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Y. Guo, M. Satpathy, G. Wilson, S. Srinivas; Benzalkonium Chloride (BAK) Induces MLC Dephosphorylation in Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):881.
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Purpose: BAK, a preservative in ophthalmic formulations, accelerates desquamation of corneal epithelial cells with a concomitant depletion of intracellular ATP. Amongst varied effects of ATP depletion, dephosphorylation of regulatory light chain of myosin II (MLC) has been reported in cells undergoing hypoxic stress. Since excessive MLC dephosphorylation is implicated in disruption of focal adhesion secondary to loss of actin contractility, we have begun to investigate if desquamation in response to BAK is a consequence of MLC dephosphorylation. In this study, as a first step, we examined the influence of BAK on ATP loss and corresponding status of MLC phosphorylation. Methods: Experiments were carried out in primarily cultured bovine corneal epithelial cells. Acute ATP release was measured by bioluminescence of Luciferrin–Luciferase reaction. MLC phosphorylation was assayed by urea glycerol gel electrophoresis and Western blotting. Deliberate ATP depletion was induced by exposure to antimycin and hypoxia as a positive control. Results:Exposure to BAK at 0.003%, which is a concentration usually found in topical drug formulations, led to MLC dephosphorylation by 54% (n=5) within 10 min and this persisted for 30 min (60%; n=5). The extent of dephosphorylation decreased with lower concentrations of BAK as 0.001% BAK led to MLC dephosphorylation by 16% (n=5) at 10 min and 40% (n=5) at 30 min. Exposure to BAK (0.003%; 0.001%) also led to a transient release of ATP which was characterized by a rapid onset and an exponential decay. Exposure to antimycin (10 µM) and hypoxia (pO2 = 1.5% for 3 hrs in the absence of glucose) led to complete MLC dephosphorylation. Conclusions: Exposure to BAK causes acute ATP release and this may underlie the reported cellular ATP depletion. Consistent with the response to antimycin and hypoxia, we suggest that BAK–induced ATP depletion leads to MLC dephosphorylation and this may form the basis for epithelial desquamation.
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