CL--HCO3- EXCHANGER IN ISOLATED RAT HEPATOCYTES - ROLE IN REGULATION OF INTRACELLULAR PH

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 (Vm). 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(o). 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.