Justyn M. Thomas

NAADP Mobilizes Ca2+ from Reserve Granules, Lysosome-Related Organelles, in Sea Urchin Eggs

Abstract Nicotinic acid adenine dinucleotide phosphate (NAADP) mobilizes Ca 2+ in many cells and species. Unlike other Ca 2+ -mobilizing messengers, NAADP mobilizes Ca 2+ from an unknown store that is not the endoplasmic reticulum, the store traditionally associated with messenger-mediated Ca 2+ signaling. Here, we demonstrate the presence of a Ca 2+ store in sea urchin eggs mobilized by NAADP that is dependent on a proton gradient maintained by an ATP-dependent vacuolar-type proton pump. Moreover, we provide pharmacological and biochemical evidence that this Ca 2+ store is the reserve granule, the functional equivalent of a lysosome in the sea urchin egg. These findings represent an unsuspected mechanism for messenger-mediated Ca 2+ release from lysosome-related organelles.

Identification of a chemical probe for NAADP by virtual screening

Research into the biological role of the Ca2+-releasing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate) has been hampered by a lack of chemical probes. To find new chemical probes for exploring NAADP signaling, we turned to virtual screening, which can evaluate millions of molecules rapidly and inexpensively. We used NAADP as the query ligand to screen the chemical library ZINC for compounds with 3D-shape and electrostatic similarity. We tested the top-ranking hits in a sea urchin egg bioassay and found that one hit, Ned-19, blocks NAADP signaling at nanomolar concentrations. In intact cells, Ned-19 blocked NAADP signaling and fluorescently labeled NAADP receptors. Moreover, we show the utility of Ned-19 as a chemical probe by using it to demonstrate that NAADP is a key causal link between glucose sensing and Ca2+ increases in mouse pancreatic beta cells.

Sperm Deliver a New Second Messenger: NAADP

NAADP is a highly potent mobilizer of Ca(2+), which in turn triggers Ca(2+)-induced Ca(2+) release pathways in a wide range of species. Nevertheless, NAADP is not presently classified as a second messenger because it has not been shown to increase in response to a physiological stimulus. We now report a dramatic increase in NAADP during sea urchin egg fertilization that was largely due to production in sperm upon contacting egg jelly. The NAADP bolus plays a physiological role upon delivery to the egg based on its ability to induce a cortical flash, a depolarization-induced activation of L-type Ca(2+) channels. Moreover, the sperm-induced cortical flash was eliminated in eggs desensitized to NAADP. We conclude that an NAADP increase plays a physiologically relevant role during fertilization and provides the first conclusive demonstration that NAADP is a genuine second messenger.

Role of NAADP and cADPR in the Induction and Maintenance of Agonist-Evoked Ca2+ Spiking in Mouse Pancreatic Acinar Cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic adenosine diphosphate ribose (cADPR) were first demonstrated to mobilize Ca2+ in sea urchin eggs. In the absence of direct measurements of these messengers, pharmacological studies alone have implicated these molecules as intracellular second messengers for specific cell surface receptor agonists. We now report that in mouse pancreatic acinar cells, cholecystokinin, but not acetylcholine, evokes rapid and transient increases in NAADP levels in a concentration-dependent manner. In contrast, both cholecystokinin and acetylcholine-mediated production of cADPR followed a very different time course. The rapid and transient production of NAADP evoked by cholecystokinin precedes the onset of the Ca2+ signal and is consistent with a role for NAADP in the initiation of the Ca2+ response. Continued agonist-evoked Ca2+ spiking is maintained by prolonged elevations of cADPR levels through sensitization of Ca2+ -induced Ca2+ -release channels. This study represents the first direct comparison of NAADP and cADPR measurements, and the profound differences observed in their time courses provide evidence in support of distinct roles of these Ca2+ -mobilizing messengers in shaping specific Ca2+ signals during agonist stimulation.

A safe lithium mimetic for bipolar disorder

Lithium is the most effective mood stabilizer for the treatment of bipolar disorder, but it is toxic at only twice the therapeutic dosage and has many undesirable side effects. It is likely that a small molecule could be found with lithium-like efficacy but without toxicity through target-based drug discovery; however, lithium’s therapeutic target remains equivocal. Inositol monophosphatase is a possible target but no bioavailable inhibitors exist. Here we report that the antioxidant ebselen inhibits inositol monophosphatase and induces lithium-like effects on mouse behaviour, which are reversed with inositol, consistent with a mechanism involving inhibition of inositol recycling. Ebselen is part of the National Institutes of Health Clinical Collection, a chemical library of bioavailable drugs considered clinically safe but without proven use. Therefore, ebselen represents a lithium mimetic with the potential both to validate inositol monophosphatase inhibition as a treatment for bipolar disorder and to serve as a treatment itself.

Analogues of the Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Antagonist Ned-19 Indicate Two Binding Sites on the NAADP Receptor

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca2+-releasing messenger. Biological data suggest that its receptor has two binding sites: one high-affinity locking site and one low-affinity opening site. To directly address the presence and function of these putative binding sites, we synthesized and tested analogues of the NAADP antagonist Ned-19. Ned-19 itself inhibits both NAADP-mediated Ca2+ release and NAADP binding. A fluorometry bioassay was used to assess NAADP-mediated Ca2+ release, whereas a radioreceptor assay was used to assess binding to the NAADP receptor (only at the high-affinity site). In Ned-20, the fluorine is para rather than ortho as in Ned-19. Ned-20 does not inhibit NAADP-mediated Ca2+ release but inhibits NAADP binding. Conversely, Ned-19.4 (a methyl ester of Ned-19) inhibits NAADP-mediated Ca2+ release but cannot inhibit NAADP binding. Furthermore, Ned-20 prevents the self-desensitization response characteristic of NAADP in sea urchin eggs, confirming that this response is mediated by a high-affinity allosteric site to which NAADP binds in the radioreceptor assay. Collectively, these data provide the first direct evidence for two binding sites (one high- and one low-affinity) on the NAADP receptor.

Bioactivity of a peptide derived from acetylcholinesterase: involvement of an ivermectin-sensitive site on the alpha 7 nicotinic receptor

A peptide fragment of 14 amino acids, derived from the C-terminus of acetylcholinesterase (AChE), might underlie the now well-established noncholinergic effects of the enzyme. This peptide is bioactive in a variety of systems including acute (brain slices) and chronic (organotypic culture) preparations of hippocampus, a pivotal area in Alzheimers disease (AD); invariably, the action of the peptide is mediated specifically via an as yet unknown receptor. In this study, the allosteric alpha 7 agent, ivermectin (IVM), had a modest inhibitory effect, whilst that of the peptide was significantly more marked. However, ivermectin rendered ineffective the toxicity of high doses of the peptide, that is, when the two were co-applied, only the smaller effects of ivermectin were seen. Ivermectin, therefore, is presumably acting at a site that is identical to, or at least strongly interactive with, the normal binding site for AChE-peptide. This observation could have important implications for eventual therapeutic targeting of the action of AChE-peptide, in neurodegeneration.

ß-Adrenergic receptor signaling increases NAADP and cADPR levels in the heart

Evidence suggests that β-Adrenergic receptor signaling increases heart rate and force through not just cyclic AMP but also the Ca(2+)-releasing second messengers NAADP (nicotinic acid adenine dinucleotide phosphate) and cADPR (cyclic ADP-ribose). Nevertheless, proof of the physiological relevance of these messengers requires direct measurements of their levels in response to receptor stimulation. Here we report that in intact Langendorff-perfused hearts β-adrenergic stimulation increased both messengers, with NAADP being transient and cADPR being sustained. Both NAADP and cADPR have physiological and therefore pathological relevance by providing alternative drug targets in the β-adrenergic receptor signaling pathway.

Spatial and Temporal Control of Calcium Signaling by NAADP

Within an individual cell Ca2+ plays many roles, some of which are seemingly contradictory. For example, in a smooth muscle cell, Ca2+ controls growth, differentiation, gene expression, and paradoxically both contraction and relaxation [1]. How can Ca2+ be linked to such a multitude of processes and maintain any specificity? For many of these processes, if not all, the answer is that Ca2+ increases are under tight control both spatially and temporally [2, 3]. The importance of the spatiotemporal aspect of a Ca2+ signal arises from necessity; Ca2+ is an atom and unlike other intracellular messengers, it cannot be created or destroyed in cells, only moved around. Spatially, localized Ca2+ increases control secretion in pancreatic acinar cells [4, 5], growth cone migration in neurones [6], and relaxation in smooth muscle cells [1], whereas global Ca2+ signals control contraction of all types of muscle cells and fertilization of eggs from marine invertebrates to mammals [7-9]. Temporally, the frequency of repetitive increases in Ca2+ (oscillations) control cell division [10] and gene expression [11, 12]. Cells decode the Ca2+ oscillations via enzymes, which exhibit different kinetics of activation and inactivation by Ca2+, effectively tuning their activity to certain Ca2+ oscillation frequencies [3, 12].