AJP - Cell Physiology, Vol 267, Issue 1 C84-C93, Copyright © 1994 by American Physiological Society
Characterization of Na(+)-H+ antiporter activity associated with human cheek epithelial cells
E. J. McMurchie, S. L. Burnard, G. S. Patten, E. J. Lee, R. A. King and R. J. Head
Division of Human Nutrition, Glenthorne Laboratory, Commonwealth Scientific and Industrial Research Organization, Australia.
Na+ transport activity was characterized in human cheek epithelial cells
obtained from normotensive adult subjects. The cells were isolated using a
mouth-wash procedure and assayed for Na+ uptake using a radioactive (22Na+)
rapid filtration assay. Cheek cells displayed proton-dependent Na+ uptake
activity that was dependent on the magnitude of the externally directed
proton gradient measured using the fluorescent probe
2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein to determine intracellular
pH. Amiloride, ethylisopropylamiloride (EIPA), 5-(N,N-dimethyl)-amiloride,
5-(N-methyl-N-isobutyl)-amiloride (MIA), and
5-(N,N-hexamethylene)-amiloride (NNHA) all inhibited proton-dependent Na+
uptake, with MIA, EIPA, and NNHA being the most potent. The Michaelis
constant (Km) for extracellular Na+ was 5.7 mM, while the maximum velocity
for Na(+)-H+ antiporter activity was 4.3 nmol Na+.mg protein-1.30s-1. The
Km for intracellular H+ was 0.17 microM, with a Hill coefficient of 0.7.
Stimulation by ouabain and inhibition by bumetanide of cheek cell
proton-dependent Na+ uptake indicated only relatively low activities of
Na(+)-K(+)-ATPase and Na(+)-K(+)-2Cl- cotransport, respectively. These
results are consistent with the presence of Na(+)-H+ antiporter activity in
cheek cells. Cheek cells therefore provide a convenient, relatively
noninvasive source of tissue for examining Na(+)-H+ antiporter activity in
human subjects.
