Solubilization of receptors for the novel Ca2+-mobilizing messenger,nicotinic acid adenine dinucleotide phosphate

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca(2+) mobilizing agent in a variety of broken and intact cell preparations. In sea urchin egg homogenates, NAADP releases Ca(2+) independently of inositol trisphosphate or ryanodine receptor activation. Little, however, is known concerning the molecular target for NAADP. Here we report for the first time solubilization of NAADP receptors from sea urchin egg homogenates. Supernatant fractions, prepared following Triton X-100 treatment, bound [(32)P]NAADP with similar affinity and selectivity as membrane preparations. Furthermore, the unusual non-dissociating nature of NAADP binding to its receptor was preserved upon solubilization. NAADP receptors could also be released into supernatant fractions upon detergent treatment of membranes prelabeled with [(32)P]NAADP. Tagged receptors prepared in this way, were readily resolved by native gel electrophoresis as a single protein target. Gel filtration and sucrose density gradient centrifugation analysis indicates that NAADP receptors are substantially smaller than inositol trisphosphate or ryanodine receptors, providing further biochemical evidence that NAADP activates a novel intracellular Ca(2+) release channel.     

Widespread distribution of binding sites for the novel Ca2+-mobilizing messenger, nicotinic acid adenine dinucleotide phosphate, in the brain

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca(2+)-mobilizing agent in invertebrate eggs that has recently been shown to be active in certain mammalian and plant systems. Little, however, is known concerning the properties of putative NAADP receptors. Here, for the first time, we report binding sites for NAADP in brain. In contrast to sea urchin egg homogenates, [(32)P]NAADP bound reversibly to multiple sites in brain membranes. The rank order of potency of NAADP, 2',3'-cyclic NAADP and 3'-NAADP in displacing [(32)P]NAADP was, however, the same in the two systems and in agreement with their ability to mobilize Ca(2+) from homogenates. These data indicate that [(32)P]NAADP likely binds to receptors mediating Ca(2+) mobilization. Autoradiography revealed striking heterogeneity in the distribution of [(32)P]NAADP binding sites throughout the brain. Our data strongly support a role for NAADP-induced Ca(2+) signaling in the brain.     

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.     

Functional ryanodine receptor expression is required for NAADP-mediated local Ca2+ signaling in T-lymphocytes

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

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.     

NAADP mediates ATP-induced Ca2+ signals in astrocytes

Intracellular Ca(2+) signals provide astrocytes with a specific form of excitability that enables them to regulate synaptic transmission. In this study, we demonstrate that NAADP-AM, a membrane-permeant analogue of the new second messenger nicotinic acid-adenine dinucleotide phosphate (NAADP), mobilizes Ca(2+) in astrocytes and that the response is blocked by Ned-19, an antagonist of NAADP signalling. We also show that NAADP receptors are expressed in lysosome-related acidic vesicles. Pharmacological disruption of either NAADP or lysosomal signalling reduced Ca(2+) responses induced by ATP and endothelin-1, but not by bradykinin. Furthermore, ATP increased endogenous NAADP levels. Overall, our data provide evidence for NAADP being an intracellular messenger for agonist-mediated calcium signalling in astrocytes.     

Role of the nicotinic acid group in NAADP receptor selectivity

Nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to be an intracellular Ca2+-releasing messenger in a wide variety of systems to date. Its actions are both potent and highly specific despite differing structurally from the endogenous cellular co-factor and its precursor, NADP, only in the substitution of a hydroxyl for the amine group at the 3' position of the pyridine ring. This substitution allows NAADP to bind to a membrane-localized binding site in sea urchin egg homogenates with an IC50 at least 1000-fold greater than that of NADP as measured by competition radioligand binding assays. This suggests that the NAADP receptor protein must include certain features in the NAADP binding site that regulate this specificity. In order to investigate this interaction, we synthesised a series of NAADP analogues differing from NAADP at the 3' position of the pyridine ring that included both simple carboxylic acid analogues as well as a series of chemical isosters. We then investigated both their affinity for the NAADP binding site in sea urchin egg homogenates and their ability to activate the NAADP sensitive Ca2+ channel. We hereby show that a negative charge at the 3' position is an important determinant of affinity but the protein displays a large tolerance for the size of the group. Furthermore, the protein does not easily accommodate multiple charged groups or large uncharged groups.     

Autocrine/Paracrine Function of Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) for Glucose Homeostasis in Pancreatic β-Cells and Adipocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger for mobilizing Ca²⁺ from intracellular stores in various cell types. Extracellular application of NAADP has been shown to elicit intracellular Ca²⁺ signals, indicating that it is readily transported into cells. However, little is known about the functional role of this NAADP uptake system. Here, we show that NAADP is effectively transported into selected cell types involved in glucose homeostasis, such as adipocytes and pancreatic β-cells, but not the acinar cells, in a high glucose-dependent manner. NAADP uptake was inhibitable by Ned-19, a NAADP mimic; dipyridamole, a nucleoside inhibitor; or NaN₃, a metabolic inhibitor or under Ca²⁺-free conditions. Furthermore, NAADP was found to be released from pancreatic islets upon stimulation by high glucose. Consistently, administration of NAADP to type 2 diabetic mice improved glucose tolerance. We propose that NAADP is functioning as an autocrine/paracrine hormone important in glucose homeostasis. NAADP is thus a potential antidiabetic agent with therapeutic relevance.     

Ca2+ release via ryanodine receptors and Ca2+ entry: major mechanisms in NAADP-mediated Ca2+ signaling in T-lymphocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+ mobilizing nucleotide essentially involved in T cell activation. Using combined microinjection and single cell calcium imaging, we demonstrate that co-injection of NAADP and the D-myo-inositol 1,4,5-trisphosphate antagonist heparin did not inhibit Ca2+ mobilization. In contrast, co-injection of the ryanodine receptor antagonist ruthenium red efficiently blocked NAADP induced Ca2+ signalling. This pharmacological approach was confirmed using T cell clones stably transfected with plasmids expressing antisense mRNA targeted specifically against ryanodine receptors. NAADP induced Ca2+ signaling was strongly reduced in these clones. In addition, inhibition of Ca2+ entry by SK&F 96365 resulted in a dramatically decreased Ca2+ signal upon NAADP injection. Gd3+, a known blocker of Ca2+ release activated Ca2+ entry, only partially inhibited NAADP mediated Ca2+ signaling. These data indicate that in T cells (i) ryanodine receptor are the major intracellular Ca2+ release channels involved in NAADP induced Ca2+ signals, and that (ii) such Ca2+ release events are largely amplified by Ca2+ entry.     

TPC2 Is a Novel NAADP-sensitive Ca2+ Release Channel, Operating as a Dual Sensor of Luminal pH and Ca2+

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a molecule capable of initiating the release of intracellular Ca²⁺ required for many essential cellular processes. Recent evidence links two-pore channels (TPCs) with NAADP-induced release of Ca²⁺ from lysosome-like acidic organelles; however, there has been no direct demonstration that TPCs can act as NAADP-sensitive Ca²⁺ release channels. Controversial evidence also proposes ryanodine receptors as the primary target of NAADP. We show that TPC2, the major lysosomal targeted isoform, is a cation channel with selectivity for Ca²⁺ that will enable it to act as a Ca²⁺ release channel in the cellular environment. NAADP opens TPC2 channels in a concentration-dependent manner, binding to high affinity activation and low affinity inhibition sites. At the core of this process is the luminal environment of the channel. The sensitivity of TPC2 to NAADP is steeply dependent on the luminal [Ca²⁺] allowing extremely low levels of NAADP to open the channel. In parallel, luminal pH controls NAADP affinity for TPC2 by switching from reversible activation of TPC2 at low pH to irreversible activation at neutral pH. Further evidence earmarking TPCs as the likely pathway for NAADP-induced intracellular Ca²⁺ release is obtained from the use of Ned-19, the selective blocker of cellular NAADP-induced Ca²⁺ release. Ned-19 antagonizes NAADP-activation of TPC2 in a non-competitive manner at 1 μm but potentiates NAADP activation at nanomolar concentrations. This single-channel study provides a long awaited molecular basis for the peculiar mechanistic features of NAADP signaling and a framework for understanding how NAADP can mediate key physiological events.     

Effect of luminal and extravesicular Ca2+ on NAADP binding and release properties

Nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to be a powerful Ca2+ release agent in numerous systems, including echinoderms, plants, and mammalian cells. NAADP has been shown to release Ca2+ via a separate mechanism to IP3 and ryanodine receptors, and specific binding sites have recently been characterised. However, functional studies have shown that there is a functional interplay between the NAADP-sensitive mechanism and the other two. In particular, it appears that activation of the NAADP receptor might act as a trigger to facilitate responses from IP3 and ryanodine receptors. To further characterise this interplay, we have investigated the effects of luminal and cytosolic Ca2+ on the NAADP receptor in sea urchin egg homogenates. We report that neither cytosolic nor luminal Ca2+ appears to influence NAADP binding. Conversely, emptying of stores significantly amplifies NAADP-induced fractional Ca2+-release, providing a mechanism of self-adjustment independent of store loading.     

Extracellular application of nicotinic acid adenine dinucleotide phosphate induces Ca2+ signaling in astrocytes in situ

Nicotinic acid adenine dinucleotide phosphate (NAADP+) has been identified as a novel second messenger triggering Ca2+ release from intracellular stores. Here we report that murine cortical astrocytes in culture and in acute slices respond with transient intracellular Ca2+ increases to extracellularly applied NAADP+ and express the NAADP+-producing enzyme CD38. The Ca2+ transients triggered by NAADP+ occurred with an average delay of 35 s as compared with ATP-triggered Ca2+ signaling, suggesting that NAADP+ may have to enter the cell to act. Blockage of connexin hemichannels (a possible entry route for NAADP+ into the cell) reduced the number of astrocytes responding to NAADP+. Disruption of lysosomes as the suggested site of NAADP+ receptors reduced the number of astrocytes responding to NAADP+ strongly. The NAADP+-triggered Ca2+ signal also depended on intact endoplasmic reticulum Ca2+ stores linked to activation of inositol 1,4,5-trisphosphate receptors and on the activity of voltage-gated Ca2+ channels. Adenosine receptor-mediated signaling contributes to the NAADP+-evoked signal, since it is strongly reduced by the adenosine receptor blocker CGS-15943. Moreover, NAADP+ triggered responses in all other cell types (cultured cerebellar neurons, microglia, and oligodendrocytes) of the central nervous system.     

Pharmacological characterization of NAADP-induced Ca2+ signals in starfish oocytes

The recently discovered second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) is central to the onset of intracellular Ca2+ signals induced by several stimuli, including fertilization. The nature of the Ca2+ pool mobilized by NAADP is still controversial. Depending on the cell type, NAADP may target either an acidic compartment with lysosomal properties or ryanodine receptors (RyRs) on endoplasmic reticulum. In addition, NAADP elicits a robust Ca2+ influx into starfish oocytes by activating a Ca2+-mediated current across the plasma membrane. In the present study, we employed the single-electrode intracellular recording technique to assess the involvement of either acidic organelles or RyRs in NAADP-elicited Ca2+ entry. We found that neither drugs which interfere with acidic compartments nor inhibitors of RyRs affected NAADP-induced depolarization. These data further support the hypothesis that a yet unidentified plasma membrane Ca2+ channel is the target of NAADP in starfish oocytes.     

Nicotinic acid adenine dinucleotide phosphate-induced Ca(2+) release. Interactions among distinct Ca(2+) mobilizing mechanisms in starfish oocytes

An intracellular mechanism activated by nicotinic acid adenine dinucleotide phosphate (NAADP(+)) contributes to intracellular Ca(2+) release alongside inositol 1,4,5-trisphosphate (Ins-P(3)) and ryanodine receptors. The NAADP(+)-sensitive mechanism has been shown to be operative in sea urchin eggs, ascidian eggs, and pancreatic acinar cells. Furthermore, most mammalian cell types can synthesize NAADP(+), with nicotinic acid and NADP(+) as precursors. In this contribution, NAADP(+)-induced Ca(2+) release has been investigated in starfish oocytes. Uncaging of injected NAADP(+) induced Ca(2+) mobilization in both immature oocytes and in oocytes matured by the hormone 1-methyladenine (1-MA). The role of extracellular Ca(2+) in NAADP(+)-induced Ca(2+) mobilization, which was minor in immature oocytes, was instead essential in mature oocytes. Thus, the NAADP(+)-sensitive Ca(2+) pool, which is known to be distinct from those sensitive to inositol 1,4,5-trisphosphate or cyclic ADPribose, apparently migrated closer to (or became part of) the plasma membrane during the maturation process. Inhibition of both Ins-P(3) and ryanodine receptors, but not of either alone, substantially inhibited NAADP(+)-induced Ca(2+) mobilization in both immature and mature oocytes. The data also suggest that NAADP(+)-induced Ca(2+) mobilization acted as a trigger for Ca(2+) release via Ins-P(3) and ryanodine receptors.     

Spatial control of Ca2+ signaling by nicotinic acid adenine dinucleotide phosphate diffusion and gradients

Intracellular Ca(2+) is able to control numerous cellular responses through complex spatiotemporal organization. Ca(2+) waves mediated by inositol trisphosphate or ryanodine receptors propagate by Ca(2+)-induced Ca(2+) release and therefore do not have an absolute requirement for a gradient in either inositol trisphosphate or cyclic ADP-ribose, respectively. In contrast, we report that although Ca(2+) increases induced by nicotinic acid adenine dinucleotide phosphate (NAADP) are amplified by Ca(2+)-induced Ca(2+) release locally, Ca(2+) waves mediated by NAADP have an absolute requirement for an NAADP gradient. If NAADP is increased such that its concentration is spatially uniform in one region of an egg, the Ca(2+) increase occurs simultaneously throughout this area, and only where there is diffusion out of this area to establish an NAADP gradient is there a Ca(2+) wave. A local increase in NAADP results in a Ca(2+) increase that spreads by NAADP diffusion. NAADP diffusion is restricted at low but not high concentrations of NAADP, indicating that NAADP diffusion is strongly influenced by binding to immobile and saturable sites, probably the NAADP receptor itself. Thus, the range of action of NAADP can be tuned by its concentration from that of a local messenger, like Ca(2+), to that of a global messenger, like IP(3) or cyclic ADP-ribose.     

Two neuropeptides recruit different messenger pathways to evoke Ca2+ signals in the same cell

Bombesin and cholecystokinin (CCK) peptides act as signalling molecules in both the central nervous system and gastrointestinal tract [1-4]. It was reported recently that nicotinic acid adenine dinucleotide phosphate (NAADP) releases Ca2+ from mammalian brain microsomes [5] and triggers Ca2+ signals in pancreatic acinar cells, where it is proposed to mediate CCK-evoked Ca2+ signals [6]. Here, for the first time, we have finely resolved bombesin-induced cytosolic Ca2+ oscillations in single pancreatic acinar cells by whole-cell patch-clamp monitoring of Ca2+-dependent ionic currents [6-8]. Picomolar concentrations of bombesin and CCK evoked similar patterns of cytosolic Ca2+ oscillations, but high, desensitising, NAADP concentrations selectively inhibited CCK, but not bombesin-evoked signals. Inhibiting inositol trisphosphate (IP3) receptors with a high concentration of caffeine blocked both types of oscillations. We further tested whether NAADP is involved in Ca2+ signals triggered by activation of the low-affinity CCK receptor sites. Nanomolar concentrations of CCK evoked non-oscillatory Ca2+ signals, which were not affected by desensitising NAADP receptors. Our results suggest that Ca2+-release channels gated by the novel Ca2+-mobilising molecule NAADP are only essential in specific Ca2+-mobilising pathways, whereas the IP3 receptors are generally required for Ca2+ signals. Thus, the same cell may use different combinations of intracellular Ca2+-releasing messengers to encode different external messages.     

PPADS is a reversible competitive antagonist of the NAADP receptor

NAADP has been shown to act as a second messenger in a wide range of systems from plants to mammalian cells. Although it had always been considered as a canonical second messenger, recent work has shown that it is also active when applied extracellularly. It has also been suggested that NAADP might have a direct action on P2 receptors, based on the action of a pharmacological agent, PPADS, on Ca2+ signals in response to extracellular NAADP. We have therefore investigated whether PPADS can act directly on the intracellular NAADP-induced Ca2+-release system in the well characterised sea urchin egg homogenate system. Indeed, PPADS, and its structural analogue PPNDS were able to compete with [32P]NAADP for the binding site and binding curves revealed that both compounds display affinities in the low micromolar range. The binding of PPADS was reversible in contrast to that of NAADP. In fluorimetric Ca2+-release experiments, PPADS was able to competitively antagonise NAADP-induced Ca2+-release with an IC50 of 20 microM, while it did not affect the other Ca2+-release channels. This is the first report of a reversible, competitive antagonist of the sea urchin NAADP receptor. Furthermore, PPADS might reveal itself as an invaluable tool to investigate NAADP signalling and is a lead compound for the synthesis of potent and specific antagonists.     

Second messenger function of nicotinic acid adenine dinucleotide phosphate revealed by an improved enzymatic cycling assay

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent activator of Ca2+ release from intracellular stores known today. Although recent reports have suggested an important function of NAADP in human T lymphocytes, direct evidence for receptor-induced formation of NAADP is yet missing in these cells. Thus, we developed a highly sensitive and specific enzyme assay capable of quantifying low fmol amounts of NAADP. In unstimulated T cells, the NAADP concentration amounted to 4.4 +/- 1.6 nm (0.055 +/- 0.028 pmol/mg of protein). Stimulation of the cells via the T cell receptor/CD3 complex resulted in biphasic elevation kinetics of cellular NAADP levels and was characterized by a bell-shaped concentration-response curve for NAADP. In contrast, the NAADP concentration was elevated neither upon activation of the ADP-ribose/TRPM2 channel Ca2+ signaling system nor by an increase of the intracellular Ca2+ concentration upon thapsigargin stimulation. T cell receptor/CD3 complex-mediated NAADP formation was dependent on the activity of tyrosine kinases because genistein completely blocked NAADP elevation. Thus, we propose a regulated formation of NAADP upon specific stimulation of the T cell receptor/CD3 complex, suggesting a function of NAADP as a Ca2+-mobilizing second messenger during T cell activation.     

Nicotinic acid adenine dinucleotide phosphate potentiates neurite outgrowth

Ca(2+) regulates a spectrum of cellular processes including many aspects of neuronal function. Ca(2+)-sensitive events such as neurite extension and axonal guidance are driven by Ca(2+) signals that are precisely organized in both time and space. These complex cues result from both Ca(2+) influx across the plasma membrane and the mobilization of intracellular Ca(2+) stores. In the present study, using rat cortical neurons, we have examined the effects of the novel intracellular Ca(2+)-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) on neurite length and cytosolic Ca(2+) levels. We show that NAADP potentiates neurite extension in response to serum and nerve growth factor and stimulates increases in cytosolic Ca(2+) from bafilomycin-sensitive Ca(2+) stores. Simultaneous blockade of inositol trisphosphate and ryanodine receptors abolished the effects of NAADP on neurite length and reduced the magnitude of NAADP-mediated Ca(2+) signals. This is the first report demonstrating functional NAADP receptors in a mammalian neuron. Interplay between NAADP receptors and more established intracellular Ca(2+) channels may therefore play important signaling roles in the nervous system.     

Involvement of PI3K in HCV-related lymphoproliferative disorders

We have investigated the role of NAADP-mediated Ca(2+) mobilization in endothelin (ET) signaling via endothelin receptor subtype A (ETA) and endothelin receptor subtype B (ETB) in rat peritubular smooth muscle cells. Microinjection and extracellular application of NAADP were both able to elicit Ca(2+) release which was blocked by inhibitory concentrations of NAADP, by impairing Ca(2+) uptake in acidic stores with bafilomycin, and by thapsigargin. Ca(2+) release in response to selective ETB stimulation was abolished by inhibition of NAADP signaling through the same strategies, while these treatments only partially impaired ETA-dependent Ca(2+) signaling, showing that transduction of the ETB signal is dependent on NAADP. In addition, we show that lipid rafts/caveolae contain ETA, ETB, and NAADP/cADPR generating enzyme CD38 and that stimulation of ETB receptors results in increased CD38 activity; interestingly, ETB- (but not ETA-) mediated Ca(2+) responses were antagonized by disruption of lipid rafts/caveolae with methyl-beta-cyclodextrin. These data demonstrate a primary role of NAADP in ETB-mediated Ca(2+) signaling and strongly suggest a novel role of lipid rafts/caveolae in triggering ET-induced NAADP signaling.