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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3172-3181
The P2X1 Receptor, an Adenosine Triphosphate-Gated
Cation Channel, Is Expressed in Human Platelets but not in Human
Blood Leukocytes
By
Erin E. Clifford,
Karen Parker,
Benjamin D. Humphreys,
Sylvia B. Kertesy, and
George R. Dubyak
From the Department of Physiology and Biophysics, School of Medicine,
Case Western Reserve University, Cleveland, OH.
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ABSTRACT |
Extracellular adenosine triphosphate (ATP) and adenosine diphosphate
(ADP) activate multiple types of P2-nucleotide receptors expressed in
platelets or leukocytes. Electrophysiological and biochemical studies
have indicated expression of the P2X1 receptor, an
ATP-gated cation channel, in human and rat platelets, rat basophilic leukemia (RBL) cells, and phorbol myristate acetate
(PMA)-differentiated HL-60 myeloid cells. Although these findings
suggest that P2X1 receptors are present in both blood
leukocytes and blood platelets, the relative levels of P2X1
receptor expression and function in human blood leukocytes and
platelets have not been directly characterized. On the basis of both
immunoblot analysis and functional assays of P2X1
receptor-mediated ionic fluxes, we report that there is significant
expression of P2X1 receptors in human platelets, but not in
neutrophils, monocytes, or blood lymphocytes. Thus, unlike platelets
and myeloid progenitor cell lines, fully differentiated human blood
leukocytes do not express functionally significant numbers of
P2X1 receptors, suggesting the downregulation of
P2X1 receptor gene expression during the differentiation of
phagocytic leukocytes. By contrast, P2X1 receptor
expression is strongly maintained during megakaryocytic differentiation
and platelet release. Immunoblot analysis indicated that the platelet
P2X1 receptor migrates as an approximately 60-kD protein
during SDS-electrophoresis under reducing or nonreducing conditions.
Treatment of platelet membranes with endoglycosidase-F causes the
P2X1 receptor band to migrate as a 46-kD protein, verifying
the highly glycosylated nature of the mature receptor protein.
Additional studies of nucleotide-induced changes in Ca2+
influx/mobilization demonstrated that the platelet P2X1
receptors are pharmacologically distinct from the well-characterized
ADP receptors of these cells. This finding suggests a unique role for
these ATP-gated ion channels during hemostasis or thrombosis.
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INTRODUCTION |
EXTRACELLULAR adenosine triphosphate
(ATP) affects the function of many tissues and cell types. ATP can be
released by cytolysis or by the regulated exocytosis of vesicles and
granules containing ATP.1 This released ATP can be rapidly
metabolized to adenosine diphosphate (ADP) or adenosine
monophosphate (AMP) or both, by ecto-ATPases and
ecto-apyrases.2,3 Biologic responses to ATP and other
extracellular nucleotides are mediated by P2-nucleotide receptors
belonging to two major classes: G-protein-coupled P2Y receptors and
nucleotide-gated ion-channel P2X receptors.4 Each class
comprises a number of pharmacologically and genetically distinct
subtypes.5
Multiple types of P2-nucleotide receptors are expressed in blood cells
and the levels of certain P2 receptors can be rapidly modulated during
activation of these cells or during differentiation of their
hematopoietic progenitors. This suggests that released nucleotides are
involved in multiple types of paracrine and autocrine signaling between
blood cells and other cell types. ADP, which is copackaged with ATP in
platelet dense granules, is a potent and well-characterized activator
of many platelet responses including adherence, shape change, granule
release, aggregation, and thromoboxane A2
(TXA2) production.6-8 The platelet receptor
that mediates these effects of ADP has been termed P2T (for
"thrombocyte") and exhibits a unique pharmacology in that ADP is
agonistic, whereas ATP is antagonistic. Despite the large number of
reports describing platelet responses to ADP, the molecular nature of
the putative P2T receptor remains undefined. Some studies suggest that
P2T receptor signaling may reflect the combined activation of two, or
more, of the already cloned P2 receptor subtypes.9,10 Leon et al9 reported that cell lines expressing recombinant
human P2Y1 receptor cDNA exhibit nucleotide-induced
increases in intracellular Ca2+ with a pharmacological
profile similar to that of P2T receptor responses in platelets.
Moreover, since P2Y1 receptor mRNA was found in both human
platelets and various megakaryocytic cell lines, these investigators
suggested that the P2Y1 receptor may be the
G-protein-coupled ADP receptor of platelets.9 The
hypothesis that this ADP receptor is likely to be a conventional
G-protein-coupled receptor is supported by recent studies of
transgenic mice that lack expression of the  -subunit of
Gq.11 Platelets from these Gq-deficient mice exhibit none
of the conventional responses to ADP.
Human platelets and rat megakaryocytes additionally express
ATP/ADP-gated ion channel receptors with properties similar to those of
the cloned P2X1 ionotropic receptor.10,12,13
These findings are consistent with recent molecular studies describing the presence of P2X1 receptor mRNA transcripts in human
platelets and megakaryocytic cell lines.14 However, the
role of these P2X1 receptor channels in the function of
blood platelets remains unclear. Although P2X1 receptors
have been studied most extensively in vascular and visceral smooth
muscle cells,15 Northern blot analysis showed very high
levels of P2X1 receptor mRNA in the total leukocyte
fraction of human blood.16 Moreover, P2X1
receptor protein and function have been characterized in phorbol
myristate acetate (PMA)-differentiated HL-60 cells and rat basophilic
leukemia (RBL) cells.17 The sequence of the
P2X1 receptor initially cloned from rat vas deferens was
found to be identical to an orphan mRNA (RP-2) upregulated in apoptotic
rat thymocytes.18 These observations suggest that
P2X1 receptors may be expressed not only in platelets, but
also in the leukocytes from human peripheral blood. To test this, we
have assayed expression of P2X1 receptor protein and P2X1 receptor function in various human blood cell types.
We report that the P2X1 receptor is not significantly
expressed in blood neutrophils, lymphocytes, or monocytes, but confirm
and extend previous studies regarding the high expression of these
ATP-gated ion channels in platelets.
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MATERIALS AND METHODS |
Isolation of platelets and leukocytes.
Venous blood (40 to 50 mL) from healthy volunteers was collected into 6 to 7 mL sterile acid-citrate-dextrose (ACD) 100 mmol/L disodium
citrate, and 128 mmol/L D-glucose, pH 5. For isolation of
platelets, the citrated whole blood was centrifuged in 50-mL polypropylene tubes at 200g for 15 minutes, and the
platelet-rich plasma was removed and centrifuged at 2,500g for
15 minutes. The pelleted platelets were resuspended and washed twice in
phosphate-buffered saline (PBS) supplemented with 13 mmol/L trisodium
citrate (PBS-citrate) and 1 mg/mL bovine serum albumin (BSA). A similar
protocol was used to obtain a mixed buffy-coat fraction containing all
leukocytes and platelets by initially centrifuging the citrated whole
blood at 2,500g for 15 minutes. The initial buffy coat was
removed, resuspended in PBS-citrate, and recentrifuged at
2,500g. For isolation of neutrophils and mononuclear
leukocytes, citrated whole blood was diluted 1:2 with PBS-citrate;
30-mL aliquots were layered over 12 mL Histopaque-1077 (Sigma, St
Louis, MO) and centrifuged at 400g for 30 minutes. Mononuclear
leukocytes banding on top of the Histopaque layer were collected into
PBS-citrate and subjected to three cycles of resuspension and
centrifugation (200g, 10 minutes). The neutrophil/erythrocyte
pellet beneath the Histopaque was suspended in 3 vol of 155 mmol/L
NH4Cl, 10 mmol/L KHCO3, 1 mmol/L EDTA, pH 7.4, and incubated on ice for 10 minutes to lyse the erythrocytes. This
resulted in a cell fraction predominated by neutrophils (>95%), as
judged by myeloperoxidase (MPO) staining.
Cell culture of hematopoietic cell lines.
HL-60 human promyelocytic leukemia cells, THP-1 human monocytic
leukemia cells, and BAC1.2F5 murine macrophage cells were cultured
using previously described protocols.19,20 HL-60 cells were
differentiated into either macrophage-like cells by treatment for 2 days with 100 nmol/L PMA or into granulocyte-like cells by treatment
for 3 days with .5 mmol/L dibutyryl cyclic AMP. THP-1 cells were
differentiated toward an inflammatory macrophage phenotype by a 2-day
treatment with either 100 nmol/L PMA or the combination of 100 ng/mL
lipopolysaccharide (LPS), (List Biologicals, Campbell, CA)
and 1,000 U/mL recombinant human interferon- (IFN-) (a gift from
Genentech, San Francisco, CA).
Generation of HEK-293 cells stably expressing recombinant
P2X1 receptors.
HEK-293 (human embryonic kidney) cells were maintained in Dulbecco's
minimal essential medium (DMEM) supplemented with penicillin (100 U/mL)
streptomycin (100 µg/mL), and 10% iron-supplemented newborn calf serum (Hyclone, Logan, UT). HEK-293 cells were stably transfected by electroporation (Bio-Rad Laboratories, Richmond, CA; 500 µF and 300 mV, time constant 7 to 10 ms) with 20 µg of the rat
P2X1 receptor cDNA (in pcBK-CMV, a generous gift from G. Buell and A. Surprenant, Glaxo Institute for Molecular
Biology, Geneva, Switzerland). Selection was accomplished with G418
(570 µg/mL) added 48 hours after transfection. After 3 weeks of
selection, approximately 500 individual, resistant colonies were
pooled, and these stably transfected cultures were maintained under
continuous G418 selection. These cells (HEK-P2X1) and the
nontransfected, parental HEK-293 line were used as positive and
negative controls for Western blot analysis and electrophysiological
studies of P2X1 receptors.
Membrane isolation and processing.
Blood cells or cultured cell lines were pretreated with PBS containing
4 mmol/L DIFP (diisopropyl fluorophosphate) for 20 minutes on ice.
Cells were washed twice and resuspended with lysis buffer (137 mmol/L
NaCl, 8.1 mmol/L Na2HPO4, 2.7 mmol/L KCl, 1.5 mmol/L KH2PO4, and 2.5 mmol/L EDTA) containing
protease inhibitors (100 µmol/L phenylmethylsulfonyl fluoride (PMSF),
10 µg/mL aprotinin, and 10 µg/mL leupeptin). Cells were lysed
either by sonication (Branson model 185 sonifier, power setting 7) for
30 seconds or by nitrogen cavitation for 20 minutes at 400 psi
(platelets at 1,200 psi). Total cell membranes were then pelleted in a
Beckman TLOPTM tabletop ultracentrifuge (100,000g for 30 minutes at 4°C). Membranes were resuspended and homogenized in lysis
buffer plus protease inhibitors and membrane protein was quantified
using Bio-Rad Protein Assay solution. Membranes were stored at
 80°C for subsequent analysis or were immediately processed for
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Membrane samples were routinely mixed with Laemmli sample buffer (final
concentrations: 62.5 mmol/L Tris-HCl (pH 6.8), 2% SDS, 10% glycerol,
5%  -mercaptoethanol) and boiled for 5 minutes. However, for all
Western blot analyses of CD61 and some P2X1 receptor analyses, membrane samples were processed under nonreducing conditions by deleting the mercaptoethanol from the sample buffer. As positive control sources of membranes containing large numbers of
P2X1 receptors, rat vas deferens or urinary bladders were
minced and Dounce homogenized in lysis buffer supplemented with
protease inhibitors. The homogenate was centrifuged at 400g for
10 minutes, the supernatant was collected, and membranes were pelleted
by ultracentrifugation, as described above.
Antibodies.
An antiserum against P2X1 receptor protein was generated in
rabbits injected with two peptides corresponding to homologous sequences within the intracellular C-terminal domains of the cloned human16 and rat15 P2X1 receptors.
These peptides (LPKRHYYKQKKFKYILYAED) and (TSSTLGLQENMRTS),
respectively, correspond to residues 357 to 373 and residues 386 to 399 of the human P2X1 receptor sequence. Peptide injection,
care, and bleeding of the rabbits were performed by a commercial
service (Quality Controlled Biochemicals, Hopkintown, MA).
Antisera were initially screened by Western blot analysis of rat
urinary bladder and vas deferens membrane proteins.
A mouse MoAb against human CD61 (MoAb MCA728 from Serotec, Raleigh,
NC) was used to assay membrane content of CD61, the GPIIIa 3 integrin expressed in platelets.21 This MoAb does not
bind to CD61 treated with sulfhydryl reducing agents. Purified
GPIIb/IIIa from human platelets (a generous gift from Dr S. D'Souza,
Cleveland Clinic Research Foundation, Cleveland, OH) was
used as a positive control source of GPIIIa. A mouse MoAb against the
-subunit of the Na+,K+-ATPase (MoAb 9A7, a
generous gift from Dr Maureen McEnery, Case Western Reserve University,
Cleveland, OH) was used to assay the relative content of
plasma membrane in the total cell membrane fractions.22
Immunoblot analyses.
Membrane proteins were separated by SDS-PAGE (12%) and transferred
electrophoretically to polyvinylidene fluoride (PVDF) membranes for 15 hours at 30 mV. PVDF membranes were rinsed in immunoblot buffer (10 mmol/L Tris, pH 7.4; 0.9% NaCl; 0.05% Tween-20; 1 mmol/L EDTA) and
blocked with milk buffer (4% nonfat dried milk (Sigma Chemical Co, St
Louis, MO) in immunoblot buffer). After washing (1 × 15 minutes
2 × 5 minutes) with immunoblot buffer, the PVDF membranes were
incubated for 1 hour with primary antibodies dissolved in immunoblot
milk buffer. Anti-P2X1 receptor antiserum was used at
either a 1:500 dilution (bleed 2 antiserum) or a 1:3,000 dilution (bleed 3 antiserum); anti-CD61 was used at a 1:1,000 dilution; anti-Na+,K+-ATPase was used at a 1:10,000
dilution. Membranes were then washed and incubated for 1 hour at room
temperature with 1:5,000 dilutions of horseradish peroxidase
(HRP)-conjugated donkey anti-rabbit antibody (Amersham, Arlington
Heights, IL) or HRP-linked sheep anti-mouse (Santa Cruz, Santa Cruz,
CA) in milk buffer. Membranes were washed and developed
with chemiluminescence reagents (SuperSignal from Pierce, Rockford, IL)
for 1 to 5 min and exposed to Kodak x-ray film (Eastman-Kodak,
Rochester, NY). In some cases, the immunoblot was stripped of bound
antibodies using conventional methods and then reprobed with different
antibodies.
Endoglycosidase F treatment of membrane proteins.
Membrane proteins were extracted and denatured by boiling for 5 minutes
in 1% SDS. Duplicate aliquots of each denatured extract were diluted
1:5 in reaction buffer (50 mmol/L KH2PO4, 25 mmol/L EDTA, 1.3% N-octylglucoside, and 1.3% -mercaptoethanol, pH
7.0), vortexed, and treated overnight at 37°C with, or without, 1 U/mL endoglycosidase F/N-glucosidase F (Boehringer, Mannheim, Germany). The extracts were then processed for electrophoresis and immunoblotting as described.
Fluorimetric analysis of cytosolic Ca2+.
Platelets, neutrophils, or mononuclear leukocytes were suspended
(5 × 106/mL for white blood cells (WBCs) or
5 × 108/mL for platelets) in a Ca-free basal salt
solution (BSS) containing 130 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L
MgCl2, 25 mmol/L Na-HEPES, pH 7.5, 5 mmol/L
D-glucose, 1 mg/mL BSA, and 0.5 U/mL apyrase (Type V,
Sigma). The suspensions were supplemented with 1 µmol/L fura 2-AM
(Molecular Probes, Eugene, OR) and incubated at room temperature for 45 minutes. The cells were pelleted, resuspended in fresh BSS containing
apyrase, and stored at room temperature for up to 3 hours before assay.
Cytosolic Ca2+ was fluorimetrically assayed using equipment
and protocols previously described.19,20 Immediately before
assay, the fura 2-loaded cell suspensions were diluted 1:3 with normal
BSS or with reduced Na-BSS in which NaCl was iso-osmotically replaced
with 130 mmol/L NMG-Cl (N-methylglucamine chloride) in thermostatted
(37°C), stirred cuvettes. Each cuvette suspension was then
supplemented with 1 mmol/L CaCl2 and an additional 2 U/mL
apyrase. After thermal equilibration, cells were stimulated by addition
of nucleotides (ATP, ADP, or -methylene-ATP) or other
Ca-mobilizing agonists (thrombin for platelets, FMLP for neutrophils,
concanavalin A [ConA] for mononuclear leukocytes).
Electrophysiological analysis of ATP-gated cation channels in
monocytes.
For electrophysiological studies of blood monocytes, washed mononuclear
leukocytes were suspended at 106 cells/mL in Iscove's
medium (supplemented with 0.1% BSA), plated in Teflon culture dishes,
and incubated at 37°C in an atmosphere of 92.5% air/7.5%
CO2. These isolated mononuclear cells were analyzed for
P2X1 receptor activity within 5 to 6 hours after isolation. Aliquots of mononuclear leukocyte suspension were removed from the
Teflon culture dish and placed in the patch clamp chamber. After about
5 minutes, the chamber was extensively washed with external solution,
during which time the lymphocytes and platelets (most cells in the
suspension) were washed away. Bath flow was constant during the
experiments. Cells that were attached to the center of the patching
chamber in the direct flow of solution and appeared flattened were
considered to be monocytes and were used in patch-clamp experiments. As
positive controls, HEK293 cells stably expressing recombinant rat
P2X1 receptors were assayed using identical patch-clamp
methods and solutions. Cells were sealed to patch electrodes with
resistances of 4 to 20 m , and the whole cell configuration was
established by suction. Monocytes were perfused internally for several
minutes (during which time they rounded up and detached) before lifting
them off the chamber floor. All cells were held at 40 mV and
perfused internally for 3 to 5 minutes before they were challenged with
ATP. Voltages were commanded and the data recorded by computer using
the pClamp series of programs and an Axopatch 1-D patch clamp with a
TL-1 DMA interface. ATP, 100 µmol/L, was applied to cells suspended in the flowing bath solution by puffer pipette as previously
described.23 External solution contained 140 mmol/L sodium
chloride, 10 mmol/L HEPES, 10 mmol/L glucose, 5 mmol/L potassium
chloride, 2.5 mmol/L calcium chloride, and 0.5 mmol/L magnesium
chloride, brought to pH 7.4 with NaOH. Internal solution contained 120 mmol/L potassium chloride, 20 mmol/L tetraethyl ammonium chloride
(TEA-Cl), 10 mmol/L EGTA, 10 mmol/L HEPES and 5 mmol/L magnesium
chloride, brought to pH 7.3 with TEA-OH.
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RESULTS |
Immunologic characterization of P2X1 receptor expression
and processing in human platelets.
All seven cloned members of the P2X receptor family are predicted to
share the basic membrane topography illustrated in Fig 1A. This receptor topography
includes intracellular N- and C-teminal segments, two
transmembrane-spanning segments, and a large, extracellular loop that
contains multiple cysteines for intramolecular disulfide bonding and
sites for N-linked glycosylation.24 The C-terminal tails
constitute the regions of least homology among the seven P2X receptor
subtypes. Accordingly, a polyclonal antiserum against two peptides
corresponding to C-terminal sequences of the human (and rat)
P2X1 receptor was used to analyze the expression and processing of P2X1 receptor protein in human platelets (Fig
1B). An intensely immunoreactive band of about 60 kD was observed in lanes containing total membrane protein from platelets; a band in the
95- to 100-kD region showed weaker but significant immunoreactivity. The 60-kD platelet protein migrated slightly faster than the major anti-P2X1 reactive protein in an adjacent lane containing
total membrane protein from rat vas deferens, a tissue known to express very high levels of P2X1 receptor (Fig 1B). Although the
amino acid sequence of the cloned human P2X1 receptor codes
for a 45-kD protein, this sequence also predicts four glycosylation
sites in the large extracellular loop.16 Previous
photoaffinity labeling studies indicated that the molecular mass of
processed P2X1 receptors in rat urinary bladder membranes
was approximately 60 kD.25 To confirm that the 60-kD
immunoreactive protein from human platelets corresponded to
glycosylated P2X1 receptors, membrane extracts were
subjected to endoglycosidase-F treatment. A 46-kD protein was the major
immunoreactive species in these deglycosylated samples and the 60-kD
product was eliminated (Fig 1C).
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| Fig 1.
Immunologic characterization of P2X1 receptor
expression and processing in human platelets. (A) The predicted
membrane topography of the P2X1 receptor showing the
extracellular glycosylation sites ( ) and the C-terminal tail
location of the antigenic sites recognized by the anti-P2X1
antiserum. (B) Anti-P2X1 receptor immunoblot of human
platelet membranes (15 µg protein) versus rat vas deferens membranes
(1 µg). (<<<, 95 kD; <<, 60 kD; < 45 kD). (C) Deglycosylation of P2X1R protein expressed in
platelets. Parallel aliquots of platelet membranes were directly
processed for electrophoresis (right lane, ) or were denatured in
1% SDS and then incubated in the absence (left lane, ) or presence
(middle lane, +) of endoglycosidase-F, as described under Materials
and Methods. Twenty-five micrograms of protein was loaded in each lane.
(<<<, 95 kD; <<, 60 kD; < 45 kD).
(D) Comparative anti-P2X1 receptor immunoblot of platelet
membranes (30 µg) versus purified GPIIb/IIIa (400 ng) that were
processed under reducing conditions. (<<<, 95 kD; <<, 60 kD). (E) Comparative anti-P2X1 receptor
immunoblot of platelet membranes (30 µg) versus purified GPIIb/IIIa
(400 ng) that were processed under nonreducing conditions.
(<<<, 95 kD; <<, 60 kD). (F)
Comparative anti-CD61 immunoblot of the same platelet membrane (30 µg) and GPIIb/IIIa (400 ng) samples used in (E) (nonreducing conditions).
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The 95- to 100-kD anti-P2X1 reactive protein is similar in
size to the subunits of certain integrins that are abundant
proteins in the plasma membranes of platelets and leukocytes. Although GPIIIa (CD61) is the major 3 integrin in platelets, several
observations indicated that GPIIIa is not the anti-P2X1
reactive protein of approximately 100 kD. No P2X1-reactive
bands were observed in lanes containing purified human GPIIb/IIIa that
were prepared and electrophoresed under reducing or nonreducing
conditions (Fig 1D and E). Parallel immunoblots using an anti-CD61 MoAb
(under nonreducing conditions) verified the presence of immunoreactive GPIIIa in the lanes containing platelet membrane proteins or purified GPIIIb/IIa (Fig 1F). By contrast, the anti-P2X1 reactive
band of approximately 100 kD was greatly diminished when these samples were processed under identical nonreducing conditions. Finally, endoglycosidase-F treatment caused no change in electrophoretic mobility of the 100-kD anti-P2X1 reactive protein (Fig 1C);
similar treatments have been reported to decrease GPIIIa mass from 105 to 90 kD.21
Immunologic characterization of P2X1 receptor expression
in myeloid cell lines and human blood leukocytes.
The same immunoblot protocol was used to compare relative levels of
P2X1 receptor protein in membranes from human myeloid cell
lines (Fig 2) and from human blood
leukocytes (Fig 3). P2X1 receptor expression was low in undifferentiated HL-60 promyelocytes but
was upregulated following PMA treatment to induce a macrophage-like phenotype. P2X1 receptor expression was also markedly
upregulated when HL-60 cells were treated with dibutyryl cAMP to induce
a neutrophil-like phenotype. In contrast to the low expression of P2X1 receptors in uninduced HL-60 cells, there was
significant expression in uninduced THP1 cells, a human leukemia line
predominated by more mature monocyte-like cells. Although treatment of
THP1 cells with PMA or LPS/interferon- (IFN-) induces expression of multiple inflammatory response genes, these latter agents did not
significantly change P2X1 receptor expression from the
levels observed in uninduced cells. These data confirmed and extended previous studies17 reporting P2X1 receptor
expression in myeloid cell lines exhibiting either monocyte/macrophage
(PMA-differentiated HL-60 cells) or granulocyte (RBL, rat basophilic
leukemia) phenotypes.
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| Fig 2.
Immunological characterization of P2X1
receptor expression in myeloid cell lines versus human blood cells.
HL60 cells or THP1 cells were untreated or were treated with 100 nmol/L
PMA (HL-60 PMA or THP1 PMA), 0.5 mmol/L dibutyryl cAMP (HL-60 cAMP), or
1,000 U/mL IFN- plus 100 ng/mL LPS (THP1 I-L) as described under
Materials and Methods. BAC1.2F5 murine macrophages (BAC1) were cultured without differentiating agents. Total cell membranes were isolated from
these cell lines and from human peripheral blood cell fractions including total WBCs plus platelets (WC/platelets), neutrophils, and
mononuclear leukocytes. Equal amounts of membrane protein (25 µg)
were loaded in each lane and probed using the P2X1R
antiserum (<<<, 95 kD; <<, 60 kD).
The cross-reactive band at about 75 kD is a serum-derived contaminant,
the intensity of which can be reduced by repeated washing of cells
before lysis. Similar patterns of P2X1 receptor expression
were observed in four other experiments.
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| Fig 3.
Comparative levels of P2X1 receptor protein
and Na+,K+-ATPase subunit in membranes from
platelets, neutrophils, and mononuclear leukocytes. Adjacent lanes were
loaded with 5, 15, or 30 µg of total membrane protein isolated from
the indicated blood cell types (platelets, neutrophils, mononuclear).
(A) The immunoblot initially probed with P2X1
receptor antiserum (<<<, 95 kD; <<, 60 kD). (B) The same blot stripped and reprobed with MoAb 9A7 against
the subunit of the Na+,K+-ATPase. ( ),
110 kD subunit.
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In contrast to the significant content of P2X1 receptors in
human myeloid cell lines, no expression of these receptors was observed
in BAC1.2F5 cells (Fig 2), a murine line that constitutively expresses
many phenotypic characteristics of mature macrophages.20 Control studies using membranes from mouse vas deferens verified that
the antibody reacted with murine P2X1 receptor protein.
Likewise, no expression was apparent in freshly isolated human
neutrophils and only low expression was observed in the mononuclear
leukocyte fraction of human blood (Figs 2 and 3). The absence of
significant P2X1 receptor protein in samples of membranes
from purified neutrophils and mononuclear leukocyte fractions
contrasted with the strong signals observed in membranes derived from
the mixed leukocyte/platelet fraction (buffy coat) of whole blood. This
indicated that most P2X1 receptor protein was derived from
platelet membranes.
The relative levels of P2X1 receptor protein in platelets
versus blood leukocytes were examined by comparing intensities of the
60-kD immunoreactive bands among lanes loaded with equivalent amounts
of total membrane protein from each cell fraction (Fig 3A). Total
membranes from these blood cell types will contain differing amounts of
plasma membrane because of the different contents of intracellular
organelles and granules within these cells. Thus, it was important to
normalize the P2X1 receptor signals relative to the plasma
membrane content of each lane. Accordingly, the anti-P2X1
immunoblot was stripped and re-probed with an antibody against the
110-kD subunit of the Na+,K+-ATPase (Fig
3B). The relative plasma membrane content (per microgram of total
membrane protein) was highest in the mononuclear leukocyte samples,
intermediate in platelets, and lowest in the neutrophil lanes. This is
consistent with the high content of primary and secondary granules in
neutrophils. Although 30 µg of total neutrophil membrane protein and
5 µg of total platelet membranes contained similar amounts of
immunoreactive Na+,K+-ATPase, there was no
appreciable P2X1 receptor signal in any lanes containing
neutrophil membranes. Although 5 µg of total mononuclear leukocyte
membranes and 30 µg of total platelet membranes contained roughly
equivalent amounts of Na+,K+-ATPase, the
P2X1 receptor signal was much stronger in the platelet membrane lane.
These observations indicate that the relative level of P2X1
receptor expression in human platelets is at least 10-fold greater than
in neutrophils or mononuclear leukocytes. Indeed, there was no
measurable amount of anti-P2X1 reactive protein in any of
five neutrophil preparations (from four different donors) that were assayed. Because the mononuclear leukocyte fraction is heterogeneous, the low (relative to platelets) amounts of P2X1 receptor
protein in this cell population may reflect its predominant expression in only certain leukocytes or residual platelet contamination of the
mononuclear leukocyte fractions. This fraction is usually composed of
about 65% T lymphocytes, 15% B lymphocytes, 15% monocytes, 5%
natural killer (NK) cells.26 Visual inspection of the
washed, mononuclear cell fractions used in our experiments indicated
approximately 20% monocytes (as assayed by rapid spreading on the
glass hemocytometer surface), 80% lymphocytes, and relatively minor
numbers of platelets (approximately 20 platelets per 100 mononuclear
cells). Given this distribution, it is unlikely that T lymphocytes
express P2X1 receptors to the level observed in platelets.
Equivalent amounts of platelet and mononuclear leukocyte membranes were
assayed for their relative contents of P2X1 receptor and
CD61 (Fig 4). Intense CD61 signals were
detected in the platelet samples, whereas lower, but significant, CD61
signals were consistently observed in the mononuclear leukocyte
samples. The relative intensities of the P2X1 receptor
signals were strongly correlated with the intensities of the CD61
signals, suggesting that residual platelets are the source of
P2X1 receptors observed in the mononuclear leukocyte samples. Because some previous reports have suggested that CD61 is
expressed by monocytes, it is possible that monocytes, like platelets,
coexpress P2X1 receptors and CD61. Mononuclear leukocyte samples were further fractionated (by adherence to plastic) into adherent monocytes and nonadherent lymphocytes. However, no significant differences were observed in the relative amounts of P2X1
receptor, CD61, or residual platelets within these fractionated
preparations of monocytes or lymphocytes (data not shown). Thus,
contamination with platelets seems to be the most likely source of the
P2X1 receptor protein observed in the mononuclear leukocyte
fraction.
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| Fig 4.
Comparative levels of P2X1 receptor protein
and CD61 protein in membranes from different blood cell fractions.
Lanes were loaded with 25 µg of total membrane protein from (left to
right): total blood leukocytes (total WC), mononuclear leukocytes,
neutrophils, platelets. (A) Anti-P2X1 receptor immunoblot
of membrane proteins prepared for electrophoresis under standard
reducing conditions. (B) Anti-CD61 immunoblot of samples from the same
membrane preparations processed under nonreducing conditions.
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Functional characterization of P2X1 receptor expression
in intact platelets and blood leukocytes.
The relative expression levels of functional P2X1 receptors
in intact blood cell types were analyzed by assaying Ca2+
influx responses to -methylene ATP (Fig
5). At micromolar concentrations, this
synthetic nucleotide selectively activates only the P2X1 and P2X3 subtypes of the P2X receptor family27
and has no significant agonistic actions on any of the P2Y family
receptors.1,4 Platelets responded to 30 µmol/L
-methylene ATP with rapid and transient twofold increases in
cytosolic [Ca2+] over the basal level of 130 nmol/L (Fig
5A). These same platelets did not respond to a second pulse of
-methylene ATP but did respond to 30 µmol/L ADP and,
subsequently, to thrombin. The peak increase of the -methylene
ATP-induced Ca2+ transient was much smaller than the 5- and
15-fold increases stimulated by ADP and thrombin, respectively.
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| Fig 5.
Comparative analysis of P2X1 receptor
function in platelets, neutrophils, and mononuclear leukocytes. The
indicated types of human blood cells were loaded with fura-2 and
assayed for agonist-induced changes in cytosolic [Ca2+]
as described under Materials and Methods. Each experiment was repeated
twice using different preparations of blood cells and yielded similar
results. For ease of graphic presentation, segments from certain
continuous records have been deleted as indicated by braces { };
the duration of each deleted segment is noted. (A) Platelets were
suspended in normal basal salt solution (BSS) containing 130 mmol/L
NaCl, 1 mmol/L CaCl2, and 2.5 U/mL apyrase. The cells were
first stimulated with 30 µmol/L -methylene ATP (me-ATP).
After about 3 minutes, the cells were rechallenged with additional
-methylene ATP (60 µmol/L final) and then serially stimulated
with 30 µmol/L ADP, followed by 1 U/mL thrombin. (B) Platelets were
suspended in reduced Na-BSS containing 40 mmol/L NaCl, 90 mmol/L
NMG-Cl, 1 mmol/L CaCl2, and 2.5 U/mL apyrase. The platelets
were serially stimulated with 30 µmol/L -methylene ATP (twice),
30 µmol/L ADP, and 1 U/mL thrombin. (C) Neutrophils were suspended in
reduced Na-BSS containing 40 mmol/L NaCl, 90 mmol/L NMG-Cl, 1 mmol/L
CaCl2, and 2.5 U/mL apyrase. They were stimulated with 30 µmol/L -methylene ATP, followed by 1 µmol/L FMLP. Another
sample (suspended in the same medium) was stimulated by 30 µmol/L
ATP. (D) Mononuclear leukocytes were suspended in reduced Na-BSS
containing 40 mmol/L NaCl, 90 mmol/L NMG-Cl, 1 mmol/L
CaCl2, and 2.5 U/mL apyrase. They were stimulated with 30 µmol/L -methylene ATP, followed by 5 µg/mL concanavalin A.
|
|
Moreover, removal of extracellular Ca2+ eliminated the
response to -methylene ATP but only marginally attenuated the
responses to ADP or thrombin (data not shown). These differences are
consistent with the ability of P2X1 receptors to trigger
only Ca2+ influx in contrast to the abilities of ADP and
thrombin to stimulate both Ca2+ mobilization from
intracellular stores and Ca2+ influx. Since
P2X1 receptors exhibit only a 5:1 selectivity for Ca2+ over Na+,15 ATP will primarily
gate Na+, rather than Ca2+, influx when
platelets are suspended in physiological medium containing 130 mmol/L
NaCl and 1 mmol/L CaCl2. When extracellular Na+
was partially replaced with NMG+, an impermeant cation, 30 µmol/L -methylene ATP triggered a more pronounced 8-fold peak
increase in cytosolic Ca2+ (Fig 5B).
Identical protocols were used to assay Ca2+ influx in
neutrophils (Fig 5C) or mononuclear leukocytes (Fig 5D) stimulated with -methylene ATP. No response to this nucleotide was observed in
neutrophils. This contrasted with the robust Ca2+
mobilization triggered by fMLP, an agonist for the myeloid-specific formyl peptide receptor, or ATP, an agonist for the G-protein-coupled P2Y2 nucleotide receptors that are expressed in neutrophils
and monocytes.19 In mononuclear leukocytes,
-methylene ATP stimulated a very modest 15 nmol/L elevation in
Ca2+ over the basal level of 170 nmol/L. By contrast,
cross-linking of T-cell receptors with concanavalin A induced a
sustained threefold increase in cytosolic Ca2+.
Owing to the high P2X1 receptor expression in platelets,
minor numbers of residual platelets could account for the small
response to -methylene observed in the fura 2-loaded mononuclear
leukocytes. Alternatively, this might reflect a low level of
P2X1 receptor-mediated Ca2+ influx channel
activity in the monocytes, which comprise only about 20% of the
mononuclear suspension. Given the significant expression of
P2X1 receptor protein and function in human monocytic cell
lines17 (Fig 2) it was important to screen for
P2X1 receptor activity in blood monocytes using sensitive
electrophysiological measurements of ATP-gated inward Na+
current. Patch-clamp assays were performed on single blood monocytes identified by their rapid adherence to, and spreading on, the plastic
floor of the recording chamber. The monocytes were also incubated with
medium containing apyrase (.5 U/mL) both before and during patch-clamp
seal formation to minimize any autocrine desensitization of
P2X1 receptors.17 However, none of the nine assayed monocytes (isolated from four donors) exhibited rapid ATP-induced inward currents (Fig 6B). As a
positive control, identical recording conditions were used to assay
P2X1 receptor function in HEK cells that stably express
recombinant rat P2X1 receptors. In this particular
experiment, four of eight patch-clamped HEK cells showed the rapid,
transient inward currents characteristic of ATP-gated P2X1
receptor channels (Fig 6A).
View larger version (9K):
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[in a new window]
| Fig 6.
Membrane currents evoked by ATP in whole-cell patch-clamp
recordings from human blood monocytes versus HEK cells expressing recombinant P2X1 receptors. Individual human monocytes or
HEK cells expressing recombinant rat P2X1
receptors (HEK-P2X1 cells) were patch-clamped as described under
Materials and Methods. The membrane potential was held at 40 mV,
while the cell was stimulated for indicated time with 100 µmol/L ATP.
(A) Representative current record from an HEK-P2X1 cell.
Similar responses were observed in four of eight patch-clamped
HEK-P2X1 cells. Since this stably transfected cell line was not generated from a single cloned cell, the
magnitude of P2X1 receptor expression and function varies considerably between individual cells. (B) Representative current record from a human monocyte. A similar absence of ATP-induced response
was observed in nine other patch-clamped monocytes.
|
|
| |
DISCUSSION |
These experiments significantly extend recent
reports10,12,14,16,17 regarding expression of
P2X1 nucleotide receptors in various types of human blood
cells. Although P2X1 receptor mRNA14 and
ATP-gated cation channels10 have been previously described
in human platelets, the studies illustrated in Figs 1 and 3 represent
the first immunological demonstration that the human P2X1
receptor is strongly expressed as a heavily glycosylated 60-kD protein
in these cells. In addition, our studies of nucleotide-induced Ca2+ transients (Fig 5) show that these P2X1
receptors are functionally and pharmacologically distinct from the
well-characterized ADP receptors of platelets. An initial exposure of
platelets to -methylene ATP triggered a transient
Ca2+ influx that was followed by a sustained
desensitization to subsequent additions of this nucleotide. However,
these -methylene ATP-desensitized platelets continued to show
strong Ca2+ mobilization responses to ADP. As noted
previously, platelets from Gq-deficient mice show no responses to
ADP,11 whereas the findings of Leon et al9
strongly suggest that the ADP receptor of platelets may be the already
cloned P2Y1 receptor. Thus, platelets express at least two
distinct types of P2 nucleotide receptors the ionotropic
P2X1 receptor and a Gq protein-coupled
P2Y1-like receptor. It is significant to note that the
P2Y1-like receptor of platelets shows an absolute
selectivity for ADP as a physiological agonist and is antagonized by
high concentrations of extracellular ATP.28 By contrast,
ATP is the most potent physiological nucleotide agonist (EC50 ~1 µmol/L) for the human P2X1
receptor, with ADP a full but less potent (EC50 ~70
µmol/L) agonist.29 These divergent nucleotide
selectivities indicate that platelets may use ATP and ADP for distinct
types of regulation. Although the roles for the P2Y1-like
ADP receptor in platelet physiology are well
established,6-8 the functions of the platelet
P2X1 receptor are undefined.
Whether platelets express additional nucleotide receptors remains to be
determined. Colman et al7,30 have used nucleotide affinity
labeling methods to identify a 100-kD platelet protein termed aggregin,
which may be part of an ADP receptor complex. The gene for this protein
has not yet been cloned and its possible relationship to either the
P2Y1-like ADP receptor or the P2X1 receptor of
platelets is unclear. Our anti-P2X1 receptor antibody labeled a 100-kD platelet protein, in addition to the major 60-kD P2X1 receptor glycoprotein. We verified that this 100-kD
protein was not CD61/GPIIIa, a major platelet membrane protein in this size range (Figs 1C and D). Because the amino acid sequence of aggregin
is unknown, we cannot ascertain whether it contains either of the two
peptide sequences recognized by the P2X1 receptor
antiserum. However, the intensity of our 100-kD band was greatly
decreased when platelet membranes were prepared in the absence of
reducing agents (Fig 1F), suggesting that this protein is normally
complexed with other platelet proteins by disulfide bonds. By contrast, affinity-labeled aggregin migrates as a 100-kD product when prepared with or without sulfhydryl reducing agents.7 The 100-kD
immunoreactive protein was not apparent in other cell types expressing
native (Figs 1-3) or recombinant (data not shown)
P2X1 receptors. Therefore, it is unlikely to be essential
for either the expression or function of these ATP-gated ion channels.
Previous studies of P2X1 receptor expression in other human
hematopoietic cell types have reported P2X1 receptor mRNA
in total human blood leukocytes16 and P2X1
receptor protein and function in PMA-differentiated HL-60 myeloid
cells.17 We also noted expression of P2X1
receptor protein in a variety of leukemic human cell lines, including
THP1 monocytes and dibutyryl cAMP-differentiated HL-60 granulocytes
(Fig 2). Thus, we expected to observe expression of P2X1
receptors in circulating human monocytes and granulocytes. However, we
measured no significant amount of P2X1 receptor protein (Fig 3) or function (Figs 5 and 6) in gradient purified granulocytes (>95% neutrophils) and only low P2X1 levels in the total
mononuclear leukocyte fraction (80% lymphocytes, 20% monocytes). The
modest levels of P2X1 receptor protein in the mononuclear
fraction were correlated with modest levels of CD61, a platelet marker
protein. We also found that the relative amounts of P2X1
receptor immunoreactivity in mononuclear leukocyte preparations varied
with the isolation protocol. Although citrated blood was used for the
experiments described in this study, our initial studies used
heparinized blood and this resulted in stronger P2X1
receptor signals and CD61 signals in the mononuclear fractions (data
not shown). Significant platelet contamination may underlie the high
levels of P2X1 receptor mRNA reported in previous Northern
blot studies of total human blood leukocytes.16 Although we
used CD61 as a marker for platelet contamination, this antigen has also
been reported on monocytic and osteoclastic cells.31-34
However, Krissansen et al34 showed that
binding of platelets or platelet fragments to monocytes can generate
false-positive CD61/CD41 signals in monocytes during fluorescence-activated cell sorter (FACS) analysis.
The absence of significant P2X1 receptor expression in
circulating neutrophils and monocytes contrasts with the strong
expression of this receptor in various human myeloid cell lines. This
observation, coupled with the high P2X1 receptor levels
noted in circulating platelets, suggests that the P2X1
receptor gene is activated in the early colony-forming unit
granulocyte, erythroid, monocyte, megakaryocyte (CFU-GEMM) cells that
are the common marrow progenitors of both megakaryocytes and the
committed granulocyte/monocyte precursors. If true, our data would
suggest that P2X1 receptor expression is maintained during
megakaryocyte development and platelet release but is repressed during
the most distal phases of phagocyte differentiation. Current
experiments are aimed at identifying the mechanisms and factors
responsible for lineage- and stage-dependent expression of this
receptor in the marrow-derived progenitor cells of the
blood.
| |
FOOTNOTES |
Submitted July 9, 1997;
accepted December 10, 1997.
Supported by National Institutes of Health (NIH) Grant No. GM36387.
E.E.C. was supported by NIH Training Grant HL07678.
Address reprint requests to George R. Dubyak, PhD,
Department of Physiology and Biophysics, School of Medicine E565, Case Western Reserve University, 10900 Euclid Ave, Cleveland, OH 44106-4970.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract