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

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Abstract
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 (pHi). In balanced media containing 25 mM HCO3-, PD averaged -32.1 .+-. 0.6 (SE) mV and pHi 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 0.85, respectively), suggesting a voltage-dependent mechanism. Removal of extracellular Na+ also decreased pHi 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+-G+ 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.

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