Characterization of H+ efflux pathways in rat hepatocytes

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 15 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 mM, (ii) inhibited by ouabain, (iii) dependent on extracellular pH and (iv) characterized by a Km of 15 +/- 3 mM Na+ and a Vmax of 9 +/- 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 CO2 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++ (less than 10 microM 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.(ABSTRACT TRUNCATED AT 250 WORDS)