Department of Physiology and Pharmacology, Institute of Medical Biology, University of Southern Denmark, J.B. Winslowsvej 21, Odense, Denmark.
Kidney Int. 2010 Jan;77(2):110-7. doi: 10.1038/ki.2009.418. Epub 2009 Nov 11.
To examine the role of the calcium/calmodulin-dependent phosphatase calcineurin in regulation of renin release, we assayed exocytosis using whole-cell patch clamp of single juxtaglomerular cells in culture. The calcineurin inhibitor, cyclosporine A (CsA), significantly increased juxtaglomerular cell membrane capacitance, an index of cell surface area and an established measure of exocytosis in single-cell assays. This effect was mimicked by intracellular delivery of a calcineurin inhibitory peptide, the calcium chelator ethylene glycol tetraacetic acid (EGTA), or the calmodulin inhibitor W-13. Simultaneous exposure to EGTA and CsA had no additive effect. The protein kinase A (PKA) blocker RpcAMPs had no effect on the CsA-induced increase in membrane capacitance. Intra- and extracellular application of tacrolimus did not alter membrane capacitance. A calmodulin antagonist (calmidazolium) and CsA, but not tacrolimus, significantly stimulated renin release from cultured juxtaglomerular cells. Juxtaglomerular cells expressed the calcineurin isoforms A-beta and A-gamma but not A-alpha. Plasma renin concentrations (PRCs) were not different in wild-type, calcineurin A-alpha, or A-beta knockout mice but increased after CsA treatment of the A-alpha knockout, while renin mRNA was suppressed. We conclude that calcineurin and calcium/calmodulin suppress exocytosis of renin from juxtaglomerular cells independent of PKA.
为了研究钙/钙调蛋白依赖性磷酸酶钙调神经磷酸酶在调节肾素释放中的作用，我们使用培养的肾小球旁细胞的全细胞膜片钳技术检测了胞吐作用。钙调神经磷酸酶抑制剂环孢素 A（CsA）显著增加了肾小球旁细胞细胞膜电容，这是细胞表面积的一个指标，也是单细胞测定中胞吐作用的一种既定测量方法。细胞内递送钙调神经磷酸酶抑制肽、钙螯合剂乙二醇四乙酸（EGTA）或钙调蛋白抑制剂 W-13 可模拟这种作用。同时暴露于 EGTA 和 CsA 没有叠加效应。蛋白激酶 A（PKA）阻断剂 RpcAMPs 对 CsA 诱导的细胞膜电容增加没有影响。他克莫司的细胞内和细胞外应用均未改变细胞膜电容。钙调蛋白拮抗剂（钙安定）和 CsA，但不是他克莫司，显著刺激培养的肾小球旁细胞释放肾素。肾小球旁细胞表达钙调神经磷酸酶同工型 A-β 和 A-γ，但不表达 A-α。野生型、钙调神经磷酸酶 A-α 或 A-β 敲除小鼠的血浆肾素浓度（PRCs）没有差异，但 CsA 处理 A-α 敲除小鼠后 PRCs 增加，而肾素 mRNA 受到抑制。我们的结论是，钙调神经磷酸酶和钙/钙调蛋白独立于 PKA 抑制肾素从肾小球旁细胞的胞吐作用。
