Inhibition by hydroxyl radicals of calcitonin gene-related peptide-mediated neurogenic vasorelaxation in isolated canine lingual artery.

Canine lingual arteries are innervated by calcitonin gene-related peptide (CGRP)-containing vasodilator nerves. Although the vascular system might be considered as the first target of oxygen-derived free radicals in some of the pathophysiological conditions, the effect of oxygen-derived free radicals on neurotransmission in CGRP nerves remains unknown. We, therefore, investigated the role of oxygen-derived free radicals generated from Fenton's reagent (3 x 10(-4) M H2O2 plus 2 x 10(-4) M FeSO4) on CGRP-mediated neurogenic relaxation of canine lingual artery ring preparations. In all experiments, endothelium-denuded preparations (which were suspended in the tissue bath for isometric tension recordings) were treated with guanethidine (5 x 10(-6) M) to block neurogenic constrictor responses. The periarterial nerve stimulation (10 V, 4-16 Hz, for 45 sec), exogenous CGRP (10(-8) M) or the ATP-sensitive K+ channel opener cromakalim (10(-6) M) produced relaxation of the rings at a stable plateau tension by the addition of norepinephrine (10(-5) M); the relaxations elicited by CGRP and cromakalim were human CGRP-(8-37)- and glibenclamide-abolishable, respectively. When the nerve stimulation, CGRP and cromakalim were given after H2O2/FeSO4 exposure (Fenton's reagent was removed from the tissue bath), the observed relaxations were markedly diminished. The effects afforded by the early exposure to H2O2/FeSO4 reaction of the preparations were significantly protected by catalase (100 U/ml, H2O2 scavenger), dimethylthiourea (1 mM, H2O2 and HO. scavenger), dimethyl sulfoxide (100 mM, HO. scavenger), deferoxamine (1 mM, a powerful iron chelator) and by a cocktail of catalase-deferoxamine. Generation of HO. from H2O2/FeSO4 was studied by electron spin resonance spectroscopy using the spintrap 5,5-dimethyl-1-pyrroline-N-oxide. We found that H2O2/ FeSO4 reaction formed a 1:2:2:1 quartet, characteristic of the HO-5,5-dimethyl-1-pyrroline-N-oxide spin adduct. After exposure to capsaicin (10(-5) M) or H2O2/FeSO4 of the artery ring preparations, the intensity of CGRP-like immunoreactivity of the periarterial nerves was reduced drastically; the relaxation caused by the nerve stimulation was nearly fully inhibited by capsaicin and H2O2/FeSO4 reaction. The relaxant response, however, to nitroglycerin (10(-5) M) in the presence of norepinephrine to induce tone was unaffected by the early H2O2/ FeSO4 exposure. The data obtained from the present study indicate that HO., rather than H2O2, is the active agent in CGRP-mediated neurogenic relaxation. It is suggested that the HO. can deplete endogenous CGRP localized prejunctionally and also damage CGRP-induced relaxation of canine lingual artery preparations that is caused by activation of ATP-sensitive K+ channels at postjunctional sites. It is also postulated that the second messenger system of the relaxation mediated, at least, by cyclic GMP may be less susceptible to HO..

The effect of hypochlorous acid and hydrogen peroxide on coronary flow and arrhythmogenesis in myocardial ischemia and reperfusion.

The purpose of this study was to investigate the effect of the oxidants hypochlorous acid (HOCl) and hydrogen peroxide (H2O2) on the vulnerability of the myocardium to reperfusion-induced arrhythmias following global ischemia. After a 15 min equilibration period with or without experimental intervention, isolated perfused rat hearts in the Langendorff mode were made globally ischemic for 5 min by cross-clamping the aortic line. No dysrhythmias were evoked upon reperfusion at the 5 min global ischemia time period. HOCl or H2O2 were added to the perfusate 5 min into the equilibration period with a total exposure of 10 min. Global ischemia was then induced for 5 min followed by 10 min of reperfusion. A dose-response curve for HOCl (50-200 microM) indicated the development of idioventricular rhythms, in a concentration-dependent way. Furthermore, coronary flow of the hearts exposed to 100 and 200 microM HOCl, at 5 min post-reperfusion, was decreased; methionine (10 microM to 1 mM), an accepted scavenger for HOCl, prevented the responses to 200 microM HOCl, in a concentration-dependent manner. All hearts exposed to 200 microM H2O2 developed ventricular dysrhythmias during the reperfusion period. Coronary flow increased after 5 min of exposure to 200 microM H2O2 and remained elevated during reperfusion. It is concluded that toxic oxygen derived products are capable of increasing the susceptibility of the myocardium to reperfusion induced arrhythmias, and that although the electrical responses to exposure to those two oxidants were similar, the effects on the vasculature were not the same.