Superoxide Generation by Nox1 in Guinea Pig Gastric Mucosal Cells Involves a Component with p67phox-Ability

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  • Yoshida Lucia Satiko
    Department of Infectious Diseases, National Research Institute for Child Health and Development, Japan
  • Nishida Satoshi
    Department of Infectious Diseases, National Research Institute for Child Health and Development, Japan
  • Shimoyama Takashi
    Department of Infectious Diseases, National Research Institute for Child Health and Development, Japan
  • Kawahara Tsukasa
    Department of Nutrition, School of Medicine, University of Tokushima
  • Kondo-Teshima Shigetada
    Department of Nutrition, School of Medicine, University of Tokushima
  • Rokutan Kazuhito
    Department of Nutrition, School of Medicine, University of Tokushima
  • Kobayashi Toshihiro
    Department of Anatomy and Cell Biology, Kochi Medical School
  • Tsunawaki Shohko
    Department of Infectious Diseases, National Research Institute for Child Health and Development, Japan

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Abstract

Nox1, a homologue of gp91phox subunit of the phagocyte NADPH oxidase, is responsible for spontaneous superoxide (O2−) generation in guinea pig gastric mucosal cells (GMC), but involvement of regulatory components (p67phox, p47phox, and Rac) which are essential in phagocytes remains unknown. Here, we aimed to figure out how Nox1 of GMC achieves an active oxidase status. GMC in primary culture show low O2− generation but acquire a 9-fold higher activity when cultured with Helicobacter pylori lipopolysaccharide (LPS), in correlation with a far increased Nox1 expression. Investigation into the O2−-generating ability of LPS-induced Nox1 in cell-free reconstitution assays showed that: 1) Nox1 is unable to generate O2− per se; 2) the combination of Nox1 with GMC cytosol is insufficient for a significant O2− generation; 3) the combination with neutrophil cytosol enables Nox1 to act like gp91phox, i.e., to produce O2− appreciably in response to myristate stimulation; 4) Nox1 prefers NADPH to NADH under the in vitro assay with neutrophil cytosol plus myristate (Km=10.4 μM); 5) substitution of neutrophil cytosol by a set of recombinant cytosolic components (rp67phox, rp47phox, Rac2) is, however, ineffective and still requires GMC cytosol. Thus, Nox1 probably requires an additional cytosolic factor(s). In contrast, GMC cytosol enables cytochrome b558 to generate plenty of O2−, on condition that rp47phox is added. This result suggests that GMC cytosol contains a component with p67phox-ability, and also Rac, but lacks p47phox. These data indicate that GMC Nox1 requires at least a p67phox counterpart and Rac to acquire NADPH oxidase activity.

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