Superoxide generation by Nox1 in guinea pig gastric mucosal cells involves a component with p67^phox-ability

Nox1, a homologue of gp91^phox 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 (p67^phox, p47^phox, 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 gp91^phox, 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 (K_m=10.4 μM); 5) substitution of neutrophil cytosol by a set of recombinant cytosolic components (rp67 ^phox, rp47^phox, 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 rp47 ^phox is added. This result suggests that GMC cytosol contains a component with p67^phox-ability, and also Rac, but lacks p47 ^phox. These data indicate that GMC Nox1 requires at least a p67 ^phox counterpart and Rac to acquire NADPH oxidase activity.