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发表于 2009-3-5 22:39 |只看该作者 |倒序浏览 |打印

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, n* M& h! N; f: L
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Correspondence to: Andreas H. Guse, University of Hamburg, Institute for Medical Biochemistry and Molecular Biology, Div. Cellular Signal Transduction, Grindelallee 117, IV, D-20146 Hamburg, Germany. Tel:49-40-42838-4276 Fax:49-40-42838-4275 E-mail:guse@uke.uni-hamburg.de.

Abstract3 X3 ~" N! V  N3 C# Y0 S  s
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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.
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Key Words: cyclic ADP-ribose, inositol 1,4,5-trisphosphate, T cell activation, signal transduction, ryanodine receptor
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Introduction% U4 ^/ Z% {8 Y2 |. e! v
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Activation of T-lymphocytes via the T cell receptor/CD3 (TCR/CD3)1 complex results in multiple intracellular signaling pathways (Kennedy et al. 1999 ). Among these pathways, an elevation of i (intracellular Ca2 -concentration) is essential for proliferation and clonal expansion (reviewed in Guse 1998 ). The increase of 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 ).6 \8 u1 s3 B3 J4 U( D
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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 Methods4 ?/ a  r' I- c3 r$ ?% H  U

Reagents" [' U' Q6 A! B# l$ t, N

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.5 S' R8 s5 {# j! S" s( J& r

Cell Culture% e" {2 U1 d  ~
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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.
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Ratiometric Ca2  Imaging6 o2 N* d8 C0 w( p$ F) P
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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.
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Microinjection Experiments" ~: K. Q" U- f1 n* H
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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., [- s1 q6 K8 Z) `- l

Results& Q  {: F8 M  w0 `7 I7 l7 S
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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., G! c2 u+ X: c$ L

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.* T4 q. Q9 p6 Y+ q; b+ P: X. D

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).

The high initial Ca2 -spike observed after microinjection of 50 nM NAADP  was massively reduced when the extracellular Ca2 -concentration was decreased to a nominal Ca2 -free buffer, indicating that Ca2 -entry is involved in the NAADP -mediated Ca2 -response (data not shown).
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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).
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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 Ca2 -release system that is targeted by NAADP  has not yet been identified, but work in other cell systems indicates that neither the Ins(1,4,5)P3 receptor (InsP3-R) nor the RyR are involved (Chini et al. 1995 ; Lee and Aarhus 1995 ). However, both of these classical intracellular Ca2 -release systems have been demonstrated to be essential parts of the Ca2 -signaling machinery of T cells (Jayaraman et al. 1995 ; Guse et al. 1999 ). Thus, the next series of experiments was designed to investigate potential interrelations between the NAADP  system on one hand and both the Ins(1,4,5)P3 and cADPR systems on the other.2 u7 i5 c+ C% u7 m3 r; ^! f1 l

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.
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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).

The specific Ins(1,4,5)P3 antagonist, Ins(1,4,6)PS3 (Guse et al. 1997 ; Murphy et al. 2000 ), was also coinjected with an optimal NAADP  concentration. Surprisingly, there was a partial reduction of the initial Ca2 -peak, but also a faster decay of this peak as compared with injection of NAADP  alone (Fig 5A and Fig F vs. B and G). Similar to the cADPR system, there was an almost complete inhibition of Ins(1,4,5)P3-mediated Ca2 -release when a desensitizing concentration of NAADP  was coinjected (Fig 5C and Fig H). Coinjection of an optimal stimulating concentration of NAADP , together with Ins(1,4,5)P3, resulted in a high initial Ca2 -peak (Fig 5D and Fig I) that was comparable to the peak observed in response to injection of NAADP  alone (Fig 5B and Fig C), whereas much less oscillatory activity of the cells after the initial peak was observed (Fig 5D and Fig I) as compared with Ins(1,4,5)P3 alone (Fig 5E and Fig J). These data also indicate that the Ins(1,4,5)P3/Ca2 -release system requires a functional nondesensitized NAADP /Ca2 -release system. Moreover, a part of the Ca2 -signal observed in response to microinjection of NAADP  alone appears to be mediated by Ins(1,4,5)P3 (Fig 5A, Fig F, Fig B, and Fig G). This may be explained by the coagonistic effect of Ca2  released by NAADP , which then acts together with basal Ins(1,4,5)P3 at the InsP3-R; this coagonistic effect of Ca2  at the InsP3-R has been demonstrated previously (Bezprozvanny et al. 1991 ).
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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).
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Both the Ins(1,4,5)P3/Ca2 - and the cADPR/Ca2 -release systems have been published to be essential parts of the Ca2 -signaling machinery of T cells upon stimulation of the TCR/CD3 complex (Jayaraman et al. 1995 ; Guse et al. 1999 ). Since the data described above indicate that a functional NAADP /Ca2 -release system is essential for both Ins(1,4,5)P3- and cADPR-mediated Ca2 -release, we investigated the effect of NAADP  on Ca2 -signaling mediated by anti-CD3 mAb OKT3 (Fig 6). Microinjection of 50 nM NAADP  before stimulation of the cells by extracellular addition of OKT3 did not significantly change the OKT3-mediated Ca2 -signal (Fig 6A and Fig B). However, there was a dramatic inhibition of OKT3-mediated Ca2 -signaling when a desensitizing concentration of NAADP  was microinjected before stimulation by OKT3 (Fig 6 C).' K- h8 f' j# k' d

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  and 10 μM ), 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.
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Discussion

The main findings of this report are: a dose-dependent and specific effect of NAADP  in T cell Ca2 -signaling; the strict dependence of both Ins(1,4,5)P3- and cADPR-mediated Ca2 -release upon a functional NAADP /Ca2 -release system; and inhibition of Ca2 -signaling mediated by ligation of the TCR/CD3 complex by prior self inactivation of the NAADP /Ca2 -release system.

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 ).; H( i& `0 R8 W/ P

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.
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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 ).
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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 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 ).+ _* E! `$ l2 L) ]0 X3 C
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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 ).- O* O% {2 u8 o- e7 P; B+ W, j
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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.
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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 .

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