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A photo-clickable analog of adenosine was devised and synthesized in which the photoactive functional group (8-azidoadenosine) and the click moiety (2'-O-propargyl-ether) were compactly combined within the structure of the adenosine nucleoside itself. We synthesized 8-N 3 -2'-O-propargyl adenosine in four steps starting from adenosine. This photo-clickable adenosine was 5'-phosphorylated and coupled to nicotinamide mononucleotide to form the NAD analog 8-N 3 -2'-O-propargyl-NAD. This NAD analog was recognized by Aplysia californica ADP-ribosyl cyclase and enzymatically cyclized producing 8-N 3 -2'-O-propargyl cyclic ADP-ribose. Photo-clickable cyclic-ADP-ribose analog was envisioned as a probe to label cyclic ADP-ribose binding proteins. The monofunctional 8-N 3 -cADPR has previously been shown to be an antagonist of cADPR-induced calcium release [T.F. Walseth et. al., J. Biol. Chem (1993) 268, 26686-26691]. 2'-O-propargyl-cADPR was recognized as an agonist which elicited Ca 2+ release when added at low concentration to sea urchin egg homogenates. The bifunctional 8-N 3 -2'-O-propargyl cyclic ADP-ribose did not elicit Ca 2+ release at low concentration or impact cyclic ADP-ribose mediated Ca 2+ release either when added to sea urchin egg homogenates or when microinjected into cultured human U2OS cells. The photo-clickable adenosine will none-the-less be a useful scaffold for synthesizing photo-clickable probes for identifying proteins that interact with a variety of adenosine nucleotides., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Cyclic adenosine diphosphate ribose (cADPR) is a second messenger involved in the Ca 2+ homeostasis. Its chemical instability prompted researchers to tune point by point its structure, obtaining stable analogues featuring interesting biological properties. One of the most challenging derivatives is the cyclic inosine diphosphate ribose (cIDPR), in which the hypoxanthine isosterically replaces the adenine. As our research focuses on the synthesis of N1 substituted inosines, in the last few years we have produced new flexible cIDPR analogues, where the northern ribose has been replaced by alkyl chains. Interestingly, some of them mobilized Ca 2+ ions in PC12 cells. To extend our SAR studies, herein we report on the synthesis of a new stable cIDPR derivative which contains the 2″S,3″R dihydroxypentyl chain instead of the northern ribose. Interestingly, the new cyclic derivative and its open precursor induced an increase in intracellular calcium concentration ([Ca 2+ ] i ) with the same efficacy of the endogenous cADPR in rat primary cortical neurons., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In spite of long-term intensive scientific research efforts, there are still many issues concerning the mechanisms of epileptogenesis and epilepsy to be resolved. Temporal lobe, in particular hippocampus, is vulnerable to epileptogenic process. Herein, electrical kindling model of temporal lobe were analyzed using proteomic approach. A dramatic decrease in nicotinamide adenine dinucleotide (NAD + ) level was exhibited during the kindling procedure in hippocampus. After stage 3, high CD38 expression was detected by qPCR, nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) and western blot analysis. An increase in expression of CD38/NADase activity was observed during the kindling procedure in hippocampus that suggest it as one of the most important NAD + degrading enzymes during epileptogenesis. Subsequently, gene expression of CD38 metabolite related proteins (Ryr2, FKBP-12.6, Chrm1, mGluR1 and Cnx43) were examined. Among them, changes in the expression level of mGluR1 was more than other genes, which was also confirmed by LC MS/MS and western blotting analysis. These findings provided valuable information about changes in the expression of CD38/cADPR signaling pathway and suggest its crucial role during epileptogenesis., (Copyright © 2021. Published by Elsevier B.V.)

Background: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is hallmarked by a significant reduction in natural killer (NK) cell cytotoxicity, a mechanism tightly regulated by calcium (Ca 2+ ). Interestingly, interleukin-2 (IL-2) increases NK cell cytotoxicity. Transient receptor potential melastatin 2 (TRPM2) ion channels are fundamental for Ca 2+ signalling in NK cells. This pilot investigation aimed to characterise TRPM2 and CD38 surface expression in vitro on NK cells in ME/CFS patients. This investigation furthermore examined the pharmaceutical effect of 8-bromoadenosine phosphoribose (8-Br-ADPR) and N 6 -Benzoyladenosine-3',5'-cyclic monophosphate (N 6 -Bnz-cAMP) on TRPM2 and CD38 surface expression and NK cell cytotoxicity between ME/CFS and healthy control (HC) participants., Methods: Ten ME/CFS patients (43.45 ± 12.36) and 10 HCs (43 ± 12.27) were age and sex-matched. Isolated NK cells were labelled with fluorescent antibodies to determine baseline and drug-treated TRPM2 and CD38 surface expression on NK cell subsets. Following IL-2 stimulation, NK cell cytotoxicity was measured following 8-Br-ADPR and N 6 -Bnz-cAMP drug treatments by flow cytometry., Results: Baseline TRPM2 and CD38 surface expression was significantly higher on NK cell subsets in ME/CFS patients compared with HCs. Post IL-2 stimulation, TRPM2 and CD38 surface expression solely decreased on the CD56 Dim CD16 + subset. 8-Br-ADPR treatment significantly reduced TRPM2 surface expression on the CD56 Bright CD16 Dim/- subset within the ME/CFS group. Baseline cell cytotoxicity was significantly reduced in ME/CFS patients, however no changes were observed post drug treatment in either group., Conclusion: Overexpression of TRPM2 on NK cells may function as a compensatory mechanism to alert a dysregulation in Ca 2+ homeostasis to enhance NK cell function in ME/CFS, such as NK cell cytotoxicity. As no improvement in NK cell cytotoxicity was observed within the ME/CFS group, an impairment in the TRPM2 ion channel may be present in ME/CFS patients, resulting in alterations in [Ca 2+ ] i mobilisation and influx, which is fundamental in driving NK cell cytotoxicity. Differential expression of TRPM2 between NK cell subtypes may provide evidence for their role in the pathomechanism involving NK cell cytotoxicity activity in ME/CFS.

The transient receptor potential melastatin-2 (TRPM2) channel belongs to the transient receptor potential channel superfamily and is a cation channel permeable to Na + and Ca 2+ . The TRPM2 ion channel is expressed in the kidney and can be activated by various molecules such as hydrogen peroxide, calcium, and cyclic adenosine diphosphate (ADP)-ribose (cADPR) that are produced during acute kidney injury. In this study, we investigated the role of 8-bromo-cyclic ADP-ribose (8-Br-cADPR; a cADPR antagonist) in renal ischemia-reperfusion injury using biochemical and histopathological parameters. CD38, cADPR, tumor necrosis factor-α, interleukin-1β, and myeloperoxidase (inflammatory markers), urea and creatinine, hydrogen peroxide (oxidant), and catalase (antioxidant enzyme) levels that increase with ischemia-reperfusion injury decreased in the groups treated with 8-Br-cADPR. In addition, renin levels were elevated in the groups treated with 8-Br-cADPR. Histopathological examination revealed that 8-Br-cADPR reduced renal damage and the expression of caspase-3 and TRPM2. Our results suggest that the inhibition of TRPM2 ion channel may be a new treatment modality for ischemic acute kidney injury., (© 2018 Wiley Periodicals, Inc.)

Cyclic adenosine 5'-diphosphate ribose (cADPR) is a ubiquitous Ca 2+ -releasing second messenger. Knowledge of its conformational landscape is an essential tool for unraveling the structure-activity relationship (SAR) in cADPR. Variable-temperature 1 H NMR spectroscopy, in conjunction with PSEUROT and population analyses, allowed us to determine the conformations and thermodynamic parameters of the furanose rings, γ-bonds (C4'-C5'), and β-bonds (C5'-O5') in the cADPR analogues 2'-deoxy-cADPR, 7-deaza-cADPR, and 8-bromo-cADPR. A significant finding was that, although the analogues are similar to each other and to cADPR itself in terms of overall conformation and population (ΔG°), there were subtle yet important differences in some of thermodynamic properties (ΔH°, ΔS°) associated with each of the conformational equilibria. These differences prompted us to propose a model for cADPR in which the interactions between the A2'-N3, A5″-N3, and H2-R5' atoms serve to fine-tune the N-glycosidic torsion angles (χ).

Background: The CD38/cADPR pathway has been found to play roles in various inflammatory conditions. However, whether CD38 plays a protective or detrimental effect in the central nervous system (CNS) is controversial. The aim of this study was to determine the effect of CD38/cADPR pathway in sepsis associated brain injury., Materials and Methods: Male Sprague-Dawley rats were undergone cecal ligation and puncture (CLP) or sham laparotomies. NAD + , cADPR and CD38 were measured in the hippocampus of septic rats at 0, 6, 12, 24, and 48h after CLP surgery. Rats were divided into the sham, CLP group, CLP+ CD38 expression lentivirus (CLP+ CD38 LV), CLP+ CD38 interference lentivirus (CLP+ CD38 Ri), CLP+ negative control lentivirus (CLP+NC) and the CLP+8-Br-cADPR groups. The Western blots of Bcl-2, Bax and iNOS, TUNEL assays, malondialdehyde (MDA) and superoxide dismutase (SOD) assays, transmission electron microscope analysis were performed in the hippocampus of rats., Results: NAD + , cADPR and CD38 levels increased significantly in the hippocampus of septic rats as early as 12-24h after CLP surgery. CD38 knockdown or blocking cADPR with 8-Br-cADPR significantly reduced apoptosis, MDA and SOD activity, iNOS expression and ultrastructural morphology damages in the hippocampus of septic rats., Conclusions: In this study, we found that the CD38/cADPR pathway was activated in sepsis associated brain injury. Blocking this pathway protected the hippocampus from apoptosis, oxidative stress and ultrastructural morphology damages in septic rats., (Copyright © 2017. Published by Elsevier B.V.)

A series of 8-substituted analogues of cyclic ADP-4-thioribose (cADPtR, 3), which is a stable equivalent of Ca 2+ -mobilizing second messenger cyclic ADP-ribose (cADPR, 1), were designed as potential pharmacological tools for studies on cADPR-modulated Ca 2+ signaling pathways. These 8-amino analogue (8-NH 2 -cADPtR, 4), 8-azido analogue (8-N 3 -cADPtR, 5), and 8-chloro analogue (8-Cl-cADPtR, 6) were efficiently synthesized, where the stereoselective N1-β-thioribosyladenine ring closure reaction via an α/β-equilibrium of the 1-aminothioribose derivative and construction of the characteristic 18-membered pyrophosphate ring by Ag + -promoted activation of a phenyl phosphorothioate type substrate were the two key steps. Although 8-NH 2 -cADPR (2) is a well-known potent antagonist against cADPR-inducing Ca 2+ -release, the 4-thioribose congener 8-NH 2 -cADPtR turned out unexpectedly to be a full agonist in sea urchin egg homogenate evaluation system. This important finding suggested that the ring-oxygen in the N1-ribose of cADPR analogues is essential for the antagonistic activity in the Ca 2+ -signaling pathway, which can contribute to clarify the structure-agonist/antagonist activity relationship.

We and others have demonstrated a protective effect of pacing postconditioning (PPC) against ischemia/reperfusion (I/R) injury. However, the mechanisms underlying this protection are not completely clear. In the present study, we evaluated the effects of calcium release from the sarcoplasmic reticulum (SR) and the novel intracellular acidic stores (AS). Isolated rat hearts (n = 6 per group) were subjected to coronary occlusion followed by reperfusion using a modified Langendorff system. Cardiac hemodynamics and contractility were assessed using a data acquisition program, and cardiac injury was evaluated by creatine kinase (CK) and lactate dehydrogenase (LDH) levels. 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. The hearts were also subjected to PPC (3 cycles of 30 s of left ventricle (LV) pacing alternated with 30 s of right atrium (RA) pacing) and/or were treated during reperfusion with agonists or antagonists of release of calcium from SR or AS. PPC significantly (P < 0.05) normalized LV, contractility, and coronary vascular dynamics and significantly (P < 0.001) decreased heart enzyme levels compared to the control treatments. The blockade of SR calcium release resulted in a significant (P < 0.01) recovery in LV function and contractility and a significant reduction in CK and LDH levels (P < 0.01) when applied alone or in combination with PPC. Interestingly, the release of calcium from AS alone or in combination with PPC significantly improved LV function and contractility (P < 0.05) and significantly decreased the CK and LDH levels (P < 0.01) compared to the control treatments. An additive effect was produced when agonism of calcium release from AS or blockade of calcium release from the SR was combined with PPC. Calcium release from AS and blockade of calcium release from the SR protect the heart against I/R. Combining calcium release from acidic stores or blockade of calcium release from the SR with PPC produced a synergistic protective effect.

Cyclic adenosine diphosphate-carbocyclic-ribose (cADPcR, 2) is a stable equivalent of cyclic adenosine diphosphate-ribose (cADPR, 1), a Ca(2+)-mobilizing second messenger. On the basis of the structure-activity relationship of cADPR-related compounds and three-dimensional structural modeling of cADPcR, we designed and synthesized cyclic-ADP-4″α-azidoethyl carbocyclic-ribose (N3-cADPcR, 3) to demonstrate that it has a highly potent Ca(2+)-mobilizing activity (EC50 = 24 nM). N3-cADPcR will be a useful precursor for the preparation of biological tools effective to investigate cADPR-mediated signaling pathways.

Background: Although the nicotinamide adenine dinucleotide (NAD(+))/CD38/cyclic ADP ribose (cADPR)/Ca(2+) signaling pathway has been shown to regulate intracellular calcium homeostasis and functions in multiple inflammatory processes, its role in sepsis remains unknown. The aim of this study was to determine whether the NAD(+)/CD38/cADPR/Ca(2+) signaling pathway is activated during sepsis and whether an inhibitor of this pathway, 8-Br-cADPR, protects the organs from sepsis-induced damage., Materials and Methods: Male Sprague-Dawley rats were subjected to cecal ligation and puncture (CLP) or sham laparotomies. NAD(+), cADPR, CD38, and intracellular Ca(2+) levels were measured in the hearts, livers, and kidneys of septic rats at 0, 6, 12, 24, and 48 h after CLP surgery. Rats were also divided into sham, CLP, and CLP+8-Br-cADPR groups, and the hearts, livers, and kidneys were hematoxylin-eosin-stained and assayed for malondialdehyde and superoxide dismutase activities., Results: NAD(+), cADPR, CD38, and intracellular Ca(2+) levels increased in the hearts, livers, and kidneys of septic rats as early as 6-24 h after CLP surgery. Treatment with 8-Br-cADPR inhibited sepsis-induced intracellular Ca(2+) mobilization, attenuated tissue injury, reduced malondialdehyde levels, and increased superoxide dismutase activity in septic rats., Conclusions: The NAD(+)/CD38/cADPR/Ca(2+) signaling pathway was activated during sepsis in the CLP rat model. Blocking this pathway with 8-Br-cADPR protected hearts, livers, and kidneys from sepsis-induced damage., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Ca2+ signaling plays a fundamental role in cardiac hypertrophic remodeling, but the underlying mechanisms remain poorly understood. We investigated the role of Ca2+-mobilizing second messengers, NAADP and cADPR, in the cardiac hypertrophy induced by β-adrenergic stimulation by isoproterenol. Isoproterenol induced an initial Ca2+ transients followed by sustained Ca2+ rises. Inhibition of the cADPR pathway with 8-Br-cADPR abolished only the sustained Ca2+ increase, whereas inhibition of the NAADP pathway with bafilomycin-A1 abolished both rapid and sustained phases of the isoproterenol-mediated signal, indicating that the Ca2+ signal is mediated by a sequential action of NAADP and cADPR. The sequential production of NAADP and cADPR was confirmed biochemically. The isoproterenol-mediated Ca2+ increase and cADPR production, but not NAADP production, were markedly reduced in cardiomyocytes obtained from CD38 knockout mice. CD38 knockout mice were rescued from chronic isoproterenol infusion-induced myocardial hypertrophy, interstitial fibrosis, and decrease in fractional shortening and ejection fraction. Thus, our findings indicate that β-adrenergic stimulation contributes to the development of maladaptive cardiac hypertrophy via Ca2+ signaling mediated by NAADP-synthesizing enzyme and CD38 that produce NAADP and cADPR, respectively.

There is little information on the effects of aging in the propagation of calcium signals and its underlying mechanisms. We studied the effects of aging on propagation of Ca(2+) signals in pancreatic acinar cells. Fura-2 loaded cells isolated from young (3-4 months old) and aged (24 months old) mouse responded to acetylcholine (ACh) and cholecystokinin (CCK) with a polarized Ca(2+) response initiated at the secretory pole before spreading to the basal one. Aging slowed down the propagation of the response to ACh but enhanced the velocity of the CCK response. This pattern can be explained by the age-induced depolarization of mitochondria, because it can be reproduced in young cells by mitochondrial inhibitors. Aging also increased the role of acidic stores in the CCK signal, as judged by the folimycin-induced suppression of the polarization in aged but not in young cells. The involvement of ryanodine receptors in the ACh response was also enhanced, as indicated by the loss of polarization after the treatment with 8Br-cyclic ADP ribose. Therefore, we conclude that aging modifies differentially the propagation of ACh and CCK-evoked Ca(2+) signals through mitochondrial depolarization and changes in the role of the acidic Ca(2+) stores and ryanodine receptors in the initiation of the signals., (© The Author 2015. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

CD38 is a multifunctional enzyme that can act as a NADase, generate cyclic adenosine diphosphate-ribose (cADPR) - a key Ca²⁺-mobilizing second messenger - and transport cADPR into cells. There have only been a small number of studies on the functions of CD38 in the central nervous system. In this study, we applied CD38 small interfering RNA (siRNA) to determine the effects of decreased CD38 on the intracellular ATP levels and survival of C6 glioma cells. Our study showed that both CD38 siRNA and 8-bromo-cADPR - a ryanodine receptor antagonist - can lead to a significant increase in the intracellular ATP levels of C6 glioma cells. We further found that the siRNA-induced CD38 reductions can induce decreases in the number of surviving cells. Our study has also suggested that P2X receptors contribute toward the CD38 siRNA-induced decreases in the cell survival. In summary, our study has provided novel evidence suggesting that CD38 mediates both the intracellular ATP levels and the survival of C6 glioma cells, suggesting that CD38 may become a therapeutic target for gliomas.

Analogues of the potent Ca(2+) releasing second messenger cyclic ADP-ribose (cADPR) with a 1,2,3-triazole pyrophosphate bioisostere were synthesised by click-mediated macrocyclisation. The ability to activate Ca(2+) release was surprisingly retained, and hydrolysis of cADPR by CD38 could also be inhibited, illustrating the potential of this approach to design drug-like signalling pathway modulators.

Extracellular β-nicotinamide adenine dinucleotide (NAD(+)) is anti-inflammatory. We hypothesized that NAD(+) would modulate the anti-inflammatory cytokine Transforming Growth Factor (TGF)-β1. Indeed, NAD(+) led to increases in both active and latent cell-associated TGF-β1 in RAW 264.7 mouse macrophages as well as in primary peritoneal macrophages isolated from both C3H/HeJ (TLR4-mutant) and C3H/HeOuJ (wild-type controls for C3H/HeJ) mice. NAD(+) acts partially via cyclic ADP-ribose (cADPR) and subsequent release of Ca(2+). Treatment of macrophages with the cADPR analog 3-deaza-cADPR or Ca(2+) ionophores recapitulated the effects of NAD(+) on TGF-β1, whereas the cADPR antagonist 8-Br-cADPR, Ca(2+) chelation, and antagonism of L-type Ca(2+) channels suppressed these effects. The time and dose effects of NAD(+) on TGF-β1 were complex and could be modeled both statistically and mathematically. Model-predicted levels of TGF-β1 protein and mRNA were largely confirmed experimentally but also suggested the presence of other mechanisms of regulation of TGF-β1 by NAD(+). Thus, in vitro and in silico evidence points to NAD(+) as a novel modulator of TGF-β1.

Novel cADPR mimics, which integrate nucleobase, northern and southern ribose modifications were synthesized. The key steps of the synthesis were a Cu(I)-catalyzed Hüisgen [3+2] cycloaddition and a microwave-assisted intramolecular pyrophosphorylation. Preliminary biological investigations showed that these cADPR mimics are membrane-permeating agonists of the calcium signaling pathway. The introduction of chlorine or fluorine at the 2'-position of the southern riboses led to a decrease of activity. The existence of a hydrophobic group on the 3'-OH of the southern riboses does not obviously alter the agonistic activity.

Here, we review the functional roles of cyclic ADP-ribose and CD38, a transmembrane protein with ADP-ribosyl cyclase activity, in mouse social behavior via the regulation of oxytocin (OXT) release, an essential component of social cognition. Herein we describe data detailing the molecular mechanism of CD38-dependent OXT secretion in CD38 knockout mice. We also review studies that used OXT, OXT receptor (OXTR), or CD38 knockout mice. Additionally, we compare the behavioral impairments that occur in these knockout mice in relation to the OXT system and CD38. This review also examines autism spectrum disorder (ASD), which is characterized by social and communication impairments, in relation to defects in the OXT system. Two single nucleotide polymorphisms (SNPs) in the human CD38 gene are possible risk factors for ASD via inhibition of OXT function. Further analysis of CD38 in relation to the OXT system may provide a better understanding of the neuroendocrinological roles of OXT and CD38 in the hypothalamus and of the pathophysiology of ASD. This article is part of a Special Issue entitled Oxytocin, Vasopressin, and Social Behavior., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Two nicotinamide adenine dinucleotide (NAD(+)) analogues modified at the 6 position of the purine ring were synthesized, and their substrate properties toward Aplysia californica ADP-ribosyl cyclase were investigated. 6-N-Methyl NAD(+) (6-N-methyl nicotinamide adenosine 5'-dinucleotide 10) hydrolyzes to give the linear 6-N-methyl ADPR (adenosine 5'-diphosphoribose, 11), whereas 6-thio NHD(+) (nicotinamide 6-mercaptopurine 5'-dinucleotide, 17) generates a cyclic dinucleotide. Surprisingly, NMR correlation spectra confirm this compound to be the N1 cyclic product 6-thio N1-cIDPR (6-thio cyclic inosine 5'-diphosphoribose, 3), although the corresponding 6-oxo analogue is well-known to cyclize at N7. In Jurkat T cells, unlike the parent cyclic inosine 5'-diphosphoribose N1-cIDPR 2, 6-thio N1-cIDPR antagonizes both cADPR- and N1-cIDPR-induced Ca(2+) release but possesses weak agonist activity at higher concentration. 3 is thus identified as the first C-6 modified cADPR (cyclic adenosine 5'-diphosphoribose) analogue antagonist; it represents the first example of a fluorescent N1-cyclized cADPR analogue and is a new pharmacological tool for intervention in the cADPR pathway of cellular signaling.

Activation of NAD(P)H oxidase has been reported to produce superoxide (O(2)(•-)) extracellularly as an autocrine/paracrine regulator or intracellularly as a signaling messenger in a variety of mammalian cells. However, it remains unknown how the activity of NAD(P)H oxidase is regulated in arterial myocytes. Recently, CD38-associated ADP-ribosylcyclase has been reported to use an NAD(P)H oxidase product, NAD(+) or NADP(+), to produce cyclic ADP-ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate, which mediates intracellular Ca(2+) signaling. This study was designed to test a hypothesis that the CD38/cADPR pathway as a downstream event exerts feedback regulatory action on the NAD(P)H oxidase activity in production of extra- or intracellular O(2)(•-) in mouse coronary arterial myocytes (CAMs). By fluorescence microscopic imaging, we simultaneously monitored extra- and intracellular O(2)(•-) production in wild-type (CD38(+/+)) and CD38 knockout (CD38(-/-)) CAMs in response to oxotremorine (OXO), a muscarinic type 1 receptor agonist. It was found that CD38 deficiency prevented OXO-induced intracellular but not extracellular O(2)(•-) production in CAMs. Consistently, the OXO-induced intracellular O(2)(•-) production was markedly inhibited by CD38 shRNA or the CD38 inhibitor nicotinamide in CD38(+/+) CAMs. Further, Nox4 siRNA inhibited OXO-induced intracellular but not extracellular O(2)(•-) production, whereas Nox1 siRNA attenuated both intracellular and extracellular O(2)(•-) production in CD38(+/+) CAMs. Direct delivery of exogenous cADPR into CAMs markedly elevated intracellular Ca(2+) and O(2)(•-) production in CD38(-/-) CAMs. Functionally, CD38 deficiency or Nox1 siRNA and Nox4 siRNA prevented OXO-induced contraction in isolated perfused coronary arteries in CD38 WT mice. These results provide direct evidence that the CD38/cADPR pathway is an important controller of Nox4-mediated intracellular O(2)(•-) production and that CD38-dependent intracellular O(2)(•-) production is augmented in an autocrine manner by CD38-independent Nox1-derived extracellular O(2)(•-) production in CAMs., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Bone marrow mesenchymal stem cells (MSCs) are a promising cell source for regenerative medicine. However, the cellular biology of these cells is not fully understood. The present study characterizes the cyclic ADP-ribose (cADPR)-mediated Ca(2+) signals in human MSCs and finds that externally applied cADPR can increase the frequency of spontaneous intracellular Ca(2+) (Ca(2+) (i) ) oscillations. The increase was abrogated by a specific cADPR antagonist or an inositol trisphosphate receptor (IP3R) inhibitor, but not by ryanodine. In addition, the cADPR-induced increase of Ca(2+) (i) oscillation frequency was prevented by inhibitors of nucleoside transporter or by inhibitors of the transient receptor potential cation melastatin-2 (TRPM2) channel. RT-PCR revealed mRNAs for the nucleoside transporters, concentrative nucleoside transporters 1/2 and equilibrative nucleoside transporters 1/3, IP3R1/2/3 and the TRPM2 channel, but not those for ryanodine receptors and CD38 in human MSCs. Knockdown of the TRPM2 channel by specific short interference RNA abolished the effect of cADPR on the Ca(2+) (i) oscillation frequency, and prevented the stimulation of proliferation by cADPR. Moreover, cADPR remarkably increased phosphorylated extracellular-signal-regulated kinases 1/2 (ERK1/2), but not Akt or p38 mitogen-activated protein kinase (MAPK). However, cADPR had no effect on adipogenesis or osteogenesis in human MSCs. Our results indicate that cADPR is a novel regulator of Ca(2+) (i) oscillations in human MSCs. It permeates the cell membrane through the nucleoside transporters and increases Ca(2+) oscillation via activation of the TRPM2 channel, resulting in enhanced phosphorylation of ERK1/2 and, thereby, stimulation of human MSC proliferation. This study delineates an alternate signalling pathway of cADPR that is distinct from its well-established role of serving as a Ca(2+) messenger for mobilizing the internal Ca(2+) stores. Whether cADPR can be used clinically for stimulating marrow function in patients with marrow disorders remains to be further studied., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

It is well established that the intracellular second messenger cADP-ribose (cADPR) activates Ca(2+) release from the sarcoplasmic reticulum through ryanodine receptors. CD38 is a multifunctional enzyme involved in the formation of cADPR in mammals. CD38 has also been reported to transport cADPR in several cell lines. Here, we demonstrate a role for extracellular cADPR and CD38 in modulating the spontaneous, but not the electrical field stimulation-evoked, release of ATP in visceral smooth muscle. Using a small-volume superfusion assay and an HPLC technique with fluorescence detection, we measured the spontaneous and evoked release of ATP in bladder detrusor smooth muscles isolated from CD38(+/+) and CD38(-/-) mice. cADPR (1 nM) enhanced the spontaneous overflow of ATP in bladders isolated from CD38(+/+) mice. This effect was abolished by the inhibitor of cADPR receptors on sarcoplasmic reticulum 8-bromo-cADPR (80 μM) and by ryanodine (50 μm), but not by the nonselective P2 purinergic receptor antagonist pyridoxal phosphate 6-azophenyl-2',4'-disulfonate (30 μM). cADPR failed to facilitate the spontaneous ATP overflow in bladders isolated from CD38(-/-) mice, indicating that CD38 is crucial for the enhancing effects of extracellular cADPR on spontaneous ATP release. Contractile responses to ATP were potentiated by cADPR, suggesting that the two adenine nucleotides may work in synergy to maintain the resting tone of the bladder. In conclusion, extracellular cADPR enhances the spontaneous release of ATP in the bladder by influx via CD38 and subsequent activation of intracellular cADPR receptors, probably causing an increase in intracellular Ca(2+) in neuronal cells., (© 2011 The Authors Journal compilation © 2011 FEBS.)

Purpose: The inflammatory response in age-related macular degeneration (AMD) is characterized by mononuclear leukocyte infiltration of the outer blood-retina barrier formed by the retinal pigment epithelium (RPE). A key mechanistic element in AMD progression is RPE dysfunction and apoptotic cell loss. The purpose of this study was to evaluate whether monocyte chemoattractant protein (MCP)-1-activated monocytes induce human RPE apoptosis and whether Ca(2+) and reactive oxygen species (ROS) are involved in this process., Methods: A cell-based fluorometric assay was used to measure intracellular Ca(2+) concentrations ([Ca(2+)](i)) in RPE cells loaded with fluorescent Ca(2+) indicator. Intracellular RPE ROS levels were measured by using the 5- and 6-chloromethyl-2',7'-dichlorodihydrofluorescence diacetate acetyl ester (CM-H(2)DCFDA) assay. RPE apoptosis was evaluated by activated caspase-3, Hoechst staining, and apoptosis ELISA., Results: MCP-1-activated human monocytes increased [Ca(2+)](i), ROS levels, and apoptosis in RPE cells, all of which were inhibited by 8-bromo-cyclic adenosine diphosphoribosyl ribose (8-Br-cADPR), an antagonist of cADPR. Although the ROS scavengers pyrrolidinedithiocarbamate (PDTC) and N-acetylcysteine (NAC) significantly inhibited ROS production and apoptosis induced by activated monocytes, they did not affect induced Ca(2+) levels. The induced Ca(2+) levels and apoptosis in RPE cells were inhibited by an antibody against cluster of differentiation antigen 14 (CD14), an adhesion molecule expressed by these cells., Conclusions: These results indicate that CD14, Ca(2+), and ROS are involved in activated monocyte-induced RPE apoptosis and that cADPR contributes to these changes. Understanding the complex interactions among CD14, cADPR, Ca(2+), and ROS may provide new insights and treatments of retinal diseases, including AMD.

Arginine vasopressin (AVP) causes increase in intracellular Ca(2+) concentration with an oscillatory pattern. Ca(2+) mobilization is required for AVP-stimulated apical exocytosis in inner medullary collecting duct (IMCD). The mechanistic basis of these Ca(2+) oscillations was investigated by confocal fluorescence microscopy and flash photolysis of caged molecules in perfused IMCD. Photorelease of caged cAMP and direct activation of ryanodine receptors (RyRs) by photorelease of caged cyclic ADP-ribose (cADPR) both mimicked the AVP-induced Ca(2+) oscillations. Preincubation of IMCD with 100 μM 8-bromo-cADPR (a competitive inhibitor of cADPR) delayed the onset and attenuated the magnitude of AVP-induced Ca(2+) oscillations. These observations indicate that the cADPR/RyR pathway is capable of supporting Ca(2+) oscillations and endogenous cADPR plays a major role in the AVP-induced Ca(2+) oscillations in IMCD. In contrast, photorelease of caged inositol 1,4,5-trisphosphate (IP(3)) induced Ca(2+) release but did not maintain sustained Ca(2+) oscillations. Removal of extracellular Ca(2+) halted ongoing AVP-mediated Ca(2+) oscillation, suggesting that it requires extracellular Ca(2+) entry. AVP-induced Ca(2+) oscillation was unaffected by nifedipine. Intracellular Ca(2+) store depletion induced by 20 μM thapsigargin in Ca(2+)-free medium triggered store-operated Ca(2+) entry (SOCE) in IMCD, which was attenuated by 1 μM GdCl(3) and 50 μM SKF-96365. After incubation of IMCD with 1 nM AVP in Ca(2+)-free medium, application of extracellular Ca(2+) also triggered Ca(2+) influx, which was sensitive to GdCl(3) and SKF-96365. In summary, our observations are consistent with the notion that AVP-induced Ca(2+) oscillations in IMCD are mediated by the interplay of Ca(2+) release from RyRs and a Ca(2+) influx mechanism involving nonselective cation channels that resembles SOCE.

Transient receptor potential melastatin 2 (TRPM2) is a Ca(2+)-permeable cation channel involved in physiological and pathophysiological processes linked to oxidative stress. TRPM2 channels are co-activated by intracellular Ca(2+) and ADP-ribose (ADPR) but also modulated in intact cells by several additional factors. Superfusion of TRPM2-expressing cells with H(2)O(2) or intracellular dialysis of cyclic ADPR (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP) activates, whereas dialysis of AMP inhibits, TRPM2 whole-cell currents. Additionally, H(2)O(2), cADPR, and NAADP enhance ADPR sensitivity of TRPM2 currents in intact cells. Because in whole-cell recordings the entire cellular machinery for nucleotide and Ca(2+) homeostasis is intact, modulators might affect TRPM2 activity either directly, by binding to TRPM2, or indirectly, by altering the local concentrations of the primary ligands ADPR and Ca(2+). To identify direct modulators of TRPM2, we have studied the effects of H(2)O(2), AMP, cADPR, NAADP, and nicotinic acid adenine dinucleotide in inside-out patches from Xenopus oocytes expressing human TRPM2, by directly exposing the cytosolic faces of the patches to these compounds. H(2)O(2) (1 mM) and enzymatically purified cADPR (10 μM) failed to activate, whereas AMP (200 μM) failed to inhibit TRPM2 currents. NAADP was a partial agonist (maximal efficacy, ∼50%), and nicotinic acid adenine dinucleotide was a full agonist, but both had very low affinities (K(0.5) = 104 and 35 μM). H(2)O(2), cADPR, and NAADP did not enhance activation by ADPR. Considering intracellular concentrations of these compounds, none of them are likely to directly affect the TRPM2 channel protein in a physiological context.

ADP-ribosyl cyclase and/or CD38 are activated after oxytocin receptor stimulation in the hypothalamus and pituitary in adult mice, leading to facilitation of oxytocin secretion. Although cyclic adenosine 5'-diphosphoribose (cADPR) primarily acts as an intracellular second messenger, it has been suggested that extracellular cADPR stimulates intracellular ryanodine receptors after internalisation via the nucleotide-transporting capacity of CD38 in fibroblasts and astrocytes. However, little is known about whether extracellular cADPR activates neurones. To address this question, we used a model neuronal cell line, NG108-15 mouse neuroblastoma x rat glioma hybrid cells possessing CD38 but not oxytocin receptors, and measured cytosolic free calcium concentrations ([Ca(2+)](i)). Extracellular application of cADPR to NG108-15 cells elevated [Ca(2+)](i) at 35 degrees C. The elevation was significantly enhanced when measured at 40 degrees C. The cADPR and heat-induced [Ca(2+)](i) increase were blocked under extracellular Ca(2+)-free conditions and by 2-aminoethoxydiphenyl borate, an antagonist of melastatin-related transient receptor potential channel 2 (TRPM2) cation channels. Reverse transcriptation-polymerase chain reaction analyses indicated that TRPM2 channels were expressed in NG108-15 cells. Application of oxytocin elevated [Ca(2+)](i) in NG108-15 cells transformed to transiently express cloned human oxytocin receptors. The oxytocin-induced [Ca(2+)](i) response was also enhanced by heat. These results indicate that the extracellular application of cADPR, together with heat, activates cation influx downstream of oxytocin receptor signalling in NG108-15 neuronal cells, and suggest the possible involvement of TRPM2 channels in oxytocin release in the mammalian brain.

We examined the role and molecular mechanism of cADPR action on Ca(2+) spark properties in mouse bladder smooth muscle. Dialysis of cADPR with patch pipettes increased frequency and amplitude of spontaneous transient out currents (STOCs) to 6.1+/-0.87 currents/min from 1.2+/-0.36 currents/min (control) and to 179.8+/-48.7pA from 36.4+/-22.6pA (control), respectively, in wildtype (WT) cells, and the effects of cADPR on STOCs were significantly blocked by JVT-591, a RYR2 stabilizer. In contrast, no significant changes were observed in FKBP12.6 null cells. Further studies indicated that Ca(2+) spark properties were altered by cADPR in WT but not FKBP12.6 null cells, namely, Ca(2+) spark frequency was increased by about 3.4-fold, peak Ca(2+) (F/F0) increased to 1.72+/-0.57 from 1.56+/-0.13, size increased to 2.86+/-0.26 microM from 1.92+/-0.14 microM, rise time and half-time decay were prolonged 1.6-fold and 2.3-fold, respectively, in WT cells. Furthermore, in the presence of thapsigargin cADPR still altered Ca(2+) spark properties, and cADPR increased F/F0 without affecting Ca(2+) spark decay time in voltage clamping cells. Dissociation studies demonstrated that application of cADPR resulted in significant removal of FKBP12.6 proteins from sarcoplasmic reticulum (SR) microsomes, and that treatment of the RyR2 immunoprecipitation complexes with cADPR or FK506 disrupted the interaction between RyR2 and FKBP12.6. Finally, cADPR altered SR Ca(2+) load in WT myocytes but not in FKBP12.6-null myocytes. Taken together, these results suggest that Ca(2+) release induced by cADPR is mediated by FKBP12.6 proteins in mouse bladder smooth muscle., (2010 Elsevier Ltd. All rights reserved.)

Cellular metabolism is required for cell proliferation. However, the way in which metabolic signals are conveyed to cell cycle decisions is unclear. Cyclic ADP-ribose (cADPR), the NAD(+) metabolite, mobilizes calcium from calcium stores in many cells. We found that dinoflagellate cells with higher metabolic rate underwent multiple fission (MF), a division mode in which cells can exceed twice their sizes at G1. A temperature shift-down experiment suggested that MF involves a commitment point at late G1. In fast-growing cells, cADPR level peaked in G(1) and increased with increasing concentrations of glucose in the medium. Addition of glycolytic poison iodoacetate inhibited cell growth, reduced cADPR levels as well as the commitment of cell cycles in fast-growing cells. Commitment of MF cell cycles was induced by a cell permeant cADPR agonist, but blocked by a specific antagonist of cADPR-induced Ca(2+) release. Our results establish cADPR as a link between cellular metabolism and cell cycle control.

Research into the biological role of the Ca(2+)-releasing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate) has been hampered by a lack of chemical probes. To find new chemical probes for exploring NAADP signaling, we turned to virtual screening, which can evaluate millions of molecules rapidly and inexpensively. We used NAADP as the query ligand to screen the chemical library ZINC for compounds with similar three-dimensional shape and electrostatic properties. We tested the top-ranking hits in a sea urchin egg bioassay and found that one hit, Ned-19, blocks NAADP signaling at nanomolar concentrations. In intact cells, Ned-19 blocked NAADP signaling and fluorescently labeled NAADP receptors. Moreover, we show the utility of Ned-19 as a chemical probe by using it to demonstrate that NAADP is a key causal link between glucose sensing and Ca(2+) increases in mouse pancreatic beta cells.

The intracellular second messenger cyclic ADP-ribose (cADPR) induces Ca(2+) release through the activation of ryanodine receptors (RyRs). Moreover, it has been suggested that cADPR may serve an additional role to modulate sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump activity, but studies have been complicated by concurrent actions on RyR. Here, we explore the actions of cADPR in Xenopus oocytes, which lack RyRs. We examined the effects of cADPR on the sequestration of cytosolic Ca(2+) following Ca(2+) transients evoked by photoreleased inositol 1,4,5-trisphosphate (InsP(3)), and by Ca(2+) influx through expressed nicotinic acetylcholine receptors (nAChR) in the oocytes membrane. In both cases the decay of the Ca(2+) transients was accelerated by intracellular injection of a non-metabolizable analogue of cADPR, 3-Deaza-cADPR, and photorelease of cADPR from a caged precursor demonstrated that this action is rapid (a few s). The acceleration was abolished by pre-treatment with thapsigargin to block SERCA activity, and was inhibited by two specific antagonists of cADPR, 8-NH(2)-cADPR and 8-br-cADPR. We conclude that cADPR serves to modulate Ca(2+) sequestration by enhancing SERCA pump activity, in addition to its well-established action on RyRs to liberate Ca(2+).

Aims: Here, we report the discovery of a small molecule inhibitor, 2,2'-dihydroxyazobenzene (DAB), of ADP ribosyl cyclase (ADPR-cyclase) and showed that this inhibitor attenuated angiotensin (Ang) II-induced hypertrophic responses., Methods: and results The intracellular concentration of free Ca(2+) [Ca(2+)](i) in adult rat cardiomyocytes was measured by using a confocal microscope. Cardiac hypertrophy was induced by the two-kidney one-clip (2K1C) method. Hypertrophy was determined by de novo protein synthesis, cell volume, echocardiography, nuclear translocation of nuclear factor of activated T-cells, and transforming growth factor-beta1 protein expression. Treatment of cardiomyocytes with Ang II generated a biphasic [Ca(2+)](i) increase that included an initial Ca(2+)peak and sustained Ca(2+) rise via inositol trisphosphate and cyclic ADP-ribose (cADPR) formation, respectively. A cADPR antagonistic analogue, 8-Br-cADPR, and an ADPR-cyclase inhibitor, DAB, blocked the sustained Ca(2+) signal, but not the initial Ca(2+) rise. Furthermore, DAB significantly inhibited Ang II-mediated cADPR formation and hypertrophic responses in vitro. Echocardiography and histological examination revealed significant cardiac hypertrophy in 2K1C rats that was potently inhibited by treatment with DAB. In addition, the hypertrophic responses induced by Ang II in vitro were significantly increased by 2K1C, and DAB treatment reversed these hypertrophic responses to the levels of sham Control., Conclusion: ADPR-cyclase is an important mediator of cardiac hypertrophy, and inhibition of ADPR-cyclase by DAB may provide a new therapeutic strategy for cardiac diseases.

The Ca(2+)-permeable TRPM2 channel is a dual function protein that is activated by intracellular ADPR through its enzymatic pyrophosphatase domain with Ca(2+) acting as a co-factor. Other TRPM2 regulators include cADPR, NAADP and H(2)O(2), which synergize with ADPR to potentiate TRPM2 activation. Although TRPM2 has been thoroughly characterized in overexpression or cell-line systems, little is known about the features of TRPM2 in primary cells. We here characterize the regulation of TRPM2 activation in human neutrophils and report that ADPR activates TRPM2 with an effective half-maximal concentration (EC(50)) of 1microM. Potentiation by Ca(2+) is dose-dependent with an EC(50) of 300nM. Both cADPR and NAADP activate TRPM2, albeit with lower efficacy than in the presence of subthreshold levels of ADPR (100nM), which significantly shifts the EC(50) for cADPR from 44 to 3muM and for NAADP from 95 to 1microM. TRPM2 activation by ADPR can be suppressed by AMP with an IC(50) of 10microM and cADPR-induced activation can be blocked by 8-Bromo-cADPR. We further show that 100microM H(2)O(2) enables subthreshold concentrations of ADPR (100nM) to activate TRPM2. We conclude that agonistic and antagonistic characteristics of TRPM2 as seen in overexpression systems are largely compatible with the functional properties of TRPM2 currents measured in human neutrophils, but the potencies of agonists in primary cells are significantly higher.

The present study was designed to determine whether the sarcoplasmic reticulum (SR) could locally produce superoxide (O2-) via NAD(P)H oxidase (NOX) in coronary arterial myocytes (CAMs) and to address whether cADPR-RyR/Ca2+ signaling pathway regulates this local O2- production from the SR. Using confocal microscopic imaging analysis in intact single CAMs, a cell-permeable indicator CM-H2DCFDA for dynamic changes in intracellular ROS (in green color) and a highly selective ER-Tracker Red dye for tracking of the SR were found co-localized. A quantitative analysis based on the intensity of different spectra demonstrated a local O2- production derived from the SR. M(1)-receptor agonist, oxotremorine (Oxo) and a Ca2+ ionophore, A23187, time-dependently increased this O2- production colocalized with the SR. NOX inhibitors, diphenylene iodonium (DPI) and apocynin (Apo), or superoxide dismutase (SOD) and catalase, and Nox4 (a major intracellular NOX subunit) siRNA all substantially blocked this local production of O2-, demonstrating an involvement of NOX. This SR-derived O2- production was also abolished by the inhibitors of cyclic ADP-ribose (cADPR)-mediated Ca2+ signaling, such as nicotinamide (Nicot, 6 mM), ryanodine (Rya, 50 muM) or 8-Br-cADPR (30 microM). However, IP3 antagonist, 2-APB (50 microM) had no effect. In CAMs transfected with siRNA of ADP-ribosyl cyclase or RyR, this SR O2- production was attenuated. Electron spin resonance (ESR) spectromic assay in purified SR also demonstrated the production of O2- that was dependent on NOX activity and Ca2+ concentrations. These results provide direct evidence that O2- could be locally produced via NOX on the SR and that this local O2- producing system is controlled by cADPR-RyR/Ca2+ signaling pathway.

The structural features needed for antagonism at the cyclic ADP-ribose (cADPR) receptor are unclear. Chemoenzymatic syntheses of novel 8-substituted 2'-deoxy-cADPR analogues, including 8-bromo-2'-deoxy-cADPR 7, 8-amino-2'-deoxy-cADPR 8, 8- O-methyl-2'-deoxy-cADPR 9, 8-phenyl-2'-deoxy-cADPR 10 and its ribose counterpart 8-phenyl-cADPR 5 are reported, including improved syntheses of established antagonists 8-amino-cADPR 2 and 8-bromo-cADPR 3. Aplysia californica ADP-ribosyl cyclase tolerates even the bulky 8-phenyl-nicotinamide adenine 5'-dinucleotide as a substrate. Structure-activity relationships of 8-substituted cADPR analogues in both Jurkat T-lymphocytes and sea urchin egg homogenate (SUH) were investigated. 2'-OH Deletion decreased antagonistic activity (at least for the 8-amino series), showing it to be an important motif. Some 8-substituted 2'-deoxy analogues showed agonist activity at higher concentrations, among which 8-bromo-2'-deoxy-cADPR 7 was, unexpectedly, a weak but almost full agonist in SUH and was membrane-permeant in whole eggs. Classical antagonists 2 and 3 also showed previously unobserved agonist activity at higher concentrations in both systems. The 2'-OH group, without effect on the Ca (2+)-mobilizing ability of cADPR itself, is an important motif for the antagonistic activities of 8-substituted cADPR analogues.

An optimized synthetic route to 7-deaza-8-bromo-cyclic adenosine 5'-diphosphate ribose (7-deaza-8-bromo-cADPR 3), an established cell-permeant, hydrolysis-resistant cyclic adenosine 5'-diphosphate ribose (cADPR) antagonist, is presented. Using NMR analysis, we found that 3 adopted a C-2' endo conformation in the N9-linked ribose and a syn conformation about the N9-glycosyl linkage, which are similar to that of cADPR. The synthetic route was also employed to produce 7-deaza-2'-deoxy-cADPR 4, a potential cell-permeant cADPR analogue. 3 and 4 were more stable to chemical hydrolysis, consistent with the observation that 7-deaza-cADPR analogues are more stable than their parent adenosine derivatives. 3 was also found to be stable to enzyme-mediated hydrolysis using CD38 ectoenzyme.

Although cyclic ADP-ribose (cADPR), a novel Ca(2+)-mobilizing mediator, is suggested to be involved in the functions of neutrophils in rodents, its role in human neutrophils remains unclear. The present study examined the ability of cADPR to mobilize Ca(2+) and mediate formyl methionyl leucyl phenylalanine (fMLP)-stimulated increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and migration in human neutrophils. cADPR induced Ca(2+) release from digitonin-permeabilized neutrophils, and the release was blocked by 8Br-cADPR, an antagonist of cADPR. Immunophilin ligands, FK506 and rapamycin, but not cyclosporine A, inhibited cADPR-induced Ca(2+) release. 8Br-cADPR partially reduced fMLP-induced [Ca(2+)](i) rise and abolished the rise in combination with 2APB, an IP(3)-receptor antagonist. Anti-CD38Ab and NADase that interfere with cADPR formation, reduced the fMLP-induced [Ca(2+)](i) rise. When beta-NAD(+), a substrate of ADP-ribosyl cyclase, and cADPR were added to the medium, the former gradually increased [Ca(2+)](i) and the latter potentiated the fMLP-induced [Ca(2+)](i) rise. The beta-NAD(+)-induced [Ca(2+)](i) rise in Ca(2+)-free medium was inhibited by anti-CD38Ab, 8Br-cADPR, FK506, ruthenium red, and thapsigargin. mRNAs of nucleoside transporter (NT), ENT1, ENT2, CNT, and CNT3 were expressed in neutrophils; and their inhibitors, inosine, uridine, and s-(4-nitrobenzyl)-6-thioinosine, reduced the [Ca(2+)](i) rise induced by beta-NAD(+) and fMLP. fMLP-timulated migration was inhibited by the removal of Ca(2+) from the medium or by the addition of 8Br-cADPR, anti-CD38Ab, NADase, and NT inhibitors. These results suggest that cADPR was synthesized extracellularly by CD38, transported into the cells through NTs, and then Ca(2+) was mobilized by FK506-binding protein-dependent process. This process may be involved in fMLP-induced intracellular Ca(2+) signaling and migration in human neutrophils.

The ectoenzyme CD38 catalyzes synthesis and degradation of cyclic ADP ribose in airway smooth muscle (ASM). The proinflammatory cytokine TNFalpha, which enhances agonist-induced intracellular Ca(2+) ([Ca(2+)](i)) responses, has been previously shown to increases CD38 expression. In the present study, we tested the hypothesis that the effects of TNFalpha on CD38 expression vs. changes in [Ca(2+)](i) regulation in ASM cells are linked. Using isolated human ASM cells, CD38 expression was either increased (transfection) or knocked down [small interfering RNA (siRNA)], and [Ca(2+)](i) responses to sarcoplasmic reticulum depletion [i.e., store-operated Ca(2+) entry (SOCE)] were evaluated in the presence vs. absence of TNFalpha. Results confirmed that TNFalpha significantly increased CD38 expression and ADP-ribosyl cyclase activity, an effect inhibited by CD38 siRNA, but unaltered by CD38 overexpression. CD38 suppression blunted, whereas overexpression enhanced, ACh-induced [Ca(2+)](i) responses. TNFalpha-induced enhancement of [Ca(2+)](i) response to agonist was blunted by CD38 suppression, but enhanced by CD38 overexpression. Finally, TNFalpha-induced increase in SOCE was blunted by CD38 siRNA and potentiated by CD38 overexpression. Overall, these results indicate a critical role for CD38 in TNFalpha-induced enhancement of [Ca(2+)](i) in human ASM cells, and potentially to TNFalpha augmentation of airway responsiveness.

The paraventricular nucleus (PVN) of the hypothalamus is important for the neural regulation of cardiovascular function. Nitric oxide (NO) increases synaptic GABA release to presympathetic PVN neurons through the cyclic guanosine monophosphate (cGMP)/protein kinase G signaling pathway. However, the downstream signaling mechanisms underlying the effect of NO on synaptic GABA release remain unclear. In this study, whole-cell voltage-clamp recordings were performed on retrograde-labeled spinally projecting PVN neurons in rat brain slices. Bath application of the NO precursor l-arginine or the NO donor S-nitroso-N-acetylpenicillamine (SNAP) significantly increased the frequency of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in labeled PVN neurons. A specific antagonist of cyclic ADP ribose, 8-bromo-cyclic ADP ribose (8-Br-cADPR), had no significant effect on l-arginine-induced potentiation of mIPSCs. Surprisingly, blocking of voltage-gated potassium channels (Kv) with 4-aminopyridine or alpha-dendrotoxin eliminated the effect of l-arginine on mIPSCs in all labeled PVN neurons tested. The membrane permeable cGMP analog mimicked the effect of l-arginine on mIPSCs, and this effect was blocked by alpha-dendrotoxin. Furthermore, the specific Kv channel blocker for Kv1.1 (dendrotoxin-K) or Kv1.2 (tityustoxin-Kalpha) abolished the effect of l-arginine on mIPSCs in all neurons tested. SNAP failed to inhibit the firing activity of labeled PVN neurons in the presence of dendrotoxin-K, Kalpha. Additionally, the immunoreactivity of Kv1.1 and Kv1.2 subunits was colocalized extensively with synaptophysin in the PVN. These findings suggest that NO increases GABAergic input to PVN presympathetic neurons through a downstream mechanism involving the Kv1.1 and Kv1.2 channels at the nerve terminals.

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

Of the three intracellular Ca2+ signalling molecules (InsP3, cADPR and NAADP) sea urchin egg homogenate has been used in the identification and characterisation of two, cADPR and NAADP. Homogenate is prepared in a Na+/Cl- substitute of N-Methyl glucamine (NMG)/gluconate. To determine how media composition affects Ca2+ release we replaced NMG with various sugars or glycine and found a dramatic improvement in InsP3 mediated Ca2+ release. Conversely the response to cADPR was diminished, whilst NAADP was unaffected. Therefore modifying media composition may be an important consideration in using homogenate to study Ca2+ release for future studies.

Nitric oxide (NO) is a gaseous intercellular messenger involved in numerous processes during development, including wiring of the nervous system. Neuronal growth cones are responsible for establishing the correct connectivity in the nervous system, but how NO might affect neuronal pathfinding is not fully understood. We have demonstrated in a previous study that local application of a NO donor, NOC-7, via micropipette onto individual growth cones from Helisoma trivolvis B5 neurons results in an increase in filopodial length, a decrease in filopodial number and an increase in the intracellular calcium concentration ([Ca(2+)](i)). Moreover, these NO-induced effects were demonstrated to be mediated via an intracellular cascade involving soluble guanylyl cyclase, protein kinase G (PKG) and cyclic adenosine diphosphate ribose (cADPR). We now demonstrate that the increase in the [Ca(2+)](i) that results from local NO application is mediated via release from ryanodine receptor (RyR)-sensitive intracellular stores. We also show that PKG and RyRs are localized within growth cones and microinjection of cADPR mimics the effects of NO, providing further support that the NO-induced effects are mediated via cADPR. Lastly, we provide evidence that calcium influx across the plasma membrane is a necessary component of the NO-induced calcium increase; however, this calcium influx is secondary to the RyR-induced calcium release from intracellular stores. This study details a signalling pathway by which NO can cause changes in growth cone morphology and thus provides a mechanism by which NO could affect neuronal wiring by acting locally on individual growth cones during the pathfinding process.

The actions of cyclic ADP-ribose (cADPR), a regulator of Ca2+-induced Ca2+ release (CICR), were investigated on Ca2+ release and sarcoplasmic reticulum (SR) Ca2+ loading in cardiac myocytes at physiological temperature. In guinea-pig ventricular cells, cADPR, applied via patch pipette or from photorelease of its caged derivative, increased contraction amplitude and whole-cell Ca2+ transients, without affecting SR Ca2+ load (measured in response to rapid caffeine application). Under voltage-clamp conditions, photorelease of caged cADPR enhanced Ca2+ transient magnitude without affecting the peak amplitude of L-type Ca2+ current or its rate of decay, indicative of an increase in CICR gain. In rat permeabilised ventricular myocytes, rapid application of cADPR increased Ca2+ spark frequency within 30 s, and this effect was maintained over a 10 min exposure. Enhancement of spark frequency was not associated with changes in SR Ca2+ load at 30 s and 3 min of exposure to cADPR; however, prolonged exposure (10 min) was associated with an increased SR Ca2+ load (32+/-7%). The observations are consistent with dual actions of cADPR: a rapid effect on CICR that does not depend on an increased SR Ca2+ load, and an additional slower effect that is associated with enhanced SR Ca2+ levels.

Nitric oxide (NO) relaxes most smooth muscle, including the circular smooth muscle (CSM) of the esophagus, whereas in the adjacent longitudinal smooth muscle (LSM), it causes contraction. The second messenger pathways responsible for this NO-induced LSM contraction are unclear, given that these opposing effects of NO are both cGMP dependent. In intestinal LSM, but not CSM, cADP ribose (cADPR)-dependent pathways participate in Ca(2+) mobilization and muscle contraction; whether similar differences exist in the esophagus is unknown. The purpose of this study was to determine whether cADPR plays a role in the NO-mediated contraction of opossum esophageal LSM. Standard isometric tension recordings were performed using both LSM and CSM strips from opossum distal esophagus that were hung in 10-ml tissue baths perfused with oxygenated Krebs solution. cADPR produced concentration-dependent contraction of LSM strips with an EC(50) of 1 nM and peak contraction of 57 +/- 18% of the 60 mM KCl-induced contraction. cADPR had no effect on CSM strips at concentrations up to 10(-6) M. The EC(50) of cADPR caused contraction (18 +/- 2% from initial resting length) of isolated LSM cells. Sodium nitroprusside (SNP; 300 muM) induced contraction of LSM strips that averaged 67 +/- 5% of the KCl response. cADPR antagonists 8-bromo-cADPR and 8-amino-cADPR, as well as ryanodine receptor antagonists ryanodine and tetracaine, significantly inhibited the SNP-induced contraction. In conclusion, in the opossum esophagus, 1) cADPR induces contraction of LSM, but not CSM, and 2) NO-induced contraction of LSM appears to involve a cADPR-dependent pathway.

Ca2+-induced Ca2+ release (CICR) occurs via activation of ryanodine receptors (RyRs) in frog motor nerve terminals after RyRs are primed for activation by repetitive Ca2+ entries, thereby contributing to synaptic plasticity. To clarify how the mechanism of CICR becomes activable by repetitive Ca2+ entries, we studied effects of a RyR modulator, cyclic ADP-ribose (cADPr), on CICR by Ca2+ imaging techniques. Use-dependent binding of fluorescent ryanodine and its blockade by ryanodine revealed the existence of RyRs in the terminals. Repetition of tetani applied to the nerve produced repetitive rises in intracellular Ca2+ ([Ca2+]i) in the terminals. The amplitude of each rise slowly waxed and waned during the course of the stimulation. These slow rises and decays were blocked by ryanodine, indicating the priming, activation and inactivation of CICR. Uncaging of caged-cADPr loaded in the terminals increased the amplitude of short tetanus-induced rises in [Ca2+]i and the amplitude, time to peak and half decay time of the slow waxing and waning rises in [Ca2+]i evoked by repetitive tetani. A cADPr blocker, 8-amino-cADPr, loaded in the terminals decreased the slow waxing and waning component of rises and blocked all the actions of exogenous cADPr. It is concluded that cADPr enhances the priming and activation of CICR. The four-state model for RyRs suggests that cADPr inhibits the inactivation of CICR and increases the activation efficacy of RyR.

Mature limpet oocytes arrested at the first metaphase (MI) of meiosis are activated by the stimulation of fertilizing sperm. The aim of the present study was to clarify the spatiotemporal property and mechanism of intracellular Ca2+ increase in limpet oocytes, which is a prerequisite signal for initiation of development at fertilization. In all of the five limpet species tested, the initial Ca2+ rising phase just after fertilization took the form of a centripetal Ca2+ wave spreading from the whole cortex to the center (cortical flash), yielding a homogeneous Ca2+ elevation throughout the oocyte. The Ca2+ level remained high during the subsequent plateau phase lasting for several minutes and then returned nearly to the original value. No additional Ca2+ increase followed the plateau phase at least by the time of first cleavage. Both rising and plateau phases of Ca2+ increase at fertilization were inhibited by removal of external Ca2+, suggesting that continuous Ca2+ entry occurs throughout the Ca2+ increase. Injection of inositol 1,4,5-trisphosphate (IP3) was effective in generating a Ca2+ increase in mature limpet oocytes arrested at MI; however, their ability to show an IP3-induced Ca2+ increase was extremely low, as compared with other animals. Responsiveness to IP3 injection in immature oocytes arrested at the first prophase (PI) was similar to that in the mature oocytes, suggesting that the IP3-induced Ca2+ release system does not develop during the process of meiotic maturation in limpet oocytes. Caffeine, cyclic adenosine diphosphate ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP), the agents known to stimulate internal Ca2+ release mechanisms distinct from an IP3-dependent pathway, had no effect on intracellular Ca2+ changes in mature limpet oocytes. Labeling of the endoplasmic reticulum (ER) with DiI revealed that cortical ER clusters are only present in the localized region around meiotic chromosomes in mature oocytes. These data strongly suggest that Ca2+ release and its propagating mechanisms are undeveloped in limpet oocytes and that Ca2+ influx is the only Ca2+-mobilizing system available and functioning at fertilization.

We previously showed that 3''-deoxy-cyclic ADP-carbocyclic-ribose (3''-deoxy-cADPcR, 4) is a stable and highly potent analogue of cyclic ADP-ribose (cADPR, 1), a Ca(2+)-mobilizing second messenger. From these results, we designed and synthesized other 3''-modified analogues of cADPcR having a substituent at the 8-position and found that this modification at the 8-position made them partial agonists. Among these compounds, 8-NH(2)-3''-deoxy-cADPcR (10) was identified as a potent partial agonist with an EC(50) value of 17 nM.

An efficient method for the total synthesis of cyclic ADP-ribose (cADPR, 1) analogues was established. In this procedure, formation of the characteristic 18membered ring was key step, which was achieved by the AgNO3-or I2-promoted condensation with the phenylthiophosphate-type substrate forming an intramolecular pyrophosphate linkage. Using this method, a variety of carbocyclic analogues of cADPR have been synthesized to investigate the structure-activity-relationship, where cyclic ADP-carbocyclic-ribose (2) was identified as a stable and cell-type selective cADPR agonist.

Background and Purpose: Recently, a number of mimics of the second messenger cyclic ADP-ribose (cADPR) with replacement of adenosine by inosine were introduced. In addition, various alterations in the molecule ranging from substitutions at C8 of the base up to full replacement of the ribose moieties still retained biological activity. However, nothing is known about the metabolic stability and cellular effects of these novel analogues., Experimental Approach: cADPR and the inosine-based analogues were incubated with CD38, ADP-ribosyl cyclase and NAD-glycohydrolase and metabolism was analysed by RP-HPLC. Furthermore, the effect of the analogues on cytokine expression and proliferation was investigated in primary T-lymphocytes and T-lymphoma cells., Key Results: Incubation of cADPR with CD38 resulted in degradation to adenosine diphosphoribose. ADP-ribosyl cyclase weakly catabolised cADPR whereas NAD-glycohydrolase showed no such activity. In contrast, N1-cyclic inosine 5'-diphosphoribose (N1-cIDPR) was not hydrolyzed by CD38. Three additional N1-cIDPR analogues showed a similar stability. Proliferation of Jurkat T-lymphoma cells was inhibited by N1-cIDPR, N1-[(phosphoryl-O-ethoxy)-methyl]-N9-[(phosphoryl-O-ethoxy)-methyl]-hypoxanthine-cyclic pyrophosphate (N1-cIDP-DE) and N1-ethoxymethyl-cIDPR (N1-cIDPRE). In contrast, in primary T cells neither proliferation nor cytokine expression was affected by these compounds., Conclusions and Implications: The metabolic stability of N1-cIDPR and its analogues provides an advantage for the development of novel pharmaceutical compounds interfering with cADPR mediated Ca2+ signalling pathways. The differential effects of N1-cIDPR and N1-cIDPRE on proliferation and cytokine expression in primary T cells versus T-lymphoma cells may constitute a starting point for novel anti-tumor drugs.

Novel analogues of cADPR with adenine as base and ether (10a) or different alkane chain (10b-d) substitutions of the northern ribose were synthesized from protected imidazole nucleoside 1 in good yields. The pharmacological activities of cyclic inosine diphosphoribose ether (cIDPRE) and the compounds (10a-d) were analyzed in intact human Jurkat T-lymphocytes. The results indicate that the analogues 10a-d permeate the plasma membrane and are weak agonists of the cADPR/ryanodine receptor signaling system in intact human Jurkat T cells. They are the first membrane-permeant and biologically active cADPR analogues that contain ether or alkane bridges instead of the northern ribose and retain adenine as its base.

The effect of the immunosuppressant drug FK506 on microsomal Ca2+ release was investigated in rat pancreatic acinar cells. When FK506 (0.1-200 microM) was added to the microsomal vesicles at a steady state of ATP-dependent 45Ca2+ uptake, FK506 caused a dose-dependent and a biphasic release of 45Ca2+. Almost 10% of total 45Ca2+ uptake was released at FK506 concentrations up to 10 microM (Km=0.47 microM), and 60% of total 45Ca2+ uptake was released at FK506 concentrations over 10 microM (Km=55 microM). Preincubation of the vesicles with cyclic ADP-ribose (cADPR, 0.5 microM) increased the FK506 (< or =10 microM)-induced 45Ca2+ release (Ozawa T, Biochim Biophys Acta 1693: 159-166, 2004). Preincubation with heparin (200 microg/ml) resulted in significant inhibition of the FK506 (30 microM)-induced 45Ca2+ release. Subsequent addition of inositol 1,4,5-trisphosphate (IP3, 5 microM) after FK506 (100 microM)-induced 45Ca2+ release did not cause any release of 45Ca2+. These results indicate that two types of FK506-induced Ca2+ release mechanism operate in the endoplasmic reticulum of rat pancreatic acinar cells: a high-affinity mechanism of Ca2+ release, which involves activation of the ryanodine receptor, and a low-affinity mechanism of Ca2+ release, which involves activation of the IP3 receptor.