PEROXYNITRITE MODULATES TYROSINE-DEPENDENT SIGNAL-TRANSDUCTION PATHWAY OF HUMAN ERYTHROCYTE BAND-3

Peroxynitrite, the product of the reaction between nitric oxide and su peroxide anion, is able to nitrate protein tyrosines. If this modifica tion occurs on phosphotyrosine kinase substrates, it can down-regulate cell signaling, We investigated the effects of peroxynitrite on band 3-mediated signal transduction of human erythrocytes, Peroxynitrite tr eatment induced two different responses, At low concentrations (10-100 mu M) it stimulated a metabolic response, leading to 1) a reversible inhibition of phosphotyrosine phosphatase activity, 2) a rise of tyros ine phosphorylation in the 22K cytoplasmic domain of band 3, 3) the re lease of glyceraldehyde 3-phosphate dehydrogenase from the membrane, a nd 4) the enhancement of lactate production, At high concentrations (2 00-1000 mu M), peroxynitrite induced 1) cross-linking of membrane prot eins, 2) inhibition of band 3 tyrosine phosphorylation, 3) nitration o f tyrosines in the 22K cytoplasmic domain of band 3, 4) binding of hem oglobin to the membrane, 5) irreversible inhibition of phosphotyrosine kinase activity, 6) massive methemoglobin production, and 7) irrevers ible inhibition of lactate production, Our results demonstrate that at concentrations that could conceivably be achieved in vivo (10-100 mu M), peroxynitrite behaves like other oxidants, i.e., it stimulates ban d 3 tyrosine phosphorylation and increases glucose metabolism, Thus, o ne plausible physiologic effect of peroxynitrite is the up-regulation of signaling through the reversible inhibition of phosphotyrosine phos phatase activity, At high concentrations of peroxynitrite, the tyrosin e phosphorylation ceases in parallel with the nitration of band 3 tyro sines, but at these concentrations phosphotyrosine kinase activity and glycolysis are also irreversibly inhibited, Thus, at least in red blo od cells, the postulated down-regulation of signaling by peroxynitrite cannot merely be ascribed to the nitration of tyrosine kinase targets .