Nitric oxide increases tumor necrosis factor production in differentiated U937 cells by decreasing cyclic AMP.
Nitric oxide (NO) increases tumor necrosis factor (TNF) synthesis in human peripheral blood mononuclear cells by a cGMP-independent mechanism. NO has been shown to inhibit adenylate cyclase in cell membranes. Since cAMP down-regulates TNF transcription, we examined the possibility that NO enhances TNF synthesis by decreasing cAMP. U937 cells were induced to differentiate using phorbol myristate acetate (100 nM for 48 h) and then were incubated for 24 h with sodium nitroprusside (SNP) or S-nitroso-N-acetylpenicillamine (SNAP). These NO donors increased TNF production (7.0- and 15.6-fold, respectively, at 500 microM) in a dose-dependent manner (p = 0.002). However, SNP and SNAP did not elevate cGMP levels in U937 cell cultures, and the cGMP analog, 8-bromo-cGMP, had no effect on TNF production. In contrast, SNP (p = 0.001) and SNAP (p = 0.009) decreased intracellular cAMP levels by up to 51.5% over 24 h and, in the presence of a phosphodiesterase inhibitor, blunted isoproterenol-stimulated increases in cAMP by 21.8% (p = 0.004) and 27.6% (p = 0.008), respectively. H89, an inhibitor of cAMP-dependent protein kinase, dose dependently increased TNF production in phorbol myristate acetate-differentiated U937 cells in the absence (6.5-fold at 30 microM; p = 0.035), but not in the presence (p = 0.77) of SNAP. Conversely, the cAMP analog dibutyryl cAMP (Bt2cAMP) blocked SNAP-induced TNF production (p = 0.001). SNP and SNAP (500 microM) increased relative TNF mRNA levels by 57.5% (p = 0.045) and 66.2% (p = 0.001), respectively. This effect was prevented by Bt2cAMP. These results indicate that NO up-regulates TNF production by decreasing intracellular cAMP.
Wang
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Critical Care Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892, USA.
Yan
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Wesley
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Danner
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D003994:Bucladesine / Q000494:pharmacology; D002454:Cell Differentiation; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D009569:Nitric Oxide / Q000494:pharmacology*; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D026423:S-Nitroso-N-Acetylpenicillamine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*; D014665:Vasodilator Agents / Q000494:pharmacology
D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D014665:Vasodilator Agents; D009569:Nitric Oxide; D003994:Bucladesine; D026423:S-Nitroso-N-Acetylpenicillamine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D010396:Penicillamine; D006152:Cyclic GMP; D013755:Tetradecanoylphorbol Acetate