Inhibition of inducible nitric oxide synthase in the human intestinal epithelial cell line, DLD-1, by the inducers of heme oxygenase 1, bismuth salts, heme, and nitric oxide donors.
BACKGROUND
The inducible isoform of nitric oxide synthase (iNOS) may be involved in the mucosal injury associated with inflammatory bowel disease (IBD). In contrast with iNOS, the inducible heme oxygenase 1 (HO-1) is considered to act as a protective antioxidant system.


OBJECTIVE
To evaluate the effects of the known HO-1 inducers, cadmium and bismuth salts, heme, and nitric oxide (NO) donors, on iNOS activity, and expression in the human intestinal epithelial cell line DLD-1.


METHODS
iNOS activity was assessed by the Griess reaction and the radiochemical L-arginine conversion assay. iNOS mRNA and iNOS protein expression were determined by northern and western blotting, respectively.


RESULTS
Cytokine exposure led to induction of iNOS activity, iNOS mRNA, and iNOS protein expression. Preincubation of DLD-1 cells with heme (1-50 microM) inhibited cytokine induced iNOS activity in a concentration dependent manner. This inhibitory effect was abolished by the HO-1 specific inhibitor tin protoporphyrin. Preincubation with NO donors sodium nitroprusside (SNP 1-1000 microM) or S-nitroso-acetyl-penicillamine (SNAP 1-1000 microM), or with the heavy metals cadmium chloride (10-40 microM), bismuth citrate, or ranitidine bismuth citrate (10-3000 microM) inhibited iNOS activity in a concentration dependent manner. Moreover, SNP and heme abolished cytokine induced iNOS protein as well as iNOS mRNA expression, whereas cadmium chloride did not modify iNOS protein expression.


CONCLUSIONS
Heme, the heavy metals cadmium and bismuth, as well as NO donors, are potent inhibitors of cytokine induced iNOS activity. Heme and NO donors act at the transcriptional level inhibiting iNOS mRNA expression. Such findings suggest the potential for interplay between the iNOS and HO-1 systems, which may modulate the progress of IBD.
Cavicchi
M
M
William Harvey Research Institute, St Bartholomew's and the Royal London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK.
Gibbs
L
L
Whittle
B J
BJ
D001729:Bismuth / Q000378:metabolism; D015152:Blotting, Northern; D015153:Blotting, Western; D019256:Cadmium Chloride / Q000494:pharmacology; D002452:Cell Count; D002470:Cell Survival; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism; D006418:Heme / Q000494:pharmacology; D006419:Heme Oxygenase (Decyclizing) / Q000096:biosynthesis*; D051547:Heme Oxygenase-1; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000166:cytology; D008565:Membrane Proteins; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000502:physiology; D052247:Nitric Oxide Synthase Type II; D009599:Nitroprusside / Q000494:pharmacology
D016207:Cytokines; D008565:Membrane Proteins; D009599:Nitroprusside; D006418:Heme; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496541:HMOX1 protein, human; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; D019256:Cadmium Chloride; D001729:Bismuth