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Volume 16, Number 17,
Issue of September 1, 1996
pp. 5393-5404
Copyright ©1996 Society for Neuroscience
Mechanisms of H+ and Na+ Changes Induced
by Glutamate, Kainate, and D-Aspartate in Rat Hippocampal
Astrocytes
Christine R. Rose1 and
Bruce R. Ransom2
1 Department of Neurology, Yale University School of
Medicine, New Haven, Connecticut 06510, and 2 Department of
Neurology, University of Washington School of Medicine, Seattle,
Washington 98195-6465
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
The excitatory transmitter glutamate (Glu), and its analogs
kainate (KA), and D-aspartate (D-Asp)
produce significant pH changes in glial cells. Transmitter-induced pH
changes in glial cells, generating changes in extracellular pH, may
represent a special form of neuronal-glial interaction. We
investigated the mechanisms underlying these changes in intracellular
H+ concentration ([H+]i) in
cultured rat hippocampal astrocytes and studied their correlation with
increases in intracellular Na+ concentration
([Na+]i), using fluorescence ratio imaging
with 2 ,7-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) or
sodium-binding benzofuran isophthalate (SBFI). Glu, KA, or
D-Asp evoked increases in [Na+]i;
Glu or D-Asp produced parallel acidifications. KA,
in contrast, evoked biphasic changes in
[H+]i, alkaline followed by acid shifts,
which were unaltered after Ca2+ removal and persisted in 0 Cl -saline, but were greatly reduced in
CO2/HCO3-free or Na+-free
saline, or during 4,4-diisothiocyanato-stilbene-2,2-disulphonic acid
(DIDS) application. The non-NMDA receptor antagonist
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) blocked KA-evoked
changes in [H+]i and
[Na+]i, indicating that they were
receptor-ionophore mediated. In contrast, CNQX increased the
[H+]i change and decreased the
[Na+]i change induced by Glu.
D-Asp, which is transported but does not act at Glu
receptors, induced [H+]i and
[Na+]i changes that were virtually unaltered
by CNQX. Our study indicates that [Na+]i
increases are not primarily responsible for Glu- or KA-induced
acidifications in astrocytes. Instead, intracellular acidifications
evoked by Glu or D-Asp are mainly caused by transmembrane
movement of acid equivalents associated with Glu/Asp-uptake into
astrocytes. KA-evoked biphasic [H+]i
changes, in contrast, are probably attributable to transmembrane ion
movements mediated by inward, followed by outward, electrogenic
Na+/HCO3 cotransport, reflecting
KA-induced biphasic membrane potential changes.
Key words:
glial cell;
glutamate;
kainate;
aspartate;
intracellular
sodium;
intracellular pH;
ion regulation;
glutamate transport;
ionotropic glutamate receptor;
Na+/HCO3
cotransport
INTRODUCTION
A well-known action of neurotransmitters in the
brain is alteration of intra- and extracellular Na+,
K+, Ca2+, and/or Cl
concentrations by activation of specific receptors or uptake
mechanisms. In addition, many transmitters, like the excitatory amino
acid L-glutamate (Glu) and its analog
D-aspartate (D-Asp) induce an acidification in
both neurons and glial cells (Bouvier et al., 1992 ; Deitmer and Munsch,
1992; Brookes and Turner, 1993; Deitmer and Schneider, 1996). Changes
in glial cell pH can influence extracellular pH and, in so doing,
potentially modulate the function of adjacent neurons (Ransom, 1992;
Kaila et al., 1993; Taira et al., 1993, Gottfried and Chesler, 1994;
Rose and Deitmer, 1994; Newman, 1996). It is, therefore, of broader
neurobiological interest to elucidate the mechanisms that cause
neurotransmitter-induced changes in glial pH.
The Glu- or D-Asp-induced intraglial acidification was
suggested to be caused by a Glu transporter that exchanges
intracellular K+ and OH with extracellular
Na+ and Glu or D-Asp (Fig.
1A) (Bouvier et al., 1992).
Surprisingly, kainate (KA), a Glu agonist, which is not a
substrate for transport-mediated Glu uptake (Kimelberg et al., 1989),
also caused an acidification in leech glial cells and cerebral
astrocytes (Deitmer and Munsch, 1992; Brune and Deitmer, 1995).
Fig. 1.
Model showing possible interrelationships between
Glu-induced ion fluxes and [H+]i regulation
in hippocampal astrocytes. Glu and
d-Asp are substrates for the electrogenic Glu
transporter (A, hatched area), which
exchanges extracellular Glu or
D-Asp and 2 Na+ for intracellular
K+ and OH. This causes cellular
depolarization and acidification. Activation of ionotropic non-NMDA
receptors by Glu or KA (indicated by arrowhead)
depolarizes the cells by influx of Na+, and sometimes
Ca2+, and efflux of K+ through cation channels
(A). Various secondary changes in
[H+]i could be caused by these actions of
Glu. Additional intracellular acidification could originate secondary
to increase in intracellular Ca2+ activating a
Ca2+/H+ pump in the plasma membrane
(B), and/or because of the increase in
[Na+]i attenuating cellular acid secretion by
Na+/H+ exchange and
Na+-dependent
Cl/HCO3 exchange
(C). Cellular depolarization could cause intracellular
alkalinization by stimulating inwardly directed electrogenic
Na+/HCO3 cotransport
(C); a late hyperpolarization follows a period of
elevated [Na+]i attributable to
Na+, K+-ATPase activity (D), and
this could activate outwardly directed
Na+/HCO3 cotransport resulting in
intracellular acidification.
[View Larger Version of this Image (73K GIF file)]
Several mechanisms could account for the KA-induced
acidification of glial cells. KA is a non-NMDA Glu-receptor
agonist that binds to ionotropic Glu receptors and opens cation
channels, generating Na+ influx, and sometimes
Ca2+ influx, and K+ efflux (Fig.
1A) (von Blankenfeld and Kettenmann, 1991). The
KA-induced acidification might be caused by influx of
H+ down its electrochemical gradient through these cation
channels (Chen and Chesler, 1992; Deitmer and Munsch, 1992). Another
hypothesis is that an increase in intracellular Ca2+
(Muller et al., 1992; Jabs et al., 1994; Munsch et al., 1994) activates
a plasma membrane Ca2+/H+-ATPase,
extruding Ca2+ in exchange for extracellular H+
(Fig. 1B) (Schwiening et al., 1993; Paalasmaa et
al., 1994).
Alternatively, the KA-induced acidification of glial cells
could involve inhibition of cellular acid extrusion after an increase
in intracellular Na+ ([Na+]i).
This hypothesis is attractive, because glial cells maintain a
relatively alkaline intracellular pH primarily by
Na+-dependent transporters including
Na+/H+ exchange,
Na+/HCO3 cotransport, and
Na+-dependent
Cl/HCO3 exchange (Fig.
1C) (Chesler, 1990; Deitmer and Rose, 1996). A reduction in
the inwardly directed Na+ gradient after increases in
[Na+]i could, therefore, lead to decreased
acid extrusion and intracellular acidification. To test this
hypothesis, we studied the correlation between changes in intracellular
H+ concentration ([H+]i) and
[Na+]i induced by Glu, KA, or
D-Asp in cultured rat hippocampal astrocytes using
fluorescence ratio imaging with BCECF-AM and SBFI-AM. We found that
[Na+]i increases are not primarily
responsible for Glu- or KA-induced acidifications in
hippocampal astrocytes. Our study confirms that the acidification
evoked by Glu or D-Asp was most likely attributable to
transport-mediated uptake and strongly suggests that KA-evoked
[H+]i changes were attributable to altered
activity of Na+/HCO3 cotransport
in response to membrane potential changes.
MATERIALS AND METHODS
Cell cultures. Cell cultures of astrocytes were
prepared as described previously (Sontheimer et al., 1991). To
summarize, newborn Sprague-Dawley rats were narcotized with
CO2 and decapitated. Hippocampi were removed and exposed to
20 U/ml Papain (Worthington, Freehold, NJ) for 30 min. After
trituration, cells were plated onto poly-D-lysine-coated
(Sigma, St. Louis, MO) coverslips at a density of 80/mm2
and grown in an incubator at 37°C and 5%
CO2/95% O2. Cells were fed every 4 d with
DMEM/F12 (Dulbecco's modified Eagle's medium) + 10% bovine serum
(JRA Scientific, Lexington, KS). These cultures contained >95%
astrocytes, as judged by immunostaining for glial fibrillary acidic
protein (GFAP). The cells grew to confluence in about 10 d, and
cells from 10-17 d in vitro were used for experiments.
For mixed neuron-glia cultures, rat hippocampi [embryonic day (E)
19] were removed and dissociated by a 10 min enzyme exposure to 10 U/ml Papain and trituration. The dissociated cells were plated onto
poly-L-ornithine/laminin-coated (Sigma) coverslips in
DMEM/F12 medium + 10% bovine serum. After 24 hr in culture, the cells
were transferred into Neurobasal medium with B27 (50:1) (Life
Technologies, Grand Island, NY), half of which was subsequently
exchanged every 5-7 d. At day 8 in culture, the mitotic
inhibitor cytosine-b-d-arabinofuranoside (ARA-C) (Sigma) was added to a
final concentration of 5 µM to inhibit glial cell
division. Measurements were made using cells between 14 and 21 d
in culture. Neurons were identified by their morphological appearance
in the light microscope and studies were limited to large pyramidal
cells with at least three processes (length  4 times the cell
diameter). Our experience is that these cells are always GFAP-negative
and have typical neuronal electrical characteristics when impaled,
including spontaneous action and synaptic potentials (our unpublished
observations).
Solutions. The standard saline contained (in
mM): 115.75 NaCl, 3 KCl, 2 MgSO4, 2 CaCl2, 1.25 NaH2PO4, 23 NaHCO3, and 10 glucose, and was continuously bubbled with
5% CO2/95% O2 resulting in a pH of 7.38. The
CO2/HCO3-free saline was titrated
to a pH of 7.4 with NaOH or HCl and contained the same amount of KCl,
MgSO4, CaCl2, NaH2PO4,
and glucose, but 126.25 mM NaCl and 25 mM
HEPES.
The fluorescent dyes for measurement of intracellular H+
(acetoxy- methylester of
2,7-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF-AM) and
Na+ (acetoxymethylester of sodium-binding benzofuran
iso- phthalate (SBFI-AM) were obtained from Teflabs (Austin, TX).
The non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione
(CNQX) was obtained from Research Biochemical Incorporated (Natick,
MA). All other drugs and chemicals were obtained from Sigma. Except for
CNQX and gramicidin, which were first dissolved in dimethyl sulfoxide
(DMSO), and monensin and nigericin, which were dissolved in ethanol,
the drugs were added directly to the saline before use.
The solutions for intracellular calibration of the BCECF ratio signal
contained (in mM): 30 Na+, 120 K+,
30 Cl, 120 gluconic acid, and 1 MgSO4, and
were adjusted to different pH with either 10 mM
piperazine-N,N-bis[2-ethane-sulfonic acid] (PIPES), for pH  6.9) or HEPES (pH 7.2). Nigericin (10 µM) and
gramicidin (3 µM) were added for equilibration of extra-
and intracellular pH. Solutions for calibration of SBFI's sensitivity
to [Na+]i contained (in mM): 150 (K+ + Na+), 30 Cl, 120 gluconic
acid, 1 MgSO4, 10 HEPES, adjusted to pH 7.2 with KOH.
Gramicidin (3 µM), monensin (10 µM), and
ouabain (1 mM), were used to equilibrate extra- and
intracellular Na+ (Rose and Ransom, 1996).
Dye loading. Cells were loaded with the AM-ester of BCECF
(20 µM) (15 min) or SBFI (10 µM) (90 min)
in CO2/HCO3-free saline at room
temperature (20-22°C). Pluronic acid (0.1%) was added to improve
dye uptake. Experiments were performed in a closed chamber, which was
perfused with CO2/HCO3-buffered
solution, warmed to 37°C, at a standard flow rate of 4 ml/min.
Because of a dead space between the switching site and the chamber,
solution exchange at standard flow rate required 20-22 sec from the
time of switching. To compensate for this fact, solution changes are
indicated 20 sec later in the figures than performed in the original
experiment.
Measurement and calibration of intracellular pH and
[Na+]. The experiments were performed with a
Nikon-Diaphot-TMD inverted epifluorescence microscope equipped with a
40× epifluorescence oil objective. Cells were excited every 5 sec with
dual digikrom 120 monochromators (CVI Laser Corporation, Albuquerque,
NM) at 440 and 490 nm (for pH measurement) or 345 and 385 nm (for
Na+ measurement); five video frames were averaged for each
excitation period. Background fluorescence was minimal (<1%),
therefore, no background subtraction was performed. Emission
fluorescence was collected above 510 nm by a GenIISys image intensifier
system connected to a video camera (MTI CCD72, Dage-MTI, Michigan City,
IN). Data were quantified by an image acquisition program from Georgia
Instruments (Roswell, GA) and analyzed by a personal computer. During
data analysis, two temporally adjacent data points were averaged to
improve signal-to-noise ratio.
To determine absolute intracelluar pH (pHi) and
intracellular H+ concentration
([H+]i) (see below), respectively, for each
individual cell, we used a one-point calibration technique as described
by Boyarski et al. (1988). Cells were perfused with a calibration
solution containing nigericin and gramicidin (see above) titrated to pH
7.2 at the end of each experiment. Data were normalized for the 440/490
nM ratio of this calibration solution and fitted to a
variant of a pH titration curve, determined for 75 cells on six
different coverslips with solutions titrated to pH 6.3, 6.6, 6.9, 7.2, 7.5, and 7.8. Every three to four experiments, we checked for the
accuracy of this calibration procedure by performing a two-point
calibration with solutions titrated to pH 7.2 and 7.4.
As described in detail by Rose and Ransom (1996), the 345/385 nm ratio
of intracellular SBFI changes monotonically in hippocampal astrocytes
with changes in [Na+]i between 0 and 50 mM. For intracellular calibration of
[Na+]i, cells were therefore perfused with
two or three calibration solutions (containing gramicidin, monensin,
and ouabain) (see above) with different Na+ concentrations
(0, 30, and 50 mM Na+); this was done at the
end of each experiment.
Each experiment with astrocyte cultures allowed analysis of 3-11
individual cells, and was repeated on at least four different
coverslips, unless stated otherwise. Data on hippocampal neurons were
obtained from four single cells on four different coverslips. Data are
presented as means ± SD and were statistically analyzed by paired
or unpaired Student's t test where appropriate
(significance level, p 0.001, unless stated
otherwise).
Intracellular H+ concentration more clearly reflects
experimental changes than intracellular pH. pH is a logarithmic
scale, introduced to conveniently handle differences in acidity over
several orders of magnitude occurring in chemical systems. When
referring to the cytosol of living cells, however, the use of a pH
scale seems neither necessary nor reasonable, because cellular acidity
usually is restricted to values between pH 7.7 and 6.0, reflecting
H+ concentrations from ~20-1000 nM. Apart
from this, the pH scale has the serious disadvantage that similar pH
changes at different baseline pH values might incorrectly suggest
similar changes in acidity to the observer.
Figure 2 illustrates this problem graphically by showing
the linearized changes in intracellular H+
([H+]i) in a hippocampal astrocyte on
switching from a HEPES-buffered to a
CO2/HCO3-buffered saline and back
(upper trace), compared with intracellular pH changes for
the same recording (lower trace). Whereas the lower trace
visually suggests that the initial acidification caused by addition of
CO2 is much smaller than the alkalinization on its removal,
the upper trace shows that the opposite is true for this cell; the
final decrease in [H+]i concentration is
actually smaller than the initial increase in [H+]. We
believe therefore that it is more sensible to illustrate cellular
acidity using a linear scale and have, for reasons of simplicity,
expressed acidity in terms of [H+]. (In actuality,
[H+] does not represent single H+ ions, which
are practically nonexistent in cells, but rather the concentration of
H3O+ molecules.)
Fig. 2.
Relationship between pHi and
intracellular H+ concentration. The distortion caused by
presenting changes in intracellular H+ concentration
([H+]i) as changes in pHi is
illustrated here. A hippocampal astrocyte was switched from a
HEPES-buffered, CO2/HCO3-free
saline to a CO2/HCO3-buffered
saline and back. The changes in [H+]i are
shown along with the changes in pHi. In the pHi
tracing, the absolute magnitude of the alkaline shift appears larger
than the acid shift, whereas the [H+]i trace
reveals just the opposite. To avoid these distortions, all subsequent
data are presented as [H+]i.
[View Larger Version of this Image (14K GIF file)]
RESULTS
Glu can activate several mechanisms in cultured astrocytes
(compare Fig. 1), such as (1) ionotropic non-NMDA receptors (also
engaged by the nondesensitizing Glu agonist KA (von
Blankenfeld and Kettenmann, 1991), (2) metabotropic Glu receptors
(e.g., trans-(±)-1-amino-(1S,3R)-cyclopentadicarboxylic acid
(t-ACPD) are selective agonists for this site) (Schoepp and
Conn, 1993), and (3) electrogenic Na+-coupled uptake of Glu
(Bouvier et al., 1992). Glu receptors of the NMDA type are not commonly
expressed in astrocytes, but have been reported (Porter and McCarthy,
1994; Steinhauser et al., 1994).
These different mechanisms can be stimulated selectively by Glu
analogs. KA activates the non-NMDA receptors but is not
transported (Kimelberg et al., 1989). D-Asp is a substrate
for the Glu transporter, but is only a very poor substrate for the
ionotropic receptor (Erecinska and Silver, 1990). To analyze the
mechanisms of Glu-induced changes in astrocytic
[H+]i, we used D-Asp in addition
to Glu and KA, to better distinguish between receptor- versus
uptake-mediated effects. We did not study the actions of
t-ACPD, because it changes intracellular Ca2+,
but not [H+]i, in astrocytes (Brune and
Deitmer, 1995). To allow uniform comparison between the actions of
different agonists, all substances were applied for 1 min at a
concentration of 1 mM by bath perfusion, unless otherwise
stated.
[H+]i changes induced by Glu,
KA, and D-Asp
Baseline intracellular H+ concentration
([H+]i) in hippocampal astrocytes was 54.6 nM in standard
CO2/HCO3-buffered saline (Table
1) corresponding to a pHi of 7.26.
Table 1.
[H+]i and
[Na+]i transients induced by Glu,
KA, or D-Asp under different
conditions
|
|
CO2/HCO3 |
CNQX
(%) |
HEPES |
|
| Baseline |
[H+]i
(nM) |
54.6 ± 14.9 (289) |
57.3 ± 12.8 (29) |
**81.7 ± 27.5 (179) |
|
[Na+]i
(mM) |
15.0 ± 4.9 (223) |
15.7 ± 5.7 (61) |
**11.9 ± 5.7 (83) |
| Glu |
[H+]i |
15.6 ± 4.2 (75) |
**138 ± 25 (29) |
**43.7 ± 25 (104) |
|
[Na+]i |
13.2 ± 4.5 (60) |
**52 ± 13 (29) |
*12.0 ± 3.7 (32) |
| KA |
[H+]i |
 9.0/10.3 ± 4.8/6.4 (179) |
**0 (39) |
**4.9 ± 4.5 (194) |
|
[Na+]i |
23.4 ± 6.1 (82) |
**0 (50) |
*21.6 ± 6.2 (82) |
| D-Asp |
[H+]i |
10.4 ± 2.9 (26) |
100 ± 23 (26) |
|
|
[Na+]i |
6.1 ± 1.0 (64) |
*89 ± 15 (52) |
|
|
|
Baseline values of intracellular H+ and
Na+ concentrations ([H+]i, in
nM; and [Na+]i, in
mM) in standard
CO2/HCO3-buffered saline, in
CO2/HCO3-buffered saline containing
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (25 µM), and
in HEPES-buffered, CO2/HCO3-free
saline. The second, third, and fourth rows list changes in
[H+]i and [Na+]i
induced by Glu, KA, or D-Asp (each applied at 1 mM for 1 min). Note that KA produces a biphasic
shift (alkaline-acid), and values for both peaks are shown. Changes in
CNQX-containing saline are expressed in % as compared with standard
CO2/HCO3-buffered saline. Shown
are means ± SD and number of cells. Negative numbers represent
decreases in [H+]i. Asterisks indicate
significant differences as compared with control values in
CO2/HCO3-buffered saline;
**p 0.001; *p 0.025.
|
|
Bath application of 1 mM Glu for 1 min elicited
[H+]i changes in every cell investigated
(n = 94). The majority of cells (80%) responded to Glu
with a pure acidification that started either directly at the onset of
the Glu perfusion or up to 30 sec later. The acid shift developed with
a maximal slope of 4.1 nM/min and reached its peak of 15.6 nM ~5-10 min after the Glu was removed from the bath
(Table 1, Fig. 3A). In 20% of the cells,
this acidification was preceded by a small alkaline shift ( 4 nM) (Fig. 3B).
Fig. 3.
[H+]i changes evoked by
Glu, KA, or D-Asp in
CO2/HCO3-containing or
CO2/HCO3-free solution.
A illustrates the typical changes in intracellular
H+ concentration ([H+]i)
caused by bath application of Glu, KA, or D-Asp in
standard, CO2/HCO3-buffered
saline. Substances were applied for 1 min (indicated by
bars) at a concentration of 1 mM. These
recordings were from three different cells. To facilitate comparison,
baseline [H+]i was set to 0 in all cells.
B, Record showing changes
[H+]i caused by 1 min bath application
(indicated by bars) of KA or Glu (1 mM) in HEPES-buffered,
CO2/HCO3-free saline, and
CO2/HCO3-buffered saline.
[View Larger Version of this Image (12K GIF file)]
KA application changed [H+]i
in 82% of the cells. The KA-induced
[H+]i changes were biphasic; a decrease in
[H+]i by 9.0 nM was followed by
an increase of 10.3 nM (Table 1). The alkaline shift
reached its peak ~40-70 sec after the onset of the
KA-perfusion, whereas the acid shift developed slowly and
peaked after 5-10 min (Fig. 3). D-Asp application elicited
a slow increase in [H+]i in all astrocytes
investigated. The D-Asp-induced increase in
[H+]i averaged 10.4 nM and had a
similar time course and maximal slope (5.3 nM/min) as the
Glu-induced acid shift (Table 1, Fig. 3A).
The Glu-induced membrane depolarization of cultured astrocytes
decreased with repetitive applications (Kettenmann and Schachner,
1985). Similar decreases in evoked changes in
[H+]i were observed in the present study.
Amplitudes and slopes of the evoked transients during second and third
applications (intervals between pulses, 15-20 min) were reduced in
comparison to the first pulse (p 0.025)
(data not shown). The amplitudes of the second and third Glu-evoked
changes, compared with the first, were 87 ± 34% (mean ± SD) and 83 ± 44%, respectively (n = 44).
Likewise, KA-induced [H+]i changes
also decremented with repetition; the second and third responses were
84 ± 10% and 82 ± 12% of the original response
(n = 47). [H+]i changes
elicited by D-Asp, however, showed virtually no attenuation
with repetitive application (97 ± 27% and 95 ± 29%,
respectively; n = 29). To facilitate comparison of
evoked changes in [H+]i before and after
various manipulations, we corrected the second and third responses for
these expected changes (i.e., in data expressed as percentage
change).
CO2/HCO3 dependence of Glu-
and Ka-induced [H+]i changes
Regulation of baseline [H+]i in glial
cells is dependent on the presence of
CO2/HCO3, partly because of its
role as a buffer system and partly because of
HCO3-carrying transporters (see Fig. 1) (Boyarski et
al., 1993; Deitmer and Rose, 1996). To investigate the influence of
CO2/HCO3 on the Glu- and
KA-induced [H+]i changes, we
performed experiments in HEPES-buffered,
CO2/HCO3-free saline. As expected
(Pappas and Ransom, 1993), baseline [H+]i
increased significantly to 81.7 nM in this saline (Table
1), corresponding to a pHi of 7.09.
In CO2/HCO3-free saline the
amplitude of the Glu-induced acid shift increased about threefold to
43.7 nM (Table 1), and its maximal slope increased to 45.0 nM/min (Fig. 3B). These effects can most likely
be attributed to the two- to threefold reduction in intracellular
buffering capacity in the absence of
CO2/HCO3 (Thomas, 1976). Cells
that showed an alkaline response in
CO2/HCO3, did not show this alkaline
shift in CO2/HCO3-free saline
(n = 13) (Fig. 3B).
In contrast to their uniform reaction in standard,
CO2/HCO3-buffered saline, the
astrocytes' response to KA was more variable in
CO2/HCO3-free saline.
Seventy-eight percent of cells responded to KA in the absence
of CO2/HCO3. Of the responders,
most (67%) acidified at KA application (Fig. 3B),
whereas 33% showed a very small alkaline shift (<2 nM)
(data not shown). The average KA-induced increase in
[H+]i was 4.9 nM (Table 1).
Surprisingly, some cells (n = 19) that did not react to
KA in HEPES-buffered saline responded with robust biphasic
[H+]i changes in standard,
CO2/HCO3-buffered saline. These
results strongly suggested that the KA-induced alkaline-acid
shifts in standard saline were attributable to HCO3
movement across the plasma membrane. Otherwise the
[H+]i changes would have increased in
CO2/HCO3-free saline because of
the amplifying effect of reduction in intracellular buffering power
(see above) (Thomas, 1976).
Origin of KA-induced [H+]i
changes in CO2-buffered saline
The amplitude of the biphasic alkaline-acid transient elicited by
KA application in
CO2/HCO3-buffered saline increased
with increasing KA concentrations from 0.1 to 2.0 mM (n = 49; application for 2 min) (Fig.
4). The KA-induced
[H+]i shifts were unaltered during
Ca2+ removal (Table 2, Fig.
5A). Replacement of extracellular
Cl by gluconic acid led to a significant decrease in
baseline [H+]i (Table 2, Fig. 5B).
This effect was probably attributable to removal of acidifying
Cl/HCO3 exchange in these
astrocytes, as reported earlier (Mellergard et al., 1993; Shrode and
Putnam, 1994). Removal of Cl also could alkalinize
astrocytes by depolarizing them and activating inward
Na+/HCO3 cotransport (Pappas and
Ransom, 1994), but astrocyte membrane potential appears to be
insensitive to the transmembrane gradient of Cl (Ransom
and Goldring, 1973). In the absence of Cl, the
KA-induced alkalinization was reversibly reduced to 52%
(p 0.0001) of the control response, and the
KA-induced acidification increased to 129% (p 0.01) (Table 2, Fig. 5B).
Fig. 4.
Concentration dependence of kainate-induced
[H+]i changes. A, The biphasic
KA-induced responses in [H+]i
increased with KA concentration. All applications were for 2 min periods; KA was applied at 0.1, 0.5, and 1 mM
(indicated by bars). B, Graphic summary
of the amplitudes of biphasic alkaline-acid transients elicited by 2 min bath applications of KA at 0.1, 0.5, 1, and 2 mM in
CO2/HCO3-buffered saline. Shown
are the mean values of 49 cells; bars indicate SD.
[View Larger Version of this Image (15K GIF file)]
Fig. 5.
Ca2+- and
Cl-dependence of kainate-induced
[H+]i changes. A,
Alkaline-acid transients elicited by 1 mM KA
applications for 1 min (indicated by bars) in standard
CO2/HCO3-buffered saline (2 mM Ca2+) and after removal of extracellular
Ca2+ (0 [Ca2+]e, solution
contained 0.5 mM EGTA). The KA response was
unchanged in the absence of [Ca2+]e.
B, Alkaline-acid transients elicited by 1 mM
KA application for 1 min (indicated by bars) in
standard CO2/HCO3-buffered saline
(containing 122.75 mM Cl) and after
replacement of extracellular Cl by gluconic acid (0 [Cl]e). Removing Cl caused an
alkaline shift probably because of reverse
Cl/HCO3 exchange. Note that the
KA response was preserved qualitatively in the absence of
[Cl]e.
[View Larger Version of this Image (12K GIF file)]
Replacement of extracellular Na+ by
N-methyl-D-glucamine (NMDG), (115.75 mM) and choline (23 mM) strongly acidified the
cells (Table 2, Fig. 6A). The
KA-induced alkaline transient was abolished in some cells and,
on average, was reversibly diminished to 18% of the control value. The
acid transient was abolished completely (Fig. 6A). Similar
effects were observed after application of the anion transport blocker
(4,4-diisothiocyanato-stilbene-2,2-disulphonic acid (DIDS) (0.5 mM). After an initial alkaline transient, baseline
[H+]i increased strongly (Table 2, Fig.
6B). The KA-evoked alkaline shift was reduced to
25% of the control value, and the acid shift was abolished (Fig.
6B). The effects of DIDS, as expected (Brune et al., 1994;
O'Connor et al., 1994), were not fully reversible.
Fig. 6.
Na+ dependence and influence of DIDS
on kainate-induced [H+]i changes.
A, Alkaline-acid transients elicited by 1 mM
KA application for 2 min (indicated by bars) in
standard CO2/HCO3-buffered saline
and after replacement of extracellular Na+ by NMDG and
choline (0 [Na+]e). Removal of
Na+ caused a marked acid shift, because it blocks or
reverses acid-exporting mechanisms. The KA-induced changes in
[H+]i were reversibly blocked in the absence
of Na+. B, Alkaline-acid transients elicited
by 1 mM KA application for 1 min (indicated by
bars) are shown in standard
CO2/HCO3-buffered saline and
during application of the anion transport blocker DIDS (0.5 mM). DIDS caused a partially reversible acid shift, because
it blocks acid- exporting mechanisms. The KA response was
largely blocked by DIDS.
[View Larger Version of this Image (11K GIF file)]
In contrast to hippocampal astrocytes, we observed only a rapid
acidification, but no alkaline shift, in cultured hippocampal neurons
after KA application. This was true in both
CO2/HCO3-buffered (Fig.
7) and
CO2/HCO3-free saline (data not
shown). In CO2/HCO3-buffered
saline, KA application rapidly increased neuronal
[H+]i by 22.8 ± 10.3 nM
from a baseline of 75.4 ± 18.6 nM (n = 4).
Fig. 7.
Neuronal [H+]i changes
induced by KA. KA application (1 mM for 1 min) (indicated by bar) rapidly increased
[H+]i in a cultured hippocampal neuron in
CO2/HCO3-buffered saline. Unlike
the situation in astrocytes, KA never evoked alkaline shifts
in neurons.
[View Larger Version of this Image (10K GIF file)]
Taken together, these results suggested that the glial-specific,
biphasic [H+]i changes after application of
KA were attributable to a
CO2/HCO3- and
Na+-dependent transport mechanism sensitive to DIDS that
can operate (although with reduced amplitude) in the absence of
external Cl.
[Na+]i changes induced by Glu,
KA, and D-Asp
In agreement with an earlier study (Rose and Ransom, 1996),
baseline [Na+]i in hippocampal astrocytes was
about 15 mM in standard saline (Table 1). Application of
either Glu, KA, or D-Asp rapidly increased
[Na+]i within 30-60 sec, followed by a
recovery to baseline within 10 min (shown for Glu in Figs.
8, 10; KA in Figs. 8, 9; D-Asp in
Fig. 11). This was true for every cell investigated, although the
absolute amplitudes of the [Na+]i signals
differed significantly between the three agonists. The average
[Na+]i increase was highest with KA
(23.4 mM), followed by Glu (13.2 mM), and
D-Asp (6.1 mM) (Table 1). The maximal slopes of
[Na+]i increase (in mM/min)
induced by these three agonists were 22.1 ± 2.4 (n = 43), 12.8 ± 9.0 (n = 30),
and 9.3 ± 5.6 (n = 26), respectively. During
repetitive applications, amplitude and slopes of the
[Na+]i transients decreased as described for
the [H+]i changes (data not shown) (see
above).
Fig. 8.
Comparison between glutamate- and kainate-induced
[Na+]i and [H+]i
transients. A, Recordings showing changes in
intracellular Na+ and H+ concentrations
([Na+]i,
[H+]i) induced by application of Glu
(1 mM for 1 min) (indicated by bars) in
CO2/HCO3-buffered saline
(solid lines) and
CO2/HCO3-free saline
(dashed lines). Although Glu-induced
[H+]i changes were greatly altered when
switching between CO2/HCO3-free
and CO2/HCO3-containing solution,
very small alterations were seen in the induced changes in
[Na+]i. B, Recordings showing
KA-induced [Na+]i and
[H+]i changes. KA (1 mM)
was applied for 1 min as indicated by the bars. Again,
the significant changes in KA-induced
[H+]i transients caused by switching from
CO2/HCO3-containing solution to
CO2/HCO3-free solution were not
associated with significant changes in the
[Na+]i transients. A,
B, Na+ and H+ measurements were
obtained from different cells.
[View Larger Version of this Image (12K GIF file)]
Fig. 10.
CNQX reduced the increase in
[Na+]i induced by Glu, but not the increase
in [H+]i. Glu-induced
[H+]i and [Na+]i
transients are shown in standard
CO2/HCO3-buffered saline and
during application of the ionotropic, non-NMDA receptor blocker CNQX
(25 µM) (solid bars). Glu (1 mM) was applied for 1 min (indicated by short
bars). The recordings were obtained from two different cells,
and two segments were deleted from the lower trace
(arrows) to enable better comparison between the
traces.
[View Larger Version of this Image (11K GIF file)]
Fig. 9.
CNQX blocked kainate-induced
[H+]i and [Na+]i
transients. KA-induced [H+]i and
[Na+]i transients are shown in standard
CO2/HCO3-buffered saline and
during application of the ionotropic, non-NMDA receptor blocker CNQX
(25 µM) (solid bars). KA (1 mM) was applied for 1 min (upper trace) or 2 min (lower trace, indicated by short
bars). The recordings were obtained from two different cells;
note the different time scales. CNQX completely blocked both the
KA-induced [H+]i and
[Na+]i transients.
[View Larger Version of this Image (10K GIF file)]
Fig. 11.
CNQX had only minor effects on aspartate-induced
[H+]i and [Na+]i
transients. [H+]i and
[Na+]i transients were induced by
D-Asp (Asp) in standard
CO2/HCO3-buffered saline and
during application of CNQX (25 µM) (solid
bars). D-Asp (1 mM) was applied for 1 min (short solid bars). The recordings were obtained
from two different cells, and two segments were deleted from the
lower trace (arrowheads) to enable better
comparison between the traces.
[View Larger Version of this Image (12K GIF file)]
In CO2/HCO3-free saline, baseline
[Na+]i decreased significantly to 11.9 mM (Table 1), indicating a contribution of inwardly
directed Na+/HCO3 cotransport
and/or Na+-dependent
Cl/HCO3 exchanger to baseline
[Na+]i in these cells under standard
conditions (Rose and Ransom, 1996). Both Glu- and KA-induced
[Na+]i increases were slightly reduced in
comparison to CO2/HCO3-buffered saline
(p 0.025) (Table 1).
Comparison of [H+]i and
[Na+]i changes
A direct comparison of the Glu- and KA-induced
[Na+]i and [H+]i
transients is shown in Figure 8. In
CO2/HCO3-buffered saline,
Glu-induced acidifications started up to 30 sec later than the
increases in [Na+]i.
[H+]i continued to rise for minutes after
[Na+]i was already decreasing and only
started to recover when [Na+]i had nearly
reached baseline again (Fig. 8A, solid traces).
The same differences also were true for D-Asp-induced
[Na+]i and [H+]i
changes (see Fig. 11).
In CO2/HCO3-free saline, in
contrast, the time course of the Glu-induced
[H+]i increase more closely matched that of
the [Na+]i transient (Fig.
8A, dashed traces). Both
[H+]i and [Na+]i
increases started at about the same time, and the acidification reached
its peak earlier than in
CO2/HCO3-buffered saline. The
[H+]i recovery to baseline, however, was
still slightly delayed compared with the
[Na+]i recovery (Fig. 8A,
dashed traces).
The time course and occurrence of KA-induced
[Na+]i and [H+]i
transients differed greatly. In
CO2/HCO3-buffered saline, the
KA-evoked [Na+]i increase was
paralleled by an alkalinization. The subsequent acidification started
about when [Na+]i reached its highest
amplitude and persisted even when [Na+]i had
reached baseline again (Fig. 8B, solid
traces). In contrast to this, KA-induced acidifications
were strongly reduced or even absent in
CO2/HCO3-free saline, whereas the
[Na+]i increases only decreased slightly
(Fig. 8B, dashed traces). This
comparison demonstrates that large increases in
[Na+]i are not necessarily associated with an
acidification of hippocampal astrocytes in
CO2/HCO3-free saline.
Effect of CNQX on [H+]i and
[Na+]i changes induced by Glu, KA,
and D-Asp
To investigate receptor-mediated effects of Glu and its agonists,
we applied CNQX (25 µM), which selectively blocks
activation of ionotropic non-NMDA receptors. CNQX perfusion did not
alter baseline [H+]i or
[Na+]i in astrocytes (Table 1), and all of
its effects were reversible.
CNQX completely blocked [H+]i and
[Na+]i transients evoked by KA in
both CO2/HCO3-free (data not
shown) and CO2/HCO3-buffered saline
(Table 1, Fig. 9), confirming that these effects of
KA were solely attributable to receptor activation.
In contrast to this, CNQX significantly increased
Glu-induced [H+]i changes to 138%, and
decreased [Na+]i changes to 52%
of control values (Table 1, Fig. 10). These results
allow several straightforward and important conclusions concerning the
mechanisms of the Glu-induced [H+]i and
[Na+]i changes. They show that about half of
the Glu-induced [Na+]i increase was
attributable to receptor activation, and, therefore, most likely caused
by Na+ influx through the receptor-ionophore complex.
Furthermore, they indicate that the observed acidification was not only
independent of receptor activation, but was in fact probably dampened
by it. In addition, this experiment demonstrated that the intraglial
acidification on Glu application was not a direct effect of the
increase in [Na+]i, confirming the impression
that [Na+]i increases are not of necessity
followed by increases in [H+]i in hippocampal
astrocytes (see above).
The D-Asp-induced acidification was virtually unaltered by
CNQX (100%) (Table 1), whereas the [Na+]i
transient was slightly reduced to 89% (p 0.025)
(Table 1, Fig. 11), consistent with the expectation
that D-Asp is a very poor substrate for the ionotropic
receptor, but is readily transported into astrocytes.
DISCUSSION
Origin of Glu- and D-Asp-evoked acidifications
In agreement with studies on cerebral astrocytes (Brookes and
Turner, 1993; Brune and Deitmer, 1995), L-Glu or
D-Asp acidified hippocampal astrocytes. Our results
indicate that these acidifications are caused primarily by a
transporter that exchanges Glu and Na+ for intracellular
OH and K+, as demonstrated for retinal glial
cells (Bouvier et al., 1992) (see Fig. 1A).
Glu-induced intracellular acidification is not a phenomenon restricted
to cell cultures; Glu uptake via this transporter also produced an
intracellular acidification in rat hippocampal slices (Amato et al.,
1994).
Our conclusion is supported by several lines of evidence. In
CO2/HCO3-free saline, Glu-induced
[H+]i increases were closely paralleled by
[Na+]i increases. The CNQX-resistant,
D-Asp-induced [H+]i increases
implied that these acidifications were not attributable to non-NMDA
receptor activation with H+ influx through receptor-coupled
ion channels. Likewise Glu-induced acidifications were resistant to
CNQX (they actually increased), demonstrating that they too were not
caused by ionotropic non-NMDA receptor activation. Glu transport is
voltage dependent (Brew and Attwell, 1987), and if CNQX partially
blocked the Glu-induced depolarization (Bowman and Kimelberg, 1984;
Kettenmann and Schachner, 1985), Glu uptake and acidification would be
increased. Reduction of Glu-induced depolarization also might have
reduced a depolarization-dependent alkalinization process, thereby
magnifying the acidification (see below).
The stoichiometry for coupled transport of
Glu/D-Asp, K+,
Na+, and OH is 1:1:2:1 (Bouvier et al., 1992)
(see Fig. 1C). Using our data, the D-Asp-induced
increase in [H+]i was slightly higher than
expected from the [Na+]i increase and this
stoichiometry. Assuming an intraglial buffering power of about 60 mM (Chesler, 1990; Deitmer, 1995) and a baseline
[H+]i of 55 nM (Table 1), the
D-Asp-induced [H+]i increase of
10.4 nM (Table 1) would correspond to a total increase in
intracellular acid by 4.2 mM, implying a
[Na+]i increase of twice this, or 8.4 mM; the average observed [Na+]i
increase, however, was only 6.1 mM (Table 1). Several
factors could account for this discrepancy. The actual inward transport
of Na+ might be higher than suggested by the net
concentration increases because of removal of Na+ by
Na+,K+-ATPase activity. Moreover, the buffering
capacity of the cells could be lower than 60 mM or the
transporter might not be the only source for the increase in
[H+]i.
Other sources of Glu/D-Asp-induced acidification could
include increased Glu metabolism by the ATP-dependent enzyme glutamine
synthetase (Sonnewald et al., 1993) or
Na+,K+-ATPase activation (Thompson and Prince,
1986; Erecinska, 1989; Pellerin and Magistretti, 1994), because in both
cases H+ is released with ATP breakdown (Hochachka and
Mommsen, 1983). Outwardly directed
Na+/HCO3 cotransport also might be
activated as discussed below. In any case, an intraglial accumulation
of Glu or D-Asp by 4-5 mM in our study
(calculated from the [H+]i increase)
corresponds reasonably well to Glu concentrations reached in the brain
(~10 mM) (Erecinska and Silver, 1990).
The Glu-induced [H+]i increase was 1.5 times
larger than the D-Asp-induced acidification, but had a
similar time course. This difference reflects the substrate specificity
of Glu uptake; D-Asp produced smaller effects than Glu in
terms of membrane current (Brew and Attwell, 1987; Barbour et al.,
1993) and pH changes (Bouvier et al., 1992; Brune and Deitmer, 1995).
Another reason for this difference might be astrocytic Glu-uptake sites
which do not accept D-Asp as substrate (Flott and Seifert,
1991).
Origin of KA-evoked
[H+]i changes
In CO2/HCO3-free saline,
where baseline [H+]i is primarily regulated
by Na+/H+-exchange (Pappas and Ransom,
1993), KA induced either no [H+]i
changes or a small acidification. At the same time, KA-induced
[Na+]i increases reduced the driving force
for Na+/H+ exchange from 24:1 to 8:1
(see also Aronson, 1985) (for ion concentrations, see Materials and
Methods and Table 1). This reduced driving force was apparently
sufficient to keep [H+]i more or less
constant, suggesting that [Na+]i increases do
not cause obligatory [H+]i increases in
hippocampal astrocytes in
CO2/HCO3-free saline. They could,
however, slow recovery from an acid load, as indicated by the delayed
[H+]i recovery compared with
[Na+]i recovery (compare Fig.
8A).
In CO2/HCO3-buffered saline, in
contrast, KA evoked alkaline-acid shifts. The ionic dependence
and pharmacology of these transients suggested that they were primarily
caused by transmembrane HCO3 fluxes mediated by
electrogenic Na+/HCO3 cotransport
(O'Connor et al., 1994; Pappas and Ransom, 1994). They were blocked by
CNQX and, therefore, dependent on activation of ionotropic non-NMDA
receptors. The [H+]i transients were
independent of extracellular Ca2+, making the contribution
of a plasma membrane Ca2+/H+ ATPase
unlikely (Schwiening et al., 1993; Paalasmaa et al., 1994).
The KA-induced transients still were seen in
Cl-free saline, although the alkaline shift was
considerably reduced in amplitude, suggesting that they were not
dependent on Cl, but possibly influenced by the
transmembrane H+ gradient. Electroneutral
Cl/HCO3 exchange and
Na+-dependent
Cl/HCO3 exchange were not
likely, therefore, to contribute significantly to the
KA-induced [H+]i changes. Removal of
[Na+]o or application of DIDS diminished the
KA-induced alkalinization by about 80% and blocked the
acidification, indicating that these [H+]i
changes were attributable to Na+-dependent anion transport.
In addition, a Na+-independent mechanism might be
responsible for a small percentage of the KA-induced
alkalinization (Grichtchenko and Chesler, 1994). Hippocampal neurons,
which do not express Na+/HCO3 cotransport
(Schwiening and Boron, 1994), only showed an acidification upon
KA application.
To understand how KA could alter
Na+/HCO3 cotransport activity in a
biphasic manner, one has to consider that this transporter is
electrogenic and, therefore, influenced by membrane potential. The
stoichiometry of Na+/HCO3
cotransport is 1:2 in hippocampal astrocytes (O'Connor et al., 1994).
With the steady-state ion concentrations given in the present study
([Na+]i = 15 mM;
[Na+]o = 140 mM;
[HCO3]i = 17.3 mM) (see
Chesler, 1990); [HCO3]o = 23 mM), its reversal potential (Erev)
is ~75 mV, which is close to the baseline membrane potential of
hippocampal astrocytes (O'Connor et al., 1994; Steinhauser et al.,
1994). Depolarization of the membrane above 75 mV should, therefore,
lead to inward Na+/HCO3
cotransport.
KA depolarizes astrocytes by 5 mV/sec (Backus et al., 1989);
the total depolarization can reach 20-25 mV (Bowman and Kimelberg,
1984; Kettenmann and Schachner, 1985). The similar time courses of
KA-induced alkalinization and depolarization (see Bowman and
Kimelberg, 1984; Kettenmann and Schachner, 1985; Backus et al., 1989)
strongly suggest that the alkalinization resulted from accelerated
inward Na+/HCO3 cotransport during
KA application (see above). KA also caused a
[Na+]i increase, with a slightly slower time
course than the depolarization (Fig.
12A,B). This
KA-induced [Na+]i increase causes a
progressive increase in the Erev of the
transporter to about 50 mV. Because the KA-induced membrane
depolarization is faster than the [Na+]i
increase, the membrane potential initially is more positive than
Erev, favoring influx of
Na+/HCO3 causing intracellular
alkalinization (Fig. 12B).
Fig. 12.
Proposed voltage shifts and transmembrane
Na+/HCO3 movements in astrocytes
during kainate application in
CO2/HCO3-buffered saline.
A, Model of KA-induced changes in membrane
potential, [Na+]i, and
Na+/HCO3-cotransporter reversal
potential in astrocytes. KA causes depolarization and
increases [Na+]i (1). This is
followed by Na+ pump stimulation leading to
hyperpolarization and normalization of [Na+]i
(2). The reversal potential of
Na+/HCO3 cotransport is altered by
the changes in [Na+]i, illustrated by the
hypothetical curves to the right. The upper
trace shows the presumed changes in membrane potential
(Em) resulting from KA
application (1 mM for 1 min) (indicated by
bars) (see also Bowman and Kimelberg, 1984; Kettenmann
and Schachner, 1985; Backus et al., 1989). The middle
trace shows the averaged KA-induced
[Na+]i change of six representative cells.
The lower trace shows changes in the reversal potential
of Na+/HCO3 cotransport
(Erev), calculated from the
average [Na+]i change in the middle
trace (see Discussion). B, Proposed mechanism of
the alkaline-acid transients seen in astrocytes resulting from
KA application (1 mM for 1 min) (indicated by
bars). The upper trace shows changes in
the Erev (solid line).
Superimposed on the Erev trace is the
presumed change in Em induced by KA
(dashed line) (see above). During the fast,
KA-induced membrane depolarization,
Em is more positive than
Erev, favoring influx of
Na+/HCO3 and, therefore,
intracellular alkalinization. During repolarization and
hyperpolarization of the membrane, Em is
more negative than Erev, because of the
relatively slower recovery of [Na+]i,
favoring efflux of Na+ and HCO3 and
intracellular acidification. The lower trace shows the
KA-induced biphasic alkaline-acid shift in
[H+]i averaged from six representative
cells.
[View Larger Version of this Image (25K GIF file)]
Further consideration of the relationship between membrane potential
and Erev can account for the KA-induced
acidification. Because of activation of
Na+,K+-ATPase secondary to increased
[Na+]i, Glu or Glu agonists induce rapid
repolarization and long-lasting hyperpolarization after their removal
(Ransom et al., 1975; Bowman and Kimelberg, 1984; Thompson and Prince,
1986; Erecinska, 1989). This produces a discrepancy between membrane
potential and Erev that is opposite to what is
seen initially, leading to reversal of
Na+/HCO3 cotransport and
acidification (Deitmer and Schneider, 1995) during periods when
Em is more negative than
Erev (Fig. 12B).
Na+/HCO3 cotransport, therefore,
links membrane potential with [H+]i and
[Na+]i homeostasis in astrocytes.
Glu and D-Asp also depolarize astrocytes (Bowman and
Kimelberg, 1984; Kettenmann and Schachner, 1985) and, therefore, also
might activate inward Na+/HCO3
cotransport. Indeed, a CO2/HCO3-dependent
alkalinization was seen in about 20% of the cells during perfusion
with Glu. In the majority of cells, however, the acidification
attributable to Glu uptake apparently obscured this alkalinizing
influence. The enhancement of the Glu-induced acid shift in
CO2/HCO3-free solution and after
CNQX, which would reduce depolarization, may be partly attributable to
removal of a superimposed alkalinization process mediated by
Na+/HCO3 cotransport.
Origin of the [Na+]i transients
Glu and its agonists evoked large [Na+]i
increases in hippocampal astrocytes, as reported earlier for leech
glial cells (Ballanyi et al., 1989), cultured cerebral astrocytes
(Kimelberg et al., 1989), and oligodendrocytes (Ballanyi and
Kettenmann, 1990). The different amplitudes of
[Na+]i increase, and CNQX sensitivity of the
tested substances mirror their substrate characteristics vis a vis Glu
receptors and Glu uptake.
The KA-induced [Na+]i increase was
dependent on activation of ionotropic non-NMDA receptors, because it
was blocked by CNQX. It was caused, therefore, by influx of
Na+ through receptor-coupled cation channels (Sontheimer et
al., 1988). Another cause of [Na+]i increase
would be inwardly directed Na+/HCO3
cotransport in these cells (see above). This was indicated by the
KA-induced alkalinization and the smaller amplitude of the
[Na+]i increase in
CO2/HCO3-free, compared with
CO2/HCO3-buffered, saline (Table 1).
In addition to receptor-mediated Na+ influx, our data
suggest that 40-50% of the Glu-induced
[Na+]i increase was caused by Na+
transport via Glu uptake, because it persisted in CNQX (Fig. 10). The
D-Asp-induced [Na+]i increases,
on the other hand, were primarily attributable to Glu uptake, because
they were only slightly altered by CNQX (Fig. 11).
Conclusions
Our study indicates that [Na+]i
increases are not primarily responsible for Glu- or KA-induced
acidifications in hippocampal astrocytes. Instead, our results imply
that the [H+]i transients are primarily
caused by Glu uptake, resulting in acidification, and by electrogenic
Na+/HCO3 cotransport activity,
resulting in biphasic alkaline-acid shifts. In the brain, the relative
influence of these two mechanisms and, therefore, the direction of
glial [H+]i changes might vary depending on
the physiological situation. During normal neuronal activity, glial
cells alkalinize after activation of inward
Na+/HCO3 cotransport, and only
blocking this transporter unmasks an acidification, which probably
results from neurotransmitter uptake (Chesler and Kraig, 1989; Rose and
Deitmer, 1995). The glial uptake of base promotes extracellular
acidification, thereby dampening neuronal excitability in a
feedback-like manner (Ransom, 1992). During pathological situations
such as stroke or epilepsy, in contrast, extracellular Glu
concentrations are increased for longer time periods, and glial cells
strongly acidify (Kraig and Chesler, 1990), probably because of maximal
Glu uptake obscuring any alkalinization.
FOOTNOTES
Received April 17, 1996; revised June 7, 1996; accepted June 14, 1996.
This study was supported by a fellowship from the Deutsche
Forschungsgemeinschaft to C.R.R. (Ro 1130/1,2), and by National
Institutes of Health Grants NS-15589 and NS-06208 to B.R.R. We thank
Dr. K. W. Kafitz for performing the antibody stainings and Dr. G. B. Richerson for helpful comments on this manuscript.
Correspondence should be addressed to Christine R. Rose, Yale School of
Medicine, Department of Neurology, 710 LCI, 333 Cedar Street, New
Haven, CT 06510.
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