PT  - JOURNAL ARTICLE
AU  - M Cavicchi
AU  - B J R Whittle
TI  - Potentiation of cytokine induced iNOS expression in the human intestinal epithelial cell line, DLD-1, by cyclic AMP
AID  - 10.1136/gut.45.3.367
DP  - 1999 Sep 01
TA  - Gut
PG  - 367--374
VI  - 45
IP  - 3
4099  - http://gut.bmj.com/content/45/3/367.short
4100  - http://gut.bmj.com/content/45/3/367.full
SO  - Gut1999 Sep 01; 45
AB  - BACKGROUND Nitric oxide production by the inducible isoform of nitric oxide synthase (iNOS) is thought to play a role in the pathogenesis of inflammatory bowel disease along with other proinflammatory mediators.AIMS To examine the effects of cAMP, an intracellular mediator of several proinflammatory mediators, on iNOS expression in the human intestinal epithelial cell line, DLD-1.METHODS iNOS activity was assessed by measuring the NO stable oxidative product NO2 −. iNOS protein expression and iNOS mRNA levels were determined by western blotting and northern blotting, respectively.RESULTS iNOS activity, protein, and mRNA were induced by a combination of interleukin 1β (0.5–5 ng/ml), interferon γ (20–200 u/ml), and tumour necrosis factor α (10–100 ng/ml). The cytokine induced NOS activity was potentiated by N6,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate and 8-bromoadenosine 3′:5′-cyclic monophosphate (0.1–1 mM), and the adenylate cyclase activator, forskolin (1–100 μM). This activity was inhibited by the selective iNOS inhibitor, 1400W (0.1–100 μM). These agents increased iNOS protein. The cAMP analogues potentiated iNOS at the transcriptional level as shown by effects of actinomycin D (5 μg/ml) and northern blot analyses; the nuclear factor (NF) κB inhibitor, pyrrolidine dithiocarbamate (10–200 μM), significantly reduced this potentiation. The cAMP potentiated iNOS activity was inhibited by the tyrosine kinase inhibitor, A25 (10–200 μM) and the Janus activated kinase 2 inhibitor, B42 (10–200 μM).CONCLUSIONS Increased intracellular cAMP is a potent stimulus of iNOS expression in combination with cytokines in DLD-1 cells, acting at the transcriptional level and involving NF-κB and the JAK-STAT pathways. Thus, proinflammatory mediators that increase cAMP levels may augment iNOS expression and NO production.8Br-cAMP8-bromoadenosine 3′:5′-cyclic monophosphate8Br-cGMP8-bromoguanosine 3′:5′-cyclic monophosphatecAMPadenosine 3′:5′-cyclic monophosphateCOX-2cyclooxygenase 2Db-cAMPN6,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphateDMEMDulbecco modified Eagle’s mediumDMSOdimethylsulphoxideIBDinflammatory bowel diseaseIBMX3-isobutyl-1-methylxanthineIFNinterferonIL-1βinterleukin 1βiNOSinducible nitric oxide synthaseJAK-2Janus activated kinase 2NF-κBnuclear factor κBNOnitric oxidePBSphosphate buffered salinePDTCpyrrolidine dithiocarbamateTNFtumour necrosis factorCREBcAMP responsive element binding proteinPKAprotein kinase AATFactivating transcription factor