(Received for publication, December 23, 1996, and in revised form, April 21, 1997)
From the Department of Medicine, Section of Pulmonary and Critical Care, the University of Chicago, Chicago, Illinois 60637
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 Vmax 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 when
N,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 Vmax
of cytochrome oxidase after hypoxic incubation. In hepatocytes loaded
with tetramethylrhodamine ethyl ester (10 nM) to quantify
mitochondrial membrane potential, acute hypoxia (<30 min) produced no
change in fluorescence, consistent with the absence of an acute change
in respiration. However, fluorescence increased during acute
reoxygenation after prolonged hypoxia, suggesting an increase in
potential. The control exhibited by NADH over mitochondrial respiration
was not altered during hypoxia. Thus, changes in the
Vmax of cytochrome oxidase during prolonged hypoxia correlate with the changes in respiration and mitochondrial potential. This suggests that the oxidase functions as an oxygen sensor
in the intact hepatocyte.
Cellular respiration is normally determined by metabolic activity and the corresponding rate of ATP utilization. Classical studies have shown that cellular oxygen uptake (VO2) remains essentially independent of oxygen tension (PO2) as long as the extracellular O2 tension exceeds a critical value ranging from 3-6 torr (1-4). Below this point, diffusion of O2 to the mitochondria begins to limit oxidative phosphorylation (5). However, in those studies the duration of hypoxia ranged from seconds to a few minutes at most, and this brief exposure may have precluded the development of an adaptive response. More recent studies have demonstrated that isolated cells undergo a significant suppression in their rate of O2 uptake when exposed to moderate hypoxia for longer periods (6, 7). This non-lethal suppression occurs at O2 tensions (PO2) between 10 and 40 torr and is rapidly reversed when the PO2 is restored to normoxic conditions. Because the decrease in respiration was not accompanied by a corresponding increase in lactate production (6, 7), the response would appear to reflect a decrease in ATP utilization rather than a mere shift in energy production from aerobic to anaerobic sources.
The mechanism responsible for the reversible decrease in respiration during moderate hypoxia has been the focus of a number of studies in our laboratory. In this regard, evidence points to a role for mitochondria as the site of cellular O2 sensing. For example, rat liver mitochondria incubated in buffer solutions at low PO2 (<2 torr) exhibited an inhibition in State 3 respiration after reoxygenation to PO2 = 20 torr, which was reversible when they were subsequently reoxygenated to PO2 = 100 torr (8). Inhibition of respiration during hypoxia also was observed when the mitochondria were given TMPD1 and ascorbate as substrates, which suggested that cytochrome oxidase function was reversibly inhibited. More recently, kinetic studies of isolated bovine heart cytochrome oxidase revealed that the apparent Vmax of the enzyme during hypoxia was reversibly inhibited by approximately 50% after incubation at PO2 = 20 torr (9). Those results suggested that O2 may interact with cytochrome oxidase, eliciting a reversible change in its catalytic function possibly through an allosteric interaction at a second binding site. Such an effect would produce a change in the reduction state of the enzyme in cells exposed to O2 tensions of approximately 20 torr for prolonged periods (i.e. >2-3 h), which in turn could allow cytochrome oxidase to function as a cellular O2 sensor 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 (10), 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 (see below).
For cells studied in suspension, approximately 1.0 × 107 cells were seeded into spinner flasks containing 250 ml of Dulbecco's modified Eagle's medium supplemented with Hepes (10 mM) and antibiotics. The media in the spinner flasks had been previously equilibrated with 21% O2, 5% CO2 or 5% O2, 5% CO2 passed through the head space in the flask. A polarographic electrode mounted in the flask was used to monitor the PO2 in the media. The spinner flasks were stirred at 60-70 rpm in an incubator regulated at 37 °C. Cellular O2 consumption rates were measured in aliquots of cells removed from the flask and studied in a magnetically stirred, water-jacketed (37 °C), anaerobic respirometer (2 ml volume) fitted with a polarographic O2 electrode. Prior to each measurement the respirometer was flushed with gas at the same PO2 used to gas the head space of the flask. Because the cell aliquots were transferred anaerobically from the spinner flask to the respirometer, contact with ambient air was prevented and the measurements of O2 uptake were obtained at approximately the same PO2 used for the incubation in the spinner flask. Mitochondrial respiration was determined as the difference between total O2 consumption and that obtained after addition of the electron transport inhibitor myxothiazol (150 ng/ml).
Changes in cellular NAD(P)H concentrations were assessed from changes
in the autofluorescence of stirred cell suspensions studied in a
spectrofluorimeter (Perkin-Elmer) at an excitation wavelength of 365 nm, an emission wavelength of 460 nm, and a slit width of 5 nm. Cells
were transferred anaerobically from the spinner flask to a quartz
cuvette (4 ml) containing a magnetic stir bar. The cuvette was filled
or emptied via a 4-cm capillary chimney and was maintained at 37 °C
in a water-jacketed holder within the spectrofluorimeter. The cuvette
had been previously flushed with 3% O2 or 15%
O2 prior to addition of cells. Different respiratory
inhibitors were added to the cell suspensions after allowing 2-5 min
for the cells to stabilize; the change in fluorescence was measured 2 min after addition. For each replicate experiment, the extent of
maximum reduction of the mitochondrial NAD(P)H pool was determined by
adding -hydroxybutyrate (20 mM) and rotenone (10 µM). Fluorescence changes are reported as percentage of
fluorescence measured during maximum mitochondrial reduction.
Cytochrome c oxidase catalytic activity was measured in whole cells in some studies and in submitochondrial particles in others. Cells or submitochondrial particles were incubated in spinner flasks at different designated PO2. Oxygen consumption rates were measured in the presence of rotenone (10 µM), myxothiazol (100 ng/ml), TMPD (0.5 mM), and ascorbate (5 mM). The former two compounds were used to inhibit autologous mitochondrial electron transport. TMPD is a electron donor which reduces cytochrome c nonenzymatically (11). Therefore, when TMPD is used as a substrate, changes in O2 uptake rates reflect changes in cytochrome oxidase activity. Ascorbate was used to reduce TMPD, which would otherwise become progressively oxidized. Submitochondrial particles were prepared from rat liver mitochondria as described previously (12). Protein concentrations were determined by the biuret procedure with bovine serum albumin as the standard.
Mitochondrial membrane potential was measured in adherent hepatocytes on glass coverslips. Approximately 105 cells in 2 ml of media were pipetted onto coverslips (25 mm) and maintained in a humidified incubator for 12 h. Coverslips were then transferred to an anaerobic flow-through chamber (Sykes-Moore, Belco Glass Co.). A thermocouple within the chamber was used to maintain the perfusate temperature at 37 °C. The chamber was clamped to a temperature-controlled block on the stage of an inverted microscope and continuously perfused with Dulbecco's modified Eagle's medium supplemented with tetramethylrhodamine ethyl ester (TMRE, 10 nM). TMRE is a cationic membrane-permeant fluorescent probe that has been shown to be distributed between cytosol and mitochondria according to Nernstian equilibrium (13). As such, it has been shown to provide a qualitative measure of mitochondrial membrane potential. Because the dye is not significantly quenched in the cell, increases in fluorescence can be used to indicate relative hyperpolarization while decreases in fluorescence can be used to indicate depolarization. Cells were superfused with the dye for 1-2 h to effect loading prior to study.
Quantitative fluorescence images of cells were acquired using a digital imaging system. An inverted microscope was equipped with a 75-watt xenon lamp for epifluorescent illumination. Changes in fluorescence were determined using an excitation wavelength of 535 nm and an emission of 610 nm. The excitation wavelength was obtained using interference and neutral density filters mounted in the light path between the source and the cells. Images were acquired with a 12-bit chilled charge-coupled device (CCD) camera (Princeton Instruments) under computer control (Metamorph, Universal Imaging).
Differences among groups were analyzed using analysis of variance, followed by Student's t statistical comparisons. Statistical significance was determined at the 0.05 level. Values are reported as means ± S.D. Replicate experiments were carried out using cells from separate isolations.
This study sought to quantify the effects of
hypoxic incubation on O2 consumption and NAD(P)H
concentrations in rat hepatocytes. Cells were maintained in suspension
in spinner flasks at PO2 = 100 or 20 torr for
5 h. Oxygen consumptions were measured in aliquots studied at
approximately the same PO2 used for incubation,
at time = 0, 1, 2, 3, and 5 h. As shown in Fig.
1, the O2 uptake at 20 torr decreased from
2.43 ± 0.82 initially to 1.39 ± 0.23 µmol/h/106 cells (p < 0.05) after 5 h at PO2 = 20 torr. By contrast, cells incubated
at PO2 = 100 torr maintained their
O2 uptake rates over the same interval. Cell viability was
unchanged from the beginning to the end of the experiment. In separate
experiments, cells were incubated at PO2 = 100 torr for 1 h, after which the oxygen tension was decreased to
PO2 = 20 torr for 3 h. An aliquot of cells
at 20 torr was then aspirated into a syringe containing 5%
CO2 and 15% O2 and rotated axially for 5 min
to restore the media PO2 to 100 torr
(acute reoxygenation). Oxygen uptake rates and NAD(P)H concentrations during that study are summarized in Fig.
2. The NAD(P)H fluorescence increased by 25.8 ± 7.0% while O2 uptake decreased from 2.57 ± 0.05 to
1.16 ± 0.31 µmol/h/106 cells during hypoxia. During
acute reoxygenation, O2 uptake and NAD(P)H fluorescence
recovered to initial values. Thus, the changes in respiratory rate and
NAD(P)H seen during prolonged hypoxia were reversible within 5 min
after reoxygenation.
Effects of Hypoxia on Cytochrome c Oxidase Catalytic Activity in Hepatocytes
This study sought to determine whether the changes in
kinetics observed for purified cytochrome oxidase (9) also occur within
intact hepatocytes during prolonged hypoxia. Hepatocytes were
maintained in suspension in spinner flasks at
PO2 = 100 or 20 torr for 5 h. Cellular
O2 uptake rates were measured at time = 0, 1, 2, 3, and 5 h at the same PO2 used for
incubation, immediately after addition of TMPD (500 µM),
ascorbate (5 mM), and rotenone (10 µM) (Fig.
3). Because TMPD supplies electrons directly to cytochrome c (1), respiration with TMPD can provide an index of maximal cytochrome oxidase activity if cytochrome c is
maximally reduced. After exposure to 20 torr for 5 h, TMPD
respiration decreased from 7.76 ± 0.57 to 4.65 ± 0.2 µmol/h/106 cells (p < 0.05). By
contrast, TMPD respiration remained unchanged in cells maintained at
100 torr for the same duration. After incubation under hypoxia for
5 h, cells were acutely reoxygenated to PO2 = 100 torr, and TMPD respiration increased to 7.39 ± 0.27, which was not different from the initial value at PO2 = 100 torr (p = not significant).
The O2 dependence of cytochrome oxidase activity was also
examined in submitochondrial particles from rat liver mitochondria. Submitochondrial particles were incubated without substrates in stirred
suspensions at different PO2 for 4 h.
Oxygen consumption rates were then assessed in aliquots studied at the
same PO2 used for incubation, after addition of
TMPD (500 µM) and ascorbate (5 mM) as
substrates. As shown in Fig. 4 (top),
submitochondrial particles incubated at PO2 < 50 torr exhibited a 43.8% decrease in cytochrome oxidase activity,
compared with those studied at higher O2 tensions. The TMPD
concentration dependence of the O2 consumption rate was
also measured in submitochondrial particles incubated at
PO2 = 100 or 20 torr for 4 h (Fig. 4,
bottom). As the concentration of TMPD was increased from 0 to approximately 100 µM there was a proportional increase
in the rate of O2 uptake, consistent with a progressive
increase in cytochrome c reduction at these low
concentrations of TMPD. At higher concentrations of TMPD the increase
in O2 uptake appeared to plateau, suggesting that
cytochrome c reduction was complete. Submitochondrial
particles incubated under hypoxia also showed a TMPD concentration
dependence of O2 uptake, but the rate was decreased by
approximately 50% at each TMPD concentration compared with the
normoxic particles (Fig. 4, bottom). Thus, cytochrome
oxidase activity in submitochondrial particles was significantly and
reversibly inhibited over a wide range of TMPD concentrations, after
exposure to prolonged hypoxia.
Effects of Prolonged Hypoxia on Mitochondrial Membrane Potential in Hepatocytes
Mitochondrial transmembrane potential reflects a balance between processes contributing to the electrochemical gradient and those tending to dissipate it. Measurement of the potential can therefore provide information regarding the relative rates of these opposing processes and insight into the processes inhibited during hypoxia. For example, if the decrease in cellular respiration during hypoxia were due solely to an inhibition of cytochrome oxidase activity without any change in ATP utilization, then a marked decrease in mitochondrial potential should occur. By contrast, if the decreases in cellular respiration were caused solely by a decrease in ATP utilization without any change in mitochondrial function then membrane potential should increase during hypoxia. To distinguish between these, we measured mitochondrial potential in intact hepatocytes superfused with normoxic or hypoxic media.
Initial studies were carried out to confirm that the fluorescent probe
TMRE behaved in the manner consistent with that expected for a
mitochondrial potentiometric dye. Hepatocytes on coverslips maintained
at 37 °C were imaged every 30 s for 12 min while the chamber
was perfused with media at PO2 = 100 torr.
Ouabain (100 µM), an inhibitor of the membrane
Na+-K+-ATPase, was added to the perfusate at
t = 5 min. Fig. 5 (top) illustrates the increase in fluorescence observed after the addition of
ouabain, consistent with the expected hyperpolarization of mitochondrial membrane potential. Fig. 5 (bottom)
illustrates the response to gramicidin D, which should decrease the
membrane potential by increasing Na+-K+-ATPase
activity and thereby increasing the supply of ADP to mitochondria. Cells given gramicidin D (1 µg/ml) at PO2 = 100 torr exhibited significant decreases in fluorescence, consistent
with mitochondrial depolarization.
A decrease in the activity of the mitochondrial ATP synthase should
result in hyperpolarization of the membrane potential. Fig.
6 (top) shows the effect of oligomycin (1 µg/ml) on TMRE fluorescence in hepatocytes during normoxia. The
increase in fluorescence that was observed is consistent with membrane
hyperpolarization. Similarly, the increase in fluorescence elicited
using carboxyatractyloside (50 µM) to block the
mitochondrial adenine transporter (Fig. 6, bottom) was
consistent with membrane hyperpolarization.
Because the mitochondrial potential is generated by electron flux, an
inhibition of electron transport would limit proton pumping and cause a
depolarization. Fig. 7 (top) shows the effect of myxothiazol (25 ng/ml) administered to cells at
PO2 = 100 torr on TMRE fluorescence. The
decrease in fluorescence is consistent with the expected decrease in
mitochondrial potential. Likewise, administration of the protonophore
carbonyl cyanide p-trifluoromethoxyphenylhydrazone (5 µM) (Fig. 7, bottom) caused a large decrease
in fluorescence, consistent with the loss of membrane potential.
Effects of Hypoxia and Reoxygenation on Mitochondrial Membrane Potential
Collectively, the above studies indicated that TMRE is
capable of providing a sensitive measure of mitochondrial potential in
hepatocytes. Accordingly, this probe was used to assess mitochondrial potential during hypoxia in two different studies. First, hepatocytes loaded with TMRE were equilibrated at PO2 = 100 torr for 1 h. The cells were then imaged every 30 s for 10 min, after which the PO2 of the perfusate was
abruptly decreased to 20 torr and the cells were imaged for an
additional 10 min (acute hypoxia). This produced no
detectable change in fluorescence (Fig. 8), suggesting that mitochondrial potential remained constant. This was consistent with the absence of change in O2 uptake or TMPD respiration
by intact hepatocytes during acute hypoxia (see Fig. 1).
By contrast, exposure to prolonged hypoxia was associated with a
decrease in O2 uptake by 3-4 h (see Fig. 1).
Unfortunately, measurement of TMRE fluorescence over the same time
frame is imprecise, due to drifts in the signal caused by additional
uptake of dye, photobleaching, or other artifacts. As an alternative,
we examined the response to acute reoxygenation to
PO2 = 100 torr in hepatocytes that had been
incubated for 3 h at PO2 = 20 torr. As
shown in Fig. 2, acute reoxygenation had been associated with a rapid
restoration of O2 consumption, indicating a reversal of the
effects of prolonged hypoxia. As shown in Fig. 9, cells
that had been perfused with media at PO2 = 20 torr for 3 h were imaged for 10 min to establish a fluorescence
baseline. Subsequently, the media PO2 was
abruptly increased to 100 torr as the cells continued to be imaged. The TMRE fluorescence increased significantly during reoxygenation, which
suggested that the mitochondrial potential had increased. Such a change
suggests that the rate of electron transport was acutely increased,
consistent with an increase in the activity of cytochrome oxidase.
Effects of Hypoxia on the Control of Mitochondrial Respiration by [NADH]
Cell processes that alter mitochondrial [NADH] produce a corresponding change in cellular O2 consumption (14). In steady state, mitochondrial [NADH] reflects a balance between "upstream" processes controlling the supply of reducing equivalents from the matrix dehydrogenases to NADH and the "downstream" steps responsible for electron transport to cytochrome oxidase and O2. The control exerted by [NADH] on cellular respiration has been described by Brown et al. (15) in terms of the relative sensitivities of the upstream and downstream arms of this metabolic pathway to changes in the [NADH]. The resulting respiratory control coefficient for NADH contains information related to the extent that [NADH] controls cellular respiration. In hepatocytes, Brown et al. (15) reported control coefficients for NADH of 0.15 to 0.30. We carried out a similar analysis to determine whether the control coefficients for NADH were altered in hepatocytes incubated at PO2 = 20 torr. Such an effect would indicate that hypoxia must influence metabolic control at multiple sites within the cell. An absence of change in the control coefficient for NADH during hypoxia would support the notion that cytochrome oxidase acts alone in this response.
To determine the sensitivity of downstream processes to [NADH],
mitochondrial [NADH] was experimentally altered by adding -hydroxybutyrate or acetoacetate, which influence mitochondrial redox states via their interconversion by the NAD+-linked
-hydroxybutyrate dehydrogenase located in the mitochondrial matrix.
Ketone bodies were added to suspended hepatocytes after incubation at
either PO2 = 100 torr or 20 torr for 4 h,
and the change in mitochondrial respiratory rate was measured. Addition of
-hydroxybutyrate to normoxic cells produced an increase in NAD(P)H fluorescence and an increase in mitochondrial respiration, whereas addition of acetoacetate produced a decrease in fluorescence and a decrease in respiration (Table I). Although cells
incubated under prolonged hypoxia exhibited increased levels of NAD(P)H fluorescence compared with normoxic cells (see Fig. 2), the responses to
-hydroxybutyrate and acetoacetate were not different between the
two groups (Table II). Thus, the suppression of cellular
respiration elicited by exposure to prolonged hypoxia did not alter the
extent to which mitochondrial [NADH]/[NAD+] ratios
control respiration at sites downstream from NADH (Fig. 10).
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An experimental intervention that alters the rate of electron transfer from NADH to O2 will elicit a change in [NADH] reflective of how the supply of reducing equivalents from the Krebs cycle to NADH is regulated. We studied this regulation in hepatocytes by measuring the changes in NADH when electron transport was partially inhibited by administering myxothiazol (50 ng/ml), or when electron transport was accelerated by adding gramicidin D (10 µg/ml) to stimulate Na+-K+-ATPase activity. The myxothiazol caused a 66% decrease in mitochondrial respiration, which elicited a 58% increase in NAD(P)H fluorescence. The gramicidin D caused a 26% increase in respiration and a 17% decrease in NAD(P)H fluorescence (Table I). The slope of the line relating change in [NAD(P)H] and the change in respiration was similar between hypoxic (0.83) and normoxic cells (0.82), suggesting that the control of electron supply to NADH was not influenced by hypoxic incubation (Fig. 10).
The above relationships were used to calculate the respiratory control
coefficient for NADH, which is the ratio of the slope relating
[NAD(P)H]/
VO2 (obtained from gramicidin
D and myxothiazol studies) to the
VO2/
[NAD(P)H] (generated by the
titration of ketone bodies). The overall control coefficient was
0.24 ± 0.03 during normoxia compared with 0.23 ± 0.03 in
hypoxia. When comparing the slope generated by myxothiazol, oligomycin,
and carboxyatractyloside to that obtained with addition of ketone
bodies, a value of 0.35 ± 0.04 was obtained during normoxia, and
a value of 0.32 ± 0.04 was found during hypoxia. Thus the overall
control coefficient of NADH over mitochondrial respiration ranged from
0.2-0.38 in both normoxic and hypoxic groups. The similar control
coefficients seen in the normoxic and hypoxic cells suggest that the
control exerted by NADH over mitochondrial respiration was not
influenced by hypoxia.
Mitochondrial respiration involves the transfer of reducing
equivalents from metabolic substrates to O2. As reducing
equivalents are shuttled through the electron transport chain, some
protons are pumped out of the mitochondrial matrix while others are
chemically consumed, thereby generating an electrochemical gradient
across the inner mitochondrial membrane consisting of a membrane
potential () and pH gradient. The ATP synthase obtains the free
energy needed for ATP synthesis from the return of protons down this gradient. Many factors are known to influence the control of
mitochondrial respiration under different conditions. For example,
models have been proposed to characterize the regulation of respiration
by [ADP] + [Pi] (16), the phosphorylation potential
(17), the supply of reducing equivalents to NADH (14), the ATP
synthase, the adenine nucleotide carrier (18), and mitochondrial
[Ca2+] (19), and the interdependence of these factors in
a multisite regulatory model (20). However, our understanding of the
regulation of respiration in intact cells during prolonged hypoxia is
still incomplete.
In the present study and in a previous report (6) we found that hepatocytes decrease their rate of O2 uptake when kept under physiological levels of hypoxia for several hours. These changes were reversed when the PO2 was restored to 100 torr and were not associated with a loss of cell viability. Moreover, lactate production by the cells did not increase significantly during prolonged hypoxia, indicating that anaerobic ATP production was not increased. Collectively, these observations suggest that hepatocytes may be capable of suppressing cellular ATP utilization at O2 tensions well above the critically low levels necessary to limit respiration. The present study sought to clarify the narrower question of whether the changes in cytochrome oxidase observed in vitro mediate the detection of O2 within the intact cell during prolonged hypoxia.
Effects of Hypoxia on the Regulation of Respiration by [NADH]To determine whether processes upstream or downstream of NADH contributed to the suppression of respiration during hypoxia, we examined the effects of hypoxia on the control exerted by NADH on mitochondrial respiration. An inhibition of the matrix dehydrogenases could decrease the supply of reducing equivalents to NADH, which could reduce O2 consumption and decrease mitochondrial membrane potential. Such a response could conceivably decrease the phosphorylation potential and thereby affect ATP synthesis. However, the data showed that NADH levels were increased in hepatocytes incubated under hypoxia, which indicated that prolonged hypoxia was associated with a change in the regulation of electron transport downstream from the NADH pool. Yet the control exerted by NADH supply over mitochondrial respiration was similar in the two groups. In both groups, the changes in [NAD(P)H] were large compared with the changes in O2 consumption, indicating that the control of respiration by [NADH] is small compared with that exerted by ATP. We therefore conclude that inhibition of matrix dehydrogenases is unlikely to contribute importantly to the hypoxic suppression of respiration. Rather, the responsible mechanism appears to reside exclusively within the mitochondrial electron transport chain downstream of NADH supply.
Mitochondrial Membrane PotentialThe mitochondrial potential provides the driving force for ATP synthesis, and its measurement can provide insight into the factors contributing to the suppression of respiration in hypoxia. Factors that increase the flux of reducing equivalents in the electron transport chain tend to increase membrane potential by augmenting proton pumping. Conversely, factors that increase the entry of protons across the inner mitochondrial membrane such as the ATP synthase or proton leaks tend to decrease mitochondrial potential. During steady state the mitochondrial potential therefore reflects a balance between the rate of electron transport and the rate of ATP utilization by the cell. If the suppression of respiration during prolonged hypoxia occurred via an inhibition of the matrix dehydrogenases or the electron transport chain without a change in the rate of ATP utilization, then mitochondrial potential would decrease. Such a depolarization could conceivably limit the rate of ATP synthesis and consequently the rate of ATP utilization. On the other hand, if hypoxia suppressed respiration by inhibiting ATP utilization at the ATPases directly, then hyperpolarization of the membrane would be predicted in response to the decrease in ADP availability.
We found that reoxygenation of cells that had been incubated under hypoxia caused an abrupt restoration in O2 uptake and a significant increase in mitochondrial potential. This suggests that the membrane potential was depolarized during hypoxia, as a consequence of a decrease in mitochondrial electron transport. The results also suggest that electron transport must have increased acutely at reoxygenation without any change in the rate of ATP utilization. Interestingly, acute hypoxia elicited no decrease in potential. However, neither basal nor TMPD respiration decreased until cells had been kept hypoxic for 2-3 h, suggesting that the changes in respiratory control required several hours of hypoxia to become apparent. These results were consistent with the changes in cytochrome oxidase function observed in submitochondrial particles, where a decrease in the apparent Vmax required 3-4 h of hypoxic incubation. These observations implicate cytochrome oxidase as a possible oxygen sensor in intact hepatocytes, through an effect of hypoxia on the kinetic activity of the enzyme. However, the significance of the changes in membrane potential as a component in the signal transduction of hypoxia is not yet known.
Cytochrome Oxidase FunctionExperiments using submitochondrial particles supplied with TMPD and ascorbate as substrates confirmed that changes in the kinetic function of the electron transport system paralleled the changes in cellular respiration and membrane potential that were observed during hypoxia and reoxygenation. In this regard, incubation of submitochondrial particles under prolonged hypoxia induced a reversible decrease in the rate of enzymatic turnover at each concentration of TMPD. Because TMPD reduces cytochrome c, measurements of respiration with TMPD reflect the activity of the cytochrome c-cytochrome oxidase complex directly. Assuming that cytochrome c reduction increases as the concentration of TMPD was increased (see Fig. 4, bottom), these results suggest that prolonged hypoxia acts on cytochrome oxidase to cause a decrease in its turnover rate at any given level of cytochrome c reduction. This observation is consistent with our previous findings demonstrating that the purified oxidase undergoes a reversible decrease in its apparent Vmax when incubated in buffer solutions at similar levels of hypoxia (9). Collectively, these observations implicate a physiological role for cytochrome oxidase in the cellular response to hypoxia and suggest a mechanism by which this oxidase can function as a cellular O2 sensor.
These results generate two important questions. First, how could
cytochrome oxidase function as an oxygen sensor at O2
concentrations much higher than its apparent Km for
oxygen? Second, why should a 50% decrease in the apparent
Vmax of the enzyme cause a decrease in
respiration if the enzyme normally functions at only a fraction of its
maximal velocity? A conceptual model that addresses these questions is
shown in Fig. 11, which is based on the experimental
data shown in Fig. 4, bottom. Note that cytochrome c and the oxidase are normally present in roughly equal
concentrations in mitochondria (11). Under normoxic conditions
(point A), cytochrome c exists in an incompletely
reduced state, and cytochrome oxidase operates well below its maximal
capacity (Vmax). Prolonged hypoxia appears to
decrease the apparent Vmax of the oxidase, as
evidenced by (a) the decrease in TMPD respiration observed
in intact cells (Fig. 3), (b) the decrease in enzymatic
turnover seen in submitochondrial particles incubated under hypoxia
(Fig. 4), or (c) the decrease in Vmax
measured for isolated cytochrome oxidase (9). If a shift to this
"hypoxic" state could occur instantaneously, O2 consumption would fall acutely (Fig. 11, top, point
B). However, continued electron flux would cause an increase in
cytochrome c reduction and tend to restore O2
uptake. Indeed, the normoxic level of O2 uptake could be
maintained if cytochrome c were sufficiently reduced.
However, a simultaneous decrease in the rate of ATP utilization by the
cell would produce the inhibited state (Fig. 11, top,
point C) seen after 3-4 h of hypoxia (see Fig. 1,
t = 3-5 h). We did not measure cytochrome c
reduction directly, but we did observe an increase in [NAD(P)H] in
cells incubated under prolonged hypoxia, which suggests that cytochrome
c reduction increased.
Reoxygenation of cytochrome oxidase appears to cause an immediate restoration of its normal Vmax, as evidenced by (a) the rapid increase in electron transport and O2 uptake observed in cells and submitochondrial particles during reoxygenation, (b) the rapid increase in cellular TMPD respiration seen at reoxygenation, and (c) the decrease in [NAD(P)H] and the increase in mitochondrial potential seen upon reoxygenation. These changes can be interpreted according to the model shown in Fig. 11, bottom. Cells adapted to hypoxia (point C) undergo a rapid increase in respiration when reoxygenated, consistent with a rapid return of the oxidase to its native state (Fig. 11, bottom, point D). The increase in O2 uptake would cause an acute decrease in cytochrome c reduction. In steady state, ATP utilization would be restored and the cell would return to its normoxic state (point A).
An assumption of the above analysis is that the decrease in respiration during prolonged hypoxia reflects a decrease in the rate of ATP utilization. In cultured cardimyocytes, prolonged moderate hypoxia caused a reversible decrease in respiration and contractile motion without a decrease in [ATP] or phosphocreatine levels (7), which was indicative of a decrease in ATP utilization. We speculate that a similar inhibition of ATP-dependent reactions occurs in hepatocytes during hypoxia. The results of the present study are supportive of that hypothesis, but additional studies will be required to test that question directly.
Under steady state conditions, ATP synthesis must match ATP utilization in the intact cell. It is unlikely that the decrease in respiration observed during prolonged hypoxia is solely a consequence of an inhibition of the oxidase, because such a limitation would limit ATP synthesis and deplete cellular ATP stores, unless ATP utilization were also inhibited. Data from the present study are consistent with the hypothesis that cytochrome oxidase acts as the O2 sensor during prolonged hypoxia. Through a signaling sequence that is not yet known, we speculate that mitochondrial responses to hypoxia initiate a reversible suppression of ATP utilization elsewhere in the cell. Reoxygenation rapidly reverses the inhibition of cytochrome oxidase, which increases mitochondrial potential by restoring electron transport while ATP utilization remains inhibited. It is conceivable that the changes in mitochondrial potential or cytochrome c reduction shown in Fig. 11 participate in this signaling sequence. However, further understanding of the details of this signaling cascade and its significance for O2 sensing in other cell types will require additional study.