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.

The ΔC splice-variant of TRPM2 is the hypertonicity-induced cation channel in HeLa cells, and the ecto-enzyme CD38 mediates its activation.

Hypertonicity-induced cation channels (HICCs) are key-players in proliferation and apoptosis but their molecular correlate remains obscure. Furthermore, the activation profile of HICCs is not well defined yet. We report here that, in HeLa cells, intracellular adenosine diphosphate ribose (ADPr) and cyclic ADPr (cADPr), as supposed activators of TRPM2, elicited cation currents that were virtually identical to the osmotic activation of HICCs. Silencing of the expression of TRPM2 and of the ecto-enzyme CD38 (as a likely source of ADPr and cADPr) inhibited HICC as well as nucleotide-induced currents and, in parallel, the hypertonic volume response of cells (the regulatory volume increase, RVI) was attenuated. Quantification of intracellular cADPr levels and the systematic application of extra- vs. intracellular nucleotides indicate that the outwardly directed gradient rather than the cellular activity of ADPr and cADPr triggers TRPM2 activation, probably along with a simultaneous biotransformation of nucleotides.Cloning of TRPM2 identified the ΔC-splice variant as the molecular correlate of the HICC, which could be strongly supported by a direct comparison of the respective Ca²âº selectivity. Finally, immunoprecipitation and high-resolution FRET/FLIM imaging revealed the interaction of TRPM2 and CD38 in the native as well as in a heterologous (HEK293T) expression system. We propose transport-related nucleotide export via CD38 as a novel mechanism of TRPM2/HICC activation. With the biotransformation of nucleotides running in parallel, continuous zero trans-conditions are achieved which will render the system infinitely sensitive.

Microinjection of recombinant p21rho induces rapid changes in cell morphology.

The rho proteins, p21rho, are ubiquitously expressed guanine nucleotide binding proteins with approximately 30% amino acid homology to p21ras, but their biochemical function is unknown. We show here that microinjection of constitutively activated recombinant rho protein (Val14rho) into subconfluent cells induces dramatic changes in cell morphology: 15-30 min after injection cells adopt a distinct and novel phenotype with a contracted cell body and finger-like processes still adherent to the substratum. Ribosylation of Val14rho with the ADP-ribosyltransferase C3 from clostridium botulinum, before microinjection, renders the protein biologically inactive, but it has no effect on either its intrinsic biochemical properties or on its interaction with the GTPase activating protein, rho GAP. Micro-injection of ribosylated normal rho, on the other hand, has a similar effect of injection of C3 transferase and induces complete rounding up of cells. We also report striking biochemical changes in actin filament organization when contact-inhibited quiescent 3T3 cells are injected with Val14rho protein. The effects induced by activation or inactivation of p21rho described here, suggest that the biological function of this protein is to control some aspect of cytoskeletal organization.

Release of Ca2+ from individual plant vacuoles by both InsP3 and cyclic ADP-ribose.

Calcium mobilization from intracellular pools couples many stimuli to responses in plant cells. Cyclic adenosine 5'-diphosphoribose (cADPR), which interacts with a ryanodine receptor in certain animal cells, was shown to elicit calcium release at the vacuolar membrane of beet storage root. The vacuolar calcium release pathway showed similarities to cADPR-gated calcium release in animal cells, including inhibition by ruthenium red, ryanodine activation, and high affinity for cADPR [Michaelis constant (Km) = 24 +/- 7 nanomolar]. Analysis by patch-clamping demonstrated that the cADPR-gated pathway in beet is voltage-dependent over the physiological range, does not spontaneously desensitize, and is colocalized with an inositol 1,4,5-trisphosphate (InsP3)-gated calcium release pathway in individual vacuoles.

Cyclic ADP-ribose in insulin secretion from pancreatic beta cells.

Inositol 1,4,5-trisphosphate (IP3) is thought to be a second messenger for intracellular calcium mobilization. However, in a cell-free system of islet microsomes, cyclic adenosine diphosphate-ribose (cADP-ribose), a nicotinamide adenine dinucleotide (NAD+) metabolite, but not IP3, induced calcium release. In digitonin-permeabilized islets, cADP-ribose and calcium, but not IP3, induced insulin secretion. Islet microsomes released calcium when combined with the extract from intact islets that had been incubated with high concentrations of glucose. Sequential additions of cADP-ribose inhibited the calcium release response to extracts from islets treated with high concentrations of glucose. Conversely, repeated additions of the islet extract inhibited the calcium release response to a subsequent addition of cADP-ribose. These results suggest that cADP-ribose is a mediator of calcium release from islet microsomes and may be generated in islets by glucose stimulation, serving as a second messenger for calcium mobilization in the endoplasmic reticulum.

Pertussis toxin B-oligomer suppresses human immunodeficiency virus-1 Tat-induced neuronal apoptosis through feedback inhibition of phospholipase C-beta by protein kinase C.

Human immunodeficiency virus (HIV)-1 Tat is a multifunctional protein involved in viral replication, inflammation and apoptosis. Tat activates phospholipase C-beta (PLC-beta), presumably via a pertussis toxin (PTX) sensitive G(i) protein, which is critical for neuronal apoptosis. In this study, we show that Tat-mediated intracellular Ca(2+) release in rat pheochromocytoma (PC-12) cells and rat primary cortical neuronal cultures was abrogated by pretreatment with either pertussis toxin and/or its B-oligomer subunit (PTX-B), devoid of ADP ribosyltransferase activity. PTX-B pretreatment also inhibited intracellular Ca(2+) release by bradykinin and 2,4,6-trimethyl-N-(m-3-trifluoromethylphenyl) benzenesulfonamide (m-3M3FBS), a director activator of phospholipase C. Activation of protein kinase C (PKC) by phorbol 12,13-dibutyrate (PdBu) mimicked the PTX-B-mediated inhibition of m-3M3FBS-stimulated intracellular Ca(2+) increase, while inhibition of PKC by bisindolylmaleimide I hydrochloride (BIM) reversed the inhibitory action of PTX-B. Functionally, PTX-B reduced Tat-induced Bax and caspase-3 proteins and reduced cell apoptosis. We conclude that PTX inhibition of Tat-mediated intracellular Ca(2+) release is independent of ADP ribosylation of the G(i) protein via the A protomer, but mediated by the B-oligomer. Furthermore, PTX-B suppresses HIV-1 Tat-mediated apoptosis by reducing its activation of PLC-beta through a PKC activation pathway.

Poly(ADP-ribose) signal in seizures-induced neuron death.

Poly(ADP-ribose) is found to be involved in many physiological or pathological processes. It is mainly modulated by poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG). Either PARP or PARG is associated with the neuronal death in a variety of neurodegenerative diseases. Cumulative data have suggested that poly(ADP-ribose) regulation might have a therapeutic value in neurotoxicity-induced neuron damage, probably due to the inhibition of apoptosis, suppressing of inflammation and activation of cell survival signaling. We hypothesize poly(ADP-ribose) play an important role in seizures-induced neuron death. Seizures can lead to neuron degeneration as for the exitotoxity of glutamate. Recently, it is indicated seizures also can trigger PARP activation. Further investigation is needed to determine whether poly(ADP-ribose) signal is a therapeutic target for seizures-induced injury.

A specific cyclic ADP-ribose antagonist inhibits cardiac excitation-contraction coupling.

BACKGROUND: Cyclic ADP-ribose (cADPR) has been shown to act as a potent cytosolic mediator in a variety of tissues, regulating the release of Ca2+ from intracellular stores by a mechanism that involves ryanodine receptors. There is controversy over the effects of cADPR in cardiac muscle, although one possibility is that endogenous cADPR increases the Ca2+ sensitivity of Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum. We investigated this possibility using 8-amino-cADPR, which has been found to antagonize the Ca2+-releasing effects of cADPR on sea urchin egg microsomes and in mammalian cells (Purkinje neurons, Jurkat T cells, smooth muscle and PC12 cells). RESULTS: In intact cardiac myocytes isolated from guinea-pig ventricle, cytosolic injection of 8-amino-cADPR substantially reduced contractions and Ca2+ transients accompanying action potentials (stimulated at 1Hertz). These reductions were not seen with injection of HEPES buffer, with heat-inactivated 8-amino-cADPR, or in cells pretreated with ryanodine (2 microM) to suppress sarcoplasmic reticulum function before injection of the 8-amino-cADPR. L-type Ca2+ currents and the extent of Ca2+ loading of the sarcoplasmic reticulum were not reduced by 8-amino-cADPR. CONCLUSIONS: These observations are consistent with the hypothesis that endogenous cADPR plays an important role during normal contraction of cardiac myocytes. One possibility is that cADPR sensitizes the CICR mechanism to Ca2+, an action antagonized by 8-amino-cADPR (leading to reduced Ca2+ transients and contractions). A direct effect of 8-amino-cADPR on CICR cannot be excluded, but observations with caffeine are not consistent with a non-selective block of release channels.

Recent advances in the regulation of plant calcium channels: evidence for regulation by G-proteins, the cytoskeleton and second messengers.

Important aspects of the regulatory properties of plant calcium channels have been discovered during the past few years. These include the control of plasma membrane-bound channels by regulatory proteins and the characterization of a plethora of intracellular calcium release channels. Deciphering the mechanisms of regulation of different Ca2+ channels and the probable co-operation of their activities in response to various stimuli is leading to a better understanding of Ca2+-signalling processes in higher plants.

Calcium transients and neurotransmitter release at an identified synapse.

It is widely accepted that the modulation of the presynaptic Ca2+ influx is one of the main mechanisms by which neurotransmitter release can be controlled. The well-identified cholinergic synapse in the buccal ganglion of Aplysia has been used to study the modulations that affect presynaptic Ca2+ transients and to relate this to quantal evoked neurotransmitter release. Three types of Ca2+ channel (L, N and P) are present in the presynaptic neurone at this synapse. Influxes of Ca2+ through N- and P-type channels trigger the release of ACh with only N-type Ca2+ channels being regulated by presynaptic neuromodulator receptors. In addition, presynaptic Ca2+ stores, via complex mechanisms of Ca2+ uptake and Ca2+ release, control the Ca2+ concentration that triggers this evoked ACh release.

Cyclic ADP-ribose is the primary trigger for hypoxic pulmonary vasoconstriction in the rat lung in situ.

Hypoxic pulmonary vasoconstriction (HPV) is unique to pulmonary arteries, and it aids ventilation/perfusion matching. However, in diseases such as emphysema, HPV can promote hypoxic pulmonary hypertension. We recently showed that hypoxia constricts pulmonary arteries in part by increasing cyclic ADP-ribose (cADPR) accumulation in the smooth muscle and, thereby, Ca(2+) release by ryanodine receptors. We now report on the role of cADPR in HPV in isolated rat pulmonary arteries and in the rat lung in situ. In isolated pulmonary arteries, the membrane-permeant cADPR antagonist, 8-bromo-cADPR, blocked sustained HPV by blocking Ca(2+) release from smooth muscle ryanodine-sensitive stores in the sarcoplasmic reticulum. Most importantly, we showed that 8-bromo-cADPR blocks HPV induced by alveolar hypoxia in the ventilated rat lung in situ. Inhibition of HPV was achieved without affecting (1) constriction by membrane depolarization and voltage-gated Ca(2+) influx, (2) the release (by hypoxia) of an endothelium-derived vasoconstrictor, or (3) endothelium-dependent vasoconstriction. Our findings suggest that HPV is both triggered and maintained by cADPR in the rat lung in situ.

[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.

Multiple calcium stores: separate but interacting.

Multiple mechanisms exist for increasing the concentration of intracellular calcium. This Perspective by Lee is one in a series on intracellular calcium release mechanisms and focuses on the calcium store operated by nicotinic acid adenine dinucleotide phosphate (NAADP). The characterization of the NAADP-operated calcium store as separate from the inositol trisphosphate (IP(3))-operated and cyclic ADP-ribose (cADPR)-operated calcium stores is discussed. Lee also addresses the role of NAADP in regulating intracellular calcium fluctuations during fertilization and hormonal activation of pancreatic acinar cells.

Cyclic ADP ribose as a calcium-mobilizing messenger.

This Perspective by Galione and Churchill is one in a series on intracellular calcium release mechanisms. The authors review the evidence for cyclic adenosine diphosphate ribose (cADPR) being a second messenger involved in regulating intracellular calcium. In addition, the physiological stimuli and responses mediated by cADPR are discussed. The Perspective is accompanied by a movie showing a calcium wave triggered by cADPR.

A pivotal role for cADPR-mediated Ca2+ signaling: regulation of endothelin-induced contraction in peritubular smooth muscle cells.

cADPR, a potent calcium-mobilizing intracellular messenger synthesized by ADP-ribosyl cyclases regulates openings of ryanodine receptors (RyR). Here we report that in the rat testis, a functional cADPR Ca2+ release system is essential for the contractile response of peritubular smooth muscle cells (PSMC) to endothelin (ET). We previously showed that this potent smooth muscle agonist elicits intracellular Ca2+ release in PSMC and seminiferous tubule contraction via activation of ETA and ETB receptors. ETB-R induces the mobilization of a thapsigargin-sensitive but IP3-independent intracellular Ca2+ pool. Stimulation of permeabilized PSMC with cADPR was found to elicit large Ca2+ releases blocked by either a selective antagonist of cADPR or a RyR blocker, but not by heparin. Western blotting and confocal fluorescence microscopy indicated the specific expression of type 2 RyR in perinuclear localization. ET was found to stimulate the activity of ADP-ribosyl cyclase. Microinjection of the selective cADPR antagonist 8NH2-cADPR completely abolished subsequent stimulation of Ca2+ signaling via ETA and ETB receptors. cADPR therefore appears to have an obligatory role for ETA-R and ETB-R-mediated calcium signaling in PSMC. However, ETB-R seem to be coupled exclusively to cADPR whereas ETA-R activation may be linked to IP3 and cADPR signaling pathways.

Cyclic ADP-ribose: a new member of a super family of signalling cyclic nucleotides.

Cyclic nucleotides are second messengers exerting their cellular effects mainly through protein phosphorylation. A new member of this family, cyclic ADP-ribose, is involved, instead, in mediating mobilization of Ca+2 from internal stores. The structure of this nucleotide has now been determined by X-ray crystallography and accumulating evidence indicates it may be an endogenous modulator of the Ca+2 induced Ca+2 release mechanism. This article summarizes the current knowledge of the structure, the mechanism of action and the metabolic enzymes of this novel nucleotide. With this new addition, the signalling functions of the cyclic nucleotide family are now extended from protein phosphorylation to Ca+2 signalling.

Mechanisms involved in alpha6beta1-integrin-mediated Ca(2+) signalling.

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.

Cyclic ADP-ribose-mediated insulin secretion and Reg, regenerating gene.

Glucose is the primary stimulus of insulin secretion in pancreatic beta-cells of the islets of Langerhans. CD38 has both ADP-ribosyl cyclase, which catalyzes the formation of cyclic ADP-ribose from NAD+, and cyclic ADP-ribose hydrolase, which converts cyclic ADP-ribose to ADP-ribose. ATP, produced by glucose metabolism, inhibits the cyclic ADP-ribose hydrolase of CD38 and therefore causes cyclic ADP-ribose accumulation in beta-cells. Then, cyclic ADP-ribose acts as a second messenger for Ca2+ mobilization from the endoplasmic reticulum to secrete insulin. The mechanism of insulin secretion as described above is completely different from the conventional hypothesis in which Ca2+ influx from extracellular sources was assumed to play a role in insulin secretion by glucose. On the other hand, strategies for influencing the replication of islet beta-cells and the growth of the beta-cell mass may be more important for ameliorating diabetes. Reg, regenerating gene, is involved in the growth of the beta-cell mass, and Reg protein has been shown to increase the beta-cell mass in a 90% depancreatized diabetic rat model, thereby ameliorating the diabetes. CD38 is involved in the formation of cyclic ADP-ribose and is essential for the glucose sensitivity of beta-cells for insulin secretion. Therefore, CD38 gene and Reg gene will become targets for genetic engineering for diabetic beta-cells.

Extracellular NAD+ regulates intracellular free calcium concentration in human monocytes.

Ca(2+) ions play a critical role in the biochemical cascade of signal transduction pathways, leading to the activation of immune cells. In the present study, we show that the exposure of freshly isolated human monocytes to NAD(+) results in a rapid concentration-dependent elevation of [Ca(2+)](i) (intracellular free Ca(2+) concentration) caused by the influx of extracellular Ca(2+). NAD(+) derivatives containing a modified adenine or nicotinamide ring failed to trigger a Ca(2+) increase. Treating monocytes with ADPR (ADP-ribose), a major degradation product of NAD(+), also resulted in a rise in [Ca(2+)](i). Selective inhibition of CD38, an NAD-glycohydrolase that generates free ADPR from NAD(+), does not abolish the effect of NAD(+), excluding the possibility that NAD(+) might act via ADPR. The NAD(+)-induced Ca(2+) response was prevented by the prior addition of ADPR and vice versa, indicating that both compounds share some mechanisms mediating the rise in [Ca(2+)](i). NAD(+), as well as ADPR, were ineffective when applied following ATP, suggesting that ATP controls events that intersect with NAD(+) and ADPR signalling.