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 pept ide (CGRP)-containing vasodilator nerves, Although the vascular system might be considered as the first target of oxygen-derived free radica ls in some of the pathophysiological conditions, the effect of oxygen- derived free radicals on neurotransmission in CGRP nerves remains unkn own, We, therefore, investigated the role of oxygen-derived free radic als 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 art ery ring preparations, In all experiments, endothelium-denuded prepara tions (which were suspended in the tissue bath for isometric tension r ecordings) were treated with guanethidine (5 x 10(-6) M) to block neur ogenic 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 ri ngs at a stable plateau tension by the addition of norepinephrine (10( -5) M); the relaxations elicited by CGRP and cromakalim were human CGR P-(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 rela xations were markedly diminished, The effects afforded by the early ex posure to H2O2/FeSO4 reaction of the preparations were significantly p rotected by catalase (100 U/ml, H2O2 scavenger), dimethylthiourea (1 m M, H2O2 and HO . scavenger), dimethyl sulfoxide (100 mM, HO . scavenge r), 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 spin-trap 5,5-dimet hyl-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-o xide spin adduct. After exposure to capsaicin (10(-6) M) or H2O2/FeSO4 of the artery ring preparations,the intensity of CGRP-like immunoreac tivity of the periarterial nerves was reduced drastically; the relaxat ion caused by the nerve stimulation was nearly fully inhibited by caps aicin and H2O2/FeSO4 reaction. The relaxant response, however, to nitr oglycerin (10(-5) M) in the presence of norepinephrine to induce tone was unaffected by the early H2O2/FeSO4 exposure, The data obtained fro m the present study indicate that HO ., rather than H2O2, is the activ e agent in CGRP-mediated neurogenic relaxation. lt is suggested that t he 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 postjunct ional sites. It is also postulated that the second messenger system of the relaxation mediated, at least, by cyclic GMP may be less suscepti ble to HO ..