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

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 5-isobutyl-1-methylxanthine. 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 kappa B (NF-kappa B) into the nucleus, and this process was abolished by forskolin, In addition, the LPS-dependent degradation of I kappa B alpha was not observed in forskolin-treated cells; the levels of the p65 subunit of NF-kappa B were minimal in the nucleus at the same time. Also, we observed that forskolin induced transcription of the I kappa B alpha gene in a time-dependent manner and in addition up-regulated LPS-induced I kappa B alpha 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 I kappa B alpha which suppresses the activation of NF-kappa B and inhibits the onset of transcription of the iNOS gene.