哇巴因对大鼠动脉平滑肌细胞分泌血管紧张素Ⅱ、心钠素的影响及厄贝沙坦的干预效果

The effects of ouabain on secretion of angiotensin Ⅱ and atrial natriuretic peptide in Aortic Smooth Muscle Cells from rats and Irbesartan intervention

目的研究哇巴因对自发性高血压大鼠(SHR)和Wistar-Kyoto(WKY)大鼠动脉平滑肌细胞(ASMCs)分泌血管紧张素Ⅱ(AngⅡ)和心钠素(ANP)的影响及其可能机制。方法用组织块种植法原代培养14周龄SHR和WKY大鼠胸主动脉平滑肌细胞,用免疫组织化学方法进行细胞鉴定,以无血清培养基培养的动脉平滑肌细胞作为对照组。先向培养液中加入终浓度为1×10^(-7)mol/L哇巴因,分别孵育1 h、6 h、24 h、48 h后收集细胞;再向培养液中加入不同终浓度的哇巴因(1×10^(-9)mol/L、1×10^(-8)mol/L、1×10^(-7)mol/L),孵育24 h后收集细胞;最后向培养液中加入不同终浓度的厄贝沙坦(1×10^(-7)mol/L、1×10^(-6)mol/L、1×10^(-5)mol/L)与SHR动脉平滑肌细胞预孵育30 min,再加入终浓度为1×10^(-7)mol/L的哇巴因共孵育24 h后收集细胞。采用放射免疫分析方法检测动脉平滑肌细胞分泌AngⅡ和ANP的水平。结果 (1)SHR动脉平滑肌细胞AngⅡ含量与WKY比较无显著差异(P>0.05),而ANP含量却显著降低(P<0.05),(2)1×10^(-7)mol/L哇巴因干预1 h、6 h、24 h、48 h,两种大鼠AngⅡ水平显著升高,WKY大鼠6 h达到峰值[(33.15±6.06)pg/10~6cells],SHR在24 h达到峰值[(64.51±8.31)pg/10~6cells];随哇巴因浓度增加,WKY大鼠动脉平滑肌细胞AngⅡ水平呈浓度依赖性升高,而SHR的AngⅡ无明显浓度依赖性。(3)1×10^(-7)mol/L哇巴因干预1 h、6 h、24 h、48 h,WKY大鼠ANP水平显著下降,SHR的ANP水平显著升高;随哇巴因浓度增高,WKY大鼠ANP下降的程度减弱,而SHR大鼠ANP水平呈浓度依赖性升高。(4)高浓度厄贝沙坦完全抑制哇巴因刺激AngⅡ分泌的作用;厄贝沙坦浓度依赖性地抑制哇巴因刺激ANP分泌的作用。结论哇巴因对局部血管活性物质AngⅡ和ANP分泌异常可能参与了高血压血管病变的发生、发展过程;AT1受体介导哇巴因对血管平滑肌细胞AngⅡ和ANP的分泌调节。

Objective To investigate the effects of ouabain on angiotensin II（ Ang II） and atrial natriuretic peptide（ ANP） secreted by aortic smooth muscle cells（ ASMCs） from spontaneously hypertensive rats（ SHR） and Wistar-Kyoto（ WKY） and its possible mechanism.Methods Primary SHR and WKY rats thoracic aorta VSMCs were cultured by tissue adherent method and identified by immunochemistry. Cultured ASMCs derived from SHR and WKY in serum-free medium were used as controls. ASMCs isolated from 14-week-old male SHR and WKYrats were incubated in different times by Oubain（ 1 × 10^（-7）mol/L）,then incubated in different doses of Ouabain（ 1 × 10^（-9）mol/L,1 × 10^（^（-8））mol/L,1 × 10^（-7）mol/L）. Finally,the addition of Irbesartan（ 1 × 10^（-7）mol/L,1 × 10^（-6）mol/L,1 × 10^（-5）mol/L） prior to ouabain incubated for30 minutes,then adding a final concentration of ouabain（ 1 × 10^（-7）mol/L） incubated for 24 hours. The contents of AngⅡand ANP in supernatant from ASMCs were measured by Radio-immunoassay. Results The AngⅡlevel of supernatant in SHR ASMCs was significantly higher than those from WKY control [（ 28. 2 ± 3. 32） pg/10~6 cells vs.（ 11. 53 ± 4. 50） pg/10~6 cells,P〈0. 05],while the content of ANP in SHR ASMCs was significantly lower than those from WKY control [（ 70. 92 ± 12. 24） pg/10~6 cells vs.（ 136. 2 ± 24. 21） pg/10~6 cells,P〈0. 05]. After using ouabain,AngⅡlevel in ASMCs of rats was remarkably higher than before,AngⅡ level in WKY rats reached the peak after 6 hours [（ 33. 15 ±6. 06） pg/10~6cells],and that in SHR reached the peak after 24 h [（ 64. 51 ± 8. 31） pg/10~6cells]. With increase in the concentration of ouabain,AngⅡlevel of ASMCs in WKY rats increased in a concentration-dependent manner. After using ouabain,the ANP level of WKY rats decreased significantly,while the ANP level in SHR was significantly increased in a concentration-dependent manner. The AngⅡ level of ASMCs in SHR stimulated by ouabain was completely blocked by high concentration of Irbesartan. The ANP level of ASMCs in SHR stimulated by ouabain was inhibited by Ibesartan in a concentration-dependent manner. Conclusion The abnormal secretion in local vasoactive substances Ang II and ANP byouabain may be involved in hypertensive vascular disease. AT1 receptor mediated the secretion of vasoactive substances in AngⅡand ANP stimulated by ouabain.