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

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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