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 PKC alpha (PKC alpha EC), PKC delta (PKC delta EC) or vector (vector control EC) cDNAs. Thrombin induced permeability changes in all EC, and induced significantly elevated rates of monolayer permeability in PKC alpha EC. Conversely, the basal level of permeability was significantly blunted in PKC delta EC, resulting in diminished thrombin-induced changes in permeability. PKC inhibitors, Gö6976 and rottlerin, reversed the effects of PKC alpha and PKC delta overexpression on permeability, respectively. Immunoblot analyses demonstrated significantly less beta-catenin associated with the cytoskeletal subcellular fraction in thrombin-treated PKC alpha EC, an effect blocked by pretreatment with Gö6976. PKC delta EC 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 PKC delta EC. 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 PKC delta EC monolayer permeability. These findings demonstrate that monolayer permeability changes are differentially regulated by PKC isoenzymes, suggesting that PKC alpha promotes endothelial barrier dysfunction and PKC delta enhances basal endothelial barrier function.
Harrington
Elizabeth O
EO
Providence VA Medical Center, Department of Medicine, Brown Medical School, Providence, RI 02908, USA. Elizabeth_Harrington@brown.edu
Brunelle
Jodi L
JL
Shannon
Christopher J
CJ
Kim
Eric S
ES
Mennella
Kirstin
K
Rounds
Sharon
S
D000098:Acetophenones / Q000494:pharmacology; D000818:Animals; D001578:Benzopyrans / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology; D002458:Cell Fractionation; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004822:Epididymis / Q000033:anatomy & histology*; D022001:Focal Adhesions / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008297:Male; D010539:Permeability; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D013917:Thrombin / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D000098:Acetophenones; D001578:Benzopyrans; D002227:Carbazoles; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D007211:Indoles; D007527:Isoenzymes; C081021:Go 6976; C085746:rottlerin; D011493:Protein Kinase C; D013917:Thrombin; D020742:rhoA GTP-Binding Protein