Expression of nitric-oxide synthase in rat Kupffer cells is regulated by cAMP

Shamimunisa B. Mustafa, Merle S. Olson

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Abstract

Treatment of cultured rat Kupffer cells with lipopolysaccharide (LPS) resulted in a time-dependent increase in the expression of the inducible isoform of nitric-oxide synthase (iNOS). Agents that elevated intracellular cAMP levels (e.g. forskolin, dibutyryl cAMP, cholera toxin, and isoproterenol) markedly decreased nitrite production and iNOS protein formation by LPS-stimulated Kupffer cells. Furthermore, inhibition of LPS- induced nitrite formation and iNOS protein levels by these agents was enhanced in the presence of the phosphodiesterase inhibitor 3-isobutyl-1- methyl-xanthine. Forskolin, the most potent inhibitor of LPS-induced nitrite formation by Kupffer cells, decreased iNOS mRNA levels in a time-dependent manner. Time course studies indicated that forskolin was most effective at inhibiting LPS-induced nitrite formation and iNOS mRNA levels by Kupffer cells when added before LPS. Message stability studies established that forskolin did not enhance the rate of decay of LPS-induced iNOS mRNA. Nuclear run-on assays revealed that forskolin decreased LPS-induced transcription of the iNOS gene. Treatment of Kupffer cells with LPS induced the translocation of the p65 subunit of nuclear factor κB (NF-κB) into the nucleus, and this process was abolished by forskolin. In addition, the LPS-dependent degradation of IκBα was not observed in forskolin-treated cells; the levels of the p65 subunit of NF-κB were minimal in the nucleus at the same time. Also, we observed that forskolin induced transcription of the IκBα gene in a time-dependent manner and in addition up-regulated LPS-induced IκBα mRNA levels. Taken together, this study indicates that the attenuation of LPS- induced iNOS formation in Kupffer cells by elevated intracellular cAMP levels occurs by preventing the degradation of IκBα which suppresses the activation of NF-κB and inhibits the onset of transcription of the iNOS gene.

Original languageEnglish (US)
Pages (from-to)5073-5080
Number of pages8
JournalJournal of Biological Chemistry
Volume273
Issue number9
DOIs
StatePublished - Feb 27 1998

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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