NAADP receptors

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a recently described Ca2+ mobilizing messenger. First described in the sea urchin egg, it has been shown to mobilize Ca2+ from intracellular stores. It is a remarkably potent molecule, and recent reports show that its cellular levels change in response to a variety of agonists confirming its role as a Ca2+ mobilizing messenger. In many cases NAADP interacts with other Ca2+ mobilizing messengers such as inositol 1,4,5 trisphosphate (IP3 and cyclic adenosine diphosphate ribose (cADPR) in shaping cytosolic Ca2+ signals. What is not clear is the molecular nature of the NAADP-sensitive Ca2+ release mechanism and its sub-cellular localization. In this review we focus on the recent progress made in sea urchin eggs, which indicates that NAADP activates a novel Ca2+ release channel distinct from the relatively well-characterized IP3 and ryanodine receptors. Furthermore, in the sea urchin egg, the NAADP-sensitive store appears to be separate from the endoplasmic reticulum (ER) and is most likely an acidic store. These findings have also been reinforced by similar findings by some in mammalian cells. Finally, we discuss ongoing strategies to characterise NAADP-binding proteins which will greatly enhance our understanding of NAADP-mediated Ca2+ signalling, and lead to the development of more selective tools to probe the role of this messenger.     

NAADP receptors are present and functional in the heart.

Alongside the well-studied inositol 1,4,5 trisphosphate and ryanodine receptors, evidence is gathering that a new intracellular release mechanism, gated by the pyridine nucleotide nicotinic acid adenine dinucleotide phosphate (NAADP), is present in numerous organisms, ranging from plant to mammalian cells (reviewed in [1]). Most cells have been shown to express at least two Ca(2+)-release mechanisms controlled by different messengers, and this can lead to redundancy, convergence, or divergence of responses. One exception appears to be muscle and heart contractile tissues. Here, it is thought that the dominant intracellular channel is the ryanodine receptor, while IP(3) receptors are poorly expressed and their role appears to be negligible. We now report that NAADP receptors are functional and abundant in cardiac microsomes. NAADP binds specifically and with high affinity (130 pM and 4 nM) to two sites on cardiac microsomes and releases Ca(2+) with an apparent EC(50) of 323 +/- 14 nM. Furthermore, binding experiments show that this receptor displays both positive and negative cooperativity, a peculiarity unique among intracellular Ca(2+) channels. Therefore, we show that the heart possesses multiple mechanisms to increase the complexity of Ca(2+) signaling and that NAADP may be integral in the functioning of this organ.     

Calcium signaling by cyclic ADP-ribose and NAADP

Ca²⁺ mobilization as a signaling mechanism has been placed on center stage with the discovery of the first Ca²⁺ messenger, inositol trisphosphate (IP₃). This article focuses on two new Ca²⁺ release activators, which mobilize internal Ca²⁺ stores via mechanisms totally independent of IP₃. They are cyclic ADP-ribose (cADPR) and nicotinic acid dinucleotide phosphate (NAADP), metabolites derived respectively from NAD and NADP. Major advances in the past decade in the understanding of these two novel signaling mechanisms are chronologically summarized.     

NAADP mobilizes Ca2+ from a thapsigargin-sensitive store in the nuclear envelope by activating ryanodine receptors

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

Innate sensing of self and non-self RNAs by Toll-like receptors

Toll-like receptors (TLRs) have an important role in innate immunity in mammals by recognizing conserved microbial components that are known as pathogen-associated molecular patterns (PAMPs). Although the majority of these receptors sense pathogen components on the cell surface, a subset of them (TLR3, TLR7, TLR8 and TLR9) senses viral and bacterial nucleic acids in endosomal compartments. Of considerable interest is the recent finding that TLR7 and TLR8 can also recognize small interfering RNA (siRNA), which is the main effector in RNA interference. This immune activation by siRNAs can be abrogated by the 2'-ribose modification of uridines. Here, we discuss the recent developments that have expanded the understanding of self-non-self discrimination of RNAs by the innate immune system, and consider future directions for therapeutic applications of these findings.     

Cyclic adenosine diphosphate ribose activates ryanodine receptors, whereas NAADP activates two-pore domain channels

The mechanism by which cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) mobilize intracellular Ca(2+) stores remains controversial. It is open to question whether cADPR regulates ryanodine receptors (RyRs) directly, as originally proposed, or indirectly by promoting Ca(2+) uptake into the sarco/endoplasmic reticulum by sarco/endoplasmic reticulum Ca(2+)-ATPases. Conversely, although we have proposed that NAADP mobilizes endolysosomal Ca(2+) stores by activating two-pore domain channels (TPCs), others suggest that NAADP directly activates RyRs. We therefore assessed Ca(2+) signals evoked by intracellular dialysis from a patch pipette of cADPR and NAADP into HEK293 cells that stably overexpress either TPC1, TPC2, RyR1, or RyR3. No change in intracellular Ca(2+) concentration was triggered by cADPR in either wild-type HEK293 cells (which are devoid of RyRs) or in cells that stably overexpress TPC1 and TPC2, respectively. By contrast, a marked Ca(2+) transient was triggered by cADPR in HEK293 cells that stably expressed RyR1 and RyR3. The Ca(2+) transient was abolished following depletion of endoplasmic reticulum stores by thapsigargin and block of RyRs by dantrolene but not following depletion of acidic Ca(2+) stores by bafilomycin. By contrast, NAADP failed to evoke a Ca(2+) transient in HEK293 cells that expressed RyR1 or RyR3, but it induced robust Ca(2+) transients in cells that stably overexpressed TPC1 or TPC2 and in a manner that was blocked following depletion of acidic stores by bafilomycin. We conclude that cADPR triggers Ca(2+) release by activating RyRs but not TPCs, whereas NAADP activates TPCs but not RyRs.     

Ca2+ release triggered by NAADP in hepatocyte microsomes

NAADP (nicotinic acid-adenine dinucleotide phosphate) is fast emerging as a new intracellular Ca2+-mobilizing messenger. NAADP induces Ca2+ release by a mechanism that is distinct from IP3 (inositol 1,4,5-trisphosphate)- and cADPR (cADP-ribose)-induced Ca2+ release. In the present study, we demonstrated that micromolar concentrations of NAADP trigger Ca2+ release from rat hepatocyte microsomes. Cross-desensitization to IP3 and cADPR by NAADP did not occur in liver microsomes. We report that non-activating concentrations of NAADP can fully inactivate the NAADP-sensitive Ca2+-release mechanism in hepatocyte microsomes. The ability of thapsigargin to block the NAADP-sensitive Ca2+ release is not observed in sea-urchin eggs or in intact mammalian cells. In contrast with the Ca2+ release induced by IP3 and cADPR, the Ca2+ release induced by NAADP was completely independent of the free extravesicular Ca2+ concentration and pH (in the range 6.4-7.8). The NAADP-elicited Ca2+ release cannot be blocked by the inhibitors of the IP3 receptors and the ryanodine receptor. On the other hand, verapamil and diltiazem do inhibit the NAADP- (but not IP3- or cADPR-) induced Ca2+ release.     

Eugenol exhibits anti-virulence properties by competitively binding to quorum sensing receptors

The primary objective of this study was to ascertain the anti-biofilm and anti-virulence properties of sub-minimum inhibitory concentration (MIC) levels of eugenol against the standard strain PAO1 and two multi-drug resistant P. aeruginosa clinical isolates utilizing quorum sensing inhibition (QSI). Eugenol at 400 μM significantly reduced biofilm formation on urinary catheters and the virulence factors (VF) including extracellular polysaccharides, rhamnolipid, elastase, protease, pyocyanin, and pyoverdine (p < 0.001). Further, eugenol exhibited a marked effect on the production of QS signals (AIs) (p < 0.001) without affecting their chemical integrity. In silico docking studies demonstrated a stable molecular binding between eugenol and QS receptor(s) in comparison with respective AIs. Investigation on reporter strains confirmed the competitive binding of eugenol to a QS receptor (LasR) as the possible QSI mechanism leading to significant repression of QS associated genes besides the VF genes (p < 0.001). This study provides insights, for the first time, into the mechanism of the anti-virulence properties of eugenol.     

Heterodimerization of calcium sensing receptors with metabotropic glutamate receptors in neurons

Calcium sensing (CaR) and Group I metabotropic glutamate receptors exhibit overlapping expression patterns in brain, and share common signal transduction pathways. To determine whether CaR and Group I metabotropic glutamate receptors (mGluRs) (mGluR1alpha and mGluR5) can form heterodimers, we immunoprecipitated CaR from bovine brain and observed co-precipitation of mGluR1alpha. CaR and mGluR1alpha co-localize in hippocampal and cerebellar neurons, but are expressed separately in other brain regions. In vitro transfection studies in HEK-293 cells established the specificity and disulfide-linked nature of the CaR:mGluR1alpha (CaR:mGluR5) interactions. CaR:mGluR1alpha (CaR:mGluR5) heterodimers exhibit altered trafficking via Homer 1c when compared with CaR:CaR homodimers. CaR becomes sensitive to glutamate-mediated internalization when present in CaR:mGluR1alpha heterodimers. These results demonstrate cross-family covalent heterodimerization of CaR with Group I mGluRs, and increase the potential role(s) for CaR in modulating neuronal function.     

Lack of effect of cADP-ribose and NAADP on the activity of skeletal muscle and heart ryanodine receptors

The calcium release channels/ryanodine receptors (RyRs) are potential/putative targets of cADPR (cyclic ADP-ribose) action in many tissue systems. In striated muscles, where RyRs predominate, cADPR action on these channels is controversial. Here cADPR modulation of cardiac and skeletal muscle RyR channels was tested. We considered factors reported as necessary for cADPR action, such as the presence of calmodulin and/or FK binding proteins (FKBPs). We found: 1) The RyR channel isoforms were insensitive to cADPR (or its metabolite NAADP [nicotinic acid adenine dinucleotide phosphate]) under all conditions examined, as studied by: 1a) single channel recordings in planar lipid bilayers; 1b) macroscopic behavior of the RyRs in sarcoplasmic reticulum (SR) microsomes (including crude microsome preparations likely to retain putative cADPR cofactors) at room temperature and at 37 degrees C (net energized Ca2+ uptake or passive Ca2+ leak); 2) [32P]cADPR did not bind significantly to SR microsomes; 3) cADPR did not affect FKBP association to SR membranes. We conclude that cADPR does not interact directly with RyRs or RyR-associated SR proteins. Our results under in vitro conditions suggest that c ADPR effects on Ca2+ signaling observed in vivo in mammalian striated muscle cells may reflect indirect modulation of RyRs or RyR-independent Ca2+ release systems.     

Calcium signalling by nicotinic acid adenine dinucleotide phosphate (NAADP)

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a recently described Ca2+ mobilizing messenger, and probably the most potent. We briefly review its unique properties as a Ca2+ mobilizing agent. We present arguments for its action in targeting acidic calcium stores rather than the endoplasmic reticulum. Finally, we discuss possible biosynthetic pathways for NAADP in cells and candidates for its target Ca2+ release channel, which has eluded identification so far.     

NAADP influences excitation-contraction coupling by releasing calcium from lysosomes in atrial myocytes

In atrial myocytes, the sarcoplasmic reticulum (SR) has an essential role in regulating the force of contraction as a consequence of its involvement in excitation-contraction coupling (ECC). Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca²⁺ mobilizing messenger that acts to release Ca²⁺ from an acidic store in mammalian cells. The photorelease of NAADP in atrial myocytes increased Ca²⁺ transient amplitude with no effect on accompanying action potentials or the L-type Ca²⁺ current. NAADP-AM, a cell permeant form of NAADP, increased Ca²⁺ spark amplitude and frequency. The effect on Ca²⁺ spark frequency could be prevented by bafilomycin A1, a vacuolar H⁺-ATPase inhibitor, or by disruption of lysosomes by GPN. Bafilomycin prevented staining of acidic stores with LysoTracker red by increasing lysosomal pH. NAADP-AM also produced an increase in the lysosomal pH, as detected by a reduction in LysoSensor green fluorescence. These effects of NAADP were associated with an increase in the amount of caffeine-releasable Ca²⁺ in the SR and may be regulated by β-adrenoceptor stimulation with isoprenaline. These observations are consistent with a role for NAADP in regulating ECC in atrial myocytes by releasing Ca²⁺ from an acidic store, which enhances SR Ca²⁺ release by increasing SR load.     

NAADP+ synthesis from cADPRP and nicotinic acid by ADP-ribosyl cyclases

ADP-ribosyl cyclases (ADPRCs) are present from lower Metazoa to mammals and synthesize the Ca2+-active (di)nucleotides cyclic ADP-ribose (cADPR), NAADP+, and ADP-ribose (ADPR), involved in the regulation of important cellular functions. NAADP+ can be synthesized by ADPRCs from NADP+ through a base-exchange reaction, which substitutes nicotinamide for nicotinic acid (NA). Here we demonstrate that ADPRCs from both lower and higher Metazoa (including human CD38) can also synthesize NAADP+ starting from 2'-phospho-cyclic ADP-ribose (cADPRP) and NA. Comparison, on the two substrates cADPRP and NADP+, of the relative rates of the reactions introducing NA and hydrolyzing/cyclizing the substrate, respectively, indicates that with all ADPRCs tested cADPRP is preferentially transformed into NAADP+, while NADP+ is preferentially cyclized or hydrolyzed to cADPRP/2'-phospho-ADP-ribose. cADPRP was detectable in retinoic acid-differentiated, CD38+ HL-60 cells, but not in undifferentiated, CD38- cells. These results suggest that cADPRP may be a NAADP+ precursor in ADPRC+ cells.     

Glucose sensing and signaling by two glucose receptors in the yeast Saccharomyces cerevisiae.

How eukaryotic cells sense availability of glucose, their preferred carbon and energy source, is an important, unsolved problem. Bakers' yeast (Saccharomyces cerevisiae) uses two glucose transporter homologs, Snf3 and Rgt2, as glucose sensors that generate a signal for induction of expression of genes encoding hexose transporters (HXT genes). We present evidence that these proteins generate an intracellular glucose signal without transporting glucose. The Snf3 and Rgt2 glucose sensors contain unusually long C-terminal tails that are predicted to be in the cytoplasm. These tails appear to be the signaling domains of Snf3 and Rgt2 because they are necessary for glucose signaling by Snf3 and Rgt2, and transplantation of the C-terminal tail of Snf3 onto the Hxt1 and Hxt2 glucose transporters converts them into glucose sensors that can generate a signal for glucose-induced HXT gene expression. These results support the idea that yeast senses glucose using two modified glucose transporters that serve as glucose receptors.     

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Degradation by Alkaline Phosphatase

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a ubiquitous second messenger providing a Ca²⁺ trigger in a wide range of cell types. However, its metabolism is not well understood. Here, we demonstrate the presence of endogenous NAADP in HeLa cells. CD38, a promiscuous enzyme described to be involved in NAADP metabolism, was not detectable in HeLa cells. In cell-free extracts of HeLa cells, NAADP was degraded to nicotinic acid adenine dinucleotide (NAAD). The enzyme was enriched in membranes (10,000 × g pellet) and displayed characteristics typical of alkaline phosphatase (AP), e.g. pH optimum at 8–9 and sensitivity to the inhibitors l-homoarginine and l-leucine. Importantly, NAADP at physiological concentrations (50–100 nm) was degraded to NAAD. Expression of AP isoenzymes was analyzed in HeLa cells. Based on the results together with inhibitor studies, the placental AP isoform emerged as the best candidate for NAADP degradation in HeLa cells. In contrast to HeLa cells, Jurkat T cells or HEK293 cells did not express any AP isoenzymes and did not display any NAADP 2′-phosphatase activity. Finally, the placental AP isoform was expressed heterologously in HEK293 cells, resulting in reconstitution of NAADP 2′-phosphatase activity in cell-free extracts. On the basis of the results, we provide evidence for AP as the metabolizing enzyme of NAADP in cells that do not express CD38.     

Thio-NADP is not an antagonist of NAADP

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a metabolite of NADP, which can release Ca²⁺ from stores that are distinct from those activated by either cyclic ADP-ribose or inositol 1,4,5-trisphosphate (IP₃). It has previously been suggested that thio-NADP is a specific antagonist of NAADP (Chini et al. [1995]J. Biol. Chem. 270, 3216–3223). Its effects in sea-urchin egg homogenates were investigated. At 50 μM, thio-NADP activates partial Ca²⁺ release and totally inhibits subsequent challenge with a saturating concentration of NAADP. Purification by HPLC eliminates the Ca²⁺ releasing activity of 50 μM thio-NADP and reduces the subsequent inhibition by 73.7±1.3%. The residual inhibitory effect is no more than that exerted by 50 μM of either NADP itself or nicotinic acid adenine dinucleotide (NAAD). These results are confirmed by³²P-NAADP binding studies. Unpurified thio-NADP inhibits the specific³²P-NAADP binding to egg microsomes with an IC₅₀ of 40 μM. After HPLC purification, only 20% inhibition is seen at a concentration as high as 50 μM, similar to the extent of inhibition effected by 40 μM NADP. These results indicate the inhibitory substance in thio-NADP is a contaminant. The partial Ca²⁺ release activity of unpurified thio-NADP suggests the contaminant is NAADP itself. This is supported by the fact that pretreatment with a subthreshold concentration of only 2 nM NAADP totally desensitizes the egg homogenates such that no Ca²⁺ response is seen with saturating NAADP. Estimation from the binding studies shows that a contamination of 0.012% of NAADP in the unpurified thio-NADP samples is sufficient to account for the inhibitory effects. These results indicate thio-NADP is not an antagonist of NAADP.     

CD38: a NAADP degrading enzyme

The role of the multifunctional enzyme CD38 in formation of the Ca(2+)-mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) was investigated. Gene silencing of CD38 did neither inhibit NAADP synthesis in intact Jurkat T cells nor in thymus or spleen obtained from CD38 knock out mice. In vitro, both NAADP formation by base-exchange and degradation to 2-phospho adenosine diphosphoribose were efficiently decreased. Thus in vivo CD38 appears to be a NAADP degrading rather than a NAADP forming enzyme, perhaps avoiding desensitizing NAADP levels in intact cells.     

Calcium sensing receptors as integrators of multiple metabolic signals

Calcium sensing receptors are critical to maintenance of organismal Ca2+ homeostasis, translating small changes in serum Ca2+ into changes in PTH secretion by the parathyroid glands and Ca2+ excretion by the kidneys. Calcium sensing receptors are also expressed in many cells and tissues not directly involved in Ca2+ homeostasis where their role(s) are less defined. Recent studies have demonstrated that calcium sensing receptors integrate a variety of metabolic signals, including polyvalent cations, pH, ionic strength, amino acids, and polypeptides, making CaR uniquely capable of generating cell- and tissue-specific responses, sensing not only Ca2+, but the local metabolic environment. The challenge for future studies is to define CaR responsiveness in each varied physiological context.