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R. Mutharasan, M. Satpathy, S.P. Srinivas; Phosphorylation of Myosin Light Chain (MLC) in Cultured Bovine Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3795.
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Purpose: The corneal epithelium (CE) forms a barrier against the influx of fluid, solutes, and pathogens from tears into stroma. When this barrier is disrupted, stromal hydration is compromised and susceptibility to pathogens is increased. The barrier integrity is influenced by a number of factors including contractility of the cortical actin cytoskeleton, which is promoted by phosphorylation of MLC (i.e., regulatory light chain of myosin II). This study has investigated MLC phosphorylation upon exposure to various agents/stresses of pathophysiologic significance to CE Methods:Cultured bovine CE were starved of serum for 12–16 hrs and then challenged with (a) ATP, UTP, and ATPγS (P2Y agonists), (b) thrombin (activates rho kinase downstream PAR–1 receptors coupled to Gα12/13), (c) LPS (agonist for Toll–like receptors), and (d) antimycin A (inhibitor of oxidative phosphorylation). Protein extracts were assayed for MLC phosphorylation in two steps: (a) separation of phosphorylated and unphosphorylated forms by urea–glycerol gel electrophoresis, and (b) their subsequent identification by immunoblotting. Results: (1) ATP, UTP, and ATPγS induced dephosphorylation by 33%, 24%, and 14% (18 min; 100 µM; n=2–4), respectively. (2) Thrombin caused phosphorylation (2 min; 26%; n=4). Pre–exposure to Y–27632 (10 µM), inhibitor of Rho kinase–1, prevented thrombin–induced phosphorylation by more than 80% (30 min; n=3), (3) Acute exposure to LPS showed phosphorylation by 44% (60 min; n=5). Antimycin A led to dephosphorylation which increased with time (max 58% by 60 min; n=2). Conclusions: ATP and its analogs, although stimulate P2Y receptors coupled to Gαq/11, induce a net dephosphorylation presumably by activation MLC phosphatase (MLCP). Thrombin–induced response is consistent with activation of rho kinase–1, an inhibitor of MLCP. Acute response to LPS is consistent with loss of barrier function reported in response to the endotoxin, and presumably mediated by p38 MAP kinase. Loss of the barrier integrity in response to ATP depletion is unrelated to contractility of the actin cytoskeleton.
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