title: Nitric-oxide synthase-2 linkage to focal adhesion kinase in neutrophils influences enzyme activity and β2 integrin function.
abstract: 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.
authors.affiliation: Department of Emergency Medicine, University of Pennsylvania Medical Center and School of Biomedical Engineering, Philadelphia, Pennsylvania 19104, USA. sthom@mail.med.upenn.edu
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Thom
authors.forename: Veena M
authors.initials: VM
authors.lastname: Bhopale
authors.forename: Tatyana N
authors.initials: TN
authors.lastname: Milovanova
authors.forename: Ming
authors.initials: M
authors.lastname: Yang
authors.forename: Marina
authors.initials: M
authors.lastname: Bogush
authors.forename: Donald G
authors.initials: DG
authors.lastname: Buerk
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry / Q000378:metabolism*; D000199:Actins / Q000737:chemistry; D000818:Animals; D018821:CD18 Antigens / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D019281:Dimerization; D005340:Fibrinogen / Q000378:metabolism; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism*; D005609:Free Radicals; D005786:Gene Expression Regulation; D005982:Glutathione Transferase / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009504:Neutrophils / Q000201:enzymology* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000737:chemistry / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D011817:Rabbits; D026361:Reactive Nitrogen Species; D012995:Solubility
chemical_list: D000199:Actins; D018821:CD18 Antigens; D005609:Free Radicals; D026361:Reactive Nitrogen Species; D005340:Fibrinogen; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D005982:Glutathione Transferase; D051416:Focal Adhesion Protein-Tyrosine Kinases; D010100:Oxygen