King's College London

Intracellular O₂ is a key regulator of NO signaling, yet most in vitro studies are conducted in atmospheric O₂ levels, hyperoxic with respect to the physiologic milieu. We investigated NO signaling in endothelial cells cultured in physiologic (5%) O₂ and stimulated with histamine or shear stress. Culture of cells in 5% O₂ (>5 d) decreased histamine-but not shear stress–stimulated endothelial (e)NOS activity. Unlike cells adapted to a hypoxic environment (1% O₂), those cultured in 5% O₂ still mobilized sufficient Ca²⁺ to activate AMPK. Enhanced expression and membrane targeting of PP2A-C was observed in 5% O₂, resulting in greater interaction with eNOS in response to histamine. Moreover, increased dephosphorylation of eNOS in 5% O₂ was Ca²⁺-sensitive and reversed by okadaic acid or PP2A-C siRNA. The present findings establish that Ca²⁺ mobilization stimulates both NO synthesis and PP2A-mediated eNOS dephosphorylation, thus constituting a novel negative feedback mechanism regulating eNOS activity not present in response to shear stress. This, coupled with enhanced NO bioavailability, underpins differences in NO signaling induced by inflammatory and physiologic stimuli that are apparent only in physiologic O₂ levels. Furthermore, an explicit delineation between physiologic normoxia and genuine hypoxia is defined here, with implications for our understanding of pathophysio-logical hypoxia.