From the Laboratory of Molecular Immunoregulation and
the § Laboratory of Experimental Immunology, Division of
Basic Sciences, NCI, National Institutes of Health, Frederick, Maryland
21702, the ¶ University of Pennsylvania, Philadelphia,
Pennsylvania 19104, and the
Science Application International
Corporation, Frederick, Maryland 21702
Received for publication, July 18, 2000, and in revised form, November 20, 2000
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ABSTRACT |
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Cytokines such as interleukin-3 (IL-3) promote
the survival of hematopoietic cells through mechanisms that are not
well characterized. Withdrawal of IL-3 from an
IL-3-dependent pro-B cell line induced early stress-related
events that preceded cell death by more than 40 h. Intracellular
pH rose above pH 7.8, peaking 2-3 h post-IL-3 withdrawal, and induced
a transient increase in mitochondrial membrane potential
( Interleukin (IL)1-3 is a
cytokine that acts on early bone marrow-derived hematopoietic
precursors, inducing their growth and differentiation into cells of the
myeloid, lymphoid, and erythoid lineages (1). Withdrawal of IL-3 from
dependent cell lines leads to cell death, with the activation of
caspases and DNA fragmentation as late events (2). Inhibition of cell
death is partly through up-regulation of anti-apoptotic proteins, such
as Bcl-2 and Bcl-XL (3, 4), and down-regulation of
pro-apoptotic proteins such as Bad (5). However, the metabolic changes
that serve as initiating conditions for cytokine withdrawal-induced
death are not fully understood.
Mitochondria play an essential role in many types of apoptotic death
(2) by releasing cytochrome c, which activates the caspase
cascade (6). In some studies, cytochrome c release preceded
the loss of Mitochondria produce energy in the form of ATP through oxidative
phosphorylation, a process coupled to electron transport. Enzymes of
the electron transport chain catalyze the transfer of electrons from
NADH to 02. The energy released through electron transport
drives protons across the mitochondrial inner membrane, generating an
electrochemical gradient. This distribution of protons produces both a
pH gradient (with alkaline pH in the matrix and neutral pH in the
cytosol) and a voltage gradient across the inner membrane or
The energy of this powerful electrochemical gradient drives the
catalytic activity of the F0F1-ATPase, a member
of the F class of ATP-powered proton pumps. Transport of protons back
into the mitochondrial matrix through the F0 component of
the F0F1-ATPase drives the conversion of ADP to
ATP, which is then exported to the cytosol in exchange for ADP by ANT.
In reverse, the F0F1-ATPase can use the energy
of ATP hydrolysis to pump protons out of the matrix through the inner
membrane, increasing the F0F1-ATPase can therefore synthesize or consume
ATP, depending on the membrane polarization and the concentrations of
ADP and ATP in a thermodynamic balance (16). In addition, a small inhibitory protein, IF1, which modulates the ATPase activity and proton
conductance (17, 18), is thought to confer additional regulation. IF1
is pH-sensitive, increasing its inhibitory activity by binding the
F0F1-ATPase in an acidic environment (19,
20).
In the current study, we examine aspects of mitochondrial bioenergetics
within the first few hours following withdrawal of IL-3 from a
dependent cell line. We identify a novel pathway in which intracellular
alkalinization induces mitochondrial hyperpolarization and total
cellular ATP loss, which represent significant metabolic stresses to
the cell and set the stage for later events in apoptotic cell death.
Cells and Reagents--
The IL-3-dependent murine
pro-B cell line, FL5.12A (a kind gift from James A. McCubrey, East
Carolina University, Greenville, SC), was maintained in RPMI 1640 supplemented with 10% fetal bovine serum (Life Technologies) and 0.4 ng/ml recombinant mouse IL-3 (PeproTech Inc). The
IL-7-dependent thymocyte cell line D1 was previously
described (21). The IL-3-dependent murine pro-B cell lines,
FL5.12, overexpressing Bcl-2, Bcl-XL, and the control
neomycin vector were previously described (22, 23). The wild-type and Bax
Cells were treated as described in the figure legends with the
following reagents: 50 µM CCCP (Sigma), 100 nM valinomycin (Molecular Probes), 1 µM
nigericin (Molecular Probes), 1 mM ouabain (Sigma), 1 µg/ml antimycin A (Sigma), 5 µg/ml oligomycin (Calbiochem), 200 µM DMA (Sigma), and 1 mM parathion (Chem
Service) in the same medium used for maintenance.
Measurement of Mitochondrial Membrane Potential, pH, ADP Import,
ATP Concentration, NADH Levels, and Caspase 3 Activity--
To measure
To measure changes in intracellular pH, cells (1 × 106 cells/ml) deprived of cytokine for various times were
treated with 1 µM 2',7'-bis-(2-carboxy ethyl)-5-(and
6)-carboxy fluorescein (BCECF)-acetomethyl ester (Molecular
Probes) as previously described (21, 27) and assayed by flow cytometry.
Dead cells were excluded by forward and side scatter gating. A pH
calibration curve was generated by preloading cells with 1 µM BCECF-acetomethyl ester followed by incubation
for 30 min in a high K-Hepes buffer (see below) at different pH values
from 6.8 to 8.0 in the presence of the permeabilizing agent nigericin
(10 µM).
ADP import was assayed as previously described (23). Briefly, cells
were mechanically lysed and mitochondria were isolated in isotonic
buffer (200 mM mannitol, 70 mM sucrose, 1 mM EDTA, 10 mM Hepes, pH adjusted to 6.5, 7.2, and 8.1) (21). Mitochondria were incubated for 10 min with 2.5 µCi of
[3H]ADP (Sigma) in the ADP import buffer (250 mM sucrose, 20 mM Hepes, 10 mM KC1,
5 mM succinate, 3 mM
KH2PO4, 1.4 mM MgCl2, 1 mM EGTA, 5 µM rotenone, pH adjusted to 6.5, 7.2, and 8.1) under different pH conditions. To inhibit ADP import and
measure background incorporation of radioactive counts, 50 µM carboxyatractyloside (CAT) was included in parallel
control samples. Reactions were stopped by addition of ADP import
buffer with 50 µM CAT. Mitochondria were washed twice,
and results were quantitated in a Wallac model 1409 liquid
scintillation counter.
ATP concentrations in cell suspensions (1 × 107cells/ml) deprived of cytokine for various times or
adjusted for pH were determined by the Luciferase-Luciferin method,
supplied in the ATP assay kit (Calbiochem), following the
manufacturer's protocol. ADP levels in mitochondrial protein lysates
(5 × 107 cells) were measured by converting ADP to
ATP with 4 mM phospho(enol)pyruvate (Sigma) and 5 units of
pyruvate kinase (Sigma) (23). Total mitochondrial ATP levels were then
assayed as described.
Total cellular NADH levels were determined by shifts in blue
fluorescence as measured by flow cytometry (28), because the reduced
form fluoresces, whereas the oxidized form does not. Cellular autofluorescence attributable to reduced pyridine nucleotide content was measured using a UV (350 nm) laser with emissions measured at 450 nm (29).
Caspase 3 activity was measured with a commercially available kit
(Calbiochem) using PhiPhiLuxG1D2, a peptide
substrate for caspase 3, which when cleaved emits green fluorescence
(excitation, 505 nm; emission, 530 nm). Results were assayed by flow
cytometry following the manufacturer's guidelines.
Manipulation of Intracellular pH--
To alter cytosolic pH in
living, viable cells, FL5.12A cells were permeabilized to adjust
intracellular pH to that of the extracellular buffers. Nigericin (1 µM) was used to permeabilize cells in a high K-Hepes
buffer (25 mM Hepes, 145 mM KCl, 0.8 mM MgCl2, 1.8 mM CaCl2,
5.5 mM glucose) at pH 7.0, 7.5, or 8.0 for 30 min at
37 °C. Cells were then analyzed for mitochondrial inner membrane
potential changes with JC-1. Where described under "Results," cells
were also pretreated with 5 µg/ml oligomycin for 1 h before adjusting intracellular pH.
Mitochondrial Protein Analysis--
Cell lysis was performed in
isotonic buffer (pH 7.2), and the mitochondrial-enriched pellets were
isolated as previously described for the ADP import assay (21). For
detection of Bax and cytochrome c oxidase protein by Western
blot, cell equivalent samples (10 µl containing ~20 µg of
protein) were separated by SDS-polyacrylamide gel electrophoresis on
12% Tris-glycine gels (Invitrogen) and transferred to 0.2 µM polyvinylidene difluoride membranes (Invitrogen). Blots were probed with a rabbit polyclonal antiserum specific for the
amino-terminal of Bax (N20, Santa Cruz) or with a mouse polyclonal
antibody specific for cytochrome c oxidase (Research Diagnostics), followed by the appropriate secondary antibodies conjugated to horseradish peroxidase (Santa Cruz) and then visualized by enhanced chemiluminescence (Pierce) following the manufacturer's protocol.
Increase in Mitochondrial Membrane Potential Following Cytokine
Withdrawal--
Respiring mitochondria generate a proton gradient
across the inner membrane, producing a pH gradient and a membrane
potential or
To quantitate the red fluorescence of cells that had taken up JC-1, we
employed flow microfluorimetry throughout this study. Initially using
fluorescent microscopy, we verified that this red fluorescence emanated
from mitochondria (not shown). Thus, in a neutral pH solution, FL5.12A
cells contained a few dots of red fluorescence against a diffuse
greenish orange fluorescence; others have correlated this pattern to a
Using the pro-B cell line FL5.12A, which is dependent on IL-3 for
survival, we examined early events following cytokine withdrawal. Deprivation of IL-3 resulted in death by 48 h with no cells
surviving beyond 72 h. Fig.
1A, a two-dimensional display
of JC-1 red fluorescence (y axis, 590 nm) versus
green fluorescence (x axis, 527 nm), illustrates the
quantitative changes in
The uncoupling agent CCCP was included as a depolarization control to
confirm the capacity of the JC-1 dye to measure
To verify that JC-1 accurately measured changes in
To determine whether alterations in Mitochondrial Hyperpolarization Is Independent of Caspases and the
Bcl-2 Family of Proteins--
We recently reported that following IL-3
or IL-7 withdrawal, intracellular alkalinization triggered the
translocation of the pro-apoptotic protein, Bax, to mitochondria (21).
To address a potential role for Bax in the observed hyperpolarization,
FL5.12A cells were treated with Bax antisense oligodeoxynucleotides
before IL-3 withdrawal; no inhibitory effect was seen on mitochondrial hyperpolarization occurring 2-3 h post-withdrawal. The same Bax antisense oligodeoxynucleotides promoted survival following cytokine withdrawal (not shown). Bax was evaluated further by isolating pro-T
cells (CD3
We next addressed whether other apoptosis regulators, caspases, Bcl-2,
or Bcl-XL, could inhibit mitochondrial hyperpolarization during cytokine withdrawal. Shown in Fig.
2A (in histograms displaying the intensity of red fluorescence) ZVAD-FMK, a caspase
inhibitor, did not prevent the increase in Intracellular Alkalinization Induces Mitochondrial
Hyperpolarization--
The early increase in
We next evaluated whether increased
Previously, we had examined the D1 and FL5.12A cells for other early
intracellular events induced by IL-7 or IL-3 withdrawal and detected a
rise in cytosolic pH (21), using the pH indicator BCECF (33). As shown
in Fig. 3B, this rise in intracellular pH measured in the
FL5.12A cells followed a parallel time course to that of the increase
in
Because these two early, transient events, the increase in
To confirm that the JC-1 dye was in fact measuring increased
An Oligomycin-sensitive Mechanism Causes Mitochondrial
Hyperpolarization in Response to Alkalinization: Implication of the
F0F1-ATPase--
To determine how alkaline pH
induced the rise in
The rise in intracellular pH mediated by cytokine withdrawal is due to
activation of the Na+/H+
exchanger,3 which is
inhibited by DMA. As shown in Fig. 4B, DMA lowered the intracellular pH to 6.5 (as measured by incorporation of BCECF) and
prevented the rise in
Having proven that oligomycin prevented alkaline-pH-induced
hyperpolarization, the results displayed in Fig. 4C indicate
that oligomycin was also effective in inhibiting the rise in
The effect of oligomycin on
The ionophore, nigericin, when added during IL-3 withdrawal, also had
no appreciable effect on Alkalinization Inhibits ADP Transport into Mitochondria--
The
increase in A Transient Decrease in ATP and Accumulation of Pyridine
Nucleotides Follows Cytokine Withdrawal--
Mitochondria are the
major source of cellular ATP. From the preceding findings, following
cytokine withdrawal, mitochondrial production of ATP should decline if
the substrate, ADP, were reduced and if the major ATP producer,
F0F1-ATPase, were hydrolyzing rather than
synthesizing ATP (17). Total intracellular ATP did decline sharply as
shown in Fig. 5A. From ~1 to 3 h after IL-3
withdrawal, there was a large decrease (4-5-fold) in ATP
concentration; this decrease coincided with the period of mitochondrial
hyperpolarization (Fig. 3A) and intracellular alkalinization
(Fig. 3B).
To determine how much of the ATP decline was due to alkaline pH, we
tested the effect of adjusting intracellular pH (in the presence of
IL-3) using the nigericin and high K+ buffer (145 mM KCl) method. This verified that alkaline intracellular pH could induce ATP depletion as shown in Fig. 5B. Note that
the ATP depletion induced by alkalinization is less severe than that induced by IL-3 withdrawal alone (Fig. 5A); this difference
could be attributed to the length of incubation time used to adjust intracellular pH with nigericin (30 min) versus withdrawal
IL-3 withdrawal (3 h). The longer incubation time would permit the effects of ADP transport inhibition to produce a larger decline in
total ATP levels. Ouabain was also tested (data not shown) because it
inhibits the plasma membrane K+/Na+-ATPase,
which can be a major consumer of ATP, but it failed to restore ATP
levels in either IL-3-withdrawal or pH-induced conditions. Treatment
with oligomycin during cytokine withdrawal could only restore the ATP
levels up to 20% of normal (data not shown); this suggests that most
of the ATP decrease could result from failure to synthesize ATP
(because of lack of ADP as a substrate), in addition to the actual
consumption of ATP by the F0F1-ATPase.
Hyperpolarization of the
Hyperpolarization of the Parathion Induces Mitochondrial Hyperpolarization and Cell Death
Independent of Bax, Bcl-2, or Caspases--
We then evaluated whether
an increase in
Because the mitochondrial translocation of Bax is an early event
induced by cytokine withdrawal (21), we tested whether mitochondrial
hyperpolarization could "attract" or "repel" Bax. Parathion,
which induced hyperpolarization, did not inhibit Bax translocation to
the mitochondria during IL-3 withdrawal and, in fact, induced Bax
translocation in the presence of IL-3 (Fig. 7B). Although
this might suggest that Bax is attracted by hyperpolarized mitochondria, other experimental approaches (not shown) suggest that
the two are unrelated; moreover, Bax is thought to insert into the
outer membrane, whereas the above effects are on the inner membrane.
After 24 h of treatment, parathion, in the presence of IL-3,
induced mitochondrial depolarization (data not shown), caspase 3 activation (Fig. 7C), and cell death (Fig.
7D).
Parathion-mediated cell death was not attributable to a mechanism
involving Bax or Bcl-2, based on its effect on cells deficient in the
former or overexpressing the latter (Fig. 7D). Bcl-2 did, however, protect from caspase 3 activation (Fig. 7C) as it
also did following IL-3 withdrawal (Fig. 2C). Therefore, the
results using parathion suggest that mitochondrial hyperpolarization
could lead to cell death, although it is also quite possible that
parathion has toxic effects in addition to induction of hyperpolarization.
In this study, we show that withdrawal of cytokines from dependent
cell lines induces several early interconnected processes. Within 2-3
h after IL-3 withdrawal, intracellular pH rose to over pH 7.8. This
alkalinization inhibited mitochondrial ADP import, which could cause a
reversal of the F0F1-ATPase, inducing
hyperpolarization of the inner mitochondrial membrane, decrease of ATP,
and the oxidation of NADH. None of these early events involved the
Bcl-2 family of proteins or caspases. These phenomena were not unique to IL-3, because similar responses (alkalinization and
hyperpolarization) occurred in IL-7-dependent cells. We
suggest that these events represent significant stresses to the cell
following cytokine withdrawal, rendering the mitochondria and other
cellular components vulnerable to damage leading to cell death.
Changes in mitochondrial membrane integrity immediately precede
apoptotic death (35). Some studies have described disruption of the
mitochondrial membrane concurrent with cytochrome c release and formation of the PT pore. The sequence of these late events include
expansion of the matrix, rupture of the outer membrane, and the release
of caspase-activating proteins (36). A previous study using rhodamine
123 concluded that a form of mitochondrial hyperpolarization shortly
preceded the late events of swelling and rupture of the outer membrane
(18 h after cytokine withdrawal). This was attributed to the energy of
the proton gradient not being dissipated by ATP synthesis, producing a
hyperpolarized state (41). Bcl-XL countered this process
(22, 36). A more recent study of p53-induced apoptosis demonstrated
that the generation of ROS led to a transient increase in
In our study, using JC-1 (and other dyes), although confirming that
mitochondria depolarize just prior to their disruption (Fig. 1) and the
activation of caspases (Fig. 2C), we now describe a very
early increase in In addition to Bcl-2 and Bcl-XL protecting mitochondria
from damage (12, 22), the pro-apoptotic protein Bax can induce formation of the PT pore and cytochrome c release (11),
perhaps through Bax associating with VDAC and ANT (40-42). However, in our experiments, we found no requirement for Bax in the early hyperpolarization of the The mitochondrial hyperpolarization and ATP depletion that we observed
following IL-3 withdrawal could be due to high intracellular pH
blocking the import of ADP, in turn causing the mitochondrial F0F1-ATPase to reverse. The
F0F1-ATPase is a protein complex comprised of
two reversible rotary motors (43). The F1 subunit can
normally synthesize ATP or, when reversed, hydrolyze ATP. An additional level of control exists in that a natural inhibitor protein, IF1, which
is pH-sensitive, also regulates the
F0F1-ATPase. IF1 can prevent the unneeded
hydrolysis of ATP, as could occur in the absence of oxygen when
glycolysis becomes the main source of ATP (17, 44). Although IF1 may
partly account for the hypothesized effect of pH on
F0F1-ATPase, the more important control could be the limited availability of ADP. ADP import (and ATP export) is
primarily through the VDAC/ANT complex. In a recent study, growth
factor withdrawal caused the VDAC/ANT complex to stop ATP/ADP exchange
after 12-18 h of IL-3 withdrawal (23). Loss of outer membrane
permeability to anions was later implicated in VDAC closure (45). That
is distinguished from the process we describe here, in that the
deficiency in ATP/ADP exchange shown in those studies was inhibited by
Bcl-XL (which also prevented disruptions in the As a result the elevated electrochemical gradient or
How do these early events during cytokine withdrawal relate to the late
events of apoptosis, such as mitochondrial disruption and caspase
activation? To address this, we induced mitochondrial hyperpolarization
with the organophosphorus compound, parathion (34). Parathion induced
cell death, with mitochondrial disruption, but this death did not
require the translocation of Bax or the activation of caspases and was
not protected by Bcl-2; thus, mitochondrial hyperpolarization per
se could lead to cell death, independent of the apoptotic cascade
(Fig. 7).
In conclusion, we show that long before the morphological changes of
apoptosis occur, cells deprived of their trophic factors have
compromised mitochondrial function. Although these processes were
transient and reversible (cells could still be rescued by readdition of
the cytokine), this disruption of essential cellular activities could
contribute in time to the sequelae ending in cell death.
m) detected using several different dyes. Similar events were observed following IL-7 withdrawal from a different
dependent cell line. Bcl-2, Bax, and caspases were unrelated to these
early events. Intracellular alkaline pH inhibited the mitochondrial
import of ADP, which would limit ATP synthesis. Total cellular ATP
sharply declined during this early period, presumably as a consequence
of suppressed ADP import. This was followed by an increase in reduced
pyridine nucleotides. The transient increase in
m was
blocked by oligomycin, an inhibitor of
F0F1-ATPase that may have undergone reversal
caused by the abnormal ADP-ATP balance within mitochondria. These
findings suggest a novel sequence of early events following trophic
factor withdrawal in which alkaline pH inhibits ADP import into
mitochondria, reversing the F0F1-ATPase, which
in turn consumes ATP and pumps out protons, raising
m.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
m (7), whereas other studies reported the
reverse, cytochrome c release following mitochondrial
swelling, outer membrane rupture, and loss of
m (8,
9). Mitochondrial swelling is related to formation of the permeability
transition (PT) pore, a complex that contains the
voltage-dependent anion channel (VDAC), and the adenine
nucleotide translocator (ANT) (9, 10) in addition to other cytosolic
and mitochondrial proteins. The Bcl-2 family of anti- and pro-apoptotic
proteins are modulaters of these processes (11, 12). However,
mitochondrial swelling, loss of
m and induction of
the PT pore were all reversible events in osteosarcoma cells (13).
Likewise in neuronal apoptosis, mitochondrial membrane depolarization
was not fatal (14) nor was it a factor in the death of HL-60 cells
induced by actinomycin-D, etoposide, or staurosporine (15). The
progression to cell death, therefore, depends on the integration of
mitochondrial processes that cannot be readily dissected.
m, the result of a net outflow of positive ions (with
negative charges inside and positive charges outside; Ref. 16). The
electrochemical proton gradient across the membrane of a respiring
mitochondrion generates a potential of 200 mV, of which 60 mV results
from the pH gradient (of about one pH unit; Ref. 16).
m.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
macrophage cell lines, maintained in Dulbecco's
modified Eagle's medium (Life Technologies, Inc.) supplemented with
10% fetal bovine serum, were derived from spleen cells isolated from
Balb/c mice and C57BL/6-Baxtm1Sjk mice (Jackson
Laboratory) and immortalized using acutely transforming retroviruses
(24).
m, cells (1 × 106 cells/ml)
deprived of cytokines for various times, treated with reagents
(described above), or adjusted for pH were resuspended in 5 µg/ml
solution of JC-1 (Molecular Probes) in phosphate buffered saline (25).
Because accumulation of the JC-1 dye is reversible, cells were
maintained in a stable concentration of the dye throughout the time of
measurement. After 20-min incubation at 37 °C, cells were
immediately analyzed by flow cytometry. Dead cells were excluded by
forward and side scatter gating. Data were accumulated by analyzing an
average population of 20,000 cells. JC-1 aggregates were detectable in the propidium iodide channel (red fluorescence, emission at 590 nm), and JC-1 monomers were detectable in the fluorescein
isothiocyanate channel (green fluorescence, emission at 527 nm)
(26).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
m. The
m represents
most of the energy of the proton gradient, and using the fluorescent
carbocyanine dye JC-1 (25, 30, 31), others have measured this
potential. Lipophilic cations such as JC-1 accumulate in the
mitochondrial matrix driven by the electrochemical gradient following
the Nernst equation. The higher the
m (the more
polarized the mitochondrial membrane), the more JC-1 dye is taken up
into the matrix. In the cytosol, the monomeric form of the dye
fluoresces green (emissions read at 527 nm), whereas highly
concentrated within mitochondrial matrix, JC-1 forms aggregates that
fluoresce red (emissions read at 590 nm) (32).
m of 140-160 mV (26). In an alkaline pH solution, we
observed a dramatic increase in punctate red fluorescence (with no
green fluorescence) reported to indicate a
m of
greater than 190mV (26). Similar studies concluded that green
fluorescing mitochondria were equivalent to a
m of
less than 100 mV (26). Thus, a higher intensity of red fluorescence
would indicate a higher
m (hyperpolarization), whereas loss of red and increased green fluorescence would indicate a
reduced
(depolarization).
m that occur following IL-3
withdrawal. At 0 h, in the presence of IL-3, cells display a
base-line level of red fluorescence that would represent normal
mitochondrial membrane polarization. However, by 3 h after IL-3
withdrawal, when cells remained >90% viable, a rise in
m was observed. This was indicated by an increase in
the number of cells in the upper two quadrants, totaling 18.8% at time
0, versus 62.8% at 3 h (Fig. 1A). This peak
of hyperpolarization was transient, in that 5 h after IL-3
withdrawal, the
m had decreased (36.2% in the
upper two quadrants) and further declined at later time
points. Note that there were no fluctuations in green fluorescence at
the 0- and 3-h time points. This is indicated by the sum of cells in the two right quadrants, 19.8% at 0 h
versus 20.8% at 3 h, showing that cellular uptake of
the JC-1 dye did not change during this period.
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Fig. 1.
Mitochondrial membrane potential detected
using JC-1 fluorescence following IL-3 or IL-7 withdrawal.
A, FL5.12A cells were incubated in medium lacking IL-3 for
0, 3, 5, 20, and 40 h or treated for 30 min with CCCP (50 µM) and assayed with JC-1 (5 µg/ml). In the top
panels, at 0 h, peak red fluorescence was 30.8; at 3 h,
peak red fluorescence was 77.4; and at 5 h, peak red fluorescence
was 40.9. In the lower panels, by 20 h, depolarization
of the mitochondrial inner membrane began (seen as increasing green
fluorescent cells), maximizing at 40 h post-IL-3 withdrawal. Cells
treated with CCCP underwent depolarization, confirming the capacity of
the JC-1 probe to detect mitochondrial membrane potential changes.
Shown is a representative example of six similar experiments.
B, D1 cells were incubated in medium lacking IL-7 for 0, 4, or 6 h, then assayed with JC-1 (5 µg/ml). An increase in the red
fluorescence emitted by the cell population was observed 4 h
post-IL-7 withdrawal. Three similar experiments were performed.
Quantification was determined by flow cytometry analysis, as described
under "Experimental Procedures." Lines defining
quadrants were arbitrarily defined to highlight changes in the
fluorescence of the cell populations. Percentages represent cell number
in each quadrant. On the y axis, in log scale from 0.1 to
1000, red fluorescence (read in the propidium iodide channel,
emissions at 590 nm) indicates the mitochondrial uptake of the JC-1 dye
and the formation of J-aggregates within the matrix. On the
x axis, in log scale from 0.1 to 1000, green fluorescence
(read in the fluorescein isothiocyanate channel, emissions at 527 nm)
indicates cytosolic retention of the JC-1 monomers.
m changes and shows that depolarized cells appear in the lower
right quadrant in this display (Fig. 1A). Following
IL-3 withdrawal, depolarized cells (lower right quadrant)
increased to 31.2% at 20 h and 61.5% at 40 h. The number of
depolarized cells approximated the number of dead cells at these time
points (30-40% at 20 h and 60-70% at 40 h). IL-3
withdrawal, therefore, resulted first in an early, transient increase
in
m (hyperpolarization) peaking at 3 h,
followed later by depolarization that began at 20 h. The early
hyperpolarization phase has not been described previously and was
examined further in this study.
m,
we evaluated other fluorescent dyes. Shown in Table
I is a representative experiment in which
the dyes DiOC6, Rhodamine 123, and JC-1 equally demonstrate
their capacity for measuring changes in
m following IL-3 withdrawal. Numbers in Table I represent the peak fluorescence intensity at the optimal emission wavelength for each dye. All three
dyes detected the increase in
m peaking 2-3 h after
IL-3 withdrawal; therefore, the rise in
m was not an
artifact of an individual dye. Because JC-1 allows dual emission
detection for both the cytosolic monomeric form of the dye and the
aggregated mitochondrial form of the dye, we favored it for subsequent
analysis.
Comparison of dyes used for the detection of changes in the
mitochondrial membrane potential
m were a general
consequence of cytokine withdrawal, we evaluated changes in
m in an IL-7-dependent thymocyte cell
line, D1. Shown in Fig. 1B, D1 cells exhibited a pattern
similar to that seen in the FL5.12A cells. Hyperpolarization of
m, observed as an increase in the red fluorescing
cell population, was a transient and early event occurring 4-5 h after
IL-7 withdrawal (Fig. 1B). In addition, mouse pro-T cells
(CD3
CD4
CD8
) responsive to
IL-7 also showed increased
m following cytokine withdrawal.2 Therefore, the
rise in
m we describe is not a phenomenon unique to
the IL-3-dependent FL5.12A cell line.
CD4
CD8
) from
Bax
/
mice and testing for changes in
m following IL-7 withdrawal. Pro-T cells lacking Bax
did indeed hyperpolarize the mitochondria.2 Therefore, we
conclude that translocation of Bax to the mitochondria is not required
for the rise in
m induced by cytokine withdrawal.
m,
although it effectively blocked the later activation (24 h) of caspase
3 (Fig. 2C). Therefore, caspases do not participate at this
early stage in the mitochondrial hyperpolarization process but are
initially activated 20-24 h post-IL-3 withdrawal (Fig. 2C).
Overexpression of the anti-apoptotic proteins Bcl-2 or
Bcl-XL also did not interfere with the
rise following IL-3 withdrawal (Fig.
2B). Hence, these anti-apoptotic proteins repress late
apoptotic events, such as mitochondrial depolarization (data not shown)
and caspase 3 activity (Fig. 2C), but not the transient
increase in
m.
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Fig. 2.
ZVAD-FMK or the overexpression of Bcl-2 and
Bcl-XL do not inhibit mitochondrial hyperpolarization, but do prevent
the later activation of caspase 3. A, FL5.12A cells
were cultured with or without IL-3 for 3 h and 100 µM ZVAD-FMK. Cells were then stained with JC-1 and
assayed by flow cytometry. Histograms display peak red fluorescence on
the x axis in log scale from 0.1 to 1000, indicating the
formation of J aggregates within the mitochondria. Shown is a
representative example of three similar experiments. B,
FL5.12A cells expressing the empty neomycin vector (vector), Bcl-2, or
Bcl-XL were cultured with or without IL-3 for 3 h.
Cells were stained with JC-1, as previously described and assayed by
flow cytometry. Results displayed in histograms as in A.
Shown is a representative example of two similar experiments.
C, FL5.12A cells expressing the empty vector
(Vector) were cultured with IL-3 (+) or without IL-3 ( ) in
the absence or presence of 100 µM ZVAD-FMK for 24 h.
Likewise, cells expressing Bcl-2 or Bcl-XL were cultured
for 24 h with (+) or without (
) IL-3. Activation of caspase 3 was measured by cleavage of fluorescent substrate, and the results were
measured by flow cytometry as described under "Experimental
Procedures." Results shown are the means and ranges of
duplicates.
m
following cytokine (IL-3 or IL-7) withdrawal has not been reported
previously; therefore, its time course was analyzed further
using the FL5.12A cell line. In Fig. 3A, mitochondrial
hyperpolarization (indicated by increased intensity of red
fluorescence) peaked 2-3 h after IL-3 withdrawal. To relate JC-1
fluorescence to the actual mitochondrial membrane potential, other
studies using JC-1 (26) reported a base-line range of 140-160 mV,
which, if extrapolated linearly, would indicate that the
hyperpolarization we observe is greater than 190 mV.
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Fig. 3.
Transient mitochondrial hyperpolarization and
intracellular alkalinization occur in parallel after IL-3
withdrawal. A, FL5.12A cells were incubated with or
without IL-3 for 6 h, and cells were assayed with JC-1 every hour
as described under "Experimental Procedures." Peak red
fluorescence, indicating hyperpolarization of the mitochondrial
membrane, occurred 2-3 h post IL-3 withdrawal. Control cells cultured
in the presence of IL-3 did not hyperpolarize. A representative example
of four similar experiments is shown. B, FL5.12A cells were
incubated with or without IL-3 for 6 h and assayed for cytosolic
pH with BCECF every hour as described under "Experimental
Procedures." Intracellular alkalinization occurred 2-3 h post-IL-3
withdrawal. Cells cultured in IL-3 maintained neutral pH. Intracellular
pH was determined by flow cytometry analysis using a pH calibration
curve generated from cells incubated with nigericin in buffers at pH 7, 7.5, and 8 (data not shown). Shown is a representative experiment; six
similar experiments were performed.
m reflected a
change in the plasma membrane polarization. However,
DiBac4, a fluorescent probe used to measure plasma membrane
potential, did not detect changes following cytokine withdrawal greater
than those observed by simply adding fresh medium (not shown).
Therefore, the rise in
m following cytokine
withdrawal does not correlate with an increase in the polarization of
the plasma membrane.
m seen in Fig. 3A. We also reported a
similar time course of alkalinization following IL-7 withdrawal from D1
cells, with a peak pH rise occurring 4-6 h post-withdrawal (21),
paralleling the rise in
m (Fig. 1B).
m and intracellular alkalinization, occurred together
following IL-3 or IL-7 withdrawal, we tested whether alkaline pH
induced mitochondrial hyperpolarization. Using nigericin (a
K+/H+ ionophore) and a high K+
buffer (145 mM KCl) to equilibrate intracellular pH to that
of the extracellular buffer, we adjusted the intracellular pH of FL5.12A cells to 7.0, 7.5, or 8.0. This method effectively fixes pH (as
confirmed by measurement of intracellular pH; data not shown) in the
presence or absence of IL-3. It should be noted that nigericin
dissipates the pH gradient of membranes and can cause a slight
hyperpolarization in response to the flux of K+ ions.
However, in our manipulation of pH with nigericin, this effect is
negligible as is demonstrated in further control experiments. As shown
in Fig. 4A (upper
panels), elevating intracellular pH to 7.5 or 8.0 induced a rise
in
m comparable with that seen during IL-3
withdrawal. No further increase in
m occurred beyond pH 8, possibly because of the effect of nigericin on plasma membrane permeability, which at higher doses limits the concentration of cytosolic JC-1 monomers (data not shown).
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Fig. 4.
Mitochondrial hyperpolarization is induced by
alkaline pH, a process inhibited by oligomycin. Additionally,
prevention of alkalinization with DMA or readdition of IL-3 also
inhibited hyperpolarization. Histograms, displaying peak red
fluorescence intensity on a log scale from 0.1 to 1000, are shown.
A, FL5.12A cells were treated for 30 min with nigericin (1 µM) in high K-Hepes buffers at pH 7.0, 7.5, and 8.0 as
described under "Experimental Procedures." Cells were stained with
JC-1 and assayed by flow cytometry. Hyperpolarization occurred only in
cells adjusted to alkaline cytosolic pH (7.5 or higher); results are
shown in the upper panels. In the lower panels,
cells were pretreated for 1 h with oligomycin (5 µg/ml), and
then the pH was adjusted as described above. Cells pretreated with
oligomycin did not hyperpolarize in response to alkaline intracellular
pH. Three similar experiments were performed. B, FL5.12A
cells were deprived of IL-3 for 3 h (( ) IL-3) or were
treated (during withdrawal) with 200 µM DMA
((
)IL-3 + DMA); results are shown in the left
panel. In the right panel, readdition of IL-3 after
2 h of withdrawal reversed the hyperpolarization measured 1 h
later. Intracellular pH was determined using the pH-sensitive
fluorescent probe, BCECF (results shown in parentheses).
Results are displayed in histograms as in A. Shown is a
representative example of two experiments performed. C,
FL5.12A cells were either maintained in IL-3 ((+) IL-3) or
deprived of IL-3 for 3 h ((
) IL-3) during which time
they were also treated with oligomycin (5 µg/ml), valinomycin (100 nM), or nigericin (1 µM). Cells were also
treated with antimycin A (1 µg/ml) for 30 min. After incubation,
cells were stained with JC-1 and assayed by flow cytometry as described
under "Experimental Procedures." Results are displayed in
histograms as in A. Shown is a representative sample of two
such experiments performed.
m versus directly responding to pH, we
assayed the effect of pH on JC-1 in a cell-free solution. Using JC-1
(300 nM) in a buffer containing mannitol, sucrose,
succinate, and Mg+-ATP (31), the effect of pH 6.5-9.0 on
fluorescence was measured by fluorimetry. The red fluorescence of JC-1
(emissions read at 590 nm) was decreased at acidic pH, was maximal at
neutral pH, and did not increase further at alkaline pH. The green
fluorescence of JC-1 was not pH-sensitive. Thus, alkaline pH does not
directly increase the red fluorescence emission of JC-1 but rather
increases the uptake of JC-1 into mitochondrial matrix in response to a heightened
m.
m, we evaluated a possible role
for the F0F1-ATPase, a protein complex spanning
the inner mitochondrial membrane. As shown in Fig. 4A (lower panels), oligomycin, a specific inhibitor of
F0F1-ATPase, completely prevented the increase
in red fluorescence or hyperpolarization induced by pH 7.5 or 8.0. This
implicated the F0F1-ATPase, in the mode of
reverse proton pumping, as the effector of the rise in
m. This experiment also served as an additional
control showing that the JC-1 dye does not directly respond to pH but
rather requires an oligomycin-sensitive process.
m. These results not only
confirmed the role of alkaline pH in altering
m, but
also demonstrated the depolarizing effect that acidifying the cells (by
inhibiting the NHE) had in contrast to alkalinization (which induces
hyperpolarization). For comparison, an experiment demonstrating that
readdition of IL-3 reverses
m hyperpolarization is
also shown (Fig. 4B).
m following IL-3 withdrawal. In the presence of IL-3
(i.e. normal cells), oligomycin produced a slight
hyperpolarization, a consequence of inhibiting the dissipation of the
proton gradient and ATP synthesis by the
F0F1-ATPase (Fig. 4C). Note that
this increase in
m caused by oligomycin was not equal
in magnitude to that induced by either alkaline pH (Fig. 4A)
or cytokine withdrawal (Fig. 4C). Thus, the increased
m we describe is not solely due to the inhibition of
ATP synthesis but requires an additional active process, likely the
F0F1-ATPase pumping protons in reverse across
the inner mitochondrial membrane.
m is distinct from that
of compounds that directly inhibit the electron transport chain (the generator of the proton gradient) and in this manner deplete ATP levels. For example, treatment with antimycin A (which blocks electron
transport) caused a rapid loss of
m or depolarization (shown by the decrease in red fluorescence) similar to that caused by
valinomycin, which induces mitochondrial swelling and depolarization (Fig. 4C), and the uncoupler, CCCP (Fig. 1A).
m (Fig. 4C) as
was shown by others (30), nor did incubation in high K+
buffers (145 mM KCl) used to adjust intracellular pH (data
not shown). Because nigericin dissipates the pH gradient but induces an
ion flux across membranes, it can cause a slight hyperpolarization when
added to healthy cells. However, this was only observed at higher
concentrations of nigericin (>10 µM; data not shown) and not at the 1 µM range we used in our experiments (Fig.
4C). This suggests that the effect of nigericin on the
mitochondrial pH gradient would be minor in comparison with the impact
high intracellular alkalinity would have following cytokine withdrawal.
m was inhibited by oligomycin, suggesting
that it was caused by the F0F1-ATPase working
in reverse, hydrolyzing ATP and pumping protons outward across the
mitochondrial membrane. However, several conditions under which the
F0F1-ATPase was known to reverse, for example
low
m or oxygen deprivation, were not observed during
IL-3 withdrawal or alkalinization. The concentrations of mitochondrial
ADP and ATP can affect the direction of
F0F1-ATPase function. Therefore, we examined
the mitochondrial levels of ADP in response to pH. Shown in Table
II, is an experiment in which mitochondrial protein lysates were extracted from cells whose intracellular pH was fixed with nigericin and high K+
buffer (145 mM KCl) at pH 6.5, 7.2, and 8.1 for 15-20 min.
Alkaline pH induced a steep decline in mitochondrial ADP and a modest
rise in mitochondrial ATP. To explain this, we next measured the effect of pH on the import of ADP into isolated mitochondria. At pH 8.1, [3H]ADP import decreased 5-fold compared with pH 6.5 and
4-fold compared with pH 7.2 (Fig.
5A). Thus, the extreme
depletion of mitochondrial ADP could explain the reversal of the
F0F1-ATPase, inducing hyperpolarization. To
evaluate whether the alkalinization effect could be mimicked by
blocking the ADP/ATP exchanger ANT, cells were treated with CAT, an
inhibitor of a high affinity ADP binding site on ANT, and assayed for
changes in
m. After 1-4 h of treatment
with CAT (dose range from 50 µM to 1 mM), no
increases in
m were observed; in fact, a decrease in
membrane polarization occurred, possibly a result of CAT inducing PT
pore formation (data not shown). Thus, although both CAT and alkalinity
block ADP import, the pH effect on membrane polarization is not
mimicked by CAT for unknown reasons. The block in ADP transport induced by alkalinity could be due to alterations in the charge of adenosine nucleotides, affecting their transport, or could result from changes in
mitochondrial protein conformation and function (i.e. VDAC channel activity). Nevertheless, by whatever means alkaline pH inhibits
ADP transport, the intramitochondrial deficit in ADP may induce the
reversal of the F0F1-ATPase.
Mitochondrial ATP and ADP levels change according to intracellular pH
adjustment
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Fig. 5.
Alkaline pH blocks ADP import into
mitochondria and results in a decrease in total cellular ATP.
A, FL5.12A cells were lysed in isotonic buffers at pH 6.5, 7.2, and 8.1, and mitochondria were isolated as described under
"Experimental Procedures." Transport of [3H]ADP into
mitochondria, incubated in import buffer at pH 6.5, 7.2, and 8.1 was
measured by incorporation of radioactivity. Backgrounds from
CAT-treated samples were subtracted. Mitochondrial import of ADP
declined at alkaline pH, whereas it increased at acidic pH.
B, FL5.12A cells were incubated with or without IL-3 for
6 h, and cells were assayed every hour for ATP by the
Lucerifase-Luciferin method. An ATP standard curve was generated using
known concentrations of ATP according to manufacturer's protocol. In
cells deprived of IL-3, ATP levels rapidly decreased 1-3 h
post-withdrawal, and increased by 4 h post-withdrawal. ATP levels
did not vary significantly in cells cultured in the presence of IL-3.
C, FL5.12A cells were treated for 30 min with nigericin (1 µM) in high K-Hepes (145 mM KCl) buffers at
pH 7.0, 7.5, and 8.0 as described under "Experiemental Procedures."
Cells were then assayed for ATP. Alkaline pH (>pH 7.5) induced
depletion of ATP levels. Results are shown in duplicate. Error
bars represent standard error of the mean. Shown are
representative examples of two similar experiments performed.
m did not result in the
immediate generation of ROS as measured by the levels of superoxide
(O
m, however, could
potentially interrupt mitochondrial respiration. To evaluate this, we
measured the levels of NADH, an early substrate used in the
electron-transport process, coupled to oxidative phosphorylation, to
synthesize ATP. The level of NADH in cells (mainly from within
mitochondria) can be determined by blue autofluorescence (450 nm). Fig.
6A shows that IL-3 withdrawal
resulted in accumulation of NADH at a time shortly following the peak
of alkalinization (Fig. 3B) and the rise in
m (Fig. 3A). NADH accumulation was also
produced by treatments with oligomycin (inhibitor of the
F0F1-ATPase) or antimycin A (electron transport
blocker) (Fig. 6B). In contrast, valinomycin (which swells
and depolarizes mitochondria) did not increase NADH levels (Fig.
6B) and in fact led to a slight reduction. Therefore, alkaline pH altered mitochondrial chemistry, producing an accumulation of reduced pyridine nucleotides, oxidation of which would have been the
critical first step of electron transport.
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Fig. 6.
IL-3 withdrawal induces a rise in cellular
NADH levels that follows increases in cytosolic pH and mitochondrial
inner membrane polarization. A, FL5.12A cells were
incubated with or without IL-3 from 0 to 6 h, and cells were
assayed every hour for blue autofluorescence, indicative of reduced
pyridine nucleotides. In cells deprived of IL-3, NADH levels increased
4-5 h post-withdrawal. In comparison, NADH levels did not vary
significantly in cells cultured in the presence of IL-3. B,
FL5.12A cells were treated with oligomycin (5 µg/ml) or valinomycin
(1 µM) for 1 h or with antimycin A (1 µg/ml) for
30 min. Cells were then assayed for increases in NADH as controls for
the time course measurements described above. Two similar experiments
were performed. Results are shown in duplicate. Error bars
represent standard errors of the mean.
m could induce cell death, and, if so,
whether the pro-apoptotic protein, Bax, was required for cell death.
The organophosphorus compound, parathion, can partition into
mitochondrial membranes and has previously been shown to induce
mitochondrial hyperpolarization in human neuroblastoma cells (34). As
shown in Fig. 7A, parathion
induced hyperpolarization of the mitochondrial inner membrane in
FL5.12A cells after 2 h in the presence of IL-3. Like the increase
in
m produced during cytokine withdrawal, this
hyperpolarization was independent of caspases (because ZVAD-FMK had no
effect), Bax (because a Bax
/
cell line hyperpolarized),
and Bcl-2 (because cell lines expressing Bcl-2 hyperpolarized) (Fig.
7A).
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Fig. 7.
Parathion induces mitochondrial
hyperpolarization, Bax translocation, caspase 3 activation, and cell
death independent of the Bcl-2 family of proteins. A,
FL5.12 cells, expressing the empty neomycin vector (Vector)
or Bcl-2 cells cultured with IL-3, and the immortalized wild-type or
Bax /
cell line were incubated with or without 1 mM parathion for 2 h. FL5.12 (Vector) cells
were also treated with 100 µM ZVAD-FMK. Cells were then
stained with JC-1 and assayed by flow cytometry. Parathion induced
mitochondrial hyperpolarization. Peak positions of red fluorescence for
cell populations are displayed. B, FL5.12 cells were treated
with or without parathion and IL-3 for 3 h. Mitochondrial protein
lysates were made as described under "Experimental Procedures," and
levels of Bax protein were assayed by Western blot. Cytochrome
c oxidase is included a control for the quality of the
mitochondrial lysates. Both IL-3 withdrawal and parathion treatment
induced the mitochondrial translocation of Bax. C and
D, FL5.12 cells, expressing the empty neomycin vector
(vector), or Bcl-2 grown in IL-3, and the immortalized wild-type or
Bax
/
cell line were cultured with or without 1 mM parathion for 2 h, and levels of caspase 3 activity
or cell viability (by
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide;
thiazolyl blue assay) were assayed 20 h later as previously
described (15). Expression of Bcl-2, but not the lack of Bax, protected
cells from caspase 3 activation but not from cell death. All results
are shown in duplicate. Error bars represent standard error
of the mean.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
m 12-24 h after induction of p53 expression, just
preceding mitochondrial depolarization (37).
m (2-3 h after cytokine
withdrawal) that does not coincide with the generation of ROS (which
occurs 20-24 later) and is not solely produced by the loss of proton gradient dissipation (Fig. 4C). Furthermore, overexpression
of Bcl-2 or Bcl-XL does not block this earlier increase in
m (Fig. 2A). In a recent report,
stimulation of the CD95/Fas/Apo-1 receptor, inducing apoptotic death in
lymphocytes, resulted in the elevation of the
m
within 20 min to 1 h of receptor engagement and preceded the later
cell death, as measured by phosphatidylserine externalization, by
several hours (38). Similarly, staurosporine treatment of Jurkat T
cells induced an increase in
m within 20 min,
preceded cytochrome c release, and was followed by a
decrease in ATP (39). Thus, induction of cell death, either by cytokine
withdrawal, the activation of a death receptor, Fas, or staurosporine
treatment is preceded by an early and transient rise in the
m. We previously observed that staurosporine
treatment induced intracellular alkalinization, suggesting that this
could be a common mechanism inducing hyperpolarization.
m (Fig. 7), nor was it
observed in p53-induced apoptosis (37). Hence, the Bcl-2 family of
proteins can regulate the later disruption of the mitochondria
(depolarization) and activation of caspase 3 (Fig. 2C),
whereas the early intracellular changes described herein
(alkalinization and hyperpolarization) do not appear to result from the
activity of Bax or the loss of Bcl-2 or Bcl-XL.
m in their studies) (23), whereas ours is not (Figs.
2 and 7). Perhaps the striking inhibitory effect of alkaline pH on ADP transport is due to changes in the conformation or charge distribution of mitochondrial proteins or substrates, altering these in such a way
that is affected by Bcl-2 family members.
m, interruption of electron transport and oxidative
phosphorylation may result. If the proton gradient is not dissipated by
ATP synthesis and a steeper proton concentration gradient results, NADH
oxidation could cease (16). It would require increased energy to move more protons across the membrane in the face of the heightened proton-motive force. Likewise, mitochondria containing NADH,
O2, and Pi but no ADP could no longer oxidize
NADH and reduce O2 (16) and could account for the
accumulation of NADH after IL-3 withdrawal (as shown in Fig.
6A).
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ACKNOWLEDGEMENTS |
---|
We thank G. Weigand for the analysis of the NADH levels and S. Zullo and J. Oppenheim for comments on the manuscript.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Section of
Cytokines and Immunity, NCI, Bldg. 560, Rm. 31-71, Frederick, MD
21702-1201. Tel.: 301-846-1545; Fax: 301-846-6720; E-mail:
durums @mail.ncifcrf.gov.
Published, JBC Papers in Press, December 1, 2000, DOI 10.1074/jbc.M006391200
2 A. R. Khaled and S. K. Durum, manuscript in preparation.
3 A. R. Khaled, K. Kim, D. K. Ferris, K. Muegge, A. N. Moor, L. Fliegel, and S. K. Durum, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: IL, interleukin; PT, permeability transition; VDAC, voltage-dependent anion channel; ANT, adenine nucleotide translocator; CCCP, carbonyl cyanide p-chlorophenylhydrazone; DMA, 5-(N,N-dimethyl)-amiloride; JD-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine iodide; BCECF, 2',7'-bis-(2-carboxy ethyl)-5-(and 6)-carboxy fluorescein (BCECF)-acetomethyl ester; CAT, carboxyatractyloside.
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