Centro Desarrollo de NanoCiencia y Nanotecnología, CEDENNA y Laboratorio de Farmacología, Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago, Alameda Lib. B. O'Higgins 3363, Estación Central, Santiago, Chile.
Purinergic Signal. 2018 Jun;14(2):121-139. doi: 10.1007/s11302-017-9599-7. Epub 2018 Jan 19.
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 Ca 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.
内皮细胞参与由机械感受器引发的细胞外 ATP 释放。为了描述调节内皮细胞 ATP 分泌的机械和化学因素之间的动态相互作用，我们评估并比较了使用大鼠肠系膜内皮细胞原代培养物参与自发（基础）和机械刺激分泌的机制。在细胞培养基中（基础）之前和之后测定和代谢物，在细胞培养基置换或作为机械刺激的皮氏培养器缓冲液喷吹之后。机械刺激增加了细胞外 ATP，其在 1 分钟内达到峰值，并在 10 分钟内衰减至基础值。囊泡转运途径的中断一致阻断了自发的 ATP 分泌。在缺乏外部 Ca 的培养基中维持的细胞引起细胞外 ATP 和腺苷的自发增加，但在机械刺激后未能引起进一步的细胞外 ATP 分泌。2-APB，一种 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 或连接蛋白阻滞剂和 Gd3+，一种 Piezo1 阻滞剂，减少了机械诱导的 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发的细胞外 ATP 分泌，而不改变自发释放。总之，目前的结果表明，自发的细胞外核苷酸分泌主要由含有 ATP 的囊泡介导，而机械诱导的分泌主要通过连接蛋白或 Pannexin1 半通道 ATP 转运发生，这一发现完全得到 Panx1 啮齿动物实验结果的支持。只有后者成分受凝血酶和相关受体激动剂的调节，突出了这种抗凝剂在血管内皮和平滑肌信号传递中的新作用。
