The mechanisms underlying nonspecific supersensitivity induced by parasympathetic denervation in rat iris sphincter

大鼠虹膜括约肌副交感神经去神经诱导非特异性超敏反应的机制

基本信息

批准号：
10672050
负责人：
WATANABE Minoru
金额：
$ 2.05万
依托单位：
NAGOYA CITY UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

The mechanisms underlying nonspecific supersensitivity induced by parasympathetic denervation were examined using RT-PCR, semi-quantitative PCR and skinned fiber techniques. It was found that microinjection of AF64A, a novel choline uptake inhibitor, into anterior eye chamber of the rat resulted in changes in iris sphincter smooth muscle similar to those elicited by surgical parasympathetic denervation by removal of ciliary gangrionectomy. The changes were characterized in vitro by reduced contractile response to nerve stimulation presumably via the decrease in acetylcholine (ACh) release from parasympathetic nerve endings and by enhanced contractile responses both specific to exogenously applied ACh and nonspecific to serotonin and high KィイD1+ィエD1. Semi-quantitative PCR analyses of muscarinic receptors in rat iris indicate that mRNAs of m2, m3 and m4 subtypes are expressed in iris and that, unexpectedly, the expression level of m4 is much higher than that of m2 and m3. The sequence homology of m2, m3 and m4 in rat iris to those reported in genome or cDNA in other tissues of the rat was over 99%, while several differences in amino acid sequence in intracellular loops were found. Following the injection of AF64A, mRNA levels of m2 and m3 increased and that of m4 decreased, respectively. The CaィイD12+ィエD1 sensitivity of the contractile machinery measured in skinned fiber was slightly but significantly increased by AF64A injection. The increase in CaィイD12+ィエD1 sensitivity was abolished by addition of H7, protein kinase C inhibitor. AF64A injection resulted in marked increase in the maximum contractile response of iris sphincter muscle. A slight increase in amount of actin and no change in those of myosin and calmodulin in iris can not explain the increase in the maximum contractile response. A novel mechanism distinctive from the changes in major contractile and related regulatory proteins may underlie the nonspecific supersensitivity induced by AF64A.

使用 RT-PCR、半定量 PCR 和剥皮纤维技术检查了副交感神经去神经引起的非特异性超敏感性的机制，发现将新型胆碱摄取抑制剂 AF64A 显微注射到大鼠眼房会导致眼部变化。虹膜括约肌平滑肌与通过切除睫状神经节切除术去除副交感神经所引起的变化相似，其特征是在体外减少。对神经刺激的收缩反应可能是通过副交感神经末梢乙酰胆碱 (ACh) 释放的减少以及对外源应用的乙酰胆碱 (ACh) 特异性和血清素非特异性的收缩反应的增强以及大鼠毒蕈碱受体的高 KiD1+D1 的半定量 PCR 分析来实现的。 iris 表明 m2、m3 和 m4 亚型的 mRNA 在 iris 中表达，并且出乎意料的是， m4的表达水平远高于m2和m3，大鼠虹膜中m2、m3和m4与大鼠其他组织基因组或cDNA中报道的序列同源性超过99%，但氨基酸存在一些差异。注射 AF64A 后，发现细胞内环中的 m2 和 m3 的 mRNA 水平分别增加，而 m4 的 mRNA 水平降低。 AF64A 注射后，CaiD12+CaiD1 敏感性的增加被消除，注射 AF64A 蛋白激酶 C 导致虹膜括约肌最大收缩反应显着增加。虹膜中肌动蛋白含量的变化以及肌球蛋白和钙调蛋白含量的变化不能解释最大收缩反应的增加，这是一种不同于这些变化的新机制。主要收缩蛋白和相关调节蛋白中的蛋白质可能是 AF64A 诱导的非特异性超敏感性的基础。