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Originally published In Press as doi:10.1074/jbc.M413085200 on March 17, 2005

J. Biol. Chem., Vol. 280, Issue 22, 21394-21399, June 3, 2005
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Functional Ryanodine Receptor Expression Is Required for NAADP-mediated Local Ca2+ Signaling in T-lymphocytes*

Werner Dammermann and Andreas H. Guse{ddagger}

From the University Hospital Hamburg-Eppendorf, Center of Experimental Medicine, Institute of Biochemistry and Molecular Biology I: Cellular Signal Transduction, Martinistrasse 52, 20246 Hamburg, Germany

Received for publication, November 19, 2004 , and in revised form, February 25, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+-mobilizing nucleotide involved in T cell Ca2+ signaling (Berg, I., Potter, B. V. L., Mayr, G. W., and Guse, A. H. (2000) J. Cell Biol. 150, 581–588). The objective of this study was to analyze whether the first subcellular Ca2+ signals obtained upon NAADP stimulation of T-lymphocytes depend on the functional expression of ryanodine receptors. Using combined microinjection and high resolution confocal calcium imaging, we demonstrate here that subcellular Ca2+ signals, characterized by amplitudes between ~30 and 100 nM and diameters of ~0.5 µm, preceded global Ca2+ signals. Co-injection of the ryanodine receptor antagonists ruthenium red and ryanodine together with NAADP abolished the effects of NAADP, whereas the D-myo-inositol 1,4,5-trisphosphate antagonist heparin and the Ca2+ entry blocker SKF&96365 were without effect. This pharmacological approach was confirmed by a molecular knock-down approach. Jurkat T cell clones with largely reduced expression of ryanodine receptors did not respond to microinjections of NAADP. Taken together, our data suggest that the Ca2+ release channel sensitive to NAADP in T-lymphocytes is the ryanodine receptor.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nicotinic acid adenine dinucleotide phosphate (NAADP)1 is an endogenous nucleotide in eukaryotic cells and to date represents the most powerful Ca2+-releasing compound. This compound was discovered by Lee and co-workers as an impurity of NADP in 1987 (1), and its structure was obtained in 1995 (2).

In contrast to the other known Ca2+-releasing compounds, D-myo-inositol 1,4,5-trisphosphate (InsP3) (for review see Ref. 3) and cyclic ADP-ribose (cADPR) (for review see Ref. 4), the Ca2+ channel sensitive to NAADP, is still a matter of debate. Pharmacological Ca2+ release data obtained in sea urchin egg homogenates suggest that neither the InsP3 receptor nor the ryanodine receptor (RyR) represents the molecular target for NAADP (2). In addition, NAADP appears not to act on the classical rapidly exchanging Ca2+ store, the endoplasmic reticulum. Indeed, a NAADP-sensitive Ca2+ pool was separated from the cADPR- and InsP3-sensitive one by stratification of sea urchin eggs (5). Subsequently, this store was identified as the reserve granule of eggs, a lysosome-related organelle (6). Recently, a lysosome-related acidic Ca2+ store sensitive to NAADP was also detected in higher eukaryotic cells, e.g. pancreatic acinar cells and the MIN6 pancreatic {beta}-cell line (7), and in rat cortical neurons (8).

Although the pharmacological characterization of NAADP-induced Ca2+ signaling in sea urchin eggs resulted in the conclusion that a novel Ca2+ channel unrelated to the known intracellular Ca2+ release channels is involved, a number of reports from heart and skeletal muscle and pancreatic acinar cells suggest that RyR are the Ca2+ channels mediating the effect of NAADP (9–11).

Accordingly, different models resulting from these conflicting data have been postulated. The two-pool model (12) consists of two separate Ca2+ pools: a lysosome-related Ca2+ pool with the novel NAADP receptor giving rise to spatiotemporally restricted trigger Ca2+ and an endoplasmic reticulum-related Ca2+ pool with InsP3 receptors and RyR, which then respond to the trigger Ca2+ by Ca2+-induced Ca2+ release (CICR). The other model reduced the number of Ca2+ channels sensitive to NAADP to the RyR but postulated different binding proteins for NAADP and cADPR, mediating their effects at the RyR (11). We have recently shown that global Ca2+ signaling induced by NAADP in human Jurkat T-lymphocytes depends on functional expression of RyR and also on Ca2+ entry (13).

Because that report was compatible with both models, the present study was conducted to analyze the very initial subcellular Ca2+ release events observed upon NAADP stimulation and, in particular, to understand whether the RyR is necessary also for these spatiotemporally restricted Ca2+ signals.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Fetal calf serum (tetracycline-free) was obtained from Biochrom (Berlin, Germany). Fura-2/AM, ionomycin, ryanodine, SKF 96365, and ruthenium red were purchased from Calbiochem. NAADP and heparin were supplied by Sigma. cADPR was obtained from Biolog (Bremen, Germany).

Cell Culture—Jurkat T-lymphocytes (clone JMP) were cultured as described previously (14). Tet-On Jurkat T cell clones stably transfected with pTRE2-enhanced green fluorescent protein/E2 (abbreviated as clone E2), pTRE2-511/25 (abbreviated as clone 25), and pTRE2-240/10 (abbreviated as clone 10) were cultured in RPMI 1640 medium supplemented with Glutamax I, 10% (v/v) fetal calf serum (free of tetracycline, Biochrom), 25 mM HEPES, 1 mM sodium pyruvate, 100 units/ml penicillin, 50 µg/ml streptomycin, 50 µg/ml hygromycin, and 400 µg/ml G418-sulfate (15).



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  		FIG. 1.
Subcellular Ca2+ signaling evoked by microinjection of NAADP in Jurkat T-lymphocytes. Jurkat T cells (subclone JMP) were loaded with Fura-2/AM and subjected to combined Ca2+ imaging and microinjection as detailed under "Experimental Procedures." A, injection of 100 nM NAADP. B, injection of 30 nM NAADP. C, injection of intracellular buffer. Data are representative of 8–14 individual cells analyzed for each condition.

 



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  		FIG. 2.
Characterization of subcellular Ca2+ signaling evoked by microinjection of NAADP in Jurkat T-lymphocytes. A and B, magnification of images from Fig. 1, A and B, to demonstrate the spatiotemporal properties of NAADP-mediated subcellular Ca2+ signals.

 
Ratiometric Ca2+ Imaging—The cells were loaded with Fura-2/AM as described by Kunerth et al. (16) and kept in the dark at room temperature until use. Thin glass coverslips (0.1 mm) were coated with bovine serum albumin (5 mg/ml) and poly-L-lysine (0.1 mg/ml). Silicon grease was used to seal small chambers consisting of a rubber O-ring on the glass coverslips. 60 µl of buffer A 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 a 40-µl cell suspension (2 x 106 cells/ml) suspended in the same buffer were added into the small chamber (16). The coverslip with cells slightly attached to the bovine serum albumin/poly-L-lysine coating was mounted on the stage of a fluorescence microscope (Leica DM IRE2).

Ratiometric Ca2+ imaging was performed as described recently (16). We used an Improvision imaging system (Tübingen, Germany) built around the Leica microscope at 100-fold magnification. Illumination at 340 and 380 nm was carried out using a monochromator system (Polychromator IV, TILL Photonics, Gräfelfing, Germany). Images were taken with a gray-scale CCD camera (type C4742-95-12ER; Hamamatsu, Enfield, United Kingdom) operated in 8-bit mode. The spatial resolution was 512 x 640 pixel at 100-fold magnification. Camera exposure times were 12 (at 340 nm) and 4 ms (at 380 nm). The acquisition rate was ~1 ratio m 160 ms. Raw data images were stored on a hard disk. Confocal Ca2+ images were obtained by off-line no-neighbor deconvolution using the volume deconvolution module of the Openlab software as described recently for 3T3 fibroblasts (17). The deconvolved images were used to construct ratio images (340/380). Finally, ratio values were converted to Ca2+ concentrations by external calibration. To reduce noise, ratio images were subjected to median filter (3 x 3) as described previously (16). Data processing was performed using Openlab software, version 1.7.8, 3.0.9, or 3.5.2 (Improvision, Tübingen, Germany).

Microinjection—Microinjections were carried out as described previously (13, 18). We used an Eppendorf system (transjector type 5246, micromanipulator type 5171, Eppendorf-Netheler-Hinz, Hamburg, Germany) with Femtotips II as pipettes. NAADP and/or ruthenium red were diluted to their final concentration in intracellular buffer (20 mM HEPES, 110 mM KCl, 2 mM MgCl2, 5 mM KH2PO4, 10 mM NaCl, pH 7.2) and filtered (0.2 µm) before use. To avoid any contamination of Ca2+ in the solution to be injected, a small amount of Chelex resin beads was added. Injections were made using the semiautomatic mode of the system with the following instrumental settings: injection pressure, 60 (clone JMP) or 40 hectoPascals (clones E2, 10, and 25); compensatory pressure, 30 (clone JMP) or 25 hectoPascals (clones E2, 10, and 25); injection time, 0.5 (clone JMP) or 0.3 s (clones E2, 10, and 25), and velocity of the pipette, 700 µm/s. Under such conditions, the injection volume was 1–1.5% cell volume (19).



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  		FIG. 3.
Inhibition of subcellular Ca2+ signals induced by NAADP by co-injection of ruthenium red. Jurkat T cells (subclone JMP) were loaded with Fura-2/AM and subjected to combined Ca2+ imaging and microinjection as detailed under "Experimental Procedures." A and B, analysis of global [Ca2+] (A) or local [Ca2+] (B) in response to injection of NAADP (30 nM), NAADP (30 nM) plus ruthenium red (RuRed; 10 µM), or intracellular buffer. Local responses were recorded in the cytosol close to the pm, in the cyt, or in the nuc and expressed as the difference between the maximal peak amplitude and the amplitude of the surrounding area. Arrows indicate time point of injection. Data are representative of 7–14 individual cells analyzed for each condition.

 



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  		FIG. 4.
Effect of various Ca2+ signaling inhibitors on the amplitude of NAADP evoked subcellular Ca2+ signals. Local responses were recorded in the cytosol close to the pm, in the cyt, or in the nuc and expressed as the difference between the maximal peak amplitude and the amplitude of the surrounding area as shown for an example in Fig. 3. Pipette concentrations of inhibitors are as follows: ruthenium red (RuRed) (10 µM); ryanodine (Rya) (20 mM); and heparin (Hep) (1 mg/ml) (note that these concentrations are diluted 1:50–1:100 in the cytosol). The cells were preincubated with SKF 96365 (30 µM) for 15 min in the extracellular medium. Peak amplitudes are given as the mean ± S.E. (n = 5–14 cells for each condition); p values were obtained from the Student's t test as indicated.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Subcellular Ca2+ signaling upon microinjection of 100 nM NAADP into Jurkat T-lymphocytes (clone JMP) remained almost unchanged for a very short, initial period of a few hundred milliseconds (Fig. 1A; time point 37.730 s). Characteristic NAADP-induced subcellular signals were observed (Fig. 1A; time point 37.903 s). These initial signals usually were localized close to the site of injection and rapidly converged into a larger signal still remaining mainly in a zone close to the plasma membrane (Fig. 1A; time point 38.077 s). The signal spread in a ring-like structure before it also arrived in the central part of the cell (Fig. 1A; time points 38.252 and 38.433 s). Thus, 100 nM NAADP in the microinjection pipette induced a very rapid global response leaving just a very limited period of time, usually <1.5 s, to analyze subcellular Ca2+ signaling events. To expand this pacemaker phase, the pipette concentration of NAADP was reduced to 30 nM, a concentration still sufficient to induce global Ca2+ signaling (20). Under these conditions, subcellular Ca2+ signals of very small diameter and amplitude were observed (Fig. 1B; time point 40.562 and 41.260 s). As for the 100 nM NAADP injections, the characteristic ring-like signaling structure became visible before global Ca2+ signaling started (Fig. 1B; time point 42.303 and 43.531 s). Importantly, when intracellular buffer, the vehicle for NAADP injections, was microinjected into Jurkat T-lymphocytes, no alterations in subcellular Ca2+ signaling were observed (Fig. 1C). These control data, together with the fact that in the very initial period after microinjection almost no changes in Ca2+ signaling were observed, suggest that (i) the subcellular signals were caused by NAADP and that (ii) the temporal resolution of image acquisition was sufficient to analyze the very initial NAADP-induced Ca2+ signals.



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  		FIG. 5.
Comparison of the spatiotemporal characteristics of subcellular Ca2+ signals induced by NAADP or by cADPR. Jurkat T cells (subclone JMP) were loaded with Fura-2/AM and subjected to combined Ca2+ imaging and microinjection as detailed under "Experimental Procedures." A, pseudocolor images displaying spatiotemporal development of Ca2+ signaling in characteristic cells injected with NAADP (30 nM) or cADPR (100 µM). B, local [Ca2+] in response to injection of NAADP (30 nM) or cADPR (100 µM) recorded in the cytosol close to the pm, in the cyt, or in the nuc and expressed as the difference between the maximal peak amplitude and the amplitude of the surrounding area. Arrows indicate time point of injection (solid for NAADP; dashed for cADPR). Data are representative of 5–9 individual cells analyzed for each condition.

 
A magnified view of the images showing NAADP-induced subcellular Ca2+ signals revealed that, in case of the 100 nM injection, the signals were more concentrated in one part of the cell, almost merging into one much bigger Ca2+ signal (Fig. 2A, arrow; diameter of the merged signal ~2 µm). However, individual small Ca2+ signals with diameters characteristic for Ca2+ quarks (diameter ~0.5 µm) were still observed in close vicinity to the bigger signal (Fig. 2A, arrowheads). Upon 30 nM NAADP microinjections, mainly the smaller Ca2+ signals were observed (Fig. 2B, arrowheads).

Very recently, we have shown that global Ca2+ signals evoked by microinjection of NAADP in T cells were completely dependent on functional expression of RyR (13). However, these data left open the possibility that NAADP may initially activate a Ca2+ channel different from the RyR, giving rise to local Ca2+ signals, which then by the process of CICR would trigger RyR, according to the 2-pool model proposed originally by Cancela et al. (12). To distinguish between the 2-pool model and a model where RyR directly (or via an additional binding protein) responds to NAADP (11), subcellular NAADP-induced Ca2+ signals as described in Figs. 1 and 2 were analyzed under further experimental conditions. Ca2+ signaling was analyzed in T cells co-injected with the following: (i) NAADP and the RyR antagonists ruthenium red and ryanodine; (ii) NAADP and the InsP3 antagonist heparin; or (iii) NAADP and the Ca2+ entry blocker SKF 96365.

As reported previously (13), the global Ca2+ signal observed upon NAADP injection was completely abolished when ruthenium red was co-injected (Fig. 3A). An analysis of small subcellular regions of interest (ROI) set either close to the plasma membrane (pm), the cytosol (cyt), or the nucleus (nuc) revealed an oscillatory behavior of the local [Ca2+] (Fig. 3B). Importantly, the amplitude of local Ca2+ signals increased with time in the presence of NAADP (Fig. 3B, black tracings). In contrast, when NAADP was co-injected with ruthenium red, the subcellular signals remained at similar amplitudes compared with buffer injections (Fig. 3B, red and blue tracings). Quantitative evaluation of an array of microinjection and preincubation data revealed the following. Co-injection of the antagonists of RyR, ruthenium red and ryanodine (the latter at a high inhibitory concentration), completely blocked subcellular Ca2+ signals evoked by NAADP, regardless of the subcellular localization of the signals (Fig. 4). In contrast, neither co-injection of the InsP3 antagonist heparin nor preincubation with the Ca2+ entry blocker SKF 96365 antagonized subcellular signals evoked by NAADP (Fig. 4). Rather, a weak stimulatory effect of both heparin microinjection and preincubation with SKF 96365 in the absence and presence of NAADP (co-)injection was observed (Fig. 4). The weak agonistic effect of heparin at the RyR and the weak Ca2+-mobilizing properties of SKF 96365 are well documented (21, 22) and thus explain the observed effects. Autoinactivation of the NAADP signaling system by injection of a high concentration of NAADP resulted in signal amplitudes comparable to buffer injections. In addition, subcellular Ca2+ signals evoked by the established RyR agonist cADPR were fully blocked by this procedure (Fig. 4).



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  		FIG. 6.
Subcellular Ca2+ signaling evoked by microinjection of NAADP in RyR knock-down T cell clones. Jurkat T cells were loaded with Fura-2/AM and subjected to combined Ca2+ imaging and microinjection as detailed under "Experimental Procedures." Injection of 30 nM NAADP into the control clone E2 (A), into pan-RyR knock-down clone 10 (B), and into type 3 RyR knock-down clone 25 (C). Data are representative of 8–11 individual cells analyzed for each condition.

 
Analysis of the spatiotemporal pattern of Ca2+ signaling evoked by NAADP compared with the pattern induced by the established RyR agonist cADPR did not result in significant differences (Fig. 5) as can be seen from the images (Fig. 5A) and the ROI tracings close to the pm, in the cyt, or in the nuc (Fig. 5B).

Although the pharmacological data and the spatiotemporal characteristics of NAADP-mediated subcellular Ca2+ signals suggest involvement of RyR, the use of inhibitors may be mis-leading due to insufficient specificity. Therefore, in a second independent approach, microinjection and imaging experiments were conducted in RyR knock-down T cell clones (13, 15, 18). These RyR knock-down Jurkat T cell clones were obtained by stable transfection with plasmids expressing antisense RNA against RyR (15). Clone 25 expressed antisense RNA specific for the type 3 RyR, whereas clone 10 expressed an antisense RNA fragment targeting all three types of RyR. As control, a Jurkat T cell clone with stable expression of an antisense fragment directed toward a protein not expressed in T cells was chosen (clone E2; fragment directed against enhanced green fluorescent protein). Largely reduced expression of RyR on the protein level was analyzed by Western blot experiments and is documented by Langhorst et al. (13) and Schwarzmann et al. (15).

Microinjection of NAADP into T cells of the control clone E2 resulted in comparable subcellular signals as described above for the Jurkat T cell clone JMP (Fig. 6A). In stark contrast, no changes in subcellular Ca2+ signaling were observed in the RyR knock-down T cells, neither in the pan knock-down clone 10 (Fig. 6B) nor in the type 3-specific knock-down clone 25 (Fig. 6C). Quantitative evaluation of individual ROI set close beneath the pm, in the cyt, or in the nuc resulted in an even clearer picture (Fig. 7) compared with the experiments described in Figs. 3 and 4. Rapidly after microinjection of NAADP, the amplitude of subcellular Ca2+ signals in all three ROIs increased in control clone E2 but remained almost unchanged in knock-down clones 10 and 25 (Fig. 7B). Quantitative evaluation of such experiments showed that NAADP induced a statistically significant increase of the signal amplitude in all three ROI in control clone E2, whereas in RyR knock-down clones 10 and 25, no significant differences to buffer injections were observed.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Here we report that the initial subcellular Ca2+ signals induced by microinjection of NAADP depend on the functional expression of RyR in human T cells. Cancela et al. (23) were the first to show that Ca2+ spiking evoked by NAADP could be efficiently blocked by an inhibitory concentration of ryanodine (23). Obviously influenced by the fact that inhibition of RyR did not affect NAADP-induced Ca2+ release in the sea urchin egg system (2), Cancela et al. (23) interpreted their data not as a direct effect but rather as inhibition of CICR via RyR secondary to the "trigger Ca2+" provided by a separate and novel receptor/Ca2+ release channel sensitive to NAADP.

In a recent report (13), we demonstrated that both RyR inhibition and the down-regulation of its expression almost completely abolished NAADP-mediated global Ca2+ signaling in T cells. These data, although pronouncing the role of RyR in NAADP-induced Ca2+ signaling in T cells, were still compatible with a separate and novel receptor/Ca2+ release channel sensitive to NAADP. Accordingly, the task of this putative channel would then be to initiate CICR via RyR by evoking local pacemaker Ca2+ signals. The alternative model is simpler and has been proposed for heart and skeletal muscle and for pancreatic acinar cells (9–11). NAADP either directly or via a specific binding protein binds to RyR to induce opening of its Ca2+ channel. In the experiments described in the current report, inhibition by the antagonists of RyR, ryanodine (at high concentration) or ruthenium red, abolished any NAADP-mediated subcellular Ca2+ signals above intrinsic background. In contrast, inhibition of InsP3 receptors by heparin or inhibition of Ca2+ entry by SKF 96365 did not block but rather enhanced the subcellular signals. Such agonistic properties of heparin on RyR are well documented (21). Likewise, already in the original publication the inventors of SKF 96365, Merrit et al. (22) stated that "in some conditions in either intact or permeabilized cells, SKF 96365 appeared to cause some discharge of intracellular Ca2+ stores" (22). Thus, the slight increase in the amplitudes of subcellular Ca2+ signals in heparin-injected or SKF 96365-preincubated cells is not unexpected. In the context of the experiments described here, these positive controls confirm the specificity of the antagonistic effects seen with high ryanodine concentrations and ruthenium red.



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  		FIG. 7.
Inhibition of subcellular Ca2+ signals induced by NAADP in RyR knock-down T cell clones. Jurkat T cells (subclones E2, 10, and 25) were loaded with Fura-2/AM and subjected to combined Ca2+ imaging and microinjection as detailed under "Experimental Procedures." Analysis of global [Ca2+] (A) or local [Ca2+] in response to injection of NAADP (30 nM) into control clone E2 or RyR knock-down clone 10 or 25 is shown. In B, local responses were recorded in the cytosol close to the pm, in the cyt, or in the nuc and expressed as the difference between the maximal peak amplitude and the amplitude of the surrounding area. Arrows indicate time point of injection. C, peak amplitudes as in B expressed as the mean ± S.E. (n = 8–11 cells for each condition); p values obtained from the Student's t test as indicated. In the condition "NAADP- -," cells were microinjected with intracellular buffer.

 
Most importantly, comparable inhibitory effects on NAADP-mediated subcellular Ca2+ signals were also obtained in RyR knock-down T cell clones. Thus, the relevance of RyR for these subcellular Ca2+ signals evoked by NAADP was confirmed by a completely independent molecular approach.

The NAADP-mediated subcellular Ca2+ signals analyzed were very small in diameter (starting from ~0.5 µm) and also small in amplitude (average between 50 and 100 nM), indicating that they may represent an opening of small clusters of RyR, as proposed recently (24). Microinjection of the established RyR agonist cADPR produced subcellular Ca2+ signals with similar spatiotemporal characteristics compared with NAADP, further indicating that both adenine nucleotides act on the same target Ca2+ channel.

Taken together, our data suggest that RyR are, in addition to their central role in the global Ca2+ signals, also responsible for the very initial subcellular Ca2+ signals evoked by NAADP in human T-lymphocytes. Our data do not rule out that NAADP-sensitive RyR may be expressed in acidic lysosomal Ca2+ stores in T-lymphocytes or that a specific binding protein for NAADP mediating its effect at the RyR exists. However, evidence for a novel NAADP-sensitive Ca2+ channel in T cells was not obtained using state-of-the-art Ca2+-imaging technology.


   FOOTNOTES
 
* This work was supported by the Werner-Otto-Foundation, Deutsche Forschungsgemeinschaft, the Wellcome Trust, and the Gemeinnützige Hertie-Stiftung. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: University Hospital Hamburg-Eppendorf, Center of Experimental Medicine, Institute of Biochemistry and Molecular Biology I: Cellular Signal Transduction, Martinistrasse 52, D-20246 Hamburg, Germany. Tel.: 49-40-42803-2828; Fax: 49-40-42803-9880; E-mail: guse{at}uke.uni-hamburg.de.

1 The abbreviations used are: NAADP, nicotinic acid adenine dinucleotide phosphate; cADPR, cyclic ADP-ribose; CICR, Ca2+-induced Ca2+ release; InsP3, D-myo-inositol 1,4,5-trisphosphate; pm, plasma membrane; cyt, cytosol; nuc, nucleus; ROI, region of interest; RyR, ryanodine receptor. Back


   ACKNOWLEDGMENTS
 
We are grateful to Matthias Langhorst for comments on the manuscript. This article is based in part on a doctoral study by W. Dammermann in the Faculty of Biology, University of Hamburg.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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