The pulmonary vasculature, particularly the endothelium, is known to be a key site responsive to both physiological and pathological changes in 02 delivery and exposure to endogenous NO. Mitochondria are critical loci of cellular respiration, biosynthesis, and metabolism of reactive oxygen species (ROS) and reactive nitrogen species (RNS). We have established that mitochondrial respiratory complexes I (NADH dehydrogenase) and Ill (cytochrome c reductase) are primary locations of irreversible inhibition by RNS and that complex IV (cytochrome c oxidase), in addition to its known cytochrome c:O2 oxidoreductase activity, may also function as an NO oxidase. Accordingly, we hypothesize that under normal physiological circumstances, the NO oxidase activity of complex IV limits formation of RNS by rapidly converting NO to the relatively innocuous nitrite ion. The efficiency of this catabolic reaction, which also consumes 02, depends upon the prevailing NO/O2 ratio. Under hyperoxic conditions, or in pathophysiological circumstances where the electron-transport chain has been compromised, RNS (and ROS) formation is not effectively suppressed. This results in irreversible inhibition of complexes I and III, which in turn exacerbates the situation by increasing the production of the damaging reactive species and ultimately, leads to cell death. In testing this broad hypothesis, we propose to determine: 1) that the NO oxidase activity of complex IV limits nitrosative stress in pulmonary endothelial cells; 2) the molecular sites at which RNS irreversibly inhibit complex I and complex III; 3) the extent to which nitrosative stress may affect the interaction of cytochrome c with complex Ill and/or complex IV; 4) the functional role of RNS-dependent modification of the interaction between cytochrome c and complexes III and/or IV in promoting the loss of cytochrome c from the intermembrane space during proapoptotic stimulation of cultured pulmonary endothelial cells.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL061411-07
Application #
7035875
Study Section
Respiratory Physiology Study Section (RESP)
Program Officer
Denholm, Elizabeth M
Project Start
2000-04-01
Project End
2008-03-31
Budget Start
2006-04-01
Budget End
2007-03-31
Support Year
7
Fiscal Year
2006
Total Cost
$253,767
Indirect Cost
Name
University of Pittsburgh
Department
Public Health & Prev Medicine
Type
Schools of Public Health
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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Pearce, Linda L; Lopez Manzano, Elisenda; Martinez-Bosch, Sandra et al. (2008) Antagonism of nitric oxide toward the inhibition of cytochrome c oxidase by carbon monoxide and cyanide. Chem Res Toxicol 21:2073-81
Basova, Liana V; Kurnikov, Igor V; Wang, Lei et al. (2007) Cardiolipin switch in mitochondria: shutting off the reduction of cytochrome c and turning on the peroxidase activity. Biochemistry 46:3423-34
Pearce, Linda L; Kanai, Anthony J; Epperly, Michael W et al. (2005) Nitrosative stress results in irreversible inhibition of purified mitochondrial complexes I and III without modification of cofactors. Nitric Oxide 13:254-63
Chen, Tracy; Pearce, Linda L; Peterson, Jim et al. (2005) Glutathione depletion renders rat hepatocytes sensitive to nitric oxide donor-mediated toxicity. Hepatology 42:598-607
Kanai, Anthony; Epperly, Michael; Pearce, Linda et al. (2004) Differing roles of mitochondrial nitric oxide synthase in cardiomyocytes and urothelial cells. Am J Physiol Heart Circ Physiol 286:H13-21
Peterson, Jim; Kanai, Anthony J; Pearce, Linda L (2004) A mitochondrial role for catabolism of nitric oxide in cardiomyocytes not involving oxymyoglobin. Am J Physiol Heart Circ Physiol 286:H55-8
Fago, Angela; Crumbliss, Alvin L; Peterson, Jim et al. (2003) The case of the missing NO-hemoglobin: spectral changes suggestive of heme redox reactions reflect changes in NO-heme geometry. Proc Natl Acad Sci U S A 100:12087-92
Peterson, Jim; Bominaar, Emile L (2002) Resolving molecular electronic spectra using magnetic linear dichroism. Anal Chem 74:527A-533A

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