Physiological and hypoxic O2 tensions rapidly regulate NO production by stimulated macrophages

Abstract

Nitric oxide (NO) production by inducible NO synthase (iNOS) is dependent on O(2) availability. The duration and degree of hypoxia that limit NO production are poorly defined in cultured cells. To investigate short-term O(2)-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 O(2) tensions (from <1 to 157 Torr) directly to a monolayer of LPS- and IFNgamma-stimulated RAW 264.7 cells while simultaneously measuring NO production via an electrochemical probe. Decreased O(2) availability rapidly (<or=30 s) and reversibly decreased NO production with an apparent K(m)O(2) of 22 (SD 6) Torr (31 microM) and a V(max) of 4.9 (SD 0.4) nmol min(-1) 10(-6) cells. To explore potential mechanisms of decreased NO production during hypoxia, we investigated O(2)-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 O(2) and NO tensions and negligible at low O(2) and NO tensions. These results are consistent with O(2) substrate limitation as a regulatory mechanism during brief hypoxic exposure. The rapid and reversible effects of physiological and pathophysiological O(2) tensions suggest that O(2) tension has the potential to regulate NO production in vivo.