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Vol. 17, Issue 8, 3378-3385, August 2006
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*MediCity Laboratory and Department of Medical Microbiology, Turku University and National Public Health Institute, FIN-20520 Turku, Finland; and Turku Centre for Biotechnology, University of Turku/Åbo Akademi University, FIN-20521 Turku, Finland
Submitted October 28, 2005;
Revised April 17, 2006;
Accepted May 10, 2006
Monitoring Editor: Guido Guidotti
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2ADP), we exploited lymphoid ecto-adenylate kinase as an intrinsic probe for accurate sensing pericellular ATP. Incubation of leukemic T- and B-lymphocytes with [3H]AMP or [
-32P]AMP induces partial nucleotide conversion into high-energy phosphoryls. This "intrinsic" AMP phosphorylation occurs in time- and concentration-dependent fashions via nonlytic supply of endogenous 
-phosphate–donating ATP, remains relatively resistant to bulk extracellular ATP scavenging by apyrase, and is diminished after lymphocyte pretreatment with membrane-modifying agents. This enzyme-coupled approach, together with confocal imaging of quinacrine-labeled ATP stores, suggests that, along with predominant ATP accumulation within cytoplasmic granules, micromolar ATP concentrations are constitutively retained on lymphoid surface without convection into bulk milieu. High basal levels of inositol phosphates in the cells transfected with ATP-selective human P2Y2-receptor further demonstrate that lymphocyte-surrounding ATP is sufficient for triggering purinergic responses both in autocrine and paracrine fashions. The ability of nonstimulated lymphocytes to maintain micromolar ATP halo might represent a novel route initiating signaling cascades within immunological synapses and facilitating leukocyte trafficking between the blood and tissues. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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); 2) nucleotide inactivation and/or interconversion by nucleotide-converting ectoenzymes (Zimmermann, 2000; Yegutkin et al., 2002); 3) interaction of the resulting adenosine with own G protein–coupled receptors (Ralevic and Burnstock, 1998); and finally, 4) adenosine uptake by the cell via equilibrative or Na+-dependent nucleoside transporters (Griffith and Jarvis, 1996).
The appearance of ATP in the external milieu represents a critical component of signaling cascade. The resting cells can release ATP at basal "constitutive" rates (Lazarowski et al., 2000), and this process is transiently intensified under various mechanical (shear stress, hypotonic swelling, stretching, hydrostatic pressure) and chemical (e.g., Ca2+-mobilizing agonists) stimuli (Joseph et al., 2003; Lazarowski et al., 2003). Current studies on ATP release involve either agitation of cell suspensions/monolayers, followed by removal of bathing medium and bioluminescent detection of ATP content, or direct addition of luciferase/luciferin reagent to the cells for sensing ATP in real time (Schwiebert and Zsembery, 2003). However, several innovative techniques provide evidence that the released ATP is spatially confined within unstirred membrane layers, and therefore, bioluminescent measurements of bulk nanomolar ATP concentrations are unlikely to reflect actual nucleotide levels.
Particularly, by using atomic force microscopy in combination with myosin ATPase-coated sensor tip, accumulation of ATP has been shown on surface of CFTR-transfected airway epithelial cells (Schneider et al., 1999). On stimulation of a single pancreatic 
-cell with glucose or glibenclamide, local ATP concentrations of over 25 µM were recorded using the patch-clamp technique with "biosensor" PC12 cell expressing P2X2 receptors (Hazama et al., 1998). Although these approaches discerned micromolar ATP in the microenvironment surrounding living cells, sophisticated detection apparatus and the necessity for three-dimensionally fine-controlled micromanipulators to bring the sensor into close proximity of the target cell limits their application for routine measurements. Moreover, correct interpretation of biosensor cell-based data is hampered by potential desensitization or self-activation of P2X receptors during simultaneous ATP release from sensor cell (Hayashi et al., 2004).
Another approach includes selective targeting of luciferase to the cell surface. The experiments of Dubyak and coworkers with membrane-attached protein A-luciferase revealed transient local release of 15–20 µM ATP from thrombin-activated platelets (Beigi et al., 1999) and further demonstrated ATP segregation in the vicinity of human astrocytoma cells upon challenging with Ca2+-mobilizing agonists (Joseph et al., 2003). By using recombinant membrane-targeted luciferase, local release of 100–200 µM ATP was also detected in HEK293 cells in response to P2X7 activation (Pellegatti et al., 2005). A limitation of these assays is the need for specially designed surface antigens or cell lines appropriate for luciferase targeting. Moreover, by measuring the relative increases in light output or second messenger production, these techniques estimated only transient local ATP release from the stimulated cells.
Our recent TLC assays revealed that adenylate kinase (AK)-positive lymphocytes slightly converted [3H]AMP into [3H]ADP/ATP even without exogenous -phosphate–donating ATP (Yegutkin et al., 2002; Henttinen et al., 2003). Given the high substrate specificity for AK for the reversible reaction ATP + AMP 2ADP (Yan and Tsai, 1999), we hypothesized that lymphoid ecto-AK may serve as an intrinsic probe for sensing cell-surrounding ATP. Here we showed that nonstimulated lymphocytes continuously retain pericellular ATP at concentrations sufficient for triggering purinergic responses. These findings provide a novel insight into the mechanism of cellular ATP turnover.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-32P]AMP (0.5–3 Ci/mmol) were from Amersham Biosciences (Little Chalfont, Buckinghamshire, United Kingdom). [-32P]ATP (3000 Ci/mmol) and myo-[2-3H]inositol (18.5 Ci/mmol) were from Perkin Elmer Life Sciences (Boston, MA). Annexin V-FITC was from Alexis (Läufelfingen, Switzerland). Alexa Fluor555 conjugate of cholera toxin subunit-B (CTB) from Vibrio cholera, 7-aminoactinomycin-D (7-AAD) and acridine orange were from Molecular Probes Europe BV (Leiden, The Netherlands). ATP kit was from BioThema AB (Haninge, Sweden). TLC plates were Alugram SIL G/UV254 and Polygram CEL-300 PEI types supplied by Macherey-Nagel (Duren, Germany). Free fatty acids were purchased from Sigma and stored at –70°C in stock 25 mM ethanol solutions. Soluble apyrase from potato (grade III), Dowex 1X8–200 ion-exchange resin, quinacrine, methyl--cyclodextrin, and all nucleotides were from Sigma (St. Louis, MO).
Cell Culture and Transfections
Human leukemic T-cell lymphoblast line Jurkat, derivative Jurkat mutant J.CaM1.6 deficient in lck kinase (JCaM), B-cell lymphoma line Namalwa and Chinese hamster ovary (CHO) cells were from ATCC (Manassas, VA). The Jurkat-CD73 transfectants (B-NT5.1) stably expressing glycosyl-phosphatidylinositol (GPI)-anchored 5'-nucleotidase/CD73 (Resta et al., 1994) were kindly provided by Dr. Linda Thompson (Oklahoma Medical Research Foundation, Oklahoma City, OK). Lymphocytes were grown in RPMI-1640 with 10% fetal calf serum (FCS), 4 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 400 µg/ml G-418 (in case of B-NT5.1 cells). The cells were also transfected with plasmid of human P2Y2 receptor (Parr et al., 1994), cloned in the pEGFP-N1 vector (gift of Dr. Wanda O’Neal, University of North Carolina, Chapel Hill, NC). Lymphocytes (5 x 106 cells) were transfected with 15 µg pEGFP-N1/P2Y2-R cDNA by electroporation (850 V/cm, 1 mFa) and, after 48 h, selected for 1–2 wk with 0.4 mg/ml G-418. CHO cells growing in six-well plates were transfected with 6 µg pEGFP-N1/P2Y2-R with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Geneticin-resistant CHO-P2Y2-R cells were selected for 3–4 wk with medium containing 1 mg/ml G-418. Peripheral blood lymphocytes (PBL) were isolated from the venous blood of healthy human volunteers using Ficoll centrifugations.
Transphosphorylation of 32P-labeled and Unlabeled Nucleotides
Jurkat and Namalwa (1 x 105 cells) were incubated for 12 min at 37°C in 80 µl RPMI-1640 containing 1 mM -glycerophosphate, 10 µM ATP with tracer [-32P]ATP, and 200 µM AMP/NDP. Aliquots of the mixture were spotted onto Polygram TLC sheets and separated with 0.75 M KH2PO4 (pH 3.5) as solvent. Lymphocytes were also incubated for 50 min at 37°C in 80 µl RPMI-1640 containing 1 mM -glycerophosphate, 0–100 µM ATP, and 10–100 µM AMP with tracer [-32P]AMP. Aliquots were spotted onto Alugram sheets, separated by TLC (Norman et al., 1974), and developed by autoradiography.
Measurement of Ectoenzymatic [3H]AMP Phosphorylation
The lymphocytes (2 x 105 cells) were incubated at 37°C in a final volume of 80 µl RPMI-1640 with 5 mM -glycerophosphate in the following two ways: 1) for evaluation of intrinsic AMP phosphorylation, cells were incubated with 1–200 µM [3H]AMP and 2) for measurement of specific AK activity, the assay buffer contained 400 µM [3H]AMP plus 800 µM ATP. After a 45-min incubation, [3H]AMP and its [3H]metabolites were separated by TLC (Norman et al., 1974) and quantified by scintillation -counting (Yegutkin et al., 2002).
Bioluminescent Quantification of Extracellular ATP
Lymphocytes (5 x 105 cells) were resuspended in 200 µl RPMI-1640 and incubated in 24-well plates for 45 min at 37°C. Aliquots of the bathing medium (40 µl) were clarified by centrifugation, and extracellular ATP was assayed by luciferin-luciferase luminometry using a TECAN-Ultra fluoropolarimeter (Yegutkin et al., 2001).
Flow-Cytometric Analysis
For viability tests, lymphocytes (5 x 105 cells) were incubated for 90 min at 37°C in 0.5 ml RPMI-1640 medium containing 100 µM AMP. The cells were then washed, resuspended in 400 µl phosphate-buffered saline (PBS) containing 2% FCS, 0.01% NaN3, 1 µl annexin V-FITC and 10 µg/ml 7-AAD, and analyzed using FACSCalibur with CellQuest-Prosoftware (Becton Dickinson, Mountain View, CA). The percentage of pEGFP-N1/P2Y2-positive cell transfectants was also determined by flow cytometry.
Inositol Phosphate Assay
Wild-type and P2Y2-transfected CHO cells were grown to confluence in 24-well tissue culture plates and radiolabeled by incubating in a humidified CO2 incubator for 22 h with 250 µl inositol-free Dulbecco’s modified Eagle’s medium (DMEM) and 0.7 µCi myo-[3H]-inositol. Exogenous nucleotide agonists and lymphoid cell suspensions were then added to the plates in 50 µl DMEM and incubated at 37°C for 5 and 30 min, respectively. The medium was aspirated, and the assay was terminated by adding 0.5 ml ice-cold 50 mM formic acid. After 20-min incubation on ice, cell extract was neutralized by 0.125 ml 150 mM NH4OH and [3H]inositol phosphates were resolved using Dowex 1X8-columns, as described previously (Lazarowski et al., 1995). Likewise, Jurkat and Namalwa cells and their P2Y2-transfectants were radiolabeled by incubating 1.5–2 x 105 lymphocytes in DMEM with 1 µCi myo-[3H]-inositol. To avoid undesirable release of intracellular ATP from the mechanically disturbed lymphocytes, formic acid was added directly to the labeling medium without washing the cells.
Confocal Microscopy
The lymphocytes (5 x 105 cells) were incubated for 10–15 min at RT in 0.5 ml PBS containing 0.2% BSA and certain fluorochrome, washed twice, resuspended in 200 µl PBS/BSA, and placed into glass-bottom microwell dishes (MatTek, Ashland, MA). A confocal spectral laser-scanning microscope (LSM-510; Zeiss, Oberkochen, Germany) equipped with Argon and HeNe lasers and 63x 1.4 oil DIC objective was used to monitor fluorescent signals. Alexa555-CTB (0.5 µg/ml) was used to label cell surface glycosphingolipids, and upon excitation with 543 nm, fluorescence was detected at >560 nm. For detection of putative ATP stores, the cells were incubated with 4 µM quinacrine, and fluorescence was detected at 500–530 nm with 477-nm excitation wavelengths. Acidic organelles were detected by using 4 µM acridine orange and fluorescence was detected at 500–530 and 650–710 nm with 477- and 488-nm excitations, respectively. The labeled lymphocytes were also pretreated for 30 min at RT with 10 mM methyl--cyclodextrin before microscopic inspection.
Data Analyses
The maximum [3H]AMP-phosphorylating capacities (Bmax) were determined using nonlinear least-squares curve fitting. Objective statistical criteria (F-test, extra sum of squares principle) were used for discriminating between one- and two-site models (Prism 4.02; GraphPad, San Diego, CA). Statistical comparisons were made using Student’s t test, and p < 0.05 was taken as significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-32P]ATP metabolism by Jurkat and Namalwa cells. The lymphocytes directly converted [-32P]ATP to 32Pi without any formation of 32PPi, thus indicating that ATP-hydrolyzing activity in lymphoid cells is represented by nucleoside triphosphate diphosphohydrolase (NTPDase) rather than by nucleotide pyrophosphatases/phosphodiesterases. EDTA markedly diminished this nucleotidase reaction, showing the Ca2+-Mg2+ dependency of lymphoid NTPDases. Lymphocyte incubation with 10 µM [-32P]ATP together with 200 µM UDP or GDP activated formation of the corresponding [-32P]NTP via backward NDP kinase reaction. Joint addition of [-32P]ATP and AMP stimulates their transphosphorylation into [-32P]ADP, which can be prevented by AK inhibitor Ap5A.
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), showing that lymphoid cells are generally characterized by "ATP-regenerating phenotype" with high ecto-AK and NDP kinase activities and relatively low or even lacking expressions of NTPDases and ecto-5'-nucleotidase.
Lymphocytes Phosphorylate Tracer AMP Even without Exogenous -Phosphate–donating ATP
The autoradiography in Figure 1B additionally elicited slight but clearly detectable formation of 3H-labeled ADP/ATP after Jurkat incubation with [3H]AMP (lane 1). A similar pattern of [3H]AMP phosphorylation was observed earlier in the experiments with Namalwa B-cells, but not with cultured human umbilical vein endothelial cells (Yegutkin et al., 2002; Henttinen et al., 2003). Because these phosphotransfers occur without exogenous -phosphate–donating ATP, this phenomenon can be reasonably designated as an "intrinsic" AMP phosphorylation by lymphocytes.
To assess the potential impact of counteracting AMP-degrading pathway on the ability of lymphocytes to phosphorylate [3H]AMP, we used Jurkat transfectants B-NT5.1 stably expressing ecto-5'-nucleotidase/CD73 (Resta et al., 1994). Unlike parental CD73-negative Jurkat, B-NT5.1 efficiently converted radiolabeled AMP via adenosine into inosine/hypoxanthine (Figure 1B, lane 4). Pretreatment of B-NT5.1 cells with known competitive inhibitors of ecto-5'-nucleotidase, ,-methylene-ADP (APCP) and ATP (Henttinen et al., 2003), completely abrogated their AMP-hydrolyzing activity (lanes 5 and 6). Strikingly, along with prominent ATP-dependent [3H]AMP phosphorylation via ecto-AK reaction (lane 6), these CD73-transfectants were still capable of slightly phosphorylating [3H]AMP alone, irrespective of high ecto-5'-nucleotidase activity (lane 4).
Because nonspecific leakage of ATP may potentially contribute to the above [3H]AMP phosphorylation, we used several independent approaches to assess this possibility. First, massive liberation of endogenous ATP was intentionally provoked by disruption of lymphocytes, accompanied by simultaneous releases of intracellular AK (Figure 1B, lanes 8 and 10) and other purinergic enzymes (Yegutkin et al., 2002). However, the extents of intrinsic [3H]AMP phosphorylation by sonicated Jurkat (Figure 1B, lane 7) and Jurkat-CD73 (lane 9) lysates were even diminished compared with intact lymphocytes (lanes 1 and 4). Second, we used a high-affinity phosphatidylserine-binding protein annexin-V that is rapidly translocated during apoptosis from the inner to the outer membrane leaflet. Flow-cytometric analysis of annexin-V staining, in combination with a marker of cell necrosis 7-AAD, confirmed that more than 95% of Jurkat remained viable after treatment with AMP (Figure 2A).
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-glycerophosphate. Most importantly, the employment of [-32P]AMP as another phosphate-accepting substrate unequivocally confirmed that lymphocytes directly phosphorylate tracer AMP into ADP in the absence of exogenous ATP, and this phosphotransfer occurs in a concentration-dependent manner, requires divalent cations, and is inhibited by Ap5A (Figure 2B). Taken together, these data show that lymphocyte activate ecto-AK–mediated phosphorylation of tracer AMP via constitutive supply of endogenous -phosphate donating ATP, which occurs not as a consequence of cell lysis or plasma membrane damage.
Evidence that Lymphocytes Maintain Micromolar Pericellular ATP without Significant Nucleotide Convection into Bulk Milieu
Further quantitative TLC analysis confirmed progressive [3H]AMP conversion into [3H]ADP/ATP by Jurkat cells, which reached a plateau value after a 45-min incubation (Figure 3A). Similar time courses for ATP-mediated [3H]AMP phosphorylation has been shown earlier for endothelial (Yegutkin et al., 2001) and lymphoid (Yegutkin et al., 2002) ecto-AK. The prolonged dynamics of [3H]AMP phosphorylation is presumably defined by the necessity for optimal binding and orientation of the phosphate acceptor relative to the phosphate being transferred via AK reaction (Yan and Tsai, 1999).
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-terminal phosphate. With this assumption, the computed Bmax values can be reasonably interpreted as total amount of cell-derived ATP transphosphorylated with tracer AMP through lymphoid ecto-AK reaction. Notably, these values significantly exceed bulk extracellular ATP measured by using conventional bioluminescent assay and equal to 42 ± 16 and 36 ± 10 nM (n = 5) for Jurkat and Namalwa, respectively. These kinetic data suggest that lymphoid cells constitutively maintain pericellular ATP at certain characteristic micromolar levels, without significant nucleotide convection into bulk milieu.
Membrane-modifying Agents Diminish the Intrinsic [3H]AMP-phosphorylating Capacity of Lymphocytes without Affecting Ecto-adenylate Kinase Activity
We then evaluated the effects of various agents on the extent of [3H]AMP phosphorylation by lymphocytes. To exclude the undesirable leakage of intracellular nucleotide kinases and/or spurious effects of certain reagents on AMP-phosphorylating capability of the treated cells, lymphoid AK was concurrently assayed by using saturating concentrations of [3H]AMP plus ATP. In support of autoradiographic data with [-32P]AMP shown in Figure 2B, cation-chelating agent EDTA and bisubstrate analogue Ap5A inhibited both intrinsic and ATP-stimulated [3H]AMP phosphorylation (Figure 4), thus confirming that these reactions occur via the same AK-mediated mechanism.
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50 µM), which is much lower than critical constant of micelle formation equal for this nonionic detergent to 0.015% (Helenius and Simons, 1975). Surprisingly, soluble apyrase only partially prevented [3H]AMP phosphorylation, thus suggesting the existence of apyrase-resistant ATP pool in the lymphocyte environment (concurrent measurement of AK was compromised by rapid scavenging of exogenous ATP by apyrase). The extent of [3H]AMP phosphorylation remained unchanged after Jurkat treatment with the inhibitors of nucleoside transport (NBT) and adenosine deaminase (EHNA; Figure 4A). Importantly, use of competitive ecto-5'-nucleotidase inhibitor APCP at concentrations sufficient to block [3H]AMP hydrolysis does not affect [3H]AMP-phosphorylating capacities of both wild-type Jurkat and CD73-transfected B-NT5.1 cells (Figures 1B and 4A). Hence, in the presence of APCP, this technique may also be applied for detecting ATP in the vicinity of various CD73-positive cells.
Lymphocytes Store Cellular ATP in Cytoplasmic Granules beneath Plasma Membrane Patches
For visualization of cellular ATP, we used fluorescent dye quinacrine that belongs to the quinoline-acridine class, binds to nucleic acids, and is considered as the preferred available marker for detecting releasable ATP stores (Mitchell et al., 1998; Sorensen and Novak, 2001). The quinacrine-labeled cells were also costained with CTB, which binds to the pentasaccharide chain of ganglioside GM1 and is widely used as a marker of glycosphingolipid-rich domains in lymphocytes (Harder et al., 1998; Janes et al., 1999). Costaining of Jurkat with quinacrine and Alexa555-CTB revealed the accumulation of green quinacrine fluorescence within granular vesicles, which significantly coincide with red CTB-labeled patches (Figure 5A). Computer reconstructions of these signals from serial confocal images along the z-axis further demonstrated that cytoplasmic quinacrine-stainable vesicles are mainly concentrated beneath CTB-labeled plasma membrane microdomains (Supplementary Video). Likewise, Namalwa also accumulated quinacrine within granular vesicles with significant colocalization of green quinacrine and red Alexa555-CTB fluorescences (Figure 5B), whereas lck-deficient JCaM cells displayed fainter quinacrine staining and more homogenous surface distribution of Alexa555-CTB (Figure 5C).
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; Janes et al., 1999). Here we show that at very low concentrations (0.5 µg/ml), Alexa555-CTB can also be concentrated to distinct patches on surface of nonstimulated lymphocytes. We also examined the patching behavior and pattern of quinacrine staining after disrupting the membrane rafts with the known cholesterol-extracting agent methyl--cyclodextrin (Harder et al., 1998; Janes et al., 1999). In comparison to control Jurkat (Figure 5A), the CTB-labeled clusters in cholesterol-depleted Jurkat were smaller and more diffusely distributed over the cell surface, whereas the granular quinacrine staining remained unchanged (Figure 5D). These findings further confirm that the visualized CTB-labeled domains can be reasonably attributed to ganglioside-GM1-enriched lipid rafts. Because quinacrine is a weak base, we used another acidotropic probe acridine orange for control labeling and tracing acidic organelles. This dual-fluorescence dye undergoes a pH-dependent shift from the green to red-orange emission and a similar shift is seen when acridine orange binds to DNA and RNA. Unlike granular quinacrine accumulation, lymphocyte staining with acridine orange revealed more even green fluorescence with a few red spots (Figure 5, A–C), thus excluding the possibility of nonspecific quinacrine partitioning into cellular acidic compartments in our assays.
Lymphocytes Maintain Pericellular ATP at Concentrations Sufficient for Activation of Purinergic Receptors
To evaluate the ATP-mediated purinergic responses, the cells were then transfected with human P2Y2 receptor, cloned in the pEGFP-N1 vector. Figure 6 confirms the moderately high expression of hP2Y2-R on surfaces of CHO (A), Namalwa (B), and to less extent, Jurkat (C) cells. Consistent with the previously reported agonist profile for human ATP/UTP-selective P2Y2 receptors (Parr et al., 1994; Lazarowski et al., 1995), treatment of CHO-P2Y2-R transfectants with the increasing micromolar concentrations of ATP caused progressive accumulation of [3H]inositol triphosphates, and similar activation was observed in the presence of 10 µM UTP, but not ADP (Figure 6D). Strikingly, the lymphoid cells were also able to increase [3H]inositol phosphates production in P2Y2-R–transfected CHO cells, presumably in an ATP-dependent manner. Furthermore, unlike CHO cells where expression of P2Y2 receptor did not affect phospholipase-C activity (Figure 6D), the basal level of [3H]inositol phosphates in Jurkat- and especially Namalwa-P2Y2-R transfectants was significantly higher than in wild-type controls (Figure 6E). Together, these functional assays support the above radio-TLC enzyme data on constitutive external ATP supply in the lymphocyte environment and further demonstrate that lymphocyte-surrounding ATP concentrations are sufficient for triggering purinergic responses both in paracrine and autocrine fashions. Noteworthy, micromolar concentrations of exogenous ATP additionally increased inositol phosphate levels in Jurkat-P2Y2–R cells, but not in Namalwa transfectants (Figure 6E). Therefore, taking into account the phenomenon of agonist-mediated desensitization of P2Y2 and other purinergic receptors (Lazarowski et al., 1995), we do not exclude that continuous "self-activation" of lymphocytes by endogenously released ATP might substantially diminish the magnitude of purinergic signaling.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Along with marked ATP-dependent nucleotide phosphorylation through sequential ecto-AK and NDP kinase reactions, the lymphocytes were able to slightly phosphorylate 3H- or 32P-labeled AMP without exogenous -phosphate–donating ATP (Figures 1 and 2). This process occurs in a time- and concentration-dependent fashions, is not accompanied by any signs of cell lysis, and is mediated via direct ectoenzymatic transphosphorylation of tracer AMP with micromolar concentrations of lymphocyte-derived ATP. Although these studies were mostly performed with CD73-negative Jurkat T-cells and Namalwa B-cells where ecto-AK represents the major AMP-converting pathway, Jurkat CD73-transfectants and human PBL also phosphorylated [3H]AMP to some extent, irrespective of the coexpression of counteracting enzyme ecto-5'-nucleotidase/CD73. Another important ectoenzyme to be taken into consideration is E-NTPDase/CD39, otherwise known as ecto-ATPase, apyrase (Zimmermann, 2000). Surprisingly, lymphocyte pretreatment with soluble apyrase only partially diminished their [3H]AMP-phosphorylating capacity (Figure 4). Probably, unlike the bulk extracellular ATP pool, surface-localized ATP is somehow protected from apyrase scavenging. Likewise, recent assays with surface-attached luciferase demonstrated that, in response to mechanical or chemical stimuli, the platelets (Beigi et al., 1999) and astrocytoma cells (Joseph et al., 2003) release ATP, which is accumulated on the cell surface and remains relatively resistant to endogenous and exogenous nucleotidases.
AK is generally considered as classical intracellular enzyme that is localized in the cytosol (AK1), mitochondrial intermembrane space (AK2), and mitochondrial matrix (AK3; Yan and Tsai, 1999). However, plasma membrane–associated isoform AK1 has also been described (Collavin et al., 1999; Ruan et al., 2002), and the existence of cell surface-associated AK activity is currently appreciated (Yegutkin et al., 2001; Picher and Boucher, 2003). Intracellular AK, in conjunction with NDP kinase, creatine kinase and glycolytic enzymes, play a critical role in energy transfer and distribution between mitochondria, cytosol, and nucleus (Dzeja and Terzic, 2003). Data on functional association of AK with ATP-sensitive potassium channels (Carrasco et al., 2001) and intrinsic AK activity of cystic fibrosis transmembrane conductance regulator that controls gating of this anion channel in vivo (Randak and Welsh, 2003) provide additional evidence for communicating phosphotransfer signals to the membrane environments. The revealed here ability of lymphocytes to retain and/or transphosphorylate significant amounts of ATP and other nucleotides in the pericellular space allows to expand the above concepts by hypothesizing the coexistence of spatially arranged intra- and extracellular phosphotransfer networks coordinating both energetic homeostasis and signal transduction pathways.
Cellular mechanisms that underlie nonlytic ATP release might include 1) exocytosis of ATP-containing vesicles; 2) facilitated diffusion by nucleotide transporters or nucleotide/nucleoside exchangers; or 3) electrodiffusional movement through ATP-permeable anion channels (Lazarowski et al., 2003; Schwiebert and Zsembery, 2003). Labeling of live lymphocytes with a marker of putative ATP stores quinacrine suggests the likely storage of ATP within cytoplasmic granules (Figure 5). Granular quinacrine staining has also been shown in experiments with rabbit ciliary epithelial cells (Mitchell et al., 1998), human vascular endothelial cells (Bodin and Burnstock, 2001), and rat pancreatic acini (Sorensen and Novak, 2001). Stimulation of ATP release by various stimuli (hypotonic stress, shear stress, cholinergic stimulation) markedly decreased fluorescence intensity in these studies, thus confirming that quinacrine labels the releasable ATP stores. The important novelty of this work is the demonstration that quinacrine-stainable granules primarily coincide beneath Alexa555-CTB–labeled lymphoid membrane patches (Figure 5 and Online Video), which are enriched with ganglioside GM1 and can be reasonably attributed to lipid rafts. Interestingly, other purinergic molecules are also associated with lipid rafts and caveolae, including ATP permeation sites (Joseph et al., 2003), P2Y (Kaiser et al., 2002) and P2X3 receptors (Vacca et al., 2004), E-NTPDase/CD39 (Koziak et al., 2000; Joseph et al., 2003), and GPI-linked ecto-5'-nucleotidase/CD73 (Resta et al., 1994).
The idea that lipid rafts may function as foci of ATP turnover is generally consistent with the important role of these membrane-specialized assemblies in signaling and membrane trafficking in lymphocytes (Janes et al., 1999; Alonso and Millan, 2001). Specifically, polyunsaturated fatty acids markedly impaired the early steps of Jurkat T-cell activation, presumably via biosynthetic incorporation into membrane microdomains and displacement of LAT and Src kinases (Stulnig et al., 2001) and coincidentally diminished the ability of lymphocytes to phosphorylate [3H]AMP in our assays (Figure 4). Concurrent disturbances of T-cell receptor activation and cellular ATP turnover provide a novel insight into membrane mechanisms underlying the beneficial effects of polyunsaturated fatty acids in a variety of autoimmune and inflammatory disorders (Stulnig, 2003). Moreover, we have shown that lck-deficient JCaM cells are characterized by lower [3H]AMP-phosphorylating capacity and fainter quinacrine staining, compared with parental Jurkat. These observations, together with data on strongly reduced amounts of tyrosine-phosphorylated proteins in these mutants (Harder and Simons, 1999), also suggest the existence of functional/structural interplay between ATP turnover and signaling machinery.
In view of the emerging proinflammatory role of ATP in the immune system, this model raises important questions regarding the physiological relevance of pericellular ATP pool. By measuring the basal and agonist-stimulated [3H]inositol phosphate accumulation in the CHO and lymphoid cells transfected with ATP-selective hP2Y2 receptor, here we also showed that lymphocytes constitutively supply external ATP at concentrations sufficient for promoting second-messenger responses both in autocrine and paracrine fashions (Figure 6). Pericellular ATP may serve as signaling loop sufficient for self-regulation of lymphocyte function and/or desensitization of purinergic receptors that particularly might explain why activation of lymphoid P2X7 and other P2X receptors occurs only at high micromolar ATP concentrations (Di Virgilio et al., 2001). Moreover, the ability of lymphocytes to retain micromolar ATP halo, in conjunction with leukocyte-governed suppression of endothelial ecto-5'-nucleotidase/CD73 (Henttinen et al., 2003), could also represent a novel form of cross-talk between adherent leukocytes and targeted endothelium, thereby facilitating leukocyte trafficking between the blood and tissues.
In conclusion, by developing a novel enzyme-coupled ATP-sensing approach, in combination with confocal microscopic imaging, we have shown that lymphocytes maintain an ATP halo at certain micromolar levels and further suggest the likely recruitment of cellular ATP via cargo-vesicle trafficking and/or exocytotic granule secretion onto specific membrane microdomains. Unlike previous techniques with membrane-targeted luciferase and external biosensors (see Introduction), this radio-TLC assay provides a simple and reliable tool to quantify total amount of ATP in the vicinity of intact nonstimulated cells, without additional steps of cell transfections or other undesirable manipulations. As for the nanomolar ATP concentrations, which can be detected luminometrically as a "purinergic tone" (Lazarowski et al., 2000), they probably reflect a minor fraction of the actual mass of cell-surrounding ATP, which is exchanged with bulk phase via dissociation-association mechanisms and can be additionally shed from the membrane layers under various stimuli. Together, these findings provide sufficient justification for reexamination of current concepts of extracellular ATP turnover.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Gennady G. Yegutkin ( gennady.yegutkin{at}utu.fi)
Abbreviations used: AK, adenylate kinase; APCP, ,-methylene-ADP; Ap5A, diadenosine pentaphosphate; CTB, cholera toxin subunit B; NTPDase, nucleoside triphosphate diphosphohydrolase; PBL, peripheral blood lymphocytes; TLC, thin-layer chromatography
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) or 400 µM [3H]AMP plus 800 µM ATP (
) for measuring the intrinsic AMP-phosphorylating capacity and specific ecto-AK activity, respectively. The amount of [3H]AMP converted into [3H]ADP/ATP after a 45-min incubation with lymphocytes was determined by TLC and is represented as percentage of controls (mean ± SEM), with the number of experiments in parentheses. * p < 0.05 compared with nontreated control cells. ND, not determined.
