Intracellular Signaling by Reactive Oxygen Species during Hypoxia in Cardiomyocytes

Cardiomyocytes suppress contraction and O2 consumption during hypoxia. Cytochrome oxidase undergoes a decrease in V max during hypoxia, which could alter mitochondrial redox and increase generation of reactive oxygen species (ROS). We therefore tested whether ROS generated by mitochondria act as second messengers in the signaling pathway linking the detection of O2 with the functional response. Contracting cardiomyocytes were superfused under controlled O2 conditions while fluorescence imaging of 2,7-dichlorofluorescein (DCF) was used to assess ROS generation. Compared with normoxia (PO2 ∼ 107 torr, 15% O2), graded increases in DCF fluorescence were seen during hypoxia, with responses at PO2 = 7 torr > 20 torr > 35 torr. The antioxidants 2-mercaptopropionyl glycine and 1,10-phenanthroline attenuated these increases and abolished the inhibition of contraction. Superfusion of normoxic cells with H2O2 (25 μm) for >60 min mimicked the effects of hypoxia by eliciting decreases in contraction that were reversible after washout of H2O2. To test the role of cytochrome oxidase, sodium azide (0.75–2 μm) was added during normoxia to reduce theV max of the enzyme. Azide produced graded increases in ROS signaling, accompanied by graded decreases in contraction that were reversible. These results demonstrate that mitochondria respond to graded hypoxia by increasing the generation of ROS and suggest that cytochrome oxidase may contribute to this O2 sensing. Cardiomyocytes suppress contraction and O2 consumption during hypoxia. Cytochrome oxidase undergoes a decrease in V max during hypoxia, which could alter mitochondrial redox and increase generation of reactive oxygen species (ROS). We therefore tested whether ROS generated by mitochondria act as second messengers in the signaling pathway linking the detection of O2 with the functional response. Contracting cardiomyocytes were superfused under controlled O2 conditions while fluorescence imaging of 2,7-dichlorofluorescein (DCF) was used to assess ROS generation. Compared with normoxia (PO2 ∼ 107 torr, 15% O2), graded increases in DCF fluorescence were seen during hypoxia, with responses at PO2 = 7 torr > 20 torr > 35 torr. The antioxidants 2-mercaptopropionyl glycine and 1,10-phenanthroline attenuated these increases and abolished the inhibition of contraction. Superfusion of normoxic cells with H2O2 (25 μm) for >60 min mimicked the effects of hypoxia by eliciting decreases in contraction that were reversible after washout of H2O2. To test the role of cytochrome oxidase, sodium azide (0.75–2 μm) was added during normoxia to reduce theV max of the enzyme. Azide produced graded increases in ROS signaling, accompanied by graded decreases in contraction that were reversible. These results demonstrate that mitochondria respond to graded hypoxia by increasing the generation of ROS and suggest that cytochrome oxidase may contribute to this O2 sensing. Alterations in oxygen tension (PO2) elicit a variety of functional responses in different cell types, including gene expression, altered metabolic function, altered ion channel activation, and release of neurotransmitters (1Bunn H.F. Poyton R.O. Physiol. Rev. 1996; 76: 839-885Crossref PubMed Scopus (1043) Google Scholar). In spontaneously contracting embryonic cardiomyocytes, we previously found significant decreases in contractile activity during prolonged moderate hypoxia (PO2 = 20 torr for >2 h) (2Budinger G.R.S. Chandel N. Shao Z.H. Li C.Q. Melmed A. Becker L.B. Schumacker P.T. Am. J. Physiol. 1996; 14: L37-L53Google Scholar). This inhibition was not associated with a depletion of ATP or phosphocreatine stores, and was reversible when normoxic conditions were restored. Similar findings of decreased contractile function during hypoxia (48 h at 1% O2) have also been seen in rat cardiac myocytes (3Silverman H.S. Wei S. Haigney M.C.P. Ocampo C.J. Stern M.D. Circ. Res. 1997; 80: 699-707Crossref PubMed Scopus (73) Google Scholar), which suggests that this response is not unique to embryonic cells. An ability to respond to changes in oxygen tension within the physiological range implies the existence of a cellular O2sensor linked to a signal transduction pathway. When activated by hypoxia, the sensor presumably would initiate a signaling cascade which ultimately leads to the functional response (e.g. diminished contractile activity). However, the O2 sensing mechanism and the subsequent signal transduction pathways involved in the cardiomyocyte responses to hypoxia are not known. A number of different potential mechanisms of cellular O2 sensing have been identified (1Bunn H.F. Poyton R.O. Physiol. Rev. 1996; 76: 839-885Crossref PubMed Scopus (1043) Google Scholar). Mitochondria are responsible for most of the O2 consumption by the cell and would seem to be well suited because their localPO2 responds to changes in the ratio of O2 supply to demand. However, the low apparentK m of cytochrome oxidase for O2 (4Cooper C.E. Biochim. Biophys. Acta. 1990; 1017: 187-203Crossref PubMed Scopus (82) Google Scholar, 5Einarsdóttir O. Biochim. Biophys. Acta. 1995; 1229: 129-147Crossref PubMed Scopus (86) Google Scholar, 6Chance B. Williams G.R. J. Biol. Chem. 1955; 217: 383-393Abstract Full Text PDF PubMed Google Scholar) would appear to render these organelles incapable of detecting changes until very low O2 concentrations are reached. Nevertheless, our studies of hypoxic cardiomyocytes (2Budinger G.R.S. Chandel N. Shao Z.H. Li C.Q. Melmed A. Becker L.B. Schumacker P.T. Am. J. Physiol. 1996; 14: L37-L53Google Scholar) and of normal rat hepatocytes (7Chandel N. Budinger G.R.S. Kemp R.A. Schumacker P.T. Am. J. Physiol. 1995; 268: L918-L925Crossref PubMed Google Scholar, 8Schumacker P.T. Chandel N. Agusti A.G.N. Am. J. Physiol. 1993; 265: L395-L402PubMed Google Scholar) have implicated mitochondria as a likely site of O2sensing underlying their metabolic and functional responses to hypoxia. In this regard, we found that cytochrome oxidase undergoes a ∼50% decrease in V max during exposure to prolonged moderate hypoxia (9Chandel N.S. Budinger G.R.S. Schumacker P.T. J. Biol. Chem. 1996; 271: 18672-18677Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). This change was manifested by decreases inN,N,N′,N′-tetramethyl-p-phenylenediamine-ascorbate respiration during hypoxia (2Budinger G.R.S. Chandel N. Shao Z.H. Li C.Q. Melmed A. Becker L.B. Schumacker P.T. Am. J. Physiol. 1996; 14: L37-L53Google Scholar), and also by increases in NAD(P)H autofluorescence (10Chandel N.S. Budinger G.R.S. Choe S.H. Schumacker P.T. J. Biol. Chem. 1997; 272: 18808-18816Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Collectively, these findings cast a new light on the possible role of mitochondria in the cellular responses to hypoxia in cardiomyocytes. The presence of a cellular O2 sensor implies the existence of a signaling pathway linking it to the targeted response. If mitochondria function as that sensor, what signaling system could be activated by a decrease in the V max of cytochrome oxidase? We hypothesized that a decrease inV max of the oxidase should increase the reduction state of mitochondrial electron carriers upstream of cytochrome aa 3. This should increase the lifetime of reduced electron carriers such as ubisemiquinone, which would increase the generation of superoxide via univalent electron transfer to O2 in the mitochondria (11Turrens J.F. Alexandre A. Lehninger A.L. Arch. Biochem. Biophys. 1985; 237: 408-414Crossref PubMed Scopus (1064) Google Scholar). Trace levels of reactive oxygen species (ROS) 1The abbreviations used are: ROS, reactive oxygen species; DCF, 2,7-dichlorofluorescein; DCFH, reduced DCF; TTFA, thenoyltrifluoroacetone. 1The abbreviations used are: ROS, reactive oxygen species; DCF, 2,7-dichlorofluorescein; DCFH, reduced DCF; TTFA, thenoyltrifluoroacetone. such as superoxide or H2O2 could then potentially act as signaling elements by activating subsequent steps in a signal transduction cascade. Indeed, numerous studies have implicated ROS as participants in a variety of intracellular signaling sequences including members of the stress- and mitogen-activated protein kinases (12Guyton K.Z. Liu Y. Gorospe M. Xu Q. Holbrook N.J. J. Biol. Chem. 1996; 271: 4138-4142Abstract Full Text Full Text PDF PubMed Scopus (1138) Google Scholar, 13Laderoute K.R. Webster K.A. Circ. Res. 1997; 80: 336-344Crossref PubMed Scopus (164) Google Scholar), the nuclear transcription factors c-Jun and NF-κB (14Flohe L. Brigelius-Flohe R. Saliou C. Traber M.G. Packer L. Free Radical. Biol. Med. 1997; 22: 1115-1126Crossref PubMed Scopus (755) Google Scholar), and other signaling systems (15Lander H.M. FASEB J. 1997; 11: 118-124Crossref PubMed Scopus (820) Google Scholar). The mechanisms responsible for intracellular oxidant generation involved in the activation of those pathways are not fully clear, but could involve mitochondrial sources during hypoxia. The present study therefore sought to test the hypothesis that ROS are generated by mitochondria during hypoxia in cardiomyocytes, and that ROS signaling is involved in coupling the O2 sensor to the contractile response to hypoxia. Embryonic chick cardiomyocytes were isolated using a method (2Budinger G.R.S. Chandel N. Shao Z.H. Li C.Q. Melmed A. Becker L.B. Schumacker P.T. Am. J. Physiol. 1996; 14: L37-L53Google Scholar) modified from Barry et al. (16Barry W.H. Pober J. Marsh J.D. Frankel S.R. Smith T.W. Am. J. Physiol. 1980; 239: H651-H657PubMed Google Scholar). Briefly, hearts of 10–11-day-old chick embryos were removed and placed in Hank's balanced salt solution lacking magnesium and calcium (Life Technologies, Inc.). Ventricular tissue was minced and the cells were dissociated using four to six cycles of trypsin (0.025%, Life Technologies, Inc.) degradation at 37 °C with gentle agitation. Trypsin digestion was halted after 8 min by transferring the cells to a trypsin inhibitor solution. After filtering (100-μm mesh), the cells were centrifuged for 5 min at 1200 rpm at 4 °C and resuspended in nutritive medium. Cells then were placed in a Petri dish in a humidified incubator (5% CO2, 95% air at 37 °C) for 45 min to promote early adherence of fibroblasts. Nonadherent cells then were counted with a hemacytometer, and their viability was measured using trypan blue (0.4%). Approximately 1 × 106cells in nutritive medium (54% Barry's solution (in mm: NaCl (116), KCl (1.3), NaHCO3 (22Petersen L.C. Biochim. Biophys. Acta. 1977; 460: 299-307Crossref PubMed Scopus (151) Google Scholar), with (Life Technologies, and were (25 × Cells were in a humidified incubator for at which of the were were on spontaneously contracting cells at or 4 after hepatocytes were isolated using the previously P.T. Chandel N. Agusti A.G.N. Am. J. Physiol. 1993; 265: L395-L402PubMed Google Scholar). contracting myocytes on were placed in a The was using to O2 the and the and on a °C) on A °C) the was used to the to O2 The of a salt solution (in mm: NaCl KCl NaHCO3 C.J. Biochem. 1996; PubMed Scopus Google Scholar), The used to the of the was by a or low was used to the to the in to O2 transfer the In in the was under conditions to those of the using method J. Biol. Chem. Full Text PDF PubMed Google Scholar, C.J. Biochem. 1996; PubMed Scopus Google Scholar) Inc.). An was for and a light a a and and and The also was with to the of contractile cell were using a were and using ROS generation in cells was using the 2,7-dichlorofluorescein (DCF) The of the DCF was added to the at a of 5 the the on the reduced 1996; PubMed Scopus Google Scholar). ROS in the cells DCFH, the DCF J. Biol. 1990; PubMed Scopus Google Scholar). studies of the of in cardiomyocytes that the is by H2O2 or but is to superoxide Li C. Shao Schumacker P.T. Becker L.B. J. 1997; Full Text PDF PubMed Scopus Google Scholar). was measured using of and of using to the was in of cells identified as of and was identified as cells or with cellular are as of after was during low light using to the cellular changes during contraction. were at on using a were and were from to the change in was These changes in were for a of in the that the that was by This was using a of in the imaging In different the cells were with the electron and The antioxidants 2-mercaptopropionyl glycine and 1,10-phenanthroline were also from embryonic cardiomyocytes on were placed in a on and with salt solution μm) under controlled O2 1 the of hypoxia at different levels of DCF fluorescence in cardiomyocytes. Compared with normoxic cells superfused with salt solution with 15% O2 (PO2 ∼ 107 cells at PO2 = or 7 torr for h significant increases in fluorescence that with the of hypoxia. of normoxia at = min was associated with a decrease in which could intracellular reduction of the or of the from the 1 the of graded hypoxia on contractile in the cells in 1 was by at under conditions for 1 decreases in contractile were seen during hypoxia, which were reversible when normoxic conditions were restored. These results were with our studies (2Budinger G.R.S. Chandel N. Shao Z.H. Li C.Q. Melmed A. Becker L.B. Schumacker P.T. Am. J. Physiol. 1996; 14: L37-L53Google Scholar). seen the decrease in contraction not but h to a of of h after of To that oxidant signaling was responsible for these the study was 20 in the presence of the 2-mercaptopropionyl glycine μm) and the 1,10-phenanthroline In the presence of these the increases in cell fluorescence were attenuated To the of during hypoxia, cells with were ∼ 20 torr O2) while DCF fluorescence were The mitochondrial electron and μm) were added to electron supply to the of This produced a decrease in DCF fluorescence during hypoxia In cardiomyocytes with and under hypoxic conditions were which the of increasing the lifetime of (11Turrens J.F. Alexandre A. Lehninger A.L. Arch. Biochem. Biophys. 1985; 237: 408-414Crossref PubMed Scopus (1064) Google Scholar). This produced a increase in as from fluorescence To test whether oxidant signaling was involved in the of contraction seen during prolonged moderate hypoxia, the antioxidants 2-mercaptopropionyl glycine μm) and 1,10-phenanthroline μm) were added to the during hypoxia in abolished the decreases in contractile seen during hypoxia, ROS in the functional response to hypoxia. superoxide generated by the mitochondrial electron system could subsequent signaling steps by a of superoxide by superoxide could which could function as the in this response. To test this contractile was in cardiomyocytes with different concentrations of normoxic 5 the of min with H2O2 (25 μm) on contractile 1 significant decreases in contractile were of H2O2 was associated with of contractile this to concentrations of H2O2 μm) not contractile during for 1 while concentrations of H2O2 μm) a decrease or of contractile activity which not during not studies of cytochrome oxidase a decrease in the V max of the during hypoxia (9Chandel N.S. Budinger G.R.S. Schumacker P.T. J. Biol. Chem. 1996; 271: 18672-18677Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). a change could alter the mitochondrial redox state and ROS generation by mitochondria during hypoxia. If then of cytochrome oxidase that reduce theV max of the during normoxia should that To test this we used sodium a inhibitor of the oxidase (22Petersen L.C. Biochim. Biophys. Acta. 1977; 460: 299-307Crossref PubMed Scopus (151) Google Scholar) to the oxidase during a the of different concentrations of azide on DCF fluorescence in cardiomyocytes at PO2 ∼ 107 torr. concentrations of azide produced graded increases in fluorescence that decreased after washout of the increases in fluorescence were with concentrations of azide which also were reversible after of the effects of azide on contractile of cardiomyocytes. decreases in contractile were during h of azide which were reversible after of the However, and decreases in contraction were seen with concentrations of azide which were of cellular To test whether decreases in cytochrome oxidase V max were responsible for the increase in ROS generation during hypoxia, rat hepatocytes were with and during normoxia h) by hypoxia h) and h) In hepatocytes were to h of hypoxia in to elicit the decrease in V max of the oxidase P.T. Chandel N. Agusti A.G.N. Am. J. Physiol. 1993; 265: L395-L402PubMed Google Scholar), the oxidase in cardiomyocytes within min of hypoxia Shao Li C. R. Schumacker P.T. Becker L.B. Am. J. Physiol. 1996; Google Scholar). ROS generation should with the changes in V max of the oxidase different cell In with this hepatocytes increase in DCF fluorescence during the of hypoxia, but demonstrate a increase at cellular fluorescence decreased cells under normoxic conditions for the increases in DCF cell respond to changes in the by activating functional or This suggests the existence of O2 sensor to a signal transduction which ultimately the functional response. a variety of O2 sensing mechanisms are to (1Bunn H.F. Poyton R.O. Physiol. Rev. 1996; 76: 839-885Crossref PubMed Scopus (1043) Google Scholar), our studies that mitochondria may act in that role in embryonic cardiomyocytes (2Budinger G.R.S. Chandel N. Shao Z.H. Li C.Q. Melmed A. Becker L.B. Schumacker P.T. Am. J. Physiol. 1996; 14: L37-L53Google Scholar, G.R.S. J. Chandel N.S. Schumacker P.T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The present study to test the hypothesis that ROS generated by mitochondria act as second messengers in the contractile response to physiological levels of hypoxia in those cells. that mitochondrial ROS generation increases as the of O2 decreases during hypoxia. Compared with normoxia O2), graded increases in were at and 1% with the levels of oxidant signaling seen at the O2 of antioxidants attenuated this oxidant signaling, and also abolished the decrease in contractile function seen during prolonged hypoxia. inhibition of cytochrome oxidase with azide produced graded increases in ROS generation during normoxia and reversible decreases in contraction. of H2O2 produced reversible decreases in contraction during normoxia that mimicked the response to hypoxia. Collectively, these suggest mitochondrial ROS generation in the signal transduction cascade linking the O2 sensor with the contractile response via the of hypoxia decreased cytochrome max mitochondrial redox mitochondrial superoxide generation H2O2 generation subsequent signaling steps decreased contraction. The suggest that ROS from mitochondria as second messengers in the signaling pathway linking the O2 sensor to the decrease in contractile function of cardiomyocytes. We that the decreases in contractile function and are a of decreased ATP G.R.S. J. Chandel N.S. Schumacker P.T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), from a signaling that activation of the ROS would appear to act at early in this the contractile response to hypoxia or of H2O2 h to and from these h for The low levels of ROS generation during hypoxia not appear to be as by the ability of the cells to contractile function after of and the of of cell viability prolonged hypoxia previously (2Budinger G.R.S. Chandel N. Shao Z.H. Li C.Q. Melmed A. Becker L.B. Schumacker P.T. Am. J. Physiol. 1996; 14: L37-L53Google Scholar). study not steps subsequent to but a number of studies role of ROS in a variety of intracellular signaling pathways (14Flohe L. Brigelius-Flohe R. Saliou C. Traber M.G. Packer L. Free Radical. Biol. Med. 1997; 22: 1115-1126Crossref PubMed Scopus (755) Google Scholar, Stern A. Free Radical. Biol. Med. 1996; PubMed Scopus Google Scholar, A. Free Radical. Biol. Med. 1997; 22: PubMed Scopus Google Scholar). et al. (12Guyton K.Z. Liu Y. Gorospe M. Xu Q. Holbrook N.J. J. Biol. Chem. 1996; 271: 4138-4142Abstract Full Text Full Text PDF PubMed Scopus (1138) Google Scholar) have that H2O2 activation of mitogen-activated protein et al. H.M. S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) have a mechanism to the protein and ROS appear to in the activation of protein kinases K.R. Webster K.A. Circ. Res. 1997; 80: 336-344Crossref PubMed Scopus (164) Google Scholar). studies be to the steps in the signal transduction cascade responsible for the inhibition of contraction. Mitochondria have been as reactive oxygen species are generated in cells. In the electron (11Turrens J.F. Alexandre A. Lehninger A.L. Arch. Biochem. Biophys. 1985; 237: 408-414Crossref PubMed Scopus (1064) Google Scholar) to be a site of superoxide generation because of for univalent electron transfer to Indeed, it been that superoxide generation for of mitochondrial O2 consumption under normal conditions A. N. B. Biochem. J. PubMed Scopus Google Scholar). of the that mitochondria were the of ROS in our electron inhibition with attenuated the ROS signal during hypoxia, inhibitor of oxidant and the of superoxide by of ubisemiquinone, A superoxide generation by increasing the lifetime of that Collectively, these results demonstrate that mitochondria function as a of ROS during hypoxia, increasing of at oxygen This could mitochondria to function as a cellular O2 sensor at physiological levels of hypoxia. different oxidase systems could contribute to the generation of ROS in cells. and identified a ROS generation in response to changes in a physiological range Am. J. Physiol. 1995; Google Scholar). of cardiac found that of not a increase in superoxide as by 1997; PubMed Scopus Google Scholar). results suggest that the oxidase is to However, their signal with and was during O2 hypoxia their signal = torr. using we found increases in mitochondrial oxidant generation at with the levels at 1% O2 (PO2 ∼ 7 the decrease in contractile during hypoxia was abolished with and the ROS generation with under hypoxia, we that the functional response to hypoxia in our study have involved increase in ROS from decreased ROS generation at from The factors that the of ROS by mitochondria are not fully but likely the reduction state of the mitochondrial electron system and of that increase the reduction state of the electron carriers appear to increase the generation of when is mitochondrial ROS generation been to during conditions when electron carriers are reduced and the cells are Li C. Shao Schumacker P.T. Becker L.B. J. 1997; Full Text PDF PubMed Scopus Google Scholar, J. 1996; PubMed Scopus Google Scholar). increases in mitochondrial redox at a appear to increase ROS generation G.R. Arch. Biochem. Biophys. PubMed Scopus Google Scholar). azide and A produced increases in reduction of during which to the increases in DCF mitochondrial superoxide generation to respond to changes in redox state at a of In our the increases in ROS during hypoxia suggest that mitochondrial reduction have as PO2 was could this we previously max of cytochrome oxidase decreases by ∼50% during hypoxia in cardiac myocytes (2Budinger G.R.S. Chandel N. Shao Z.H. Li C.Q. Melmed A. Becker L.B. Schumacker P.T. Am. J. Physiol. 1996; 14: L37-L53Google Scholar), normal rat hepatocytes P.T. Chandel N. Agusti A.G.N. Am. J. Physiol. 1993; 265: L395-L402PubMed Google Scholar, N.S. Budinger G.R.S. Choe S.H. Schumacker P.T. J. Biol. Chem. 1997; 272: 18808-18816Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), rat mitochondria (7Chandel N. Budinger G.R.S. Kemp R.A. Schumacker P.T. Am. J. Physiol. 1995; 268: L918-L925Crossref PubMed Google Scholar), (10Chandel N.S. Budinger G.R.S. Choe S.H. Schumacker P.T. J. Biol. Chem. 1997; 272: 18808-18816Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), and in isolated cytochrome oxidase (9Chandel N.S. Budinger G.R.S. Schumacker P.T. J. Biol. Chem. 1996; 271: 18672-18677Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). We suggest that the increase in mitochondrial reduction by the decrease in cytochrome oxidase V max increase in mitochondrial superoxide generation. A second mechanism to increase mitochondrial reduction during hypoxia was by and measured reduction as PO2 was reduced from torr J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. J. Biol. PubMed Scopus Google Scholar). to their increases in cytochrome reduction at physiological levels of hypoxia, which to a electron O2 consumption until low levels of are reached. the increases in reduction were during physiological hypoxia, may have been to superoxide generation. To the of these we the ROS response in cardiac myocytes to that in normal rat In their studies and increases in reduction within after the of hypoxia in a variety of cell J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. C. Arch. Biochem. Biophys. PubMed Scopus Google Scholar, C. M. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar). We previously that cytochrome oxidase cell with to the of hypoxia to elicit a decrease in V The oxidase in embryonic cardiac myocytes to hypoxia within min (2Budinger G.R.S. Chandel N. Shao Z.H. Li C.Q. Melmed A. Becker L.B. Schumacker P.T. Am. J. Physiol. 1996; 14: L37-L53Google Scholar), rat hepatocytes under hypoxia for 1 to h to elicit a change in V max P.T. Chandel N. Agusti A.G.N. Am. J. Physiol. 1993; 265: L395-L402PubMed Google N.S. Budinger G.R.S. Choe S.H. Schumacker P.T. J. Biol. Chem. 1997; 272: 18808-18816Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). If the decrease in V max were responsible for the increase in ROS signal during hypoxia, then DCF fluorescence should not in hepatocytes until been hypoxic for In increases in mitochondrial redox by and were the increases in ROS would in cell in hepatocytes with and superfused at O2 increase in fluorescence that not until h after the of hypoxia, and which decreased after of increases in DCF fluorescence were after min of hypoxia in the cardiomyocytes. We that changes in theV max of cytochrome oxidase during hypoxia, changes in mitochondrial reduction state J. Biol. Chem. Full Text PDF PubMed Google Scholar), have been responsible for the increase in ROS signaling during hypoxia. a inhibitor of cytochrome oxidase (22Petersen L.C. Biochim. Biophys. Acta. 1977; 460: 299-307Crossref PubMed Scopus (151) Google Scholar), azide effects that on concentrations it is possible that azide could mitochondrial respiration by cytochrome However, concentrations should the effects of hypoxia by cytochrome This should the V max of the oxidase via inhibition during Indeed, the increase in reduction of electron carriers a graded increase in ROS generation by DCFH, which a and reversible of contractile However, studies of the effects of O2 on cytochrome oxidase V max that the oxidase function in of (9Chandel N.S. Budinger G.R.S. Schumacker P.T. J. Biol. Chem. 1996; 271: 18672-18677Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). when hypoxia a decrease in cytochrome oxidase V a increase in superoxide generation should be In this the graded increases in oxidant signaling seen at levels would seem because mitochondrial redox should while for superoxide generation would These suggest that factors may the of superoxide generation at different levels of moderate hypoxia. studies be to fully the mechanisms superoxide generation in the cell at graded levels of hypoxia.