Inhibition of NADPH oxidase 5 activity by NO through S‐nitrosylation

The NADPH oxidases (Nox) are a family of multi‐subunit enzymes that produce superoxide and other reactive oxygen species. Nox5 is a calcium‐dependent enzyme that does not depend on accessory subunits for activation and in comparison to other Nox isoforms. Recently we and others have documented the expression of Nox5 in human blood vessel. The goal of current study was to determine whether NO can modulate Nox5 activity and the underlying mechanism. iNOS was a potent inhibitor of basal and both ionomycin and PMA‐stimulated activity. This action of iNOS was reversible with chronic, but not acute exposure to L‐NAME and was due to the diffusion of NO, as iNOS was able to inhibit Nox5 when transfected in separate cells. Nox5 activity was also inhibited by eNOS and PM‐targeted eNOS albeit to a less extent. The ability of NO to inhibit Nox5 activity was due to a direct modification of enzyme activity as Nox5 activity was inhibited in a disrupted/cell free assay replete with excess calcium and NADPH. Co‐expression of iNOS and Nox5 did not increase the levels of ONOO‐ or induce the tyrosine nitration of Nox5. In contrast, there was evidence for the increased nitrosylation of Nox5 using the biotin‐switch assay. Also, de‐nitrosylation enzymes, thioredoxin and GSNO reductase, can reverse the inhibition of Nox5 activity by NO. Collectively, these results suggests that endogenous NO can directly nitrosylate and inhibit the activity of Nox5.