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Nicotinic acid adenine dinucleotide phosphate (NAADP), identified as one of the most potent calcium-mobilizing second messengers, has been studied in different eukaryotic cell types, including lymphocytes. Although aspects of NAADP-mediated calcium release in lymphocytes are still under debate, the organelles pertaining to NAADP-mediated calcium release are often characterized as acidic and related to lysosomes. Although NAADP-mediated calcium release in different subsets of T cells, including naïve, effector and natural regulatory T cells, has been studied, it has not been widely studied in memory CD4 + T cells, which show a different calcium flux profile. Using a pharmacological approach, the effect of Ned-19, an NAADP pathway antagonist, on the involvement of NAADP in TCR activation in murine memory CD4 + T cells and their downstream effector functions, such as proliferation and cytokine production, was studied. According to this study, Ned-19 inhibited TCR-mediated calcium flux and its downstream effector functions in primary memory CD4 + T cells. The study also revealed that both extracellular and intracellular calcium stores, including endoplasmic reticulum and lysosome-like acidic calcium stores, contribute to the TCR-mediated calcium flux in memory CD4 + T cells. NAADP-AM, a cell permeable analogue of NAADP, was shown to release calcium in memory CD4 + T cells and calcium flux was inhibited by Ned-19.

The discovery of NAADP-evoked Ca 2+ release in sea urchin eggs and then as a ubiquitous Ca 2+ mobilizing messenger has introduced several novel paradigms to our understanding of Ca 2+ signalling, not least in providing a link between cell stimulation and Ca 2+ release from lysosomes and other acidic Ca 2+ storage organelles. In addition, the hallmark concentration-response relationship of NAADP-mediated Ca 2+ release, shaped by striking activation/desensitization mechanisms, influences its actions as an intracellular messenger. There has been recent progress in our understanding of the molecular mechanisms underlying NAADP-evoked Ca 2+ release, such as the identification of the endo-lysosomal two-pore channel family of cation channels (TPCs) as their principal target and the identity of NAADP-binding proteins that complex with them. The NAADP/TPC signalling axis has gained recent prominence in pathophysiology for their roles in such disease processes as neurodegeneration, tumorigenesis and cellular viral entry., (© 2022. Springer Nature Switzerland AG.)

In recent years, our understanding of the structure, mechanisms and functions of the endo-lysosomal TPC (two-pore channel) family have grown apace. Gated by the second messengers, NAADP and PI(3,5)P2, TPCs are an integral part of fundamental signal-transduction pathways, but their array and plasticity of cation conductances (Na+, Ca2+, H+) allow them to variously signal electrically, osmotically or chemically. Their relative tissue- and organelle-selective distribution, together with agonist-selective ion permeabilities provides a rich palette from which extracellular stimuli can choose. TPCs are emerging as mediators of immunity, cancer, metabolism, viral infectivity and neurodegeneration as this short review attests., (© 2022 The Author(s).)

Two-pore channels TPC1 and TPC2 are ubiquitously expressed pathophysiologically relevant proteins that reside on endolysosomal vesicles. Here, we review the electrophysiology of these channels. Direct macroscopic recordings of recombinant TPCs expressed in enlarged lysosomes in mammalian cells or vacuoles in plants and yeast demonstrate gating by the Ca 2+ -mobilizing messenger NAADP and/or the lipid PI(3,5)P 2 . TPC currents are regulated by H + , Ca 2+ , and Mg 2+ (luminal and/or cytosolic), as well as protein kinases, and they are impacted by single-nucleotide polymorphisms linked to pigmentation. Bisbenzylisoquinoline alkaloids, flavonoids, and several approved drugs demonstrably block channel activity. Endogenous TPC currents have been recorded from a number of primary cell types and cell lines. Many of the properties of endolysosomal TPCs are recapitulated upon rerouting channels to the cell surface, allowing more facile recording through conventional electrophysiological means. Single-channel analyses have provided high-resolution insight into both monovalent and divalent permeability. The discovery of small-molecule activators of TPC2 that toggle the ion selectivity from a Ca 2+ -permeable (NAADP-like) state to a Na + -selective (PI(3,5)P 2 -like) state explains discrepancies in the literature relating to the permeability of TPCs. Identification of binding proteins that confer NAADP-sensitive currents confirm that indirect, remote gating likely underpins the inconsistent observations of channel activation by NAADP.

Calcium ions play a central role in determining the timing and magnitude of the pumping action of heart muscle in a process which couples electrical activity of action potentials to muscle contraction. Regulation of this excitation-contraction coupling is achieved by Ca 2+ signalling mechanisms that include activation of Ca 2+ mobilising agents which influence the movement of Ca 2+ between intracellular membrane-bound compartments. Research discussed here concerns endolysosomes, which play diverse signalling roles throughout the body. In the heart, a population of endolysosomes is strategically placed close to two other important membrane bound organelles, sarcoplasmic reticulum (SR) and mitochondria. In each case this proximity provides a structural basis for highly localised Ca 2+ signalling in nanodomains between endolysosomes and the organelle. Ca 2+ is released from endolysosomes via at least two varieties of two-pore domain channels (TPCs) in mammalian cardiac cells, TPC1 determining the interaction with mitochondria, while TPC2 controls the influence on SR. Ca 2+ release via both TPC1 and TPC2 is enhanced by the Ca 2+ mobilising agent, nicotinic acid adenine dinucleotide phosphate (NAADP) which is synthesised in the heart primarily by CD38. In normal physiology, NAADP plays an important regulatory role in which Ca 2+ is released from endolysosomes via TPC2 channels into a nanodomain next to SR, and an amplification mechanism resulting from Ca 2+ activation of CaMKII enhances SR Ca 2+ uptake by the enzyme SERCA to increase the amplitude of the Ca 2+ transient accompanying action potentials. A separate mechanism underlies pathology associated with reperfusion after ischaemia, when NAADP-mediated endolysosomal calcium release via TPC1 acts on nearby mitochondria resulting in abnormal SR Ca 2+ release and extreme disruption to the normal excitation-contraction coupling process, causing muscle damage. There are different roles for PKA in the two pathways dependant on TPC1 or TPC2. Oxidising conditions during reperfusion following ischaemia promote disulphide bond formation in PKAIalpha causing accumulation of PKAI holoenzyme in endolysosomes and cardioprotective inhibition of TPC1 channels. In the case of TPC2, PKAII actions are thought to enhance NAADP synthesis by CD38 therefore promoting the endolysosomal influence on SR Ca 2+ . Excessive activation of this pathway leads to cardiac arrhythmias and hypertrophy., (Copyright © 2022. Published by Elsevier Ltd.)

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a universal Ca 2+ mobilizing second messenger essential for initiation of Ca 2+ signaling. Recently, novel molecular mechanisms of both its rapid formation upon receptor stimulation and its mode of action were discovered. Dual NADPH oxidase 2 (DUOX2) and hematological and neurological expressed 1-like protein (HN1L)/Jupiter microtubule-associated homolog 2 (JPT2) were discovered as NAADP-forming enzyme and NAADP receptor/binding protein-the new kids on the block. These novel aspects are reviewed and integrated into the previous view of NAADP signaling.

Many viruses exploit host-cell Ca 2+ signaling processes throughout their life cycle. This is especially relevant for viruses that translocate through the endolysosomal system, where cellular infection is keyed to the microenvironment of these acidic Ca 2+ stores and Ca 2+ -dependent trafficking pathways. As regulators of the endolysosomal ionic milieu and trafficking dynamics, two families of endolysosomal Ca 2+ -permeable cation channels - two pore channels (TPCs) and transient receptor potential mucolipins (TRPMLs) - have emerged as important host-cell factors in viral entry. Here, we review: (i) current evidence implicating Ca 2+ signaling in viral translocation through the endolysosomal system, (ii) the roles of these ion channels in supporting cellular infection by different viruses, and (iii) areas for future research that will help define the potential of TPC and TRPML ligands as progressible antiviral agents., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

CD38 is a single-pass transmembrane enzyme catalyzing the synthesis of two nucleotide second messengers, cyclic ADP-ribose (cADPR) from NAD and nicotinic acid adenine dinucleotide phosphate (NAADP) from NADP. The former mediates the mobilization of the endoplasmic Ca 2+ -stores in response to a wide range of stimuli, while NAADP targets the endo-lysosomal stores. CD38 not only possesses multiple enzymatic activities, it also exists in two opposite membrane orientations. Type III CD38 has the catalytic domain facing the cytosol and is responsible for producing cellular cADPR. The type II CD38 has an opposite orientation and is serving as a surface receptor mediating extracellular functions such as cell adhesion and lymphocyte activation. Its ecto-NADase activity also contributes to the recycling of external NAD released by apoptosis. Endocytosis can deliver surface type II CD38 to endo-lysosomes, which acidic environment favors the production of NAADP. This article reviews the rationale and evidence that have led to CD38 as a paradigm for membrane topology defining distinct functions of proteins. Also described is the recent discovery of a hitherto unknown cADPR-synthesizing enzyme, SARM1, ushering in a new frontier in cADPR-mediated Ca 2+ -signaling., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Nicotinic acid adenine dinucleotide 2'-phosphate (NAADP) is a naturally occurring nucleotide that has been shown to be involved in the release of Ca 2+ from intracellular stores in a wide variety of cell types, tissues and organisms. Current evidence suggests that NAADP may function as a trigger to initiate a Ca 2+ signal that is then amplified by other Ca 2+ release mechanisms. A fundamental question that remains unanswered is the identity of the NAADP receptor. Our recent studies have identified HN1L/JPT2 as a high affinity NAADP binding protein that is essential for the modulation of Ca 2+ channels., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Walseth and Guse.)

Second trimester foetal human amniotic fluid-derived stem cells (hAFS) have been shown to possess remarkable cardioprotective paracrine potential in different preclinical models of myocardial injury and drug-induced cardiotoxicity. The hAFS secretome, namely the total soluble factors released by cells in their conditioned medium (hAFS-CM), can also strongly sustain in vivo angiogenesis in a murine model of acute myocardial infarction (MI) and stimulates human endothelial colony-forming cells (ECFCs), the only truly recognized endothelial progenitor, to form capillary-like structures in vitro. Preliminary work demonstrated that the hypoxic hAFS secretome (hAFS-CM Hypo ) triggers intracellular Ca 2+ oscillations in human ECFCs, but the underlying mechanisms and the downstream Ca 2+ -dependent effectors remain elusive. Herein, we found that the secretome obtained by hAFS undergoing hypoxic preconditioning induced intracellular Ca 2+ oscillations by promoting extracellular Ca 2+ entry through Transient Receptor Potential Vanilloid 4 (TRPV4). TRPV4-mediated Ca 2+ entry, in turn, promoted the concerted interplay between inositol-1,4,5-trisphosphate- and nicotinic acid adenine dinucleotide phosphate-induced endogenous Ca 2+ release and store-operated Ca 2+ entry (SOCE). hAFS-CM Hypo -induced intracellular Ca 2+ oscillations resulted in the nuclear translocation of the Ca 2+ -sensitive transcription factor p65 NF-κB. Finally, inhibition of either intracellular Ca 2+ oscillations or NF-κB activity prevented hAFS-CM Hypo -induced ECFC tube formation. These data shed novel light on the molecular mechanisms whereby hAFS-CM Hypo induces angiogenesis, thus providing useful insights for future therapeutic strategies against ischaemic-related myocardial injury., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

The emergence of endo-lysosomes as ubiquitous Ca 2+ stores with their unique cohort of channels has resulted in their being implicated in a growing number of processes in an ever-increasing number of cell types. The architectural and regulatory constraints of these acidic Ca 2+ stores distinguishes them from other larger Ca 2+ sources such as the ER and influx across the plasma membrane. In view of recent advances in the understanding of the modes of operation, we discuss phagocytosis as a template for how endo-lysosomal Ca 2+ signals (generated via TPC and TRPML channels) can be integrated in multiple sophisticated ways into biological processes. Phagocytosis illustrates how different endo-lysosomal Ca 2+ signals drive different phases of a process, and how these can be altered by disease or infection., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Lysosomes are acidic Ca 2+ stores often mobilised in conjunction with endoplasmic reticulum (ER) Ca 2+ stores. Glycyl-L-phenylalanine 2-naphthylamide (GPN) is a widely used lysosomotropic agent that evokes cytosolic Ca 2+ signals in many cells. However, whether these signals are the result of a primary action on lysosomes is unclear in light of recent evidence showing that GPN mediates direct ER Ca 2+ release through changes in cytosolic pH. Here, we show that GPN evoked rapid increases in cytosolic pH but slower Ca 2+ signals. NH 4 Cl evoked comparable changes in pH but failed to affect Ca 2+ The V-type ATPase inhibitor, bafilomycin A1, increased lysosomal pH over a period of hours. Acute treatment modestly affected lysosomal pH and potentiated Ca 2+ signals evoked by GPN. In contrast, chronic treatment led to more profound changes in luminal pH and selectively inhibited GPN action. GPN blocked Ca 2+ responses evoked by the novel nicotinic acid adenine dinucleotide phosphate-like agonist, TPC2-A1-N. Therefore, GPN-evoked Ca 2+ signals were better correlated with associated pH changes in the lysosome compared to the cytosol, and were coupled to lysosomal Ca 2+ release. We conclude that Ca 2+ signals evoked by GPN most likely derive from acidic organelles., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Ca 2+ -mobilizing adenine nucleotide second messengers cyclic adenosine diphosphoribose, (cADPR), nicotinic acid adenine dinucleotide phosphate (NAADP), adenosine diphosphoribose (ADPR), and 2'deoxy-ADPR were discovered since the late 1980s. They either release Ca 2+ from endogenous Ca 2+ stores, e.g., endoplasmic reticulum or acidic organelles, or evoke Ca 2+ entry by directly activating a Ca 2+ channel in the plasma membrane. For 25 years, Professor Barry Potter has been one of the major medicinal chemists in this topical area, designing and contributing numerous analogues to develop structure-activity relationships (SAR) as a basis for tool development in biochemistry and cell biology and for lead development in proof-of-concept studies in disease models. With this review, I wish to acknowledge our 25-year-long collaboration on Ca 2+ -mobilizing adenine nucleotide second messengers as a major part of Professor Potter's scientific lifetime achievements on the occasion of his retirement in 2020.

Two-pore channels (TPCs) are cation-permeable channels located on endolysosomal membranes and important mediators of intracellular Ca 2+ signalling. TPCs are involved in various pathophysiological processes, including cell growth and development, metabolism, and cancer progression. Most studies of TPCs have used TPC -/- cell or whole-animal models, or Ned-19, an indirect inhibitor. The TPC activation mechanism remains controversial, which has made it difficult to develop selective modulators. Recent studies of TPC structure and their interactomes are aiding the development of direct pharmacological modulators. This process is still in its infancy, but will facilitate future research and TPC targeting for therapeutical purposes. Here, we review the progress of current research into TPCs, including recent insights into their structures, functional roles, mechanisms of activation, and pharmacological modulators., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Ca 2+ signals are probably the most common intracellular signaling cellular events, controlling an extensive range of responses in virtually all cells. Many cellular stimuli, often acting at cell surface receptors, evoke Ca 2+ signals by mobilizing Ca 2+ from intracellular stores. Inositol trisphosphate (IP 3 ) was the first messenger shown to link events at the plasma membrane to release Ca 2+ from the endoplasmic reticulum (ER), through the activation of IP 3 -gated Ca 2+ release channels (IP 3 receptors). Subsequently, two additional Ca 2+ mobilizing messengers were discovered, cADPR and NAADP. Both are metabolites of pyridine nucleotides, and may be produced by the same class of enzymes, ADP-ribosyl cyclases, such as CD38. Whilst cADPR mobilizes Ca 2+ from the ER by activation of ryanodine receptors (RyRs), NAADP releases Ca 2+ from acidic stores by a mechanism involving the activation of two pore channels (TPCs). In addition, other pyridine nucleotides have emerged as intracellular messengers. ADP-ribose and 2'-deoxy-ADPR both activate TRPM2 channels which are expressed at the plasma membrane and in lysosomes.

Second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) triggers Ca 2+ release via two-pore channels (TPCs) localized in endolysosomal vesicles. The aim of the present work is to evaluate the role of TPCs in the action of norepinephrine (NE), angiotensin II (AngII), vasopressin (AVP), and 5-hydroxytriptamine (5-HT) on free cytoplasmic calcium concentration ([Ca 2+ ] i ) in smooth muscle cells (SMCs) isolated from rat aorta and on aorta contraction. To address this issue, the NAADP structural analogue and inhibitor of TPCs, NED 19, was applied. We have demonstrated a high degree of colocalization of the fluorescent signals of cis -NED 19 and endolysosmal probe LysoTracker in SMCs. Both cis - or trans -NED 19 inhibited the rise of [Ca 2+ ] i in SMCs induced by 100 μM NE by 50-60%. IC 50 for cis - and trans -NED 19 were 2.7 and 8.9 μM, respectively. The inhibition by NED 19 stereoisomers of the effects of AngII, AVP, and 5-HT was much weaker. Both forms of NED 19 caused relaxation of aortic rings preconstricted by NE, with relative potency of cis -NED 19 several times higher than that of trans -NED 19. Inhibition by cis -NED 19 of NE-induced contraction was maintained after intensive washing and slowly reversed within an hour of incubation. Cis - and trans -NED 19 did not cause decrease in the force of aorta contraction in response to Ang II and AVP, and only slightly relaxed aorta preconstricted by 5-HT and by KCl. Suppression of TPC1 in SMCs with siRNA caused a 40% decrease in [Ca 2+ ] i in response to NE, whereas siRNA against TPC2 did not change NE calcium signaling. These data suggest that TPC1 is involved in the NE-stimulated [Ca 2+ ] i rise in SMCs. Inhibition of TPC1 activity by NED 19 could be the reason for partial inhibition of aortic rings contraction in response to NE.

Endothelial cells (ECs) constitute the innermost layer that lines all blood vessels from the larger arteries and veins to the smallest capillaries, including the lymphatic vessels. Despite the histological classification of endothelium of a simple epithelium and its homogeneous morphological appearance throughout the vascular system, ECs, instead, are extremely heterogeneous both structurally and functionally. The different arrangement of cell junctions between ECs and the local organization of the basal membrane generate different type of endothelium with different permeability features and functions. Continuous, fenestrated and discontinuous endothelia are distributed based on the specific function carried out by the organs. It is thought that a large number ECs functions and their responses to extracellular cues depend on changes in intracellular concentrations of calcium ion ([Ca 2+ ] i ). The extremely complex calcium machinery includes plasma membrane bound channels as well as intracellular receptors distributed in distinct cytosolic compartments that act jointly to maintain a physiological [Ca 2+ ] i , which is crucial for triggering many cellular mechanisms. Here, we first survey the overall notions related to intracellular Ca 2+ mobilization and later highlight the involvement of this second messenger in crucial ECs functions with the aim at stimulating further investigation that link Ca 2+ mobilization to ECs in health and disease.

Sea urchin eggs have been extensively used to study Ca 2+ release through intracellular Ca 2+ -permeable channels. Their amenability to homogenization yields a robust, cell-free preparation that was central to establishing the Ca 2+ mobilizing actions of cyclic ADP-ribose and NAADP. Egg homogenates have continued to provide insight into the basic properties and pharmacology of intracellular Ca 2+ release channels. In this chapter, we describe methods for the preparation of egg homogenates and monitoring Ca 2+ release using fluorimetry and radiotracer flux., (© 2019 Elsevier Inc. All rights reserved.)

Trichoderma filamentous fungi are increasingly used as biocontrol agents and plant biostimulants. Growing evidence indicates that part of the beneficial effects is mediated by the activity of fungal metabolites on the plant host. We have investigated the mechanism of plant perception of HYTLO1, a hydrophobin abundantly secreted by Trichoderma longibrachiatum , which may play an important role in the early stages of the plant-fungus interaction. Aequorin-expressing Lotus japonicus suspension cell cultures responded to HYTLO1 with a rapid cytosolic Ca 2+ increase that dissipated within 30 min, followed by the activation of the defence-related genes MPK3 , WRK33 , and CP450 . The Ca 2+ -dependence of these gene expression was demonstrated by using the extracellular Ca 2+ chelator EGTA and Ned-19, a potent inhibitor of the nicotinic acid adenine dinucleotide phosphate (NAADP) receptor in animal cells, which effectively blocked the HYTLO1-induced Ca 2+ elevation. Immunocytochemical analyses showed the localization of the fungal hydrophobin at the plant cell surface, where it forms a protein film covering the plant cell wall. Our data demonstrate the Ca 2+ -mediated perception by plant cells of a key metabolite secreted by a biocontrol fungus, and provide the first evidence of the involvement of NAADP-gated Ca 2+ release in a signalling pathway triggered by a biotic stimulus.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is currently the most potent endogenous Ca 2+ mobilizing second messenger. Upon specific extracellular stimulation, rapid production of NAADP has been observed in different cell types from sea urchin eggs to mammalian cells. More than 20 years after the discovery of NAADP, there is still controversy surrounding its metabolism and target receptors/ion channels and organelles. This article briefly reviews recent developments in the NAADP field. Besides the metabolism of NAADP, this review focuses on assumed organelles and putative targets, e.g. ion channels, with special emphasis on ryanodine receptor type 1 (RyR1) and two-pore channels (TPCs). The role of NAADP as a Ca 2+ trigger is also discussed and the importance of NAADP in the formation of initial Ca 2+ microdomains is highlighted., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)

During the development of the early spinal circuitry in zebrafish, spontaneous Ca 2+ transients in the primary motor neurons (PMNs) are reported to transform from being slow and uncorrelated, to being rapid, synchronized and patterned. In this study, we demonstrated that in intact zebrafish, Ca 2+ release via two-pore channel type 2 (TPC2) from acidic stores/endolysosomes is required for the establishment of synchronized activity in the PMNs. Using the SAIGFF213A;UAS:GCaMP7a double-transgenic zebrafish line, Ca 2+ transients were visualized in the caudal PMNs (CaPs). TPC2 inhibition via molecular, genetic or pharmacological means attenuated the CaP Ca 2+ transients, and decreased the normal ipsilateral correlation and contralateral anti-correlation, indicating a disruption in normal spinal circuitry maturation. Furthermore, treatment with MS-222 resulted in a complete (but reversible) inhibition of the CaP Ca 2+ transients, as well as a significant decrease in the concentration of the Ca 2+ mobilizing messenger, nicotinic acid adenine diphosphate (NAADP) in whole embryo extract. Together, our new data suggest a novel function for NAADP/TPC2-mediated Ca 2+ signaling in the development, coordination, and maturation of the spinal network in zebrafish embryos., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Membrane contact sites are regions of close apposition between organelles that facilitate information transfer. Here, we reveal an essential role for Ca 2+ derived from the endo-lysosomal system in maintaining contact between endosomes and the endoplasmic reticulum (ER). Antagonizing action of the Ca 2+ -mobilizing messenger NAADP, inhibiting its target endo-lysosomal ion channel, TPC1, and buffering local Ca 2+ fluxes all clustered and enlarged late endosomes/lysosomes. We show that TPC1 localizes to ER-endosome contact sites and is required for their formation. Reducing NAADP-dependent contacts delayed EGF receptor de-phosphorylation consistent with close apposition of endocytosed receptors with the ER-localized phosphatase PTP1B. In accord, downstream MAP kinase activation and mobilization of ER Ca 2+ stores by EGF were exaggerated upon NAADP blockade. Membrane contact sites between endosomes and the ER thus emerge as Ca 2+ -dependent hubs for signaling., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Autonomic neural activation of intracellular Ca 2+ release in parotid acinar cells induces the secretion of the fluid and protein components of primary saliva critical for maintaining overall oral homeostasis. In the current study, we profiled the role of acidic organelles in shaping the Ca 2+ signals of parotid acini using a variety of imaging and pharmacological approaches. Results demonstrate that zymogen granules predominate as an apically polarized population of acidic organelles that contributes to the initial Ca 2+ release. Moreover, we provide evidence that indicates a role for the intracellular messenger NAADP in the release of Ca 2+ from acidic organelles following elevation of cAMP. Our data are consistent with the "trigger" hypothesis where localized release of Ca 2+ sensitizes canonical intracellular Ca 2+ channels to enhance signals from the endoplasmic reticulum. Release from acidic stores may be important for initiating saliva secretion at low levels of stimulation and a potential therapeutic target to augment secretory activity in hypofunctioning salivary glands., (Copyright © 2016 the American Physiological Society.)

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca(2+) mobilizing second messenger that belongs to the superfamily of regulatory adenine nucleotides. Though NAADP has been known since 20 years, several aspects of its metabolism and molecular mode of action are still under discussion. Though the importance of the type 1 ryanodine receptor was discovered and published already in 2002 Hohenegger et al. (2002 Oct 15), recent data re-emphasize these original findings in pancreatic acinar cells and in T-lymphocytes. Here we review recent developments in NAADP formation and metabolism, putative target Ca(2+) channels for NAADP with special emphasis on the type 1 ryanodine receptor, and NAADP binding proteins. The latter are basis for a unifying hypothesis for NAADP action. Finally, the role of NAADP in T cell Ca(2+) signaling and activation is discussed. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Extracellular stimuli evoke the synthesis of intracellular second messengers, several of which couple to the release of Ca(2+)from Ca(2+)-storing organelles via activation of cognate organellar Ca(2+)-channel complexes. The archetype is the inositol 1,4,5-trisphosphate (IP3) and IP3receptor (IP3R) on the endoplasmic reticulum (ER). A less understood, parallel Ca(2+)signalling cascade is that involving the messenger nicotinic acid adenine dinucleotide phosphate (NAADP) that couples to Ca(2+)release from acidic Ca(2+)stores [e.g. endo-lysosomes, secretory vesicles, lysosome-related organelles (LROs)]. NAADP-induced Ca(2+)release absolutely requires organellar TPCs (two-pore channels). This review discusses how ER and acidic Ca(2+)stores physically and functionally interact to generate and shape global and local Ca(2+)signals, with particular emphasis on the two-way dialogue between these two organelles., (© 2016 Authors; published by Portland Press Limited.)

The Sidt2 global knockout mouse (Sidt2(-/-)) has impaired insulin secretion. The aim of this study was to assess the role of SIDT2 protein in glucose-induced insulin secretion in primary cultured mouse β-cells. The major metabolic and electrophysiological steps of glucose-induced insulin secretion of primary cultured β-cells from Sidt2(-/-) mice were investigated. The β-cells from Sidt2(-/-) mice had normal NAD(P)H responses and KATP and KV currents. However, they exhibited a lower [Ca(2+)]i peak height when stimulated with 20mM glucose compared with those from WT mice. Furthermore, it took a longer time for the [Ca(2+)]i of β-cell from Sidt2(-/-) mice to reach the peak. Pretreatment with ryanodine or 2-aminoethoxydiphenyl borate (2-APB) did not change [Ca(2+)]i the response pattern to glucose in Sidt2(-/-) cells. Extraordinarily, pretreatment with bafilomycin A1(Baf-A1) led to a comparable [Ca(2+)]i increase pattern between these two groups, suggesting that calcium traffic from the intracellular acidic compartment is defective in Sidt2(-/-) β-cells. Bath-mediated application of 50nM nicotinic acid adenine dinucleotide phosphate (NAADP) normalized the [Ca(2+)]i response of Sidt2(-/-) β-cells. Finally, glucose-induced CD38 expression increased to a comparable level between Sidt2(-/-) and WT islets, suggesting that Sidt2(-/-) islets generated NAADP normally. We conclude that Sidt2 is involved in NAADP-mediated release of calcium from insulin secretory granules and thus regulates insulin secretion., (© 2016 Society for Endocrinology.)

Embryonic stem cells (ESCs) are promising resources for both scientific research and clinical regenerative medicine. With regards to the latter, ESCs are especially useful for treating several neurodegenerative disorders. Two significant characteristics of ESCs, which make them so valuable, are their capacity for self-renewal and their pluripotency, both of which are regulated by the integration of various signaling pathways. Intracellular Ca(2+) signaling is involved in several of these pathways. It is known to be precisely controlled by different Ca(2+) channels and pumps, which play an important role in a variety of cellular activities, including proliferation, differentiation and apoptosis. Here, we provide a review of the recent work conducted to investigate the function of Ca(2+) signaling in the self-renewal and the neural differentiation of ESCs. Specifically, we describe the role of intracellular Ca(2+) mobilization mediated by RyRs (ryanodine receptors); by cADPR (cyclic adenosine 5'-diphosphate ribose) and CD38 (cluster of differentiation 38/cADPR hydrolase); and by NAADP (nicotinic acid adenine dinucleotide phosphate) and TPC2 (two pore channel 2). We also discuss the Ca(2+) influx mediated by SOCs (store-operated Ca(2+) channels), TRPCs (transient receptor potential cation channels) and LTCC (L-type Ca(2+) channels) in the pluripotent ESCs as well as in neural differentiation of ESCs. Moreover, we describe the integration of Ca(2+) signaling in the other signaling pathways that are known to regulate the fate of ESCs., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Ca(2+) signals are central to the stimulation of insulin secretion from pancreatic β-cells by glucose and other agents, including glucagon-like peptide-1 (GLP-1). Whilst Ca(2+) influx through voltage-gated Ca(2+) channels on the plasma membrane is a key trigger for glucose-stimulated secretion, mobilisation of Ca(2+) from acidic stores has been implicated in the control of more localised Ca(2+) changes and membrane potential. Nicotinic acid adenine dinucleotide phosphate (NAADP), generated in β-cells in response to high glucose, is a potent mobiliser of these stores, and has been proposed to act through two pore channels (TPC1 and TPC2, murine gene names Tpcn1 and Tpcn2). Whilst the role of TPC1 in the control of Ca(2+) mobilisation and insulin secretion was recently confirmed, conflicting data exist for TPC2. Here, we used the selective and efficient deleter strain, Ins1Cre to achieve β-cell selective deletion of the Tpcn2 gene in mice. βTpcn2 KO mice displayed normal intraperitoneal and oral glucose tolerance, and glucose-stimulated Ca(2+) dynamics and insulin secretion from islets were similarly normal. GLP-1-induced Ca(2+) increases involved an increase in oscillation frequency from 4.35 to 4.84 per minute (p=0.04) at 8mM glucose, and this increase was unaffected by the absence of Tpcn2. The current data thus indicate that TPC2 is not absolutely required for normal glucose- or incretin-stimulated insulin secretion from the β-cell. Our findings suggest that TPC1, whose expression tended to increase in Tpcn2 null islets, might be sufficient to support normal Ca(2+) dynamics in response to stimulation by nutrients or incretins., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Endo-lysosomes are acidic organelles that besides the role in macromolecules degradation, act as intracellular Ca(2+) stores. Nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent Ca(2+)-mobilizing second messenger, produced in response to agonist stimulation, activates Ca(2+)-releasing channels on endo-lysosomes and modulates a variety of cellular functions. NAADP-evoked signals are amplified by Ca(2+) release from endoplasmic reticulum, via the recruitment of inositol 1,4,5-trisphosphate and/or ryanodine receptors through a Ca(2+)-induced Ca(2+)- release (CICR) mechanism. The endo-lysosomal Ca(2+) channels activated by NAADP were recently identified as the two-pore channels (TPCs). In addition to TPCs, endo-lysosomes express another distinct family of Ca(2+)- permeable channels, namely the transient receptor potential mucolipin (TRPML) channels, functionally distinct from TPCs. TPCs belong to the voltage-gated channels, resembling voltage-gated Na(+) and Ca(2+) channels. TPCs have important roles in vesicular fusion and trafficking, in triggering a global Ca(2+) signal and in modulation of the membrane excitability. Depletion of acidic Ca(2+) stores has been shown to activate store-operated Ca(2+) entry in human platelets and mouse pancreatic β-cells. In human platelets, Ca(2+) influx in response to acidic stores depletion is facilitated by the tubulin-cytoskeleton and occurs through non-selective cation channels and transient receptor potential canonical (TRPC) channels. Emerging evidence indicates that activation of intracellular receptors, situated on endo-lysosomes, elicits canonical and non-canonical signaling mechanisms that involve CICR and activation of non-selective cation channels in plasma membrane. The ability of endo-lysosomal Ca(2+) stores to modulate the Ca(2+) release from other organelles and the Ca(2+) entry increases the diversity and complexity of cellular signaling mechanisms.

Neurons possess an elaborate system of endolysosomes. Recently, endolysosomes were found to have readily releasable stores of intracellular calcium; however, relatively little is known about how such 'acidic calcium stores' affect calcium signaling in neurons. Here we demonstrated in primary cultured neurons that calcium released from acidic calcium stores triggered calcium influx across the plasma membrane, a phenomenon we have termed "acidic store-operated calcium entry (aSOCE)". aSOCE was functionally distinct from store-operated calcium release and calcium entry involving endoplasmic reticulum. aSOCE appeared to be governed by N-type calcium channels (NTCCs) because aSOCE was attenuated significantly by selectively blocking NTCCs or by siRNA knockdown of NTCCs. Furthermore, we demonstrated that NTCCs co-immunoprecipitated with the lysosome associated membrane protein 1 (LAMP1), and that aSOCE is accompanied by increased cell-surface expression levels of NTCC and LAMP1 proteins. Moreover, we demonstrated that siRNA knockdown of LAMP1 or Rab27a, both of which are key proteins involved in lysosome exocytosis, attenuated significantly aSOCE. Taken together our data suggest that aSOCE occurs in neurons, that aSOCE plays an important role in regulating the levels and actions of intraneuronal calcium, and that aSOCE is regulated at least in part by exocytotic insertion of N-type calcium channels into plasma membranes through LAMP1-dependent lysosome exocytosis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Acidic organelles such as lysosomes serve as non-canonical Ca(2+) stores. The Ca(2+) mobilising messenger NAADP is thought to trigger local Ca(2+) release from such stores. These events are then amplified by Ca(2+) channels on canonical ER Ca(2+) stores to generate physiologically relevant global Ca(2+) signals. Coupling likely occurs at microdomains formed at membrane contact sites between acidic organelles and the ER. Molecular analyses and computational modelling suggest heterogeneity in the composition of these contacts and predicted Ca(2+) microdomain behaviour. Conversely, acidic organelles might also locally amplify and temper ER-evoked Ca(2+) signals. Ca(2+) microdomains between distinct Ca(2+) stores are thus likely to be integral to the genesis of complex Ca(2+) signals., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Nicotinamide adenine dinucleotide (NAD) has been known since a long period of time as co-factor of oxidoreductases. However, in the past couple of decades further roles have been assigned to NAD. Here, metabolism of NAD to the Ca²⁺ mobilizing second messengers cyclic adenosine diphosphoribose, nicotinic acid adenine dinucleotide phosphate and adenosine diphosphoribose is reviewed. Moreover, the mechanisms of Ca²⁺ mobilization by these adenine nucleotides and their putative target Ca²⁺ channels, ryanodine receptors and transient receptor potential channels are discussed. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The second messenger NAADP triggers Ca(2+) release from endo-lysosomes. Although two-pore channels (TPCs) have been proposed to be regulated by NAADP, recent studies have challenged this. By generating the first mouse line with demonstrable absence of both Tpcn1 and Tpcn2 expression (Tpcn1/2(-/-)), we show that the loss of endogenous TPCs abolished NAADP-dependent Ca(2+) responses as assessed by single-cell Ca(2+) imaging or patch-clamp of single endo-lysosomes. In contrast, currents stimulated by PI(3,5)P2 were only partially dependent on TPCs. In Tpcn1/2(-/-) cells, NAADP sensitivity was restored by re-expressing wild-type TPCs, but not by mutant versions with impaired Ca(2+)-permeability, nor by TRPML1. Another mouse line formerly reported as TPC-null likely expresses truncated TPCs, but we now show that these truncated proteins still support NAADP-induced Ca(2+) release. High-affinity [(32)P]NAADP binding still occurs in Tpcn1/2(-/-) tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca(2+)-permeable channels indispensable for NAADP signalling., (© 2015 The Authors. Published under the terms of the CC BY 4.0 license.)

Endolysosomes are emerging as key players that generate as well as respond to intracellular Ca(2+) signals. The role of Ca(2+) in modulating acidic organelle function has long been recognized, but it is now emerging that acidic organelles also act as intracellular Ca(2+) stores; they actively sequester Ca(2+) in their lumina and release it to the cytosol upon activation of endolysosomal Ca(2+) channels. This local Ca(2+) signal is crucial for endolysosomal function and/or global Ca(2+) signaling. Importantly, defects in endolysosomal Ca(2+) are associated with disease. This chapter discusses several complimentary approaches to monitor endolysosomal Ca(2+), with particular emphasis on the inherent pitfalls that can plague the unwary., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Egg activation at fertilization is an excellent process for studying calcium regulation. Nicotinic acid adenine dinucleotide-phosphate (NAADP), a potent calcium messenger, is able to trigger calcium release, likely through two-pore channels (TPCs). Concomitantly, a family of ectocellular enzymes, the ADP-ribosyl cyclases (ARCs), has emerged as being able to change their enzymatic mode from one of nucleotide cyclization in formation of cADPR to a base-exchange reaction in the generation of NAADP. Using sea star oocytes we gain insights into the functions of endogenously expressed TPCs and ARCs in the context of the global calcium signals at fertilization. Three TPCs and one ARC were found in the sea star (Patiria miniata) that were localized in the cortex of the oocytes and eggs. PmTPCs were localized in specialized secretory organelles called cortical granules, and PmARCs accumulated in a different, unknown, set of vesicles, closely apposed to the cortical granules in the egg cortex. Using morpholino knockdown of PmTPCs and PmARC in the oocytes, we found that both calcium regulators are essential for early embryo development, and that knockdown of PmTPCs leads to aberrant construction of the fertilization envelope at fertilization and changes in cortical granule pH. The calcium signals at fertilization are not significantly altered when individual PmTPCs are silenced, but the timing and shape of the cortical flash and calcium wave are slightly changed when the expression of all three PmTPCs is perturbed concomitantly, suggesting a cooperative activity among TPC isoforms in eliciting calcium signals that may influence localized physiological activities., (© 2014. Published by The Company of Biologists Ltd.)

Strong β-adrenergic stimulation induced spontaneous diastolic Ca(2+) transients (SCTs) in electrically paced murine cardiac myocytes [28]. To obtain further insights into the underlying mechanism, we developed a method for a simultaneous analysis, in which the free luminal Ca(2+) concentration in the sarcoplasmic reticulum (SR) ([Ca(2+)]SR) and the free cytosolic Ca(2+) concentration ([Ca(2+)]i) were measured in parallel in the same cell. Each spontaneous diastolic Ca(2+) transient was exactly mirrored by a decrease of [Ca(2+)]SR. Since antagonism of the Ca(2+) mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) was shown to block SCTs in single cardiac myocytes [28], we analyzed the effect of the novel ADP-ribosyl cyclase inhibitor SAN4825 on both cytosolic and intra-luminal Ca(2+) transients upon strong β-adrenergic stimulation. A strong antagonist effect of SAN4825 on SCTs at low micromolar concentrations was observed. Our results suggest that the underlying mechanism of spontaneous diastolic Ca(2+) transients observed upon strong β-adrenergic stimulation is sensitization of type 2 ryanodine receptor by the Ca(2+) releasing activity of the products of ADP-ribosyl cyclase activity., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Acidic organelles form an important intracellular Ca(2+) pool that can drive global Ca(2+) signals through coupling with endoplasmic reticulum (ER) Ca(2+) stores. Recently identified lysosome-ER membrane contact sites might allow formation of Ca(2+) microdomains, although their size renders observation of Ca(2+) dynamics impractical. Here, we generated a computational model of lysosome-ER coupling that incorporated a previous model of the inositol trisphosphate (IP3) receptor as the ER Ca(2+) 'amplifier' and lysosomal leaks as the Ca(2+) 'trigger'. The model qualitatively described global Ca(2+) responses to the lysosomotropic agent GPN, which caused a controlled but substantial depletion of small solutes from the lysosome. Adapting this model to physiological lysosomal leaks induced by the Ca(2+) mobilising messenger NAADP demonstrated that lysosome-ER microdomains are capable of driving global Ca(2+) oscillations. Interestingly, our simulations suggest that the microdomain [Ca(2+)] need not be higher than that in the cytosol for responses to occur, thus matching the relatively high affinity of IP3 receptors for Ca(2+). The relative distribution and overall density of the lysosomal leaks dictated whether microdomains triggered or modulated global signals. Our data provide a computational framework for probing lysosome-ER Ca(2+) dynamics., (© 2014. Published by The Company of Biologists Ltd.)

Ca(2+) signaling in spermatozoa plays a crucial role during processes such as capacitation and release of the acrosome, but the underlying molecular mechanisms still remain unclear. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca(2+)-releasing second messenger in a variety of cellular processes. The presence of a NAADP synthesizing enzyme in sea urchin sperm has been previously reported, suggesting a possible role of NAADP in sperm Ca(2+) signaling. In this work we used in vitro enzyme assays to show the presence of a novel NAADP synthesizing enzyme in human sperm, and to characterize its sensitivity to Ca(2+) and pH. Ca(2+) fluorescence imaging studies demonstrated that the permeable form of NAADP (NAADP-AM) induces intracellular [Ca(2+)] increases in human sperm even in the absence of extracellular Ca(2+). Using LysoTracker, a fluorescent probe that selectively accumulates in acidic compartments, we identified two such stores in human sperm cells. Their acidic nature was further confirmed by the reduction in staining intensity observed upon inhibition of the endo-lysosomal proton pump with Bafilomycin, or after lysosomal bursting with glycyl-l-phenylalanine-2-naphthylamide. The selective fluorescent NAADP analog, Ned-19, stained the same subcellular regions as LysoTracker, suggesting that these stores are the targets of NAADP action., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The endoplasmic reticulum is a major organelle in all eukaryotic cells which performs multiple functions including protein and lipid synthesis and sorting, drug metabolism, and Ca(2+) storage and release. The endoplasmic reticulum, and its specialized muscle counterpart the sarcoplasmic reticulum, is the largest and most extensive of Ca(2+) storage organelle in eukaryotic cells, often occupying in excess of 10% of the cell volume. There are three major components of Ca(2+) storage organelles which mediate their major functions: Ca(2+) uptake, mediated by pumps and exchangers; storage enhanced by luminal Ca(2+) binding proteins, and Ca(2+) mobilization mediated by specific ion channels. Ca(2+) mobilization from the endoplasmic reticulum plays a central role in Ca(2+) signaling. Through Ca(2+) release channels in its membrane, the pervading and plastic structure of the endoplasmic reticulum allows Ca(2+) release to be rapidly targeted to specific cytoplasmic sites across the whole cell. That several endoplasmic reticulum Ca(2+) release channels are also activated by Ca(2+) itself, contributes to endoplasmic reticulum membrane excitability which is the principal basis for generating spatio-temporal complex cellular Ca(2+) signals, allowing specific processes to be regulated by this universal messenger. In addition, the endoplasmic reticulum forms discrete junctions with the plasma membrane and membranes of organelles such as mitochondria and lysosomes, forming nanodomains at their interfaces that play critical roles in Ca(2+) signaling during key cellular processes such as cellular bioenergetics, apoptosis and autophagy. At these junctions key Ca(2+) transport and regulatory processes come into play, and a recurring theme in this review is the often tortuous paths in identifying these mechanisms unequivocally. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Introduction: CD38 is a multifunctional enzyme with a potent Ca2+ mobilizing effect, cyclic ADP-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP). Here, we aimed to demonstrate the role of CD38 in platelets via protein kinase C (PKC)-mediated internalization and activation. Methods: Mouse platelets were used in this study. Thrombin, an agonist of platelet function, provoked a prompt and long-lasting increase in intracellular Ca2+ concentration ([Ca2+]i), resulting from an interplay of multifold Ca2+ mobilizing messengers.The signaling pathway was delineated using different inhibitors and techniques such as platelet aggregation assay, intracellular calcium measurements, immunoprecipitation, immunoblotting, and flow cytometry. Results: We observed a sequential formation of cADPR and NAADP through CD38 activation by PKC of non-muscle myosin heavy chain IIA (MHCIIA), resulting in phospholipase C (PLC) activation in the thrombin-stimulated platelets. These findings reveal that PKC is fundamental in activating CD38 and elicits a physiological response in the murine platelets. Conclusion: PKC is involved in many signaling pathways. Specifically, PKC is involved in the internalization of CD38 via MHCIIA in CD38+/+ wild-type (WT) and CD38-/- knockout mice (KO). CD38 generates calcium-mobilizing agents that act on specific receptors of the calcium stores. Calcium triggered platelet aggregation while serving as a secondary messenger. [ABSTRACT FROM AUTHOR]

Introduction: CD38 is a multifunctional enzyme with a potent Ca2+ mobilizing effect, cyclic ADP-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP). Here, we aimed to demonstrate the role of CD38 in platelets via protein kinase C (PKC)-mediated internalization and activation. Methods: Mouse platelets were used in this study. Thrombin, an agonist of platelet function, provoked a prompt and long-lasting increase in intracellular Ca2+ concentration ([Ca2+]i), resulting from an interplay of multifold Ca2+ mobilizing messengers.The signaling pathway was delineated using different inhibitors and techniques such as platelet aggregation assay, intracellular calcium measurements, immunoprecipitation, immunoblotting, and flow cytometry. Results: We observed a sequential formation of cADPR and NAADP through CD38 activation by PKC of non-muscle myosin heavy chain IIA (MHCIIA), resulting in phospholipase C (PLC) activation in the thrombin-stimulated platelets. These findings reveal that PKC is fundamental in activating CD38 and elicits a physiological response in the murine platelets. Conclusion: PKC is involved in many signaling pathways. Specifically, PKC is involved in the internalization of CD38 via MHCIIA in CD38+/+ wild-type (WT) and CD38-/- knockout mice (KO). CD38 generates calcium-mobilizing agents that act on specific receptors of the calcium stores. Calcium triggered platelet aggregation while serving as a secondary messenger.

NAADP is one of the most potent calcium mobilizing second messengers. Only recently, two NAADP-binding proteins have been identified: HN1L/JPT2 and LSM12. Further, ASPDH was suggested as a less selective binding partner. Apart from this newly uncovered link, little is known about the shared mechanisms between these proteins. The aim of this review is to assess potential functional connections between NAADP and its binding proteins. We here give a description of two major links. For one, HN1L/JPT2 and LSM12 both have potent oncogenic functions in several cancer types. Second, they are involved in similar cellular pathways in both cancer and immunity. • HN1L/JPT2 and LSM12 are the two currently known NAADP-binding proteins. • HN1L/JPT2 and LSM12 are involved in NAADP-mediated Ca2+ signaling. • HN1L/JPT2 and LSM12 incorporate an oncogenic function in different cancer types. • HN1L/JPT2, LSM12 and NAADP regulate similar signaling pathways in immunity. [ABSTRACT FROM AUTHOR]