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目的观察血管紧张素Ⅱ(AngⅡ)刺激心肌细胞,导致经典瞬时受体电位(TRPC)通道1表达的改变情况,并探讨其机制。方法分离和培养原代乳鼠心室肌细胞,运用Western blot法检测细胞TRPC1、TRPC3和TRPC6蛋白的表达,3H-亮氨酸掺入法测定细胞蛋白质合成速率。结果 AngⅡ能刺激心肌细胞TRPC1表达显著上调(P<0.01),心肌细胞TRPC1的表达随AngⅡ浓度增加而逐渐明显上调;但AngⅡ刺激并未明显增高TRPC3和TRPC6蛋白的表达(P>0.05)。给予AT1受体拮抗剂氯沙坦、磷脂酶C(PLC)抑制剂U73122、钙池操作性钙内流阻滞剂SKF96365或钙调神经磷酸酶抑制剂环孢素A预处理后,都能抑制AngⅡ引起的TRPC1高表达(均P<0.05),但AT2受体拮抗剂PD123319却不能。SKF96365预处理在能明显抑制AngⅡ引起的TRPC1高表达的同时,也能抑制AngⅡ诱导的细胞肥大。结论 AngⅡ诱导大鼠心室肌细胞TRPC1表达上调并呈现剂量依赖性,这种上调是通过AT1R/PLC信号传导,激活钙调神经磷酸酶途径来实现的。
Objective To observe the effects of Angiotensin Ⅱ (Ang Ⅱ) on cardiomyocytes and the changes of the expression of classical transient receptor potential (TRPC) channel 1, and to explore its mechanism. Methods Primary neonatal rat ventricular myocytes were isolated and cultured. Western blot was used to detect the expression of TRPC1, TRPC3 and TRPC6. 3H-leucine incorporation assay was used to determine the rate of cellular protein synthesis. Results Ang Ⅱ stimulated the expression of TRPC1 in cardiomyocytes significantly (P <0.01). The expression of TRPC1 in cardiomyocytes increased gradually with the increase of Ang Ⅱ concentration. However, the expression of TRPC3 and TRPC6 protein did not increase significantly with Ang Ⅱ stimulation (P> 0.05). Administration of AT1 receptor antagonist losartan, phospholipase C (PLC) inhibitor U73122, calcium pool-operated calcium blockade SKF96365 or calcineurin inhibitor cyclosporin A pretreatment inhibited Ang Ⅱ induced high expression of TRPC1 (all P <0.05), but the AT2 receptor antagonist PD123319 did not. SKF96365 pretreatment can significantly inhibit Ang Ⅱ induced TRPC1 high expression, but also can inhibit Ang Ⅱ induced cell hypertrophy. CONCLUSION Ang Ⅱ induced upregulation of TRPC1 expression in rat ventricular myocytes in a dose-dependent manner. This upregulation is mediated by the activation of calcineurin via AT1R / PLC signaling.