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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.

采用RT-PCR、半定量PCR和皮肤纤维技术研究了去副交感神经诱导的非特异性超敏反应的机制。结果发现，微量注射AF 64 A，一种新的胆碱摄取抑制剂，到大鼠的前房导致虹膜括约肌平滑肌的变化类似于通过去除睫状神经节切除术的手术副交感神经去神经引起的。这些变化的特点是在体外减少收缩反应神经刺激可能通过减少乙酰胆碱（ACh）释放副交感神经末梢和增强收缩反应特异性外源性应用乙酰胆碱和非特异性血清素和高钾通道D1+乙酰胆碱D1。大鼠虹膜中毒蕈碱受体的半定量PCR分析表明，m2、m3和m4亚型的mRNA在虹膜中表达，并且出乎意料地，m4的表达水平远高于m2和m3的表达水平。m2、m3和m4在大鼠虹膜中的序列与基因组或其它组织cDNA中报道的序列同源性均在99%以上，但在胞内环中发现了几个氨基酸序列的差异，注射AF 64 A后，m2和m3的mRNA水平分别升高，m4的mRNA水平降低。在皮肤纤维中测量的收缩机械的Ca ~（2+）D_12 + Ca ~（2+）D_1敏感性通过AF 64 A注射轻微但显著地增加。加入蛋白激酶C抑制剂H7可消除Ca ~（2+）D_1 ~（12+）D_1敏感性的增加。注射AF 64 A后，虹膜括约肌的最大收缩反应明显增加。肌动蛋白含量的轻微增加和肌球蛋白和钙调蛋白含量的无变化不能解释最大收缩反应的增加。一种新的机制，从主要的收缩和相关的调节蛋白的变化不同，可能是AF 64 A诱导的非特异性超敏反应的基础。