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COMPARATIVE AND EVOLUTIONARY PHYSIOLOGY

Canine erythrocytes express the P2X₇ receptor: greatly increased function compared with human erythrocytes

¹Department of Medicine, Nepean Clinical School, University of Sydney, Penrith; ²Phospholipid Biology Group, The Garvan Institute of Medical Research, Sydney; ³Department of Medicine, University of New South Wales, St Vincent's Hospital, Sydney; and ⁴Faculty of Veterinary Science, University of Sydney, New South Wales, Australia

Submitted 6 March 2007 ; accepted in final form 20 August 2007

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 that was 20 times greater than that from human erythrocytes. The P2X₇ agonist 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-trisphosphate (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. 2-Methylthioadenosine 5'-triphosphate and adenosine 5'-O-(3-thiotriphosphate) induced a smaller ⁸⁶Rb⁺ efflux from erythrocytes, whereas 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 uptake of choline⁺ into canine erythrocytes that was 60 times greater than that into human erythrocytes. Overnight incubation of canine erythrocytes with ATP and BzATP induced phosphatidylserine exposure in >80% of cells and caused up to 20% hemolysis. In contrast, <30% of human erythrocytes showed phosphatidylserine exposure after overnight incubation with ATP and BzATP, and hemolysis was negligible. Flow cytometric measurements of ATP-induced ethidium⁺ uptake showed that P2X₇ function was three times 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.

purinergic receptor; P2Z receptor; extracellular adenosine 5'-triphosphate; red blood cell; dog