Köhler R, Brakemeier S, Kühn M, Degenhardt C, Buhr H, Pries A, Hoyer J
Department of Endocrinology and Nephrology, Medical Center Benjamin Franklin, Freie Universität Berlin, Berlin, Germany.
Cardiovasc Res. 2001 Jul;51(1):160-8. doi: 10.1016/s0008-6363(01)00281-4.
Ca(2+) mobilization plays an important role in endothelial function by stimulating Ca(2+)-dependent synthesis of vasodilating factors. In addition to inositol-1,4,5-trisphosphate (InsP(3)) mediated Ca(2+) mobilization, Ca(2+) release from ryanodine-sensitive pools and Ca(2+)-influx through TRP channels have been suggested to be important in endothelial Ca(2+)-signaling. However, the function and molecular identity of TRP channels and ryanodine receptors in human endothelium in situ are still elusive. We hypothesized that expression of ryanodine-receptors (RyR) and TRP channels differs between human endothelium in situ and in cultured cells.
By combining single-cell RT-PCR and patch-clamp techniques, expression of RyR and TRP channels was determined in situ in endothelial cells of human mesenteric artery (HMAECs) obtained from patients undergoing bowel resection and in the endothelial cell line EA.hy926.
At the single cell level, expression of RyR 3 was detected in 25 and 5% of HMAECs and EA.hy926 samples, respectively. Expression of the RyR 1 and 2 was not detected in either HMAECs or EA.hy926. In patch-clamp experiments in HMAECs, applications of caffeine (0.5 mM) induced sustained hyperpolarization mediated by activation of Ca(2+)-activated K channels. In EA.hy926, caffeine-induced hyperpolarization was not detected. Single HMAECs expressed the TRP genes, TRP1 and TRP3, but not TRP 4 and 6. The TRP1 was the predominantly expressed TRP gene in HMAECs in situ whereas TRP3 expression was rarely detected. EA.hy926 expressed only TRP1. In patch clamp experiments in HMAECs, Ca(2+)-store depletion activated non-selective cation currents leading to Ca(2+) entry.
Our findings suggest that, in addition to InsP(3) mediated Ca(2+) release, Ca(2+) release from ryanodine-sensitive stores mediated by RyR3 and Ca(2+) entry through TRP1 might represent important components of endothelial Ca(2+) signaling in situ and thereby of endothelial function in intact human blood vessels.
钙离子动员通过刺激血管舒张因子的钙离子依赖性合成在内皮功能中发挥重要作用。除了肌醇-1,4,5-三磷酸（InsP(3)）介导的钙离子动员外，从兰尼碱敏感池释放钙离子以及通过瞬时受体电位（TRP）通道的钙离子内流在内皮细胞钙离子信号传导中也被认为很重要。然而，人原位内皮细胞中TRP通道和兰尼碱受体的功能及分子特性仍不清楚。我们推测人原位内皮细胞与培养细胞中兰尼碱受体（RyR）和TRP通道的表达存在差异。
通过结合单细胞逆转录聚合酶链反应（RT-PCR）和膜片钳技术，测定从接受肠切除手术患者获取的人肠系膜动脉内皮细胞（HMAECs）原位以及内皮细胞系EA.hy926中RyR和TRP通道的表达。
在单细胞水平，分别在25%的HMAECs样本和5%的EA.hy926样本中检测到RyR 3的表达。在HMAECs或EA.hy926中均未检测到RyR 1和2的表达。在HMAECs的膜片钳实验中，应用咖啡因（0.5 mM）可诱导由钙离子激活钾通道激活介导的持续超极化。在EA.hy926中，未检测到咖啡因诱导的超极化。单个HMAECs表达TRP基因TRP1和TRP3，但不表达TRP 4和6。TRP1是原位HMAECs中主要表达的TRP基因，而TRP3表达很少被检测到。EA.hy926仅表达TRP1。在HMAECs的膜片钳实验中，钙离子储存耗竭激活非选择性阳离子电流导致钙离子内流。
我们的研究结果表明，除了InsP(3)介导的钙离子释放外，由RyR3介导的从兰尼碱敏感储存池中释放钙离子以及通过TRP1的钙离子内流可能是人原位内皮细胞钙离子信号传导的重要组成部分，进而也是完整人体血管内皮功能的重要组成部分。
