Peroxynitrite inhibits inducible (Type 2) nitric oxide synthase in murine lung epithelial cells in vitro

Peroxynitrite, formed by nitric oxide (NO) and superoxide, can alter protei n function by nitrating amino acids such as tyrosine, cysteine, trytophan, or methionine. Inducible nitric oxide synthase (Type 2 NOS or iNOS) convert s arginine to citrulline, releasing NO. We hypothesized that peroxynitrite could function as a negative feedback modulator of NO production by nitrati on of iNOS. Confluent cultures of the murine lung epithelial cell line, LA- 4 were stimulated with cytokines to express iNOS, peroxynitrite was added, and the flasks sealed, After 3 h, NO in the headspace above the culture was sampled. Peroxynitrite caused a concentration-dependent decrease in NO. Si milar results were obtained when 3-morpholinosydnonimine (SIN-1), a peroxyn itrite generator, was added to the flasks. PAPA-NONOate, the NO generator, did not affect the headspace NO. Nitration of the: iNOS was confirmed by de tection of 3-nitrotyrosine by Western blotting. These data suggest a mechan ism for inhibition of NO synthesis at inflammatory sites where iNOS, NO, an d superoxide would be expected. (C) 2001 Elsevier Science Inc.