Tnf-[alpha] induces peroxynitrite-mediated depletion of lung
endothelial glutathione via protein kinase c.
Phelps, Douglas T., Thomas J. Ferro, Paul J. Higgins, Ravi Shankar,
Dawn M. Parker, and Arnold Johnson.
Research Service, Stratton Veterans Affairs Medical Center, and the
Departments of Microbiology and Immunology, Medicine, and Physiology
and Cell Biology, The Albany Medical College, Albany, NY 12208
APStracts 2:0106L, 1995.
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 (1000
U/ml) for 6 and 18 hours. The PAEM were assayed for ONOO- dependent
changes in the concentration of luminol, free glutathione (Gfree;
i.e., reduced glutathione [GSH] and oxidized glutathione [GSSG]) and
GSSG. TNF treatment decreased luminol and Gfree, and increased GSSG
and GSSG/Gfree compared to 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 myristate acetate (PMA, 1 [mu]M)
induced similar alterations in luminol and glutathione as TNF. TNF
and PMA induced a protein of similar molecular weight ( 90 kD) in the
focal contact rich fraction of PAEM lysate. TNF and PMA-induced
effects were prevented with the specific PKC inhibitor calphostin C
(1 [mu]M). The data indicate that TNF-induced PKC activation mediates
ONOO- generation which results in the oxidation and depletion of
glutathione in PAEM.
Received 19 December 1994; accepted in final form 1 June 1995.
APS Manuscript Number L364-4.
Article publication pending Am. J. Physiol. (Lung Cell. Mol.
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 11 July 1995.