A fast and simple screening test to search for specific inhibitors of the plasma membrane calcium pump.
No specific inhibitors of the plasma membrane Ca(2+) pump have been found to date, limiting research on the particular contribution of this pump to the Ca(2+) homeostasis of animal cells. The search for Ca(2+) pump inhibitors may have been hampered by the lack of an efficient screening method to measure pump activity that would provide an alternative to the lengthy and costly adenosine triphosphatase or Ca(2+)-flux measurements. We propose here a novel screening method in which Ca(2+) pump inhibition is translated into easily measurable cell dehydration. Intact human red cells, suspended in Ca(2+)-containing, low-K(+) buffers were exposed to sequential additions of (1) ionophore A23187 (t = 0) to load the cells with Ca(2+); (2) CoCl(2) (t = 1 minute) to block ionophore-mediated Ca(2+) transport and to allow complete extrusion of the Ca(2+) load by the pump in less than 5 minutes; and (3) NaSCN (t = 6 minutes) to accelerate cell dehydration via Ca(2+)-sensitive K(+) channels when the Ca(2+) load is retained as a result of Ca(2+) pump inhibition. Samples were taken at 10 to 25 minutes after ionophore addition and delivered into hypotonic media containing about 45 mmol/L NaCl. Non-dehydrated cells-with normal, uninhibited pumps-instantly underwent lysis, whereas dehydrated cells-with inhibited pumps-resisted lysis, resulting in translucent or opaque samples, respectively, which were quantifiable by light-absorption measurements. Vanadate was used as a test substance to assess the effect of putative pump inhibitors. This method offers a cost-efficient and easily automated alternative for testing large numbers of natural or synthetic agents.
Tiffert
T
T
Physiological Laboratory, University of Cambridge, United Kingdom.
Daw
N
N
Perdomo
D
D
Lew
V L
VL
D000755:Anemia, Sickle Cell / Q000175:diagnosis / Q000201:enzymology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002462:Cell Membrane / Q000201:enzymology*; D003681:Dehydration / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006706:Homeostasis / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D007476:Ionophores / Q000494:pharmacology; D008403:Mass Screening / Q000379:methods*; D015221:Potassium Channels / Q000378:metabolism; D011189:Potassium Chloride / Q000493:pharmacokinetics; D012966:Sodium Cyanide / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology
D004791:Enzyme Inhibitors; D007476:Ionophores; D015221:Potassium Channels; D000001:Calcimycin; D014638:Vanadates; D011189:Potassium Chloride; D000252:Calcium-Transporting ATPases; D012966:Sodium Cyanide; D002118:Calcium