Protein kinase C-alpha mediates endothelial barrier dysfunction induced by TNF-alpha.
We tested the hypothesis that protein kinase C-alpha (PKC-alpha) mediates tumor necrosis factor-alpha (TNF-alpha)-induced alterations in permeability of pulmonary microvessel endothelial monolayers (PEM). The permeability of PEM was assessed by the clearance rate of Evans blue-labeled albumin. PEM lysates were analyzed for PKC-alpha mRNA (Northern cDNA blot), protein (Western immunoblot), and activity (translocation and phosphorylation of myristoylated arginine-rich C kinase substrate). Incubation of PEM with TNF-alpha (1,000 U/ml) for 4 h resulted in increases in 1) PKC-alpha protein, 2) cytoskeletal-associated PKC-alpha, 3) PKC-alpha activity, and 4) permeability to albumin. The TNF-alpha-induced increase in PKC-alpha protein, PKC-alpha activity, and permeability was prevented by a 4-h pretreatment with PKC-alpha antisense oligonucleotide but not by the scrambled nonsense oligonucleotide. The TNF-alpha-induced increase in permeability to albumin was prevented by myristoylated protein kinase C inhibitor (an inhibitor of PKC-alpha/beta, 100 microM) and calphostin (an inhibitor of the classic and novel PKC isotypes, 200 nM). The treatment with calphostin from 0.5 to 3.0 h after TNF-alpha still prevented barrier dysfunction induced by 4 h of TNF-alpha treatment. The data indicate that prolonged activation of PKC-alpha, maintained by a translation-dependent pool of PKC-alpha protein, mediates TNF-alpha-induced increases in endothelial permeability in PEM.
Ferro
T
T
Research Service, Stratton Veterans Affairs Medical Center, Albany Medical College, New York 12208, USA.
Neumann
P
P
Gertzberg
N
N
Clements
R
R
Johnson
A
A
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002199:Capillary Permeability / Q000187:drug effects*; D002417:Cattle; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000503:physiopathology*; D004789:Enzyme Activation; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism; D008833:Microcirculation / Q000187:drug effects; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051571:Protein Kinase C-alpha; D011652:Pulmonary Circulation / Q000187:drug effects*; D012709:Serum Albumin / Q000493:pharmacokinetics; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D000099221:Protein Kinase C zeta
D003598:Cytoskeletal Proteins; D007527:Isoenzymes; D008565:Membrane Proteins; D016376:Oligonucleotides, Antisense; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D012709:Serum Albumin; D014409:Tumor Necrosis Factor-alpha; D000099221:Protein Kinase C zeta; C410302:PKM-alpha protein, Bos taurus