pH regulation in hepatoma cells: roles for Na-H exchange, Cl-HCO3 exchange, and Na-HCO3 cotransport.
Regulation of intracellular pH (pHi) was studied in Fu5, a rat hepatoma cell line that maintains a variety of differentiated functions. Microspectrofluorimetry of the pH-sensitive dye 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) was used to measure pHi in 10-15 cells growing on cover glasses that were mounted in a flow-through chamber on the stage of a microscope. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solutions, pHi was 7.14, and intrinsic buffer capacity was inversely related to pHi. Amiloride (0.1 mM) caused pHi to decrease by 0.33 pH units in 4 min. Recovery from an acid load (using either NH4 prepulse technique or Na-free solutions) was completely blocked by amiloride. In HCO3-CO2-buffered solutions, pHi was 7.15, and buffer capacity was relatively insensitive to pHi between pHi of 6.6 and 7.2. Amiloride caused pHi to decrease by only 0.09 units. Recovery from an acid load was Na dependent, occurred in Cl-free solutions, and was totally blocked by the combination of amiloride plus 0.5 mM dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (H2DIDS); recovery occurred when either amiloride or H2DIDS was removed. Removal of external Cl caused a rapid, H2DIDS-blockable alkalinization that was faster in HCO3-CO2 than in HEPES. The apparent Km for Clout for relaxation of Cl-free alkalinization was 4.5 mM. Rate of HCO3 transport during Cl-free treatment increased at alkaline resting pHi. It is concluded that Fu5 cells have two Na-dependent base-loading mechanisms and an acid-loading Cl-HCO3 exchanger. In solutions containing HCO3-CO2, the Na-H exchanger accounts for approximately 40% of recovery from an acid load, and a Na-HCO3 cotransporter accounts for the remainder. Recovery from an alkaline load appears to occur through the activity of the Cl-HCO3 exchanger.
Weintraub
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WH
Department of Physiology/Anatomy, University of California, Berkeley 94720.
Machen
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D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D002352:Carrier Proteins / Q000502:physiology*; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology; D002463:Cell Membrane Permeability; D027963:Chloride-Bicarbonate Antiporters; D005452:Fluoresceins; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000032:analysis / Q000166:cytology / Q000378:metabolism; D008114:Liver Neoplasms, Experimental / Q000378:metabolism / Q000473:pathology* / Q000503:physiopathology; D051381:Rats; D012964:Sodium / Q000378:metabolism / Q000493:pharmacokinetics; D027982:Sodium-Bicarbonate Symporters; D017923:Sodium-Hydrogen Exchangers; D014407:Tumor Cells, Cultured / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology
D015415:Biomarkers; D002352:Carrier Proteins; D027963:Chloride-Bicarbonate Antiporters; D005452:Fluoresceins; D027982:Sodium-Bicarbonate Symporters; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium