Abstract
Canine erythrocytes are known to undergo a reversible increase in cation permeability when incubated with extracellular ATP. We have examined the expression and function of P2X receptors on human erythrocytes using confocal microscopy and a panel of anti-P2X(1-7) antibodies and have measured monovalent cation fluxes in the presence of various nucleotide agonists. Human erythrocytes expressed P2X7 receptors on all cells examined from eight of eight subjects, as well as P2X2 at a far lower staining intensity in six of eight subjects. ATP stimulated the efflux of 86Rb+ (K+) from human erythrocytes in a dose-dependent fashion with an EC50 of approximately 95 microM. Other nucleotides also induced an efflux of 86Rb+ from erythrocytes with an order of agonist potency of 2'- and 3'-O(4-benzoylbenzoyl) ATP (BzATP) > ATP > 2-methylthio-ATP (2MeSATP) > adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), whereas ADP or UTP had no effect. ATP-induced efflux of 86Rb+ from erythrocytes was inhibited by extracellular Na+ and oxidized ATP, as well as by KN-62, an antagonist specific for the human P2X7 receptor. When erythrocytes were incubated in isotonic KCl medium, the addition of ATP stimulated an 86Rb+ influx approximately equal in magnitude to ATP-stimulated 86Rb+ efflux from the same cells. BzATP also stimulated the influx of 22Na+ into erythrocytes incubated in isotonic NaCl medium. Both ATP-induced efflux and influx of 86Rb+ and 22Na+ were impaired in erythrocytes from subjects who had inherited loss-of-function polymorphisms in the P2X7 receptor. These results suggest that the reversible permeabilization of erythrocytes by extracellular ATP is mediated by the P2X7 receptor.
Highlights
Many of our concepts of cellular Naϩ and Kϩ homeostasis were based on experiments in the erythrocyte, a cell type in which intracellular Naϩ and Kϩ concentrations could be readily changed and the subsequent effect on ion transport could be measured
BzATP stimulated the influx of 22Na؉ into erythrocytes incubated in isotonic NaCl medium. Both ATP-induced efflux and influx of 86Rb؉ and 22Na؉ were impaired in erythrocytes from subjects who had inherited loss-offunction polymorphisms in the P2X7 receptor. These results suggest that the reversible permeabilization of erythrocytes by extracellular ATP is mediated by the P2X7 receptor
Our results showed that functional P2X7 receptors are present on human erythrocytes at lower abundance than on human lymphocytes
Summary
Reagents—Bovine serum albumin (BSA), D-glucose, glycerol gelatin mounting medium, saponin, ATP, BzATP, ADP, UTP, 2MeSATP, ATP␥S, OxATP, Me2SO, ouabain, furosemide, and Drabkin’s reagent (used according to the manufacturer’s instructions) were from Sigma. For time-course studies, 86Rbϩ-loaded erythrocyte suspensions (10 ml) were incubated in the absence or presence of 1 mM ATP for 4 h, with 1-ml samples collected at 30 min intervals as above. Statistical Analyses—The differences in 86Rbϩ release were compared using either the unpaired Student’s t test for single comparisons to control samples (see Figs. 2 and 4) or analysis of variance for multiple comparisons (see Figs. 5 and 6 and Table I) using SPSS 11.5 for Windows (SPSS Inc, Chicago, IL) with p Ͻ 0.05 considered significant
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