A fast and simple screening test to search for specific inhibitors of the plasma membrane calcium pump

No specific inhibitors of the plasma membrane Ca2+ pump have been found to date, limiting research on the particular contribution of this pump to the Ca2+ homeostasis of animal cells. The search for Ca2+ pump inhibitors may h ave been hampered by the lack of an efficient screening method to measure p ump activity that would provide an alternative to the lengthy and costly ad enosine triphosphatase or Ca2+-flux measurements. We propose here a novel s creening method in which Ca2+ pump inhibition is translated into easily mea surable cell dehydration. Intact human red cells, suspended in Ca2+-contain ing, low-K+ buffers were exposed to sequential additions of (1) ionophore A 23187 (t = 0) to load the cells with Ca2+; (2) CoCl2 (t = 1 minute) to bloc k ionophore-mediated Ca2+ transport and to allow complete extrusion of the Ca2+ load by the pump in less than 5 minutes; and (3) NaSCN (t = 6 minutes) to accelerate cell dehydration via Ca2+-sensitive K+ channels when the Ca2 + load is retained as a result of Ca2+ pump inhibition. Samples were taken at 10 to 25 minutes after ionophore addition and delivered into hypotonic m edia containing about 45 mmol/L NaCl, Non-dehydrated cells-with normal, uni nhibited pumps-instantly underwent lysis, whereas dehydrated cells-with inh ibited pumps-resisted lysis, resulting in translucent or opaque samples, re spectively, which were quantifiable by light-absorption measurements, Vanad ate was used as a test substance to assess the effect of putative pump inhi bitors. This method offers a cost-efficient and easily automated alternativ e for testing large numbers of natural or synthetic agents.