Connexin 40 and ATP-dependent intercellular calcium wave in renal glomerular endothelial cells
Connexin 40 and ATP-dependent intercellular calcium wave in renal glomerular endothelial cells
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DOI：
10.1152/ajpregu.00489.2007
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发表时间：
2008-06-01
影响因子：
2.8
通讯作者：
Peti-Peterdi, Janos
中科院分区：
文献类型：
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作者：
Toma, Ildiko;Bansal, Eric;Peti-Peterdi, Janos
内皮细胞内钙（[Ca（2+）]（i））在肾小球血管系统的功能中起着重要作用。本研究旨在确定培养的肾小球内皮细胞（GENC）中内皮钙波的存在及其分子基础。用Fluo-4/Fura red装载玻璃盖玻片上的GENCs，并使用荧光共聚焦显微镜进行比率[Ca（2+）]（i）成像。单个GENC的机械刺激导致[Ca（2+）]（i）增加9倍，其在>400 μ m的距离内以再生方式（没有幅度、动力学和速度的降低）在整个单层中从细胞到细胞增殖（7.9 +/- 0.3 μ m/s）。用硝苯地平抑制电压依赖性钙通道对上述参数没有影响，但去除细胞外钙使Delta[Ca（2+）]（i）降低50%。重要的是，差距连接解偶联剂α-大黄酸或通过用Cx40短干扰RNA（siRNA）抑制GENC来敲低连接蛋白40（Cx40）几乎完全消除了Delta[Ca（2+）]（i）和钙波。使用清道夫鸡尾酒（腺苷三磷酸双磷酸酶和己糖激酶）或非选择性抑制嘌呤能P2受体与苏拉明细胞外ATP的故障，有类似的阻断作用。沿沿着刮除细胞消除了细胞之间的物理接触，但不影响钙波的传播。使用ATP生物传感器技术，我们检测到在钙波传播过程中细胞外ATP的显著升高（Delta = 76 +/- 2 μ M），这被Cx40 siRNA处理（Delta = 6 +/- 1 μ M）所消除。这些研究表明，连接蛋白40半通道和细胞外ATP是肾小球内皮钙波的关键分子元件，可能发挥重要的肾小球功能。
Endothelial intracellular calcium ([Ca(2+)](i)) plays an important role in the function of the juxtaglomerular vasculature. The present studies aimed to identify the existence and molecular elements of an endothelial calcium wave in cultured glomerular endothelial cells (GENC). GENCs on glass coverslips were loaded with Fluo-4/Fura red, and ratiometric [Ca(2+)](i) imaging was performed using fluorescence confocal microscopy. Mechanical stimulation of a single GENC caused a nine-fold increase in [Ca(2+)](i), which propagated from cell to cell throughout the monolayer (7.9 +/- 0.3 mu m/s) in a regenerative manner (without decrement of amplitude, kinetics, and speed) over distances >400 mu m. Inhibition of voltage-dependent calcium channels with nifedipine had no effect on the above parameters, but the removal of extracellular calcium reduced Delta[Ca(2+)](i) by 50%. Importantly, the gap junction uncoupler alpha-glycyrrhetinic acid or knockdown of connexin 40 (Cx40) by transfecting GENCs with Cx40 short interfering RNA (siRNA) almost completely eliminated Delta[Ca(2+)](i) and the calcium wave. Breakdown of extracellular ATP using a scavenger cocktail (apyrase and hexokinase) or nonselective inhibition of purinergic P2 receptors with suramin, had similar blocking effects. Scraping cells off along a line eliminated physical contact between cells but did not effect calcium wave propagation. Using an ATP biosensor technique, we detected a significant elevation in extracellular ATP (Delta = 76 +/- 2 mu M) during calcium wave propagation, which was abolished by Cx40 siRNA treatment (Delta = 6 +/- 1 mu M). These studies suggest that connexin 40 hemichannels and extracellular ATP are key molecular elements of the glomerular endothelial calcium wave, which may serve important juxtaglomerular functions.