adrenoceptor agonist. Both effects of acetylcholine were blocked by compounds A A C O C F 3 and U73122, inhibitors of phospholipase A 2 and C, respectively. Vascular homogenates, or microsomes converted 1 4[C]-arachidonic acid to EETs (determined by H P L C profiles) in the presence of N A D P H and molecular oxygen. Infusion of acetylcholine, but not pinacidil or sodium nitroprusside, to constricted blood vessels released a substance identified positively by H P L C - M S - M S as an E E T . Authentic 5,6-EET elicited vasodilatation, which like that to acetylcholine, was attenuated by tetrabutylammonium, apamin plus charybdotoxin, or by 80 m M K + . These data strongly suggest that an E E T is the mediator of the nitric oxide-/prostanoid-independent dilatation in response to acetylcholine, and thus that an E E T probably is E D H F in the rat mesenteric vascular bed.
