Tissue Processing of Nitrite in Hypoxia
2008; Elsevier BV; Volume: 283; Issue: 49 Linguagem: Inglês
10.1074/jbc.m806654200
ISSN1083-351X
AutoresMartin Feelisch, Bernadette Fernandez, Nathan S. Bryan, Maria F. Garcia-Saura, Selena Bauer, David R. Whitlock, Peter C. Ford, David R. Janero, Juan Rodríguez, Houman Ashrafian,
Tópico(s)Hemoglobin structure and function
ResumoAlthough nitrite ( NO2-) and nitrate ( NO3-) have been considered traditionally inert byproducts of nitric oxide (NO) metabolism, recent studies indicate that NO2- represents an important source of NO for processes ranging from angiogenesis through hypoxic vasodilation to ischemic organ protection. Despite intense investigation, the mechanisms through which NO2- exerts its physiological/pharmacological effects remain incompletely understood. We sought to systematically investigate the fate of NO2- in hypoxia from cellular uptake in vitro to tissue utilization in vivo using the Wistar rat as a mammalian model. We find that most tissues (except erythrocytes) produce free NO at rates that are maximal under hypoxia and that correlate robustly with each tissue's capacity for mitochondrial oxygen consumption. By comparing the kinetics of NO release before and after ferricyanide addition in tissue homogenates to mathematical models of NO2- reduction/NO scavenging, we show that the amount of nitrosylated products formed greatly exceeds what can be accounted for by NO trapping. This difference suggests that such products are formed directly from NO2-, without passing through the intermediacy of free NO. Inhibitor and subcellular fractionation studies indicate that NO2- reductase activity involves multiple redundant enzymatic systems (i.e. heme, iron-sulfur cluster, and molybdenum-based reductases) distributed throughout different cellular compartments and acting in concert to elicit NO signaling. These observations hint at conserved roles for the NO2--NO pool in cellular processes such as oxygen-sensing and oxygen-dependent modulation of intermediary metabolism. Although nitrite ( NO2-) and nitrate ( NO3-) have been considered traditionally inert byproducts of nitric oxide (NO) metabolism, recent studies indicate that NO2- represents an important source of NO for processes ranging from angiogenesis through hypoxic vasodilation to ischemic organ protection. Despite intense investigation, the mechanisms through which NO2- exerts its physiological/pharmacological effects remain incompletely understood. We sought to systematically investigate the fate of NO2- in hypoxia from cellular uptake in vitro to tissue utilization in vivo using the Wistar rat as a mammalian model. We find that most tissues (except erythrocytes) produce free NO at rates that are maximal under hypoxia and that correlate robustly with each tissue's capacity for mitochondrial oxygen consumption. By comparing the kinetics of NO release before and after ferricyanide addition in tissue homogenates to mathematical models of NO2- reduction/NO scavenging, we show that the amount of nitrosylated products formed greatly exceeds what can be accounted for by NO trapping. This difference suggests that such products are formed directly from NO2-, without passing through the intermediacy of free NO. Inhibitor and subcellular fractionation studies indicate that NO2- reductase activity involves multiple redundant enzymatic systems (i.e. heme, iron-sulfur cluster, and molybdenum-based reductases) distributed throughout different cellular compartments and acting in concert to elicit NO signaling. These observations hint at conserved roles for the NO2--NO pool in cellular processes such as oxygen-sensing and oxygen-dependent modulation of intermediary metabolism. Nitric oxide (NO) 3The abbreviations used are: NO, nitric oxide; ALDH2 (mtALDH2), mitochondrial aldehyde dehydrogenase 2; DPI, diphenyleneiodonium; EthOx, ethoxyresorufin; Hb, hemoglobin; i.p., intraperitoneal; NaNO2, sodium nitrite; NO2-, nitrite; NO3-, nitrate; RBC, red blood cells; RNNO, N-nitrosamines; RSNO, S-nitrosothiols; SNO-Hb, S-nitrosohemoglobin; XOR, xanthine oxidoreductase; PBS, phosphate-buffered saline. 3The abbreviations used are: NO, nitric oxide; ALDH2 (mtALDH2), mitochondrial aldehyde dehydrogenase 2; DPI, diphenyleneiodonium; EthOx, ethoxyresorufin; Hb, hemoglobin; i.p., intraperitoneal; NaNO2, sodium nitrite; NO2-, nitrite; NO3-, nitrate; RBC, red blood cells; RNNO, N-nitrosamines; RSNO, S-nitrosothiols; SNO-Hb, S-nitrosohemoglobin; XOR, xanthine oxidoreductase; PBS, phosphate-buffered saline. is the archetypal effector of redox-regulated signal transduction throughout phylogeny, from microorganisms to plants and animals (1Schmidt H.H. Walter U. Cell. 1994; 78: 919-925Abstract Full Text PDF PubMed Scopus (1495) Google Scholar). The conserved influences of NO extend from the regulation of basic cellular processes such as intermediary metabolism (2Erusalimsky J.D. Moncada S. Arterioscler. Thromb. Vasc. Biol. 2007; 27: 2524-2531Crossref PubMed Scopus (264) Google Scholar), cellular proliferation (3Lancaster Jr., J.R. Xie K. Cancer Res. 2006; 66: 6459-6462Crossref PubMed Scopus (90) Google Scholar), and apoptosis (4Li C.Q. Wogan G.N. Cancer Lett. 2005; 226: 1-15Crossref PubMed Scopus (189) Google Scholar) to systemic processes such as hypoxic vasoregulation (5Edmunds N.J. Moncada S. Marshall J.M. J. Physiol. 2003; 546: 521-527Crossref PubMed Scopus (40) Google Scholar). Mammalian NO production has been attributed to the enzymatic activity of NO synthases, nitrate ( NO3-)/nitrite ( NO2-) reductases and non-enzymatic NO2- reduction (6Lundberg J.O. Weitzberg E. Gladwin M.T. Nat. Rev. Drug Discov. 2008; 7: 156-167Crossref PubMed Scopus (1776) Google Scholar). The NO produced is believed to act directly as a signaling molecule by binding to the heme of soluble guanylyl cyclase or nitrosating peptide/protein cysteine residues (7Stamler J.S. Lamas S. Fang F.C. Cell. 2001; 106: 675-683Abstract Full Text Full Text PDF PubMed Scopus (1113) Google Scholar). More recently, it has become apparent that NO2-, previously considered an inert byproduct of NO metabolism present in plasma (50–500 nm) and tissues (0.5–25 μm), is, under some conditions, also a source of NO/nitrosothiol signaling (6Lundberg J.O. Weitzberg E. Gladwin M.T. Nat. Rev. Drug Discov. 2008; 7: 156-167Crossref PubMed Scopus (1776) Google Scholar, 8Bryan N.S. Fernandez B.O. Bauer S.M. Garcia-Saura M.F. Milsom A.B. Rassaf T. Maloney R.E. Bharti A. Rodriguez J. Feelisch M. Nat. Chem. Biol. 2005; 1: 290-297Crossref PubMed Scopus (408) Google Scholar). 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Our findings reveal that multiple heme, iron-sulfur cluster, and molybdenum-based reductases, distributed among distinct subcellular compartments, act in a cooperative manner to convert NO2- to NO and related signaling products. The correlation between NO2- reductase activity and oxidative phosphorylation capacity across organs suggests that NO2- serves a cell regulatory role (e.g. the modulation of intermediary metabolism) beyond its capacity to elicit hypoxic vasodilatation.EXPERIMENTAL PROCEDURESAnimals and ReagentsMale Wistar rats (250–350 g) from Harlan (Indianapolis, IN) were allowed food (2018 rodent diet, Harlan) and water ad libitum and were maintained on a regular 12 h light/12 h dark cycle with at least 10 days of local vivarium acclimatization prior to experimental use. All protocols were approved by the Institutional Animal Care and Use Committee of the Boston University School of Medicine. All gasses and chemicals were of the highest available grade (see supplemental information for details).Biological Sample Harvest and PreparationHeparinized (0.07 units/g body weight, intraperitoneal) rats were anesthetized with diethylether and euthanized by cervical dislocation. Whole blood (3–5 ml) was collected from the inferior vena cava into EDTA-containing tubes (1.8 mg/mL) and processed as detailed (23Bryan N.S. Rassaf T. Maloney R.E. Rodriguez C.M. Saijo F. Rodriguez J.R. Feelisch M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4308-4313Crossref PubMed Scopus (356) Google Scholar, 24Feelisch M. Rassaf T. Mnaimneh S. Singh N. Bryan N.S. Jourd'Heuil D. Kelm M. FASEB J. 2002; 16: 1775-1785Crossref PubMed Scopus (341) Google Scholar) to obtain RBC and plasma. Isolated, packed RBC (1 blood vol-equivalent) were immediately lysed hypotonically with 3 vol of water. After thoracotomy, a catheter was inserted into the infrarenal portion of the abdominal aorta, and organs were flushed free of blood by retrograde in situ perfusion at a rate of 10 ml/min with air-equilibrated PBS before extirpation. NO2- reductase activity was assessed by measuring the NO generated immediately after addition of 100 μl of a 20 mm NaNO2 stock solution to tissue samples (200 μm final [ NO2-]) using gas-phase chemiluminescence.Liver Homogenate PreparationHepatic tissue was acquired by standard techniques (see supplemental information). Liver homogenate was diluted 180-fold in LHM and preincubated at 37 °C for 4 min with either PBS (vehicle control) or a variety of inhibitors (supplemental Table S1) in a light-protected reaction vessel continuously purged with nitrogen. For analysis of inhibitor effects, the amount of NO generated within the first 4 min of incubation in the presence of test compound was compared with a parallel sample treated only with the respective inhibitor vehicle.Nitrite Reductase Activity in Subcellular FractionsHepatic subcellular fractions were obtained by differential centrifugation (25Rickwood D. Preparative Centrifugation: A Practical Approach. Oxford University Press, New York1993Google Scholar). Low-speed centrifugation (1,000 × g, 10 min) of whole-liver homogenate (S0) was used to remove undisrupted tissue, nuclei, and particulate debris into the resulting pellet (P1), which was discarded. The supernatant (S1) was recovered and re-centrifuged (10,000 × g, 10 min) to obtain a mitochondrial fraction (P2). The corresponding supernatant (S2) was subjected to a final centrifugation (105,000 × g, 60 min) to obtain microsomal (P3) and cytosolic (S3) fractions. For analysis of NO2- reductase activity across fractions, each mitochondrial (P2) and microsomal (P3) pellet was re-suspended in LHM to the same dilution as that of the supernatant fractions from which they were derived and analyzed as described above. The contribution of each subcellular fraction with respect to total hepatic NO2- reductase activity was calculated by accounting for the individual fractional product yield (6.2% for P2; 1.3% for P3; 77.8% for S3, with 14.7% discarded as P1 debris). The influence of NAD(P)H (100 μm) on the conversion of NO2- to NO by the subcellular fractions was determined as the relative NO2- reductase activity in the absence or presence of pyridine nucleotide and expressed as percent of control (with no exogenous pyridine nucleotide added). NO2- interaction with microsomal cytochrome P450 was assessed by difference spectrophotometry and chemiluminescence (see supplemental information).In Vitro StudiesNO2- Uptake by RBCs and Tissues—Rats were anesthetized with diethylether, and 5 ml of arterial blood was collected via cardiac puncture. Blood was immediately centrifuged at 1,400 × g (8 min, 4 °C). The supernatant was discarded, and the RBC pellet was placed on ice. Blood-free heart and liver tissue was obtained as described above and also kept on ice until use. Tissue (0.5 g) was minced into 2-mm pieces and placed into 2 ml of air-saturated or "hypoxic" (15 min bubbling with air or argon, respectively) PBS and incubated at 37 °C. Following addition of 10 μm NaNO2, aliquots of 50 μl were removed after 1, 3, 5, and 10 min and centrifuged briefly (60 s at 16,100 × g) to clarify samples prior to NO2-/NO3- analysis. For uptake measurements by RBC, 4 ml of PBS were added to 1 ml of packed RBC pellet and processed as above. No changes in the concentrations of NO2-/NO3- were observed in the absence of cells/tissue.Tissue Homogenization and Incubation—Blood-free tissue samples were homogenized in chilled PBS (1:5 w/v) using a Polytron (PT10–35) homogenizer. Just prior to use, samples were brought with PBS to a 6-fold final dilution (v/v) for tissues and a 4-fold final dilution (v/v, equivalent to 2.5% hematocrit) for lysed RBC. For incubation, an equivalent tissue/RBC sample volume was placed into the light-protected reaction vessel of an ozone-chemiluminescence NO analyzer (CLD 77sp, EcoPhysics). The vessel was maintained at 37 °C, and samples were purged sequentially with either medical-grade air (normoxia) or nitrogen (hypoxia/near-anoxia). NO generation was continuously monitored for 5 min following addition of 200 μm NaNO2 (final conc.). This NO2- concentration has been established as sufficient to support vasorelaxation in vitro, regional vasodilation in vivo, and nitrosylation and S-nitrosation of Hb in vitro and in vivo (10Cosby K. Partovi K.S. Crawford J.H. Patel R.P. Reiter C.D. Martyr S. Yang B.K. Waclawiw M.A. Zalos G. Xu X. Huang K.T. Shields H. Kim-Shapiro D.B. Schechter A.N. Cannon III, R.O. Gladwin M.T. Nat. Med. 2003; 9: 1498-1505Crossref PubMed Scopus (1413) Google Scholar). Aliquots of samples were collected for protein determination, and all values were normalized to total protein.In Vivo StudiesRats were administered 1.0 mg/kg NaNO2, intraperitoneal This dosing regimen ensures that NO2- equilibrates rapidly across all major organ systems, RBC, and plasma to reach a global steady-state prior to tissue sampling (8Bryan N.S. Fernandez B.O. Bauer S.M. Garcia-Saura M.F. Milsom A.B. Rassaf T. Maloney R.E. Bharti A. Rodriguez J. Feelisch M. Nat. Chem. Biol. 2005; 1: 290-297Crossref PubMed Scopus (408) Google Scholar). Three min after NO2- injection acute systemic hypoxia was induced by cervical dislocation and maintained for 2 min. Normoxic controls were examined in parallel without subjecting animals to global hypoxia. In both cases, animals were sacrificed 5 min after NO2- administration. The same in situ retrograde perfusion technique was performed as for the in vitro studies above except that the perfusate was air-equilibrated PBS supplemented with NEM/EDTA (10 mm/2.5 mm) to eliminate interference by exogenous NO2-. NO-related metabolites following NO2- administration were profiled using previously validated methods (23Bryan N.S. Rassaf T. Maloney R.E. Rodriguez C.M. Saijo F. Rodriguez J.R. Feelisch M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4308-4313Crossref PubMed Scopus (356) Google Scholar, 24Feelisch M. Rassaf T. Mnaimneh S. Singh N. Bryan N.S. Jourd'Heuil D. Kelm M. FASEB J. 2002; 16: 1775-1785Crossref PubMed Scopus (341) Google Scholar). NO metabolites include S-nitrosothiols (RSNO), N-nitrosamines (RNNO), and iron-nitrosyls (NO-heme). Quantification of these species employed group-specific derivatization, denitrosation, and gas-phase chemiluminescence. NO2- and NO3- were quantified by ion chromatography (ENO-20, Eicom).Data Analysis, Presentation, and ModelingUnless otherwise noted, data are averages ± range from n = 3 individual experiments or means ± S.E. from n ≥ 3, as specified. Where appropriate, statistical analysis was performed by one-way analysis of variance with the Bonferroni post-hoc test. Least-squares regression analysis was used to characterize the slope and goodness-of-fit of model linear associations between data sets. Spearman rank correlation was applied to evaluate data co-variation. Statistical significance was set at p < 0.05. Origin 7.0 and Graph Pad Prism 4.0 were used for the statistical analyses. Modeling data were obtained through numeric integration using Mathematica, with a working precision of 20 digits.RESULTSTissues Readily Take Up NO2- in an Oxygen-independent Manner— NO2- uptake by RBCs, heart, and liver tissue was assessed under normoxic and hypoxic conditions by measuring its disappearance from an external medium containing 10 μm NO2-. Because NO2- concentrations in these tissues are ≪10 μm (8Bryan N.S. Fernandez B.O. Bauer S.M. Garcia-Saura M.F. Milsom A.B. Rassaf T. Maloney R.E. Bharti A. Rodriguez J. Feelisch M. Nat. Chem. Biol. 2005; 1: 290-297Crossref PubMed Scopus (408) Google Scholar, 23Bryan N.S. Rassaf T. Maloney R.E. Rodriguez C.M. Saijo F. Rodriguez J.R. Feelisch M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4308-4313Crossref PubMed Scopus (356) Google Scholar, 24Feelisch M. Rassaf T. Mnaimneh S. Singh N. Bryan N.S. Jourd'Heuil D. Kelm M. FASEB J. 2002; 16: 1775-1785Crossref PubMed Scopus (341) Google Scholar), the initial disappearance of NO2- from PBS should reflect the flux of this anion into the cells/tissues. Indeed, NO2- is taken up at similar rates by RBC and heart tissue (0.31 μm/min and 0.24 μm/min, respectively), and rates are roughly comparable under hypoxic conditions (heart: 0.16 μm/min; RBC: 0.28 μm/min; see supplemental Fig. S1). Similar data were obtained with liver (not shown). No NO3- accumulation was seen in the extracellular medium.Hypoxia Markedly Potentiates Tissue NO Production from NO2- in Vitro—Under aerobic conditions, NO production by RBC and blood-free tissues was minimal even in the presence of 200 μM NO2- (Fig. 1A). Hypoxia/near-anoxia (achieved by N2 purging) dramatically enhanced NO formation from NO2-, particularly by heart, liver, and vascular tissue. NO production by RBC lysate peaked after ∼3 min, whereas NO production by liver homogenate increased to reach maximal levels after 50–60 min of hypoxia following a brief lag. Near-anoxic, NO2--dependent NO production (standardized as mg protein/min) in all tissues surpassed that of RBC (Fig. 1B). The 2-fold increase in RBC NO2- reduction from normoxic to near-anoxic conditions is consistent with data previously reported (10Cosby K. Partovi K.S. Crawford J.H. Patel R.P. Reiter C.D. Martyr S. Yang B.K. Waclawiw M.A. Zalos G. Xu X. Huang K.T. Shields H. Kim-Shapiro D.B. Schechter A.N. Cannon III, R.O. Gladwin M.T. Nat. Med. 2003; 9: 1498-1505Crossref PubMed Scopus (1413) Google Scholar). No NO formation was observed when 200 μm NO2- was substituted for NO3- (not shown).NO Generation from NO2- Is Profoundly Oxygen-dependent—The oxygen-dependence of tissue NO2- reductase activity was investigated by adding 200 μm NaNO2 to liver and heart homogenates and RBC lysate incubated at various oxygen concentrations (21, 10, 5, 1, 0.5, and 0%) and monitoring of the resulting NO formation (Fig. 1C). NO production from NO2- was maximal under anoxia. Oxygen proved to be a potent inhibitor of NO2- reduction in liver and heart with >80% inhibition at 0.5% oxygen. Less pronounced changes in oxygen dependence were observed with RBC lysate, with maximal rates of NO formation occurring at ∼1% oxygen.Acute Hypoxia Potentiates NO2--dependent NO Metabolite Formation in Vivo—To extend the above observations to a more physiological context and to expand on existing observations that hypoxia reduces endogenous brain NO2- stores coincident with RSNO formation (23Bryan N.S. Rassaf T. Maloney R.E. Rodriguez C.M. Saijo F. Rodriguez J.R. Feelisch M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4308-4313Crossref PubMed Scopus (356) Google Scholar), the in vivo impact of NO2- on NO-related metabolite formation was assessed. To ensure initial NO2- equilibration across all compartments studied, we followed our previous protocol (8Bryan N.S. Fernandez B.O. Bauer S.M. Garcia-Saura M.F. Milsom A.B. Rassaf T. Maloney R.E. Bharti A. Rodriguez J. Feelisch M. Nat. Chem. Biol. 2005; 1: 290-297Crossref PubMed Scopus (408) Google Scholar) and administered to rats a single intraperitoneal bolus of 1.0 mg/kg NaNO2 3 min prior to inducing 2 min of global hypoxia. Acute global hypoxia attenuated NO2- concentrations in heart, liver, kidney, lung, and aorta (Fig. 2A) and enhanced NO metabolite formation in a tissue- and product-selective manner (Fig. 2, B–D). Tissue nitroso/nitrosyl products originated from NO2-, since their levels under identical hypoxic conditions were far less (<0.1%) without the supplied NO2- (23Bryan N.S. Rassaf T. Maloney R.E. Rodriguez C.M. Saijo F. Rodriguez J.R. Feelisch M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4308-4313Crossref PubMed Scopus (356) Google Scholar). Although each tissue examined was capable of hypoxia-induced, NO2--dependent NO metabolite formation, it was most pronounced in liver, heart, and the RBC. The tissue-specificity of these responses to systemic hypoxia accords with previous demonstrations that endogenous substrates and the turnover of NO-related oxidative and nitrosative metabolites vary greatly among tissues (23Bryan N.S. Rassaf T. Maloney R.E. Rodriguez C.M. Saijo F. Rodriguez J.R. Feelisch M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4308-4313Crossref PubMed Scopus (356) Google Scholar, 26Rodriguez J. Maloney R.E. Rassaf T. Bryan N.S. Feelisch M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 336-341Crossref PubMed Scopus (187) Google Scholar). Although there is limited NO production from NO2- by hypoxic RBC in vitro (Fig. 1), RBC exhibited the greatest relative RSNO formation during acute hypoxia in vivo. This finding is consistent with the S-nitrosation and nitrosylation of hemoglobin (Hb) by NO2- to form both, SNO-Hb and Hb-NO in vitro and in vivo (10Cosby K. Partovi K.S. Crawford J.H. Patel R.P. Reiter C.D. Martyr S. Yang B.K. Waclawiw M.A. Zalos G. Xu X. Huang K.T. Shields H. Kim-Shapiro D.B. Schechter A.N. Cannon III, R.O. Gladwin M.T. Nat. Med. 2003; 9: 1498-1505Crossref PubMed Scopus (1413) Google Scholar); indeed, SNO-Hb is probably generated from circulating NO2- without the intermediate liberation of NO within the RBC (27Angelo M. Singel D.J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8366-8371Crossref PubMed Scopus (184) Google Scholar).FIGURE 2Increased formation of tissue NO metabolites from NO2- during acute global hypoxia in vivo. Blood and tissues utilize NO2- during global hypoxia in vivo (A) to generate NO-related products including S-nitrosothiols (RSNO) (B), N-nitrosamines (RNNO) (C), and iron nitrosyls (NO-heme) (D). (n = 3); *, p < 0.05 versus normoxia.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Kinetics and Concentration-dependence of NO2- Reduction to NO—Liver effectively generates NO from NO2- (Fig. 1), has a multiplicity of potential reductases that can be pharmacologically investigated, and is readily available and easily perfused free of blood. Accordingly, liver was used to characterize more comprehensively the kinetics and chemistry of NO production from NO2- in tissue. The NO2- reductase activity of anoxic whole liver homogenate was linearly dependent upon the concentration of supplied NO2- (25–1000 μm) and, to a lesser extent, the amount of tissue protein employed (Fig. 3A). Because the generation of NO was measured over 4 min in each case and therefore represents the initial rate of NO formation, this observation suggests that whole liver homogenate grossly conforms to first order kinetics with respect to NO2- and, to a lesser extent, tissue protein concentration. In contrast to the results obtained with whole liver homogenate, no strict linearity was evident in the cytosolic fraction (S3) between NO formation and NO2- concentration, although the dependence upon protein concentration was similar to that in whole liver homogenate (Fig. 3B).FIGURE 3Correlation between NO formation from NO2- in different tissues and putative NO2- reductase activity. A, apparent linear dependence of NO2- to NO conversion by hypoxic whole liver homogenate (25–1000 μm NO2-). Inset, hypoxic NO production from 200 μm NO2- is not as linearly dependent upon hepatic protein. B, NO2- and protein dependence deviate from linearity with the cytosolic fraction. C, significant correlation exists between hypoxic NO2- reductase activity and tissue XOR activity (r = 0.892, p = 0.042). The correlation between hypoxic NO2- reductase activity and mitochondrial inner surface area (D) is more striking, except for kidney (r = 0.9998, p = 0.0001).View Large Image Figure ViewerDownload Hi-res image Download (PPT)NO2- Reduction to NO Requires Enzymatic Activity—To probe the involvement of enzymatic processes in hypoxic NO formation, aliquots of liver homogenate were subjected to heat pretreatment. Exposure of liver homogenate for 60 min to 56 or 80 °C inhibited NO2- reductase activity by 72 and >90%, respectively, relative to control, unheated tissue samples. These results are consistent with the conclusion that thermolabile enzymes mediate the majority of hypoxic NO2- reduction to NO, either directly or indirectly (e.g. by modulating reducing equivalent supply).Putative Cellular NO2- Reductases—To gain insight into potential mechanisms of tissue NO2- reduction, NO2--dependent NO production was quantified as a function of reported literature values
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