Osmotic activation of Na(+)-H+ exchange in human endothelial cells.
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.
Escobales
N
N
Department of Physiology, University of Puerto Rico, San Juan 00936.
Longo
E
E
Cragoe
E J
EJ
Danthuluri
N R
NR
Brock
T A
TA
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009994:Osmolar Concentration; D012964:Sodium / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; C039465:5-dimethylamiloride; D000584:Amiloride; D012964:Sodium; C039614:ethylisopropylamiloride