Inhaled isobutyl nitrite inhibited macrophage inducible nitric oxide by blocking NFkappaB signaling and promoting degradation of inducible nitric oxide synthase-2.
We previously reported that inhaled isobutyl nitrite inhibited macrophage tumoricidal activity by inhibiting inducible nitric oxide (NO) production. In the present study, a much shorter inhalant exposure regimen (five daily exposures) inhibited inducible NO and the NO synthase (NOS2). One of the ways in which NO and NOS2 are regulated is by ubiquitin-dependent NOS2 degradation. Immunoprecipitated NOS2 showed increased poly-ubiquitination, following exposure to the inhalant. In addition, Western blots of macrophage nuclear extracts for the NFkappaB subunit, p65, showed that exposure to the inhalant inhibited NFkappaB signaling, necessary for induction of NOS2. The inhalant blocked phosphorylation of the NFkappaB inhibitor, IkappaBalpha. The inhibition of NFkappaB signaling following inhalant exposure was confirmed using mice transgenic for the kappaB-dependent promoter of the HIV 5' LTR linked to luciferase. The data suggested that inhalant exposure likely inhibited macrophage NO production by blocking NFkappaB-mediated activation signaling and promoting poly ubiquitination of NOS2.
Ponnappan
Usha
U
Department of Microbiology and Immunology, College of Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham, Little Rock, AR 72205, USA.
Yull
Fiona E
FE
Soderberg
Lee S F
LS
D000280:Administration, Inhalation; D000818:Animals; D015153:Blotting, Western; D002073:Butanes / Q000008:administration & dosage*; D005260:Female; D020672:I-kappa B Proteins / Q000378:metabolism; D047468:Immunoprecipitation; D017737:Macrophages, Peritoneal / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000502:physiology; D009566:Nitrates / Q000008:administration & dosage*; D009569:Nitric Oxide / Q000037:antagonists & inhibitors; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D010766:Phosphorylation; D015398:Signal Transduction / Q000187:drug effects*; D025801:Ubiquitin / Q000378:metabolism
D002073:Butanes; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606205:Nfkbia protein, mouse; D009566:Nitrates; D025801:Ubiquitin; D000072000:NF-KappaB Inhibitor alpha; D009569:Nitric Oxide; C069055:isobutyl nitrate; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse