TNF-alpha induces peroxynitrite-mediated depletion of lung endothelial glutathione via protein kinase C.
We tested the hypothesis that tumor necrosis factor-alpha (TNF) induces a peroxynitrite (ONOO-)-mediated depletion of glutathione via a protein kinase C (PKC)-dependent mechanism in pulmonary artery endothelial monolayers (PAEM). PAEM were incubated with TNF (1,000 U/ml) for 6 and 18 h. The PAEM were assayed for ONOO(-)-dependent changes in the concentration of luminol, free glutathione [Gfree; i.e., reduced glutathione and oxidized glutathione (GSSG)] and GSSG. TNF treatment decreased luminol and Gfree, and increased GSSG and GSSG/Gfree, compared with treatment with control media. The TNF-induced effects were prevented by co-incubation with the nitric oxide synthase inhibitors NG-monomethyl-L-arginine (1 mM), NG-nitro-L-arginine methyl ester (1 mM), or NG-nitro-L-arginine (1 mM). In addition, the TNF-induced effects were prevented by superoxide dismutase (10 U/ml), which removes O2-, and by urate (0.5 mM) and L-cysteine (3 mM), putative scavengers of ONOO-. The treatment of PAEM with the PKC activator phorbol 12-myristate 13-acetate (PMA, 1 microM) induced similar alterations in luminol and glutathione as TNF. TNF and PMA induced a protein of similar molecular weight (approximately 90 kDa) in the focal contact-rich fraction of PAEM lysate. TNF- and PMA-induced effects were prevented with the specific PKC inhibitor calphostin C (1 microM). The data indicate that TNF-induced PKC activation mediates ONOO- generation, which results in the oxidation and depletion of glutathione in PAEM.
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D003545:Cysteine / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D008165:Luminol / Q000378:metabolism; D009281:Naphthalenes / Q000494:pharmacology; D009566:Nitrates / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D010084:Oxidation-Reduction; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011651:Pulmonary Artery / Q000166:cytology / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D009281:Naphthalenes; D009566:Nitrates; D014409:Tumor Necrosis Factor-alpha; C039642:peroxynitric acid; D008165:Luminol; D019001:Nitric Oxide Synthase; D013482:Superoxide Dismutase; D011493:Protein Kinase C; D005978:Glutathione; C058819:calphostin C; D003545:Cysteine