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

冠状动脉痉挛性心绞痛发生的临床、分子和生物学研究

基本信息

批准号：
18590758
负责人：
OKUMURA Ken
金额：
$ 2.5万
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：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18590758/
关键词：
coronary spastic angina vascular smooth muscle skin fibroblast phospholipase C-δ1 p122 protein Rho A intracellular Ca ion

项目摘要

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

磷脂酶C（PLC）-δ1可被Ca^2+激活，并诱导[Ca^2+] i进一步增加，增强对收缩肌刺激的反应。我们先前报道了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活性与对照受试者相比增强（J Am科尔Cardiol，2000）。通过对编码PLC-δ1的cDNA的序列分析，我们发现在核苷酸位置864处鸟嘌呤转化为腺嘌呤（A），导致精氨酸257被组氨酸（R257 H）取代（Circulation，2002）。该变体PLC-δ1蛋白的活性是野生型蛋白的2倍。在对乙酰胆碱的反应中，具有变异PLC-δ1的细胞中[Ca^<2+>] i的峰值增加大于具有野生型的细胞。因此，PLC-δ1基因中的R257 H变异可能是增强冠状动脉血管运动的新机制。然而，R257 H变异体仅在 ...更多信息 10%的患者。PLC-δ1受RhoA负调控，受p122蛋白正调控。结果表明，CSA患者中RhoA蛋白表达与对照组（n=4）相似，而CSA患者中p122蛋白表达较对照组（n=9）增加约3倍（p<0.0001）。CSA组p122基因表达较对照组明显增加（P<0.01）。在转染p122的人胚肾293细胞中，细胞内钙离子浓度（[Ca ^<2 +>] i）的基线值和乙酰胆碱引起的[Ca^<2+] i的峰值增加值均高于未转染p122的细胞。对编码p122启动子区（-1599至+1）的基因组DNA的序列分析显示了8个核苷酸转换。我们检测了144名CSA患者和148名对照者的DNA序列，发现男性CSA患者（8/91）中-228位鸟嘌呤转化为腺嘌呤的频率高于男性对照者（1/62）（p<0.05）。与野生型相比，转染-228G/A变体启动子构建体的细胞中的荧光素酶活性显著增加（p<0.001）。结论：CSA患者p122蛋白表达上调，其上调可能通过增加[Ca^<2+>] i参与冠状动脉舒缩功能的增强。-228G/A多态性是p122蛋白表达上调的机制之一。少