Jiang Chun-Ming, Han Li-Ping, Li Hong-Zhu, Qu Ying-Bo, Zhang Zhuo-Ran, Wang Rui, Xu Chang-Qing, Li Wei-min
Department of Pathophysiology, Harbin Medical University, Baojian Street, Harbin 150086, China.
Cell Biol Int. 2008 Jul;32(7):792-800. doi: 10.1016/j.cellbi.2008.03.009. Epub 2008 Mar 29.
Calcium-sensing receptors (CaSRs) are G-protein coupled receptors which regulate systemic calcium homeostasis and also participate in cell proliferation, differentiation and apoptosis. We have previously shown that CaSR can induce apoptosis in isolated rat adult hearts and in normal rat neonatal cardiomyocytes. However, no knowledge exists concerning the role of CaSR in apoptosis induced by ischemia and reperfusion in neonatal cardiac myocytes. Therefore, in the present study, we incubated primary neonatal rat ventricular cardiomyocytes in ischemia-mimetic solution for 2h, then re-incubated them in a normal culture medium for 24h to establish a model of simulated ischemia/reperfusion (I/R). We assayed the apoptotic ratio of the cardiomyocytes by flow cytometry; observed morphological alterations by transmission electron microscope; analyzed the expression of caspase-3, Bcl-2, CaSR, extracellular signal-regulated protein kinase (ERK), and Fas/Fas ligand (FasL) by Western blotting; and measured the concentration of intracellular calcium by Laser Confocal Scanning Microscopy. The results showed that simulated I/R increased the expression of CaSR and cardiomyocyte apoptosis. GdCl3, a specific activator of CaSR, further enhanced CaSR expression, along with increases in intracellular calcium and apoptosis in cardiomyocytes during I/R. Activation of CaSR down-regulated Bcl-2 expression, up-regulated caspase-3 and Fas/FasL expression and stimulated ERK1/2 phosphorylation. In summary, CaSR is involved in I/R injury and apoptosis of neonatal rat ventricular cardiomyocytes by inhibiting Bcl-2, inducing calcium overload and activating the Fas/FasL death receptor pathway.
钙敏感受体（CaSRs）是G蛋白偶联受体，可调节全身钙稳态，还参与细胞增殖、分化和凋亡。我们之前已经表明，CaSR可在离体成年大鼠心脏和正常大鼠新生心肌细胞中诱导凋亡。然而，关于CaSR在新生心肌细胞缺血再灌注诱导的凋亡中的作用尚无相关研究。因此，在本研究中，我们将原代新生大鼠心室心肌细胞在模拟缺血溶液中孵育2小时，然后在正常培养基中再孵育24小时，以建立模拟缺血/再灌注（I/R）模型。我们通过流式细胞术检测心肌细胞的凋亡率；通过透射电子显微镜观察形态学改变；通过蛋白质免疫印迹法分析半胱天冬酶-3、Bcl-2、CaSR、细胞外信号调节蛋白激酶（ERK）和Fas/Fas配体（FasL）的表达；并通过激光共聚焦扫描显微镜测量细胞内钙浓度。结果表明，模拟I/R增加了CaSR的表达和心肌细胞凋亡。CaSR的特异性激活剂GdCl3进一步增强了CaSR的表达，同时在I/R期间心肌细胞内钙和凋亡增加。CaSR的激活下调了Bcl-2的表达，上调了半胱天冬酶-3和Fas/FasL的表达，并刺激了ERK1/2的磷酸化。总之，CaSR通过抑制Bcl-2、诱导钙超载和激活Fas/FasL死亡受体途径参与新生大鼠心室心肌细胞的I/R损伤和凋亡。
