Polarity of alveolar epithelial cell acid-base permeability.
We investigated acid-base permeability properties of electrically resistive monolayers of alveolar epithelial cells (AEC) grown in primary culture. AEC monolayers were grown on tissue culture-treated polycarbonate filters. Filters were mounted in a partitioned cuvette containing two fluid compartments (apical and basolateral) separated by the adherent monolayer, cells were loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and intracellular pH was determined. Monolayers in HCO-free Na(+) buffer (140 mM Na(+), 6 mM HEPES, pH 7.4) maintained a transepithelial pH gradient between the two fluid compartments over 30 min. Replacement of apical fluid by acidic (6.4) or basic (8.0) buffer resulted in minimal changes in intracellular pH. Replacement of basolateral fluid by acidic or basic buffer resulted in transmembrane proton fluxes and intracellular acidification or alkalinization. Intracellular alkalinization was blocked > or =80% by 100 microM dimethylamiloride, an inhibitor of Na(+)/H(+) exchange, whereas acidification was not affected by a series of acid/base transport inhibitors. Additional experiments in which AEC monolayers were grown in the presence of acidic (6.4) or basic (8.0) medium revealed differential effects on bioelectric properties depending on whether extracellular pH was altered in apical or basolateral fluid compartments bathing the cells. Acid exposure reduced (and base exposure increased) short-circuit current from the basolateral side; apical exposure did not affect short-circuit current in either case. We conclude that AEC monolayers are relatively impermeable to transepithelial acid/base fluxes, primarily because of impermeability of intercellular junctions and of the apical, rather than basolateral, cell membrane. The principal basolateral acid exit pathway observed under these experimental conditions is Na(+)/H(+) exchange, whereas proton uptake into cells occurs across the basolateral cell membrane by a different, undetermined mechanism. These results are consistent with the ability of the alveolar epithelium to maintain an apical-to-basolateral (air space-to-blood) pH gradient in situ.
Joseph
Dilip
D
Division of Pulmonary and Critical Care Medicine, Will Rogers Institute Pulmonary Research Center, University of Southern California, Keck School of Medicine, 2011 Zonal Avenue, Los Angeles, CA 90033, USA.
Tirmizi
Omar
O
Zhang
Xiao-Ling
XL
Crandall
Edward D
ED
Lubman
Richard L
RL
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000136:Acid-Base Equilibrium / Q000502:physiology*; D000138:Acidosis / Q000378:metabolism; D000471:Alkalosis / Q000378:metabolism; D000818:Animals; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D016764:Cell Polarity / Q000502:physiology*; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D015633:Extravascular Lung Water / Q000378:metabolism; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008297:Male; D011650:Pulmonary Alveoli / Q000166:cytology* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020545:Respiratory Mucosa / Q000166:cytology* / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism
D005452:Fluoresceins; D005456:Fluorescent Dyes; D017923:Sodium-Hydrogen Exchangers; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid