血管紧张素Ⅱ对大鼠血管平滑肌细胞T型钙通道及其信号转导通路的影响

Influence of Ang Ⅱ on Ttype Calcium Channels and Signal Transduction Pathway in Rat Vascular Smooth Muscle Cells

目的研究血管紧张素Ⅱ(AngⅡ)对血管平滑肌细胞Cav3.1表达的影响及其作用机制。方法酶消化法原代培养大鼠主动脉血管平滑肌细胞,应用第5代传代细胞,实验随机分为7组:对照组(不加任何药物)、AngⅡ组[加入AngⅡ(0.1μmol/L)培养24 h]、AngⅡ+氯沙坦钾组[加入氯沙坦钾(1μmol/L)培养1 h,再加入AngⅡ(0.1μmol/L)共同培养24 h]、AngⅡ+PD98059组[加入PD98059(10μmol/L)预刺激1 h,再加入AngⅡ(0.1μmol/L)共同培养24 h]、PD98059组[加入PD98059(10μmol/L)培养1 h]、AngⅡ+氯沙坦钾+PD98059组[先加入氯沙坦钾(1μmol/L),PD98059(10μmol/L)培养1 h,再加入AngⅡ(0.1μmol/L)共同培养24 h]、氯沙坦钾组[加入氯沙坦钾(1μmol/L)培养4 h]。RT-PCR、Western blot方法测定各组T型钙通道的表达。结果PCR结果显示:与对照组(1.000±0.315)比较,AngⅡ组Cav3.1表达量(17.930±0.954)明显增加(P<0.01);PD98059组、氯沙坦钾组Cav3.1表达量为0.928±0.262、0.978±0.292,与对照组比较差异无统计学意义(P>0.05);AngⅡ+氯沙坦钾组、AngⅡ+PD98059组、AngⅡ+氯沙坦钾+PD98059组的Cav3.1表达量分别为0.125±0.007、0.066±0.015、0.109±0.018,明显低于AngⅡ组(P<0.01)。Western blot结果显示:与对照组比较,AngⅡ+氯沙坦钾组、AngⅡ+PD98059组及AngⅡ+氯沙坦钾+PD98059组Cav3.1蛋白表达明显降低(P<0.05);而PD98059组及氯沙坦钾组Cav3.1蛋白表达与对照组比较差异无统计学意义(P>0.05)。结论 AngⅡ通过Ras/PKCζ/MEK/ERK1/2路径增加血管平滑肌细胞Cav3.1的表达。

Objective To investigate the effects of Ang Ⅱ on T-type calcium channels in rat vascular smooth muscle cells. Methods Primary. cul- ture of rat aortic vascular smooth muscle cells was established by enzyme digestion method, and the fifth generation cells were used in the study. Cells were randomly divided into 7 groups ： ①control group ： without any drugs ; ②Ang Ⅱ group ： Ang Ⅱ 0.1 μmol/L for 24 hours ; ③Ang Ⅱ ＋losartan po- tassium group： losartan potassium 1 μmol/L for 1 hour, then add Ang Ⅱ 0.1 μmol/L and cultures for another 24 hours ; ④Ang Ⅱ ＋PD98059 group ： adds 10 μmol/L PD98059 and stimulus for 1 hour, then adds 0.1 μmol/L Ang Ⅱ and cultures for 24 hours together; ⑤PD98059 group ： adds 10 μmol/L PD98059 and cultures for 1 hour; ⑥Ang Ⅱ ＋ losartan potassium ＋ PD98059 group： adds 1 μmol/L losartan potassium and 10 μmol/L PD98059 cultures for 1 hour, then adds 0.1 μmol/L Ang Ⅱ and cultures for 24 hours together; ⑦losartan potassium group ： adds losartan potassium and cultures for 4 hours. The expression of T-type calcium channel of every group was determined by PT-PCR and Western blot. Results ①PCR results showed that the expression of Cav3.1 in Ang Ⅱ group （ 17.930±0.954 ） increased significantly compared with control group （1.000±0.315 ） （P 〈 0.05 ）, which were not observed in PD98059 group （2^-△△Ct ： 0.928±0.262 ） and losartan potassium group （2^-△△Ct： 0.978±0.292） ; the expression of Cav3.1 in Ang Ⅱ ＋losartan potassium group （0.125±0.007 ）, Ang Ⅱ ＋PD98059 group （0.066±0.015 ） and Ang Ⅱ ＋ losartan potassium ＋ PD98059 group （0.109±0.018） significantly lower than Ang Ⅱ group （ 1.000±0.053 ） （all P 〈 0.01 ）.②Western blot result showed that the expression of Cav3.1 in Ang Ⅱ ＋losartan potassium group, Ang Ⅱ ＋ losartan potassium ＋ PD98059 group and Ang Ⅱ ＋PD98059 group is significantly reduced compared with the control group ; while no obvious statistical significance was found between PD98059 group and losartan potassium group （P 〉 0.05 ）. Conclusion Angiotensin Ⅱ can lead excessive expression of T-type calcium channel in rat vascular smooth muscle cells ; However, there is no statistically significant differencescompamd with the control group.