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首页> 美国卫生研究院文献>The Journal of Biological Chemistry >Thrombin Promotes Release of ATP from Lung Epithelial Cells through Coordinated Activation of Rho- and Ca2+-dependent Signaling Pathways
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Thrombin Promotes Release of ATP from Lung Epithelial Cells through Coordinated Activation of Rho- and Ca2+-dependent Signaling Pathways

机译：凝血酶通过Rho-和Ca2 +依赖性信号通路的协同激活促进肺上皮细胞释放ATP。

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Extracellular ATP controls key aspects of lung function via activation of epithelial cell purinergic receptors, but how ATP is released from cells remains poorly understood. To identify mechanistic components upstream of ATP release, we examined the effect of selected G protein coupled-receptor activation on ATP release from lung epithelial cells. The protease-activated receptor (PAR) agonist thrombin elicited a rapid Ca2+-dependent release of ATP from A549 cells. In contrast, the P2Y2 receptor agonist UTP caused negligible ATP release, despite promoting a robust Ca2+ response. Agonist-elicited ATP release was associated with Rho activation and was reduced in cells transfected with dominant negative mutants of p115-Rho GEF or RhoA, and by inhibitors of Rho kinase (ROCK). However, RhoA activation alone did not promote ATP release if temporally separated from Ca2+ mobilization. PAR3 was the only PAR subtype detected in A549 cells by reverse transcription-PCR. Transfection of cells with human PAR3 cDNA increased thrombin-promoted ATP release, inositol phosphate formation, and RhoA activation. Conversely, small interference RNA against PAR3 diminished thrombin-evoked responses. Thrombin-elicited ATP release was accompanied by an enhanced cellular uptake of propidium iodide in a Ca2+- and ROCK-dependent manner and was inhibited by connexin/pannexin hemichannel blockers. Our data suggest that thrombin promotes ATP release from A549 cells via Rho- and Ca2+-dependent activation of connexin/pannexin hemichannels. The relevance of these findings is highlighted by the observation that exposure of primary cultures of well differentiated human bronchial epithelial cells to thrombin resulted in robust ATP release, which was inhibited by ROCK inhibitors and by connexin/pannexin hemichannel blockers.
机译：细胞外ATP通过激活上皮细胞嘌呤能受体来控制肺功能的关键方面，但是如何从细胞释放ATP仍然知之甚少。为了确定ATP释放上游的机械成分，我们检查了所选G蛋白偶联受体激活对肺上皮细胞ATP释放的影响。蛋白酶激活受体（PAR）激动剂凝血酶引起A549细胞快速Ca 2 + 依赖性的ATP释放。相比之下，尽管P2Y2受体激动剂UTP促进了强烈的Ca 2 + 反应，但其ATP的释放却微不足道。激动剂引起的ATP释放与Rho活化有关，并且在用p115-Rho GEF或RhoA的显性负突变体以及Rho激酶（ROCK）的抑制剂转染的细胞中减少。然而，如果暂时与Ca 2 + 动员分开，单独的RhoA激活并不能促进ATP的释放。 PAR3是通过逆转录PCR检测到的A549细胞中唯一的PAR亚型。用人PAR3 cDNA转染细胞会增加凝血酶促进的ATP释放，磷酸肌醇形成和RhoA激活。相反，针对PAR3的小干扰RNA减少了凝血酶诱发的反应。凝血酶引起的ATP释放伴随以Ca 2 + -和ROCK依赖性方式增加碘化丙啶的细胞摄取，并被连接蛋白/泛神经毒素半通道阻滞剂抑制。我们的数据表明，凝血酶通过连接蛋白/泛素新半通道的Rho-和Ca 2 + 依赖性激活促进A549细胞的ATP释放。这些发现的相关性通过以下观察而突出显示：高度分化的人支气管上皮细胞原代培养物暴露于凝血酶会导致ATP释放强劲，而ROCK抑制剂和连接蛋白/泛神经毒素半通道阻滞剂抑制了ATP的释放。

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