Department of Physiology and Neuroscience and Department of Neurosurgery, New York University School of Medicine, New York, New York 10016
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ABSTRACT |
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Smith, S. E. and
M. Chesler.
Effect of Divalent Cations on AMPA-Evoked Extracellular Alkaline
Shifts in Rat Hippocampal Slices.
J. Neurophysiol. 82: 1902-1908, 1999.
The generation of activity-evoked
extracellular alkaline shifts has been linked to the presence of
external Ca2+ or Ba2+. We further investigated
this dependence using pH- and Ca2+-selective
microelectrodes in the CA1 area of juvenile, rat hippocampal slices. In
HEPES-buffered media, alkaline transients evoked by pressure ejection
of RS--amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)
averaged ~0.07 unit pH and were calculated to arise from the
equivalent net addition of ~1 mM strong base to the interstitial space. These alkaline responses were correlated with a mean decrease in
[Ca2+]o of ~300 µM. The alkalinizations
were abolished reversibly in zero-Ca2+ media, becoming
indiscernible at a [Ca2+]o of 117 ± 29 µM. Addition of as little as 30-50 µM Ba2+ caused the
reappearance of an alkaline response. In approximately one-fourth of
slices, a persistent alkaline shift of ~0.03 unit pH was observed in
zero-Ca2+ saline containing EGTA. In HEPES media, addition
of 300 µM Cd2+, 100 µM Ni2+, or 100 µM
nimodipine inhibited the alkaline shifts by roughly one-half,
one-third, and one-third, respectively, whereas Cd+ and
Ni2+ in combination fully blocked the response. In
bicarbonate media, by contrast, Cd+ and Ni2+
blocked only two-thirds of the response. In the presence of
bicarbonate, Ni2+ caused an unexpected enhancement of the
alkalinization by ~150%. However, when the extracellular carbonic
anhydrase was blocked by benzolamide, addition of Ni2+
reduced the alkaline shift. These results suggested that
Ni2+ partially inhibited the interstitial carbonic
anhydrase and thereby increased the alkaline responses. These data
indicate that an activity-dependent alkaline shift is largely dependent
on the entry of Ca2+ or Ba2+ via voltage-gated
calcium channels. However, sizable alkaline transients still can be
generated with little or no external presence of these ions.
Implications for the mechanism of the activity-dependent alkaline shift
are discussed.
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INTRODUCTION |
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Neuronal activity is accompanied by rapid
extracellular alkalinizations that can arise from at least two sources
(Chesler and Kaila 1992). Activation of GABA-A receptors
leads to the efflux of bicarbonate across the anion channel and a
consequent rise in extracellular pH (pHo)
(Chen and Chesler 1990
, 1992a
; Kaila and
Voipio 1987
; Kaila et al. 1990
). In addition,
there is a bicarbonate-independent alkaline shift that can be elicited
by activation of ionotropic glutamate receptors (Chen and
Chesler 1992a
; Chesler and Chan 1988
) or by
direct electrical stimulation of a neuronal population (Chen and
Chesler 1992b
; Grichtchenko and Chesler 1996
;
Paalasmaa and Kaila 1996
; Tong and Chesler
1999
). Speculation on the mechanism of the
bicarbonate-independent alkaline transient has centered on the role of
calcium ions.
In giant nerve cells of the snail, alkaline shifts in surface pH have
been linked to Ca2+ entry and its subsequent extrusion by a
plasmalemmal CaATPase (Schwiening et al. 1993). This
transporter imports two protons per extruded Ca2+
(Niggli et al. 1982
) and therefore may account for the
bicarbonate-independent alkaline shift in vertebrate preparations.
Observations consistent with this hypothesis have been noted in
mammalian brain slices. Extracellular alkaline shifts evoked by
glutamate agonists or by direct electrical stimulation were abolished
in the absence of external Ca2+ (Grichtchenko and
Chesler 1996
; Paalasmaa and Kaila 1996
;
Paalasmaa et al. 1994
; Smith et al.
1994
). In addition, cytoplasmic acid shifts sensitive to
CaATPase inhibitors have been linked to neuronal Ca2+ entry
(Trapp et al. 1996
). On the other hand,
Ba2+, which is thought to be poorly extruded by this
transporter (Kwan et al. 1990
; Zhao and Dhalla
1988
), also was able to support these alkaline shifts
(Grichtchenko and Chesler 1996
; Tong and Chesler 1999
).
The relationship between extracellular Ca2+ shifts and the
alkaline transient, and the dependence of this pH response on the external concentration of Ca2+ and Ba2+, has
not been investigated. In this study, we used pH- and
Ca2+-selective microelectrodes, and discrete concentrations
of external Ba2+, to more fully examine these
relationships, employing focal injections of AMPA to elicit
extracellular alkaline shifts. In this context, the effect of divalent
Ca2+ channel blockers on the alkaline shift also was
studied. Ion substitution and divalent blocker experiments confirmed a
large dependence on Ca2+ or Ba2+ ions. However,
our data indicate that significant alkaline shifts can be elicited
reliably in the presence of micromolar external Ba2+ and
often can be evoked in the absence of either Ba2+ or
Ca2+. Some of these results have appeared in an abstract
(Smith et al. 1996).
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METHODS |
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Procedures were carried out with approval of the NYU Medical Center Institutional Animal Care and Use Committee. All efforts were made to minimize animal suffering and reduce the number of animals used to the minimum required for our study. Long Evans rat pups, bred in-house (P12-18 of either sex), were anesthetized and rapidly decapitated. Hippocampal slices (300-400 µm) were prepared on a Vibratome in ice-cold Ringer, then incubated at room temperature until use. Experiments were conducted at 28-32°C in a submersion-style chamber with constant superfusion of the Ringer solution at 3-5 ml per min.
Normal Ringer solution contained (in mM) 124 NaCl, 26 NaHCO3, 3 KCl, 3 CaCl2, 1.5 MgCl2, 1 NaH2PO4, and 10 glucose,
gassed with 95% O2-5% CO2
(pH 7.4). Ringer solution buffered with 26 mM
N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
(HEPES) had NaHCO3 omitted and was titrated to pH
7.4 with NaOH. In some experiments, the Ca2+ was
reduced to 1 mM to allow for greater resolution of
Ca2+ shifts detected with ion-selective
microelectrodes. Zero-Ca2+ Ringer had
CaCl2 omitted and contained 1 or 5 mM ethylene
glycol bis (beta-aminoethyl ether)-N, N,N',N'-tetraacetic
acid (EGTA). Solutions containing 5 mM EGTA were prepared with 35 mM
NaHCO3 to titrate the acidity of the EGTA and had
NaCl reduced accordingly. All experiments were conducted in saline
containing 100 µM picrotoxin (PiTX; Sigma Chemical). Solutions
containing CdCl2 or NiCl2
had NaH2PO4 omitted. The
pressure ejection pipettes had tip diameters of ~5 µm and contained
100 µM RS--amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
(AMPA) dissolved in 150 mM NaCl. In a small number of experiments, the
ejection pipette was filled with 10 mM sodium glutamate, and the Ringer
contained 50 µM DL-2-amino-5-phosphonovaleric acid (APV)
to block activation of N-methyl-D-aspartate
receptors. APV and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were
purchased from Tocris Cookson. Benzolamide was a gift of Dr. T. H. Maren and Lederle Laboratories, Pearl River, NY.
Double-barreled pH electrodes were fabricated as previously described
(Chesler and Chan 1988). Reference barrels were
back-filled with 1 M NaCl. The silanized pH-sensitive barrels were
back-filled with 150 mM NaCl plus phosphate buffer (pH 6.87) and
front-filled with a pH-sensitive liquid cocktail (Fluka 95291). The pH
microlectrodes, calibrated in standard phosphate buffers, exhibited a
55- to 60-mV response per unit pH. Ion and reference signals were
recorded via high-impedance (>1013
)
operational amplifiers wired for unity gain. Extracellular DC potential
was monitored and subtracted from the signal on the ion-sensitive barrel.
The pH electrode and the pressure ejection pipette were mounted on a Narashige dual micromanipulator with their tips ~50 µm apart. The array was lowered to a tissue depth of ~150 µm in the CA1 stratum pyramidale or s. radiatum. The choice of region was governed only by the ability of the tissue to generate sizable, repeatable alkaline responses. As no consistent differences were noted between the behavior of pHo responses in s. pyramidale versus s. radiatum, the results from these regions were pooled. To avoid movement of the electrode tips relative to one another, the array was not moved after being positioned in the tissue. Therefore baseline pHo was checked only at the start and at the end of experiments when the array was moved between tissue and bath. The pH of the extracellular space was more acid than the bath, typically ranging between 7.0 and 7.20.
To elicit pHo shifts, a Picospritzer (General Valve) was used to deliver pressure pulses (40 psi) of AMPA at regular intervals (1-5 min.). The duration of the agonist pulse was adjusted until a consistent alkaline shift >0.05 unit pH was obtained in normal Ringer. In a few experiments, pHo shifts were evoked by antidromic stimulation (50 Hz, 5 s) of the CA1 pyramidal neurons using a bipolar stimulating electrode placed in the alveus. The unfiltered signals were measured as a change in voltage on a strip chart recorder and archived to video tape. Means were presented with standard errors. Values of n referred to the number of slices. Amplitudes of the alkaline shifts were compared using a paired t-test.
In bicarbonate Ringer, the interstitial buffering of alkaline shifts
depends on the extracellular carbonic anhydrase activity and
assumptions about the CO2 partial pressure
(Chesler 1990). Because these factors are uncertain,
experiments requiring estimates of interstitial buffering capacity were
conducted in HEPES Ringer. The buffering capacity (
) of the
extracellular fluid was estimated as
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(1) |
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(2) |
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RESULTS |
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Baseline pHo was similar in 26 mM
bicarbonate verses 26 mM HEPES-buffered media as has been previously
reported (Gottfried and Chesler 1994). In a sample of 10 slices in bicarbonate saline, the baseline pHo
was 7.15 ± 0.019 (range 7.08-7.23). In another 10 slices studied
in 26 mM HEPES Ringer, the baseline pHo was 7.13 ± 0.015 (range 7.06-7.20).
Simultaneous measurement of pHo and [Ca2+]o
In seven slices, we studied evoked pH shifts in 26 mM
HEPES-buffered Ringer. A pH- and a Ca2+-selective
microelectrode were used to record simultaneous ion shifts (Fig.
1, A and B) evoked
by AMPA (n = 5) or glutamate (n = 2, in
50 µM APV). The bath Ca2+ was reduced from 3 to
1 mM in these experiments to increase the sensitivity of the
Ca2+-selective microelectrode. For all seven
slices, the peak alkaline pHo shifts averaged
0.072 ± 0.015 unit pH and were associated with a peak fall in
[Ca2+]o of 295 ± 47 µM. Assuming an extracellular pH buffering capacity of 14.7 mM (see
METHODS), the alkaline shifts were associated with a change
in the extracellular strong ion difference (SID) equivalent to the
addition of 1.06 ± 0.22 mM strong base. Over the seven slices,
the ratio of the
SID:
[Ca2+]o ranged
from 2.1-5.5 and averaged 3.65 ± 0.44.
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Alkaline shifts during washout of extracellular Ca2+
In solutions containing EGTA and no added
Ca2+, the agonist-evoked alkaline shift was
abolished reversibly in 41 slices (34 slices bathed in bicarbonate
saline and 7 slices bathed in HEPES saline). In many cases, transition
to the zero-Ca2+/EGTA Ringer caused the alkaline
shift to be superseded by a rapid acid shift (Fig. 1C) as
noted previously (Smith et al. 1994). This acid
transient could be abolished by CNQX (not shown), indicating that it
was triggered by the activation of AMPA receptors and was not a
mechanical or electrical artifact. The mechanism of this acid transient
was not explored.
To better resolve the relationship between the pHo changes and [Ca2+]o, adjacent recordings of the ion transients were made in slices during the washout and return of extracellular Ca2+. Superfusion of zero-Ca2+, HEPES-buffered Ringer (with 1 mM EGTA) caused a fall in extracellular [Ca2+]o to micromolar levels within a few minutes (Fig. 2). During the Ca2+ washout, AMPA was intermittently pressure-ejected from a nearby micropipette to follow the effect on the evoked alkaline shift. As shown in Fig. 2, the reduction of [Ca2+]o caused a decrease in the amplitude of the alkaline shifts, accompanied by the appearance of an initial acid-going transient that eventually superseded the alkalinization. The acid transient remained during much of the return of extracellular Ca2+. These observations suggested that a persistent component of the alkaline shift could have been obscured by the early acid transient (see following text). In eight slices, the alkaline shifts became indiscernible at an average [Ca2+]o of 117 ± 29 µM, over a [Ca2+]o range from 1 to 281 µM.
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Evoked alkaline shifts in zero-Ca2+ media
In a subset of experiments (15 of 59 slices), a component of the alkaline shift persisted in the nominal absence of extracellular calcium. These extracellular Ca2+-independent alkalinizations occurred in both bicarbonate (Fig. 3A) and HEPES-buffered Ringer (Fig. 3B) in which the measured [Ca2+]o was <1 µM. In bicarbonate buffer, these alkaline shifts averaged 0.032 ± 0.004 unit pH (n = 9) and were 42 ± 8% of the paired control responses in normal [Ca2+]o. In six slices studied in HEPES Ringer, alkaline shifts noted in the absence of external Ca2+ averaged 0.027 ± 0.006 unit pH and were 28 ± 3% of the paired controls. These persistent responses were abolished by CNQX (not shown), indicating that they were not electrical or mechanical artifacts.
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Effect of Ca2+ channel blockers in bicarbonate-free Ringer
In HEPES-buffered Ringer, addition of 300 µM Cd2+ was able to reversibly reduce the AMPA-evoked alkaline shift. The mean decrease of the response was 55 ± 2% (n = 5, P < 0.001; Fig. 4A). Addition of 100 µM Ni2+ alone reduced the alkaline shift by 36 ± 4% (n = 7, P < 0.001; Fig. 4B). When 300 µM Cd2+ and 100 µM Ni2+ were applied together, the AMPA-evoked alkalinization was blocked completely in four slices (Fig. 4C).
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To better define the Ca2+ entry pathway associated with activation of the alkaline shift, we tested the effect of nimodipine, an L-type calcium channel antagonist. In seven slices, addition of 10 µM nimodipine to HEPES- buffered Ringer inhibited the AMPA-evoked alkaline shift by 30 ± 7% (P < 0.05). When 100 µM Ni2+ was added to the nimodipine-containing Ringer, the alkaline shifts were further reduced to 51 ± 3% of control (n = 5, P < 0.001; not shown).
Effect of Cd2+ and Ni2+ in bicarbonate Ringer
In bicarbonate-buffered Ringer, addition of 300 µM
Cd2+ alone (Fig.
5A) reduced the AMPA-evoked
alkaline shift by 67 ± 4% (n = 4, P < 0.05). Whereas addition of
Cd2+ plus Ni2+ abolished
the response in HEPES Ringer, Ni2+ produced no
additional block in bicarbonate Ringer (Fig. 5A). In the
presence of 300 µM Cd2+ plus 100 µM
Ni2+, the AMPA-evoked alkaline shift was reduced
65 ± 5% (n = 4, P < 0.02),
which was not significantly different from the effect of
Cd2+ alone (n = 4, P = 0.7). When 10 µM benzolamide was added in
addition to the Cd2+ and
Ni2+, the remaining alkaline shift was amplified
three- to sevenfold (Fig. 5B). The residual response
therefore originated from a proton sink, typical of alkaline shifts
activated by ionotropic glutamate receptors (Chen and Chesler
1992c).
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Further dissection of these effects revealed a paradoxical action of Ni2+ when applied alone. In 11 slices, addition of 50-100 µM Ni2+ increased the AMPA-evoked alkaline shift by 154 ± 9% (P < 0.001; Fig. 6A). The enhancement by Ni2+ was not specific to the response evoked by AMPA receptors. In four slices, alkaline transients were evoked in the absence of synaptic transmission (in 20 µM CNQX, 50 µM APV, and 100 µM PiTX) by repetitive (50 Hz) antidromic activation of the CA1 pyramidal neurons. After addition of 100 µM Ni2+, the antidromic alkaline shifts were increased by 171 ± 12% (n = 4, P < 0.05; Fig. 6B).
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This enhancement of the alkaline shift would be expected if Ni2+ had an inhibitory effect on the interstitial carbonic anhydrase. To test this hypothesis, we prevented the possibility of such inhibition by bathing slices in benzolamide (10-20 µM). With the extracellular carbonic anhydrase inhibited, a comparatively small AMPA ejection pulse was used to elicit an average alkaline shift of 0.10 ± 0.02 unit pH (n = 7). Under these conditions, Ni2+ inhibited the alkaline responses. After addition of 50-100 µM Ni2+ to the benzolamide-containing saline, the AMPA-evoked alkaline shifts were reduced to 73 ± 11% of controls, (P < 0.05) averaging 0.078 ± 0.02 unit pH (Fig. 6C).
Replacement of Ca2+ with Ba2+
Although the AMPA-evoked alkaline shift was blocked in media containing 1-5 mM EGTA and no added Ca2+, a component of the response could be restored by adding Ba2+. In these experiments, Ca2+ first was removed from the interstitial space by superfusion with Ringer containing 1 mM EGTA and no added Ca2+. Before addition of Ba2+, the EGTA was washed out of the slice. Periodic ejection of AMPA confirmed that the alkaline shift remained blocked. With subsequent addition of Ba2+, a significant component of the alkalinization returned. In HEPES (n = 12; Fig. 7A) and bicarbonate media (n = 13; Fig. 7B), the alkaline shift elicited by 500 µM Ba2+ was, respectively, 40 ± 3 and 51 ± 5% of the responses obtained in 3 mM Ca2+.
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Like the control responses in normal Ca2+, the
alkalinizations elicited in 500 µM Ba2+ were
amplified by benzolamide (273 ± 34%, n = 4; Fig.
7B), indicating that they also arose from a net proton sink
(Chen and Chesler 1992c). In addition, the control and
the Ba2+ responses were similarly affected by
solutions containing Cd2+ (300-500 µM) and
Ni2+ (100 µM). Addition of both
Cd2+ and Ni2+ only
partially blocked the Ba2+-dependent alkaline
shift in bicarbonate Ringer (50 ± 4%, n = 3;
Fig. 8A) but completely
abolished the responses when added to HEPES-buffered solutions
(n = 2; Fig. 8B).
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In a previous study, Ba2+-dependent alkaline
shifts evoked by field stimulation required millimolar concentrations
of this cation (Grichtchenko and Chesler 1996). By
contrast, the AMPA responses obtained in 500 µM
Ba2+ were blocked consistently (n = 6) when the Ba2+ concentration was raised to 2 mM (not shown) and sizable AMPA-evoked alkaline shifts could be
elicited by Ba2+ at concentrations of
50 µM
(Fig. 9). Combining data from bicarbonate (n = 4; Fig. 9A) and HEPES-buffered media
(n = 3), the alkaline shift evoked in 50 µM
Ba2+ averaged 0.030 ± 0.004 unit pH, which
was 32 ± 4% of the control responses in 3 mM
Ca2+. To further ensure that these responses
were unrelated to extracellular Ca2+, we used a
Ca2+-free bicarbonate Ringer that contained 2 mM
Ba2+ plus 5 mM EGTA to provide a calculated
free-Ba2+ concentration of 37 µM. In five
slices bathed in this solution, the AMPA-evoked alkaline shift was
0.025 ± 0.01 unit pH and averaged 41 ± 10% of the control
responses in 3 mM Ca2+ (Fig. 9B).
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DISCUSSION |
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Relationship between divalent cation shifts and the alkaline transient
Simultaneous recording of the alkaline shift and the
[Ca2+]o transient allowed
comparisons of the net ionic changes after AMPA ejection. If the
measured fall in [Ca2+]o
represented a cellular Ca2+ load, which
subsequently was extruded in entirety by a plasmalemmal CaATPase, a
SID:
[Ca2+]o ratio
of 2:1 would be anticipated (Niggli et al. 1982
). Using a HEPES buffering power of 15 mM, the mean AMPA-evoked alkaline shift
(0.072 unit pH) corresponded to a change in the strong ion difference
(
SID) of ~1 mM (equivalent to the addition of this concentration
of strong base to the extracellular fluid). Because the corresponding
measured fall in [Ca2+]o
averaged ~300 µM, the
SID:
[Ca2+]o ratio
would range between 3 and 4. However, the limited response times of the
pH and Ca2+ electrodes and the diffusion time
between pressure ejection and ion electrodes sites would have distorted
these measurements. It is therefore likely that both the
SID and the
cellular Ca2+ load were greater. Thus, order of
magnitude comparisons of Ca2+ and pH responses
are probably the best that can be expected using these techniques.
However, it should be noted that that evoked intracellular
Ca2+ load was likely handled by mechanisms in
addition to the CaATPase, such as
Na+-Ca2+ exchange
(Blaustein 1988
), uptake into endoplasmic reticulum (Markram et al. 1995
), or sequestration by mitochondria
(Wang and Thayer 1996
). To the extent that a
plasmalemmal CaATPase did not fully handle the intracelluar
Ca2+ load, the calculated ratios would have been greater.
In a previous study, electrically evoked alkaline shifts could be
supported by Ba2+ but required millimolar
concentrations of this ion (Grichtchenko and Chesler
1996). Using AMPA microejection to evoke the pH response, micromolar concentrations of Ba2+ were
sufficient. The ability of 50 µM Ba2+ to
support the alkaline shift can be considered in the context of
H+ antiport. If the entire extracellular
concentration of 50 µM Ba2+ had entered cells
and was extruded in exchange for protons (in a ratio of 2 H+:1Ba2+), then the
expected extracellular
SID would have been 100 µM. With a mean
measured alkaline shift of ~0.03 unit pH and a calculated buffering
capacity of 15 mM, the experimental
SID would be ~450 µM. In
view of the limited response time of the pH electrodes, the actual
SID is likely to have been larger. To generate the observed alkaline
shifts using the plasmalemmal CaATPase therefore would have required
efficient extrusion of Ba2+ ions by this
transporter and cycling of Ba2+, whereby ions
entered cells and were extruded multiple times in exchange for protons.
Alkaline shifts in the absence of extracellular Ca2+
In one-fourth of the slices, alkaline shifts were elicited in the
nominal absence of external Ca2+ or
Ba2+. These shifts averaged ~0.03 unit pH,
corresponding to a SID of 450 µM. Although these alkalinizations
were not noted in the majority of slices, the appearance of an acid
transient at low [Ca2+]o
may have obscured such changes, as suggested by Fig. 2. For the
plasmalemmal CaATPase hypothesis to be considered in this context, the
extruded Ca2+ would have had to originate from
internal stores. This possibility has not been excluded by the present experiments.
Effect of Ca2+ channel blockers on the alkaline shift
In HEPES-buffered solution, simultaneous addition of
Cd2+ and Ni2+ completely
blocked the AMPA-evoked alkaline shift, consistent with a requirement
for Ca2+ entry via voltage-gated channels. In
isolation, these blockers may roughly distinguish between low- and
high-voltage-activated Ca2+ channels (Bean
1989; Fox et al. 1987
). The block of roughly
one-third of the alkaline shift by Ni2+ was
similar to the effect of this cation on spike-induced
Ca2+ entry in postnatal CA1 pyramidal cells
(Christie et al. 1995
) and indicates that both low- and
high-voltage-activated Ca2+ channels can trigger
the pH response. Nimodipine was about half as effective as
Cd2+ alone, suggesting that L-type
Ca2+ channels were responsible for triggering
approximately one-fourth of the alkaline shift. Nimodipine blocked a
similar fraction of somatic Ca2+ entry in
postnatal CA1 pyramidal neurons (Christie et al. 1995
). Neuronal entry of Ca2+ via AMPA receptors is
likely to have played a minor role because the AMPA receptor subtype
with significant Ca2+ permeability is confined to
the less prominent GABAergic interneurons (Geige et al.
1995
; Racca et al. 1996
).
In bicarbonate Ringer, Cd2+ and
Ni2+ did not fully abolish the alkaline shift.
Partial binding of these cations by bicarbonate ions might have
contributed to the incomplete inhibition. In addition, because
Ni2+ alone had a paradoxical enhancing effect,
the combination of Ni2+ and
Cd2+ would be less effective than anticipated.
The Ni2+-mediated enhancement of the alkaline
shift was dependent on bicarbonate and was occluded by benzolamide.
These observations are consistent with a partial reduction of
extracellular carbonic anhydrase activity mediated by
Ni2+. Inhibition of this enzyme by a variety of
divalent cations has been reported (Maren 1967;
Puscas et al. 1989
). These effects of
Ni2+ on activity-dependent
pHo shifts may warrant consideration in other
studies where this divalent is used in brain slices.
In summary, the present data indicate that the influx of Ca2+ or Ba2+ across voltage-gated Ca2+ channels is an important component in the steps that lead to extracellular alkalinization after activation of AMPA receptors. The results are in agreement with other studies of activity-dependent alkaline shifts and can be interpreted to support the involvement of a plasmalemmal CaATPase. However, alkaline responses could be obtained at extremely low concentrations of external Ba2+ and in many instances occurred in the absence of extracellular Ca2+. These results indicate that the entry of Ca2+ and its subsequent extrusion by the plasmalemmal CaATPase cannot explain all of the properties of activity-dependent alkaline shifts. Involvement of internal Ca2+ stores or alternative pathways for transmembrane H+ movement are possibilities that have not been excluded.
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ACKNOWLEDGMENTS |
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We thank C. K. Tong for assistance.
This research was supported by National Institute of Neurological Disorders and Stroke Grant NS-32123. S. E. Smith was supported by the Medical Scientist Training Program under National Institute of General Medical Sciences Grant 5 T32 GM-07308.
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FOOTNOTES |
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Address for reprint requests: M. Chesler, Dept. of Physiology and Neuroscience, New York University Medical Center, 550 First Ave., New York, NY 10016.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 2 March 1999; accepted in final form 18 May 1999.
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REFERENCES |
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