NAADP Mobilizes Calcium from the Endoplasmic Reticular Ca2+ Store in T-lymphocytes
2007; Elsevier BV; Volume: 282; Issue: 26 Linguagem: Inglês
10.1074/jbc.m610925200
ISSN1083-351X
AutoresMareike Steen, Tanja Kirchberger, Andreas H. Guse,
Tópico(s)Piperaceae Chemical and Biological Studies
ResumoThe target calcium store of nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent endogenous calcium-mobilizing compound known to date, has been proposed to reside in the lysosomal compartment or in the endo/sarcoplasmic reticulum. This study was performed to test the hypothesis of a lysosomal versus an endoplasmic reticular calcium store sensitive to NAADP in T-lymphocytes. Pretreatment of intact Jurkat T cells with glycyl-phenylalanine 2-naphthylamide largely reduced staining of lysosomes by LysoTracker Red and abolished NAADP-induced Ca2+ signaling. However, the inhibitory effect was not specific since Ca2+ mobilization by d-myo-inositol 1,4,5-trisphosphate and cyclic ADP-ribose was abolished, too. Bafilomycin A1, an inhibitor of the lysosomal H+-ATPase, did not block or reduce NAADP-induced Ca2+ signaling, although it effectively prevented labeling of lysosomes by LysoTracker Red. Further, previous T cell receptor/CD3 stimulation in the presence of bafilomycin A1, assumed to block refilling of lysosomal Ca2+ stores, did not antagonize subsequent NAADP-induced Ca2+ signaling. In contrast to bafilomycin A1, emptying of the endoplasmic reticulum by thapsigargin almost completely prevented Ca2+ signaling induced by NAADP. In conclusion, in T-lymphocytes, no evidence for involvement of lysosomes in NAADP-mediated Ca2+ signaling was obtained. The sensitivity of NAADP-induced Ca2+ signaling toward thapsigargin, combined with our recent results identifying ryanodine receptors as the target calcium channel of NAADP (Dammermann, W., and Guse, A. H. (2005) J. Biol. Chem. 280, 21394–21399), rather suggest that the target calcium store of NAADP in T cells is the endoplasmic reticulum. The target calcium store of nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent endogenous calcium-mobilizing compound known to date, has been proposed to reside in the lysosomal compartment or in the endo/sarcoplasmic reticulum. This study was performed to test the hypothesis of a lysosomal versus an endoplasmic reticular calcium store sensitive to NAADP in T-lymphocytes. Pretreatment of intact Jurkat T cells with glycyl-phenylalanine 2-naphthylamide largely reduced staining of lysosomes by LysoTracker Red and abolished NAADP-induced Ca2+ signaling. However, the inhibitory effect was not specific since Ca2+ mobilization by d-myo-inositol 1,4,5-trisphosphate and cyclic ADP-ribose was abolished, too. Bafilomycin A1, an inhibitor of the lysosomal H+-ATPase, did not block or reduce NAADP-induced Ca2+ signaling, although it effectively prevented labeling of lysosomes by LysoTracker Red. Further, previous T cell receptor/CD3 stimulation in the presence of bafilomycin A1, assumed to block refilling of lysosomal Ca2+ stores, did not antagonize subsequent NAADP-induced Ca2+ signaling. In contrast to bafilomycin A1, emptying of the endoplasmic reticulum by thapsigargin almost completely prevented Ca2+ signaling induced by NAADP. In conclusion, in T-lymphocytes, no evidence for involvement of lysosomes in NAADP-mediated Ca2+ signaling was obtained. The sensitivity of NAADP-induced Ca2+ signaling toward thapsigargin, combined with our recent results identifying ryanodine receptors as the target calcium channel of NAADP (Dammermann, W., and Guse, A. H. (2005) J. Biol. Chem. 280, 21394–21399), rather suggest that the target calcium store of NAADP in T cells is the endoplasmic reticulum. Nicotinic acid adenine dinucleotide phosphate (NAADP) 3The abbreviations used are: NAADP, nicotinic acid adenine dinucleotide phosphate; GPN, glycyl-phenylalanine 2-naphthylamide; cADPR, cyclic ADP-ribose; InsP3, d-myo-inositol 1,4,5-trisphosphate; RyR, ryanodine receptor(s); TCR-CD3, T cell receptor-CD3 complex; ER, endoplasmic reticulum; SR, sarcoplasmic reticulum; HPLC, high pressure liquid chromatography; RP-HPLC, reverse phase-HPLC. 3The abbreviations used are: NAADP, nicotinic acid adenine dinucleotide phosphate; GPN, glycyl-phenylalanine 2-naphthylamide; cADPR, cyclic ADP-ribose; InsP3, d-myo-inositol 1,4,5-trisphosphate; RyR, ryanodine receptor(s); TCR-CD3, T cell receptor-CD3 complex; ER, endoplasmic reticulum; SR, sarcoplasmic reticulum; HPLC, high pressure liquid chromatography; RP-HPLC, reverse phase-HPLC. was discovered by Lee et al. (1Lee H.C. Aarhus R. J. Biol. Chem. 1995; 270: 2152-2157Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar) as endogenous nucleotide with Ca2+-mobilizing properties. In comparison with the Ca2+-mobilizing second messengers d-myo-inositol 1,4,5-trisphosphate (InsP3) and cyclic ADP-ribose (cADPR) that require micromolar concentrations for activity in mammalian cells (2Guse A.H. da Silva C.P. Emmrich F. Ashamu G.A. Potter B.V.L. Mayr G.W. J. Immunol. 1995; 155: 3353-3359PubMed Google Scholar, 3Cancela J.M. Van Coppenolle F. Galione A. Tepikin A.V. Petersen O.H. EMBO J. 2002; 21: 909-919Crossref PubMed Scopus (157) Google Scholar), NAADP acts at low nanomolar concentrations and displays a bell-shaped concentration-response curve in many different eukaryotic cell types (4Harmer A.R. Gallacher D.V. Smith P.M. Biochem. J. 2001; 353: 555-560Crossref PubMed Scopus (17) Google Scholar, 5Johnson J.D. Misler A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14566-14571Crossref PubMed Scopus (115) Google Scholar, 6Berg I. Potter B.V.L. Mayr G.W. Guse A.H. J. Cell Biol. 2000; 150: 581-588Crossref PubMed Scopus (148) Google Scholar). Although InsP3 and cADPR are firmly established as second messengers, receptor-mediated formation of NAADP has yet been shown in a limited number of cellular systems only (7Masgrau R. Churchill G.C. Morgan A.J. Ashcroft S.J. Galione A. Curr. Biol. 2003; 13: 247-251Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 8Yamasaki 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, 9Gasser A. Bruhn S. Guse A.H. J. Biol. Chem. 2006; 281: 16906-16913Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The search for the target organelle and the molecular receptor for NAADP has attracted much attention in the last years. Lee et al. (10Lee H.C. Aarhus R. J. Cell Sci. 2000; 113: 4413-4420Crossref PubMed Google Scholar) demonstrated different subcellular localizations of the NAADP-sensitive Ca2+ pool versus the InsP3- and cADPR-sensitive Ca2+ pools in stratified sea urchin eggs. This NAADP-sensitive Ca2+ pool was identified as the reserve granule in sea urchin eggs, an organelle related to lysosomes (11Churchill 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). In addition, pharmacological and partial biochemical characterization suggests expression of a novel NAADP receptor/Ca2+ channel in sea urchin eggs (1Lee H.C. Aarhus R. J. Biol. Chem. 1995; 270: 2152-2157Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar, 12Patel S. Churchill G.C. Galione A. Biochem. J. 2000; 352: 725-729Crossref PubMed Scopus (51) Google Scholar, 13Berridge G. Dickinson G. Parrington J. Galione A. Patel S. J. Biol. Chem. 2002; 277: 43717-43723Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 14Dickinson G.D. Patel S. Biochem. J. 2003; 375: 805-812Crossref PubMed Scopus (35) Google Scholar, 15Churamani D. Dickinson G.D. Patel S. Biochem. J. 2005; 386: 497-504Crossref PubMed Scopus (7) Google Scholar). In a few higher eukaryotic cell types, evidence for the involvement of ryanodine receptors (RyR) in NAADP-mediated Ca2+ signaling has been published. NAADP activated purified RyR from heart and skeletal muscle in lipid planar bilayers (16Mojzisova A. Krizanova O. Zacikova L. Kominkova V. Ondrias K. Pfluegers Arch. Eur. J. Physiol. 2001; 441: 674-677Crossref PubMed Scopus (58) Google Scholar, 17Hohenegger M. Suko J. Gscheidlinger R. Drobny H. Zidar A. Biochem. J. 2002; 367: 423-431Crossref PubMed Scopus (107) Google Scholar). In addition, NAADP-induced Ca2+ release from the nuclear envelope of pancreatic acinar cells and from permeabilized pancreatic acinar cells was blocked by RyR antagonists (18Gerasimenko 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, 19Gerasimenko 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). In human Jurkat T-lymphocytes, we have demonstrated: (i) TCR-CD3-mediated formation of NAADP (9Gasser A. Bruhn S. Guse A.H. J. Biol. Chem. 2006; 281: 16906-16913Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), (ii) Ca2+ signaling induced by microinjection of NAADP in a concentration-dependent fashion (6Berg I. Potter B.V.L. Mayr G.W. Guse A.H. J. Cell Biol. 2000; 150: 581-588Crossref PubMed Scopus (148) Google Scholar), (iii) that NAADP-induced Ca2+ signaling consists of both Ca2+ release and Ca2+ entry (6Berg I. Potter B.V.L. Mayr G.W. Guse A.H. J. Cell Biol. 2000; 150: 581-588Crossref PubMed Scopus (148) Google Scholar, 20Langhorst M.F. Schwarzmann N. Guse A.H. Cell. Signal. 2004; 16: 1283-1289Crossref PubMed Scopus (63) Google Scholar), and (iv) involvement of RyR in both local and global Ca2+ signaling (20Langhorst M.F. Schwarzmann N. Guse A.H. Cell. Signal. 2004; 16: 1283-1289Crossref PubMed Scopus (63) Google Scholar, 21Dammermann W. Guse A.H. J. Biol. Chem. 2005; 280: 21394-21399Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Here we confirm that lysosomes can be functionally destroyed using either hypertonic swelling and physical destruction by glycyl-phenylalanine 2-naphthylamide (GPN) or inhibition of the lysosomal H+-ATPase using bafilomycin A1 (11Churchill 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). Although GPN pretreatment resulted in loss of Ca2+-mobilizing activity of all three second messengers, NAADP, cADPR, and InsP3, there was no inhibitory effect of bafilomycin A1 on any of the Ca2+-mobilizing compounds, even when endogenous Ca2+ stores were previously emptied by TCR-CD3 stimulation in the presence of bafilomycin A1. In contrast, emptying the endoplasmic reticular (ER) Ca2+ store by thapsigargin almost completely prevented Ca2+ signaling induced by NAADP. Taken together, our data indicate a major role for the ER, but not for lysosomes, in NAADP-mediated Ca2+ signaling in T cells. Materials—NAADP, GPN, GdCl3, and bafilomycin A1 (lot 1) were supplied from Sigma-Aldrich (Deisenhofen, Germany). cADPR was obtained from Biolog (Bremen, Germany). Fura-2/AM, bafilomycin A1 (lots 2 and 3), cathepsin inhibitor 1, and thapsigargin were purchased from Calbiochem. InsP3 was from Biomol (Hamburg, Germany). The anti-CD3 mouse monoclonal antibody OKT3 was purified from hybridoma supernatant on protein G-Sepharose FF (GE Healthcare, Freiburg, Germany). LysoTracker Red DND-99 was obtained from Molecular Probes (Leiden, The Netherlands). Cell Culture—Jurkat T-lymphocytes (subclone JMP) were cultured as described previously (22Guse A.H. Roth E. Emmrich F. Biochem. J. 1993; 291: 447-451Crossref PubMed Scopus (93) Google Scholar). Ratiometric Ca2+ Imaging—The cells were loaded with Fura-2/AM as described (22Guse A.H. Roth E. Emmrich F. Biochem. J. 1993; 291: 447-451Crossref PubMed Scopus (93) Google Scholar) and kept in the dark at room temperature until use. Thin glass coverslips (0.1 mm) were coated first with bovine serum albumin (5 mg/ml) and subsequently with poly-l-lysine (0.1 mg/ml). Silicon grease was used to seal small chambers consisting of a rubber O-ring on the glass coverslip. Then, 60 μl of buffer A containing 140 mm NaCl, 5 mm KCl, 1 mm MgSO4, 1 mm CaCl2, 1 mm Na2HPO4, 5.5 mm glucose, and 20 mm HEPES (pH 7.4), and 40 μl of cell suspension (2 × 106 cells/ml) suspended in the same buffer were added into the small chamber (6Berg I. Potter B.V.L. Mayr G.W. Guse A.H. J. Cell Biol. 2000; 150: 581-588Crossref PubMed Scopus (148) Google Scholar), and the coverslip was mounted on the stage of a fluorescence microscope (Leica DMIRE2). In some experiments, GdCl3 (10 or 100 μm) was added to buffer A to block capacitative Ca2+ entry; in these experiments, buffer A without Na2HPO4 was used to prevent precipitation of GdPO4. Ratiometric Ca2+ imaging was performed as described recently (23Kunerth S. Mayr G.W. Koch-Nolte F. Guse A.H. Cell. Signal. 2003; 15: 783-792Crossref PubMed Scopus (34) Google Scholar). We used an Improvision imaging system (Tübingen, Germany) build around the Leica microscope at 100-fold magnification. Illumination at 340 and 380 nm was carried out using a monochromator system (Polychrom IV, TILL Photonics, Gräfelfing, Germany). Images were taken with a gray-scale CCD camera (type C 4742-95-12ER; Hamamatsu, Enfield, United Kingdom) operated in 8-bit mode. The spatial resolution was 512 × 640 pixels at 100-fold magnification. Camera exposure times were 12 ms (at 340 nm) and 4 ms (at 380 nm). The acquisition rate was adjusted to ∼14 ratios/min. Raw data images were stored on a hard disk. Confocal Ca2+ images were obtained by off-line deconvolution (no-neighbor algorithm) using the volume deconvolution module of the Openlab software as described recently for 3T3 fibroblasts (24Bruzzone S. Kunerth S. Zocchi E. De Flora A. Guse A.H. J. Cell Biol. 2003; 163: 837-845Crossref PubMed Scopus (25) Google Scholar). The deconvolved images were used to construct ratio images (340/380 nm) pixel by pixel. Finally, ratio values were converted into Ca2+ concentrations by external calibration (23Kunerth S. Mayr G.W. Koch-Nolte F. Guse A.H. Cell. Signal. 2003; 15: 783-792Crossref PubMed Scopus (34) Google Scholar). Data processing was performed using Openlab software, version 3.0.8 or 1.7.8 (Improvision). Microinjection—Microinjections were carried out as described (25Guse A.H. Berg I. da Silva C.P. Potter B.V.L. Mayr G.W. J. Biol. Chem. 1997; 272: 8546-8550Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). We used an Eppendorf system (transjector type 5246, micromanipulator type 5171, Eppendorf-Netheler-Hinz, Hamburg, Germany) with Femtotips I and II as pipettes. NAADP, InsP3, or cADPR were diluted to their final concentration in intracellular buffer (20 mm HEPES, 110 mm KCl, 10 mm NaCl, pH 7.2) and filtered (0.2 μm) before use. Injections were made using the semiautomatic mode of the system with following instrumental settings: injection pressure 60–90 hectopascals, compensatory pressure 30–50 hectopascals, injection time 0.3–0.5 s, and velocity of the pipette 700 μm/s. Staining of Lysosomes—Lysosomes were labeled by incubation of cells with 75 nm LysoTracker Red DND-99 for 30 min at room temperature. Cells were added into small chambers (see above), and fluorescence was determined at excitation and emission wavelengths of 575 and 590 nm using the Improvision imaging system as described above. Then, cells were treated either with 50 μm GPN, 10 μm cathepsin inhibitor 1, plus 50 μm GPN or with Me2SO (0.1% v/v or 0.2% v/v) for 15 min at room temperature. To analyze the effect of bafilomycin A1 on LysoTracker staining, untreated cells were incubated with different concentrations of bafilomycin A1 or 0.1% v/v Me2SO as control for 15 min at room temperature, and afterward, lysosomes were labeled with Lyso-Tracker Red DND-99 as described above (26Sun-Wada G-H. Imai-Senga Y. Yamamoto A. Murata Y. J. Biol. Chem. 2006; 277: 18098-18105Abstract Full Text Full Text PDF Scopus (97) Google Scholar). HPLC Analysis of Bafilomycin A1—RP-HPLC analysis of bafilomycin A1 was performed on a 250 × 4.6-mm Multohyp BDS C18-5μ column (CS Chromatographie Service, Langerwehe, Germany) equipped with a 4.0 × 3.0-mm guard cartridge containing a C18 (ODS) filter element (Phenomenex, Aschaffenburg, Germany). The separation was performed at a flow rate of 1 ml/min with phosphate buffer (20 mm KH2PO4, pH 6) containing increasing amounts of methanol. The gradient used for separation was as follows (number in parentheses represents the percentage of methanol): 0 min (5), 2 min (5), 16 min (100), 20 min (100), 22 min (5), 25 min (5). Bafilomycin A1 was detected using a Diode Array Detector (Agilent, Santa Clara, CA) at 246 nm. To analyze whether in Jurkat T-lymphocytes lysosomes represent a Ca2+ store sensitive to NAADP, we treated intact cells with GPN, a substrate of cathepsin C. Via osmotic lysis, the metabolic products of GPN selectively disrupt lysosomal membranes (11Churchill 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, 27Jadot M. Comant C. Wattiaux-de Conick S. Wattiaux R. Biochem. J. 1984; 219: 965-970Crossref PubMed Scopus (97) Google Scholar). The resulting significant loss of lysosome staining by LysoTracker Red was confirmed in Jurkat T cells (Fig. 1A). GPN increased the intracellular Ca2+ concentration by lysing the lysosomes (Fig. 1, B–D, right panels). As described for sea urchin eggs (11Churchill 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), GPN also abolished NAADP-mediated Ca2+ release in T cells (Fig. 1B, right panel). However, Ca2+ release by InsP3 or cADPR was eliminated, too (Fig. 1, C and D, right panels). Therefore, we used cathepsin inhibitor 1, which is known to reduce the damaging effect of GPN by inhibition of cathepsin B, L, and S and also papain released from the lysosomes. Since cathepsin inhibitor 1 does not inhibit cathepsin C, the disruption of lysosomes by GPN should be unaffected (19Gerasimenko 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). This was indeed confirmed since the intensity of lysosomes labeled with LysoTracker Red after incubation with cathepsin inhibitor 1 (10 μm) plus GPN (50 μm) was reduced similarly as compared with the control (Fig. 1A). However, treatment with cathepsin inhibitor 1 and GPN not only blocked NAADP-mediated Ca2+ release, but also, Ca2+ release by InsP3 or cADPR was abolished in Jurkat T-lymphocytes (data not shown) as shown above for GPN alone. Thus, although GPN appears to be a powerful tool to destroy lysosomes, the release of lysosomal proteases or other hydrolases seems to interfere with all Ca2+ release systems present in T cells. Since cADPR and InsP3 are supposed to target the ER, the data obtained with GPN do not allow a clear-cut decision as to whether NAADP solely acts on lysosomes. As a second approach, we investigated the effect of bafilomycin A1 on global Ca2+ signals induced by NAADP. Bafilomycin A1 is an inhibitor of vacuolar H+-pumps powered by ATP (28Docampo R. Moreno S.N. Parasitol. Today. 1999; 15: 443-448Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), prevents organelle acidification, and blocks the Ca2+ reuptake into lysosomes via Ca2+/H+-exchanger since this transport protein requires a proton gradient (11Churchill 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). As LysoTracker Red DND-99 is a weak base and accumulates in acidic compartments, preincubation with bafilomycin A1 concentration-dependently reduced or abolished labeling of lysosomes with LysoTracker Red DND-99 (26Sun-Wada G-H. Imai-Senga Y. Yamamoto A. Murata Y. J. Biol. Chem. 2006; 277: 18098-18105Abstract Full Text Full Text PDF Scopus (97) Google Scholar), a result that was confirmed in Jurkat T cells (Fig. 2A). For our experiments, we used different lots of bafilomycin A1 purchased from different suppliers. Identity, purity, and content were confirmed by HPLC (Fig. 2B). Then, cells were preincubated with increasing concentrations of bafilomycin A1 or vehicle (Me2SO 0.1% v/v) and microinjected with 100 nm NAADP (Fig. 2, C–G). No reduction of NAADP-mediated Ca2+ signaling was observed after preincubation with bafilomycin A1 at concentrations up to 1 μm (Fig. 2, C–G). In contrast, a slight increase of the NAADP-mediated Ca2+ peak at 250 nm bafilomycin A1 was detected as compared with the control (Fig. 2, E and G). Bafilomycin A1 at a concentration that almost completely abolished the lysosome H+ gradient did not affect InsP3- or cADPR-mediated Ca2+ signaling (Fig. 3). Since InsP3 and cADPR have been shown to release Ca2+ from the ER, this control experiment suggests that bafilomycin A1, in contrast to GPN (Fig. 1), in principle can be used to analyze the role of lysosomes in T cell Ca2+ signaling.FIGURE 3Effect of bafilomycin A1 (Baf) on Ca2+ release by InsP3 and cADPR in intact Jurkat T-lymphocytes. A–D, Jurkat T cells were loaded with Fura-2 and subjected to combined Ca2+ imaging and microinjection as detailed under “Experimental Procedures.” The time points of microinjections are indicated by arrows. Cells were preincubated with Me2SO (DMSO) (0.1% v/v, A and C) or 12.5 nm bafilomycin A1 (B and D) and microinjected with 4 μm InsP3 (A and B) or 100 μm cADPR (C and D).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Lysosomes are supposed to be very tight Ca2+ stores, with almost no passive leak of Ca2+ ions into the cytosol (11Churchill 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). In Jurkat T-lymphocytes, TCR-CD3 stimulation is known to increase intracellular NAADP levels (9Gasser A. Bruhn S. Guse A.H. J. Biol. Chem. 2006; 281: 16906-16913Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). If lysosomes are the organelles targeted by NAADP, stimulation via the TCR-CD3 complex in the presence of bafilomycin A1 should lead to Ca2+ depletion of lysosomal stores. As bafilomycin A1 inhibits the Ca2+ reuptake in lysosomes, a second increase of the NAADP level via microinjection should show a reduced or abolished NAADP-mediated Ca2+ release. However, Ca2+ signaling by NAADP after preincubation with bafilomycin A1 and subsequent stimulation via TCR-CD3 was not different from untreated controls, indicating no major role for bafilomycin A1-sensitive compartments in NAADP-mediated Ca2+ signaling (Fig. 4). Since no evidence for an involvement for lysosomes in NAADP-induced Ca2+ signaling was obtained, the alternative hypothesis (the ER as NAADP-sensitive Ca2+ stores) was taken into account. Although the specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+ ATPases (SERCA), thapsigargin (29Thastrup O. Cullen P.J. Drobak B.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (2978) Google Scholar), is an effective and specific tool to deplete ER/SR-type Ca2+ stores, in intact, electrically non-excitable cells, there is often the problem of capacitative Ca2+ entry being switched on immediately (30Gouy H. Cefai D. Christensen S.B. Debre P. Bismuth G. Eur. J. Immunol. 1990; 20: 2269-2275Crossref PubMed Scopus (88) Google Scholar, 31Hoth M. Penner R. Nature. 1992; 355: 353-356Crossref PubMed Scopus (1471) Google Scholar). Thus, to avoid any effects of capacitative Ca2+ entry, all further experiments were conducted in the presence of GdCl3 in the extracellular buffer. Under these conditions, microinjection of 100 nm NAADP resulted in transient elevations of [Ca2+]i with most of the cells returning to basal values within a few hundred seconds (Fig. 5A). Also, the mean amplitude of the initial peak induced by NAADP was significantly decreased in the absence of Ca2+ entry (compare Fig. 5A, right panel, with Fig. 2C, right panel). Thapsigargin also induced a transient elevation of [Ca2+]i, indicating rapid depletion of the ER and no concomitant activation of capacitative Ca2+ entry (Fig. 5B). A second activation of thapsigargin confirmed that the ER Ca2+ stores have indeed been depleted (Fig. 5B). Then, ER Ca2+ stores were depleted by thapsigargin, and NAADP was subsequently microinjected (Fig. 5C); almost none of the cells responded to NAADP (7 out of 9 cells). In a complimentary approach, the intracellular store sensitive to NAADP was partially depleted by microinjection of NAADP in the presence of GdCl3; then, the filling states of both the ER and the lysosomes were analyzed by the addition of either thapsigargin or GPN (Fig. 6). When compared with controls (injections of intracellular buffer instead of NAADP), the filling state of the ER was decreased slightly (Fig. 6, A, B, and E), whereas the content of the lysosomal store was unaffected (Fig. 6, C–E), indicating that NAADP releases Ca2+ from the ER rather than from the lysosomes.FIGURE 6Effect of NAADP on Ca2+ release by thapsigargin (Tg) or GPN in intact Jurkat T-lymphocytes. Jurkat T cells were loaded with Fura-2 and subjected to combined Ca2+ imaging and microinjection as detailed under “Experimental Procedures.” All experiments were conducted in the presence of GdCl3 (10 μm) to block capacitative Ca2+ entry. The time points of microinjections and additions of thapsigargin or GPN are indicated by arrows; the time points of thapsigargin or GPN additions were synchronized. After microinjection of 100 nm NAADP or intracellular buffer (IC) as control, thapsigargin (A and B; 1 μm) or GPN (C and D; 50 μm) was added. A quantitative comparison of mean [Ca2+]i at the peak after the addition of thapsigargin or GPN is shown in E; data represent mean values ± S.E. from the time point 400–550 s (mean values and S.E. were calculated from each individual tracing as shown in the overlays).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Although not confirmed in a great variety of cells, NAADP has been proposed to be an important second messenger in glucose-stimulated pancreatic β-cells (7Masgrau R. Churchill G.C. Morgan A.J. Ashcroft S.J. Galione A. Curr. Biol. 2003; 13: 247-251Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), in cholecystokinin-mediated Ca2+ signaling in pancreatic acinar cells (8Yamasaki 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 in Ca2+ signaling in Jurkat T cells stimulated via the TCR-CD3 complex (9Gasser A. Bruhn S. Guse A.H. J. Biol. Chem. 2006; 281: 16906-16913Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Thus, at least in these selected cell types, the NAADP/Ca2+ signaling system may play an important role for cellular function. Therefore, important modules of this signaling system are the target organelle and target Ca2+ channel of NAADP. There has been much debate as to whether NAADP acts on a novel receptor/Ca2+ channel or whether RyRs respond to NAADP directly or indirectly, e.g. via a separate binding protein (reviewed in Ref. 32Galione A. Petersen O. Mol. Interv. 2005; 5: 73-79Crossref PubMed Scopus (88) Google Scholar). For T cells, we recently demonstrated that functional RyR are required for both local and global Ca2+ signals induced by NAADP (20Langhorst M.F. Schwarzmann N. Guse A.H. Cell. Signal. 2004; 16: 1283-1289Crossref PubMed Scopus (63) Google Scholar, 21Dammermann W. Guse A.H. J. Biol. Chem. 2005; 280: 21394-21399Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Although RyR are assumed to be localized at the ER (or SR), suggesting that the NAADP-sensitive Ca2+ pool is located there, convincing data from other cell systems, including sea urchin eggs (10Lee H.C. Aarhus R. J. Cell Sci. 2000; 113: 4413-4420Crossref PubMed Google Scholar, 11Churchill 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), pancreatic acinar- and β-cells (33Yamasaki M. Masgrau R. Morgan A.J. Churchill G.C. Patel S. Ashcroft S.J. Galione A. J. Biol. Chem. 2004; 279: 7234-7240Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar), neurosecretory PC12 cells (34Brailoiu E. Churamani D Pandey V. Brailoiu G.C. Tuluc F. Patel S. Dun N.J. J. Biol. Chem. 2006; 281: 15923-15928Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), and guinea pig cardiac myocytes (35Macgregor A. Yamasaki M. Rakovic S. Sanders L. Parkesh R. Churchill G.C. Galione A. Terrar D.A. J. Biol. Chem. 2007; 10.1074/jbc.M611167200PubMed Google Scholar), prompted us to investigate involvement of a lysosomal Ca2+ store in NAADP-mediated Ca2+ signaling in T cells. The first approach, lysis of lysosomes using the cathepsin substrate GPN, has successfully been used in sea urchin eggs (11Churchill 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) and pancreatic acinar cells (33Yamasaki M. Masgrau R. Morgan A.J. Churchill G.C. Patel S. Ashcroft S.J. Galione A. J. Biol. Chem. 2004; 279: 7234-7240Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar); in sea urchin eggs, GPN-mediated bursting of lysosomes selectively abolished Ca2+ signaling by NAADP but not by InsP3 or cADPR (11Churchill 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). This result could not be reproduced in T cells; the Ca2+-mobilizing effects of all three messengers, NAADP, InsP3, or cADPR, were massively reduced. Since bursting of lysosomes is assumed to release proteases and other hydrolases into the cytosol, it is well imaginable that proteolytic attack of the InsP3 receptor or the RyR is the reason for the lack of effect of InsP3 or cADPR. Thus, although the method may work well in other cell types, such as sea urchin eggs, it appears to be not suitable for T cells. The second approach, bafilomycin A1, yielded an even better loss of staining of the lysosomes in intact Jurkat T cells (compare Fig. 1A with Fig. 2A), indicating that the H+ gradient across the lysosomal membrane was effectively destroyed. Lack of lysosomal staining was almost completed at 25 nm bafilomycin A1. However, NAADP-mediated Ca2+ signaling was not affected by up to 1000 nm bafilomycin A1 in T cells. In contrast, in sea urchin eggs, bafilomycin A1 inhibited Ca2+ release induced by NAADP in experiments where NAADP was liberated from caged NAADP subsequently two times. Interestingly, the Ca2+ release induced by the first uncaging of NAADP was not influenced, whereas the second one was largely reduced (11Churchill 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). Churchill et al. (11Churchill 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) suggested, “that the lysosomal Ca2+ store is replete and non-leaky in resting cells, and that bafilomycin sensitivity is only revealed once the store is mobilized, making its inhibition use-dependent.” However, in higher eukaryotic cell types, e.g. in pancreatic acinar cells, bafilomycin A1 inhibited cholecystokinin-induced Ca2+ spiking without prior emptying of stores (33Yamasaki M. Masgrau R. Morgan A.J. Churchill G.C. Patel S. Ashcroft S.J. Galione A. J. Biol. Chem. 2004; 279: 7234-7240Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Similarly, glucose-induced Ca2+ signaling in pancreatic β-cells (MIN6 cells) was abrogated by bafilomycin A1 (33Yamasaki M. Masgrau R. Morgan A.J. Churchill G.C. Patel S. Ashcroft S.J. Galione A. J. Biol. Chem. 2004; 279: 7234-7240Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Thus, in higher eukaryotic cells, the NAADP-sensitive Ca2+ stores obviously are not very “non-leaky.” Consequently, the lack of inhibitory effect of bafilomycin A1 on NAADP-induced Ca2+ signals in T cells (Fig. 2) strongly indicates that the NAADP-sensitive Ca2+ store of T cells is not the lysosomal compartment. To be on the safe side, and assuming that T cells are more similar to sea urchin eggs than to pancreatic cells concerning a non-leaky NAADP-sensitive Ca2+ store, a further crucial experiment was performed (Fig. 4); T cells were first stimulated via the TCR-CD3 complex in the presence of bafilomycin A1, and then NAADP was microinjected. TCR-CD3 stimulation elevates NAADP in a biphasic manner (9Gasser A. Bruhn S. Guse A.H. J. Biol. Chem. 2006; 281: 16906-16913Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), and thus, Ca2+ present in NAADP-sensitive stores was released. Due to the presence of bafilomycin A1, any reuptake of Ca2+ into acidic stores was inhibited. Nevertheless, there was no inhibition of subsequent Ca2+ signaling induced by microinjection of NAADP, ruling out the possibility that T cells possess a very tight (= non-leaky) NAADP-sensitive lysosomal Ca2+ store. In contrast to the idea that pancreatic acinar cells exclusively possess a lysosomal Ca2+ store sensitive to NAADP, Gerasimenko et al. obtained evidence for NAADP-mediated Ca2+ release from the nuclear envelope (18Gerasimenko 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) and partially from both the ER and acidic organelles (19Gerasimenko 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). In a similar approach in which the activation of capacitative Ca2+ entry was blocked by GdCl3, we emptied ER Ca2+ stores by the addition of thapsigargin. Since subsequent microinjection of NAADP did not induce Ca2+ release in most of the cells investigated, it is very likely that NAADP indeed acts on an endoplasmic reticular Ca2+ store in T cells. In contrast, in the complimentary experiment (prior microinjection of NAADP followed by the addition of thapsigargin), the effect of thapsigargin was reduced as compared with prior microinjection of intracellular buffer (Fig. 6). These data indicate that indeed the filling state of the ER was decreased by approximately 35% by prior injection of NAADP. A similar experiment carried out using GPN instead of thapsigargin to analyze the filling state of the lysosomes resulted in no difference regardless of whether the cells had been microinjected by NAADP or intracellular buffer. Taken together, our results demonstrate a major role for the ER, but not for lysosomes, in NAADP-mediated Ca2+ signaling in T cells.
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