Nicotinic Acid Adenine Dinucleotide Phosphate Receptors Research Articles

Diazirine-AIOC-NAADP, a Clickable-Photoactive NAADP Analog for Sea Urchin NAADP Binding Proteins.

Calcium ions (Ca2+) play a vital role as intracellular messengers, regulating essential cellular processes. Nicotinic acid adenine dinucleotide phosphate (NAADP) serves as a potent second messenger, responsible for releasing Ca2+ in both mammals and echinoderms. Despite identification of two human NAADP receptor proteins, their counterparts in sea urchins remain elusive. Sea urchin NAADP binding proteins are important due to their unique identities and NAADP binding properties which may illuminate new signaling modalities in other species. Consequently, the development of new photoactive and clickable NAADP analogs with specificity for binding targets in sea urchin egg homogenates is a priority. We designed and synthesized diazirine-AIOC-NAADP, a photoactive and "clickable" NAADP analog, to specifically label and identify sea urchin NAADP receptors. This analog, synthesized using a chemo-enzymatic approach, induced Ca2+ release from sea urchin egg homogenates at low-micromolar concentrations. The ability of diazirine-AIOC-NAADP to mobilize Ca2+ in cultured human cells was investigated by microinjection of the probe into U2OS cells. Microinjected NAADP elicited a robust Ca2+ release, but even 6000-fold higher concentrations of diazirine-AIOC-NAADP were unable to release Ca2+. Our results indicate that our new probe is specifically recognized at low concentration by sea urchin egg NAADP receptors but not by the NAADP receptors in a human cultured cell line.

Lsm12 is an NAADP receptor and a two-pore channel regulatory protein required for calcium mobilization from acidic organelles

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+-mobilizing second messenger which uniquely mobilizes Ca2+ from acidic endolysosomal organelles. However, the molecular identity of the NAADP receptor remains unknown. Given the necessity of the endolysosomal two-pore channel (TPC1 or TPC2) in NAADP signaling, we performed affinity purification and quantitative proteomic analysis of the interacting proteins of NAADP and TPCs. We identified a Sm-like protein Lsm12 complexed with NAADP, TPC1, and TPC2. Lsm12 directly binds to NAADP via its Lsm domain, colocalizes with TPC2, and mediates the apparent association of NAADP to isolated TPC2 or TPC2-containing membranes. Lsm12 is essential and immediately participates in NAADP-evoked TPC activation and Ca2+ mobilization from acidic stores. These findings reveal a putative RNA-binding protein to function as an NAADP receptor and a TPC regulatory protein and provides a molecular basis for understanding the mechanisms of NAADP signaling.

Essential requirement for JPT2 in NAADP-evoked Ca2+ signaling.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca2+ from acidic organelles through the activation of two-pore channels (TPCs) to regulate endolysosomal trafficking events. NAADP action is mediated by NAADP-binding protein(s) of unknown identity that confer NAADP sensitivity to TPCs. Here, we used a "clickable" NAADP-based photoprobe to isolate human NAADP-binding proteins and identified Jupiter microtubule-associated homolog 2 (JPT2) as a TPC accessory protein required for endogenous NAADP-evoked Ca2+ signaling. JPT2 was also required for the translocation of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus through the endolysosomal system. Thus, JPT2 is a component of the NAADP receptor complex that is essential for TPC-dependent Ca2+ signaling and control of coronaviral entry.

Chemo-enzymatic synthesis of adenine substituted nicotinic acid adenine dinucleotide phosphate (NAADP) analogs

Nicotinamide adenine dinucleotide phosphate (NADP) is an indispensable metabolic co-substrate and nicotinic acid adenine dinucleotide phosphate (NAADP) is an important Ca2+ releasing intracellular second messenger. Exploration of the NADP and NAADP interactome often requires the synthesis of NADP derivatives substituted on the adenosine nucleoside. The introduction of the 2′-phosphate of NADP makes the synthesis of substituted NADP derivatives difficult. We have employed recombinant human NAD kinase expressed in E. coli as an enzymatic reagent to convert readily available synthetic NAD derivatives to NADP analogs, which were subsequently transformed into NAADP derivatives using enzyme catalyzed pyridine base exchange. 8-Ethynyl-NADP, 8-ethynyl-NAADP and 5-N3-8-ethynyl-NAADP were synthesized starting from a protected 8-ethynyladenosine using a combination of chemical and enzymatic steps and the NAADP derivatives shown to be recognized by the sea urchin NAADP receptor at low concentration. Our methodology will enable researchers to produce mono- and bi-substituted NADP and NAADP analogs that can be applied in proteomic studies to identify NADP and NAADP binding proteins.

Quantitative Proteomic Analysis of the Putative NAADP-TPC Signaling Complex

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent known Ca2+-mobilizing second messenger which uniquely mobilizes Ca2+ from acidic endolysosomal organelles. Despite the importance of NAADP-evoked Ca2+ signaling, the molecular identities of the NAADP receptors and Ca2+-release channels involved in this process have yet to be unequivocally defined. Accumulated evidence indicates that the poorly understood two-pore channels (TPCs) participate in NAADP-evoked Ca2+ release. However, how TPCs are involved in NAADP-signaling remains largely unknown or debatable. A more unified hypothesis is that TPCs are not directly involved in NAADP binding but are part of the NAADP receptor/Ca2+-release channel complex, in which the molecular identities of other key proteins remain unknown. To dissect the molecular components of the putative NAADP-TPC signaling complex, we performed affinity purification using both TPCs and NAADP as baits to purify the signaling complex in HEK-293 and SKBR3 cells. We employed stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomic approach to specifically identify the TPC and NAADP interacting proteins in these cells. Upon systematic investigation, we have identified large and comprehensive datasets of TPC-interactome and NAADP-interactome.

TPC: the NAADP discovery channel?

The Ca2+-mobilizing second messenger, NAADP (nicotinic acid adenine dinucleotide phosphate), has been with us for nearly 20years and yet we still cannot fully agree on the identity of its target Ca2+-release channel. In spite of some recent robust challenges to the idea that two-pore channels (TPCs) represent the elusive "NAADP receptor", evidence continues to accumulate that TPCs are important for NAADP-mediated responses. This article will briefly outline the background and review more recent work pertaining to the TPC story.

Abstract 537: Rgulation of ALG-2 in NAADP-induced Ca2+ Release via TRP-ML1 Channel in Mouse Coronary Arterial Myocytes

We have demonstrated that nicotinic acid adenine dinucleotide phosphate (NAADP) sensitive Ca2+ release channel is characteristic of lysosomal TRP-ML1 in coronary arterial myocytes (CAMs). The present study hypothesized that NAADP induces Ca2+ release from TRP-ML1 channels via apoptosis-linked gene 2 (ALG-2), which was reported to act as a Ca2+ sensor that modulates the function of TRP-ML1. By RT-PCR and Western blot analysis, we demonstrated that ALG-2 is abundantly expressed in the cytosol of mouse CAMs. Confocal microscopy found that FasL and ET-1, well-known NAADP stimulators, increased the colocalization coefficient of ALG-2-GFP with TRP-ML1-RFP in CAMs from 0.17±0.033 to 0.3±0.044 and 0.3±0.09, respectively. In addition, the TRP-ML1 level was significantly increased in FasL or ET-1-treated CAMs after immunoprecipitation with anti-ALG-2, confirming that the interaction of ALG-2 with TRP-ML1 was enhanced in response to NAADP production. Furthermore, NAADP competitively inhibited recombinant ALG-2 protein binding with tis-NED-19, which fluorescently labels NAADP receptors. Importantly, fluorescent microscopic image analysis showed that NAADP-induced Ca2+ release was reduced from 380±42.03 nM to 216.7±14.7 nM after the transfection with ALG-2 siRNA in CAMs, which was also found after the treatment with FasL or ET-1. Our findings support the view that ALG-2 can directly bind with NAADP, which mediates the action of NAADP through lysosomal TRP-ML1 channels. Such results throw light on NAADP-TRP-ML1 working model in vascular cells.

Calcium and IP3 dynamics in cardiac myocytes: experimental and computational perspectives and approaches

Calcium plays a crucial role in excitation-contraction coupling (ECC), but it is also a pivotal second messenger activating Ca2+-dependent transcription factors in a process termed excitation-transcription coupling (ETC). Evidence accumulated over the past decade indicates a pivotal role of inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release in the regulation of cytosolic and nuclear Ca2+ signals. IP3 is generated by stimulation of plasma membrane receptors that couple to phospholipase C (PLC), liberating IP3 from phosphatidylinositol 4,5-bisphosphate (PIP2). An intriguing aspect of IP3 signaling is the presence of the entire PIP2-PLC-IP3 signaling cascade as well as the presence of IP3Rs at the inner and outer membranes of the nuclear envelope (NE) which functions as a Ca2+ store. The observation that the nucleus is surrounded by its own putative Ca2+ store raises the possibility that nuclear IP3-dependent Ca2+ release plays a critical role in ETC. This provides a potential mechanism of regulation that acts locally and autonomously from the global cytosolic Ca2+ signal underlying ECC. Moreover, there is evidence that: (i) the sarcoplasmic reticulum (SR) and NE are a single contiguous Ca2+ store; (ii) the nuclear pore complex is the major gateway for Ca2+ and macromolecules to pass between the cytosol and the nucleoplasm; (iii) the inner membrane of the NE hosts key Ca2+ handling proteins including the Na+/Ca2+ exchanger (NCX)/GM1 complex, ryanodine receptors (RyRs), nicotinic acid adenine dinucleotide phosphate receptors (NAADPRs), Na+/K+ ATPase, and Na+/H+ exchanger. Thus, it appears that the nucleus represents a Ca2+ signaling domain equipped with its own ion channels and transporters that allow for complex local Ca2+ signals. Many experimental and modeling approaches have been used for the study of intracellular Ca2+ signaling but the key to the understanding of the dual role of Ca2+ mediating ECC and ECT lays in quantitative differences of local [Ca2+] in the nuclear and cytosolic compartment. In this review, we discuss the state of knowledge regarding the origin and the physiological implications of nuclear Ca2+ transients in different cardiac cell types (adult atrial and ventricular myocytes) as well as experimental and mathematical approaches to study Ca2+ and IP3 signaling in the cytosol and nucleus. In particular, we focus on the concept that highly localized Ca2+ signals are required to translocate and activate Ca2+-dependent transcription factors (e.g., nuclear factor of activated T-cells, NFAT; histone deacetylase, HDAC) through phosphorylation/dephosphorylation processes.

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.

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 Ca(2+) from intracellular stores in various cell types. Extracellular application of NAADP has been shown to elicit intracellular Ca(2+) 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 Ca(2+)-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.

N-terminal tagging of two-pore channels interferes with NAADP action

NAADP (nicotinic acid-adenine dinucleotide phosphate) is the most potent Ca2+-releasing second messenger known to date. Since its discovery in 1995 identifying the NAADP receptor protein/Ca2+ channel has been a major persuit of the Ca2+ signalling community. In their paper 'The N-terminal region of two-pore channel 1 regulates trafficking and activation by NAADP' published in this issue of the Biochemical Journal Patel and colleagues describe that the N-terminus of one of the NAADP receptor protein/Ca2+ channel candidates, TPC1 (two-pore channel 1), is crucial for protein targeting and for sensitivity to NAADP.

Characterization of NAADP‐Induced Global and Local Calcium Signals in Rat Pulmonary Arterial Smooth Muscle Cells

Ca2+ signaling in vascular smooth muscle cells involves global and local interactions between multiple Ca2+ influx and release pathways. Ca2+ release pathways include IP3 receptor‐, ryanodine receptor‐, and nicotinic acid adenine dinucleotide phosphate (NAADP) receptor‐mediated mechanisms. Recent studies demonstrated that the two‐pore channels (TPC1 and TPC2) are NAADP receptors located in the endo‐/lysosomes. Here we characterized the NAADP‐mediated Ca2+ signals in rat pulmonary arterial smooth muscle cells (PASMCs). Application of the membrane permeable NAADP‐AM (0.25–1 μM) to PASMCs elicited concentration‐dependent increase in global [Ca2+]i. It was blocked by the NAADP antagonist NED‐19 or the acidic vacuolar H+‐ATPase bafilomycin A, suggesting Ca2+ release from the lysosomal Ca2+ stores. The Ca2+ response was independent of extracellular Ca2+ influx, and was unaffected by the IP3 receptor blocker xestospongin C; but was partially inhibited by ryanodine or thapsigargin. Moreover, NAADP caused dramatic increase in local Ca2+ release events, which was inhibited by ryanodine or NED‐19. The temporal and spatial properties of the NAADP‐induced local release events were comparable to the spontaneous Ca2+ sparks, the elementary Ca2+ events mediated via the ryanodine receptors. Our results, hence, show that the NAADP‐sensitive release channels are functionally expressed and Ca2+ signals from the NAADP channel‐gated lysosomal stores cross‐activate ryanodine receptors to amplify Ca2+ release in PASMCs.

NAADP-Induced Calcium Release in Pulmonary Arterial and Aortic Smooth Muscle Cells is Mediated by Cross-Activation of NAADP-Receptor and Ryanodine Receptor-Gated Calcium Stores

Ca2+ signaling in vascular smooth muscle cells involves dynamic interactions between multiple Ca2+ influx and release pathways. Ca2+ release pathways include the IP3 receptor-, ryanodine receptor-, and nicotinic acid adenine dinucleotide phosphate (NAADP) receptor-gated Ca2+ stores. Recent studies demonstrated that the two-pore channels (TPC1 and TPC2) are NAADP receptors located in the endo-/lysosomes. Here we quantified the expression of TPC channels and characterized the NAADP-mediated Ca2+ signals in vascular tissues. Western blot analysis and real-time RT-PCR detected TPC1 and TPC2 protein and mRNA respectively in aorta, pulmonary, mesenteric, renal, femoral, tail, and cerebral artery of rat, with TPC1 mRNA level five to ten-fold higher than that of TPC2. Application of the membrane permeable NAADP-AM to pulmonary arterial smooth muscle cells (PASMCs) elicited concentration-dependent increase in global [Ca2+]i. The Ca2+ response was independent of extracellular Ca2+ influx, and was unaffected by the IP3 receptor blocker xestospongin C. In contrast, the NAADP-induced Ca2+ response was partially inhibited by ryanodine or thapsigargin, and was abolished by the NAADP antagonist NED-19 or the acidic vacuolar H+-ATPase bafilomycin A. Moreover, NAADP caused dramatic increase in local Ca2+ release events, which was inhibited by ryanodine or NED-19. The temporal and spatial properties of the NAADP-induced local release events were comparable to the spontaneous Ca2+ sparks. Similar observations were also made in aortic smooth muscle cells. Our results, hence, show that the NAADP channels, TPC1 and TPC2, are functionally expressed in vascular smooth muscle; and Ca2+ signals from the NAADP channel-gated lysosomal stores cross-activate ryanodine receptors to amplify Ca2+ release in pulmonary arterial and aortic smooth muscle cells.

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.

Photoaffinity Labeling of High Affinity Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-Binding Proteins in Sea Urchin Egg

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Recent studies have identified two-pore channels (TPCs) as endolysosomal channels that are regulated by NAADP; however, the nature of the NAADP receptor binding site is unknown. To further study NAADP binding sites, we have synthesized and characterized [(32)P-5-azido]nicotinic acid adenine dinucleotide phosphate ([(32)P-5N(3)]NAADP) as a photoaffinity probe. Photolysis of sea urchin egg homogenates preincubated with [(32)P-5N(3)]NAADP resulted in specific labeling of 45-, 40-, and 30-kDa proteins, which was prevented by inclusion of nanomolar concentrations of unlabeled NAADP or 5N(3)-NAADP, but not by micromolar concentrations of structurally related nucleotides such as NAD, nicotinic acid adenine dinucleotide, nicotinamide mononucleotide, nicotinic acid, or nicotinamide. [(32)P-5N(3)]NAADP binding was saturable and displayed high affinity (K(d) ∼10 nM) in both binding and photolabeling experiments. [(32)P-5N(3)]NAADP photolabeling was irreversible in a high K(+) buffer, a hallmark feature of NAADP binding in the egg system. The proteins photolabeled by [(32)P-5N(3)]NAADP have molecular masses smaller than the sea urchin TPCs, and antibodies to TPCs do not detect any immunoreactivity that comigrates with either the 45-kDa or the 40-kDa photolabeled proteins. Interestingly, antibodies to TPC1 and TPC3 were able to immunoprecipitate a small fraction of the 45- and 40-kDa photolabeled proteins, suggesting that these proteins associate with TPCs. These data suggest that high affinity NAADP binding sites are distinct from TPCs.

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.

Leucine-rich repeat kinase 2 regulates autophagy through a calcium-dependent pathway involving NAADP

Mutations in the leucine-rich repeat kinase-2 (LRRK2) gene cause late-onset Parkinson’s disease, but its physiological function has remained largely unknown. Here we report that LRRK2 activates a calcium-dependent protein kinase kinase-β (CaMKK-β)/adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway which is followed by a persistent increase in autophagosome formation. Simultaneously, LRKR2 overexpression increases the levels of the autophagy receptor p62 in a protein synthesis-dependent manner, and decreases the number of acidic lysosomes. The LRRK2-mediated effects result in increased sensitivity of cells to stressors associated with abnormal protein degradation. These effects can be mimicked by the lysosomal Ca2+-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) and can be reverted by an NAADP receptor antagonist or expression of dominant-negative receptor constructs. Collectively, our data indicate a molecular mechanism for LRRK2 deregulation of autophagy and reveal previously unidentified therapeutic targets.

Transient Receptor Potential Mucolipin 1 (TRPML1) and Two-pore Channels Are Functionally Independent Organellar Ion Channels

NAADP is a potent second messenger that mobilizes Ca(2+) from acidic organelles such as endosomes and lysosomes. The molecular basis for Ca(2+) release by NAADP, however, is uncertain. TRP mucolipins (TRPMLs) and two-pore channels (TPCs) are Ca(2+)-permeable ion channels present within the endolysosomal system. Both have been proposed as targets for NAADP. In the present study, we probed possible physical and functional association of these ion channels. Exogenously expressed TRPML1 showed near complete colocalization with TPC2 and partial colocalization with TPC1. TRPML3 overlap with TPC2 was more modest. TRPML1 and to some extent TRPML3 co-immunoprecipitated with TPC2 but less so with TPC1. Current recording, however, showed that TPC1 and TPC2 did not affect the activity of wild-type TRPML1 or constitutively active TRPML1(V432P). N-terminally truncated TPC2 (TPC2delN), which is targeted to the plasma membrane, also failed to affect TRPML1 and TRPML1(V432P) channel function or TRPML1(V432P)-mediated Ca(2+) influx. Whereas overexpression of TPCs enhanced NAADP-mediated Ca(2+) signals, overexpression of TRPML1 did not, and the dominant negative TRPML1(D471K) was without affect on endogenous NAADP-mediated Ca(2+) signals. Furthermore, the single channel properties of NAADP-activated TPC2delN were not affected by TRPML1. Finally, NAADP-evoked Ca(2+) oscillations in pancreatic acinar cells were identical in wild-type and TRPML1(-/-) cells. We conclude that although TRPML1 and TPCs are present in the same complex, they function as two independent organellar ion channels and that TPCs, not TRPMLs, are the targets for NAADP.

NAADP mediates ATP-induced Ca2+ signals in astrocytes

Intracellular Ca2+ 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 Ca2+ 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 Ca2+ 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.

NAADP links histamine H1 receptors to secretion of von Willebrand factor in human endothelial cells

AbstractA variety of endothelial agonist–induced responses are mediated by rises in intracellular Ca2+, suggesting that different Ca2+ signatures could fine-tune specific inflammatory and thrombotic activities. In search of new intracellular mechanisms modulating endothelial effector functions, we identified nicotinic acid adenine dinucleotide phosphate (NAADP) as a crucial second messenger in histamine-induced Ca2+ release via H1 receptors (H1R). NAADP is a potent intracellular messenger mobilizing Ca2+ from lysosome-like acidic compartments, functionally coupled to the endoplasmic reticulum. Using the human EA.hy926 endothelial cell line and primary human umbilical vein endothelial cells, we show that selective H1R activation increases intracellular NAADP levels and that H1R-induced calcium release involves both acidic organelles and the endoplasmic reticulum. To assess that NAADP links H1R to Ca2+-signaling we used both microinjection of self-inactivating concentrations of NAADP and the specific NAADP receptor antagonist, Ned-19, both of which completely abolished H1R-induced but not thrombin-induced Ca2+ mobilization. Interestingly, H1R-mediated von Willebrand factor (VWF) secretion was completely inhibited by treatment with Ned-19 and by siRNA knockdown of 2-pore channel NAADP receptors, whereas thrombin-induced VWF secretion failed to be affected. These findings demonstrate a novel and specific Ca2+-signaling mechanism activated through H1R in human endothelial cells, which reveals an obligatory role of NAADP in the control of VWF secretion.