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 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.
Robinson
V K
VK
Southern Arizona Veterans Health Care System and the Departments of Medicine and Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85723, USA.
Sato
E
E
Nelson
D K
DK
Camhi
S L
SL
Robbins
R A
RA
Hoyt
J C
JC
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
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