Pharmacological study on the mechano-sensitive molecules involved in vascular smooth muscle and endothelial cells.

1)拉伸激活阳离子通道（SAC）的分子身份和激活机制仍然知之甚少。我们发现，TRPM 4样阳离子通道被激活的大鼠脑动脉肌细胞（CAMs）的膜拉伸。也就是说，负压激活孤立CAM中的单通道。这些通道对Na^+和Cs^+具有渗透性，并被Gd^<3+>和DIDS抑制。负压的影响被膜切除所消除，但随后在膜的胞内侧施加Ca^<2+>（> 100 nM）恢复了与SAC无法区分的单通道活性。在HEK 293细胞中过表达hTRPM 4 B导致出现了可被负压和Ca^<2+>激活的阳离子通道，与CAM中的SAC相比，其具有非常相似的生物物理和药理学特性。这些结果表明，CAMs中的TRPM 4样通道可以被膜拉伸激活，这可能有助于细胞的去极化。 ...更多信息 机械刺激后完整脑动脉的收缩和伴随的血管收缩。2)低渗应激（HTS）和溶血磷脂酸（LPA）通过依次激活RhoA/Rho激酶和粘着斑激酶F-肌动蛋白（FAK）/桩蛋白（paxillin）诱导人脐静脉内皮细胞（HUVECs）ATP释放和肌动蛋白瞬时重组。已知LPA通过其特异性受体诱导Rho A活化，但HTS启动这些细胞内信号的机制尚不清楚。我们发现抗整合素α5β1抗体（Ab），而不是抗整合素α2、α6、αv或β4抗体，抑制HTS诱导的RhoA易位、FAK和桩蛋白的酪氨酸磷酸化、ATP释放和肌动蛋白重组。然而，LPA诱导的ATP释放和肌动蛋白重组不被任何这些抗整合素抗体抑制，表明整合素α5 β1在HTS诱导的反应中起关键作用，但在LPA诱导的反应中不起关键作用。因此，可以合理地假设这种特定的整合素亚型参与HUVEC中由机械刺激诱导的反应的起始。少

1)The molecular identity and activation mechanisms of Stretch-activated cation channels (SACs) remains poorly understood. We found that TRPM4-like cation channels are activated by membrane stretch in rat cerebral artery myocytes (CAMs). Namely, negative pressure activated single channels in isolated CAMs. These channels were permeable to Na^+ and Cs^+ and inhibited by Gd^<3+> and DIDS. The effect of negative pressure was abolished by membrane excision, but subsequent application of Ca^<2+> (>100nM) to the intracellular side of the membrane restored single channel activity that was indistinguishable from SACs. Overexpression of hTRPM4B in HEK293 cells resulted in the appearance of cation channels which were activated by both negative pressure and Ca^<2+> and which had very similar biophysical and pharmacological properties as compared with SACs in CAMs. These results indicate that TRPM4-like channels in CAMs can be activated by membrane stretch, and this may contribute to the depolariza … More tion and concomitant vasoconstriction of intact cerebral arteries following mechanical stimulation. 2)Both hypotonic stress (HTS) and lysophosphatidic acid (LPA) induce ATP release and a transient reorganization of actin through sequential activation of RhoA/Rho-kinase and focal adhesion kinase F-actin (FAK)/paxillin in human umbilical cord vein endothelial cells (HUVECs). LPA is known to induce the activation of Rho A via its specific receptors, but the mechanisms by which HTS initiates these intracellular signals are not known. We found anti-integrin α5β1 antibody(Ab), but not anti-integrin α2,α6,αv, or β4 antibodies, inhibited HTS-induced RhoA translocation, tyrosine phosphorylation of FAK and paxillin, ATP release, and actin reorganization. However, the LPA-induced ATP release and actin reorganization were not inhibited by any of these anti-integrin antibodies, indicating the integrin α5 β1 plays a pivotal role in the HTS-induced but not in the LPA-induced responses. It is therefore reasonable to assume that this particular subtype of integrin is involved in the initiation of the responses induced by mechanical stimuli in HUVECs. Less