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The Journal of Neuroscience, April 15, 1998, 18(8):2982-2990
Regulation of Presynaptic NMDA Responses by External and
Intracellular pH Changes at Developing Neuromuscular Synapses
Yu-Hwa
Chen1,
Mei-Lin
Wu2, and
Wen-Mei
Fu1
Departments of 1 Pharmacology and
2 Physiology, College of Medicine, National Taiwan
University, Taipei, Taiwan 100, Republic of China
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ABSTRACT |
NMDA receptors play important roles in synaptic plasticity and
neuronal development. The functions of NMDA receptors are modulated by
many endogenous substances, such as external pH
(pHe), as well as second messenger systems. In the
present study, the nerve-muscle cocultures of Xenopus
embryos were used to investigate the effects of both external and
intracellular pH (pHi) changes on the functional responses of presynaptic NMDA receptors. Spontaneous synaptic currents
(SSCs) were recorded from innervated myocyte using whole-cell recordings. Local perfusion of NMDA at synaptic regions increased the
SSC frequency via the activation of presynaptic NMDA receptors. A
decrease in pHe from 7.6 to 6.6 reduced NMDA responses to
23% of the control, and an increase in pHe from 7.6 to 8.6 potentiated the NMDA responses in increasing SSC frequency. The effect
of NMDA on intracellular Ca2+ concentration
([Ca2+]i) was also affected by
pHe changes: external acidification inhibited and
alkalinization potentiated [Ca2+]i
increases induced by NMDA. Intracellular pH changes of single soma were
measured by ratio fluorometric method using 2,7-bis (carboxyethyl)-5,6-carboxyfluorescein (BCECF). Cytosolic acidification was used in which NaCl in Ringer's solution was replaced with weak
organic acids. Acetate and propionate but not methylsulfate substitution caused intracellular acidification and potentiated NMDA
responses in increasing SSC frequency, intracellular free Ca2+ concentration, and NMDA-induced currents. On
the other hand, cytosolic alkalinization with NH4Cl did not
significantly affect these NMDA responses. These results suggest that
the functions of NMDA receptors are modulated by both pHe
and pHi changes, which may occur in some physiological or
pathological conditions.
Key words:
NMDA receptor; NMDA-induced current; intracellular
alkalinization; cytosolic acidification; extracellular pH change; developing motoneuron
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INTRODUCTION |
L-Glutamate plays an
important role as an excitatory synaptic neurotransmitter in the
mammalian CNS (Watkins and Evans, 1981 ; Foster and Fagg, 1984).
Evidence has accumulated that at least three types of ionotropic
receptors for glutamate exist on neuronal postsynaptic membranes: AMPA,
kainate, and NMDA receptors (Monaghan et al., 1989). NMDA receptors
possess high Ca2+ permeability and serve many
functions in neuronal development, synaptic plasticity, and acquisition
of memory (Hollmann and Heinemann, 1994; McBain and Mayer, 1994).
However, activation of these receptors also can contribute to the
pathology of epilepsy (Dingledine et al., 1990; Meldrum, 1992), stroke
(Choi, 1990, 1992), and neurodegenerative disorders such as
Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's
disease (Beal, 1992). Therefore, the regulation of NMDA receptor
activity is important in the CNS.
NMDA receptors are controlled by many endogenous substances as well as
second messenger systems (Hollmann and Heinemann, 1994; McBain and
Mayer, 1994). Among the ionotropic glutamate receptors, the NMDA
receptor displays a unique sensitivity to extracellular pH
(pHe). In mammalian central neurons, NMDA-evoked
currents were significantly enhanced by extracellular alkaline shifts
in pH of a few tenths. On the contrary, a decrease in pHe
was shown to reduce NMDA receptor-mediated cation conductance
dramatically in cultured central neurons (Tang et al., 1990; Vyklicky
et al., 1990; Traynelis and Cull-Candy, 1991; Takahashi and Copenhagen, 1996). Both extracellular and intracellular pH are modulated in many
physiological and pathological conditions. Excitatory synaptic transmission in the CNS has been reported to be associated with a rapid
alkalinization of the extracellular space (Chesler, 1990; Chesler and
Kaila, 1992). On the other hand, under normal conditions the
pHi is decreased after prolonged neuronal activity (Chesler and Kaila, 1992). In addition, NMDA is reported to reduce neuronal pHi in central neurons and in spinal motoneurons (Edres et
al., 1986; Dixon et al., 1993; Hartley and Dubinsky, 1993; Irwin et al., 1994). Whether the NMDA receptor is modulated by intracellular pH
shifts is still unknown. Recently, we found that glutamate, which is
also reported to be co-released from some cholinergic nerve terminals
(Vyas and Bradford, 1987; Israel et al., 1993; Meister et al., 1993),
markedly increased the frequency of spontaneous synaptic currents
(SSCs) at embryonic neuromuscular synapses via the activation of NMDA
and non-NMDA receptors (Fu et al., 1995). SSC frequency increased
markedly in response to the local perfusion of glutamate at the
synaptic regions, whereas only a slight increase was observed when
perfusion was performed at the soma. Furthermore, local perfusion of
NMDA to the growth cone induced an inward current (our unpublished
observation). These results indicate the existence of NMDA receptors at
the developing motor nerve terminals. In this study, we further
investigated the functional regulation of presynaptic NMDA receptors by
both pHe and pHi changes. It was found that
intracellular acidification potentiated the responses to NMDA in
increasing SSC frequency, intracellular free Ca2+
concentration, and NMDA-induced currents, whereas intracellular alkalinization did not significantly affect these responses.
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MATERIALS AND METHODS |
Cell culture. Xenopus nerve-muscle
cultures were prepared as reported previously (Tabti and Poo, 1991).
Briefly, the neural tube and the associated myotomal tissues of 1-d-old
Xenopus embryos (stages 20-22) were dissociated in
Ca2+- and Mg2+-free Ringer's
solution supplemented with EDTA. The cells were plated onto clean glass
coverslips and were used for experiments after 24 hr at room
temperature (20-22°C). The culture medium consisted of 50% (v/v)
Ringer's solution (115 mM NaCl, 2 mM
CaCl2, 1.5 mM KCl, 10 mM
HEPES, pH 7.6), 49% L-15 Leibovitz medium (Sigma, St. Louis, MO), 1%
fetal bovine serum (Life Technologies, Grand Island, NY), and
antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin).
Electrophysiology. Whole-cell patch-clamp recording methods
followed those described by Hamill et al. (1981). Patch pipettes were
pulled with a two-stage electrode puller (pp-83, Narishige, Tokyo,
Japan), and the tips were polished immediately before the experiment
using a microforge (MF-83, Narishige). SSCs were recorded from
innervated myocytes by whole-cell recording in the voltage-clamp mode.
Recordings were made at room temperature in the Ringer's solution. For
whole-cell recordings of myocytes, the solution inside the recording
pipette contained 150 mM KCl, 1 mM NaCl, 1 mM MgCl2, and 10 mM HEPES,
pH 7.2. The extent of the potentiation was measured by the frequency
ratio of SSCs, which is defined as the ratio of SSC frequency at peak
level observed during application of drugs compared with the mean
frequency observed before drug treatment. NMDA-induced currents were
recorded from soma. The solution inside the recording pipette contained
145 mM CsCl, 0.1 mM CaCl2, 3 mM MgCl2, 5 mM EGTA, 5 mM HEPES, pH 7.2, and 1 mM amphotericin B was
added to internal solution to get perforated patch. The membrane
currents passing through the patch pipette were recorded with a
patch-clamp amplifier (Dagan 8900). The data were digitized using
Neuro-corder (Neuro Data DR 390) and stored on a videotape for later
playback onto a storage oscilloscope (Tektronix 5113, Beaverton, OR) or
an oscillographic recorder (Gould RS3200, Valley View, OH). The Data
6100 waveform analyzer (Data Precision, Danvers, MA) was used to
analyze the frequency of SSCs. For analysis of spontaneous events at
high frequency when overlaps of current events occurred, the events
were counted visually from chart records of an oscillographic recorder
driven at high chart speeds (25-100 mm/sec). Data are expressed as
mean ± SEM. Statistical significance was evaluated by Student's
t test.
Measurement of pHi. Measurement of
pHi has been described in detail elsewhere (Wu et al.,
1994). In brief, cultures were loaded with 5 µM 2,7-bis
(carboxyethyl)-5,6-carboxyfluorescein (BCECF-AM, Molecular Probes,
Eugene, OR) for 5-10 min at room temperature in Ringer's solution and
then washed with the same solution three times. A single soma was
isolated by adjusting the slit width of the photomultiplier tube and
excited by alternate flashes of 490 and 440 nm wavelength light, using
a filter wheel (Cairn Research, Kent, England) rotating at 32 Hz. The
excitation light was transmitted to the cell under study using a 510 nm
dichroic mirror under the microscope nosepiece, and the resulting
fluorescence was collected via a 40× magnification oil-immersion lens.
The overall sampling rate was 0.5 Hz. The 490/440 emission ratio from
the intracellular BCECF was calculated and converted to a linear pH
scale using in situ calibration data obtained at the end of
the experiment by the nigericin technique. A calibration curve similar
to that used by Wu et al. (1994) was established by measuring the
fluorescence ratio values between pHi 4.5 and 9.5. Between
pHi 5.5 and 9.0, the response is linear and fits the
equation pHi-pK + log
[(Rmax R)/(R  Rmin] + log(F440min/F440max),
where R is the ratio of 530 nm fluorescence at 490 nm
excitation to 530 nm fluorescence at 440 nm excitation.
Rmax and Rmin are the
maximum and minimum ratio values from the data curve, and K
is the dissociation constant for the dye, taken as 55 nM.
Measurement of intracellular Ca2+ levels.
The intracellular concentration of free Ca2+
([Ca2+]i) in the soma was
measured using the Ca2+-sensitive fluorescent dye
fura-2. Cultures plated onto a 25 mm round coverslip (No. 1001, Assistent) were loaded with 2 µM fura-2 AM (Molecular
Probes) in Ringer's solution for 1 hr at room temperature. The cells
were then washed with the same solution. The fluorescence of a single
soma was measured as indicated above except that the cell was excited
by alternate flashes of 340 and 380 nm wavelength. The 340/380 emission
ratio from the intracellular fura-2 was calculated and converted to a
linear Ca2+ scale by in situ calibration
at the end of the experiment using the Ca2+
ionophore ionomycin (5 µM, Sigma). The following equation
(Grynkiewicz et al., 1985) was used to convert the fluorescence ratio
into the intracellular Ca2+ concentration
[Ca2+]i = Kd
[(R Rmin)/Rmax R](Sf2/Sb2),
where R is the ratio of 510 nm fluorescence at 340 nm
excitation to 510 nm fluorescence at 380 nm excitation,
Rmax (10 mM Ca2+)
and Rmin (10 mM EGTA in
Ca2+-free Ringer's solution) are the maximum and
minimum ratio values from the data curve, Kd is
the dissociation constant for the dye, taken as 135 nM at
room temperature, and
Sf2/Sb2 is the
ratio of the 380 nm signals determined at Rmin
and Rmax.
Because Ca2+ and H+ are
presumably competitive, we have tested whether the 340/380 signal was
affected by the change of pHi because of the possible pH
sensitivity of fura-2. Three levels of pH (6.8, 7.2, and 7.8) were
tested in Ca2+-free Ringer's solution supplemented
with 2 mM EGTA using the free acid form of fura-2, and we
found that there was little change in the 340/380 ratio when the pH was
changed (6.8, 7.8, and 7.2); the first two points were the two extreme
levels of pHi we have observed in our experiments. We used
Ca2+-free Ringer's solution supplemented with 2 mM EGTA in the above test to mimic a similar level of
resting [Ca2+]i (~70 nM)
in Xenopus cultured spinal neurons.
Chemicals. The following chemicals were used:
NH4Cl and sodium acetate (Sigma), sodium methylsulfate
(Aldrich, Milwaukee, WI), sodium propionate (Hayashi, Osaka, Japan),
and NMDA (RBI, Natick, MA).
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RESULTS |
Regulation of NMDA responses in increasing SSC frequency by
pHe changes
In nerve-muscle cultures prepared from 1-d-old Xenopus
embryos, synaptic contacts were established between dissociated spinal neurons and myocytes within the first day of culture. SSCs were readily
detectable from the innervated myocytes by whole-cell voltage-clamp
recording. These currents have been shown to be caused by spontaneous
ACh secretion from the neuron, because they are abolished by bath
application of D-tubocurarine and unaffected by
tetrodotoxin (Xie and Poo, 1986). It was found recently that there are
NMDA receptors in the nerve terminals of developing motoneurons (Fu et
al., 1995; Liou et al., 1996). We thus used these nerve-muscle
cocultures to investigate the functional regulation of presynaptic NMDA
receptors by pH changes. Figure 1 shows
the recording of SSCs from an innervated myocyte. Local application of
NMDA by pressure at synaptic regions with another glass microelectrode as indicated in the inset (pipette opening, 9 µm; pipette NMDA concentration, 150 µM) reversibly increased the SSC
frequency (SSC frequency ratio was 13.7 ± 3.4; n = 21). The effects of changes in pHe on the NMDA responses
were investigated by replacing normal Ringer's solution with
pHe 6.6 or 8.6 Ringer's solution. The pH was adjusted
before the experiment with 1N HCl or NaOH, respectively, and the pH
value remained unchanged throughout the experiment (within 1 hr). The
change in extracellular pH from 7.6 to 6.6 or 8.6 did not by itself
significantly affect the frequency of spontaneous ACh secretion (SSC
frequency ratio was 1.2 ± 0.1, n = 5, and
0.9 ± 0.1, n = 6, respectively). The mean SSC
amplitude in pH 6.6 and 8.6 Ringer's solution also showed no
significant change before NMDA application (146.5 ± 35.0 pA,
n = 5, and 154.7 ± 19.3 pA, n = 4, respectively; control SSC amplitude was 167.3 ± 14.3 pA,
n = 21). However, changes in pHe range
between 6.6 and 8.6 had different effects on the SSC potentiating
action of NMDA. Alkalinization of external pH from 7.6 to 8.6 potentiated the NMDA effect by 195 ± 34% of the control (SSC
frequency ratio was 26.5 ± 4.7, n = 4) (Figs. 1,
2A). On the other hand,
acidification of external medium from pH 7.6 to 6.6 antagonized the
NMDA action to 23 ± 12% of the control (SSC frequency ratio was
3.2 ± 1.7, n = 4) (Figs. 1,
2A). The time course-response curves of the NMDA action in different external pH are shown in Figure
2B.
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Figure 1.
Effect of external pH changes on the
SSC-increasing action of NMDA. Shown is continuous tracing of SSCs
recorded from an innervated muscle cell in 1-d-old
Xenopus culture. The myocyte was voltage-clamped at a
potential of 60 mV. Downward deflections are SSCs (filtered at 150 Hz). The top right panel shows the relative position of
patch pipette (R) and NMDA pipette
(NMDA). Local perfusion of NMDA at the synaptic region
with another micropipette increased SSC frequency. NMDA responses were
potentiated in pH 8.6 Ringer's solution and antagonized in pH 6.6 Ringer's solution. Samples of superimposed SSCs in 1 sec before and
after NMDA treatment are shown at higher time resolution. Scale bar, 40 µm. M, Myocyte; N, neuron.
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Figure 2.
Summary of the effect of external pH changes on
the SSC-increasing action of NMDA. Note that NMDA increased SSC
frequency in normal pH 7.6 Ringer's solution and the change of
external pH to 8.6 potentiated NMDA action, whereas the reduction of
external pH to 6.6 antagonized the SSC-increasing action of NMDA
(A). B, Time course-response
curves in response to NMDA application. Data are presented as mean ± SEM (n). *p < 0.05 compared with control.
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Intracellular acidification enhances the responses to NMDA in
increasing SSC frequency
Cytosolic acidification was induced by weak organic acid
substitution. Weak organic acid with pKa values
in the range of 4-6 might allow selective acidification of cells. The
protonated forms of such weak acids are more membrane permeable than
their charged anionic forms and then dissociate intracellularly to
liberate H+. Exposure of cells to weak acids is thus
expected to result in an acute acid load to the cytoplasm. For these
experiments, all the extracellular NaCl was replaced by the sodium
salts of organic acids in normal Ringer's solution. Cytosolic pH in
neuronal soma was monitored by the BCECF fluorescence method. The
baseline pHi value was 7.17 ± 0.03 (n = 46). The change of external solution from pHe 7.6 to 6.6 or 8.6 caused only a slight cytosolic acidification of 0.14 ± 0.02 units (n = 6) or intracellular alkalinization of 0.11 ± 0.04 units (n = 4), respectively (Fig.
3A,D). However, a rapid
intracellular acidification occurred after exposure with either acetate
or propionate at a fixed pH of external medium (pHe 7.6)
(Fig. 3B,D). The pHi reached peak values of
6.86 ± 0.12 (n = 8) with acetate and 6.78 ± 0.15 (n = 7) with propionate. The intracellular fluid
revealed an acidic shift of 0.2-0.4 pH units. Removal of acetate or
propionate results in a rebound cytosolic alkalinization. On the other
hand, the peak value of pHi after exposure to methylsulfate
did not show significant change (pHi was 7.09 ± 0.03;
n = 6). The frequency and amplitude of SSC did not show
significant change after exposure to organic acids alone for 1-2 min
(SSC frequency ratio was 1.3 ± 0.2, n = 6, with
acetate; 1.4 ± 0.3, n = 6, with propionate; and
1.1 ± 0.2, n = 4, with methylsulfate,
respectively; mean SSC amplitude was 150.9 ± 21.6 pA with
acetate; 186.3 ± 46.9 pA with propionate; and 142.2 ± 13.5 pA with methylsulfate, respectively). However, substitution with either
acetate or propionate markedly potentiated SSC frequency, increasing
the action of NMDA (SSC frequency ratio was 35.5 ± 7.2, n = 6, and 34.8 ± 10.9, n = 5, respectively) (Figs.
4A,B, 5A), while substitution of
NaCl with strong organic acids such as methylsulfate resulted in no
significant effect on the NMDA response (SSC frequency ratio was
8.5 ± 2.6, n = 4) (Fig. 5A). We
further investigated the effect of intracellular alkalinization on the
NMDA responses. Exposure of the soma to 15 mM
NH4Cl resulted in a rapid increase of pHi
( pH = 0.77 ± 0.04; n = 8), and the pHi in peak value was 7.67 ± 0.14 (Fig.
3C,D). The alkalinization seen during the first moments of
exposure to NH4+ is presumably caused by
the rapid, passive entry of NH3 and its subsequent
hydration to form NH4+ and
OH (Roos and Boron, 1981). Cytosolic
alkalinization with NH4Cl alone for 2 min also did not
significantly affect SSC frequency and amplitude (SSC frequency ratio
was 0.9 ± 0.2; mean SSC amplitude was 145.4 ± 28.5 pA;
n = 6). As shown in Figures 4C and
5A, the NMDA responses were not affected after intracellular
alkalinization (SSC frequency ratio was 12.6 ± 3.6, n = 6). The time course-response curves of the NMDA
action on the cytosolic acidification and alkalinization are
shown in Figure 5B.
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Figure 3.
Cytosolic pH changes induced by organic acids and
NH4Cl in neuronal soma. One-day-old Xenopus
cultures were loaded with BCECF-AM, and ratio fluorometric measurements
of intracellular pH were made on single soma. A,
Top panel: the area of single soma for the measurement
of pHi was outlined by the dotted box. Scale
bar, 20 µm. Bottom panel: change of external pH
slightly affected the intracellular pH. B, Replacement
of all NaCl in Ringer's solution with sodium acetate resulted in a
marked cytosolic acidification. C, Addition of
NH4Cl to the bath caused cytosolic alkalinization.
D, Summarized effect on cytosolic pH by the changes of
external pH or by the addition of organic acids or NH4Cl.
Data are presented as mean ± SEM (n = 4-8).
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Figure 4.
Effect of intracellular pH changes on the
SSC-increasing action of NMDA. Shown is a continuous tracing of SSCs
recorded from an innervated muscle cell in 1-d-old Xenopus
laevis culture. The myocyte was voltage-clamped at a potential
of 60 mV. Downward deflections are SSCs (filtered at 150 Hz).
Perfusion of NMDA at synaptic region with another micropipette
increased SSC frequency. NMDA responses were potentiated by cytosolic
acidification with organic acid substitution and not significantly
affected by cytosolic alkalinization with NH4Cl. Samples of
superimposed SSCs in 1 sec before and after NMDA treatment are shown at
higher time resolution.
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Figure 5.
Summary of the effects of intracellular pH changes
on the SSC-increasing action of NMDA. Note that NMDA increased SSC
frequency in normal Ringer's solution, and intracellular acidification
with either acetate or propionate potentiated NMDA responses
(A). B, Time course-response
curves of the NMDA action on the cytosolic pH changes. Data are
presented as mean ± SEM (n).
# p < 0.05 compared with control.
*p < 0.05 compared with NMDA in normal Ringer's
solution.
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Modulation of NMDA effects on neuronal cytoplasmic
Ca2+ by the changes of pHe and
pHi
The intracellular concentration of free Ca2+
([Ca2+]i) in the soma was
measured using the Ca2+-sensitive fluorescent dye
fura-2. The basal neuronal [Ca2+]i
value was 70.2 ± 4.6 nM (n = 32).
Superfusion with 100 µM NMDA in pH 7.6 Ringer's solution
increased [Ca2+]i by 131.3 ± 26.9 nM (n = 27). Responses to NMDA were
affected by changing the pH of external medium. Lowering the
pHe from 7.6 to 7.0 or 6.6 decreased the
[Ca2+]i responses of NMDA to 64 ± 10% and 23 ± 7% of the control (n = 4 for
each), respectively (Fig.
6A,B), whereas
increasing the pHe to 8.6 potentiated the
[Ca2+]i response of NMDA to 156 ± 18% of the control (n = 4) (Fig. 6A,B). By contrast, intracellular acidification by
either acetate or propionate substitution enhanced the
[Ca2+]i response to NMDA. NMDA
response was enhanced concentration-dependently by acetate
substitution. Two-thirds of acetate substitution (two-thirds of the
NaCl in Ringer's solution was replaced with equimolar sodium acetate)
nonsignificantly increased NMDA response to 134 ± 15% of control
(n = 7), whereas 100% acetate substitution
significantly increased the NMDA response to 168 ± 28%
(n = 8) (Fig.
7A,D). Substitution with
another weak acid propionate (100% substitution) also increased NMDA
response to 200 ± 50% (n = 5) (Fig.
7B,D). However, intracellular alkalinization by 15 mM NH4Cl had no significant effect (Fig.
7C,D).
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Figure 6.
Effect of external pH changes on the
[Ca2+]i-increasing action of NMDA.
A, Xenopus nerve-muscle cultures were
loaded with fura-2 AM, and ratio fluorometric measurement of
intracellular Ca2+ was made on a single soma.
Superfusion with NMDA increased
[Ca2+]i, and this response was
potentiated in pH 8.6 Ringer's solution and antagonized in pH 6.6 Ringer's solution. B, Summary of the effect of external
pH changes on the
[Ca2+]i-increasing action of NMDA.
Data are presented as mean ± SEM (n = 4).
*p < 0.05 compared with control.
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Figure 7.
Effect of intracellular pH changes on the
[Ca2+]i-increasing action of NMDA.
Xenopus nerve-muscle cultures were loaded with fura
2-AM, and ratio fluorometric measurement of intracellular
Ca2+ was made on a single soma. Superfusion with
NMDA increased [Ca2+]i, and this
response was enhanced by cytosolic acidification with either acetate
(A) or propionate (B) but
not affected by cytosolic alkalinization with NH4Cl
(C). D, Summary of the effects of
intracellular pH changes on the
[Ca2+]i-increasing action of NMDA.
Data are presented as mean ± SEM (n = 3-22).
*p < 0.05 compared with control.
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Potentiation of NMDA-induced currents by
intracellular acidification
To determine directly the effect of intracellular acidification on
the NMDA receptor, the soma of isolated spinal neurons was whole-cell
voltage-clamped at 60 mV by using perforated patch. Local perfusion
of NMDA with another micropipette (intrapipette concentration, 150 µM), as indicated in the right panel of Figure 8A, induced an inward
current in the soma. Intracellular acidification by the substitution of
all NaCl of Ringer's solution with either sodium acetate (Fig.
8A) or sodium propionate (Fig. 8B)
enhanced the NMDA-induced currents (134.6 ± 6.4% and 162.6 ± 14.0% of the control, respectively; n = 7 for
each). On the other hand, cytosolic alkalinization with
NH4Cl did not significantly affect the NMDA-induced currents (96.1 ± 4.8% of the control, n = 6)
(Fig. 8C). However, changes of external pH from 7.6 to 8.6 or 6.6 enhanced and inhibited the NMDA-induced currents, respectively
[149.4 ± 8.5% (n = 6) and 37.1 ± 6.6%
(n = 4) of the control, respectively].
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Figure 8.
Potentiation of NMDA-induced current by
intracellular acidification. A, The soma of an isolated
neuron was whole-cell voltage-clamped at 60 mV. Local perfusion with
NMDA from a glass micropipette (NMDA) as indicated in
the right panel induced an inward current. NMDA-induced
current was enhanced by intracellular acidification with either acetate
(A) or propionate (B) but
not affected by cytosolic alkalinization (C).
Scale bar, 10 µm. R, Patch pipette.
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DISCUSSION |
Many studies have revealed that second messengers such as
Ca2+, cyclic nucleotides, and G-proteins modulate
voltage-gated ion channels and ligand-gated receptor channels. Here we
show that NMDA receptor-mediated synaptic potentiation, increase of
intracellular Ca2+ concentration, and NMDA-induced
currents are significantly influenced by the changes of either external
or intracellular pH. We have observed that external acidification
inhibited but intracellular acidification enhanced NMDA responses. On
the other hand, external alkalinization potentiated the NMDA action,
whereas intracellular alkalinization had no significant effect. The
modulation of NMDA responses by both external and internal pH changes
might mediate some of the physiological and pathological actions of
glutamate.
Our results are different from those reported by Tang et al. (1990),
who showed that NMDA-activated current was rather insensitive to the
change in internal pH. The intracellular pH was altered in their study
by filling the pipette with an internal solution of different values of
pH. The pH of intracellular fluid may be different from that of patch
pipette as a result of the buffering capacity of cytoplasm and improper
diffusion of patch pipette solution. We here acidified intracellular
fluid by using a weak organic acid substitution. It has long been known
that weak acids can readily cross cell membranes in their uncharged
forms and then dissociate, thereby acidifying the cell interior (Karuri et al., 1993). Once inside the cell, the pHi is well above
pKa, leading to the dissociation of the
protonated form and intracellular acidification. Therefore, the anions
of weak acids such as acetate (pKa 4.75) or
propionate (pKa 4.87) caused larger internal
acidification (pHi 6.8), and the anions of strong acids
such as methylsulfate (pKa < 1.0) induced
little pHi change of the cells (Chesler, 1990).
The potentiation of NMDA action was in parallel with the levels of
cytosolic acidification by these organic acids. We found no effect on
NMDA response by methylsulfate substitution, ruling out the likelihood
that the Cl substitution itself was affecting NMDA
receptor. Modulation of the NMDA-activated current by external pH
changes resulted primarily from changes in the number of channel
openings. The single-channel conductance of NMDA receptor was not
significantly affected (Tang et al., 1990; Vyklicky et al., 1990;
Traynelis and Cull-Candy, 1991). We here confirmed previous reports
that external alkalinization enhances and external acidosis inhibits
NMDA receptor activation (Tang et al., 1990; Vyklicky et al., 1990;
Traynelis and Cull-Candy, 1991; Takahashi and Copenhagen, 1996). A
lowering of pHe from 7.6 to 6.6 inhibits the functional
activation of NMDA receptors, whereas a rise of pHe from
7.6 to 8.6 significantly enhanced NMDA responses. However, we provide
additional evidence that the function of the NMDA receptor can also be
regulated by the changes of intracellular pH. The potentiation of SSC
and an increase in intracellular free Ca2+ by NMDA
are regulated in parallel. We found previously that a larger decrease
of pHi (~1.4-1.6 pH units) is required for SSC frequency-increasing effect by weak organic acid substitution in
pHe 6.6 Ringer's solution (Chen et al., 1998). In the
current study, the intracellular fluid revealed an acidific shift in pH of only 0.2-0.4 units by weak organic acid substitution in normal Ringer's solution. Therefore, the potentiation effect on SSC frequency can be fully accounted for by changes in the NMDA-induced
[Ca2+]i increase. Furthermore, direct
evidence is shown in Figure 8 that NMDA-induced inward currents were
increased in the presence of acetate or propionate. On the other hand,
intracellular alkalinization had no significant effect on these NMDA
responses. Because the NMDA receptor is activated in the absence of
glycine, the modulation of intracellular H+ appears
not to be related to the glycine recognition site of the NMDA receptor.
How the internal H+ modulates the gating of the NMDA
channel needs further investigation. Although pHi was
reduced by 0.1 units after the pHe was lowered from 7.6 to
6.6, no potentiation of NMDA responses was observed, probably because
of the larger inhibitory effect on NMDA receptors by external
acidification and the insufficient intracellular acidification.
Changes in the concentration of H+ have been clearly
shown to serve important biological roles by regulating the properties of protein macromolecules. Accumulating evidence suggests that extracellular and intracellular pH can change significantly under not
only pathological but also physiological circumstances. The local pH
shifts could be large in synaptic cleft and subsynaptic regions under
these conditions. Repetitive neuronal activity may lower intracellular
pH through several mechanisms. These include the metabolic production
of CO2 and lactic acid, the release of protons from
internal sites in response to elevation of free intracellular Ca2+ concentration, and the net entry of acid
through ligand- or voltage-gated channels (Chesler and Kaila, 1992).
Brief exposure of hippocampal neurons to NMDA results in a
concentration-dependent pHi reduction (0.2-0.4 pH units)
(Irwin et al., 1994). Acute focal electrical stimulation evoked an
intracellular acidosis of approximately pH 6.8 in cortical tissue
(Yaksh and Anderson, 1987). Changes in pHi and
pHe have been known to modify several types of
voltage-gated ion channels (Takahashi and Copenhagen, 1996). Modulation
of excitatory transmission by pH shifts may be especially pertinent to
forms of long-term potentiation (LTP) that have been linked to NMDA receptor activation (Blanton et al., 1989). Experimentally, LTP is
often induced by trains of high frequency stimulation. Both the
intracellular and external pH shifts may thus influence the relationship between LTP induction and stimulus frequency.
Pathologically, hypoxia and glucose depletion in hippocampal slices
have also been shown to result in a decrease in pHi within
5 min of exposure (Chesler, 1990). Furthermore, brain ischemia has been
reported to lower brain pHe and pHi (Nedergaard
et al., 1991; Fujiwara et al., 1992). Recent evidence suggests that
mild extracellular acidosis protects central neurons from injury
induced by oxygen and glucose deprivation (Giffard et al., 1990;
Tombaugh and Sapolsky, 1990). Extracellular acidosis prevents the
activation of NMDA receptors, which play a major role in mediating
ischemic neuronal injury. By contrast, extracellular alkalosis can
induce epileptiform activity, which is blocked by NMDA antagonists in
mammalian brain slices (Aram and Lodge, 1987). Our results showed that
intracellular acidic shift in pH of only 0.2-0.4 units enhanced NMDA
responses. Therefore, intracellular acidosis may enhance NMDA responses
in some physiological conditions and NMDA neurotoxicity in certain pathological diseases. The proton sensitivity of NMDA receptors could
influence synaptic plasticity and seizure generation and may be
relevant for glutamate-induced neurotoxicity during ischemia. These
results add another level of complexity to the regulation of NMDA
synaptic events by endogenous factors. Intracellular
H+, acting as a second messenger, may influence the
neuronal excitability via modulation of NMDA channel activity.
| |
FOOTNOTES |
Received Oct. 20, 1997; revised Jan. 27, 1998; accepted Jan. 29, 1998.
This work was supported by research Grant NSC 87-2314-B002-303 from
the National Science Council. We thank Mr. I. S. Peng for help in
preparing this manuscript.
Correspondence should be addressed to Dr. Wen-Mei Fu, Department of
Pharmacology, College of Medicine, National Taiwan University, Taipei,
Taiwan 100, Republic of China.
| |
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Top
Abstract
Introduction
Compound via MeSH
