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Nicotinamide adenine dinucleotide (NAD(+)) is the universal currency of energy metabolism and electron transfer. Recent studies indicate that apart from its role as a coenzyme, NAD(+) and its metabolites also function in cell signaling pathways; for example, they are substrates for nucleotide-metabolizing enzymes and ligands for extra- and intracellular receptors and ion channels. Moreover, the NAD(+) and NAD(+) phosphate metabolites adenosine 5'-diphosphoribose (ADP-ribose), cyclic ADP-ribose, and nicotinic acid adenine dinucleotide phosphate (NAADP) have emerged as key second messengers in Ca(2+) signaling. A symposium in Hamburg, Germany, brought together 120 researchers from various fields, who were all engaged in the molecular characterization of the key players of NAD(+) signaling (www.NAD2008.de).

Cells possess multiple calcium ion (Ca2+) stores and multiple messenger molecules to mobilize them. These include d-myo-inositol 1,4,5-trisphosphate (IP(3)), cyclic adenosine diphosphoribose (cADPR), and the most recently identified Ca2+-mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), which acts on a wide spectrum of cells, from plant cells to mammalian cells. Accumulating evidence indicates that NAADP targets both acidic (lysosome-like) Ca2+ stores and endoplasmic reticular stores. Recent studies in invertebrate and mammalian cells suggest that NAADP provides an initiating Ca2+ signal, which is amplified by cADPR- or IP(3)-dependent mechanisms (or both) through Ca2+-induced Ca2+ release. Diverse stimuli activate a rapid rise of endogenous NAADP concentration, resulting in severalfold increases of NAADP over basal values within seconds. The enzyme CD38 can catalyze both the synthesis and hydrolysis of NAADP, making it ideal for effecting the rapid metabolism of NAADP. The crystal structure of CD38 and the structures of its various substrate complexes have now been determined, clarifying the mechanism of its multifunctional catalysis. We anticipate that these advances will lead to the unmasking of all the key components of the Ca2+ signaling pathway mediated by NAADP.

In the present study, effects of increased IP3K-A [Ins(1,4,5) P 3 3-kinase-A] expression were analysed. H1299 cells overexpressing IP3K-A formed branching protrusions, and under three-dimensional culture conditions, they exhibited a motile fibroblast-like morphology. They lost the ability to form actin stress fibres and showed increased invasive migration in vitro . Furthermore, expression levels of the mesenchymal marker proteins vimentin and N-cadherin were increased. The enzymatic function of IP3K-A is to phosphorylate the calcium-mobilizing second messenger Ins(1,4,5) P 3 to (Ins(1,3,4,5) P 4 . Accordingly, cells overexpressing IP3K-A showed reduced calcium release and altered concentrations of Ins P s, with decreasing concentrations of Ins(1,4,5) P 3 , Ins P 6 and Ins(1,2,3,4,5) P 5 , and increasing concentrations of Ins(1,3,4,5) P 4 . However, IP3K-A-induced effects on cell morphology do not seem to be dependent on enzyme activity, since a protein devoid of enzyme activity also induced the formation of branching protrusions. Therefore we propose that the morphological changes induced by IP3K-A are mediated by non-enzymatic activities of the protein.

Cyclic adenosine diphosphoribose (cADPR) is an endogenous Ca 2+ -mobilizing second messenger found in cells of animals, plants, and protozoans. It is formed by a specific class of enzymes, the ADP-ribosyl cyclases. cADPR stimulates Ca 2+ release by means of ryanodine receptors located in the sarcoplasmic and endoplasmic reticulum. Recently, a role for cADPR has been demonstrated in the obligate intracellular protozoan pathogen Toxoplasma gondii . In T. gondii , stress conditions evoked synthesis of the plant hormone abscisic acid by the apicoplast, a remnant organelle of an algal endosymbiont of T. gondii . Abscisic acid in turn activated formation of cADPR within T. gondii , resulting in Ca 2+ release and secretion of proteins involved in egress of T. gondii from its host cell. Evidence for a synthetic pathway of plant origin was obtained with the ABA synthesis inhibitor fluridone, which antagonized cellular egress and induced differentiation of long-lived semidormant cystic forms of T. gondii. Moreover, fluridone protected mice from toxoplasmosis.

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.

The ectoenzyme CD38 catalyzes the production of cyclic ADP-ribose (cADPR) and ADP-ribose (ADPR) from its substrate, NAD+. Both products of the CD38 enzyme reaction play important roles in signal transduction, as cADPR regulates calcium release from intracellular stores and ADPR controls cation entry through the plasma membrane channel TRPM2. We previously demonstrated that CD38 and the cADPR generated by CD38 regulate calcium signaling in leukocytes stimulated with some, but not all, chemokines and controls leukocyte migration to inflammatory sites. However, it is not known whether the other CD38 product, ADPR, also regulates leukocyte trafficking In this study we characterize 8-bromo (8Br)-ADPR, a novel compound that specifically inhibits ADPR-activated cation influx without affecting other key calcium release and entry pathways. Using 8Br-ADPR, we demonstrate that ADPR controls calcium influx and chemotaxis in mouse neutrophils and dendritic cells activated through chemokine receptors that rely on CD38 and cADPR for activity, including mouse FPR1, CXCR4, and CCR7. Furthermore, we show that the calcium and chemotactic responses of leukocytes are not dependent on poly-ADP-ribose polymerase 1 (PARP-1), another potential source of ADPR in some leukocytes. Finally, we demonstrate that NAD+ analogues specifically block calcium influx and migration of chemokine-stimulated neutrophils without affecting PARP-1-dependent calcium responses. Collectively, these data identify ADPR as a new and important second messenger of mouse neutrophil and dendritic cell migration, suggest that CD38, rather than PARP-1, may be an important source of ADPR in these cells, and indicate that inhibitors of ADPR-gated calcium entry, such as 8Br-ADPR, have the potential to be used as anti-inflammatory agents.

TRPM4b is a Ca(2+)-activated, voltage-dependent monovalent cation channel that has been shown to act as a negative regulator of Ca(2+) entry and to be involved in the generation of oscillations of Ca(2+) influx in Jurkat T-lymphocytes. Transient overexpression of TRPM4b as an enhanced green fluorescence fusion protein in human embryonic kidney (HEK) cells resulted in its localization in the plasma membrane, as demonstrated by confocal fluorescence microscopy. The functionality and plasma membrane localization of overexpressed TRPM4b was confirmed by induction of Ca(2+)-dependent inward and outward currents in whole cell patch clamp recordings. HEK-293 cells stably overexpressing TRPM4b showed higher ionomycin-activated Ca(2+) influx than wild-type cells. In addition, analysis of the membrane potential using the potentiometric dye bis-(1,3-dibutylbarbituric acid)-trimethine oxonol and by current clamp experiments in the perforated patch configuration revealed a faster initial depolarization after activation of Ca(2+) entry with ionomycin. Furthermore, TRPM4b expression facilitated repolarization and thereby enhanced sustained Ca(2+) influx. In conclusion, in cells with a small negative membrane potential, such as HEK-293 cells, TRPM4b acts as a positive regulator of Ca(2+) entry.

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

Stimulation of Jurkat T cells by high concentrations of concanavalin A (ConA) induced an elevation of the endogenous adenosine diphosphoribose (ADPR) concentration and an inward current significantly different from the Ca2+ release-activated Ca2+ current (I(CRAC)). Electrophysiological characterization and activation of a similar current by infusion of ADPR indicated that the ConA-induced current is carried by TRPM2. Expression of TRPM2 in the plasma membrane of Jurkat T cells was demonstrated by reverse transcription-PCR, Western blot, and immunofluorescence. Inhibition of ADPR formation reduced ConA-mediated, but not store-operated, Ca2+ entry and prevented ConA-induced cell death of Jurkat cells. Moreover, gene silencing of TRPM2 abolished the ADPR- and ConA-mediated inward current. Thus, ADPR is a novel second messenger significantly involved in ConA-mediated cell death in T cells.

We previously developed cyclic ADP-carbocyclic ribose (cADPcR, 2) as a stable mimic of cyclic ADP-ribose (cADPR, 1), a Ca(2+)-mobilizing second messenger. A series of the N1-ribose modified cADPcR analogues, designed as novel stable mimics of cADPR, which were the 2"-deoxy analogue 3, the 3"-deoxy analogue 4, the 3"-deoxy-2"-O-(methoxymethyl) analogue 5, the 3"-O-methyl analogue 6, the 2",3"-dideoxy analogue 7, and the 2",3"-dideoxydidehydro analogue 8, were successfully synthesized using the key intramolecular condensation reaction with phenylthiophosphate-type substrates. We investigated the conformations of these analogues and of cADPR and found that steric repulsion between both the adenine and N9-ribose moieties and between the adenine and N1-ribose moieties was a determinant of the conformation. The Ca(2+)-mobilizing effects were evaluated systematically using three different biological systems, i.e., sea urchin eggs, NG108-15 neuronal cells, and Jurkat T-lymphocytes. The relative potency of Ca(2+)-mobilization by these cADPR analogues varies depending on the cell-type used: e.g., 3"-deoxy-cADPcR (4)cADPcR (2)cADPR (1) in sea urchin eggs; cADPR (1)cADPcR (2) approximately 3"-deoxy-cADPcR (4) in T-cells; and cADPcR (2)cADPR (1)3"-deoxy-cADPcR (4) in neuronal cells, respectively. These indicated that the target proteins and/or the mechanism of action of cADPR in sea urchin eggs, T-cells, and neuronal cells are different. Thus, this study represents an entry to cell-type selective cADPR analogues, which can be used as biological tools and/or novel drug leads.

Cyclic ADP-ribose (cADPR) is an endogenous Ca(2+)-mobilizing second messenger in many cell types and organisms. Although the biological activity of several modified analogues of cADPR has been analyzed, most of these structures were still very similar to the original molecule. Recently, we have introduced simplified analogues in which the northern ribose (N(1)-linked ribose) was replaced by an ether strand. Here we also demonstrate that the southern ribose (N(9)-linked ribose) can be replaced by an ether strand resulting in N(1)-[(phosphoryl-O-ethoxy)-methyl]-N(9)-[(phosphoryl-O-ethoxy)-methyl]-hypoxanthinecyclic pyrophosphate (cIDP-DE). This minimal structural analogue of cyclic ADP-ribose released Ca(2+) from intracellular stores of permeabilized Jurkat T lymphocytes. In intact T lymphocytes initial subcellular Ca(2+) release events, global Ca(2+) release, and subsequent global Ca(2+) entry were observed. Cardiac myocytes freshly prepared from mice responded to cIDP-DE by increased recruitment of localized Ca(2+) signals and by global Ca(2+) waves.

N1-[(5' '-O-Phosphorylethoxy)methyl]-5'-O-phosphorylinosine 5',5''-cyclicpyrophosphate (cIDPRE 2a) and the 8-substituted derivatives 8-bromo-, 8-azido-, 8-amino-, and 8-Cl-cIDPRE (2b-e) were synthesized from N1-[(5''-acetoxyethoxy)methyl]-2',3'-O-isopropylideneinosine (5) in good yields. The pharmacological activities of cIDPRE and the 8-substituted derivatives (2a-e) were analyzed in intact and permeabilized human Jurkat T-lymphocytes. The results indicate that cIDPRE permeates the plasma membrane, releases Ca2+ from an intracellular, cADPR-sensitive Ca2+ store, and subsequently initiates Ca2+ release-activated Ca2+ entry. The Ca(2+)-releasing activity of cIDPRE was confirmed directly in permeabilized cells. Using time-resolved confocal Ca2+ imaging at the single cell level, the development of global Ca2+ signals starting from local small Ca2+ signals evoked by cIDPRE was observed. 8-N3-cIDPRE 2c and 8-NH2-cIDPRE 2d were similarly effective in their agonistic activity as compared to cIDPRE 2a, showing almost indistinguishable concentration-response curves for 2a, 2c, and 2d and very similar kinetics of Ca2+ signaling. In contrast, the halogenated derivatives 8-Br- and 8-Cl-cIDPRE (2b and 2e) did not significantly elevate [Ca2+]i. Therefore, cIDPRE 2a, 8-N3-cIDPRE 2c, and 8-NH2-cIDPRE 2d are novel membrane permeant cADPR mimic and may provide important novel tools to study cADPR-mediated Ca2+ signaling in intact cells.

Ca2+ ions are involved in the regulation of many diverse functions in animal and plant cells, e.g. muscle contraction, secretion of neurotransmitters, hormones and enzymes, fertilization of oocytes, and lymphocyte activation and proliferation. The intracellular Ca2+ concentration can be increased by different molecular mechanisms, such as Ca2+ influx from the extracellular space or Ca2+ release from intracellular Ca2+ stores. Release from intracellular Ca2+ stores is accomplished by the small molecular compounds D-myo-inositol 1,4,5-trisphosphate (InsP3), cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). This review will focus on the effects of cADPR in different cells and tissues, the mechanisms of cADPR-mediated Ca2+ release and Ca2+ entry, extracellular effects of cADPR, and the role of cADPR in a cell system studied in detail, human T-lymphocytes.

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.

Ligation of the T-cell receptor/CD3 complex results in global Ca 2+ signals that are essential for T-cell activation. We have recently reported that these global Ca 2+ signals are preceded by localized pacemaker Ca 2+ signals. Here, we demonstrate for the first time for human T cells that an increase in signal frequency of subcellular pacemaker Ca 2+ signals at sites close to the plasma membrane, in the cytosol and in the nucleus depends on the type 3 ryanodine receptor (RyR) and its modulation by cyclic ADP-ribose. The spatial distribution of D- myo -inositol 1,4,5-trisphosphate receptors and RyRs indicates a concerted action of both of these receptors/Ca 2+ channels in the generation of initial pacemaker signals localized close to the plasma membrane. Inhibition or knockdown of RyRs resulted in significant decreases in (1) the frequency of initial pacemaker signals localized close to the plasma membrane, and (2) the frequency of localized pacemaker Ca 2+ signals in the inner cytosol. Moreover, upon microinjection of cyclic ADP-ribose or upon extracellular addition of its novel membrane-permeant mimic N -1 - ethoxymethyl-substituted cyclic inosine diphosphoribose, similarly decreased Ca 2+ signals were observed in both type 3 RyR-knockdown cells and in control cells microinjected with the RyR antagonist Ruthenium Red. Taken together, our results show that, under physiological conditions in human T cells, RyRs play crucial roles in the local amplification and the spatiotemporal development of subcellular Ca 2+ pacemaker signals.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+ mobilizing nucleotide essentially involved in T cell activation. Using combined microinjection and single cell calcium imaging, we demonstrate that co-injection of NAADP and the D-myo-inositol 1,4,5-trisphosphate antagonist heparin did not inhibit Ca2+ mobilization. In contrast, co-injection of the ryanodine receptor antagonist ruthenium red efficiently blocked NAADP induced Ca2+ signalling. This pharmacological approach was confirmed using T cell clones stably transfected with plasmids expressing antisense mRNA targeted specifically against ryanodine receptors. NAADP induced Ca2+ signaling was strongly reduced in these clones. In addition, inhibition of Ca2+ entry by SK&F 96365 resulted in a dramatically decreased Ca2+ signal upon NAADP injection. Gd3+, a known blocker of Ca2+ release activated Ca2+ entry, only partially inhibited NAADP mediated Ca2+ signaling. These data indicate that in T cells (i) ryanodine receptor are the major intracellular Ca2+ release channels involved in NAADP induced Ca2+ signals, and that (ii) such Ca2+ release events are largely amplified by Ca2+ entry.

The cellular prion protein (PrPc) resides in lipid rafts, yet the type of raft and the physiological function of PrPc are unclear. We show here that cross-linking of PrPc with specific antibodies leads to 1) PrPc capping in Jurkat and human peripheral blood T cells; 2) to cocapping with the intracellular lipid raft proteins reggie-1 and reggie-2; 3) to signal transduction as seen by MAP kinase phosphorylation and an elevation of the intracellular Ca2+ concentration; 4) to the recruitment of Thy-1, TCR/CD3, fyn, lck and LAT into the cap along with local tyrosine phosphorylation and F-actin polymerization, and later, internalization of PrPc together with the reggies into limp-2 positive lysosomes. Thus, PrPc association with reggie rafts triggers distinct transmembrane signal transduction events in T cells that promote the focal concentration of PrPc itself by guiding activated PrPc into preformed reggie caps and then to the recruitment of important interacting signaling molecules.

Cyclic ADP-ribose, a metabolite of NAD+ evokes Ca2+ release from intracellular stores in different cells. We have determined the activity of cADPr-producing enzymes (ADP-ribosyl cyclases) in different cellular fractions prepared from isolated pancreatic acinar cells by measuring the conversion of the beta-NAD+ analogs 1,N6-etheno-NAD and nicotinamide guanine dinucleotide to the fluorescent products 1,N6-etheno-cADPr and cyclic GDP-ribose, respectively. Substrate/product analyses were carried out by reverse-phase high pressure liquid chromatography. In all subcellular fractions examined (cytosol, mitochondria, plasma, and intracellular membranes), ADP-ribosyl cyclase activity was detected except in zymogen granular membranes. Western blot analysis and immunoprecipitation experiments revealed the presence of the ADP-ribosyl cyclase CD38 in both plasma membranes and mitochondria but not in the cytosol. Hormonal stimulation of intact acinar cells for 1 min with acetylcholine (ACh), cholecystokinin (CCK), or a membrane-permeant analog of cGMP increased ADP-ribosyl cyclase activity in the cytosol by 1.8-, 1.6-, and 1.9-fold, respectively, as compared with the control but had no effect in any other fraction. Both ACh and CCK also increased accumulation of cGMP in the cells by about 2-fold. Bombesin had no significant effect on either ADP-ribosyl cyclase activity or cGMP accumulation within this short period of stimulation. We conclude that at least two types of ADP-ribosyl cyclases are present in pancreatic acinar cells: membrane-bound CD38 and a cytosolic enzyme different from CD38. Stimulation of pancreatic acinar cells with CCK or ACh results in exclusive activation of the cytosolic ADP-ribosyl cyclase activity, most likely mediated by cGMP.

Ca2+ signaling in T-lymphocytes involves Ca2+ release from intracellular stores as well as Ca2+entry from the extracellular space [reviewed in 1–3]. Ten years ago many researchers believed that Ca2+ release in T cells is exclusively operated by D-myo-inositol 1,4,5-trisphosphate (InsP3; reviewed in ref. 4) while the capacitative mechanism of Ca2+ entry proposed by Putney [5] was thought to play a central role in the influx process.

Cyclic ADP-ribose (cADPR), a natural metabolite of beta-NAD(+), is a second messenger for Ca(2+) signaling in T cells. As a tool for purification and identification of ADP-ribosyl cyclase(s) in T cells, a sensitive and specific enzymatic assay using 1,N(6)-etheno-NAD(+) as substrate was developed. A major problem-the sensitivity of 1,N(6)-etheno-cADPR toward the extraction medium perchloric acid-was solved by replacing the perchloric acid extraction procedure of nucleotides by a filtration step. Standard compounds for the HPLC analysis of ADP-ribosyl cyclases and NAD(+)-glycohydrolases, e.g., 1,N(6)-etheno-cADPR, 1,N(6)-etheno-ADPR, and 1,N(6)-etheno-AMP, were produced by ADP-ribosyl cyclase from Aplysia californica and dinucleotide pyrophosphatase. The assay was applied to subcellular fractions prepared from human Jurkat T cells. As a result ADP-ribosyl cyclase and NAD(+)-glycohydrolase activity could be detected and precisely quantified in different subcellular fractions indicating the presence of different isoenzymes in T cells.

Contact of Jurkat T-lymphocytes with the extracellular matrix (ECM) protein laminin resulted in long-lasting alpha6beta1-integrin-mediated Ca(2+) signalling. Both Ca(2+) release from thapsigargin-sensitive Ca(2+) stores and capacitative Ca(2+) entry via Ca(2+) channels sensitive to SKF 96365 constitute important parts of this process. Inhibition of alpha6beta1-integrin-mediated Ca(2+) signalling by (1) the src kinase inhibitor PP2, (2) the PLC inhibitor U73122, and (3) the cyclic adenosine diphosphoribose (cADPR) antagonist 7-deaza-8-Br-cADPR indicate the involvement of src tyrosine kinases and the Ca(2+)-releasing second messengers D-myo-inositol 1,4,5-trisphosphate (InsP3) and cADPR.

The ryanodine receptor of Jurkat T lymphocytes was phosphorylated on tyrosine residues upon stimulation of the cells via the T cell receptor/CD3 complex. The tyrosine phosphorylation was transient, reaching a maximum at 2 min, and rapidly declined thereafter. In co-immunoprecipitates of the ryanodine receptor, the tyrosine kinases p56(lck) and p59(fyn) were detected. However, only p59(fyn) associated with the ryanodine receptor in a stimulation-dependent fashion. Both tyrosine kinases, recombinantly expressed as glutathione S-transferase (GST) fusion proteins, phosphorylated the immunoprecipitated ryanodine receptor in vitro. In permeabilized Jurkat T cells, GST-p59(fyn), but not GST-p56(lck), GST-Grb2, or GST alone, significantly and concentration-dependently enhanced Ca(2+) release by cyclic ADP-ribose. The tyrosine kinase inhibitor PP2 specifically blocked the effect of GST-p59(fyn). This indicates that intracellular Ca(2+) release via ryanodine receptors may be modulated by tyrosine phosphorylation during T cell activation.

The elevation of the cytosolic and nuclear Ca 2+ concentration is a fundamental signal transduction mechanism in almost all eukaryotic cells. Interestingly, three Ca 2+ -mobilising second messengers, d - myo -inositol 1,4,5-trisphosphate (InsP 3 ), cyclic adenosine diphosphoribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP + ) were identified in a phylogenetically wide range of different organisms. Moreover, in an as yet very limited number of cell types, sea urchin eggs, mouse pancreatic acinar cells, and human Jurkat T-lymphocytes, all three Ca 2+ -mobilising ligands have been shown to be involved in the generation of Ca 2+ signals. This situation raises the question why during evolution all three messengers have been conserved in the same cell type. From a theoretical point of view the following points may be considered: (i) redundant mechanisms ensuring intact Ca 2+ signalling even if one system does not work, (ii) the need for subcellularly localised Ca 2+ elevations to obtain a certain physiological response of the cell, and (iii) tight control of a physiological response of the cell by a temporal sequence of Ca 2+ signalling events. These theoretical considerations are compared to the current knowledge regarding the three messengers in sea urchin eggs, mouse pancreatic acinar cells, and human Jurkat T lymphocytes.

Cyclic ADP-ribose (cADPR) is a natural compound that mobilizes calcium ions in several eukaryotic cells1,2,3. Although it can lead to the release of calcium ions in T lymphocytes4,5,6,7, it has not been firmly established as a second messenger in these cells. Here, using high-performance liquid chromatography analysis8, we show that stimulation of the T-cell receptor/CD3 (TCR/CD3) complex results in activation of a soluble ADP-ribosyl cyclase and a sustained increase in intracellular levels of cADPR. There is a causal relation between increased cADPR concentrations, sustained calcium signalling and activation of T cells, as shown by inhibition of TCR/CD3-stimulated calcium signalling, cell proliferation and expression of the early- and late-activation markers CD25 and HLA-DR by using cADPR antagonists9. The molecular target for cADPR, the type-3 ryanodine receptor/calcium channel, is expressed in T cells. Increased cADPR significantly and specifically stimulates the apparent association of [3H]ryanodine with the type-3 ryanodine receptor, indicating a direct modulatory effect of cADPR on channel opening. Thus we show the presence, causal relation and biological significance of the major constituents of the cADPR/calcium-signalling pathway in human T cells.

Ca2+ signaling in response to antigenic stimulation is essential for proliferation of T cells and therefore is one of the important early events in T-lymphocyte signal transduction. Several aspects of T cell receptor/CD3 complex stimulated Ca2+ signaling are reviewed: generation, metabolism, function, and intracellular targets of the Ca(2+)-mobilizing second messengers inositol 1,4,5-trisphosphate and cyclic ADP-ribose, the mechanism of Ca2+ entry, and the generation of Ca2+ oscillations on the single cell level. In addition, Ca2+ signaling induced by further stimuli is discussed, including other T-lymphocyte surface receptors (e.g., CD4 or beta 1-integrins, lipids, and physical stimuli).

Inositol 1,2,3,5,6-pentakisphosphate (Ins(1,2,3,5,6)P5) 5-phosphatase present in fetal calf thymus has been partially purified. This enzyme was inhibited dose-dependently by different thiol modifiers likeN-ethylmaleimide (NEM),p-chloromercuribenzene sulfonate (PCMBS), diamide, and phenylarsine oxide (PAO). The inhibition by PCMBS and diamide was protected by preincubation with dithiothreitol (DTT) and the phosphatase substrate, Ins(1,2,3,5,6)P5. Diamide, a compound that specifically modifies vicinal thiol groups, also blocked the 5-phosphatase dose-dependently. Specificity of this blockade was proven by using dimercaptopropanol (DMP), a compound known to protect vicinal thiol groups. DMP prevented the enzyme from inhibition by diamide. These data suggest that vicinal thiols are involved in Ins(1,2,3,5,6)P55-phosphatase activity.

1-(5-Phospho-beta-D-ribosyl)2'-phosphoadenosine 5'-phosphate cyclic anhydride [2'-phospho-cyclic ADP-ribose, cAdo(2')P(5')PP-Rib] was prepared enzymatically from NADP+ using ADP-ribosyl-cyclase from Aplysia californica. The product was purified by HPLC and characterized by NMR and mass spectroscopy, by conversion to 1-(5-phospho-beta-D-ribosyl)adenosine 5'-phosphate cyclic anhydride (cADP-Rib) by alkaline phosphatase and by resistance to snake venom phosphodiesterase. cAdo-(2')P(5')PP-Rib dose-dependently released Ca2+ from an intracellular, non-endoplasmic reticular Ca2+ pool of permeabilized Jurkat and HPB. ALL T-lymphocytes. In contrast, the closely related compounds 1-(5-phospho-beta-D-ribosyl)3'phosphoadenosine 5'-phosphate cyclic anhydride and 1-(5-phospho-beta-D-ribosyl)cyclic 2',3'-phosphoadenosine 5'-phosphate cyclic anhydride did not induce Ca2+-release from permeabilized T cells. The Ca2+ pool sensitive to cAdo(2')P(5')PP-Rib partially overlapped with the Ca2+ pool sensitive to cADP-Rib recently described in T cells [Guse, A. H., da Silva, C. P., Emmrich, F., Ashamu, G. A., Potter, B. V. L.Mayr, G. W. (1995) Characterization of cyclic adenosine diphosphate-ribose-induced Ca2+-release in T-lymphocyte cell lines, J. Immunol. 155, 3353-3359]. Control experiments suggest that the results were neither due to Ca2+ contaminations in the cADP-Rib preparation nor to catabolism of cAdo(2')P(5')PP-Rib to cADP-Rib.

Cyclic ADP-ribose (cADPr) is a potent Ca2+-mobilizing natural compound (Lee, H. C., Walseth, T. F., Bratt, G. T., Hayes, R. N., and Clapper, D. L. (1989) J. Biol. Chem. 264, 1608-1615) which has been shown to release Ca2+ from an intracellular store of permeabilized T-lymphocytes (Guse, A. H., Silva, C. P., Emmrich, F., Ashamu, G., Potter, B. V. L., and Mayr, G. W. (1995) J. Immunol. 155, 3353-3359). Microinjection of cADPr into intact single T lymphocytes dose dependently induced repetitive but irregular Ca2+ spikes which were almost completely dependent on the presence of extracellular Ca2+. The Ca2+ spikes induced by cADPr could be blocked either by co-injection of cADPr with the specific antagonist 8-NH2-cADPr, by omission of Ca2+ from the medium, or by superfusion of the cells with Zn2+ or SK-F 96365. Ratiometric digital Ca2+ imaging revealed that single Ca2+ spikes were initiated at several sites ("hot spots") close to the plasma membrane. These hot spots then rapidly formed a circular zone of high Ca2+ concentration below the plasma membrane which subsequently propagated like a closing optical diaphragm into the center of the cell. Taken together these data indicate a role for cADPr in Ca2+ entry in T-lymphocytes.

Intracellular Ca2+-signals belong to the major events transducing extracellular signals into living cells. The discovery of (i) a caffeine-sensitive intracellular Ca2+-pool in Jurkat T-lymphocytes [1] and (ii) cyclic adenosine diphosphoribose (cADPR) as an agent that mobilizes Ca2+ from a caffeine- and ryanodine sensitive Ca2+-store in sea urchin egg homogenates [2] prompted us to investigate the potential role of this compound in T-lymphocyte Ca2+-signalling. cADPR, as well as its 2-phosphorylated derivative, 2′-phospho-cADPR (2-P-cADPR), released Ca2+ in a dose-dependent, specific manner from intracellular, non-endoplasmic reticular stores of permeabilized Jurkat and HPB.ALL T cells. In addition, attempts were made to prove the presence of endogenous cADPR and 2-P-cADPR by HPLC. Several HPLC protocols, including microbore-HPLC were tested resulting in the detection of endogenous cADPR by sequential separation on strong-anion exchange HPLC and reverse-phase ion-pair HPLC.

As a ubiquitous second messenger, the Ca2+ mobilizing activity of cyclic ADP-ribose (cADPR) has been observed in many different cell types. The measurement of Ca2+ release evoked by cADPR comprises several practical challenges. At physiological pH, cADPR has a net negative charge and it therefore cannot cross the cell membrane in cells that lack a suitable cADPR-transporting system. Thus, either the plasma membrane must be permeabilized or microinjection must be used to deliver cADPR to the cytosol. In this article, two methods for cADPR delivery (using permeabilized cells or microinjection) are explained step-by-step. Because most of our work has been performed using the Jurkat T-lymphoma cell line, the methods are tailored for this specific cell type. For other cell types, the procedures may need to be adapted.

cADP-ribose (cADPr) has recently been shown to release Ca2+ from an intracellular store of permeabilized T lymphocyte cell lines (Guse, A. H., da Silva, C. P., Emmrich, F., Ashamu, G. A., Potter, B. V. L., and Mayr, G. W. (1995) J. Immunol. 155, 3353-3359). Using permeabilized Jurkat and HPB. ALL T lymphocytes, the effects of varying concentrations of inorganic phosphate and Mg2+ on cADPr-induced Ca2+ release were investigated. cADPr-induced Ca2+ release was dependent on the concentration of inorganic phosphate, showing very low Ca2+ release activity between 0.5 and 2 mM inorganic phosphate. At 4 to 5 mM inorganic phosphate, the cADPr-induced Ca2+ release was much more pronounced, reaching maximal values at 10 mM inorganic phosphate. The underlying mechanism for this stimulatory effect was an increased loading of the cADPr-sensitive Ca2+ store, which was demonstrated by enhanced resequestration of Ca2+ selectively into the cADPr-sensitive Ca2+ store. The free Mg2+ concentration also influenced cADPr-induced Ca2+ release in permeabilized cells: at 0 and 8.58 mM the release was nearly completely abolished, whereas at 1.06 mM maximal Ca2+ release by cADPr was observed. High performance liquid chromatographic analysis of exogenously added cADPr revealed that the catabolism of cADPr at varying Mg2+ and Pi concentrations had only minor relevance for the modulatory effects observed. To correlate the effects of inorganic phosphate and Mg2+ on cADPr-induced Ca2+ release observed in the permeabilized cell preparations, measurements of these ions in intact Jurkat T lymphocytes were carried out. Intact Jurkat T cells stimulated via the T cell receptor middle dotCD3 complex did not respond with significant elevation of the free intracellular Mg2+ concentration. In contrast, stimulation via the T cell receptor middle dotCD3 complex resulted in an increase in the intracellular inorganic phosphate concentration. These data indicate a role for the intracellular inorganic phosphate concentration in the regulation of cADPr-mediated Ca2+ release in T lymphocytes.

A microbore high-performance liquid chromatographic (HPLC) method is presented allowing rapid and sensitive mass analysis of inositol phosphates from cells and tissues. An analysis starting from inorganic phosphate up to inositol hexakisphosphate displaying a similar isomer selectivity as compared to the standard metal-dye detection system takes about 15 min. The detection sensitivity was about 15 pmol for inositol trisphosphate, about 10 pmol for inositol tetrakisphosphate, about 5 pmol for inositol pentakisphosphate and less than 5 pmol for inositol hexakisphosphate. The method was validated regarding day-to-day variations and variations at the same day of retention times and peak areas of standard inositol phosphates. Standard deviations of retention times ranged from 0.25 to 0.62% (same day) and from 0.64 to 1.61% (day-to-day variations). Ranges of standard deviations of peak areas were between 2.24% and 3.91% (same day) and 6.13% and 13.8% (day-to-day variations). Linearity of the post-column complexometric metal-dye detection system was demonstrated in the range of a few picomoles and at least 800 pmol. The method was applied to the analysis of inositol phosphates in Jurkat T-lymphocytes and assays from minute amounts of enzymes interconverting inositol phosphates. While measurements of inositol phosphates from cell extracts are now possible using significantly reduced cell numbers, micro-enzyme assays are feasible in reasonable repeated analysis times and with sufficient isomer selectivity. In conclusion, a substantial improvement towards speed of analysis and detection sensitivity of inositol phosphate mass analysis was achieved by microbore metal-dye detection HPLC.

Murine class III receptors for IgG (mFc gamma RIII) are composed of an IgG-binding alpha chain associated with a gamma subunit dimer. These receptors have been shown to trigger the release of serotonin and tumor necrosis factor-alpha [Daëron, M., Latour, S., Hückel, C., Bonnerot, C.Fridman, W. H. (1992) Immunobiology 185, 159-174], and are involved in endocytosis and phagocytosis [Daëron, M., Malbec, O., Bonnerot, C., Latour, S., Segal, D. M.Fridman, W. H. (1994) J. Immunol. 152, 783-792]. Using a transfection model where the cDNA encoding mFc gamma RIII was stably transfected into the rat basophilic leukemia cell line RBL-2H3, we found that the functional efficiency of mFc gamma RIII is correlated with its ability to increase the intracellular Ca2+ concentration and to stimulate inositol phosphate metabolism. The deletion of intracellular sequences of the alpha subunit did not alter the ability of mFc gamma RIII to trigger the Ca2+ and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] response. After substitution of the intracellular domain of mFc gamma RIII for that of mFc gamma RIII gamma, but not that of mFc gamma RIII alpha, the chimeric receptor was also able to trigger Ca2+ and PtdIns(4.5)P2 responses. In contrast, all transfected receptors induced protein kinase C translocation. Furthermore, dimerization of the receptor was sufficient for the initiation of this protein kinase C translocation while a further crosslinking was necessary for the induction of the Ca2+ and PtdIns(4,5)P2 responses. Protein kinase C translocation therefore can be dissociated from Ca2+ mobilization, PtdIns(4,5)P2 turnover and mast cell secretory responses induced by murine Fc gamma RIII.

A concise method for the formation of cyclopyrophosphate of cIDPRE as well as sulfur and selenium-substituted pyrophosphate cIDPRE analogues (P(1)(S)-cIDPRE, P(1)(Se)-cIDPRE, P(2)(S)-cIDPRE and P(2)(Se)-cIDPRE) was reported and one of the P(S)-diastereoisomers, P(1)(S)-cIDPRE-1, is a novel membrane-permeant cADPR antagonist.

Clinical improvement after treatment with anti-CD4 antibodies has been documented in patients suffering from rheumatoid arthritis. This observation has stimulated the interest in effects induced by the in vivo application of anti-CD4 antibodies. Here, we have investigated features of CD4 modulation during and after anti-CD4 therapy with the monoclonal anti-body MAX.16H5. Depletion of circulating helper T cells was accompanied by modulation of the CD4 molecule down to 30% of the initial antigen density 1 hr after antibody infusion. However, despite the reappearance of CD4 + cells in the circulation CD4 remained down-modulated for up to 28 days without a significant residual anti-CD4 binding. Depletion of CD4 + cells as well as CD4 modulation were observed to a similar extent both in responders and non-responders to anti-CD4 therapy. Modulation of CD4 was more effective in vivo than in vitro with a mean reduction of CD4 density down to 46% in vitro . It was induced in varying degrees by all anti-CD4 antibodies investigated except for OKT4 and required viable monocytes in the case of MAX.16H5 and most of the anti-CD4 antibodies investigated. Supernatants from LPS-activated monocytes or the addition of monocytes that were freeze-fractioned or fixed monocytes did not substitute for this requirement. The effect was Fc-receptor dependent since F(ab) 2 fragments of MAX.16H5 did not induce CD4 modulation. No significant co-modulation was found for a variety of T-cell surface antigens including CD2, CD3, CD8, CD45R, CD45RO, CD25, CDw29, and HLA-DR. In order to test functional effects, the influence of CD4 modulation on the increase of free cytosuite Ca 2+ concentration ([Ca 2+ ] 1 ) stimulated via the T-cell receptor complex by an anti-CD3 antibody was studied. A significant inhibition was observed upon direct binding of anti-CD4 to its ligand. However, a diminished CD4 density alone as induced by in vivo modulation did not reduce, but rather enhanced the T cell receptor-mediated mobilization of [Ca 2+ ]1 in T cells of the patients. Taken together, no evidence was found that CD4 modulation per se could explain the beneficial effects of anti-CD4 therapy.

A purine nucleobase-simplified cyclic ADP ribose (cADPR) analogue 6b was synthesized, in which a 1,2,3-triazole-4-amide was constructed, instead of a purine moiety, and the northern ribose was replaced by an ether strand. Compound 6b exhibits calcium release activity in intact T-lymphocytes and indicates that it is a membrane-permeable cADPR mimic. Thus, the cADPR analogue containing 1,2,3-triazole-4-amide provides a novel template for further designing cADPR analogues and elucidating their structure–activity relationships.

Exclusive stimulation of the alpha 1-adrenoceptor in cultured cardiac myocytes led to a time-dependent increase of multiple inositol phosphate isomers. However, under physiological conditions, alpha 1-adrenergic stimulation is accompanied by beta-adrenergic stimulation as noradrenaline is a ligand for both receptors. Thus, the aim of our study was to investigate the cross-talk between the alpha 1- and the beta-adrenoceptor on the post-receptor level. When both the alpha 1- and the beta-adrenoceptor were stimulated with noradrenaline for 30 s, a diminished increase in all inositol phosphates and a concomitant increase in cAMP was observed. Similar results were obtained by pre-incubation of the myocytes with forskolin for 2 min and subsequent stimulation of the alpha 1-adrenoceptor for 30 s. In contrast, after 15 min the inhibitory effect of simultaneous beta-stimulation as well as of forskolin pre-treatment upon the alpha 1-mediated inositol phosphate response was abolished, although the concentration of cAMP was still elevated. These data suggest that beta-adrenoceptor- and forskolin-induced cAMP production or a cAMP-activated subsequent signal have a short-term, transient, inhibitory effect on alpha 1-adrenoceptor-mediated inositol phosphate response.

Microinjection of inositol 1,3,4,5-tetrakisphosphate or inositol 1,4,5-trisphosphate induced distinct chloride membrane currents in defolliculated Xenopus laevis oocytes. To decide whether these Cl − -currents were due to the injected compounds or their metabolic products, [ 3 H]Ins(1,3,4,5)P 4 or [ 3 H]Ins(1,4,5)P 3 were injected into oocytes and their metabolites were analyzed by HPLC. Our results indicate that Ins(1,3,4,5)P 4 itself or its metabolite Ins(1,3,4,6)P 4 is able to induce Cl − -membrane currents, most likely by increasing the cytosolic Ca ++ -concentration.