Physiological and hypoxic O2tensions rapidly regulate NO production by stimulated macrophages

Abstract

Nitric oxide (NO) production by inducible NO synthase (iNOS) is dependent on O2availability. The duration and degree of hypoxia that limit NO production are poorly defined in cultured cells. To investigate short-term O2-mediated regulation of NO production, we used a novel forced convection cell culture system to rapidly (response time of 1.6 s) and accurately (±1 Torr) deliver specific O2tensions (from <1 to 157 Torr) directly to a monolayer of LPS- and IFNγ-stimulated RAW 264.7 cells while simultaneously measuring NO production via an electrochemical probe. Decreased O2availability rapidly (≤30 s) and reversibly decreased NO production with an apparent KmO2of 22 (SD 6) Torr (31 μM) and a Vmaxof 4.9 (SD 0.4) nmol·min−1·10−6cells. To explore potential mechanisms of decreased NO production during hypoxia, we investigated O2-dependent changes in iNOS protein concentration, iNOS dimerization, and cellular NO consumption. iNOS protein concentration was not affected ( P = 0.895). iNOS dimerization appeared to be biphasic [6 Torr ( P = 0.008) and 157 Torr ( P = 0.258) >36 Torr], but it did not predict NO production. NO consumption was minimal at high O2and NO tensions and negligible at low O2and NO tensions. These results are consistent with O2substrate limitation as a regulatory mechanism during brief hypoxic exposure. The rapid and reversible effects of physiological and pathophysiological O2tensions suggest that O2tension has the potential to regulate NO production in vivo.