NAADP receptors

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a recently described Ca2+ mobilizing messenger. First described in the sea urchin egg, it has been shown to mobilize Ca2+ from intracellular stores. It is a remarkably potent molecule, and recent reports show that its cellular levels change in response to a variety of agonists confirming its role as a Ca2+ mobilizing messenger. In many cases NAADP interacts with other Ca2+ mobilizing messengers such as inositol 1,4,5 trisphosphate (IP3 and cyclic adenosine diphosphate ribose (cADPR) in shaping cytosolic Ca2+ signals. What is not clear is the molecular nature of the NAADP-sensitive Ca2+ release mechanism and its sub-cellular localization. In this review we focus on the recent progress made in sea urchin eggs, which indicates that NAADP activates a novel Ca2+ release channel distinct from the relatively well-characterized IP3 and ryanodine receptors. Furthermore, in the sea urchin egg, the NAADP-sensitive store appears to be separate from the endoplasmic reticulum (ER) and is most likely an acidic store. These findings have also been reinforced by similar findings by some in mammalian cells. Finally, we discuss ongoing strategies to characterise NAADP-binding proteins which will greatly enhance our understanding of NAADP-mediated Ca2+ signalling, and lead to the development of more selective tools to probe the role of this messenger.     

Time sensing by NAADP receptors

NAADP (nicotinic acid-adenine dinucleotide phosphate) is a newly described intracellular messenger molecule that mediates Ca2+ increases in a variety of cells. However, little is known of the mechanism whereby ligand binding regulates the target protein. We report in the present paper that NAADP receptors from sea urchin eggs undergo an unusual stabilization process that appears to be dependent upon the time during which receptors are exposed to their ligand. We demonstrate that receptors 'tagged' with NAADP for short periods were more readily dissociated following subsequent delipidation than those labelled for longer. Stabilization of NAADP receptors by their ligand was delayed relative to ligand association taking on the order of minutes to develop at picomolar concentrations. The stabilizing effects of NAADP did not require cytosolic factors or the continued presence of NAADP and persisted upon solubilization. NAADP receptors, however, failed to stabilize at reduced temperature. We conclude that NAADP receptors possess a simple molecular memory endowing them with the remarkable ability to detect the duration of their activation.     

NAADP receptors are present and functional in the heart.

Alongside the well-studied inositol 1,4,5 trisphosphate and ryanodine receptors, evidence is gathering that a new intracellular release mechanism, gated by the pyridine nucleotide nicotinic acid adenine dinucleotide phosphate (NAADP), is present in numerous organisms, ranging from plant to mammalian cells (reviewed in [1]). Most cells have been shown to express at least two Ca(2+)-release mechanisms controlled by different messengers, and this can lead to redundancy, convergence, or divergence of responses. One exception appears to be muscle and heart contractile tissues. Here, it is thought that the dominant intracellular channel is the ryanodine receptor, while IP(3) receptors are poorly expressed and their role appears to be negligible. We now report that NAADP receptors are functional and abundant in cardiac microsomes. NAADP binds specifically and with high affinity (130 pM and 4 nM) to two sites on cardiac microsomes and releases Ca(2+) with an apparent EC(50) of 323 +/- 14 nM. Furthermore, binding experiments show that this receptor displays both positive and negative cooperativity, a peculiarity unique among intracellular Ca(2+) channels. Therefore, we show that the heart possesses multiple mechanisms to increase the complexity of Ca(2+) signaling and that NAADP may be integral in the functioning of this organ.     

Cyclic adenosine diphosphate ribose activates ryanodine receptors, whereas NAADP activates two-pore domain channels

The mechanism by which cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) mobilize intracellular Ca(2+) stores remains controversial. It is open to question whether cADPR regulates ryanodine receptors (RyRs) directly, as originally proposed, or indirectly by promoting Ca(2+) uptake into the sarco/endoplasmic reticulum by sarco/endoplasmic reticulum Ca(2+)-ATPases. Conversely, although we have proposed that NAADP mobilizes endolysosomal Ca(2+) stores by activating two-pore domain channels (TPCs), others suggest that NAADP directly activates RyRs. We therefore assessed Ca(2+) signals evoked by intracellular dialysis from a patch pipette of cADPR and NAADP into HEK293 cells that stably overexpress either TPC1, TPC2, RyR1, or RyR3. No change in intracellular Ca(2+) concentration was triggered by cADPR in either wild-type HEK293 cells (which are devoid of RyRs) or in cells that stably overexpress TPC1 and TPC2, respectively. By contrast, a marked Ca(2+) transient was triggered by cADPR in HEK293 cells that stably expressed RyR1 and RyR3. The Ca(2+) transient was abolished following depletion of endoplasmic reticulum stores by thapsigargin and block of RyRs by dantrolene but not following depletion of acidic Ca(2+) stores by bafilomycin. By contrast, NAADP failed to evoke a Ca(2+) transient in HEK293 cells that expressed RyR1 or RyR3, but it induced robust Ca(2+) transients in cells that stably overexpressed TPC1 or TPC2 and in a manner that was blocked following depletion of acidic stores by bafilomycin. We conclude that cADPR triggers Ca(2+) release by activating RyRs but not TPCs, whereas NAADP activates TPCs but not RyRs.     

5-Azido-8-ethynyl-NAADP: A bifunctional,clickable photoaffinity probe for the identification of NAADP receptors

Nicotinic acid adenine dinucleotide phosphate is an evolutionarily conserved second messenger, which mobilizes Ca²⁺ from acidic stores. The molecular identity of the NAADP receptor has yet to be defined. In pursuit of isolating and identifying NAADP-binding proteins, we synthesized and characterized a bifunctional probe that incorporates both a photoactivatable crosslinking azido moiety at the 5-position of the nicotinic ring and a ‘clickable’ ethynyl moiety to the 8-adenosyl position in NAADP. Microinjection of this 5N₃-8-ethynyl-NAADP into cultured U2OS cells induced robust Ca²⁺ responses. Higher concentrations of 5N₃-8-ethynyl were required to elicit Ca²⁺ release or displace ³²P-NAADP in radioligand binding experiments in sea urchin egg homogenates. In human cell extracts, incubation of ³²P-5N₃-8-ethynyl-NAADP followed by UV irradiation resulted in selective labeling of 23 kDa and 35 kDa proteins and photolabeling of these proteins was prevented when incubated in the presence of unlabeled NAADP. Compared to the monofunctional ³²P-5N₃-NAADP, the clickable ³²P-5N₃-8-ethynyl-NAADP demonstrated less labeling of the 23 kDa and 35 kDa proteins (~3-fold) but provided an opportunity for further enrichment through the ‘clickable’ ethynyl moiety. No proteins were specifically labeled by ³²P-5N₃-8-ethynyl-NAADP in sea urchin egg homogenate. These experiments demonstrate that 5N₃-8-ethynyl-NAADP is biologically active and selectively labels putative NAADP-binding proteins in mammalian systems, evidencing a ‘bifunctional’ probe with utility for isolating NAADP-binding proteins.     

Lack of effect of cADP-ribose and NAADP on the activity of skeletal muscle and heart ryanodine receptors

The calcium release channels/ryanodine receptors (RyRs) are potential/putative targets of cADPR (cyclic ADP-ribose) action in many tissue systems. In striated muscles, where RyRs predominate, cADPR action on these channels is controversial. Here cADPR modulation of cardiac and skeletal muscle RyR channels was tested. We considered factors reported as necessary for cADPR action, such as the presence of calmodulin and/or FK binding proteins (FKBPs). We found: 1) The RyR channel isoforms were insensitive to cADPR (or its metabolite NAADP [nicotinic acid adenine dinucleotide phosphate]) under all conditions examined, as studied by: 1a) single channel recordings in planar lipid bilayers; 1b) macroscopic behavior of the RyRs in sarcoplasmic reticulum (SR) microsomes (including crude microsome preparations likely to retain putative cADPR cofactors) at room temperature and at 37 degrees C (net energized Ca2+ uptake or passive Ca2+ leak); 2) [32P]cADPR did not bind significantly to SR microsomes; 3) cADPR did not affect FKBP association to SR membranes. We conclude that cADPR does not interact directly with RyRs or RyR-associated SR proteins. Our results under in vitro conditions suggest that c ADPR effects on Ca2+ signaling observed in vivo in mammalian striated muscle cells may reflect indirect modulation of RyRs or RyR-independent Ca2+ release systems.     

NAADP mobilizes Ca2+ from a thapsigargin-sensitive store in the nuclear envelope by activating ryanodine receptors

Ca2+ release from the envelope of isolated pancreatic acinar nuclei could be activated by nicotinic acid adenine dinucleotide phosphate (NAADP) as well as by inositol 1,4,5-trisphosphate (IP3) and cyclic ADP-ribose (cADPR). Each of these agents reduced the Ca2+ concentration inside the nuclear envelope, and this was associated with a transient rise in the nucleoplasmic Ca2+ concentration. NAADP released Ca2+ from the same thapsigargin-sensitive pool as IP3. The NAADP action was specific because, for example, nicotineamide adenine dinucleotide phosphate was ineffective. The Ca2+ release was unaffected by procedures interfering with acidic organelles (bafilomycin, brefeldin, and nigericin). Ryanodine blocked the Ca2+-releasing effects of NAADP, cADPR, and caffeine, but not IP3. Ruthenium red also blocked the NAADP-elicited Ca2+ release. IP3 receptor blockade did not inhibit the Ca2+ release elicited by NAADP or cADPR. The nuclear envelope contains ryanodine and IP3 receptors that can be activated separately and independently; the ryanodine receptors by either NAADP or cADPR, and the IP3 receptors by IP3.     

Activity of nicotinic acid substituted nicotinic acid adenine dinucleotide phosphate (NAADP) analogs in a human cell line: Difference in specificity between human and sea urchin NAADP receptors

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca²⁺ mobilizing second messenger that has been identified. We have previously shown that NAADP analogs substituted at the 5-position of nicotinic acid were recognized by the sea urchin receptor at low concentration, whereas the 4- substituted analogs were not as potent. However, to date the structure–activity relationship (SAR) of these analogs has not been addressed in mammalian systems. Thus, we asked whether these structurally modified analogs behave similarly in an NAADP-responsive mammalian cell line (SKBR3) using microinjection and single cell fluorescent imaging methods. Novel “caged” 4- and 5-substituted NAADP analogs that were activated inside the cell by flash photolysis resulted in Ca²⁺ mobilizing activity in SKBR3 cells in a concentration dependent manner, but with reduced effectiveness compared to unmodified NAADP. The SAR in mammalian SKBR3 cells was quite different from that of sea urchin and may suggest that there are differences between NAADP receptors in different species or tissues. Importantly, these data indicate that modifications at the 4- and 5-position of the nicotinic acid ring may lead to the development of functional photoaffinity labels that could be used for receptor localization and isolation in mammalian systems.     

NAADP regulates human platelet function

Platelets play a vital role in maintaining haemostasis. Human platelet activation depends on Ca2+ release, leading to cell activation, granule secretion and aggregation. NAADP (nicotinic acid-adenine dinucleotide phosphate) is a Ca2+-releasing second messenger that acts on acidic Ca2+ stores and is used by a number of mammalian systems. In human platelets, NAADP has been shown to release Ca2+ in permeabilized human platelets and contribute to thrombin-mediated platelet activation. In the present study, we have further characterized NAADP-mediated Ca2+ release in human platelets in response to both thrombin and the GPVI (glycoprotein VI)-specific agonist CRP (collagen-related peptide). Using a radioligand-binding assay, we reveal an NAADP-binding site in human platelets, indicative of a platelet NAADP receptor. We also found that NAADP releases loaded 45Ca2+ from intracellular stores and that total platelet Ca2+ release is inhibited by the proton ionophore nigericin. Ned-19, a novel cell-permeant NAADP receptor antagonist, competes for the NAADP-binding site in platelets and can inhibit both thrombin- and CRP-induced Ca2+ release in human platelets. Ned-19 has an inhibitory effect on platelet aggregation, secretion and spreading. In addition, Ned-19 extends the clotting time in whole-blood samples. We conclude that NAADP plays an important role in human platelet function. Furthermore, the development of Ned-19 as an NAADP receptor antagonist provides a potential avenue for platelet-targeted therapy and the regulation of thrombosis.     

Thio-NADP is not an antagonist of NAADP

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a metabolite of NADP, which can release Ca²⁺ from stores that are distinct from those activated by either cyclic ADP-ribose or inositol 1,4,5-trisphosphate (IP₃). It has previously been suggested that thio-NADP is a specific antagonist of NAADP (Chini et al. [1995]J. Biol. Chem. 270, 3216–3223). Its effects in sea-urchin egg homogenates were investigated. At 50 μM, thio-NADP activates partial Ca²⁺ release and totally inhibits subsequent challenge with a saturating concentration of NAADP. Purification by HPLC eliminates the Ca²⁺ releasing activity of 50 μM thio-NADP and reduces the subsequent inhibition by 73.7±1.3%. The residual inhibitory effect is no more than that exerted by 50 μM of either NADP itself or nicotinic acid adenine dinucleotide (NAAD). These results are confirmed by³²P-NAADP binding studies. Unpurified thio-NADP inhibits the specific³²P-NAADP binding to egg microsomes with an IC₅₀ of 40 μM. After HPLC purification, only 20% inhibition is seen at a concentration as high as 50 μM, similar to the extent of inhibition effected by 40 μM NADP. These results indicate the inhibitory substance in thio-NADP is a contaminant. The partial Ca²⁺ release activity of unpurified thio-NADP suggests the contaminant is NAADP itself. This is supported by the fact that pretreatment with a subthreshold concentration of only 2 nM NAADP totally desensitizes the egg homogenates such that no Ca²⁺ response is seen with saturating NAADP. Estimation from the binding studies shows that a contamination of 0.012% of NAADP in the unpurified thio-NADP samples is sufficient to account for the inhibitory effects. These results indicate thio-NADP is not an antagonist of NAADP.     

CD38: a NAADP degrading enzyme

The role of the multifunctional enzyme CD38 in formation of the Ca(2+)-mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) was investigated. Gene silencing of CD38 did neither inhibit NAADP synthesis in intact Jurkat T cells nor in thymus or spleen obtained from CD38 knock out mice. In vitro, both NAADP formation by base-exchange and degradation to 2-phospho adenosine diphosphoribose were efficiently decreased. Thus in vivo CD38 appears to be a NAADP degrading rather than a NAADP forming enzyme, perhaps avoiding desensitizing NAADP levels in intact cells.     

Calcium signaling by cyclic ADP-ribose and NAADP

Ca²⁺ mobilization as a signaling mechanism has been placed on center stage with the discovery of the first Ca²⁺ messenger, inositol trisphosphate (IP₃). This article focuses on two new Ca²⁺ release activators, which mobilize internal Ca²⁺ stores via mechanisms totally independent of IP₃. They are cyclic ADP-ribose (cADPR) and nicotinic acid dinucleotide phosphate (NAADP), metabolites derived respectively from NAD and NADP. Major advances in the past decade in the understanding of these two novel signaling mechanisms are chronologically summarized.     

Synthesis of NAADP and cADPR in mitochondria.

Here we investigated whether cADPR and NAADP are synthesized in mitochondria. We found that ADPR-cyclase activity is present in mitochondria. In addition, we describe for the first time synthesis of NAADP in this intracellular organelle. ADPR-cyclase activities (V(MAX)) and NAADP synthesis in mitochondria were about 4-fold lower than that in plasma membranes. Otherwise, ADPR-cyclases in mitochondria and in plasma membranes have similar catalytic properties in terms of apparent K(m) for the substrate NGD and K(i) values for inhibition by dithiotreitol, beta-NAD, and nicotinamide. ADPR-cyclase in plasma membranes and to a lesser degree mitochondrial enzyme, was inhibited by Zn(2+) and Cu(2+); ADPR-cyclase from mitochondria was more stable upon thermal inactivation. CD38 antigen, determined by Western blot, was well-expressed in plasma membranes but was far less so (17-fold less) in mitochondria. The major difference between ADPR-cyclase activity in mitochondria and plasma membranes is that mitochondrial cyclase activity was increased by incubation with nonionic detergents. Conversely, the incubation with phosphatidylinositol-specific phosphodiesterase C (PI-PLC) released ADPR-cyclase activity from plasma membranes, but not from mitochondria. We conclude that ADPR-cyclase in mitochondria and in plasma membranes are both multifunctional enzymes with similar catalytic properties; however, the two ADPR-cyclases differ in the mode of anchoring to the membrane: by glycosylphosphoinositol anchor in plasma membranes and by hydrophobic interactions in mitochondria. In addition, synthesis of NAADP can also be found in intracellular organelles via mitochondria. We propose that independent mitochondrial cADPR and NAADP systems may have an intracrine signaling function that is not dependent on direct input by extracellular hormonal stimuli, but rather responds to changes of intermediary cellular metabolism.     

Coordination of Ca2+ signalling by NAADP

Nicotinic acid adenine dinucleotide phosphate (NAADP) mobilizes intracellular Ca2+ stores in several cell types. Ample evidence suggests that NAADP activates intracellular Ca2+ channels distinct from those that are sensitive to inositol trisphosphate and ryanodine/cyclic ADP-ribose. Recent studies in intact cells have demonstrated functional coupling ('channel chatter') between Ca2+ release pathways mediated by NAADP, inositol trisphosphate and cyclic ADP-ribose. Thus, NAADP is probably an important determinant in shaping cytosolic Ca2+ signals.     

Fluorescent analogs of NAADP with calcium mobilizing activity

Nicotinic acid adenine dinucleotide phosphate (NAADP) mobilizes Ca²⁺ through a mechanism totally independent of cyclic ADP-ribose or inositol trisphosphate. Fluorescent analogs of NAADP were synthesized in this study to facilitate further characterization of this novel Ca²⁺ release mechanism. The base-exchange reaction catalyzed by ADP-ribosyl cyclase was utilized to convert nicotinamide 1,N⁶-ethenoadenine dinucleotide phosphate to a fluorescent product, nicotinic acid 1,N⁶-ethenoadenine dinucleotide phosphate (etheno-NAADP). The excitation spectrum of the product showed two maxima at 275 nm and 300 nm and an emission maximum at 410 nm. An aza derivative of etheno-NAADP was also synthesized by sequential treatments with NaOH and nitrite. The product, nicotinic acid 1,N⁶-etheno-2-aza-adenine dinucleotide phosphate (etheno-aza-NAADP) had excitation maxima at 280 nm and 360 nm and an emission maximum at 470 nm. The fluorescence of both analogs was sensitive to polarity and exhibited a 3–4-fold enhancement going from an aqueous buffer to an organic solvent. Proton-NMR measurements confirmed the presence of the etheno ring in both analogs. In the aza derivative the proton at the 2-position of the adenine ring was absent, consistent with the conversion of the 2-carbon to a nitrogen. Both analogs could activate Ca²⁺ release from sea urchin egg homogenates and the half-maximal concentrations for etheno-aza-NAADP and etheno-NAADP were at about 2.5 μM and 5 μM, respectively. At sub-threshold concentrations, both analogs could also function as antagonists, inactivating the NAADP-sensitive Ca²⁺ release with a half-maximal concentration of 60–80 nM. Microinjection of etheno-aza-NAADP into live eggs activated Ca²⁺ increase and triggered a cortical exocytotic reaction confirming its effectiveness in vivo. These fluorescent analogs are potentially useful for visualizing the novel Ca²⁺ stores that are sensitive to NAADP in live cells.     

Analogues of the Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Antagonist Ned-19 Indicate Two Binding Sites on the NAADP Receptor

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca²⁺-releasing messenger. Biological data suggest that its receptor has two binding sites: one high-affinity locking site and one low-affinity opening site. To directly address the presence and function of these putative binding sites, we synthesized and tested analogues of the NAADP antagonist Ned-19. Ned-19 itself inhibits both NAADP-mediated Ca²⁺ release and NAADP binding. A fluorometry bioassay was used to assess NAADP-mediated Ca²⁺ release, whereas a radioreceptor assay was used to assess binding to the NAADP receptor (only at the high-affinity site). In Ned-20, the fluorine is para rather than ortho as in Ned-19. Ned-20 does not inhibit NAADP-mediated Ca²⁺ release but inhibits NAADP binding. Conversely, Ned-19.4 (a methyl ester of Ned-19) inhibits NAADP-mediated Ca²⁺ release but cannot inhibit NAADP binding. Furthermore, Ned-20 prevents the self-desensitization response characteristic of NAADP in sea urchin eggs, confirming that this response is mediated by a high-affinity allosteric site to which NAADP binds in the radioreceptor assay. Collectively, these data provide the first direct evidence for two binding sites (one high- and one low-affinity) on the NAADP receptor.     

NAADP triggers the fertilization potential in starfish oocytes

In invertebrates oocytes or eggs, the fertilization or activation potential establishes the fast electrical block to polyspermy and, in some species, provides the Ca2+ influx which contributes to the following intracellular Ca2+ wave. In echinoderms, the molecule triggering the activation potential is still unknown. The aim of this study was to assess whether nicotinic acid-adenine dinucleotide phosphate (NAADP) elicited the fertilization potential in starfish oocytes. The changes in membrane potential induced by the sperm were measured in oocytes held at a low resting potential, so that the Ca2+-action potential was inactivated and only the initial slower depolarization caused by the sperm could be studied. Decreasing extracellular Na+ concentration did not prevent the onset of the fertilization potential, while removal of external Ca2+ abolished it. The pre-incubation with SK&F 96365 and verapamil and the pre-injection of BAPTA inhibited the fertilization potential, while the injection of heparin only reduced its duration. The biophysical and pharmacological properties of the sperm-elicited depolarization were similar to those displayed by the NAADP-activated Ca2+-mediated current recently described in starfish oocytes. Indeed, the desensitization of NAADP-receptors prevented the onset of the fertilization potential. Taken together, these data suggest that NAADP could trigger the fertilization potential in starfish oocytes.     

Roles of cADPR and NAADP in pancreatic cells

Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are Ca(2+)-mobilizing nucleotides that were discovered in the late 1980s. Two decades of investigations have built up a considerable understanding about these two molecules that are related because both are derived from pyridine nucleotides and known to be generated by CD38/ADP-ribosyl cyclases. cADPR has been shown to target the ryanodine receptors in the endoplasmic reticulum whereas NAADP stimulates the two-pore channels in the endo-lysosomes. Accumulating results indicate that cADPR and NAADP are second messenger molecules mediating Ca(2+) signaling activated by a wide range of agonists. This article reviews what is known about these two molecules, especially regarding their signaling roles in the pancreatic cells.     

Sperm deliver a new second messenger: NAADP

NAADP is a highly potent mobilizer of Ca(2+), which in turn triggers Ca(2+)-induced Ca(2+) release pathways in a wide range of species. Nevertheless, NAADP is not presently classified as a second messenger because it has not been shown to increase in response to a physiological stimulus. We now report a dramatic increase in NAADP during sea urchin egg fertilization that was largely due to production in sperm upon contacting egg jelly. The NAADP bolus plays a physiological role upon delivery to the egg based on its ability to induce a cortical flash, a depolarization-induced activation of L-type Ca(2+) channels. Moreover, the sperm-induced cortical flash was eliminated in eggs desensitized to NAADP. We conclude that an NAADP increase plays a physiologically relevant role during fertilization and provides the first conclusive demonstration that NAADP is a genuine second messenger.