Potentiation of cytokine induced iNOS expression in the human intestinal epithelial cell line, DLD-1, by cyclic AMP

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 1 beta (0.5-5 ng/ml), interferon gamma (20-200 u/ml), and tumour necrosis factor cr (10-100 ng/ml). The cytokine induced NOS activity was potentiated by N-6,2'-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 mu M). This activity was inhibited by the selective iNOS inhibitor, 1400W (0.1-100 mu M) These agents increased iNOS protein. The cAMP analogues potentiated iNOS at the transcriptional level as shown by effects of actinomycin D (5 mu g/ml) and northern blot analyses; the nuclear factor (NF) KB inhibitor, pyrrolidine dithiocarbamate (10-200 mu M), significantly reduced this potentiation. The cAMP potentiated iNOS activity was inhibited by the tyrosine kinase inhibitor, A25 (10-200 mu M) and the Janus activated kinase 2 inhibitor, B42 (10-200 mu M). 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-KB and the JAK-STAT pathways. Thus, proinflammatory mediators that increase cAMP levels may augment iNOS expression and NO production.