Am J Physiol Cell Physiol AJP: Lung Cellular and Molecular Physiology
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Am J Physiol Cell Physiol 294: C1079-C1087, 2008. First published February 13, 2008; doi:10.1152/ajpcell.00469.2007
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CELLULAR METABOLISM

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

Mary A. Robinson,1,2 James E. Baumgardner,3,4 Virginia P. Good,2 and Cynthia M. Otto1,2

1Department of Clinical Studies-Philadelphia, School of Veterinary Medicine, 2Center for Sleep and Respiratory Neurobiology, and 3Department of Anesthesiology and Critical Care, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; and 4Oscillogy, Folsom, Pennsylvania

Submitted 6 October 2007 ; accepted in final form 7 February 2008

Nitric oxide (NO) production by inducible NO synthase (iNOS) is dependent on O2 availability. 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 O2 tensions (from <1 to 157 Torr) directly to a monolayer of LPS- and IFN{gamma}-stimulated RAW 264.7 cells while simultaneously measuring NO production via an electrochemical probe. Decreased O2 availability rapidly (≤30 s) 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–1·10–6 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 it 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 physiological and pathophysiological O2 tensions suggest that O2 tension has the potential to regulate NO production in vivo.

inducible nitric oxide synthase; substrate limitation; nitric oxide consumption


Address for reprint requests and other correspondence: C. M. Otto, Dept. of Clinical Studies-Philadelphia, School of Veterinary Medicine, Univ. of Pennsylvania, Philadelphia, PA 19104 (e-mail: cmotto{at}vet.upenn.edu)






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