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

Abstract

Micromolar concentrations of extracellular β-NAD+ (NADe+) 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 NADe+ 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 NADe+-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 NADe+ 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 NADe+-induced intracellular increases of [Ca2+]i and chemotaxis. These results demonstrate that β-NADe+ 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. Micromolar concentrations of extracellular β-NAD+ (NADe+) 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 NADe+ 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 NADe+-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 NADe+ 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 NADe+-induced intracellular increases of [Ca2+]i and chemotaxis. These results demonstrate that β-NADe+ 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. Extracellular NAD+ (NADe+) 2The abbreviations used are: NADe+, extracellular NAD+; [Ca2+]i, intracellular calcium concentration; ADPRC, ecto-ADP-ribosyl cyclase; cADPR, cyclic ADP-ribose; PCA, perchloric acid; ATPe, 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. 2The abbreviations used are: NADe+, extracellular NAD+; [Ca2+]i, intracellular calcium concentration; ADPRC, ecto-ADP-ribosyl cyclase; cADPR, cyclic ADP-ribose; PCA, perchloric acid; ATPe, 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. is known to increase intracellular calcium concentrations ([Ca2+]i) in different cell types and by different mechanisms (1De Flora A. 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KochNolte F. Schott W.H. Leiter E.H. Dennert G. J. Immunol. 2005; 174: 1971-1979Crossref PubMed Scopus (55) Google Scholar, 11Krebs C. Adriouch S. Braasch F. Koestner W. Leiter E.H. Seman M. Lund F.E. Oppenheimer N. Haag F. Koch-Nolte F. J. Immunol. 2005; 174: 3298-3305Crossref PubMed Scopus (76) Google Scholar). In human monocytes, NADe+ and ADPR trigger influx of extracellular Ca2+, but neither CD38 nor P2X7-induced pore formation are involved (8Gerth A. Nieber K. Oppenheimer N.J. Hauschildt S. Biochem. J. 2004; 382: 849-856Crossref PubMed Scopus (29) Google Scholar). Recently, we demonstrated that NADe+ behaves as a proinflammatory cytokine targeting human polymorphonuclear granulocytes (12Bruzzone S. Moreschi I. Guida L. Usai C. Zocchi E. De Flora A. Biochem. J. 2006; 393: 697-704Crossref PubMed Scopus (57) Google Scholar). Exposure of granulocytes to micromolar concentrations of NADe+ (either the naturally occurring β or the α 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+ (12Bruzzone S. Moreschi I. Guida L. Usai C. Zocchi E. De Flora A. Biochem. J. 2006; 393: 697-704Crossref PubMed Scopus (57) Google Scholar). Increases in [Ca2+]i are known to be responsible for activation of human granulocytes (13Davies E.V. Hallett M.B. Int. J. Mol. Med. 1998; 1: 485-490PubMed Google Scholar). Indeed, NADe+-induced [Ca2+]i elevation triggered increased O2- and NO generation and enhanced chemotaxis toward NADe+. Therefore, the results obtained with NADe+-stimulated granulocytes (12Bruzzone S. Moreschi I. Guida L. Usai C. Zocchi E. De Flora A. Biochem. J. 2006; 393: 697-704Crossref PubMed Scopus (57) Google Scholar) support a key role of cADPR in control of receptor-mediated chemotaxis in murine and human granulocytes, as previously demonstrated both in vivo (14Partida-Sa´nchez S. Cockayne D.A. Monard S. Jacobson E.L. Oppenheimer N. Garvy B. Kusser K. Goodrich S. Howard M. Harmsen A. Randall T.D. Lund F.E. Nat. Med. 2001; 7: 1209-1216Crossref PubMed Scopus (376) Google Scholar) and in vitro (15Partida-Sa´nchez S. Iribarren P. Moreno-Garcia M.E. Gao J.L. Murphy P.M. Oppenheimer N. Wang J.M. Lund F.E. J. Immunol. 2004; 172: 1896-1906Crossref PubMed Scopus (86) Google Scholar). The rapid increase of [cAMP]i in granulocytes that follows NADe+ exposure (12Bruzzone S. Moreschi I. Guida L. Usai C. Zocchi E. De Flora A. Biochem. J. 2006; 393: 697-704Crossref PubMed Scopus (57) Google Scholar) suggested that NADe+ 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 NADe+. 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) NADe+-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 NADe+ 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 Gq and Gs (16Communi D. Govaerts C. Parmentier M. Boeynaems J.M. J. Biol. Chem. 1997; 272: 31969-31973Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 17Qi A.-D. Kennedy C. Harden T.K. Nicholas R. Br. J. Pharmacol. 2001; 132: 318-326Crossref PubMed Scopus (55) Google Scholar, 18Torres B. Zambon A.C. Insel P.A. J. Biol. Chem. 2002; 277: 7761-7765Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Results obtained with hP2Y11-transfected 1321N1 astrocytoma cells reveal that β-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 NADe+-induced activation of human granulocytes. 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 (19Ullmann H. Meis S. Hongwiset D. Marzian C. Wiese M. Nickel P. Communi D. Boeynaems J.M. Wolf C. Hausmann R. Schmalzing G. Kassack M.U. J. Med. Chem. 2005; 48: 7040-7048Crossref PubMed Scopus (77) Google Scholar). All other chemicals were obtained from Sigma (Milan, Italy). High-performance Liquid Chromatography Analyses of Nucleotides and Chromatographic Purification of α- and β-NAD+—High-performance liquid chromatography analyses of nucleotides and purification of α- and β-NAD+ were performed as described previously (12Bruzzone S. Moreschi I. Guida L. Usai C. Zocchi E. De Flora A. Biochem. J. 2006; 393: 697-704Crossref PubMed Scopus (57) Google Scholar). 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 (12Bruzzone S. Moreschi I. Guida L. Usai C. Zocchi E. De Flora A. Biochem. J. 2006; 393: 697-704Crossref PubMed Scopus (57) Google Scholar). Cell Culture—Control and hP2Y11-transfected 1321N1 astrocytoma cell lines (17Qi A.-D. Kennedy C. Harden T.K. Nicholas R. Br. J. Pharmacol. 2001; 132: 318-326Crossref PubMed Scopus (55) Google Scholar) 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 × 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 × 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 (12Bruzzone S. Moreschi I. Guida L. Usai C. Zocchi E. De Flora A. Biochem. J. 2006; 393: 697-704Crossref PubMed Scopus (57) Google Scholar). Control and hP2Y11-transfected 1321N1 cells were seeded in 96-well plates (2 × 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 × 106/ml) were incubated for 0, 15, and 60 min at 25 °C in the absence (control) or in the presence of 1 mm α-NAD+. At each time point, a 500-μl aliquot of the cell suspension was withdrawn and centrifuged at 5,000 × 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 (20Graeff R. Lee H.C. Biochem. J. 2002; 361: 379-384Crossref PubMed Scopus (153) Google Scholar). cADPR levels were expressed as picomoles/106 cells. Control and hP2Y11-transfected 1321N1 cells were seeded in 35 × 10-mm dishes (2.4 × 105 cells/dish). After 48 h, the medium was removed and HBSS was added (0.6 ml). ATP or α-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 × 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 α- or β-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 (20Graeff R. Lee H.C. Biochem. J. 2002; 361: 379-384Crossref PubMed Scopus (153) Google Scholar). 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 × 10 mm dishes (5 × 105 cells/dish). After 24 h, the medium was removed and HBSS was added (0.6 ml). Cells were then challenged with ATP or α-or β-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 × 106/ml) and challenged with 1 mm β-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 (20Graeff R. Lee H.C. Biochem. J. 2002; 361: 379-384Crossref PubMed Scopus (153) Google Scholar), 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 × 10-mm dishes (5 × 105 cells/dish). After 24 h, the medium was removed and HBSS was added (0.6 ml). Cells were challenged with ATP or α-or β-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 × 106/ml) were incubated at 25 °C in the absence (control) or in the presence of 0.1 mm α-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 β-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 (20Graeff R. Lee H.C. Biochem. J. 2002; 361: 379-384Crossref PubMed Scopus (153) Google Scholar). The protein content in each sample was determined by a Bradford assay (21Bradford M. Anal. Biochem. 1976; 72: 248-252Crossref PubMed Scopus (216178) Google Scholar). 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 × 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 × 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 30 of primers mixture μm each of and and μl of (Bio-Rad). The program of each to °C and to 60 respectively. were at the of each the of the a was after each were to of the real-time PCR was obtained using the iQ5 System on the which in expression of the to and to a cells to in the absence of siRNA). of and were determined by are as hours after transfection, a 300-μl aliquot of granulocytes from each was centrifuged at × g for 10 s, and cells were resuspended at × in chemotaxis (HBSS, and 5% were performed using 96-well system with a pore α-or β-NAD+ (10 were in chemotaxis and added in the Granulocytes μl) were on of the and incubated for 60 min at 37 the cells were evaluated as previously described (12Bruzzone S. Moreschi I. Guida L. Usai C. Zocchi E. De Flora A. Biochem. J. 2006; 393: 697-704Crossref PubMed Scopus (57) Google Scholar). The results were expressed as chemotaxis of cells toward of cells toward were by were NADe+ and Ca2+ in Human of freshly isolated granulocytes with extracellular β-NAD+ concentrations from 1 to μm in a sustained elevation of [Ca2+]i (12Bruzzone S. Moreschi I. Guida L. Usai C. Zocchi E. De Flora A. Biochem. J. 2006; 393: 697-704Crossref PubMed Scopus (57) Google Scholar). in 1 and a different Ca2+ response was observed granulocytes with 1 mm Under these conditions, an and elevation of the [Ca2+]i was followed by a sustained increase after 15 min from the addition of the 1 mm β-NADe+ was added to granulocytes in the presence of mm extracellular in Ca2+-free HBSS, the and the of the [Ca2+]i increase were to in a medium that the increase in Ca2+ levels from release from intracellular and not from extracellular the sustained Ca2+ elevation triggered by millimolar β-NADe+ was in the presence of that influx of extracellular Ca2+ is responsible for the sustained Ca2+ elevation at times to was observed with micromolar β-NADe+ concentrations (12Bruzzone S. Moreschi I. Guida L. Usai C. Zocchi E. De Flora A. Biochem. J. 2006; 393: 697-704Crossref PubMed Scopus (57) Google Scholar). 1 mm the same two-step of Ca2+ response as β-NADe+ Moreover, were observed in the Ca2+ by or of IP3 and cADPR in and Ca2+ of Granulocytes to of granulocytes with the phospholipase C inhibitor 5 the [Ca2+]i elevation promoted by β-NADe+ or the same of the to be the sustained increase in [Ca2+]i observed with α- and β-NADe+ was not by of the granulocytes with or These demonstrate that α- and β-NADe+ at 1 mm a rapid Ca2+ release from intracellular the role of cADPR in the Ca2+ response by millimolar as was previously demonstrated with micromolar β-NADe+ concentrations (12Bruzzone S. Moreschi I. Guida L. Usai C. Zocchi E. De Flora A. Biochem. J. 2006; 393: 697-704Crossref PubMed Scopus (57) Google Scholar), granulocytes were to β-NADe+ following with (100 μm), a cADPR T.F. Lee H.C. Biophys. PubMed Scopus Google Scholar), or at a that Ca2+ release from H.C. and and Google Scholar). neither the [Ca2+]i both and by the sustained [Ca2+]i elevation and These results demonstrate that the sustained Ca2+ increase triggered by 1 mm NADe+ is mediated by cADPR, to was observed at micromolar Ca2+ increase is abrogated in the presence of extracellular Intracellular IP3, cADPR, and cAMP in NADe+-stimulated of granulocytes to β-NADe+ promoted a rapid increase in IP3 Following a incubation with the intracellular IP3 levels increased by from a of to cells Micromolar concentrations of β-NADe+ not in IP3 levels not These demonstrate the of IP3 generation in the [Ca2+]i increase in response to millimolar β-NADe+ in cADPR levels promoted by millimolar NADe+, we used as an agonist (12Bruzzone S. Moreschi I. Guida L. Usai C. Zocchi E. De Flora A. Biochem. J. 2006; 393: 697-704Crossref PubMed Scopus (57) Google Scholar), which to be as as β-NADe+ in a leading to stimulation of intracellular cADPR synthesis from (12Bruzzone S. Moreschi I. Guida L. Usai C. Zocchi E. De Flora A. Biochem. J. 2006; 393: 697-704Crossref PubMed Scopus (57) Google Scholar). was used to interference of the of intracellular cADPR levels by extracellular cADPR from β-NADe+ (12Bruzzone S. Moreschi I. Guida L. Usai C. Zocchi E. De Flora A. Biochem. J. 2006; 393: 697-704Crossref PubMed Scopus (57) Google Scholar). After a exposure of granulocytes to 1 mm cADPR levels increased to 300 levels