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M. Shivanna, F. Wang, M. Satpathy, S. Srinivas; Thrombin–Induced MLC Phosphorylation Results in Barrier Dysfunction in Retinal Pigment Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3325.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: MLC phosphorylation increases the contractility of the actin cytoskeleton. This effect on the cortical actin leads to a loss of barrier integrity in cellular monolayers. This mechanism of barrier dysfunction, noted prominently in response to inflammatory stress in vascular endothelium, has been examined in retinal pigment epithelium (RPE) by activating proteinase–activated receptor–1 (PAR–1) receptors. Methods: Experiments were performed with ARPE–19 cell line and in primary cultures of human fetal RPE. MLC phosphorylation was induced by thrombin–mediated activation of PAR–1. Expression of MLC kinase (MLCK), a Ca2+/calmodulin–dependent protein kinase that phosphorylates MLC, was determined by RT–PCR. Expression of RhoA, and Rho kinase 1/2 (effectors of RhoA) was also ascertained by RT–PCR. Influence of Rho Kinase was characterized by using its selective inhibitor Y–27632 and through inhibition of its activation by elevated cAMP. The barrier integrity of ARPE–19 monolayer grown on porous filters was assayed as permeability to horseradish peroxidase (HRP). Results: (a) RT–PCR, with total RNA from ARPE–19, showed expression of MLCK, Rho kinase 1/2, and RhoA. (b) Exposure to thrombin (2 U/ml; 2 min) led to mono– and di–phosphorylation of MLC in both ARPE and human fetal RPE. Each of these appeared as doublets. The non–phosphorylated MLC also appeared as a doublet consistent with the appearance of its phosphorylated forms. (c) Both basal and thrombin–induced MLC phosphorylations were suppressed by co–treatment with Y–27632 (10 µM; 30 min) or forskolin (10 µM; 10 min). In resting cells, phalloidin staining of F–actin indicated organization of the cortical actin cytoskeleton into a thick band. This organization was disrupted by thrombin and led to formation of inter–endothelial gaps. Exposure to thrombin also increased permeability to HRP. Conclusions: The appearance of MLC as doublets indicates expression of two isoforms of MLC as demonstrated recently in the renal epithelium of rat (Chou et al., J Biol. Chem, 279, 47, 2004). Thrombin induces MLC phosphorylation in RPE. Consequent increase in the contractility of the actin cytoskeleton produces a centripetal force leading to inter–endothelial gaps and a breakdown of the barrier integrity.
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