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.


OBJECTIVE
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 NO(2)(-). 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 1beta (0.5-5 ng/ml), interferon gamma (20-200 u/ml), and tumour necrosis factor alpha (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 microM). This activity was inhibited by the selective iNOS inhibitor, 1400W (0.1-100 microM). These agents increased iNOS protein. The cAMP analogues potentiated iNOS at the transcriptional level as shown by effects of actinomycin D (5 microgram/ml) and northern blot analyses; the nuclear factor (NF) kappaB inhibitor, pyrrolidine dithiocarbamate (10-200 microM), significantly reduced this potentiation. The cAMP potentiated iNOS activity was inhibited by the tyrosine kinase inhibitor, A25 (10-200 microM) and the Janus activated kinase 2 inhibitor, B42 (10-200 microM).


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-kappaB and the JAK-STAT pathways. Thus, proinflammatory mediators that increase cAMP levels may augment iNOS expression and NO production.
Cavicchi
M
M
The William Harvey Research Institute, St Bartholomew's and the Royal London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK.
Whittle
B J
BJ
D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D000242:Cyclic AMP / Q000494:pharmacology*; D016207:Cytokines / Q000494:pharmacology*; D004357:Drug Synergism; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D012333:RNA, Messenger / Q000378:metabolism; D020032:Tyrphostins / Q000494:pharmacology
D016207:Cytokines; D012333:RNA, Messenger; D020032:Tyrphostins; D000242:Cyclic AMP; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II