TITLE

Superoxide production and expression of NAD(P)H oxidases by transformed and primary human colonic epithelial cells

AUTHOR(S)
Perner, A.; Andresen, L.; Pedersen, G.; Rask-Madsen, J.
PUB. DATE
February 2003
SOURCE
Gut;Feb2003, Vol. 52 Issue 2, p231
SOURCE TYPE
Academic Journal
DOC. TYPE
Article
ABSTRACT
Background: Superoxide (O[sup -, sub 2]) generation through the activity of reduced nicotinamide dinucleotide (NADH) or reduced nicotinamide dinucleotide phosphate (NADPH) oxidases has been demonstrated in a variety of cell types, but not in human colonic epithelial cells. Aims: To measure O[sup -, sub 2] production and effects of modulators of NAD(P)H oxidase activity and inhibitors of potential O[sup -, sub 2] generating enzymes in cultures of human colonic epithelial cells. Expression of the catalytic subunits of NAD(P)H oxidase, Nox1 and gp91[sub phox] (phox, phagocytic oxidase), and the membrane bound subunit p22[sup phox] was assessed. Methods: The transformed colonic epithelial cell lines (DLD-1, HT-29, and Caco-2) were studied at subconfluence, confluence, and after differentiation. Primary colonic epithelial cells were isolated from mucosal biopsies from the normal human colon. Extracellular O[sup -, sub 2] production was measured by the cytochrome c reduction assay or luminal enhanced luminescence. Nox1, gp91[sup phox], and p22[sup phox] mRNA expression was assessed in colonic epithelial cells and blood neutrophils by reverse transcriptasepolymerase chain reaction. Results: Production rates of O[sup -, sub 2] were higher in subconfluent transformed cells (mean (SEM) 35.8 (4.2) nmol/mg of protein/h) and primary cells (40.4 (5.9)) than in confluent transformed cells (6.0 (0.9); p<0.01). The oxidoreductase inhibitor diphenylene iodonium significantly inhibited O[sup -, sub 2] production whereas NADPH and NADH increased production rates. In contrast, O[sup -, sub 2] was unaffected by phorbol myristate ester, N[sup G]-nitro-L-arginine methyl ester, indomethacin, or allopurinol. Nox1 mRNA was expressed in all colonic epithelial cells whereas gp91[sup phox] was detected only in HT-29 cells and neutrophils, p22[sup phox] was expressed in all cell types. Conclusions: Cultures of transformed and primary epithelial cells from human colon may produce...
ACCESSION #
9704713

 

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