Submitted on October 6, 2007
Accepted on February 7, 2008
Physiologic and Hypoxic O2 Tensions Rapidly Regulate NO production by Stimulated Macrophages
Mary A Robinson1, James E. Baumgardner2, Virginia P. Good3, and Cynthia M. Otto1*
1 Department of Clinical Studies-Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania, United States; Center for Sleep and Respiratory Neurobiology, University of Pennsylvania, Philadelphia, Pennsylvania, United States
2 Department of Anesthesiology and Critical Care, University of Pennsylvania, Philadelphia, Pennsylvania, United States
3 Center for Sleep and Respiratory Neurobiology, University of Pennsylvania, Philadelphia, Pennsylvania, United States
* To whom correspondence should be addressed. E-mail: cmotto{at}vet.upenn.edu.
NO production by inducible nitric oxide synthase (iNOS) is dependent on O2 availability. The duration and degree of hypoxia which 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 = 1.6 seconds) and accurately (± 1 Torr) deliver specific O2 tensions (from < 1 to 157 Torr) directly to a monolayer of LPS and IFN
stimulated RAW 264.7 cells while simultaneously measuring NO production via an electrochemical probe. Decreased O2 availability rapidly (
30 seconds) and reversibly decreased NO production with an apparent KmO2 of 22 (SD 6) Torr (31 µM) and a Vmax of 4.9 (SD 0.4) nmol/min·106 cells. 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 did not predict NO production. NO consumption was minimal at high O2 and NO tensions and negligible at low O2 and NO tensions. These results are consistent with O2 substrate limitation as a regulatory mechanism during brief hypoxic exposure. The rapid and reversible effects of physiologic and pathophysiologic O2 tensions suggest that O2 tension has the potential to regulate NO production in vivo.
