Cellular Respiration during Hypoxia

We previously reported that hepatocytes exhibit a reversible suppression of respiration during prolonged hypoxia (PO2 = 20 torr for 3–5 h). Also, isolated bovine heart cytochrome c oxidase undergoes a reversible decrease in apparent V max when incubated under similar conditions. This study sought to link the hypoxia-induced changes in cytochrome oxidase to the inhibition of respiration seen in intact cells. Hepatocytes incubated at PO2 = 20 torr exhibited decreases in respiration and increases in NAD(P)H after 2–3 h that were reversed upon reoxygenation (PO2 = 100 torr). Respiration during hypoxia was also inhibited whenN,N,N′,N′-tetramethyl-p-phenylenediamine (0.5 mm) and ascorbate (5 mm) were used to reduce cytochrome c, suggesting that cytochrome oxidase was partially inhibited. Similarly, liver submitochondrial particles revealed a 44% decrease in the apparent V maxof cytochrome oxidase after hypoxic incubation. In hepatocytes loaded with tetramethylrhodamine ethyl ester (10 nm) to quantify mitochondrial membrane potential, acute hypoxia (2–3 h), which in turn could allow cytochrome oxidase to function as a cellular O2sensor in the physiological range of hypoxia. The present study sought to determine whether the kinetic changes in cytochrome oxidase observed with the purified enzyme also contribute to the hypoxic responses in the intact cell. First, we assessed the function of cytochrome oxidase in intact cells and in submitochondrial particles to determine whether the changes in the oxidase were consistent with the changes in cellular respiration. Second, we studied the effects of hypoxia on mitochondrial transmembrane potential to assess the balance between electron flux and ATP consumption. Finally, we measured the respiratory control coefficients for NADH to determine whether processes controlling the supply and utilization of NADH were also affected by prolonged hypoxia. The results support the hypothesis that changes in the kinetic properties of cytochrome oxidase allow the enzyme to function as an O2 sensor in the intact cell, participating in the reversible inhibition of respiration during hypoxia. Male Harlan Sprague Dawley rats weighing 225 ± 25 g were provided with food and water ad libitum. Hepatocytes were isolated by collagenase digestion of livers using methodology similar to that previously described (10Seglen P. Exp. Cell. Res. 1972; 74: 450-454Crossref PubMed Scopus (395) Google Scholar), followed by Percoll centrifugation. This procedure yielded hepatocytes with >90% viability, as determined by trypan blue (0.4%) exclusion. Cells then were studied either in suspension or adhered to glass coverslips coated with 0.1% collagen For cells studied in approximately cells were of with (10 mm) and The in the been previously with or through the in the in the was used to the PO2 in the The were at in an at Cellular O2 were measured in of cells from the and studied in a anaerobic with a O2 to the was with at the PO2 used to the of the Because the were from the to the with was and the of O2 uptake were at approximately the PO2 used for the incubation in the respiration was determined as the between O2 and that after of the electron in cellular were assessed from changes in the of studied in a at an of an of and a of Cells were from the to a a The was or a and was at in a the The been previously with O2 or O2 to of cells. respiratory were to the after for the cells to the change in fluorescence was measured after For the of reduction of the mitochondrial was determined by mm) and (10 changes reported as of fluorescence measured during mitochondrial c oxidase catalytic activity was measured in cells in studies and in submitochondrial particles in Cells or submitochondrial particles were incubated in at were measured in the of (10 TMPD (0.5 and ascorbate (5 The were used to mitochondrial electron TMPD is a electron which cytochrome c PubMed Scopus Google Scholar). when TMPD is used as a changes in reflect changes in cytochrome oxidase was used to reduce which would particles were from rat liver mitochondria as described previously F. F. P. O. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). were determined by the procedure with bovine as the membrane potential was measured in hepatocytes on glass cells in of were coverslips mm) and in a for were then to an anaerobic the was used to the at The was to a on the of an and with with tetramethylrhodamine ethyl ester 10 is a that has been shown to between and mitochondria to P. J. Full Text PDF PubMed Scopus Google Scholar). has been shown to a of mitochondrial membrane potential. Because the is not in the cell, increases in fluorescence used to decreases in fluorescence used to Cells were with the for h to effect to fluorescence of cells were using a was with a for in fluorescence were determined using an of and an of The was using and in the between the and the cells. were with a under control were using of followed by was determined at the reported as ± were using cells from This study sought to quantify the effects of hypoxic incubation on O2 and in rat Cells were in suspension in at PO2 = 100 or 20 torr for were measured in studied at approximately the PO2 used for at = and shown in the O2 uptake at 20 torr from ± to ± cells after h at PO2 = 20 cells incubated at PO2 = 100 torr their O2 uptake over the was from the to the of the In cells were incubated at PO2 = 100 torr for after which the oxygen tension was toPO2 = 20 torr for 3 of cells at 20 torr was then a and O2 and for to the PO2 to 100 torr uptake and during that study in The fluorescence increased by ± O2 uptake from ± to ± cells during hypoxia. acute O2 uptake and fluorescence to Thus, the changes in respiratory rate and seen during prolonged hypoxia were reversible after rate and fluorescence during exposure to PO2 = 100 torr = 20 torr h), = 100 torr fluorescence changes reported as of mitochondrial between = ± This study sought to determine whether the changes in observed for purified 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 also intact hepatocytes during prolonged hypoxia. Hepatocytes were in suspension in = 100 or 20 torr for Cellular O2 uptake were measured at = and h at the PO2 used for after of TMPD ascorbate (5 and (10 Because TMPD to cytochrome c (1Wilson D.F. Rumsey W.L. Green T.J. Vanderkooi J.M. J. Biol. Chem. 1988; 263: 2712-2718Abstract Full Text PDF PubMed Google Scholar), respiration with TMPD an of cytochrome oxidase activity cytochrome c is exposure to 20 torr for TMPD respiration from ± to ± cells TMPD respiration in cells at 100 torr for the incubation under hypoxia for cells were reoxygenated to 100 torr, and TMPD respiration increased to ± which was not from the value at 100 torr = not The O2 of cytochrome oxidase activity was also in submitochondrial particles from rat liver particles were incubated in at PO2 for were then assessed in studied at the PO2 used for after of TMPD and ascorbate (5 mm) as shown in submitochondrial particles incubated at PO2 torr exhibited a decrease in cytochrome oxidase with those studied at O2 The TMPD of the O2 rate was also measured in submitochondrial particles incubated = 100 or 20 torr for h the of TMPD was increased from to approximately 100 was a increase in the rate of O2 consistent with a increase in cytochrome c reduction at of of TMPD the increase in O2 uptake to suggesting that cytochrome c reduction was particles incubated under hypoxia also a TMPD of O2 the rate was by approximately 50% at TMPD with the normoxic particles Thus, cytochrome oxidase activity in submitochondrial particles was and reversibly inhibited over a range of TMPD after exposure to prolonged hypoxia. transmembrane potential a balance between processes to the and those to of the potential the of processes and the processes inhibited during hypoxia. For example, the decrease in cellular respiration during hypoxia were to an inhibition of cytochrome oxidase activity change in ATP then a decrease in mitochondrial potential the decreases in cellular respiration were by a decrease in ATP utilization change in mitochondrial function then membrane potential increase during hypoxia. between we measured mitochondrial potential in intact hepatocytes with normoxic or hypoxic studies were to that the in the consistent with that for a mitochondrial Hepatocytes on coverslips at were for the was with at PO2 = 100 an of the membrane was to the = the increase in fluorescence observed after the of consistent with the of mitochondrial membrane potential. the response to which decrease the membrane potential by activity and the supply of to Cells given at PO2 = 100 torr exhibited significant decreases in fluorescence, consistent with mitochondrial decrease in the activity of the mitochondrial ATP in of the membrane potential. the effect of on fluorescence in hepatocytes during The increase in fluorescence that was observed is consistent with membrane Similarly, the increase in fluorescence using to the mitochondrial was consistent with membrane Because the mitochondrial potential is by electron an inhibition of electron would limit and a the effect of to cells = 100 torr on The decrease in fluorescence is consistent with the decrease in mitochondrial potential. of the (5 a decrease in fluorescence, consistent with the of membrane potential. the studies that is of a of mitochondrial potential in this was used to assess mitochondrial potential during hypoxia in First, hepatocytes loaded with were at PO2 = 100 torr for The cells were then for 10 after which the PO2 of the was to 20 torr and the cells were for an 10 This produced no change in fluorescence suggesting that mitochondrial potential This was consistent with the absence of change in O2 uptake or TMPD respiration by intact hepatocytes during acute hypoxia exposure to prolonged hypoxia was with a decrease in O2 uptake by h of fluorescence over the is to in the by uptake of or an we the response to acute reoxygenation toPO2 = 100 torr in hepatocytes that been incubated for 3 h at PO2 = 20 shown in acute reoxygenation been with a of O2 a of the effects of prolonged hypoxia. shown in cells that been with at PO2 = 20 torr for 3 h were for 10 to a fluorescence the PO2 was increased to 100 torr as the cells to The fluorescence increased during which suggested that the mitochondrial potential Such a change suggests that the rate of electron was consistent with an increase in the activity of cytochrome processes that mitochondrial produce a corresponding change in cellular O2 J. Biochem. PubMed Scopus Google Scholar). In mitochondrial a balance between processes controlling the supply of from the to NADH and the responsible for electron to cytochrome oxidase and The control by on cellular respiration has been described by J. Biochem. PubMed Scopus Google in of the of the and of this metabolic to changes in the The control for NADH to the that cellular respiration. In J. Biochem. PubMed Scopus Google reported control coefficients for NADH of to We a similar to determine whether the control coefficients for NADH were altered in hepatocytes incubated at PO2 = 20 Such an effect would that hypoxia metabolic control at the cell. absence of change in the control for NADH during hypoxia would support the that cytochrome oxidase in this response. determine the of processes to mitochondrial was altered by or which mitochondrial their by the in the mitochondrial were to hepatocytes after incubation at either PO2 = 100 torr or 20 torr for and the change in mitochondrial respiratory rate was of to normoxic cells produced an increase in fluorescence and an increase in mitochondrial of produced a decrease in fluorescence and a decrease in respiration cells incubated under prolonged hypoxia exhibited increased of fluorescence with normoxic cells the responses to and were not between the Thus, the suppression of cellular respiration by exposure to prolonged hypoxia not the to which mitochondrial control respiration at from NADH control in hepatocytes incubated at 20 torr for ± ± ± ± (10 ± ± ± ± (5 ± ± ± ± were exposed to hypoxia (PO2 = 20 torr) for 3 Cells were for and changes and mitochondrial respiration were For the of reduction of the mitochondrial was determined by 20 and 10 changes reported as of mitochondrial respiration was by the respiration from the cellular respiration ± = in a control in hepatocytes incubated at 100 torr for ± ± ± ± (10 ± ± ± ± (5 ± ± ± ± were exposed to (PO2 = 100 torr) for 3 Cells were for and changes and mitochondrial respiration were For the of reduction of the mitochondrial was determined by 20 and 10 changes reported as of mitochondrial respiration was by the respiration from the cellular respiration ± = in a Cells were exposed to hypoxia (PO2 = 20 torr) for 3 Cells were for and changes and mitochondrial respiration were For the of reduction of the mitochondrial was determined by 20 and 10 changes reported as of mitochondrial respiration was by the respiration from the cellular respiration ± = Cells were exposed to (PO2 = 100 torr) for 3 Cells were for and changes and mitochondrial respiration were For the of reduction of the mitochondrial was determined by 20 and 10 changes reported as of mitochondrial respiration was by the respiration from the cellular respiration ± = that the rate of electron from NADH to O2 a change in of the supply of from the to NADH is We studied this in hepatocytes by the changes in NADH when electron was partially inhibited by or when electron was by (10 to The a decrease in mitochondrial which a increase in The a increase in respiration and a decrease in fluorescence The of the change in NAD(P)H and the change in respiration was similar between hypoxic and normoxic cells suggesting that the control of electron supply to NADH was not by hypoxic incubation The were used to the respiratory control for which is the of the from and to the by the of The control was ± during with ± in hypoxia. the by and to that with of a value of ± was during and a value of ± was during hypoxia. the control of NADH over mitochondrial respiration ranged from in normoxic and hypoxic The similar control coefficients seen in the normoxic and hypoxic cells that the control by NADH over mitochondrial respiration was not by hypoxia. respiration the of from metabolic to through the electron of the mitochondrial an the mitochondrial membrane of a membrane potential and The ATP the energy for ATP from the of this to the control of mitochondrial respiration under conditions. For example, have been to the of respiration by J. Biol. Chem. Full Text PDF PubMed Google Scholar), the phosphorylation potential D.F. J. Biol. PubMed Scopus Google Scholar), the supply of to NADH J. Biochem. PubMed Scopus Google Scholar), the ATP the J. Biol. PubMed Scopus Google Scholar), and mitochondrial J. 1995; PubMed Scopus Google Scholar), and the of in a D.P. PubMed Scopus Google Scholar). However, our of the of respiration in intact cells during prolonged hypoxia is In the present study and in a (6Schumacker P.T. Chandel N. Agusti A.G.N. Am. J. Physiol. 1993; 265: L395-L402PubMed Google we that hepatocytes decrease their rate of O2 uptake when under physiological of hypoxia for changes were reversed when the PO2 was restored to 100 torr and were not with a of lactate production by the cells not increase during prolonged hypoxia, that anaerobic ATP production was not that hepatocytes may of cellular ATP utilization at O2 tensions the to limit respiration. The present study sought to the of whether the changes in cytochrome oxidase observed in the of O2 the intact during prolonged hypoxia. determine whether processes or of NADH to the suppression of respiration during hypoxia, we the effects of hypoxia on the control by NADH on mitochondrial respiration. inhibition of the could decrease the supply of to which could reduce O2 and decrease mitochondrial membrane potential. Such a response could decrease the phosphorylation potential and ATP However, the that NADH were increased in hepatocytes incubated under hypoxia, which that prolonged hypoxia was with a change in the of electron from the NADH the control by NADH supply over mitochondrial respiration was similar in the In the changes in NAD(P)H were with the changes in O2 that the control of respiration by is with that by We that inhibition of is to contribute to the hypoxic suppression of respiration. the responsible mechanism to the mitochondrial electron of NADH The mitochondrial potential the for ATP and its the to the suppression of respiration in hypoxia. that increase the flux of in the electron to increase membrane potential by that increase the of the mitochondrial membrane as the ATP or to decrease mitochondrial potential. state the mitochondrial potential a balance between the rate of electron and the rate of ATP utilization by the cell. the suppression of respiration during prolonged hypoxia an inhibition of the or the electron a change in the rate of ATP then mitochondrial potential would Such a could limit the rate of ATP and the rate of ATP utilization. the hypoxia respiration by ATP utilization at the then of the membrane would in response to the decrease in We that reoxygenation of cells that been incubated under hypoxia an in O2 uptake and a significant increase in mitochondrial potential. This suggests that the membrane potential was during hypoxia, as a of a decrease in mitochondrial electron The results also that electron have increased at reoxygenation change in the rate of ATP utilization. acute hypoxia no decrease in potential. However, TMPD respiration cells been hypoxic for 2–3 suggesting that the changes in respiratory control of hypoxia to results were consistent with the changes in cytochrome oxidase function observed in submitochondrial a decrease in the apparent V max h of hypoxic incubation. cytochrome oxidase as a oxygen sensor in intact through an effect of hypoxia on the kinetic activity of the However, the of the changes in membrane potential as a in the of hypoxia is not using submitochondrial particles with TMPD and ascorbate as that changes in the kinetic function of the electron the changes in cellular respiration and membrane potential that were observed during hypoxia and In this regard, incubation of submitochondrial particles under prolonged hypoxia a reversible decrease in the rate of at of Because TMPD cytochrome c, of respiration with TMPD reflect the activity of the cytochrome oxidase that cytochrome c reduction increases as the of TMPD was increased results that prolonged hypoxia on cytochrome oxidase to a decrease in its rate at given of cytochrome c This is consistent with our that the purified oxidase undergoes a reversible decrease in its max when incubated in buffer solutions at similar of 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 physiological role for cytochrome oxidase in the cellular response to hypoxia and a mechanism by which this oxidase function as a cellular O2 results First, could cytochrome oxidase function as an oxygen sensor at than its apparent for Second, a 50% decrease in the max of the enzyme a decrease in respiration the enzyme normally functions at a of its that is shown in which is on the shown in that and the oxidase normally present in in mitochondria PubMed Scopus Google Scholar). normoxic cytochrome c in an and cytochrome oxidase its hypoxia to decrease the apparent V max of the oxidase, as by the decrease in TMPD respiration observed in intact cells the decrease in seen in submitochondrial particles incubated under hypoxia or the decrease in V for 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). a shift to this state could would However, electron flux would an increase in cytochrome c reduction and to the normoxic of O2 uptake could cytochrome c were However, a decrease in the rate of ATP utilization by the would produce the inhibited state seen after h of hypoxia = 3–5 h). We not cytochrome we an increase in NAD(P)H in cells incubated under prolonged hypoxia, which suggests that reduction of cytochrome oxidase to an of its V as by the increase in electron and O2 uptake observed in cells and submitochondrial particles during the increase in cellular TMPD respiration seen at and the decrease in NAD(P)H and the increase in mitochondrial potential seen upon changes to the shown in Cells to hypoxia undergo a increase in respiration when consistent with a of the oxidase to its state The increase in O2 uptake would an acute decrease in cytochrome c In ATP utilization would restored and the would to its normoxic state of the is that the decrease in respiration during prolonged hypoxia a decrease in the rate of ATP utilization. In prolonged moderate hypoxia a reversible decrease in respiration and a decrease in or 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), which was of a decrease in ATP utilization. We that a similar inhibition of occurs in hepatocytes during hypoxia. The results of the present study of that studies to that state ATP ATP utilization in the intact cell. is that the decrease in respiration observed during prolonged hypoxia is a of an inhibition of the oxidase, a would limit ATP and cellular ATP ATP utilization were also inhibited. from the present study consistent with the hypothesis that cytochrome oxidase as the O2 sensor during prolonged hypoxia. a that is not we that mitochondrial responses to hypoxia a reversible suppression of ATP utilization in the cell. rapidly the inhibition of cytochrome oxidase, which increases mitochondrial potential by electron ATP utilization remains inhibited. is that the changes in mitochondrial potential or cytochrome c reduction shown in in this However, of the of this and its for O2 in