Bicarbonate-dependent and -independent intracellular pH regulatory mechanisms in rat hepatocytes. Evidence for Na+-HCO3- cotransport

Abstract

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(i)) regulation in the presence (+HCO3-) and absence (-HCO3-) of bicarbonate. Baseline pH(i) was higher (7.28 ± 09) in +HCO3- than in -HCO3- (7.16 ± 0.14). Blocking Na+/H+ exchange with amiloride had no effect on pH(i) 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 inhibitable. The recovery of pH(i) from an intracellular acid load (maximum H+ efflux rate) was 50% higher in +HCO3- than in -HCO3-. Amiloride inhibited H+ efflux(max) by 75% in -HCO3- but by only 27% in +HCO3-. The amiloride-independent pH(i) 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 inhibitable, findings consistent with electroneutral HCO3- transport. We conclude that Na+-HCO3- cotransport, in addition to Na+/H+ exchange, is an important regulator of pH(i) in rat hepatocytes.