title: Reduction of high glucose and phorbol-myristate-acetate-induced endothelial cell permeability by protein kinase C inhibitors LY379196 and hypocrellin A.
abstract: Endothelial barrier dysfunction plays a pivotal role in the pathogenesis of diabetic vascular complications. Although recent studies have established a link between protein kinase C (PKC) pathway and hyperglycaemic-induced vascular permeability, it is unclear which PKC isoforms involve increased endothelial cell permeability. In the present study, we investigated whether high glucose induced endothelial hyperpermeability via distinct PKC isoforms in human umbilical vein endothelial cells (HUVECs) and whether increased endothelial permeability could be substantially reversed by PKC inhibitors LY379196 and hypocrellin A (HA). High glucose (20 mM) and phorbol-myristate-acetate (PMA)-induced endothelial hyperpermeability was almost abolished by 150 nM HA and partially reduced by 30 nM PKC beta inhibitor (LY379196). LY379196 and HA inhibited the membrane fraction of PKC activity in a dose-dependent manner. Western blot analysis revealed high-glucose-induced overexpression of PKC alpha and PKC beta2 in the membrane fraction of HUVECs. LY379196 (30 and 150 nM) selectively inhibited PKC beta2 with no significant effect on PKC alpha expression. HA (150 nM) significantly reduced PKC alpha expression with no inhibitory effect on PKC beta2. At higher concentrations (300 nM), both LY379196 and HA were no longer selective for PKC beta or alpha, respectively. This study showed that both PKC alpha and beta2 contributed to endothelial hyperpermeability. Since reduction of endothelial hyperpermeability was greater with inhibition of PKC alpha rather than PKC beta2, we conclude that PKC alpha may be a major isoform involved in endothelial permeability in HUVECs, and that PKC alpha-mediated endothelial permeability was significantly reversed by the PKC inhibitor HA.
authors.affiliation: Department of Health Sciences, University of Technology, Sydney, P.O. Box 123, Broadway, NSW 2007, Australia.
authors.forename: Lei
authors.initials: L
authors.lastname: Dang
authors.forename: J Paul
authors.initials: JP
authors.lastname: Seale
authors.forename: Xianqin
authors.initials: X
authors.lastname: Qu
mesh_terms: D001692:Biological Transport / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D004347:Drug Interactions; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008698:Mesylates / Q000494:pharmacology*; D010569:Perylene / Q000031:analogs & derivatives* / Q000494:pharmacology*; D019800:Phenol; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011758:Pyrroles / Q000494:pharmacology*; D011809:Quinones / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: C434605:5,21 - 12,17-dimetheneo-18H-dibenzo(i,o)pyrrolo(3,4-1)(1,8)diazacyclohexandecine-18,10(19H)dione,8((dimethylamino)methyl)-6,7,8,9,10,11-hexahydro,monomethanesulfonate; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008698:Mesylates; D011758:Pyrroles; D011809:Quinones; D019800:Phenol; D010569:Perylene; D011493:Protein Kinase C; D005947:Glucose; D013755:Tetradecanoylphorbol Acetate; C048388:hypocrellin A