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J. Biol. Chem., Vol. 281, Issue 42, 31419-31429, October 20, 2006

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Extracellular NAD⁺ Is an Agonist of the Human P2Y₁₁ Purinergic Receptor in Human Granulocytes^*

Iliana Moreschi, Santina Bruzzone¹, Robert A. Nicholas, Floriana Fruscione, Laura Sturla, Federica Benvenuto^¶, Cesare Usai^||, Sabine Meis^**, Matthias U. Kassack^**, Elena Zocchi, and Antonio De Flora

From the Department of Experimental Medicine, Section of Biochemistry, and Center of Excellence for Biomedical Research (CEBR), University of Genova, Viale Benedetto XV/1, 16132 Genova, Italy, the Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599-7365, the ^¶Neuroimmunology Unit, Department of Neurosciences, Ophthalmology and Genetics, and CEBR, University of Genova, Genova, Italy, the ^||Institute of Biophysics, National Research Council, Via De Marini 6, 16149 Genova, Italy, and the ^**Pharmaceutical Institute, University of Bonn, An der Immenburg 4, Bonn D-53121, Germany

Received for publication, July 12, 2006 , and in revised form, August 9, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Micromolar concentrations of extracellular -NAD⁺ () activate human granulocytes (superoxide and NO generation and chemotaxis) by triggering: (i) overproduction of cAMP, (ii) activation of protein kinase A, (iii) stimulation of ADP-ribosyl cyclase and overproduction of cyclic ADP-ribose (cADPR), a universal Ca²⁺ mobilizer, and (iv) influx of extracellular Ca²⁺. Here we demonstrate that exposure of granulocytes to millimolar rather than to micromolar generates both inositol 1,4,5-trisphosphate (IP₃) and cAMP, with a two-step elevation of intracellular calcium levels ([Ca²⁺]_i): a rapid, IP₃-mediated Ca²⁺ release, followed by a sustained influx of extracellular Ca²⁺ mediated by cADPR. Suramin, an inhibitor of P2Y receptors, abrogated -induced intracellular increases of IP₃, cAMP, cADPR, and [Ca²⁺]_i, suggesting a role for a P2Y receptor coupled to both phospholipase C and adenylyl cyclase. The P2Y₁₁ receptor is the only known member of the P2Y receptor subfamily coupled to both phospholipase C and adenylyl cyclase. Therefore, we performed experiments on hP2Y₁₁-transfected 1321N1 astrocytoma cells: micromolar promoted a two-step elevation of the [Ca²⁺]_i due to the enhanced intracellular production of IP₃, cAMP, and cADPR in 1321N1-hP2Y₁₁ but not in untransfected 1321N1 cells. In human granulocytes NF157, a selective and potent inhibitor of P2Y₁₁, and the down-regulation of P2Y₁₁ expression by short interference RNA prevented -induced intracellular increases of [Ca²⁺]_i and chemotaxis. These results demonstrate that is an agonist of the P2Y₁₁ purinoceptor and that P2Y₁₁ is the endogenous receptor in granulocytes mediating the sustained [Ca²⁺]_i increase responsible for their functional activation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Extracellular NAD⁺ ()² is known to increase intracellular calcium concentrations ([Ca²⁺]_i) in different cell types and by different mechanisms (1–8). In cells expressing the ecto-ADP-ribosyl cyclase (ADPRC) CD38, e.g. in CD38-transfected fibroblasts and HeLa cells, as well as in native astrocytes, retinal Muller cells and osteoblasts, direct conversion of NAD⁺ to the Ca²⁺ mobilizer cyclic ADP-ribose (cADPR) (9) has been implicated as the principal mechanism leading to increases in [Ca²⁺]_i in response to (1, 3–6). In murine T lymphocytes, is the substrate of the ecto-enzyme mono-ADP-ribosyltransferase, which ADP-ribosylates the purinoceptor P2X7 or a P2X7-associated protein, leading to Ca²⁺ influx, formation of large pores, and cell death (7, 10, 11). In human monocytes, and ADPR trigger influx of extracellular Ca²⁺, but neither CD38 nor P2X7-induced pore formation are involved (8).

Recently, we demonstrated that behaves as a proinflammatory cytokine targeting human polymorphonuclear granulocytes (12). Exposure of granulocytes to micromolar concentrations of (either the naturally occurring or the form) triggered the following cascade of causally related events: (i) activation of adenylyl cyclase and a rapid increase of intracellular cAMP levels, (ii) PKA-mediated stimulation of ADPRC activity and elevation of intracellular cADPR levels, and (iii) sustained [Ca²⁺]_i rise, due to influx of extracellular Ca²⁺ (12). Increases in [Ca²⁺]_i are known to be responsible for activation of human granulocytes (13). Indeed, -induced [Ca²⁺]_i elevation triggered increased and NO generation and enhanced chemotaxis toward . Therefore, the results obtained with -stimulated granulocytes (12) support a key role of cADPR in control of receptor-mediated chemotaxis in murine and human granulocytes, as previously demonstrated both in vivo (14) and in vitro (15).

The rapid increase of [cAMP]_i in granulocytes that follows exposure (12) suggested that interacts with an unidentified receptor, which activates the signaling cascade involving cADPR and increased [Ca²⁺]_i that eventually results in enhanced respiratory burst and chemotaxis. In the present study, we challenged granulocytes with millimolar . Under these conditions, we observed a qualitatively different Ca²⁺ response, i.e. a biphasic one, with an initial inositol 1,4,5-trisphosphate (IP₃)-mediated peak of [Ca²⁺]_i caused by release from intracellular stores, followed by sustained influx of extracellular Ca²⁺. Given that (i) NAD⁺ is a nucleotide and (ii) -induced intracellular increases of IP₃, cAMP, cADPR, and [Ca²⁺]_i were abrogated by suramin, a relatively non-selective inhibitor of P2Y receptors, we focused on the possibility that the signaling activities promoted by were the result of activation of a G protein-coupled P2Y receptor. The signaling properties of P2Y receptors characterized to date suggested the P2Y₁₁ receptor as a putative NAD⁺ receptor, because its activation increases both IP₃ and cAMP levels by virtue of its dual coupling to G_q and G_s (16–18). Results obtained with hP2Y₁₁-transfected 1321N1 astrocytoma cells reveal that -NAD⁺ is an agonist of the P2Y₁₁ purinoceptor. In addition, the use of NF157 (a recently synthesized, selective inhibitor of P2Y₁₁) and the down-regulation of P2Y₁₁ expression by short interfering RNA (siRNA) demonstrate that endogenous P2Y₁₁ is responsible for the -induced activation of human granulocytes.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—FLUO-3AM and FURA-2AM were obtained from Calbiochem (Milan, Italy). The [³H]cAMP and [³H]IP₃ assay systems and Ficoll-Paque Plus were purchased from Amersham Biosciences. A5 peptide (sequence: H₂N-SLLWLT-CRPWEAM-OH) was obtained from New England Peptide, Inc. (Gardner, MA). Dulbecco's modified Eagle's medium and RPMI cell culture medium were purchased from Cambrex Bio Science Milano (Bergamo, Italy). NF157 was synthesized as described (19). All other chemicals were obtained from Sigma (Milan, Italy).

High-performance Liquid Chromatography Analyses of Nucleotides and Chromatographic Purification of - and -NAD⁺—High-performance liquid chromatography analyses of nucleotides and purification of - and -NAD⁺ were performed as described previously (12).

Isolation of Human Granulocytes—Buffy coats, prepared from freshly drawn blood of healthy volunteers, were provided by Galliera Hospital, Genova, Italy. Granulocytes were isolated from the buffy coats as described before (12).

Cell Culture—Control and hP2Y₁₁-transfected 1321N1 astrocytoma cell lines (17) were cultured in Dulbecco's modified Eagle's medium supplemented with fetal calf serum (10%), penicillin (50 units/ml), and streptomycin (50 µg/ml) in a humidified 5% CO₂ atmosphere at 37 °C.

Fluorometric Measurements of [Ca²⁺]_i—Freshly prepared granulocytes (10 x 10⁶/ml) were loaded with 10 µM FLUO-3AM for 45 min at 25 °C in RPMI medium, washed with Hanks' balanced salt solution (HBSS, cat. no. H8264, Sigma), and resuspended in the same solution or in Ca²⁺-free HBSS (cat. no. H6648, Sigma) at 5 x 10⁶ cells/ml. [Ca²⁺]_i measurements were performed in 96-well plates (10⁶ cells/well), and fluorescence (excitation, 485 nm; emission, 520 nm) was measured every 3 s with a fluorescence plate reader (Fluostar Optima, BMG Labtechnologies GmbH, Offenburg, Germany). The intensity of emitted light was plotted as a function of time. For [Ca²⁺]_i calibration, granulocytes were loaded with 10 µM FURA-2AM for 45 min at 25 °C in RPMI medium, and measurements were performed as described in a previous study (12).

Control and hP2Y₁₁-transfected 1321N1 cells were seeded in 96-well plates (2 x 10⁴ cells/well). After 24 h, cells were loaded with 10 µM FLUO-3AM for 45 min at 25 °C in complete medium (Dulbecco's modified Eagle's medium) and washed once with 0.2 ml of HBSS. The indicated concentrations of nucleotides in HBSS (0.1 ml) were then added, and [Ca²⁺]_i measurements were performed using the fluorescence plate reader, as described above.

Determination of Intracellular cADPR Levels—Granulocytes (40 x 10⁶/ml) were incubated for 0, 15, and 60 min at 25 °C in the absence (control) or in the presence of 1 mM -NAD⁺. At each time point, a 500-µl aliquot of the cell suspension was withdrawn and centrifuged at 5,000 x g for 15 s, and the resulting cell pellets were lysed at 4 °C with 500 µl of 0.6 M perchloric acid (PCA). After centrifugation to remove precipitated proteins, the cADPR content was measured on the neutralized PCA cell extracts by a highly sensitive enzymatic cycling assay (20). cADPR levels were expressed as picomoles/10⁶ cells.

Control and hP2Y₁₁-transfected 1321N1 cells were seeded in 35 x 10-mm dishes (2.4 x 10⁵ cells/dish). After 48 h, the medium was removed and HBSS was added (0.6 ml). ATP or -NAD⁺ was added: the incubation was stopped after 15 min by removal of HBSS and addition of 300 µl if ice-cold PCA (0.6 M), and cells were removed by scraping. Cell extracts were centrifuged to remove precipitated proteins, and the cADPR content was measured on the neutralized cell extracts as described above.

Determination of Intracellular cAMP Levels—Granulocytes were suspended in HBSS or in Ca²⁺-free HBSS (30 x 10⁶/ml), preincubated for 5 min at 25 °C in the presence of the cAMP phosphodiesterase inhibitor 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one (Ro 20-1724, Sigma, cat. no. B8279) (10 µM), and then challenged with vehicle or 1 mM - or -NAD⁺. At different times (0, 20, 40, 60, 150, and 300 s), a 300-µl aliquot of the suspension was withdrawn, and the reaction was stopped by adding 20 µl of 9 M PCA at 4 °C. PCA was removed as described (20). Intracellular cAMP levels (expressed as picomoles of cAMP/10⁶ cells) were determined by radioimmunoassay according to the manufacturer's protocol.

Control and hP2Y₁₁-transfected 1321N1 cells were seeded in 35 x 10 mm dishes (5 x 10⁵ cells/dish). After 24 h, the medium was removed and HBSS was added (0.6 ml). Cells were then challenged with ATP or -or -NAD⁺, and at different times the incubation mixtures were stopped by removal of HBSS and addition of 200 µl of ice-cold PCA (0.6 M). Cells were removed by scraping, the cell extracts were centrifuged to remove the proteins, and the cAMP levels in the neutralized cell extracts were measured as described above.

Determination of Intracellular IP₃ Levels—Granulocytes were resuspended in HBSS (40 x 10⁶/ml) and challenged with 1 mM -NAD⁺. Aliquots of the suspensions (500 µl) were withdrawn at different times (0, 30, and 90 s), and the reaction was stopped by adding 30 µl of 9 M PCA at 4 °C. Following removal of PCA (20), intracellular IP₃ levels were determined by radioimmunoassay. Results were expressed as picomoles of IP₃/10⁶ cells.

Control and hP2Y₁₁-transfected 1321N1 cells were seeded in 35 x 10-mm dishes (5 x 10⁵ cells/dish). After 24 h, the medium was removed and HBSS was added (0.6 ml). Cells were challenged with ATP or -or -NAD⁺, and at various times (0, 30, 90, and 900 s) HBSS was removed and 300 µl of ice-cold PCA (0.6 M) was added. The cells were scraped, the precipitated proteins were removed by centrifugation, and IP₃ levels were measured on the supernatants of the neutralized cell extracts as described above.

Assays of ADP-ribosyl Cyclase Activity—hP2Y₁₁-transfected 1321N1 cells (4 x 10⁶/ml) were incubated at 25 °C in the absence (control) or in the presence of 0.1 mM -NAD⁺ or ATP for 10 min. After addition of 1:500 protease inhibitor mixture (Sigma, cat. no. P8340) and 1:100 phosphatase inhibitor mixture (Sigma, cat. no. P2850), control and stimulated cells were lysed by sonication in ice for 1 min at 3 watts (Heat-System Ultrasonics, W380, New York). ADP-ribosyl cyclase activity was measured at 37 °C on cell lysates by adding 0.4 mM -NAD⁺. Aliquots (100 µl) were withdrawn at various times (0, 10, and 30 min), the reactions were stopped by addition of 220 µl of 0.9 M PCA to each aliquot, and the cADPR concentrations were measured by the enzymatic cycling assay (20). The protein content in each sample was determined by a Bradford assay (21).

siRNA Transfection—Transfection of human granulocytes was performed using the Nucleofector System (Amaxa GmbH, Cologne, Germany). Preliminary experiments were carried out with pmaxGFP to select the cell concentration, the Nucleofector solution, and program yielding the highest percentage of cell transfection, which was monitored by measuring GFP-positive cells. Moreover, viability of freshly isolated granulocytes was estimated at 24, 48, and 72 h measuring propidium iodide-positive cells by flow cytometry: the corresponding figures of cells viability were 78%, 49 and 10%, respectively. Thus, the following protocols were chosen as optimal. Freshly isolated granulocytes (20 x 10⁶ cells) were transfected without (control), or with 2 µM duplex short interference RNA (siRNA) or with 2 µg of pmaxGFP, using the Cell Line Nucleofector Kit T according to the manufacturer's instructions (Nucleofector program X-005). The control siRNA was obtained from Ambion (Austin, TX, negative control #1 siRNA). The P2Y₁₁-targeting siRNA was obtained from Invitrogen (P2RY11-HSS143212: 5'-UAUGUCUGCAAAGCUCGGGCAGCGG-3'). Immediately after transfection, cells were resuspended in 2.5 ml of RPMI supplemented with fetal calf serum (10%), penicillin (50 units/ml), and streptomycin (50 µg/ml) and incubated in a humidified 5% CO₂ atmosphere at 37 °C for 24 h. After 24 h, GFP-positive cells were evaluated by using FACS-Canto flow cytometer (BD Biosciences), and data, expressed as percentage of alive, propidium iodide-negative cells, were analyzed by using DIVA software.

Real-time PCR—Twenty-four hours after transfection, total RNA was extracted from cells (2 x 10⁶ cells) using the RNeasy mini kit (Qiagen) according to the manufacturer's instructions and reverse transcribed into cDNA using the Superscript III first strand synthesis system (Invitrogen). The cDNA was used as template for real-time PCR analysis: reactions were performed in an iCycler iQ5 real-time PCR detection system (Bio-Rad). The human P2Y₁₁-specific primers were designed by using Beacon Designer 2.0 software (Bio-Rad), and their sequences were as follows: 5'-CGTGAGCTGAGCCAATGATGTG-3' (forward) and 5'-GGGTGGGAAAGGCGACTGC-3' (reverse). Each sample was assayed in triplicate in a 25-µl amplification reaction, containing 30 ng of cDNA, primers mixture (0.4 µM each of sense and antisense primers), and 12.5 µl of 2x iQ SYBR Green Supermix Sample (Bio-Rad). The amplification program included 40 cycles of two steps, each comprising heating to 95 °C and to 60 °C, respectively. Fluorescent products were detected at the last step of each cycle. To verify the purity of the products, a melting curve was produced after each run. Values were normalized to -actin mRNA expression. Statistical analysis of the quantitative real-time PCR was obtained using the iQ5 Optical System Software version 1.0 (Bio-Rad) based on the 2^–Ct method, which calculated relative changes in gene expression of the target (P2Y₁₁) normalized to -actin and relative to a calibrator ("control," cells subjected to electroporation in the absence of siRNA). Amplification efficiencies of target and reference genes were determined by generating standard curves. Data are presented as means ± S.D.

Chemotaxis—Twenty-four hours after transfection, a 300-µl aliquot of granulocytes from each condition was centrifuged at 5000 x g for 10 s, and cells were resuspended at 7 x 10⁶/ml in chemotaxis buffer (HBSS, phosphate-buffered saline, and 5% albumin, 39:16:1). Chemotaxis assays were performed using 96-well ChemoTx system microplates (Neuro Probe, Inc., Gaithersburg, MD) with a 3-µm pore size polycarbonate filter. -or -NAD⁺ (10 µM) were diluted in chemotaxis buffer and added in the bottom wells. Granulocytes (25 µl) were placed on top of the filter and incubated for 60 min at 37 °C: the transmigrated cells were evaluated as previously described (12). The results were expressed as chemotaxis index (CI): CI = number of cells migrated toward chemoattractant/number of cells migrated toward medium.

Statistical Analyses—All parameters were tested by paired t test. p values <0.05 were considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Millimolar Induces Both Transient and Sustained Ca²⁺ Responses in Human Granulocytes—Incubation of intact, freshly isolated granulocytes with extracellular -NAD⁺ () concentrations ranging from 1 to 100 µM resulted in a slowly developing, sustained elevation of [Ca²⁺]_i (12). As shown in Fig. 1 (A and B), a markedly different Ca²⁺ response was consistently observed upon incubating granulocytes with 1 mM . Under these conditions, an immediate and transient elevation of the [Ca²⁺]_i (from 40 ± 6to105 ± 9nM, n = 5) was followed by a sustained increase (to 145 ± 28 after 15 min from the addition of the nucleotide, n = 5).

When 1 mM was added to granulocytes in the presence of 0.3 mM extracellular EGTA in Ca²⁺-free HBSS, the kinetics and the extent of the immediate [Ca²⁺]_i increase were superimposable to those recorded in a Ca²⁺-containing medium (Fig. 1C), indicating that the transient increase in Ca²⁺ levels arises from release from intracellular stores and not from extracellular influx. By contrast, the sustained Ca²⁺ elevation triggered by millimolar was markedly decreased (80%) in the presence of EGTA, demonstrating that influx of extracellular Ca²⁺ is responsible for the sustained Ca²⁺ elevation at longer times (Fig. 1C), similar to what was observed with micromolar concentrations (12).

View larger version (21K): [in this window] [in a new window]   FIGURE 1. Kinetics of - and -induced [Ca2+]i increase in human granulocytes. Fluo-3AM-loaded (A) or FURA-2AM-loaded (B) granulocytes were treated at 25 °C with 100 µM (open triangles) or 1 mM -NAD+ (black squares). Fluo-3AM-loaded granulocytes were treated at 25 °C with: C, 1 mM -NAD+ in calcium buffer (HBSS, black squares) or in HBSS containing 0.3 mM EGTA (open squares); D, 1 mM -NAD+ in calcium buffer (HBSS, black squares) or in HBSS containing 0.3 mM EGTA (open squares). [Ca2+]i changes were measured in parallel using a fluorescence plate reader (A, C, and D), or with the system described in Ref. 12 for [Ca2+]i calibration (B). Nucleotide was added at 50 s in A and B and at 70 s in C and D. Representative traces are shown (A and C, n = 11; B, n = 5; D, n = 7); B, each point is the mean of three [Ca2+]i values, recorded every 9 s (S.D. 15% of the mean value).

Distinct Roles of IP₃ and cADPR in Transient and Sustained Ca²⁺ Responses of Intact Granulocytes to —Preincubation of intact granulocytes with the membrane-permeant phospholipase C (PLC) inhibitor (U73122 [GenBank] , 5 µM) abolished the early transient [Ca²⁺]_i elevation promoted by either (Fig. 2A), or (not shown), whereas the same concentration of the inactive analog U73343 [GenBank] proved to be ineffective (Fig. 2A). Conversely, the sustained increase in [Ca²⁺]_i observed with - and was not affected by pretreatment of the granulocytes with either U73122 [GenBank] or U73343. [GenBank] These findings demonstrate that - and at 1 mM evoke a rapid Ca²⁺ release from IP₃-responsive intracellular stores.

To confirm the role of cADPR in the long lasting Ca²⁺ response induced by millimolar , as was previously demonstrated with micromolar concentrations (12), intact granulocytes were exposed to (1 mM) following preincubation with either 8-Br-cADPR (100 µM), a membrane-permeant cADPR antagonist (22), or ryanodine at 50 µM, a concentration that inhibits Ca²⁺ release from cADPR-responsive stores (23). Although neither treatment affected the first, transient [Ca²⁺]_i increase, both 8-Br-cADPR and ryanodine significantly reduced (approximately by 80%) the sustained [Ca²⁺]_i elevation (Fig. 2, B and C). These results demonstrate that the sustained Ca²⁺ increase triggered by 1 mM is mediated by cADPR, similarly to what was observed at micromolar dinucleotide concentrations. This cADPR-dependent Ca²⁺ increase is abrogated in the presence of extracellular EGTA (Fig. 1).

Intracellular IP₃, cADPR, and cAMP Concentrations in -stimulated Granulocytes—Exposure of granulocytes to 1mM promoted a rapid increase in IP₃ levels. Following a 60-s incubation with the dinucleotide, intracellular IP₃ levels increased by 2-fold, from a basal (unstimulated) value of 0.25 ± 0.03 pmol/10⁶ to 0.54 ± 0.08 pmol/10⁶ cells (n = 3, p < 0.005, Fig. 3A). Micromolar (10–100) concentrations of did not promote significant changes in IP₃ levels (data not shown). These data demonstrate the causal involvement of IP₃ generation in the initial, transient [Ca²⁺]_i increase in response to millimolar (Fig. 2A).

To investigate changes in cADPR levels promoted by millimolar , we used as an agonist (12), which proved to be as effective as in triggering a pathway leading to stimulation of intracellular cADPR synthesis from -NAD_i+ (12). was used to avoid possible interference of the measurement of intracellular cADPR levels by extracellular cADPR generated from (12). After a 15-min exposure of granulocytes to 1 mM , cADPR levels increased to 300 ± 80% (n = 12, p < 0.001) over basal levels (19.11 ± 4.01 pmol/10⁹ cells, Fig. 3B), in agreement with the concentration-dependent effects observed at micromolar (12).

View larger version (26K): [in this window] [in a new window]   FIGURE 2. Role of IP3 and cADPR in the -induced [Ca2+]i increase in human granulocytes. A, Fluo-3AM-loaded granulocytes were preincubated at 25 °C for 10 min in HBSS in the presence of 5 µM U73122 (open triangles) or U73343 (black squares). B and C, granulocytes were preincubated for 50 min in HBSS (control, black squares), or in HBSS containing 100 µM 8-Br-cADPR (open squares, B) or 50 µM ryanodine (open triangles, C) and then loaded for further 40 min with Fluo-3-AM. -NAD+ (1 mM) was then added, and [Ca2+]i was measured in parallel using a fluorescence plate reader, as described under "Experimental Procedures." Nucleotide was added at 40 s in A–C. Representative traces are shown (n 3 for each condition).

Role of Purinergic Receptors in the -induced Ca²⁺ Responses of Intact Granulocytes—Recently, a role for purinergic receptors in nucleotide-triggered Ca²⁺ responses or Ca²⁺-regulated cell functions in blood cells (24), dendritic cells (25), and T lymphocytes (7, 10, 11) has emerged. To address the type of receptors that mediate the -induced Ca²⁺ responses in human granulocytes, intact cells were preincubated with suramin, a widely used antagonist of both G protein-coupled P2Y and ion channel-forming P2X receptors (24, 26). To investigate the specificity of suramin as a P2Y receptor inhibitor in our cell system, granulocytes were first incubated in the presence of suramin (100 µM for 1 h) and then incubated with either 100 µM ATP, 1 µM fMLP, or 1 µM A5 peptide (15). Indeed, suramin strongly inhibited (87%, n = 3) the ATP-induced Ca²⁺ increase, whereas little to no changes in the P2Y-unrelated Ca²⁺ responses to fMLP and A5 peptide were observed (15 and 0% inhibition, respectively, n = 3, data not shown).

View larger version (15K): [in this window] [in a new window]   FIGURE 3. Intracellular IP3, cADPR, and cAMP levels in -stimulated human granulocytes. After addition of 1 mM -NAD+ to control, untreated granulocytes (black squares), or to cells preincubated for 1 h with 100 µM suramin (open squares), IP3 (A), cADPR (B), and cAMP (C) levels were determined at the times indicated, as described under "Experimental Procedures." Results are the mean ± S.D. of at least three different experiments for each assay.

View larger version (21K): [in this window] [in a new window]   FIGURE 4. Effect of suramin on the -induced [Ca2+]i increase in human granulocytes. Control, untreated (black squares) or suramin-treated (100 µM for 1 h, open triangles) granulocytes were loaded with Fluo-3AM and exposed to 1 mM -NAD+ at 25 °C. [Ca2+]i changes were measured in parallel using a fluorescence plate reader, as described under "Experimental Procedures." Nucleotide was added at 40 s. Representative traces are shown (n = 5).

Interestingly, the slow, sustained elevation of [Ca²⁺]_i elicited by 1 mM was also strongly inhibited (80 ± 5%) by suramin (Fig. 4). Thus, we explored the effect of suramin on cAMP levels in granulocytes stimulated with 1 mM . Indeed, preincubation with suramin resulted in complete inhibition of cAMP generation (Fig. 3C). Likewise, preincubation of granulocytes with suramin resulted in almost complete inhibition of -promoted increases in cADPR (Fig. 3B), suggesting that a suramin-inhibitable receptor, triggered by 1 mM , lies upstream of the activation of AC and of ADPRC. Taken together, these data demonstrate that in human granulocytes a suramin-inhibitable purinoceptor mediates both the rapid, transient, and IP₃-dependent elevation of [Ca²⁺]_i and the subsequent slow, sustained cAMP/PKA/cADPR-dependent elevation of [Ca²⁺]_i promoted by millimolar .

Extracellular NAD⁺ Increases the [Ca²⁺]_i of P2Y₁₁-1321N1 Astrocytoma Cells—Among the human G protein-coupled P2Y receptor (hP2Y) subtypes cloned to date, only the hP2Y₁₁ receptor couples to both PLC and adenylyl cyclase (16). To establish whether the P2Y₁₁ receptor is activated by , we investigated the effect of the dinucleotide on [Ca²⁺]_i in 1321N1 human astrocytoma cells stably expressing the hP2Y₁₁ receptor (1321N1-hP2Y₁₁ cells) (17). To rule out possible interferences afforded by metabolic by-products of (e.g. ADPR, AMP, and adenosine), we investigated the stability of - and during incubation with both native 1321N1 and 1321N1-hP2Y₁₁ cells. Importantly, no significant degradation of either dinucleotide form (100 µM) was observed over 60 min at 25 °C.

Addition of 100 µM to 1321N1-hP2Y₁₁ cells promoted a rapid and transient increase of [Ca²⁺]_i, followed by a slow, sustained [Ca²⁺]_i rise. Conversely, did not induce any Ca²⁺ response on control 1321N1 cells (Fig. 5A), indicating that the P2Y₁₁ receptor was responsible for the -promoted transient and sustained increases of [Ca²⁺]. In agreement with results obtained with human granulocytes, the rapid and transient -promoted elevation in [Ca²⁺]_i was due to release from intracellular stores, because it was not abrogated in the presence of extracellular EGTA. In contrast, the sustained [Ca²⁺]_i rise elicited by was completely inhibited by extracellular EGTA, implicating Ca²⁺ influx (data not shown). Ca²⁺ mobilization was also observed in 1321N1-hP2Y₁₁ cells challenged with extracellular ATP. The kinetics of the Ca²⁺ response was similar to those observed with , although consistently more robust (Fig. 5A).

Preincubation of 1321N1-hP2Y₁₁ cells with either the PLC inhibitor, U73122 [GenBank] , or with suramin completely abrogated the rapid and transient increase in [Ca²⁺]_i (Fig. 5, B and C), confirming the involvement of the PLC-coupled P2Y₁₁ receptor in triggering the rapid [Ca²⁺]_i rise. Suramin also abrogated the slow and sustained [Ca²⁺]_i elevation, whereas the PLC inhibitor was ineffective (Fig. 5, B and C). These results indicate that the sustained [Ca²⁺]_i rise is also triggered by the P2Y₁₁ receptor, yet via a PLC-independent pathway. The sustained Ca²⁺ increase, which was due to extracellular Ca²⁺ influx, was inhibited by preincubation of 1321N1-hP2Y₁₁ cells with 8-BrcADPR (Fig. 5D), in agreement with previous data obtained for human granulocytes (12).

Challenging 1321N1-hP2Y₁₁ cells with ATP (100 µM) instead of NAD⁺ had no effect on the action of the various inhibitors on the biphasic Ca²⁺ response. Thus, suramin inhibited almost completely (80 ± 5%, n = 3) both the rapid and sustained increases in [Ca²⁺]_i, whereas U73122 [GenBank] abrogated only the rapid, transient phase (n = 3) and 8-Br-cADPR strongly reduced (73 ± 8%, n = 3) only the slowly developing sustained phase of ATP-promoted [Ca²⁺]_i increase (data not shown). The sustained [Ca²⁺]_i increases induced by both (100 µM) and ATP (100 µM) in 1321N1-hP2Y₁₁ cells were completely inhibited by SKF96365 (10 µM, 1-min preincubation), an inhibitor of store-operated calcium channels (27).

Extracellular NAD⁺ Increases Intracellular IP₃, cAMP, and cADPR Levels in 1321N1-hP2Y₁₁ Cells—The results described above clearly indicate the activation by of an IP₃- and a cAMP/cADPR-dependent pathway leading to discrete Ca²⁺ responses in hP2Y₁₁-1321N1 cells. The hP2Y11 receptor expressed by 1321N1 cells is indeed known to be coupled to both PLC and AC (16).

View larger version (21K): [in this window] [in a new window]   FIGURE 5. and ATP-induced [Ca2+]i increase in hP2Y11-1321N1 cells. A, Fluo-3AM-loaded hP2Y11-1321N1 cells were treated with 100 µM (black squares) or with 100 µM ATPe (open rhombus, inset); control 1321N1 cells were exposed to 100 µM (open squares); B, untreated (black squares) or U73122 preincubated (5 µM for 10 min, open triangles) hP2Y11-1321N1 cells were incubated with 100 µM ; C, untreated (black squares) or suramin preincubated (100 µM for 1 h, open triangles) hP2Y11-1321N1 cells were challenged with 100 µM ; D, untreated (black squares) or 8-Br-cADPR preincubated (100µM for 90 min, open triangles) hP2Y11-1321N1 cells were incubated with 100 µM . [Ca]i changes were measured in parallel at 25 °C using a fluorescence plate reader, as described under "Experimental Procedures." Nucleotide was added at 70 s in A–C and at 40 s in D. Representative traces are shown (A, n = 8; B–D, n = 5).

Challenge of 1321N1-hP2Y₁₁ cells with 10 and 100 µM caused a steady elevation of IP₃ levels, reaching 134 and 163% of basal values, respectively, recorded in unstimulated cells (Table 1) and measured 30 s after addition of agonist. By contrast, the same concentration of ATP induced a 3-fold increase in IP₃ levels in 1321N1-hP2Y₁₁ cells. In native 1321N1 cells, the basal value of [IP₃]_i (8.79 ± 1.32 pmol/10⁶ cells) was not significantly modified by incubation with 100 µM .

View this table: [in this window] [in a new window]   TABLE 1 -induced intracellular IP3, cAMP, and cADPR increases in hP2Y11-1321N1 cells -NAD+ was added at the concentrations indicated to hP2Y11-1321N1 cells, IP3 levels were determined after 30 s from the addition of (n = 4), cAMP levels were determined after 15 min from the addition of (n = 6), and cADPR levels were measured in lysates from cells incubated for 15 min with -NAD+ (n = 5), as described under "Experimental Procedures." Results are the mean ± S.D.

We next investigated whether elevations in cAMP in NAD⁺-stimulated 1321N1-hP2Y₁₁ cells are responsible for the slowly developing, sustained [Ca²⁺]_i rise through the -triggered signaling pathway described in human granulocytes (12). For this purpose, Fluo-3AM-loaded 1321N1-hP2Y₁₁ cells were incubated in the presence of the cell-permeant PKA activator, 8-Br-cAMP (500 µM). As shown in Fig. 6, a sustained [Ca²⁺]_i increase was recorded within 4 min of 8-Br-cAMP addition (n = 3), similar to that previously described in human granulocytes (12). A comparable sustained [Ca²⁺]_i increase was also obtained upon addition of 500 µM 8-Br-cAMP to control 1321N1 cells (not shown).

View larger version (21K): [in this window] [in a new window]   FIGURE 6. 8-Br-cAMP-induced [Ca2+]i increase in hP2Y11-1321N1 cells. Traces shown, representative of three different experiments, were obtained upon addition (at 300 s) of 500 µM 8-Br-cAMP (black squares) to Fluo-3AM-loaded hP2Y11-1321N1 cells. Open triangles indicate the same cells without addition of 8-Br-cAMP. [Ca2+]i changes were measured in parallel at 25 °C using a fluorescence plate reader.

Likewise, following a 15-min addition of 100 µM to 1321N1-hP2Y₁₁ cells, the levels of cADPR increased to 145 ± 22% of basal value (0.23 ± 0.05 pmol/10⁶ cells) (Table 1). Similar results were obtained when 1321N1-hP2Y₁₁ cells were exposed for 15 min to 100 µM ATP (increase to 0.33 ± 0.08 pmol/10⁶ cells, n = 4). Conversely, cADPR levels in control 1321N1 cells (basal value, 0.22 ± 0.05 pmol/10⁶ cells, n = 3) were not significantly increased by 100 µM .

Role of Endogenous P2Y₁₁ in NAD⁺-induced Activation of Human Granulocytes—To demonstrate that endogenous P2Y₁₁ is the receptor responsible for the NAD⁺-induced Ca²⁺ response leading to activation of human granulocytes, we followed two different experimental approaches. First, cells were preincubated with 1 µM NF157, a selective and potent P2Y₁₁ receptor antagonist: as reported by Ullmann et al. (19), 1µM NF157 does not affect the P2Y₂ purinoceptor, i.e. the receptor involved in the ATP-induced activation of human granulocytes (29). Second, down-regulation of P2Y₁₁ expression in human granulocytes was achieved by means of specific siRNA transfection.

Preincubation of human granulocytes with NF157 resulted in the abrogation of the sustained [Ca²⁺]_i increase induced by 100 µM -NAD⁺ (not shown). Moreover, the presence of NF157 completely prevented the transient [Ca²⁺]_i elevation triggered by 1 mM -NAD⁺ and strongly inhibited (81 ± 5%) the sustained [Ca²⁺]_i increase induced by 1 mM -NAD⁺ (Fig. 7A). At the same time, the presence of NF157 abrogated the -promoted increases in IP₃ and cAMP levels (Fig. 7, B and C). Similar results were obtained upon stimulation of granulocytes with -NAD⁺ (not shown). In addition, as shown in Fig. 7D, preincubation of cells with NF157 strongly inhibited chemotaxis of human granulocytes toward 10 µM - or -NAD⁺, the concentration triggering the maximal chemotactic response (12).

Next, human granulocytes were transfected with specific siRNA for P2Y₁₁: after 24 h, P2Y₁₁ mRNA levels were decreased to 20% compared with control cells (electroporated in the absence of siRNA), as confirmed by real-time PCR analysis (Fig. 8A). The presence of a negative control siRNA did not induce any significant modification of P2Y₁₁ mRNA levels in human granulocytes (Fig. 8A). The -induced Ca²⁺ responses were then measured at 24 h after transfection: as shown in Fig. 8B, down-regulation of P2Y₁₁ was accompanied by the almost complete inhibition of both Ca²⁺ responses triggered by 1 mM -NAD⁺, i.e. the IP₃-related rapid increase and the cADPR-dependent sustained rise. Comparable results were obtained upon stimulation of cells with 1 mM -NAD⁺ (not shown). Finally, P2Y₁₁ siRNA-transfected, negative control siRNA-transfected, and control granulocytes were comparatively challenged to migrate toward 10 µM - or -NAD⁺: as shown in Fig. 8C, transfection with specific siRNA for P2Y₁₁ completely prevented chemotaxis of granulocytes.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The recent discovery of NAD⁺ as an extracellular agonist eliciting functional activation of human granulocytes (12) highlighted a new mechanism for this dinucleotide, which was distinct from its action in other mammalian cell types (1–8). In astrocytes, osteoblasts, CD38-transfected fibroblasts, and HeLa cells, CD38, an ecto-ADPRC, converts into the intracellular calcium mobilizer cADPR (1, 3, 5, 6). Subsequent translocation of extracellularly generated cADPR into the cytoplasm, where it can cause release of Ca²⁺ stores by binding to the ryanodine receptor, is performed either by CD38 itself or by nucleoside transporters (1). In murine T lymphocytes, the addition of promotes Ca²⁺ influx secondary to -mediated ADP ribosylation of the P2X₇ purinoceptor or of a P2X-associated protein (7, 10, 11).

In contrast, the [Ca²⁺]_i increase in human granulocytes promoted by micromolar occurs through (i) overproduction of cAMP, (ii) activation of PKA, (iii) stimulation of ADPRC activity and consequent overproduction of cADPR, and (iv) influx of extracellular Ca²⁺ (12). Functional equivalence between and , which is not a substrate of either ADPRCs (28) or ADP-ribosyl transferases (30), suggested that activates one or more plasma membrane receptors coupled to a signaling pathway that result in granulocyte activation. However, identification of such receptor(s) has remained elusive (12).

The results obtained in this study demonstrate for the first time that is an endogenous agonist of the P2Y₁₁ purinoceptor and that engagement of P2Y₁₁ by extracellular NAD⁺ triggers a cADPR-dependent Ca²⁺ signaling eventually leading to activation of human granulocytes. Both conclusions are supported by several biochemical, pharmacological, and molecular lines of evidence. A first clue came from the finding of different patterns of [Ca²⁺]_i variations induced by micromolar and millimolar concentrations. The two steps of Ca²⁺ mobilization proved to involve independent, not causally interrelated mechanisms, because the fast, IP₃-mediated increase in [Ca²⁺]_i was not necessary to induce the subsequent sustained [Ca²⁺]_i rise. These results indicated that the role of the cAMP/cADPR pathway is predominant at micromolar over the PLC/IP₃ cascade. Activation of both signaling pathways by millimolar concentrations suggested two alternative explanations: (a) binding of high [NAD⁺]_e to two different plasma membrane receptors, each of which couples to a distinct signaling cascade and (b) interaction of with a single receptor coupling to both pathways of the Ca²⁺ response.

View larger version (12K): [in this window] [in a new window]   FIGURE 7. Effect of NF157 on the -induced [Ca2+]i, [IP3]i and [cAMP]i increases and on NAD+-directed chemotaxis in human granulocytes. A, control, untreated (black squares) or NF157-treated (1 µM for 1 h, open triangles) granulocytes were loaded with Fluo-3AM and exposed to 1 mM -NAD+ at 25 °C. [Ca2+]i changes were measured in parallel using a fluorescence plate reader, as described under "Experimental Procedures." Nucleotide was added at 70 s. Representative traces are shown (n = 3). After addition of 1 mM -NAD+ to control, untreated granulocytes (black squares), or to cells preincubated for 1 h with 1µM NF157 (open squares), both IP3 (B) and cAMP (C) levels were determined at the times indicated, as described under "Experimental Procedures." Results are the mean ± S.D. of at least three different experiments for each assay. D, migration of granulocytes preincubated, or not (control), with NF157 (1 µM for 40 min) through 3-µm pore membranes toward a medium containing, or not, 10 µM -NAD+ (black bars) or -NAD+ (white bars), was measured in ChemoTx chambers and the chemotaxis index (CI) was calculated. Migration of NF157-pretreated granulocytes toward the medium in the absence of NAD+ was not significantly different from that of control, untreated cells. Results shown are the mean ± S.D. of four experiments (p < 0.005).

View larger version (17K): [in this window] [in a new window]   FIGURE 8. Effect of siRNA for P2Y11 on the -induced [Ca2+]i increases and NAD+-directed chemotaxis in human granulocytes. 24 h after transfection, control (electroporated in the absence of siRNA), negative control siRNA-transfected, or P2Y11 siRNA-transfected granulocytes were treated as follows. A, subjected to total RNA extraction: real-time PCR for P2Y11 expression was performed as described under "Experimental Procedures" (n = 3); B, loaded with Fluo-3AM and exposed to 1 mM -NAD+ at 25 °C (control, black squares; control siRNA, open triangles; P2Y11 siRNA, open squares). [Ca2+]i changes were measured in parallel using a fluorescence plate reader, as described under "Experimental Procedures." Nucleotide was added at 70 s. Representative traces are shown (n = 3); C, challenged to migrate toward a medium containing, or not, 10 µM -NAD+ (black bars) or -NAD+ (white bars). Migration was measured in ChemoTx chambers, and the chemotaxis index (CI) was calculated. Migration of transfected granulocytes toward the medium in the absence of NAD+ was not significantly different from that of control cells. Results shown are the mean ± S.D. of three experiments (p < 0.001).

The abrogation of all the -NAD⁺-promoted effects by suramin, a non-selective inhibitor of the family of P2 receptors (24, 26), suggested the possibility that was an agonist at one or more P2Y receptors; indeed, P2Y receptors are involved in PLC and/or AC activation (26). Human granulocytes express P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁ receptor subtypes, as revealed by reverse transcription-PCR analysis (29). P2Y₂, P2Y₄, and P2Y₆ receptors are coupled to PLC, resulting in the formation of IP₃ and mobilization of [Ca²⁺]_i, as well as activation of PKC (31). Notably, distinct from other members of the P2Y subfamily, the human P2Y₁₁ receptor is functionally coupled to both PLC and AC (16).

The unambiguous association of any P2Y receptor subtype with a specific physiological effect is hampered by the lack of subtype-selective agonists and antagonists (17). Thus, the pharmacological selectivities of these receptors can be optimally defined by expressing individual subtypes of cloned P2Y receptors in null cells. In our study, an astrocytoma cell line (1321N1) expressing the human P2Y₁₁ (17) was exploited to determine the possible activation of this receptor by . Indeed, native 1321N1 astrocytoma cells lack expression of any known P2 receptor subtypes (32). triggers both the cAMP/cADPR and the PLC/IP₃ pathways in 1321N1-hP2Y₁₁ cells but not in native 1321N1 cells.

The fact that the hP2Y₁₁ receptor is the only purinoceptor coupled to AC, together with its known expression in granulocytes, implicated the hP2Y₁₁ receptor as the putative receptor responsive to in these cells. This view received experimental support by use of NF157, a suramin-related P2Y₁₁-selective antagonist, which was recently synthesized and characterized (19). Indeed, 1 µM NF157 abrogated in granulocytes both Ca²⁺ responses elicited by millimolar (Fig. 7A).

Final evidence for the identification of P2Y₁₁ as the NAD⁺-binding purinoceptor that mediates granulocyte activation was provided by specific siRNA transfection (Fig. 8). A significant decrease in P2Y₁₁ mRNA levels, observed 24 h after transfection with specific siRNA, indicates a relatively high turnover of P2Y₁₁ in human granulocytes (Fig. 8A). The experiments with P2Y₁₁-specific siRNA unequivocally identified P2Y₁₁ as the receptor interacting with .

Previous studies performed on 1321N1-hP2Y₁₁ astrocytoma cells provided information on the pharmacological properties of this purinoceptor, demonstrating that it couples to AC and PLC with different efficiencies depending on the used agonist (16, 17). Specifically, previous data demonstrated that coupling of P2Y₁₁ with AC was triggered by ADP with a lower potency than by ATP as an agonist (17), whereas UTP stimulation did not lead to accumulation of IP₃, under conditions in which ATP gave a robust effect (33).

From the standpoint of agonist activity, it is of potential biological interest that, like , micromolar ATP_e also evoked both an initial IP₃-dependent Ca²⁺ spike and a cADPR-dependent sustained [Ca²⁺]_i rise in 1321N1-hP2Y₁₁ cells. The ATP-promoted sustained rise in intracellular Ca²⁺ was significantly higher than that evoked by (Fig. 5A); this difference might be accounted for by the greater depletion of intracellular Ca²⁺ stores elicited by ATP via IP₃ overproduction (34), as suggested by experiments with SKF96365 (see "Results"). cADPR was involved in the slowly developing sustained rise in [Ca²⁺]_i promoted by both and ATP_e, as demonstrated by the substantial inhibition afforded by 8-BrcADPR. Previous reports have postulated the occurrence of a cADPR-mediated activation of store-operated calcium channels (12, 14, 35–38). In recent years, growing evidence has accumulated that strongly implicates the TRPM2 channels as responsible for cADPR- and ADPR-mediated Ca²⁺ influx (39, 40).

The causal role of cADPR in the sustained [Ca²⁺]_i rise induced by both and ATP_e in the hP2Y₁₁-132N1 cells is well established. However, it is somewhat surprising that the increase of intracellular cADPR in the hP2Y₁₁⁺ cells was not much greater with ATP than with (see "Results"). This seems to contrast with the different extents of [cAMP]_i elevation in response to and ATP_e, respectively, with ATP being far more potent and efficacious in stimulating cAMP overproduction. A possible explanation for this quantitative discrepancy might be the attainment of near-maximal activation of PKA at the [cAMP]_i levels induced by .

In conclusion, the present study demonstrates that is an endogenous agonist of the P2Y₁₁ purinergic receptor, acting as a pro-inflammatory cytokine on human granulocytes. In this respect, increased levels of and ATP expected at sites of inflammation, as a consequence of cell lysis (11) or of regulated release (41, 42), are likely to be sufficient to trigger functional responses in granulocytes, particularly enhanced chemotaxis, that are causally related to the cADPR/Ca²⁺ system (12). Specifically, as far as concentrations are concerned, these average 50–100 nM in human blood plasma (1), thus close to those resulting in enhanced chemotaxis (12). In vivo, the extent of cell lysis as a source of at specific sites can only be postulated, but hardly estimated. Conversely, the established capacity of increased [Ca²⁺]_i of down-regulating intracellular NAD⁺ release via PKC-mediated phosphorylation of Cx43 hemichannels (43) might suggest the opposite process, i.e. opening of these hemichannels via dephosphorylation and consequently enhanced efflux of NAD⁺ from cells.

	FOOTNOTES

 
^* This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro, the Italian Ministry of Education, University and Scientific Research (Grants MIUR-PRIN 2003, MIUR FIRB RBAUO19A3C, MIUR FIRB RBNE01ERXR, and MIUR FIRB RBLA039LSF), the University of Genova, and the Fondazione Cassa di Risparmio di Genova e Imperia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV/1, 16132 Genova, Italy. Tel.: 39-010-353-8158; Fax: 39-010-354-415; E-mail: santina.bruzzone{at}unige.it.

² The abbreviations used are: , extracellular NAD⁺; [Ca²⁺]_i, intracellular calcium concentration; ADPRC, ecto-ADP-ribosyl cyclase; cADPR, cyclic ADP-ribose; PCA, perchloric acid; ATP_e, extracellular ATP; IP₃, inositol 1,4,5-trisphosphate; [cADPR]_i, intracellular cADPR concentration; [cAMP]_i, intracellular cAMP concentration; AC, adenylate cyclase; PLC, phospholipase C; PKA, cAMP-activated protein kinase; siRNA, short interference RNA; CI, chemotaxis index; fMLP, formylmethionylleucylphenylalanine; HBSS, Hanks' balanced salt solution; GFP, green fluorescent protein; h2PY, human G protein-coupled P2Y receptor.