Guinea pig gastric mucosal cells produce abundant superoxide anion through an NADPH oxidase-like system  Shigetada Teshima, Kazuhito Rokutan, Takeshi Nikawa, Kyoichi Kishi  Gastroenterology  Volume 115, Issue 5, Pages 1186-1196 (November 1998) DOI: 10.1016/S0016-5085(98)70090-3 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Characterization of O2−-generating system in cultured gastric mucosal cells. Cells were cultured in 24-well culture plates for 2 days in RPMI 1640 containing 10% FCS. After washing the confluent cells with HBSS, they were incubated for 1 hour at 37°C in HBSS containing 80 μmol/L cytochrome c in the absence or presence of the indicated concentration of a possible substrate (xanthine, NADP+, or NADPH), stimulator (PMA or GTPγ-S), or inhibitor (allopurinol, azide, DPI, L-NAME, or L-NMMA). The amount of O2− was determined by measuring the SOD-inhibitable reduction of cytochrome c as described in Materials and Methods. The results were expressed as nmol O2− · mg protein−1 · h−1. Values are expressed as means ± SD (n = 12). Gastroenterology 1998 115, 1186-1196DOI: (10.1016/S0016-5085(98)70090-3) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 O2− production in a reconstitution cell-free system. The cytosolic and membrane fractions were prepared from guinea pig neutrophils and gastric mucosal cells as described in Materials and Methods. Ten micrograms of protein of neutrophil (PMN) membrane, 60 μg protein of gastric mucosal cell (GMC) membrane, and 80 μg protein of neutrophil or gastric mucosal cell cytosol were used. The membrane, cytosol, or membrane plus cytosol was incubated in the reaction buffer for 3 minutes at 37°C, as shown in A–H. NADPH (0.2 mmol/L) was added at the time point as indicated by arrows. The rates of SOD-inhibitable reduction of cytochrome c were monitored. SOD (50 μg/mL) was added as indicated by arrows in A and E. The results shown are representative of those obtained in 3 experiments. Gastroenterology 1998 115, 1186-1196DOI: (10.1016/S0016-5085(98)70090-3) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Expression of p47-phox and p67-phox proteins in gastric mucosal cells. Whole-cell proteins were extracted from human peripheral blood neutrophils (lanes 1 and 4), guinea pig peritoneal neutrophils (lanes 2 and 5), or guinea pig cultured gastric mucosal cells (lanes 3 and 6) as described previously.18 An equal amount of cell protein (40 μg of protein per lane) was subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. Immunoblot analysis with a polyclonal antibody against p47-phox or p67-phox was performed as described in Materials and Methods. p47-phox or p67-phox is indicated by an arrow. Molecular mass standards (MWSTD; in kilodaltons) are shown on the left. Gastroenterology 1998 115, 1186-1196DOI: (10.1016/S0016-5085(98)70090-3) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Characterization of gastric cell preparation and identification of O2−-producing cells. Gastric mucosal cells were cultured in a chamber slide for 2 days. Cells were fixed with 3.5% formaldehyde in PBS and stained (A) by galactose oxidase–Schiff reaction, (B) by paradoxical concanavalin A reaction, or (C) by immunoreaction with monoclonal antibodies against β subunit of H+,K+-adenosine triphosphatase and (D) against vimentin or with polyclonal antibodies against (F) p47-phox and (G) p67-phox. (E) Gastric mucosal fibroblasts, prepared as described in Materials and Methods, were immunostained with antivimentin antibody. After cells, cultured in a chamber slide, were incubated for 2 hours with 100 μmol/L NBT in HBSS in the (H) absence or (I) presence of 40 μg/mL SOD, they were fixed with 3.5% formaldehyde in PBS. Blue formazan particles precipitated on cell surface are shown in H. (A) The majority of the cell preparation was composed of pit cells that contained large red granules positive for galactose oxidase–Schiff reaction. Parietal cells are shown by arrows in A, C, D, F, G, and H, and a cell containing granules positive for paradoxical concanavalin A reaction (mucous neck cell) is shown by an open arrow. The composition of cell preparations did not change during the first 4 days of culture. Original magnification 100×. Gastroenterology 1998 115, 1186-1196DOI: (10.1016/S0016-5085(98)70090-3) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of H. pylori LPS or E. coli LPS on O2− release from gastric mucosal cells. (A and B) Cells were treated with H. pylori LPS (●) or E. coli LPS (■) at different concentrations for 18 hours in RPMI 1640 containing 10% FCS. (C and D) Cells were untreated (2) or treated with control vehicle (○), 0.5 ng/mL of H. pylori LPS (●), or 10 ng/mL of E. coli LPS (■) for the indicated hours in RPMI 1640 containing 10% FCS. FCS was removed by washing the cells with HBSS, and they were incubated in HBSS containing 80 μmol/L cytochrome c. The amounts of O2− production for 1 hour were measured, as described in Figure 1. Values are expressed as means ± SD (n = 12). Gastroenterology 1998 115, 1186-1196DOI: (10.1016/S0016-5085(98)70090-3) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Activation of NF-κB in H. pylori LPS– or E. coli LPS–primed cells. Gastric mucosal cells were untreated (lanes 1 and 9) or treated with 0.5 ng/mL H. pylori LPS (lanes 2–8 and 16–18) or 10 ng/mL E. coli LPS (lanes 10–15) for the indicated hours in RPMI 1640 containing 10% FCS. After washing with Eagle minimum essential medium, the cells were further incubated in Eagle minimum essential medium without (lanes 2–6, 10–14, and 16–18) or with (lanes 7, 8, and 15) 50 μg/mL SOD and 3000 U/mL catalase (Cat.). After incubating for 15 minutes, nuclear extracts were prepared, and gel mobility shift assay was performed with a [32P]κB oligonucleotide, as described previously.11,19 Lane 16, marked nonself, contained 100-fold molar excess of unlabeled AP-1 oligonucleotide; lane 17, marked self, contained 100-fold molar excess of unlabeled κB oligonucleotide; and lane 18, marked mutant, contained 100-fold molar excess of unlabeled mutant κB oligonucleotide. Interaction shown by arrows was specific κB-binding activity. Results were similar in 3 separate experiments. Gastroenterology 1998 115, 1186-1196DOI: (10.1016/S0016-5085(98)70090-3) Copyright © 1998 American Gastroenterological Association Terms and Conditions