title: Peroxynitrite inhibits inducible (type 2) nitric oxide synthase in murine lung epithelial cells in vitro.
abstract: Peroxynitrite, formed by nitric oxide (NO) and superoxide, can alter protein function by nitrating amino acids such as tyrosine, cysteine, trytophan, or methionine. Inducible nitric oxide synthase (Type 2 NOS or iNOS) converts arginine to citrulline, releasing NO. We hypothesized that peroxynitrite could function as a negative feedback modulator of NO production by nitration 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. Similar results were obtained when 3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, was added to the flasks. PAPA-NONOate, the NO generator, did not affect the headspace NO. Nitration of the iNOS was confirmed by detection of 3-nitrotyrosine by Western blotting. These data suggest a mechanism for inhibition of NO synthesis at inflammatory sites where iNOS, NO, and superoxide would be expected.
authors.affiliation: Southern Arizona Veterans Health Care System and the Departments of Medicine and Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85723, USA.
authors.forename: V K
authors.initials: VK
authors.lastname: Robinson
authors.forename: E
authors.initials: E
authors.lastname: Sato
authors.forename: D K
authors.initials: DK
authors.lastname: Nelson
authors.forename: S L
authors.initials: SL
authors.lastname: Camhi
authors.forename: R A
authors.initials: RA
authors.lastname: Robbins
authors.forename: J C
authors.initials: JC
authors.lastname: Hoyt
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology; D015870:Gene Expression / Q000187:drug effects; D006834:Hydrazines / Q000494:pharmacology; D008168:Lung / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D051379:Mice; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D009566:Nitrates / Q000494:pharmacology*; D009569:Nitric Oxide / Q000096:biosynthesis / Q000494:pharmacology; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D016877:Oxidants / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D017931:DNA Primers; D004791:Enzyme Inhibitors; D006834:Hydrazines; D009566:Nitrates; D020030:Nitric Oxide Donors; D016877:Oxidants; C112653:PAPA NONOate; D012333:RNA, Messenger; C039642:peroxynitric acid; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; C002385:linsidomine; D008981:Molsidomine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse