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© The Rockefeller University Press, 0021-9525/2000//581 $5.00
The Journal of Cell Biology, Volume 150, Number 3, , 2000 581-588

Nicotinic Acid Adenine Dinucleotide Phosphate (Naadp⁺) Is an Essential Regulator of T-Lymphocyte Ca²⁺-Signaling

^a Institute of Medical Biochemistry and Molecular Biology, Division of Cellular Signal Transduction, University of Hamburg, D-20146 Hamburg, Germany
^b Wolfson Laboratory for Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom
University of Hamburg, Institute for Medical Biochemistry and Molecular Biology, Div. Cellular Signal Transduction, Grindelallee 117, IV, D-20146 Hamburg, Germany.49-40-42838-427549-40-42838-4276

guse{at}uke.uni-hamburg.de

Microinjection of human Jurkat T-lymphocytes with nicotinic acid adenine dinucleotide phosphate (NAADP⁺) dose-dependently stimulated intracellular Ca²⁺-signaling. At a concentration of 10 nM NAADP⁺ evoked repetitive and long-lasting Ca²⁺-oscillations of low amplitude, whereas at 50 and 100 nM, a rapid and high initial Ca²⁺-peak followed by trains of smaller Ca²⁺-oscillations was observed. Higher concentrations of NAADP⁺ (1 and 10 µM) gradually reduced the initial Ca²⁺-peak, and a complete self-inactivation of Ca²⁺-signals was seen at 100 µM. The effect of NAADP⁺ was specific as it was not observed with nicotinamide adenine dinucleotide phosphate. Both inositol 1,4,5-trisphosphate– and cyclic adenosine diphosphoribose–mediated Ca²⁺-signaling were efficiently inhibited by coinjection of a self-inactivating concentration of NAADP⁺. Most importantly, microinjection of a self-inactivating concentration of NAADP⁺ completely abolished subsequent stimulation of Ca²⁺-signaling via the T cell receptor/CD3 complex, indicating that a functional NAADP⁺ Ca²⁺-release system is essential for T-lymphocyte Ca²⁺-signaling.

Key Words: cyclic ADP-ribose • inositol 1,4,5-trisphosphate • T cell activation • signal transduction • ryanodine receptor

© 2000 The Rockefeller University Press

	Introduction

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Activation of T-lymphocytes via the T cell receptor/CD3 (TCR/CD3) complex results in multiple intracellular signaling pathways (Kennedy et al. 1999). Among these pathways, an elevation of [Ca²⁺]_i (intracellular Ca²⁺-concentration) is essential for proliferation and clonal expansion (reviewed in Guse 1998). The increase of [Ca²⁺]_i in T cells consists of calcium release from intracellular stores, and, as a major source for the long-lasting Ca²⁺-signal observed in T cells, subsequent entry of calcium through specific calcium channels in the plasma membrane (reviewed in Guse 1998). Ca²⁺-release is activated by the calcium mobilizing second messengers D-myo-inositol 1,4,5-trisphosphate(Ins(1,4,5)P₃) and cyclic ADP-ribose (cADPR). Recent work indicates that Ins(1,4,5)P₃ primarily acts during the initial phase of Ca²⁺-signaling in T cells, whereas cADPR is essentially involved in the sustained phase of Ca²⁺-signaling (Guse et al. 1999).

Besides Ins(1,4,5)P₃ and cADPR, another Ca²⁺-mobilizing natural compound, nicotinic acid adenine dinucleotide phosphate (NAADP⁺) was introduced (Chini et al. 1995; Lee and Aarhus 1995). NAADP⁺ was originally discovered as a contaminant of commercial nicotinamide adenine dinucleotide phosphate (NADP⁺) preparations; such preparations could also be enriched in NAADP⁺ content by alkaline treatment (Clapper et al. 1987). Very low concentrations of NAADP⁺ in the range of 10–50 nM were shown to effectively release Ca²⁺ from intracellular stores of selected invertebrate and mammalian cell types, such as sea urchin eggs (Lee and Aarhus 1995), ascidian oocytes (Albrieux et al. 1998), and mouse pancreatic acinar cells (Cancela et al. 1999). NAADP⁺-mediated Ca²⁺-release was not sensitive to the cADPR antagonist, 8-NH₂-cADPR; the Ins(1,4,5)P₃ antagonist, heparin (Lee and Aarhus 1995); or to the antagonists of ryanodine receptors (RyR), procaine or ruthenium red (Chini et al. 1995). Together, with the lack of cross-desensitization observed between the NAADP⁺/Ca²⁺-release system on one hand, and the cADPR/- or the Ins(1,4,5)P₃/Ca²⁺-release systems on the other hand (Chini et al. 1995; Lee and Aarhus 1995), these data indicate that the NAADP⁺-dependent Ca²⁺-release system is different from the two others. Although the receptor for NAADP⁺ has not yet been identified, unspecific effects of NAADP⁺ are largely unlikely since concrete structural requirements for NAADP⁺-mediated Ca²⁺-release were demonstrated in sea urchin eggs, e.g., the NH₂-group at position 6 of the adenine ring or the phosphate group at the 2'-position of the ribose are necessary (Lee and Aarhus 1997). The latter can be replaced by a 3'-phosphate or a 2',3'-cyclic phosphate, but this alteration resulted in a weaker Ca²⁺-release activity (Lee and Aarhus 1997). Further characteristic properties of NAADP⁺-mediated Ca²⁺-release in sea urchin eggs are a unique self inactivation or self desensitization process (Aarhus et al. 1996; Genazzani et al. 1996), and Ca²⁺-release from a thapsigargin-insensitive Ca²⁺-pool (Genazzani and Galione 1996).

The fact that recent reports indicated a role for NAADP⁺ in Ca²⁺-signaling of mammalian cells (Bak et al. 1999; Cancela et al. 1999) prompted us to study its effects in human Jurkat T cells. We report here that NAADP⁺ specifically and dose-dependently stimulated Ca²⁺-signaling when microinjected into intact Jurkat T cells. Furthermore, we show that self inactivation of the NAADP⁺/Ca²⁺-release system almost completely inhibited Ins(1,4,5)P₃- or cADPR-mediated Ca²⁺-signaling. Most importantly, we demonstrate that self inactivation of the NAADP⁺/Ca²⁺-release also completely antagonized Ca²⁺-signaling mediated by ligation of the TCR/CD3-complex.

	Materials and Methods

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Reagents
cADPR, 8-OCH₃-cADPR, and D-myo-inositol 1,4,6-trisphosphorothioate (Ins(1,4,6)PS₃) were synthesized exactly as described (Ashamu et al. 1995; Murphy et al. 2000), purified by anion-exchange chromatography on Q-Sepharose, and used as their triethylammonium salts. Purity of ligands was assessed by ¹H and ³¹P NMR spectroscopy, mass spectroscopy, and, when appropriate, HPLC. NAADP⁺ and NADP⁺ were purchased from Sigma-Aldrich. The purity of NAADP⁺ was described by the manufacturer to be 95%; this was confirmed by reverse phase HPLC using the method of da Silva et al. 1998. Fura 2/AM was obtained from Calbiochem. Anti-CD3 mAb OKT3 was purified from hybridoma supernatant on protein G–Sepharose.

Cell Culture
Jurkat T lymphocytes (subclone JMP) were cultured in RPMI 1640 medium containing the following additions: glutamax I, Hepes (20 mM, pH 7.4), NCS (7.5%), penicillin (100 U/ml), and streptomycin (50 µg/ml; all obtained from Life Technologies). The cells were cultured at 37°C in a humidified atmosphere in the presence of 5% CO₂.

Ratiometric Ca²⁺ Imaging
Batches of 10⁷ Jurkat T cells were loaded with Fura2/AM as described (Guse et al. 1993). Fura2-loaded cells (10⁷ cells/5 ml) were kept at room temperature until use. Glass coverslips were coated first with BSA (5 mg/ml), and then with poly-L-lysine (0.1 mg/ml). Small chambers consisting of a rubber O-ring were sealed on the coverslips by silicon grease. Then, 90 µl of extracellular buffer (140 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 1 mM CaCl₂, 20 mM Hepes, 1 mM NaH₂PO₄, 5.5 mM glucose, pH 7.4) was added, followed by addition of 10 µl cell suspension. The coverslip was mounted on the stage of an inverted microscope (Axiovert 100, ZEISS). Ratiometric Ca²⁺ imaging was performed using a PhotoMed/Photon Technology (Wedel) digital imaging system built around the Axiovert 100 microscope. Illumination at 340 and 380 nm was carried out using a chopper/optical filter system. Images were captured by an intensified CCD camera (type C2400-77; spatial resolution: 525 x 487 pixel; Hamamatsu) and stored as individual 340 and 380 images on hard disk. Sampling rate was usually 5 s for a pair of images (340 and 380 nm) using 100-fold magnification. Data analysis was performed off-line using PhotoMed/Photon Technology (Wedel) Image master analysis software. Ratio images (340/380) were constructed pixel by pixel, and changes in the ratio over time were measured by applying regions-of-interest on individual cells. Finally, ratio values were converted to Ca²⁺-concentrations by external calibration.

Microinjection Experiments
Parallel Ca²⁺ imaging and microinjection experiments require a firm attachment of the Jurkat T cells without preactivation of intracellular Ca²⁺-signaling. This was achieved by the above mentioned coating procedure of the glass coverslips, as detailed earlier (Guse et al. 1997). The cells were kept in a small chamber (100 µl vol) in extracellular buffer (140 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 1 mM CaCl₂, 20 mM Hepes, 1 mM NaH₂PO₄, 5.5 mM glucose, pH 7.4). Compounds to be microinjected were cleared from particles by either filtration through 0.45-µm filters, by centrifugation in an Eppendorf centrifuge at maximal speed for 10 min, or by both. Femtotips II (Eppendorf) were filled with 5 µl of reagent solution and inserted into the semiautomatic microinjection system (Transjector 5246, Micromanipulator 5171, Eppendorf). Injection parameters were: injection pressure, 80 hPa; compensatory pressure, 40 hPa; duration of injection. 0.5 s; velocity of pipette, 700 µm/s; pipette angle, 45°. Injections were performed into the upper part of the cell.

	Results

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Microinjection of NAADP⁺ at a pipette concentration as low as 10 nM stimulated repetitive, long-lasting Ca²⁺-spiking of low amplitude in intact Jurkat T cells, whereas injection of intracellular buffer alone had no effect (Fig. 1A, Fig. B, Fig. E, and Fig. F). Microinjection of 0.1 or 1 nM NAADP⁺ was without effect in most of the cells (Fig. 1C and Fig. D, and data not shown). At a pipette concentration of 50 nM NAADP⁺, an initial, rapidly occurring Ca²⁺-peak with a high amplitude was observed which turned into gradually lowering oscillations during the first 350–400 s. After this time period, the calcium response changed into a low, but sustained, plateau phase with very small oscillations (Fig. 1G and Fig. H). At pipette concentrations of 100 nM and 1 µM, similar responses were observed (Fig. 1, I–L). However, the peak amplitude of the initial Ca²⁺-spike declined with increasing NAADP⁺ concentrations (Fig. 1J and Fig. L), and the decay of the Ca²⁺-signal was accelerated (Fig. 1 H, J, and L). At 10 µM NAADP⁺, the Ca²⁺-response appeared similar to the one at 1 nM (Fig. 1M and Fig. N), whereas at 100 µM NAADP⁺, no signal was detectable (Fig. 1O). The dose response relationship shows a bell-shaped curve for the initial Ca²⁺-peak with an optimal NAADP⁺ concentration at 100 nM (Fig. 2 A). However, only minor changes of the long-lasting Ca²⁺-signal as measured at 400 s were observed in response to 100 nM NAADP⁺ (Fig. 2 B). These data indicate that, similar to the few other cellular systems investigated so far (Chini et al. 1995; Lee and Aarhus 1995; Albrieux et al. 1998; Cancela et al. 1999), NAADP⁺ at low nanomolar concentrations activates Ca²⁺-signaling in T cells, whereas micromolar concentrations of NAADP⁺ rapidly cause self inactivation of the Ca²⁺-release system.

View larger version (42K): [in this window] [in a new window] [Download PPT slide]   Figure 1 Concentration-response curves of Ca2+ signals in single intact T-lymphocytes microinjected with NAADP+. Jurkat T-lymphocytes were loaded with Fura2/AM and Ca2+ was measured as detailed in Materials and Methods. Cells were injected as described in Materials and Methods in the presence of 1 mM extracellular Ca2+. Data are presented as overlays of single tracings of individual cells (left). The right shows the corresponding averages from these measurements (number of cells displayed, n = 5–17). As a control, vehicle buffer without NAADP+ was injected (A and B). The time points of microinjection are indicated by arrows.

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   Figure 2 Dose-response curve for NAADP+ in Jurkat T cells. Data from Fig. 1 are shown as mean values (n = 5–17) from time point 80 s (Ca2+-peak; A) or 400 s (Ca2+-plateau; B).

To prove the specificity of the effect of NAADP⁺ on intracellular Ca²⁺-signaling in T cells, NADP⁺ was used in parallel microinjection experiments. NADP⁺ is a structurally similar molecule, bearing a nicotinamide group instead of the nicotinic acid group. In contrast to NAADP⁺, microinjection of NADP⁺ (50 nM) was completely without effect on Ca²⁺-signaling (Fig. 3A and Fig. B).

View larger version (14K): [in this window] [in a new window] [Download PPT slide]   Figure 3 NADP+ does not mediate Ca2+-signaling. Jurkat T-lymphocytes were loaded with Fura2/AM and ratiometric Ca2+ imaging, and parallel microinjection in the presence of 1 mM extracellular Ca2+ was carried out as detailed under Materials and Methods. Cells were injected with 50 nM NAADP+ (A) or NADP+ (B). Shown are the averages from 13 (A) and 5 (B) cells. The time points of microinjection are indicated by arrows.

The specific cADPR antagonist 8-OCH₃-cADPR (Guse et al. 1999), when coinjected with an optimal NAADP⁺ concentration, did not significantly affect NAADP⁺-mediated Ca²⁺-signaling (Fig. 4A and Fig. F vs. B and G). However, when a self-desensitizing concentration of NAADP⁺ (10 µM) was coinjected with a stimulating concentration of cADPR (10 µM), a massive decrease of the cADPR-mediated Ca²⁺-signal was observed (Fig. 4C and Fig. H vs. E and J). On the other hand, an optimal stimulating concentration of NAADP⁺ (50 nM) microinjected together with cADPR (10 µM) did not significantly change the Ca²⁺-signals (Fig. 4D and Fig. I vs. E and J). These data indicate that a functional, nondesensitized NAADP⁺/Ca²⁺-release system is necessary for cADPR-mediated Ca²⁺-release.

View larger version (45K): [in this window] [in a new window] [Download PPT slide]   Figure 4 Influence of cADPR and its antagonist 8-OCH3-cADPR on NAADP+-mediated Ca2+-signaling. Jurkat T-lymphocytes were loaded with Fura2/AM, and ratiometric Ca2+ imaging and parallel microinjection in the presence of 1 mM extracellular Ca2+ was carried out as detailed under Materials and Methods. Left, Overlays of single tracings of individual cells after injection (A–E); right, demonstrates the corresponding averages from these overlays (F–J). Shown are (n = number of experiments): A/F, coinjection of NAADP (50 nM) and 8-OCH3-cADPR (100 µM; n = 7); B/G, injection of NAADP+ (50 nM, n = 10); C/H, coinjection of NAADP+ (10 µM) and cADPR (10µM; n = 7); D/I, coinjection of NAADP+ (50 nM) and cADPR (10 µM; n = 5); and E/J, injection of cADPR (10 µM, n = 5).

View larger version (44K): [in this window] [in a new window] [Download PPT slide]   Figure 5 Influence of Ins(1,4,5)P3 and its antagonist Ins(1,4,6)PS3 on NAADP+-mediated Ca2+-signaling. Jurkat T-lymphocytes were loaded with Fura2/AM, and ratiometric Ca2+ imaging and parallel microinjection in the presence of 1 mM extracellular Ca2+ was carried out as detailed under Materials and Methods. Left, Overlays of single tracings of individual cells after injection (A–E); right, demonstrates the corresponding averages from these overlays (F–J). Shown are: A/F, coinjection of NAADP+ (50 nM) and Ins(1,4,6)PS3 (40 µM; n = 7); B/G, injection of NAADP+ (50 nM; n = 10); C/H, coinjection of NAADP+ (10 µM) and Ins(1,4,5)P3 (4 µM; n = 9); D/I, coinjection of NAADP+ (50 nM) and Ins(1,4,5)P3 (4 µM; n = 3); and E/J injection of Ins(1,4,5)P3 (4 µM; n = 8).

View larger version (32K): [in this window] [in a new window] [Download PPT slide]   Figure 6 Effect of NAADP+ on OKT3-induced Ca2+-signaling in single Jurkat T-lymphocytes. Jurkat T-lymphocytes were loaded with Fura2/AM, and ratiometric Ca2+ imaging and parallel microinjection in the presence of 1 mM extracellular Ca2+ was carried out as detailed under Materials and Methods. The cells were injected with different concentrations of NAADP+ and then OKT3 (10 µg/ml) was added. Injection of intracellular buffer (A), NAADP+ (50 nM [B] and 10 µM [C]), and addition of OKT3 is indicated by arrows. Data are presented as a typical tracing from one individual cell; for each condition at least three experiments were carried out.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In sea urchin eggs, Ca²⁺-release by NAADP⁺ was half-maximal between 16 and 30 nM, and showed saturation between 100 and 400 nM (Chini et al. 1995; Lee and Aarhus 1995). In ascidian oocytes and mouse pancreatic acinar cells, effective concentrations between 10 and 50 nM were observed (Albrieux et al. 1998; Cancela et al. 1999), although in brain microsomes 1 µM NAADP⁺ was necessary (Bak et al. 1999). These data fit very well to our current data in Jurkat T cells, the first human cell system where an effect of NAADP⁺ is reported. Ca²⁺-mobilizing concentrations were in the range between 10 and 100 nM (Fig. 1 and Fig. 2), whereas at concentrations 1 µM, partial or complete self inactivation was observed (Fig. 1 and Fig. 2).

The self inactivation properties of the NAADP⁺/Ca²⁺-release system, at least in sea urchin eggs, appear to be unique as compared with the known Ca²⁺-mobilizing second messengers, Ins(1,4,5)P₃ and cADPR (Aarhus et al. 1996; Genazzani et al. 1996). Especially the fact that subthreshold concentrations of NAADP⁺ (2 to 4 nM) almost completely inhibited subsequent Ca²⁺-release by a high concentration of NAADP⁺ (Aarhus et al. 1996; Genazzani et al. 1996) indicates that activation of the NAADP⁺/Ca²⁺-release system followed by its rapid inactivation can supply the cell with a short pulse of elevated Ca²⁺ only, and that the basal endogenous concentration of NAADP⁺ must be below a concentration that would permanently inactivate the system, e.g., in sea urchin eggs below 0.1 nM (Aarhus et al. 1996) or even less (Genazzani et al. 1996). Data in mammalian cell types, pancreatic acinar cells (Cancela et al. 1999), and T cells, however, indicate that low concentrations of NAADP⁺ do not substantially self-inactivate the system, e.g., microinjections of 10 nM NAADP⁺ in the majority of cases stimulated long-lasting trains of low-amplitude Ca²⁺-spikes in T cells (Fig. 1 E), and infusion of 50 nM NAADP⁺ into acinar cells evoked sustained Ca²⁺-spiking (Cancela et al. 1999).

To completely unravel the role of NAADP⁺, mainly to verify (or to falsify) its status as a second messenger, measurement of the endogenous concentration of NAADP⁺ would be helpful. However, regarding the theoretically expected concentrations of 0.1 nM in unstimulated cells and 50–100 nM NAADP⁺ in stimulated cells, it might be very difficult to develop an analytical system to measure these low concentrations. Our recently developed HPLC systems for the mass determination of Ins(1,4,5)P₃ (Guse et al. 1995) and cADPR (da Silva et al. 1998) require 0.5–1 x 10⁸ cells per sample to measure these compounds in the low micromolar range. To measure basal NAADP⁺ concentrations a 1,000-fold more sensitive analytical method would be required. Potential methods to achieve this may include labeling of NAADP⁺ by a fluorescent dye (pre- or postcolumn derivatization) combined with a very sensitive fluorescence detector, e.g., laser-induced fluorescence detection (Rahavendran and Karnes 1993). Alternatively, a competitive protein binding assay based on a high affinity binding protein for NAADP⁺ may also be sufficient.

As discussed above, regarding the NAADP⁺/Ca²⁺-release system, there are similarities between mouse pancreatic acinar cells and human T cells, e.g., a very similar dose-response relationship for NAADP⁺, and the fact that self inactivation of the NAADP⁺/Ca²⁺-release renders both cell types insensitive to physiological stimulation. However, there are also at least three clear differences between the two cell systems: inhibition of either the cADPR/Ca²⁺-release system or the Ins(1,4,5)P₃/Ca²⁺-release system in pancreatic acinar cells completely blocked NAADP⁺-mediated Ca²⁺-signaling (Cancela et al. 1999), whereas similar inhibition protocols were without or almost without effect in T cells; self inactivation of the NAADP⁺/Ca²⁺-release system did not influence Ca²⁺-signaling mediated by infusion of cADPR or Ins(1,4,5)P₃ in acinar cells (Cancela et al. 1999), whereas in T cells such self-inactivation of the NAADP⁺/Ca²⁺-release system almost completely inhibited subsequent signaling by cADPR or Ins(1,4,5)P₃; and in acinar cells, the sustained phase of Ca²⁺-spiking induced by infusion of cADPR could be blocked by the Ins(1,4,5)P₃ antagonist heparin (Thorn et al. 1994), whereas in T cells there was no effect of the Ins(1,4,5)P₃ antagonist, Ins(1,4,6)PS₃, on cADPR-mediated Ca²⁺-signaling (Guse et al. 1997).

Using the data obtained from pancreatic acinar cells, Petersen and Cancela 1999 developed a model with the following sequence of events: stimulation of acinar cells by the brain–gut peptide, cholecystokinin, in first instance elevates NAADP⁺ to nanomolar concentrations. Ca²⁺ released by NAADP⁺ then serves as a trigger for the Ca²⁺-induced Ca²⁺-release mechanism at the RyR. This mechanism, in addition to the stimulatory effect of cADPR on RyR, then amplifies the Ca²⁺-signal. The increased [Ca²⁺]_i in concert with Ins(1,4,5)P₃ then releases more Ca²⁺ via the InsP₃-R (Petersen and Cancela 1999). Only this last element is measurable as a Ca²⁺-spike, whereas the trigger and amplifier element, provided by NAADP⁺ and the Ca²⁺-induced Ca²⁺-release mechanism modulated by cADPR, appear to be too small to be detected by patch clamp measurements of the Ca²⁺-dependent currents (Petersen and Cancela 1999).

In contrast to acinar cells, in Jurkat T cells NAADP⁺ produced a substantial Ca²⁺-spike, even if cADPR- and Ins(1,4,5)P₃-antagonists were present (Fig. 4A and Fig. F, and Fig. 5A and Fig. F). The second main difference to acinar cells was that in Jurkat T cells, Ca²⁺-signaling by cADPR and Ins(1,4,5)P₃ depended on a functional NAADP⁺/Ca²⁺-release system (Fig. 4C and Fig. H, and Fig. 5C and Fig. H). Although two different methods were used to detect the Ca²⁺-spikes: patch clamp measurements of the Ca²⁺-dependent currents vs. single cell Ca²⁺ imaging using Fura2-loaded cells, this is unlikely to be the reason for the differences observed. Thus, the model developed for the acinar cells (Petersen and Cancela 1999) needs some modification to fit to the data obtained in Jurkat T cells. In accordance with the acinar cell model, NAADP⁺ appears to act first in sequence providing trigger-calcium needed for the two other Ca²⁺-release systems. Because of the experimental difficulties to measure nanomolar concentrations of NAADP⁺ in cells as discussed above, it is unclear whether NAADP⁺ concentrations in fact do increase upon stimulation, or whether NAADP⁺ stays unaltered in the low nanomolar range keeping the T cell in an excitable state. Experimental evidence for the latter may be obtained by high temporal and spatial resolution Ca²⁺ imaging experiments in Jurkat T cells; preliminary data indicate a basal Ca²⁺-signaling activity of very low amplitude in nonstimulated cells (Guse, A.H., and S. Heidbrink, unpublished results). However, trigger-calcium provided by NAADP⁺ further acts in concert with Ins(1,4,5)P₃, which is rapidly, but transiently, formed in the first minutes of T cell activation (Brattsand et al. 1990; Ng et al. 1990), and then with cADPR, which is elevated during the sustained phase of T cell Ca²⁺-signaling (Guse et al. 1999).

However, despite these differences between acinar and T cells the dependence of cholecystokinin receptor–mediated Ca²⁺-signaling on a Ca²⁺-trigger supplied by an initial NAADP⁺-mediated Ca²⁺-release event to integrate the Ca²⁺-amplifier, cADPR, and the Ca²⁺-oscillator, Ins(1,4,5)P₃ (Petersen and Cancela 1999), exactly mirrors the situation observed in Jurkat T cells. As shown in Fig. 6, if the NAADP⁺/Ca²⁺-release system was inactivated by high NAADP⁺ concentrations subsequent quasiphysiological stimulation of the T cell by anti-CD3 mAb did not result in any Ca²⁺-signaling. This result is in complete accordance with the inhibition of cADPR- or Ins(1,4,5)P₃-mediated Ca²⁺-signaling by coinjection of NAADP⁺ (Fig. 4C and Fig. H, and Fig. 5C and Fig. H) since Ca²⁺-signaling in T cells critically depends on these two second messengers (Jayaraman et al. 1995; Guse et al. 1999).

More generalized, if the NAADP⁺/Ca²⁺-release system acts as the Ca²⁺-providing trigger, the complex behavior of activation and inactivation opens a multitude of regulatory possibilities: simply by changing their endogenous NAADP⁺ concentration cells might regulate the status of the NAADP⁺/Ca²⁺-release system; e.g., by increasing NAADP⁺ the NAADP⁺/Ca²⁺-release system will become inactivated, and Ca²⁺-signaling will in turn be completely unresponsive. For T-lymphocytes, such behavior of unresponsiveness to antigenic or mitogenic stimulation is well known as anergy (Jenkins et al. 1987); however, it is less clear which intracellular mechanism is responsibly involved. Our data indicate that the NAADP⁺/Ca²⁺-release system with its complex inactivation/activation properties might be such a mechanism underlying anergy in T cells.

From an evolutionary point of view, it is of particular interest that both a very similar dose-response relationship and the self inactivation property of the NAADP⁺/Ca²⁺-release have been conserved between sea urchin eggs, ascidian oocytes, and higher eukaryotic cells from pancreas and lymphocytes. This indicates that these two characteristic features are of outstanding importance for the regulation of intracellular Ca²⁺-signaling in general. One of the important future aspects will be the identification of the molecular target for NAADP⁺. In addition to the model for pancreatic acinar cells (Cancela et al. 1999; Petersen and Cancela 1999) in which a separate NAADP⁺ receptor has been suggested, it might also be possible that NAADP⁺ acts as a comessenger at the known intracellular Ca²⁺-release channels, the Ins(1,4,5)P₃ receptor and/or the RyR. Along these lines, the fluorescent 1,N⁶-etheno-NAADP⁺ has been shown to release Ca²⁺ in sea urchin eggs (Lee and Arhus, 1998), and thus, may serve as a tool to identify the receptor for NAADP⁺.

	Acknowledgments

 
This project was supported by grants from the Deutsche Forschungsgemeinschaft (grant nos. Gu 360/5-1, Gu 360/2-4, and Gu 360/2-5 to A.H. Guse and G.W. Mayr), a Wellcome Trust Research Collaboration grant (no. 051326 to A.H. Guse and B.V.L. Potter), and a Wellcome Trust Programme Grant (045491 to B.V.L. Potter).

Submitted: 19 April 2000

Revised: 8 June 2000

Accepted: 15 June 2000

Abbreviations used in this paper: [Ca²⁺]_i, intracellular Ca²⁺ concentration; cADPR, cyclic ADP-ribose; Ins(1,4,5)P₃, D-myo-inositol 1,4,5-trisphosphate; InsP₃-R, Ins(1,4,5)P₃ receptor; Ins(1,4,6)PS₃, D-myo-inositol 1,4,6-trisphosphorothioate; NAADP⁺, nicotinic acid adenine dinucleotide phosphate; NADP⁺, nicotinamide adenine dinucleotide phosphate; RyR, ryanodine receptor(s); TCR/CD3, T cell receptor/CD3.

	References

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