Calcium mobilization by nicotinic acid adenine dinucleotide phosphate (NAADP) in rat astrocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to release intracellular Ca(2+) in several types of cells. We have used Ca(2+)-sensitive fluorescent dyes (Fura-2, Fluo-4) to measure intracellular Ca(2+) in astrocytes in culture and in situ. Bath-applied NAADP elicited a reversible and concentration-dependent Ca(2+) rise in up to 90% of astrocytes in culture (EC(50)=7 microM). The NAADP-evoked Ca(2+) rise was maintained in the absence of extracellular Ca(2+), but was suppressed after depleting the Ca(2+) stores of the ER with ATP (20 microM), with cyclopiazonic acid (10 microM) or with ionomycin (5 microM). P(2) receptor antagonist pyridoxalphosphate-6-azophenyl-2'4'-disulfonic acid (PPADS, 100 microM), IP(3) receptor blocker 2-aminoethoxydiphenyl borate (2-APB, 100 microM) and PLC inhibitor U73122 (10 microM) also reduced or suppressed the NAADP-evoked Ca(2+) rise. NAADP still evoked a Ca(2+) response after application of glycyl-l-phenylalanine-beta-naphthylamide (GPN, 200 microM), which permeabilizes lysosomes, or preincubation with H(+)-ATPase inhibitor bafilomycin A1 (4 microM) and of p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP, 2 microM), that impairs mitochondrial Ca(2+) handling. In acute brain slices, NAADP (10 microM) evoked Ca(2+) transients in cerebellar Bergmann glial cells and in hippocampal astrocytes. Our results suggest that NAADP recruits Ca(2+) from inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores in mammalian astrocytes, at least partly by activating metabotropic P(2)Y receptors.     

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.     

Production of NAADP and its role in Ca2+ mobilization associated with lysosomes in coronary arterial myocytes

The present study was designed to determine the production of nicotinic acid adenine dinucleotide phosphate (NAADP) and its role associated with lysosomes in mediating endothelin-1 (ET-1)-induced vasoconstriction in coronary arteries. HPLC assay showed that NAADP was produced in coronary arterial smooth muscle cells (CASMCs) via endogenous ADP-ribosyl cyclase. Fluorescence microscopic analysis of intracellular Ca2+ concentration ([Ca2+]i) in CASMCs revealed that exogenous 100 nM NAADP increased [Ca2+]i by 711 +/- 47 nM. Lipid bilayer experiments, however, demonstrated that NAADP did not directly activate ryanodine (Rya) receptor Ca2+ release channels on the sarcoplasmic reticulum. In CASMCs pretreated with 100 nM bafilomycin A1 (Baf), an inhibitor of lysosomal Ca2+ release and vacuolar proton pump function, NAADP-induced [Ca2+]i increase was significantly abolished. Moreover, ET-1 significantly increased NAADP formation in CASMCs and resulted in the rise of [Ca2+]i in these cells with a large increase in global Ca2+ level of 1,815 +/- 84 nM. Interestingly, before this large Ca2+ increase, a small Ca2+ spike with an increase in [Ca2+]i of 529 +/- 32 nM was observed. In the presence of Baf (100 nM), this ET-1-induced two-phase [Ca2+]i response was completely abolished, whereas Rya (50 microM) only markedly blocked the ET-1-induced large global Ca2+ increase. Functional studies showed that 100 nM Baf significantly attenuated ET-1-induced maximal constriction from 82.26 +/- 4.42% to 51.80 +/- 4.36%. Our results suggest that a lysosome-mediated Ca2+ regulatory mechanism via NAADP contributes to ET-1-induced Ca2+ mobilization in CASMCs and consequent vasoconstriction of coronary arteries.     

Regulation of TRPM2 by extra- and intracellular calcium

TRPM2 is a calcium-permeable nonselective cation channel that is opened by the binding of ADP-ribose (ADPR) to a C-terminal nudix domain. Channel activity is further regulated by several cytosolic factors, including cyclic ADPR (cADPR), nicotinamide adenine dinucleotide phosphate (NAADP), Ca(2+) and calmodulin (CaM), and adenosine monophosphate (AMP). In addition, intracellular ions typically used in patch-clamp experiments such as Cs(+) or Na(+) can alter ADPR sensitivity and voltage dependence, complicating the evaluation of the roles of the various modulators in a physiological context. We investigated the roles of extra- and intracellular Ca(2+) as well as CaM as modulators of ADPR-induced TRPM2 currents under more physiological conditions, using K(+)-based internal saline in patch-clamp experiments performed on human TRPM2 expressed in HEK293 cells. Our results show that in the absence of Ca(2+), both internally and externally, ADPR alone cannot induce cation currents. In the absence of extracellular Ca(2+), a minimum of 30 nM internal Ca(2+) is required to cause partial TRPM2 activation with ADPR. However, 200 microM external Ca(2+) is as efficient as 1 mM Ca(2+) in TRPM2 activation, indicating an external Ca(2+) binding site important for proper channel function. Ca(2+) facilitates ADPR gating with a half-maximal effective concentration of 50 nM and this is independent of extracellular Ca(2+). Furthermore, TRPM2 currents inactivate if intracellular Ca(2+) levels fall below 100 nM irrespective of extracellular Ca(2+). The facilitatory effect of intracellular Ca(2+) is not mimicked by Mg(2+), Ba(2+), or Zn(2+). Only Sr(2+) facilitates TRPM2 as effectively as Ca(2+), but this is due to Sr(2+)-induced Ca(2+) release from internal stores rather than a direct effect of Sr(2+) itself. Together, these data demonstrate that cytosolic Ca(2+) regulates TRPM2 channel activation. Its facilitatory action likely occurs via CaM, since the addition of 100 microM CaM to the patch pipette significantly enhances ADPR-induced TRPM2 currents at fixed [Ca(2+)](i) and this can be counteracted by calmidazolium. We conclude that ADPR is responsible for TRPM2 gating and Ca(2+) facilitates activation via calmodulin.     

NAADP: a new second messenger for glucose-induced Ca2+ responses in clonal pancreatic beta cells

Important questions remain concerning how elevated blood glucose levels are coupled to insulin secretion from pancreatic beta cells and how this process is impaired in type 2 diabetes. Glucose uptake and metabolism in beta cells cause the intracellular Ca(2+) concentration ([Ca(2+)](i)) to increase to a degree necessary and sufficient for triggering insulin release. Although both Ca(2+) influx and Ca(2+) release from internal stores are critical, the roles of inositol 1,4,5-trisphosphate (IP(3)) and cyclic adenosine dinucleotide phosphate ribose (cADPR) in regulating the latter have proven equivocal. Here we show that glucose also increases [Ca(2+)](i) via the novel Ca(2+)-mobilizing agent nicotinic acid adenine dinucleotide phosphate (NAADP) in the insulin-secreting beta-cell line MIN6. NAADP binds to specific, high-affinity membrane binding sites and at low concentrations elicits robust Ca(2+) responses in intact cells. Higher concentrations of NAADP inactivate NAADP receptors and attenuate the glucose-induced Ca(2+) increases. Importantly, glucose stimulation increases endogenous NAADP levels, providing strong evidence for recruitment of this pathway. In conclusion, our results support a model in which NAADP mediates glucose-induced Ca(2+) signaling in pancreatic beta cells and are the first demonstration in mammalian cells of the presence of endogenous NAADP levels that can be regulated by a physiological stimulus.     

Prolonged inactivation of nicotinic acid adenine dinucleotide phosphate-induced Ca2+ release mediates a spatiotemporal Ca2+ memory

Although numerous extracellular stimuli are coupled to increases in intracellular Ca(2+), different stimuli are thought to achieve specificity by eliciting different spatiotemporal Ca(2+) increases. We investigated the effect of nicotinic acid adenine dinucleotide phosphate (NAADP) inactivation on spatiotemporal Ca(2+) signals in intact sea urchin eggs. The photorelease of NAADP but not inositol 1,4,5-trisphosphate or cyclic ADP-ribose resulted in self-inactivation. When NAADP was released first locally and subsequently globally, the spatial pattern of the first response shaped that of the second. Specifically, the local release of NAADP created a Ca(2+) gradient that was reversed during the subsequent global release of NAADP. Neither cyclic ADP-ribose nor inositol 1,4,5-trisphosphate showed a similar effect. In contrast to homogenates, NAADP inactivation was reversible in intact eggs with resensitization occurring in approximately 20 min. Because initial NAADP responses affect later responses, NAADP can serve as a mechanism for a Ca(2+) memory that has both spatial and temporal components. This NAADP-mediated Ca(2+) memory provides a novel mechanism for cells to control spatiotemporal Ca(2+) increases.     

Reconstitution of lysosomal NAADP-TRP-ML1 signaling pathway and its function in TRP-ML1(-/-) cells

It is well known that the mutation of TRP-ML1 (transient receptor potential-mucolipin-1) causes mucolipidosis IV, a lysosomal storage disease. Given that lysosomal nicotinic acid adenine dinucleotide phosphate (NAADP)-Ca(2+) release channel activity is associated with TRP-ML1, the present study was designed to test the hypothesis that NAADP regulates lysosome function via activation of TRP-ML1 channel activity. Using lysosomal preparations from wild-type (TRP-ML1(+/+)) human fibroblasts, channel reconstitution experiments demonstrated that NAADP (0.01-1.0 μM) produced a concentration-dependent increase in TRP-ML1 channel activity. This NAADP-induced activation of TRP-ML1 channels could not be observed in lysosomes from TRP-ML1(-/-) cells, but was restored by introducing a TRP-ML1 transgene into these cells. Microscopic Ca(2+) fluorescence imaging showed that NAADP significantly increased intracellular Ca(2+) concentration to 302.4 ± 74.28 nM (vs. 180 ± 44.13 nM of the basal) in TRP-ML1(+/+) cells, but it had no effect in TRP-ML1(-/-) cells. If a TRP-ML1 gene was transfected into TRP-ML1(-/-) cells, the Ca(2+) response to NAADP was restored to the level comparable to TRP-ML1(+/+) cells. Functionally, confocal microscopy revealed that NAADP significantly enhanced the dynamic interaction of endosomes and lysosomes and the lipid delivery to lysosomes in TRP-ML1(+/+) cells. This functional action of NAADP was abolished in TRP-ML1(-/-) cells, but restored after TRP-ML1 gene was rescued in these cells. Our results suggest that NAADP increases lysosomal TRP-ML1 channel activity to release Ca(2+), which promotes the interaction of endosomes and lysosomes and thereby regulates lipid transport to lysosomes. Failure of NAADP-TRP-ML1 signaling may be one of the important mechanisms resulting in intracellular lipid trafficking disorder and consequent mucolipidosis.     

NAADP mobilizes Ca(2+) from reserve granules,lysosome-related organelles,in sea urchin eggs

Nicotinic acid adenine dinucleotide phosphate (NAADP) mobilizes Ca(2+) in many cells and species. Unlike other Ca(2+)-mobilizing messengers, NAADP mobilizes Ca(2+) from an unknown store that is not the endoplasmic reticulum, the store traditionally associated with messenger-mediated Ca(2+) signaling. Here, we demonstrate the presence of a Ca(2+) store in sea urchin eggs mobilized by NAADP that is dependent on a proton gradient maintained by an ATP-dependent vacuolar-type proton pump. Moreover, we provide pharmacological and biochemical evidence that this Ca(2+) store is the reserve granule, the functional equivalent of a lysosome in the sea urchin egg. These findings represent an unsuspected mechanism for messenger-mediated Ca(2+) release from lysosome-related organelles.     

An enzymatic cycling assay for nicotinic acid adenine dinucleotide phosphate using NAD synthetase

Nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to mobilize Ca(2+) from intracellular stores in a wide variety of organisms, ranging from plants to humans. We have developed a novel enzyme cycling assay for NAADP that involves coupled reactions catalyzed by four enzymes. In this system, NAADP is first converted into nicotinic acid adenine dinucleotide (NAAD) by alkaline phosphatase, after which the NAAD is converted to NAD, AMP, and PPi by NAD synthetase (NADS) in the presence of ATP and ammonia. The NAD is then amplified using an enzyme cycling system driven by glucose dehydrogenase and diaphorase. The resultant formation of formazan dye is measured spectrophotometrically based on the increase in absorbance at 450 nm. Using this method, NAADP (20-400 nM) was assayed, and a highly linear correlation was obtained between the NAADP concentration and the increase in absorbance at 450 nm. The cycling rate was approximately 95 cycles/min. In addition, the within-run coefficients of variation (CVs) for 25, 50, and 100 nM NAADP solutions were 9.33, 4.86, and 3.13%, respectively. Interference by NAD analogs (e.g., NAAD, NADP) in the sample was eliminated prior to running the assay by treating the sample with NADS and NAD nucleosidase (NADase). In sum, our findings indicate this enzyme cycling assay to be readily applicable for determination for NAADP in a variety of biological samples and to be particularly appropriate for use with an autoanalyzer.     

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.     

Nicotinic acid adenine dinucleotide phosphate (NAADP) regulates autophagy in cultured astrocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca(2+)-mobilizing messenger that in many cells releases Ca(2+) from the endolysosomal system. Recent studies have shown that NAADP-induced Ca(2+) mobilization is mediated by the two-pore channels (TPCs). Whether NAADP acts as a messenger in astrocytes is unclear, and downstream functional consequences have yet to be defined. Here, we show that intracellular delivery of NAADP evokes Ca(2+) signals from acidic organelles in rat astrocytes and that these signals are potentiated upon overexpression of TPCs. We also show that NAADP increases acidic vesicular organelle formation and levels of the autophagic markers, LC3II and beclin-1. NAADP-mediated increases in LC3II levels were reduced in cells expressing a dominant-negative TPC2 construct. Our data provide evidence that NAADP-evoked Ca(2+) signals mediated by TPCs regulate autophagy.     

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 controls cross-talk between distinct Ca2+ stores in the heart

In cardiac muscle the sarcoplasmic reticulum (SR) plays a key role in the control of contraction, releasing Ca(2+) in response to Ca(2+) influx across the sarcolemma via voltage-gated Ca(2+) channels. Here we report evidence for an additional distinct Ca(2+) store and for actions of nicotinic acid adenine dinucleotide phosphate (NAADP) to mobilize Ca(2+) from this store, leading in turn to enhanced Ca(2+) loading of the SR. Photoreleased NAADP increased Ca(2+) transients accompanying stimulated action potentials in ventricular myocytes. The effects were prevented by bafilomycin A (an H(+)-ATPase inhibitor acting on acidic Ca(2+) stores), by desensitizing concentrations of NAADP, and by ryanodine and thapsigargin to suppress SR function. Bafilomycin A also suppressed staining of acidic stores with Lysotracker Red without affecting SR integrity. Cytosolic application of NAADP by means of its membrane permeant acetoxymethyl ester increased myocyte contraction and the frequency and amplitude of Ca(2+) sparks, and these effects were inhibited by bafilomycin A. Effects of NAADP were associated with an increase in SR Ca(2+) load and appeared to be regulated by beta-adrenoreceptor stimulation. The observations are consistent with a novel role for NAADP in cardiac muscle mediated by Ca(2+) release from bafilomycin-sensitive acidic stores, which in turn enhances SR Ca(2+) release by increasing SR Ca(2+) load.     

Fertilization and nicotinic acid adenine dinucleotide phosphate induce pH changes in acidic Ca(2+) stores in sea urchin eggs

The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) releases Ca(2+) from the acidic Ca(2+) stores of many organisms, including those of the sea urchin egg. We investigated whether the pH within the lumen of these acidic organelles changes in response to stimuli. Fertilization activates the egg by Ca(2+) release dependent upon NAADP, and accordingly, we report that fertilization also alters organellar pH in a spatio-temporally complex manner. Upon sperm fusion, vesicles deep in the egg center slowly acidify, whereas cortical vesicles undergo a rapid alkalinization. The cortical vesicle alkalinization is independent of exocytosis and cytosolic pH but coincides with the NAADP-dependent fertilization Ca(2+) wave. Microinjection of NAADP mimicked the fertilization cortical response, suggesting that it occurred within NAADP-sensitive acidic Ca(2+) stores. Our data show that NAADP and physiological stimuli alter the pH within intracellular organelles and suggest that NAADP signals through pH as well as Ca(2+).     

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.     

NAADP: a new second messenger comes of age

Nicotinic acid adenine dinucleotide phosphate (NAADP) is one of the most potent stimulators of intracellular Ca(2+) mobilization in a variety of cell types. Its role in physiological processes is increasingly demonstrated by NAADP increases following cellular stimulation. As a second messenger NAADP shows unique features such as the ability to mobilize Ca(2+) from stores that are physically distinct from those connected to the Ca(2+) channels located in the endoplasmic reticulum, namely, the inositol-1,4,5-trisphosphate and the cyclic-ADP-ribose/ ryanodine receptors. Furthermore, the NAADP-induced self-inactivation mechanism is suggestive of an irreversible binding of NAADP to its putative receptor.     

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.     

NAADP mobilizes calcium from the endoplasmic reticular Ca(2+) store in T-lymphocytes

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 Ca(2+) signaling. However, the inhibitory effect was not specific since Ca(2+) 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 Ca(2+) 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 Ca(2+) stores, did not antagonize subsequent NAADP-induced Ca(2+) signaling. In contrast to bafilomycin A1, emptying of the endoplasmic reticulum by thapsigargin almost completely prevented Ca(2+) signaling induced by NAADP. In conclusion, in T-lymphocytes, no evidence for involvement of lysosomes in NAADP-mediated Ca(2+) signaling was obtained. The sensitivity of NAADP-induced Ca(2+) 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.     

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.     

Photoaffinity labeling of nicotinic acid adenine dinucleotide phosphate (NAADP) targets in mammalian cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) is an agonist-generated second messenger that releases Ca(2+) from intracellular acidic Ca(2+) stores. Recent evidence has identified the two-pore channels (TPCs) within the endolysosomal system as NAADP-regulated Ca(2+) channels that release organellar Ca(2+) in response to NAADP. However, little is known about the mechanism coupling NAADP binding to calcium release. To identify the NAADP binding site, we employed a photoaffinity labeling method using a radioactive photoprobe based on 5-azido-NAADP ([(32)P-5N(3)]NAADP) that exhibits high affinity binding to NAADP receptors. In several systems that are widely used for studying NAADP-evoked Ca(2+) signaling, including sea urchin eggs, human cell lines (HEK293, SKBR3), and mouse pancreas, 5N(3)-NAADP selectively labeled low molecular weight sites that exhibited the diagnostic pharmacology of NAADP-sensitive Ca(2+) release. Surprisingly, we were unable to demonstrate labeling of endogenous, or overexpressed, TPCs. Furthermore, labeling of high affinity NAADP binding sites was preserved in pancreatic samples from TPC1 and TPC2 knock-out mice. These photolabeling data suggest that an accessory component within a larger TPC complex is responsible for binding NAADP that is unique from the core channel itself. This observation necessitates critical evaluation of current models of NAADP-triggered activation of the TPC family.