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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inosines, in the last few years we have produced new flexible cIDPR analogues, where the northern ribose has been replaced by alkyl chains.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.)

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.

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.

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.)

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.)

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.

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.

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.

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.

Store-operated Ca2+ entry plays an important role in Ca2+ homeostasis in cells but the mechanisms of control of these channels are not completely understood. We describe an investigation of the role of the CD38-cyclic-ADP-ribose (cADPR)-ryanodine-channel (RyR) signaling pathway in store-operated Ca2+ entry in human smooth muscle. We observed that human myometrial cells have a functional store-operated Ca2+ entry mechanism. Furthermore, we observed the presence of transient receptor potential 1, 3, 4, 5, and 6 ion channels in human myometrial cells. Store-operated Ca2+ transient was inhibited by at least 50-70% by several inhibitors of the RyR, including ryanodine (10 microM), dantrolene (10 microM), and ruthenium red (10 microM). Furthermore, the cell permeable inhibitor of the cADPR-system, 8-Br-cADPR (100 microM), is a potent inhibitor of the store-operated entry, decreasing the store operated entry by 80%. Pre-incubation of cells with 100 microM cADPR and the hydrolysis-resistant cADPR analog 3-deaza-cADPR (50 microM), but not with ADP-ribose (ADPR) leads to a 1.6-fold increase in the store-operated Ca2+ transient. In addition, we observed that nicotinamide (1-10 mM), an inhibitor of cADPR synthesis, also leads to inhibition of the store-operated Ca2+ transient by 50-80%. Finally, we observed that the transient receptor potential channels, RyR, and CD38 can be co-immunoprecipitated, indicating that they interact in vivo. Our observations clearly implicate the CD38-cADPR-ryanodine signaling pathway in the regulation of store-operated Ca2+ entry in human smooth muscle cells.

Cyclic ADP-ribose (cADPR) is a novel Ca(2+) mobilizing second messenger, which is capable of inducing Ca(2+) release from the sarcoplasmic reticulum (SR) via activation of ryanodine receptors (RyR) in vascular cells. This signaling nucleotide has also been reported to participate in generation or modulation of intracellular Ca(2+) sparks, Ca(2+) waves or oscillations, Ca(2+)- induced Ca(2+) release (CICR) and spontaneous transient outward currents (STOCs) in vascular smooth muscle cells (VSMCs). With respect to the role of cADPR-mediated signaling in mediation of vascular responses to different stimuli, there is accumulating evidence showing that cADPR is importantly involved in the Ca(2+) response of vascular endothelial cells (ECs) and VSMCs to various chemical factors such as vasoactive agonists acetylcholine, oxotremorine, endothelin, and physical stimuli such as stretch, electrical depolarization and sheer stress. This cADPR-RyR-mediated Ca(2+) signaling is now recognized as a fundamental mechanism regulating vascular function. Here we reviewed the literature regarding this cADPR signaling pathway in vascular cells with a major focus on the production of cADPR and its physiological roles in the control of vascular tone and vasomotor response. We also summarized some publish results that unveil the underlying mechanisms mediating the actions of cADPR in vascular cells. Given the importance of Ca(2+) in the regulation of vascular function, the results summarized in this brief review will provide new insights into vascular physiology and circulatory regulation.

Mechanical loads can profoundly alter cell growth and cell proliferation. The dinoflagellates are especially sensitive to mechanical stimulation. Many species will be arrested in cell cycle in response to turbulence or shear stress. We demonstrate here that mechanical shaking and caffeine, the ryanodine-receptor agonist, induced an elevation of cytosolic calcium in the dinoflagellate Crypthecodinium cohnii. Dantrolene, a ryanodine-receptor antagonist, dose-dependently inhibited both shaking-induced and caffeine-induced calcium release. Similar to the effect of mechanical shaking, caffeine alone dose-dependently and reversibly induced cell cycle arrest in dinoflagellates. Prolonged shaking substantially abolished the magnitude of caffeine-induced calcium release and vice-versa, suggesting that both agents released calcium from similar stores through ryanodine receptors. Fluorescence-conjugated ryanodine gave positive labeling, which could be blocked by ryanodine, in the cortice of C. cohnii cells. In addition, caffeine or shaking mobilized intracellular chlortetracycline (CTC)-positive membrane-bound calcium, which could be similarly depleted by t-BuBHQ, a SERCA pump inhibitor. Prior treatment with shaking or caffeine also inhibited the ability of the other agent in mobilizing CTC-positive calcium. CTC-positive microsomal fractions could also be induced to release calcium by caffeine and cADPR, the ryanodinee receptor modulator. t-BuBHQ, but not calcium ionophores, induced cell cycle arrest, and the calcium chelator BAPTA-AM was unable to rescue caffeine-induced cell cycle arrest. These data culminate to suggest that mobilization or depletion of caffeine-sensitive calcium stores, but not calcium elevation per se, is involved in the induction of cell cycle arrest by mechanical stimulation. The present study establishes the role of caffeine-sensitive calcium stores in the regulation of cell cycle progression.

Functions and possible mechanisms of action of adenosine diphosphate ribose (ADP-ribose) and its cyclic analogue--cycle-ADP-ribose (cADP-ribose)-- substances pretend to role of novel second messengers are reviewed. Possible mechanisms of ADP-ribose and cADP-ribose synthesis regulation and metabolism are analyzed. Prospective mechanisms of ADP-ribose and cADP-ribose transportation into the cell are described. ADP-ribose and cADP-ribose are considered as endogenous regulators of ionic balance (in particular regulators of calcium balance) in different tissues. Effects of ADP-ribose on isolated heart of frog and warm-blooded animal (rat), as well as on rat heart in vivo are discussed. The role of ionic channels and receptors, gated by ADP- and cADP-ribose in regulation of cardiomyocyte ionic balance in normal and pathological condition are analyzed. The possible role of purine receptors in ADP-ribose and cADP-ribose effects on heart are discussed.

We previously showed that 3"-deoxy-cyclic ADP-carbocyclic-ribose (3"-deoxy-cADPcR, 3) is a stable and highly potent analogue of cyclic ADP-ribose (cADPR, 1), a Ca2+ -mobilizing second messenger. From these results, we newly designed another 3"-modified analogues of cADPcR and identified the N1-"xylo"-type carbocyclic analogue, i.e., cADPcX (4), as one of the most potent cADPR-related compounds reported so far.

The ability of O(2) levels to regulate Ca(2+) signalling in non-excitable cells is poorly understood, yet crucial to our understanding of Ca(2+)-dependent cell functions in physiological and pathological situations. Here, we demonstrate that hypoxia mobilizes Ca(2+) from an intracellular pool in primary cultures of cortical astrocytes. This pool can also be mobilized by bradykinin, which acts via phospholipase C and inositol trisphosphate production. By contrast, hypoxic Ca(2+) mobilization utilizes ryanodine receptors, which appear to be either present on the same intracellular pool, or on a separate but functionally coupled pool. Hypoxic activation of ryanodine receptors requires formation of cyclic ADP ribose, since hypoxic Ca(2+) mobilization was fully prevented by nicotinamide (which inhibits ADP ribosyl cyclase) or by 8-Br-cADP ribose, an antagonist of cyclic ADP ribose. Our results demonstrate for the first time the involvement of cyclic ADP ribose in hypoxic modulation of Ca(2+) signalling in the central nervous system, and suggest that this modulator of ryanodine receptors may play a key role in the function of astrocytes under conditions of fluctuating O(2) levels.

Cyclic-ADP-ribose (cADPR) has been reported to serve as a second messenger to mobilize intracellular Ca2+ independent of IP3 in a variety of mammalian cells. This cADPR-mediated Ca2+ signaling pathway importantly participates in the regulation of various cell functions. The present study determined the role of endogenous cADPR in mediating ryanodine-sensitive Ca2+-induced Ca2+ release (CICR) in vascular myocytes from small renal arteries and vasomotor response of these arteries. In freshly-isolated renal arterial myocytes, addition of CaCl2 (0.01, 0.1, and 1 mM) into the Ca2+-free bath solution produced a rapid Ca2+ release response from the sarcoplasmic reticulum (SR), with a maximal increase of 237+/-25 nM at 1 mM CaCl2. This CaCl2 response was significantly blocked by a cell-membrane permeant cADPR antagonist, 8-bromo-cADP-ribose (8-br-cADPR) (30 microM) or ryanodine (50 microM). Caffeine, a classical CICR or ryanodine receptor activator was found to stimulate the SR Ca2+ release (Delta[Ca2+]i: 253+/-35 nM), which was also attenuated by 8-br-cADPR or ryanodine. Using isolated and pressurized small renal arteries bathed with Ca2+-free solution, both CaCl2 and caffeine-induced vasoconstrictions were significantly attenuated by either 8-br-cADPR or ryanodine. Biochemical analyses demonstrated that CaCl2 and caffeine did not increase cADPR production in these renal arterial myocytes, but confocal microscopy showed that a dissociation of the accessory protein, FK506 binding protein 12.6 (FKBP12.6) from ryanodine receptors was induced by CaCl2. We conclude that cADPR importantly contributes to CICR and vasomotor responses of small renal arteries through enhanced dissociation of ryanodine receptors from their accessory protein.

We synthesized analogs modified in the ribose unit (ribose linked to N1 of adenine) of cyclic ADP-ribose (cADPR), a Ca2+-mobilizing second messenger. The biological activities of these analogs were determined in NG108-15 neuroblastoma x glioma hybrid cells that were pre-loaded with fura-2 acetoxymethylester and subjected to whole-cell patch-clamp. Application of the hydrolysis-resistant cyclic ADP-carbocyclic-ribose (cADPcR) through patch pipettes potentiated elevation of the cytoplasmic free Ca2+ concentration ([Ca2+]i) at the depolarized membrane potential. The increase in [Ca2+]i evoked upon sustained membrane depolarization was significantly larger in cADPcR-infused cells than in non-infused cells and its degree was equivalent to or significantly greater than that induced by cADPR or beta-NAD+. 8-Chloro-cADPcR and two inosine congeners (cyclic IDP-carbocyclic-ribose and 8-bromo-cyclic IDP-carbocyclic-ribose) did not induce effects similar to those of cADPcR or cADPR. Instead, 8-chloro-cADPcR together with cADPR or cADPcR caused inhibition of the depolarization-induced [Ca2+]i increase as compared with either cADPR or cADPcR alone. These results demonstrated that our cADPR analogs have agonistic or antagonistic effects on the depolarization-induced [Ca2+]i increase and suggested the presence of functional reciprocal coupling between ryanodine receptors and voltage-activated Ca2+ channels via cADPR in mammalian neuronal cells.

Exposure of pheochromocytoma (PC 12) cells to a time-varying 1.51 T magnetic field inhibited an increase in the intracellular Ca2+ concentration ([Ca2+]i) induced by addition of caffeine to Ca(2+)-free medium. This inhibition occurred after a 15-min exposure and was maintained for at least 2 h. [Ca2+]i sharply increased in cells loaded with cyclic ADP-ribose, and 2-h exposure significantly suppressed the increase. Addition of ATP induced a transient increase in intracellular Ca2+ release mediated by IP3 receptor, and this increase was strongly inhibited by the exposure. Results indicated that the magnetic field exposure strongly inhibited Ca2+ release mediated by both IP3 and ryanodine receptors in PC 12 cells. However, thapsigargin-induced Ca2+ influx (capacitative Ca2+ entry) across the cell membrane was unaffected. The ATP content was maintained at the normal level during the 2-h exposure, suggesting that ATP hydrolysis was unchanged. Therefore, Mg2+ which is known to be released by ATP hydrolysis and inhibit intracellular Ca2+ release may not relate the exposure-caused inhibition. Eddy currents induced in culture medium appear to change cell membrane properties and indirectly inhibit Ca2+ release from endoplasmic reticulum and other Ca2+ stores in PC 12 cells.

Analogues of cyclic ADP-ribose (cADPR) incorporating a methylenebisphosphonate linkage in the place of the pyrophosphate have been synthesized from nicotinamide adenine dinucleotide analogues enzymatically using Aplysia californica ADP-ribosyl cyclase. Methylenebisphosphonate cyclic ADP-ribose (cADPR[CH(2)]) and methylenebisphosphonate cyclic 3-deaza-ADP-ribose (3-deaza-cADPR[CH(2)]) showed full agonist activity for release of Ca(2+) ions from sea urchin egg homogenates. The EC(50) for cADPR[CH(2)] was 856 nM and that for 3-deaza-cADPR[CH(2)] was 300 nM, about 15- and 5-fold less potent than cADPR, respectively.

The involvement of intracellular Ca(2+) stores and their regulatory mechanisms in mediating pituitary adenylate cyclase-activating polypeptide (PACAP) stimulation of growth hormone (GH) and maturational gonadotrophin (GTH-II) secretion from goldfish pituitary cells was investigated using a cell column perifusion system. Pretreatment with caffeine abolished the GH and GTH-II responses to PACAP. Dantrolene attenuated PACAP-elicited GTH-II release but did not affect the GH response, whereas ryanodine and 8-bromo-cADP ribose did not alter PACAP-induced GH and GTH-II release. Two endoplasmic/sarcoplasmic reticulum Ca(2+) ATPase (SERCA) inhibitors, thapsigargin and cyclopiazonic acid, augmented PACAP-induced GTH-II release; similarly, thapsigargin elevated GH responses to PACAP. Treatment with carbonyl cyanide m-chlorophenylhydrazone, a mitochondrial uncoupler, reduced PACAP-stimulated GH release; however, inhibition of the mitochondrial Ca(2+) uniport by Ru360 did not affect GH and GTH-II responses. The phosphatidyl inositol (PI)-specific phospholipase C (PLC) inhibitor ET-18-OCH(3) inhibited, whereas the phosphatidyl-choline (PC)-specific PLC inhibitor D609 enhanced, PACAP-stimulated GH and GTH-II responses. On the other hand, the IP(3) receptor blocker xestospongin D had no effect on PACAP-induced GTH-II response and potentiated the GH response. These results suggest that, despite some differences between GH and GTH-II cells, PACAP actions in both cell types generally rely on a caffeine-sensitive, but a largely ryanodine receptor-independent, mechanism. PC-PLC and some SERCA negatively modulate PACAP actions but mitochondrial Ca(2+) stores per se are not important. A novel PI-PLC mechanism, which does not involve the traditional IP(3)/Ca(2+) pathway, is also suggested.

We have clarified, for the first time, the spatiotemporal patterns of intracellular Ca(2+) increases at fertilization and the Ca(2+)-mobilizing mechanisms in eggs of hydrozoan jellyfish, which belong to the evolutionarily old diploblastic phylum, Cnidaria. An initial Ca(2+) increase just after fertilization took the form of a Ca(2+) wave starting from one cortical region of the egg and propagating to its antipode in all of four hydrozoan species tested: Cytaeis uchidae, Cladonema pacificum, Clytia sp., and Gonionema vertens. The initiation site of the Ca(2+) wave was restricted to the animal pole, which is known to be the only area of sperm-egg fusion in hydrozoan eggs, and the wave propagating velocity was estimated to be 4.2-5.9 mum/s. After a Ca(2+) peak had been attained by the initial Ca(2+) wave, the elevated Ca(2+) gradually declined and returned nearly to the resting value at 7-10 min following fertilization. Injection of inositol 1,4,5-trisphosphate (IP(3)), an agonist of IP(3) receptors (IP(3)R), was highly effective in inducing a Ca(2+) increase in unfertilized eggs; IP(3) at a final intracellular concentration of 12-60 nM produced a fully propagating Ca(2+) wave equivalent to that observed at fertilization. In contrast, a higher concentration of cyclic ADP-ribose (cADPR), an agonist of ryanodine receptors (RyR), only generated a localized Ca(2+) increase that did not propagate in the egg. In addition, caffeine, another stimulator of RyR, was completely without effect. Sperm-induced Ca(2+) increases in Gonionema eggs were severely affected by preinjection of heparin, an inhibitor of Ca(2+) release from IP(3)R. These results strongly suggest that there is a well-developed IP(3)R-, but not RyR-mediated Ca(2+) release mechanism in hydrozoan eggs and that the former system primarily functions at fertilization. Our present data also demonstrate that the spatial characteristics and mechanisms of Ca(2+) increases at fertilization in hydrozoan eggs resemble those reported in higher triploblastic animals.

A rapid superfusion system measuring the amounts, kinetics, and Ca dependencies of released 45Ca, was used to examine the effects of ryanodine (RY), caffeine (CF), and cyclic ADP ribose (cADPr) on sea urchin egg homogenates. The RY-sensitive compartment had more than twice the Ca release capacity of the CF-sensitive or cADPr-sensitive compartment. cADPr-stimulated 45Ca release required calcium with half-maximal activation at approximately 0.2 to 0.6 microM [Ca2+]. K(1/2) for cADPr activation was approximately 100 nM, and in spite of the Ca requirement for cADPr-stimulated release, the cADPr affinity was not affected by [Ca2+]. Peak 45Ca release rate with cADPr (3 microM) was greater than with CF (20 mM), yet the release amounts were similar and both were [Ca2+]-dependent. When activated with CF and cADPr simultaneously, 45Ca release was large and, no longer [Ca2+]-dependent. Mg competitively inhibited the Ca activation site(s), yet did not inhibit the activation with CF-plus-cADPr. Pre-release of 45Ca by cADPr with low (approximately 0.1 microM) [Ca2+] right-shifted the [Ca2+] dependence of the remaining cADPr-response. These data suggest that (a) only a portion of RY-sensitive compartments empty when stimulated with cADPr or CF, (b) Ca and cADPr act on non-interacting sites, and (c) cADPr-sensitive compartments represent a heterogeneous population with different [Ca2+] dependencies.

Background: Patients with chronic renal failure may develop muscle weakness and fatigability due to disorders of skeletal muscle function, collectively known as the uremic myopathy. Cyclic adenosine diphosphate-ribose (cADPR), an endogenous metabolite of beta-NAD+, activates Ca2+ release from intracellular stores in vertebrate and invertebrate cells. The current study investigated the possible role of cADPR in uremic myopathy., Methods: We have examined the effect of cADPR on myoplasmic resting Ca2+ concentration ([Ca2+]i) in skeletal muscle obtained from control subjects and uremic patients (UP). [Ca2+]i was measured using double-barreled Ca2+-selective microelectrodes in muscle fibers, prior to and after microinjections of cADPR., Results: Resting [Ca2+]i was elevated in UP fibers compared with fibers obtained from control subjects. Removal of extracellular Ca2+, or incubation of cells with nifedipine, did not modify [Ca2+]i in UP or control fibers. Microinjection of cADPR produced an elevation of [Ca2+]i in both groups of cells. This elevation was not mediated by Ca2+ influx, or inhibited by heparin or ryanodine. [cADPR]i was determined to be higher in muscle fibers from UP compared to those from the control subjects. Incubation of cells with 8-bromo-cADPR, a cADPR antagonist, partially reduced [Ca2+]i in UP muscle fibers and blocked the cADPR-elicited elevation in [Ca2+]i in both groups of muscle cells., Conclusion: Skeletal muscles of the UP exhibit chronic elevation of [Ca2+]i that can be partially reduced by application of 8-bromo-cADPR. cADPR was able to mobilize Ca2+ from intracellular stores, by a mechanism that is independent of ryanodine or inositol trisphosphate receptors. It can be postulated that an alteration in the cADPR-signaling pathway may exist in skeletal muscle of the patients suffering from uremic myopathy.

4'',6''-Unsaturated cyclic ADP-carbocyclic-ribose (3) was designed as a novel stale mimic of cyclic ADP-ribose, an intracellular Ca2+-mobilizing second messenger, based on the previous SAR studies, and was successfully synthesized.

In mammals, endothelin receptors are sub-classified into ET(A) receptors (ET(A)R), which are purely constrictive in vascular smooth muscle (VSM), and ET(B)R, which may produce constriction in VSM or dilatation by stimulating the production of nitric oxide (NO) from endothelial cells. In contrast, previous studies suggested that shark VSM is stimulated exclusively by ET(B)R. The Ca(2+) signaling pathways utilized by shark VSM in response to stimulation by endothelin-1 (ET-1) have not previously been investigated. We measured cytosolic Ca(2+) concentration ([Ca(2+)](i)) in fura-2-loaded VSM of anterior mesenteric artery of Squalus acanthias and show that the ET(B)R agonists IRL 1620 and sarafotoxin S6c (SRX) increase [Ca(2+)](i) in VSM to the same extent as ET-1 and ET(B)R appears to be the only ETR subtype in sharks. To investigate the participation of the inositol trisphosphate (IP(3)) receptors (IP(3)R), we utilized two inhibitors of the mammalian IP(3)R, TMB-8 and 2-APB. In Ca(2+)-free Ringer, these agents inhibit the response to ET(B)R agonist stimulation by 71%. The ryanodine-sensitive receptor (RyR) may be activated by low concentrations of ryanodine, by abrupt local increases of [Ca(2+)](i), (calcium-induced calcium release) or by cyclic adeninediphosphate ribose (cADPR). We employed three inhibitors of activation of the RyR, Ruthenium Red, 8-Br cADPR and high concentrations of ryanodine; these agents blocked the [Ca(2+)](i) response to ET(B)R agonist stimulation by a mean of 39%. These data show for the first time that in VSM of the shark, ET(B)R activation stimulates both IP(3)R and RyR, and that cADPR is involved in RyR activation.

Cyclic ADP-ribose (cADPR), a putative Ca(2+)-mobilizing second messenger, has been reported to operate in several mammalian cells. To investigate whether cADPR is involved in electrolyte secretion from airway glands, we used a patch-clamp technique, the measurement of microsomal Ca(2+) release, quantification of cellular cADPR, and RT-PCR for CD38 mRNA in human and feline tracheal glands. cADPR (>6 microM), infused into the cell via the patch pipette, caused ionic currents dependent on cellular Ca(2+). Infusions of lower concentrations (2-4 microM) of cADPR or inositol 1,4,5-trisphosphate (IP(3)) alone were without effect on the baseline current, but a combined application of cADPR and IP(3) mimicked the cellular response to low concentrations of acetylcholine (ACh). Microsomes derived from the isolated glands released Ca(2+) in response to both IP(3) and cADPR. cADPR released Ca(2+) from microsomes desensitized to IP(3) or those treated with heparin. The mRNA for CD38, an enzyme protein involved in cADPR metabolism, was detected in human tissues, including tracheal glands, and the cellular content of cADPR was increased with physiologically relevant concentrations of ACh. We conclude that cADPR, in concert with IP(3), operates in airway gland acinar cells to mobilize Ca(2+), resulting in Cl(-) secretion.

Cyclic adenosine diphosphoribose (cADPR) is an endogenous Ca2+ mobilizing nucleotide in many cell types and different species covering protozoa, plants and animals, including humans. cADPR is formed by ADP-ribosyl cyclases from nicotinamide adenine dinucleotide (NAD). Since at least some of the ADP-ribosyl cyclases are under the control of receptors for exogenous ligands, cADPR is regarded as a second messenger for Ca2+ signaling. The main intracellular target for cADPR is the ryanodine receptor, but it is unclear whether cADPR elicits Ca2+ release by direct binding or via a binding protein. Derivatives of NAD and cADPR are potent ADP-ribosyl cyclase inhibitors and cADPR antagonists. Since Ca2+ ions are regulators of many diverse cell functions, e.g. muscle contraction, secretion of neurotransmitters, hormones and enzymes, fertilization of oocytes, and lymphocyte activation and proliferation, the cADPR signaling pathway may become a valuable target for pharmaceutical intervention.

Human myometrial contraction plays a fundamental role in labor. Dysfunction of uterine contraction is an important cause of labor progression failure. Although the mechanisms controlling uterine contraction are not completely understood, intracellular Ca2+ mobilization plays an important role during uterine contraction. Several mechanisms of intracellular Ca2+ mobilization are present in smooth muscle, but in the human uterus, only 1,4,5-trisphosphate-induced Ca2+ release has been studied extensively. Ryanodine receptor channels are present in myometrium. We determined the role of the cyclic ADP-ribose (cADPR)-signaling pathway in oxytocin-induced intracellular Ca2+ [(Ca2+)i] transients in human myometrial cells. We found that oxytocin-induced Ca2+ transient is dependent on several sources of Ca2+, including extracellular Ca2+ and intracellular Ca2+ stores. In addition, we found that both the 1,4,5-trisphosphate- and the cADPR-induced Ca2+ releasing systems are important for the induction of [Ca2+]i transients by oxytocin in human myometrial cells. Furthermore, we investigated TNFalpha regulation of oxytocin-induced [Ca2+]i transients, CD38 cyclase activity, and CD38 expression in human myometrial cells. We found that oxytocin-induced [Ca2+]i transients were significantly increased by 50 ng/ml TNF. Similarly, CD38 mRNA levels, CD38 expression, and cyclase activity were increased by TNFalpha, thus increasing cADPR levels. We propose that a complex interaction between multiple signaling pathways is important for the development of intracellular Ca2+ transients induced by oxytocin and that TNFalpha may contribute for the myometrium preparation for labor by regulating the cADPR-signaling pathway. The observation that the cADPR-signaling pathway is important for the development of intracellular Ca2+ transients in human myometrial cells raises the possibility that this signaling pathway could serve as a target for the development of new therapeutic strategies for abnormal myometrial contraction observed during pregnancy.

Ca2+ release from the envelope of isolated pancreatic acinar nuclei could be activated by nicotinic acid adenine dinucleotide phosphate (NAADP) as well as by inositol 1,4,5-trisphosphate (IP3) and cyclic ADP-ribose (cADPR). Each of these agents reduced the Ca2+ concentration inside the nuclear envelope, and this was associated with a transient rise in the nucleoplasmic Ca2+ concentration. NAADP released Ca2+ from the same thapsigargin-sensitive pool as IP3. The NAADP action was specific because, for example, nicotineamide adenine dinucleotide phosphate was ineffective. The Ca2+ release was unaffected by procedures interfering with acidic organelles (bafilomycin, brefeldin, and nigericin). Ryanodine blocked the Ca2+-releasing effects of NAADP, cADPR, and caffeine, but not IP3. Ruthenium red also blocked the NAADP-elicited Ca2+ release. IP3 receptor blockade did not inhibit the Ca2+ release elicited by NAADP or cADPR. The nuclear envelope contains ryanodine and IP3 receptors that can be activated separately and independently; the ryanodine receptors by either NAADP or cADPR, and the IP3 receptors by IP3.

Vesl/Homer proteins physically link proteins that mediate cellular signaling [Curr. Opin. Neurobiol. 10 (2000) 370; Trends Neurosci. 23 (2000) 80; J. Cell Sci. 113 (2000) 1851] and thereby influence cellular function [Nat. Neurosci. 4 (2001) 499; Nature 411 (2001) 962]. A previous study reported that Vesl-1L/Homer-1c (V-1L) controls the gain of the intracellular calcium activated calcium channel ryanodine receptor type 1 (RyR1) channel [J. Biol Chem. 277 (2002) 44722]. Here, we show that the function of RyR1 is differentially regulated by two isoforms of Vesl-1/Homer-1, V-1L and Vesl-1S/Homer-1a (V-1S). V-1L increases the activity of RyR1 while important regulatory functions and pharmacological characteristics are preserved. V-1S alone had no effect on RyR1, even though, like V-1L, it is directly bound to the channel. However, V-1S dose-dependently decreased the effects of V-1L on RyR1, providing a novel mechanism for the regulation of intracellular calcium channel activity and calcium homeostasis by changing expression levels of Vesl/Homer proteins.

Cyclic ADP-ribose (cADPr) is a second messenger that regulates intracellular free [Ca2+] ([Ca2+](i)) in a variety of cell types, including immature oocytes from the starfish Astropecten auranciacus. In this study, we employed confocal laser scanning microscopy and voltage clamp techniques to investigate the source of the cADPr-elicited Ca2+ wave originating from the cortical Ca2+ patches we have described previously. The Ca2+ swing was accompanied by a membrane current with a reversal potential of approximately +20 mV. Decreasing external Na+ almost abolished the current without affecting the Ca2+ response. Removal of extracellular Ca2+ altered neither the Ca2+ transient nor the ionic current, nor did the holding potential exert any effect on the Ca2+ wave. Both the Ca2+ response and the membrane current were abolished when BAPTA, ruthenium red or 8-NH(2)-cADPr were preinjected into the oocytes, while perfusion with ADPr did not elicit any [Ca2+](i) increase or ionic current. However, elevating [Ca2+](i) by uncaging Ca2+ from nitrophenyl- (NP-EGTA) or by photoliberating inositol 1,4,5-trisphosphate (InsP(3)) induced an ionic current with biophysical properties similar to that elicited by cADPr. These results suggest that cADPr activates a Ca2+ wave by releasing Ca2+ from intracellular ryanodine receptors and that the rise in [Ca2+](i) triggers a non-selective monovalent cation current that does not seem to contribute to the global Ca2+ elevation.

Nicotinic acid-adenine dinucleotide phosphate (NAADP) is a recently described potent intracellular Ca(2+)-mobilizing messenger active in a wide range of diverse cell types. In the present study, we have investigated the interaction of NAADP with other Ca(2+)-mobilizing messengers in the release of transmitter at the frog neuromuscular junction. We show, for the first time, that NAADP enhances neurosecretion in response to inositol 1,4,5-trisphosphate (IP(3)), cADP-ribose (cADPR) and sphingosine 1-phosphate (S1P), but not sphingosylphosphorylcholine. Thapsigargin was without effect on transmitter release in response to NAADP, but blocked the responses to subsequent application of IP(3), cADPR and S1P and their potentiation by NAADP. Asynchronous neurotransmitter release may therefore involve functional coupling of endoplasmic reticulum Ca(2+) stores with distinct Ca(2+) stores targeted by NAADP.

In olfactory receptor cells, it is well established that cyclic AMP (cAMP) and inositol-1,4,5-trisphosphate (IP(3)) act as second messengers during odor responses. In previous studies, we have shown that cAMP-increasing odorants induce odor responses even after complete desensitization of the cAMP-mediated pathway. These results suggest that at least one cAMP-independent pathway contributes to the generation of odor responses. In an attempt to identify a novel second messenger, we investigated the possible role of cyclic ADP-ribose (cADPR) in olfactory transduction. Turtle olfactory receptor cells were isolated using an enzyme-free procedure and loaded with fura-2/AM. The cells responded to dialysis with cADPR with an inward current and an increase of the intracellular Ca(2+) concentration, [Ca(2+)](i). Flooding of cells with 100 microM cADPR from the pipette also induced an inward current without changes in [Ca(2+)](i) in Na(+)-containing and Ca(2+)-free Ringer solution. In an Na(+)-free and Ca(2+)-containing Ringer solution, cADPR induced only a small inward current with a concomitant increase in [Ca(2+)](i). Inward currents and increases in [Ca(2+)](i) induced by cADPR were completely inhibited by removal of both Na(+) and Ca(2+) from the outer solution. The experiments suggest that cADPR activates a cation channel at the plasma membrane, allowing inflow of Na(+) and Ca(2+) ions. The magnitudes of the inward current responses to cAMP-increasing odorants were greatly reduced by prior dialyses of a high concentration of cADPR or 8-bromo-cyclic ADP-ribose (8-Br-cADPR), an antagonist. It is possible that the cADPR-dependent pathway contributes to the generation of olfactory responses.

The properties of the Na(+)-Ca(2+) exchanger in isolated crypts from rat colon were studied using the Fura-2 imaging technique. The transport mode of the exchanger was reversed by replacing extracellular Na(+) by the impermeable cation, N-methyl-D-glucamine (NMDG(+)), so that the transporter mediated a Ca(2+) influx into the cells. Depletion of intracellular Ca(2+) stores by inhibitors of sarcoplasmatic endoplasmatic calcium ATPases (SERCA), i.e., cyclopiazonic acid (10(-5) mol l(-1)) or thapsigargin (10(-6) mol l(-1)), reduced the increase in [Ca(2+)](i) evoked by superfusion with NMDG(+), suggesting a cross-talk between the exchanger and the Ca(2+) stores. However, measurement of Ca(2+) influx with the Mn(2+) quench technique revealed that the activity of the exchanger was independent of the filling state of the stores. Instead, the obvious inhibition of the [Ca(2+)](i)response by SERCA blockers was due to a reduction of Ca(2+)-induced Ca(2+) release after inhibition of store-refilling. The functional presence of ryanodine receptors was demonstrated by the increase in [Ca(2+)](i)evoked by ryanodine (10(-7) to 3x10(-4) mol l(-1)) in a concentration-dependent manner. This effect was mimicked by cADP ribose (10(-5) mol l(-1)) in crypts permeabilized with saponin (10 mg l(-1)). Ruthenium red (5x10(-5) mol l(-1)) or high concentrations of ryanodine (3x10(-4) mol l(-1)) inhibited this response. In Ussing chamber experiments ruthenium red (5x10(-4) mol l(-1)) or a high concentration of ryanodine (10(-3) mol l(-1)) inhibited the increase in short-circuit current evoked by the cholinergic agonist, carbachol (5x10(-5) mol l(-1)). Consequently, Ca(2+)-induced Ca(2+) release may act as kind of amplifier during Ca(2+)-dependent Cl(-) secretion in order to maintain a long-lasting increase in the intracellular Ca(2+) concentration.

Cyclic ADP-ribose (cADPR) is not only a potent endogenous calcium modulator but also a second messenger. However, studies on the mechanism of cADPR action were limited due to its instability and lack of available structural modifications in the N1-glyosyl unit of cADPR. In the present work, a series of N1-glycosyl mimics with different configurational glycosyls or an ether strand were designed and synthesized mimicking the furanose ring. S(N)2 substitutions were carried out between the protected inosine and glycosyl triflates to form the N1-glycosylinosine derivatives, accompanied with some O6-glycosyl-substituted as side products. The intramolecular cyclization was followed the strategy described by Matsuda et al. It was found that the 8-unsubstituted substrate could also be used to construct the intramolecular cyclic pyrophosphate. The activities of N1-glycosyl-substituted cADPR mimics were evaluated by induced Ca2+ release in rat brain microsomes and HeLa cells. It was found that the configuration of the N1-glycosyl moiety in cADPR is not a critical structural factor for retaining the activity of mobilizing Ca2+ release. More interestingly, the N1-acyclic analogue 6 exhibited strong activity by inducing Ca2+ release in both rat brain microsomes and HeLa cells. It constitutes a useful tool for further studies.

In voltage-clamped guinea-pig chromaffin cells, muscarine (50 microM) or caffeine (30 mM) produced a transient intracellular Ca(2+) concentration ([Ca(2+)](i)) increase, catecholamine release and an outward K(+) current mediated through Ca(2+) released from internal Ca(2+) stores at a holding potential of -40 mV. Caffeine followed by muscarine failed to evoke these responses, while muscarine followed by caffeine was effective in producing about 30% of [Ca(2+)](i) increase and catecholamine secretion. In cells dialyzed with inositol 1,4,5-trisphosphate (IP(3)), caffeine failed to produce the [Ca(2+)](i) increase. Intracellular application of cyclic adenosine 5'-diphosphate-ribose (cADP-ribose) or 8-bromo cADP-ribose exerted no effect on the resting [Ca(2+)](i) and the caffeine-induced [Ca(2+)](i) increase. These results suggest that IP(3)-sensitive stores are functionally divided into two subpopulations, sensitive and insensitive to caffeine, and it is unlikely that cADP-ribose plays a role as a Ca(2+) releaser in guinea-pig adrenal chromaffin cells.