血管紧张素Ⅱ调节肾脏球旁器颗粒细胞合成肾素的机制研究

Mechanism of angiotensin Ⅱ in regulation of renin synthesis in juxtaglomerular granular cells

目的观察血管紧张素Ⅱ（AngⅡ）对肾脏球旁器颗粒细胞肾素分泌的调节作用。方法密度梯度离心原代分离小鼠肾小球球旁器颗粒细胞（JGC）。实时定量PCR法检测JGC内血管紧张素转换酶（ACE）、AngⅡ受体（AT）1型和2型（AT1和AT2）mRNA表达。不同浓度的AngⅡ与球旁器细胞共孵育后，放射免疫法测定上清中。肾素活性；实时定量PCR法测定细胞内肾素mRNA表达；化学发光法测定细胞内、外环磷酸腺苷（cAMP）水平的剂量和时间效应。改变细胞内钙离子浓度后，观察JGC内和上清中cAMP浓度改变。实时定量PCR法检测AngⅡ对JGC内腺苷酸环化酶（AC）5和AC6mRNA表达的影响。结果成功分离的JGC存在ACE、AT基因表达。AngⅡ可抑制JGC肾素分泌[（370．6：e36．9）比（299．6＋25．7）ngAngI·ml^-1·h^-1，P=0．014]，并能抑制基础状态和前列腺素E2、去甲肾上腺素诱导的肾素mRNA表达。AngⅡ剂量依赖地降低JGC内和上清中cAMP水平。内质网上的Ca—ATP泵抑制剂毒胡萝b内酯和环盐酸吗甲吡嗪酸均能剂量依赖地降低cAMP水平。细胞内钙离子螯合剂BAPTA—AM可降低细胞内钙离子浓度，进而使细胞内cAMP水平显著升高[（11．09＋0．48）比（3．55±0．47）nmol／L，P〈0．01]。AngⅡ能通过减少42．12％的AC5mRNA表达，进而抑制肾素分泌。结论AngⅡ直接抑制肾素分泌可能是通过影响AC5，进而抑制cAMP表达来实现的。JGC内调节肾素分泌过程中，钙离子途径和Gsd-cAMP两大信号传导途径中存在交联对话。

Objective To elucidate the mechanism of angiotensin （Ang）Ⅱ in regulation of renin synthesis and secretion in primary cultured juxtaglomerular granular cells （JGCs）. Methods Mice JGCs were isolated and cultured as described before. Real-time PCR was used to demonstrate the expression of angiotensin converting enzyme （CE） and angiotencin receptor （AT1, AT2） in JGCs. Ang Ⅱ was co-cultured with JGCs stimulated by PGE2 and isoproternol or not. Renin activity in supernatant was detected by radioimmunoassay and renin mRNA expression was examined by real-time PCR. Different concentrations of Ang Ⅱ were co-cultured with JGCs with different time （1 h, 4 h and 24 h）, cAMP in cell lysate and supernatant was measured by Cayman Cyclic AMP EIA Kit. The cytoplasmic calcium was decreased by BAPTA-AM, and increased by thapsigargin and cyclopiazonic acid, which could be used to observe the cAMP concentration affected by calcium. Adenylyl cyelase type 5, 6 （ACS, AC6） mRNA expression of JGCs co-cultured with Ang Ⅱ was measured by real-time PCR. Results Real-time PCR confirmed the expression of ACE and AT mRNA in JGCs of WT mice. Angiotensin Ⅱ reduced renin secretion in primary cultures of JGCs [（370.6±36.9） vs （299.6±25.7） ng Ang·ml^-1·h-1, P=0.014]. Angiotensin Ⅱ dose-dependently down-regulated forskolin-stimulated cAMP production in JGCs. Thapsigargin and cyclopiazonic acid decreased cAMP level. BAPTA-AM increased cAMP production obviously [（11.09±0.48） vs （3.55±0.47） nmol/L, P〈0.01]. Ang lI inhibited AC5 mRNA expression by 42.12%, but did not inhibit AC6 mRNA expression. Conclusions Angiotensin Ⅱ can directly inhibit renin synthesis and secrestion maybe through reducing AC-stimulated cAMP levels. Crosstalk between calcium and cAMP system may exist in precise regulation of renin in JGCs.