MINIREVIEW Calcium signalling by nicotinic acid adenine dinucleotide phosphate (NAADP) Michiko Yamasaki, Grant C. Churchill and Antony Galione Department of Pharmacology, University of Oxford, UK Intracellular Ca 2+ signals are coordinated to elicit spa- tiotemporal patterns. These include repetitive Ca 2+ transients, which may be localized or propagated as regenerative waves that may also pass into neighbour- ing cells [1–3]. d-myo-Inositol 1,4,5-trisphosphate (InsP 3 ) is a well-established intracellular Ca 2+ mobil- izing messenger in many cell types [3], and is a para- digm for additional molecules that release Ca 2+ from intracellular Ca 2+ stores. Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) were first discovered in the sea urchin egg as novel Ca 2+ mobilizing agents [4–6]. In this cell, cADPR was shown to target ryanodine receptors (RyRs) to release Ca 2+ from the endoplasmic reticu- lum (ER), and now has been established as an intracel- lular messenger in several cell types [7,8]. In contrast, NAADP was found to activate a Ca 2+ release mech- anism distinct from those activated by InsP 3 and cADPR, based on pharmacology and self-induced inactivation of the different Ca 2+ release mechanisms. It has thus been of great interest to investigate the physiology, enzymology and pharmacology of the NAADP signalling pathway. Recent reports have shown increases in NAADP levels in response to cellular stim- uli fulfilling a major criterion for the classification of NAADP as a second messenger not only in sea urchin eggs but also in mammalian cells [9–12]. Here we focus on the Ca 2+ mobilizing properties of NAADP and compare them with the actions of InsP 3 and cADPR. Distinct properties of NAADP Since the discovery of NAADP as a Ca 2+ mobilizing molecule in sea urchin egg homogenates, the sea urchin egg has remained an important system in which to study the actions of NAADP. NAADP has an ability to release Ca 2+ from intracellular Ca 2+ stores and is the most potent Ca 2+ mobilizing agent described so Keywords acidic stores; cADPR; endoplasmic reticulum; InsP 3 ; NAADP Correspondence A. Galione, Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK Fax: +44 1865 271853 Tel: +44 1865 271633 E-mail: antony.galione@pharm.ox.ac.uk (Received 28 April 2005, accepted 30 June 2005) doi:10.1111/j.1742-4658.2005.04860.x Nicotinic acid adenine dinucleotide phosphate (NAADP) is a recently described Ca 2+ mobilizing messenger, and probably the most potent. We briefly review its unique properties as a Ca 2+ mobilizing agent. We present arguments for its action in targeting acidic calcium stores rather than the endoplasmic reticulum. Finally, we discuss possible biosynthetic pathways for NAADP in cells and candidates for its target Ca 2+ release channel, which has eluded identification so far. Abbreviations cADPR, cyclic ADP-ribose; CICR, Ca 2+ -induced Ca 2+ release; ER, endoplasmic reticulum; InsP 3 , D-myo-inositol 1,4,5-trisphosphate; NAADP, nicotinic acid adenine dinucleotide phosphate; RyR, ryanodine receptor. 4598 FEBS Journal 272 (2005) 4598–4606 ª 2005 FEBS far. Perhaps the most intriguing property of NAADP is its profound self-desensitization mechanism that is unparalleled by any other intracellular messenger. Sub- threshold concentrations of NAADP inactivate the NAADP evoked Ca 2+ release that normally shows a robust Ca 2+ release response [13–15]. Although similar effects have been seen in plant cell preparations [16], in intact mammalian cells only high concentrations of NAADP cause such self-desensitization [10,11,17–20], which is also interesting as this occurs in the apparent absence of any Ca 2+ release (Fig. 1). In sea urchin eggs and egg homogenates, heparin (an InsP 3 receptor antagonist), ruthenium red, pro- caine and 8-NH 2 -cADPR (ryanodine or cADPR recep- tor antagonists), inhibit InsP 3 - and cADPR-induced Ca 2+ signals, whilst the NAADP-evoked Ca 2+ release persists. In these preparations, thapsigargin, an ER Ca 2+ -ATPase inhibitor, depletes InsP 3 and ryanodine sensitive Ca 2+ stores, resulting in the inhibition of InsP 3 and cADPR responses. However, NAADP- induced Ca 2+ release remains [21]. Similar results were seen in intact sea urchin eggs when photolysing caged derivatives of these messengers. Both photoreleased InsP 3 and cADPR failed to evoke Ca 2+ release in thapsigargin-treated cells, whilst the response to photo- released NAADP remained unaffected [22,23] (Fig. 2). Pharmacological analyses extended to mammalian preparations have also confirmed the distinct nature of the NAADP-sensitive Ca 2+ release mechanism from those regulated by InsP 3 or cADPR, particularly in brain [24], and cardiac microsomes [25] as well as in arterial smooth muscle cells [26]. Furthermore, in sea urchin eggs, NAADP-sensitive Ca 2+ stores can be separated physically from thapsigargin-sensitive stores sensitive to InsP 3 and cADPR by cell fraction- ation of egg homogenates or intact egg stratification [6,27,28]. The pharmacology of NAADP-induced Ca 2+ release in sea urchin egg homogenates has been found to be different from known Ca 2+ release channels. For example, it is sensitive to l-type Ca 2+ channel inhibi- tors, such as dihydropyridines, D600 and diltiazem, and to certain K + channel blockers, without affecting Ca 2+ release via either InsP 3 or ryanodine receptors [14,21,24]. Furthermore, NAADP-mediated Ca 2+ release is neither potentiated by Ca 2+ or Sr 2+ , nor inhibited by Mg 2+ [14,21,29]. Therefore in contrast to Fig. 1. Inactivation properties of NAADP-induced release. (A) Sea urchin eggs: The top panel illustrates the unusual phenomenon whereby in sea urchin egg homogenates, a low concentration of NAADP (1 n M) that induces no apparent Ca 2+ release (right hand trace), fully desensiti- zes the NAADP receptor mechanism so that a subsequent application of a maximal concentration of NAADP (500 n M, see left hand trace) is now without effect [13,14]. (B) Mammalian cells: NAADP-induced Ca 2+ release in MIN6 cells. Percentage of the increase in normalized fluor- escence (F ⁄ F 0 %) demonstrated for each caged NAADP concentration. The inset bar symbolizes the least significant difference (LSD) that was calculated from observed errors. The numbers in brackets over the bars present number of replicates. Data are means ± SEM. The graph shows a bell-shaped concentration-response curve which is typical in mammalian systems. Higher concentrations of NAADP inactivate release by this messenger under conditions where little if any release occurs. Modified from [10]. Fig. 2. NAADP-induced Ca 2+ release from a thapsigargin-insensitive Ca 2+ store. Effect of thapsigargin on the initial response to photo- release of an InsP 3 -cADPR mixture and NAADP. Eggs were treated with thapsigargin (2 l M) for > 30 min and then exposed to UV. The final intracellular concentrations were (l M): Oregon green 488 BAPTA Dextran, 10; caged NAADP, 0.5; and both caged cADPR and caged InsP 3 , 5. Modified from [22]. M. Yamasaki et al. Calcium signalling by NAADP FEBS Journal 272 (2005) 4598–4606 ª 2005 FEBS 4599 Ca 2+ release channels modulated by either InsP 3 or cADPR that participate in Ca 2+ -induced Ca 2+ release mechanism (CICR), the NAADP-sensitive Ca 2+ release mechanism is unlikely to do so directly. The apparent inability of NAADP to induce regenerative Ca 2+ signals itself implies a role in initiating localized Ca 2+ signals, which may then be propagated by recruiting CICR mechanisms. Additional interactions of NAADP signalling pathways with Ca 2+ signals may arise since the metabolism of NAADP to inactive NAAD is regu- lated by a Ca 2+ -dependent 2¢-phosphatase [30]. Radioligand binding studies employing [ 32 P]NAADP support the idea that NAADP acts on a fundamentally different Ca 2+ releasing channel from those gated by InsP 3 or cADPR. Binding of radiolabelled NAADP to sea urchin egg homogenate membranes is highly speci- fic [13,31,32] and is unaffected by InsP 3 or cADPR [13,31]. Binding studies have revealed another peculiar property of the NAADP receptor where NAADP binds to its receptor in an essentially irreversible man- ner in the sea urchin egg homogenates [13,31,32]. In mammalian systems, however, [ 32 P]NAADP binding to membrane preparations from rat brain [31], rat heart [25] and MIN-6 cells [10] is reversible. The apparent irreversibility of NAADP binding in sea urchin egg preparations is dependent on high K + concentrations in the binding medium routinely used [15]. Ca 2+ mobilizing messengers and multiple stores Studies of the Ca 2+ mobilizing effects of NAADP in intact cells have revealed that this Ca 2+ release mech- anism rarely operates in isolation. Rather the resultant Ca 2+ signals evoked by this molecule are often boos- ted by Ca 2+ release by RyRs, InsP 3 Rs or both. Inter- actions between different Ca 2+ release mechanisms are critical for shaping Ca 2+ signals in response to agon- ists in many different cell types [33]. The effects of NAADP on Ca 2+ release are often abolished or attenuated by both heparin and 8-NH 2 -cADPR, anta- gonists for InsP 3 and cADPR receptors, indicating that the different Ca 2+ release channels are tightly coupled functionally. In sea urchin eggs, ascidian oocytes, and arterial smooth muscle, antagonists of InsP 3 Rs or RyRs reduce responses to NAADP [26,34,35], whereas in T-lymphocytes, starfish oocytes and pancreatic aci- nar cells, little effect of NAADP is seen in the presence of these inhibitors [18,36–39]. To explain these phe- nomena two models have currently been proposed. The first describes a single pool, the ER, expressing InsP 3 Rs and RyRs. Here NAADP interacts either directly with RyRs or via a separate protein that may indirectly activate RyRs [40,41]. This model accounts for the apparent complete abolition of NAADP evoked release by either RyR blockers or thapsigargin. A direct action of NAADP on RyRs is also supported by the findings that NAADP was shown to activate isolated RyRs reconstituted in lipid bilayers from rabbit skeletal muscle (RyR1) [42] and cardiac micro- somes (RyR2) [43]. A second model, the two pool or trigger hypothesis, is based on the idea that there is a distinct NAADP-sensitive storage organelle, possibly an thapsigargin-insensitive acidic store [28], that is responsible for a localized signal which is amplified by InsP 3 Rs and RyRs the on the ER by CICR [22,34,36,38]. This model accounts for the finding in some cells that localized NAADP-induced signals per- sist in the presence of InsP 3 Rs and RyR antagonists or thapsigargin, but are abolished by agents that dissipate storage of by acidic organelles, such as the vacuolar H + pump inhibitor, bafilomycin A1. This has been most clearly demonstrated in the sea urchin egg [28], but also extended to several mammalian cell types [11,44–46]. Two types of pharmacological manipula- tion of acidic stores have been investigated with regard to NAADP-evoked release. Glycyl-phenylalanyl-naph- thylamide (GPN) is an agent that penetrates cellu- lar membranes but is a substrate for the luminal lysosomal enzyme cathepsin C trapping membrane impermeant products within lysosomes resulting in disruption of lysosomal-related organelles by osmotic lysis [47]. The other approach is aimed at collapsing proton gradients thought to power Ca 2+ uptake into acidic stores by Ca 2+ ⁄ H + exchange, such as bafilo- mycin A1, FCCP and NH 3 [48]. These agents selec- tively inhibit NAADP-induced Ca 2+ release, whilst having little effect on the effects of either InsP 3 or cADPR [11,28,45,46]. Changes in endogenous levels of NAADP Only recently have NAADP levels been measured directly by using a radioreceptor assay with the NAADP binding protein from sea urchin eggs [9– 12,49] and shown to change in response to extracellu- lar stimuli [9–12]. This provided the final piece of evidence required to classify NAADP as a second mes- senger. NAADP levels have been shown to change in sea urchin sperm during activation before fertilization [9], in pancreatic beta cells in response to glucose [10], in smooth muscle cells in response to endothelin [11], and in pancreatic acinar cells in response to gut-peptide cholecystokinin [12], which has been the most detailed study so far. As outlined above, mouse Calcium signalling by NAADP M. Yamasaki et al. 4600 FEBS Journal 272 (2005) 4598–4606 ª 2005 FEBS pancreatic acinar cells have been an important system in which investigate mechanisms for the generation of intracellular Ca 2+ signals. It has been suggested that interactions between a subset and all three messengers are used to generate specific Ca 2+ signatures in response to extracellular agonists such as cholecysto- kinin and neurotransmitter acetylcholine [20,38,39,50– 52]. In this cell type, it has been proposed that an initial increase in NAADP in response to cholecysto- kinin triggers a primary Ca 2+ release, followed by recruitment of InsP 3 Rs and RyRs by CICR. Although there is much circumstantial evidence from physiologi- cal and pharmacological studies that cholecystokinin increases NAADP and cADPR levels, changes in the levels of NAADP or cADPR had not been character- ized in response to this agonist until recently. We have recently provided the strong evidence to establish NAADP as a second messenger in pancreatic acinar cells [12]. Significant elevations of both NAADP and cADPR levels in response to a specific agonist, chole- cystokinin, in a concentration-dependent manner were reported (Fig. 3). Cholecystokinin A receptors, expres- sed on mouse pancreatic acinar cells, possess two bind- ing sites for cholecystokinin, high and low-affinity binding sites [53–55]. Concentration-response data sug- gest that production of NAADP and cADPR can be Fig. 3. Effects of cholecystokinin on NAADP and cADPR production in pancreatic acinar cells. (A) Time course of cholecystokinin induced NAADP (r). Data were normalized to the maximum obtained with each individual time-course experiment. The NAADP levels reach a maxi- mum within 10 s and return to resting levels in about 60 s (n ¼ 12). (B) Concentration-response curve for cholecystokinin-induced NAADP increases (d). The data were filled to the Hill equation with two-sites (EC 50 s of 11.0 ± 3.0 pM and 830 ± 6.6 pM). Lorglumide, a cholecysto- kinin A receptor agonist, was present at 10 l M (n ¼ 3–6) (open triangles). (C) The time course of cADPR production (d) was determined in the presence of physiological concentration of cholecystokinin (10 p M). The production of cAPDR showed prolonged elevations comparing that of NAADP. Lorglumide inhibited cADPR production (m). (D) Cholecystokinin-induced cADPR elevations (j) occur in a concentration- dependent manner. Data are mean ± SEM. Modified from [12]. M. Yamasaki et al. Calcium signalling by NAADP FEBS Journal 272 (2005) 4598–4606 ª 2005 FEBS 4601 activated through both high and low-affinity sites on cholecystokinin A receptor. This same study also dem- onstrated receptor specificity for the production of NAADP and cADPR, whereby increases in cADPR levels via stimulation of acetylcholine muscarinic recep- tors as well as cholecystokinin A receptors, whereas NAADP increased only through the activation of chol- ecystokinin A. Intriguingly, the striking difference seen in time courses between NAADP and cADPR pro- duction, where the increase in NAADP was rapid and transient, whereas the increase in cADPR was much prolonged, strongly supports the proposed hypothesis that NAADP provides a localized Ca 2+ trigger signal at the apical region where InsP 3 Rs, RyRs and NAADP-sensitive Ca 2+ stores coexist [45,56–62] (Fig. 4), and subsequently this localized Ca 2+ signal is amplified by a CICR mechanism via sensitization of RyRs throughout of the cell [20,38,39,50–52,60–63]. Cell surface receptors are predominantly located in the basolateral membrane, however, agonist-induced Ca 2+ signals initiate at the apical pole before propagating into the basolateral domain by the CICR mechanism. The abundance of RyRs in the basolateral region together with the slow rise in the cADPR levels dem- onstrated in our recent report [12] may also contribute greatly to such spatiotemporal heterogeneities of Ca 2+ signals. Although there are few reports of direct NAADP measurements, an interesting correlation is emerging. Inhibition of agonist-evoked signalling by inactivating NAADP concentrations or bafilomycin A1 correlates well with receptors whose stimulation leads to eleva- tions in NAADP levels, whereas those that are not sensitive to these pharmacological manipulations are not [11,12,45]. Outstanding questions in NAADP signalling pathways There are still several important aspects of the NAADP signalling pathway that are unclear. Foremost is the nature of the NAADP receptor. Studies from the sea urchin egg system have suggested that NAADP probably acts on a distinct protein that is pharmacolo- gically different from IsnP 3 Rs of RyRs [64], although direct activation of RyRs has also been proposed [64]. The kinetics of Ca 2+ release evoked by NAADP are consistent with the gating of a Ca 2+ release channel rather than a transporter protein [65]. Preliminary biochemical characterization of [ 32 P]NAADP binding proteins from sea urchin eggs have shown that such proteins are likely to be integral membrane proteins, and probably smaller than either InsP 3 Rs or RyRs [30]. However, it is possible that the NAADP binding pro- teins may not form a pore themselves but rather inter- act with and modulate other channels. Further, in line with the multiplicity of InsP 3 Rs and RyR isoforms, it is also possible that multiple isoforms of NAADP ‘recep- tors’ exist which may go some way in reconciling con- flicting pharmacological data from different systems [64]. Perhaps the most detailed study of the functional properties of NAADP receptors, in the absence of their molecular isolation, has come from the study of NAADP signalling in starfish oocytes [37,66]. Here much emphasis has been placed on the ability of NAADP to gate a cation influx in addition to release from internal stores. In contrast to the situation in sea urchin eggs where NAADP induces a brief Ca 2+ influx (the ‘cortical flash’), followed by a more substantial mobilization [9], the starfish oocytes exhibits a pro- found Ca 2+ influx of in response to NAADP. It has been proposed that NAADP receptors may be expressed at the plasma membrane of these cells, and thus electrophysiological analyses have been employed to characterize such NAADP-induced currents [67]. An interesting question is whether these currents arise from direct activation of NAADP receptors on the plasma membrane or activation of a plasma membrane channel via calcium released from cortical NAADP-sensitive stores. Taken together these observations imply the widespread distribution of NAADP receptors and multiple roles of NAADP. However, the ultimate Fig. 4. Localization of NAADP-induced Ca 2+ signals in mouse pan- creatic acinar cells. Pancreatic acinar cells were injected with Ore- gon Green 488 BAPTA Dextran and caged NAADP (estimated final concentration: 100 n M). In response photoreleased NAADP, the local Ca 2+ spikes were confined to the apical pole (blue trace), but not to the basal pole (red trace) (n ¼ 8). These localized Ca 2+ spikes become progressively amplified. Modified from [45]. Calcium signalling by NAADP M. Yamasaki et al. 4602 FEBS Journal 272 (2005) 4598–4606 ª 2005 FEBS resolution of many these questions will require isolation of NAADP-binding proteins. It may come as some surprise that the biosynthetic pathway for NAADP synthesis is still unknown. Endogenous levels have been reported in several cell types and in some of these changes in levels in response to agonists have been reported. A favoured pathway for synthesis involves enzymes known as ADP-ribosyl cyclases (Fig. 5). As the name suggests these where first described as activities and then char- acterized as membrane proteins that cyclized NAD to form cADPR. In mammalian systems the best-charac- terized cyclases so far are CD38 and CD157 (BST-1) (Fig. 5), although other membrane-bound and soluble forms have been reported [68]. However, in vitro, these multifunctional enzymes can utilize NADP as an alter- native substrate, and at acidic pH and in the presence of nicotinic acid, they can catalyse NAADP synthesis by a mechanism involving base exchange (Fig. 5). Whether CD38 or CD157 are responsible for agonist- promoted NAADP synthesis in mammalian cells remains to be demonstrated. Two potential problems may confound this possibility. The first is that both CD38 and CD157 are largely ectoenzymes, although several reports suggest intracellular localizations as well. The second is that large, and perhaps nonphysio- logical, concentrations of nicotinic acid are required for any appreciable NAADP production by these enzymes, although high localized concentrations may be postulated. One possibility that has not been explored is that synthesis of NAADP may occur inside intracellular vesicles, or even extracellularly (as has been proposed for cADPR [69]), with the products then being transported back into the cytoplasm where it can interact with its putative targets. If these vesicles were also the acidic stores proposed as major sites for NAADP-evoked release [28], then the pH of the lumi- nal environment would also promote NAADP synthe- sis, and could also be a site for the accumulation of nicotinic acid. However, endogenous changes in NAADP levels have been reported in several systems, and we are now in a position to elucidate the mecha- nisms of NAADP production. For example, an investi- gation of agonist-induced NAADP changes in cells and tissues from CD38 ⁄ CD157 knockout mice may be informative. Other possibilities for NAADP synthetic pathways also deserving investigation are phosphory- lation of NAAD, better known as a biosynthetic pre- cursor of NAD, or a direct deamination reaction of NADP. The identification of the enzymes involved in NAADP synthesis with the recent identification of various agonists that stimulate increases in cellular NAADP levels may also lead to an understanding of the coupling mechanisms between cell surface receptors and NAADP production. Both activation of the chole- cystokinin A receptor and the ET-1 receptor have been shown to couple to NAADP synthesis. As these recep- tors are G protein coupled it is possible that G protein subunits may directly regulate enzymes catalysing NAADP production. In addition, the finding that cAMP stimulates the NAADP synthesis in the pres- ence of sea urchin membranes [70] may indicate an involvement of downstream regulators (Fig. 5). Summary NAADP has been reported to be an endogenous and potent Ca 2+ mobilizing agent in several cell types of many different organisms. NAADP evokes localized signals, which may be amplified by recruiting InsP 3 Rs and RyRs through CICR mechanisms. Changes in NAADP levels are linked to the activation of several cell surface receptors. All the criteria have now been satisfied for its recognition as an intracellular messen- ger, however, further studies required in the future are to establish the cellular mechanisms for the regulation of NAADP synthesis and metabolism as well as the molecular mechanisms mediating NAADP-induced Ca 2+ release. Fig. 5. Putative synthesis pathway for NAADP. In the presence of b-NADP, ADP- ribosyl cyclase catalyses the synthesis of NAADP by a base exchange reaction with an optimum pH of 4 [71]. CD38 and CD157 have been shown to be capable of forming NAADP under the same condition [71–73]. cAMP is a stimulator of NAADP synthesis via ADP-ribosyl cyclase [70]. M. Yamasaki et al. Calcium signalling by NAADP FEBS Journal 272 (2005) 4598–4606 ª 2005 FEBS 4603 Acknowledgements AG is a Wellcome Trust Senior Fellow in Basic Bio- medical Science; MY is a Wellcome Trust Prize Stu- dent. Work in AG and GCC’s laboratories is funded by the Wellcome Trust. References 1 Berridge MJ (1993) Inositol trisphosphate and calcium signalling. Nature 361, 315–325. 2 Thomas AP, Bird GST, Hajnoczky G, Robb-Gaspers LD & Putney J (1996) Spatial and temporal aspects of cellular calcium signaling. FASEB J 10, 1505–1517. 3 Berridge MJ, Lipp P & Bootman MD (2000) The versa- tility and universality of calcium signalling. Nat Mol Cell Biol Rev 1, 11–21. 4 Clapper DL, Walseth TF, Dargie PJ & Lee HC (1987) Pyridine nucleotide metabolites stimulate calcium release from sea urchin egg microsomes desensitized to inositol trisphosphate. J Biol Chem 262 , 9561–9568. 5 Lee HC, Walseth TF, Bratt GT, Hayes RN & Clapper DL (1989) Structural determination of a cyclic metabo- lite of NAD with intracellular calcium-mobilizing activ- ity. J Biol Chem 264, 1608–1615. 6 Lee HC, Aarhus R & Graeff RM (1995) Sensitization of calcium-induced calcium release by cyclic ADP-ribose and calmodulin. J Biol Chem 270, 9060–9066. 7 Galione A (2002) Regulation of synthesis of cADPR and NAADP. In Cyclic ADP-Ribose and NAADP: Structures, Metabolism and Functions. (Lee HC, ed), pp. 45–64. Kluwer, Dordrecht, the Netherlands. 8 Galione A & Churchill GC (2000) Cyclic ADP ribose as a calcium-mobilizing messenger. Sci STKE 2000, PE1. 9 Churchill GC, O’Neill JS, Masgrau R, Patel S, Thomas JM, Genazzani AA & Galione A (2003) Sperm deliver a new second messenger. NAADP. Curr Biol 13, 125–128. 10 Masgrau R, Churchill GC, Morgan AJ, Ashcroft SJ & Galione A (2003) NAADP. A new second messenger for glucose-induced Ca 2+ responses in clonal pancreatic beta cells. Curr Biol 13, 247–251. 11 Kinnear NP, Boittin FX, Thomas JM, Galione A & Evans AM (2004) Lysosome-sarcoplasmic reticulum junctions: a trigger zone for calcium signalling by NAADP and endothelin-1. J Biol Chem 279, 54319– 54326. 12 Yamasaki M, Thomas JM, Churchill GC, Garnham C, Lewis AM, Cancela J-M, Patel S & Galione A (2005) Role of NAADP and cADPR in the induction and maintenance of agonist-evoked Ca 2+ spiking in mouse pancreatic acinar cells. Curr Biol 15, 874–878. 13 Aarhus R, Dickey DM, Graeff RM, Gee KR, Walseth TF & Lee HC (1996) Activation and inactivation of Ca 2+ release by NAADP + . J Biol Chem 271 , 8513– 8516. 14 Genazzani AA, Empson RM & Galione A (1996) Unique inactivation properties of NAADP-sensitive Ca 2+ release. J Biol Chem 271, 11599–11602. 15 Dickinson GD & Patel S (2003) Modulation of NAADP receptors by K + ions: evidence for multiple NAADP receptor conformations. Biochem J 375, 805– 812. 16 Navazio L, Bewell MA, Siddiqua A, Dickinson GD, Galione A & Sanders D (2000) Calcium release from the endoplasmic reticulum of higher plants elicited by the NADP metabolite nicotinic acid adenine dinucleo- tide phosphate. Proc Natl Acad Sci USA 97, 8693–8698. 17 Johnson JD & Misler S (2002) Nicotinic acid-adenine dinucleotide phosphate-sensitive calcium stores initiate insulin signaling in human beta cells. Proc Natl Acad Sci USA 99, 14566–14571. 18 Berg I, Potter BV, Mayr GW & Guse AH (2000) Nico- tinic acid adenine dinucleotide phosphate (NAADP + )is an essential regulator of T-lymphocyte Ca 2+ -signaling. J Cell Biol 150, 581–588. 19 Lee HC (2001) Physiological functions of cyclic ADP- ribose and NAADP as calcium messengers. Annu Rev Pharmacol Toxicol 41, 317–345. 20 Patel S (2004) NAADP-induced Ca 2+ release – a new signalling pathway. Biol Cell 9, 19–28. 21 Genazzani AA, Mezna M, Dickey DM, Michelangeli F, Walseth TF & Galione A (1997) Pharmacological prop- erties of the Ca 2+ -release mechanism sensitive to NAADP in the sea urchin egg. Br J Pharmacol 121, 1489–1495. 22 Churchill GC & Galione A (2001) NAADP induces Ca 2+ oscillations via a two-pool mechanism by priming IP 3 - and cADPR-sensitive Ca 2+ stores. EMBO J 20, 2666–2671. 23 Churchill GC, Patel S, Thomas JM & Galione A (2002) Spatial and temporal control of Ca 2+ signalling by NAADP. In Cyclic ADP-Ribose and NAADP: Struc- tures, Metabolism and Functions. (Lee HC, ed), pp. 199– 215. Kluwer, Dordrecht, the Netherlands. 24 Bak J, White P, Timar G, Missiaen L, Genazzani AA & Galione A (1999) Nicotinic acid adenine dinucleotide phosphate triggers Ca 2+ release from brain microsomes. Curr Biol 9, 751–754. 25 Bak J, Billington RA, Timar G, Dutton AC & Genaz- zani AA (2001) NAADP receptors are present and func- tional in the heart. Curr Biol 11, 987–990. 26 Boittin FX, Galione A & Evans AM (2002) Nicotinic acid adenine dinucleotide phosphate mediates Ca 2+ sig- nals and contraction in arterial smooth muscle via a two-pool mechanism. Circ Res 91, 1168–1175. 27 Lee HC & Aarhus R (2000) Functional visualization of the separate but interacting calcium stores sensitive to Calcium signalling by NAADP M. Yamasaki et al. 4604 FEBS Journal 272 (2005) 4598–4606 ª 2005 FEBS NAADP and cyclic ADP-ribose. J Cell Sci 113, 4413– 4420. 28 Churchill GC, Okada Y, Thomas JM, Genazzani AA, Patel S & Galione A (2002) NAADP mobilizes Ca 2+ from reserve granules, a lysosome-related organelle, in sea urchin eggs. Cell 111, 703–708. 29 Chini EN & Dousa TP (1996) Nicotinate-adenine dinu- cleotide phosphate-induced Ca 2+ -release does not behave as a Ca 2+ -induced Ca 2+ -release system. Bio- chem J 316, 709–711. 30 Berridge G, Dickinson G, Parrington J, Galione A & Patel S (2002) Solubilization of receptors for the novel Ca 2+ -mobilizing messenger, nicotinic acid adenine di- nucleotide phosphate. J Biol Chem 277, 43717–43723. 31 Patel S, Churchill GC & Galione A (2000) Unique kinetics of nicotinic acid–adenine dinucleotide phos- phate (NAADP) binding enhance the sensitivity of NAADP receptors for their ligand. Biochem J 352 Part 3, 725–729. 32 Billington RA & Genazzani AA (2000) Characterization of NAADP + binding in sea urchin eggs. Biochem Bio- phys Res Commun 276, 112–116. 33 Galione A & Churchill GC (2002) Interactions between calcium release pathways: multiple messengers and mul- tiple stores. Cell Calcium 32, 343–354. 34 Churchill GC & Galione A (2000) Spatial control of Ca 2+ signaling by nicotinic acid adenine dinucleotide phosphate diffusion and gradients. J Biol Chem 275, 38687–38692. 35 Albrieux M, Lee HC & Villaz M (1998) Calcium signal- ing by cyclic ADP-ribose, NAADP, and inositol tri- sphosphate are involved in distinct functions in ascidian oocytes. J Biol Chem 273, 14566–14574. 36 Cancela JM, Churchill GC & Galione A (1999) Coordi- nation of agonist-induced Ca 2+ -signalling patterns by NAADP in pancreatic acinar cells. Nature 398, 74–76. 37 Santella L, Kyozuka K, Genazzani AA, De Riso L & Carafoli E (2000) Nicotinic acid adenine dinucleotide phosphate-induced Ca 2+ release. Interactions among distinct Ca 2+ mobilizing mechanisms in starfish oocytes. J Biol Chem 275, 8301–8306. 38 Cancela JM, Gerasimenko OV, Gerasimenko JV, Tepi- kin AV & Petersen OH (2000) Two different but con- verging messenger pathways to intracellular Ca 2+ release: the roles of nicotinic acid adenine dinucleotide phosphate, cyclic ADP-ribose and inositol trisphos- phate. EMBO J 19, 2549–2557. 39 Cancela JM, Van Coppenolle F, Galione A, Tepikin AV & Petersen OH (2002) Transformation of local Ca 2+ spikes to global Ca 2+ transients: the combinato- rial roles of multiple Ca 2+ releasing messengers. EMBO J 21, 909–919. 40 Gerasimenko JV, Maruyama Y, Yano K, Dolman NJ, Tepikin AV, Petersen OH & Gerasimenko OV (2003) NAADP mobilizes Ca 2+ from a thapsigargin-sensitive store in the nuclear envelope by activating ryanodine receptors. J Cell Biol 163, 271–282. 41 Dammermann W & Guse AH (2005) Functional ryano- dine receptor expression is required for NAADP- mediated local Ca 2+ signaling in T-lymphocytes. J Biol Chem 280, 21394–21399. 42 Hohenegger M, Suko J, Gscheidlinger R, Drobny H & Zidar A (2002) Nicotinic acid adenine dinucleotide phosphate, NAADP, activates the skeletal muscle rya- nodine receptor. Biochem J 367, 423–431. 43 Mojzisova A, Krizanova O, Zacikova L, Kominkova V & Ondrias K (2001) Effect of nicotinic acid adenine dinucleotide phosphate on ryanodine calcium release channel in heart. Pflu ¨ g Arch 441, 674–677. 44 Mitchell KJ, Lai FA & Rutter GA (2003) Ryanodine receptor type I and nicotinic acid adenine dinucleotide phosphate (NAADP) receptors mediate Ca 2+ release from insulin-containing vesicles in living pancreatic beta-cells (MIN6). J Biol Chem 278, 11057–11064. 45 Yamasaki M, Masgrau R, Morgan AJ, Churchill GC, Patel S, Ashcroft SJ & Galione A (2004) Organelle selection determines agonist-specific Ca 2+ signals in pancreatic acinar and beta cells. J Biol Chem 279, 7234– 7240. 46 Brailoiu E, Hoard JL, Filipeanu CM, Brailoiu GC, Dun SL, Patel S & Dun NJ (2005) NAADP potentiates neur- ite outgrowth. J Biol Chem 280, 5646–5650. 47 Jadot M, Andrianaivo F, Dubois F & Wattiaux R (2001) Effects of methylcyclodextrin on lysosomes. Eur J Biochem 268, 1392–1399. 48 Docampo R & Moreno SN (1999) Acidocalcisome: a novel Ca 2+ storage compartment in trypanosomatids and apicomplexan parasites. Parasitol Today 15, 443– 448. 49 Billington RA, Ho A & Genazzani AA (2002) Nicotinic acid adenine dinucleotide phosphate (NAADP) is pre- sent at micromolar concentrations in sea urchin sperma- tozoa. J Physiol 544, 107–112. 50 Patel S, Churchill GC & Galione A (2001) Coordination of Ca 2+ signalling by NAADP. Trends Biochem Sci 26, 482–489. 51 Cancela JM (2001) Specific Ca 2+ signaling evoked by cholecystokinin and acetylcholine: the roles of NAADP, cADPR, and IP 3 . Annu Rev Physiol 63, 99–117. 52 Cancela JM, Charpentier G & Petersen OH (2003) Co- ordination of Ca 2+ signalling in mammalian cells by the new Ca 2+ -releasing messenger NAADP. Pflu ¨ g Arch 446, 322–327. 53 Yule DI & Williams JA (1994) Stimulus-secretion cou- pling in the pancreatic acinus. Physiology of the Gastro- intestinal Tract (Johnson, L R, ed.), pp. 221–242. Raven Press, New York. 54 Jensen RT (1994) Receptors on pancreatic acinar cells. In Physiology of the Gastrointestinal Tract (Johnson LR, ed), pp. 1377–1446. Raven Press, New York, USA. M. Yamasaki et al. Calcium signalling by NAADP FEBS Journal 272 (2005) 4598–4606 ª 2005 FEBS 4605 55 Matozaki T, Goke B, Tsunoda Y, Rodriguez M, Marti- nez J & Williams JA (1990) Two functionally distinct cholecystokinin receptors show different modes of action on Ca 2+ mobilization and phospholipid hydro- lysis in isolated rat pancreatic acini: studies using a new cholecystokinin analog, JMV-180. J Biol Chem 15, 6247–6254. 56 Nathanson MH, Fallon MB, Padfield PJ & Maranto AR (1994) Localization of the type 3 inositol 1,4,5-tris- phosphate receptor in the Ca 2+ wave trigger zone of pancreatic acinar cells. J Biol Chem 269, 4693–4696. 57 Gerasimenko OV, Gerasimenko JV, Belan PV & Petersen OH (1996) Inositol trisphosphate and cyclic ADP-ribose-mediated release of Ca2+ from single isolated pancreatic zymogen granules. Cell 84, 473– 480. 58 Yule DI, Ernst SA, Ohnishi H & Wojcikiewicz RJ (1997) Evidence that zymogen granules are not a phy- siologically relevant calcium pool: defining the distribu- tion of inositol 1,4,5-trisphosphate receptors in pancreatic acinar cells. J Biol Chem 272, 9093–9098. 59 Lee MG, Xu X, Zeng W, Diaz J, Wojcikiewicz RJ, Kuo TH, Wuytack F, Racymaekers L & Muallem S (1997) Polarized expression of Ca 2+ channels in pancreatic and salivary gland cells: correlation with initiation and pro- pagation of [Ca 2+ ] i waves. J Biol Chem 272, 15765– 15770. 60 Leite MF, Dranoff JA, Gao L & Nathanson MH (1999) Expression and subcellular localization of the ryanodine receptor in rat pancreatic acinar cells. Bio- chem J 337, 305–309. 61 Leite MF, Burgstahler AD & Nathanson MH (2002) Ca 2+ waves require sequential activation of inositol tris- phosphate receptors and ryanodine receptors in pancre- atic acini. Gastroenterology 122, 415–427. 62 Fitzsimmons TJ, Gukovsky I, McRoberts JA, Rodri- guez E, Lai FA & Pandol SJ (2000) Multiple isoforms of the ryanodine receptor are expressed in rat pancreatic acinar cells. Biochem J 351, 265–271. 63 Straub SV, Giovannucci DR & Yule DI (2000) Calcium wave propagation in pancreatic acinar cells: functional interaction of inositol 1,4,5-trisphosphate receptors, ryanodine receptors, and mitochondria. J Gen Physiol 116, 547–560. 64 Galione A & Petersen OH (2005) The NAADP recep- tor: new receptors or new regulation? Mol Interv 5, 73–79. 65 Genazzani AA, Mezna M, Summerhill RJ, Galione A & Michelangeli F (1997) Kinetic properties of nicotinic acid adenine dinucleotide phosphate-induced Ca 2+ release. J Biol Chem 272, 7669–7675. 66 Lim D, Kyozuka K, Gragnaniello G, Carafoli E & San- tella L (2001) NAADP + initiates the Ca 2+ response during fertilization of starfish oocytes. FASEB J 15, 2257–2267. 67 Moccia F, Lim D, Kyozuka K & Santella L (2004) NAADP triggers the fertilization potential in starfish oocytes. Cell Calcium 36, 515–524. 68 Berger F, Ramirez-Hernandez MH & Ziegler M (2004) The new life of a centenarian: signalling functions of NAD(P). Trends Biochem Sci 29, 111–118. 69 De Flora A, Zocchi E, Guida L, Franco L & Bruzzone S (2004) Autocrine and paracrine calcium signaling by the CD38 ⁄ NAD+ ⁄ Cyclic ADP-ribose system. Ann NY Acad Sci 1028, 176–191. 70 Wilson HL & Galione A (1998) Differential regulation of nicotinic acid-adenine dinucleotide phosphate and cADP-ribose production by cAMP and cGMP. Biochem J 331, 837–843. 71 Aarhus R, Graeff RM, Dickey DM, Walseth TF & Lee HC (1995) ADP-ribosyl cyclase and CD38 catalyze the synthesis of a calcium- mobilizing metabolite from NADP. J Biol Chem 270, 30327–30333. 72 Hirata Y, Kimura N, Sato K, Ohsugi Y, Takasawa S, Okamoto H, Ishikawa J, Kaisho T, Ishihara K & Hir- ano T (1996) ADP ribosyl cyclase activity of a novel bone marrow stromal cell surface molecule, BST-1. FEBS Lett 356, 244–248. 73 Itoh M, Ishihara K, Hiroi T, Lee BO, Maeda H, Iijima H, Yanagita M, Kiyono H & Hirano T (1998) Deletion of bone marrow stromal cell antigen-1 (CD157) gene impaired systemic thymus independent-2 antigen- induced IgG3 and mucosal TD antigen-elicited IgA responses. J Immunol 161, 3974–3983. Calcium signalling by NAADP M. Yamasaki et al. 4606 FEBS Journal 272 (2005) 4598–4606 ª 2005 FEBS . MINIREVIEW Calcium signalling by nicotinic acid adenine dinucleotide phosphate (NAADP) Michiko Yamasaki, Grant C. Churchill. of Ca 2+ signaling by nicotinic acid adenine dinucleotide phosphate diffusion and gradients. J Biol Chem 275, 38687–38692. 35 Albrieux M, Lee HC & Villaz M (1998) Calcium