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

Abstract

Nox1, a homologue of gp91(phox) subunit of the phagocyte NADPH oxidase, is responsible for spontaneous superoxide (O(2)(-)) 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 O(2)(-) 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 O(2)(-)-generating ability of LPS-induced Nox1 in cell-free reconstitution assays showed that: 1) Nox1 is unable to generate O(2)(-) per se; 2) the combination of Nox1 with GMC cytosol is insufficient for a significant O(2)(-) generation; 3) the combination with neutrophil cytosol enables Nox1 to act like gp91(phox), i.e., to produce O(2)(-) 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 microM); 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 b(558) to generate plenty of O(2)(-), 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.