Cytokines activate inducible nitric oxide synthase gene transcription in inner medullary collecting duct cells.
The effects of lipopolysaccharide (LPS) and/or inflammatory cytokines on the expression of inducible nitric oxide synthase (iNOS) were studied in mIMCD-3 cells, derived from the murine inner medullary collecting duct. Under basal conditions, the production of nitrite, a stable metabolite of NO, was negligible; however, incubation with tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IF-gamma) for 24 h resulted in a 12-fold increase in nitrite synthesis and the appearance of abundant iNOS mRNA and protein. The induction of nitrite production and iNOS mRNA was time dependent, requiring approximately 8 h for expression of significant levels of nitrite or iNOS mRNA. Coincubation with the transcription inhibitor actinomycin D or the translation inhibitor cycloheximide prevented the cytokine induction of iNOS mRNA and NO production, indicating that synthesis of intermediary proteins stimulated transcription of the iNOS gene. Nuclear run-on transcription demonstrated that the iNOS gene was transcriptionally inactive under basal conditions, but was markedly induced by TNF-alpha and IF-gamma. These results indicate that inflammatory cytokines stimulate NO production in mIMCD-3 cells by activating iNOS gene transcription in a process that requires new protein synthesis.
Mohaupt
M G
MG
Department of Medicine, University of Florida College of Medicine, Gainesville 32610-0224, USA.
Schwöbel
J
J
Elzie
J L
JL
Kannan
G S
GS
Kone
B C
BC
D000594:Amino Acid Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D016207:Cytokines / Q000494:pharmacology*; D004790:Enzyme Induction / Q000187:drug effects; D007679:Kidney Medulla; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000502:physiology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D019001:Nitric Oxide Synthase; D014158:Transcription, Genetic
D016207:Cytokines; D008070:Lipopolysaccharides; D003513:Cycloheximide; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases