Bicarbonate-dependent and -independent intracellular pH regulatory mechanisms in rat hepatocytes. Evidence for Na+-HCO3- cotransport.
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 (pHi) regulation in the presence (+HCO3-) and absence (-HCO3-) of bicarbonate. Baseline pHi was higher (7.28 +/- 09) in +HCO3- than in -HCO3- (7.16 +/- 0.14). Blocking Na+/H+ exchange with amiloride had no effect on pHi 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- alos caused acidification which was amiloride independent but DIDS inhibitible. The recovery of pHi from an intracellular acid load (maximum H+ efflux rate) was 50% higher in +HCO3- than in -HCO3-. Amiloride inhibited H+ effluxmax by 75% in -HCO3- but by only 27% in +HCO3-. The amiloride-independent pHi 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 pHi in rat hepatocytes.
Gleeson
D
D
Liver Center, Yale University School of Medicine, New Haven, Connecticut 06510.
Smith
N D
ND
Boyer
J L
JL
D000584:Amiloride / Q000494:pharmacology; D000641:Ammonia / Q000494:pharmacology; D000818:Animals; D001639:Bicarbonates / Q000494:pharmacology*; D001692:Biological Transport; D002352:Carrier Proteins; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000378:metabolism*; D008297:Male; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D027982:Sodium-Bicarbonate Symporters; D017923:Sodium-Hydrogen Exchangers
D001639:Bicarbonates; D002352:Carrier Proteins; D027982:Sodium-Bicarbonate Symporters; D017923:Sodium-Hydrogen Exchangers; D000641:Ammonia; D000584:Amiloride; D011188:Potassium