Sepsis is now the 11th leading cause of death in the US. Dysregulation of perfusion and tissue oxygenation is a hallmark of sepsis. Hypoxia is an important intracellular signal that alters cell function and gene expression. Tissue macrophages are critical in the initiation and perpetuation of the inflammatory response and are a major source of nitric oxide (NO). NO has been implicated as a mediator of hypotension and tissue injury in sepsis, however nonspecific inhibition of NO and genetic deletion of the inducible nitric oxide synthase (iNOS) fail to improve survival. Recent evidence suggests that hypoxia alters the macrophage inflammatory response, particularly NO synthesis. Hypoxic dysregulation of NO synthesis may contribute to the pathology of sepsis and represents an important link between the microcirculation and the immune system in sepsis. Most prior in vitro studies of NO production have been performed at 148 Torr. Tissue macrophages in vivo, however, exist at 5-80 Torr of oxygen. Shock states further decrease tissue PO2, and thus tissue macrophage PO2. Under carefully controlled culture conditions, NO production has been shown to be dependent on PO2. iNOS protein and to a lesser extent activity is reduced in hypoxic culture. Hypoxia does not, however, inhibit iNOS mRNA expression. Based on these findings and recent studies demonstrating hypoxic inhibition of iNOS dimerization, reductions in culture PO2 decrease NO production by three potential mechanisms: 1) decreased substrate availability, 2) increased protein degradation and 3) impaired enzyme function. The proposed work will determine the mechanisms for inhibition of NO synthesis in low PO2. This study will evaluate the NO pathway in lipopolysaccharide/interferon-gamma stimulated RAW 264.7 cells (a mouse macrophage cell line) cultured under standard (148 Torr) and physiologic oxygen tensions (8, 24, 40, and 80 Torr) and hypoxia (1 Torr).
Aim 1 : On-line NO production in response to rapid changes in culture a) PO2 and b) arginine will be used to determine the role of substrate limitation in the hypoxic inhibition of NO synthesis.
Aim 2 : In order to define the mechanism of decreased iNOS protein accumulation in hypoxia, iNOS mRNA stability and enzyme degradation will be evaluated.
Aim 3 : The effects of PO2 on factors necessary for iNOS dimerization and activity (tetrahydrobiopterin, heme, arginine, and HSP90) will be evaluated. The amount and functional state of iNOS can influence the progression of inflammatory diseases and development of tissue injury. The results of this study will provide definitive information regarding the role of oxygen in controlling the synthesis of NO, information that is essential for developing in vivo therapeutic targets. It will also reinforce the importance of culture conditions on the translation of in vitro NO studies to in vivo hypoxia and inflammation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM064486-03
Application #
6710082
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Somers, Scott D
Project Start
2002-03-01
Project End
2007-02-28
Budget Start
2004-03-01
Budget End
2005-02-28
Support Year
3
Fiscal Year
2004
Total Cost
$213,975
Indirect Cost
Name
University of Pennsylvania
Department
Other Clinical Sciences
Type
Schools of Veterinary Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Robinson, Mary A; Baumgardner, James E; Good, Virginia P et al. (2008) Physiological and hypoxic O2 tensions rapidly regulate NO production by stimulated macrophages. Am J Physiol Cell Physiol 294:C1079-87
Otto, Cynthia M; Markstaller, Klaus; Kajikawa, Osamu et al. (2008) Spatial and temporal heterogeneity of ventilator-associated lung injury after surfactant depletion. J Appl Physiol 104:1485-94
Syring, Rebecca S; Otto, Cynthia M; Spivack, Rebecca E et al. (2007) Maintenance of end-expiratory recruitment with increased respiratory rate after saline-lavage lung injury. J Appl Physiol 102:331-9
Baumgardner, James E; Otto, Cynthia M (2003) In vitro intermittent hypoxia: challenges for creating hypoxia in cell culture. Respir Physiol Neurobiol 136:131-9