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Role of Protein Kinase C Isoforms in Rat Epididymal Microvascular Endothelial Barrier Function

Pulmonary Vascular Biology Research Laboratory, Providence Veterans Affairs Medical Center, Department of Medicine, Brown Medical School, Providence, Rhode Island

Address correspondence to: Elizabeth O. Harrington, Ph.D., Providence VA Medical Center, Research Services, 151, 830 Chalkstone Avenue, Providence, RI 02908. E-mail: Elizabeth_Harrington{at}brown.edu

Endothelial barrier dysfunction is involved in a variety of diseased states. We investigated the role of protein kinase C (PKC) in monolayer permeability using endothelial cells (EC) overexpressing PKC (PKCEC), PKC (PKCEC) or vector (vector control EC) cDNAs. Thrombin induced permeability changes in all EC, and induced significantly elevated rates of monolayer permeability in PKCEC. Conversely, the basal level of permeability was significantly blunted in PKCEC, resulting in diminished thrombin-induced changes in permeability. PKC inhibitors, Gö6976 and rottlerin, reversed the effects of PKC and PKC overexpression on permeability, respectively. Immunoblot analyses demonstrated significantly less ß-catenin associated with the cytoskeletal subcellular fraction in thrombin-treated PKCEC, an effect blocked by pretreatment with Gö6976. PKCEC contained significantly greater numbers of focal contacts per cell. Thrombin enhanced RhoA GTPase activity in all EC; with a 3-fold greater level of activity in PKCEC. Rottlerin significantly blunted RhoA GTPase activity in all EC. Overexpression of RhoA dominant-negative cDNA diminished the size and number of focal contacts in EC, and significantly enhanced the basal rate of PKCEC monolayer permeability. These findings demonstrate that monolayer permeability changes are differentially regulated by PKC isoenzymes, suggesting that PKC promotes endothelial barrier dysfunction and PKC enhances basal endothelial barrier function.

Abbreviations: endothelial cell(s), EC • focal adhesion kinase, FAK • glycogen synthase kinase 3ß, GSK-3ß • horseradish peroxidase, HRP • protein kinase C, PKC • sodium dodecyl sulfate–polyacrylamide gel electrophoresis, SDS-PAGE

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