Blood Cells, Molecules, and Diseases 2012-02-15

Vanadate-induced Ca(2+) and Co(2+) uptake in human red blood cells.

Poul Bennekou, Henrik Harbak, Lars Ole Simonsen

Index: Blood Cells Mol. Dis. 48(2) , 102-9, (2012)

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Abstract

The vanadate-induced increase in passive uptake of calcium and cobalt and their interference were studied in human red cells using (45)Ca and (57)Co as tracers. Vanadate is a potent inhibitor of the Ca-pump in red cells, although in fed cells a residual pump activity remains that is highly significant compared to the passive influx, and even in cells that are both ATP-depleted and vanadate-treated the pump arrest is not complete. In the presence of vanadate the Ca(2+) uptake is increased due to inhibition of Ca-pump extrusion, but is further increased due to a vanadate-induced increment in passive influx. In order to measure the vanadate-induced increment in Ca(2+) influx, the total uptake in vanadate-treated cells is corrected for the basal influx, as recorded in ATP-depleted cells in the presence of tetrathionate (5mM) that has been shown to eliminate the residual Ca-pump activity in ATP-depleted cells. The (57)Co uptake is also increased by vanadate. (57)Co is not transported by the Ca-pump, and hence the uptake in vanadate-treated cells can be directly compared to the basal uptake, both in fed and in ATP-depleted cells. The vanadate effect shows rapid onset and appears to be irreversible. The vanadate-induced increment in uptake of both (45)Ca and (57)Co is reduced by about 50% in ATP-depleted cells compared to fed cells, suggesting a metabolism- or SH-group-dependent component. The influx of both (45)Ca (in ATP-depleted cells) and (57)Co (in fed cells) increases with the vanadate concentration, with a similar K(½) (0.4 and 0.3mM, respectively), and is nearly maximal at 5mM vanadate. The vanadate-induced increment in influx of both (45)Ca and (57)Co increases with the extracellular concentration as a saturable function, with K(½) estimated at, respectively, 700 and 80μM. In the case of (57)Co K(½) is similar in fed and in ATP-depleted cells. The vanadate-induced uptake of (45)Ca and of (57)Co shows interference. The uptake of (45)Ca is inhibited by Co(2+), and the uptake of (57)Co is inhibited by Ca(2+), although with an unexplained time course. The vanadate-induced uptake of (45)Ca and (57)Co are both inhibited, and to a similar degree, by the 1,4-dihydropyridine Ca(2+)-channel blocker nifedipine, although only at concentrations much higher than IC(50) for classical Ca-channels. The vanadate-induced increment in (57)Co uptake is electroneutral, in contrast to the basal uptake that is at least partially electrogenic. In experiments with resealed ghosts a vanadate-induced (57)Co uptake could not be detected. The vanadate-induced increment in (57)Co uptake amounts to nearly half the increment in (45)Ca uptake, both in fed and in ATP-depleted cells. It is speculated that the vanadate-induced Ca(2+) and Co(2+) uptake could be mediated by a putative common transporter, which appears to be separate and distinct from the putative common transporter for basal Ca(2+) and Co(2+) uptake.Copyright © 2011 Elsevier Inc. All rights reserved.