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Molecular and Biological Approaches to the Genesis of Coronary Spastic Angina : With special reference to the intracellular signaling pathway participating coronary artery contraction

冠状动脉痉挛性心绞痛发生的分子和生物学方法：特别参考参与冠状动脉收缩的细胞内信号通路

基本信息

批准号：
15590712
负责人：
OKAMURA Ken
金额：
$ 2.18万
依托单位：
Hirosaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

Phospholipase C (PLC)-d1, an isozyme of phospholipase C that constitutes a key enzyme for the mechanism for cellular contraction, is activated by Ca^<++> and induces a further increment in the [Ca^<2+>]_i and enhanced response to the constrictor stimuli. We previously reported that PLC activity in the membrane fraction of the cultured skin fibroblasts obtained from the patients with coronary spastic angina is enhanced compared with that of control subjects and PLC activity shows a positive correlation with the basal tone of the coronary artery and its response to the constrictor stimulus (J Am Coll Cardiol,2000). By the sequence analysis of the cDNA coding for PLC-d1 obtained from fibroblasts, we recently found a conversion of guanine to adenine (A) at nucleotide position 864, resulting in the amino acid replacement of arginine 257 by histidine (R257H)(Circulation,2002). The incidence of 864A/A in genomic DNA was greater in patients with CSA than in male control subjects. The activity … More of the variant PLC-d1 protein was 2-fold higher than that of the wild-type protein. The peak increase in [Ca^<2+>]_i in response to acetylcholine was greater in the cells with the variant PLC-d1 than in those with the wild type. Thus, R257H variant in the PLC-d1 gene is associated with enhancement of enzyme activity, and can be a novel mechanism for the enhanced coronary vasomotility. However, R257H variant was detected only in 10% of the patients and we have sought other mechanisms for enhanced activity of PLC-d1.It is known that small G protein Rho A suppresses PLC-d1 activity whereas P122 protein enhances the activity. We compared the protein expressions of Rho A and P122 between the patient groups with (n=11) and without coronary spastic angina (n=9) by Western blot analysis. Although there was no difference in the expression of Rho A protein between the 2 groups, the expression of P122 was markedly enhanced in the groups of coronary spastic angina compared with the group without spastic angina. The protein level of PLC-d1 was not different between the 2 groups. Thus, enhanced expression of P122 protein may explain the enhanced PLC-d1 activity in the patients with coronary spastic angina. We are now investigating the molecular mechanism for the enhanced expression of P122 protein. Less

磷脂酶C(PLC)-D_1是磷脂酶C的同工酶，是细胞收缩机制中的一个关键酶，它被钙激活，导致[Ca^&lt；2+&gt；]_i进一步升高，对收缩肌刺激的反应增强。我们曾报道冠状动脉痉挛型心绞痛患者培养的皮肤成纤维细胞膜上的PLC活性高于对照组，并且PLC活性与冠状动脉的基础张力及其对收缩刺激的反应呈正相关(J Am Coll Medicol，2000)。通过对从成纤维细胞中获得的PLC-D1编码基因的序列分析，我们最近发现在864位核苷酸上鸟嘌呤转变为腺嘌呤(A)，导致精氨酸257的氨基酸被组氨酸(R257H)取代(循环，2002)。CsA患者基因组DNA中864A/A的发生率高于男性对照组。活动…变异型PLC-D1蛋白是野生型蛋白的2倍。[Ca~(2+)~(2+)]_i对乙酰胆碱的反应峰值在变异的PLC-D_1细胞中大于野生型。因此，PLC-D1基因的R257H变异与酶活性增强有关，可能是冠脉血管动力增强的新机制。然而，只有10%的患者检测到R257H突变，我们还寻找了其他机制来增强PLC-d1的活性。众所周知，小G蛋白Rho A抑制PLC-d1的活性，而P122蛋白增强PLC-d1的活性。通过Western印迹分析比较11例冠脉痉挛心绞痛患者和9例非冠脉痉挛心绞痛患者Rho A和P122的蛋白表达。Rho A蛋白的表达在冠脉痉挛心绞痛组与非痉挛心绞痛组比较差异无统计学意义，但冠脉痉挛心绞痛组P122的表达明显高于非痉挛心绞痛组。两组间PLC-D1蛋白表达水平差异无统计学意义。因此，P122蛋白表达增强可能是冠脉痉挛型心绞痛患者PLC-D1活性增强的原因之一。我们正在研究P122蛋白表达增强的分子机制。较少