These studies have employed various experimental preparations including rabbit aorta in tissue baths and cultured bovine aorta endothelial cells in order to study the formation and mechanism of action of endothelium-derived relaxing factor utilizing a number of different physiological and biochemical measurements.
In a prototypical study, for example, rings of rabbit thoracic aorta, precontracted with phenylephrine, were used to show that 6-anilino-5,8-quinolinedione (LY83583) inhibited the endothelium-dependent relaxation produced by either acetylcholine or the calcium ionophore A23187. The production of the superoxide ion was monitored employing a split-beam spectrophotometer in a continuous assay, as superoxide dismutase inhibitable ferricytochrome c reduction. This showed that superoxide accumulated in the culture fluid of LY83583-treated bovine endothelial cells or of incubated rabbit aorta.
Other studies suggested that LY83583 was reduced to a semiquinone or hydroquinone which then produced superoxide ions by reacting with molecular oxygen. This was consistent with the knowledge that the superoxide ion is an inhibitor of the relaxation of aortic rings by nitric oxide, and that LY83583 inhibits nitric oxide-mediated relaxation of precontracted rings of aorta as effectively as it inhibits EDRF-induced relaxation.
Thus, the studies were consistent with the idea that endothelium-derived relaxing factor might be identical to nitric oxide, and that the inhibitory action of LY83583 is the result of the formation of the superoxide ion. Other studies, like the one described here, are being used to provide additional information about the identity, formation, and reactivity of EDRF.
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