Search

Melanosomal pH is important for the synthesis of melanin as the rate‐limiting enzyme, tyrosinase, is very pH‐sensitive. The soluble adenylyl cyclase (sAC) signaling pathway was recently identified as a regulator of melanosomal pH in melanocytes; however, the melanosomal proteins critical for sAC‐dependent regulation of melanosomal pH were undefined. We now systematically examine four well‐characterized melanosomal membrane proteins to determine whether any of them are required for sAC‐dependent regulation of melanosomal pH. We find that OA1, OCA2, and SLC45A2 are dispensable for sAC‐dependent regulation of melanosomal pH. In contrast, TPC2 activity is required for sAC‐dependent regulation of melanosomal pH and melanin synthesis. In addition, activation of TPC2 by NAADP‐AM rescues melanosomal pH alkalinization and reduces melanin synthesis following pharmacologic or genetic inhibition of sAC signaling. These studies establish TPC2 as a critical melanosomal protein for sAC‐dependent regulation of melanosomal pH and pigmentation. [ABSTRACT FROM AUTHOR]

Two‐pore channels and TRP mucolipins are ubiquitous endo‐lysosomal cation channels of pathophysiological relevance. Both are Ca2+‐permeable and regulated by phosphoinositides, principally PI(3,5)P2. Accumulating evidence has uncovered synergistic channel activation by PI(3,5)P2 and endogenous metabolites such as the Ca2+ mobilizing messenger NAADP, synthetic agonists including approved drugs and physical cues such as voltage and osmotic pressure. Here, we provide an overview of this coordination. [ABSTRACT FROM AUTHOR]

Two-pore channel (TPC) is an endolysosomal calcium channel implicated in numerous physiological processes and pathologies. Since no bibliometric studies have been conducted to evaluate the typical characteristics of TPC's research, this study sought to pinpoint TPC's global research trends and hotspots. The Scopus database was searched for TPC-related articles from 2000 to 2022. The data were analysed using Harzing's Publish, Open Refine, and VOS viewer. In total, 292 TPC-related articles written in English were analysed. This study demonstrates that the publication rate of TPC has increased since their discovery in 2000 and will continue to increase until 2022. Over the past 22 years, research on TPC has focused on elucidating its function in calcium signalling, revealing its significance in a variety of physiological and pathological conditions. Yet, there are some research prospects for TPC that are currently understudied and warrant attention, notably in the context of autophagy, cancer, and COVID-19. [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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Bafilomycin A1 inhibits V-type H+ ATPases on the molecular level, which acidifies endo-lysosomes. The main objective of the study was to assess the effect of bafilomycin A1 on Ca2+ content, NAADP-induced Ca2+ release, and ATPase activity in rat hepatocytes and human colon cancer samples. Chlortetracycline (CTC) was used for a quantitative measure of stored calcium in permeabilized rat hepatocytes. ATPase activity was determined by orthophosphate content released after ATP hydrolysis in subcellular post-mitochondrial fraction obtained from rat liver as well as from patients' samples of colon mucosa and colorectal cancer samples. In rat hepatocytes, bafilomycin A1 decreased stored Ca2+ and prevented the effect of NAADP on stored Ca2+. This effect was dependent on EGTA–Ca2+ buffers in the medium. Bafilomycin A1 significantly increased the activity of Ca2+ ATPases of endoplasmic reticulum (EPR), but not plasma membrane (PM) Ca2+ ATPases in rat liver. Bafilomycin A1 also prevented the effect of NAADP on these pumps. In addition, bafilomycin A1 reduced Na+/K+ ATPase activity and increased basal Mg2+ ATPase activity in the subcellular fraction of rat liver. Concomitant administration of bafilomycin A1 and NAADP enhanced these effects. Bafilomycin A1 increased the activity of the Ca2+ ATPase of EPR in the subcellular fraction of normal human colon mucosa and also in colon cancer tissue samples. In contrast, it decreased Ca2+ ATPase PM activity in samples of normal human colon mucosa and caused no changes in colon cancer. Bafilomycin A1 decreased Na+/K+ ATPase activity and increased basal Mg2+ ATPase activity in normal colon mucosa samples and in human colon cancer samples. It can be concluded that bafilomycin A1 targets NAADP-sensitive acidic Ca2+ stores, effectively modulates ATPase activity, and assumes the link between acidic stores and EPR. Bafilomycin A1 may be useful for cancer therapy. [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.

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.

Ischemia and perfusion (I/R) induce inflammation and oxidative stress, which play a notable role in tissue damage. The aim of this study was to investigate the role of an NADPH oxidase inhibitor (apocynin) in the protection of the heart from I/R injury. Hearts isolated from Wistar rats (n = 8 per group) were perfused with a modified Langendorff preparation. Left ventricular (LV) contractility and cardiovascular hemodynamics were evaluated by a data acquisition program, and infarct size was evaluated by 2,3,5-Triphenyl-2H-tetrazolium chloride (TTC) staining. Furthermore, the effect of apocynin on the pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and anti-inflammatory cytokine (IL-10) was evaluated using an enzyme linked immunosorbent assay (ELISA). Hearts were subjected to 30 min of regional ischemia, produced by ligation of the left anterior descending (LAD) coronary artery, followed by 30 min of reperfusion. Hearts were infused with apocynin before ischemia, during ischemia or at reperfusion. To understand the potential pathways of apocynin protection of the heart, a nitric oxide donor (S-nitroso-N-acetylpenicillamine, SNAP), nitric oxide blocker (N (gamma)-nitro-L-arginine methyl ester, L-Name), nicotinic acid adenine dinucleotide phosphate (NAADP) inhibiter (Ned-K), cyclic adenosine diphosphate ribose (cADPR) agonist, or CD38 blocker (Thiazoloquin (az)olin (on)e compound, 78c) was infused with apocynin. Antioxidants were evaluated by measuring superoxide dismutase (SOD) and catalase (CAT) activity. Apocynin infusion before ischemia or at reperfusion protected the heart by normalizing cardiac hemodynamics and decreasing the infarct size. Apocynin treatment resulted in a significant (p < 0.05) decrease in pro-inflammatory cytokine levels and a significant increase (p < 0.05) in anti-inflammatory and antioxidant levels. Apocynin infusion protected the heart by improving LV hemodynamics and coronary vascular dynamics. This treatment decreased the infarct size and inflammatory cytokine levels and increased anti-inflammatory cytokine and antioxidant levels. This protection follows a pathway involving CD38, nitric oxide and acidic stores. [ABSTRACT FROM AUTHOR]

Huntington's disease (HD) is a progressive neurodegenerative disease characterized by mutations in the huntingtin gene (mHtt), causing an unstable repeat of the CAG trinucleotide, leading to abnormal long repeats of polyglutamine (poly-Q) in the N-terminal region of the huntingtin, which form abnormal conformations and aggregates. Alterations in Ca2+ signaling are involved in HD models and the accumulation of mutated huntingtin interferes with Ca2+ homeostasis. Lysosomes are intracellular Ca2+ storages that participate in endocytic and lysosomal degradation processes, including autophagy. Nicotinic acid adenine dinucleotide phosphate (NAADP) is an intracellular second messenger that promotes Ca2+ release from the endo-lysosomal system via Two-Pore Channels (TPCs) activation. Herein, we show the impact of lysosomal Ca2+ signals on mHtt aggregation and autophagy blockade in murine astrocytes overexpressing mHtt-Q74. We observed that mHtt-Q74 overexpression causes an increase in NAADP-evoked Ca2+ signals and mHtt aggregation, which was inhibited in the presence of Ned-19, a TPC antagonist, or BAPTA-AM, a Ca2+ chelator. Additionally, TPC2 silencing revert the mHtt aggregation. Furthermore, mHtt has been shown co-localized with TPC2 which may contribute to its effects on lysosomal homeostasis. Moreover, NAADP-mediated autophagy was also blocked since its function is dependent on lysosomal functionality. Taken together, our data show that increased levels of cytosolic Ca2+ mediated by NAADP causes mHtt aggregation. Additionally, mHtt co-localizes with the lysosomes, where it possibly affects organelle functions and impairs autophagy. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide (NAD) and its 2′-phosphorylated cousin NADP are precursors for the enzymatic formation of the Ca2+-mobilizing second messengers adenosine diphosphoribose (ADPR), 2′-deoxy-ADPR, cyclic ADPR, and nicotinic acid adenine dinucleotide phosphate (NAADP). The enzymes involved are either NAD glycohydrolases CD38 or sterile alpha toll/interleukin receptor motif containing-1 (SARM1), or (dual) NADPH oxidases (NOX/DUOX). Enzymatic function(s) are reviewed and physiological role(s) in selected cell systems are discussed. [ABSTRACT FROM AUTHOR]

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a signaling molecule that can induce calcium release from intracellular acidic stores. However, proteins that bind to NAADP are understudied. Here, we identify aspartate dehydrogenase domain-containing protein (ASPDH) as an NAADP-binding protein through biochemical purification from pig livers. Isothermal titration calorimetry (ITC) experiment using the recombinantly expressed protein shows a 1:1 binding stoichiometry and a Kd of 455 nM between NAADP and mouse ASPDH. In contrast, recombinantly expressed Jupiter microtubule-associated homolog 2 (JPT2) and SM-like protein LSM12, two proteins previously identified as NAADP-receptors, show no binding in ITC experiments. [Display omitted] • A non-radioactive NAADP binding assay has been developed. • ASPDH is identified as an NAADP-binding protein in livers. • Recombinant LSM12 and JPT2 do not bind NAADP. [ABSTRACT FROM AUTHOR]

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 Ca2+-mobilizing messenger NAADP and/or the lipid PI(3,5)P2. TPC currents are regulated by H+, Ca2+, and Mg2+ (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 Ca2+-permeable (NAADP-like) state to a Na+-selective (PI(3,5)P2-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. [ABSTRACT FROM AUTHOR]

The recent elegant study by Y. Yuan and colleagues examined functional relationships between the lysosomal two-pore channels 2 (TPC2) and IP3 receptors (IP3Rs) located in the endoplasmic reticulum [1]. The findings of this study suggest functional coupling of these channels and receptors. The study also describes interesting novel phenomena, which may indicate an additional coupling mechanism. Illustrates our interpretation of findings by Y. Yuan and colleagues [1]. NAADP was not used in the study; the putative effects of NAADP and its sensors Jupiter Microtubule Associated Homolog 2 (JPT2) and LSm12 (Sm-like protein 12) are, therefore shown by dashed arrows (for further information about this pathway see recent review [3]). The study extensively utilised TPC2-A1-N, which stimulates Ca2+ conductance of TPC2 (shown on the Figure). In some experiments TPC2-A1-P, which preferentially increases Na+ conductance, was also used (not shown). [Display omitted] [ABSTRACT FROM AUTHOR]

Ischemia and perfusion (I/R) induce inflammation and oxidative stress, which play a notable role in tissue damage. The aim of this study was to investigate the role of an NADPH oxidase inhibitor (apocynin) in the protection of the heart from I/R injury. Hearts isolated from Wistar rats (n = 8 per group) were perfused with a modified Langendorff preparation. Left ventricular (LV) contractility and cardiovascular hemodynamics were evaluated by a data acquisition program, and infarct size was evaluated by 2,3,5-Triphenyl-2H-tetrazolium chloride (TTC) staining. Furthermore, the effect of apocynin on the pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and anti-inflammatory cytokine (IL-10) was evaluated using an enzyme linked immunosorbent assay (ELISA). Hearts were subjected to 30 min of regional ischemia, produced by ligation of the left anterior descending (LAD) coronary artery, followed by 30 min of reperfusion. Hearts were infused with apocynin before ischemia, during ischemia or at reperfusion. To understand the potential pathways of apocynin protection of the heart, a nitric oxide donor (S-nitroso-N-acetylpenicillamine, SNAP), nitric oxide blocker (N (gamma)-nitro-L-arginine methyl ester, L-Name), nicotinic acid adenine dinucleotide phosphate (NAADP) inhibiter (Ned-K), cyclic adenosine diphosphate ribose (cADPR) agonist, or CD38 blocker (Thiazoloquin (az)olin (on)e compound, 78c) was infused with apocynin. Antioxidants were evaluated by measuring superoxide dismutase (SOD) and catalase (CAT) activity. Apocynin infusion before ischemia or at reperfusion protected the heart by normalizing cardiac hemodynamics and decreasing the infarct size. Apocynin treatment resulted in a significant (p < 0.05) decrease in pro-inflammatory cytokine levels and a significant increase (p < 0.05) in anti-inflammatory and antioxidant levels. Apocynin infusion protected the heart by improving LV hemodynamics and coronary vascular dynamics. This treatment decreased the infarct size and inflammatory cytokine levels and increased anti-inflammatory cytokine and antioxidant levels. This protection follows a pathway involving CD38, nitric oxide and acidic stores.

Huntington’s disease (HD) is a progressive neurodegenerative disease characterized by mutations in the huntingtin gene (mHtt), causing an unstable repeat of the CAG trinucleotide, leading to abnormal long repeats of polyglutamine (poly-Q) in the N-terminal region of the huntingtin, which form abnormal conformations and aggregates. Alterations in Ca2+ signaling are involved in HD models and the accumulation of mutated huntingtin interferes with Ca2+ homeostasis. Lysosomes are intracellular Ca2+ storages that participate in endocytic and lysosomal degradation processes, including autophagy. Nicotinic acid adenine dinucleotide phosphate (NAADP) is an intracellular second messenger that promotes Ca2+ release from the endo-lysosomal system via Two-Pore Channels (TPCs) activation. Herein, we show the impact of lysosomal Ca2+ signals on mHtt aggregation and autophagy blockade in murine astrocytes overexpressing mHtt-Q74. We observed that mHtt-Q74 overexpression causes an increase in NAADP-evoked Ca2+ signals and mHtt aggregation, which was inhibited in the presence of Ned-19, a TPC antagonist, or BAPTA-AM, a Ca2+ chelator. Additionally, TPC2 silencing revert the mHtt aggregation. Furthermore, mHtt has been shown co-localized with TPC2 which may contribute to its effects on lysosomal homeostasis. Moreover, NAADP-mediated autophagy was also blocked since its function is dependent on lysosomal functionality. Taken together, our data show that increased levels of cytosolic Ca2+ mediated by NAADP causes mHtt aggregation. Additionally, mHtt co-localizes with the lysosomes, where it possibly affects organelle functions and impairs autophagy.

Nicotinamide adenine dinucleotide (NAD) and its 2′-phosphorylated cousin NADP are precursors for the enzymatic formation of the Ca2+-mobilizing second messengers adenosine diphosphoribose (ADPR), 2′-deoxy-ADPR, cyclic ADPR, and nicotinic acid adenine dinucleotide phosphate (NAADP). The enzymes involved are either NAD glycohydrolases CD38 or sterile alpha toll/interleukin receptor motif containing-1 (SARM1), or (dual) NADPH oxidases (NOX/DUOX). Enzymatic function(s) are reviewed and physiological role(s) in selected cell systems are discussed.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a universal Ca2+ mobilizing second messenger essential for initiation of Ca2+ 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. [ABSTRACT FROM AUTHOR]

Ca2+ signalling plays an essential role in T cell activation, which is a key step to start an adaptive immune response. During the transition from a quiescent to a fully activated state, Ca2+ microdomains of reduced spatial and temporal extents develop in the junctions between the plasma membrane and the endoplasmic reticulum (ER). These microdomains rely on Ca2+ entry from the extracellular medium, via the ORAI1/STIM1/STIM2 system that mediates store operated Ca2+ entry Store operated calcium entry. The mechanism leading to local store depletion and subsequent Ca2+ entry depends on the activation state of the cells. The initial, smaller microdomains are triggered by D-myo-inositol 1,4,5-trisphosphate (IP3) signalling in response to T cell adhesion. T cell receptor (TCR)/CD3 stimulation then initiates nicotinic acid adenine dinucleotide phosphate signalling, which activates ryanodine receptors (RYR). We have recently developed a mathematical model to elucidate the spatiotemporal Ca2+ dynamics of the microdomains triggered by IP3 signalling in response to T cell adhesion (Gil et al., 2021). This reaction-diffusion model describes the evolution of the cytosolic and endoplasmic reticulum Ca2+ concentrations in a three-dimensional ER-PM junction and was solved using COMSOL Multiphysics. Modelling predicted that adhesion-dependent microdomains result from the concerted activity of IP3 receptors and pre-formed ORAI1-STIM2 complexes. In the present study, we extend this model to include the role of RYRs rapidly after TCR/CD3 stimulation. The involvement of STIM1, which has a lower KD for Ca2+ than STIM2, is also considered. Detailed 3D spatio-temporal simulations show that these Ca2+ microdomains rely on the concerted opening of ∼7 RYRs that are simultaneously active in response to the increase in NAADP induced by T cell stimulation. Opening of these RYRs provoke a local depletion of ER Ca2+ that triggers Ca2+ flux through the ORAI1 channels. Simulations predict that RYRs are most probably located around the junction and that the increase in junctional Ca2+ concentration results from the combination between diffusion of Ca2+ released through the RYRs and Ca2+ entry through ORAI1 in the junction. The computational model moreover provides a tool allowing to investigate how Ca2+ microdomains occur, extend and interact in various states of T cell activation.

Nicotinic acid adenine dinucleotide 2'-phosphate (NAADP) is a naturally occurring nucleotide that has been shown to be involved in the release of Ca2+ 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 Ca2+ signal that is then amplified by other Ca2+ 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 Ca2+ channels. [ABSTRACT FROM AUTHOR]

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‐CMHypo) triggers intracellular Ca2+ oscillations in human ECFCs, but the underlying mechanisms and the downstream Ca2+‐dependent effectors remain elusive. Herein, we found that the secretome obtained by hAFS undergoing hypoxic preconditioning induced intracellular Ca2+ oscillations by promoting extracellular Ca2+ entry through Transient Receptor Potential Vanilloid 4 (TRPV4). TRPV4‐mediated Ca2+ entry, in turn, promoted the concerted interplay between inositol‐1,4,5‐trisphosphate‐ and nicotinic acid adenine dinucleotide phosphate‐induced endogenous Ca2+ release and store‐operated Ca2+ entry (SOCE). hAFS‐CMHypo‐induced intracellular Ca2+ oscillations resulted in the nuclear translocation of the Ca2+‐sensitive transcription factor p65 NF‐κB. Finally, inhibition of either intracellular Ca2+ oscillations or NF‐κB activity prevented hAFS‐CMHypo‐induced ECFC tube formation. These data shed novel light on the molecular mechanisms whereby hAFS‐CMHypo induces angiogenesis, thus providing useful insights for future therapeutic strategies against ischaemic‐related myocardial injury. [ABSTRACT FROM AUTHOR]

Nicotinic acid adenine dinucleotide 2’-phosphate (NAADP) is a naturally occurring nucleotide that has been shown to be involved in the release of Ca2+ 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 Ca2+ signal that is then amplified by other Ca2+ 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 Ca2+ channels.

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 Ca2+-mobilizing messenger NAADP and/or the lipid PI(3,5)P2. TPC currents are regulated by H+, Ca2+, and Mg2+ (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 Ca2+-permeable (NAADP-like) state to a Na+-selective (PI(3,5)P2-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.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a universal Ca2+ mobilizing second messenger essential for initiation of Ca2+ 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.

Emerging evidence hints in favor of a life-threatening link between severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and the cardiovascular system. SARS-CoV-2 may result in dramatic cardiovascular complications, whereas the severity of COronaVIrus Disease 2019 (COVID-19) and the incidence of fatalities tend to increase in patients with pre-existing cardiovascular complications. SARS-CoV-2 is internalized into the host cells by endocytosis and may then escape the endolysosomal system via endosomes. Two-pore channels drive endolysosomal trafficking through the release of endolysosomal Ca2+. Recent evidence suggested that the pharmacological inhibition of TPCs prevents Ebola virus and Middle East Respiratory Syndrome COronaVirus (MERS-CoV) entry into host cells. In this perspective, we briefly summarize the biophysical and pharmacological features of TPCs, illustrate their emerging role in the cardiovascular system, and finally present them as a reliable target to treat cardiovascular complications in COVID-19 patients. [ABSTRACT FROM AUTHOR]

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most recently discovered Ca2+‐releasing messenger that increases the intracellular Ca2+ concentration by mobilizing the lysosomal Ca2+ store through two‐pore channels 1 (TPC1) and 2 (TPC2). NAADP‐induced lysosomal Ca2+ release regulates multiple endothelial functions, including nitric oxide release and proliferation. A sizeable acidic Ca2+ pool endowed with TPC1 is also present in human endothelial colony‐forming cells (ECFCs), which represent the only known truly endothelial precursors. Herein, we sought to explore the role of the lysosomal Ca2+ store and TPC1 in circulating ECFCs by harnessing Ca2+ imaging and molecular biology techniques. The lysosomotropic agent, Gly–Phe β‐naphthylamide, and nigericin, which dissipates the proton gradient which drives Ca2+ sequestration by acidic organelles, caused endogenous Ca2+ release in the presence of a replete inositol‐1,4,5‐trisphosphate (InsP3)‐sensitive endoplasmic reticulum (ER) Ca2+ pool. Likewise, the amount of ER releasable Ca2+ was reduced by disrupting lysosomal Ca2+ content. Liposomal delivery of NAADP induced a transient Ca2+ signal that was abolished by disrupting the lysosomal Ca2+ store and by pharmacological and genetic blockade of TPC1. Pharmacological manipulation revealed that NAADP‐induced Ca2+ release also required ER‐embedded InsP3 receptors. Finally, NAADP‐induced lysosomal Ca2+ release was found to trigger vascular endothelial growth factor‐induced intracellular Ca2+ oscillations and proliferation, while it did not contribute to adenosine‐5′‐trisphosphate‐induced Ca2+ signaling. These findings demonstrated that NAADP‐induced TPC1‐mediated Ca2+ release can selectively be recruited to induce the Ca2+ response to specific cues in circulating ECFCs. [ABSTRACT FROM AUTHOR]

Two-pore channels (TPCs) constitute a small family of cation channels that are localized in membranes of endosomal and lysosomal compartments. Although their roles for vesicular fusion and endolysosomal trafficking have been investigated, our knowledge on their expression pattern and higher order functions in the murine brain is still limited. Western blot analysis indicated a broad expression of TPC1 in the neocortex, cerebellum and hippocampus. In order to investigate the consequences of the genetic inactivation of TPC1, we performed a set of behavioural studies with TPC1−/− mice. TPC1−/− mice were analysed for an altered motor coordination and grip-strength, exploratory drive and anxiety as well as learning and memory. TPC1−/− mice did not show any differences in their exploratory drive or in their anxiety levels. There were also no differences in spontaneous activity or motor performance. However, the Morris water maze test uncovered a deficit in spatial learning and memory in TPC1−/− mice. [ABSTRACT FROM AUTHOR]

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that evokes calcium release from intracellular organelles by the engagement of calcium release channels, including members of the Transient Receptor Potential (TRP) family, such as TRPML1, the (structurally) related Two Pore Channel type 1 (TPC1) and TPC2 channels as well as Ryanodine Receptors type 1 (RYR1; Guse, 2012). NAADP evokes calcium release from acidic calcium stores of many cell types (Guse, 2012), and NAADP-sensitive Ca2+ stores have been described in hippocampal neurons of the rat (Bak et al., 1999; McGuinness et al., 2007). Glutamate triggers Ca2+-mediated neuronal excitotoxicity in inflammation-induced neurodegenerative pathologies such as Multiple Sclerosis (MS; Friese et al., 2014), and when applied extracellularly to neurons glutamate can elevate NAADP levels in these cells. Accordingly, glutamate-evoked Ca2+ signals from intracellular organelles were inhibited by preventing organelle acidification (Pandey et al., 2009). Analysis of reported RNA sequencing experiments of cultured hippocampal neurons revealed the abundance of Mcoln1 (encoding TRPML1), Tpcn1, and Tpcn2 (encoding TPC1 and TPC2, respectively) as potential NAADP target channels in these cells. Transcripts encoding Ryr1 were not found in contrast to Ryr2 and Ryr3. To study the contribution of NAADP signaling to glutamate-evoked calcium transients in murine hippocampal neurons we used the NAADP antagonists Ned-19 (Naylor et al., 2009) and BZ194 (Dammermann et al., 2009). Our results show that both NAADP antagonists significantly reduce glutamate-evoked calcium transients. In addition to extracellular glutamate application, we studied synchronized calcium oscillations in the cells of the neuronal cultures evoked by addition of the GABAA receptor antagonist bicuculline. Pretreatment with Ned-19 (50 μM) or BZ194 (100 μM) led to an increase in the frequency of bicuculline-induced calcium oscillations at the cost of calcium transient amplitudes. Interestingly, Ned-19 triggered a rise in intracellular calcium concentrations 25 min after bicuculline stimulation, leading to the question whether NAADP acts as a neuroprotective messenger in hippocampal neurons. Taken together, our results are in agreement with the concept that NAADP signaling significantly contributes to glutamate evoked Ca2+ rise in hippocampal neurons and to the amplitude and frequency of synchronized Ca2+ oscillations triggered by spontaneous glutamate release events.

Ion selectivity is a defining feature of a given ion channel and is considered immutable. Here we show that ion selectivity of the lysosomal ion channel TPC2, which is hotly debated (Calcraft et al., 2009; Guo et al., 2017; Jha et al., 2014; Ruas et al., 2015; Wang et al., 2012), depends on the activating ligand. A high-throughput screen identified two structurally distinct TPC2 agonists. One of these evoked robust Ca2+-signals and non-selective cation currents, the other weaker Ca2+-signals and Na+-selective currents. These properties were mirrored by the Ca2+-mobilizing messenger, NAADP and the phosphoinositide, PI(3,5)P2, respectively. Agonist action was differentially inhibited by mutation of a single TPC2 residue and coupled to opposing changes in lysosomal pH and exocytosis. Our findings resolve conflicting reports on the permeability and gating properties of TPC2 and they establish a new paradigm whereby a single ion channel mediates distinct, functionally-relevant ionic signatures on demand.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+ mobilizing agent and its inhibition proved to inhibit T-cell activation. However, the impact of the NAADP signaling on CD4+ T-cell differentiation and plasticity and on the inflammation in tissues other than the central nervous system remains unclear. In this study, we used an antagonist of NAADP signaling, trans-Ned 19, to study the role of NAADP in CD4+ T-cell differentiation and effector function. Partial blockade of NAADP signaling in naïve CD4+ T cells in vitro promoted the differentiation of Th17 cells. Interestingly, trans-Ned 19 also promoted the production of IL-10, co-expression of LAG-3 and CD49b and increased the suppressive capacity of Th17 cells. Moreover, using an IL-17A fate mapping mouse model, we showed that NAADP inhibition promotes conversion of Th17 cells into regulatory T cells in vitro and in vivo. In line with the results, we found that inhibiting NAADP ameliorates disease in a mouse model of intestinal inflammation. Thus, these results reveal a novel function of NAADP in controlling the differentiation and plasticity of CD4+ T cells.

In tumor cells with defects in apoptosis, autophagy allows prolonged survival. Autophagy leads to an accumulation of damaged mitochondria by autophagosomes. An acidic environment is maintained in compartments of cells, such as autophagosomes, late endosomes, and lysosomes; these organelles belong to the “acid store” of the cells. Nicotinic acid adenine dinucleotide phosphate (NAADP) may affect the release of Ca2+ from these organelles and affect the activity of Ca2+ ATPases and other ion transport proteins. Recently, a growing amount of evidence has shown that the variations in the expression of calcium channels or pumps are associated with the occurrence, disease-presentation, and the prognosis of colorectal cancer. We hypothesized that activity of ATPases in cancer tissue is higher because of intensive energy metabolism of tumor cells. The aim of our study was to ascertain the effect of NAADP on ATPase activity on tissue samples of colorectal cancer patients’ and healthy individuals. We tested the effect of NAADP on the activity of Na+/K+ ATPase; Ca2+ ATPase of endoplasmic reticulum (EPR) and plasma membrane (PM) and basal ATPase activity. Patients’ colon mucus cancer samples were obtained during endoscopy from cancer and healthy areas (control) of colorectal mucosa of the same patients. Results. The mean activity of Na+/K+ pump in samples of colorectal cancer patients (n = 5) was 4.66 ± 1.20 μmol Pi/mg of protein per hour, while in control samples from healthy tissues of the same patient (n = 5) this value was 3.88 ± 2.03 μmol Pi/mg of protein per hour. The activity of Ca2+ ATPase PM in control samples was 6.42 ± 0.63 μmol Pi/mg of protein per hour and in cancer −8.50 ± 1.40 μmol Pi/mg of protein per hour (n = 5 pts). The mean activity of Ca2+ ATPase of EPR in control samples was 7.59 ± 1.21 μmol Pi/mg versus 7.76 ± 0.24 μmol Pi/mg in cancer (n = 5 pts). Basal ATPase activity was 3.19 ± 0.87 in control samples versus 4.79 ± 1.86 μmol Pi/mg in cancer (n = 5 pts). In cancer samples, NAADP reduced the activity of Na+/K+ ATPase by 9-times (p < 0.01) and the activity of Ca2+ ATPase EPR about 2-times (p < 0.05). NAADP caused a tendency to decrease the activity of Ca2+ ATPase of PM, but increased basal ATPase activity by 2-fold vs. the mean of this index in cancer samples without the addition of NAADP. In control samples NAADP caused only a tendency to decrease the activities of Na+/K+ ATPase and Ca2+ ATPase EPR, but statistically decreased the activity of Ca2+ ATPase of PM (p < 0.05). In addition, NAADP caused a strong increase in basal ATPase activity in control samples (p < 0.01). Conclusions: We found that the activity of Na+/K+ pump, Ca2+ ATPase of PM and basal ATPase activity in cancer tissues had a strong tendency to be higher than in the controls. NAADP caused a decrease in the activities of Na+/K+ ATPase and Ca2+ ATPase EPR in cancer samples and increased basal ATPase activity. In control samples, NAADP decreased Ca2+ ATPase of PM and increased basal ATPase activity. These data confirmed different roles of NAADP-sensitive “acidic store” (autophagosomes, late endosomes, and lysosomes) in control and cancer tissue, which hypothetically may be connected with autophagy role in cancer development. The effect of NAADP on decreasing the activity of Na+/K+ pump in cancer samples was the most pronounced, both numerically and statistically. Our data shows promising possibilities for the modulation of ion-transport through the membrane of cancer cells by influence on the “acidic store” (autophagosomes, late endosomes and lysosomes) as a new approach to the treatment of colorectal cancer.

Macrophages clear pathogens by phagocytosis and lysosomes that fuse with phagosomes are traditionally regarded as to a source of membranes and luminal degradative enzymes. Here, we reveal that endo‐lysosomes act as platforms for a new phagocytic signalling pathway in which FcγR activation recruits the second messenger NAADP and thereby promotes the opening of Ca2+‐permeable two‐pore channels (TPCs). Remarkably, phagocytosis is driven by these local endo‐lysosomal Ca2+ nanodomains rather than global cytoplasmic or ER Ca2+ signals. Motile endolysosomes contact nascent phagosomes to promote phagocytosis, whereas endo‐lysosome immobilization prevents it. We show that TPC‐released Ca2+ rapidly activates calcineurin, which in turn dephosphorylates and activates the GTPase dynamin‐2. Finally, we find that different endo‐lysosomal Ca2+ channels play diverse roles, with TPCs providing a universal phagocytic signal for a wide range of particles and TRPML1 being only required for phagocytosis of large targets. Synopsis: A role for early Ca2+ signals in phagocytosis remains controversial. Here local Ca2+‐enriched nanodomains formed by two‐pore channels (TPCs) on endolysosomes, rather than global cytoplasmic or ER calcium signals, are shown to promote phagocytosis in murine macrophages. TPCs promote FcR‐mediated phagocytosis and clearance of infective pathogens from endolysosomes in murine bone marrow‐derived macrophages.Phagocytosis is driven by local, TPC‐generated Ca2+ nanodomains, but not by global Ca2+ signals generated through IP3R/ORAI.TPCs activate calcineurin and the GTPase dynamin‐2.TPC‐mediated Ca2+ nanodomains provide a universal phagocytic signal, whereas TRPML1‐mediated Ca2+ signals are only required for the phagocytosis of large particles.Phagocytic uptake of live bacteria requires TPC1 and TPC2. [ABSTRACT FROM AUTHOR]

Lysosomes and the endoplasmic reticulum (ER) are Ca2+ stores mobilized by the second messengers NAADP and IP 3 , respectively. Here, we establish Ca2+ signals between the two sources as fundamental building blocks that couple local release to global changes in Ca2+. Cell-wide Ca2+ signals evoked by activation of endogenous NAADP-sensitive channels on lysosomes comprise both local and global components and exhibit a major dependence on ER Ca2+ despite their lysosomal origin. Knockout of ER IP 3 receptor channels delays these signals, whereas expression of lysosomal TPC2 channels accelerates them. High-resolution Ca2+ imaging reveals elementary events upon TPC2 opening and signals coupled to IP 3 receptors. Biasing TPC2 activation to a Ca2+-permeable state sensitizes local Ca2+ signals to IP 3. This increases the potency of a physiological agonist to evoke global Ca2+ signals and activate a downstream target. Our data provide a conceptual framework to understand how Ca2+ release from physically separated stores is coordinated. [Display omitted] • Lysosomal TPC2 channels evoke local and global Ca2+ signals involving the ER • ER-localized IP 3 receptor channels locally couple to TPC2 • TPC2 sensitizes IP 3 receptors in an agonist-selective manner • Crosstalk regulates physiological Ca2+ signals and Ca2+-dependent function Yuan et al. investigate organelle crosstalk in the genesis of cytosolic Ca2+ signals. They resolve a continuum of Ca2+ transients from local elementary events driven by lysosomal TPC2 channels, through Ca2+-coupled events driven by IP 3 receptors on the neighboring endoplasmic reticulum, to functionally relevant global signals during physiological cell stimulation. [ABSTRACT FROM AUTHOR]

• In mice, TPC1 & TPC2 deletion alters left ventricular (LV) systolic function. • In cardiomyocytes, TPC1 & TPC2 deletion reduces the sarcoplasmic reticulum (SR) Ca2+ content and the expression of various key proteins regulating Ca2+ stores and down regulates two main regulators of the energy metabolism. • Tpcn1/2−/− mice display poor performances in a treadmill exhaustion test despite normal skeletal muscle strength. • Expression of TPC1 and TPC2 increase in two mouse models of heart hypertrophy. Cardiac hypertrophy is reduced by TPC1/2 deletion. Ca2+ signaling is essential for cardiac contractility and excitability in heart function and remodeling. Intriguingly, little is known about the role of a new family of ion channels, the endo-lysosomal non-selective cation "two-pore channel" (TPCs) in heart function. Here we have used double TPC knock-out mice for the 1 and 2 isoforms of TPCs (Tpcn1/2−/−) and evaluated their cardiac function. Doppler-echocardiography unveils altered left ventricular (LV) systolic function associated with a LV relaxation impairment. In cardiomyocytes isolated from Tpcn1/2−/- mice, we observed a reduction in the contractile function with a decrease in the sarcoplasmic reticulum Ca2+ content and a reduced expression of various key proteins regulating Ca2+ stores, such as calsequestrin. We also found that two main regulators of the energy metabolism, AMP-activated protein kinase and mTOR, were down regulated. We found an increase in the expression of TPC1 and TPC2 in a model of transverse aortic constriction (TAC) mice and in chronically isoproterenol infused WT mice. In this last model, adaptive cardiac hypertrophy was reduced by Tpcn1/2 deletion. Here, we propose a central role for TPCs and lysosomes that could act as a hub integrating information from the excitation-contraction coupling mechanisms, cellular energy metabolism and hypertrophy signaling. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] [ABSTRACT FROM AUTHOR]

Abstract Background Although malaria is a preventable and curable human disease, millions of people risk to be infected by the Plasmodium parasites and to develop this illness. Therefore, there is an urgent need to identify new anti-malarial drugs. Ca2+ signalling regulates different processes in the life cycle of Plasmodium falciparum, representing a suitable target for the development of new drugs. Results This study investigated for the first time the effect of a highly specific inhibitor of nicotinic acid adenine dinucleotide phosphate (NAADP)-induced Ca2+ release (Ned-19) on P. falciparum, revealing the inhibitory effect of this compound on the blood stage development of this parasite. Ned-19 inhibits both the transition of the parasite from the early to the late trophozoite stage and the ability of the late trophozoite to develop to the multinucleated schizont stage. In addition, Ned-19 affects spontaneous intracellular Ca2+ oscillations in ring and trophozoite stage parasites, suggesting that the observed inhibitory effects may be associated to regulation of intracellular Ca2+ levels. Conclusions This study highlights the inhibitory effect of Ned-19 on progression of the asexual life cycle of P. falciparum. The observation that Ned-19 inhibits spontaneous Ca2+ oscillations suggests a potential role of NAADP in regulating Ca2+ signalling of P. falciparum.