Intracellular pH in the OK cell. I. Identification of H+ conductance and observations on buffering capacity.
The regulation of intracellular pH (pHi) in the opossum kidney (OK) cell line was studied in vitro using the pH-sensitive excitation ratio of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Recovery from an NH4Cl acid load disclosed a Na-dependent component blocked by amiloride and a smaller Na-independent component. The Na-independent recovery rate was proportional to the H+ gradient from cell to buffer and was zero in the absence of an electrochemical gradient. The Na-independent recovery was not affected by N-ethylmaleimide, dicyclohexylcarbodiimide, HCO3, phloretin, or ZnCl2 but was accelerated in depolarized cells and by membrane-fluidizing drugs and was inhibited by glutaraldehyde. The apparent cellular buffering capacity changed in proportion to this H+ conductance. Consistent with an electrogenic H+ leak, steady-state cell pH alkalinized with depolarization and acidified with hyperpolarization. Removal of buffer Na+ produced a profound acidification, as did amiloride. In 0-Na+ buffers, extremely large cell-to-buffer H+ gradients were present and proportional to buffer pH. 4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid had no effect on steady-state pHi. Measurements of intracellular buffering capacity were derived from the change of cell pH induced by withdrawing NH4Cl. This buffering capacity was increased threefold in Na-free buffers, whereas the value measured by direct titration of cell lysate was the same or less than that of control cells. The NH4Cl-derived buffering capacity varied in direct proportion to the magnitude of the H+ leak. Drugs that changed H+ permeability produced the apparent changes of the measured buffering capacity within a few minutes. We conclude that, in HCO3-free buffer, the OK cell uses two membrane acid-base transport pathways: a Na-H antiporter active at physiological pH and a substantial passive H+ conductance. The results also reveal that the NH4Cl-derived buffering capacity is subject to artifacts, possibly due to a finite leak of ionic NH4+.
Graber
M
M
Veterans Affairs Medical Center, Northport 11768.
DiPaola
J
J
Hsiang
F L
FL
Barry
C
C
Pastoriza
E
E
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002021:Buffers; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005452:Fluoresceins; D006863:Hydrogen-Ion Concentration; D007672:Kidney Cortex / Q000201:enzymology / Q000378:metabolism*; D008564:Membrane Potentials; D009893:Opossums; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011522:Protons; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers
D002021:Buffers; D002352:Carrier Proteins; D005452:Fluoresceins; D011522:Protons; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium; D006180:Proton-Translocating ATPases; D005033:Ethylmaleimide; D004121:Dimethyl Sulfoxide