尿促皮素诱导乳大鼠心肌细胞肥大的作用及信号传导机制

Effect and mechanism of signal transduction pathway in urocortin-induced cardiomyocytes hypertrophy in neonatal rat

目的探讨尿促皮素(urocortin)诱导大鼠心肌细胞肥大的作用及其信号传导机制。方法实验分8组,正常对照组、尿促皮素0.1μmol.L-1组、星形孢菌素(Sta)1μmo.lL-1、H890.1μmol.L-1和维拉帕米(Ver)1μmol.L-1组及尿促皮素分别加Sta,H89和Ver组。采用体外培养的乳大鼠心肌细胞,应用尿促皮素0.1μmol.L-1诱导心肌肥大,观察Sta1μmol.L-1,H890.1μmo.lL-1和Ver1μmol.L-1的作用,进一步探讨尿促皮素0.1μmol.L-1诱导心肌肥厚的作用机制。用消化分离法及计算机图像分析系统检测心肌细胞直径;[3H]亮氨酸掺入法测定心肌细胞蛋白质的合成;用Lowry法检测心肌细胞蛋白质含量;用Western蛋白印迹法测定心房钠尿肽(ANP)表达;采用Till阳离子测定系统,以Fura-2/AM为荧光探针,观察心肌细胞[Ca2+]i瞬间变化。结果尿促皮素使心肌细胞直径、蛋白质合成、蛋白质含量和ANP表达分别增加30.9%,36.3%,35.5%和34.7%;尿促皮素+Sta组使心肌细胞直径、蛋白质合成、蛋白质含量和ANP表达分别降低了16.5%,22.1%,18.1%和21.3%;尿促皮素+H89组使心肌细胞直径、蛋白质合成、蛋白质含量和ANP表达分别降低了16.6%,21.5%,19.5%和20.6%;尿促皮素+Ver组使心肌细胞直径、蛋白质合成、蛋白质含量和ANP表达分别降低了17.1%,20.9%,17.9%及19.9%;尿促皮素能够使心肌细胞[Ca2+]i瞬间变化水平增高,Sta,H89和Ver能够降低尿促皮素引起的心肌细胞[Ca2+]i瞬间变化升高。结论尿促皮素可能通过蛋白激酶C和蛋白激酶A信号途径影响L-型Ca2+通道,进而影响细胞[Ca2+]i瞬间变化水平,诱导乳大鼠心肌细胞肥大。

AIM To investigate the effects and mechanism of cardiomyocyte hypertrophy induced by urocortin.METHODS The cardiomyocytes were divided into 8 groups：normal control,urocortin,staurosporine（Sta）,verapamil（Ver）,H89,urocortin＋Sta,urocortin＋Ver,and urocortin＋H89 groups.The cardiomyocytes diameter was measured by computer photograph analysis system.The protein synthetic rate was obtained through measuring the incorporation of [3H]-leucine into myocyte protein by liquid scintillation method.The total protein content was assayed by Lowry method.The expression of atrial natriuretic peptide（ANP） was determined by Western blot.[Ca2＋]i transient was measured by Till image system by cell loading Fura-2/AM.RESULTSUrocortin group enhanced cardiomyocyte volume,protein synthesis,total protein content and expression of ANP by 30.9%,36.3%,35.5% and 34.7%;urocortin＋Sta group decreased cardiomyocyte diameter,protein synthesis,total protein content and expression of ANP by 16.5%,22.1%,18.1% and 21.3%;urocortin＋H89 group decreased the cardiomyocyte diameter,the protein synthesis,total protein content and expression of ANP by 16.6%,21.5%,19.5% and 20.6%;urocortin＋Ver decreased the cardiomyocyte diameter,the protein synthesis,total protein content and the expression of ANP by 17.1%,20.9%,17.9% and 19.9%;Sta,H89 and Ver could decrease the [Ca2＋]i transient induced by urocortin.CONCLUSION The hypertrophic effect of urocortin in rat neonatal cardiomyocytes is mediated via activation of protein kinase C and protein kinase A pathway and L-type calcium channels.