血管紧张素Ⅱ受体基因敲除对跨膜钙内流的作用

Effect of knockout AT_(1a) receptor gene on transplasmamembrane calcium influx in aortic smooth muscle cells

目的 观察血管紧张素Ⅱ受体亚型 (AT1a)基因敲除对跨膜钙内流的影响。方法 培养AT1a基因敲除及其野生型对照小鼠的主动脉血管平滑肌细胞 (VSMC)和应用荧光倒置显微镜及钙荧光指示剂Fura 2 /AM动态观测VSMC的钙离子 [Ca2 +]i变化。结果 在血管紧张素Ⅱ (AngⅡ )刺激下钙内流显著增加 ,AT1a 敲除组VSMC钙净增值为 ( 2 0 4± 2 2 )nmol/L、基础 [Ca2 +]i为 ( 10 8± 9)nmol/L野生型对照VSMC钙净增值为 [( 194± 19)nmol/L ,基础 [Ca2 +]i为 ( 110± 7)nmol/L](P <0 0 1) ,应用钙通道阻滞剂硝苯啶和三磷酸鸟苷 γs能明显抑制AngⅡ介导的细胞钙增加 ,但G抑制蛋白的阻断剂百日咳毒素却能明显增强AngⅡ介导的细胞钙增加。结论 AT1a基因敲除后 ,AngⅡ介导的钙内流主要通过L 型钙通道 ,并受百日咳毒素的调节。

Objective To investigate the mechanism of angiotensin Ⅱ receptor subtype (AT 1a ) mutant on angiotensin Ⅱ ( Ang Ⅱ) mediated transplasmamembrane calcium influx in aortic smooth muscle cells (SMCs) from AT 1a knockout and wild type mice Method Aortic SMCs were isolated and cultured Intracellular free calcium concentration [Ca 2+ ]i was measured using Fura 2/AM fluorescentc technique Results Ang Ⅱ caused a marked increase in [Ca 2+ ]i in both cell types [AT 1a group: (204±22) nmol/L vs (108±9) nmol/L]; Control: [(194±19) nmol/L vs (110±7) nmol/L] Administration of both calcium channel blocker, nifedipine and GTP γs significantly inhibited Ang Ⅱ effect, in contrast, application of pertussin toxin (PTX) activated the Ang Ⅱ mediated calcium influx The response of AT 1a knockout cells was more sensitive to nifedipine and were enhanced by PTX Conclusion Calcium influx induced by Ang Ⅱ can be regulated by PTX insensitive and non Gi protein in AT 1a knockout cells