Characterization of Na(+)-H+ antiporter activity associated with human cheek epithelial cells

Abstract

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.