Blood Pressure Control by Intracellular Ca2_+-Modulatory Factors in Vascular Endothelial and Smooth Muscle Cells

我们之前的研究表明，与WKY等其他正常血压大鼠的基因相比，SHR中编码磷脂酰肌醇特异性磷脂酶C (PLC-delta1) δ 1异构体的基因在编码催化结构域的区域有两个缺失碱基替换。由于许多报道都涉及到PLC活性的增强以及细胞Ca^<2+>水平的增加或SHR的许多细胞类型的生长速度，因此澄清PLC-delta1是否对这些现象负责，以及SHR衍生的PLC-delta1是否在酶活性和/或二级或三级结构上与正常血压大鼠不同是很重要的。利用克隆的编码shrr -PLC-delta1和WKY-PLC-delta1的cdna，我们在大肠杆菌中将这两个分子作为谷胱甘肽s -转移酶的融合蛋白表达，发现每个细菌产物都具有较高的PLC活性。我们没有发现亲和纯化的shry型酶和wky型酶在比活性、底物特异性、Ca^<2+>需要量和pH依赖性方面有显著差异。与携带WKY PLC-delta1基因的细胞相比，携带SHR PLC-delta1基因的酵母细胞表现出更快的细胞生长。转化的酵母细胞（SHR PLC-delta1）也显示出细胞内钙水平的增加，通过两种独立的方法进行测量，即通过紫外激光扫描显微镜监测纳入细胞的吲哚葡聚糖的吸光度，并采用重组水芹蛋白（Ca^<2+>-特异性指示剂）测量钙。两株酵母细胞亲和纯化的PLC-delta1蛋白的酶活性几乎相同，而携带SHR PLC-delta1基因的细胞全提取物的酶活性高于携带WKY PLC-delta1基因的酵母细胞。研究结果表明，SHR PLC-delta1 x结构域的两个氨基酸替换与突变蛋白酶活性的增加有关，可能涉及与未知调节剂的相互作用。由此导致的细胞生长加速和[Ca^<2+>] i的增加可能是高血压相关现象（如SHR中观察到的增生）的主要原因。少

Our previous study has revealed that, in SHR,the gene encoding the delta1 isoform of phosphatidylinositol-specific phospholipase C (PLC-delta1) has two missence base replacements in the region encoding catalytic domainwhen compared with genes from other normotensive rats such as WKY.Since a number of reports concern augmented PLC activities together with increased cellular Ca^<2+> levels or the growth rate of many cell types of SHR,it is important to clarify whether PLC-delta1 is responsible for these phenomena, and whether SHR-derived PLC-delta1 differs in enzymatic activity and/or in the secondary or the tertiary structure from that of normotensive rats. Using cloned cDNAs encodnig SHR-PLC-delta1 and WKY-PLC-delta1, we expressed each molecule in E.coli as a fusion protein with glutathione S-transferase, and found that each bacteriallysate showed high PLC activity. We were not able to detect significant difference in the specific activity, substrate specificity, Ca^<2+> requirement or … More pH dependency between affinity-purified SHR-and WKY-type enzymes. Yeast cells carrying the PLC-delta1 gene of SHR displayd accelerated cell growth in comparison to cells carrying the PLC-delta1 gene of WKY.The transformed yeast cells (SHR PLC-delta1) also showed increased levels of intracellular calcium when measured by two independent methods, namely by monitoring the absorbance ratio of indo dextran incorporated into cells by UV-laser scan microscopy and employing recombinant aequorin, a Ca^<2+>-specific indicator, to measure calcium. Affinity purified PLC-delta1 proteins from the two mutated strains of yeast cells showed nearly the same enzymic activity, whereas whole extract of cells carrying the PLC-delta1 of SHR showed higher enzymic activity than yeast cells carrying the PLC-delta1 gene of WKY.The results of the study suggest that the two amino acid replacements in the x domain of PLC-delta1 of SHR is related to the increased enzymic activity of the mutant protein, possibly involving an interaction with an unidentified modulator. It is proposed that the resultant accelerated cell growth and increased [Ca^<2+>] i may be the major cause of hypertension-related phenomena, such as hyperplasia, observed in SHR. Less