Novel role of the Ca2+-ATPase
in NMDA-induced intracellular acidification
Mei-Lin
Wu1,
Jeng-Haur
Chen1,
Wei-Hao
Chen2,
Yu-Jen
Chen3, and
Kuan-Chou
Chu1
Departments of 1 Physiology and
3 Toxicology, College of
Medicine, 2 Internal Medicine,
National Taiwan University Hospital, Taipei, Taiwan
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ABSTRACT |
The mechanism involved in
N-methyl-D-glucamine
(NMDA)-induced Ca2+-dependent
intracellular acidosis is not clear. In this study, we investigated in
detail several possible mechanisms using cultured rat cerebellar
granule cells and microfluorometry [fura 2-AM or 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM].
When 100 µM NMDA or 40 mM KCl was added, a marked increase in the
intracellular Ca2+ concentration
([Ca2+]i)
and a decrease in the intracellular pH were seen. Acidosis was
completely prevented by the use of
Ca2+-free medium or
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, suggesting that it resulted from an influx of extracellular Ca2+. The following four
mechanisms that could conceivably have been involved were excluded:
1)
Ca2+ displacement of intracellular
H+ from common binding sites;
2) activation of an acid loader or inhibition of acid extruders; 3)
overproduction of CO2 or lactate; and 4) collapse of the mitochondrial
membrane potential due to Ca2+
uptake, resulting in inhibition of cytosolic
H+ uptake. However,
NMDA/KCl-induced acidosis was largely prevented by glycolytic
inhibitors (iodoacetate or deoxyglucose in glucose-free medium) or by
inhibitors of the Ca2+-ATPase
(i.e.,
Ca2+/H+
exchanger), including La3+,
orthovanadate, eosin B, or an extracellular pH of 8.5. Our results therefore suggest that Ca2+-ATPase
is involved in NMDA-induced intracellular acidosis in granule cells. We
also provide new evidence that NMDA-evoked intracellular acidosis
probably serves as a negative feedback signal, probably with the
acidification itself inhibiting the NMDA-induced
[Ca2+]i increase.
N-methyl-D-glucamine; intracellular calcium ion; intracellular pH; calcium
ion-adenosinetriphosphatase; cerebellar granule cells
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INTRODUCTION |
N-methyl-d-glucamine (NMDA)
receptor/channels possess high
Ca2+ permeability and serve many
physiological or pathophysiological functions, such as synaptic
plasticity, neuronal development, and excitotoxicity. Although the
exact cellular mechanism(s) underlying the excitotoxicity is not clear,
there is ample evidence that it is mediated, in part, by an
NMDA-mediated rise in intracellular Ca2+ concentration
([Ca2+]i;
see Refs. 15 and 33). Changes in the extracellular pH (pHo 6.6-8.0) have been shown
to modulate the NMDA-evoked currents in hippocampal neurons (48). In
cerebellar granule cells, NMDA currents are reversibly inhibited by
extracellular protons, with a IC50
of pHo 7.3 (52), close to the
physiological pH. The NMDA current in hippocampal neurons has been
shown to be rather insensitive to changes in intracellular pH
(pHi) over the range of
6.3-8.0 (48). In Xenopus
motoneurons, however, internal acidosis potentiates the NMDA current
(11).
Both pHo and
pHi are dynamically modulated
under physiological or pathological states (14). For example,
excitatory synaptic transmission is reported to be associated with
rapid alkalinization of the extracellular space (12, 14, 39). An
NMDA-evoked [Ca2+]i-dependent
pHi decrease (~0.2 pH unit) in
neurons has also been described (17, 26, 28), although the
H+ source was not identified. It
is therefore important to understand possible interactions between
intracellular Ca2+
(Ca2+i) and
H+. Several mechanisms have been
proposed to account for
Ca2+-induced intracellular
acidosis in neurons and other types of cells.
1) One popular hypothesis is that
Ca2+i can displace
H+ from a common binding site
(26). This hypothesis has also been suggested in cardiac and smooth
muscle cells (4, 54).
2) Mitochondria in neurons show a
high capacity for Ca2+ uptake when
there is a Ca2+ overload (55). The
consequence of Ca2+ uptake, via a
Ca2+ uniporter, is that the
negative potential across the mitochondrial inner membrane collapses,
i.e., for each Ca2+ that enters
the matrix, two H+ fail to enter
via ATP synthase, resulting in cytosolic acidification (55, 57).
3) In cerebellar granule cells, a
rise in CO2 production can be
induced either by addition of 50 mM KCl or by depolarization of the
membrane potential (40); thus, another possible cause of
Ca2+-induced acidosis is
CO2 overproduction. During the
increase in Ca2+i levels, activation of
energy-utilizing processes in the plasma membrane, e.g., increased
activity of the plasma membrane
Ca2+-ATPase, may also increase
glycolysis (18), resulting in lactic acid overproduction.
4) Evidence for a plasmalemma
Ca2+-ATPase (i.e.,
Ca2+/H+
exchange) involving depolarization-induced intracellular acidosis has recently been found in snail neurons (45) and in hippocampal (38, 51)
or brain stem slices (50).
The mechanism involved in NMDA-induced intracellular acidosis in
neurons is not clear. We have used cultured rat cerebellar granule
cells, the largest population of neurons in the brain, to investigate
possible mechanisms of NMDA activation-induced intracellular acidosis.
When intracellular H+ and
Ca2+ levels were measured using
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM and fura 2-AM, respectively, a rise in
Ca2+ levels on NMDA addition was
found to result in a pHi decrease. Our results show that this phenomenon is mainly caused by activation of
plasmalemma Ca2+-ATPase. Moreover,
in contrast to the findings in hippocampal neurons and
Xenopus motoneurons (11, 48), internal
acidification may significantly inhibit the NMDA-induced
[Ca2+]i increase.
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MATERIALS AND METHODS |
Chemicals and solutions. The
experiment was performed at 37°C in HEPES-buffered modified Tyrode
solution at pHo 7.4, containing (in mM) 118 NaCl, 4.7 KCl, 1.2 MgCl2, 2.0 CaCl2, 10 glucose, and 10 HEPES.
When chemicals were added at concentrations higher than 5 mM, the NaCl
concentration was reduced by a similar amount to compensate for the
osmolarity change in the solution. Unless otherwise stated, all
reagents were purchased from Sigma. HOE-694 was a generous gift from
Dr. H.-J. Lang (Hoechst Aktiengesellschaft).
Primary culture of cerebellar granule
cells. Cerebellar granule cells were prepared and
cultured essentially as described by Gallo et al. (22). In brief,
8-day-old Wistar rats were killed by cervical dislocation and then
decapitated. The cerebella were removed, minced into 0.4-mm cubes, and
dissociated with 0.025% trypsin for 15 min at 37°C. The
dissociated cells were suspended in basal modified Eagle's medium
containing 10% FCS, 2 mM glutamine, 50 µg/ml gentamycin, and 25 mM
KCl and were plated on poly-L-lysine-coated cover glasses.
They were then maintained in this depolarizing medium in a humidified
5% CO2 incubator. Cytosine
arabinoside (10 M) was added 24 h after plating to arrest the
replication of, and kill, nonneuronal cells, especially astrocytes. The
cells were used for Ca2+ or pH
measurements after 6-8 days in culture, at which time the purity
of the granule cells is >90%.
Measurement of
Ca2+i
levels.
The method for measuring Ca2+i levels,
via microfluorometry, was similar to that used in a previous study (58). In brief, cells were loaded for 60 min at room temperature with
fura 2-AM (5 µM; Molecular Probes) and then excited alternately using
340- and 380-nm wavelength light. The excitation light was transmitted
to a small group of cells (~5-10 cells) for each experiment, and
the resulting mean fluorescence was collected using a ×40 oil-immersion lens. The ratio of the emission at 510 nm with the excitation wavelengths, respectively, of 340 and 380 nm was calculated and converted to
[Ca2+]i
using the following equation (25)
where
R is the ratio of the 510 nm fluorescence at 340 nm excitation over
that at 380 nm. Sf2/Sb2 is the ratio of the
510-nm emission at 380-nm excitation determined at Rmin and
Rmax, respectively. Calibration constants are obtained by
addition of 5 µM ionomycin in solutions containing either 1.8 mM
Ca2+
(Rmax) or no added
Ca2+ plus 10 mM EGTA
(Rmin). A
Kd of 224 nM was
used (25).
Measurement of pHi.
Measurement of the pHi, via
microfluorometry, has been described in detail elsewhere (59). Cells
were loaded for 10 min at room temperature with 5 µM BCECF-AM
(Molecular Probes) in HEPES-buffered solution and excited alternately
using 490- and 440-nm wavelength light. The 530-nm emission ratio
resulting from 490/440 excitation (from 5-10 cells) was calculated
and converted to a linear pH scale (see below) by means of in situ
calibration at pH 4.5 and 9.5, carried out when it is necessary, using
the nigericin technique (42). Being aware of the possibility that
nigericin may contaminate solution lines (41), we used a completely
different tubing system for the calibration procedure and changed the
cell bath before the next experiment. A calibration curve was prepared
by measuring the fluorescence ratio values between pH 4.5 and 9.5, with
measurements being made every 0.5 pH unit. The following equation was
then used to convert the fluorescence ratio to
pH
where
R is the ratio of the 530 nm fluorescence at 490 nm excitation over the
530 nm fluorescence at 440 nm excitation,
Rmax and
Rmin are the maximum and minimum
ratio values, respectively, of the data curve (not shown), and
pKd (
log of
Kd) is 7.16. F440min and
F440max are the minimum and
maximum fluorescence at 440 nm, respectively.
Intracellular ATP measurements. The
intracellular ATP content of granule cells was measured by the
luciferin-luciferase bioluminescent assay, as described previously
(53). In brief, ATP was extracted from granule cells using 1 ml of 100 mM Tris-EDTA buffer (pH 7.75) at 100°C for 10 min. After
centrifugation, the ATP content of 0.2 ml of the supernatant was
measured in a LKB 1251 luminometer (LKB-Wallac, Tarku, Finland). The
sensitivity of the assay is ~1 pmol of ATP, and ATP standards were
used for calibration. Total protein levels were measured, and the
results are expressed as nanomoles of ATP per milligram of protein.
Lactate assay. Lactate production in
granule cells was assessed by measuring the lactate concentration in
the supernatants collected before (control group) and after 15 min of
treatment with 40 mM KCl or 100 µM NMDA, respectively. Lactate was
assayed spectrophotometrically from the change in absorbance of NAD at 340 nm in the presence of excess lactic dehydrogenase, according to
well-established procedures (Sigma).
Experimental design. The experimental
design to test the involvement of four possible mechanisms in
NMDA-induced acidosis (see the Introduction) is illustrated in Figs.
1 and 2. First, we tested
whether addition of NMDA or KCl activated intracellular acidosis as a
result of an increase in
[Ca2+]i
in granules (Fig. 1), and, if so, what mechanism(s) might be involved
(Fig. 2).

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Fig. 1.
Does an intracellular Ca2+
concentration
([Ca2+]i)
increase induce an increase in intracellular
H+ concentration
([H+]i) [or intracellular pH
(pHi) decrease]? MK-801 and
nifedipine are inhibitors, respectively, of
N-methyl-D-glucamine
(NMDG) channels and voltage-dependent (Voltage-dep.)
Ca2+ channels (see
RESULTS for details).
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Fig. 2.
Four potential mechanisms (i to iv) involved in the
[Ca2+]i-induced
[H+]i
increase were tested. The chemicals in front of the dashed arrows are
inhibitors of the different mechanisms. Na-free,
Na+-free medium; NHE,
Na+/H+
exchanger; NCBE, Na+-dependent
Cl/HCO3 exchanger; AE, anion
exchanger (i.e., Cl/HCO3
exchanger).
Ca2+/H+
interactions: Ca2+ and proton ions
compete for common binding sites. The possible role of the two
Ca2+-ATPases in the plasmalemma or
the endoplasmic reticulum (ER) membrane was tested. EIPA,
ethylisopropyl amiloride; CPA, cyclopiazonic acid.
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Statistical method. All data are
expressed as means ± SE of n
experiments (dishes). Statistical comparison was by the nonpaired Student's t-test; a
P value of <0.05 was considered
statistically significant.
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RESULTS |
Effect of addition of NMDA or KCl on pHi.
The resting
[Ca2+]i
and resting intracellular H+
concentration
([H+]i)
in these cells are, respectively, 35 ± 10 nM
(n = 32) and 79 ± 11 nM
(pHi = 7.10 ± 0.03;
n = 19), similar to values reported in
other studies (6, 16). In 2 mM
Ca2+-containing
Mg2+-free solutions, two
successive additions of 100 µM NMDA (+ 10 µM glycine) caused two
successive
[Ca2+]i
increases (Fig.
3A)
mediated via NMDA channels, since they are known to be blocked by 10 µM MK-801 (10). In a parallel experiment (Fig.
3B), a reversible
pHi decrease was also produced by
NMDA addition (additions 1 and
2 in Table
1). In
Ca2+-free medium, however, NMDA (+ glycine) had no effect on either parameter (Fig. 3,
A and
B).

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Fig. 3.
Addition of NMDA or KCl induces an increase in
[Ca2+]i
and a decrease in pHi.
A and
B: addition of NMDA (100 µM) in
Mg2+-free medium containing 10 µM glycine induces an increase in
[Ca2+]i
and a decrease in pHi.
C:
[Ca2+]i
undergoes a reversible increase during 2 successive additions of 40 mM
KCl. D:
pHi undergoes a reversible
decrease when 40 mM KCl is added. E
and F: nifedipine (10 µM) inhibits
the KCl-induced
[Ca2+]i
and pHi responses.
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We then checked whether a depolarization-induced
pHi decrease occurred in granule
cells. For convenience, 40 mM KCl solution (isosmotic replacement of 40 mM NaCl; Fig. 3C) was added to
depolarize the membrane potential. A biphasic
Ca2+ response consisting of an
initial peak followed by a fall to a noninactivating plateau (Fig.
3C) was seen. A further addition of
40 mM KCl resulted in a significantly smaller
Ca2+ peak and a plateau of similar
amplitude to the first (Table 1). In a parallel experiment (Fig.
3D), two successive additions of 40 mM KCl resulted in reversible acidosis of similar magnitude (Table 1).
We then tested whether an increase in
[Ca2+]i
was a prerequisite for the pHi
decrease. The Ca2+ and pH
responses were almost entirely eliminated by the use of Ca2+-free medium (see above)
or by pretreatment with the Ca2+i chelator
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA)-AM (200 µM; Table 1), indicating that an increase in [Ca2+]i
is required for acidosis to occur. Furthermore, nifedipine (10 µM;
Fig. 3, E and
F) completely prevented the
KCl-induced changes in
[Ca2+]i
and pHi, indicating that the
intracellular acidosis resulted from an influx of extracellular
Ca2+, probably via
voltage-dependent Ca2+ channels.
The above results also suggest that the steady state of the
Ca2+ plateau (Fig. 3,
C and
D), but not the initial
Ca2+ peak induced by KCl addition,
is important for the amplitude of acidosis, as the two acidosis
plateaus were similar while the initial peaks were significantly
different (Table 1).
Effect of inhibition of pHi regulation on
Ca2+-induced
acidosis.
Three pHi regulators, the
Na2+/H+
exchanger, the Na2+-dependent
Cl
/HCO3
exchanger, and the
Cl
/HCO3
exchanger, are present in neurons under nominally
HCO3-free conditions (e.g.,
HEPES-buffered medium; see Refs. 6 and 45); the first two are acid
extruders, whereas the
Cl
/HCO3
exchanger is an alkaline extruder (or an acid loader). If these
transporters were to be inhibited or stimulated by a rise in
Ca2+ levels, a
pHi change would occur. Because
Ca2+-induced acidosis also occurs
in HEPES-buffered medium, for convenience, we tested whether it
occurred in HEPES-buffered medium that was Na2+ free (blocking the
Na2+-dependent acid extruders) or
contained 0.1 mM DIDS (blocking the
Cl
/HCO3 exchanger).
Under Na2+-free conditions (Fig.
4A) or
in the presence of either 10 µM ethylisopropyl amiloride (EIPA, an
Na2+/H+
exchanger blocker; Fig. 4B) or 0.1 mM DIDS (Fig. 4C), KCl-induced intracellular acidosis was not inhibited (Table
2), suggesting that the
Ca2+-induced acidosis is not
mediated by either inhibition of acid extrusion or stimulation of acid
loading.

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Fig. 4.
pHi regulators are not involved in
the Ca2+-induced acidosis.
A: all of the extracellular
Na2+ was replaced by NMDG in the
Na2+-free conditions.
B and
C: concentrations of EIPA and DIDS
were 10 µM and 0.1 mM, respectively.
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Is
Ca2+-induced
acidosis due to the sharing of common binding sites by
Ca2+i
and intracellular
H+?
This possibility was tested using a weak base or acid to change
[H+]i
(pHi) and noting whether a
change in
[Ca2+]i
occurred. Using successive additions of
NH4Cl (ammonium prepulse; see Ref.
6) and sodium propionate (isosmotic replacement of extracellular NaCl),
it is possible to change the
[H+]i
without affecting the pHo.
The results are shown in Fig. 5, which
shows pHi changes as changes in
[H+]i
to compare
[H+]i
and
[Ca2+]i
changes more directly. An ammonium prepulse
(NH4Cl addition, then wash out)
caused a
[H+]i
increase of ~400 nM (i.e., a pHi
decrease from the basal level of 7.10 to 6.32; 0.78 ± 0.06 pH
units, n = 4). A smaller internal acid
loading was then induced by subsequent treatment with 40 mM sodium
propionate
([H+]i = 95 nM, the pHi decreasing from
7.1 to 6.76;
0.34 ± 0.02 pH units,
n = 4). A parallel experiment showed a
much smaller increase in
[Ca2+]i
(25 ± 5 and 15 ± 5 nM, n = 4, respectively; Fig. 5B) during the
acid load. Assuming two H+ are
displaced by one Ca2+ from the
common binding site, a much larger increase in
[Ca2+]i
would be expected. Another explanation for this small rise in
[Ca2+]i
could be that, in the resting state, the "common binding site" is
not saturated with Ca2+ or
H+, and therefore no significant
changes in
[Ca2+]i
are seen. We tested this possibility by saturating the
Ca2+i/intracellular H+ binding sites using 40 mM KCl
(Fig. 5, C and
D,
right); under these conditions, an
additional acid loading (
0.40 ± 0.05 pH units,
n = 4; i.e., from
pHi 6.78 to 6.38, [H+]i = 250 nM) was induced by the ammonium prepulse (Fig.
5C), but, again, a much smaller
change in
[Ca2+]i
was seen (75 ± 15 nM, n = 4; see Fig. 5D), indicating
that the sharing of a common binding site by
Ca2+ and
H+ was probably not the main cause
of the acidosis. A small
[Ca2+]i
increase (10 ± 5 nM, n = 4) was
seen during intracellular alkalosis on
NH4Cl addition (Fig.
5B), probably due to alkalization
promoting Ca2+ release from
internal stores, as demonstrated in vascular smooth muscle cells (4).

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Fig. 5.
Increase in intracellular H+
concentration
([H+]i)
does not induce a corresponding change in
[Ca2+]i.
A and
C:
[H+]i.
B and
D:
[Ca2+]i.
Concentrations of NH4Cl and sodium
propionate were 10 and 30 mM, respectively. Dashed line in
D is the
[Ca2+]i
before the ammonium prepulse.
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Is intracellular acidosis caused by increased mitochondrial
Ca2+ uptake or
CO2 overproduction?
Another possibility could be that a large
Ca2+ uptake by the mitochondria
might inhibit the proton channel in mitochondrial ATP synthase, with
each Ca2+ entering the matrix
resulting in H+ failing to enter
via ATP synthase, thus causing intracellular acidosis (55, 57). When
oligomycin (10 µg/ml, 15-min treatment), an ATP synthase inhibitor,
was used, there was little effect on either KCl-induced
[Ca2+]i
increase or acidosis (Table 3).
CO2 overproduction due to activation of the tricarboxylic acid cycle in the mitochondrion could
be another explanation for
Ca2+-induced acidosis. However, in
the presence of potent tricarboxylic acid cycle inhibitors,
fluoroacetate (1 mM; 15-min treatment) or malonate (10 mM; 15-min
treatment),
[Ca2+]i-induced
acidosis was not significantly altered (Table 3). The above results
therefore suggest that neither
Ca2+ uptake nor
CO2 overproduction in the
mitochondria is involved.
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Table 3.
Effects of metabolic inhibitors on the
[Ca2+]i or pHi changes
induced by addition of 100 µM NMDA or 40 mM KCl
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Is
Ca2+-ATPase
involved in
Ca2+-induced
acidosis?
Two glycolytic inhibitors, 0.5 mM iodoacetate (IAA; ~10-min
treatment; Fig.
6A) and
10 mM deoxyglucose (DOG)/glucose-free medium (~30-min treatment),
were used to lower intracellular ATP levels. Under either of these
conditions, the
[Ca2+]i
increase seen on addition of 40 mM KCl (Fig.
6A and Table 3) or 100 µM NMDA (in
glycine/Mg2+-free medium; Fig.
6C and Table 3) was greatly enhanced.
Furthermore, IAA or DOG/glucose-free medium also completely inhibited
the KCl (Fig. 6B)- or NMDA (Fig.
6D)-evoked
pHi decrease, suggesting that the
Ca2+/H+
exchanger (i.e., Ca2+-ATPase) is
involved. These results are also further evidence that
Ca2+ displacement of
H+ from common binding sites is
very unlikely, since a marked elevation in
[Ca2+]i
with little change in
[H+]i
was observed (Fig. 6). IAA (or DOG) itself may cause acidosis, probably
due to hydrolysis of intracellular ATP, with the release of protons
(1). However, the inhibitory effect of IAA on NMDA- or KCl-induced
acidosis should not be due to the
pHi being very acidic, because, in
the Na2+-free/EIPA medium,
KCl-induced acidosis still occurred under very acidified conditions
(Fig. 4, A and
B).

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Fig. 6.
Intracellular ATP depletion by a metabolic inhibitor [iodoacetate
(IAA)] causes an increased
[Ca2+]i
response (A and
C), whereas
Ca2+-induced acidosis on addition
of 40 mM KCl (B) and 100 µM NMDA
(D) is inhibited. Concentration of
IAA was 0.5 mM.
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Because all of the above metabolic inhibitors (oligomycin,
fluoroacetate, IAA, or DOG) presumably also lowered intracellular ATP
concentrations
([ATP]i), it was of
interest to determine why only the glycolytic inhibitors (IAA and
DOG/glucose-free medium; see Table 3) had an inhibitory effect. When
the level of [ATP]i was measured under similar treatments to those used in the above experiments, we found that, although the
[ATP]i was indeed
significantly decreased by all of the inhibitors used (Table
4), the magnitude of the reduction in
[ATP]i was different,
being much greater in the presence of the glycolytic inhibitors.
These metabolic inhibitors have also been shown to inhibit
intracellular lactate acid production. However, lactate production was
not increased by treatment with either 100 µM NMDA or 40 mM KCl
(NMDA: 6.1 ± 0.2 vs. 6.5 ± 0.1 mg/dl; KCl: 7.0 ± 0.2 vs.
6.8 ± 0.3 mg/dl, P > 0.05, paired t-test), showing that
lactate overproduction was not the cause of these effects.
Ca2+-ATPase inhibitors were then
used to directly test involvement of the pump.
La3+ (1 mM) has been clearly shown
to effectively inhibit both the plasmalemma and endoplasmic reticulum
(ER) Ca2+-ATPases (2, 21, 27, 44,
47). In its presence, the KCl- or NMDA-evoked
[Ca2+]i
response was greatly enhanced (Fig. 7,
A and
C), and intracellular acidosis was
completely inhibited (Fig. 7, B and
D). The smaller KCl-induced
Ca2+ peak seen after pretreatment
with La3+ (Fig.
7A) is probably due to inhibition of
voltage-dependent Ca2+ channels.
In the presence of glycolytic inhibitors (Fig.
6B) or
La3+ (Fig.
7B), however, KCl induced a small
intriguing pHi increase (~0.1 pH
unit), rather than a decrease, and the intracellular acidosis induced
by 100 µM NMDA was completely blocked in the presence of IAA (Fig.
6D) or
La3+ (Fig.
7D), and no alkalosis was seen.
Possibly, a K+-dependent or an
electrogenic acid transporter is exposed by blocking the
Ca2+i-induced acidosis, since we often
observe K+-dependent
pHi changes in granule cells
(unpublished observations).

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Fig. 7.
Inhibition of Ca2+-ATPase by
La3+ inhibits
Ca2+-induced acidosis
(A-D). Concentrations of
La3+ and NMDA were 1 mM and 100 µM, respectively. E and
F: CPA (15 µM) and ryanodine (10 µM), ER Ca2+-ATPase inhibitors,
cannot inhibit the KCl-induced acidosis. Concentration of KCl was 40 mM.
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Because both the plasmalemma and ER
Ca2+-ATPases are known to be
Ca2+/H+
exchangers (20, 30, 35, 46) and because
La3+ completely blocked
Ca2+-induced acidosis (Fig. 7,
B and
D), we tested whether the
Ca2+-ATPase in the ER membrane was
involved. After its selective inhibition (cytosolic
Ca2+ reuptake inhibited) by
addition of 15 µM cyclopiazonic acid (CPA; see Ref. 32) and depletion
of the ER Ca2+ store by 10 µM
ryanodine (Figs. 7, E and
F), the plateau levels for
[Ca2+]i
and the acidosis induced by 40 mM KCl showed little change (Table
5), suggesting that only the plasmalemma
Ca2+-ATPase is involved. A
vacuolar-type H+-ATPase may
also be expressed in granule cells; however, bafilomycin A1 (10 nM; a vacuolar-type H+-ATPase
inhibitor) had no inhibitory effect on the KCl-induced pHi decrease (Table 5), suggesting
that this pump is not involved.
Vanadate is a p-type Ca2+-ATPase
inhibitor (9, 44). The apparent affinity of
Ca2+-ATPase for vanadate is
thought to be higher in the plasmalemma than in the ER membrane (44).
Sodium orthovanadate (10 mM) had no effect on the resting level of
[Ca2+]i
(Fig.
8C), but
the
[Ca2+]i
response to KCl or NMDA was markedly enhanced (Fig. 8,
A and C), indicating that
Ca2+-ATPase in granule cells is
indeed blocked by orthovanadate. The KCl- or NMDA-induced
pHi decrease was also blocked
(Fig. 8, B and
D).

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Fig. 8.
Orthovanadate inhibits KCl- or NMDA-induced acidosis.
A and
C: KCl- or NMDA-induced
[Ca2+]i
increase is markedly potentiated in the presence of 10 mM
orthovanadate. B and
D:
pHi decrease produced by KCl or
NMDA is markedly inhibited in the presence of 10 mM orthovanadate.
Concentrations of KCl and NMDA were 40 mM and 100 µM, respectively.
|
|
To strengthen the idea that plasmalemma
Ca2+-ATPase was indeed involved in
the NMDA-evoked pHi decrease, 20 µM eosin B (3, 19, 23, 24) or a
pHo of 8.5 (5, 38), two known
plasmalemma Ca2+-ATPase
inhibitors, were used, and the NMDA-induced
Ca2+ response was found to be
markedly enhanced and the acidosis inhibited (Table 5).
Possible involvement of intracellular acidification in the
NMDA-activated
[Ca2+]i
increase.
An NMDA-induced increase in
[Ca2+]i
results in excitotoxicity (15, 33). It was therefore of interest to
determine whether there was any possible physiological involvement of
intracellular acidosis in the NMDA-induced
[Ca2+]i
increase, as the pHi decreased by
0.2-0.25 pH units on NMDA addition (see Table 1). We used 40 mM
propionic acid (replacing 40 mM NaCl) to acidify the
pHi by ~0.2-0.25 pH units
and maintained the pHo at 7.4, at
the same time inhibiting the acid extrusion mechanism, the
Na2+/H+
exchanger, by addition of 60 µM HOE-694 (Fig.
9, A and
B) and found that both the
NMDA-induced
[Ca2+]i
and pHi changes were significantly
inhibited by 50 and 65% (Table 5), respectively (see first and second
dashed lines, respectively, in Fig. 9,
A and
B). Similar results were obtained
using another weak acid, sodium acetate (30 mM; see Table 5).

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|
Fig. 9.
Intracellular acidosis inhibits NMDA- or KCl-induced
[Ca2+]i
and pHi responses. Dashed lines
indicate the net
[Ca2+]i
increase or pHi decrease induced
by addition of NMDA or KCl. Concentrations of NMDA, KCl, propionate,
and HOE-694 were 100 µM, 40 mM, 40 mM, and 60 µM, respectively.
|
|
To determine whether the inhibitory effect of acidosis on the
NMDA-induced Ca2+ increase only
affected the NMDA channel, we examined possible effects on the
voltage-dependent Ca2+ channel.
When propionate (Fig. 9, C and
D) or EIPA was used to induce an
internal acid load of ~0.2-0.25 pH units, there was a lower
(~15 ± 3%, n = 4, see dashed
line in Fig. 5D, or ~25% in last
column in Table 5) but statistically significant (P < 0.05, paired t-test) inhibitory effect on
the KCl-induced Ca2+ plateau.
Therefore, this result suggested that the voltage-dependent Ca2+ channel was partially
inhibited by the acidosis. A similar inhibitory effect is also seen in
CA1 neurons (49). The smaller change (
) in
pHi seen in Fig. 9,
B and
D, is probably due to a lower Ca2+/H+
exchange, due to a smaller change in
[Ca2+]i
(Fig. 9, A and
C). Because EIPA had an inhibitory
effect (~15%) on the
[Ca2+]i
plateau (dashed line in Fig. 5D),
its lack of effect on the amplitude of the
pHi (second KCl addition in
Fig. 4B) is probably due to
inhibition of acid extrusion by EIPA (an
Na2+/H+
exchanger inhibitor), resulting in more
H+ accumulation in the cytosol.
 |
DISCUSSION |
Changes in either
[Ca2+]i
or pHi have important
physiological or pathophysiological functions. In neurons, there is
evidence that the plasmalemma
Ca2+-ATPase
(Ca2+/H+
exchange) is involved in electrical stimulation-induced intracellular acidosis (38, 46, 50, 51). However, other mechanisms for this effect
have also been suggested (see Introduction). To study these phenomena
in a more detailed manner in granule cells, we first confirmed that
depolarization induced Ca2+-evoked
intracellular acidosis and that the acidosis was due to activation of
the plasmalemma Ca2+-ATPase.
Having first ruled out several other possible mechanisms for the
Ca2+i-induced intracellular acidosis, namely pHi regulators,
Ca2+ and
H+ sharing common binding sites,
overproduction of CO2/lactate, or
inhibition of Ca2+ uptake by the
mitochondria, we have provided new evidence that the NMDA-induced
intracellular acidosis also results from activation of the plasmalemma
Ca2+-ATPase. Our major evidence is
as follows.
1) Severe depletion of
[ATP]i by two
glycolytic inhibitors (IAA and DOG/glucose-free medium), which resulted
in an ~90-96% decrease in
[ATP]i (see Table 4),
caused a marked elevation of the KCl/NMDA-induced
Ca2+ response (suggesting that the
Ca2+-ATPase was blocked) and
completely inhibited the acidosis (Fig. 6,
C and
D).
Although a significant, but smaller, reduction in
[ATP]i was also
produced by addition of oligomycin or fluoroacetate (~35-55%; see Table 4), there was little inhibitory effect on either the Ca2+ plateau or the
pHi (Table 3). Possible
explanations for this are as follows. Assuming the normal
[ATP]i is 4-7 mM
(1), a 35-55% reduction in
[ATP]i gives an
[ATP]i of
~1.8-4.6 mM. Because the
Km
(Ca2+i) of the plasmalemma
Ca2+-ATPase is low
(~0.2-2.5 µM; see Refs. 7 and 9), a more marked
[ATP]i depletion, as
seen using IAA or DOG/glucose-free medium (Table 4), is probably
required to inhibit this pump. A similar differential reduction in
[ATP]i by different
metabolic inhibitors has also been demonstrated in other studies (29, 31). An alternative possibility is that metabolic compartmentation may
result in the preferential use of glycolysis-derived ATP, rather than
mitochondrial ATP, for certain membrane activities (56), including the
plasmalemma Ca2+-ATPase (37).
2) Two structurally unrelated known
Ca2+-ATPase blockers,
La3+ and orthovanadate,
potentiated the
[Ca2+]i
increase and inhibition of
Ca2+-induced acidosis (see Figs. 7
and 8). However, neither CPA (+ ryanodine; Table 5) nor thapsigargin
(unpublished observations), two selective ER
Ca2+-ATPase blockers, had any
effect on the Ca2+ plateau (Fig.
7E) or the
Ca2+-induced
pHi decrease (Fig.
7F).
In the presence of La3+ (Fig. 7)
or orthovanadate (Fig. 8), the basal
pHi increased (Figs. 7 and 8,
B and
D) with little change in the basal
level of
[Ca2+]i
(Figs. 7A and
8C); therefore, the alkalosis should
not be due to inhibition of the
Ca2+-ATPase. The reason for the
little or no change in the basal level of
[Ca2+]i
on addition of these Ca2+-ATPase
inhibitors is not clear. One possible explanation is that, since the
Km
(Ca2+i) of the ER
Ca2+-ATPase is as low
(~0.1-0.3 µM; see Ref. 7) as that of the plasmalemma
Ca2+-ATPase (~0.2-2.5 µM;
see Refs. 7 and 9), when the plasmalemma enzyme is blocked, the ER
enzyme may uptake this background
Ca2+ influx during the resting
state. The pHi increase induced by addition of either La3+ or
orthovanadate seems more complicated but is probably due to nonspecific
inhibition of background acid load, including a reduction in
extracellular H+ influx or
metabolic acid production; however, other possibilities cannot be excluded.
3) In terms of the results obtained
using either eosin B or a pHo of
8.5, both of these treatments, known to be more selective and potent
inhibitors of the plasmalemma
Ca2+-ATPase, acting by two
different mechanisms, had a similar inhibitory effect on the
NMDA-induced pHi decrease (Table
5). However, neither of these agents affected the basal level of
[Ca2+]i,
whereas pHo 8.5, but not eosin B,
caused an increase in pHi (for
possible reasons, see above). BCECF is also an eosin, and high
concentrations of this chemical may therefore inhibit the plasmalemma
Ca2+-ATPase (23). Due to the much
higher IC50 of BCECF compared with
other eosin analogs (100 vs. 0.2 µM) for this pump (23), the
possibility of pHi changes being
underestimated in the presence of BCECF needs further investigation.
Possible role of intracellular acidosis in the NMDA-activated
Ca2+ influx.
It has been clearly demonstrated that the NMDA-induced current is very
sensitive to a change in pHo
within a physiological range. Decreasing the
pHo suppresses, and increasing the
pHo enhances, the NMDA-induced
Ca2+ current in hippocampal
neurons (48); in the same study, however, there was little, or no,
evidence that the pHi also
modulates this current in mammalian neurons. However, it has been shown that an internal acid load does potentiate the NMDA current in Xenopus motoneurons (11). It is
especially interesting that we found that decreasing the
pHi had a marked inhibitory effect (~50%) on the NMDA-evoked
[Ca2+]i
response in granule cells, since this type of neuron represents the
largest population of neurons in the brain. The difference between our
results and those described previously in hippocampal neurons (48) may
result from the cytosolic pH being different from the pH of the pipette
solutions applied to change the
pHi in the previous study.
Although the modulatory site of the internal proton ions on the NMDA
channel is unknown, the
pHi-sensitive NMDA-induced
Ca2+ response might mediate some
of the physiological and pathophysiological actions of glutamate. For
example, NMDA-induced acidosis may act as a negative feedback signal
for the NMDA-activated Ca2+
influx, in which case the NMDA-induced
[Ca2+]i
increase would have been even larger if the
pHi had not been changed.
Moreover, in addition to the effect of the
pHo on the NMDA current,
intracellular acidosis might also play a protective role during brain
ischemia, a condition in which glutamate accumulates in the
extracellular space and both pHo
and pHi are markedly decreased.
 |
ACKNOWLEDGEMENTS |
We thank the National Science Council of the Republic of China for
financial support.
 |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M.-L. Wu, Dept.
of Physiology, College of Medicine, National Taiwan Univ., No. 1, Sec.
1, Jen-Ai Rd., Taipei, Taiwan (E-mail:
mlw{at}ha.mc.ntu.edu.tw).
Received 2 February 1999; accepted in final form 17 June 1999.
 |
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