Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus : II. Electrogenic Na/HCO₃ Cotransport

Address correspondence to Walter F. Boron, Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520. Fax: 203-785-4951; E-mail: [email protected]

Portions of this work have been previously published in preliminary form (Bevensee, M.O., W.F. Boron, and M. Apkon. 1995. FASEB J. 9: A308.).

Abbreviations used in this paper: BCECF, 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein; DIA, depolarization-induced alkalinization; DIDS, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid; MQAE, N-(6-methoxyquinolyl)acetoethyl ester; V_m, plasma membrane voltage.

The sum of pH_i increases in Fig. 5, ab, cd, and ef was 0.51. β_T is the sum of β_I (12.7 mM [pH unit]⁻¹) and β_HCO3⁻ (12.1 mM [pH unit]⁻¹), or 24.8 mM (pH unit)⁻¹ at an average pH_i of 6.79. 0.51 pH units × 24.8 mM (pH unit)⁻¹ × 0.75 (DIDS-sensitive fraction, as described in the accompanying paper; Bevensee et al., 1997) = 9.5 mM HCO₃⁻ moved into the cell by the Na-driven HCO₃⁻ transporter. For a Na-driven Cl-HCO₃ exchanger, two HCO₃⁻ ions would move into the cell in exchange for one Cl⁻ ion out of the cell. Therefore, the movement of 9.5 mM HCO₃⁻ into the cell by this exchanger would result in the movement of 4.7 mM Cl⁻ out of the cell.

Removing external Na⁺ in CO₂/HCO₃⁻ elicited a HCO₃⁻ efflux of 101 μM s⁻¹, of which 90% or 91 μM s⁻¹ was DIDS sensitive. This DIDS-sensitive HCO₃⁻ efflux corresponds to the net movement of 4.55 C (liter cell vol)⁻¹ s⁻¹ for a Na/HCO₃ cotransporter that moves one charge equivalent per two HCO₃⁻ ions. We calculated the average cell volume of an astrocyte to be 1.6 pl, assuming the geometry of an astrocyte to be a right-angled cone (V = [area × h]/3) with a height (h) of 5 μm (O'Connor et al., 1993). We imaged BCECF-loaded astrocytes to obtain an average cell area of 973 ± 70 μm² (n = 17). Therefore, the current flow through the Na/HCO₃ cotransporter in a typical astrocyte would be 7.3 pA. Given an average input resistance for hippocampal astrocytes of 236 MΩ (see below), this 7.3-pA current would depolarize astrocytes by 1.7 mV.

We sometimes observed such abrupt depolarizations in experiments in which we monitored only V_m, as in Fig. 10,A. Conversely, we sometimes observed slower depolarizations in experiments in which we monitored V_m and pH_i simultaneously. One would expect such depolarizations due to electrogenic Na/HCO₃ cotransport to be instantaneous under conditions in which the bath solution could be instantaneously changed. However, to maintain seals in our patch-clamp experiments performed at 37°C, we were often forced to reduce our solution flow rate. During such experiments, we often observed the slower depolarizations in response to Na⁺ removal (e.g., Fig. 10 A).

35.8 μM s⁻¹ corresponds to the net movement of 1.79 C (liter cell vol)⁻¹ s⁻¹ for a Na/HCO₃ cotransporter that moves one charge equivalent per two HCO₃⁻ ions. The current flow through the Na/HCO₃ cotransporter in a typical astrocyte with a volume of 1.6 pl would be 2.9 pA. This current flow would depolarize hippocampal astrocytes with an input resistance of 236 MΩ by only 0.7 mV.