Clinical, Molecular and Biological Studies to the Genesis of Coronary Spastic Angin

磷脂酶C(PLC)-δ1被Ca^<2+>激活，诱导[Ca^<2+>]_i进一步增加，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（J Am Coll Cardiol, 2000）。通过对PLC-δ1编码的cDNA序列分析，我们发现在864核苷酸位置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）。该变异体PLC-δ1蛋白的活性比野生型高2倍。变异PLC-δ1细胞的[Ca^<2+>] i响应于乙酰胆碱的峰值增加大于野生型细胞。因此，PLC-δ1基因的R257H变异可能是冠状动脉血管运动性增强的新机制。而R257H变异仅占1%，超过10%。PLC-δ1受RhoA负调控，p122蛋白正调控。因此，我们研究了RhoA和P122蛋白在CSA中PLC-δ1活性增强中的可能作用。CSA患者（n=5）与对照组（n=4）的RhoA蛋白表达相似，而CSA患者（n=11）的p122蛋白表达较对照组（n=9）增加约3倍（p<0.0001）。与对照组相比，CSA中p122基因表达量显著增加（p<0.01）。转染p122的人胚胎肾293细胞的细胞内钙基线浓度（[Ca^<2+>]_i）和[Ca^<2+>]_i在乙酰胆碱作用下的峰值升高均高于未转染p122的人胚胎肾293细胞。对编码p122启动子区的基因组DNA（-1599至+1）进行序列分析，发现该核苷酸发生了8次转化。我们检测了144例CSA患者和148例对照患者的DNA序列，男性CSA患者（8/91）比男性对照组（1/62）更频繁地在-228位点上鸟嘌呤转化为腺嘌呤（p<0.05）。转染-228G/A变异启动子的细胞荧光素酶活性显著高于野生型（p<0.001）。综上所述，p122蛋白在CSA患者中表达上调，其表达增强可能通过[Ca^<2+>]_i升高参与冠状动脉血管运动增强。A -228G/A多态性是p122蛋白上调的机制之一。少

Phospholipase C(PLC)-δ1 is activated by Ca^<2+> 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 (J Am Coll Cardiol, 2000). By the sequence analysis of the cDNA coding for PLC-δ1, we 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 activity of this variant PLC-δ1 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-δ1 than in those with the wild type. Thus, R257H variantin the PLC-δ1 gene can be a novel mechanism for the enhanced coronary vasomotility. However, R257H variant was detected only i … More n 10% of the patients. PLC-δ1 is negatively regulated by RhoA and positively by p122 protein. We therefore examined the possible roles of RhoA and P122 protein in the enhanced PLC-δ1 activity in CSA.Protein expression of RhoA was similar between CSA (n=5) and control patients (n=4), while that of p122 was increased in CSA (n=11) by about three-fold compared with control (n=9) (p<0.0001). Gene expression of p122 was also increased in CSA compared with control (p<0.01). Baseline intracellular calcium concentration ([Ca^<2+>]_i) and the peak increase in [Ca^<2+>]_i in response to acetylcholine were both higher in the human embryonic kidney 293 cells trans fected with p122 than in those without p122. Sequence analysis of the genomic DNA coding the promoter region of p122 (-1599 through +1) revealed 8 conversions of the nucleotide. We examined the DNA sequence in 144 CSA and 148 control patients, and one conversion of guanine to adenine at the position of -228 was more frequent in male CSA patients (8/91) than in male controls (1/62) (p<0.05). The luciferase activity in the cells transfected with the -228G/A variant promoter construct was significantly increased compared with that of wild type (p<0.001). In conclusion, p122 protein is upregulated in CSA patients, and its enhancement may be involved in the increased coronary vasomotility via the increase in [Ca^<2+>]_i. A -228G/A polymorphism is one mechanism for the upregulated p 122 protein. Less