Polarity and kinetics of Na(+)-H+ exchange in cultured opossum kidney cells.
Opossum kidney (OK) cells (an established cell line) were loaded with 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF; a fluorescent dye with a pH-sensitive spectrum), and intracellular pH (pHi) was examined by microfluorometry. Single cells, within a confluent monolayer and grown on a permeant support, were examined for the mechanism of recovery from an acid load as imposed by exposure to ammonium chloride (NH4 prepulse). The Na(+)-dependent recovery of pHi from an acid load (Na(+)-H+ exchange) is examined in terms of the Na+ activation kinetics of the recovery and the polarity of the response. In 80% of the cells examined (33/41), both apical and basolateral Na+ cause recovery from an acid load. The response of cells to apical Na+ is well fit by Michaelis-Menten kinetics [Kt(Na) = 35 mM], but the response to basolateral Na+ is not. The response to basolateral Na+ addition is modeled in terms of variable transepithelial leak of Na+ and variable amounts of basolateral Na(+)-H+ exchange. Despite an average response to basolateral (145 mM) Na+ that is 34% of the response to apical Na+, modeling suggests that basolateral Na(+)-H+ exchange must be less than 10% of the cellular total to fit the basolateral Na+ activation kinetics. The model, and experiments using ordered addition of Na+ from the apical vs. basolateral medium, also suggest that transepithelial leak (of basolateral Na+ to the apical compartment) is required to explain the pHi recovery observed due to addition of basolateral Na+. Direct estimation of (basolateral to apical) transepithelial leak demonstrates that the response due to basolateral Na+ addition is explained by transepithelial leak and a Na(+)-H+ exchange that is expressed solely in the apical membrane.
Montrose
M H
MH
Institute of Physiology, University of Zurich, Switzerland.
Murer
H
H
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008954:Models, Biological; D009893:Opossums; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism* / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers; D013050:Spectrometry, Fluorescence
D002352:Carrier Proteins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D000644:Quaternary Ammonium Compounds; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium; C027917:tetramethylammonium; C039614:ethylisopropylamiloride