ELECTROGENIC NA+ K+-TRANSPORT IN HUMAN ENDOTHELIAL-CELLS/

Na+/K+ pump currents were measured in endothelial cells from human umb ilical cord vein using the whole-cell or nystatin-perforated-patch-cla mp 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 holdi ng potential of 0 mV. This current was nearly completely blocked by 10 0 mumol/l dihydroouabain (DHO) and was therefore identified as a Na+/K + pump current. During block and reactivation of the Na+/K+ pump no ch anges in Ca2+!i could be observed. Pump currents were blocked concent ration dependently by DHO. The concentration for half-maximal inhibiti on was 21 mumol/l. This value is larger than that reported for other t issues 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 abo ut 60 mmol/l. The concentration for half-maximal activation by extrace llular 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-se nsitive current was obtained by applying linear voltage ramps. Its rev ersal potential was more negative than - 150 mV. Pump currents measure d with the conventional whole-cell technique were about four times sma ller than pump currents recorded with the nystatin-perforated-patch me thod. If however 100 mumol/l guanosine 5'-O-(3-thiotriphosphate) (GTPg ammaS) were added to the pipette solution, the currents measured in th e ruptured-whole-cell-mode were not significantly different from the c urrents measured with the perforated-patch technique. We suppose that the use of the perforated-patch technique prevents wash out of a guani ne nucleotide-binding protein (G-protein)-connected intracellular regu lator that is necessary for pump activation.