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B. J. Himpens, R. Ponsaerts, C. D'hondt, F. Hertens, J. Vereecke, G. Bultynck; RhoA Activation Participates in Thrombin-Induced Inhibition of Hemichannel-Responses in Bovine Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2833.
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Thrombin inhibits connexin-hemichannel responses in bovine corneal endothelial cells (BCEC) via PAR-1 activation. This inhibition involves the activation of the actomyosin cytoskeleton by sensitization towards intracellular [Ca2+] and is mediated by enhanced phosphorylation of myosin light chain 2 (MLC). We have previously shown that Rho-associated kinase plays an important role in this MLC-phosphorylation. Here, we investigated the role of RhoA activation in the thrombin-induced hemichannel inhibition in BCEC.
RhoA activity was assessed via GST-Rhotekin-RBD protein agarose beads (Cytoskeleton) as well as RhoA G-lisaTM Activation assay (Cytoskeleton). Inhibition of RhoA activity was achieved by pre-treating cells with C3 toxin of Clostridium botulinum. Hemichannel responses were assayed as intercellular Ca2+-wave propagation and ATP release in response to mechanical stimulation (MS) and as LY-dye uptake (2.5 %; 5 min) in response to Ca2+-free medium. MS-induced intercellular Ca2+-wave propagation, mainly driven by Cx43-mediated paracrine signaling, was measured in BCEC loaded with Fluo4-AM and the maximal spreading was quantified (active area). ATP release was measured via a luciferin/luciferase assay.
Within less than 1 min, treatment of BCEC with thrombin (2U/ml; 5 min) induced a rapid and transient activation of RhoA via a GDP/GTP exchange mechanism. This RhoA activation was prevented by pre-treating the cells with C3 toxin (1 mg/ml; 3 hours). C3 toxin by itself did not affect the hemichannel responses in BCEC. Furthermore, C3 toxin abolished the inhibition of Cx43-hemichannel responses by thrombin in BCEC, as observed in intracellular Ca2+-wave propagation (p<0.001), ATP release (p<0.001) and restored LY-dye uptake.
RhoA is rapidly and transiently activated upon thrombin treatment of BCEC. Since pharmacological inhibition of Rho-GTPase activity significantly overcomes the inhibitory effects of thrombin, it is likely that RhoA activation is a key player in the thrombin-induced inhibition of Cx43-hemichannel activity in BCEC.
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