Second Messenger Function of Nicotinic Acid Adenine Dinucleotide Phosphate Revealed by an Improved Enzymatic Cycling Assay
2006; Elsevier BV; Volume: 281; Issue: 25 Linguagem: Inglês
10.1074/jbc.m601347200
ISSN1083-351X
AutoresAndreas Gasser, Sören Bruhn, Andreas H. Guse,
Tópico(s)Piperaceae Chemical and Biological Studies
ResumoNicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent activator of Ca2+ release from intracellular stores known today. Although recent reports have suggested an important function of NAADP in human T lymphocytes, direct evidence for receptor-induced formation of NAADP is yet missing in these cells. Thus, we developed a highly sensitive and specific enzyme assay capable of quantifying low fmol amounts of NAADP. In unstimulated T cells, the NAADP concentration amounted to 4.4 ± 1.6 nm (0.055 ± 0.028 pmol/mg of protein). Stimulation of the cells via the T cell receptor/CD3 complex resulted in biphasic elevation kinetics of cellular NAADP levels and was characterized by a bell-shaped concentration-response curve for NAADP. In contrast, the NAADP concentration was elevated neither upon activation of the ADP-ribose/TRPM2 channel Ca2+ signaling system nor by an increase of the intracellular Ca2+ concentration upon thapsigargin stimulation. T cell receptor/CD3 complex-mediated NAADP formation was dependent on the activity of tyrosine kinases because genistein completely blocked NAADP elevation. Thus, we propose a regulated formation of NAADP upon specific stimulation of the T cell receptor/CD3 complex, suggesting a function of NAADP as a Ca2+-mobilizing second messenger during T cell activation. Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent activator of Ca2+ release from intracellular stores known today. Although recent reports have suggested an important function of NAADP in human T lymphocytes, direct evidence for receptor-induced formation of NAADP is yet missing in these cells. Thus, we developed a highly sensitive and specific enzyme assay capable of quantifying low fmol amounts of NAADP. In unstimulated T cells, the NAADP concentration amounted to 4.4 ± 1.6 nm (0.055 ± 0.028 pmol/mg of protein). Stimulation of the cells via the T cell receptor/CD3 complex resulted in biphasic elevation kinetics of cellular NAADP levels and was characterized by a bell-shaped concentration-response curve for NAADP. In contrast, the NAADP concentration was elevated neither upon activation of the ADP-ribose/TRPM2 channel Ca2+ signaling system nor by an increase of the intracellular Ca2+ concentration upon thapsigargin stimulation. T cell receptor/CD3 complex-mediated NAADP formation was dependent on the activity of tyrosine kinases because genistein completely blocked NAADP elevation. Thus, we propose a regulated formation of NAADP upon specific stimulation of the T cell receptor/CD3 complex, suggesting a function of NAADP as a Ca2+-mobilizing second messenger during T cell activation. During a rise of the intracellular Ca2+ concentration, Ca2+ either enters the cell from the extracellular space or is released from intracellular stores. The latter process is regulated by an expanding group of intracellular messengers (1Lee H.C. Curr. Mol. Med. 2004; 4: 227-237Crossref PubMed Scopus (110) Google Scholar, 2Bootman M.D. Berridge M.J. Roderick H.L. Curr. Biol. 2002; 12: R563-R565Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar) including d-myo-inositol 1,4,5-trisphosphate (InsP3), 3The abbreviations used are: InsP3, d-myo-inositol 1,4,5-trisphosphate; ADPR, ADP-ribose; ADPRC, ADP-ribosyl cyclase; BSA, bovine serum albumin; cADPR, cyclic ADP-ribose; NAADP, nicotinic acid adenine dinucleotide phosphate; NADase, NAD glycohydrolase; RyR, ryanodine receptors; TCR, T cell receptor; TRPM2, melastatin-related transient receptor potential channel 2; NAAD, nicotinic acid adenine dinucleotide; HPLC, high pressure liquid chromatography. 3The abbreviations used are: InsP3, d-myo-inositol 1,4,5-trisphosphate; ADPR, ADP-ribose; ADPRC, ADP-ribosyl cyclase; BSA, bovine serum albumin; cADPR, cyclic ADP-ribose; NAADP, nicotinic acid adenine dinucleotide phosphate; NADase, NAD glycohydrolase; RyR, ryanodine receptors; TCR, T cell receptor; TRPM2, melastatin-related transient receptor potential channel 2; NAAD, nicotinic acid adenine dinucleotide; HPLC, high pressure liquid chromatography. cyclic ADP-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP). In addition, Ca2+ itself co-modulates Ca2+ release via both InsP3 receptors and ryanodine receptors (RyR). Clapper et al. (3Clapper D.L. Walseth T.F. Dargie P.J. Lee H.C. J. Biol. Chem. 1987; 262: 9561-9568Abstract Full Text PDF PubMed Google Scholar) reported a Ca2+ releasing activity of a contamination in commercially available NADP preparations. Nearly a decade later, this contamination was identified as NAADP, which turned out to be the most potent Ca2+-mobilizing compound in sea urchin egg homogenates (4Lee H.C. Aarhus R. J. Biol. Chem. 1995; 270: 2152-2157Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). NAADP is active in a variety of cells, ranging from plants to animals (for reviews, see Refs. 5Guse A.H. Curr. Mol. Med. 2002; 2: 273-282Crossref PubMed Scopus (49) Google Scholar, 6Yamasaki M. Churchill G.C. Galione A. FEBS J. 2005; 272: 4598-4606Crossref PubMed Scopus (46) Google Scholar, 7Lee H.C. J. Biol. Chem. 2005; 280: 33693-33696Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Interestingly the functional properties of NAADP-induced Ca2+ release in sea urchin egg homogenates are different from the systems activated by InsP3 or cADPR (4Lee H.C. Aarhus R. J. Biol. Chem. 1995; 270: 2152-2157Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). Moreover because extracellular stimulation induces increases in intracellular NAADP levels, a second messenger function for NAADP has been proposed in a few experimental systems (for reviews, see Refs. 8Lee H.C. Curr. Biol. 2003; 13: R186-R188Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar and 9Rutter G.A. Biochem. J. 2003; 373: e3-e4Crossref PubMed Google Scholar), including murine pancreatic beta cells (10Masgrau R. Churchill G.C. Morgan A.J. Ashcroft S.J.H. Galione A. Curr. Biol. 2003; 13: 247-251Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), murine pancreatic acinar cells (11Yamasaki M. Thomas J.M. Churchill G.C. Garnham C. Lewis A.M. Cancela J. Patel S. Galione A. Curr. Biol. 2005; 15: 874-878Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), and arterial smooth muscle cells (12Kinnear N.P. Boittin F. Thomas J.M. Galione A. Evans A.M. J. Biol. Chem. 2004; 279: 54319-54326Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). However, the determination of NAADP is still difficult because the data published so far were obtained by a radioligand binding assay requiring sea urchin egg homogenates as well as 32P-labeled NAADP (10Masgrau R. Churchill G.C. Morgan A.J. Ashcroft S.J.H. Galione A. Curr. Biol. 2003; 13: 247-251Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 11Yamasaki M. Thomas J.M. Churchill G.C. Garnham C. Lewis A.M. Cancela J. Patel S. Galione A. Curr. Biol. 2005; 15: 874-878Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 12Kinnear N.P. Boittin F. Thomas J.M. Galione A. Evans A.M. J. Biol. Chem. 2004; 279: 54319-54326Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 13Billington R.A. Ho A. Genazzani A.A. J. Physiol. 2002; 544: 107-112Crossref PubMed Scopus (46) Google Scholar, 14Churamani D. Carrey E.A. Dickinson G.D. Patel S. Biochem. J. 2004; 380: 449-454Crossref PubMed Scopus (35) Google Scholar). The molecular identity of the NAADP receptor is still controversial. Although a specific receptor has been proposed in sea urchin eggs (4Lee H.C. Aarhus R. J. Biol. Chem. 1995; 270: 2152-2157Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar, 15Genazzani A.A. Mezna M. Dickey D.M. Michelangeli F. Walseth T.F. Galione A. Br. J. Pharmacol. 1997; 121: 1489-1495Crossref PubMed Scopus (90) Google Scholar, 16Churchill G.C. Galione A. J. Biol. Chem. 2000; 275: 38687-38692Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), arterial smooth muscle cells (17Boittin F. Galione A. Evans A.M. Circ. Res. 2002; 91: 1168-1175Crossref PubMed Scopus (102) Google Scholar), and microsomes from brain (18Bak J. White P. Timar G. Missiaen L. Genazzani A.A. Galione A. Curr. Biol. 1999; 9: 751-754Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) or heart (19Bak J. Billington R.A. Timar G. Dutton A.C. Genazzani A.A. Curr. Biol. 2001; 11: 987-990Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), a direct activation of RyR by NAADP has been reported upon reconstitution in artificial membranes (20Hohenegger M. Suko J. Gscheidlinger R. Drobny H. Zidar A. Biochem. J. 2002; 367: 423-431Crossref PubMed Scopus (107) Google Scholar, 21Mojzisova A. Krizanova O. Zacikova L. Kominkova V. Ondrias K. Pfluegers Arch. Eur. J. Physiol. 2001; 441: 674-677Crossref PubMed Scopus (58) Google Scholar). Furthermore NAADP-induced Ca2+ release via RyR has been described for MIN6 cells (22Mitchell K.J. Lai F.A. Rutter G.A. J. Biol. Chem. 2003; 278: 11057-11064Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), pancreatic acinar cells (23Gerasimenko J.V. Maruyama Y. Yano K. Dolman N.J. Tepikin A.V. Petersen O.H. Gerasimenko O.V. J. Cell Biol. 2003; 163: 271-282Crossref PubMed Scopus (192) Google Scholar, 24Gerasimenko J.V. Sherwood M. Tepikin A.V. Petersen O.H. Gerasimenko O.V. J. Cell Sci. 2006; 119: 226-238Crossref PubMed Scopus (126) Google Scholar), and human T cells (25Langhorst M.F. Schwarzmann N. Guse A.H. Cell. Signal. 2004; 16: 1283-1289Crossref PubMed Scopus (63) Google Scholar, 26Dammermann W. Guse A.H. J. Biol. Chem. 2005; 280: 21394-21399Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Galione and co-workers (10Masgrau R. Churchill G.C. Morgan A.J. Ashcroft S.J.H. Galione A. Curr. Biol. 2003; 13: 247-251Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 12Kinnear N.P. Boittin F. Thomas J.M. Galione A. Evans A.M. J. Biol. Chem. 2004; 279: 54319-54326Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 27Churchill G.C. Okada Y. Thomas J.M. Genazzani A.A. Patel S. Galione A. Cell. 2002; 111: 703-708Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 28Yamasaki M. Masgrau R. Morgan A.J. Churchill G.C. Patel S. Ashcroft S.J.H. Galione A. J. Biol. Chem. 2004; 279: 7234-7240Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar) recently reported the release of Ca2+ by NAADP from a lysosomal compartment. Thus, both a "one pool-one receptor" mechanism (NAADP directly activates RyR) and a "two-pool" mechanism (NAADP releases Ca2+ from a lysosomal store, which than induces Ca2+ release by RyR) may be used by different cell types (for a review, see Ref. 29Galione A. Petersen O.H. Mol. Interv. 2005; 5: 73-79Crossref PubMed Scopus (88) Google Scholar). In T lymphocytes an important function of NAADP in the process of Ca2+ signaling induced by stimulation of the T cell receptor (TCR)/CD3 complex has been reported (30Berg I. Potter B.V. Mayr G.W. Guse A.H. J. Cell Biol. 2000; 150: 581-588Crossref PubMed Scopus (148) Google Scholar). Thus, an easy, sensitive, and reliable method to quantify cellular NAADP concentrations would be highly important to analyze a second messenger function in this cell type. In the present study, we describe an improved enzymatic cycling assay for the accurate determination of NAADP, allowing the quantification of low fmol amounts. All enzymes used for this assay are commercially available, and neither sea urchin egg homogenates nor radio-actively labeled NAADP are required. This method was used for the determination of cellular NAADP concentrations in T cells. Upon stimulation of the cells via the TCR/CD3 complex, we found a biphasic elevation of NAADP levels. A bell-shaped concentration-response curve was obtained upon stimulation of the TCR/CD3 complex. In contrast, no NAADP elevation was detectable upon stimulation of the ADP-ribose (ADPR)/TRPM2 Ca2+ signaling pathway (31Gasser A. Glassmeier G. Fliegert R. Langhorst M.F. Meinke S. Hein D. Kruger S. Weber K. Heiner I. Oppenheimer N. Schwarz J.R. Guse A.H. J. Biol. Chem. 2006; 281: 2489-2496Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) or upon an increase of [Ca2+]i induced by thapsigargin. Therefore, we propose a second messenger function of NAADP in T cells. Materials—Resazurin (R2127), ADP-ribosyl cyclase (A8950), diaphorase (D5540), glucose-6-phosphate dehydrogenase (G8164), NAD glycohydrolase (N9629), nucleotides, and other standard compounds were purchased from Sigma (catalog numbers are in parentheses) or from Roche Applied Science. Trichloroacetic acid, diethyl ether, and trifluoroacetic acid were obtained from Merck, and Q-Sepharose Fast Flow was from GE Healthcare. Charcoal Norit A, obtained from Serva (Heidelberg, Germany), was activated by treatment with 1 m HCl by boiling for 30 min and washed with water until the pH value was neutral. PP2 and genistein were from Calbiochem. All other chemicals were of the highest purity available. SeralPur water (Seral, Ransbach, Germany) was used during all experiments. Cell Culture—Jurkat T cells (subclone JMP) were cultured as described previously (32Guse A.H. Roth E. Emmrich F. Biochem. J. 1993; 291: 447-451Crossref PubMed Scopus (93) Google Scholar). Briefly the cells were kept in RPMI 1640 medium with 25 mm HEPES, pH 7.4, and Glutamax I supplemented with 100 units/ml penicillin, 50 μg/ml streptomycin, and 7.5% (v/v) newborn calf serum (Biochrom, Berlin, Germany) at a density of 0.3–1.0 × 106 cells/ml. Counting of the cells and determination of the cell volume were performed using a CASY TT 1 system (Schärfe System, Reutlingen, Germany). To quantify cellular protein content, the cells were lysed in 25 mm HEPES, pH 7.2, in the presence of Complete protease inhibitor mixture (Roche Applied Science) by incubation for 10 min on ice followed by ultrasonic disruption for 30 s. Thereafter, the protein concentration was determined with Bradford reagent (Bio-Rad) using BSA as standard. Extraction of Cellular NAADP—1.6 × 108 Jurkat cells were harvested by centrifugation (500 × g for 5 min at room temperature), resuspended in 16 ml of buffer containing 140 mm NaCl, 5 mm KCl, 1 mm MgSO4, 1 mm CaCl2, 1 mm NaH2PO4, 5.5 mm glucose, and 20 mm HEPES, pH 7.4, and kept at room temperature for 40 min. After this incubation, the cell suspension was divided into two identical halves ("twin samples"), and the volume was adjusted to 10 ml with the same buffer as above. The cells in both parts of the twin samples were then stimulated for different periods of time with either different concentrations of OKT3, a vehicle control, 100 μg/ml concanavalin A, or 100 nm thapsigargin. In some experiments, 1 μm PP2 was preincubated with cells for 5 min, or 80 μg/ml genistein was preincubated with cells for 10 min; then the cells were stimulated for 5 min with 5 μg/ml OKT3. After rapid centrifugation (960 × g for 1 min at 10°C), the cell pellets were resuspended in 1 ml of ice-cold trichloroacetic acid solution (20%, w/v) and lysed using a Potter-Elvehjem homogenizer (five strokes at 1500 rpm). Thereafter the samples were frozen in liquid nitrogen and thawed at 37°C two times, cell debris and precipitated proteins were removed by centrifugation (4400 × g for 10 min), and the supernatants were collected. One of each of a pair of twin samples was spiked by addition of 15 pmol of NAADP to calculate the recovery of the extraction procedure. Then the samples were repeatedly extracted with 5 volumes of water-saturated diethyl ether until the pH value was nearly neutral and freeze-dried for 60 min to remove traces of ether. In some experiments artificial samples were used consisting of 0.5 ml of BSA solution (5 mg/ml in water) and 15 pmol of NAADP and/or 375 pmol of purified NADP (see below). These samples were processed in the same way as described for the cell extract samples. Purification of Cell Extracts by Gravity-fed Anion-exchange Chromatography—For the anion-exchange chromatography of neutralized trichloroacetic acid extracts, plastic filtration tubes (Supelco, Bellefonte, PA) were packed with 500 μl of Q-Sepharose Fast Flow, cleaned in place with 5 ml of 150 mm trifluoroacetic acid, and equilibrated with 15 ml of 10 mm Tris/HCl, pH 8.0. The cell extracts were diluted with 10 ml of 10 mm Tris/HCl, pH 8.0, and applied to the columns in aliquots of 1 ml. The columns were then washed with 10 ml of 10 mm Tris/HCl, pH 8.0. For the elution of NAADP, trifluoroacetic acid solutions of increasing concentrations (each 1.5 ml of 10, 12.5, 15, 17.5, 20, and 25 mm) were used. The eluate fractions were rapidly diluted with 3.5 ml of ice-cold water and freeze-dried. These samples were stored at –80°C and analyzed as soon as possible. To determine the elution profile of NAADP, artificial samples consisting of BSA with or without 15 pmol of NAADP were extracted as detailed above. In this case, all eluate fractions were separately cleaved with NAD glycohydrolase (NADase) and then analyzed by the cycling assay (see below). As standard procedure the 17.5–25 mm trifluoroacetic acid fractions containing the NAADP were pooled and further processed. Removal of Interfering Compounds by Cleavage with NAD Glycohydrolase—NAADP standards or other compounds were dissolved in 1.5 ml of buffer containing 1 mm Tris/HCl and 2.5 mm MgCl2, pH 7.3. For the determination of cellular NAADP amounts, the 17.5–25 mm pool of trifluoroacetic acid fractions was used. This sample was reconstituted in 1.5 ml of the same buffer as above. To remove interfering nucleotides, 0.1 unit of the purified NADase was added to the samples. After incubation for 4 h at 37°C, the enzyme was inactivated for 20 min at 95°C, and the samples were cooled on ice and then directly used for the cycling assay. Purification of NADP—NADP was purified by gravity-fed anion-exchange chromatography because commercially available NADP preparations always contain a contamination of NAADP. About 10 μmol of NADP (Roche Applied Science) were dissolved in 20 ml of 1 mm Tris/HCl, pH 8.0, and applied to a column of 1 ml of Q-Sepharose Fast Flow that was prepared as detailed above. NADP was then eluted using increasing concentrations of trifluoroacetic acid (4 ml of each 2, 4, 6, 8, 10, 12, and 15 mm). The NADP concentration in each fraction was determined by measuring the UV absorption at 260 nm (and initially also by HPLC). After freeze-drying, the purified NADP, which eluted mainly in the 10 mm trifluoroacetic acid fraction, was dissolved at a concentration of 10 mm, and the purity was additionally checked by HPLC. Purification of Enzymes—To purify commercially available NADase, 2.9 mg (1.6 units) of the lyophilized enzyme was dissolved in 320 μl of buffer containing 1 mm Tris/HCl and 2.5 mm MgCl2, pH 7.0; mixed with 320 μl of BSA solution (5 mg/ml in water) and 640 μl of a suspension (2%, w/v) of activated charcoal in 150 mm NaCl and 20 mm NaH2PO4, pH 8.0 adjusted with NaOH; and incubated for 30 min at 37°C with vigorous shaking. The charcoal was removed by centrifugation at 11,000 × g for 10 min at 4°C, and the supernatant was transferred to a Centricon YM-10 device (cutoff, 10 kDa; Millipore). The enzyme solution was concentrated (4400 × g for 20 min at 4°C) to a volume of 150 μl and stored at –20°C. Glucose-6-phosphate dehydrogenase and diaphorase were purified as follows. 100 μl of each enzyme solution (1 mg/ml glucose-6-phosphate dehydrogenase and 12 mg/ml diaphorase, both in 50 mm NaH2PO4, pH 8.0 adjusted with NaOH) were mixed with 200 μl of BSA solution (5 mg/ml in water) and 1.2 ml of a suspension (2%, w/v) of activated charcoal in 50 mm NaCl and 20 mm NaH2PO4, pH 8.0 adjusted with NaOH. After incubation at 37°C for 30 min, the charcoal was removed by centrifugation (11,000 × g for 10 min at 4°C), and the supernatant was used directly for the cycling assay. Cycling Assay for NAADP—In the first reaction of the enzymatic cycling assay, NAADP was converted into NADP (see Fig. 1A). Because all measurements were done in triplicate, six wells of a microplate were filled with each 200 μl of the samples, which had been incubated with the NADase before, or with standard NAADP in the same buffer. In some experiments, standard NAADP was incubated with 10 units/ml alkaline phosphatase in the same buffer at 37°C overnight, and the enzyme was subsequently removed using a Centricon YM-10 device (4400 × g for 20 min at 4°C). To three of the wells, 11 μl of a solution containing 0.55 m nicotinamide, 36 mm sodium acetate, pH 4.2, and 9.1 μg/ml ADP-ribosyl cyclase (ADPRC) were added. 11 μl of the same solution containing BSA instead of the enzyme were added to each of the three remaining wells. The microplate was sealed with Parafilm and incubated for 60 min in the dark at room temperature. Then the amplification and indicator reaction (see Fig. 1A) were started by addition of 59 μl of cycling mixture to each well. The cycling mixture contained (all solutions in water except the purified enzymes) 1 μl of 1 m nicotinamide, 20 μl of 500 mm NaH2PO4, pH 8.0 adjusted with NaOH, 1 μl of 1 mm flavin mononucleotide, 2 μl of 5 mg/ml BSA, 0.1 μl of 10 mm resazurin, 5 μl of 100 mm glucose 6-phosphate, and 30 μl of the purified enzymes (see above). Directly after addition of the cycling mixture, the fluorescence (excitation, 544 nm; emission, 590 nm) was measured for each well by a microplate reader (Victor 4120, Wallac, Freiburg, Germany). The microplate was then sealed with Parafilm and incubated for 12 h in the dark at room temperature. The fluorescence was measured again, and the differences between end and start of the reaction were determined for each well. To correct the obtained values for the background due to the presence of NADP or unspecific oxidation of resazurin, each rise in fluorescence in the presence of ADPRC was corrected for the fluorescence increase in the absence of the enzyme (see Fig. 1B). The NAADP content in the samples that were purified by anion-exchange chromatography was obtained by addition of the pooled 17.5–25 mm trifluoroacetic acid fraction. For each pair of twin samples, the recovery was determined by calculating the difference between the halves of the sample with and without spike. Finally the NAADP amount in the twin sample without exogenous NAADP was corrected for the respective recovery. Measurement of [Ca2+]i—Loading of Jurkat T cells with Fura2/AM (Molecular Probes) and determination of [Ca2+]i was performed as described previously (32Guse A.H. Roth E. Emmrich F. Biochem. J. 1993; 291: 447-451Crossref PubMed Scopus (93) Google Scholar, 33Schwarzmann N. Kunerth S. Weber K. Mayr G.W. Guse A.H. J. Biol. Chem. 2002; 277: 50636-50642Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The cells were stimulated with different concentrations of OKT3 or a vehicle control. For the quantification of the increase in [Ca2+]i (see Fig. 3), both the peak and the plateau (500 s after stimulation) were corrected for the basal Ca2+ concentration. Statistical Analysis—For determination of statistical significance, data were tested using a one-way analysis of variance in combination with a Student-Newman-Keuls test. Statistical significance was considered for p < 0.05. The principle of the cycling assay for NAADP is depicted in Fig. 1A. The assay consists of three enzymatic reactions. In the first reaction (Fig. 1A, upper panel), NAADP is converted into NADP using the ADPRC from Aplysia californica. This reaction was conducted at an acidic pH of 4.2 and in the presence of a high excess of nicotinamide, thus reversing the enzymatic formation of NAADP from NADP by ADPRCs (34Aarhus R. Graeff R.M. Dickey D.M. Walseth T.F. Lee H.C. J. Biol. Chem. 1995; 270: 30327-30333Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). The NADP produced by the ADPRC is then cycled by two enzymatic reactions (Fig. 1A, lower right panel, amplification). In the first reaction, glucose-6-phosphate dehydrogenase from Saccharomyces cerevisiae reduces NADP to NADPH, which is then reoxidized to NADP by diaphorase from Clostridium kluyveri. Simultaneously to the reoxidation of NADPH, diaphorase catalyzes the conversion of resazurin into the highly fluorescent resorufin (Fig. 1A, lower left panel, indicator reaction). The major advantage of this kind of cyclic enzyme assay is the strong amplification of the initial signal because the presence of one NADP molecule results in the formation of many molecules of resorufin, thus allowing the detection of minute amounts of NAADP. The first step to establish the assay described above was to adopt the cycling reaction for NADP from the literature (35Stephon R.L. Niedbala R.S. Schray K.J. Heindel N.D. Anal. Biochem. 1992; 202: 6-9Crossref PubMed Scopus (7) Google Scholar, 36Wang W. Inoue N. Nakayama T. Ishii M. Kato T. Anal. Biochem. 1995; 227: 274-280Crossref PubMed Scopus (31) Google Scholar) and to optimize it. For this purpose, the cycling reaction was performed in the presence of NADP without prior conversion of NAADP into NADP (data not shown). A very crucial point was the quality of the enzymes used in the cycling reaction: because the enzymes commercially available were not sufficiently pure (which resulted in a high fluorescence background even in the absence of any substrate in the cycling reaction), both enzymes were purified using activated charcoal in the presence of BSA (see "Experimental Procedures"). After this purification, only a very small increase in the resorufin fluorescence was detected in the absence of NADP (data not shown). The complete conversion of NAADP to NADP by the ADPRC is essential to establish an assay that indirectly quantifies NAADP after conversion into NADP. The ADPRC from A. californica catalyzes the synthesis of NAADP from NADP at an acidic pH and in the presence of high concentrations of nicotinic acid (34Aarhus R. Graeff R.M. Dickey D.M. Walseth T.F. Lee H.C. J. Biol. Chem. 1995; 270: 30327-30333Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). Different conditions to reverse this reaction were tested and analyzed by HPLC separation of the reaction products (data not shown). At an acidic pH value, the ADPRC converted NAADP into NADP at room temperature, whereas no reaction was observed at 10°C. Besides NADP, which was the main product of the reaction, also small amounts of 2′-phospho-ADP-ribose were generated. Next the formation of NADP from NAADP was coupled to the enzymatic cycling reaction. The reaction conditions of the NAADP formation from NADP were extensively optimized, i.e. different buffers, pH values, concentrations of nicotinamide, amounts of ADPRC, temperatures, and incubation times were tested. The optimal condition to convert NAADP into NADP was an incubation for 60 min at 23°C in the presence of 29 mm nicotinamide, 1.88 mm sodium acetate, pH 4.2, and 0.474 μg/ml ADPRC. After this optimization was completed, a calibration of the cycling assay was performed using different amounts of NAADP standards in the range from 25 to 1000 fmol (data not shown). A linear correlation between NAADP amount and fluorescence increase after 12 h was found (r2 = 0.9996), and the detection limit was 25 fmol at a signal/noise ratio of >3. Another important point to consider is the presence of NADP in cell extracts because NADP would also be detected by the cycling reaction, resulting in a false positive signal. To remove endogenous NADP, both gravity-fed anion-exchange chromatography and an enzymatic digest using an NADase from Neurospora crassa were used. A large portion of NADP was eluted at 10 mm trifluoroacetic acid, whereas NAADP eluted in fractions 17.5–25 mm (Fig. 1E). The remaining contamination of NADP was removed by NADase digest. This enzyme hydrolyzes NAD to ADPR and nicotinamide but is also capable of cleaving NADP into 2′-phospho-ADP-ribose and nicotinamide. In contrast, NAADP is resistant to NADase treatment (37Graeff R. Lee H.C. Biochem. J. 2002; 367: 163-168Crossref PubMed Google Scholar). Because it was not known whether the enzymatic removal of NADP using the NADase would be complete, we used the following procedure to correct for a possible background of NADP. After the cleavage using the NADase, all samples were incubated either with or without ADPRC in the first reaction of the assay. Then the enzymatic cycling was performed with both types of samples, and the fluorescence increase was quantified after 12 h. Because NAADP cannot be converted into NADP in the absence of the ADPRC, the NADP background was quantified by the reactions without ADPRC. Thus, the fluorescence increase in the presence of ADPRC was corrected for the increase in the absence of the enzyme for each sample. The use of the NADase resulted in a decrease in the sensitivity of the assay. This may be due to a co-precipitation of NAADP with the denatured enzyme during the heat inactivation of the NADase. However, the correlation between the amount of NAADP and the fluorescence increase was still linear as depicted in Fig. 1C. Under these conditions, the detection limit for NAADP was 50 fmol at a signal/noise ratio of >3. Obviously the correction of the fluorescence difference in the presence of ADPRC (Fig. 1B, black bars) for the fluorescence increase in the absence of the enzyme (Fig. 1B, light gray bars) resulted in a complete correction of the background (see blank value in Fig. 1B). Possibly this background in the absence of NADP was due to partial spontaneous oxidation of resazurin. To investigate the specificity of the assay, NAADP was incubated with alkaline phosphatase, leading to the degradation of NAADP to NAAD (Fig. 1D) (37Graeff R. Lee H.C. Biochem. J. 2002; 367: 163-168Crossref PubMed Google Scholar). In this case, no fluorescence inc
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