Barrier integrity of the corneal endothelium: regulation by contractility of the actin cytoskeleton

Abstract

Purpose: This study demonstrates endothelial cell signalling, influencing its barrier integrity through actin cytoskeleton contraction. Methods: In cultured bovine corneal endothelia, myosin light chain (MLC) phosphorylation was assayed by Westens blotting after urea-glycerol gel electrophoresis. Actin cytoskeleton reorganisation was evaluated by phalloidin staining. Barrier integrity was assayed as tracer solute (HRP and /or carboxyfluorescein) permeability and transendothelial electrical resistance (TER) changes. Results: Resting corneal endothelial cells show a hexagonal morphology with a thick band of cortical actin. The PAR-1 agonist thrombin (2 U/ml) induced MLC phosphorylation, cortical actin disruption and appearance of interendothelial gaps. HRP permeability increased several fold. Responses were inhibited by Y-27632 or adenosine. Histamine caused MLC phosphorylation, a significan tincrease in carboxyfluorescein permeability and actin cytoskeleton disruption. Responses were inhibited by adenosine, by ML-7 (an MLCK inhibitor) or chelerythrine (PKC inhibitor). Adenosine increased TER and its inhibitory effects were mimicked by forskolin. Conclusions: MLC phosphorylation is inhibited by PKA but stimulated by Rho kinase. These results provide further understanding how corneal endothelium regulates stromal hydration through active modulation of its barrier integrity.