AJP - Cell Physiology, Vol 259, Issue 1 C121-C133, Copyright © 1990 by American Physiological Society
Polarity and kinetics of Na(+)-H+ exchange in cultured opossum kidney cells
M. H. Montrose and H. Murer
Institute of Physiology, University of Zurich, Switzerland.
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.
