The P2X(7) receptor mediates the uptake of organic cations in canine erythrocytes and mononuclear leukocytes: comparison to equivalent human cell types.

We previously demonstrated that canine erythrocytes express the P2X(7) receptor, and that the function and expression of this receptor is greatly increased compared with human erythrocytes. Using (86)Rb(+) (K(+)) and organic cation flux measurements, we further compared P2X(7) in erythrocytes and mononuclear leukocytes from these species. Concentration response curves of BzATP- and ATP-induced (86)Rb(+) efflux demonstrated that canine P2X(7) was less sensitive to inhibition by extracellular Na(+) ions compared to human P2X(7). In contrast, canine and human P2X(7) showed a similar sensitivity to the P2X(7) antagonists KN-62 and Mg(2+). KN-62 and Mg(2+) also inhibited ATP-induced choline(+) uptake into canine and human erythrocytes. BzATP and ATP but not ADP or NAD induced ethidium(+) uptake into canine monocytes, T- and B-cells. ATP-induced ethidium(+) uptake was twofold greater in canine T-cells compared to canine B-cells and monocytes. KN-62 inhibited the ATP-induced ethidium(+) uptake in each cell type. P2X(7)-mediated uptake of organic cations was 40- and fivefold greater in canine erythrocytes and lymphocytes (T- and B-cells), respectively, compared to equivalent human cell types. In contrast, P2X(7) function was threefold lower in canine monocytes compared to human monocytes. Thus, P2X(7) activation can induce the uptake of organic cations into canine erythrocytes and mononuclear leukocytes, but the relative levels of P2X(7) function differ to that of equivalent human cell types.

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

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 P2X(7) receptor on canine erythrocytes. P2X(7) receptors were detected on canine erythrocytes by immunocytochemistry and immunoblotting. Extracellular ATP induced (86)Rb(+) (K(+)) efflux from canine erythrocytes that was 20 times greater than that from human erythrocytes. The P2X(7) agonist 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-trisphosphate (BzATP) was more potent than ATP, and both stimulated (86)Rb(+) efflux from erythrocytes in a dose-dependent fashion with EC(50) values of approximately 7 and approximately 309 microM, respectively. 2-Methylthioadenosine 5'-triphosphate and adenosine 5'-O-(3-thiotriphosphate) induced a smaller (86)Rb(+) efflux from erythrocytes, whereas ADP, AMP, UTP, or adenosine had no effect. ATP-induced (86)Rb(+) efflux from erythrocytes was inhibited by oxidized ATP, KN-62, and Brilliant blue G, known P2X(7) 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 P2X(7) 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 P2X(7) receptor channel/pore.