*
From the * Department of Cellular and Molecular Physiology, and Department of Pediatrics, Yale University School of Medicine, New
Haven, Connecticut 06520
In the preceding paper (Bevensee, M.O., R.A. Weed, and W.F. Boron. 1997. J. Gen. Physiol. 110:
453-465.), we showed that a Na+-driven influx of HCO3 causes the increase in intracellular pH (pHi) observed
when astrocytes cultured from rat hippocampus are exposed to 5% CO2/17 mM HCO3
. In the present study, we
used the pH-sensitive fluorescent indicator 2
,7
-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and the perforated patch-clamp technique to determine whether this transporter is a Na+-driven Cl-HCO3 exchanger, an electrogenic Na/HCO3 cotransporter, or an electroneutral Na/HCO3 cotransporter. To determine if the transporter
is a Na+-driven Cl-HCO3 exchanger, we depleted the cells of intracellular Cl
by incubating them in a Cl
-free solution for an average of ~11 min. We verified the depletion with the Cl
-sensitive dye N-(6-methoxyquinolyl)acetoethyl ester (MQAE). In Cl
-depleted cells, the pHi still increases after one or more exposures to CO2/HCO3
.
Furthermore, the pHi decrease elicited by external Na+ removal does not require external Cl
. Therefore, the
transporter cannot be a Na+-driven Cl-HCO3 exchanger. To determine if the transporter is an electrogenic Na/
HCO3 cotransporter, we measured pHi and plasma membrane voltage (Vm) while removing external Na+, in the
presence/absence of CO2/HCO3
and in the presence/absence of 400 µM 4,4
-diisothiocyanatostilbene-2,2
-disulphonic acid (DIDS). The CO2/HCO3
solutions contained 20% CO2 and 68 mM HCO3
, pH 7.3, to maximize
the HCO3
flux. In pHi experiments, removing external Na+ in the presence of CO2/HCO3
elicited an equivalent HCO3
efflux of 281 µM s
1. The HCO3
influx elicited by returning external Na+ was inhibited 63% by
DIDS, so that the predicted DIDS-sensitive Vm change was 3.3 mV. Indeed, we found that removing external Na+
elicited a DIDS-sensitive depolarization that was 2.6 mV larger in the presence than in the absence of CO2/
HCO3
. Thus, the Na/HCO3 cotransporter is electrogenic. Because a cotransporter with a Na+:HCO3
stoichiometry of 1:3 or higher would predict a net HCO3
efflux, rather than the required influx, we conclude that
rat hippocampal astrocytes have an electrogenic Na/HCO3 cotransporter with a stoichiometry of 1:2.
As described in the accompanying paper (Bevensee et
al., 1997), exposing rat hippocampal astrocytes to CO2/
HCO3
causes pHi to decrease initially, due to the influx
of CO2, and then generally to increase to a value higher
than the initial one prevailing in the nominal absence of
CO2/HCO3
. Because this pHi increase is blocked by the
HCO3
-transport inhibitors 4,4
-diisothiocyanatostilbene-2,2
-disulphonic acid (DIDS)1 and 4-acetamido-4
-isothiocyanatostilbene-2,2
-disulfonic acid (SITS), and requires
external Na+, we concluded that the astrocytes have a
Na+-driven HCO3
transporter. The data are consistent
with the presence of one or more of three HCO3
transporters known to exist in other cells (Fig. 1): (a) a
Na+-driven Cl-HCO3 exchanger, (b) an electroneutral
Na/HCO3 cotransporter with a Na+:HCO3
stoichiometry of 1:1, and (c) an electrogenic Na/HCO3 cotransporter with a Na+:HCO3
stoichiometry of 1:2. Theoretically, a fourth possibility is the electrogenic NaHCO3
cotransporter with a 1:3 stoichiometry, as exists in renal
proximal tubules (Boron and Boulpaep, 1983
; Soleimani et al., 1987
). However, given the ion and voltage
gradients likely to prevail in an astrocyte, this 1:3 cotransporter would almost certainly mediate net HCO3
efflux, and not the influx necessary to account for the observed CO2/HCO3
-induced alkalinization.
Compared with the 1:1 and 1:2 Na/HCO3 cotransporters, the Na+-driven Cl-HCO3 exchanger is unique
in requiring internal Cl. Because the Na+-driven Cl-HCO3 exchanger normally moves Na+ and HCO3
into
a cell and Cl
out, transport in its normal ("forward")
direction should be inhibited by depleting cells of internal Cl
. However, because the K1/2 of Cl
for the
Na+-driven Cl-HCO3 exchanger in mammalian cells is
not known, even low levels of intracellular Cl
([Cl
]i)
might still support the exchanger in cells preincubated
in a Cl
-free solution. Indeed, depleting [Cl
]i has proven
difficult in some cells. For example, renal mesangial cells
had to be incubated in a Cl
-free solution for 1-2 h to
achieve substantial inhibition of their Na+-driven
Cl-HCO3 exchanger (Boyarsky et al., 1988b
). In pyramidal neurons from rat hippocampus, the Na+-driven
Cl-HCO3 exchanger was active even after the cells were preincubated in a Cl
-free solution for up to 4 h (Schwiening and Boron, 1994
). Therefore, one must be cautious
when interpreting results from experiments designed
to assess the effect of acute extracellular Cl
removal
on transporters requiring intracellular Cl
. In the present
study, we use the Cl
-sensitive dye N -(6-methoxyquinolyl)
acetoethyl ester (MQAE) to study the time course of depletion of [Cl
]i, finding that [Cl
]i falls to very low levels
when astrocytes are incubated in a Cl
-free solution for as
few as ~11 min. Under these conditions, the Na+-driven
HCO3
transporter still operates in the forward direction, and moves HCO3
into cells exposed to CO2/
HCO3
. Furthermore, in the absence of external Cl
, the
transporter still operates in the reverse direction, and moves HCO3
out of cells exposed to a Na+-free solution.
The other possibilities shown in Fig. 1 are two Na/
HCO3 cotransporters, one that is electroneutral and
has a Na+:HCO3 stoichiometry of 1:1, and one that is
electrogenic and has a stoichiometry of 1:2. In the
present study, we distinguish between these two by using the perforated patch-clamp technique to record plasma membrane voltage (Vm) in astrocytes, and determine whether the movement of net negative charge
accompanies the movement of HCO3
. We found that
removing Na+ elicits a DIDS-sensitive depolarization
that is larger in the presence than in the absence of
CO2/HCO3
. Comparing the size of the DIDS-sensitive,
HCO3
-dependent depolarizations with the magnitude
of the DIDS-sensitive HCO3
effluxes measured under
similar conditions, we conclude that the Na+-driven
HCO3
transporter in hippocampal astrocytes is an
electrogenic Na/HCO3 transporter, presumably with a
Na+:HCO3
stoichiometry of 1:2.
Solutions
The solutions used in the present study, with the exception of the
ones buffered with 20% CO2/68 mM HCO3, are described in
the accompanying paper (Bevensee et al., 1997
). These 20% CO2
solutions were made by isotonically substituting 68 mM HCO3
salt for HEPES and a Cl salt. The patch-pipette filling solution
contained (mM): 105 KCl, 45 NaCl, 1.0 MgCl2, 0.2 CaCl2, 10 EGTA, and 1.0 HEPES. The pH of the patch-pipette solution
(pHpip) was adjusted to 7.4 with Tris. pHpip was buffered with only
1 mM HEPES to minimize the effect of pHpip on the pHi of a
patched cell. In some early experiments, we used 120 KCl and 30 NaCl in the pipette solution. Pipette solutions contained either
nystatin or amphotericin B (80-300 µM). MQAE was obtained
from Molecular Probes, Inc. (Eugene, OR). Bumetanide, nystatin, and amphotericin B were obtained from Sigma Chemical Co.
(St. Louis, MO). All other reagents were obtained as described in
the accompanying paper (Bevensee et al., 1997
).
Measurement of [Cl]i in Astrocytes
Handling of cells.
We measured [Cl]i in astrocytes using the Cl-sensitive indicator MQAE, developed by Verkman et al. (1989)
.
Astrocytes were grown to confluence, as described in the accompanying paper (Bevensee et al., 1997
), and passaged onto 8.5 × 8.5-mm glass coverslips. 2-8.25 h (average 4.7 ± 0.3 h, n = 23)
before each experiment, a coverslip with confluent astrocytes was
transferred to a HEPES-buffered solution containing 5 mM MQAE.
Optics. After cells were exposed to dye, the coverslip was placed into a quartz cuvette designed to fit into a SPEX Fluorolog-2 spectrofluorometer (CM1T10E; Spex Industries, Inc., Edison, NJ). The coverslip was mounted at a 30° angle to the excitation light. Solutions flowed through the cuvette from bottom to top through Tygon® or stainless-steel tubing. Both the tubing and the cuvette were maintained at 37°C by a water jacket. The temperature of the cuvette was monitored continually during an experiment with a thermistor placed at the base of the cuvette. During an experiment, we used only one of the excitation monochromators of the dual-beam spectrofluorometer, continuously exciting with light at 320 nm (1.89-nm bandwidth). A photomultiplier tube mounted on an emission monochromator monitored the emitted fluorescence intensity (I320) at 460 nm (4.71-nm bandwidth). During each 3.0-s data collection cycle, the spectrofluorometer integrated the emitted intensity for 1.0 s, and corrected for fluctuations in the arc-lamp intensity (continuously monitored). The background fluorescence of cells containing no dye was measured daily, and subtracted from the total emitted fluorescence. The background fluorescence averaged 10.4 ± 1.5% (n = 23) of total I320 at the beginning of an experiment.
Spectral properties of MQAE.
The fluorescence of the quinolinium dye MQAE is quenched at progressively higher concentrations of halides such as Cl. Fig. 2 A shows the fluorescence excitation and emission spectra of 5 µM MQAE in HEPES-buffered solutions of different Cl
concentrations. The spectra were obtained
at 37°C; all solutions were maintained at a constant ionic strength
of ~305 mosmol by replacing Cl
with cyclamate. The excitation
spectra (Fig. 2 A, left) were obtained by measuring the emitted
fluorescence at 460 nm while exciting the dye with light from 270 to 420 nm in intervals of 1 nm. For each of the seven excitation
spectra, there was a major peak at ~320 nm and a minor peak at
~350 nm. The emission spectra (Fig. 2 A, right) were obtained by
exciting the dye at 350 nm and measuring the emitted fluorescence from 375 to 600 nm in intervals of 1 nm. For each of the
seven emission spectra, the peak was at ~460 nm. These spectral
data are similar to those previously reported (Verkman et al.,
1989
), except that our 320-nm peaks in the excitation spectra
were more prominent.
From the emission spectra shown in Fig. 2 A, we plotted the emitted fluorescence as a function of [Cl
Intracellular calibration of MQAE.
In experiments on astrocytes
loaded with MQAE, we calibrated the dye using the high K+/nigericin/tributyltin chloride technique (Chao et al., 1989). At the end
of an experiment, astrocytes were exposed to a solution containing (a) 105 mM K+ and 10 µM nigericin to force pHi to approach
pHo (Thomas et al., 1979
), and (b) 5 µM tributyltin chloride, an
ionophore that exchanges Cl
and OH
, and thus forces [Cl
]i
to approach [Cl
]o. By exposing astrocytes to a high K+/nigericin/tributyltin chloride solution and changing external Cl
, we
determined the Stern-Volmer relationship (see above) for intracellular MQAE in each experiment. The average KCl of MQAE in
hippocampal astrocytes was 8.7 ± 0.8 M
1 (n = 16). Intracellular
values in the range of 5.3 to 25 M
1 have been reported for the
KCl of MQAE in other cell types (Lancer et al., 1990
; Engblom
and Akerman, 1993
; Lau et al., 1994
; Koncz and Daugirdas, 1994
;
Martínez-Zaguilán et al., 1994
).
Measurement of pHi in Single Astrocytes
We measured pHi using the pH-sensitive dye 2,7
-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) in astrocytes cultured from the hippocampus of the rat, as described in the accompanying
paper (Bevensee et al., 1997
).
Electrophysiology
Vm was recorded with a patch-clamp amplifier (PC-501A; Warner
Instruments Corp., Hamden, CT), using the perforated whole-cell recording technique in current-clamp mode (Horn and
Marty, 1988). Recordings were made on the stage of a microscope equipped for epi-fluorescence (IM-35; Carl Zeiss, Inc.,
Thornwood, NY), as described in the accompanying paper (Bevensee et al., 1997
). Signals were filtered at 1 kHz with a 4-pole
Bessel filter. Vm was digitized on-line at 50 Hz using an analog-to-digital converter board (Labmaster TL-1; Scientific Solutions
Inc., Solon, OH) interfaced with a personal computer (Dell
Computers, Austin, TX) based on an Intel-80486 microprocessor. The control of data acquisition and the analysis of data were
performed with either a custom-modified version of WinClamp
(Indec System, Inc., Capitola, CA) or pClamp (Axon Instruments, Inc., Foster City, CA). LG16 borosilicate glass capillaries (Dagan Corp., Minneapolis, MN) were pulled (PP-83; Narishige Scientific Instruments, Tokyo, Japan) and fire polished (MF-83; Narishige Scientific Instruments, Toyko, Japan) to make patch pipettes with resistances of 2-6 M
. In some experiments, the patch pipettes were coated with Sylgard® (Dow Corning Corp., Midland, MI) before being fire polished to minimize noise. Vm recordings were
usually made at 37°C, and in no case <34°C.
Statistics
Data are reported as mean ± SEM. Means were compared using
the paired and unpaired forms of the Student's t test (one-tail). P < 0.05 is considered significant. The best-fit line to the I0/I vs.
[Cl] data plotted in Fig. 2 B was determined using a least-squares method. Rates of MQAE loss from cells was determined
by fitting a line to the fluorescence vs. time data using a least-squares method. Rates of change in pHi (dpHi/dt) were determined by fitting a line to pHi vs. time data using a least-squares
method.
Using MQAE to Measure Intracellular Cl in
Hippocampal Astrocytes
In 16 experiments in which we measured
[Cl]i using the Cl
-sensitive dye MQAE, astrocytes had
a resting [Cl
]i of 36 ± 4 mM. This is similar to values
in the range 25-30 mM, reported by Walz and Hertz
(1983)
for mouse astrocytes cultured from the cortex.
The average resting [Cl
]i of 36 mM is well above the
equilibrium value of 4.0 mM that is predicted from the
mean Vm of ~
88 mV that we observed (see below) for
astrocytes in a HEPES-buffered solution containing 130 mM Cl
. Therefore, Cl
must be actively transported
into the cells. In mouse astrocytes, a furosemide-sensitive Na/K/Cl cotransporter is responsible for maintaining intracellular Cl
well above the value predicted if
Cl
were passively distributed (Walz and Hertz, 1983
). In
the experiment shown in Fig. 3 A, we determined
whether a similar bumetanide-sensitive Na/K/Cl cotransporter contributes to the high resting [Cl
]i in rat hippocampal astrocytes. The [Cl
]i time course shown in
Fig. 3 A is the result of a series of steps, outlined in Appendix, for converting MQAE fluorescence into [Cl
]i
values. The astrocytes in this experiment had an initial
[Cl
]i of ~18 mM before point a (Fig. 3 A, a). When we
exposed the cells to a solution containing 1 µM bumetanide, [Cl
]i decreased to ~8 mM (Fig. 3 A, ab). Returning the cells to a bumetanide-free solution elicited
an increase in [Cl
]i to a value similar to that at the
start of the experiment (Fig 3 A, bc). Because 1 µM bumetanide fluoresces when excited at 320 nm, we subtracted the signal due to bumetanide from the total I320. This correction resulted in offsets in the [Cl
]i vs.
time trace at the points where bumetanide was applied
and removed (Fig. 3 A, arrows). In four experiments,
bumetanide caused [Cl
]i to decrease from 23 ± 3 to
15 ± 3 mM (P < 0.005). A bumetanide-insensitive Cl
uptake mechanism could also contribute to the elevated
[Cl
]i in the presence of bumetanide. We conclude that
a bumetanide-sensitive Na/K/Cl cotransporter contributes to the high resting [Cl
]i in hippocampal astrocytes.
Others have found that applying furosemide or bumetanide to cultured hippocampal astrocytes causes a decrease in intracellular Na+ ([Na+]i), suggesting that the
Na/K/Cl cotransporter also helps maintain a high resting [Na+]i (Rose and Ransom, 1996
).
Removing extracellular Cl
In Fig. 3 B, we show the record of [Cl]i in an experiment in which astrocytes were exposed to a solution in
which Cl
was replaced with cyclamate. The cells in this
experiment had an initial [Cl
]i of ~43 mM before
point a (Fig. 3 B, a). Removing external Cl
elicited a
sharp decrease in [Cl
]i to ~2 mM (Fig. 3 B, ab). Returning external Cl
caused [Cl
]i to increase to its initial value (Fig. 3 B, bc). In 11 experiments similar to that
shown in Fig. 3 B, removing external Cl
caused [Cl
]i
to decrease from an average resting value of 40 ± 5 to
2.8 ± 1.5 mM in 10.7 ± 1.9 min. [Cl
]i decreased at an
initial rate of 277 ± 71 µM s
1 (n = 11).
Na+-driven HCO3 Influx: Testing the Intracellular
Cl
Dependence
If the CO2/HCO3-induced alkalinization were
mediated by a Na+-driven Cl-HCO3 exchanger, then
the alkalinization should require the efflux of substantial amounts of Cl
. In the following experiments, we
monitored pHi in single astrocytes, using the pH-sensitive dye BCECF. Our first approach was to acid load the
astrocytes repeatedly, using the NH4+ prepulse technique (Boron and De Weer, 1976
), with 0.9 mM
amiloride present to inhibit Na-H exchange. Because
the amiloride was added as the Cl
salt, [Cl
]o was ~ 0.9
mM. After each acid load, we added CO2/HCO3
to activate the Na+-driven Cl-HCO3
uptake mechanism.
From the magnitude of the pHi recovery and from the
known
T, we can compute how much internal Cl
would have to be transported out of the cell to achieve
each pHi recovery.
In the experiment of Fig. 4, we acid loaded the astrocytes seven times, the first time in the presence of 130 mM Cl. As shown in the inset, applying and removing
20 mM NH3/NH4+ caused the usual series of pHi
changes (Fig. 4, abcd), as noted in the accompanying
study (Bevensee et al., 1997
). In the absence of CO2/
HCO3
, the pHi recovery was blocked by amiloride
(Fig. 4, de). However, adding CO2/HCO3
caused pHi
to increase rapidly (Fig. 4, ef ). Removing the CO2/ HCO3
caused a transient pHi increase (Fig. 4, fg), due
to CO2 efflux, followed by a slower decline (Fig. 4, ghi).
At point h, we reduced [Cl
]o to ~0.9 mM and did not
return [Cl
]o to 130 mM until the recovery from the sixth
NH4+ pulse. Each of the seven CO2/HCO3
-induced pHi
recoveries is indicated by an arrow in Fig. 4. Two technical points are noteworthy. First, after the recovery
from the fifth NH4+ pulse, we continued the experiment on a nearby cell on the same cover slip, because
of evidence of dye loss from the original astrocyte. Similarly, we switched to a third cell after the sixth NH4+
pulse. We used this third cell, as well as two neighbors,
for the calibration of intracellular BCECF. Second, there
was a general tendency for the rates of pHi recovery in
the presence of CO2/HCO3
to decrease somewhat over
the course of this very long experiment (>4 h). Indeed,
the rate of the seventh and final pHi recovery (Cl
present) was ~1/3 less than that of the first pHi recovery (Cl
present).
The CO2/HCO3-induced alkalinization after the
second NH4+ pulse in Fig. 4 (i.e., the first in the absence of Cl
) would require the efflux of 4.5 mM Cl
, if
it were mediated by Na+-driven Cl-HCO3 exchange.
Similarly, the third through sixth CO2/HCO3
-induced
alkalinizations would require effluxes of 4.9, 4.9, 5.5, and 2.5 mM Cl
. However, based on the results from
experiments similar to Fig. 3 B, the [Cl
]i just before
the addition of CO2/HCO3
in Fig. 4 was probably only
~3 mM. Therefore, it appears unlikely that the pHi recoveries in 0 Cl
could be mediated by a Na+-driven
Cl-HCO3 exchanger unless substantial amounts of Cl
could recycle back into the cell from the 0.9 mM Cl
in
the external solution. However, for the first pHi recovery in the absence of extracellular Cl
, the maximal
rate of HCO3
influx would have required a Cl
efflux
of 2.7 mM min
1. Because the Na/K/Cl cotransporter
in primary cultures of rat astrocytes has a Km for external Cl
of 40 mM (Tas et al., 1987
), it is unlikely that recycling could keep up with the Cl
depletion caused by
HCO3
transport. Thus, [Cl
]i during the CO2/HCO3
-induced pHi recovery (i.e., Cl
efflux) was probably
substantially less than the initial ~3 mM mentioned
above. Even if Cl
recycling were substantial, the five
pHi recoveries under low [Cl
]o conditions could have
been mediated by a Na+-driven Cl-HCO3 exchanger
only if the hypothetical Na+-driven Cl-HCO3 exchanger
had an extremely high affinity for intracellular Cl
. In
squid axons, the only preparation in which the Km for intracellular Cl
has been determined for the Na+-driven
Cl-HCO3 exchanger, Km (Cl
) is about the same as the
resting [Cl
]i (Boron and Russell, 1983
). Even if the Km
(Cl
) for a hypothetical Na+-driven Cl-HCO3 exchanger
in astrocytes were substantially less than the resting
[Cl
]i of 36 mM, this Km (Cl
) would still be several
times higher than the likely [Cl
]i during the CO2/
HCO3
-induced pHi increase.
Our second approach for assessing
the Cl dependence of the CO2/HCO3
-induced alkalinization was to determine if depleting astrocytes of
Cl
slows this alkalinization. Before the start of the experiment in Fig. 5, the coverslip containing astrocytes
was incubated in a Cl
-free solution for 30 min, enough
time to reduce [Cl
]i to an average of ~3 mM (Fig. 3
B). The Cl
-free solution also contained 1 mM isoguvacine, which stimulates the GABAA-activated Cl
channels known to be present in astrocytes in rat hippocampal slices (MacVicar et al., 1989
). Thus, with isoguvacine accelerating the Cl
loss, [Cl
]i should have been
extremely low by the outset of the experiment.
In Fig. 5, the astrocyte remained in Cl-free solutions
throughout the experiment. The initial pHi was ~6.7 in
the standard HEPES solution. We then exposed the astrocyte to a CO2/HCO3
-buffered solution three times.
With each exposure, pHi decreased transiently, due to
CO2 influx (Fig. 5, a, c, and e), and then increased rapidly to a value higher than in the HEPES solution, due
to Na+-driven HCO3
influx (Fig. 5, b, d, and f ). The
three pHi increases were of similar speed, and had a
magnitude close to that observed in the accompanying
paper (Bevensee et al., 1997
) under control conditions (i.e., presence of extracellular Cl
). If the alkalinizations ab, cd, and ef were due to a Na+-driven Cl-HCO3
exchanger, then each alkalinization would further deplete the cell of intracellular Cl
. From the sum of the
pHi increases in Fig. 5, ab, cd, and ef, and the average
buffering power (
T), we calculate that, at a minimum,
9.5 mM HCO3
was transported into the cell. If this
HCO3
transport were mediated by a Na+-driven Cl-HCO3 exchanger moving two HCO3
ions for each Na+
and Cl
ion, then 4.7 mM Cl
would be required.2 As
summarized in Table I, the computed Cl
loss was 8.8 and 12.3 in two other experiments. Because it is unlikely that these cells could have had >~3 mM intracellular Cl
at the outset, it would appear that there was
insufficient Cl
to support the CO2/HCO3
-induced
pHi increases, if the alkalinizations were mediated by a
Na+-driven Cl-HCO3 exchanger.
Table I.
Intracellular Cl |
Na+-driven HCO3 Efflux: Testing the Extracellular
Cl
Dependence
The data in Figs. 4
and 5 suggest that the Na+-driven HCO3 transporter
in astrocytes does not require intracellular Cl
to operate in the forward direction (i.e., HCO3
influx). We
also tested the hypothesis that the transporter does not
require extracellular Cl
to operate in the reverse direction (i.e., HCO3
efflux). This assay has the advantage that one can be reasonably confident that the Cl
has been rapidly and completely removed.
Our first step was to determine if the Na+-driven
HCO3 transporter in astrocytes can be forced to operate in the reverse direction by removing external Na+,
thereby causing the net movement of Na+ and HCO3
out of the cells, and decreasing pHi. Fig. 6 illustrates an
experiment on a single astrocyte with a steady state pHi
of ~7.0 in a HEPES solution. Exposing the cell to a solution in which Li+ replaced Na+ as the major cation
caused the pHi to decrease by ~0.1 (Fig. 6, ab), presumably due to reversal of Na-H exchange. The pHi decrease with Li+ as the replacement cation was substantially less than that observed with NMDG+, because Li+,
in contrast to NMDG+, can partially substitute for Na+
on the Na-H exchanger (Bevensee et al., 1997
). Returning the cell to the Na+-containing solution caused pHi
to increase (Fig. 6, bc) and actually overshoot the initial
value (Fig. 6, c vs. a). This overshoot could be due to
stimulation of Na-H exchange, inasmuch as the exchanger is stimulated by intracellular Li+ in barnacle
muscle fibers (Davis et al., 1992
). When the astrocyte in
Fig. 6 was then switched to a solution buffered with 5%
CO2/17 mM HCO3
, the pHi decreased initially (Fig. 6,
cd), and then increased slowly due, in part, to the
Na+-driven HCO3
transporter (Fig. 6, de). Removing
external Na+ (replaced with Li+) in the presence of
CO2/HCO3
elicited a decrease in pHi (Fig. 6, ef) that
was faster and larger than in HEPES (Fig. 6, ab), presumably because the Na+-driven HCO3
transporter
was operating in the reverse direction and moving HCO3
out of the cell. As shown in the previous manuscript, Li+ is a poor substitute for Na+ on the Na+-driven HCO3
transporter in hippocampal astrocytes
(Bevensee et al., 1997
). After its initial decrease, pHi
began to increase slowly (Fig. 6, fg), presumably because external Li+ exchanged with internal H+ via the
Na-H exchanger. Returning the astrocyte to the Na+-containing solution caused the pHi to increase (Fig. 6,
gh) and overshoot the initial value (Fig. 6, h vs. e). In 14 experiments on astrocytes with an average pHi of 6.98 ±
0.04 in a HEPES solution, removing external Na+ elicited
an average initial acid influx (
L) of 26.0 ± 3.7 µM s
1.
However, in 27 experiments on astrocytes with a similar
average pHi of 6.95 ± 0.02 in a CO2/HCO3
-buffered
solution, removing external Na+ elicited a mean
L of
102 ± 6 µM s
1. Therefore,
L is approximately fourfold greater when Na+ is removed in the presence than
in the absence of CO2/HCO3
, consistent with Na/
HCO3 efflux.
DIDS inhibits the increase in pHi when astrocytes are switched from Li+ to Na+ in the presence of CO2/HCO3
Initially, we attempted to determine if DIDS could
block the HCO3-dependent pHi decrease observed
when astrocytes are exposed to a Na+-free solution.
However, we found that exposing cells to DIDS in the
presence of CO2/HCO3
causes a decrease in steady
state pHi (not shown), presumably, in part, because
DIDS inhibits the Na-dependent HCO3
transporter
that is active in the steady state. Because pHi was lower
in the presence of DIDS, it was not possible to compare rates of acidification at similar pHi values in the presence and absence of DIDS. Therefore, we compared
rates of alkalinization when astrocytes were reexposed
to external Na+ in the presence and absence of DIDS.
At the start of the experiment in Fig. 7, the astrocyte
had a steady state pHi of ~7.0 in a HEPES-buffered solution (before point a). Exposing the cell to CO2/
HCO3 caused the usual changes in pHi (Fig. 7, abc).
Replacing the Na+ with Li+ in the presence of CO2/
HCO3
elicited a rapid decrease in pHi (Fig. 7, cd), followed by a slow increase (Fig. 7, de), as seen in Fig. 6.
Returning Na+ to the external solution caused pHi to
increase (Fig. 7, ef ) to a value higher than that prevailing in the presence of CO2/HCO3
(Fig. 7, f vs. c). After we replaced Na+ with Li+ a second time, and observed the fall and partial recovery of pHi (Fig. 7, fgh),
we added 400 µM DIDS. This addition had little effect
on pHi (Fig. 7, hi). When we now returned external
Na+ in the presence of DIDS, the acceleration of the
pHi recovery (Fig. 7, ij vs. hi) was much smaller than in
the absence of DIDS (Fig. 7, ef vs. de). Removing DIDS
from the external solution allowed pHi to increase even
faster (Fig. 7, jk), indicating that DIDS was partially reversible. Removing external Na+ a third time in the absence of DIDS elicited the expected decrease in pHi
(Fig. 7, kl). However, DIDS may not have been entirely reversible, inasmuch as the rate of the pHi decrease
(Fig. 7, k) was slower than at Fig. 7, c and f. Similarly,
the pHi recovery elicited by returning Na+ the third
time (Fig. 7, lm) was slower than for the first time (Fig.
7, ef ).
Summarizing data from four such experiments, we
found that returning Na+ in the presence of DIDS was
associated with an acid extrusion rate (E) of 13 ± 9 µM s
1 at a pHi of 6.83 ± 0.03. In paired experiments
in the absence of DIDS,
E was 127 ± 15 µM s
1 at a
slightly lower pHi of 6.75 ± 0.03. Therefore, DIDS inhibits ~90% of the acid extrusion when astrocytes in
CO2/HCO3
are returned to a solution containing Na+
(P < 0.0003). The effect of DIDS is also CO2/HCO3
dependent: returning external Na+ to astrocytes bathed
in a HEPES solution containing 400 µM DIDS elicited
a
E of 23 ± 9 µM s
1 at a pHi of 6.82 ± 0.07 (n = 6). In
paired experiments at a similar pHi,
E in a HEPES solution in the absence of DIDS was nearly the same (25 ± 8 µM s
1, P = 0.26).
If a Na+-driven
Cl-HCO3 exchanger were responsible for the CO2/
HCO3-dependent decrease in pHi when astrocytes are
exposed to a Na+-free solution, then the pHi decrease
should require external Cl
(Fig. 8, inset). In the experiment shown in Fig. 8, the astrocyte at the start of the
experiment had a pHi of ~6.85 in a HEPES solution. Switching to CO2/HCO3
caused pHi to decrease (Fig.
8, ab), and then to increase promptly (Fig. 8, bc) to a
value ~0.1 higher than in the HEPES solution (Fig. 8, c
vs. a). Removing external Na+ elicited a rapid and reversible pHi decrease (Fig. 8, cde), similar to that seen
in Figs. 6 and 7. When the astrocyte was exposed to a
Cl
-free solution, the pHi decreased slowly (Fig. 8, ef ).
If the cells had either a Na+-driven Cl-HCO3 exchanger
or a Cl-HCO3 exchanger, one might have expected pHi
to increase in the absence of external Cl
, due to exchange of internal Cl
for external HCO3
. In the continued absence of external Cl
, removing external Na+
elicited a rapid decrease in pHi (Fig. 8, fg) that was of
the same rate as observed in the presence of external
Cl
(Fig. 8, arrows). Returning the astrocyte to a solution containing Na+, but not Cl
caused pHi to increase to its initial value (Fig. 8, gh). Reintroducing Cl
had little effect on pHi (Fig. 8, hi). Finally, switching
back to the HEPES solution elicited a sharp increase in
pHi (Fig. 8, ij), followed by a rapid decrease (Fig. 8, jk)
to the pHi prevailing at the start of the experiment
(Fig. 8, k vs. a). In a total of eight experiments similar
to that in Fig. 8, the
L caused by Na+ removal in the
presence of external Cl
(Fig. 8, cd) was 101 ± 5 µM s
1
at a pHi 6.91 ± 0.03.
L in the absence of external Cl
(Fig. 8, fg) was 96 ± 10 µM s
1 (P = 0.32, paired t test)
at the same pHi of 6.91 ± 0.03. As noted above, 75% of
the acid influx (
L) observed during removal of extracellular Na+ in the presence of CO2/HCO3
is due to
reversal of the Na+-driven HCO3
transporter. Because
L was unaffected by removing external Cl
, the Na+-driven HCO3
transporter could not have been a Na+-driven Cl-HCO3 exchanger.
Na/HCO3 Cotransport in 20% CO2 /68 mM HCO3
Because the Na+-driven HCO3 transporter in hippocampal astrocytes does not require intracellular Cl
to
operate in the normal forward direction, nor extracellular Cl
to operate in the reverse direction, we conclude that the transporter cannot be a Na+-driven Cl-HCO3 exchanger. By default (see INTRODUCTION), the Na+-driven HCO3
transporter must be a Na/HCO3
cotransporter with a Na+:HCO3
stoichiometry of either 1:1 (electroneutral) or 1:2 (electrogenic). If the
transporter were electrogenic, then the acid influx mediated by the transporter operating in reverse (Fig. 8,
cd) should be associated with an inward current that depolarizes the astrocyte. From the initial rate of pHi decrease during Fig. 8, cd, we calculate3 that a 1:2 Na/
HCO3 cotransporter would produce a DIDS-sensitive depolarization of 1.7 mV. Because such a small depolarization could be difficult to detect, we attempted
first to enhance the HCO3
efflux, and thus the predicted depolarization.
We performed experiments (not shown) identical to that in Fig. 7, except that the CO2/HCO3 solutions contained 20%
CO2 and 68 mM HCO3
, pH 7.3. The HCO3
efflux
from astrocytes should be greater when the cells, at the same pHi, are incubated in this "high CO2/HCO3
" solution because the fourfold increase in [HCO3
]i
would increase the efflux (if the cotransporter were not
already saturated). In the continued presence of high
CO2/HCO3
, removing external Na+ elicited a rapid
decrease in pHi that was reversed upon returning Na+.
In paired experiments, returning external Na+ in the
presence of DIDS elicited a pHi increase that was substantially slower than in the absence of DIDS. Summarizing the HCO3
-influx data for astrocytes bathed in
20% CO2/68 mM HCO3
, DIDS caused
E (measured
when we returned Na+) to decrease from 239 ± 48 to
87 ± 15 µM s
1 at a pHi of ~6.7 (n = 6; P < 0.008).
Thus, DIDS inhibited acid extrusion less in 20% CO2
(63%) than in 5% CO2 (90%), suggesting that HCO3
may compete with DIDS.
Summarizing HCO3-efflux data in 20% CO2, we
found that removing Na+ was associated with a
L of
281 ± 31 µM s
1, at a pHi of 6.96 ± 0.03 (n = 14). This
efflux is 2.8-fold greater than the
L of 101 µM s
1 observed in 5% CO2. Assuming that 63% of the HCO3
efflux in 20% CO2 is DIDS sensitive, the predicted DIDS-sensitive depolarization would be ~3.3 mV, approximately twice as large as in 5% CO2. In the next section,
we describe experiments in which we attempted to detect this predicted ~3.3-mV depolarization.
Electrogenic Na/HCO3 Cotransport
Astrocytes exposed to 20% CO2/68 mM HCO3The average resting Vm
of astrocytes in a HEPES solution was 87.5 ± 0.8 mV
(range:
72.0 to
97.7, n = 52), whereas the average resting Vm of astrocytes in a 20% CO2/68 mM HCO3
-buffered solution was
92.0 ± 0.7 mV (range:
80.8 to
99.1, n = 40). In 28 experiments in which we switched
from HEPES to CO2/HCO3
, Vm became more negative by an average of 4.3 ± 0.5 mV (P < 0.0001; paired
t test). At least qualitatively, this hyperpolarization is
consistent with the movement of net negative charge
into the cell via electrogenic Na/HCO3 cotransport.
The input resistance of astrocytes was computed at
the beginning of some experiments from the current
step (I) in response to a 5-ms, 5-mV depolarizing
pulse (
V) from a holding potential of ~
80 mV. The
average input resistance (
V/
I) was 236 ± 32 M
(n = 18). In other experiments, we computed total resistance at each sample point from the change in Vm in
response to current injections. Total resistance did not
appear to change when astrocytes were switched from a
HEPES- to a CO2/HCO3
-buffered solution or vice versa.
Because the pH of the patch-pipette solution was only weakly buffered (1 mM HEPES),
and because a perforated patch limits the diffusion of
solutes between cell and pipette, we hoped that patched
astrocytes subjected to manipulations of the extracellular solution would display the same pHi changes as unpatched cells. In the experiment shown in Fig. 9, we used
a digital-imaging system to monitor pHi in four astrocytes,
one of which was attached to the patch pipette (thick
trace). In the presence of 5% CO2/17 mM HCO3, all
cells underwent similar (and expected) pHi changes in
response to replacing extracellular Na+ with Li+.
Removing external Na+ elicits an HCO3
In the experiment shown
in Fig. 10 A, the astrocyte had a resting Vm of ~82 mV
in the nominal absence of CO2/HCO3
(Fig. 10 A, a).
Replacing external Na+ with Li+ elicited a depolarization to ~
76 mV (Fig. 10 A, ab) that was completely reversed when we returned the Na+ (Fig. 10 A, bc). Some
of this depolarization may be due to the inhibition of a
Na-dependent K+ conductance (Martin and Dryer,
1989
). Subsequently, exposing the cell to 20% CO2/68
mM HCO3
elicited an initial hyperpolarization to
~
87 mV (Fig. 10 A, cd), followed by a slower depolarization to ~
84 mV (Fig. 10 A, de). As noted above, the
initial hyperpolarization is qualitatively consistent with
the hypothesis that a Na/HCO3 cotransporter moves net negative charge into the cell. When the astrocyte
was exposed to a Na+-free solution in the presence of
CO2/HCO3
, the cell depolarized to ~
73 mV (Fig.
10 A, ef ). The depolarization in the presence of CO2/
HCO3
was ~6 mV greater than in the absence of
CO2/HCO3
. Moreover, compared with the depolarization in the nominal absence of CO2/HCO3
, the one in
the presence of CO2/HCO3
started at a more negative
Vm and finished at a more positive Vm, ruling out the
possibility that the difference in depolarizations was
due to the voltage dependence of the conductances.
When Na+ was returned to the external solution, the
astrocyte hyperpolarized towards the initial resting Vm
in the presence of CO2/HCO3
(Fig. 10 A, fg). In a separate experiment, the changes in Vm produced by removing and returning external Na+ in the presence of
20% CO2/68 mM HCO3
were greatly reduced in the
presence of DIDS (Fig. 10 A, e ' f ' g ' ).
Fig. 10 B summarizes the results from 14 experiments
similar to that shown in Fig. 10 A. In each case, the astrocyte was exposed to a Na+-free solution in the presence and absence of 20% CO2/68 mM HCO3. The
magnitude of the depolarizations in either the presence or absence of CO2/HCO3
are plotted as a function of the resting Vm before the Na+ removal. In general, the
Vm in the presence of CO2/HCO3
(
) is
larger than in HEPES (
). In the presence of CO2/
HCO3
(average resting Vm =
93.4 ± 1.5 mV), removing external Na+ elicited a mean depolarization of
6.6 ± 0.7 mV (Fig. 11). In the absence of CO2/HCO3
(average resting Vm =
89.0 ± 1.7 mV), removing external Na+ caused a statistically smaller mean depolarization of 4.0 ± 0.5 mV. In the presence of DIDS, removing external Na+ in CO2/HCO3
elicited a depolarization of only 2.8 ± 0.7 from a resting Vm of
93.3 ± 2.6. Thus, the depolarization in CO2/HCO3
is larger than
in either HEPES or CO2/HCO3
plus DIDS, and the
depolarization in HEPES is similar to that in CO2/
HCO3
plus DIDS.
Na/HCO3 cotransport elicited simultaneously recorded changes in pHi and Vm in single astrocytes.
Fig. 12 illustrates an experiment in which we recorded pHi and Vm simultaneously in a single astrocyte. The cell had an initial pHi
of ~7.2 in a HEPES solution. When the astrocyte was
exposed to a solution buffered with 5% CO2/17 mM
HCO3, the pHi decreased (Fig. 12, ab), and then increased rapidly to a value well above the initial pHi
(Fig. 12, bc). Replacing bath Na+ with Li+ elicited a
rapid pHi decrease (Fig. 12, cd) that was reversible (Fig.
12, de). The resting Vm was ~
98 mV when the astrocyte was returned to the Na+-containing solution at Fig.
12, d'. The Vm recording before Fig. 12, d' was unstable
and is therefore not shown. When Na+ was removed
from the bath a second time, pHi again decreased (Fig.
12, ef ). From the speed of the pHi decrease, we predict a
Vm of 8.1 mV for an electrogenic Na/HCO3 cotransporter with a 1:2 stoichiometry. The onset of the pHi decrease (Fig. 12, e) coincided with an abrupt depolarization4 of ~7 mV (Fig. 12, e'). When Na+ was returned
to the bath, the pHi increased as expected (Fig. 12, fg).
The onset of the pHi increase (Fig. 12, f ) coincided with
the return of Vm to the initial resting value (Fig. 12, f '). When the astrocyte was switched to a HEPES-buffered
solution, the pHi increased rapidly (Fig. 12, gh), and
then decreased (Fig. 12, hi). Switching to a Na+-free solution, now in the nominal absence of CO2/HCO3
,
caused a further decrease in pHi (Fig. 12, ij), but only a
small depolarization (Fig. 12, i '). When Na+ was returned to the bath before the pHi stabilized, pHi began to increase (Fig. 12, jk), but the cell hyperpolarized
only slightly (Fig. 12, j '). The simultaneous recording
of pHi and Vm in hippocampal astrocytes demonstrates
that removing/returning external Na+ in the presence
of CO2/HCO3
elicits pHi changes that coincide with
Vm changes.
Evidence Against Na+-driven Cl-HCO3 Exchange in Hippocampal Astrocytes
The transporter can move HCO3In the accompanying paper (Bevensee et al., 1997), we demonstrate that cultured hippocampal astrocytes exposed to CO2/HCO3
display, on
average, an increase in their steady state pHi. The pHi
increase is mediated, in part, by a HCO3
transporter
that requires Na+ and is blocked by the stilbene derivatives DIDS and 4-acetamido-4
-isothiocyanatostilbene-2,2
-disulfonic acid (SITS). In the present study, we show
that this transporter does not require intracellular Cl
to operate in the normal, forward direction. Our approach was to deplete cells of intracellular Cl
, and then
monitor the pHi increase in the cells exposed multiple times to CO2/HCO3
. Using the Cl
-sensitive indicator
MQAE, we found that hippocampal astrocytes could be
nearly depleted of intracellular Cl
by incubating the
cells in a Cl
-free solution for an average of ~11 min.
This Cl
depletion was probably mediated, at least in
part, by a bumetanide-sensitive Na/K/Cl cotransporter.
In the absence of external Cl
, cells exposed to CO2/
HCO3
still displayed an increase in pHi similar to that
observed in the presence of Cl
. In fact, astrocytes exposed multiple times to CO2/HCO3
in the absence of
external Cl
displayed multiple increases in pHi. It is
unlikely that these pHi increases could have been mediated by a Na+-driven Cl-HCO3 exchanger, inasmuch
as such an exchanger would have required substantial
amounts of intracellular Cl
.
We also tested the Cl dependence
of the transporter using a simpler and briefer protocol
in which we drove the transporter in the reverse (i.e.,
HCO3
outward) direction by removing external Na+
in the presence of CO2/HCO3
. Under such conditions, we found the rate of pHi decrease to be the same
in both the presence and absence of external Cl
. If
the transporter were a Na+-driven Cl-HCO3 exchanger,
which would require external Cl
to operate in the reverse direction, the pHi decrease should have been substantially reduced in the absence of external Cl
.
Evidence for Electrogenic Na/HCO3 Cotransport in Hippocampal Astrocytes
Switching from HEPES to CO2/HCO3Because the Na+-driven HCO3 transporter is
not coupled to Cl
, it presumably is either a 1:1 (electroneutral) or a 1:2 (electrogenic) Na/HCO3 cotransporter. If this astrocyte cotransporter were electrogenic, then it should simultaneously move net negative
charge and HCO3
in the same direction. In the accompanying study (Bevensee et al., 1997
), we show that
hippocampal astrocytes exposed to 5% CO2/HCO3
at
37°C first acidify due to CO2 influx, and then display a
maximum net acid extrusion (i.e., HCO3
uptake) rate
of 47.7 µM s
1, of which 75% is inhibited by DIDS. Therefore, the DIDS-sensitive HCO3
influx is 35.8 µM s
1.
From the average cell volume and input resistance of
astrocytes, we predict5 that such a HCO3
influx would
generate a current of only 2.9 pA and a hyperpolarization of only 0.7 mV. Such small CO2/HCO3
-induced
currents and Vm changes would be difficult to measure.
Our measured net acid extrusion, after the addition
of CO2/HCO3, could have underestimated the unidirectional DIDS-sensitive HCO3
influx if the actual
HCO3
influx were partially masked by an acid-loading
process (e.g., Cl-HCO3 exchange). Imagine that the
DIDS-sensitive HCO3
influx was in fact 360 µM s
1, 10-fold higher than we suspect, but that this was opposed
by a Cl-HCO3 exchange of 324 µM s
1. In this case, the
net flux would be only 36 µM s
1, as observed. Whereas
masking of a substantial HCO3
influx is theoretically
possible, we think that it is unlikely inasmuch as astrocytes seem to lack significant Cl-HCO3 exchange (see
Fig. 8). The potential complexity of the pHi changes associated with exposing a cell to CO2/HCO3
underscores
the difficulty of using the application of CO2/HCO3
as
an assay for electrogenic Na/HCO3 cotransport.
Brune et al. (1994), working on cultured rat cerebellar astrocytes, observed a mean hyperpolarization threefold larger than our prediction, 2.3 mV. O'Connor et
al. (1994)
, working on cultured rat hippocampal astrocytes, observed hyperpolarizations ranging from ~0
mV (Vm
75 mV) to ~25 mV (Vm
40 mV). Because our calculations show that a depolarization of 0.7 mV would be coupled to a DIDS-sensitive acid-extrusion
rate of 35.8 µM s
1, we can conclude that a depolarization of ~25 mV would be coupled to a DIDS-sensitive
acid-extrusion rate of 35.8 × (25/0.7)
1,280 µM s
1.
This figure is substantially greater than any transporter-mediated acid-base flux of which we are aware. For example, it is about eightfold greater than the flux of 160 µM s
1 mediated jointly by Na-H and Na-dependent Cl-HCO3 exchangers in acid-loaded renal mesangial cells
(Boyarsky et al., 1988a
), or the flux of 170 µM s
1 mediated by two similar transporters in acid-loaded pyramidal neurons from rat hippocampus (Bevensee et al.,
1996
), or the flux of 170 µM s
1 induced by basolateral
CO2/HCO3
addition in rabbit S3 proximal tubules
(Nakhoul et al., 1993
). One could argue that, under
the depolarized conditions (~40 mV) at which the data
from O'Connor et al. (1994)
were obtained, the electrogenic Na/HCO3 cotransporter influx may have been
substantially higher than in our cells (mean Vm
88
mV). Therefore, we measured the net HCO3
influx after addition of CO2/HCO3
in astrocytes depolarized
with 2 or 5 mM Ba2+ (Vm
56 mV). We found no significant difference (not shown) between the net
HCO3
influx under normally polarized and depolarized conditions. The large hyperpolarization observed
upon first exposing astrocytes to CO2/HCO3
may primarily reflect processes other than electrogenic Na/
HCO3 cotransport, such as the influx of HCO3
through
channels (see Bevensee et al., 1997
, INTRODUCTION). This large hyperpolarization may, in fact, inhibit the electrogenic influx of Na/HCO3 after cells are exposed to
CO2/HCO3
.
In addition to the large magnitudes of some of the
previously observed CO2/HCO3-induced hyperpolarizations, one could argue that the very assay (i.e., an exposure to CO2/HCO3
) is not sufficiently specific for
identifying an electrogenic Na/HCO3 cotransporter.
As discussed in the accompanying paper (Bevensee et
al., 1997
, INTRODUCTION), CO2 not only lowers pHi rapidly in the unstirred layer on the inner surface of the
cell membrane, but also reacts with susceptible amino
groups on proteins to form carbamino compounds
(Morrow et al., 1974
), thereby shifting their net charge
in a negative direction. Moreover, adding HCO3
could induce HCO3
currents, such as those observed
through GABAA-receptor channels in cultured rat astrocytes (Kaila et al., 1991
). The sensitivity of CO2/
HCO3
-induced electrical changes to DIDS, which is
not specific for Na-coupled HCO3
transporters, or
their sensitivity to Na+ removal, which changes a wide
range of cellular parameters, could be due to the nonspecific effects of these treatments.
Our approach for assaying for the electrogenicity of the cotransporter in hippocampal astrocytes was to expose cells to a Na+-free
solution in the presence vs. the absence of CO2/
HCO3, and in the presence vs. the absence of DIDS.
This was the assay used for the initial identification of
the electrogenic Na/HCO3 cotransporter in proximal-tubule cells (Boron and Boulpaep, 1983
), and for expression cloning the transporter in Xenopus oocytes (Romero et al., 1997
). The advantages of this assay,
over the less specific addition of CO2/HCO3
, are discussed in the accompanying paper (Bevensee et al.,
1997
). In our initial experiments, we removed Na+ in
the continued presence of 5% CO2 and 17 mM HCO3
,
pH 7.3. Under such conditions, however, removing external Na+ elicited an acid influx that predicts only
small currents and Vm changes. To enhance the predicted responses to Na+ removal/readdition, we performed experiments in solutions buffered with 20%
CO2 and 68 mM HCO3
, pH 7.3. Under these conditions, DIDS inhibited 63% of the acid extrusion caused
by Na+ readdition. Assuming that DIDS inhibits, to the
same extent, the acid loading elicited by Na+ removal,
then the DIDS-sensitive flux elicited by Na+ removal
was 281 µM s
1 × 0.63 = 177 µM s
1. This flux predicts
a
Vm of 3.3 mV, assuming a Na+:HCO3
stoichiometry
of 1:2. Indeed, we found that the depolarization elicited by removing external Na+ was 2.6 mV larger in the
presence vs. the absence of CO2/HCO3
. In the presence of 20% CO2/68 mM HCO3
, the DIDS-sensitive
Vm was 4.8 mV (paired experiments). Thus, because
the predicted and observed Vm changes were similar
quantitatively, our Na+-removal data are consistent with
the idea that the Na/HCO3 cotransporter in hippocampal astrocytes is electrogenic.
One would expect that removing Na+ may also alter
Vm by influencing the activity of the Na-K pump. Initially, removing external Na+ should drive the pump in
its normal forward direction, thereby leading to a hyperpolarization, not a depolarization. In our experiments, we replaced Na+ with Li+, a cation known to
promote active efflux of Na+ via the Na-K pump in human red blood cells (McConaghey and Maizels, 1962;
Maizels, 1968
; Beaugé and Del Campillo, 1976
), presumably by substituting for extracellular K+. Therefore,
replacing external Na+ with Li+ may actually stimulate
the Na-K pump in astrocytes, and elicit a hyperpolarization (J.F. Hoffman, personal communication). In the
continual absence of external Na+, however, the cells
would eventually become depleted of internal Na+,
thereby leading to inhibition of the Na-K pump and a
gradual, but continual depolarization. To avoid this inhibition, we added Na+ to the patch pipette solution in
experiments in which we measured Vm using the perforated patch-clamp technique.
Because
the Na/HCO3 cotransporter we have identified in hippocampal astrocytes is electrogenic, it is reasonable to
expect it to be modulated by Vm. When leech glial cells
are depolarized by an increase in [K+]o, the electrogenic Na/HCO3 cotransporter drives HCO3 into the
cells and elicits an increase in pHi (Deitmer and Szatkowski, 1990
). Such a depolarization-induced alkalinization (DIA) was first described in the proximal tubule
of the salamander kidney, where ~50% of the DIA is
believed to be mediated by the electrogenic Na/HCO3
cotransporter, and the remainder is due to stimulation of one of two apparently electroneutral lactate transporters (Siebens and Boron, 1989
).
As in leech glial cells and salamander proximal-tubule
cells, the pHi of rat forebrain astrocytes (Boyarsky et al.,
1993) and several other mammalian astrocyte preparations also increases when the cells are depolarized. An
exception is C6 glioma cells (Shrode and Putnam, 1994
).
The DIA generally is larger in the presence than in the
absence of CO2/HCO3
. In cultured astrocytes from
the cerebral cortex of the mouse (Brookes and Turner,
1994
), astrocytes in gliotic hippocampal slices from the
rat (Grichtchenko and Chesler, 1994
) and cultured astrocytes from the hippocampus of the rat (Pappas and
Ransom, 1994
), the DIA was partially or completely inhibited by removing external Na+, and unaffected by
acutely removing external Cl
. Stilbene derivatives partially inhibited the DIA in astrocytes from the mouse
cortex and the rat hippocampus, but not the DIA in astrocytes from the gliotic hippocampal slices. Thus, the
electrogenic Na/HCO3 cotransporter described in the
present studies could be responsible for at least a part
of the DIAs in these mammalian astrocytes.
Address correspondence to Walter F. Boron, Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520. Fax: 203-785-4951; E-mail: boronwf{at}maspo3.mas.yale.edu
Received for publication 24 January 1997 and accepted in revised form 30 June 1997.
Portions of this work have been previously published in preliminary form (Bevensee, M.O., W.F. Boron, and M. Apkon. 1995. FASEB J. 9: A308.).Ratiometric Correction for a Nonratiometric Indicator:
Application to the Cl-sensitive Dye MQAE
Fig. 13 A is the time course of
the raw MQAE fluorescence used to generate the [Cl]i
vs. time trace shown in Fig. 3 A. At the outset of the experiment, I320 decreased slowly (Fig. 13 A, ab) in the astrocytes, which were bathed in the standard HEPES-buffered solution. This slow decrease probably represents both dye loss from cells, as well as loss of cells that
become dislodged from the coverslip. When we exposed the cells to 1 µM of bumetanide, an inhibitor of
the Na/K/Cl cotransporter, I320 increased (Fig. 13 A,
bc), presumably because intracellular Cl
decreased
and relieved the quenching of intracellular MQAE. Removing bumetanide caused I320 to decrease (Fig. 13 A,
cd) to a level below that before removing external Cl
(Fig. 13 A, d vs. b). Subsequently, I320 continued to decline (Fig. 13 A, de) at about the same rate as in Fig. 13
A, ab. Exposing the cells to a high K+/nigericin/tributyltin calibration solution containing 65 mM Cl
caused
I320 to decrease rapidly at first (Fig. 13 A, ef ), and then
more slowly (Fig. 13 A, fg). Conversely, exposing the
cells to a calibration solution without Cl
caused I320
first to increase rapidly (Fig. 13 A, gh), and then to decline more slowly (Fig. 13 A, hi).
In the experiment shown in Fig. 13 A, the cells were
probably losing dye at different rates in each of three
different time periods. (a) During the main part of the
experiment (Fig. 13 A, a-e), dye was lost at a relatively
low rate. Notice that a single line (Fig. 13 A) fits the data
before and after the application of bumetanide. (b) During the first part of the calibration (Fig. 13 A, e-g), the
rate of dye loss was somewhat higher. (c) During the second part of the calibration (Fig. 13 A, g-i), when [Cl]o
was zero, the rate of dye loss was greatest.
We corrected for dye loss separately in each of these
three segments using a novel approach that compensates not only for dye loss per se at a particular [Cl]i,
but also for the change in quenching that occurs as
[Cl
]i changes. For example, during Fig. 13 A, ab and
de, when [Cl
]i is presumably at some fixed initial
value, the time-dependent decrease in I320 represents
only a linear loss of dye. To correct for this simple dye
loss, it would be reasonable to determine the rate of I320
decrease by fitting a line simultaneously to the data in
Fig. 13 A, ab and de (broken line). Starting from the original or "raw" I320 values (I traw) at each time t in Fig. 13 A,
one could thus obtain corrected I320 values (Itcorr) by
adding a fraction of the final difference between the intensities at Fig. 13 A, a and e (I araw - I eraw), assuming
that this difference increased linearly with time:
![]() |
(A1) |
However, a problem with this simplistic approach is
that, at any given time, the computed increment in I320
is independent of [Cl]i. For example, to compensate
for the dye loss during the bumetanide exposure in Fig.
13 A, bc in which [Cl
]i is decreasing, there must be two
components to the total increment I320. First, we must
increment I320 to compensate for dye loss per se, as in
Eq. A1. Second, we must increment I320 by an additional amount because the dye that was lost would have
been sensing a lower than normal [Cl
]i, and thus
would have been quenched to a lesser extent. Therefore, we did not use Eq. A1. Rather, we introduced an
approach in which we simultaneously compensate for
both components by multiplying Itraw by a time-dependent correction as follows.
Imagine that there had been no dye loss in Fig. 13 A.
We will assume that at Fig. 13 A, e, where [Cl]i has the
value x, I320 is 50 arbitrary fluorescence units. We also
will assume that when [Cl
]i is zero, I320 will be 100. Thus,
at Fig. 13 A, e, the [Cl
]i of x would have produced a
Stern-Volmer ratio of I0/I = 100/50 = 2. Now imagine
that, in fact, half the dye had been lost between Fig. 13
A, a and e. The Stern-Volmer ratio at e would thus be
I0/I = 50/25 = 2. If we were to compensate for dye loss
by simply adding 25 to both I0 and I (as in Eq. A1),
then I0/I would be 75/50 = 1.5, and we would underestimate [Cl
]i. Thus, to compensate for having lost half
the dye by the end of the time interval, we must multiply both I0 and I by 2 at Fig. 13 A, e. At the beginning of
the interval, we would have to multiply both I0 and I by
1; between Fig. 13 A, a and e, we would have to multiply
by a factor between 1 and 2. The general, time-dependent ratiometric correction is given by the following
equation:
![]() |
(A2) |
Applying this formula to Fig. 13 A, a-e yields the corrected curve ae' in Fig. 13 B. Notice that the uncorrected segment e-i in Fig. 13 B is now discontinuous with the corrected segment ae'.
To correct for the dye loss in Fig. 13 A, e-g, we applied the same formula (Eq. A2) that we used to correct for the dye loss in Fig. 13 A, a-e. This yielded the
partially corrected curve ef'g' shown in Fig. 13 C. Notice
that this curve is discontinuous with both Fig. 13 C, ae'
and g-i. To reestablish continuity, one might imagine
adding the same correction to all points in Fig. 13 C, ef'g'.
Although such an addition would yield the correct [Cl]i
for the transformed point e, the computed [Cl
]i values
at all other points would be incorrect. The reason is that
Stern-Volmer relationship deals with ratios of intensities. All [Cl
]i values in the transformed segment can be correct only if the transformation maintains its relative ratios. Therefore, we reestablished the continuity of Fig. 13
C, ae' and ef'g' by scaling all the points in segment ef'g'
by a factor that made I320 identical at Fig. 13 C, e and e' :
![]() |
(A3) |
The result, shown in Fig. 13 D, is that the ratio e'/g" is
the same as the ratio e/g' in Fig. 13 C. Thus, the [Cl]i
computed at point g" is the same as that computed at g'.
To compensate for dye loss in Fig. 13 D, g-i, we applied the same two-step correction that we did in Fig.
13 B, e-g. The result is shown as segment g"h"i" in Fig.
13 E. Finally, we used the Stern-Volmer relationship to
convert the fully corrected fluorescence data in Fig. 13
E to the [Cl]i time course shown in Fig. 13 F.
The greatest potential source of error in the time-dependent ratiometric correction described above is in assuming a constant rate of dye loss, especially when a solution change is made during the period of the correction. This assumption of constant dye loss can sometimes be verified directly. For example, in Fig. 13 A, a single best-fit line passes through a and b (before addition of bumetanide) as well as through d and e (after recovery from bumetanide withdrawal). Thus, it is reasonable to conclude that MQAE disappeared at a constant rate throughout the entire segment Fig. 13 A, a-e. On the other hand, we cannot directly verify that the rate of dye loss during Fig. 13 B, ef is the same as during fg, nor that the rate during Fig. 13 B, gh is the same as during hi.
Our ratiometric-correction technique is not well
suited to compensate for changes in the dye signal arising from factors other than dye loss or changes in
[Cl]i. For example, our approach would not properly
compensate for changes in the dye signal due to cell
shrinkage or swelling.
The time-dependent ratiometric correction described above for MQAE could be applied to other nonratiometric dyes, such as the Ca2+ indicator Fluo-3. In fact, our ratiometric correction could, in principle, be applied to any indicator whose fluorescence (a) is proportional to dye concentration, and (b) changes with substrate binding. For example, in experiments in which pHi is measured with BCECF, one could apply our ratiometric-correction technique to the I490 (i.e., pH sensitive) signal to arrive at a corrected I490 vs. time record that compensates for dye loss. Thus, without any knowledge of I440, one could use this corrected I490 to arrive at a pHi vs. time record, similar to that obtained using the standard I490/I440 ratiometric approach.
We thank Dr. W. Knox Chandler for evaluating the manuscript and providing useful suggestions. We also thank Dr. Fred J. Sigworth for useful discussions about our patch-clamp data.
This work was supported by National Institutes of Health Program Project grant PO1HD32573. M.O. Bevensee was supported by a predoctoral training grant (5-T32-GM0752718). M. Apkon was supported by a Physician Postdoctoral Fellowship Award by the Howard Hughes Medical Institute and is an established investigator of the Society for Critical Care Medicine.
1. | Beaugé, L.A., and E. Del Campillo. 1976. The ATP dependence of a ouabain-sensitive sodium efflux activated by external sodium, potassium and lithium in human red cells. Biochim. Biophys. Acta 433: 547-554 [Medline]. |
2. |
Bevensee, M.O.,
R.W. Weed, and
W.F. Boron.
1997.
Intracellular
pH regulation in cultured astrocytes from rat hippocampus. I. Role of HCO3![]() |
3. | Bevensee, M.O., T.R. Cummins, G.G. Haddad, W.F. Boron, and G. Boyarsky. 1996. pH regulation in single CA1 neurons acutely isolated from the hippocampi of immature and mature rats. J. Physiol. 494: 315-328 [Abstract]. |
4. |
Boron, W.F., and
E.L. Boulpaep.
1983.
Intracellular pH regulation
in the renal proximal tubule of the salamander: basolateral
HCO3![]() |
5. | Boron, W.F., and P. De Weer. 1976. Intracellular pH transients in squid giant axons caused by CO2, NH3 and metabolic inhibitors. J. Gen. Physiol. 67: 91-112 [Abstract]. |
6. | Boron, W.F., and J.M. Russell. 1983. Stoichiometry and ion dependencies of the intracellular-pH-regulating mechanism in squid giant axons. J. Gen. Physiol. 81: 373-399 [Abstract]. |
7. |
Boyarsky, G.,
M.B. Ganz,
B. Sterzel, and
W.F. Boron.
1988a.
pH regulation in single glomerular mesangial cells. I. Acid extrusion in
absence and presence of HCO3.
Am. J. Physiol.
255:
C844-C856
|
8. |
Boyarsky, G.,
M.B. Ganz,
B. Sterzel, and
W.F. Boron.
1988b.
pH regulation in single glomerular mesangial cells. II. Na-dependent
and -independent Cl-HCO3 exchangers.
Am. J. Physiol.
255:
C857-C869
|
9. | Boyarsky, G., B. Ransom, W.-R. Schlue, M.B.E. Davis, and W.F. Boron. 1993. Intracellular pH regulation in single cultured astrocytes from rat forebrain. Glia 8: 241-248 [Medline]. |
10. |
Brookes, N., and
R.J. Turner.
1994.
K+-induced alkalinization in
mouse cerebral astrocytes mediated by reversal of electrogenic
Na+-HCO3![]() |
11. | Brune, T., S. Fetzer, K.H. Backus, and J.W. Deitmer. 1994. Evidence for electrogenic Na-HCO3 cotransport in cultured rat cerebellar astrocytes. Pflugers Archiv. 429: 64-71 [Medline]. |
12. | Chao, A.C., J.A. Dix, M.C. Sellers, and A.S. Verkman. 1989. Fluorescent measurement of chloride transport in monolayer cultured cells. Biochem. J. 56: 1071-1081 . |
13. |
Davis, B.A.,
E.M. Hogan, and
W.F. Boron.
1992.
Activation of Na-H
exchange by intracellular lithium in barnacle muscle fibers.
Am.
J. Physiol.
263:
C246-C256
|
14. | Deitmer, J.W., and M. Szatkowski. 1990. Membrane potential dependence of intracellular pH regulation by identified glial cells in the leech central nervous system. J. Physiol. 421: 617-631 [Abstract]. |
15. | Engblom, A.C., and K.E.O. Akerman. 1993. Determination of the intracellular free chloride concentration in rat brain synaptoneurosomes using a chloride-sensitive fluorescent indicator. Biochim. Biophys. Acta 1153: 262-266 [Medline]. |
16. | Grichtchenko, I.I., and M. Chesler. 1994. Depolarization-induced alkalinization of astrocytes in gliotic hippocampal slices. Neuroscience 62: 1071-1078 [Medline]. |
17. | Horn, R., and A. Marty. 1988. Muscarinic activation of ionic currents measured by a new whole-cell recording method. J. Gen. Physiol. 92: 145-159 [Abstract]. |
18. | Kaila, K., P. Panula, T. Karhunen, and E. Heinonen. 1991. Fall in intracellular pH mediated by GABAA receptors in cultured rat astrocytes. Neurosci. Lett. 126: 9-12 [Medline]. |
19. |
Koncz, C., and
J.T. Daugirdas.
1994.
Use of MQAE for measurement of intracellular [Cl![]() |
20. |
Lancer, W.I.,
P. Weyer,
A.S. Verkman,
D. Ausiello, and
D. Brown.
1990.
FITC-dextran as a probe for endosome function and localization in kidney.
Am. J. Physiol.
258:
C309-C317
|
21. |
Lau, K.R.,
R.L. Evans, and
R.M. Case.
1994.
Intracellular Cl![]() |
22. | Lin, A., G. Krockmalnic, and S. Penman. 1990. Imaging cytoskeleton-mitochondrial membrane attachments by embedment-free electron microscopy of saponin-extracted cells. Proc. Natl. Acad. Sci. USA 87: 8565-8569 [Abstract]. |
23. |
MacVicar, B.A.,
F.W.Y. Tse,
S.A. Crichton, and
H. Kettenmann.
1989.
GABA-activated Cl![]() |
24. | Maizels, M.. 1968. Effect of sodium content on sodium efflux from human red cells suspended in sodium-free media containing potassium, rubidium, caesium or lithium chloride. J. Physiol. 195: 657-679 [Medline]. |
25. | Martin, A.R., and S.E. Dryer. 1989. Potassium channels activated by sodium. Quart. J. Exp. Physiol 74: 1033-1041 [Medline]. |
26. |
Martínez-Zaguilán, R.,
R.J. Gillies, and
S. Sánchez-Armass.
1994.
Regulation of pH in rat brain synaptosomes. II. Role of Cl![]() |
27. | McConaghey, P.D., and M. Maizels. 1962. Cation exchanges of lactose-treated human red cells. J. Physiol. 162: 485-509 . |
28. | Morrow, J.S., R.S. Gurd, and F.R.N. Gurd. 1974. The chemical basis and possible role of carbamino homeostatic mechanisms. In Peptides, Polypeptides, and Proteins. E.R. Blout, F.A. Bovey, M. Goodman, and N. Lotan, editors. John Wiley and Sons Inc., New York. 594-604. |
29. |
Nakhoul, N.L.,
L.K. Chen, and
W.F. Boron.
1993.
Effect of basolateral CO2/HCO3![]() |
30. |
O'Connor, E.,
H. Sontheimer, and
B.R. Ransom.
1994.
Rat hippocampal astrocytes exhibit electrogenic sodium-bicarbonate cotransport.
J. Neurophysiol.
72:
2580-2589
|
31. |
O'Connor, E.R.,
H.K. Kimelberg,
C.R. Keese, and
I. Giaever.
1993.
Electrical resistance method for measuring volume changes in
monolayer cultures applied to primary astrocyte cultures.
Am. J. Physiol.
264:
C471-C478
|
32. |
Pappas, C.A., and
B.R. Ransom.
1994.
Depolarization-induced alkalinization (DIA) in rat hippocampal astrocytes.
J. Neurophysiol.
72:
2816-2826
|
33. |
Romero, M.F.,
M.A. Hediger,
E.L. Boulpaep, and
W.F. Boron.
1997.
Expression cloning and characterization of a renal electrogenic Na+/HCO3![]() |
34. | Rose, C.R., and B.R. Ransom. 1996. Intracellular sodium homeostasis in rat hippocampal astrocytes. J. Physiol. 491: 291-305 [Abstract]. |
35. |
Schwiening, C.J., and
W.F. Boron.
1994.
Regulation of intracellular
pH in pyramidal neurones from the rat hippocampus by Na+-
dependent Cl![]() ![]() |
36. | Shrode, L.D., and R.W. Putnam. 1994. Intracellular pH regulation in primary rat astrocytes and C6 glioma cells. Glia 12: 196-210 [Medline]. |
37. |
Siebens, A.W., and
W.F. Boron.
1989.
Depolarization-induced alkalinization in proximal tubules. I. Characteristics and dependence
on Na+.
Am. J. Physiol.
256:
F342-F353
|
38. |
Soleimani, M.,
S.M. Grassl, and
P.S. Aronson.
1987.
Stoichiometry
of Na+-HCO3![]() |
39. |
Tas, P.W.L.,
P.T. Massa,
H.G. Kress, and
K. Koschel.
1987.
Characterization of an Na+/K+/Cl![]() |
40. | Thomas, J.A., R.N. Buchsbaum, A. Zimniak, and E. Racker. 1979. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 81: 2210-2218 . |
41. | Verkman, A.S., M.C. Sellers, A.C. Chao, T. Leung, and R. Ketcham. 1989. Synthesis and characterization of improved chloride-sensitive fluorescent indicators for biological applications. Anal. Biochem. 178: 355-361 [Medline]. |
42. | Walz, W., and L. Hertz. 1983. Comparison between fluxes of potassium and of chloride in astrocytes in primary culture. Brain Res. 227: 321-328 . |