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Barrier integrity of the corneal endothelium: regulation by contractility of the actin cytoskeleton
Author(s) -
Jans Danny J,
Mahesh Shivanna,
Ramachandran Charanya,
Jalimarada Supriya,
Srinivas Sangly P
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a913-b
Subject(s) - myosin light chain kinase , cytoskeleton , microbiology and biotechnology , corneal endothelium , actin cytoskeleton , phalloidin , chemistry , phosphorylation , chelerythrine , actin , protein kinase c , adenosine , biology , endothelium , biochemistry , cell , endocrinology
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 Western blotting after urea‐glycerol gel electrophoresis. Actin cytoskeleton reorganization 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 significant increase 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.