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 m M , (ii) inhibited by ouabain, (iii) dependent on extracellular pH and (iv) characterized by a K m of 15 ± 3 m M 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 CO 2 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 ++ (<10 μ M and 1.0 m M ). 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 results 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.