Sb. Mustafa et Ms. Olson, EXPRESSION OF NITRIC-OXIDE SYNTHASE IN RAT KUPFFER CELLS IS REGULATEDBY CAMP, The Journal of biological chemistry, 273(9), 1998, pp. 5073-5080
Treatment of cultured rat Kupffer cells with lipopolysaccharide (LPS)
resulted in a time-dependent increase in the expression of the inducib
le isoform of nitric-oxide synthase (iNOS). Agents that elevated intra
cellular cAMP levels (e.g, forskolin, dibutyryl cAMP, cholera toxin, a
nd isoproterenol) markedly decreased nitrite production and iNOS prote
in formation by LPS-stimulated Kupffer cells, Furthermore, inhibition
of LPS-induced nitrite formation and iNOS protein levels by these agen
ts was enhanced in the presence of the phosphodiesterase inhibitor 5-i
sobutyl-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 forsko
lin was most effective at inhibiting LPS-induced nitrite formation and
iNOS mRNA levels by Kupffer cells when added before LPS. Message stab
ility studies established that forskolin did not enhance the rate of d
ecay of LPS-induced iNOS mRNA Nuclear run-on assays revealed that fors
kolin 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 pro
cess was abolished by forskolin, In addition, the LPS-dependent degrad
ation 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 nucleu
s at the same time. Also, we observed that forskolin induced transcrip
tion of the I kappa B alpha gene in a time-dependent manner and in add
ition up-regulated LPS-induced I kappa B alpha mRNA levels. Taken toge
ther, this study indicates that the attenuation of LPS-induced iNOS fo
rmation 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 th
e iNOS gene.