title: Elevated intracellular Ca2+ reveals a functional membrane nucleotide pool in intact human red blood cells.
abstract: Elevated intracellular calcium generates rapid, profound, and irreversible changes in the nucleotide metabolism of human red blood cells (RBCs), triggered by the adenosine triphosphatase (ATPase) activity of the powerful plasma membrane calcium pump (PMCA). In the absence of glycolytic substrates, Ca(2+)-induced nucleotide changes are thought to be determined by the interaction between PMCA ATPase, adenylate kinase, and AMP-deaminase enzymes, but the extent to which this three-enzyme system can account for the Ca(2+)-induced effects has not been investigated in detail before. Such a study requires the formulation of a model incorporating the known kinetics of the three-enzyme system and a direct comparison between its predictions and precise measurements of the Ca(2+)-induced nucleotide changes, a precision not available from earlier studies. Using state-of-the-art high-performance liquid chromatography, we measured the changes in the RBC contents of ATP, ADP, AMP, and IMP during the first 35 min after ionophore-induced pump-saturating Ca(2+) loads in the absence of glycolytic substrates. Comparison between measured and model-predicted changes revealed that for good fits it was necessary to assume mean ATPase V(max) values much higher than those ever measured by PMCA-mediated Ca(2+) extrusion. These results suggest that the local nucleotide concentrations generated by ATPase activity at the inner membrane surface differed substantially from those measured in bulk cell extracts, supporting previous evidence for the existence of a submembrane microdomain with a distinct nucleotide metabolism.
authors.affiliation: Department of Physiology, Development, and Neuroscience, University of Cambridge, Cambridge CB2 3EG, England, UK.
authors.forename: Teresa
authors.initials: T
authors.lastname: Tiffert
authors.forename: Virgilio L
authors.initials: VL
authors.lastname: Lew
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism*; D006019:Glycolysis; D006801:Humans; D008954:Models, Biological; D009711:Nucleotides / Q000097:blood* / Q000378:metabolism
chemical_list: D009711:Nucleotides; D002118:Calcium