The Journal of General Physiology, Vol 95, 369-392, Copyright © 1990 by The Rockefeller University Press
Apical membrane Na+/H+ exchange in Necturus gallbladder epithelium. Its dependence on extracellular and intracellular pH and on external Na+ concentration
GA Altenberg and L Reuss
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77550.
Intracellular microelectrode techniques and extracellular pH measurements
were used to study the dependence of apical Na+/H+ exchange on mucosal and
intracellular pH and on mucosal solution Na+ concentration ([Na+]o). When
mucosal solution pH (pHo) was decreased in gallbladders bathed in
Na(+)-containing solutions, aNai fell. The effect of pHo is consistent with
titration of a single site with an apparent pK of 6.29. In Na(+)-depleted
tissues, increasing [Na+]o from 0 to values ranging from 2.5 to 110 mM
increased aNai; the relationship was well described by Michaelis-Menten
kinetics. The apparent Km was 15 mM at pHo 7.5 and increased to 134 mM at
pHo 6.5, without change in Vmax. In Na(+)-depleted gallbladders, elevating
[Na+]o from 0 to 25 mM increased aNai and pHi and caused acidification of a
poorly buffered mucosal solution upon stopping the superfusion; lowering
pHo inhibited both apical Na+ entry and mucosal solution acidification.
Both effects can be ascribed to titration of a single site; the apparent
pK's were 7.2 and 7.4, respectively. Diethylpyrocarbonate (DEPC), a
histidine- specific reagent, reduced mucosal acidification by 58 +/- 4 or
39 +/- 6% when exposure to the drug was at pHo 7.5 or 6.5, respectively.
Amiloride (1 mM) did not protect against the DEPC inhibition, but reduced
both apical Na+ entry and mucosal acidification by 63 +/- 5 and 65 +/- 9%,
respectively. In the Na(+)-depleted tissues mean pHi was 6.7. Cells were
alkalinized by exposure to mucosal solutions containing high concentrations
of nicotine or methylamine. Estimates of apical Na+ entry at varying pHi,
upon increasing [Na+]o from 0 to 25 mM, indicate that Na+/H+ exchange is
active at pHi 7.4. Intracellular H+ stimulated apical Na+ entry by
titration of more than one site (apparent pK 7.1, Hill coefficient 1.7).
The results suggest that external Na+ and H+ interact with one site of the
Na+/H+ exchanger and that cytoplasmic H+ acts on at least two sites. The
external titratable group seems to be an imidazolium, which is apparently
different from the amiloride- binding site. The dependence of Na+ entry on
pHi supports the notion that the Na+/H+ exchanger is operational under
normal transport conditions.
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