Electrophysiological evidence for Na⁺-coupled bicarbonate transport in cultured rat hepatocytes

Recent observations suggest that hepatocytes exhibit basolateral electrogenic Na⁺-coupled HCO₃^- transport. In these studies, we have further investigated this transport mechanism in primary culture of rat hepatocytes using intracellular microelectrodes to measure membrane potential difference (PD) and the pH-sensitive fluorochrome 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein to measure intracellular pH (pH(i)). In balanced media containing 25 mM HCO₃^-, PD averaged -32.1 ± 0.6 (SE) mV and pH(i) averaged 7.22 ± 0.03. PD became more negative (hyperpolarized) when extracellular [HCO₃^-] was increased and less negative (depolarized) when extracellular HCO₃^- was decreased. Acute replacement of extracellular Na⁺ by choline also resulted in membrane depolarization of 18.0 ± 1.6 mV, suggesting net transfer of negative charge. This decrease in PD upon Na⁺ removal was HCO₃^--dependent, amiloride insensitive, and inhibited by the disulfonic stilbene 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). PD also decreased upon acute exposure to SITS. The degree of depolarization seen with removal of Na⁺ or HCO₃^- correlated directly with resting PD (r = 0.81 and 0.95, respectively), suggesting a voltage-dependent mechanism. Removal of extracellular Na⁺ also decreased pH(i) to 7.06 ± 0.02, and this acidification was decreased in the absence of HCO₃^- or in the presence of SITS or amiloride. These studies provide direct evidence for electrogenic Na⁺-coupled HCO₃^- transport in rat hepatocytes. Further, they suggest that it represents a major pathway for conductive movement of Na⁺ across the membrane and that it contributes, along with Na⁺-H⁺ exchange, to the intracellular acidification observed upon removal of extracellular Na⁺. Thermodynamic considerations as well as the acute effect of SITS on PD suggest that this mechanism may mediate influx of Na⁺, HCO₃^-, and net negative charge under basal conditions and that it may be regulated in part by the membrane PD.