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

磷脂酶C(PLC)-δ1被钙激活，引起[Ca^&lt；2+]_i进一步升高，并增强对收缩肌刺激的反应。我们先前报道，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(J Am Coll Medicol，2000)。通过对编码PLC-δ1的基因的序列分析，我们发现在第864位发生了鸟嘌呤到腺嘌呤(A)的转换，导致精氨酸257的氨基酸被组氨酸(R257H)取代(循环，2002年)。该突变蛋白的活性是野生型蛋白的2倍以上。[Ca~(2+)~(2+)]_(gt；)i对乙酰胆碱的反应峰值在携带变异的PLC-δ_1的细胞中大于野生型。因此，PLC-δ1基因的R257H变异可能是增强冠脉血管动力的一种新机制。然而，仅检测到I…的R257H变异超过10%的患者。PLC-δ1受RhoA负调控，受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。在CsA组(n=5)和对照组(n=4)中，RhoA的蛋白表达相似，而在CsA组(n=11)中P122的蛋白表达比对照组(n=9)增加了约三倍(p&lt；0.0001)。CsA组P122基因表达也明显高于对照组(p&lt；0.01)。P122基因转染人胚肾293细胞的基础细胞内钙离子浓度([Ca^&lt；2+&gt；]i)和乙酰胆碱引起的细胞内[Ca^&lt；2+&gt；]i峰值升高均高于未转染组。对编码P122启动子区域(-1599至+1)的基因组DNA进行了序列分析，发现该核苷酸发生了8次转换。我们检测了144名CsA患者和148名对照组的DNA序列，发现男性CsA患者中-228位鸟嘌呤转换为腺嘌呤的频率(8/91)高于男性对照组(1/62)(p&lt；0.05)。与野生型相比，转染-228G/A变异型启动子的细胞中荧光素酶活性显著增强(p&lt；0.001)。综上所述，CsA患者P122蛋白表达上调，其增强可能与[Ca^&lt；2+&gt；]_i升高有关，A-228G/A基因多态性是P122蛋白表达上调的机制之一。较少

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