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S. Srinivas, C. D’ hondt, R. Ponsaerts, B. Himpens, J. Vereecke; Thrombin Inhibits Connexin Hemichannels in Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2814.
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Connexin hemichannels, which are permeable to molecules up to 1 kDa, are implicated in ATP release during intercellular Ca2+ wave elicited by a point mechanical stimulus. In this study, we have investigated the activity of hemichannels in cultured bovine corneal epithelial cells (BCEC) and their sensitivity to altered myosin light chain (MLC) phosphorylation, which is a trigger for actin contractility.
Ca2+ wave was elicited by applying a point mechanical stimulus to a confluent monolayer of BCEC loaded with Ca2+-sensitive dye Fluo-4. Confocal [Ca2+]i images were employed to calculate the spread of the Ca2+ wave (called active area, AA). Functional state of hemichannels was also assayed by LY uptake (2.5%, 5 min) in Ca2+-free medium. MLC phosphorylation was induced by exposure to thrombin (2 U/ml; 5 min).
(1) In untreated BCEC, exogenous apyrase [Apyrase 6 (5 U/ml for 30 min) + Apyrase 7 (5 U/ml for 30 min)], flufenamic acid (inhibits hemichannels; 50 µM; 30 min), and Gap26 (inhibitor of Cx43 hemichannels; 300 µM for 30 min) reduced AA (>80%) indicating involvement of hemichannels and ATP-dependent paracrine mechanism in the Ca2+ wave propagation. (2) LY uptake, which occurrs in a Ca2+-free medium, was blocked by Gap26. (3) Thrombin blocked LY uptake. This effect could be surmounted by pre-treatment with adenosine (200µM, 30 min).
Connexin hemichannels are involved in Ca2+ wave propagation in BCEC. Thrombin blocks connexin hemichannels by MLC phosphorylation. Inhibition of this effect by adenosine could be possibly through inhibition of MLC phosphorylation as in bovine corneal endothelial cells [Srinivas et al., Exp Eye Res. 79(4):543-51 (2004)].
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