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..