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

我们以前的研究发现，在自发性高血压大鼠中，编码磷脂酰肌醇特异性磷脂酶C的Delta1亚型(PLC-Delta1)的基因与其他正常血压大鼠(如WKY)的基因相比，在催化结构域的编码区有两个碱基缺失替换。由于许多报告涉及PLC活性增强以及细胞内Ca~(2+)水平的增加或许多细胞类型的生长速度，因此弄清PLC-Delta1是否与这些现象有关，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，这一点很重要。利用克隆的编码SHR-PLC-Delta1和WKY-PLC-Delta1的基因，在大肠杆菌中以谷胱甘肽S转移酶融合蛋白的形式表达，发现每种细菌裂解产物都具有较高的PLC活性。我们没有检测到比活性、底物特异性、Ca~(2+)需求或…的显著差异亲和纯化的SHR-和WKY-型酶之间的pH依赖性更强。与携带WKY的PLC-Delta1基因的酵母细胞相比，携带SHR PLC-Delta1基因的酵母细胞显示出更快的细胞生长。转化的酵母细胞(SHR PLC-Delta1)也显示出细胞内钙水平的增加，这两种独立的方法是通过紫外激光扫描显微镜监测掺入细胞中的吲哚葡聚糖的吸光度比，以及使用重组Aequorin(一种钙离子特异性指示剂)来测量钙。亲和纯化的两株突变酵母细胞的PLC-Delta1蛋白显示出几乎相同的酶活性，而携带SHR PLC-Delta1基因的酵母细胞的整个提取液显示出比携带WKY的PLC-Delta1基因的酵母细胞更高的酶活性。研究结果表明，SHR PLC-Delta1基因x域上的两个氨基酸替换与突变蛋白的酶活性增加有关，可能与一种未知的调节子相互作用。由此产生的细胞生长加速和[Ca~(2+)]i升高可能是SHR高血压相关现象的主要原因。较少

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