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

磷脂酶C（PLC）-d1是磷脂酶C的一种同工酶，是细胞收缩机制的关键酶，它被Ca ^++激活，并诱导[Ca ^2 +] i进一步增加，增强对收缩刺激的反应。我们先前报道，与对照受试者相比，从冠状动脉痉挛性心绞痛患者获得的培养皮肤成纤维细胞的膜部分中的PLC活性增强，并且PLC活性与冠状动脉的基础张力及其对缩窄刺激的反应呈正相关（J Am科尔Cardiol，2000）。通过对从成纤维细胞获得的编码PLC-d1的cDNA的序列分析，我们最近发现在核苷酸位置864处鸟嘌呤转化为腺嘌呤（A），导致精氨酸257被组氨酸（R257H）取代（Circulation，2002）。CSA患者基因组DNA中864 A/A的发生率高于男性对照组。活动 关于我们 PLC-d1蛋白的表达量是野生型蛋白的2倍。与野生型相比，具有变异型PLC-d1的细胞中响应乙酰胆碱的[Ca ^<2 +>] i的峰值增加更大。因此，PLC-d1基因中的R257 H变体与酶活性的增强相关，并且可能是增强冠状动脉血管运动性的新机制。然而，仅在10%的患者中检测到R257 H变异，我们还寻找了PLC-d1活性增强的其他机制。已知小G蛋白Rho A抑制PLC-d1活性，而P122蛋白增强PLC-d1活性。采用Western blot方法检测11例有冠状动脉痉挛性心绞痛患者和9例无冠状动脉痉挛性心绞痛患者的Rho A和P122蛋白表达。两组间Rho A蛋白表达差异无统计学意义，但痉挛性心绞痛组P122蛋白表达明显高于非痉挛性心绞痛组。PLC-d1的蛋白水平在两组之间没有差异。P122蛋白表达增强可能是痉挛型心绞痛患者PLC-d1活性增强的原因。我们正在研究P122蛋白表达增强的分子机制。少

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