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（SACs）的分子特性和激活机制尚不清楚。我们发现trpm4样阳离子通道在大鼠脑动脉肌细胞（CAMs）中被膜拉伸激活。也就是说，负压激活了孤立cam中的单个通道。这些通道可渗透Na^+和Cs^+，并受到Gd^<3+>和DIDS的抑制。膜切除可以消除负压的影响，但随后将Ca^<2+> (>100nM)应用于膜的细胞内侧，恢复了与SACs难以区分的单通道活性。HEK293细胞中hTRPM4B的过表达导致阳离子通道的出现，这些阳离子通道被负压和Ca^<2+>激活，与CAMs中的SACs相比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trpm4样通道可以被膜拉伸激活，这可能有助于机械刺激后完整脑动脉的去极化和随之的血管收缩。2)低渗应激（HTS）和溶血磷脂酸（LPA）均通过依次激活人脐带静脉内皮细胞（HUVECs）的RhoA/ rho激酶和focal adhesion kinase F-actin (FAK)/paxillin诱导ATP释放和肌动蛋白的短暂重组。已知LPA通过其特异性受体诱导Rho A的激活，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），而非抗整合素α2、α6、αv和β4抗体，能够抑制hts诱导的RhoA易位、FAK和paxillin的酪氨酸磷酸化、ATP释放和肌动蛋白重组。然而，这些抗整合素抗体均未抑制lpa诱导的ATP释放和肌动蛋白重组，这表明整合素α5 β1在hts诱导的反应中起关键作用，而在lpa诱导的反应中不起作用。因此，我们可以合理地假设，这种特殊的整合素亚型参与了HUVECs中机械刺激引起的反应的启动。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