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

Open Access
Publisher Website
Google Scholar
Abstract
Background: Superoxide (O2−) 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 O2− production and effects of modulators of NAD(P)H oxidase activity and inhibitors of potential O2− generating enzymes in cultures of human colonic epithelial cells. Expression of the catalytic subunits of NAD(P)H oxidase, Nox1 and gp91phox (phox, phagocytic oxidase), and the membrane bound subunit p22phox 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 O2− production was measured by the cytochrome c reduction assay or luminol enhanced luminescence. Nox1, gp91phox, and p22phox mRNA expression was assessed in colonic epithelial cells and blood neutrophils by reverse transcriptase-polymerase chain reaction. Results: Production rates of O2− 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); p2− production whereas NADPH and NADH increased production rates. In contrast, O2− was unaffected by phorbol myristate ester, NG-nitro-l-arginine methyl ester, indomethacin, or allopurinol. Nox1 mRNA was expressed in all colonic epithelial cells whereas gp91phox was detected only in HT-29 cells and neutrophils. p22phox was expressed in all cell types. Conclusions: Cultures of transformed and primary epithelial cells from human colon may produce extracellular O2− through an NAD(P)H oxidase expressing Nox1 and p22phox.