Electrogenic Na+/K(+)-transport in human endothelial cells.

Abstract

Na+/K+ pump currents were measured in endothelial cells from human umbilical cord vein using the whole-cell or nystatin-perforated-patch-clamp technique combined with intracellular calcium concentration ([Ca2+]i) measurements with Fura-2/AM. Loading endothelial cells through the patch pipette with 40 mmol/l [Na+] did not induce significant changes of [Ca2+]i. Superfusing the cells with K(+)-free solutions also did not significantly affect [Ca2+]i. Reapplication of K+ after superfusion of the cells with K(+)-free solution induced an outward current at a holding potential of 0 mV. This current was nearly completely blocked by 100 mumol/l dihydroouabain (DHO) and was therefore identified as a Na+/K+ pump current. During block and reactivation of the Na+/K+ pump no changes in [Ca2+]i could be observed. Pump currents were blocked concentration dependently by DHO. The concentration for half-maximal inhibition was 21 mumol/l. This value is larger than that reported for other tissues and the block was practically irreversible. Insulin (10-1000 U/l) did not affect the pump currents. An increase of the intracellular Na+ concentration ([Na+]i) enhanced the amplitude of the pump current. Half-maximal activation of the pump current by [Na+]i occurred at about 60 mmol/l. The concentration for half-maximal activation by extracellular K+ was 2.4 +/- 1.2 mmol/l, and 0.4 +/- 0.1 and 8.7 +/- 0.7 mmol/l for Tl+ and NH4+ respectively. The voltage dependence of the DHO-sensitive current was obtained by applying linear voltage ramps. Its reversal potential was more negative than -150 mV. Pump currents measured with the conventional whole-cell technique were about four times smaller than pump currents recorded with the nystatin-perforated-patch method.(ABSTRACT TRUNCATED AT 250 WORDS)