维拉帕米对KCNJ5突变型H295R细胞醛固酮分泌功能的影响

Effects of verapamil on aldosterone secretion in KCNJ5-mutated human adrenocortical carcinoma H295R cells

目的探讨维拉帕米对KCNJ5突变型(G151R、de1I157)人肾上腺皮质腺癌细胞(H295R)醛固酮分泌功能的影响。方法采用脂质体LipofectamineTM 3000转染H295R细胞,将其分为野生型KCNJ5组、突变型KCNJ5 G151R组、突变型KCNJ5 delI157组和对照组。转染H295R细胞成功后,更换含血管紧张素Ⅱ或维拉帕米的培养液培养36 h,应用放射免疫技术检测各组细胞中醛固酮浓度,实时荧光定量PCR技术检测细胞中11-β羟化酶(CYP11B1)、醛固酮合酶(CYP11B2)基因的表达。结果在血管紧张素Ⅱ刺激后,野生型KCNJ5、转染突变型KCNJ5 G151R和KCNJ5 delI157的H295R细胞中CYP11B1、CYP11B2 mRNA的表达及醛固酮分泌显著增加(P<0.05)。在维拉帕米作用下,转染突变型KCNJ5 G151R和KCNJ5 delI157的H295R细胞中CYP11B1、CYP11B2 mRNA的表达及醛固酮分泌下降(P<0.05)。结论钙离子通道阻滞剂——维拉帕米对KCNJ5突变型H295R细胞的醛固酮分泌具有抑制作用。

Objective To investigate the effects of verapamil-stimulated aldosterone secretion on KCNJ5 G151R/delI157-mutanted human adrenocortical carcinoma H295R cells.Methods By using Lipofectamine3000,H295R cells were transfected with wild-type KCNJ5,mutant KCNJ5 G151R,KCNJ5 delI157 or control vector plasmid as indicated.Twenty-four hours after the transfection,the cell medium was replaced by new medium supplemented with angiotensinⅡor verapamil and incubated for 36 hours.The aldosterone was determined by radioimmunoassay,and the mRNA expression level of the 11-βhydroxylase(CYP11B1)and aldosterone synthase(CYP11B2)gene was detected by real-time fluorescence quantitative PCR.Results The mRNA expression of CYP11B1/CYP11B2 and the secretion of aldosterone were all significantly increased in wild-type KCNJ5,KCNJ5 G151R-and KCNJ5 delI157-transfected H295R cells after angiotensinⅡstimulation.After verapamil treatment,the mRNA expression of CYP11B1/CYP11B2 and the secretion of aldosterone were all signifcantly decreased in KCNJ5 G151R-and KCNJ5 delI157-transfected H295R cells.Conclusions Calcium channel blocker-verapamil can inhibit the secretion of aldosterone in KCNJ5 mutated H295R cells.