INDUCIBLE NITRIC-OXIDE SYNTHASE ACTIVITY AND EXPRESSION IN A HUMAN COLONIC EPITHELIAL-CELL LINE, HT-29

被引:76
作者
KOLIOS, G
BROWN, Z
ROBSON, RL
ROBERTSON, DAF
WESTWICK, J
机构
[1] UNIV BATH, DEPT PHARM, BATH BA2 7AY, AVON, ENGLAND
[2] ROYAL UNITED HOSP, DEPT GASTROENTEROL, BATH BA1 3NG, AVON, ENGLAND
基金
英国惠康基金;
关键词
NITRIC OXIDE; INDUCIBLE NITRIC OXIDE SYNTHASE; HT-29; CELLS; COLONIC EPITHELIAL CELLS; ULCERATIVE COLITIS; INFLAMMATORY BOWEL DISEASE;
D O I
10.1111/j.1476-5381.1995.tb15938.x
中图分类号
R9 [药学];
学科分类号
1007 ;
摘要
1 We have determined which cytokines regulate the expression of human inducible nitric oxide synthase (iNOS) mRNA and nitrite generation in the human colonic epithelial cell line HT-29. 2 Growth arrested cell cultures were stimulated with the human recombinant cytokines interleukin-1 alpha (IL-1 alpha), tumour necrosisfactor-alpha: (TNF-alpha), interferon gamma (IFN-gamma) or vehicle added alone or in combination. Human iNOS mRNA was determined by Northern blot analysis and nitrite generation by the use of a fluorometric assay. 3 Unstimulated cells produced a small time-dependent increase in nitrite generation of 50 +/- 4, 75 +/- 8, and 103 +/- 8 nM per 10(6) cells at 24 h, 48 h, and 72 h respectively. This nitrite generation was unaffected by cycloheximide (5 mu g ml(-1)) pretreatment and iNOS mRNA was not detected. 4 None of cytokines alone induced either iNOS mRNA expression or an increase in nitrite generation. The combination of IL-1 alpha/IFN-gamma produced a highly significant (P<0.001) 4 fold increase in nitrite production at 48 h, compared to basal values, while no other pair of cytokines was effective. 5 Time course studies with IL-1 alpha/IFN-gamma combination revealed significant (P<0.001) increases in nitrite at 24 h (153 +/- 7), 48 h (306 +/- 24), and 72 h (384 +/- 15) compared to basal values of 50 +/- 4, 75 +/- 8, and 103 +/- 8 nM per 10(6) cells respectively. 6 Studies with IL-1 alpha/IFN-gamma combination demonstrated a time dependent expression of iNOS mRNA, first observed at 6 h, peaked at 24 h and was undetectable by 72 h. IL-1 alpha (0.3-10 ng ml(-1)) and IFN-gamma (10-300 u ml(-1)) in combination induced a concentration-dependent expression of iNOS mRNA at 24 h. 7 Pretreatment with cycloheximide before IL-1 alpha/IFN-gamma stimulation reduced nitrite levels to basal values. These data suggest that the IL-1 alpha/IFN-gamma-induced nitrite production by HT-29 cells is dependent on de novo protein synthesis, probably the iNOS enzyme. 8 The addition of TNF-alpha produced a significant (P<0.001) 3 fold increase of IL-1 alpha/IFN-gamma-induced nitrite generation. In marked contrast the presence of TNF-alpha had no effect on IL-1 alpha/IFN-gamma-induced iNOS mRNA expression by HT-29 cells. These findings suggest that the up-regulation by TNF-alpha of IL-1 alpha/IFN-gamma-induced nitrite generation is at the post-transcriptional level. 9 These data suggest that pro-inflammatory cytokines induce NO production in colonic epithelial cells probably due to the induction of iNOS and these cells may be a major source of NO generation in inflammatory bowel disease.
引用
收藏
页码:2866 / 2872
页数:7
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