CYTOKINES ACTIVATE INDUCIBLE NITRIC-OXIDE SYNTHASE GENE-TRANSCRIPTIONIN INNER MEDULLARY COLLECTING DUCT CELLS

The effects of lipopolysaccharide (LPS) and/or inflammatory cytokines on the expression of inducible nitric oxide synthase (NOS) were studie d in mIMCD-3 cells, derived from the murine inner medullary collecting duct. Under basal conditions, the production of nitrite, a stable met abolite of NO, was negligible; however, incubation with tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IF-gamma) for 24 h res ulted in a 12-fold increase in nitrite synthesis and the appearance of abundant iNOS mRNA and protein. The induction of nitrite production a nd iNOS mRNA was time dependent, requiring similar to 8 h for expressi on of significant levels of nitrite or iNOS mRNA. Coincubation with th e transcription inhibitor actinomycin D or the translation inhibitor c ycloheximide prevented the cytokine induction of iNOS mRNA and NO prod uction, indicating that synthesis of intermediary proteins stimulated transcription of the iNOS gene. Nuclear run-on transcription demonstra ted that the iNOS gene was transcriptionally inactive under basal cond itions, but was markedly induced by TNF-alpha and IF-gamma. These resu lts indicate that inflammatory cytokines stimulate NO production in mI MCD-3 cells by activating iNOS gene transcription in a process that re quires new protein synthesis.