Calcium mobilizing second messengers derived from NAD.

Nicotinamide adenine dinucleotide (NAD) has been known since a long period of time as co-factor of oxidoreductases. However, in the past couple of decades further roles have been assigned to NAD. Here, metabolism of NAD to the Ca²âº mobilizing second messengers cyclic adenosine diphosphoribose, nicotinic acid adenine dinucleotide phosphate and adenosine diphosphoribose is reviewed. Moreover, the mechanisms of Ca²âº mobilization by these adenine nucleotides and their putative target Ca²âº channels, ryanodine receptors and transient receptor potential channels are discussed. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

Regulation of ion channels by pyridine nucleotides.

Recent research suggests that in addition to their role as soluble electron carriers, pyridine nucleotides [NAD(P)(H)] also regulate ion transport mechanisms. This mode of regulation seems to have been conserved through evolution. Several bacterial ion-transporting proteins or their auxiliary subunits possess nucleotide-binding domains. In eukaryotes, the Kv1 and Kv4 channels interact with pyridine nucleotide-binding ß-subunits that belong to the aldo-keto reductase superfamily. Binding of NADP(+) to Kvß removes N-type inactivation of Kv currents, whereas NADPH stabilizes channel inactivation. Pyridine nucleotides also regulate Slo channels by interacting with their cytosolic regulator of potassium conductance domains that show high sequence homology to the bacterial TrkA family of K(+) transporters. These nucleotides also have been shown to modify the activity of the plasma membrane K(ATP) channels, the cystic fibrosis transmembrane conductance regulator, the transient receptor potential M2 channel, and the intracellular ryanodine receptor calcium release channels. In addition, pyridine nucleotides also modulate the voltage-gated sodium channel by supporting the activity of its ancillary subunit-the glycerol-3-phosphate dehydrogenase-like protein. Moreover, the NADP(+) metabolite, NAADP(+), regulates intracellular calcium homeostasis via the 2-pore channel, ryanodine receptor, or transient receptor potential M2 channels. Regulation of ion channels by pyridine nucleotides may be required for integrating cell ion transport to energetics and for sensing oxygen levels or metabolite availability. This mechanism also may be an important component of hypoxic pulmonary vasoconstriction, memory, and circadian rhythms, and disruption of this regulatory axis may be linked to dysregulation of calcium homeostasis and cardiac arrhythmias.

Pyridine nucleotide metabolites and calcium release from intracellular stores.

Ca(2+) signals are probably the most common intracellular signaling elements, controlling an extensive range of responses in virtually all cells. Many cellular stimuli, often acting at cell surface receptors, evoke Ca(2+) signals by mobilizing Ca(2+) from intracellular stores. Inositol trisphosphate (IP3) was the first messenger shown to link events at the plasma membrane to release of Ca(2+) from the endoplasmic reticulum (ER), through activation of IP3-gated Ca(2+) release channels (IP3 receptors). Subsequently, two additional Ca(2+) mobilizing messengers were discovered, cADPR and NAADP. Both are metabolites of pyridine nucleotides, and may be produced by the same class of enzymes, ADP-ribosyl cyclases, such as CD38. Whilst cADPR mobilizes Ca(2+) from the ER by activation of ryanodine receptors (RyRs), NAADP releases Ca(2+) from acidic stores by a mechanism involving the activation of two pore channels (TPCs).

Roles of cADPR and NAADP in pancreatic cells.

Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are Ca(2+)-mobilizing nucleotides that were discovered in the late 1980s. Two decades of investigations have built up a considerable understanding about these two molecules that are related because both are derived from pyridine nucleotides and known to be generated by CD38/ADP-ribosyl cyclases. cADPR has been shown to target the ryanodine receptors in the endoplasmic reticulum whereas NAADP stimulates the two-pore channels in the endo-lysosomes. Accumulating results indicate that cADPR and NAADP are second messenger molecules mediating Ca(2+) signaling activated by a wide range of agonists. This article reviews what is known about these two molecules, especially regarding their signaling roles in the pancreatic cells.

Proteomic profiling of the rat hippocampus from the kindling and pilocarpine models of epilepsy: potential targets in calcium regulatory network.

Herein proteomic profiling of the rat hippocampus from the kindling and pilocarpine models of epilepsy was performed to achieve new potential targets for treating epileptic seizures. A total of 144 differently expressed proteins in both left and right hippocampi by two-dimensional electrophoresis coupled to matrix-assisted laser desorption-mass spectrometry were identified across the rat models of epilepsy. Based on network analysis, the majority of differentially expressed proteins were associated with Ca2+ homeostasis. Changes in ADP-ribosyl cyclase (ADPRC), lysophosphatidic acid receptor 3 (LPAR3), calreticulin, ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), synaptosomal nerve-associated protein 25 (SNAP 25) and transgelin 3 proteins were probed by Western blot analysis and validated using immunohistochemistry. Inhibition of calcium influx by 8-Bromo-cADP-Ribose (8-Br-cADPR) and 2-Aminoethyl diphenylborinate (2-APB) which act via the ADPRC and LPAR3, respectively, attenuated epileptic seizures. Considering a wide range of molecular events and effective role of calcium homeostasis in epilepsy, polypharmacy with multiple realistic targets should be further explored to reach the most effective treatments.

Surface-enhanced Raman spectroscopy as a tool for detecting Ca2+ mobilizing second messengers in cell extracts.

Understanding of calcium signaling pathways in cells is essential for elucidating the mechanisms of both normal cell function and cancer development. Calcium messengers play the crucial role for intracellular Ca(2+) release. We propose a new approach to detecting the calcium second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) in cell extracts using surface-enhanced Raman spectroscopy (SERS). Currently available radioreceptor binding and enzymatic assays require extensive sample preparation and take more than 12 h. With a SERS sensor, NAADP can be detected in less than 1 min without any special sample preparation. To the best of our knowledge, this is the first demonstration of using SERS for calcium signaling applications.

Generation of specific Ca(2+) signals from Ca(2+) stores and endocytosis by differential coupling to messengers.

It remains unclear how different intracellular stores could interact and be recruited by Ca(2+)-releasing messengers to generate agonist-specific Ca(2+) signatures. In addition, refilling of acidic stores such as lysosomes and secretory granules occurs through endocytosis, but this has never been investigated with regard to specific Ca(2+) signatures. In pancreatic acinar cells, acetylcholine (ACh), cholecystokinin (CCK), and the messengers cyclic ADP-ribose (cADPR), nicotinic acid adenine dinucleotide phosphate (NAADP), and inositol 1,4,5-trisphosphate (IP(3)) evoke repetitive local Ca(2+) spikes in the apical pole. Our work reveals that local Ca(2+) spikes evoked by different agonists all require interaction of acid Ca(2+) stores and the endoplasmic reticulum (ER), but in different proportions. CCK and ACh recruit Ca(2+) from lysosomes and from zymogen granules through different mechanisms; CCK uses NAADP and cADPR, respectively, and ACh uses Ca(2+) and IP(3), respectively. Here, we provide pharmacological evidence demonstrating that endocytosis is crucial for the generation of repetitive local Ca(2+) spikes evoked by the agonists and by NAADP and IP(3). We find that cADPR-evoked repetitive local Ca(2+) spikes are particularly dependent on the ER. We propose that multiple Ca(2+)-releasing messengers determine specific agonist-elicited Ca(2+) signatures by controlling the balance among different acidic Ca(2+) stores, endocytosis, and the ER.

Cyclic ADP-ribose-mediated expansion and stimulation of human mesenchymal stem cells by the plant hormone abscisic acid.

Abscisic acid (ABA) is a phytohormone involved in fundamental processes in higher plants. Endogenous ABA biosynthesis occurs also in lower Metazoa, in which ABA regulates several physiological functions by activating ADP-ribosyl cyclase (ADPRC) and causing overproduction of the Ca(2+)-mobilizing second messenger cyclic ADP-ribose (cADPR), thereby enhancing intracellular Ca(2+) concentration ([Ca(2+)](i)). Recently, production and release of ABA have been demonstrated to take place also in human granulocytes, where ABA behaves as a proinflammatory hormone through the same cADPR/[Ca(2+)](i) signaling pathway described in plants and in lower Metazoa. On the basis of the fact that human mesenchymal stem cells (MSC) express ADPRC activity, we investigated the effects of ABA and of its second messenger, cADPR, on purified human MSC. Both ABA and cADPR stimulate the in vitro expansion of MSC without affecting differentiation. The underlying mechanism involves a signaling cascade triggered by ABA binding to a plasma membrane receptor and consequent cyclic AMP-mediated activation of ADPRC and of the cADPR/[Ca(2+)](i) system. Moreover, ABA stimulates the following functional activities of MSC: cyclooxygenase 2-catalyzed production of prostaglandin E(2) (PGE(2)), release of several cytokines known to mediate the trophic and immunomodulatory properties of MSC, and chemokinesis. Remarkably, ABA proved to be produced and released by MSC stimulated by specific growth factors (e.g., bone morphogenetic protein-7), by inflammatory cytokines, and by lymphocyte-conditioned medium. These data demonstrate that ABA is an autocrine stimulator of MSC function and suggest that it may participate in the paracrine signaling among MSC, inflammatory/immune cells, and hemopoietic progenitors. Disclosure of potential conflicts of interest is found at the end of this article.

Investigating cADPR and NAADP in intact and broken cell preparations.

The body of literature characterizing cyclic adenosine diphosphoribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) as Ca2+-mobilizing second messengers is growing apace. However, their unique properties may, for the uninitiated, make them difficult to work with. This article reviews many of the available techniques (and associated pitfalls) for investigating these nucleotide messengers, predominantly focusing upon optical techniques using fluorescent reporters to measure Ca2+ in the cytosol as well as Ca2+ or pH within the lumen of intracellular organelles.

Localization of the cyclic ADP-ribose-dependent calcium signaling pathway in bovine rod outer segments.

PURPOSE: Calcium ions play a pivotal role in phototransduction. In this study, the presence and functional role of the adenosine diphosphoribosyl (ADPR)-cyclase-cyclic ADP-ribose (cADPR) system in bovine retinal rod outer segments (ROS) was investigated. METHODS: A Ca(2+) release from osmotically intact ROS discs elicited by cADPR was studied in the presence of the Ca(2+) tracer fluo-3. Endogenous cyclic guanosine diphosphate ribose (cGDPR) formation in discs was investigated by spectrophotometric detection of its synthesis from nicotinamide guanine dinucleotide (NGD(+)). ADPR-cyclase was also investigated at a structural level on mildly denaturing SDS-PAGE by production of cyclic inosine diphosphate ribose from nicotinamide hypoxantine dinucleotide (NHD(+)). Western immunoblot analysis with a specific antibody was conducted to verify the presence of ryanodine-sensitive Ca(2+) channels (RyRs) in ROS discs. RESULTS: cADPR-dependent Ca(2+) release was a linear function of extravesicular free Ca(2+) concentration, between 200 and 900 nM Ca(2+). When free Ca(2+) was 203 +/- 10 nM the mean Ca(2+) release was 23 +/- 3 pmol/mL per milligram protein. The average rate of cGDPR production was 13 +/- 2 nmol cGDPR/min per milligram protein, by a putative enzyme with an apparent molecular mass of 53 +/- 1 kDa. ROS ADPR-cyclase was localized in the membranous fraction. No nicotinamide adenine dinucleotide glycohydrolase (NADase) activity was detected. The presence of RyR channels in pure disc preparations was confirmed by confocal laser scanning microscopy. CONCLUSIONS: A cADPR metabolism may be present in retinal ROS discs, which may be Ca(2+) stores operated by cADPR. A model is proposed for the physiological role of cADPR-mediated Ca(2+) release in bovine ROS.

[Evolution of mechanisms of Ca2+ -signaling. Significance of Ca2+-messenger systems during transition of organisms to multicellularity].

The review considers Ca2+ -messenger systems in primitive multicellulars (sponges and hydrozoa organisms). Analysis is performed of Ca2+ participation in regulation of early development of the organisms, their mobility, metamorphosis, chemoreception, and some other functions.

Ca2+ microdomains in smooth muscle.

In smooth muscle, Ca(2+) controls diverse activities including cell division, contraction and cell death. Of particular significance in enabling Ca(2+) to perform these multiple functions is the cell's ability to localize Ca(2+) signals to certain regions by creating high local concentrations of Ca(2+) (microdomains), which differ from the cytoplasmic average. Microdomains arise from Ca(2+) influx across the plasma membrane or release from the sarcoplasmic reticulum (SR) Ca(2+) store. A single Ca(2+) channel can create a microdomain of several micromolar near (approximately 200 nm) the channel. This concentration declines quickly with peak rates of several thousand micromolar per second when influx ends. The high [Ca(2+)] and the rapid rates of decline target Ca(2+) signals to effectors in the microdomain with rapid kinetics and enable the selective activation of cellular processes. Several elements within the cell combine to enable microdomains to develop. These include the brief open time of ion channels, localization of Ca(2+) by buffering, the clustering of ion channels to certain regions of the cell and the presence of membrane barriers, which restrict the free diffusion of Ca(2+). In this review, the generation of microdomains arising from Ca(2+) influx across the plasma membrane and the release of the ion from the SR Ca(2+) store will be discussed and the contribution of mitochondria and the Golgi apparatus as well as endogenous modulators (e.g. cADPR and channel binding proteins) will be considered.

Emerging role of cyclic ADP-ribose (cADPR) in smooth muscle.

Cyclic adenosine diphosphate ribose (cADPR) is a naturally occurring cyclic nucleotide and represents a novel class of endogenous Ca(2+) messengers implicated in the regulation of the gating properties of ryanodine receptors (RyRs). This action of cADPR occurs independently from the inositol-1,4,5-trisphosphate (IP(3)) receptor. The regulation of intracellular Ca(2+) release is a fundamental element of cellular Ca(2+) homeostasis since a number of smooth muscle functions (tone, proliferation, apoptosis, and gene expression) are modulated by intracellular Ca(2+) concentration ([Ca(2+)](i)). There has been a surge in the efforts aimed at understanding the mechanisms of cADPR-mediated Ca(2+) mobilization and its impact on smooth muscle function. This review summarizes the proposed roles of cADPR in the regulation of smooth muscle tone.

Pyridine nucleotides and calcium signalling in arterial smooth muscle: from cell physiology to pharmacology.

It is generally accepted that the mobilisation of intracellular Ca2+ stores plays a pivotal role in the regulation of arterial smooth muscle function, paradoxically during both contraction and relaxation. However, the spatiotemporal pattern of different Ca2+ signals that elicit such responses may also contribute to the regulation of, for example, differential gene expression. These findings, among others, demonstrate the importance of discrete spatiotemporal Ca2+ signalling patterns and the mechanisms that underpin them. Of fundamental importance in this respect is the realisation that different Ca2+ storing organelles may be selected by the discrete or coordinated actions of multiple Ca2+ mobilising messengers. When considering such messengers, it is generally accepted that sarcoplasmic reticulum (SR) stores may be mobilised by the ubiquitous messenger inositol 1,4,5 trisphosphate. However, relatively little attention has been paid to the role of Ca2+ mobilising pyridine nucleotides in arterial smooth muscle, namely, cyclic adenosine diphosphate-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). This review will therefore focus on these novel mechanisms of calcium signalling and their likely therapeutic potential.

[ADP-ribose and cADP-ribose--endogenous regulators of cellular ionic balance. Cardiotropic effects of ADP-ribose].

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.

Two intracellular pathways mediate metabotropic glutamate receptor-induced Ca2+ mobilization in dopamine neurons.

Activation of metabotropic glutamate receptors (mGluRs) causes membrane hyperpolarization in midbrain dopamine neurons. This hyperpolarization results from the opening of Ca(2+)-sensitive K(+) channels, which is mediated by the release of Ca(2+) from intracellular stores. Neurotransmitter-induced mobilization of Ca(2+) is generally ascribed to the action of inositol 1,4,5-triphosphate (IP(3)) in neurons. Here we show that the mGluR-mediated Ca(2+) mobilization in dopamine neurons is caused by two intracellular second messengers: IP(3) and cyclic ADP-ribose (cADPR). Focal activation of mGluRs, attained by synaptic release of glutamate or iontophoretic application of aspartate, induced a wave of Ca(2+) that spread over a distance of approximately 50 microm through dendrites and the soma. Simultaneous inhibition of both IP(3)- and cADPR-dependent pathways with heparin and 8-NH(2)-cADPR was required to block the mGluR-induced Ca(2+) release, indicating a redundancy in the signaling mechanism. Activation of ryanodine receptors was suggested to mediate the cADPR-dependent pathway, because ruthenium red, an antagonist of ryanodine receptors, inhibited the mGluR response only when the cADPR-dependent pathway was isolated by blocking the IP(3)-dependent pathway with heparin. Finally, the mGluR-mediated hyperpolarization was shown to induce a transient pause in the spontaneous firing of dopamine neurons. These results demonstrate that an excitatory neurotransmitter glutamate uses multiple intracellular pathways to exert an inhibitory control on the excitability of dopamine neurons.

Role of the second-messenger cyclic-adenosine 5'-diphosphate-ribose on adrenocorticotropin secretion from pituitary cells.

We examined the role of the second-messenger cyclic-ADP-ribose (cADPR) on the regulation of ACTH secretion using AtT20 corticotroph tumor cell line. We found that the cADPR antagonist, 8-Br-cADPR, substantially diminished the secretion of ACTH induced by CRH and potassium in these cells, whereas xestospongin C, an inositol 1,4,5-triphosphate receptor antagonist, had no effect. In addition, the cADPR agonist, 3-deaza-cADPR, augmented ACTH secretion. The presence of the components of the cADPR system, namely ryanodine receptor, CD38, and cADPR itself, was determined in AtT20 cells. Furthermore, we observed that antagonists of the ryanodine channel and cADPR system can decrease the potassium-induced Ca2+ transients in these cells. These results suggest that cADPR is a second messenger in pituitary cells and regulates ACTH secretion by a mechanism dependent on activation of the ryanodine channel by extracellular Ca2+.

Regulation of intracellular Ca2+ stores by multiple Ca2+-releasing messengers.

Although glucose-elicited insulin secretion depends on Ca(2+) entry through voltage-gated Ca(2+) channels in the surface cell membrane of the pancreatic beta-cell, there is also ample evidence for an important role of intracellular Ca(2+) stores, particularly in relation to hormone- or neurotransmitter-induced insulin secretion. There is now direct evidence for Ca(2+) entry-induced release of Ca(2+) from the endoplasmic reticulum in neurons, but with regard to glucose stimulation of beta-cells, there is conflicting evidence about the operation of such a process. This finding suggests that the sensitivity of the Ca(2+) release channels in the endoplasmic reticulum membrane varies under different conditions and therefore is regulated. Recent evidence from studies of pancreatic acinar cells has revealed combinatorial roles of multiple messengers in setting the sensitivity of the endoplasmic reticulum for Ca(2+) release. Here we focus on the possible combinatorial roles of inositol 1,4,5-trisphosphate, cyclic ADP-ribose, and nicotinic acid adenine dinucleotide phosphate in beta-cell function.

Recent advances in the Okamoto model: the CD38-cyclic ADP-ribose signal system and the regenerating gene protein (Reg)-Reg receptor system in beta-cells.

Twenty years ago, we first proposed our hypothesis on beta-cell damage and its prevention (the Okamoto model), according to which poly(ADP-ribose) synthetase/polymerase (PARP) activation is critically involved in the consumption of NAD(+), leading to energy depletion and cell death by necrosis. Recently, the model was reconfirmed by results using PARP knockout mice and has been recognized as providing the basis for necrotic death of various cells and tissues. Based on the model, we proposed two signal systems in beta-cells: one is the CD38-cyclic ADP-ribose (cADPR) signal system for insulin secretion, and the other is the regenerating gene protein (Reg)-Reg receptor system for beta-cell regeneration. The physiological and pathological significance of the two signal systems in a variety of cells and tissues as well as in pancreatic beta-cells has recently been recognized. Here, we describe the Okamoto model and its descendents, the CD38-cADPR signal system and the Reg-Reg receptor system, focusing on recent advances and how their significance came to light. Because PARP is involved in Reg gene transcription to induce beta-cell regeneration, and the PARP activation reduces the cellular NAD(+) to decrease the formation of cADPR (a second messenger for insulin secretion) and further to cause necrotic beta-cell death, PARP and its inhibitors have key roles in the induction of beta-cell regeneration, the maintenance of insulin secretion, and the prevention of beta-cell death.

Second messenger function and the structure-activity relationship of cyclic adenosine diphosphoribose (cADPR).

Cyclic ADP-ribose (cADPR) is a Ca2+ mobilizing second messenger found in various cell types, tissues and organisms. Receptor-mediated formation of cADPR may proceed via transmembrane shuttling of the substrate NAD and involvement of the ectoenzyme CD38, or via so far unidentified ADP-ribosyl cyclases located within the cytosol or in internal membranes. cADPR activates intracellular Ca2+ release via type 2 and 3 ryanodine receptors. The exact molecular mechanism, however, remains to be elucidated. Possibilities are the direct binding of cADPR to the ryanodine receptor or binding via a separate cADPR binding protein. In addition to Ca2+ release, cADPR also evokes Ca2+ entry. The underlying mechanism(s) may comprise activation of capacitative Ca2+ entry and/or activation of the cation channel TRPM2 in conjunction with adenosine diphosphoribose. The development of novel cADPR analogues revealed new insights into the structure-activity relationship. Substitution of either the northern ribose or both the northern and southern ribose resulted in much simpler molecules, which still retained significant biological activity.