Using the pH-sensitive dye 2,7-bis(carboxyethyl)-5(6)-carboxy-fluorescein and a continuously perfused subconfluent hepatocyte monolayer cell culture system, we studied rat hepatocyte intracellular pH (pH,) regulation in the presence (+HCO3) and absence (-HCO3) of bicarbonate. Baseline pH, was higher (7.28±09) in +HCO3 than in -HCO3 (7.16±0.14). Blocking Na+/H' exchange with amiloride had no effect on pH1 in +HCO3 but caused reversible 0.1-0.2-U acidification in -HCO3 or in +HCO3 after preincubation in the anion transport inhibitor 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). Acute Na' replacement in +HCO3 also caused acidification which was amiloride independent but DIDS inhibitible.The recovery of pH, from an intracellular acid load (maximum H+ efilux rate) was 50% higher in +HCO3 than in -HCO3. Amiloride inhibited H+ efflux..M by 75% in -HCOj but by only 27% in +HCO3. The amiloride-independent pH, recovery in +HCO3 was inhibited 50-63% by DIDS and 79% by Na+ replacement but was unaffected by depletion of intracellular Cl-, suggesting that Cl-/HCO3 exchange is not involved. Depolarization of hepatocytes (raising external K+ from 5 to 25 mM) caused reversible 0.05-0.1-U alkalinization, which, however, was neither Na+ nor HCO3 dependent, nor DIDS inhibitible, findings consistent with electroneutral HCO3 transport.We conclude that Na'-HCO3 cotransport, in addition to Na+/H+ exchange, is an important regulator of pH1 in rat hepatocytes.

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