AJP - Cell Physiology, Vol 259, Issue 4 C640-C646, Copyright © 1990 by American Physiological Society
Osmotic activation of Na(+)-H+ exchange in human endothelial cells
N. Escobales, E. Longo, E. J. Cragoe Jr, N. R. Danthuluri and T. A. Brock
Department of Physiology, University of Puerto Rico, San Juan 00936.
Regulation of intracellular pH (pHi) via a Na(+)-H+ exchange-dependent
mechanism was studied in cultured human umbilical vein endothelial cells
(HEC) using the pH-sensitive fluorescent dye
2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, as well as measuring 22Na
influx. Basal pHi of HEC incubated in a bicarbonate-free Na+ medium was
6.99 +/- 0.03. In HEC that had been acid-loaded using nigericin or a NH4Cl
prepulse, pHi recovery occurred via a Na(+)-dependent mechanism that was
inhibited by 5-(N-ethyl-N-isopropyl)amiloride (EIPA). The potency of
amiloride derivatives to inhibit 22Na influx was EIPA greater than
5-(N,N-dimethyl)amiloride greater than amiloride [Ki (extracellular Na = 30
mM) = 17 nM, 150 nM, and 8.8 microM, respectively]. EIPA-sensitive 22Na
influx in acid-loaded HEC was a saturable function of the external Na+
concentration (0-130 mM), exhibiting an approximate Km and Vmax of 19.70
+/- 0.14 mM and 34.01 +/- 2.2 nmol.10(6) cells-1.min-1, respectively. H+
efflux was also dependent on external Na+ and blocked by EIPA. At resting
pHi, HEC Na(+)-H+ exchange was slightly stimulated by increases in medium
osmolality. However, when HEC were acid-loaded in the presence of
hypertonic (sucrose) medium, Na(+)-H+ exchange activity (22Na influx or pHi
recovery) increased markedly. Overall, these data indicate that pHi in
cultured HEC can be regulated by a Na(+)-H+ exchanger and that its activity
can be markedly influenced by osmolality at acidic pHi.
