This investigation was to elucidate the basis for augmentation of
nitric-oxide synthesis in neutrophils exposed to hyperbaric
oxygen.
Hyperoxia increases synthesis of reactive species leading to S-nitrosylation of β-actin, which causes temporary inhibition of β(2)
integrin adherence. Impaired β(2)
integrin function and actin S-nitrosylation do not occur in neutrophils from mice lacking type-2
nitric-oxide synthase (iNOS) or when incubated with 1400W, an iNOS inhibitor. Similarly, effects of
hyperoxia were abrogated in cells depleted of
focal adhesion kinase (FAK) by treatment with small inhibitory
RNA and those exposed to a specific FAK inhibitor concurrent with
hyperoxia.
Nitric oxide production doubles within 10 min exposure to
hyperoxia but declines to approximately half-maximum production over an additional 10 min. Elevated
nitric oxide production did not occur after FAK depletion or inhibition, or when filamentous actin formation was inhibited by
cytochalasin D. Intracellular content of iNOS triples over the course of a 45-min exposure to
hyperoxia and iNOS dimers increase in a commensurate fashion. Confocal microscopy and immunoprecipitation demonstrated that co-localization/linkage of FAK, iNOS, and filamentous actin increased within 15 min exposure to
hyperoxia but then decreased below the control level. Using isolated
enzymes in ex vivo preparations an association between iNOS and filamentous actin mediated by FAK could be demonstrated and complex formation was impeded when actin was S-nitrosylated. We conclude that iNOS activity is increased by an FAK-mediated association with actin filaments but peak
nitric oxide production is transient due to actin S-nitrosylation during exposure to
hyperoxia.