1 Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4970; and 2 Department of Bioengineering, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan
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ABSTRACT |
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We have developed a method for measuring the local concentration of ATP at the extracellular surface of live cells. This method relies on the specific attachment to the cell surface of a chimeric protein that consists of the IgG-binding domain of Staphylococcus aureus protein A fused in-frame with the complete sequence for firefly luciferase (proA-luc). Expression of proA-luc in Escherichia coli and its one-step affinity purification are straightforward. Attachment to cells is demonstrated to be specific and antibody dependent using several suspended and adherent cell types. Light production by cell surface-attached luciferase is continuous, linearly related to ATP concentration, and sufficient to provide nanomolar sensitivity. The spatial resolution of this method enables the observation of strictly local changes in extracellular ATP during its secretion from activated platelets. Furthermore, the activity of cell-attached luciferase is relatively refractory to the inclusion of nucleotidases in the medium, arguing for its effectiveness in cell systems possessing potent ecto-ATPases.
purinergic signal transduction; cell surface microenvironment
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INTRODUCTION |
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THE APPEARANCE OF nucleotides, particularly ATP, in extracellular spaces is known to modulate many functions, including cardiovascular performance, neurotransmission, epithelial secretion, skeletal and smooth muscle contraction, endocrine/exocrine secretion, leukocyte adhesion, and immune, inflammatory, and thrombotic reactions (9). These effects are mediated by P2 nucleotide and P1 nucleoside receptors that are expressed in multiple subtypes on a wide array of cells and tissues (4, 9). It is recognized that ATP efflux from diverse cell types may be elicited by a variety of stimuli, including hypoxia, acidosis, mechanical deformation, hyposmotic shock, receptor stimulation, fluid shear stress, and membrane depolarization (9). Additionally, recent work has demonstrated mechanically induced release of UTP from human astrocytes (18).
There are three general mechanisms by which intracellular ATP can be released to extracellular spaces. The first involves direct, nonspecific cytolysis of healthy cells by physical and biological traumas. Given the presence of 3-5 mM ATP within the cytoplasm of all cells, any cell type can be considered a potential source of this nonselectively released ATP. A second mechanism involves the release of ATP compartmentalized within exocytosis-competent granules or vesicles. ATP is copackaged in the secretory granules of many neuronal, neuroendocrine, or endocrine cells and is rapidly released during synaptic neurotransmission and other types of regulated exocytosis. In some cases, nonlytic, stress-induced ATP release can be modulated by drugs known to alter the function of certain ATP-binding cassette type transport proteins (29). Thus a third potential route for ATP release is the efflux of cytosolic ATP via plasma membrane transport proteins.
Several factors complicate the straightforward analysis of physiologically relevant ATP release. The ubiquitous presence of ATP in all cell types makes it difficult to determine whether extracellular ATP is derived from particular, intact cell types via selective release mechanisms or whether this ATP is derived from the nonselective cytolysis of cells during experimental manipulations. In addition, most cells express ecto-nucleotidases that rapidly hydrolyze extracellular ATP present at the cell surface (26, 33). Released nucleotides may be spatially confined between cells or diluted by diffusion and dissipated by convection. For these reasons, the concentration of ATP in the proximity of cell surfaces is expected to be dynamic, and the measurement of ATP in bulk extracellular fluids may greatly underestimate the amount of ATP actually released at the cell surface. This may be particularly true in cells or tissues with substantial unstirred surface layers. It is therefore likely that ATP release in amounts that are sufficient to activate cells locally (via autocrine or paracrine mechanisms) cannot be adequately measured using bulk solution samples. Taken together, these factors confound the direct measurement of ATP efflux during most experimental manipulations and contribute to substantial disagreement between different groups (1, 15, 27).
Firefly luciferase is widely used as an extraordinarily sensitive bioluminescent ATP sensor protein (7, 19). Under suitable conditions, this assay system can detect ATP concentrations ranging from picomolar to micromolar levels. This method has been primarily used in offline experiments, wherein cell-free aliquots of extracellular medium are periodically sampled following stimulation of cells or tissue and then mixed with standard solutions of luciferase and D-luciferin. Luciferase-luciferin has also been used for online assays of ATP release from cell types such as platelets or adrenal chromaffin cells, which are readily studied as cell suspensions and which release large amounts of ATP by exocytosis of ATP-containing granules (8, 28). Unfortunately, such studies rely on the analysis of bulk solution samples that, as described above, do not accurately represent the dynamic ATP concentrations localized in the immediate vicinity of cell surfaces.
In principle, the sensitivity and utility of luciferase as a sensor of ATP release into localized or restricted extracellular spaces might be improved by selectively targeting the luciferase to the extracellular surface of a cell type being considered as a potential source of ATP. In this study, we report the use of a protein A-luciferase chimera (proA-luc) that can be stably adsorbed onto the surface of intact cells via interactions with primary IgG antibodies directed against native surface antigens. We show that local extracellular ATP levels can be continuously assayed with high sensitivity and excellent temporal and spatial resolution using either suspended cells or adherent cells that are coated with proA-luc. With this method, we are able to observe cell surface-localized transient ATP concentrations during secretion from activated platelets. This reagent and method should prove useful for the online detection of low-level ATP release from cells, such as epithelia, that utilize highly localized nucleotide release for autocrine signaling or tissues, such as endothelia, that express very high levels of ecto-nucleotidase activity.
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METHODS |
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Materials
Apyrase grade I and lysozyme were from Sigma. Luciferin was purchased from Molecular Probes. 1,4-Dithiothreitol (DTT), leupeptin, and aprotinin were obtained from Boehringer Mannheim. Luria-Bertani (LB) medium components were supplied by DIFCO Laboratories. Unless explicitly stated otherwise, all other reagents were from Sigma.Transformation of Bacteria
pMALU5 plasmid DNA (120 ng) was incubated with 100 µl of competent JM109 Escherichia coli for transformation by heat shock. Transformants were selected on ampicillin plates, grown to saturation in LB medium (30) containing 50 µg/ml ampicillin, and stored as 15% glycerol stocks.Growth of ProA-luc-Transformed Bacteria and Preparation of Bacterial Lysates
Transformed E. coli (500 µl) were added to 500 ml of LB medium in the presence of 50 µg/ml ampicillin and grown to mid-log phase at 37°C, at which time isopropylPurification of Recombinant ProA-luc Protein
The soluble fraction of bacterial lysates was filtered through 0.2-µm Acrodisks (Gelman Sciences). ProA-luc was purified at room temperature by affinity chromatography on immobilized bovine IgG-agarose beads (Sigma). Filtered samples (3 ml) were diluted 1:1 with Immunopure Gentle Ag/Ab binding buffer (Pierce) and incubated end-over-end with 1.4 ml of bovine IgG-agarose beads for 1 h at room temperature. The suspended beads were packed into a 12-ml plastic column, allowed to settle, and then washed with 15 column volumes of binding buffer. ProA-luc was eluted with 8.5 ml of Immunopure Gentle Ag/Ab elution buffer (Pierce) and allowed to drop directly into 1 ml of 10× STE [containing 1.5 M NaCl, 1 M Tris · HCl (pH 8.5), and 50 mM EDTA] to rapidly neutralize the pH of the eluate. The eluate was applied to a HiTrap desalting column (Pharmacia) and was buffer exchanged into 1× STE [containing (in mM) 150 NaCl, 100 Tris · HCl (pH 7.8), and 5 EDTA]. Fractions from this column were tested for luciferase activity, and those containing the peaks of the activity were pooled and concentrated 25-fold using Ultrafree centrifugal filters (Biomax 10k, Millipore). After addition of DTT to 1 mM, the purified proA-luc was stored at 4°C.Luciferase Activity Assays
The activity of proA-luc during purification was assayed by diluting samples in an optimized solution (FL-AAB, Sigma) containing MgSO4, DTT, EDTA, BSA, and tricine buffer salts. Luminescence was measured with a Berthold Lumat LB 9501 luminometer set to measure integrated light intensity in 5.0-s intervals. Assays were performed by mixing typically 5-20 µl of proA-luc fractions with 20 µl of 1.5 mM luciferin and FL-AAB buffer in 12 × 75-mm polystyrene tubes (Falcon 2008, Becton Dickinson) and then recording background luminescence. Twenty microliters of 10 µM ATP were added (1 µM ATP final), and the maximum light output during subsequent integration periods was recorded in arbitrary units. Final volumes were 200 µl. To measure specific luciferase activities, protein content was determined using the Bio-Rad protein assay and used to normalize light output by a given sample. Specific activities were expressed as relative light units (RLU) per mg protein per µM ATP.For all experiments with intact cells, we designed a luminometer apparatus that permits continuous recording of light output. This instrument consists of a photomultiplier tube (Thorn EMI 9924B) operated at 800 V and surrounded by a light-tight machined stainless-steel housing and a sample holder that can accommodate either 12 × 75-mm tubes (for suspended cells) or 35-mm tissue culture plates (for adherent cells). A shutter between the sample holder and the surface of the photomultiplier protects the tube from stray light during reagent addition. Output from the instrument was filtered at 10 Hz and recorded in analog fashion on chart paper. Light production measured with this apparatus was expressed as relative light output (RLO) to distinguish this arbitrary scale from that utilized by the Berthold luminometer (RLU).
SDS-PAGE and Western Blotting
SDS-PAGE (9% acrylamide) was performed under denaturing conditions in the presence ofCell Culture
HL-60 promyelocytes and Bac-1.2F5 murine macrophages were maintained as previously described (6, 24). HL-60 cells were used 36-48 h after passage by pelleting and resuspension to a density of 30 × 106/ml in balanced salt solution (BSS) containing (in mM) 130 NaCl, 5 KCl, 25 HEPES (pH 7.5), 1.5 CaCl2, 1 MgSO4, 5 NaHCO3, 1.5 KH2PO4, 10 dextrose, and 3.5 mg/ml BSA. Bac-1.2F5 macrophages were passaged for 36-48 h before experiments and reseeded onto 35 × 10-mm sterile tissue culture dishes (Corning).Preparation of Washed Human Platelets
All steps of the preparation were at room temperature. Blood was collected from healthy volunteers into acid-citrate-dextrose anticoagulant (100 mM trisodium citrate and 128 mM dextrose, pH 5) at a final ratio of 6:1. The collected blood was diluted 1:1 in 50-ml Falcon tubes with PBS containing (in mM) 9.1 Na2HPO4, 1.7 NaH2PO4, and 150 NaCl (pH 7.4); 25-ml aliquots were then layered onto 13 ml of Histopaque-1077 (Sigma) and centrifuged at 400 g for 30 min. The resultant mononuclear layer at the Histopaque-plasma interface was collected using a sterile transfer pipette, diluted to 50 ml with PBS-citrate (containing 13 mM trisodium citrate, pH 7.4) and then centrifuged at 250 g for 10 min to pellet monocytes and lymphocytes. Suspended platelets were transferred to a new Falcon tube, counted, and then centrifuged at 1,200 g for 15 min. After aspiration of the supernatant, the pelleted platelets were resuspended at a working density of 15 × 108/ml in platelet buffer (BSS lacking CaCl2, supplemented with 50 U/ml heparin, 600 nM indomethacin, and 0.5 U/ml apyrase) and allowed to incubate at room temperature for at least 15 min before being subjected to further manipulation.Antibodies
To remove azide from storage solutions, all monoclonal antibodies used for attachment to intact cells were diluted at least 20-fold into BSS or platelet buffer (lacking glucose and BSA) before each experiment and reconcentrated to the desired concentration using Ultrafree centrifugal filters (Millipore). For studies with HL-60 human promyelocytes, we used mouse anti-human monomorphic human leukocyte antigen (HLA)-ABC monoclonal IgG2a antibody (MAB1280, Chemicon). For studies with Bac-1.2F5 murine macrophages, we used either mouse anti-mouse CD45.2 monoclonal IgG2a (09611D, PharMingen) or mouse anti-mouse H-2Kd major histocompatibility complex (MHC) class I monoclonal IgG2a (06091D, PharMingen) antibodies. For human platelets we used mouse anti-human CD41 monoclonal IgG2b (MCA 468S, Serotec) or anti-HLA-ABC (see above) antibodies.Cell Surface Attachment of ProA-luc
Cells were incubated in 1.5-ml microcentrifuge tubes. In practice, we found that cell loss during washing was minimized by conducting all steps in a single tube.Platelets.
The cells were maintained at room temperature throughout the following
procedure that provided 12 individual samples for testing. Aliquots
(600 µl) of platelets at 15 × 108/ml were incubated for 45 min
with 200 µl of buffer-exchanged anti-CD41 or anti-HLA-ABC antibodies
at various dilutions. Platelets were washed twice to remove unbound
antibody by adding platelet buffer to 1.5 ml, centrifuging at 1,200 g for 15 min, resuspending in 1 ml of
buffer, and recentrifuging. The pellet was then resuspended in 600 µl
of platelet buffer plus 200 µl of proA-luc at an initial concentration of roughly 100 µg/ml (corresponding to a specific activity of roughly 4 × 109
RLU · mg
protein1 · µM
ATP
1). After a further
45-min incubation, the cells were washed once in platelet buffer and
twice in platelet buffer lacking heparin, apyrase, and indomethacin and
were finally resuspended in 1,560 µl of the latter buffer containing
1.5 mM CaCl2. Aliquots (120 µl)
were used for bioluminescence assays.
HL-60 promyelocytes. Aliquots (40 µl) of cells at 30 × 106/ml were incubated for 1 h on ice with 10 µl of buffer-exchanged anti-HLA-ABC antibody at various dilutions. The cells were subsequently washed twice to remove unbound antibody by diluting with 1 ml ice-cold BSS, centrifuging for 1 min at 2,000 g, and aspirating the supernatant. After the second wash, cells were resuspended in 180 µl of BSS plus 20 µl of proA-luc at an initial concentration of ~100 µg/ml and incubated on ice for 1 h. After three washes with ice-cold buffer (as before), cells were finally resuspended in 150 µl of BSS and stored on ice until they were used for bioluminescence assays. Aliquots (140 µl) were used for the assays.
Bac-1.2F5 macrophages. Adherent cells on 35-mm plates were washed twice with 1-ml portions of ice-cold BSS and then allowed to incubate for 1 h on ice in 1 ml of BSS plus 16-50 µl of either anti-CD45.2 or anti-H-2Kd antibodies at various dilutions. After this incubation, the adherent cells were washed three times and then allowed to incubate for another 1 h on ice in 1 ml of BSS plus 30 µl of proA-luc (at roughly 100 µg/ml). Subsequently, the cells were washed four times and then allowed to incubate on ice in 1 ml of BSS until they were used for luminescence assays.
Cell-Attached Luciferase Assays
Cells (prepared as above) were supplemented with 150 µM luciferin (added from 15 mM stock) and tested at room temperature. HL-60 cells and platelets were assayed without continuous stirring in 12 × 75-mm polystyrene tubes. Bac-1.2F5 cells were tested in the 35-mm dishes in which they were initially plated. For tests involving the addition of exogenous ATP, HL-60 and platelet suspensions were brought to a final volume of 200 µl. Bac-1.2F5 cells were tested in 1 ml of buffer.All assays were performed by placing prepared cells in the luminometer and opening the shutter to record background luminescence. The shutter was manually closed during addition of various reagents and then reopened after mixing (within 3-4 s) to record light output. In practice, we found that this small time delay did not introduce significant errors. Added reagents were mixed by agitation of the tubes (suspended cells) or dishes (adherent cells) before they were placed in the luminometer.
For some tests (see Fig. 8), we utilized a Chronolog 460-VS lumiaggregometer with continuous stirring at 37°C, which permitted the simultaneous measurement of activated platelet ATP release and aggregation.
Figure Preparation
Pen recordings of the photomultiplier tube output were traced onto white paper and scanned using a Scanjet 4C (Hewlett-Packard) and Adobe Photoshop 3.0. Figures were prepared using Adobe Illustrator 7.0 or Microsoft Excel 97. ![]() |
RESULTS |
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Cell Attachment Strategy
In a cell-attached configuration, proA-luc is ideally situated to detect surface-localized ATP as it is released from cells and to compete for this ATP with ectoenzymes that catalyze its hydrolysis (Fig. 1A). Attachment is achieved by sequential incubations with primary antibodies against a particular surface epitope and then proA-luc. The protein A domain of proA-luc interacts strongly with the exposed Fc domains on individual antibody molecules, leading to its attachment. Cells are washed extensively afterwards to remove unbound luciferase.
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Engineering of pMALU5
The previously constructed expression vector pMALU2 (16) lacked twelve residues corresponding to the amino-terminus of luciferase, which caused the luminescence behavior of the expressed protein to differ from that of wild-type luciferase. Therefore, a new plasmid was constructed (Fig. 1B) that contains the four IgG-binding domains of protein A (17, 23) fused in-frame with the complete coding sequence for firefly luciferase. The chimeric DNA is under the control of a tac promoter and is inducible by IPTG at room temperature (Fig. 2A). Ampicillin resistance is conferred by the inclusion of a gene for
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Expression and Purification of ProA-luc
A significant fraction of recombinant proA-luc was found in the insoluble pellet from lysed cells (not shown). However, the relative inducible expression of proA-luc in the soluble phase was substantially increased at temperatures below 30°C (not shown; Ref. 21). Overproduction of the protein in JM109 E. coli was confirmed by the presence of conspicuous bands in the region of its predicted molecular mass (Fig. 2A). Luciferase activity in the different lysates correlated with the relative intensities of the bands (not shown). Western blotting confirmed the presence of protein A activity (Fig. 2B), which also correlated with the relative band intensities.Purification of proA-luc from E. coli lysates was achieved using affinity chromatography (Fig. 2) at room temperature. Eluted protein was desalted and thereby exchanged into STE buffer at pH 7.8. Luciferase activities were determined in arbitrary light units and normalized to the protein content; specific activities were thus expressed as relative light units (RLU) per mg protein per µM ATP (see Luciferase Activity Assays). Greater than 90% of the luciferase activity was retained by the beads as judged by the activity of the flow-through fraction (Table 1). A modest increase in specific activity was observed after purification of the enzyme (Table 1). It is clear that a large loss in enzyme activity occurred during elution of the protein from the affinity column. Indeed, an estimate of the percent luciferase activity recovered relative to an equimolar amount of wild-type luciferase indicates that at least 75% of the purified molecules were inactive for light production (see DISCUSSION).
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To assess the nature of the purified luciferase activity, wild-type luciferase (Sigma) was diluted to the point at which its light production in response to a given bolus of ATP was equal to that of an equal-volume sample of proA-luc. At all concentrations of ATP used, the two enzymes exhibited identical peak light production and decay characteristics (Fig. 3A), demonstrating the integrity of the luciferase domain when fused with protein A. Peak light output by the two enzymes was linear over the tested range of ATP concentrations (Fig. 3B), which extended into the subnanomolar and high-micromolar levels (not shown). The lower limit of reliable ATP measurement was dictated by the noise-to-signal ratio, which improved from 33% at 100 pM ATP to 8% at 320 pM to 3% at 1 nM and which continued to decrease with increasing ATP concentration.
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Specificity and Antibody Dependence of Cell Attachment
Attachment of proA-luc to cell surfaces was studied using three different suspended and adherent cell types (Fig. 4). The choice of antibodies for cell surface attachment was made with several considerations in mind: 1) antibodies must be against extracellular epitopes, 2) epitopes should be in relatively high copy number per cell, and 3) antibodies should be of subtypes that possess relatively high affinity for protein A. Among mouse subtypes, binding strengths to protein A follow the order IgG2b > IgG2a
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To measure cell-attached luciferase activity, cells were prepared as described in Cell-Attached Luciferase Assays and challenged with exogenous ATP in the presence of 150 µM luciferin. Only those cells preincubated with primary antibodies retained a significant amount of luciferase activity, demonstrating the specificity of cell attachment (Fig. 4A). The dependence of attached activity on primary antibody concentration was studied by incubating identical cell preparations with different concentrations of antibodies. In all cases, the amount of surface-attached luciferase increased monotonically with primary antibody concentration (Fig. 4, B-D); saturation of surface binding sites was observed with anti-HLA-ABC antibodies on platelets and HL-60 cells. The increased retention of proA-luc by anti-CD41-coated platelets vs. anti-HLA-ABC-coated platelets presumably reflects differences in the copy number of these respective surface antigens.
ATP Dependence of Cell-Attached Luciferase Activity
ProA-luc-coated cells were prepared and identical samples were tested for their response to different concentrations of exogenous ATP. Peak light production from coated HL-60 cells and from coated platelets was linear with respect to added ATP over the expected range of physiological concentrations from high-micromolar to mid-nanomolar levels (not shown). Light production was additionally proportional to the total proA-luc concentration, as demonstrated by using dilutions of coated cells (not shown). At high-micromolar concentrations of ATP, light output by proA-luc exhibited the well-characterized "flash" kinetics that are due to product inhibition (Fig. 3A, top). This behavior could be eliminated by performing assays in the presence of CoA (Fig. 5). With at least 100 µg/ml CoA in the sample, light output by coated platelets in response to exogenous ATP was sustained and responded linearly to ATP concentrations (not shown). Consistent with this observation is the idea that consumption of ATP by cell-attached luciferase is negligible on the time scale of these experiments.
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Light Transients From Activated Platelets
Secretion from platelets can be elicited by a variety of stimuli, including thrombin, collagen, von Willebrand factor, ADP, vasopressin, platelet-activating factor, and Ca2+ ionophores (31). ATP is copackaged in platelet dense granules with serotonin, Ca2+, and ADP (31) and is therefore secreted when the cells are activated. We examined light production by soluble vs. cell-attached luciferase during platelet ATP secretion elicited by thrombin (Fig. 6A). The experiment with cell-attached luciferase produced a light transient that was uniquely sensitive to the local concentration of ATP at platelet cell surfaces during the secretion process (Fig. 6A, top trace), whereas an equivalent amount of soluble luciferase (25 ng/ml) was unable to yield this information (Fig. 6A, middle trace). Even at a much higher concentration (100 µg/ml), soluble luciferase was unable to detect the ATP transient near platelet cell surfaces (Fig. 6A, inset). In steady state, presumably after released ATP had diffused evenly throughout the sample volume, equivalent amounts of soluble or cell-attached luciferases produced equal amounts of light (Fig. 6A, top and middle traces). To control for washout of unbound proA-luc, the experiment was repeated using platelets that had been preincubated in the absence of primary antibody (Fig. 6A, bottom trace). Platelet secretion was also studied using platelets coated with primary antibodies against the HLA-ABC complex, in the absence or presence of CoA. As shown in Fig. 6B, the essential features of the light transients in these proA-luc-coated platelets were similar. This shows that local product inhibition of adsorbed proA-luc was not the cause of the light transients observed in thrombin-activated platelets (see DISCUSSION).
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Many of the cell types known to participate in purinergic signaling possess potent ecto-ATPases that rapidly degrade extracellular ATP (26, 33). To study how extracellular ATPases might modulate the luciferase signals, platelet degranulation was triggered in the presence of a high concentration of soluble apyrase (10 U/ml). The magnitude of the light signal produced by cell-attached proA-luc was only moderately attenuated by apyrase addition (Fig. 7A). In contrast, the light signal produced by soluble luciferase was nearly eliminated in the presence of apyrase (Fig. 7B). Whereas one might conclude from the soluble luciferase experiment that ecto-ATPase activity can completely scavenge the secreted ATP before it could initiate signal transduction, the cell-attached observations demonstrate the existence of a significant local ATP concentration at the platelet surface, consistent with the potential for autocrine/paracrine stimulation of P2 receptors.
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To assess the effects of stirring and temperature on the observed light and to measure another index of normal platelet function, we measured ATP secretion and aggregation simultaneously using a Chronolog lumiaggregometer. As shown in Fig. 8A, the essential characteristics of the light transients produced by surface-attached luciferase were qualitatively unaltered by stirring or an increase in temperature to 37°C. Furthermore, proA-luc-coated platelets remained competent for aggregation despite ligation of a fraction of their CD41 molecules (Fig. 8B). A subsequent addition of 10 µM ATP for calibration of the light signal indicated that the peak ATP concentration at the surface of stirred, coated platelets was in the 15-20 µM range, consistent with the data from unstirred platelets (Fig. 6A).
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DISCUSSION |
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The relatively recent classification of nucleotide receptor subtypes with respect to function, pharmacology, and primary structure (13) has been crucial to an understanding of nucleotide-based signaling. The study of ATP-based signaling has also been facilitated by the extremely sensitive and specific assay for released extracellular ATP in the form of the luciferase-catalyzed reaction. However, by the nature of the assay, it is restricted to the determination of ATP in the bulk solution in which cells are bathed. This is generally unsatisfactory because many factors (e.g., ecto-ATPases, unstirred layers) may cause the concentration of ATP in the bulk medium to be different from that in the local microenvironment of cell surfaces. It is therefore important to be able to measure extracellular ATP at the site of receptor stimulation.
Our observations of ATP secretion from platelets (Figs. 6, 7A, and 8A) demonstrate that cell-attached luciferase monitors the local rise of ATP as it is deposited in the extracellular space and the decay in this concentration as ATP diffuses into the bulk solution. Significantly, even much higher (4,000×) amounts of soluble luciferase in the medium were incapable of recording this type of "ATP transient" (Fig. 6A, inset). Although the signal from soluble luciferase was acutely sensitive to the presence of soluble apyrase, that from cell-attached luciferase was relatively refractory to such treatment (Fig. 7). The latter result is consistent with that of Aflalo (2), who monitored local ATP at the surface of respiring mitochondria using recombinant luciferase targeted to the cytoplasmic face of the mitochondrial outer membrane.
Surface-localized ATP may be transiently sequestered from the bulk solution by unstirred layer effects, even during rapid stirring of the suspended platelets (Fig. 8A). Alternatively, the shape change that accompanies platelet activation (31) leads to the invagination and sequestration of membrane areas from the bulk solution. This could explain the protection of surface-localized ATP from exogenous apyrase (Fig. 7), as well as the inability of soluble luciferase to detect the surface-localized transient (Fig. 6A). However, the equality of steady-state light production by soluble and cell-attached luciferases (Fig. 6A) does not support this interpretation. Furthermore, our data using coated platelets in the presence of exogenous apyrase (Fig. 7A) argue against the complete sequestration of extracellular ATP, given the decay of the cell-attached luciferase signal to baseline levels (presumably due to consumption of the released ATP by apyrase). Escolar et. al. (10) demonstrated that CD41 moves from the periphery toward platelet centers during thrombin stimulation; however, complete sequestration of a fraction of the total CD41 did not become apparent until 3-5 min after activation.
Tethering of proA-luc to platelet surfaces greatly reduces the effective volume in which it is confined, ensuring that it will be responsive only to ATP that is in the same microregion. In contrast, soluble luciferase is freely diffusible throughout the entire assay volume. Under such conditions, the enzyme is unable to discriminate between surface-localized and bulk ATP. This feature is independent of the concentration of soluble luciferase, consistent with the inability of concentrated luciferase to detect the surface-localized transient (Fig. 6A, inset).
This method provides information about platelet secretion that has previously been unavailable. Under the conditions of these experiments (Figs. 6, 7A, 8A), we were able to observe a transient peak ATP concentration on the order of 15-20 µM, which is sufficient to initiate autocrine stimulation of platelet P2X1 receptors (5). This estimate assumes that all platelets degranulate with identical time courses. Numerical deconvolution of the light output from proA-luc-coated platelets should yield a time-dependent kinetic profile for the average platelet secretory response under various conditions, which should permit the testing of this assumption. The local concentration of secreted ADP is not directly measurable by this technique. However, the ATP-to-ADP ratio in human platelet dense granules is approximately two (32). If it is assumed that dense granule ATP and ADP undergo secretion with the same kinetics, our data suggest that the surface concentration of ADP following thrombin stimulation will transiently reach 7-10 µM. This is sufficient for maximal activation of the platelet P2Y1 receptor (20) and consistent with feed-forward autocrine/paracrine activation of platelet responses (31).
Firefly luciferase catalyzes the production of 562-nm light with high
quantum yield when incubated with its substrates,
D-luciferin and MgATP, in the
presence of molecular oxygen (7, 19). Light output by the enzyme in
response to ATP concentrations
>106 M shows flash
kinetics that include a rapid rise (~300 ms) to a peak followed by a
rapid decay (~0.5 s), presumably due to product inhibition (19). A
variety of compounds are known to stimulate turnover by the enzyme,
including CoA (11), which effectively removes the product inhibition
and results in constant light output (even for ATP concentrations
>10
4 M). We repeated
platelet secretion studies using cell-attached luciferase in the
presence of 250 µg/ml CoA to ensure that the observed light transient
was not due to product inhibition of proA-luc (Fig.
6B). Peak heights observed during
platelet secretion were reduced slightly in the presence of CoA (Fig.
6B,
left), which may be due to somewhat
slower activation of luciferase under these conditions (Fig.
5A,
right).
Protein A is a component of the cell wall of
Staphylococcus
aureus that is bound to peptidoglycan
at its carboxy terminus and is distinguished by the presence of four
tandem IgG-binding domains in its amino-terminal portion (17, 23). It
has been widely used as an immunoaffinity reagent (14) and as a fusion partner to facilitate the purification of recombinant proteins (23).
Recently developed applications include the construction of novel
bioluminescent immunoassay reagents (12, 16, 25). IgG-subtype
specificity of protein A binding played an important role in the
purification of proA-luc. In general, antibody binding strength by
protein A is species specific and follows the order human = rabbit = guinea pig > mouse > cow goat, rat, chicken, and hamster (30).
Although we first attempted to purify proA-luc using rabbit
IgG-agarose, we found that the buffers required to elute proA-luc from
this resin caused significant loss of luciferase activity, due to
irreversible denaturation. The intermediate affinity of protein A for
bovine IgG-agarose was sufficient for depletion of proA-luc from
bacterial lysates while facilitating its elution by relatively mild
buffers. Under these conditions, we typically recovered 20% of the
original luciferase activity (Table 1). However, affinity purification
of proA-luc based on interactions with the protein A domain does
present a few disadvantages. Proteolytic fragments of proA-luc copurify
due to their possession of an active protein A domain (Fig. 2,
A and
B). These species may compete for
cell surface binding sites, occupying them nonproductively. Additionally, it is likely that a significant portion of purified proA-luc consists of active protein A moieties coupled to luciferase that is denatured during elution from the affinity column.
A potential complication of this method is that antibody binding to some cell surface epitopes can cause activation of the cells. In this preliminary study, we have not investigated the functional consequences of antibody attachment. However, consistent secretion profiles from activated platelets were obtained using either anti-HLA-ABC or anti-CD41 antibodies (Fig. 6). For cell types in which it may be difficult to select an appropriate membrane marker on which to tether proA-luc, transfection of cDNAs engineered to express intermembrane proteins that lack signaling function may provide a practical alternative.
It should be relatively straightforward to adapt this method to many different cell types, both suspended and adherent. Nanomolar changes in extracellular ATP can be observed (not shown) with excellent spatial and temporal resolution (Figs. 6-8). It should also be possible to study local ATP release in mixed populations of cell types by using antibodies against cell-specific surface antigens. Another potential application is to adapt the method for use with cooled charge-coupled device (CCD) cameras for observation of ATP secretion at the single-cell level. The feasibility of this application is supported by the findings of Oker-Blom and colleagues (25), who recorded cooled CCD bioluminescence images of protein A-luciferase attached to individual IgG-Sepharose beads.
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ACKNOWLEDGEMENTS |
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We are indebted to Erin Clifford, Ulrich Hopfer, Ben Humphreys, Masao Ikeda-Saito, Emil Negrescu, and Karen Parker for helpful discussions, to Richard Eckert for access to the Berthold luminometer, and to Joan Fox for access to the Chronolog lumiaggregometer. José Whittembury built the homemade luminometer we used for continuous light recording. Sylvia Kertesy provided excellent technical assistance.
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FOOTNOTES |
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This work was supported in part by National Institutes of Health (NIH) Grant GM-36387 (to G. R. Dubyak), by NIH Cystic Fibrosis Feasibility Grant DK-27651 (to G. R. Dubyak), and by NIH Training Grant HL-07415 (to R. Beigi).
Portions of this work appeared in poster and abstract form at the 1998 Biophysical Society Meeting (Kansas City, MO).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: G. R. Dubyak, Dept. of Physiology and Biophysics, School of Medicine, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-4970.
Received 12 June 1998; accepted in final form 17 August 1998.
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REFERENCES |
---|
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---|
1.
Abraham, E. H.,
P. Okunieff,
S. Scala,
P. Vos,
M. J. S. Oosterveld,
A. Y. Chen,
B. Shrivastav,
and
G. Guidotti.
Cystic fibrosis transmembrane conductance regulator and adenosine triphosphate.
Science
275:
1324-1325,
1997
2.
Aflalo, C.
Localized firefly luciferase probes ATP at the surface of mitochondria.
J. Bioenerg. Biomembr.
29:
549-559,
1997[Medline].
3.
Ball, E. D.,
P. M. Guyre,
J. M. Glynn,
W. F. C. Rigby,
and
M. W. Fanger.
Modulation of class I HLA antigens on HL-60 promyelocytic leukemia cells by serum-free medium: re-induction by -IFN and 1,25-dihydroxyvitamin D3 (calcitriol).
J. Immunol.
132:
2424-2428,
1984
4.
Bruns, R. F.
Adenosine receptors: roles and pharmacology.
Ann. NY Acad. Sci.
603:
211-226,
1990[Medline].
5.
Clifford, E. E.,
K. Parker,
B. D. Humphreys,
S. B. Kertesy,
and
G. R. Dubyak.
The P2X1 receptor, an adenosine triphosphate-gated cation channel, is expressed in human platelets but not in human blood leukocytes.
Blood
91:
3172-3181,
1998
6.
Cowen, D. S.,
M. Berger,
L. Nuttle,
and
G. R. Dubyak.
Chronic treatment with P2-purinergic receptor agonists induces phenotypic modulation of the HL-60 and U937 human myelogenous leukemia cell lines.
J. Leukoc. Biol.
50:
109-122,
1991[Abstract].
7.
DeLuca, M.,
and
W. D. McElroy.
Purification and properties of firefly luciferase.
Methods Enzymol.
57:
3-15,
1978.
8.
Detwiler, T. C.,
and
R. D. Feinman.
Kinetics of the thrombin-induced release of adenosine triphosphate by platelets. Comparison with release of calcium.
Biochemistry
12:
2462-2468,
1973[Medline].
9.
Dubyak, G. R.,
and
C. El-Moatassim.
Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides.
Am. J. Physiol.
265 (Cell Physiol. 34):
C577-C606,
1993
10.
Escolar, G.,
E. Leistikow,
and
J. G. White.
The fate of the open canalicular system in surface and suspension-activated platelets.
Blood
74:
1983-1988,
1989[Abstract].
11.
Ford, S. R.,
M. S. Hall,
and
F. R. Leach.
Enhancement of firefly luciferase activity by cytidine nucleotides.
Anal. Biochem.
204:
283-291,
1992[Medline].
12.
Frank, L. A.,
V. A. Illarionova,
and
E. S. Vysotski.
Use of proZZ-obelin fusion protein in bioluminescent immunoassay.
Biochem. Biophys. Res. Commun.
219:
475-479,
1996[Medline].
13.
Fredholm, B. B.,
M. P. Abbracchio,
G. Burnstock,
G. R. Dubyak,
T. K. Harden,
K. A. Jacobson,
U. Schwabe,
and
M. Williams.
Towards a revised nomenclature for P1 and P2 receptors.
Trends Pharmacol. Sci.
18:
79-82,
1997[Medline].
14.
Goding, J. W.
Use of staphylococcal protein A as an immunological reagent.
J. Immunol. Methods
20:
241-253,
1978[Medline].
15.
Grygorczyk, R.,
and
J. W. Hanrahan.
Cystic fibrosis transmembrane conductance regulator and adenosine triphosphate.
Science
275:
1325-1326,
1997.
16.
Kobatake, E.,
T. Iwai,
Y. Ikariyama,
and
M. Aizawa.
Bioluminescent immunoassay with a protein A-luciferase fusion protein.
Anal. Biochem.
208:
300-305,
1993[Medline].
17.
Langone, J. J.
Protein A of Staphylococcus aureus and related immunoglobulin receptors produced by streptococci and pneumonococci.
Adv. Immunol.
32:
157-252,
1982[Medline].
18.
Lazarowski, E. R.,
L. Homolya,
R. C. Boucher,
and
T. K. Harden.
Direct demonstration of mechanically induced release of cellular UTP and its implication for uridine nucleotide receptor activation.
J. Biol. Chem.
272:
24348-24354,
1997
19.
Leach, F. R.
ATP determination with firefly luciferase.
J. Appl. Biochem.
3:
473-517,
1981.
20.
Léon, C.,
B. Hechler,
C. Vial,
C. Leray,
J.-P. Cazenave,
and
C. Gachet.
The P2Y1 receptor is an ADP receptor antagonized by ATP and expressed in platelets and megakaryoblastic cells.
FEBS Lett.
403:
26-30,
1997[Medline].
21.
Leont'eva, O.,
G. Kutuzova,
E. Skripkin,
and
N. Ugarova.
The stress-related production of the active Photinus pyralis and Luciola mingrelica firefly luciferases in Escherichia coli.
Appl. Biochem. Biotechnol.
61:
109-122,
1996[Medline].
22.
McEver, R. P.,
N. L. Baenziger,
and
P. W. Majerus.
Isolation and quantitation of the platelet membrane glycoprotein deficient in thrombasthenia using a monoclonal hybridoma antibody.
J. Clin. Invest.
66:
1311-1318,
1980[Medline].
23.
Nilsson, B.,
and
L. Abrahmsén.
Fusions to staphylococcal protein A.
Methods Enzymol.
185:
144-161,
1990[Medline].
24.
Nuttle, L.,
and
G. R. Dubyak.
Differential activation of cation channels and non-selective pores by macrophage P2z purinergic receptors expressed in Xenopus oocytes.
J. Biol. Chem.
269:
13988-13996,
1994
25.
Oker-Blom, C.,
A.-M. Suomalainen,
K. Åkerman,
Z. Qi,
C. Lindqvist,
A. Kuusisto,
and
M. Karp.
A baculovirus-expressed fusion protein containing the antibody-binding domain of protein A and insect luciferase.
Biotechniques
14:
800-809,
1993[Medline].
26.
Plesner, L.
Ecto-ATPases: identities and functions.
Int. Rev. Cytol.
158:
141-214,
1995[Medline].
27.
Reddy, M. M.,
P. M. Quinton,
C. Haws,
J. J. Wine,
R. Grygorczyk,
J. A. Tabcharani,
J. W. Hanrahan,
K. L. Gunderson,
and
R. R. Kopito.
Cystic fibrosis transmembrane conductance regulator and adenosine triphosphate.
Science
275:
1325,
1997.
28.
Rojas, E.,
H. B. Pollard,
and
E. Heldman.
Real-time measurements of acetylcholine-induced release of ATP from bovine medullary chromaffin cells.
FEBS Lett.
185:
323-327,
1985[Medline].
29.
Roman, R. M.,
Y. Wang,
S. D. Lidofsky,
A. P. Feranchak,
N. Lomri,
B. F. Scharschmidt,
and
J. G. Fitz.
Hepatocellular ATP-binding cassette protein expression enhances ATP release and autocrine regulation of cell volume.
J. Biol. Chem.
272:
21970-21976,
1997
30.
Sambrook, J.,
E. F. Fritsch,
and
T. Maniatis.
Molecular Cloning. A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Press, 1989, p. 17.13, 18.11, 18.47-18.58, and A.1.
31.
Siess, W.
Molecular mechanisms of platelet activation.
Physiol. Rev.
69:
58-178,
1989
32.
Weiss, H. J.,
L. D. Witte,
K. L. Kaplan,
B. A. Lages,
A. Chernoff,
H. L. Nossel,
D. S. Goodman,
and
H. R. Baumgartner.
Heterogeneity in storage pool deficiency: studies on granule-bound substances in 18 patients including variants deficient in -granules, platelet factor 4,
-thromboglobulin, and platelet-derived growth factor.
Blood
54:
1296-1319,
1979[Medline].
33.
Zimmermann, H.
5'-Nucleotidase: molecular structure and functional aspects.
Biochem. J.
285:
345-365,
1992[Medline].