Vanadate-induced Ca2 + and Co2 + uptake in human red blood cells

Abstract

The vanadate-induced increase in passive uptake of calcium and cobalt and their interference were studied in human red cells using 45Ca and 57Co 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 Ca2+ 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 Ca2+ 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 57Co uptake is also increased by vanadate. 57Co 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 45Ca and 57Co 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 45Ca (in ATP-depleted cells) and 57Co (in fed cells) increases with the vanadate concentration, with a similar K½ (0.4 and 0.3mM, respectively), and is nearly maximal at 5mM vanadate. The vanadate-induced increment in influx of both 45Ca and 57Co increases with the extracellular concentration as a saturable function, with K½ estimated at, respectively, 700 and 80μM. In the case of 57Co K½ is similar in fed and in ATP-depleted cells. The vanadate-induced uptake of 45Ca and of 57Co shows interference. The uptake of 45Ca is inhibited by Co2+, and the uptake of 57Co is inhibited by Ca2+, although with an unexplained time course. The vanadate-induced uptake of 45Ca and 57Co are both inhibited, and to a similar degree, by the 1,4-dihydropyridine Ca2+-channel blocker nifedipine, although only at concentrations much higher than IC50 for classical Ca-channels. The vanadate-induced increment in 57Co uptake is electroneutral, in contrast to the basal uptake that is at least partially electrogenic. In experiments with resealed ghosts a vanadate-induced 57Co uptake could not be detected. The vanadate-induced increment in 57Co uptake amounts to nearly half the increment in 45Ca uptake, both in fed and in ATP-depleted cells. It is speculated that the vanadate-induced Ca2+ and Co2+ uptake could be mediated by a putative common transporter, which appears to be separate and distinct from the putative common transporter for basal Ca2+ and Co2+ uptake.