Nature of Endothelium-Derived Relaxing Factor: Are There Two Relaxing Mediators?

The role of arachidonic acid in forming endothelium-derived relaxing factor remains controversial.This controversy may be explained if more than one factor exists.To test this hypothesis, the effects of various inhibitors of arachidonic acid metabolism were studied.The perfusate from canine femoral arteries with endothelium was bioassayed with coronary artery rings without endothelium.Treatment of the perfused segment (but not the bioassay ring) with inhibitors of phospholipase A 2 (quinacrine) or cytochrome P 45 0 (metyrapone) had no effect on the basal relaxing activity of the effluent; treatment with the inhibitor of lipoxygenase, nordihydroguaiaretic acid, significantly depressed it.With increasing concentrations of acetylcholine, a biphasic concentration-relaxation curve was obtained; the enzyme inhibitors depressed or prevented the first phase but did not affect the second phase.Infusion of arachidonic acid or soybean llpoxldase directly on the bioassay ring did not cause relaxation; together, they evoked concentration-dependent relaxations.These data suggest that acetylcholine can trigger the release of two chemically different relaxing mediators from the endothelium of the canine femoral artery.One factor may be a product of lipoxygenase (or epoxigenase).The second factor is not a metabolite of arachidonic acid and may be released under basal conditions.The existence of two (or more) chemically different endothelium-derived mediators may help to explain the controversial data regarding the nature of the factor(s).(Circulation Research 1987;61(suppl II):II-61-II-67) E ndothelial cells release a labile relaxing sub- stance when stimulated by acetylcholine.'~7 To differentiate from other vasoactive substances, the term endothelium-derived relaxing factor 8 was introduced.Anoxia and various inhibitors of phospholipase A 2 (the enzyme facilitating the release of arachidonic acid from membrane phospholipids) and lipoxygenase prevent endothelium-dependent relaxation to acetylcholine in a variety of blood vessels from different species.8 " 10 Thus, it has been postulated that endothelium-derived relaxing factor may be a product of lipoxygenase generated during the oxidative metabolism of arachidonic acid.8 However, bioassay studies on the rabbit aorta have demonstrated that inhibitors of lipoxygenase do not prevent the intraluminal release of endothelium-derived relaxing factor but chemically inactivate the released mediator in the perfusate.2 Furthermore, cultured endothelial cells release a relaxing factor that is hydrophylic 6 but not lipophylic as are the metabolites of arachidonic acid.These contradictory findings concerning the involvement of product(s) of the metabolism of arachidonic acid in endothelium-dependent inhibitory responses may be explained if there were multiple