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M. Satpathy, V. Anandan, S. Srinivas; PKC–Mediated Regulation of the Barrier Integrity Associated With Corneal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2204.
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Purpose: Phosphorylation of the regulatory light chain of myosin II (i.e., myosin light chain or MLC) increases the contractility of actin cytoskeleton leading to a loss in the barrier integrity of corneal endothelial cells. This study has investigated the role of PKC on MLC phosphorylation in cultured bovine corneal endothelial cells (BCEC). Methods: PKC was activated by an acute exposure to PMA as well as by exposure to agonists of Gαq/11–coupled receptors. PKC was inhibited by its selective inhibitor chelerythrine. MLC phosphorylation was quantified by urea–glycerol gel electrophoresis and Western blotting. Localization of phosphorylated MLC was determined by immunofluorescence. Actin cytoskeleton was visualized by phalloidin staining. The barrier integrity of cells grown on porous filters was assayed as permeability to horseradish peroxidase (HRP). Results: (a) Transcript for CPI–17, known to inhibit PPC1Δ (catalytic subunit of MLC phosphatase) upon phosphorylation by PKC, was expressed in BCEC. (b) Exposure to agonists for PAR–1 (thrombin) and H1 (histamine) receptors induced phosphorylation of CPI–17 and also led to phosphorylation of MLC. Furthermore, phosphorylated MLC was found concentrated in punctate form proximal to ZO–1 locale, and the cortical actin, usually organized as a dense band, was disrupted with a concomitant appearance of inter–endothelial gaps. (c) Both MLC phosphorylation and its punctate disposition along the locus of ZO–1 could be reproduced using PMA as well. (d) Chelerythrine inhibited MLC phosphorylation by histamine, thrombin, and PMA, each of which independently led to breakdown of the barrier integrity. Conclusions: (a) MLC phosphorylation is stimulated by PKC. This may involve phosphorylation of CPI–17. (b) Localization of phosphorylated MLC in proximity to tight junctional complex and the development of interendothelial gaps are consistent with the breakdown of the barrier integrity by opposing the tethering forces which facilitate interactions of TJ proteins. Taken together, this study provides further understanding as to how corneal endothelium regulates stromal hydration through an active modulation of its barrier integrity.
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