Depolarization-induced alkalinization (DIA) in rat hippocampal astrocytes

1. Depolarization of glial cells causes their intracellular pH (pH(i)) to increase. To more completely characterize this depolarization-induced alkalinization (DIA) in mammalian astrocytes, we studied DIA in cultured rat hippocampal astrocytes. Astrocytes were loaded with the fluorescent pH indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF), and pH(i) was monitored with the use of an imaging system. Cells were studied ~24 h after removing them from serum-containing culture medium. In HCO₃/^-- buffered solution containing 3 mM K⁺, mean baseline pH(i) was 7.14 ± 0.14 (mean ± SD). 2. Astrocyte pH(i) rapidly and reversibly alkalinized when bath [K⁺] was increased from 3 to 12 mM. In HCO₃/^--buffered solution, mean DIA amplitude was 0.16 ± 0.01 pH units, and mean rate of pH(i) change was 0.076 ± 0.03 pH units/min. In contrast, DIA elicited in nominally HCO₃/^--free, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution was much smaller (0.03 ± 0.01 pH units, 0.04 ± 0.01 pH units/min; P < 0.0001), indicating that DIA was, in large part, a HCO₃/^--dependent process. Subsequent experiments were carried out in HCO₃/^--buffered solution. 3. The relationship between DIA and variable changes in bath [K⁺] was examined. Increasing bath [K⁺] from 3 to 6 mM produced a DIA of 0.07 ± 0.04 pH units, and lowering [K⁺] to 0.5 mM resulted in an acid shift of 0.08 ± 0.05 pH units. The effects of these changes in [K⁺] on membrane potential were measured in separate experiments by whole cell patch-clamp recording. On the basis of these data, it was possible to construct a relationship between V(m) and pH(i); shifting membrane potential ~10 mV resulted in a pH(i) shift of ~0.07. 4. Application of 0.5 mM Ba²⁺ depolarized V(m) and elicited DIA in astrocytes. This indicated that depolarization, in the absence of an increase in [K⁺], could cause DIA. Application of Ba²⁺ also blocked K⁺- induced DIA, presumably because blockade of K⁺ channels prevented any depolarization by K⁺. 5. Cells with more alkaline baseline pH(i)s exhibited larger and more rapidly developing DIAs. The mechanism of this effect is not known. 6. The timing of serum removal affected astrocyte DIA. Cells studied ~24 h after serum removal always exhibited robust DIA (mean = 0.16 ± 0.01 pH units). In cells removed from serum just before experimentation, only 40% showed DIA, and the response amplitude was small (mean pH(i) = 0.05 ± 0.02; P < 0.0001). The mechanism of this serum-related suppression of DIA was not determined. 7. Removal of extracellular Na⁺ significantly decreased DIA, suggesting that DIA is, at least in part, a Na⁺-dependent process. Removal of extracellular Cl^- did not reduce DIA and seemed to increase its initial rate, possibly by removing an opposing acid loader such as the Cl^-/HCO₃/^- antiporter. 8. The stilbene 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and the anion channel blocker diphenylamine-2-carboxylic acid (DPC), both blockers of anion transport, markedly reduced DIA in the presence or absence of Na⁺. 9. DIA in rat hippocampal astrocytes was Na⁺ dependent, HCO₃/^- dependent, and DIDS sensitive; it did not depend on Cl^-. These features strongly suggest that DIA was primarily mediated by the electrogenic Na⁺/HCO₃/^- co-transporter, a transport mechanism known to be present in these cells (O'Connor et al. 1994). 10. DIA was elicited by small increases in [K⁺](o) (i.e., 3-6 mM), as might be seen with neural activity in vivo. Astrocytic pH(i) would be expected to fluctuate with neural activity, therefore, and to encode the intensity of this activity in the duration and amplitude of the evoked alkaline shifts.