Role of protein kinase C isoforms in rat epididymal microvascular endothelial barrier function

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 PKCalpha (PKCalphaEC), PKCdelta (PKCdeltaEC) or vector (vector control EC) cDNAs. Thrombin induced permeability changes in all EC, and induced significantly elevated rates of monolayer permeability in PKCalphaEC. Conversely, the basal level of permeability was significantly blunted in PKCBEC, resulting in diminished thrombin-induced changes in permeability. PKC inhibitors, Go6976 and rottlerin, reversed the effects of PKCalpha and PKCdelta overexpression on permeability, respectively. Immunoblot analyses demonstrated significantly less beta-catenin associated with the cytoskeletal subcellular fraction in thrombin-treated PKCaEC, an effect blocked by pretreatment with Go6976. PKCdeltaEC 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 PKCdeltaEC. 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 PKCdeltaEC monolayer permeability. These findings demonstrate that monolayer permeability changes are differentially regulated by PKC isoenzymes, suggesting that PKCalpha promotes endothelial barrier dysfunction and PKCdelta enhances basal endothelial barrier function.