A pH-stat method was used to characterize H' efflux pathways in hepatocytes in order to determine if Na+/ H+ and Ca++/H+ exchange are involved in H+ efflux from hepatocytes under basal conditions and if cyclic AMP analogs affect Na+/H+ exchange. Total H' efflux of freshly prepared hepatocytes ranged from 10 to 16 nmoles per min per mg protein. A part of total H+ efflux (35 to 50%) was dependent on extracellular Na'. This Na+-dependent H+ efflux was (i) inhibited by amiloride with a half-maximal effect at 0.3 mlM, (ii) inhibited by ouabain, (iii) dependent on extracellular pH and (iv) characterized by a K, of 15 2 3 mM Na+ and a Vmax of 9 f 0.07 nmoles per min per mg protein. Amiloride, ouabain and replacement of Na+ by choline also decreased intracellular pH determined from equilibrium distribution of dimethyloxazolidinedione. Li+ could partially substitute for Na+ in Na+-dependent H+ efflux and in maintaining intracellular pH. Efflux of COz and lactic acid from hepatocytes represented 80% of Na+-independent H' efflux. Efflux of H+ in the presence and absence of Na+ was not significantly altered by extracellular Ca" (<lo pM and 1.0 mM). Thus, Ca+'/H+ exchange is unlikely to contribute significantly to total H' efflux from hepatocytes. Cyclic AMP analogs, dibutyryl cyclic AMP and 8-bromo cyclic AMP, inhibited amiloride-sensitive Na+-dependent H+ efflux, and dibutyryl cyclic AMP decreased intracellular pH. These re- sults indicate that (i) both Na'-independent and Na+dependent mechanisms are involved in H' efflux from hepatocytes, (ii) Na+-dependent H' efflux represents Na+/H+ exchange and is involved in maintaining intracellular pH and (iii) cyclic AMP analogs decrease intracellular pH by inhibiting Na+/H' exchange.
Metabolically active cells like hepatocytes produce H+ which must be removed from the cell in order to maintain intracellular pH. Studies with plasma membranes isolated from rat livers have identified different transport processes that may be involved in the regulation of intracellular pH (pHi). A Na+/H+ exchange mechanism is located at the sinusoidal membrane (1, 2). Studies on Ca++ transport in the presence of a pH gradient indicate the presence of a Ca++/H+ exchange mechanism (3).