© 1997 American Heart Association, Inc.
Articles |
Functional Expression of a P2T ADP Receptor in Xenopus Oocytes Injected With Megakaryocyte (CMK 11-5) RNA
From the Platelet Biology Department, The American Red Cross, Jerome Holland Laboratory, Rockville, Md.
Correspondence to Nicholas J. Greco, PhD, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. E-mail greco{at}usa.redcross.org
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Abstract |
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Abstract Since the P2T purinergic (ADP) receptor is unique to the megakaryocytic/platelet lineage, cells of this lineage were screened for the relative effects of ADP and ATP in intracellular Ca2+ levels. Like platelets, CMK 11-5 cells responded with an increase in intracellular Ca2+ mobilization in response to ADP but not to ATP or adenosine. In contrast, both nucleotides increased intracellular Ca2+ mobilization in the megakaryoblastic cell lines MO7E and Meg-01, indicating that they contain P2Y receptors or a mixed complement of purinergic receptors. Pharmacological responsiveness of CMK 11-5 cells to nucleotides paralleled those of platelets, in which ADP and ADP-
-S are active as agonists and ATP and ATP--S are inactive as agonists but act as antagonists. [3H]ADP and 35S-ATP--S bound to CMK 11-5 cells at a high-affinity site (Kd1 and Ki1, 262 and 125 nmol/L, respectively) and a low-affinity site (Kd2 and Ki2, 10 100 and 5400 nmol/L, respectively) with 2x106 to 6x106 sites per cell. ADP bound at both sites was competed with ADP, ATP, and ATP--S with affinities in a rank order similar to that found for platelets (ATP--S
ATPADP
ADP-ß-Sadenosine), suggesting the presence of a P2T receptor on CMK 11-5 cells. Photoaffinity labeling of intact CMK 11-5 cells with 35S-ATP--S resulted in the labeling of the -subunit of GP IIb as found with platelets, although this was confirmed to be independent of ADP receptors. After RNA from CMK 11-5 cells was microinjected into Xenopus oocytes, only ADP and ADP--S stimulated 45Ca2+ efflux, which was not observed with ATP, 2-methylthio-ATP, ,ß-methylene-ATP, ATP-
-S, ATP--S, or adenosine. In addition, incubation of RNA-injected oocytes with ATP or ATP--S but not adenosine blocked the 45Ca2+ response to ADP. These experiments demonstrate that a nascent receptor that responded specifically to ADP but not to other P1, P2Y, P2X, and P2U agonists was expressed in functional form on Xenopus oocytes.
Key Words: Xenopus oocyte • ADP • P2T purinergic receptor
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Introduction |
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Purinergic receptors have been classified as P1-purinoreceptors, which bind adenosine, and P2-purinoreceptors, which bind ADP and ATP.1 2 3 Six P2 nucleotide receptor subtypes, D, T, Z, Y, U, and X, have been defined on the basis of pharmacological studies. The P2T subtype has been described only in cells of the megakaryocytic/platelet lineage, for which the distinguishing characteristic is that ADP acts as an agonist and ATP acts as an antagonist. Although the P2T (platelet), P2D, and P2Z receptors have not been cloned, a number of seven-transmembrane G protein–coupled purinergic receptors (P2Y and P2U) have been identified in addition to P2X (ATP) receptors with a proposed topography of two transmembrane domains that have been postulated to be Ca2+/cation channels (for review see Website http://mgddk1.niddk.nih.gov:8000/). A G protein–coupled P2 purinoceptor (P2Y3) from chicken brain is activated preferentially by nucleotide diphosphates but does not appear to be identical to the platelet ADP receptor.4
Putative P2T ADP platelet receptors have been extensively evaluated by pharmacological and equilibrium binding techniques, but characterization of the receptors at the molecular level and its signal-transducing mechanism has not been reported.
Platelet P2T receptor occupancy is associated with numerous postreceptor events in platelets, including Na+/H+ exchange and intracellular pH changes,5 aggregation by ADP itself 6 and concurrently with other agonists,7 inhibition of adenylyl cyclase activity,8 Ca2+ mobilization,9 10 and the activation of protein kinase C coupled to signal-transducing G proteins.11 The involvement of the cellular signal transduction messenger inositol triphosphate generated by the hydrolysis of phosphatidyl inositol 4,5-bisphosphate12 in the activation of platelets by ADP is debated.13 14 15 Platelet aggregation by ADP plays a key role in the development of arterial thrombosis,6 the deposition of platelets onto collagen under flow conditions,16 and collagen-induced aggregation, because platelets from patients lacking ADP in dense granules (Chediak-Higashi syndrome) do not respond to collagen.17 Activation of platelets for primary hemostasis involves ADP, which influences the growth of thrombi on subendothelium and/or maintains thrombus stability.7 18 These observations emphasize the importance of endogenous ADP in thrombogenesis by itself and as a modulator of the action of other endogenous agonists.19
Modifications in the structure of ADP, whether in the purine, ribose, or phosphate moieties, result in reduction or loss of agonist activity at P2T receptors. An exception to this strict structural requirement for agonists is if substitution occurs in the C-2 position of the adenine ring. Because of this strict structural requirement, the absence of specific high-affinity nonhydrolyzable ligands and probes has contributed to the limited characterization of P2T receptors. Attempts to identify the platelet P2T ADP receptor by photoaffinity-labeling techniques have been constrained by this structural limitation and have yielded uncertain results. For example, 2-azidoadenosine-5'-diphosphate labeled several proteins in intact platelets, but none were competed by ADP, suggesting that nonspecific labeling had occurred.20 Affinity analogues 5'-fluorosulfonylbenzoyladenosine and 8-bromo-dioxobutylthio-ADP inhibit ADP-induced shape change and aggregation and label a 100-kD protein (aggregin),21 22 but the amino acid sequence of the labeled protein has not been established, although its association with GP IIb/IIIa has been suggested.23 A new photoprobe, 2-(p-azidophenyl)-ethylthioadenosine 5'-phosphate, labels a 43-kD protein and is proposed to be the ADP receptor through which adenylate cyclase is inhibited.8 This labeled protein is in the expected range of G protein–coupled receptors but has not as yet been further characterized.
We have shown that 1-thiopurine and thiopyrimidine nucleotides such as ATP--S serve as antagonists for platelet activation and for equilibrium nucleotide binding24 : both unmodified purine and pyrimidine nucleotides are selectively photoaffinity incorporated into the integrin IIbß3 (GP IIb/IIIa).25 26 27 Glanzmann's thrombasthenic platelets, despite the absence of GP IIb/IIIa, show binding of ADP and ATP--S similar to that of control platelets, normal ADP-induced Ca2+ influx, platelet shape change, and inhibition of cAMP levels,28 29 30 although their ability to synthesize thromboxane B2 is reduced.31 These latter studies unequivocally demonstrate that GP IIb is neither the site measured in the steady-state binding assays nor the receptor for ADP but is a target for photoinsertion. These results suggest that P2T receptors may be in close proximity to GP IIb/IIIa. Because the numbers of nucleotide binding sites are similar, this may suggest a stoichiometric ratio between the number of high-affinity ADP binding sites and the number of GP IIb/IIIa complexes on the surface of normal platelets.
The human megakaryocytic cell line CMK 11-5 expresses the platelet antigens GP Ib, GP IIb/IIIa, and GP IV.32 This cell line releases platelet-like particles that bind fibrinogen after but not before exposure to ADP; in addition, these particles bind to aortic subendothelium similarly to activated platelets.33 PMA treatment induces expression of megakaryocytic/platelet cell surface antigens and increases the mRNA levels for glycoproteins GP IIb/IIIa, GP Ib, platelet factor 4, thrombospondin, and GMP 140 (P selectin).32 In addition, CMK 11-5 cells respond to platelet agonists ADP, collagen, and -thrombin.33 Taken together, these observations indicate a similarity of receptor characteristics between platelets and the CMK 11-5 megakaryocytic cell line.
The present studies demonstrate that CMK 11-5 cells respond to ADP in a manner pharmacologically appropriate for the P2T receptor; that is, ADP functions as an agonist and ATP functions as an antagonist for ADP-induced activation. Furthermore, ATP--S photoaffinity labels GP IIb of CMK 11-5 cells identically to the pattern seen in platelets. It is concluded that the CMK 11-5 cells contain P2T receptors as found on platelets. Finally, microinjection of megakaryocytic CMK 11-5 cell RNA into Xenopus oocytes results in the expression of nascent P2T ADP receptors.
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Materials
Meg-01 and MO7E cells were obtained through Dr Allan Mufson (American Red Cross) and maintained in RPMI 1640 medium (Gibco BRL) with 10% heat-inactivated FCS; medium for the MO7E cells was supplemented with 5 ng/mL recombinant IL-3. The CMK and CMK 11-5 cell lines were generous gifts of Dr T. Sato (Chiba University, Chiba, Japan) and Drs H. Ogino and R. Deguchi (Mochida Pharmaceutical Co, Ltd, Tokyo, Japan). Both cell lines were maintained in RPMI 1640 medium containing 2 mmol/L glutamine, 10% FCS, 50 U/mL penicillin G, and 50 µg/mL streptomycin and grown at 37°C in a humidified atmosphere of 5% CO2 in air.34 Since these autocrine cells produce IL-6 and respond to IL-3, IL-6, and GM-CSF,34 35 cells were maintained at densities of 0.5x105 to 8x105/mL by dilution of the medium, and cells were used for experiments during exponential growth. Flow cytometry (FACScan, Becton Dickinson) of 1% paraformaldehyde–treated cells was used to confirm the presence of platelet antigens on the CMK 11-5 cell line: GP Ib was determined by the binding of monoclonal antibody TM60, and GP IIb/IIIa was determined by the binding of monoclonal antibody AP-2 (kind gifts of Drs Naomasa Yamamoto, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan, and Thomas Kunicki, Scripps Research Institute, La Jolla, Calif, respectively).
Measurement of Intracellular Ca2+ Mobilization
Meg-O1, MO7E, CMK, and CMK 11-5 cells were isolated (200g for 5 minutes) from RPMI 1640 media and resuspended in modified Tyrode's-HEPES buffer (pH 6.5) containing 5 U/mL apyrase (type I, Sigma Chemical Co) and 0.35% BSA. Cells were incubated with 2.5 µmol/L fura 2-AM dissolved in dimethyl sulfoxide (Molecular Probes) for 30 to 45 minutes at 37°C. Cells were recovered by centrifugation (200g for 5 minutes) and washed once in phosphate wash buffer (pH 6.5)25 containing 5 U/mL apyrase before resuspension at 4x105 cells/mL in modified Tyrode's-HEPES buffer (pH 7.4). Addition of apyrase is required to maintain the sensitivity of the cells to ADP: in the absence of apyrase, concentrations of 300 µmol/L ADP were unable to cause Ca2+ mobilization. Measurement of intracellular Ca2+ mobilization was identical to methods used for platelets, and fura 2–loaded cells were maintained at 15°C to limit dye leakage.36
The nucleotides used in this study are as follows: radiolabeled ATP--S, guanosine 5'-(1-thiotriphosphate) (GTP--S) and uridine 5'-(1-thiotriphosphate) (UTP--S) (DuPont–New England Nuclear), ATP--S and adenosine 5'-(2-thiodiphosphate) (Boehringer Mannheim), adenosine, ATP, and ATP--S (Sigma), and 2-MeS-ATP and ,ß-Me-ATP (Research Biochemicals International). Nucleotides were diluted into modified Tyrode's-HEPES buffer from 10 mmol/L stocks in water.
Nucleotide Binding to Platelets and CMK 11-5 Cells
Formaldehyde-fixed cells were used to achieve steady-state binding and avoid the complications of nucleotide metabolism. Furthermore, it should be noted that the binding of von Willebrand factor,37 thrombin,38 antibodies,39 fibrinogen,40 and collagen41 to fixed platelets yields steady-state binding data similar to those found with unfixed samples. We have observed similar binding results using platelets fixed with 4% formalin for 48 hours at 4°C or after fixation with 1% formalin for 30 to 60 minutes, as routinely used in flow cytometric evaluation of platelet antigens, suggesting that significant further modification has not occurred during the more vigorous fixation procedure.30 CMK 11-5 cells were isolated by centrifugation at 200g for 5 minutes, washed once in phosphate wash buffer (pH 6.5),25 and resuspended in PBS containing a final concentration of 1% paraformadehyde. After fixation for 1 hour, cells were washed three times with PBS before final resuspension in Tyrode's-HEPES buffer (pH 7.4). Platelets were isolated and fixed with 1% paraformaldehyde as described.30
Photoaffinity Labeling of CMK 11-5 Cells
Washed CMK 11-5 cells (2.5x105 cells) in Tyrode's-HEPES buffer (without BSA) were mixed with 50 nmol/L 35S-ATP--S in a total volume of 200 µL as previously described.25
Injection of mRNA Into Xenopus laevis Oocytes
Ovarian tissue was resected from Tribucane-anesthetized (ICN Biochemicals) Xenopus laevis toads (Nasco) and manually defolliculated or treated with collagenase (Sigma, type IV, 2 mg/mL for 3 hours at 22°C) in modified Barth's solution (in mmol/L: NaCl 88, KCl 1, NaHCO3 2.4, MgSO4 0.82, HEPES 10; pH 7.4) containing 50 U/mL penicillin and 50 µg/mL streptomycin without Ca2+. After a 15- to 20-hour recovery period, oocytes were microinjected via a Narishige IM-200 microinjector (5 to 15 kPa, 0.3- to 1.0-ms duration) with 
50 nL of RNase-free sterile water or CMK 11-5 total RNA (80 ng) or mRNA (3 ng) dissolved in sterile water.42 43 For the isolation of total and poly (A)+ RNA, CMK 11-5 cells (3x108) were solubilized with guanidinium-isothiocyanate-sarcosyl, and RNA was isolated by isopropanol precipitation and oligo (dT) cellulose chromatography. To screen for translationally competent oocytes, mRNA encoding for SEAP/RNA (ATCC) and identified by a p-nitrophenylphosphate chromogenic substrate at 405 nm was used.44 The plasmid pGEM4Z (ATCC) containing a 2-kb insert for SEAP was transformed in E coli HB101 and amplified, and the plasmid was purified by cesium chloride ultracentrifugation and linearized by digestion with HindIII. After plasmid transcription with SP6 polymerase45 in the presence of m7G(5')ppp(5')G (Pharmacia), purified SEAP/RNA was dissolved in sterile water for injection, and 1 to 5 ng (2 to 10 nL) was coinjected with total or poly (A)+ CMK 11-5 RNA. Only injected oocytes having an elevated SEAP in the medium (typically 20 to 100 relative optical density units above background) were used.
45Ca2+ Efflux Measurements
RNA-injected oocytes were incubated for 2 hours at 18°C to 22°C individually in V-bottom assay plates (Rainin) in 50 µL of modified Barth's solution containing 50 µCi/mL [45Ca]CaCl2. Labeled oocytes were washed thoroughly with Barth's buffer (5 times with 0.3 mL), and finally, 150 µL of modified Barth's solution including Ca2+ was added to the well. To determine basal levels of 45Ca and subsequent leakage, if any, from the oocytes in the absence of added agonist, two samples (20 µL) of extracellular medium were withdrawn at "-5 minutes" and at the zero time point. Only samples that showed comparable counts at -5 minutes and the zero time were used for the determination of the effect of added agonists. Nucleotides were then added (10 µmol/L) at the zero time, three subsequent samples (20 µL) were withdrawn at 5, 10, and 20 minutes after the addition of the nucleotides, and all samples were subjected to ß-counting. In separate experiments, oocytes were emulsified in the presence of 0.2 mL 20% SDS and counted by liquid scintillation to determine the average total 45Ca2+ content. Labeling of individual oocytes with 45Ca2+ varied considerably, with from 300 to 900 000 cpm incorporated per oocyte. Therefore, after each experiment, the total associated radioactivity in each oocyte was determined to confirm a sufficient content of releasable 45Ca2+.
Nucleotide Binding to Oocytes
In the presence of 25 nmol/L 35S-ATP--S (2.2 µCi), collagenase-treated unfixed oocytes bound 11 495±7256 cpm of nucleotide (±SD; range, 875 to 18 375 cpm; n=16 oocytes), and this binding was reduced with the coincubation of 25 µmol/L ATP--S to 4626±3772 cpm (range, 375 to 8625 cpm; n=16 oocytes): in these experiments, an average of 0.24% of the added nucleotide bound to the oocytes and coincubation of a 1000-fold molar excess of ATP--S reduced the mean value 60%.
Since the binding of 35S-ATP--S to unfixed oocytes varied considerably around mean binding values and nucleotides are unstable when coincubated with intact oocytes (see next section), 1% paraformaldehyde-treated oocytes were used because they demonstrated reduced variation in the range of binding values: paraformaldehyde fixation of oocytes allows the direct identification of superficial oocyte morphology without structural changes.46 Collagenase-treated oocytes were treated with 1% paraformaldehyde dissolved in modified Barth's buffer for 18 hours at 4°C and used for all further experiments.46 After fixation, oocytes were washed five times with 5 mL modified Barth's buffer to remove paraformaldehyde before binding experiments were performed. Single oocytes were added to individual wells of a 96-well V-shaped bottom assay plate (Rainin): binding buffer (modified Barth's buffer) contained either 10 nmol/L 35S-ATP--S (0.88 µCi, 1.95x106 cpm) or 10 nmol/L [3H]ADP (0.15 µCi, 150 000 cpm) in a total volume of 0.1 mL. After a 10-minute incubation with the nucleotide, unbound radioligand was removed by aspiration and oocytes were washed rapidly three times with 0.3 mL MBS. Oocyte-associated radioactivity was measured by ß-counting of oocytes emulsified in 0.2 mL 20% SDS.
Metabolism of [3H]ADP by Xenopus Oocytes
Single oocytes were added to 500 µL of modified Barth's buffer containing 10 nmol/L [3H]ADP (0.15 µCi, 150 000 cpm); 75-µL aliquots were removed at 1, 5, 10, 20, and 30 minutes and immediately frozen in liquid nitrogen. Before analysis, samples were rapidly thawed and mixed with an equal volume of 10 mmol/L NaH2PO4, and the metabolism of [3H]ADP was analyzed with an HPLC apparatus (Beckman) equipped with an Altex Ultrasil SAX column. To each sample was added 10 µg each of adenosine, AMP, ADP, and ATP as carriers for the radiolabeled nucleotide, and injected samples were eluted with a linear gradient of 10 to 700 mmol/L NaH2PO4 over 10 minutes at 2 mL/min.47 Fractions corresponding to the nucleoside and nucleotides were detected by absorption at 254 nm, collected, and counted.
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Cell Characterization
Megakaryocytic CMK and CMK 11-5 cells, megakaryoblastic cells Meg-01 and MO7E, and platelets were evaluated for reactivity to ADP and ATP by measurement of intracellular Ca2+ mobilization using the photoprobe fura 2 (Fig 1
).With platelets and CMK 11-5 cells, only ADP and ADP--S (data not shown) induced a comparable increase in intracellular Ca2+ mobilization. The absolute levels of Ca2+ mobilized in responses to ADP in CMK 11-5 cells are similar to those in platelets (Fig 1) and, moreover, are consistent with results when other megakaryoblastic48 49 and megakaryocytic cell lines are used.50 In addition, CMK 11-5 cells were unresponsive to ATP--S and ATP--S at concentrations to 100 µmol/L, as were platelets. The rate and extent of Ca2+ mobilization induced by ADP was dose-dependently inhibited by ATP and ATP--S, whereas GDP and adenosine were inactive as antagonists. Mobilization of Ca2+ was not observed in the presence of 100 µmol/L ADP after CMK 11-5 cells were treated with 10 nmol/L PMA for 48 hours (binding data shown in the Table). In contrast to these results, both ADP and ATP elicited approximately equal levels of Ca2+ mobilization with MO7E and Meg-01 cell lines and the parental cell line CMK, indicating that these cells either have non-P2T receptors or a mixed complement of purinergic receptors.
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Nucleotide Binding Isotherms
The binding of ADP to paraformaldehyde-treated CMK 11-5 cells was evaluated by incubation with 10 nmol/L [3H]ADP in the presence or absence of increasing concentrations of unlabeled ADP or unlabeled ATP--S (Fig 2). For each homologous ([3H]ADP versus ADP) or heterologous ([3H]ADP versus ATP--S) binding isotherm (Fig 2A and 2C, respectively), two experiments were completed using 21 duplicate point determinations over at least four log concentrations of competing ligand. The augmented ability of ATP--S to displace [3H]ADP from surface binding sites relative to ADP is shown by the leftward shift of the binding isotherm (compare Fig 2A and 2C). The binding isotherms were transformed by Scatchard analysis and plotted, and [3H]ADP binding sites were identified (Fig 2B and 2D). For experiments shown in this figure, the duplicate binding isotherms were coanalyzed by the LIGAND program to increase the accuracy of the nonlinear line fitting for a two-site model25 : both a single-site and a triple-site model were also examined for representation of the binding data, but in both cases, least-squares analysis results indicated greater divergence than for the two-site model. A comparison of binding parameters between CMK 11-5 cells and platelets is shown in the Table
. In a single experiment performed with quadruplicate determinations, coincubation of [3H]ADP with 500 µmol/L ATP, ADP--S, or adenosine reduced [3H]ADP binding by 100%, 93%, and 18%, respectively, showing that a P2T antagonist and agonist, respectively, but not a P1 receptor agonist, influenced the binding of [3H]ADP to the CMK 11-5 cells. Treatment of CMK 11-5 cells with PMA at a concentration of 10 nmol/L, which induces the appearance of a number of platelet antigens,32 resulted in a twofold to threefold increase in the binding site affinities for [3H]ADP at both sites and a twofold to threefold increase in the total number of binding sites.
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Photoaffinity Labeling
35S-ATP--S at 50 nmol/L was directly and specifically photoincorporated into the -subunit of GP IIb (122 kD) of CMK 11-5 cells, as shown by electrophoresis of photoaffinity-labeled material on 8% SDS-PAGE gels under reducing and nonreducing (not shown) conditions and its specific retention on an anti–GP IIb/IIIa (AP-2)-immunoaffinity column25 (n=3 experiments) (Fig 3). The radiolabeled protein of 122 kD was not retained on an IgG2a-conjugated protein-A Sepharose column (data not shown). In the presence of 500 µmol/L ADP or ATP--S, photoincorporation of 35S-ATP--S into GP IIb was not observed, thus demonstrating specific interactions with the precautions used as described.25 26 In contrast to platelets,25 CMK 11-5 cells were not photolabeled by either 60 nmol/L 35S-GTP--S or 140 nmol/L 35S-UTP--S.
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Binding of Nucleotides to Oocytes
Paraformaldehyde-treated oocytes were incubated with either [3H]ADP or 35S-ATP--S to evaluate their binding characteristics (Fig 4). Although both nucleotides bound to the oocyte surface, only a portion was specific binding, because coincubation with nonlabeled nucleotide at a 1000-fold concentration excess reduced total binding from 40% to 60% (Fig 4). In homologous experiments in the presence of 10 nmol/L 35S-ATP--S, individual oocytes (n=15 oocytes) bound 8310±1466 (±SD) cpm, with a range of 6250 to 11 500 cpm, and this bound amount was reduced to 3884±764 cpm (±SD), with a range of 3150 to 6250 cpm in the presence of 10 µmol/L ATP--S, with an average decrease of 53%. In the presence of 10 nmol/L [3H]ADP, individual oocytes (n=15) bound 5362±1268 cpm (range, 3894 to 7493 cpm), and these values were reduced to 2495±566 (range, 1888 to 3600 cpm) in the presence of a 1000-fold excess of unlabeled ligand. Values were adjusted for specific activity differences between the ligands, and results indicate that comparable amounts of ADP and ATP--S are bound to Xenopus oocytes. Comparable results were obtained in heterologous experiments, that is, those using [3H]ADP in the presence of an excess of ATP--S or 35S-ATP--S in the presence of an excess of ADP, thereby demonstrating competition for binding sites (data not shown).
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Metabolism of Exogenous Nucleotides
To determine whether ADP would be altered by ectoenzymes on the intact oocyte, 10 nmol/L [3H]ADP was incubated with individual oocytes, and the appearance of nucleotide/nucleoside degradation products was determined by SAX-HPLC analysis. In the presence of oocytes, ADP was converted 60% to adenosine within 1 minute, and this conversion was >85% by 10 minutes (Fig 5). The radioactivity recovered in the supernatant accounted for 98% to 99% of the added ligand-associated radioactivity. From the binding studies, which indicate that only 0.5% to 1.5% of the added nucleotide binds to the oocyte, sampling of the oocyte buffer is a valid representation of the status of added nucleotide.
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), ADP (
), ATP (
), and adenosine (
) as described in "Methods." This figure is average of two experiments assayed in duplicate.
Efflux of 45Ca2+ From Oocytes After Injection of RNA
Oocytes were injected with RNase-free water, total RNA (80 ng), or mRNA (3 ng) isolated from CMK 11-5 cells: comparable results were observed between oocytes injected with total RNA and mRNA. After 48 hours at 18°C to 20°C, radiolabeled (45Ca2+) oocytes were exposed to 10 µmol/L of ADP, ADP--S, ATP, ATP--S, ATP--S, 2-MeS-ATP, ,ß-Me-ATP, or adenosine, and the ability of the nucleotide to cause an efflux of 45Ca2+ was determined (Fig 6). A consistent and significant efflux of 45Ca2+ was observed only in the presence of 10 µmol/L ADP (Fig 6, solid circles) and ADP--S and not in the presence of other nucleotides or nucleosides. Increasing the concentration of ADP to 100 µmol/L did not significantly increase the amount of Ca2+ effluxed from the oocytes, nor did increasing the ATP and ATP--S concentrations to 100 µmol/L result in efflux. In a limited number of experiments, RNA-injected oocytes were exposed to 1 µmol/L ADP, but Ca2+ efflux results were variable compared with data observed with 10 µmol/L ADP. These activating concentrations are in reasonable agreement with the dissociation constants of ADP binding sites observed, considering the rapid metabolism of ADP in the presence of intact oocytes. In the absence of injected RNA, ADP at concentrations to 100 µmol/L was unable to effect an increase of 45Ca2+ into the assay medium in parallel experiments (n=25 oocytes). To demonstrate that specific efflux resulted from the nascent appearance of a P2T receptor, pretreatment (1 minute) of mRNA-injected oocytes with 100 µmol/L ATP or ATP--S blocked the ability of 10 µmol/L ADP to cause an efflux of 45Ca2+, whereas adenosine was ineffective as a blocker (Fig 6, inverted triangles).
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, 2-MeS-ATP (P2Y agonist); 
, 
, ATP/ADP; and |
Discussion |
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The present report demonstrates that (1) ADP but not ATP or adenosine causes a rapid onset of Ca2+ mobilization in CMK 11-5 cells but not in other megakaryocytic and megakaryoblastic cell lines tested, and (2) microinjection of total RNA or mRNA derived from these cells into Xenopus oocytes results in the translation of functional receptors that respond to ADP with Ca2+ efflux, which is blocked by ATP and ATP-
-S. This response is not observed in uninjected oocytes or with CMK 11-5 RNA injected oocytes in the presence of the P2X agonist ,ß-Me-ATP, the P2Y agonist 2-MeS-ATP, or the P1 agonist adenosine. These results are consistent with the translation of CMK 11-5 cell RNA into a functional receptor with the pharmacological characteristics of the platelet P2T ADP receptor. Although CMK 11-5 cells possess only P2T purinergic receptors, other megakaryocytic and megakaryoblastic cell lines do not give unequivocal evidence of this restricted presence of P2T receptors: for example, both the purine nucleotides ADP and ATP and the pyrimidine nucleotide UTP cause mobilization of Ca2+ in Dami50 and HEL49 cells, whereas in the present report, both of the megakaryoblastic MO7E and Meg-O1 cells were shown to mobilize Ca2+ in response to ATP and ADP. These results suggest the coexistence of multiple purinergic receptors and/or the existence of P2Y receptors in these four cell types. Rat promegakaryoblasts respond to ADP but not to ATP, suggesting the presence of P2T receptors, but these cells are contraindicated as a model for the human system because they lack responses to several other agonists active in the platelet.51 K562 erythroleukemic cells respond to ADP with an increase in intracellular Ca2+, whereas ATP acts as antagonist,48 but this cell line fails to shed platelet-like particles that are released from CMK 11-5 cells.52 Compared with the CMK 11-5 cell line, both ADP and ATP mobilize Ca2+ in the parental CMK cell line. The parental CMK cell line exhibits reduced surface expression of GP Ib (CD42b) and GP IIb/IIIa (CD61/CD41) as well as elevated expression of erythroid markers Leu3a (CD4) and Leu M1(CD16).53
Pharmacological binding studies demonstrated the specific binding of [3H]ADP to CMK 11-5 cells, which was competed with nonlabeled ADP, ADP--S, ATP, and ATP--S and is consistent with findings from studies using platelets.25 In addition, neither GDP nor adenosine competed significantly with the binding of ADP to CMK 11-5 cells, indicating the absence of other purinergic receptors. Ligand affinities approximate those of platelets,25 26 whereas the number of binding sites is substantially greater, presumably because of the increase in cell surface. The number of ADP binding sites on CMK 11-5 cells is consistent with studies using formaldehyde-fixed cell lines, in which 4x105 to 1x106 sites have been measured on rat promegakaryoblasts and HEL, U937, and K562 cells.51 In addition, the measured high-affinity binding (Kd 300 nmol/L) is consistent with the minimal concentrations of ADP required for the mobilization of Ca2+ in CMK 11-5 cells (1 µmol/L), the concentration of ATP--S required to inhibit ADP-induced Ca2+ mobilization, and the concentration of 35S-ATP--S resulting in specific incorporation into GP IIb. The tumor promoter PMA induces the differentiation of CMK 11-5 cells toward a more megakaryocytic phenotype32 34 and increases both the affinity and number of binding sites for [3H]ADP. This increased number of binding sites may represent an increased translation of P2T receptors, although subsequent addition of ADP to PMA-treated CMK 11-5 cells does not result in Ca2+ mobilization, perhaps because of PMA-activated protein kinase C terminating signal transduction and exerting negative feedback control over receptor signaling pathways.54
By use of intact platelets, the purine nucleotides [35S]-ATP--S and [35S]-GTP--S and the pyrimidine nucleotide [35S]-UTP--S were previously shown to label the -subunit of GP IIb,25 26 whereas the photoaffinity reagent 8-azido-[32P]ATP labels both GP IIb and GP IIIa27 30 : with CMK 11-5 cells, photoaffinity labeling with 35S-ATP--S also resulted in its specific photoincorporation into the -subunit of GP IIb in a manner identical to the labeling of platelets.25 Both of these results suggest that the P2T receptor in these cells is in close proximity to the GP IIb/IIIa complex,30 but we have concluded that the binding of ADP to its receptors is distinct from GP IIb/IIIa and that GP IIb/IIIa is not an ADP receptor.30 One explanation for the absence of photoincorporation of GTP--S and UTP--S into GP IIb/IIIa could be that the binding sites for these nucleotides on the CMK 11-5 cells may be more specific in binding because of differences in its proximity to the receptor or to an altered nucleotide binding conformation of the receptor.
It has been suggested previously that surface nucleoside diphosphokinases may function as ADP receptors.55 The action of these surface enzymes on ADP may lead to the formation of ATP, which is a competitive antagonist for activation by ADP. In addition, ATP itself may directly interact with receptors on the platelet surface leading to alterations in cAMP levels.56 However, oocytes that were not injected with CMK 11-5 RNA did not respond to nucleotides, suggesting that binding to these endogenous sites does not generate Ca2+ efflux. From the amount of specific 35S-ATP--S and [3H]ADP bound, it was calculated that there are 2x109 to 8x109 endogenous binding sites per oocyte, assuming a stoichiometry of one nucleotide per site. Although [3H]ADP is rapidly metabolized when incubated with intact oocytes, these sites do not appear to have a role in ADP-induced Ca2+ mobilization in this system. Nonetheless, the presence of these endogenous nucleotide binding sites suggests that caution should be exercised in the use of hydrolyzable nucleotides, which could influence the assignment of rank order of nucleotide agonists in evaluation of cloned receptors using the Xenopus oocyte expression system.
This study demonstrates the presence of P2T receptors on CMK 11-5 cells and validates their use as a source of P2T ADP receptor mRNA for cloning studies. CMK 11-5 cell–derived mRNA is translated and expressed in Xenopus oocytes after microinjection, and therefore these cells should allow an expression cloning approach for the identification and characterization of this important receptor.57 58
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Acknowledgments |
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This study was supported by US Public Health Service Merit Award HL-39438 (to Dr G.A. Jamieson). I am indebted to Dr Jamieson for his interest and for providing support. I thank Dr Narendra Tandon and Barrington Jackson for their assistance with binding studies. I also thank Dr Robert Friesel for access to the Narishige microinjector and Drs Friesel, Tim Hla, and Eric Ackerman for their instrumental advice in the microinjection of Xenopus oocytes. The anti–GP IIb/IIIa monoclonal antibody AP-2 was kindly provided by Dr Thomas J. Kunicki, Scripps Research Institute, La Jolla, Calif.
Received October 20, 1995; accepted January 23, 1997.
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