Document Detail

Intracellular pH in the OK cell. I. Identification of H+ conductance and observations on buffering capacity.
MedLine Citation:
PMID:  1662906     Owner:  NLM     Status:  MEDLINE    
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+.
M Graber; J DiPaola; F L Hsiang; C Barry; E Pastoriza
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.    
Journal Detail:
Title:  The American journal of physiology     Volume:  261     ISSN:  0002-9513     ISO Abbreviation:  Am. J. Physiol.     Publication Date:  1991 Dec 
Date Detail:
Created Date:  1992-02-14     Completed Date:  1992-02-14     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0370511     Medline TA:  Am J Physiol     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  C1143-53     Citation Subset:  IM    
Veterans Affairs Medical Center, Northport 11768.
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MeSH Terms
Amiloride / pharmacology
Carrier Proteins / metabolism*
Cell Line
Cell Membrane Permeability / drug effects
Dimethyl Sulfoxide / pharmacology
Ethylmaleimide / pharmacology
Hydrogen-Ion Concentration
Kidney Cortex / enzymology,  metabolism*
Membrane Potentials
Proton-Translocating ATPases / antagonists & inhibitors,  metabolism*
Sodium / metabolism
Sodium-Hydrogen Antiporter
Reg. No./Substance:
0/Buffers; 0/Carrier Proteins; 0/Fluoresceins; 0/Protons; 0/Sodium-Hydrogen Antiporter; 128-53-0/Ethylmaleimide; 2609-46-3/Amiloride; 67-68-5/Dimethyl Sulfoxide; 7440-23-5/Sodium; 85138-49-4/2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; EC ATPases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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