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

血管内皮细胞和平滑肌细胞内 Ca2_ 调节因子对血压的控制

• 批准号: 05837020
• 负责人: YAGISAWA Hitoshi
• 金额: $ 1.15万
• 依托单位: Himeji Institute of Technology(HIT)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05837020/
• 关键词: Hypertension; Vascular cells; Calcium ion; Phospholipase; Spontaneously Hypertensive Rat; Gene expression; Linkage analysis; PH domain; 高血圧自然発症ラット; 血管内皮・平滑筋細胞; 遺伝子クローニング; 遺伝子連鎖解析

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

我们之前的研究表明，在SHR中，与来自其他正常血压大鼠（例如WKY）的基因相比，编码磷脂酰肌醇特异性磷脂酶C（PLC-delta1）的delta1亚型的基因在编码催化结构域的区域中有两个错义碱基替换。因为许多报道涉及PLC活性的增强以及细胞Ca^2+水平或生长速率的增加 SHR 的许多细胞类型中，重要的是要澄清 PLC-delta1 是否与这些现象有关，以及 SHR 衍生的 PLC-delta1 在酶活性和/或二级或三级结构方面是否与正常血压大鼠不同。使用编码 SHR-PLC-delta1 和 WKY-PLC-delta1 的克隆 cDNA，我们在大肠杆菌中将每个分子表达为与谷胱甘肽 S-转移酶的融合蛋白，并发现每种细菌裂解物都显示出高 PLC 活性。我们无法检测到亲和纯化的 SHR 型酶和 WKY 型酶之间的比活性、底物特异性、Ca^2+ 要求或 pH 依赖性方面的显着差异。与携带 WKY 的 PLC-delta1 基因的细胞相比，携带 SHR PLC-delta1 基因的酵母细胞显示出加速的细胞生长。当通过两种独立的方法（即通过紫外激光扫描显微镜监测掺入细胞的吲哚葡聚糖的吸光率并采用 重组水母发光蛋白，一种Ca 2+ 特异性指示剂，用于测量钙。亲和纯化的两种突变酵母细胞株的PLC-delta1蛋白显示出几乎相同的酶活性，而携带SHR的PLC-delta1的细胞的全提取物显示出比携带WKY的PLC-delta1基因的酵母细胞更高的酶活性。研究结果表明SHR的PLC-delta1的x结构域中的两个氨基酸替换与SHR的PLC-delta1的酶活性增加有关。 突变蛋白，可能涉及与未知调节剂的相互作用。提出由此产生的细胞生长加速和[Ca 2+ ] i 增加可能是SHR中观察到的高血压相关现象例如增生的主要原因。较少的

项目成果

Yoko-o,T.et al.: "The putative phosphoinositide-specific phospholipase C gene,PLC1,of the yeast Saccharomyces cerevisiae is important for cell growth" Proc.Natl.Acad.Sci.U.S.A.90. 1804-1808 (1993)

Yoko-o,T.et al.：“酿酒酵母假定的磷酸肌醇特异性磷脂酶 C 基因 PLC1 对于细胞生长很重要”Proc.Natl.Acad.Sci.U.S.A.90。

Kamata,H.et al.: "Amphiphilic peptides enhances the efficiency of liposome-mediated DNA transfection" Nucleic Acid Res.22. 536-537 (1994)

Kamata, H.et al.：“两亲性肽增强脂质体介导的 DNA 转染的效率”Nucleic Acid Res.22。

Hirata,M.et al.: "D-myo-lnositol 1,4,5-Trisphosphate Binding Domain of Phospholipase C-δ1." Biochem,Biophys.Res.Comm.204. 1563-1571 (1994)

Hirata, M. 等人：“磷脂酶 C-δ1 的 D-肌醇 1,4,5-三磷酸结合域。”Biochem，Biophys.Res.Comm.204（1994）。

Hirata, M.et al.: "Modulation of Phospholipase C-delta1." Seikagaku. 67(in press). (1995)

Hirata, M.et al.：“磷脂酶 C-delta1 的调节”。

Yagisawa, H.et al.: "Expression and Characterization of an Inositol 1,4,5-Trisphosphate Binding Domain of Phosphatidylinositol-specific Phospholipase C-delta1." J.Biol.Chem.269. 20179-20188 (1994)

Yagisawa, H.et al.：“磷脂酰肌醇特异性磷脂酶 C-delta1 的肌醇 1,4,5-三磷酸结合域的表达和表征”。