Superoxide Generation by Nox1 in Guinea Pig Gastric Mucosal Cells Involves a Component with p67^phox-Ability

抄録

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.