Pumping ions: Regulation of intracellular pH in hepatocytes

In rat hepatocytes, basolateral Na⁺‐H⁺ exchange and Na⁺‐HCO₃⁻ cotransport function as acid extruders. To assess mechanisms of acid loading, intracellular pH (pH_i) recovery from an alkaline load was analyzed in short‐term cultured rat hepatocyte monolayers using the pH‐sensitive dye BCECF. Electrophysiological techniques were also used to assess the role of the membrane potential (V_m). Cells were alkaline loaded by suddenly reducing external CO₂ and HCO₃⁻ (from 10% and 50 mM, respectively, to 5% and 25 mM) at constant pH. After this maneuver, pH_i rapidly rose by 0.13 ± 0.03 pH units (pH_u) and recovered to baseline at an initial rate of 0.026 ± 0.009 pH_u/min. Intracellular buffering power was estimated from the dependence of pH_i on [NH₄⁻]_o and varied between 70 and 10.5 mM/pH_u in a pH_i range of 6.5‐7.6. Initial pH_i recovery corresponded to a rate of OH⁻ efflux (J_OH) of 1.76 ± 0.71 mM/min and was blocked by 0.5 mM DIDS (0.003 ± 0.002; J_OH = 0.18 ± 0.06) or by 1 mM H₂DIDS (0.001 ± 0.002; J_OH = 0.26 ± 0.08) and by removal of [Cl⁻]_o (0.003 ± 0.007; J_OH = 0.28 ± 0.07). The dependence of J_OH on [Cl⁻]_o exhibited saturation kinetics with an apparent K_m for [Cl₋]_o of 5.1 mM. pH_i recovery was Na⁺ independent and was not inhibited by substitution of Na⁺ with NMDG (0.045 ± 0.09; J_OH = 2.94 ± 0.59). During an alkaline load, cell V_m hyperpolarized from –33.4 ± 1.8 to –43.4 ± 2.8 mV, mainly due to an increase in K⁺ conductance by a factor of 2.8 ± 0.3. Ba²⁺ blocked these changes and depolarized V_m by 12.1 ± 1.2 mV but had no effect on pH_i recovery (0.025 ± 0.007; J_OH = 1.46 ± 0.60), excluding that V_m functions as a major force for HCO₃⁻ extrusion after an alkaline load. The rate of J_OH was directly proportional to the pH_i reached after the alkaline load and varied fourfold over the pH_i range of 7.25‐7.46. These data indicate that pH_i recovery from an alkaline load in rat hepatocytes is mediated by an electroneutral Na⁺‐independent Cl⁻‐HCO₃⁻ exchanger.