cADPR-induced Ca2 Research Articles

Direct Gating of the TRPM2 Channel by cADPR via Specific Interactions with the ADPR Binding Pocket

cADPR is a well-recognized signaling molecule by modulating the RyRs, but considerable debate exists regarding whether cADPR can bind to and gate the TRPM2 channel, which mediates oxidative stress signaling in diverse physiological and pathological processes. Here, we show that purified cADPR evoked TRPM2 channel currents in both whole-cell and cell-free single-channel recordings and specific binding of cADPR to the purified NUDT9-H domain of TRPM2 by surface plasmon resonance. Furthermore, by combining computational modeling with electrophysiological recordings, we show that the TRPM2 channels carrying point mutations at H1346, T1347, L1379, S1391, E1409, and L1484 possess distinct sensitivity profiles for ADPR and cADPR. These results clearly indicate cADPR is a bona fide activator at the TRPM2 channel and clearly delineate the structural basis for cADPR binding, which not only lead to a better understanding in the gating mechanism of TRPM2 channel but also shed light on a cADPR-induced RyRs-independent Ca2+ signaling mechanism.

Identifying Glyceraldehyde 3-Phosphate Dehydrogenase as a Cyclic Adenosine Diphosphoribose Binding Protein by Photoaffinity Protein-Ligand Labeling Approach.

Cyclic adenosine diphosphoribose (cADPR), an endogenous nucleotide derived from nicotinamide adenine dinucleotide (NAD+), mobilizes Ca2+ release from endoplasmic reticulum (ER) via ryanodine receptors (RyRs), yet the bridging protein(s) between cADPR and RyRs remain(s) unknown. Here we synthesized a novel photoaffinity labeling (PAL) cADPR agonist, PAL-cIDPRE, and subsequently applied it to purify its binding proteins in human Jurkat T cells. We identified glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as one of the cADPR binding protein(s), characterized the binding affinity between cADPR and GAPDH in vitro by surface plasmon resonance (SPR) assay, and mapped cADPR's binding sites in GAPDH. We further demonstrated that cADPR induces the transient interaction between GAPDH and RyRs in vivo and that GAPDH knockdown abolished cADPR-induced Ca2+ release. However, GAPDH did not catalyze cADPR into any other known or novel compound(s). In summary, our data clearly indicate that GAPDH is the long-sought-after cADPR binding protein and is required for cADPR-mediated Ca2+ mobilization from ER via RyRs.

Carvedilol inhibits cADPR- and IP3-induced Ca2+ release.

Spontaneous Ca2+ waves, also termed store-overload-induced Ca2+ release (SOICR), in cardiac cells can trigger ventricular arrhythmias especially in failing hearts. SOICR occurs when RyRs are activated by an increase in sarcoplasmic reticulum (SR) luminal Ca2+. Carvedilol is one of the most effective drugs for preventing arrhythmias in patients with heart failure. Furthermore, carvedilol analogues with minimal β-blocking activity also block SOICR showing that SOICR-inhibiting activity is distinct from that for β-block. We show here that carvedilol is a potent inhibitor of cADPR-induced Ca2+ release in sea urchin egg homogenate. In addition, the carvedilol analog VK-II-86 with minimal β-blocking activity also suppresses cADPR-induced Ca2+ release. Carvedilol appeared to be a non-competitive antagonist of cADPR and could also suppress Ca2+ release by caffeine. These results are consistent with cADPR releasing Ca2+ in sea urchin eggs by sensitizing RyRs to Ca2+ involving a luminal Ca2+ activation mechanism. In addition to action on the RyR, we also observed inhibition of inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release by carvedilol suggesting a common mechanism between these evolutionarily related and conserved Ca2+ release channels.

Regulation of Angiogenic Functions by Angiopoietins through Calcium-Dependent Signaling Pathways

Angiopoietins are vascular factors essential for blood vessel assembly and correct organization and maturation. This study describes a novel calcium-dependent machinery activated through Angiopoietin-1/2-Tie receptor system in HUVECs monolayer. Both cytokines were found to elicit intracellular calcium mobilization. Targeting intracellular Ca2+ signaling, antagonizing IP3 with 2-APB or cADPR with 8Br-cADPR, was found to modulate in vitro angiogenic responses to Angiopoietins in a specific way. 2-APB and 8Br-cADPR impaired the phosphorylation of AKT and FAK induced by Ang-1 and Ang-2. On the other hand, phosphorylation of ERK1/2 and p38, as well as cell proliferation, was not affected by either inhibitor. The ability of ECs to migrate following Angs stimulation, evaluated by “scratch assay,” was reduced by either 2-APB or 8Br-cADPR following Ang-2 stimulation and only slightly affected by 2-APB in cells stimulated with Ang-1. These results identify a novel calcium-dependent machinery involved in the complex interplay regulating angiogenic processes showing that IP3- and cADPR-induced Ca2+ release specifically regulates distinct Angs-mediated angiogenic steps.

A Novel Fluorescent Cell Membrane-permeable Caged Cyclic ADP-ribose Analogue

Cyclic adenosine diphosphate ribose is an endogenous Ca(2+) mobilizer involved in diverse cellular processes. A cell membrane-permeable cyclic adenosine diphosphate ribose analogue, cyclic inosine diphosphoribose ether (cIDPRE), can induce Ca(2+) increase in intact human Jurkat T-lymphocytes. Here we synthesized a coumarin-caged analogue of cIDPRE (Co-i-cIDPRE), aiming to have a precisely temporal and spatial control of bioactive cIDPRE release inside the cell using UV uncaging. We showed that Co-i-cIDPRE accumulated inside Jurkat cells quickly and efficiently. Uncaging of Co-i-cIDPRE evoked Ca(2+) release from endoplasmic reticulum, with concomitant Ca(2+) influx in Jurkat cells. Ca(2+) release evoked by uncaged Co-i-cIDPRE was blocked by knockdown of ryanodine receptors (RyRs) 2 and 3 in Jurkat cells. The associated Ca(2+) influx, on the other hand, was abolished by double knockdown of Stim1 and TRPM2 in Jurkat cells. Furthermore, Ca(2+) release or influx evoked by uncaged Co-i-cIDPRE was recapitulated in HEK293 cells that overexpress RyRs or TRPM2, respectively, but not in wild-type cells lacking these channels. In summary, our results indicate that uncaging of Co-i-cIDPRE incites Ca(2+) release from endoplasmic reticulum via RyRs and triggers Ca(2+) influx via TRPM2.

Cyclic ADP-ribose links metabolism to multiple fission in the dinoflagellate Crypthecodinium cohnii

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.

Cyclic ADP-Ribose Mediates Formyl Methionyl Leucyl Phenylalanine (fMLP)-Induced Intracellular Ca2+Rise and Migration of Human Neutrophils

Although cyclic ADP-ribose (cADPR), a novel Ca2+-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 Ca2+and mediate formyl methionyl leucyl phenylalanine (fMLP)-stimulated increase in cytosolic free Ca2+concentration ([Ca2+]i) and migration in human neutrophils. cADPR induced Ca2+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 Ca2+release. 8Br-cADPR partially reduced fMLP-induced [Ca2+]irise and abolished the rise in combination with 2APB, an IP3-receptor antagonist. Anti-CD38Ab and NADase that interfere with cADPR formation, reduced the fMLP-induced [Ca2+]irise. When β-NAD+, a substrate of ADP-ribosyl cyclase, and cADPR were added to the medium, the former gradually increased [Ca2+]iand the latter potentiated the fMLP-induced [Ca2+]irise. The β-NAD+–induced [Ca2+]irise in Ca2+-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 [Ca2+]irise induced by β-NAD+and fMLP. fMLP-stimulated migration was inhibited by the removal of Ca2+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 Ca2+was mobilized by FK506-binding protein–dependent process. This process may be involved in fMLP-induced intracellular Ca2+signaling and migration in human neutrophils.

The role of cyclic-ADP-ribose-signaling pathway in oxytocin-induced Ca2+ transients in human myometrium cells.

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.

Calmodulin Dissociation Mediates Desensitization of the cADPR-Induced Ca2+ Release Mechanism

Ryanodine receptor (RyR) activation by cyclic ADP-ribose (cADPR) is followed by homologous desensitization [1–3]. Though poorly understood, this “switching off” process has provided a key experimental tool for determining the pathway through which cADPR mediates Ca2+ release [4]. Moreover, desensitization is likely to play an important role in shaping the complexities of Ca2+ signaling involving cADPR, for example, localized release events [5, 6] and propagated waves [7–9]. Using the sea urchin egg, we unmask a role of calmodulin, a component of the RyR complex [10, 11] and a key cofactor for cADPR activity [12–14], during RyR/cADPR desensitization. Recovery from desensitization in calmodulin-depleted purified endoplasmic reticulum (microsomes) is severely impaired compared to that in crude egg homogenates. An active, soluble factor, identified as calmodulin, is required to restore the capacity of microsomes to recover from desensitization. Calmodulin mediates recovery in a manner that tightly parallels its time course of association with the RyR. Conversely, direct measurement of calmodulin binding to microsomes reveals a loss of specific binding during cADPR, but not IP3, desensitization. Our results support a mechanism in which cycles of calmodulin dissociation and reassociation to an endoplasmic reticulum protein, most likely the RyR itself, mediate RyR/cADPR desensitization and resensitization, respectively.

Interactions between intracellular Ca2+ stores: Ca2+ released from the NAADP pool potentiates cADPR-induced Ca2+ release.

Cells possess multiple intracellular Ca2+-releasing systems. Sea urchin egg homogenates are a well-established model to study intracellular Ca2+ release. In the present study the mechanism of interaction between three intracellular Ca2+ pools, namely the nicotinic acid adenine dinucleotide phosphate (NAADP), the cyclic ADP-ribose (cADPR) and the inositol 1',4',5'-trisphosphate (IP3)-regulated Ca2+ stores, is explored. The data indicate that the NAADP Ca2+ pool could be used to sensitize the cADPR system. In contrast, the IP3 pool was not affected by the Ca2+ released by NAADP. The mechanism of potentiation of the cADPR-induced Ca2+ release, promoted by Ca2+ released from the NAADP pool, is mediated by the mechanism of Ca2+-induced Ca2+ release. These data raise the possibility that the NAADP Ca2+ store may have a role as a regulator of the cellular sensitivity to cADPR.

Molecular cloning and characterization of ryanodine receptor from unfertilized sea urchin eggs.

Unfertilized eggs of sea urchins (Hemicentrotus pulcherrimus) demonstrated cyclic ADP-ribose (cADPR)-induced Ca(2+) release and caffeine-induced Ca(2+) release, both of which were considered to be mediated through the ryanodine receptor (RyR). We cloned cDNAs for sea urchin egg RyR (suRyR), which encode a 597-kDa protein of 5,317 amino acids. suRyR shares common structural features with known RyRs: the well-conserved COOH-terminal domain, which forms a functional Ca(2+) channel, and a large hydrophilic NH2-terminal domain. suRyR shows amino acid sequence identity (43-45%) similar to the three mammalian RyR isoforms. Phylogenetic analysis indicates that suRyR branched from three isoforms of vertebrates before they diverged, suggesting that suRyR may be the only RyR isoform in the sea urchin. Four in-frame insertions were found in suRyR cDNAs, one of which was novel and unique, in that it had a cluster of serine residues. The transcripts with and without these insertions were found in the egg RNA. These results suggest that suRyR may be expressed as a functional Ca(2+)-induced Ca(2+) release channel, which might also be involved in cADPR-induced Ca(2+) release.

Differential mechanisms of Ca(2+) release from vascular smooth muscle cell microsomes.

The release of Ca(2+) from intracellular stores is a fundamental element of signaling pathways involved in regulation of vascular tone, proliferation, apoptosis, and gene expression. Studies of sea urchin eggs have led to the identification of three functionally distinct Ca(2+) signaling pathways triggered by IP3, cADPR, and NAADP. The coexistence and functional relevance of these distinct intracellular Ca(2+) release systems has only been described in a few mammalian cell types. The purpose of this study was to determine whether the IP3, cADPR, and NAADP Ca(2+) release systems coexist in smooth muscle cells (SMC) and to determine the specificity of these intracellular Ca(2+) release pathways. Microsomes were prepared from rat aortic SMC (VSMC) and were loaded with 45Ca(2+). cADPR, NAADP, and IP3 induced Ca(2+) release from VSMC microsomes in a dose-dependent fashion. Heparin blocked only IP3-mediated Ca(2+) release, whereas the ryanodine channel inhibitors 8-Br-cADPR and ruthenium red blocked only cADPR-induced Ca(2+) release. Nifedipine, an L-type Ca(2+) channel blocker, inhibited NAADP elicited Ca(2+) release, but had no effect on IP3- or cADPR-mediated Ca(2+) release. An increase in pH from 7.2 to 8.2 inhibited cADPR-mediated Ca(2+) release, but had no effect on IP3- or NAADP-induced Ca(2+) release. By RT-PCR, VSMC expressed ryanodine receptor types 1, 2, and 3. Ca(2+)-dependent binding of [3H]-ryanodine to VSMC microsomes was enhanced by the ryanodine receptor agonists 4-chloro-methyl-phenol (CMP) and caffeine, but was inhibited by ruthenium red and cADPR. We conclude that VSMC possess at least three functionally distinct pathways that promote intracellular Ca(2+) release. IP3-, cADPR-, and NAADP-induced intracellular Ca(2+) release may play a critical role in the maladaptive responses of VSMC to environmental stimuli that are characteristically associated with hypertension and/or atherogenesis.

Calcium-induced calcium release in neurosecretory insect neurons:fast and slow responses

The dynamics of intracellular free Ca2+([Ca2+]i) changes were investigated in dorsal unpaired median (DUM) neurons of the cockroach Periplaneta americana. Activation of voltage-gated Ca2+channels caused a steep increase in [Ca2+]i. Depolarizations lasting for < 100ms led to Ca2+release from intracellular stores as is indicated by the finding that the rise of [Ca2+]iwas greatly reduced by the antagonists of ryanodine receptors, ryanodine and ruthenium red. There is a resting Ca2+current which is potentiated on application of a neuropeptide, Neurohormone D (NHD), a member of the adipokinetic hormone family. Ca2+influx enhanced in this way again caused a rise of [Ca2+]isensitive to ryanodine and ruthenium red. Such rises developed and relaxed much more slowly than the depolarization-induced signals. Ca2+responses similar to those induced by NHD were obtained with the ryanodine receptor agonists caffeine (20mM) and cADP-ribose (cADPR, 100nM). These Ca2+responses, however, varied considerably in size and kinetics, and part of the cells did not respond at all to caffeine or cADPR. Such cells, however, produced Ca2+rises after having been treated with NHD. Thus, the variability of Ca2+signals might be caused by different filling states of Ca2+stores, and the resting Ca2+current seems to represent a source to fill empty Ca2+stores. In line with this notion, block of the endoplasmic Ca2+pump by thapsigargin (1μM) produced either no or largely varying Ca2+responses. The Ca2+signals induced by caffeine and cADPR displayed different sensitivity to ryanodine receptor blockers. cADPR failed to elicit any response when ryanodine or ruthenium red were present. By contrast, the response to caffeine, in the presence of ryanodine, was only reduced by about 50% and, in the presence of ruthenium red, it was not at all reduced. Thus, there may be different types of Ca2+release channels. Block of mitochondrial Ca2+uptake with carbonyl cyanidem -chlorophenylhydrazone (CCCP, 1μM) completely abolished cADPR-induced Ca2+signals, but it did not affect the caffeine-induced signals. Taken together our findings seem to indicate that there are different stores using different Ca2+uptake pathways and that some of these pathways involve mitochondria.

Commentary

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Selected contribution: effect of volatile anesthetics on cADP-ribose-induced Ca(2+) release system.

Volatile anesthetics have multiple actions on intracellular Ca(2+) homeostasis, including activation of the ryanodine channel (RyR) and sensitization of this channel to agonists such as caffeine and ryanodine. Recently it has been described that the nucleotide cADP-ribose (cADPR) is the endogenous regulator of the RyR in many mammalian cells, and cADPR has been proposed to be a second messenger in many signaling pathways. I investigated the effect of volatile anesthetics on the cADPR signaling system, using sea urchin egg homogenates as a model of intracellular Ca(2+) stores. Ca(2+) uptake and release were monitored in sea urchin egg homogenates by using the fluo-3 fluorescence technique. Activity of the ADP-ribosyl cyclase was monitored by using a fluorometric method using nicotinamide guanine dinucleotide as a substrate. Halothane in concentrations up to 800 microM did not induce Ca(2+) release by itself in sea urchin egg homogenates. However, halothane potentiates the Ca(2+) release mediated by agonists of the ryanodine channel, such as ryanodine. Furthermore, other volatile anesthetics such as isoflurane and sevoflurane had no effect. Halothane also potentiated the activation of the ryanodine channel mediated by the endogenous nucleotide cADPR. The half-maximal concentration for cADPR-induced Ca(2+) release was decreased about three times by addition of 800 microM halothane. The reverse was also true: addition of subthreshold concentrations of cADPR sensitized the homogenates to halothane. In contrast, all the volatile anesthetics used had no effect on the activity of the enzyme that synthesizes cADPR. I propose that the complex effect of volatile anesthetics on intracellular Ca(2+) homeostasis may involve modulation of the cADPR signaling system.

ADP-Ribosyl Cyclase in Rat Salivary Glands

Both the Ca2+-releasing mechanism induced by cyclic ADP-ribose (cADPR) and the ADP-ribosyl cyclase (ADPRC) activity that converts NAD+ to cADPR were observed in a variety of cell types. We studied the ADPRC activity in rat major salivary glands that include parotid gland (PG), submandiblar gland (SMG), and sublingual gland (SLG). The enzyme activity responsible for cADPR synthesis was detected by spectrofluorometric assay using NGD+ as a substrate. The enzyme activities in SLG, SMG, and PG were about 400, 30, and 40 nmol/min/g tissue, respectively, in 5-week-old rats. The highest value was observed in SLG and this value was higher than those in other tissues; e.g., spleen (200 nmol/min/g tissue). The enzyme activity in SLG increased gradually after birth and showed a maximum value at 3 weeks. On the other hand, the enzyme activities almost did not change in both PG and SMG between 0 and 9 weeks. In spite of the high ADPRC activity in SLG, we could not detect the cADPR-induced Ca2+-release from SLG microsomes. These results suggest that the ADPRC in SLG does not function through Ca2+-release observed in various tissues.

Differential Effect of Glycolytic Intermediaries upon Cyclic ADP-ribose-, Inositol 1′,4′,5′-Trisphosphate-, and Nicotinate Adenine Dinucleotide Phosphate-Induced Ca2+ Release Systems

We investigated the effect of glycolytic pathway intermediaries upon Ca2+ release induced by cyclic ADP-ribose (cADPR), inositol 1′,4′,5-trisphosphate (IP3), and nicotinate adenine dinucleotide phosphate (NAADP) in sea urchin egg homogenate. Fructose 1,6,-diphosphate (FDP), at concentrations up to 8 mM, did not induce Ca2+ release by itself in sea urchin egg homogenate. However, FDP potentiates Ca2+ release mediated by agonists of the ryanodine channel, such as ryanodine, caffeine, and palmitoyl-CoA. Furthermore, glucose 6-phosphate had similar effects. FDP also potentiates activation of the ryanodine channel mediated by the endogenous nucleotide cADPR. The half-maximal concentration for cADPR-induced Ca2+ release was decreased approximately 3.5 times by addition of 4 mM FDP. The reverse was also true: addition of subthreshold concentrations of cADPR sensitized the homogenates to FDP. The Ca2+ release mediated by FDP in the presence of subthreshold concentrations of cADPR was inhibited by antagonists of the ryanodine channel, such as ruthenium red, and by the cADPR inhibitor 8-Br-cADPR. However, inhibition of Ca2+ release induced by IP3 or NAADP had no effect upon Ca2+ release induced by FDP in the presence of low concentrations of cADPR. Furthermore, FDP had inhibitory effects upon Ca2+ release induced by both IP3 and NAADP. We propose that the state of cellular intermediary metabolism may regulate cellular Ca2+ homeostases by switching preferential effects from one intracellular Ca2+ release channel to another.

Potentiation of cADPR-induced Ca(2+)-release by methylxanthine analogues.

Caffeine and other methylxanthines are known to induce Ca(2+)-release from intracellular stores via the ryanodine receptor. In the present work, a range of caffeine analogues, in which methyl groups at the 1 and 7 positions were replaced with alkyl chains containing different functional groups (oxo, hydroxyl, propargyl, ester, and acids), were synthesized. These compounds were then screened for their ability to potentiate Ca(2+)-release induced by cADPR (an endogenous modulator of ryanodine receptors) in sea urchin egg homogenates. Two of the synthesized methylxanthines, 1, 3-dimethyl-7-(7-hydroxyoctyl)xanthine (37) and 3-methyl-7-(7-oxooctyl)-1-propargylxanthine (66), were shown to be more potent than caffeine in potentiating cADPR-induced Ca(2+)-release, while 1,3-dimethyl-7-(5-ethylcarboxypentyl)xanthine (14) was shown to be more efficacious. The development of new methylxanthine analogues may lead to a better understanding of ryanodine receptor function and could possibly provide novel therapeutic agents.

Cyclic ADP-ribose induces Ca2+ release from caffeine-insensitive Ca2+ pools in canine salivary gland cells.

Cyclic ADP-ribose (cADPR), a novel putative messenger of the ryanodine receptor, was examined regarding its ability to mobilize Ca2+ from intracellular Ca2+ stores in isolated cells of parotid and submandibular glands of the dog. cADPR induced a rapid and transient Ca2+ release in the digitonin-permeabilized cells of salivary glands. cADPR-induced Ca2+ release was inhibited by ryanodine receptor antagonists ruthenium red, ryanodine, benzocaine, and imperatoxin inhibitor but not by the inositol 1,4,5-trisphosphate (IP3)-receptor antagonist heparin. Thapsigargin, at a concentration of 3 to 30 microM, inhibited IP3-induced Ca2+ release, while higher concentrations were required to inhibit cADPR-induced Ca2+ release. Cross-potentiation was observed between cADPR and ryanodine or SrCl2, suggesting that cADPR sensitizes the Ca2+-induced Ca2+ release mechanism. Cyclic AMP plays a stimulatory role on cADPR- and IP3-induced Ca2+ release in digitonin-permeabilized cells. Calmodulin also potentiated cADPR-induced Ca2+ release, but inhibited IP3-induced Ca2+ release. Acetylcholine and ryanodine caused the rise in intracellular free Ca2+ concentration ([Ca2+]i) in intact submandibular and parotid cells. Caffeine did not produce any increase in Ca2+ release or [Ca2+]i rise in any preparation. ADP-ribosyl cyclase activity was found in the centrifuged particulate fractions of the salivary glands. These results suggest that cADPR serves as an endogenous modulator of Ca2+ release from Ca2+ pools through a caffeine-insensitive ryanodine receptor channel, which are different from IP3-sensitive pools in canine salivary gland cells. This system is positively regulated by cyclic AMP and calmodulin.

U73122 Blocked the cGMP-Induced Calcium Release in Sea Urchin Eggs

U73122, a phospholipase C inhibitor, dose dependently blocks the cGMP-induced Ca 2+release in sea urchin eggs and homogenates. U73122 inhibition was prevented by cotreatment with dithiothreitol (DTT), but DTT is ineffective when eggs or homogenates were pretreated with U73122. U73122 action is different from the other sulfhydryl reagents, thimerosal and N-ethylmaleimide, which cause Ca 2+release in egg homogenates at high concentration, but at lower concentration have no significant effect on cGMP-induced Ca 2+release. Histone, a reported NAD glycohydrolase (NADase) activator, was found to induce Ca 2+release in egg homogenates via the same pathway as the cGMP response, since histone-induced Ca 2+release is blocked by Rp-8-pCPT-cGMPS, a cGMP-dependent protein kinase (PKG) inhibitor, and nicotinamide, a NADase inhibitor. Histone-induced Ca 2+release is similarly blocked by U73122. The aminosteroid U73122 does not inhibit cADPR-induced Ca 2+release, which is significantly reduced by PKG inhibitors. Furthermore, U73122 has no significant effect on phorbol 12-myristate 13-acetate induced-cytoplasmic alkalinization in intact eggs, which depends on protein kinase C activity. These results suggest that U73122 does not act as a general serine–threonine protein kinase inhibitor, and the aminosteroid inhibition of the cGMP-induced Ca 2+release may interfere with ADP ribosyl cyclase activity.