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Article

I. Role of HCO₃^–

Mark O. Bevensee, Regina A. Weed, and Walter F. Boron

From the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520

	ABSTRACT

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We studied the regulation of intracellular pH (pH_i) in single cultured astrocytes passaged once from the hippocampus of the rat, using the dye 2',7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) to monitor pH_i. Intrinsic buffering power (β_I) was 10.5 mM (pH unit)^–1 at pH_i 7.0, and decreased linearly with pH_i; the best-fit line to the data had a slope of –10.0 mM (pH unit)^–2. In the absence of HCO₃^–, pH_i recovery from an acid load was mediated predominantly by a Na-H exchanger because the recovery was inhibited 88% by amiloride and 79% by ethylisopropylamiloride (EIPA) at pH_i 6.05. The ethylisopropylamiloride-sensitive component of acid extrusion fell linearly with pH_i. Acid extrusion was inhibited 68% (pH_i 6.23) by substituting Li⁺ for Na⁺ in the bath solution. Switching from a CO₂/HCO₃^–-free to a CO₂/HCO₃^–-containing bath solution caused mean steady state pH_i to increase from 6.82 to 6.90, due to a Na⁺-driven HCO₃^– transporter. The HCO₃^–-induced pH_i increase was unaffected by amiloride, but was inhibited 75% (pH_i 6.85) by 400 µM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and 65% (pH_i 6.55–6.75) by pretreating astrocytes for up to 6.3 h with 400 µM 4-acetamide-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS). The CO₂/HCO₃^–-induced pH_i increase was blocked when external Na⁺ was replaced with N-methyl-D-glucammonium (NMDG⁺). In the presence of HCO₃^–, the Na⁺-driven HCO₃^– transporter contributed to the pH_i recovery from an acid load. For example, HCO₃^– shifted the plot of acid-extrusion rate vs. pH_i by 0.15–0.3 pH units in the alkaline direction. Also, with Na-H exchange inhibited by amiloride, HCO₃^– increased acid extrusion 3.8-fold (pH_i 6.20). When astrocytes were acid loaded in amiloride, with Li⁺ as the major cation, HCO₃^– failed to elicit a substantial increase in pH_i. Thus, Li⁺ does not appear to substitute well for Na⁺ on the HCO₃^– transporter. We conclude that an amiloride-sensitive Na-H exchanger and a Na⁺-driven HCO₃^– transporter are the predominant acid extruders in astrocytes.

Key Words: H⁺ concentration • acid–base transport • glia • nervous system • Na-H exchanger

	INTRODUCTION

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It is well established that the pH of the brain extracellular fluid (pH_ECF)¹ can influence neuronal activity (for reviews, see Chesler and Kaila, 1992; Ransom, 1992), especially because many ion channels are sensitive to changes in extracellular pH (pH_o) (for reviews see Moody, 1984; Chesler, 1990). For example, the open probability of the N-methyl-D-aspartate-activated channel decreases at progressively lower pH_o values, with an apparent pK in the range 6.7–7.3 (Tang et al., 1990; Traynelis and Cull-Candy, 1990). The relationship between changes in pH_ECF and neuronal activity is complicated, however, because electrical activity itself can alter the pH of the brain extracellular fluid. The changes in pH_ECF elicited by neuronal firing are caused by the transport of acid–base equivalents across the plasma membrane of neurons and/or glia cells. This acid–base transport will have two effects. First, it obviously will affect the pH_i of the cells doing the transport, as well as the pH_ECF in the microenvironment. Second, because acid–base transporters on neighboring cells generally are sensitive to such pH_ECF changes, there will be indirect effects on the pH_i of these neighboring cells.

pH_ECF is thus in the position to mediate a complex interaction among neurons and glial cells. Indeed, the glial cells are thought to play a key role in regulating ECF composition, including pH_ECF, and the acid–base transporters of both invertebrate and mammalian glial cells are capable of modulating pH_ECF (see review by Deitmer and Rose, 1996). One such transporter thought to play an important role in regulating pH_ECF is the electrogenic Na/HCO₃ cotransporter. A similar transporter was first described in the cells of the proximal tubule of salamander kidney (Boron and Boulpaep, 1983), where it moves Na⁺ and HCO₃^– across the basolateral membrane from the cell to the blood, with a Na⁺: HCO₃^– stoichiometry of 1:3 (Soleimani et al., 1987). The electrogenic Na/HCO₃ cotransporter is independent of Cl^–, but is inhibited by disulfonic stilbene derivatives, such as 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Electrogenic Na/HCO₃ cotransporters were identified subsequently in a number of glial cells, including giant neuropile glial cells (Deitmer and Schlue, 1987, 1989) and connective glial cells (Szatkowski and Schlue, 1992) of the leech, astrocytes in the optic nerve of Necturus (Astion and Orkand, 1988), and Müller cells from the salamander retina (Newman, 1991; Newman and Astion, 1991). In the Müller cells, the electrogenic Na/HCO₃ cotransporter has a Na⁺:HCO₃^– stoichiometry of 1:3, and moves HCO₃^– out of the cells (Newman, 1991; Newman and Astion, 1991). On the other hand, in leech neuropile glial cells, and perhaps others as well, it is thought that the cotransporters have a Na⁺:HCO₃^– stoichiometry of 1:2, and move HCO₃^– into the cells (Deitmer and Schlue, 1989; Deitmer, 1992; Munsch and Deitmer, 1994). It is not known whether the electrogenic Na/ HCO₃ cotransporters with stoichiometries of 1:3 and 1:2 are different proteins.

There is also evidence that a Na/HCO₃ cotransporter might exist in mammalian glial cells. In primary cultures of astrocytes from the cerebral cortex of the mouse, the pH_i recovery from an acid load depends on Na⁺ and HCO₃^–, is unaffected by acute Cl^– removal, but is inhibited by SITS (Chow et al., 1991). In astrocytes cultured from rat cerebellum, exposure to CO₂/ HCO₃^– at 25°C causes a transient decrease in pH_i, followed by a sustained pH_i increase that has properties similar to those of the above mouse astrocytes (Brune et al., 1994). Both the mouse and rat data are consistent with mammalian astrocytes' having a Na/HCO₃ cotransporter that moves HCO₃^– into the cell and has a stoichiometry of either 1:2 (electrogenic) or 1:1 (electroneutral).

Two groups have used the whole-cell patch-clamp technique, one in rat astrocytes cultured from the cerebellum (Brune et al., 1994) and the other from the hippocampus (O'Connor et al., 1994), to address the question of whether Na/HCO₃ cotransporters in mammalian astrocytes are electrogenic. These authors recorded membrane voltage (V_m) and current (I_m) in astrocytes, while exposing them to CO₂/HCO₃^– at room temperature. They found that the exposure to CO₂/ HCO₃^– elicited hyperpolarizations and outward currents that were at least partially inhibited by removing external Na⁺ or applying DIDS, but were not inhibited by acutely removing external Cl^–. These data are consistent, but as discussed below, do not prove the hypothesis that introducing CO₂/HCO₃^– initiates the influx of HCO₃^– and negative charge via a DIDS-sensitive, electrogenic Na/HCO₃ cotransporter.

In this and the accompanying paper on cultured rat hippocampal astrocytes, we examine whether mammalian astrocytes do indeed possess an electrogenic Na/ HCO₃ cotransporter. Our approach is significant in three ways. First, we perform the experiments at 37°C, where the activity of the electrogenic Na/HCO₃ cotransporter is likely to be greater than at room temperature. Second, we use a more specific assay for the cotransporter than monitoring V_m while adding CO₂/HCO₃^– under three conditions (control, DIDS, 0 Na⁺). A potential difficulty with focusing on the DIDS-sensitive electrical changes elicited by exposing the cells to CO₂/ HCO₃^– is that both DIDS and CO₂ are rather nonspecific amino-reactive agents. The isothiocyano groups on DIDS interact with amino groups via the Edman reaction. CO₂ interacts with susceptible free amines of proteins, resulting in the formation of carbamino compounds (Morrow et al., 1974), a classic example of which is the formation of carbamino hemoglobin. Moreover, the influx of CO₂ into the cell will rapidly lower pH_i, especially in the microenvironment on the inner surface of the membrane, and potentially change pH_i-sensitive ionic conductances. An additional problem is that switching to a HCO₃^–-containing solution could evoke HCO₃^– currents per se. In cultured rat astrocytes, GABA_A-receptor channels can mediate HCO₃^– currents (Kaila et al., 1991).

Therefore, we have chosen to monitor pH_i and V_m while executing a maneuver (i.e., Na⁺ removal) designed to elicit a response (i.e., a rapid depolarization) that is far more specific for the cotransporter than is the addition of CO₂/HCO₃^–. Although removing Na⁺ per se is not specific, the resulting rapid depolarization can be attributed to only a few causes: inhibiting a Na⁺-dependent K⁺ conductance, inhibiting the Na-K pump, and forcing the electrogenic Na/HCO₃ cotransporter to move in the outward direction. Other Na⁺-coupled transporters would either directly produce rapid V_m changes of the wrong sign, or indirectly produce slow V_m changes by changing cell composition. The Na⁺- removal assay can be made even more specific by requiring that the rapid depolarization also depend on CO₂/HCO₃^– and be inhibited by DIDS.

Our third significant approach for identifying an electrogenic Na/HCO₃ cotransporter in hippocampal astrocytes is to determine whether the changes in V_m and pH_i are quantitatively consistent with electrogenic Na/HCO₃ cotransport. Thus, we compare the magnitude of the V_m change (from which we can compute the flux of charge) to the observed rate of pH_i change (from which we can compute the flux of HCO₃^–).

In the first of our two papers, we use a fluorescent pH-sensitive dye to evaluate the acid–base transporters responsible for regulating pH_i in both the presence and absence of CO₂/HCO₃^–. We found that the astrocytes have both a previously identified amiloride-sensitive Na-H exchanger, as well as a CO₂/HCO₃^–-dependent transporter that requires external Na⁺ and can be inhibited by the stilbene derivatives, DIDS and SITS. In the second paper (Bevensee et al., 1997), we provide evidence that the CO₂/HCO₃^–-dependent transporter does not require Cl^–. This rules out a Na⁺-driven Cl-HCO₃ exchanger. In addition, we used the perforated patch-clamp technique to record changes in V_m during the aforementioned Na⁺-removal protocol, observing V_m changes nearly identical to those predicted from the rates of pH_i change. Our data indicate that hippocampal astrocytes have an electrogenic 1:2 Na/HCO₃ cotransporter.

	METHODS

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Solutions
The standard HEPES-buffered solution contained (mM): 125 NaCl, 3 KCl, 1 CaCl₂, 1.2 MgSO₄, 2 NaH₂PO₄, 32 HEPES, and 10.5 glucose, titrated to pH 7.3 at 37°C with NaOH. 5% CO₂/17 mM HCO₃^–-buffered solutions were made by substituting 17 mM NaHCO₃ for HEPES and adding 8.4 mM NaCl to maintain constant ionic strength. In NH₃/NH₄⁺-containing solutions, NaCl was replaced with equimolar NH₄Cl. In 0-Na⁺ solutions, the Na⁺ substitute was either N-methyl-D-glucammonium (NMDG⁺) or Li⁺. In 0-Cl^– solutions, the Cl^– substitute was cyclamate, and total [Ca²⁺] was increased threefold to compensate for Ca²⁺ chelation by the anion substitute. In the 10-µM nigericin solution, KCl was 105 mM and the remainder of the Na⁺ was replaced with NMDG⁺. The acetoxymethyl ester of the pH-sensitive dye 2',7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF-AM) was obtained from Molecular Probes, Inc. (Eugene, OR). Ethylisopropyl-amiloride (EIPA) was obtained from either E.J. Cragoe, Jr. (Nacogdoches, TX) or Research Biochemicals, Inc. (Natick, MA). DIDS was obtained from either Fluka Chemical Corp. (Ronkon-koma, NY) or Sigma Chemical Co. (St. Louis, MO). All other chemicals were obtained from Sigma Chemical Co.

Cell Isolation and Culturing
Astrocytes were isolated and cultured from the hippocampi of 2–3-d-old rats using a modified version of the procedure described by McCarthy and de Vellis (1980). Rats were decapitated and the brains placed in chilled Dulbecco's PBS supplemented with 33 mM glucose. The hippocampi were removed from each brain by microdissection and placed in fresh, chilled PBS + 33 mM glucose. The hippocampi were triturated with pipettes of decreasing pore diameters until the solution was cloudy in appearance and contained only small hippocampal fragments. A final concentration of 0.01% trypsin (GIBCO BRL, Life Technologies Inc., Gaithersburg, MD) was then added to the solution to digest the fragments further. After 1 min, a final concentration of 25% fetal calf serum (Gemini Bioproducts, Inc., Calabasas, CA, or GIBCO BRL, Life Technologies Inc.) was added to the solution to inhibit further digestion by the trypsin. The solution was centrifuged (TJ-6; Beckman Instruments, Inc., Fullerton, CA) at 400 g for 5 min, and the cell pellet was resuspended in PBS + 33 mM glucose. This new suspension was again centrifuged and the pellet resuspended in the standard cell culture medium, which consisted of MEM supplemented with 28 mM glucose, 2 mM L-glutamine, 100 U ml^–1 penicillin/streptomycin (GIBCO BRL, Life Technologies Inc.), and 10% fetal calf serum (Gemini Bioproducts, Inc. or GIBCO BRL, Life Technologies Inc.). After a final centrifugation, the pellet was resuspended in the culture medium and the cell suspension was plated into cell culture flasks. The cells were grown in a 5% CO₂, 37°C incubator, and the culture medium was changed every 3–5 d. To minimize the growth of oligodendrocytes in the cell culture, the flasks were agitated on a variable rotator (R4140; American Hospital Supply Corp., Miami, FL) at 100 rpm for 2 h before the me33dia was changed on the fourth day after cell plating. 10–12 d after cell plating, the flasks were again agitated at 100 rpm for 18–24 h before the confluent monolayer of cells was treated with trypsin-EDTA (GIBCO BRL, Life Technologies Inc.) and passaged onto 22 x 22–mm glass coverslips. Before cell plating, the coverslips were washed with RadiacWash^® (Biodex Medical Systems, Shirley, NY), and then 70–100% ethanol several times to rid the glass of grease and optimize cell attachment and growth. Experiments were performed on the cells 1–6 wk after the cells were passaged onto coverslips. The cells grown on the coverslips were immunocytochemically stained routinely with an antibody to the astrocyte-specific glial fibrillary acidic protein (GFAP). Over 95% of the cells stained positive for GFAP.

Measurement of pH_i in Single Astrocytes
Coverslips with attached astrocytes were first washed in a standard HEPES-buffered solution before being mounted in a perfusion chamber. The coverslip comprised the floor of the chamber. The chamber was then filled with a HEPES-buffered solution containing 10 µM BCECF-AM. Cells were incubated in this BCECF-AM solution, equilibrated with room air, for 10–20 min at 37°C. The perfusion chamber was then secured to the stage of a microscope equipped for epifluorescence (IM-35; Carl Zeiss, Inc., Thornwood, NY), and perfused for a minimum of 5 min with a HEPES-buffered solution to remove any unhydrolyzed BCECF-AM. Because the optical technique and the data acquisition used to measure pH_i have been described extensively elsewhere (Boyarsky et al., 1988), they will be only briefly summarized here. A 63x oil-immersion objective was used to choose a single astrocyte that was typically 20–40 µm in diameter, star shaped, and had a homogeneous distribution of dye inside the cytoplasm. pH_i recordings were obtained on cells that were nearly always surrounded by other cells. Dye was excited in an 20-µm diameter area in the center of the cell; the light source was a 100-W halogen bulb. Approximately every 8 s, the dye was alternatively excited for 200 ms by light at wavelengths of 490 and 440 nm. The emitted fluorescence at a wavelength of 530 nm was amplified by a photomultiplier tube and recorded as I₄₉₀ and I₄₄₀. Because I₄₉₀ is pH dependent and I₄₄₀ is relatively pH independent, the ratio of the two (I₄₉₀/I₄₄₀) is mainly a function of pH. Normalized I₄₉₀/I₄₄₀ values were converted to pH_i using a high K⁺/nigericin technique (Thomas et al., 1979), as modified for a single-point calibration by Boyarsky et al. (1988). Background I₄₉₀ and I₄₄₀ levels in the absence of dye were subtracted from the total I₄₉₀ and I₄₄₀ values.

In some experiments, pH_i was measured using a fluorescence imaging system in which the photomultiplier tube mounted on the microscope was replaced with an intensified CCD camera (ICCD-350F; Video Scope Int., Ltd., Sterling, VA). The system software included custom program macros written by Dr. Michael Apkon (Department of Pediatrics, Yale University, New Haven, CT) for OPTIMAS (Optimas Co., Edmonds, WA).

In nine experiments on hippocampal astrocytes loaded with BCECF, applying 0.01% saponin caused the fluorescent signal to decrease to 4.3 ± 0.6% of the signal at the start of the experiment. Because 0.01% saponin is thought to permeabilize only the plasma membrane (Lin et al., 1990), we conclude that 96% of intracellular BCECF is located in the cytoplasm.

Intracellular Buffering Power
From experiments in which pH_i recovered from acute acid loads, we computed acid extrusion as the product of the rate of change in pH_i (dpH_i/dt) and total intracellular buffering power (β_T). Acid-extrusion rate (_E) thus has the units of moles per unit volume of cytoplasm per unit time (e.g., µM s^–1). In the subsequent manuscript (Bevensee et al., 1997), _L refers to acid loading and has similar units. β_T is the sum of the buffering power due to CO₂/HCO₃^– (β_HCO3^–) and the buffering power due to intrinsic intracellular buffers (β_I). We computed the theoretical β_HCO3^– as 2.3 x [HCO₃^–]. As recently confirmed by our laboratory (Zhao et al., 1995), the actual β_HCO3^– is indistinguishable from the theoretical value. We calculated β_I in hippocampal astrocytes from the increases in pH_i elicited by increasing the external concentration of the weak base NH₃, as described by Roos and Boron (1981).

An example of an experiment for determining β_I in a single hippocampal astrocyte is shown in Fig. 1 A. We added 0.9 mM amiloride to all solutions to inhibit the Na-H exchanger previously identified in these cells (Pappas and Ransom, 1993), and removed the Cl^– to minimize potential Cl-base exchange (Vaughan-Jones, 1982). We acid loaded the astrocyte using the NH₄⁺-prepulse technique (Boron and De Weer, 1976). Briefly, exposing the cell to a solution containing 20 mM NH₃/NH₄⁺ caused pH_i to increase rapidly as NH₃ entered the cell and consumed H⁺ to form NH₄⁺ (Fig. 1 A, ab). In the continued presence of the NH₃/NH₄⁺ solution, the pH_i decreased more slowly due to NH₄⁺ influx and/or stimulation of acid-loading mechanisms (Fig. 1 A, bc). When the NH₃/NH₄⁺ solution was removed, the pH_i decreased rapidly to a value lower than at the start of the experiment (Fig. 1 A, cd). As discussed below, pH_i normally recovers rapidly from such an acid load due to Na-H exchange. In Fig. 1 A, the pH_i recovery was greatly slowed by amiloride (Fig. 1 A, de). Exposing the cell sequentially to solutions containing 1, 2.5, and 5 mM NH₃/NH₄⁺ caused pH_i to increase in a stepwise fashion (Fig. 1 A, ef, gh, and ij, respectively). However, especially at higher pH_i values, the rapid increase in pH_i in response to elevations of external NH₃/NH₄⁺ was often followed by a slower decrease in pH_i (e.g., Fig. 1 A, jk). Therefore, we back extrapolated the pH_i vs. time record (see Fig. 1 A, f', h', and j') to determine more accurately the pH_i changes due to the changes in NH₃/ NH₄⁺ (Aickin and Thomas, 1977). In Fig. 1 B, we plotted the results from five experiments similar to that shown in Fig. 1 A. β_I is plotted as a function of the average pH_i before and after a step change in NH₃/NH₄⁺. β_I is 10.5 mM (pH unit)^–1 at pH_i 7.0. The best-fit line to the data has a slope of –10.0 mM (pH unit)^–2. Our β_I values are similar to those reported by Pappas and Ransom (1994), although they did not observe a dependence of β_I on pH_i.

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Figure 1 NH3/NH4+ solutions can be used to calculate the pHi dependence of intrinsic buffering power (βI) in cultured astrocytes from the rat hippocampus. (A) The effect of step changes in [NH3/NH4+] on pHi. A single astrocyte was bathed in a Cl–-free, HEPES-buffered solution containing 0.9 mM amiloride. After the cell was acid loaded by a brief exposure to 20 mM NH3/NH4+ (a–d), the cell was subsequently exposed to solutions containing 1, 2.5, and 5 mM NH3/NH4+. (B) pHi dependence of intrinsic buffering power (βI) in rat hippocampal astrocytes.

	RESULTS

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Initial Steady State pH_i in the Absence and Presence of CO₂/HCO₃^–
The astrocytes in this study, cultured from the rat hippocampus, had an average initial steady state pH_i of 6.83 ± 0.01 (n = 180) when exposed to a nominally CO₂/HCO₃^–-free, HEPES-buffered solution (pH 7.3). The frequency distribution of these pH_i values is slightly skewed to more alkaline values (Fig. 2). For 63 of these 180 astrocytes, we switched the extracellular solution to one buffered with 5% CO₂/17 mM HCO₃^– (pH 7.3). In the presence of CO₂/HCO₃^–, these astrocytes had a mean steady state pH_i of 6.90 ± 0.01 (n = 63), 0.08 pH units higher, on average, than for the same cells in the absence of CO₂/HCO₃^– (6.82 ± 0.02). As indicated by the inset to Fig. 2, the frequency distribution of steady state pH_i for the 63 astrocytes in the presence of CO₂/ HCO₃^– (solid bars) is shifted toward more alkaline pH_i values, and has a smaller standard deviation than for cells in the absence of CO₂/HCO₃^– (hatched bars).

View larger version (45K): [in this window] [in a new window] [Download PPT slide]   Figure 2 The distribution of steady state pHi is bell-shaped for 180 hippocampal astrocytes bathed in a nominally CO2/HCO3–-free, HEPES-buffered solution. The bin width is 0.1 pH unit. (inset) The distribution of steady state pHi of 63 astrocytes first exposed to HEPES is alkaline shifted when the cells are subsequently exposed to 5% CO2/17 mM HCO3–.

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   Figure 3 CO2/HCO3– stimulates the pHi recovery from an acid load when the Na-H exchanger is inhibited. (A) Effect of CO2/HCO3– on the pHi recovery from an acid load in the presence of amiloride. The single astrocyte was acid loaded twice by a brief exposure to a solution containing 20 mM NH4Cl at a constant pHO of 7.3 (a–d and e–h). During the indicated periods, 0.9 mM amiloride was present (hj), and 5% CO2/17 mM HCO3– was present (ij). (B) The pHi dependence of acid extrusion in a HEPES-buf-fered solution in the absence (circles) and presence (open diamonds) of 0.9 mM amiloride. In principle, the gap between the two groups of diamonds would have been filled had it been practical, in experiments such as that shown in A, to monitor the segment-hi pHi recovery (which generated the diamonds near pHi 6) until pHi had reached the second group of diamonds (near pHi 6.6). (inset) The pHi dependence of the amiloride-sensitive acid extrusion (closed diamonds), obtained by subtracting the acid extrusion rate in the presence from that in the absence of amiloride.

At a pH_i of 6.05, _HEPES averaged 173 ± 12 µM s^–1 (n = 19), whereas _Amil averaged only 20.1 ± 10.1 µM s^–1 (n = 4). Therefore, at this pH_i, 88% of _HEPES was amiloride sensitive in the nominal absence of CO₂/ HCO₃^–. The pH_i dependence of the amiloride-sensitive acid extrusion rate (_Amil-sens), defined as _HEPES – _Amil, is shown in the inset to Fig. 3 B.

In six experiments in which pH_i recovered in the presence of both amiloride and CO₂/HCO₃^– (e.g., Fig. 3 A, ij), acid extrusion increased 3.8-fold from 32.0 ± 12.9 µM s^–1 (e.g., just before i) to 121 ± 29.2 µM s^–1 (e.g., just after i) at a pH_i of 6.20 ± 0.05 (P = 0.003). The higher β_T in the presence of CO₂/HCO₃^– is factored into the above calculations. The data in Fig. 3 thus suggest that hippocampal astrocytes have as many as three acid-extrusion mechanisms: (a) an amiloride-sensitive Na-H exchanger (Fig. 3 A, de vs. hi); (b) possibly an amiloride-insensitive mechanism (e.g., H⁺ pump) that functions in the nominal absence of CO₂/HCO₃^– (Fig. 3 A, hi); and (c) a CO₂/HCO₃^–-dependent mechanism that can be observed even in the presence of amiloride (Fig. 3 A, ij).

If astrocytes possess both a Na-H exchanger and a HCO₃^– transporter, then the acid-extrusion rate should be greater in the presence than in the absence of CO₂/ HCO₃^–. In the experiment shown in Fig. 4 A, we acid loaded an astrocyte twice in a HEPES-buffered solution, using NH₄⁺ prepulses (Fig. 4 A, a–d and e–h). As we removed the NH₃/NH₄⁺ the second time, we simultaneously introduced CO₂/HCO₃^–. Although the rates of pH_i change do not appear substantially different for the two pH_i recoveries, recall that β_T is substantially higher in the presence of CO₂/HCO₃^–. Moreover, it is clear from Fig. 4 A that pH_i recovered to a higher value in the presence vs. the absence of CO₂/HCO₃^– (compare points i and e). For six experiments similar to that shown in Fig. 4 A, we determined the pH_i dependence of acid extrusion both in the absence (_HEPES, open circles) and presence (_HCO3, closed circles) of CO₂/HCO₃^– (Fig. 4 B). Over a wide range of pH_i values, _E was as much as 80 µM s^–1 higher in the presence than in the absence of CO₂/HCO₃^–. Viewed differently, CO₂/ HCO₃^– shifted the _E vs. pH_i curve 0.15–0.3 pH units in the alkaline direction.

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Figure 4 During the recovery from an acid load, acid extrusion is greater in the presence than in the absence of CO2/HCO3–. (A) Effect of CO2/HCO3– on the pHi recovery from an acid load. An astrocyte was acid loaded twice by applying and withdrawing 20 mM NH3/NH4+ (a–d and e–h). When NH3/NH4+ was removed the second time, the cell was exposed to CO2/HCO3– simultaneously (g–i). (B) The pHi dependence of acid extrusion in the absence (open circles) and presence (closed circles) of CO2/HCO3–. The data were taken from experiments similar to those in A. (inset) The open symbols are a replot of the data obtained in HEPES. The closed triangles represent the HCO3–-dependent component of the flux, obtained by subtracting the open circles from the closed circles in the main panel.

Further Characteristics of Na-H Exchange
EIPA also inhibits the Na-H exchanger.
Na-H exchange isoforms can be distinguished by their level of sensitivity to both amiloride and amiloride analogues such as EIPA. To characterize the pharmacology of the Na-H exchanger present in rat hippocampal astrocytes further, we examined how EIPA affects the pH_i dependence of acid extrusion in the nominal absence of CO₂/HCO₃^–. In the experiment represented in Fig. 5 A, we acid loaded an astrocyte by pulsing with 20 mM NH₃/NH₄⁺ (Fig. 5 A, a–d), and then monitored the pH_i recovery with the cell exposed to 10 µM EIPA (Fig. 5 A, de). Using the segment-de pH_i recoveries from 13 similar EIPA experiments and 26 control experiments, we plotted the pH_i dependence of acid extrusion in the presence (_EIPA, Fig. 5 B, open squares) and absence (_HEPES, Fig. 5 B, circles) of EIPA. At a pH_i of 6.05, EIPA decreased _E from 173 ± 11.7 µM s^–1 (n = 19) to 35.9 ± 25.7 µM s^–1 (n = 7). Therefore, at a pH_i of 6.05, 79% of acid extrusion was inhibited by EIPA. In the four experiments discussed in Fig. 3 B, analyzed at this same pH_i, amiloride decreased _E to 20.1 ± 10.1 µM s^–1 (n = 4), a reduction of 88%. Thus, it seems that amiloride is similar to EIPA in inhibiting Na-H exchange in cultured hippocampal astrocytes. The inset to Fig. 5 B, a plot of the EIPA-sensitive component of _E (_EIPA-sens), confirms that Na-H exchange activity is highest at low pH_i values and falls to nearly zero at a pH_i of 6.8.

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Figure 5 The pHi recovery from an acid load in the nominal absence of CO2/HCO3– is sensitive to EIPA. (A) Effect of EIPA on the pHi recovery from an acid load. An astrocyte was acid loaded by applying and withdrawing 20 mM NH3/NH4+ (a–d). EIPA (10 µM) was present during the pHi recovery (de). (B) The pHi dependence of acid extrusion in the absence (circles) and presence (open squares) of 10 µM EIPA. (inset) The pHi dependence of the EIPA-sensitive component of acid extrusion (closed squares). These data were obtained by subtracting the open squares in the main panel from the open circles.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 6 Li+ can exchange with H+ on the Na-H exchanger. (A) Effect of replacing Na+ with Li+ on the pHi recovery from an acid load. An astrocyte was acid loaded twice by applying and withdrawing 20 mM NH3/NH4+ (a–d and f–i). During the indicated times (c–e and h–k), Li+ instead of Na+ was the major extracellular cation. Amiloride (0.9 mM) was added immediately after the NH3/ NH4+ was removed (h–j). (B) The pHi dependence of acid extrusion when Na+ (circles) or Li+ (open triangles) is the major cation. (inset). The pHi dependence of the "Li+-dependent" acid extrusion (closed triangles). These data were obtained by subtracting the open triangles in the main panel from the open circles.

CO₂/HCO₃^–-induced Alkalinization
CO₂/HCO₃^– elicits an increase in the mean steady state pH_i of astrocytes.
Because the pH_i to which astrocytes recover after an acid load is higher in the presence than in the absence of CO₂/HCO₃^– (Fig. 4), one would predict that simply applying CO₂/HCO₃^– should increase steady state pH_i. As shown in the experiment in Fig. 7 A, when a single astrocyte was switched from a HEPES-buffered to a CO₂/HCO₃^–-buffered solution, the pH_i transiently fell, due to the influx of CO₂ and production of intracellular H⁺ and HCO₃^– (Fig. 7 A, ab), and then increased to a value higher than the initial one (Fig. 7 A, bc). When the astrocyte was returned to a HEPES-buffered solution, the opposite pH_i changes occurred (Fig. 7 A, c–e). The mechanism of the segment- bc pH_i increase is the subject of the rest of this paper, as well as the accompanying one (Bevensee et al., 1997). The mechanism of the segment-de decrease in Fig. 7 A is presently unknown. Some have suggested that a similar pH_i decrease in other cells could be caused by Cl-HCO₃ exchange, with the HCO₃^– being provided by intracellular metabolism. However, as discussed in the accompanying paper (Bevensee et al., 1997), hippo-campal astrocytes do not appear to have appreciable Cl-HCO₃ exchange activity. Another possibility is that the Fig. 7 A, de pH_i decrease is mediated by a K/HCO₃ cotransporter, similar to that described in squid axons (Hogan et al., 1995).

View larger version (14K): [in this window] [in a new window] [Download PPT slide]   Figure 7 Mean steady state pHi increases when hippocampal astrocytes are switched from HEPES to CO2/HCO3–. (A) Effect of CO2/HCO3– on steady state pHi. During the indicated period, we switched to an extracellular solution that was buffered with 5% CO2/17 mM HCO3– rather than HEPES (a–c). (B) The change in steady state pHi caused by adding CO2/HCO3–, plotted as a function of steady state pHi in the HEPES-buffered solution.

Amiloride does not inhibit the CO₂/HCO₃^–-induced alkalinization.
One possible explanation for the CO₂/ HCO₃^–-induced alkalinization in Fig. 7 A, bc is that CO₂/HCO₃^– stimulates Na-H exchange, as has been postulated for the proximal tubule (Chen and Boron, 1995a, 1995b). In six experiments in which the astrocytes had an average initial pH_i of 6.78 ± 0.06 in a HEPES-buffered solution, adding 0.9 mM amiloride caused pH_i to decrease to 6.66 ± 0.05 (data not shown), presumably because amiloride inhibited Na-H exchange that actively extruded acid in the steady state (see Fig. 3 B, inset). In contrast, adding 10 µM EIPA had no effect on the steady state pH_i (n = 7, data not shown). Introducing CO₂/HCO₃^– in the continued presence of amiloride elicited a rapid, initial decrease in pH_i, followed by a sustained increase (data not shown). In five experiments, the maximum acid extrusion rate was 89.3 ± 11.4 µM s^–1 at a pH_i of 6.60 ± 0.03 during the CO₂/HCO₃^–-induced alkalinization in the presence of amiloride. This average _E in the presence of amiloride is similar to _E in three day-matched control cells in the absence of amiloride (73.2 ± 4.8 µM s^–1; P = 0.13) at a similar pH_i of 6.67 ± 0.02. Therefore, even when Na-H exchange is substantially inhibited with amiloride, CO₂/HCO₃^– elicits an alkalinization in hippocampal astrocytes that have relatively low initial pH_i.

Stilbene derivatives inhibit the CO₂/HCO₃^–-induced alkalinization.
Because the most likely explanation for the CO₂/HCO₃^–-induced alkalinization is stimulation of a HCO₃^–-dependent acid extrusion mechanism, we tried inhibiting the alkalinization with stilbene derivatives that block HCO₃^– transporters in other preparations. Fig. 8 A shows an experiment on a single hippocampal astrocyte exposed to CO₂/HCO₃^– in the presence of the stilbene derivative DIDS. Applying 400 µM DIDS in the nominal absence of CO₂/HCO₃^– elicited a sustained decrease in pH_i (Fig. 8 A, ab). The mechanism of this DIDS-stimulated acidification is unknown. In principle, some of the pH_i decrease could have been caused by inhibition of a HCO₃^–-dependent acid extruder capable of using the small amount of HCO₃^– (100 µM) present in the nominally CO₂/HCO₃^–-free solution. However, it seems unlikely that such a mechanism, when inhibited, could produce the rapid segment-ab decrease in pH_i (Fig. 8 A). In the presence of DIDS, exposing the cell to CO₂/HCO₃^– caused a further acidification (Fig. 8 A, bc), followed by a slow recovery of pH_i (Fig. 8 A, cd) that is probably mediated by the Na-H exchanger. In the continued presence of CO₂/HCO₃^–, removing DIDS relieved most of the inhibition, causing pH_i to increase (Fig. 8 A, de). Removing the CO₂/HCO₃^– caused the usual series of pH_i changes (Fig. 8 A, e–g). Summarizing mean data, switching astrocytes from HEPES to CO₂/HCO₃^– in the presence of 400 µM DIDS resulted in a mean _E of 12.8 ± 3.2 µM s^–1 at a pH_i of 6.79 ± 0.06 (n = 7). In day-matched control experiments in the absence of DIDS, _E was 51.0 ± 6.5 µM s^–1 at a similar pH_i of 6.85 ± 0.06 (n = 7). Therefore, 400 µM DIDS inhibited the CO₂/ HCO₃^–-induced acid extrusion by 75% (P < 0.0004).

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Figure 8 Stilbene derivatives partially inhibit the alkalinization when astrocytes are exposed to CO2/HCO3–. (A) Inhibition by DIDS. At the indicated time, 400 µM DIDS was present in the extracellular solution (a–d). Between b and e, we switched the extracellular buffer from HEPES to 5% CO2/17 mM HCO3–. (B) Inhibition by SITS. One of the two astrocytes was preincubated in 400 µM SITS for 4.8 h before the start of the experiment. Both cells were then exposed to CO2/HCO3– (a–c and a–c'). (C) Maximum acid extrusion (i.e., at point b in B) as a function of the length of time astrocytes were preincubated in 400 µM SITS. All the experiments for this protocol were performed over a 2-d period. The best-fit line to the data has a slope of –5.5 µM s–1 h–1 and a y-intercept of 53.1 µM s–1.

CO₂/HCO₃^–-induced alkalinization requires external Na⁺.
Because several HCO₃^–-dependent acid extruders also require Na⁺, we performed experiments similar to those shown in Fig. 9 A to determine if Na⁺ is required for the CO₂/HCO₃^–-induced alkalinization in single hippocampal astrocytes. When we replaced external Na⁺ with NMDG⁺ in a HEPES-buffered solution, pH_i rapidly decreased (Fig. 9 A, ab), presumably due at least in part to inhibition and/or reversal of Na-H exchange. Reversal of Na-Ca exchange, by elevating intracellular Ca²⁺ and displacing H⁺ from Ca²⁺ buffers, might also cause pH_i to decrease (Meech and Thomas, 1977). Switching to a CO₂/HCO₃^– buffer in the continued absence of external Na⁺ caused pH_i to decrease further by 0.06 (Fig. 9 A, bc). This pH_i decrease is small compared with Fig. 7, ab because the low pH_i in 0 Na⁺ is closer to the pK of carbonic acid. However, there was no CO₂/HCO₃^–-induced alkalinization (Fig. 9 A, cd). Switching the cell to a solution containing both Na⁺ and CO₂/HCO₃^– elicited a rapid increase in pH_i to a value above that in the HEPES-buffered solution (compare Fig. 9 A, e and a). In five of six such experiments, removing extracellular Na⁺ (Fig. 9 A, ab) caused pH_i to decrease from an average of 6.84 ± 0.08 to 6.50 ± 0.14. In one of the six experiments, the pH_i did not change appreciably when the cell was exposed to the Na⁺-free solution. Exposing the six cells to CO₂/ HCO₃^– in the continued absence of external Na⁺ (Fig. 9 A, bc) caused a further decrease in the mean pH_i to 6.45 ± 0.11. The mean _E computed for Fig. 9 A, cd was only 4.0 ± 4.4 µM s^–1, compared with the maximum _E of 47.7 ± 2.4 µM s^–1 at a pH_i of 6.74 ± 0.02 (n = 63) in experiments in which we added the CO₂/HCO₃^– in the presence of Na⁺ (e.g., Fig. 7 A, bc). Because removing external Na⁺ elicited a large decrease in pH_i, it was not possible to compare _E values in the presence and absence of Na⁺ at the same pH_i. However, because other HCO₃^–-dependent acid extruders are stimulated by low pH_i, we would have expected _E to be even larger at lower pH_i values prevailing in the presence of external Na⁺. Therefore, the CO₂/HCO₃^–-induced alkalinization requires external Na⁺.

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Figure 9 The HCO3– transporter is blocked when Na+ is replaced with NMDG+ or Li+. (A) The effect of replacing Na+ with NMDG+ on the CO2/HCO3–-induced alkalinization. Between a and d, we replaced the extracellular Na+ with NMDG+. During b–e, we switched the extracellular solution to one buffered with 5% CO2/17 mM HCO3–. (B) The effect of replacing Na+ with Li+ on the CO2/HCO3–-induced pHi recovery from an acid load in the presence of amiloride. We exposed the astrocyte to 20 mM NH3/ NH4+ during a–c. Amiloride (0.9 mM) was present between c and g. Between c and f, Li+ replaced extracellular Na+. Finally, between e and g, the extracellular buffer was 5% CO2/17 mM HCO3–.

	DISCUSSION

Top ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Properties of the Na-H Exchanger in Hippocampal Astrocytes
Na-H exchanger mediates the pH_i recovery from an acid load and contributes to the steady state pH_i.
In this manuscript, we have investigated the major acid extruders responsible for regulating pH_i in astrocytes cultured from the hippocampus of the rat. Although we have focused predominantly on a HCO₃^–-dependent acid extruder, we have also further characterized the Na-H exchanger that mediates almost the entire pH_i recovery from an acid load in the nominal absence of CO₂/HCO₃^– (Pappas and Ransom, 1993). We found that, at pH_i 6.05, amiloride inhibits 88% of acid extrusion during the pH_i recovery from an acid load. Moreover, our observation that applying amiloride leads to a decrease in steady state pH_i implies that the Na-H exchanger contributes to maintaining the steady state pH_i of the astrocytes, at least in the nominal absence of CO₂/HCO₃^–. In three experiments (not shown) on cells exposed to CO₂/ HCO₃^–, we found that removing amiloride caused pH_i to increase by 0.03 ± 0.01, consistent with a modest role for the Na-H exchanger in maintaining steady state pH_i even in the presence of CO₂/HCO₃^–. Recently, Pizzonia et al. (1996) demonstrated that rat hippocampal astrocytes express the Na-H exchanger, NHE-1.

Although 10 µM EIPA inhibits the pH_i recovery from an acid load, it does not lower steady state pH_i.
Because Na-H exchangers typically are sensitive to amiloride analogues such as EIPA, we studied the effect of EIPA both on the pH_i recovery from an acid load and on the steady state pH_i in hippocampal astrocytes. At very low pH_i values (i.e., 6.05), 10 µM EIPA inhibited the pH_i recovery from an acid load about as well as amiloride (79 vs. 88%). On the other hand, at progressively higher pH_i values, the amiloride-sensitive flux (Fig. 3 B, inset) fell towards zero more gradually than the EIPA-sensitive flux (Fig. 5 B, inset). Indeed, applying amiloride to a naive cell consistently elicited a pH_i decrease, presumably because it inhibited Na-H exchange and unmasked background acid loading, whereas applying EIPA did not. One explanation for these findings is that the astrocytes have two Na-H exchangers, one of which is less EIPA sensitive, particularly at high pH_i. Another explanation is that a single Na-H exchanger has a differential sensitivity to amiloride and EIPA that becomes especially apparent at high pH_i. A third possibility is that, at high pH_i, 10 µM EIPA simultaneously blocks Na-H exchange and alkalinizes the cell by an independent mechanism. Indeed, high levels of EIPA (i.e., 50 µM) elicit paradoxical alkalinizations in NIH-3T3 fibroblasts (Kaplan and Boron, 1994), rat osteoclasts (Ravesloot et al., 1995), and rat hippocampal CA1 neurons (Bevensee et al., 1996), as well as cultured astrocytes from the forebrain (Boyarsky et al., 1993; Bevensee, M.O., G. Frey, and W.F. Boron, unpublished data) and hippocampus (Pizzonia et al., 1996).

Li⁺ can substitute for Na⁺ and exchange with H⁺ on the Na-H exchanger.
The astrocyte Na-H exchanger appears to exchange Na⁺ for H⁺ approximately fourfold faster than it exchanges Li⁺ for H⁺. In apical membranes of renal proximal tubules, the V_max for Li-H exchange is less than for Na-H exchange, although the affinity for Li-H exchange is higher (see review by Aronson, 1985). In NN and C6 glioma cells, the K_1/2 for Na⁺ activation of the EIPA-sensitive ²²Na⁺ uptake was 17 and 50 mM, respectively. However, the K_1/2 for Li⁺ inhibition of the EIPA-sensitive ²²Na⁺ uptake was only 5 and 9 mM, respectively (Jean et al., 1986).

Evidence for a Na⁺-driven, HCO₃^–-dependent Acid Extruder in Hippocampal Astrocytes
Five observations suggest that hippocampal astrocytes possess a Na⁺-driven, HCO₃^–-dependent acid extruder. (a) Acid extrusion during the recovery from an acid load is greater in the presence than in the absence of a CO₂/HCO₃^–-buffered solution (Fig. 4). (b) When the pH_i recovery from an acid load is inhibited by amiloride in the nominal absence of CO₂/HCO₃^–, adding CO₂/HCO₃^– stimulates pH_i recovery (Fig. 3 A). (c) When astrocytes are switched from a solution buffered with HEPES to one buffered with CO₂/HCO₃^–, the average steady state pH_i increases (Fig. 7). (d) The CO₂/ HCO₃^–-induced alkalinization is inhibited by the HCO₃^–-transport inhibitors DIDS (Fig. 8 A) and SITS (Fig. 8, B and C), but not by the Na-H exchange inhibitor amiloride. (e) The CO₂/HCO₃^–-induced alkalinization requires external Na⁺ (Fig. 9). These data are consistent with the Na⁺-driven HCO₃^– transporter being either a Na⁺-driven Cl-HCO₃ exchanger, or a Na/ HCO₃ cotransporter. We address these possibilities in the subsequent manuscript (Bevensee et al., 1997).

¹ Abbreviations used in this paper: BCECF-AM, acetoxymethyl ester of the pH-sensitive dye 2',7'-biscarboxyethyl-5,6-carboxyfluorescein; DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; EIPA, ethylisopropylamiloride; NMDG⁺, N-methyl-D-glucammonium; pH_ECF, pH of the brain extracellular fluid; SITS, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid.

² We noted that SITS-treated astrocytes assumed a more spherical shape, rather than the flat polygonal shape that characterized the other cells in this study.

	ACKNOWLEDGMENTS

 
We thank Dr. W. Knox Chandler for evaluating the manuscript and providing useful suggestions.

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).

Submitted: 24 January 1997
Accepted: 30 June 1997

	REFERENCES

Top ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 

Aickin CC & Thomas RC. Microelectrode measurement of the intracellular pH and buffering power of mouse soleus muscle fibres, J Physiol, 1977, 267, 791–810.[Abstract/Free Full Text]

Aronson PS. Kinetic properties of the plasma membrane Na⁺-H⁺exchanger, Annu Rev Physiol, 1985, 47, 545–560.[Medline]

Astion ML & Orkand RK. Electrogenic Na⁺/HCO₃^–cotransport in neuroglia, Glia, 1988, 1, 355–357.[Medline]

Bevensee MO, Apkon M & Boron WF. Intracellular pH regulation in cultured astrocytes from rat hippocampus. II. Electrogenic Na/HCO₃cotransport, J Gen Physiol, 1997, 110, 000–000.

Bevensee MO, Cummins TR, Haddad GG, Boron WF & Boyarsky G. pH regulation in single CA1 neurons acutely isolated from the hippocampi of immature and mature rats, J Physiol, 1996, 494, 315–328.[Abstract/Free Full Text]

Boron WF & Boulpaep EL. Intracellular pH regulation in the renal proximal tubule of the salamander: basolateral HCO₃^–transport, J Gen Physiol, 1983, 81, 53–94.[Abstract/Free Full Text]

Boron WF & De Weer P. Intracellular pH transients in squid giant axons caused by CO₂, NH₃, and metabolic inhibitors, J Gen Physiol, 1976, 67, 91–112.[Abstract/Free Full Text]

Boyarsky G, Ganz MB, Sterzel B & Boron WF. pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of HCO₃^–, Am J Physiol, 1988, 255, C844–C856.[Medline]

Boyarsky G, Ransom B, Schlue W-R, Davis MBE & Boron WF. Intracellular pH regulation in single cultured astrocytes from rat forebrain, Glia, 1993, 8, 241–248.[Medline]

Brune T, Fetzer S, Backus KH & Deitmer JW. Evidence for electrogenic Na/HCO₃cotransport in cultured rat cerebellar astrocytes, Pflügers Archiv, 1994, 429, 64–71.[Medline]

Chen LK & Boron WF. Acid extrusion in S3 segment of rabbit proximal tubule I. Effect of bilateral CO₂/HCO₃^–, Am J Physiol, 1995a, 268, F179–F192.[Medline]

Chen LK & Boron WF. Acid extrusion in S3 segment of rabbit proximal tubule. II. Effect of basolateral CO₂/HCO₃^–, Am J Physiol, 1995b, 268, F193–F203.[Medline]

Chesler M. The regulation and modulation of pH in the nervous system, Prog Neurobiol (Oxf), 1990, 34, 401–427.[Medline]

Chesler M & Kaila K. Modulation of pH by neuronal activity, TINS (Trends Neurosci), 1992, 15, 396–402.[Medline]

Chow SY, Yen-Chow YC, White HS & Woodbury DM. pH regulation after acid load in primary cultures of mouse astrocytes, Dev Brain Res, 1991, 60, 69–78.[Medline]

Deitmer JW. Bicarbonate-dependent changes of intracellular sodium and pH in identified leech glial cells, Pflügers Arch, 1992, 420, 584–589.[Medline]

Deitmer JW & Rose CR. pH regulation and proton signalling by glial cells, Prog Neurobiol (Oxf), 1996, 48, 73–103.[Medline]

Deitmer JW & Schlue W-R. The regulation of intracellular pH by identified glial cells and neurones in the central nervous system of the leech, J Physiol (Camb), 1987, 388, 261–283.[Abstract/Free Full Text]

Deitmer JW & Schlue W-R. An inwardly directed electrogenic sodium-bicarbonate cotransport in leech glial cells, J Physiol (Camb), 1989, 411, 179–194.[Abstract/Free Full Text]

Dixon, S.J., and J.X. Wilson. 1995. Fluorescence measurement of cytosolic pH in cultured rodent astrocytes. In Methods in Neurosciences. Vol. 27. J. Kraicer and S.J. Dixon, editors. Academic Press, Inc., San Diego, CA. 196–213.

Hogan EM, Cohen MA & Boron WF. K⁺ and HCO₃^–-dependent acid–base transport in squid giant axons: base efflux, J Gen Physiol, 1995, 106, 821–844.[Abstract/Free Full Text]

Jean T, Frelin C, Vigne P & Lazdunski M. The Na/H exchange system in glial cell lines, Eur J Biochem, 1986, 160, 211–219.[Medline]

Kaila K, Panula P, Karhunen T & Heinonen E. Fall in intracellular pH mediated by GABA_Areceptors in cultured rat astrocytes, Neurosci Lett, 1991, 126, 9–12.[Medline]

Kaplan D & Boron WF. Long-term expression of c-ras stimulates Na-H and Na⁺-dependent Cl^–-HCO₃^–exchange in NIH-3T3 fibroblasts, J Biol Chem, 1994, 269, 4116–4124.[Abstract/Free Full Text]

Kraig RP & Chesler M. Astrocytic acidosis in hyperglycemic and complete ischemia, J Cereb Blood Flow Metab, 1990, 10, 104–114.[Medline]

Lin A, Krockmalnic G & Penman S. Imaging cytoskeleton-mitochondrial membrane attachments by embedment-free electron microscopy of saponin-extracted cells, Proc Natl Acad Sci USA, 1990, 87, 8565–8569.[Abstract/Free Full Text]

McCarthy KD & de Vellis J. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissues, J Cell Biol, 1980, 85, 890–902.[Abstract/Free Full Text]

Meech RW & Thomas RC. The effect of calcium injection on the intracellular sodium and pH of snail neurones, J Physiol (Oxf), 1977, 265, 867–879.[Abstract/Free Full Text]

Moody W Jr. Effects of intracellular H⁺on the electrical properties of excitable cells, Annu Rev Neurosci, 1984, 7, 257–278.[Medline]

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 & Sons Inc., New York. 594–604.

Munsch T & Deitmer JW. Sodium-bicarbonate cotransport current in identified leech glial cells, J Physiol (Oxf), 1994, 474, 43–53.[Abstract/Free Full Text]

Newman EA. Sodium-bicarbonate cotransport in retinal Müller (glial) cells of the salamander, J Neurosci, 1991, 11, 3972–3983.[Abstract]

Newman EA & Astion ML. Localization and stoichiometry of electrogenic sodium bicarbonate cotransport in retinal glial cells, Glia, 1991, 4, 424–428.[Medline]

O'Connor ER, Sontheimer H & Ransom BR. Rat hippocampal astrocytes exhibit electrogenic sodium-bicarbonate co-transport, J Neurophysiol, 1994, 72, 2580–2589.[Abstract/Free Full Text]

Pappas CA & Ransom BR. A depolarization-stimulated, bafilomycin-inhibitable H⁺pump in hippocampal astrocytes, Glia, 1993, 9, 280–291.[Medline]

Pappas CA & Ransom BR. Depolarization-induced alkalinization (DIA) in rat hippocampal astrocytes, J Neurophysiol, 1994, 72, 2816–2826.[Abstract/Free Full Text]

Pizzonia JH, Ransom BR & Pappas CR. Characterization of Na⁺/H⁺exchange activity in cultured rat hippocampal astrocytes, J Neurosci Res, 1996, 44, 191–198.[Medline]

Ransom, B.R. 1992. Glial modulation of neural excitability mediated by extracellular pH: a hypothesis. In Progress Brain Research. Vol. 94. A. Yu, L. Hertz, M. Norenberg, E. Sykova, and S. Waxman, editors. Elsevier Science, Amsterdam, Netherlands. 37– 46.

Ravesloot JH, Eisen T, Baron R & Boron WF. Role of Na-H exchangers and vacuolar H⁺pumps in intracellular pH regulation in neonatal rat osteoclasts, J Gen Physiol, 1995, 105, 177–207.[Abstract/Free Full Text]

Roos A & Boron WF. Intracellular pH, Physiol Rev, 1981, 61, 296–434.[Free Full Text]

Soleimani M, Grassl SM & Aronson PS. Stoichiometry of Na⁺-HCO₃^–cotransport in basolateral membrane vesicles isolated from rabbit renal cortex, J Clin Invest, 1987, 79, 1276–1280.[Medline]

Szatkowski M & Schlue W-R. Mechanisms of pH recovery from intracellular acid loads in the leech connective glial cell, Glia, 1992, 5, 193–200.[Medline]

Tang C-M, Dichter M & Morad M. Modulation of the N-methyl-D-aspartate channel by extracellular H⁺, Proc Natl Acad Sci USA, 1990, 87, 6445–6449.[Abstract/Free Full Text]

Thomas JA, Buchsbaum RN, Zimniak A & Racker E. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ, Biochemistry, 1979, 81, 2210–2218.

Traynelis SF & Cull-Candy SG. Proton inhibition of N-methyl-D-aspartate receptors in cerebellar neurons, Nature (Lond), 1990, 345, 347–350.[Medline]

Vaughan-Jones, R.D. 1982. Chloride-bicarbonate exchange in the sheep cardiac Purkinje fibre. In Intracellular pH: Its Measurement, Regulation and Utilization in Cellular Function. Vol. 15. R. Nuccitelli and D.W. Deamer, editors. Alan R. Liss, Inc., New York. 239–252.

Wuttke WA & Walz W. Sodium- and bicarbonate-independent regulation of intracellular pH in cultured mouse astrocytes, Neurosci Lett, 1990, 117, 105–110.[Medline]

Zhao J, Hogan EM, Bevensee MO & Boron WF. Out-of-equilibrium CO₂/HCO₃^–- solutions and their use in characterizing a new K/HCO₃cotransporter, Nature (Lond), 1995, 374, 636–639.[Medline]

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				A Schwab, H Rossmann, M Klein, P Dieterich, B Gassner, C Neff, C Stock, and U Seidler Functional role of Na+-HCO3- cotransport in migration of transformed renal epithelial cells J. Physiol., October 15, 2005; 568(2): 445 - 458. [Abstract] [Full Text] [PDF]

				D. B. Kintner, A. Look, G. E. Shull, and D. Sun Stimulation of astrocyte Na+/H+ exchange activity in response to in vitro ischemia depends in part on activation of ERK1/2 Am J Physiol Cell Physiol, October 1, 2005; 289(4): C934 - C945. [Abstract] [Full Text] [PDF]

				S. Horita, H. Yamada, J. Inatomi, N. Moriyama, T. Sekine, T. Igarashi, Y. Endo, M. Dasouki, M. Ekim, L. Al-Gazali, et al. Functional Analysis of NBC1 Mutants Associated with Proximal Renal Tubular Acidosis and Ocular Abnormalities J. Am. Soc. Nephrol., August 1, 2005; 16(8): 2270 - 2278. [Abstract] [Full Text] [PDF]

				M. Hegde, J. Roscoe, P. Cala, and F. Gorin Amiloride Kills Malignant Glioma Cells Independent of Its Inhibition of the Sodium-Hydrogen Exchanger J. Pharmacol. Exp. Ther., July 1, 2004; 310(1): 67 - 74. [Abstract] [Full Text] [PDF]

				D. B. Kintner, G. Su, B. Lenart, A. J. Ballard, J. W. Meyer, L. L. Ng, G. E. Shull, and D. Sun Increased tolerance to oxygen and glucose deprivation in astrocytes from Na+/H+ exchanger isoform 1 null mice Am J Physiol Cell Physiol, July 1, 2004; 287(1): C12 - C21. [Abstract] [Full Text] [PDF]

				J. S. Erlichman, A. Cook, M. C. Schwab, T. W. Budd, and J. C. Leiter Heterogeneous patterns of pH regulation in glial cells in the dorsal and ventral medulla Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2004; 286(2): R289 - R302. [Abstract] [Full Text] [PDF]

				M. CHESLER Regulation and Modulation of pH in the Brain Physiol Rev, October 1, 2003; 83(4): 1183 - 1221. [Abstract] [Full Text] [PDF]

				P. Bouyer, Y. Zhou, and W. F. Boron An increase in intracellular calcium concentration that is induced by basolateral CO2 in rabbit renal proximal tubule Am J Physiol Renal Physiol, October 1, 2003; 285(4): F674 - F687. [Abstract] [Full Text] [PDF]

				R. Daskalopoulos, J. Korcok, P. Farhangkhgoee, M. Karmazyn, A. W. Gelb, and J. X. Wilson Propofol Protection of Sodium-Hydrogen Exchange Activity Sustains Glutamate Uptake During Oxidative Stress Anesth. Analg., November 1, 2001; 93(5): 1199 - 1204. [Abstract] [Full Text] [PDF]

				D. Stakisaitis, M. S. Lapointe, and D. Batlle Mechanisms of chloride transport in thymic lymphocytes Am J Physiol Renal Physiol, February 1, 2001; 280(2): F314 - F324. [Abstract] [Full Text] [PDF]

				G. Su, R. A. Haworth, R. J. Dempsey, and D. Sun Regulation of Na+-K+-Cl- cotransporter in primary astrocytes by dibutyryl cAMP and high [K+]o Am J Physiol Cell Physiol, December 1, 2000; 279(6): C1710 - C1721. [Abstract] [Full Text] [PDF]

				X. Q. Gu, H. Yao, and G. G. Haddad Effect of Extracellular HCO3- on Na+ Channel Characteristics in Hippocampal CA1 Neurons J Neurophysiol, November 1, 2000; 84(5): 2477 - 2483. [Abstract] [Full Text] [PDF]

				B. M. Schmitt, U. V. Berger, R. M. Douglas, M. O. Bevensee, M. A. Hediger, G. G. Haddad, and W. F. Boron Na/HCO3 Cotransporters in Rat Brain: Expression in Glia, Neurons, and Choroid Plexus J. Neurosci., September 15, 2000; 20(18): 6839 - 6848. [Abstract] [Full Text] [PDF]

				H. K. Patton, Z.-H. Zhou, J. K. Bubien, E. N. Benveniste, and D. J. Benos gp120-induced alterations of human astrocyte function: Na+/H+ exchange, K+ conductance, and glutamate flux Am J Physiol Cell Physiol, September 1, 2000; 279(3): C700 - C708. [Abstract] [Full Text] [PDF]

				M. O. Bevensee, B. M. Schmitt, I. Choi, M. F. Romero, and W. F. Boron An electrogenic Na+-HCO-3 cotransporter (NBC) with a novel COOH-terminus, cloned from rat brain Am J Physiol Cell Physiol, June 1, 2000; 278(6): C1200 - C1211. [Abstract] [Full Text] [PDF]

				L. A. McLean, J. Roscoe, N. K. Jorgensen, F. A. Gorin, and P. M. Cala Malignant gliomas display altered pH regulation by NHE1 compared with nontransformed astrocytes Am J Physiol Cell Physiol, April 1, 2000; 278(4): C676 - C688. [Abstract] [Full Text] [PDF]

				M. O. Bevensee, E. Bashi, W.-R. Schlue, G. Boyarsky, and W. F. Boron Shrinkage-induced activation of Na+/H+ exchange in rat renal mesangial cells Am J Physiol Cell Physiol, March 1, 1999; 276(3): C674 - C683. [Abstract] [Full Text] [PDF]

				M. O. Bevensee, M. Apkon, and W. F. Boron Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus: II. Electrogenic Na/HCO3 Cotransport J. Gen. Physiol., October 1, 1997; 110(4): 467 - 483. [Abstract] [Full Text] [PDF]

				R. A. Colvin pH dependence and compartmentalization of zinc transported across plasma membrane of rat cortical neurons Am J Physiol Cell Physiol, February 1, 2002; 282(2): C317 - C329. [Abstract] [Full Text] [PDF]

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