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M. Satpathy, P. Gallagher, S.P. Srinivas; Extracellular ATP Opposes Thrombin–Induced Myosin Light Chain Phosphorylation in Corneal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4798.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:MLC phosphorylation induces contractility of the actin cytoskeleton. Such an effect on the cortical actin results in break down of the cellular barrier integrity. This type of barrier dysfunction, in response to cytokines and inflammatory mediators, has been demonstrated in bovine corneal endothelial cells (BCEC) through thrombin–induced MLC phosphorylation (Satpathy et al., ARVO, 2002). In this study, we have investigated if extracellular ATP can overcome MLC phosphorylation in the thrombin model of hyperpermeability in CE. Methods: BCEC grown on Petri dishes were used for all experiments. Cells, serum starved for ∼12 hrs, were challenged with ATP, [ATP + Thrombin], or other agents. Protein extracts were then assayed for MLC phosphorylation using a protocol urea–glycerol gel electrophoresis and immunoblotting. Densitometry of the gels led to quantification of MLC phosphorylation. Reorganization of the cortical cytoskeleton was imaged after phalloidin staining of actin. Results:(a) ATP induced MLC dephosphorylation in a time dependent manner (MLC–P = 47% at 10 min; n = 9), (b) UTP (a potent P2Y2 agonist) and ATPgS (a non–hydrolysable P2Y2 agonist), also induced significant MLC dephosphorylation (UTP:76%; n=5; ATPgS:74.8%; n=3),at t= 18min (c) Exposure to thrombin alone induced MLC phosphorylation (222%; n=4). When cells were exposed to thrombin and ATP together, MLC–P was only 128% (n=4) at t = 10 min. In contemporaneous trials, ATP led to MLC dephosphorylation (49%; n=4) as in the initial set, (d) Immunostaining of actin indicated that thrombin disrupted the thick band of cortical actin prominently noticeable in resting cells and led to appearance of a number of inter–endothelial gaps. These characteristic morphologic responses to thrombin were prevented by concomitant exposure to ATP. Conclusions:(1) ATP and other P2Y2 agonists induce MLC dephosphorylation. Since P2Y2 receptors are coupled to Gaq/11 G proteins, ATP effect is likely to be through modulation of MLC phosphatase. This is consistent with report by Noll et al, Am J Physiol (2000).(2) Activation of MLC phosphatase by ATP is antagonistic to the effect of thrombin which mediates its effect through inhibition of the phosphatase downstream of activaed rho kinase. Support: NIH EY11107 (SPS)
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