Rat hippocampal astrocytes exhibit electrogenic sodium-bicarbonate co-transport

Abstract

1. We probed for the expression of electrogenic Na+/HCO3- co-transport in cultured mammalian astrocytes by recording voltage and current changes induced by bath application of HCO3-, with the use of patch-clamp electrophysiology. Application of 25 mM HCO3-, at a constant pHo, to astrocytes bathed in a nominally HCO3(-)-free solution, produced an abrupt and reversible change in membrane potential ranging from +3 to -30 mV [-11.8 +/- 9.34 (SD) mV]; 55% of cells showed relatively large hyperpolarizing responses (-18.8 +/- 6.23 mV), whereas 45% showed only small shifts in membrane potential (range of -5 to +3 mV; -1.9 +/- 1.96 mV). 2. The size of the HCO3(-)-induced hyperpolarization was strongly related to the cell's initial resting membrane potential in HCO3(-)-free solution; the larger responses were seen in cells with relatively low resting membrane potentials (-48.5 +/- 9.4 mV), and the smaller responses were seen in cells with more negative potentials (-68.1 +/- 6.5 mV). The membrane potentials of hippocampal astrocytes were highly variable in HCO3(-)-free solution (range -38 to -80 mV; -60.9 +/- 12.53); this variability was greatly reduced in HCO3(-)-containing solution (range -59 to -82 mV; -68.5 +/- 4.8). 3. The magnitude of the HCO3(-)-induced response was less strongly correlated with cell input resistance, which was higher in the larger responder cells than in the small responders. However, the differences in input resistance were insufficient to account for the different HCO3(-)-induced responses observed. 4. In the presence of extracellular Ba2+, which by blocking K+ conductance depolarized cells by 30-50 mV, cells that initially showed a small response, showed a large and completely reversible hyperpolarization (-18.4 +/- 6.13 mV) to application of 25 mM HCO3-. In Na(+)-free solution, the HCO3(-)-induced hyperpolarization was reduced by 66%, and the response was not sustained, as in Na(+)-containing solution. Removal of extracellular Cl- had no effect on the HCO3- response The stilbene derivative 4,4'-diisothiocyano-2,2'-stilbene disulfonate (DIDS), a blocker of anion transport, eliminated the HCO3(-)-induced hyperpolarization. Blockers of Na+/K+ ATPase and Na(+)-H+ exchange were without effect. These observations indicated the presence of an electrogenic Na+/HCO3- co-transporter in hippocampal astrocytes. 6. Voltage-clamp recording demonstrated that the HCO3(-)-induced hyperpolarization was caused by outward currents averaging 335 +/- 104 pA. The reversal potential of the HCO3(-)-induced current ranged between -80 and -99 mV with an average = -86.1 +/- 6.2 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Heterogeneity of Astrocyte Resting Membrane Potentials and Intercellular Coupling Revealed by Whole-Cell and Gramicidin-Perforated Patch Recordings from Cultured Neocortical and Hippocampal Slice Astrocytes

“…In agreement with O'Connor et al (1994), whole-cell recordings revealed that by switching from a HEPES-buffered solution to HEPES plus CO 2 /HCO 3 Ϫ media, a hyperpolarization of cultured astrocytes occurred; this response is consistent with the activation of the inward electrogenic Na ϩ /2HCO 3 Ϫ transporter current. More depolarized cells had a larger hyperpolarizing response to CO 2 /HCO 3 Ϫ (Fig.…”

“…6 B). Because no bicarbonate was added to the intracellular pipette solution, the extrapolated reversal potential for the transporter was more negative than the previously reported value of Ϫ95 mV (O'Connor et al, 1994). The hyperpolarizing response to HCO 3 Ϫ was rapidly reversed on returning to HEPES-buffered solution (no HCO 3 Ϫ ).…”

“…Because of its electrogenic nature (1Na ϩ for 2HC O 3 Ϫ taken up by the cell), this co-transport mechanism may be involved in the regulation of glial RMP. O'Connor et al (1994) have shown that some of the variability of cultured astrocyte resting membrane potential detected in HEPES-buffered recording solution is lost in CO 2 /HCO 3 Ϫ -buffered solution because of the voltage-dependent hyperpolarizing effect of the Na ϩ /2HC O 3 Ϫ co-transporter. To determine how much of the RM P heterogeneity found in our experiments was attributable to a lack of Na ϩ /2HCO 3 Ϫ transporter activity in H EPES-buffered solution, we performed experiments similar to those by O'Connor et al; we thus initially used the whole-cell patch-clamp recording configuration (Fig.…”

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

“…6B). The effects of DIDS, as expected (Brune et al, 1994;O'Connor et al, 1994), were not fully reversible.…”

“…In CO 2 / HCO 3 Ϫ -buffered saline, in contrast, K A evoked alkaline-acid shifts. The ionic dependence and pharmacology of these transients suggested that they were primarily caused by transmembrane HCO 3 Ϫ fluxes mediated by electrogenic Na ϩ / HCO 3 Ϫ 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.…”