Center for the Study of Nervous System Injury and Department of
Neurology, Washington University School of Medicine, St Louis, MO 63110,
USA
* Merck Research Labs, West Point, PA 19486, USA
Department of Pharmaceutical Sciences, School of Pharmacy, Medical University
of South Carolina, 280 Calhoun Street, Charleston, SC 29425 USA
Author for correspondence (e-mail:
yusp{at}musc.edu)
Accepted 29 January 2003
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Summary |
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Key words: Na+, K+-ATPase, Apoptosis, Potassium homeostasis, Neuron
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Introduction |
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Excessive K+ efflux and intracellular K+ depletion
are thought to be critical steps in cell body shrinkage and apoptotic death
(Yu et al., 1997;
Dallaporta et al., 1998
;
Bortner and Cidlowski, 1999
). A
significant reduction in intracellular K+ concentration may be a
prerequisite for key apoptotic events including caspase-3 cleavage and
endonuclease activation (Dallaporta et
al., 1998
; Bortner and
Cidlowski, 1999
). The pro-apoptotic K+ efflux may be
mediated by voltage-gated K+ channels in neurons
(Yu et al., 1997
;
Colom et al., 1998
;
Nadeau et al., 2000
); and
other cells (Nietsch et al.,
2000
; Wang et al.,
1999
; Diem et al.,
2001
; Krick et al.,
2001
). In addition, K+ loss may occur through NMDA or
AMPA/kainate receptor channels (Yu et al.,
1999a
; Xiao et al.,
2001
). Theoretically, K+ homeostasis may not be altered
if K+ efflux can be balanced by sufficient K+ uptake.
Since the Na+, K+-pump is the only major mechanism for
K+ uptake, we hypothesized that cells undergoing apoptosis, in
addition to an enhanced K+ efflux, might additionally suffer from
dysfunction of the Na+, K+-pump. To test this
hypothesis, we identified the membrane currents associated with the
Na+, K+-pump activity in cortical neurons, examined the
effects of several apoptotic insults on the Na+, K+-pump
current and modeled the putative role of the Na+,
K+-pump in neuronal apoptosis. This work was partly presented in an
abstract (Wang et al.,
2001
).
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Materials and Methods |
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Electrophysiological recordings of Na+, K+-pump
currents
The 35-mm culture dish containing cortical neurons was placed on the stage
of an inverted microscope, and membrane currents were recorded by whole-cell
configuration or perforated patch using an EPC-9 amplifier (List-Electronic,
Germany). Recording electrodes of 8-10 M (fire-polished) were pulled
from Corning Kovar Sealing #7052 glass pipettes (PG52151-4, WPI, USA) by a
Flaming-Brown micropipette puller (P-80/PC, Sutter Instrument Co., USA). For
perforated patches, gramicidin D was dissolved in DMSO (10 mg/ml) and freshly
diluted to a final concentration of 50 µg/ml with the internal solution.
After formation of a gigaohm seal, brief voltage steps of 10 mV were
applied to monitor the changes in input resistance and capacitance for 15-20
minutes before the formation of the perforated patch. Series resistance
compensation was routinely applied during recordings. Current and voltage
signals were displayed on a computer monitor and collected by a data
acquisition/analysis program PULSE (HEKE, Lambrect, Germany). Currents were
digitally sampled at 0.33 kHz and filtered at 3 Hz by a 3-pole Bessel
filter.
An inward current representing the tonic Na+,
K+-ATPase activity was generated by application of the selective
blocker ouabain (1 mM) or strophanthidin (10-1000 µM). Experimental testing
solutions were locally applied to the cell surface using the DAD-12 drug
delivery system (Adams & List, New York, NY). As a reversible inhibitor,
strophanthidin could be repeatedly applied with intervals of 2 minutes.
The extracellular solution contained (in mM): NaCl 125, KCl 3,
MgCl2 2, CaCl2 2, Na-HEPES 10, Glucose 10, and 0.01
µM TTX. The electrode solution contained (in mM): Cs-acetate 60, NaCl 20,
N-methyl-D-glucamine 100, Mg-ATP 5, BAPTA 1, TEA 10, and HEPES 10. To
record an outward current at 70 mV associated with activation of the
Na+, K+-pump, cells were first exposed to a
K+-free solution (10 seconds) to minimize the pump activity and
then to a solution containing 4 mM K+ (5 seconds) to activate the
pump. Gadolinium (1 µM) was applied into the external solution to prevent
openings of voltage-gated Ca2+ channels and stretch-sensitive
channels. In the experiment examining outward currents, TTX concentration was
raised to 0.1 µM to ensure complete block of Na+ channels.
For effects of acute treatments (30 minutes) on the pump current,
whole-cell or perforated patch recordings was performed in the same cells
before and after a treatment; group studies in different cells were used for
chronic treatments (hours). Recordings were performed at room temperature
(21±1°C); all solutions had a pH of 7.3-7.4.
Assessments of cell death
Neuronal cell death was assessed in 24-well plates by measuring lactate
dehydrogenase (LDH) released into the bathing medium (MEM + 20 mM glucose and
30 mM NaHCO3) using a multiple plate reader (Molecular Devices,
Sunnyvale, CA), and confirmed by staining DNA with propidium iodide (PI)
followed by quantification using a fluorometric plate reader (PerSeptive
Biosystems, Framingham, MA). Neuronal loss is expressed as either a percentage
of LDH release or fluorescence measured in each experimental condition
normalized to the negative control (sham wash) and positive control (complete
neuronal death induced by 24-hour exposure to 300 µM NMDA or cell death
induced by ouabain alone). There was no significant glial death detected by
Trypan Blue exclusion in these injury paradigms. In serum deprivation
experiments, the NMDA receptor antagonist MK-801 (1 µM) was included in the
serum-free medium to block excitotoxicity
(Yu et al., 1997).
ATP and ADP assays
For ATP determinations, neurons in 24-well plates were washed with ice cold
PBS, scraped off with 0.125 ml of 5% trichloroacetic acid per well and
collected into tubes left on ice for 5 minutes and centrifuged for 5 minutes.
A fraction (0.4 ml) of the supernatant was mixed with 1.5 ml of diethylether,
and the ether phase containing trichloroacetic acid was discarded. This step
was repeated three times to ensure complete elimination of trichloroacetic
acid. The extracts were then diluted with 0.4 ml of a buffer (buffer A)
containing 20 mM HEPES and 3 mM MgCl2, adequate KOH was added to
adjust pH to 7.75.
ATP and ADP were assayed by a modified luminometric method
(Detimary et al., 1996). For
measurements of the sum of ATP + ADP, ADP was first converted into ATP by
mixing 100 µl of the diluted extract with 300 µl of buffer A
supplemented with 1.5 mM phosphoenolpyruvate and 2.3 units/ml pyruvate kinase,
and incubated at room temperature for 15 minutes. Samples with known amounts
of ADP but without ATP were run in parallel to check that the transformation
was complete. An ATP assay kit (Sigma, St Louis, MO) was used and the emitted
light was measured in a luminometer. For the measurements of ATP, the same
procedure was followed except that the first incubation step was performed in
the absence of pyruvate kinase. ADP levels were calculated by subtracting the
concentration of ATP in the parallel lysate from this ATP + ADP value. Blanks
and ATP standards were run through the entire procedure, including the
extraction steps. For each measurement, the sample was collected from six
culture wells; at least three different culture batches were used for each
task. Protein concentrations were determined in the pellet after
solubilization with 0.1 M NaOH by a protein assay kit (Bio-Rad, Hercules, CA)
using bovine serum albumin as the standard.
Confocal imaging of fluorescence measurement of ROS
The fluorescent dye dihydroethidium (DHE) was used for detecting the
production of superoxide anion (O2-) during apoptosis.
DHE was prepared as a 10 µg/ml stock solution, packed under N2,
and stored in 80°C; working stocks consisted of 1 µg/ml
dilutions made in DMSO. A fresh aliquot was used for each experiment. Other
drugs and test solutions were made in regular saline; all solutions contained
(in mM): NaCl 144, HEPES 10, CaCl2 2, MgCl2 1, KCl 5,
and D-glucose 10 (measured osmolarity=312 mOsm, pH 7.4 adjusted with
NaOH).
Cells were loaded with 1 µg/ml DHE for 1 hour, culture dishes were then
placed on the stage of the confocal fluorescence microscope (Olympus IX-70,
Japan). Fields of cells were randomly selected, and fluorescence images were
obtained using excitation =488 nm and emission
>590 nm.
Frame-averaged confocal images were digitized at 640x480 pixels using
the Fluoview image acquisition software (Olympus, Japan). Fluorescence
intensity was calculated as fluorescence pixel intensity for each cell.
Mitochondrial membrane potential determination
We examined mitochondrial membrane potential by monitoring mitochondrial
uptake and distribution of rhodamine 123 (R123), a lipophilic cationic
indicator that, driven by mitochondrial membrane potential, accumulates in
mitochondrial matrix where it undergoes quenching
(Scaduto and Grotyohann,
1999). Mitochondrial membrane depolarization results in the R123
release into the cytosol, unquenching and rapid fluorescence increase, which
reflects the amount of dye taken up by the mitochondria, and the mitochondrial
membrane potential, before depolarization.
Cells were loaded with 1 µM rhodamine 123 (Molecular Probes, Eugene, OR) by 20-minute incubation in HBBSS. After washing the dye off with HBBSS, the fluorescence of the dye was recorded for 10 minutes before and after depolarizing mitochondria with 10 µM carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP). The cells were visualized at 530 nm emission filter with a cooled CCD camera (Cooke, Auburn Hills, MI) on a Nikon Diaphot inverted microscope (40x, 1.3 NA oil lens, 75 W xenon arc lamp). The images were collected at 485 nm at 120-second intervals and digitized. After subtracting background, the fluorescence was divided by the average rhodamine fluorescence before FCCP application for each individual cell. The MetaFluor software system (Universal Imaging, West Chester, PA) was used for image acquisition and analysis. Experiments were performed in 11-78 neurons per group from at least two separate cultures.
Protein phosphorylation assay
Immunoprecipitation
Cortical cultures in 24-well dishes were washed in ice-cold PBS and lysed
for 10 minutes in 0.5 ml immunoprecipitation buffer containing 20 mM Tris-HCl,
2 mM EGTA, 2 mM EDTA, 30 mM NaF, 30 mM
Na4P2O7, 2 mM Na3VO4, 1
mM 4-(2-aminoethyl)-benzenesulfonylfluoride, 10 µg/ml leupeptin, 4 µg/ml
aprotinin,and 1% Triton X-100 (pH=7.45). After centrifugation (12,000
g) and determination of protein concentration in the
supernatants by the bicinchoninic acid method (BCA assay), equal amounts of
protein from supernatants were incubated overnight at 4°C with antibodies.
Antibodies were subsequently bound to a saturating amount of Protein
A-Sepharose beads at 4°C for 4 hours. Centrifugation (12,000
g) was performed twice in 1 ml ice-cold immunoprecipitation
buffer and then once in TBS. After adding sample buffer (2% SDS, 50 mM
Tris-Cl, 100 mM DTT, 10% glycerol, 0.1% Bromphenol Blue), the samples were
heated at 65°C for 15 minutes and loaded onto SDS-PAGE gels.
Phosphorylated proteins were immunoprecipitated with rabbit poly-clonal
anti-pan phosphorylated protein antibody (5 µg for 200 µg protein)
(Zymed Laboratories, CA) or with rabbit polyclonal
anti-Na+,K+-ATPase 3-subunit antibody (5 µg
for 200 µg protein) (Upstate Biotechnology, Lake Placid, NY).
Western blotting
Proteins were separated by electrophoresis on 6% SDS-PAGE and transferred
to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). Membranes
were blocked with 3% BSA in Tris-buffered saline (TBS) containing 150 mM NaCl,
50 mM Tris, and 0.2% Tween 20 (pH=7.5) for 1 hour at room temperature. After
three washes in TBS-Tween, the membranes were incubated overnight at 4°C
with rabbit poly-clonal Na+,K+-ATPase 3-subunit
antibody (1 µg/ml) (Upstate Biotechnology, Lake placid, NY) in TBS-Tween
containing 1% BSA. The membranes were then washed three times and incubated
for 1 hour at room temperature in TBS-Tween containing 1% BSA and anti-rabbit
alkaline phosphatase-conjunated second antibody (Promega, WI) at dilution of
1:5000 (v/v). Protein bands were quantified under conditions of linearity by
integration of the density of the total area of each band using the MetaMorph
software (Universal Imaging, West Chester, PA). Protein A band was used as
intra-control. Results were expressed as percentage ± s.e.m. of the
intra-control optical density.
Chemicals
The caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-FMK)
was purchased from Enzyme Systems Products (Dublin, CA), MK-801 was purchased
from RBI (Natick, MA), rhodamine 123 and dihydroethidium were from Molecular
Probes (Eugene, OR). Other chemicals including ouabain, strophanthidin,
pyruvic acid, succinate acid, C2-ceramide, and oxaloacetate were
purchased from Sigma.
Statistics analysis
Student's two-tailed t-test was used for comparison of two
experimental groups; multiple comparisons were done using one-way ANOVA test
followed by Tukey test for multiple pairwise tests. Changes were identified as
significant if P value was less than 0.05. Mean values were reported
together with the standard error of mean (s.e.m.). The statistical analysis
was performed using SigmaPlot or the statistical software SigmaStat (SPSS,
Chicago, IL).
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Results |
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The Na+, K+-pump maintains its tonic activity in the
presence of normal concentrations of intracellular Na+ and
extracellular K+. This activity was revealed by an inward current
Ipump upon application of the specific pump inhibitor
ouabain (1.0 mM) or strophanthidin (500 µM) at the holding potential of
60 mV (Fig. 1A). The
effect of strophanthidin was reversible and concentration-dependent with an
IC50 of 76 µM. At 500 µM, strophanthidin triggered an
Ipump of 29.37±1.70 pA (n=56 cells); the
current density was 0.39±0.03 pA/pF, which is comparable to that
reported in other neurons, cardiac myocytes, and epithelial cells
(Gao et al., 1995;
Senatorov et al., 1997
;
Gao et al., 2000
;
Hansen et al., 2000
). The pump
current had an estimated reversal potential of 133 mV, consistent with
the voltage-dependent nature of the pump; the reversal potential was highly
sensitive to changes in extracellular K+, intracellular
Na+, and intracellular ATP levels
(Fig. 1B-D).
|
In neocortical cultures, a typical apoptotic insult such as serum deprivation usually commits cells to die after about 10 hours; widespread cell death can be detected after 24-48 hours. To delineate activity changes of the Na+, K+-pump in cells undergoing the early stage of apoptosis, we examined Ipump in viable cells challenged by the classic apoptotic insult serum deprivation. Ipump progressively declined a few hours after serum withdrawal; the current density of Ipump was only 26±14% of controls after 9-hour serum deprivation (P<0.05, n=16) (Fig. 2A). Staurosporine is another widely used apoptotic insult and a pan inhibitor of protein kinases. When tested by acute application, Ipump was not altered by a 30-minute exposure to 0.1 µM staurosporine (Fig. 2B). Exposure to staurosporine (0.1 µM) for several hours, however, gradually downregulated Ipump. After 5 and 12 hours in staurosporine, the current density of Ipump was 42±5% and 24±3% of controls, respectively (P<0.05 for both tests, n=8) (Fig. 2B,D). C2-ceramide is a synthetic cell-permeable apoptotic signal mimicking the effect of endogenous ceramide; in the presence of C2-ceramide (25 µM) Ipump was time-dependently inhibited too; the current density was 63±9% of controls after 20-hour exposure to C2-ceramide (P<0.05, n=14) (Fig. 2C).
|
|
Preservation of the Na+, K+-ATPase activity by
pyruvate and succinate in apoptotic cells
To understand whether a cellular energy failure might be responsible for
the diminished Ipump during the apoptotic process, we
studied the effect of intracellular ATP dialysis on Ipump
in apoptotic cells. Supporting the ATP-dependence of the pump activity,
intracellular dialysis of 10 mM ATP in the whole-cell configuration increased
Ipump in apoptotic cells although the recovery was not
complete (Fig. 2D). In the
following experiments, pyruvate or succinate were tested during an apoptotic
treatment. Ipump was recorded using the perforated patch
configuration, so as not to interfere with the intracellular ATP level.
Pyruvate is the product of glycolysis and is transported into mitochondria for
the citric acid cycle. When 5 mM pyruvate was included in the serum-free
medium or added together with 0.1 µM staurosporine,
Ipump remained at normal levels even 12 hours after the
onset of exposures (Fig. 2A,B).
Succinate, the middle product of the citric acid cycle, also prevented the
Ipump depression induced by serum-free medium or
staurosporine (Fig. 2A,B).
Pyruvate and succinate, nevertheless, could not preserve the Na+,
K+-pump current depressed by C2-ceramide
(Fig. 2C), probably as a result
of a ceramide-induced direct damage to mitochondrial respiratory chain
(Gudz et al., 1997).
Apoptosis, Na+, K+-pump activity and ATP
metabolism
The experiments reported above suggested that intracellular ATP level is
relevant but not fully responsible for the pump failure. To further delineate
the relationship between ATP production and activity of the Na+,
K+-pump during apoptotic process, we measured ATP levels in control
cells and cells treated with the serum-free medium or staurosporine for 9-10
hours. In agreement with the reduced Na+, K+-ATPase
activity, diminished cellular ATP levels were found in both apoptotic settings
(Fig. 3). By contrast, pyruvate
(5 mM) and succinate (20 mM) increased ATP levels during serum deprivation;
ATP levels were also markedly increased by succinate co-applied with
staurosporine (Fig. 3).
Pyruvate, however, failed to prevent staurosporine-induced ATP depletion
(Fig. 3C). To understand why
pyruvate improved Ipump without increasing the ATP level,
we examined the effect of pyruvate on cellular ADP/ATP ratio, which is an
indicator for ATP hydrolysis or ATP use. The ADP/ATP ratio decreased either by
serum deprivation or staurosporine, consistent with a diminished ATP
metabolism and dysfunction of the Na+, K+-ATPase (Figs
2,
3). The ADP/ATP ratio was
raised by co-applied pyruvate or succinate
(Fig. 3B,D), implying an
enhanced ATP consumption that might be favorable for preservation of the
Na+, K+-pump activity.
|
Phosphorylation state of the Na+, K+-pump in
cells undergoing apoptosis
Phosphorylation is a primary regulatory mechanism for activities of the
Na+, K+-pump
(Borghini et al., 1994;
Feraille et al., 1997
;
Feschenko et al., 1997
). To
understand whether the reduced ATP level might affect the phosphorylation
state of the Na+, K+-pump, its phosphorylation status
was assessed using the antibody (anti-pan) recognizing serine, threonine, and
tyrosine phosphorylated proteins, and the antibody against the pump
3
subunit that is commonly and abundantly expressed in brain neurons
(Juhaszova and Blaustein,
1997
; Habiba et al.,
2000
). After a 9-hour incubation in serum-free medium or 0.1 µM
staurosporine, the
3 subunit phosphorylation level was reduced
(Fig. 4). Phosphorylation of
the
1 subunit, regarded as a `housekeeping' subunit in different cells
(Juhaszova and Blaustein,
1997
; Crambert et al.,
2000
), was not tested.
|
Reactive oxygen species (ROS) and Na+, K+-pump
activity
Since above results implied a more complex mechanism underlying the
apoptotic pump failure, we hypothesized that, in addition to ATP level and
ADP/ATP ratio, additional factors especially the ROS production might
contribute to the pump failure. In neurons treated by serum deprivation or
staurosporine-enhanced production of superoxide radical anion
(O2-) was indeed detected by the fluorescent dye
dihydroethidium (DHE) 9-10 hours after the onset of exposure
(Fig. 5). Superoxide production
was much higher upon exposure to staurosporine than to serum deprivation,
correlating with a more severe block of Ipump by
staurosporine at this time point (Fig.
2). Pyruvate (5 mM) completely prevented the superoxide production
induced either by staurosporine or by serum deprivation
(Fig. 5B,C). Succinate (20 mM)
also attenuated the superoxide production induced by apoptotic insults
(Fig. 5B,C).
|
To understand whether the ROS production was mitochondria associated, we used the fluorescent indicator, R123, to determine whether conditions affecting the pump activity involved loss of mitochondrial membrane potential. During staurosporine exposure, the mitochondrial indicator uptake was initially (at 5 hours) over 30% larger (n=66 cells, P<0.05) but later (9 hour) 30% smaller (n=84, P<0.05) than that in untreated controls (n=60), indicating an early mitochondrial membrane hyperpolarization followed by depolarization after prolonged incubation with staurosporine. Serum deprivation in pure neuronal cultures reduced the ability of mitochondria to accumulate R123 by 30% at 5 hours (n=41, P<0.05) but not at 9 hours (n=34), suggesting an initial drop and subsequent recovery of mitochondrial membrane potential. At each time point, the cells maintained some mitochondrial membrane potential as they accumulated R123, as demonstrated by indicator release upon full mitochondrial membrane depolarization with 10 µM FCCP (data not shown).
To verify the ROS effect on the Na+, K+-pump activity and further delineate the antagonizing mechanism of pyruvate and succinate, oxidative stress was generated by exogenous hydrogen peroxide (H2O2). H2O2 (0.25 mM, 10 minutes) blocked more than 50% of Ipump; the marked acute effect of H2O2 was attenuated by the ROS scavenger catalase (250 unit/ml) and by pyruvate (5 mM), applied 5 minutes before and during H2O2 exposure (Fig. 6A). The acute H2O2 suppression of Ipump, however, was not prevented by succinate even at the high concentration of 20 mM (Fig. 6A). We then examined the effect of endogenous (O2-) induced by menadione or 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) on Ipump. Menadione (20 µM, 10 minutes) blocked about 50% of the pump current; the menadione effect was prevented by co-applied SOD (25 unit/ml) (Fig. 6B). Similarly to the experiment with H2O2, the effect of menadione on Ipump was fully prevented by 5 mM pyruvate but not by 20 mM succinate (Fig. 6B). In chronic experiments, exposure to 8 µM menadione for 9-10 hours suppressed Ipump, the inhibitory effect was prevented by pyruvate (Fig. 6C). Interestingly, the menadione-induced chronic suppression of Ipump was also attenuated by succinate (Fig. 6C), suggesting that succinate might diminish an oxidant stress via slow or indirect means. As shown in Fig. 5, succinate indeed reduced ROS production during the apoptotic process of several hours. DMNQ is an intracellular superoxide- and H2O2-forming compound metabolized in the mitochondria; a 20-minute exposure to 5 or 20 µM DMNQ caused dose-dependent block of Ipump (Fig. 6D). Pyruvate (5 mM) and succinate (20 mM) largely prevented the acute DMNQ effect on the pump activity (Fig. 6D). In chronic experiments of 15-20-hour incubations, the DMNQ inhibitory effect on Ipump was again attenuated by co-applied pyruvate or succinate (Fig. 6D). These results confirmed an inhibitory effect of endogenous ROS on the Na+, K+-pump and its antagonism by pyruvate and succinate.
|
Protective effects of pyruvate and succinate against neuronal
apoptosis
We next examined the hypothesis that a failure of the Na+,
K+-pump was not only a consequence of apoptotic pathophysiology,
but also affected the fate of neurons subjected to an apoptotic offense.
Consistent with their effects of retaining Ipump in cells
undergoing apoptosis, pyruvate or succinate added into serum-free medium
attenuated apoptotic cell death 24-30 hours after the onset of the exposure
(Fig. 7). The neuroprotective
effect was concentration dependent; the effective concentrations were
consistent with their effects on preserving the Na+,
K+-pump activity. The protective effect persisted even when
application of pyruvate was delayed for up to 4 hours after serum withdrawal
(Fig. 7B), consistent with the
observation that Ipump was not noticeably suppressed
during the first 4 hours of serum deprivation
(Fig. 2A). -Cyano-4-hydroxycinnamate (4-CIN), an inhibitor of the monocarboxylic
acid transporter and mitochondrial pyruvate carrier, blocked the
neuroprotective ability of pyruvate, suggesting the involvement of
mitochondria in the protection (Fig.
7B). In support of the notion that succinate may antagonize
apoptotic damage via its downstream pathway, its metabolite oxaloacetate (5
mM) exhibited similar anti-apoptotic effect
(Fig. 7C). As pyruvate failed
to prevent the Na+, K+-pump failure induced by
C2-ceramide, it could not prevent C2-ceramide-induced
apoptosis (Fig. 7D).
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Discussion |
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It is well established that blocking the Na+, K+-pump
diminishes intracellular K+ concentration
(Archibald and White, 1974;
Lijnen et al., 1986
;
Xiao et al., 2002
). This event
has been linked to apoptotic cell shrinkage in Jurkat cells
(Nobel et al., 2000
) and
cortical neurons (Xiao et al.,
2002
). In our previous studies, we showed that serum deprivation,
staurosporine, and C2-ceramide augmented the outward delayed
rectifier K+ current after 36-hour exposures
(Yu et al., 1997
;
Yu et al., 1999b
). Based on
previous and present data, we now propose that an excessive K+
efflux mediated by upregulated K+ channels and a belated
Na+, K+-pump failure are the joined forces contributing
to intracellular K+ depletion. Considering the key contribution of
the Na+, K+-pump in K+ uptake against the
large K+ gradient across the plasma membrane, the Na+,
K+-pump failure may play an even more decisive role in the
disruption of ion homeostasis and cell death.
The Na+, K+-ATPase can be stimulated by external
K+ in a voltage-dependent manner
(Vasilets et al., 1991;
Omay and Schwarz, 1992
). It is
expected that following an excessive K+ efflux and accumulation of
extracellular K+, the Na+, K+-pump would be
stimulated at least during some early period of apoptosis. In the present
study, however, no such augmented pump activity (i.e. enhanced
Ipump) was observed. This is perhaps mainly due to the huge
dilution of escaped K+ by extracellular solutions in the recording
dish. Accumulations of extracellular K+ and intracellular
Na+ do occur in the brain under pathological conditions such as
cerebral ischemia (Siesjo,
1992
) and hypoxia (Haddad,
1997
); it is thus predicted that an enhanced Na+,
K+-pump activity could take place under such conditions in vivo.
Although a high activity of the Na+, K+-pump with
sufficient ATP supply can be beneficial, in the event of mitochondrial damage
and lack of continuous ATP production, such a transient increase of pump
activity may magnify the energy crisis and exacerbate the cell injury.
The intracellular ATP level may be a factor influencing the fate of cells
in the direction of either apoptosis or necrosis. Depletion of intracellular
ATP prevents Fas/Apo-1-stimulated apoptosis and induces necrotic death in
Jurkat cells; replenishment of ATP restores the ability of these cells to
undergo apoptosis (Eguchi et al.,
1997; Leist et al.,
1997
). This is consistent with the finding that cytochrome c
release and perhaps caspase activation are ATP-dependent
(Li et al., 1997
;
Volbracht et al., 1999
).
Therefore, during the early phase of apoptosis depletion of ATP can preclude
caspase activation and consequently switch execution of cells towards necrosis
(Nicotera and Lipton, 1999
).
By contrast, mitochondrial damage and energy insufficiency are shown in
apoptotic cells (Fiskum,
2000
). A reduction in ATP production by mitochondria (caused by
hypoxia or mutations in genes encoding mitochondrial proteins of the electron
transport chain) can induce apoptosis in neurons or increase their sensitivity
to apoptosis (Gorman et al.,
2000
). It is specifically hypothesized that the cellular ATP level
is an important determinant for apoptosis; a cell stays alive as long as a
certain ATP level is maintained (Richter
et al., 1996
). When ATP falls below this level, apoptosis ensues
provided adequate ATP is still available for energy-requiring apoptotic
processes. However, the exact machinery that is responsible for the
ATP-dependent control of apoptosis was ambiguous. Recent studies suggest that
binding of ATP to the ATP-binding domain in an ATPase can protect it from ROS
injury (Wei and Richardson,
2001
). The present investigation provides novel evidence that
dysfunction of the Na+, K+-ATPase is probably one of the
main mechanisms mediating the ATP deficiency-induced apoptosis or exacerbating
other insult-induced apoptotic injury. Thus reversing the consequences of ATP
depletion in neurons prevents apoptosis. This idea is further supported by
results demonstrating that retaining Ipump during
serum-deprivation by stimulating endogenous ATP synthesis attenuated apoptosis
and that pyruvate could not prevent Na+, K+-pump failure
nor apoptosis induced by C2-ceramide.
Mitochondrial dysfunction has been strongly implicated in mediating both
apoptosis and necrosis (Kroemer et al.,
1998). We observed the mitochondrial potential changes in
apoptotic cells although the change pattern was different following different
insults. Interestingly, mitochondria were hyperpolarized after 5-hour
staurosporine exposure when the Na+, K+-pump current was
already substantially blocked (Fig.
2B); and, despite the pump failure, only transient mitochondrial
depolarization took place during 9-hour serum deprivation. Comparing these
data, it appears that the observed pump failure does not necessarily require
loss of mitochondrial membrane polarization and that the ROS production
related to the pump failure may be mediated by mechanisms mostly independent
of the depolarization of the mitochondrial membrane. These observations are
generally in agreement with recent reports that staurosporine induced
mitochondria hyperpolarization at an earlier stage of apoptosis
(Poppe et al., 2001
) and that
mitochondrial depolarization may not be required for neuronal apoptosis
(Krohn et al., 1999
).
In addition to its role in ATP synthesis, H2O2 was
shown to uncouple the Na+, K+-ATPase from ATP hydrolysis
(Garner et al., 1983). The
failure of ATP consumption and the inability to provide adequate ADP
(unbalanced ATP and ADP ratio) for the adenine nucleotide transporter during
oxidative stress may promote cytochrome c release and initiate apoptosis
(Kantrow et al., 2000
). It is
conceivable that both ATP and ADP levels have to be considered so the effects
of pyruvate and succinate on energy metabolism (e.g. ATP synthesis, available
ATP and ADP, ATP consumption, and ADP/ATP ratio) will be evaluated. In the
present investigation, both pyruvate and succinate increased the ADP/ATP ratio
concurrently with improvements in Na+, K+-pump currents,
supporting the hypothesis that an increased ATP hydrolysis along with
sufficient ATP production is essential for a more balanced ADP/ATP ratio and
may act as a potential anti-apoptotic mechanism.
The Na+, K+-pump activity is highly sensitive to
oxidant stress and production of free radicals
(Kourie, 1998;
Shattock and Matsuura, 1993
).
Previously observed anti-apoptotic effects of ROS scavengers
(Lieberthal et al., 1998
;
Pong et al., 2001
) are
consistent with the preserved Na+, K+-pump function
observed in this study. Pyruvate is well-known for its direct antioxidant
effect of reacting with H2O2 to form water and carbon
dioxide (Crestanello et al.,
1998
; Varma et al.,
1998
); it may also possess a hydroxyl radical scavenging property
(Dobsak et al., 1999
) or
stimulate NADPH-dependent peroxide scavenging systems
(Cavallini et al., 1990
). In
our study both exogenous H2O2 and endogenous ROS induced
by menadione, DMNQ, or apoptotic insults inhibited Ipump.
Succinate showed little effect against the acute effect of menadione on
Ipump, however, it did antagonize acute and chronic
inhibitory effects of DMNQ. Its metabolite oxaloacetate showed a
neuroprotective effect, presumably due to the oxidation of the carbonyl carbon
and carboxyl groups in the structure of oxaloacetate, which reduces the
production of hydrogen peroxide (Ramsay,
1949
).
Information from this and previous investigations suggests that failure of
the Na+, K+-pump can either be causative or contributory
in neuronal injury, depending on whether the Na+,
K+-pump is the original target of the insult. For example,
increased endogenous ouabain in some pathological conditions can be the causal
factor for disruptions of the ionic homeostasis and hybrid cell death
(Budzikowski et al., 1998;
Ferrandi and Manunta, 2000
;
Xiao et al., 2002
); while a
secondary destruction of the Na+, K+-pump may occur
following energy deficiency and ROS production induced by apoptotic and other
insults. Based on available knowledge and our data, it also appears
conceivable that pyruvate and succinate preserve the pump activity through
multiple actions as the primary mechanism of promoting cell survival. We
propose that the Na+, K+-ATPase can be a therapeutic
target for neuroprotection in apoptosis-related diseases.
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