Background-Peroxynitrite generated from nitric oxide (NO) and superoxide (O 2 Ϫ ) contributes to ischemia/reperfusion (I/R) injury. Feedback inhibition of endothelial NO synthase by NO may inhibit O 2 Ϫ production generated also by endothelial NO synthase at diminished local L-arginine concentrations accompanying I/R. Methods and Results-During hindlimb I/R (2.5 hours/2 hours), in vivo NO was monitored continuously (porphyrinic sensor), and high-energy phosphates, reduced and oxidized glutathione (chromatography), and I/R injury were measured intermittently. Rabbits receiving human serum albumin (HSA) (controls) were compared with those receiving S-nitroso human serum albumin (S-NO-HSA) beginning 30 minutes before reperfusion for 1 hour or 30 minutes before ischemia for 3.5 hours (0.1 mol · kg Ϫ1 · h Ϫ1 ). The onset of ischemia led to a rapid increase of NO from its basal level (50Ϯ12 nmol/L) to 120Ϯ20 and 220Ϯ15 nmol/L in the control and S-NO-HSA-treated groups, respectively. In control animals, NO dropped below basal levels at the end of ischemia and to undetectable levels (Ͻ1 nmol/L) during reperfusion. In S-NO-HSA-treated animals, maximal NO levels never decreased below basal concentration and on reperfusion were 100Ϯ15 nmol/L (S-NO-HSA preischemia group, 175Ϯ15 nmol/L). NO supplementation by S-NO-HSA led to partial and in the preischemia group to total preservation of high-energy phosphates and glutathione status in reperfused muscle (eg, preischemia groups: ATP, 30.23Ϯ5.02 mol/g versus control, 15.75Ϯ4.33 mol/g, PϽ0.0005; % oxidized glutathione, 4.49Ϯ1.87% versus control, 22.84Ϯ6.39%, PϽ0.0001). S-NO-HSA treatment in all groups led to protection from vasoconstriction and reduced edema formation after reperfusion (eg, preischemia groups: interfiber area, 12.94Ϯ1.36% versus control, 27.83Ϯ1.95%, PϽ0.00001). Conclusions-Long

1 The effects of hydrogen peroxide (H202, 0.1-1 mM) on the tone of the rings of rabbit aorta precontracted with phenylephrine (0.2-0.3 gAM) were studied.2 H202 induced a concentration-dependent relaxation of both the intact and endothelium-denuded rings. However, in the presence of intact endothelium, H202-induced responses were 2-3 fold larger than in its absence, demonstrating the existence of endothelium-independent and endothelium-dependent components of the vasorelaxant action of H202. 3 The endothelium-dependent component of H202-induced relaxation was prevented by N0-nitro-Larginine methyl ester (L-NAME, 30 gM) or N0-monomethyl-L-arginine (300 JAM), inhibitors of nitric oxide synthase (NOS), in a manner that was reversible by L-, but not by D-arginine (2 mM). The inhibitors of NOS did not affect the responses of denuded rings.4 Methylene blue (10 JAM), an inhibitor of soluble guanylate cyclase, blocked H202-induced relaxation of both the intact and denuded rings. 5 H202 (1 mM) enhanced the efflux of cyclic GMP from both the endothelium-intact and denuded rings. The effect of H202 was 4 fold greater in the presence of intact endothelium and this endotheliumdependent component was abolished after the inhibition of NOS by L-NAME (30 JAM). 6 In contrast to the effects of H202, the vasorelaxant action of stable organic peroxides, tert-butyl hydroperoxide or cumene hydroperoxide, did not have an endothelium-dependent component. Moreover, they did not potentiate the efflux of cyclic GMP from the rings of rabbit aorta. 7 Exogenous donors of NO, specifically, 3-morpholinosydnonimine (SIN-1), glyceryl trinitrate or sodium nitroprusside were used to decrease the tone of denuded rings to the level induced by endogenous NO released from intact endothelium. This procedure did not influence the vasorelaxant activity of H202, showing that H202 does not potentiate the vasorelaxant action of NO within the smooth muscle. 8 Thus, H202-induced relaxation in the rabbit aorta has both endothelium-dependent and independent components. The endothelium-dependent component of the relaxant action of H202 is due to enhanced endothelial synthesis of NO.

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