Norisue M, Todoki K, Okabe E
Department of Pharmacology, Kanagawa Dental College, Japan.
J Pharmacol Exp Ther. 1997 Jan;280(1):492-500.
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..
犬舌动脉由含降钙素基因相关肽（CGRP）的血管舒张神经支配。尽管在某些病理生理条件下，血管系统可能被视为氧衍生自由基的首要靶点，但氧衍生自由基对CGRP神经中神经传递的影响仍不清楚。因此，我们研究了由芬顿试剂（3×10⁻⁴M H₂O₂加2×10⁻⁴M FeSO₄）产生的氧衍生自由基对犬舌动脉环制备物中CGRP介导的神经源性舒张的作用。在所有实验中，用胍乙啶（5×10⁻⁶M）处理去内皮的制备物（将其悬浮于组织浴中进行等长张力记录）以阻断神经源性收缩反应。动脉周围神经刺激（10V，4 - 16Hz，持续45秒）、外源性CGRP（10⁻⁸M）或ATP敏感性钾通道开放剂克罗卡林（10⁻⁶M）通过添加去甲肾上腺素（10⁻⁵M）使环在稳定的平台张力下舒张；CGRP和克罗卡林引起的舒张分别可被人CGRP -（8 - 37）和格列本脲消除。当在暴露于H₂O₂/FeSO₄（从组织浴中去除芬顿试剂）后给予神经刺激、CGRP和克罗卡林时，观察到的舒张明显减弱。过氧化氢酶（100U/ml，H₂O₂清除剂）、二甲基硫脲（1mM，H₂O₂和HO·清除剂）、二甲基亚砜（100mM, HO·清除剂）、去铁胺（1mM，一种强力铁螯合剂）以及过氧化氢酶 - 去铁胺混合物可显著保护制备物早期暴露于H₂O₂/FeSO₄反应所产生的影响。使用自旋捕获剂5,5 - 二甲基 - 1 - 吡咯啉 - N - 氧化物通过电子自旋共振光谱研究了H₂O₂/FeSO₄产生HO·的情况。我们发现H₂O₂/FeSO₄反应形成了一个1:2:2:1的四重峰，这是HO - 5,5 - 二甲基 - 1 - 吡咯啉 - N - 氧化物自旋加合物的特征。在动脉环制备物暴露于辣椒素（10⁻⁵M）或H₂O₂/FeSO₄后，动脉周围神经的CGRP样免疫反应强度急剧降低；神经刺激引起 的舒张几乎完全被辣椒素和H₂O₂/FeSO₄反应抑制。然而，在存在去甲肾上腺素以诱导张力的情况下，对硝酸甘油（10⁻⁵M）的舒张反应不受早期H₂O₂/FeSO₄暴露的影响。本研究获得的数据表明，在CGRP介导的神经源性舒张中，起作用的是HO·而非H₂O₂。提示HO·可在节前消耗内源性CGRP，并且还可损害CGRP诱导的犬舌动脉制备物的舒张，这种舒张是由节后位点ATP敏感性钾通道的激活所引起的。还推测至少由环鸟苷酸介导的舒张的第二信使系统可能对HO·不太敏感。
