Yan Ling, Zhu Tiebing, Sun Tingting, Wang Liansheng, Pan Shiyang, Tao Zhengxian, Yang Zhijian, Cao Kejiang
Department of Cardiology, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China.
J Biomed Res. 2010 Jul;24(4):301-7. doi: 10.1016/S1674-8301(10)60042-5.
Calcium-sensing receptors (CaSRs) are G-protein coupled receptors which maintain systemic calcium homeostasis and participate in hormone secretion, activation of ion channels, cell apoptosis, proliferation, and differentiation. Previous studies have shown that CaSRs induce apoptosis in isolated adult rat heart and in normal neonatal rat cardiomyocytes by G-protein-PLC-IP3 signaling transduction. However, little knowledge is presently available concerning the role of CaSRs in the apoptosis induced by ischemia and reperfusion in neonatal cardiomyocytes.
Primary neonatal rat ventricular cardiomyocytes were incubated in ischemiamimetic solution for 2 h, and then re-incubated in normal culture medium for 24 h to establish a model of simulated ischemia/reperfusion (I/R). Cardiomyocyte apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The expression of CaSRs mRNA was detected by real-time reverse transcription polymerase chain reaction (RT-PCR). In addition, the expressions of caspase-3 and Bcl-2 were analyzed by western blot.
The simulated I/R enhanced the expression of CaSRs and cardiomyocyte apoptosis. GdCl3, a specific activator of CaSRs, further increased the expression of CaSRs and cardiomyocyte apoptosis, along with up-regulation of caspase-3 and down-regulation of Bcl-2.
CaSRs are associated with I/R injury and apoptosis in neonatal rat ventricular cardiomyocytes via suppressing Bcl-2 and promoting caspase-3 expression.
钙敏感受体（CaSRs）是G蛋白偶联受体，可维持全身钙稳态，并参与激素分泌、离子通道激活、细胞凋亡、增殖及分化过程。既往研究表明，CaSRs通过G蛋白-PLC-IP3信号转导诱导成年大鼠离体心脏及正常新生大鼠心肌细胞凋亡。然而，目前关于CaSRs在新生心肌细胞缺血再灌注诱导的凋亡中的作用知之甚少。
将原代新生大鼠心室肌细胞置于缺血模拟溶液中孵育2小时，然后再置于正常培养基中孵育24小时，以建立模拟缺血/再灌注（I/R）模型。采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法（TUNEL）检测心肌细胞凋亡。通过实时逆转录聚合酶链反应（RT-PCR）检测CaSRs mRNA的表达。此外，采用蛋白质免疫印迹法分析caspase-3和Bcl-2的表达。
模拟I/R增强了CaSRs的表达及心肌细胞凋亡。CaSRs的特异性激活剂GdCl3进一步增加了CaSRs的表达及心肌细胞凋亡，同时上调了caspase-3的表达并下调了Bcl-2的表达。
CaSRs通过抑制Bcl-2并促进caspase-3表达，与新生大鼠心室肌细胞的I/R损伤及凋亡相关。
