Osmotic activation of Na(+)-H+ exchange in human endothelial cells

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Abstract
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.