Vol. 280, Issue 1, 492-500, 1997

Inhibition by Hydroxyl Radicals of Calcitonin Gene-Related Peptide-Mediated Neurogenic Vasorelaxation in Isolated Canine Lingual Artery¹

Masahiro Norisue, Kazuo Todoki and Eiichiro Okabe

Department of Pharmacology, Kanagawa Dental College, 82 Inaoka-Cho, Yokosuka, Kanagawa 238, Japan

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 × 10⁴ M H₂O₂ plus 2 × 10⁴ 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 × 10⁶ M) to block neurogenic constrictor responses. The periarterial nerve stimulation (10 V, 4-16 Hz, for 45 sec), exogenous CGRP (10⁸ M) or the ATP-sensitive K⁺ channel opener cromakalim (10⁶ M) produced relaxation of the rings at a stable plateau tension by the addition of norepinephrine (10⁵ 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 spin-trap 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 (10⁶ 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 (10⁵ 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·.