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的同工酶，是细胞收缩机制的关键酶，可被Ca^<++>激活，诱导[Ca^<2+>]_i进一步增加，并增强对收缩刺激的反应。我们之前报道过，与对照组相比，冠状动脉痉挛性心绞痛患者培养的皮肤成纤维细胞膜部分的PLC活性增强，PLC活性与冠状动脉基底张力及其对收缩刺激的反应呈正相关（J Am Coll Cardiol,2000）。通过对从成纤维细胞中获得的PLC-d1编码的cDNA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864A/A的发生率高于男性对照组。变异PLC-d1蛋白的活性比野生型高2倍。变异PLC-d1细胞的[Ca^<2+>]_i在乙酰胆碱作用下的峰值增幅大于野生型细胞。因此，PLC-d1基因的R257H变异与酶活性增强有关，可能是冠状动脉血管运动性增强的新机制。然而，R257H变异仅在10%的患者中检测到，我们正在寻找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方法比较11例冠心病患者（n=11）和9例冠心病患者（n=9） Rho A和P122蛋白的表达情况。虽然两组间Rho A蛋白表达无差异，但P122在冠脉性痉挛性心绞痛组的表达明显高于非痉挛性心绞痛组。PLC-d1蛋白水平在两组间无显著差异。因此，P122蛋白表达的增强可能解释了冠心病痉挛性心绞痛患者PLC-d1活性的增强。我们目前正在研究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