Guinea pig gastric mucosal cells produce abundant superoxide anion through an NADPH oxidase-like system

Background & Aims: Superoxide anion (O-2(-)) plays an important role in gastric pathophysiology. The aims of this study were to identify O-2(-)-producing activity in gastric mucosal cells and to elucidate its possible roles in inflammatory responses of the cells. Methods: The amount of O-2(-) was measured by the reduction of cytochrome c, and O-2(-)-producing cells were visualized by nitroblue tetrazolium reaction, Cytosolic components of the phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were detected by immunoblotting and immunocytochemical analyses with antibodies against p47-phox and p67-phox, Results: Gastric pit cells, but not parietal cells, spontaneously released O-2(-) at 50 nmol . mg protein(-1) . h(-1). NADPH or guanosine 5'-O-(3-thiotriphosphate) increased the release more than threefold, whereas diphenylene iodonium inhibited it. A reconstituted cell-free system showed that both membrane fraction and neutrophil-related cytosolic components were required for the activity, p47-phox and p67-phox were expressed in the cells. Live Helicobacter pylori organisms and their culture supernatants significantly increased the O-2(-) release. Furthermore, H. pylori lipopolysaccharide could enhance the release more effectively than Escherichia coli lipopolysaccharide. The O-2(-)-dependent activation of nuclear factor kappa B occurred in these primed cells. Conclusions: Gastric pit cells may actively regulate inflammatory responses of gastric mucosa through a phagocyte NADPH oxidase-like activity,