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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

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
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

The P2X₇ receptor is predominately expressed on mononuclear leukocytes but can be present on other cell types (5). Our laboratory has shown that human erythrocytes express relatively low levels of P2X₇ receptors compared with mononuclear leukocytes (52) and that activation of this receptor mediates cation fluxes (52) and PS exposure in these cells (54). Some 35 years ago, Parker and Snow reported in this journal that canine erythrocytes undergo an increase in cation permeability when incubated with extracellular ATP but not other nucleotides (43). The gene coding the P2X₇ receptor has been identified in the canine genome (36), and P2X₇ transcripts have been detected in canine brain (35), indicating that the P2X₇ receptor may be present within the dog.

Our studies confirm that the magnitude of the effect of ATP on cation permeability in canine erythrocytes is at least 20 times greater than that observed in human erythrocytes. Many features of this ATP-induced cation permeability in canine erythrocytes were characteristic of functional P2X₇ receptors, and the presence of these receptors was directly shown by immunocytochemistry and immunoblotting. Finally, we demonstrate that overnight incubation with ATP or BzATP induces PS exposure in the majority of canine erythrocytes, with 17% of cells on average undergoing hemolysis. The ATP- or BzATP-induced PS exposure on canine erythrocytes was at least five times greater than that of human erythrocytes, and hemolysis of human erythrocytes was negligible.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Reagents. BSA, D-glucose, glycerol gelatin mounting medium, saponin, ATP, BzATP, ATPS, ADP, AMP, adenosine, UTP, ATP-2',3'-dialdehyde (oxidized ATP, OxATP), Brilliant blue G (BBG), DMSO, Drabkin's reagent, EDTA, dithiothreitol, rabbit anti-actin antibody, and other general reagent-grade chemicals were obtained from Sigma (St. Louis, MO). 2MeSATP was acquired from Tocris Biosciences (Ellisville, MO). KN-62 was obtained from Biomol Research Laboratories (Plymouth Meeting, PA). Brij 35 was obtained from Merck (Darmstadt, Germany). H₂O₂ was acquired from Fronine Laboratory Supplies (Riverstone, New South Wales, Australia). HEPES, normal horse serum (NHS), and NuPAGE SDS running and transfer buffers were obtained from Invitrogen (Carlsbad, CA). Ficoll-Paque PLUS was obtained from GE Healthcare Biosciences (Uppsala, Sweden). Rubidium-86 (⁸⁶Rb⁺), SOLVABLE tissue solubilizer, and Ultima Gold were acquired from PerkinElmer Life Sciences (Boston, MA). [Methyl-¹⁴C]choline-Cl was obtained from GE Healthcare UK (Little Chalfont, UK). Di-n-butyl phthalate and di-isooctyl phthalate (BDH Chemicals, Poole, UK) were blended 80:20 (vol/vol) to give an oil mixture of density 1.03 g/ml. Sheep polyclonal antibody raised against an extracellular epitope of human P2X₇ was kindly provided by Dr. Julian A. Barden (University of Sydney, Sydney, New South Wales, Australia) (61). This antibody binds P2X₇-transfected but not nontransfected HEK-293 cells as assessed by confocal microscopy (61), as well as a band at the predicted 75-kDa region in P2X₇-transfected but not nontransfected HEK-293 cells as determined by Western blotting (20). Cy3-conjugated donkey anti-sheep IgG antibody was obtained from Jackson ImmunoResearch (West Grove, PA). FITC-conjugated murine anti-human CD14 MAb (clone TUK4) and horseradish peroxidase-conjugated rabbit anti-sheep Ig antibody were obtained from Dako (Carpinteria, CA). Horseradish peroxidase-conjugated goat anti-rabbit Ig antibody and SuperSignal West Pico chemiluminescent substrate were acquired from Pierce (Rockford, IL). Phenylmethylsulfonyl fluoride and annexin V-FLUOS were obtained from Roche Diagnostics (Pensberg, Germany).

Erythrocytes and mononuclear cells. The study was approved by the Westmead Hospital Animal Ethics Committee (Westmead, New South Wales, Australia) and the Sydney West Area Health Service Human Ethics Committee (Penrith, New South Wales, Australia). Peripheral blood was collected in heparin-containing vacutainer tubes from four adult English springer spaniels, as well as from five healthy human volunteers. Blood was centrifuged at 580 g for 15 min, the plasma and platelets were discarded, and leukocytes and the upper 10% of erythrocytes were collected to obtain buffy coats. The remaining erythrocytes were washed twice in NaCl medium (147.5 mM NaCl, 2.5 mM KCl, 5 mM D-glucose, 0.1% BSA, and 20 mM HEPES, pH 7.5) at 450 g for 5 min. To isolate mononuclear cells, buffy coats were diluted in five volumes of NaCl medium, underlaid with Ficoll-Paque PLUS, and centrifuged at 580 g for 30 min. Isolated mononuclear cells were washed in NaCl medium at 450 g for 10 min.

Immunofluorescence staining and confocal microscopy. Erythrocytes were resuspended in NaCl medium (5 x 10⁷ cells/ml), and 20 µl were smeared onto glass slides, air-dried for 30 min, and fixed with acetone-methanol for 2 min. The slides were washed four times over 10 min with phosphate-buffered saline (PBS; pH 7.2) and blocked with PBS-20% NHS-0.1% BSA for 20 min before incubation for 45 min with anti-P2X₇ antibody diluted in PBS-0.2% NHS. The slides were washed three times over 30 min with PBS and incubated for 45 min with Cy3-conjugated secondary antibody diluted in PBS-0.2% NHS. The slides were again washed before being embedded in glycerol gelatin mounting medium. Cells were visualized using a Leica Microsystems (Wetzlar, Germany) laser scanning confocal microscope as described previously (23).

Immunoblotting. Hemoglobin (Hb)-free ghost membranes were prepared as previously described (9) using 20 mM phosphate buffer (pH 7.4) containing 1 mM EDTA and 0.2 mM phenylmethylsulfonyl fluoride. Protein concentrations were determined using the DC protein assay (Bio-Rad, Hercules, CA) with BSA as a standard. Proteins of ghost membranes (20 µg/well) were separated under reducing conditions (100 mM dithiothreitol) using 4–12% gradient NuPAGE Novex Bis-Tris mini gels (Invitrogen) and transferred to nitrocellulose membranes (Bio-Rad) using NuPAGE transfer buffer. Nitrocellulose membranes were blocked overnight at 4°C with 5% skim milk powder in Tris-buffered saline containing Tween 20 (TBST; 250 mM NaCl, 50 mM Tris, and 0.2% Tween 20, pH 7.5) and then incubated for 2 h at room temperature with either anti-P2X₇ or anti-actin antibody diluted in TBST containing 5% skim milk powder. Membranes were washed three times over 30 min with TBST and then incubated for 1 h at room temperature with either horseradish peroxidase-conjugated anti-sheep or anti-rabbit Ig antibody, respectively, diluted in TBST containing 5% skim milk powder. Membranes were washed, incubated with chemiluminescent substrate, and visualized using a Gel Doc system and Quantity One software (Bio-Rad).

⁸⁶Rb⁺ efflux measurements. Erythrocytes were loaded with ⁸⁶Rb⁺ (5 µCi/ml) at a hematocrit of 40% in NaCl medium for 4 h at 37°C. Cells were then washed three times with ice-cold NaCl medium at 4°C (1,800 g for 3 min) and resuspended in either NaCl medium or KCl medium (150 mM KCl, 5 mM D-glucose, 0.1% BSA, and 20 mM HEPES, pH 7.5) at a final hematocrit of 5%. ⁸⁶Rb⁺-loaded erythrocyte suspensions were incubated in the absence or presence of nucleotide for up to 40 min at 37°C. At 0, 2, 12, 20, or 40 min, 1-ml samples were overlaid on 300 µl of phthalate oil mixture and centrifuged at 8,000 g for 30 s. For studies using the P2X₇ antagonist OxATP (40), ⁸⁶Rb⁺-loaded erythrocytes in NaCl medium were preincubated in the absence or presence of 300 µM OxATP for 60 min at 37°C, washed once with NaCl medium, and resuspended in NaCl medium before incubation in the absence or presence of ATP for 12 min. For studies using the P2X₇ antagonists KN-62 (17) and BBG (28), ⁸⁶Rb⁺-loaded erythrocytes resuspended in NaCl medium were preincubated in the presence or absence of 1 µM KN-62, 1 µM BBG, or an equal volume of DMSO vehicle for 5 min at 37°C before incubation in the absence or presence of ATP for 12 min. The level of radioactivity in cell lysates (lysed with an equal volume of 0.4% saponin) and supernatants was measured using a Wallac (Turku, Finland) 1480 Wizard 3" automatic gamma counter. Hemolysis (Hb release) was determined spectrophotometrically on cell-free supernatants with Drabkin's reagent (containing 0.015% Brig 35) according to the manufacturer's instructions.

Choline⁺ uptake measurements. Erythrocytes were washed once with choline-Cl medium (150 mM choline-Cl, 5 mM D-glucose, 0.1% BSA, 20 mM HEPES, pH 7.5). Erythrocyte suspensions (2 ml) at a final hematocrit of 5% in choline Cl medium containing [methyl-¹⁴C]choline⁺ (1 µCi/ml) were incubated in the absence or presence of 1 mM ATP for up to 40 min at 37°C. At 2, 20, or 40 min, cells were washed three times with ice-cold isotonic saline containing 1 mM choline-Cl (1,800 g for 60 s). To determine the level of [¹⁴C]choline⁺ uptake, 50-µl aliquots of erythrocyte pellets were incubated with 1 ml of SOLVABLE tissue solubilizer for 1 h at 60°C, followed by incubation with 100 µl of 100 mM EDTA and 500 µl of 30% H₂O₂ for 30 min at room temperature and then for a further 30 min at 60°C before the addition of 10 ml of Ultima Gold. The level of radioactivity was measured using a Packard (Meriden, CT) Tri-Carb 2100TR liquid scintillation analyzer. The Hb content of erythrocyte pellets (50 µl) was measured spectrophotometrically using Drabkin's reagent. The Hb content was used to determine the level of choline⁺ uptake per milliliter of canine and human erythrocytes based on mean cell Hb concentrations of 5.4 and 5.2 µmol Hb/ml, respectively.

Ethidium⁺ uptake measurements. Mononuclear cells were prelabeled with FITC-conjugated anti-CD14 MAb and resuspended in 1 ml of KCl medium at 37°C. At 0 s, 25 µM ethidium⁺ was added, followed 40 s later by the addition of ATP as indicated. In some experiments, cells were preincubated for 5 min at 37°C in the presence of KN-62 or an equal volume of DMSO vehicle. Data were collected using a Becton Dickinson (San Jose, CA) FACScalibur flow cytometer over 6 min at 37°C with constant stirring using a Cytek (Fremont, CA) Time Zero module. The linear mean channel of ethidium⁺ fluorescence intensity for each gated population over successive 5-s intervals was analyzed using WinMDI 2.8 software developed by Joseph Trotter (http://www.scripps.edu) and plotted against time. Nucleotide-induced ethidium⁺ uptake was quantified as the difference in arbitrary units of area under the uptake curves in the presence and absence of nucleotide in the first 5 min of incubation.

PS exposure assay. Erythrocytes, resuspended in NaCl medium (without BSA) at a final hematocrit of 2%, were added to 96-well U-bottom plates (200 µl/well) and incubated in the absence or presence ATP or BzATP for 24 h at 37°C and 95% air-5% CO₂. Erythrocytes (20 µl) were washed in 2 ml of annexin V-binding buffer (NaCl medium without BSA but containing 5 mM CaCl₂) and centrifuged at 450 g for 3 min. Erythrocytes were incubated at room temperature with 100 µl annexin V-FLUOS diluted 1:100 in annexin V-binding buffer. After 15 min, 400 µl of annexin V-binding buffer were added, and fluorescence intensity was measured using flow cytometry and analyzed using CellQuest Pro software (Becton Dickinson).

Hb release measurements. Erythrocytes were incubated in the absence or presence of ATP or BzATP as for the PS exposure assay. Plates were then centrifuged (1,800 g for 5 min), and 100 µl of cell-free supernatants were stored at –30°C for 2 h. Cells and the remaining supernatant in wells were also stored at –30°C for 2 h (cell lysates). The Hb content of supernatants and cell lysates was assayed spectrophotometrically using Drabkin's reagent, and the percent hemolysis was determined.

Statistical analyses. Results are means (SD). Differences between treatments were compared using either the unpaired Student's t-test for single comparisons to control samples or ANOVA for multiple comparisons (with Bonferroni post hoc test), using SPSS 14.0 for Windows (SPSS, Chicago, IL) with P < 0.05 considered significant.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
P2X₇ receptors are present on canine erythrocytes. Our laboratory (52) has previously shown, using an anti-human P2X₇ antibody and confocal microscopy, that P2X₇ receptors are present on human erythrocytes. Using this same procedure, we examined whether P2X₇ receptors also are present on canine erythrocytes. Confocal microscopy showed P2X₇ to be present on erythrocytes obtained from two different dogs with a punctate pattern of labeling on all cells present (Fig. 1A and results not shown). As a positive control and for comparison, erythrocytes from a human subject were examined and showed P2X₇ receptor immunoreactivity (Fig. 1B). Labeling in the absence of anti-P2X₇ antibody was negligible (results not shown).

View larger version (69K): [in this window] [in a new window]   Fig. 1. Expression of P2X7 receptors on canine and human erythrocytes by confocal microscopy. Fixed smears of canine (A) or human erythrocytes (B) were incubated in the presence (A and B) or absence (not shown) of sheep anti-P2X7 polyclonal antibody and then with Cy3-conjugated anti-sheep IgG antibody before examination by confocal microscopy. Labeling in the absence of anti-P2X7 antibody was negligible. The calibration bar is 4 µm.

View larger version (25K): [in this window] [in a new window]   Fig. 2. Expression of P2X7 receptors on canine and human erythrocytes by immunoblotting. Proteins of Hb-free ghost membranes were separated by gel electrophoresis and transferred to nitrocellulose membranes. Nitrocellulose membranes were incubated with anti-P2X7 or anti-actin antibody and then with horseradish peroxidase-conjugated anti-sheep or anti-rabbit Ig antibody, respectively, before visualization with chemiluminescent substrate. Actin served as a loading control. Representative results of 2 experiments are shown.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Time course of ATP-induced 86Rb+ efflux from canine and human erythrocytes. 86Rb+-loaded canine or human erythrocytes resuspended in NaCl (A) or KCl medium (B) were incubated at 37°C for up to 40 min in the absence or presence of 1 mM ATP. 86Rb+ efflux was calculated as the difference in percent release between 0 and 2, 20, or 40 min. Results are means (SD) (n = 3).

P2X₇ agonists induce ⁸⁶Rb⁺ efflux from canine erythrocytes. The dose effect of ATP and the most potent P2X₇ agonist, BzATP (16, 57), on ⁸⁶Rb⁺ efflux from canine erythrocytes was then studied. Cells loaded with ⁸⁶Rb⁺ were resuspended in NaCl medium and incubated at 37°C with varying concentrations of ATP or BzATP for 12 min. The efflux of ⁸⁶Rb⁺ from erythrocytes after 12 min of incubation ranged from 2.4% (SD 0.7) in the absence of nucleotide to 61.9% (SD 1.8) with 2 mM ATP and 71.8% (SD 3.4) with 200 µM BzATP (Fig. 4). EC₅₀ values of 309 µM (SD 14) for ATP and 7 µM (SD 1) for BzATP were obtained from these dose-response curves, which are comparable to respective EC₅₀ values for BzATP-and ATP-induced cation fluxes mediated by recombinant murine, rat, and human P2X₇ receptors (7).

View larger version (20K): [in this window] [in a new window]   Fig. 4. Dose-response curves for ATP- and 2'(3')-O-(4-benzoylbenzoyl) adenosine 5'-trisphosphate (BzATP)-induced 86Rb+ efflux from canine erythrocytes. 86Rb+-loaded erythrocytes resuspended in NaCl medium were incubated at 37°C for 12 min in the presence of varying ATP or BzATP concentrations as indicated. 86Rb+ release, calculated as the difference in percent release between 0 and 12 min, was used to determine the percentage of maximal response to 2 mM ATP or 200 µM BzATP. Results are means (SD) (n = 3).

View larger version (19K): [in this window] [in a new window]   Fig. 5. P2X7 agonists induce 86Rb+ efflux from human erythrocytes. 86Rb+-loaded erythrocytes resuspended in NaCl medium were incubated at 37°C for 12 min in the absence (control) or presence of 1 mM nucleotide or adenosine (as indicated) except for BzATP (100 µM). 86Rb+ efflux was calculated as the difference in percent release between 0 and 60 min. *P < 0.01; **P = 0.001 compared with control. Results are means (SD) from 3 different animals. ATPS, adenosine 5'-O-(3-thiotriphosphate); 2MeSATP, 2-methylthioadenosine 5'-triphosphate.

P2X₇ antagonists inhibit ATP-induced ⁸⁶Rb⁺ efflux from canine erythrocytes. To confirm that ATP-induced ⁸⁶Rb⁺ efflux from canine erythrocytes was mediated by P2X₇ activation, we preincubated ⁸⁶Rb⁺-loaded cells in the absence or presence of OxATP, KN-62, or BBG, antagonists of human, mouse, and rat P2X₇ (17, 28, 40), and the ATP-induced ⁸⁶Rb⁺ effluxes were measured. Incubation with 300 µM OxATP, 1 µM KN-62, or 1 µM BBG significantly inhibited 1 mM ATP-induced ⁸⁶Rb⁺ efflux by 98.51% (SD 1.4) (P < 0.001), 92.7% (SD 3.5) (P < 0.001), and 94.2% (SD 5.9) (P = 0.003), respectively (Fig. 6). Neither OxATP nor KN-62 altered the basal release of ⁸⁶Rb⁺ from erythrocytes incubated in the absence of ATP. In contrast, BBG increased the basal release of ⁸⁶Rb⁺ in the absence of ATP compared with erythrocytes incubated without both BBG and ATP; however, this failed to reach significance (P = 0.058).

View larger version (11K): [in this window] [in a new window]   Fig. 6. P2X7 antagonists impair ATP-induced 86Rb+ efflux from canine erythrocytes. 86Rb+-loaded erythrocytes resuspended in NaCl medium were preincubated at 37°C for 60 min in the absence or presence of 300 µM oxidized ATP (OxATP; A) or for 5 min in the absence or presence of DMSO vehicle or 1 µM KN-62 (B) or 1 µM Brilliant blue G (BBG; C) and then incubated at 37°C for 12 min in the absence (control) or presence of ATP. 86Rb+ efflux was calculated as the difference in percent release between 0 and 12 min. *P < 0.001 compared with control. P < 0.01; P < 0.001 compared with corresponding ATP-treated sample. Results are means (SD) (n = 3).

View larger version (18K): [in this window] [in a new window]   Fig. 7. ATP induces choline+ uptake into canine and human erythrocytes. Canine or human erythrocytes resuspended in 150 mM choline-Cl medium containing [methyl-14C]choline+ (1 µCi/ml) were incubated at 37°C for up to 40 min in the absence or presence of 1 mM ATP. The level of choline+ uptake at each time point was determined as µmol of choline+ per milliliter of cells. Results are means (SD) (n = 4).

View larger version (23K): [in this window] [in a new window]   Fig. 8. ATP induces ethidium+ uptake into canine and human monocytes. Canine or human mononuclear cells were prelabeled with FITC-CD14 MAb 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). The mean channel of cell-associated fluorescence was measured using time-resolved flow cytometry for CD14+ monocytes incubated in the absence or presence of ATP. Representative results from 3 experiments are shown.

View larger version (18K): [in this window] [in a new window]   Fig. 9. A P2X7 antagonist impairs ATP-induced ethidium+ uptake into canine monocytes. Canine mononuclear cells were prelabeled with FITC-CD14 MAb and resuspended in KCl medium at 37°C. Cells were preincubated at 37°C for 5 min in the absence or presence of DMSO vehicle or 1 µM KN-62. Ethidium+ (25 µM) was then added, followed 40 s later by the addition of 1 mM ATP (arrow). The mean channel of cell-associated fluorescence was measured using time-resolved flow cytometry for CD14+ monocytes incubated in the absence (basal) or presence of ATP. Representative results from 3 experiments are shown.

View larger version (16K): [in this window] [in a new window]   Fig. 10. Flow cytometric profiles of phosphatidylserine (PS) exposure on canine and human erythrocytes incubated with P2X7 agonists. Canine or human erythrocytes resuspended in NaCl medium (without BSA) were incubated at 37°C and 95% air-5% CO2 for 24 h in the absence (control) or presence of 1 mM ATP or 100 µM BzATP. Cells were labeled with annexin V-FLUOS, and fluorescence intensity was measured using flow cytometry. The percentage of erythrocytes binding annexin V-FLUOS was calculated from the marker region as shown. Representative results from 6 experiments are shown.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Over 30 years ago, Parker and Snow demonstrated that extracellular ATP but not other nucleotides could increase the permeability of canine erythrocytes to small cations (43), and this effect was confirmed by other investigators (11, 49). Although the molecular target of ATP was never identified in this process, the discovery of the P2X receptor family (42), and in particular the discovery of the P2X₇ receptor on human erythrocytes (52), suggests the involvement of the canine homolog of P2X₇. The present data demonstrate that immunoreactive P2X₇ receptors are present in canine erythrocytes and that their function, as assessed using cation fluxes, is at least 20 times greater than that of human erythrocytes. Immunocytochemistry and immunoblotting with an anti-human P2X₇ polyclonal antibody (61) demonstrated P2X₇ on canine erythrocytes (Figs. 1 and 2). This antibody recognizes a 17-amino acid epitope within the extracellular domain of the human P2X₇ receptor (KVKGIAEVKEEIVENGVK), which differs by four residues (underlined) from the homologous epitope in the canine P2X₇ receptor (KVKGTAEVKMEILENGIK) as reported by the Canine Genome Project (GenBank accession no. XM_534669) (36). The effect of extracellular ATP on ⁸⁶Rb⁺ (K⁺) efflux from canine erythrocytes also was examined as previously described for human erythrocytes (52). ATP increased the ⁸⁶Rb⁺ efflux from canine erythrocytes to an extent similar (Fig. 3) to that reported previously (43, 49), with cation equilibration reached by 40 min. The effect of ATP and other nucleotides was characteristic of P2X₇ receptors found in other species. First, BzATP was more potent than ATP in causing ⁸⁶Rb⁺ efflux from canine erythrocytes with EC₅₀ values of 7 and 309 µM, respectively (Fig. 4), values that are similar to those for recombinant human and rodent P2X₇ receptors (7, 46, 57). Second, the rank order of agonist potency (BzATP > ATP > 2MeSATP > ATPS) (Fig. 5) was typical of other mammalian P2X₇ receptors (16, 57). Third, ATP-induced ⁸⁶Rb⁺ efflux was inhibited by OxATP, KN-62, and BBG (Fig. 6), antagonists of human and/or rodent P2X₇ receptors (17, 28, 40). Fourth, P2X₇ activation induces the uptake of choline⁺ into murine macrophages (10) and human B lymphocytes (15), and we similarly observed that ATP induced choline⁺ uptake into canine erythrocytes (Fig. 7). Finally, both BzATP and ATP induced PS exposure (Fig. 10) and hemolysis (cytolysis) in canine erythrocytes, two well-known downstream events of P2X₇ activation in leukocytes (8).

Comparison of P2X₇ function in canine and human erythrocytes in this study, as well as between previous studies (43, 52), demonstrates that P2X₇ function as determined by ATP-induced ⁸⁶Rb⁺ efflux and choline⁺ uptake is at least 20 times greater in canine erythrocytes than in human erythrocytes (Figs. 3 and 7). Moreover, ATP and BzATP induce at least five times the amount of PS externalization in canine erythrocytes compared with human erythrocytes (Fig. 10), and these agonists also induce significant hemolysis of canine but not human erythrocytes. The level of ATP- and BzATP-induced PS exposure in human erythrocytes, as well as the absence of nucleotide-induced hemolysis, was similar to that previously reported (54). The differences between the two species in regard to P2X₇ function is specific to erythrocytes and is not due to canine P2X₇ per se, since P2X₇ function in canine monocytes, measured as ATP-induced ethidium⁺ uptake, was three times lower than the P2X₇ function in human monocytes tested in this study (Fig. 8) and within the lower end of the range for wild-type human monocytes obtained in previous studies in our laboratory (21, 53). Immunoblotting with an anti-human P2X₇ antibody revealed a more intense band at 75 kDa, the predicted size of P2X₇, in canine erythrocytes compared with human erythrocytes despite equal protein loading and similar levels of actin (Fig. 2). This indicates that a difference in P2X₇ expression is the most likely cause for differences in P2X₇ function between canine and human erythrocytes. We cannot, however, exclude differences in expression or function of secondary messengers and/or molecules thought to be responsible for P2X₇ pore formation (13). In this regard, pannexin-1, which is present in human erythrocytes (37), was recently implicated as the pore-forming unit of the P2X₇ receptor (38, 45); thus relatively higher levels of pannexin-1 in canine erythrocytes may also explain the high P2X₇ function in this cell type. Differences in levels of P2X₇, pannexin-1, or secondary messengers in canine and human erythrocytes may be due to variations in the proteolytic systems responsible for maturation-associated degradation in reticulocytes (2), which exist in both canine and human red blood cells (25, 47).

It is unlikely that variations in protein structure account for the differences observed between canine and human erythrocyte P2X₇ function. Comparison between the protein sequences for the canine (GenBank accession no. XM_534669) and human P2X₇ receptors (GenBank accession no. Y09561) revealed an overall sequence homology of 85%.¹ Canine P2X₇, like human P2X₇, comprises 595 amino acids, although an additional residue (asparagine at position 282)² is found in the extracellular loop in close proximity to the putative ATP-binding site (20, 48), whereas a valine residue (position 538) is absent from the intracellular carboxyl terminus. The canine P2X₇ receptor contains several conserved residues or motifs important in the expression or function of human and rodent P2X receptors. Residues (K64, F198/T199, N293/F/R295 and R308) important in the binding of ATP to P2X receptors (20, 48) are conserved within canine P2X₇. Also, cysteine residues within the extracellular domain and an intracellular motif (Y384/X/X/X/K388) are both important in the trafficking of P2X receptors (6, 12). In addition, the canine P2X₇ receptor, like other mammalian P2X₇ receptors, contains a putative phosphorylation site at Y344 (32) and a threonine-serine cluster (T358/Y/S/S361) (14), both of which regulate function (32, 51). Finally, the canine P2X₇ retains conserved residues at those polymorphic sites known to alter P2X₇ function or expression in humans (20, 21, 51, 53, 61) and mice (1).

ATP-induced ⁸⁶Rb⁺ efflux from canine erythrocytes was inhibited by >90% by three commonly used inhibitors of mammalian P2X₇ receptors, OxATP, KN-62, and BBG (Fig. 6). OxATP at concentrations of 100 and 300 µM was originally shown to completely block native P2X₇ in murine macrophages (40) and human lymphocytes (60), respectively. Subsequently, OxATP was also shown to inhibit recombinant human, murine, and rat P2X₇ receptors (22). KN-62 at 100 nM can completely block native P2X₇ in human lymphocytes (17), whereas 10-fold higher concentrations are required to completely inhibit recombinant and native murine P2X₇ (7, 22, 50). In contrast, several studies have repeatedly shown that KN-62 is a poor inhibitor of the rat P2X₇ receptor (7, 22, 24). BBG blocks both rat and murine P2X₇ with about 10-fold greater efficacy than human P2X₇, completely blocking the receptor at concentrations of 100 nM (22, 28). In our studies, 1 µM BBG caused a small amount of nonspecific lysis of canine erythrocytes, which was not apparent at 100 nM; however, at this concentration BBG only blocked ATP-induced ⁸⁶Rb⁺ efflux by 50% (results not shown). In summary, although we did not determine the relative potency of OxATP, KN-62, and BBG against the canine P2X₇ receptor, our results demonstrate that these antagonists would be useful in establishing the presence of native P2X₇ receptors in other canine cell types.

The physiological significance of P2X₇ on canine erythrocytes, as well as on human erythrocytes, is currently unknown. We have previously shown that P2X₇ activation induces PS exposure on human erythrocytes (54), whereas in this study 24-h incubation of canine erythrocytes with either ATP or BzATP also induced PS exposure (Fig. 10), with 17% of canine erythrocytes on average undergoing hemolysis. PS exposure on erythrocytes can lead to their recognition and removal by splenic macrophages and Kupffer cells, as well as adherence to endothelial cells (3, 34). Thus P2X₇ activation may play a role in the destruction of erythrocytes after their approximate 120-day life spans, a hypothesis consistent with the P2X₇-mediated hemolysis of canine erythrocytes. Alternatively, P2X₇ activation may lead to PS-exposing erythrocytes adhering to endothelium and contributing to vaso-occlusion (3, 34). Conversely, erythrocyte P2X₇ may play a role in vasodilation or antithrombotic functions, given that a recent study demonstrated that P2X₇ activation induces the release of epoxyeicosatrienoic acids from rat erythrocytes (27). P2X₇ activation also may play a role in vascular dysfunction, since hydrolysis of PS-exposing erythrocytes by secretory phospholipase A₂ can generate lysophosphatidic acid, which can increase loss of vascular integrity and eventual tissue damage (41). Finally, pannexin-1 is thought to mediate ATP release from erythrocytes (37); thus, if P2X₇ couples to pannexin-1 in these cells, it may trigger the release of ATP, which in turn may activate purinergic receptors on erythrocytes and endothelium. Further studies are required to determine the physiological role of P2X₇ receptors in erythrocytes and whether differences in P2X₇ function between species is of physiological significance, as well as whether P2X₇ couples to pannexin-1 in erythrocytes.

Canine erythrocytes also may express P2Y₁ and P2Y₁₃ receptors, as found in human erythrocytes (58, 59). P2Y₁ receptor activation is involved in the induction of an osmolyte permeability in malaria-infected and oxidized erythrocytes (58), whereas ADP activation of P2Y₁₃ receptors impairs ATP release from erythrocytes (59). Thus the current physiological roles of these P2Y receptors and the P2X₇ receptor in erythrocytes appear quite distinct; however, the possibility remains that these receptors may interact. Activation of erythrocyte P2Y₁ and possibly P2X₇ receptors by autocrine ATP may be limited by P2Y₁₃ receptor signaling induced by ADP resulting from the hydrolysis of ATP. Moreover, activation of erythrocyte P2Y receptors may inhibit P2X₇ receptors in these cells similarly to that observed with P2Y receptors and P2X₃ receptors in dorsal root ganglion neurons (18) and transfected cell systems (19). Further studies investigating whether canine erythrocytes express other purinergic receptors and the potential interactions between these receptors in erythrocytes are warranted.

In conclusion, we have demonstrated that canine erythrocytes express functional P2X₇ receptors and that this receptor is responsible for the long-known ability of extracellular ATP to mediate large increases in cation permeability in these cells. Identification of the canine P2X₇ receptor may help in providing a better understanding of P2X₇ receptor expression and function and of cation transport in erythrocytes.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by the National Health and Medical Research Council of Australia, Cure Cancer Australia, and Nepean Medical Research Foundation.

	FOOTNOTES

 

Address for reprint requests and other correspondence: R. Sluyter, Dept. of Medicine, Univ. of Sydney, Level 5 South Block, Nepean Hospital, Penrith, New South Wales, Australia (e-mail: rons{at}med.usyd.edu.au)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Comparison was made using Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi).

² Numbering of amino acid positions cited in this and subsequent instances are based on the canine P2X₇ receptor sequence (GenBank accession no. XM_534669).

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