pH homeostasis in promyelocytic leukemic HL60 cells.
By measuring the membrane potential using the influx of the lipophilic cation tetraphenylphosphonium and intracellular pH using 2,7-biscarboxy-ethyl-5(6)-carboxyfluorescein and the distribution of the weak acid 5,5-dimethyl-2,4-oxazolidinedione, we have determined that intracellular pH is 0.9-1.1 pH units above electrochemical equilibrium in undifferentiated HL60 cells, indicating that these cells actively extrude proton equivalents. The Na/H exchanger is not the system responsible for keeping the pH above the electrochemical equilibrium, since adding inhibitors of this transport system (dimethylamiloride and ethylisopropylamiloride) or removing the extracellular sodium has no effect on intracellular pH. In contrast, the addition of the Cl/HCO3 exchange inhibitors H2 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) or pentachlorophenol (PCP) causes a drop in intracellular pH, and the removal of extracellular chloride in the presence of bicarbonate leads to a large intracellular alkalinization, which indicates a role for the anion exchanger in pH homeostasis in these cells. In addition, we find that the intracellular chloride concentration is about one order of magnitude above electrochemical equilibrium. We conclude that an H2DIDS and PCP inhibitable system, probably the Cl/HCO3 exchanger, is at least partially responsible for keeping intracellular pH above electrochemical equilibrium in HL60 cells under resting conditions. We also find no change in intracellular pH when cells differentiate along the granulocytic pathway (having been induced by the addition of dimethylsulfoxide or of retinoic acid), which indicates that changes in intracellular pH are not causally related to cell differentiation.
Restrepo
D
D
Department of Biophysics, University of Rochester School of Medicine, New York 14642.
Kozody
D J
DJ
Spinelli
L J
LJ
Knauf
P A
PA
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000031:analogs & derivatives; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D027963:Chloride-Bicarbonate Antiporters; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D006706:Homeostasis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015473:Leukemia, Promyelocytic, Acute / Q000503:physiopathology*; D008564:Membrane Potentials; D010416:Pentachlorophenol / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers; D013757:Tetraethylammonium Compounds / Q000494:pharmacology
D002352:Carrier Proteins; D027963:Chloride-Bicarbonate Antiporters; D017923:Sodium-Hydrogen Exchangers; D013757:Tetraethylammonium Compounds; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C018564:dihydro-DIDS; D010416:Pentachlorophenol; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D004121:Dimethyl Sulfoxide