Reconstitution of the basal calcium transport in resealed human red blood cell ghosts

Abstract

The 45Ca 2+ influx into right-side-out resealed ghosts (RG) prepared from human red blood cells (RBC) was measured. The 45Ca 2+ equilibration occurred with t 1/2 = 2.5 min and the steady-state was reached after 17 min with the level of 22 ± 2 μmol/L packed cells at 37 °C. The rate of the influx was 97 ± 17 μmol/L packed cells h. The 45Ca 2+ influx was saturated with [Ca 2+] 0 at 4 mmol/L and was optimal at pH 6.5 and 30 °C. Divalent cations (10 −4–10 −6 mol/L), nifedipine (10 −5–10 −4 mol/L), DIDS (up to 10 −4 mol/L), and quinidine (10 −4–10 −3 mol/L), inhibited the 45Ca 2+ influx while uncoupler (10 −6–10 −5 mol/L) stimulated it. In contrast to intact RBC, vanadate inhibited the 45Ca 2+ influx when added to the external medium, however, the stimulation was observed when vanadate was present in media during both lysis and resealing. PMA had no effect under conditions found to stimulate the Ca 2+ influx in intact RBC. The results show that the Ca 2+ influx into RG is a carrier-mediated process but without control by protein kinase C and that the influx and efflux of Ca 2+ are coupled via the H + homeostasis similarly as in intact RBC but with modified mechanism.