Detection and separation of human red cells with different calcium contents following uniform calcium permeabilization

1. The human red cell, permeabilized to calcium with the ionophore A23187, is extensively used to study Ca2+ transport and the effects of intracellular Ca2+ on transport and metabolism. The interpretation of results with calcium-permeabilized cells, in general, has depended on the implicit assumption that the ionophore-induced calcium distribution among the cells is uniform. 2. To establish whether or not calcium permeabilization with the ionophore A23187 generated a uniform calcium distribution in normal-ATP red cells, a method was developed to detect and separate calcium-permeabilized red cells with different calcium contents. For the method to uncover pre-existing heterogeneity without itself inducing it, it was essential to preserve the calcium distribution which existed at the time of sampling. The method was based (i) on the ability of cytoplasmic Ca2+ to activate K+-selective channels in the membrane, and (ii) on the demonstration here that thiocyanate (SCN-) is a non-limiting co-ion for rapid net KSCN efflux and cell shrinkage in the cold. 3. Calcium-permeabilized cells in pump-leak steady state were washed free of ionophore using ice-cold, albumin-containing media. Subsequent incubation at 0 degrees C in low-K+ media with 45-75 mM-SCN- generated dense-cell fractions (H cells) in less than 10 min. These could be separated from the remaining light cells (L cells) by either centrifugation over phthalate oils, or differential osmotic haemolysis, with conservation of the mean total cell calcium. 4. Analysis of the calcium content of H and L cell fractions revealed striking differences in their calcium content, with 70-99% of the mean total cell calcium in the H cell fraction. 5. The ionophore content of density-separated cells, processed with omission of the ionophore removal step, was similar for cells with high- and low-calcium. Magnesium loss from ionophore-treated red cells suspended in magnesium-free media followed single exponentials. Thus ionophore distribution and induced permeability were uniform, and the unequal cell calcium content must be due to factors affecting active calcium extrusion.