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Abstract：
Endothelial cells participate in extracellular ATP release elicited by mechanosensors. To characterize the dynamic interactions between mechanical and chemical factors that modulate ATP secretion by the endothelium, we assessed and compared the mechanisms participating in the spontaneous (basal) and mechanically stimulated secretion using primary cultures of rat mesentery endothelial cells. ATP/metabolites were determined in the cell media prior to (basal) and after cell media displacement or a picospritzer buffer puff used as mechanical stimuli. Mechanical stimulation increased extracellular ATP that peaked within 1 min, and decayed to basal values in 10 min. Interruption of the vesicular transport route consistently blocked the spontaneous ATP secretion. Cells maintained in media lacking external Ca2+ elicited a spontaneous rise of extracellular ATP and adenosine, but failed to elicit a further extracellular ATP secretion following mechanical stimulation. 2-APB, a TRPV agonist, increased the spontaneous ATP secretion, but reduced the mechanical stimulation-induced nucleotide release. Pannexin1 or connexin blockers and gadolinium, a Piezo1 blocker, reduced the mechanically induced ATP release without altering spontaneous nucleotide levels. Moreover, thrombin or related agonists increased extracellular ATP secretion elicited by mechanical stimulation, without modifying spontaneous release. In sum, present results allow inferring that the spontaneous, extracellular nucleotide secretion is essentially mediated by ATP containing vesicles, while the mechanically induced secretion occurs essentially by connexin or pannexin1 hemichannel ATP transport, a finding fully supported by results from Panx1-/- rodents. Only the latter component is modulated by thrombin and related receptor agonists, highlighting a novel endothelium-smooth muscle signaling role of this anticoagulant.
摘要：
内皮细胞参与机械传感器引起的细胞外ATP释放。为了表征调节内皮ATP分泌的机械和化学因素之间的动态相互作用，我们使用大鼠肠系膜内皮细胞的原代培养物评估并比较了参与自发（基础）和机械刺激分泌的机制。在细胞培养基置换之前（基础）和之后或在用作机械刺激的picospritzer缓冲液粉扑之后，在细胞培养基中测定ATP/代谢物。机械刺激增加细胞外ATP，在1分钟内达到峰值，并在10分钟内衰减至基础值。囊泡运输途径的中断始终阻止了自发的ATP分泌。维持在缺乏外部Ca2+的培养基中的细胞引起细胞外ATP和腺苷的自发上升，但在机械刺激后未能引起进一步的细胞外ATP分泌。2-APB,TRPV激动剂，增加了自发的ATP分泌，但减少了机械刺激诱导的核苷酸释放。Pannexin1或连接蛋白阻断剂和钆，Piezo1阻断剂，减少机械诱导的ATP释放而不改变自发核苷酸水平。此外，凝血酶或相关激动剂增加了机械刺激引起的细胞外ATP分泌，不改变自发释放。总之,目前的结果可以推断，自发的，细胞外核苷酸分泌基本上是由含ATP的囊泡介导的，而机械诱导的分泌基本上是通过连接蛋白或pannexin1半通道ATP转运发生的，这一发现得到了Panx1-/-啮齿动物的结果的完全支持。只有后者的成分受凝血酶和相关受体激动剂的调节，强调了这种抗凝剂的一种新的内皮-平滑肌信号作用。
