Submitted on March 6, 2007
Accepted on August 20, 2007
Canine erythrocytes express the P2X7 receptor: greatly increased function compared to human erythrocytes
Ronald Sluyter1*, Anne N Shemon1, William E Hughes2, Ryan O Stevenson1, Jennifer G Georgiou1, Guy D Eslick1, Rosanne M Taylor3, and James S Wiley1
1 Medicine, University of Sydney, Penrith, New South Wales, Australia
2 Phospholipid Biology Group, The Garvan Institute of Medical Research, Sydney, New South Wales, Australia; Medicine, University of New South Wales, Sydney, New South Wales, Australia
3 Veterinary Science, University of Sydney, Sydney, New South Wales, Australia
* To whom correspondence should be addressed. E-mail: rons{at}med.usyd.edu.au.
Over three decades ago Parker and Snow (Am J Physiol 223: 888-893, 1972) demonstrated that canine erythrocytes undergo an increase in cation permeability when incubated with extracellular ATP. In this study we examined the expression and function of the channel/pore-forming P2X7 receptor on canine erythrocytes. P2X7 receptors were detected on canine erythrocytes by immunocytochemistry and immunoblotting. Extracellular ATP induced 86Rb+ (K+) efflux from canine erythrocytes which was 20-fold greater than from human erythrocytes. The P2X7 agonist, BzATP, was more potent than ATP and both stimulated 86Rb+ efflux from erythrocytes in a dose-dependent fashion with EC50 values of ~7 and ~309 µM respectively. 2MeSATP and ATP
S induced a smaller 86Rb+ efflux from erythrocytes, while ADP, AMP, UTP or adenosine had no effect. ATP-induced 86Rb+ efflux from erythrocytes was inhibited by oxidized ATP, KN-62 and Brilliant Blue G, known P2X7 antagonists. ATP also induced the uptake of choline+ into canine erythrocytes which was 60-fold greater than into human erythrocytes. Overnight incubation of canine erythrocytes with ATP and BzATP induced phosphatidylserine exposure in over 80% of cells and caused up to 20% hemolysis. In contrast less than 30% of human erythrocytes exposed phosphatidylserine after overnight incubation with ATP and BzATP, while hemolysis was negligible. Flow cytometric measurements of ATP-induced ethidium+ uptake showed that P2X7 function was three-fold lower in canine monocytes than in human monocytes. These data show that the massive cation permeability increase induced by extracellular ATP in canine erythrocytes results from activation and opening of the P2X7 receptor channel/pore.
