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

Abstract

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.

Fig. 1

BzATP and ATP induce ⁸⁶Rb⁺ efflux from canine and human erythrocytes in a concentration-dependent fashion. ⁸⁶Rb⁺ loaded (a) canine or (b) human erythrocytes were incubated at 37 C for 4 or 60 min respectively in KCl or NaCl medium containing varying concentrations of BzATP or ATP as indicated. ⁸⁶Rb⁺ release, calculated as the difference in percentage release between 0 and 4 or 60 min, respectively, was used to determine the percentage of maximal response to 200 μM BzATP. Results are expressed as the mean (SEM; n = 3)

Fig. 2

KN-62 and Mg²⁺ inhibit ATP-induced ⁸⁶Rb⁺ efflux from canine and human erythrocytes in a concentration-dependent fashion. ⁸⁶Rb⁺ loaded canine or human erythrocytes in KCl medium were pre-incubated at 37 C for 5 min with varying concentrations of a KN-62 and b Mg²⁺ as indicated. The canine and human erythrocytes were then incubated at 37 C for 4 min with 250 μM ATP or 60 min with 100 μM ATP, respectively. ⁸⁶Rb⁺ release, calculated as the difference in percentage release between 0 and 4 or 60 min, respectively, in the absence and presence of either antagonist was used to determine the percent inhibition relative to the maximum ATP response. Results are expressed as the mean (SEM; n = 3)

Fig. 3

KN-62 and Mg²⁺ inhibit ATP-induced choline⁺ uptake into canine and human erythrocytes. a, b Canine and c, d human erythrocytes resuspended in 150 mM choline Cl medium containing [methyl-¹⁴C]choline⁺ (1 μCi/ml) were pre-incubated at 37 C for 5 min with a, c DMSO or 1 μM KN-62, or b, d H₂O or 10 mM Mg²⁺. The canine and human erythrocytes were then incubated at 37 C for 12 or 60 min, respectively, in the absence or presence of 1 mM ATP. The level of choline⁺ uptake at each time point was determined as nanomole choline⁺ per millilitre of cells per minute. Results are expressed as the mean (SEM; n = 3). *P < 0.01, **P < 0.001 to Control; ^†P < 0.01, ^††P < 0.001 to corresponding ATP-treated sample. Results are expressed as the mean (SEM; n = 3)

Fig. 4

ATP induces ethidium⁺ uptake into canine and human mononuclear leukocytes. a Canine or b human mononuclear leukocytes were pre-labelled with cell-specific monoclonal antibodies and resuspended in KCl medium at 37 C. Ethidium⁺ (25 μM) was added, followed 40 s later by the addition of 1 mM ATP (arrow). Mean channel of cell-associated fluorescence was measured by time-resolved flow cytometry for monocytes, T- and B-cells incubated in the absence or presence of ATP. Representative results from three experiments are shown

Fig. 5

BzATP but not ADP nor NAD induce ethidium⁺ uptake into canine mononuclear leukocytes. Canine a monocytes, b T- and c B-cells were pre-labelled with cell-specific monoclonal antibodies and resuspended in KCl medium at 37 C. Ethidium⁺ (25 μM) was added, followed 40 s later by the addition of 200 μM BzATP, 1 mM ADP or 300 μM NAD (arrow). Mean channel of cell-associated fluorescence was measured by time-resolved flow cytometry for monocytes, T- and B-cells incubated in the absence or presence of agonist. Representative results from three experiments are shown

Fig. 6

KN-62 inhibits ATP-induced ethidium⁺ uptake into canine mononuclear leukocytes. Canine a monocytes, b T- and c B-cells were pre-labelled with cell-specific mAb and resuspended in KCl medium at 37 °C. Cells were pre-incubated at 37 °C for 5 min with DMSO or 1 μM KN-62. Ethidium⁺ (25 μM) was added, followed 40 s later by the addition of 1 mM ATP (arrow). Mean channel of cell-associated fluorescence was measured by time-resolved flow cytometry for monocytes, T- and B-cells incubated in the absence or presence of ATP. Representative results from three experiments are shown