TNF-ALPHA INDUCES PEROXYNITRITE-MEDIATED DEPLETION OF LUNG ENDOTHELIAL GLUTATHIONE VIA PROTEIN-KINASE-C

We tested the hypothesis that tumor necrosis factor-alpha (TNF) induce s a peroxynitrite (ONOO-)-mediated depletion of glutathione via a prot ein 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 concen tration of luminol, free glutathione [G(free); i.e., reduced glutathio ne and oxidized glutathione (GSSG)] and GSSG. TNF treatment decreased luminol and G(free), and increased GSSG and GSSG/G(free), compared wit h treatment with control media. The TNF-induced effects were prevented by co-incubation with the nitric oxide synthase inhibitors N-G-monome thyl-L-arginine (1 mM), N-G-nitro-L-arginine methyl ester (1 mM), or N -G-nitro-L-arginine (1 mM). In addition, the TNF-induced effects were prevented by superoxide dismutase (10 U/ml), which removes O-2(-), 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 mu M) induced similar alterations in luminol and glut athione as TNF. TNF and PMA induced a protein of similar molecular wei ght (similar to 90 kDa) in the focal contact-rich fraction of PAEM lys ate. TNF- and PMA-induced effects were prevented with the specific PKC inhibitor calphostin C (1 mu M). The data indicate that TNF-induced P KC activation mediates ONOO- generation, which results in the oxidatio n and depletion of glutathione in PAEM.