BACKGROUND: Superoxide (O(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(2)(-) production and effects of modulators of NAD(P)H oxidase activity and inhibitors of potential O(2)(-) generating enzymes in cultures of human colonic epithelial cells. Expression of the catalytic subunits of NAD(P)H oxidase, Nox1 and gp91(phox) (phox, phagocytic oxidase), and the membrane bound subunit p22(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(2)(-) production was measured by the cytochrome c reduction assay or luminol enhanced luminescence. Nox1, gp91(phox), and p22(phox) mRNA expression was assessed in colonic epithelial cells and blood neutrophils by reverse transcriptase-polymerase chain reaction. RESULTS: Production rates of O(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(2)(-) production whereas NADPH and NADH increased production rates. In contrast, O(2)(-) was unaffected by phorbol myristate ester, N(G)-nitro-L-arginine methyl ester, indomethacin, or allopurinol. Nox1 mRNA was expressed in all colonic epithelial cells whereas gp91(phox) was detected only in HT-29 cells and neutrophils. p22(phox) was expressed in all cell types. CONCLUSIONS: Cultures of transformed and primary epithelial cells from human colon may produce extracellular O(2)(-) through an NAD(P)H oxidase expressing Nox1 and p22(phox).
BACKGROUND:Superoxide (O(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(2)(-) production and effects of modulators of NAD(P)H oxidase activity and inhibitors of potential O(2)(-) generating enzymes in cultures of human colonic epithelial cells. Expression of the catalytic subunits of NAD(P)H oxidase, Nox1 and gp91(phox) (phox, phagocytic oxidase), and the membrane bound subunit p22(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(2)(-) production was measured by the cytochrome c reduction assay or luminol enhanced luminescence. Nox1, gp91(phox), and p22(phox) mRNA expression was assessed in colonic epithelial cells and blood neutrophils by reverse transcriptase-polymerase chain reaction. RESULTS: Production rates of O(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(2)(-) production whereas NADPH and NADH increased production rates. In contrast, O(2)(-) was unaffected by phorbol myristate ester, N(G)-nitro-L-arginine methyl ester, indomethacin, or allopurinol. Nox1 mRNA was expressed in all colonic epithelial cells whereas gp91(phox) was detected only in HT-29 cells and neutrophils. p22(phox) was expressed in all cell types. CONCLUSIONS: Cultures of transformed and primary epithelial cells from human colon may produce extracellular O(2)(-) through an NAD(P)H oxidase expressing Nox1 and p22(phox).
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