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-indu ced alkalinization (DIA) in mammalian astrocytes, we studied DIA in cu ltured rat hippocampal astrocytes. Astrocytes were loaded with the flu orescent pH indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein ( BCECF), and pH(i) was monitored with the use of an imaging system. Cel ls were studied similar to 24 h after removing them from serum-contain ing culture medium. In HCO3--buffered solution containing 3 mM K+, mea n baseline pH(i) was 7.14 +/- 0.14 (mean +/- SD). 2. Astrocyte pH(i) r apidly 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 uni ts/min. In contrast, DIA elicited in nominally HCO3--free, N-2-hydroxy ethylpiperazine-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 pro cess. Subsequent experiments were carried out in HCO3--buffered soluti on. 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 c ell patch-clamp recording. On the basis of these data, it was possible to construct a relationship between V-m and pH(i) shifting membrane p otential similar to 10 mV resulted in a pH(i) shift of similar to 0.07 . 4. Application of 0.5 mM Ba2+ depolarized V-m and elicited DIA in as trocytes. This indicated that depolarization, in the absence of an inc rease in [K+], could cause DIA. Application of Ba2+ also blocked K+-in duced DIA, presumably because blockade of K+ channels prevented any de polarization by K+. 5. Cells with more alkaline baseline pH(i)s exhibi ted larger and more rapidly developing DIAs. The mechanism of this eff ect is not known. 6. The timing of serum removal affected astrocyte DI A. Cells studied similar to 24 h after serum removal always exhibited robust DIA (mean = 0.16 +/- 0.01 pH units). In cells removed from seru m just before experimentation, only 40% showed DIA, and the response a mplitude was small (mean pH(i) = 0.05 +/- 0.02; P < 0.0001). The mecha nism of this serum-related suppression of DIA was not determined. 7. R emoval of extracellular Na+ significantly decreased DIA, suggesting th at DIA is, at least in part, a Na+-dependent process. Removal of extra cellular Cl- did not reduce DIA and seemed to increase its initial rat e, 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 w as Na+ dependent, HCO3- dependent, and DIDS sensitive; it did not depe nd on Cl-. These features strongly suggest that DIA was primarily medi ated by the electrogenic Na+/HCO3- co-transporter, a transport mechani sm 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 inten sity of this activity in the duration and amplitude of the evoked alka line shifts.