Nitric oxide synthase activity, viability and cyclic GMP levels in rat colonic epithelial cells: effect of endotoxin challenge.
The present study has examined whether epithelial cells could be a source of the inducible form of nitric oxide synthase (iNOS) in the colon of rats challenged with E. coli lipopolysaccharide and whether such excessive endogenous nitric oxide (NO) or exogenous NO released from NO donors could affect their viability. Epithelial cells were isolated from rat colon, and cell viability was determined by trypan blue exclusion. The appearance of a calcium-independent iNOS determined by the conversion of radiolabeled L-arginine to citrulline was observed in cells harvested from lipoplysaccharide (3 mg/kg for 4 hr)-treated rats, with a 10-fold increase in total NOS activity compared with control. This was accompanied by a 3-fold decrease in epithelial cell viability. Levels of iNOS and of cellular injury were not significantly affected in rats made neutropenic by the administration of antineutrophil serum. Induction of NOS was also associated with an increase in epithelial cyclic guanylate monophosphate levels. Both iNOS activity and cell injury were inhibited by in vivo pretreatment with dexamethasone (1 mg/kg i.v., for 4 hr) or the NOS inhibitor NG-nitro-L-arginine methyl ester (10 mg/kg s.c.) in a dose that itself reduced viability. The incubation of epithelial cells with the NO donors, nitroprusside, S-nitroso-N-acetyl penicillamine or S-nitroso-N-glutathione (0.1-1 mM) produced concentration-dependent cytotoxicity. These findings indicate that the induction of NOS in colonic epithelial cells is not dependent upon infiltration of neutrophils and is accompanied by a reduction in cellular viability, an effect mimicked in vitro by NO donors.(ABSTRACT TRUNCATED AT 250 WORDS)
Tepperman
B L
BL
Department of Pharmacology, Wellcome Research Laboratories, Beckenham Kent, United Kingdom.
Brown
J F
JF
Korolkiewicz
R
R
Whittle
B J
BJ
D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis*; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D003106:Colon / Q000187:drug effects* / Q000201:enzymology; D006152:Cyclic GMP / Q000096:biosynthesis*; D004790:Enzyme Induction / Q000187:drug effects; D004848:Epithelium / Q000187:drug effects / Q000201:enzymology; D007106:Immune Sera / Q000276:immunology; D008070:Lipopolysaccharides / Q000633:toxicity*; D008297:Male; D019331:NG-Nitroarginine Methyl Ester; D009504:Neutrophils / Q000502:physiology; D019001:Nitric Oxide Synthase; D051381:Rats; D017208:Rats, Wistar
D007106:Immune Sera; D008070:Lipopolysaccharides; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D006152:Cyclic GMP; D019331:NG-Nitroarginine Methyl Ester