ATP-independent calcium net movements in human red cell ghosts.

Abstract

Net Ca movements in metabolically depleted red cell ghosts were measured under the influence of different inwardly and outwardly directed Ca concentration gradients. Four variants of potassium-rich ghosts were prepared by reversal of osmotic hemolysis: (1) OCa-ghosts (hemolyzing medium (HM) contained no Ca); (2) OCa-EGTA-ghosts (HM contained 2 to 4mm EGTA1 and no Ca); (3) Ca-ghosts (HM contained 0.1 to 4.0mm Ca); and (4) Ca-EGTA-ghosts [HM contained Ca and EGTA in various proportions ([Ca]<[EGTA])]. Ca uptake in OCa- and OCa-EGTA-ghosts at any particular extracellular concentration ([Ca]0) within 60 min reached a steady state when cellular Ca and [Ca]0 were still far from equilibration. About half of the total Ca taken up by OCa-EGTA-ghosts penetrated into the cell interior whereas the other half seemed to be bound to membrane sites. The amount of cellular Ca uptake (i.e., uptake into the cell interior as well as binding to the membrane) decreased with increasing internal concentration of ionized Ca. With 10−6m internal free Ca, the uptake was nearly completely inhibited. In OCa-EGTA-ghosts the amount of Ca taken up into the intracellular space was markedly increased by the addition of mersalyl (0.05mm) to the external medium. Ca outflow under outward gradients was larger than inflow under inward concentration gradients of similar magnitude and no steady state was reached within 180 min. The incorporation of more than 5mm Ca into red cell ghosts led to a general increase in membrane permeability and finally to hemolysis of the ghosts. Lanthanum was found to inhibit Ca uptake in OCa-and OCa-EGTA-ghosts. The experimental results suggest that membrane-bound Ca regulates the Ca permeability of the red cell membrane. In the absence of ATP the uptake of Ca into red cell ghosts in contrast to outflow is a self-limiting process. This mechanism, in addition to active outward Ca pumping, may help to keep the free internal Ca in red cells at a low level.