Mechanisms of H⁺ and Na⁺ Changes Induced by Glutamate, Kainate, and d-Aspartate in Rat Hippocampal Astrocytes

[H⁺]_i changes induced by Glu, KA, and d-Asp

Baseline intracellular H⁺ concentration ([H⁺]_i) in hippocampal astrocytes was 54.6 nm in standard CO₂/HCO⁻₃-buffered saline (Table1) corresponding to a pH_i of 7.26. 

Bath application of 1 mm Glu for 1 min elicited [H⁺]_i changes in every cell investigated (n = 94). The majority of cells (80%) responded to Glu with a pure acidification that started either directly at the onset of the Glu perfusion or up to 30 sec later. The acid shift developed with a maximal slope of 4.1 nm/min and reached its peak of 15.6 nm ∼5–10 min after the Glu was removed from the bath (Table 1, Fig. 3A). In 20% of the cells, this acidification was preceded by a small alkaline shift (≤ 4 nm) (Fig. 3B).

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Fig. 3.

[H⁺]_i changes evoked by Glu, KA, or d-Asp in CO₂/HCO⁻₃-containing or CO₂/HCO⁻₃-free solution.A illustrates the typical changes in intracellular H⁺ concentration (Δ[H⁺]_i) caused by bath application of Glu, KA, or d-Asp in standard, CO₂/HCO⁻₃-buffered saline. Substances were applied for 1 min (indicated bybars) at a concentration of 1 mm. These recordings were from three different cells. To facilitate comparison, baseline [H⁺]_i was set to 0 in all cells.B, Record showing changes [H⁺]_i caused by 1 min bath application (indicated by bars) of KA or Glu (1 mm) in HEPES-buffered, CO₂/HCO⁻₃-free saline, and CO₂/HCO⁻₃-buffered saline.

KA application changed [H⁺]_iin 82% of the cells. The KA-induced [H⁺]_i changes were biphasic; a decrease in [H⁺]_i by 9.0 nm was followed by an increase of 10.3 nm (Table 1). The alkaline shift reached its peak ∼40–70 sec after the onset of the KA-perfusion, whereas the acid shift developed slowly and peaked after 5–10 min (Fig. 3). d-Asp application elicited a slow increase in [H⁺]_i in all astrocytes investigated. The d-Asp-induced increase in [H⁺]_i averaged 10.4 nm and had a similar time course and maximal slope (5.3 nm/min) as the Glu-induced acid shift (Table 1, Fig. 3A).

The Glu-induced membrane depolarization of cultured astrocytes decreased with repetitive applications (Kettenmann and Schachner, 1985). Similar decreases in evoked changes in [H⁺]_i were observed in the present study. Amplitudes and slopes of the evoked transients during second and third applications (intervals between pulses, 15–20 min) were reduced in comparison to the first pulse (p ≤ 0.025) (data not shown). The amplitudes of the second and third Glu-evoked changes, compared with the first, were 87 ± 34% (mean ± SD) and 83 ± 44%, respectively (n = 44). Likewise, KA-induced [H⁺]_i changes also decremented with repetition; the second and third responses were 84 ± 10% and 82 ± 12% of the original response (n = 47). [H⁺]_i changes elicited by d-Asp, however, showed virtually no attenuation with repetitive application (97 ± 27% and 95 ± 29%, respectively; n = 29). To facilitate comparison of evoked changes in [H⁺]_i before and after various manipulations, we corrected the second and third responses for these expected changes (i.e., in data expressed as percentage change).

CO₂/HCO⁻₃ dependence of Glu- and Ka-induced [H⁺]_i changes

Regulation of baseline [H⁺]_i in glial cells is dependent on the presence of CO₂/HCO⁻₃, partly because of its role as a buffer system and partly because of HCO⁻₃-carrying transporters (see Fig. 1) (Boyarski et al., 1993; Deitmer and Rose, 1996). To investigate the influence of CO₂/HCO⁻₃ on the Glu- and KA-induced [H⁺]_i changes, we performed experiments in HEPES-buffered, CO₂/HCO⁻₃-free saline. As expected (Pappas and Ransom, 1993), baseline [H⁺]_iincreased significantly to 81.7 nm in this saline (Table1), corresponding to a pH_i of 7.09.

In CO₂/HCO⁻₃-free saline the amplitude of the Glu-induced acid shift increased about threefold to 43.7 nm (Table 1), and its maximal slope increased to 45.0 nm/min (Fig. 3B). These effects can most likely be attributed to the two- to threefold reduction in intracellular buffering capacity in the absence of CO₂/HCO⁻₃ (Thomas, 1976). Cells that showed an alkaline response in CO₂/HCO⁻₃, did not show this alkaline shift in CO₂/HCO⁻₃-free saline (n = 13) (Fig. 3B).

In contrast to their uniform reaction in standard, CO₂/HCO⁻₃-buffered saline, the astrocytes’ response to KA was more variable in CO₂/HCO⁻₃-free saline. Seventy-eight percent of cells responded to KA in the absence of CO₂/HCO⁻₃. Of the responders, most (67%) acidified at KA application (Fig. 3B), whereas 33% showed a very small alkaline shift (<2 nm) (data not shown). The average KA-induced increase in [H⁺]_i was 4.9 nm (Table 1). Surprisingly, some cells (n = 19) that did not react to KA in HEPES-buffered saline responded with robust biphasic [H⁺]_i changes in standard, CO₂/HCO⁻₃-buffered saline. These results strongly suggested that the KA-induced alkaline-acid shifts in standard saline were attributable to HCO⁻₃movement across the plasma membrane. Otherwise the [H⁺]_i changes would have increased in CO₂/HCO⁻₃-free saline because of the amplifying effect of reduction in intracellular buffering power (see above) (Thomas, 1976).

Origin of KA-induced [H⁺]_ichanges in CO₂-buffered saline

The amplitude of the biphasic alkaline-acid transient elicited by KA application in CO₂/HCO⁻₃-buffered saline increased with increasing KA concentrations from 0.1 to 2.0 mm (n = 49; application for 2 min) (Fig.4). The KA-induced [H⁺]_i shifts were unaltered during Ca²⁺ removal (Table 2, Fig.5A). Replacement of extracellular Cl⁻ by gluconic acid led to a significant decrease in baseline [H⁺]_i (Table 2, Fig. 5B). This effect was probably attributable to removal of acidifying Cl⁻/HCO⁻₃ exchange in these astrocytes, as reported earlier (Mellergard et al., 1993; Shrode and Putnam, 1994). Removal of Cl⁻ also could alkalinize astrocytes by depolarizing them and activating inward Na⁺/HCO⁻₃ cotransport (Pappas and Ransom, 1994), but astrocyte membrane potential appears to be insensitive to the transmembrane gradient of Cl⁻ (Ransom and Goldring, 1973). In the absence of Cl⁻, the KA-induced alkalinization was reversibly reduced to 52% (p ≤ 0.0001) of the control response, and the KA-induced acidification increased to 129% (p ≤ 0.01) (Table 2, Fig. 5B).

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Fig. 4.

Concentration dependence of kainate-induced [H⁺]_i changes. A, The biphasic KA-induced responses in [H⁺]_iincreased with KA concentration. All applications were for 2 min periods; KA was applied at 0.1, 0.5, and 1 mm(indicated by bars). B, Graphic summary of the amplitudes of biphasic alkaline-acid transients elicited by 2 min bath applications of KA at 0.1, 0.5, 1, and 2 mm in CO₂/HCO⁻₃-buffered saline. Shown are the mean values of 49 cells; bars indicate SD.

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Fig. 5.

Ca²⁺- and Cl⁻-dependence of kainate-induced [H⁺]_i changes. A, Alkaline-acid transients elicited by 1 mm KA applications for 1 min (indicated by bars) in standard CO₂/HCO⁻₃-buffered saline (2 mm Ca²⁺) and after removal of extracellular Ca²⁺ (0 [Ca²⁺]_e, solution contained 0.5 mm EGTA). The KA response was unchanged in the absence of [Ca²⁺]_e.B, Alkaline-acid transients elicited by 1 mmKA application for 1 min (indicated by bars) in standard CO₂/HCO⁻₃-buffered saline (containing 122.75 mm Cl⁻) and after replacement of extracellular Cl⁻ by gluconic acid (0 [Cl⁻]_e). Removing Cl⁻ caused an alkaline shift probably because of reverse Cl⁻/HCO⁻₃ exchange. Note that the KA response was preserved qualitatively in the absence of [Cl⁻]_e.

Replacement of extracellular Na⁺ byN-methyl-d-glucamine (NMDG), (115.75 mm) and choline (23 mm) strongly acidified the cells (Table 2, Fig. 6A). The KA-induced alkaline transient was abolished in some cells and, on average, was reversibly diminished to 18% of the control value. The acid transient was abolished completely (Fig. 6A). Similar effects were observed after application of the anion transport blocker (4,4′-diisothiocyanato-stilbene-2,2′-disulphonic acid (DIDS) (0.5 mm). After an initial alkaline transient, baseline [H⁺]_i increased strongly (Table 2, Fig.6B). The KA-evoked alkaline shift was reduced to 25% of the control value, and the acid shift was abolished (Fig.6B). The effects of DIDS, as expected (Brune et al., 1994;O’Connor et al., 1994), were not fully reversible.

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Fig. 6.

Na⁺ dependence and influence of DIDS on kainate-induced [H⁺]_i changes.A, Alkaline-acid transients elicited by 1 mmKA application for 2 min (indicated by bars) in standard CO₂/HCO⁻₃-buffered saline and after replacement of extracellular Na⁺ by NMDG and choline (0 [Na⁺]_e). Removal of Na⁺ caused a marked acid shift, because it blocks or reverses acid-exporting mechanisms. The KA-induced changes in [H⁺]_i were reversibly blocked in the absence of Na⁺. B, Alkaline-acid transients elicited by 1 mm KA application for 1 min (indicated bybars) are shown in standard CO₂/HCO⁻₃-buffered saline and during application of the anion transport blocker DIDS (0.5 mm). DIDS caused a partially reversible acid shift, because it blocks acid- exporting mechanisms. The KA response was largely blocked by DIDS.

In contrast to hippocampal astrocytes, we observed only a rapid acidification, but no alkaline shift, in cultured hippocampal neurons after KA application. This was true in both CO₂/HCO⁻₃-buffered (Fig.7) and CO₂/HCO⁻₃-free saline (data not shown). In CO₂/HCO⁻₃-buffered saline, KA application rapidly increased neuronal [H⁺]_i by 22.8 ± 10.3 nmfrom a baseline of 75.4 ± 18.6 nm (n = 4).

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Fig. 7.

Neuronal [H⁺]_i changes induced by KA. KA application (1 mm for 1 min) (indicated by bar) rapidly increased [H⁺]_i in a cultured hippocampal neuron in CO₂/HCO⁻₃-buffered saline. Unlike the situation in astrocytes, KA never evoked alkaline shifts in neurons.

Taken together, these results suggested that the glial-specific, biphasic [H⁺]_i changes after application of KA were attributable to a CO₂/HCO⁻₃- and Na⁺-dependent transport mechanism sensitive to DIDS that can operate (although with reduced amplitude) in the absence of external Cl⁻.

[Na⁺]_i changes induced by Glu, KA, and d-Asp

In agreement with an earlier study (Rose and Ransom, 1996), baseline [Na⁺]_i in hippocampal astrocytes was about 15 mm in standard saline (Table 1). Application of either Glu, KA, or d-Asp rapidly increased [Na⁺]_i within 30–60 sec, followed by a recovery to baseline within 10 min (shown for Glu in Figs.8, 10; KA in Figs. 8, 9; d-Asp in Fig. 11). This was true for every cell investigated, although the absolute amplitudes of the [Na⁺]_i signals differed significantly between the three agonists. The average [Na⁺]_i increase was highest with KA (23.4 mm), followed by Glu (13.2 mm), andd-Asp (6.1 mm) (Table 1). The maximal slopes of [Na⁺]_i increase (in mm/min) induced by these three agonists were 22.1 ± 2.4 (n = 43), 12.8 ± 9.0 (n = 30), and 9.3 ± 5.6 (n = 26), respectively. During repetitive applications, amplitude and slopes of the [Na⁺]_i transients decreased as described for the [H⁺]_i changes (data not shown) (see above).

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Fig. 8.

Comparison between glutamate- and kainate-induced [Na⁺]_i and [H⁺]_itransients. A, Recordings showing changes in intracellular Na⁺ and H⁺ concentrations (Δ[Na⁺]_i, Δ[H⁺]_i) induced by application of Glu (1 mm for 1 min) (indicated by bars) in CO₂/HCO⁻₃-buffered saline (solid lines) and CO₂/HCO⁻₃-free saline (dashed lines). Although Glu-induced [H⁺]_i changes were greatly altered when switching between CO₂/HCO⁻₃-free and CO₂/HCO⁻₃-containing solution, very small alterations were seen in the induced changes in [Na⁺]_i. B, Recordings showing KA-induced [Na⁺]_i and [H⁺]_i changes. KA (1 mm) was applied for 1 min as indicated by the bars. Again, the significant changes in KA-induced [H⁺]_i transients caused by switching from CO₂/HCO⁻₃-containing solution to CO₂/HCO⁻₃-free solution were not associated with significant changes in the [Na⁺]_i transients. A,B, Na⁺ and H⁺ measurements were obtained from different cells.

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Fig. 10.

CNQX reduced the increase in [Na⁺]_i induced by Glu, but not the increase in [H⁺]_i. Glu-induced [H⁺]_i and [Na⁺]_itransients are shown in standard CO₂/HCO⁻₃-buffered saline and during application of the ionotropic, non-NMDA receptor blocker CNQX (25 μm) (solid bars). Glu (1 mm) was applied for 1 min (indicated by short bars). The recordings were obtained from two different cells, and two segments were deleted from the lower trace(arrows) to enable better comparison between the traces.

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Fig. 9.

CNQX blocked kainate-induced [H⁺]_i and [Na⁺]_itransients. KA-induced [H⁺]_i and [Na⁺]_i transients are shown in standard CO₂/HCO⁻₃-buffered saline and during application of the ionotropic, non-NMDA receptor blocker CNQX (25 μm) (solid bars). KA (1 mm) was applied for 1 min (upper trace) or 2 min (lower trace, indicated by short bars). The recordings were obtained from two different cells; note the different time scales. CNQX completely blocked both the KA-induced [H⁺]_i and [Na⁺]_i transients.

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Fig. 11.

CNQX had only minor effects on aspartate-induced [H⁺]_i and [Na⁺]_itransients. [H⁺]_i and [Na⁺]_i transients were induced byd-Asp (Asp) in standard CO₂/HCO⁻₃-buffered saline and during application of CNQX (25 μm) (solid bars). d-Asp (1 mm) was applied for 1 min (short solid bars). The recordings were obtained from two different cells, and two segments were deleted from thelower trace (arrowheads) to enable better comparison between the traces.

In CO₂/HCO⁻₃-free saline, baseline [Na⁺]_i decreased significantly to 11.9 mm (Table 1), indicating a contribution of inwardly directed Na⁺/HCO⁻₃ cotransport and/or Na⁺-dependent Cl⁻/HCO⁻₃ exchanger to baseline [Na⁺]_i in these cells under standard conditions (Rose and Ransom, 1996). Both Glu- and KA-induced [Na⁺]_i increases were slightly reduced in comparison to CO₂/HCO⁻₃-buffered saline (p ≤ 0.025) (Table 1).

Comparison of [H₊]_i and [Na^+]i changes

A direct comparison of the Glu- and KA-induced [Na⁺]_i and [H⁺]_itransients is shown in Figure 8. In CO₂/HCO⁻₃-buffered saline, Glu-induced acidifications started up to 30 sec later than the increases in [Na⁺]_i. [H⁺]_i continued to rise for minutes after [Na⁺]_i was already decreasing and only started to recover when [Na⁺]_i had nearly reached baseline again (Fig. 8A, solid traces). The same differences also were true for d-Asp-induced [Na⁺]_i and [H⁺]_ichanges (see Fig. 11).

In CO₂/HCO⁻₃-free saline, in contrast, the time course of the Glu-induced [H⁺]_i increase more closely matched that of the [Na⁺]_i transient (Fig.8A, dashed traces). Both [H⁺]_i and [Na⁺]_iincreases started at about the same time, and the acidification reached its peak earlier than in CO₂/HCO⁻₃-buffered saline. The [H⁺]_i recovery to baseline, however, was still slightly delayed compared with the [Na⁺]_i recovery (Fig. 8A,dashed traces).

The time course and occurrence of KA-induced [Na⁺]_i and [H⁺]_itransients differed greatly. In CO₂/HCO⁻₃-buffered saline, the KA-evoked [Na⁺]_i increase was paralleled by an alkalinization. The subsequent acidification started about when [Na⁺]_i reached its highest amplitude and persisted even when [Na⁺]_i had reached baseline again (Fig. 8B, solid traces). In contrast to this, KA-induced acidifications were strongly reduced or even absent in CO₂/HCO⁻₃-free saline, whereas the [Na⁺]_i increases only decreased slightly (Fig. 8B, dashed traces). This comparison demonstrates that large increases in [Na⁺]_i are not necessarily associated with an acidification of hippocampal astrocytes in CO₂/HCO⁻₃-free saline.

Effect of CNQX on [H⁺]_i and [Na⁺]_i changes induced by Glu, KA, and d-Asp

To investigate receptor-mediated effects of Glu and its agonists, we applied CNQX (25 μm), which selectively blocks activation of ionotropic non-NMDA receptors. CNQX perfusion did not alter baseline [H⁺]_i or [Na⁺]_i in astrocytes (Table 1), and all of its effects were reversible.

CNQX completely blocked [H⁺]_i and [Na⁺]_i transients evoked by KA in both CO₂/HCO⁻₃-free (data not shown) and CO₂/HCO⁻₃-buffered saline (Table 1, Fig. 9), confirming that these effects of KA were solely attributable to receptor activation.

In contrast to this, CNQX significantly increasedGlu-induced [H⁺]_i changes to 138%, anddecreased [Na⁺]_i changes to 52% of control values (Table 1, Fig. 10). These results allow several straightforward and important conclusions concerning the mechanisms of the Glu-induced [H⁺]_i and [Na⁺]_i changes. They show that about half of the Glu-induced [Na⁺]_i increase was attributable to receptor activation, and, therefore, most likely caused by Na⁺ influx through the receptor–ionophore complex. Furthermore, they indicate that the observed acidification was not only independent of receptor activation, but was in fact probably dampened by it. In addition, this experiment demonstrated that the intraglial acidification on Glu application was not a direct effect of the increase in [Na⁺]_i, confirming the impression that [Na⁺]_i increases are not of necessity followed by increases in [H⁺]_i in hippocampal astrocytes (see above).

The d-Asp-induced acidification was virtually unaltered by CNQX (100%) (Table 1), whereas the [Na⁺]_itransient was slightly reduced to 89% (p ≤ 0.025) (Table 1, Fig. 11), consistent with the expectation that d-Asp is a very poor substrate for the ionotropic receptor, but is readily transported into astrocytes.

Origin of Glu- and d-Asp-evoked acidifications

In agreement with studies on cerebral astrocytes (Brookes and Turner, 1993; Brune and Deitmer, 1995), l-Glu ord-Asp acidified hippocampal astrocytes. Our results indicate that these acidifications are caused primarily by a transporter that exchanges Glu and Na⁺ for intracellular OH⁻ and K⁺, as demonstrated for retinal glial cells (Bouvier et al., 1992) (see Fig. 1A). Glu-induced intracellular acidification is not a phenomenon restricted to cell cultures; Glu uptake via this transporter also produced an intracellular acidification in rat hippocampal slices (Amato et al., 1994).

Our conclusion is supported by several lines of evidence. In CO₂/HCO⁻₃-free saline, Glu-induced [H⁺]_i increases were closely paralleled by [Na⁺]_i increases. The CNQX-resistant,d-Asp-induced [H⁺]_i increases implied that these acidifications were not attributable to non-NMDA receptor activation with H⁺ influx through receptor-coupled ion channels. Likewise Glu-induced acidifications were resistant to CNQX (they actually increased), demonstrating that they too were not caused by ionotropic non-NMDA receptor activation. Glu transport is voltage dependent (Brew and Attwell, 1987), and if CNQX partially blocked the Glu-induced depolarization (Bowman and Kimelberg, 1984;Kettenmann and Schachner, 1985), Glu uptake and acidification would be increased. Reduction of Glu-induced depolarization also might have reduced a depolarization-dependent alkalinization process, thereby magnifying the acidification (see below).

The stoichiometry for coupled transport of Glu⁻/d-Asp⁻, K⁺, Na⁺, and OH⁻ is 1:1:2:1 (Bouvier et al., 1992) (see Fig. 1C). Using our data, the d-Asp-induced increase in [H⁺]_i was slightly higher than expected from the [Na⁺]_i increase and this stoichiometry. Assuming an intraglial buffering power of about 60 mm (Chesler, 1990; Deitmer, 1995) and a baseline [H⁺]_i of 55 nm (Table 1), thed-Asp-induced [H⁺]_i increase of 10.4 nm (Table 1) would correspond to a total increase in intracellular acid by 4.2 mm, implying a [Na⁺]_i increase of twice this, or 8.4 mm; the average observed [Na⁺]_iincrease, however, was only 6.1 mm (Table 1). Several factors could account for this discrepancy. The actual inward transport of Na⁺ might be higher than suggested by the net concentration increases because of removal of Na⁺ by Na⁺,K⁺-ATPase activity. Moreover, the buffering capacity of the cells could be lower than 60 mm or the transporter might not be the only source for the increase in [H⁺]_i.

Other sources of Glu/d-Asp-induced acidification could include increased Glu metabolism by the ATP-dependent enzyme glutamine synthetase (Sonnewald et al., 1993) or Na⁺,K⁺-ATPase activation (Thompson and Prince, 1986; Erecinska, 1989; Pellerin and Magistretti, 1994), because in both cases H⁺ is released with ATP breakdown (Hochachka and Mommsen, 1983). Outwardly directed Na⁺/HCO⁻₃ cotransport also might be activated as discussed below. In any case, an intraglial accumulation of Glu or d-Asp by 4–5 mm in our study (calculated from the [H⁺]_i increase) corresponds reasonably well to Glu concentrations reached in the brain (∼10 mm) (Erecinska and Silver, 1990).

The Glu-induced [H⁺]_i increase was 1.5 times larger than the d-Asp-induced acidification, but had a similar time course. This difference reflects the substrate specificity of Glu uptake; d-Asp produced smaller effects than Glu in terms of membrane current (Brew and Attwell, 1987; Barbour et al., 1993) and pH changes (Bouvier et al., 1992; Brune and Deitmer, 1995). Another reason for this difference might be astrocytic Glu-uptake sites which do not accept d-Asp as substrate (Flott and Seifert, 1991).

Origin of KA-evoked [H⁺]_i changes

In CO₂/HCO⁻₃-free saline, where baseline [H⁺]_i is primarily regulated by Na⁺/H⁺-exchange (Pappas and Ransom, 1993), KA induced either no [H⁺]_ichanges or a small acidification. At the same time, KA-induced [Na⁺]_i increases reduced the driving force for Na⁺/H⁺ exchange from 24:1 to 8:1 (see also Aronson, 1985) (for ion concentrations, see Materials and Methods and Table 1). This reduced driving force was apparently sufficient to keep [H⁺]_i more or less constant, suggesting that [Na⁺]_i increases do not cause obligatory [H⁺]_i increases in hippocampal astrocytes in CO₂/HCO⁻₃-free saline. They could, however, slow recovery from an acid load, as indicated by the delayed [H⁺]_i recovery compared with [Na⁺]_i recovery (compare Fig.8A).

In CO₂/HCO⁻₃-buffered saline, in contrast, KA evoked alkaline-acid shifts. The ionic dependence and pharmacology of these transients suggested that they were primarily caused by transmembrane HCO⁻₃ fluxes mediated by electrogenic Na⁺/HCO⁻₃ cotransport (O’Connor et al., 1994; Pappas and Ransom, 1994). They were blocked by CNQX and, therefore, dependent on activation of ionotropic non-NMDA receptors. The [H⁺]_i transients were independent of extracellular Ca²⁺, making the contribution of a plasma membrane Ca²⁺/H⁺ ATPase unlikely (Schwiening et al., 1993; Paalasmaa et al., 1994).

The KA-induced transients still were seen in Cl⁻-free saline, although the alkaline shift was considerably reduced in amplitude, suggesting that they were not dependent on Cl⁻, but possibly influenced by the transmembrane H⁺ gradient. Electroneutral Cl⁻/HCO⁻₃ exchange and Na⁺-dependent Cl⁻/HCO⁻₃ exchange were not likely, therefore, to contribute significantly to the KA-induced [H⁺]_i changes. Removal of [Na⁺]_o or application of DIDS diminished the KA-induced alkalinization by about 80% and blocked the acidification, indicating that these [H⁺]_ichanges were attributable to Na⁺-dependent anion transport. In addition, a Na⁺-independent mechanism might be responsible for a small percentage of the KA-induced alkalinization (Grichtchenko and Chesler, 1994). Hippocampal neurons, which do not express Na⁺/HCO⁻₃ cotransport (Schwiening and Boron, 1994), only showed an acidification upon KA application.

To understand how KA could alter Na⁺/HCO⁻₃ cotransport activity in a biphasic manner, one has to consider that this transporter is electrogenic and, therefore, influenced by membrane potential. The stoichiometry of Na⁺/HCO⁻₃cotransport is 1:2 in hippocampal astrocytes (O’Connor et al., 1994). With the steady-state ion concentrations given in the present study ([Na⁺]_i = 15 mm; [Na⁺]_o = 140 mm; [HCO⁻₃]_i = 17.3 mm) (seeChesler, 1990); [HCO⁻₃]_o = 23 mm), its reversal potential (E_rev) is ∼−75 mV, which is close to the baseline membrane potential of hippocampal astrocytes (O’Connor et al., 1994; Steinhauser et al., 1994). Depolarization of the membrane above −75 mV should, therefore, lead to inward Na⁺/HCO⁻₃cotransport.

KA depolarizes astrocytes by 5 mV/sec (Backus et al., 1989); the total depolarization can reach 20–25 mV (Bowman and Kimelberg, 1984; Kettenmann and Schachner, 1985). The similar time courses of KA-induced alkalinization and depolarization (see Bowman and Kimelberg, 1984; Kettenmann and Schachner, 1985; Backus et al., 1989) strongly suggest that the alkalinization resulted from accelerated inward Na⁺/HCO⁻₃ cotransport during KA application (see above). KA also caused a [Na⁺]_i increase, with a slightly slower time course than the depolarization (Fig.12A,B). This KA-induced [Na⁺]_i increase causes a progressive increase in the E_rev of the transporter to about −50 mV. Because the KA-induced membrane depolarization is faster than the [Na⁺]_iincrease, the membrane potential initially is more positive thanE_rev, favoring influx of Na⁺/HCO⁻₃ causing intracellular alkalinization (Fig. 12B).

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Fig. 12.

Proposed voltage shifts and transmembrane Na⁺/HCO⁻₃ movements in astrocytes during kainate application in CO₂/HCO⁻₃-buffered saline.A, Model of KA-induced changes in membrane potential, [Na⁺]_i, and Na⁺/HCO⁻₃-cotransporter reversal potential in astrocytes. KA causes depolarization and increases [Na⁺]_i (1). This is followed by Na⁺ pump stimulation leading to hyperpolarization and normalization of [Na⁺]_i(2). The reversal potential of Na⁺/HCO⁻₃ cotransport is altered by the changes in [Na⁺]_i, illustrated by the hypothetical curves to the right. The upper trace shows the presumed changes in membrane potential (E_m) resulting from KA application (1 mm for 1 min) (indicated bybars) (see also Bowman and Kimelberg, 1984; Kettenmann and Schachner, 1985; Backus et al., 1989). The middle trace shows the averaged KA-induced [Na⁺]_i change of six representative cells. The lower trace shows changes in the reversal potential of Na⁺/HCO⁻₃ cotransport (E_rev), calculated from the average [Na⁺]_i change in the middle trace (see Discussion). B, Proposed mechanism of the alkaline-acid transients seen in astrocytes resulting from KA application (1 mm for 1 min) (indicated bybars). The upper trace shows changes in the E_rev (solid line). Superimposed on the E_rev trace is the presumed change in E_m induced by KA (dashed line) (see above). During the fast, KA-induced membrane depolarization,E_m is more positive thanE_rev, favoring influx of Na⁺/HCO⁻₃ and, therefore, intracellular alkalinization. During repolarization and hyperpolarization of the membrane, E_m is more negative than E_rev, because of the relatively slower recovery of [Na⁺]_i, favoring efflux of Na⁺ and HCO⁻₃ and intracellular acidification. The lower trace shows the KA-induced biphasic alkaline-acid shift in [H⁺]_i averaged from six representative cells.

Further consideration of the relationship between membrane potential and E_rev can account for the KA-induced acidification. Because of activation of Na⁺,K⁺-ATPase secondary to increased [Na⁺]_i, Glu or Glu agonists induce rapid repolarization and long-lasting hyperpolarization after their removal (Ransom et al., 1975; Bowman and Kimelberg, 1984; Thompson and Prince, 1986; Erecinska, 1989). This produces a discrepancy between membrane potential and E_rev that is opposite to what is seen initially, leading to reversal of Na⁺/HCO⁻₃ cotransport and acidification (Deitmer and Schneider, 1995) during periods whenE_m is more negative thanE_rev (Fig. 12B). Na⁺/HCO⁻₃ cotransport, therefore, links membrane potential with [H⁺]_i and [Na⁺]_i homeostasis in astrocytes.

Glu and d-Asp also depolarize astrocytes (Bowman and Kimelberg, 1984; Kettenmann and Schachner, 1985) and, therefore, also might activate inward Na⁺/HCO⁻₃cotransport. Indeed, a CO₂/HCO⁻₃-dependent alkalinization was seen in about 20% of the cells during perfusion with Glu. In the majority of cells, however, the acidification attributable to Glu uptake apparently obscured this alkalinizing influence. The enhancement of the Glu-induced acid shift in CO₂/HCO⁻₃-free solution and after CNQX, which would reduce depolarization, may be partly attributable to removal of a superimposed alkalinization process mediated by Na⁺/HCO⁻₃ cotransport.

Origin of the [Na⁺]_i transients

Glu and its agonists evoked large [Na⁺]_iincreases in hippocampal astrocytes, as reported earlier for leech glial cells (Ballanyi et al., 1989), cultured cerebral astrocytes (Kimelberg et al., 1989), and oligodendrocytes (Ballanyi and Kettenmann, 1990). The different amplitudes of [Na⁺]_i increase, and CNQX sensitivity of the tested substances mirror their substrate characteristics vis a vis Glu receptors and Glu uptake.

The KA-induced [Na⁺]_i increase was dependent on activation of ionotropic non-NMDA receptors, because it was blocked by CNQX. It was caused, therefore, by influx of Na⁺ through receptor-coupled cation channels (Sontheimer et al., 1988). Another cause of [Na⁺]_i increase would be inwardly directed Na⁺/HCO⁻₃cotransport in these cells (see above). This was indicated by the KA-induced alkalinization and the smaller amplitude of the [Na⁺]_i increase in CO₂/HCO⁻₃-free, compared with CO₂/HCO⁻₃-buffered, saline (Table 1).

In addition to receptor-mediated Na⁺ influx, our data suggest that 40–50% of the Glu-induced [Na⁺]_i increase was caused by Na⁺transport via Glu uptake, because it persisted in CNQX (Fig. 10). Thed-Asp-induced [Na⁺]_i increases, on the other hand, were primarily attributable to Glu uptake, because they were only slightly altered by CNQX (Fig. 11).

Conclusions

Our study indicates that [Na⁺]_iincreases are not primarily responsible for Glu- or KA-induced acidifications in hippocampal astrocytes. Instead, our results imply that the [H⁺]_i transients are primarily caused by Glu uptake, resulting in acidification, and by electrogenic Na⁺/HCO⁻₃ cotransport activity, resulting in biphasic alkaline-acid shifts. In the brain, the relative influence of these two mechanisms and, therefore, the direction of glial [H⁺]_i changes might vary depending on the physiological situation. During normal neuronal activity, glial cells alkalinize after activation of inward Na⁺/HCO⁻₃ cotransport, and only blocking this transporter unmasks an acidification, which probably results from neurotransmitter uptake (Chesler and Kraig, 1989; Rose and Deitmer, 1995). The glial uptake of base promotes extracellular acidification, thereby dampening neuronal excitability in a feedback-like manner (Ransom, 1992). During pathological situations such as stroke or epilepsy, in contrast, extracellular Glu concentrations are increased for longer time periods, and glial cells strongly acidify (Kraig and Chesler, 1990), probably because of maximal Glu uptake obscuring any alkalinization.