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
the p2x receptor family (P2X1 to P2X7) of ligand-gated cation channels are emerging as important molecules in health and disease (31). The P2X7 receptor has been cloned and expressed from rat (57), human (46), mouse (7), Xenopus (44), zebrafish (33), and seabream (39). Like other P2X receptor members, the P2X7 receptor has intracellular amino and carboxyl termini with two hydrophobic membrane-spanning domains, separated by a long, glycosylated extracellular loop containing the proposed ATP-binding sites (42). The P2X7 receptor differs from other P2X receptors, having a relatively low affinity for ATP and a rank order of agonist potency of 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-trisphosphate (BzATP) > ATP > 2-methylthioadenosine 5′-trisphosphate (2MeSATP) > adenosine 5′-O-(3-thiotriphosphate) (ATPγS) (7, 46, 57). Activation of P2X7 opens a cation-selective channel, allowing an influx of Ca2+ and Na+ and efflux of K+ (7, 46, 57). Prolonged exposure to ATP induces a second permeability state, which allows the uptake of larger cations such as choline+, ethidium+, and Yo-Pro-12+ (7, 10, 15, 46, 57). Whether this second permeability state is due to dilatation of the P2X7 channel or activation/recruitment of a second molecule is the subject of much debate (29, 42, 55), although recent data indicate that pannexin-1 is the pore-forming unit of the P2X7 receptor (45). A range of downstream events follow P2X7 activation, including phosphatidylserine (PS) exposure and cell lysis (8).
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
BSA, d-glucose, glycerol gelatin mounting medium, saponin, ATP, BzATP, ATPγS, 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 × 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).
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.
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.
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).
To confirm the presence of P2X7 on canine erythrocytes and to compare its relative expression to P2X7 on human erythrocytes, we performed immunoblotting of erythrocyte ghost membranes isolated from both species. Immunoblotting with the anti-P2X7 antibody revealed the presence of major band at 75 kDa, the predicted size of glycosylated P2X7 (Fig. 2). This 75-kDa band was repeatedly stronger in canine erythrocytes compared with human erythrocytes despite similar levels of actin (Fig. 2). Bands also were evident with the anti-P2X7 antibody at 220 and 72 kDa (Fig. 2), which may represent trimeric P2X7 and differences in P2X7 glycosylation, respectively.
ATP induces 86Rb+ efflux from canine erythrocytes.
Extracellular ATP induced a near complete loss of 42K+ from canine erythrocytes within 40 min (43); therefore, the effect of extracellular ATP on 86Rb+ (a surrogate of K+) efflux in canine erythrocytes was studied as previously carried out for human erythrocytes (52). 86Rb+-loaded cells resuspended in NaCl medium were incubated at 37°C with 1 mM ATP for up to 40 min with samples collected at 2, 20, and 40 min. ATP induced a significant increment in 86Rb+ efflux from canine erythrocytes at all three time points compared with cells incubated in the absence of ATP for the same length of time (P = 0.008, P < 0.001, and P < 0.001, respectively) (Fig. 3A). Comparison of human erythrocytes demonstrates that ATP induced a far smaller increment in 86Rb+ efflux (Fig. 3A), which only attained significance at longer times of incubation (52).
Similar results were observed when canine erythrocytes were incubated in a KCl medium, with ATP inducing a significant increment in 86Rb+ efflux from cells at 2, 20, and 40 min compared with cells incubated in the absence of ATP for the same length of time (all P < 0.001) (Fig. 3B). In human erythrocytes, ATP induced a small increment in 86Rb+ efflux at 2, 20, and 40 min (P = 0.016 at 40 min), which was far smaller than that observed with canine erythrocytes (Fig. 3B).
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).
In addition to ATP and BzATP, the partial P2X7 agonists (16, 57) 2MeSATP and ATPγS (1 mM) induced a smaller but significant 86Rb+ efflux of 16.4% (SD 2.2) (P = 0.005) and 14.6% (SD 1.8) (P = 0.008), respectively (Fig. 5). Agonists of P1 and other P2 receptors, adenosine, AMP, ADP, and UTP (1 mM), resulted in a release of 86Rb+ similar to that of control values (Fig. 5). Thus, for canine erythrocytes, the ability of nucleotides to induce 86Rb+ efflux follows a rank order of BzATP > ATP > 2MeSATP > ATPγS, which is identical to that observed for cation fluxes mediated by rat and human P2X7 receptors (16, 57). UTP, ADP, AMP, and adenosine were ineffective, thus excluding a role for many of the P2Y and adenosine receptors, some of which are found in mammalian and avian erythrocytes (4, 56, 58, 59).
The percent release of Hb after 12 min of incubation with BzATP or ATP was measured using Drabkin's reagent. Hb release in the absence of nucleotide was 1.1% (0.5 SD) (n =3). BzATP but not ATP induced a small but significant release of Hb from erythrocytes: 3.7% (SD 11.7) (P = 0.024) and 2.7% (SD 1.1) (P = 0.084), respectively (n =3). Thus the ATP- and the majority of the BzATP-induced 86Rb+ release was a result of efflux rather than hemolysis.
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).
ATP induces choline+ uptake into canine and human erythrocytes.
To determine whether the difference in P2X7 function between canine and human erythrocytes was also apparent with larger, more complex cations and thus directly implicating the P2X7 pore, we measured the ability of ATP to induced choline+ uptake into these cells. Erythrocytes resuspended in 150 mM choline-Cl medium containing [14C]choline+ were incubated at 37°C with 1 mM ATP for up to 40 min with samples collected at 2, 20, and 40 min. ATP induced a significant increment in choline+ uptake into canine erythrocytes at all three time points compared with cells incubated in the absence of ATP for the same length of time (all P < 0.001) (Fig. 7). Comparison of human erythrocytes showed that ATP induced a far smaller increment in choline+ uptake (Fig. 7), which only reached significance at 40 min (P = 0.043).
ATP induces ethidium+ uptake into canine monocytes.
Collectively, the above data indicate that functional P2X7 receptors are expressed on canine erythrocytes and that P2X7 function in this cell type is at least 20 times higher than in human erythrocytes. Therefore, to determine whether this difference was limited to canine erythrocyte P2X7 or canine P2X7 per se, we compared the ability of ATP to induce ethidium+ uptake in canine monocytes with that in CD14+ human monocytes. Mononuclear cells isolated from dogs or humans were prelabeled with FITC-conjugated anti-human CD14 MAb (clone TUK4), and the ability of 1 mM ATP to induce ethidium+ uptake over 5 min was measured using time-resolved flow cytometry as described previously (30). The anti-human CD14 MAb binds to canine monocytes, but not canine lymphocytes or granulocytes (26), as well as to human monocytes. ATP induced the uptake of ethidium+ into CD14+ canine monocytes with a mean uptake of 11,251 arbitrary units of area under the curve (SD 1,199) (Fig. 8). The ATP-induced uptake of ethidium+ into CD14+ human monocytes was on average three times higher (P < 0.001) than that of canine monocytes, with a mean uptake of 36,177 arbitrary units of area under the curve (SD 2,264) (Fig. 8). The uptake of ethidium+ into CD14+ canine and human monocytes in the absence of ATP was minimal (Fig. 8).
The effect of the P2X7 antagonist KN-62 on the ATP-induced ethidium+ uptake into CD14+ canine monocytes was measured. ATP induced ethidium+ uptake into CD14+ canine monocytes preincubated with DMSO vehicle with a mean uptake of 18,925 arbitrary units of area under the curve (SD 362) (Fig. 9). Preincubation of cells with 1 μM KN-62 significantly inhibited the ATP-induced ethidium+ uptake by 89.3% (SD 2.0) (Fig. 9). Collectively, these results demonstrate that ATP can induce the uptake of ethidium+ into CD14+ canine monocytes via activation of the P2X7 receptors and that canine monocytes do not share the very high P2X7 function of erythrocytes from this species.
ATP induces PS exposure and hemolysis in canine erythrocytes.
We have recently shown that P2X7 activation induces PS exposure in human erythrocytes (54). Therefore, we compared ATP- or BzATP-induced PS exposure in canine and human erythrocytes. Erythrocytes were incubated with 1 mM ATP or 100 μM BzATP for 24 h, and PS exposure was measured as annexin V-FLUOS binding. Annexin V-FLUOS binding on canine erythrocytes following incubation in the absence of nucleotide was 1.0% (SD 0.6) (Fig. 10). Both ATP and BzATP induced massive PS exposure with 88.6% (SD 8.7) and 98.8% (SD 1.1) of erythrocytes binding annexin V-FLUOS, respectively (both P < 0.001) (Fig. 10). Similar to our previous findings (54), ATP and BzATP induced a much smaller PS exposure in human erythrocytes with 13.7% (SD 5.8) and 21.3% (SD 7.0) of cells binding annexin V-FLUOS, respectively (Fig. 10). Annexin V-FLUOS binding on human erythrocytes following incubation in the absence of nucleotide (4.9%, SD 1.1) was significantly lower in response to either agonist (both P < 0.001) (Fig. 10).
In our previous study (54), overnight incubation with either ATP or BzATP failed to induce significant hemolysis of human erythrocytes; however, it became apparent during the PS exposure assays that both ATP and BzATP caused hemolysis of some canine erythrocytes. Spectrophotometric measurements of Hb using Drabkin's reagent demonstrated that 24-h incubation with ATP or BzATP induced significant hemolysis of canine erythrocytes with a Hb release of 16.4% (SD 3.6) and 17.2% (SD 4.4), respectively (n = 6) (both P < 0.001). In contrast, Hb release from canine erythrocytes incubated in the absence of nucleotide was 1.0% (0.6) (n = 6). Again as previously observed (54), Hb release from human erythrocytes following 24-h treatment with ATP and BzATP was negligible with values of 0.6% (0.5) and 0.8% (0.7) respectively, which were not significantly different from control values of 0.5% (0.5) (n = 6) (P = 0.759 and 0.343, respectively).
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 > ATPγS) (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.
This work was supported by the National Health and Medical Research Council of Australia, Cure Cancer Australia, and Nepean Medical Research Foundation.
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