NAADP-AM, A New Player in Calcium Signaling Pathways

Nicotinic acid adenine dinucleotide phosphate (NAADP, Cat# 20999) is a secondary messenger that plays a key role in calcium signaling pathways. First discovered in the early 1980s, this dinucleotide has become the focus of intense research in recent years. It has been proposed as a pharmacological target for a variety of diseases affecting the pancreas, heart and nervous system. Experiments with NAADP have shown it to be an extremely potent calcium mobilizer as well as a modulating agent for other cellular pathways, such as those involving inositol trisphosphate (IP₃). The strong interest in NAADP has also led to the discovery and development of several key research tools, namely, NED-19 (a NAADP antagonist) and NAADP-AM (a cell-permeable form of NAADP).

IP₃, cADPR and NAADP

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Without question, calcium ions (Ca²⁺) are one of the most important secondary messengers in biology. They are crucial not only for cellular events such as apoptosis and proliferation, but also for macroscopic functions such as muscle contraction and neuron signaling. Their importance has prompted much investigative effort, as researchers attempt to understand the mechanisms and pathways with which calcium ions exert their influence on cells.

One of the early breakthroughs in this regard was the discovery of inositol triphosphate (IP₃). While the precursor of IP₃, phosphatidylinositol (PIP₂), was first studied in the 1950s, it took an additional 30 years before the link between PIP₂, IP₃ and Ca²⁺ mobilization was understood. In the early 1980s, extensive experimentation showed that extracellular PIP₂ was catabolized by a membrane bound protein called phospholipase C (PLC). This resulted in the formation of intracellular IP₃, which acted as a secondary messenger and stimulated Ca²⁺ release from endoplasmic reticulum (ER) stores. This discovery was particularly notable because it demonstrated a mechanism whereby external stimuli (ie. PIP₂) could influence intracellular calcium levels, and by extension, the activation of various protein kinases.



With the explication of the IP₃-Ca²⁺ relationship, many researchers were satisfied with their understanding of calcium pathways. It was amongst a great deal of surprised individuals then that Hon Cheung Lee and colleagues published their research in 1987, which proposed not one, but two, additional calcium mobilizing secondary messengers. While studying sea urchin eggs, Lee and colleagues discovered that two previously unknown nucleotides, cADPR and NAADP, could stimulate calcium release in cells. Their pivotal study placed much of what was known about calcium pathways at that time into question, as researchers once again scrambled to decipher the complexities of cellular calcium signaling.

NAADP Structure

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While Lee and colleagues first discovered the presence of NAADP in 1987, it was not until 1995, almost a decade later, that its structure was determined. Through a combination of high pressure liquid chromatography (HPLC) and proton NMR, it was discovered that NAADP is in fact a derivative of the co-factor NADP, wherein the nicotinamide group is replaced with a nicotinic acid group. This structural determination was somewhat expected, as NAADP was originally discovered as a contaminant in commercial NADP sources. What surprised many researchers, however, was that cellular NAADP did not result from a simple de-amination of existing NADP. Rather, it is a metabolite formed by a base-exchange reaction catalyzed by the same enzyme which forms cyclic ADP-ribose (cADPR). This was a particularly surprising discovery as cADPR and NAADP have very distinct structural and functional properties.

 

NAADP Formation

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In humans, a glycoprotein called CD38 (cluster of differentiation 38) is responsible for both the formation of cADPR and of NAADP. This discovery, however, was surprising for two reasons.

First, cADPR and NAADP are very different structurally; NAADP is a linear compound whereas cADPR is cyclical. This is an important distinction because it indicates that CD38 catalyzes two discrete reactions, a base-exchange reaction for NAADP and a cyclization reaction for cADPR. While certainly not a unique occurrence in biological systems, CD38 is rather uncommon not only for its discrete catalytic abilities, but also for the specificity with which those catalytic processes occur. That is, despite two different starting compounds, CD38 recognizes and converts NAD to cADPR and NADP to NAADP with a high degree of consistency.

Second, cADPR and NAADP are very different functionally.

NAADP Targets

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As a calcium mobilizer, NAADP is functionally distinct from cADPR and IP₃. Unlike the latter, NAADP does not mobilize calcium stores in the ER. Rather, it mobilizes recently discovered acidic calcium stores located throughout the cytoplasm. These acidic calcium stores include subcellular compartments such as endosomes, lysosomes, secretory granules and Golgi bodies. More specifically, recent research suggests that NAADP targets a family of membrane bound ion-channels, called two-pore channels (TPC), in order to stimulate calcium release. The specific mechanisms behind this interaction, however, are still not well understood.

NAADP Mechanism of Action

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There are currently two hypothesis regarding NAADP's role in calcium signaling pathways.

The first proposes that NAADP serves as a signaling primer. The hypothesis is that NAADP causes calcium release from acidic stores, which are then taken up by ER stores. In this way, a cell is primed for an enhanced response upon later stimuli. Research into excitation-contraction coupling (EEC) in atrial myocyte suggests this may be the case, as the presence of NAADP leads to the release of Ca²⁺ from acidic stores, followed by an increase in sarcoplasmic reticulum (SR) Ca²⁺ release.

The second hypothesis is that NAADP acts as a calcium signal amplifier. In this model, NAADP stimulates Ca²⁺ ion release from acidic stores, calcium ions which then interact with calcium sensitive targets to effectuate further calcium mobilization, for example, from the ER. The proposal suggests this can be achieved if the initial calcium release, from acidic stores, can influence additional calcium mobilizing secondary messengers such as cADPR and IP₃. Indeed, this appears to be the case in a study conducted on pancreatic acinar cells, in which cADPR and IP₃ antagonists were able to block NAADP-dependent Ca²⁺response.

Advancements in NAADP Research

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As interest in it deepens, scientists have begun looking for better tools to study NAADP. In recent years, the research process has been significantly aided by the development of two separate compounds: NED-19 and NAADP-AM (Cat# 20998).

NED-19 is a NAADP antagonist that was first developed through virtual chemical screening of NAADP analogs. It acts specifically to block both NAADP-mediated Ca²⁺response as well as NAADP binding. Interestingly enough, however, by using NED-19 analogs, researchers have been able to show that these two antagonistic effects can be teased apart. Using NED-20, researchers were able to specifically block NAADP binding, while leaving NAADP-mediated Ca²⁺ release untouched. On the other hand, when NED-19.4 was used, NAADP binding could occur, but NAADP-mediated Ca²⁺ release was inhibited. This result suggests that there are actually two different binding sites on NAADP receptors and two different sites with which NED-19 can interact with.

The second important development in the study of NAADP is the synthesis of a cell permeable NAADP analog, NAADP-AM. Prior to its development, studies with NAADP had to utilize invasive cellular techniques such as microinjections or electroporation in order to load NAADP into cells. There are several well-documented problems with these methods. At the very least, normal cellular function is disrupted due to the disruption of the cell membrane. In the case of microinjections, the process is very time-intensive as it is limited to single cells. For electroporation, common problems include low loading efficiency and high rates of cell death.

The usage of acetoxymethyl esters (AM esters) resolves many of the problems faced by prior loading techniques. This is particularly true in the case of NAADP because, as a compound, it is negatively charged. What this means is that while NAADP is well-retained in cells, it has an especially difficult time passing through cell membranes. But by chemically adding AM esters to it, thus synthesizing NAADP-AM, NAADP not only loses its negative charge but also becomes hydrophobic. This change in chemical properties allows NAADP-AM to easily pass through the phospholipid membrane of cells. Once inside, the AM ester is cleaved by intracellular esterases, thus returning the compound to its original NAADP form. In this manner, through the use of AM esters, NAADP can be easily loaded into a population of cells without the need for invasive cellular techniques.

 

Current Research

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NAADP has been the focus of study in a wide range of fields. For example, NAADP has been suggested to play an important role in the onset and progression of diabetes. Studies in pancreatic β cells show that NAADP is involved in glucose-induced Ca²⁺ signaling pathways related to the regulation and release of insulin. In these studies, it was found that intracellular NAADP levels fluctuate as a function of glucose addition. Furthermore, it was found that NAADP acts as a sensitizing agent in pancreatic β cells. That is, at concentrations lower than 100 nM, NAADP leads to an enhancement of cellular calcium responses while levels greater than 1 µM leads to the inactivation of NAADP receptors and downstream pathways.

Another active area of investigation is NAADP's role in Parkinson's disease. In these studies, it is again suggested that NAADP acts as a sensitizing agent, enhancing cell response to additional stressors and stimuli. In particular, focus was placed on the de-regulation of the leucine-rich repeat kinase-2 (LRRK2) gene, wherein mutations are thought to cause late-onset Parkinson's disease characterized by increased autophagy and abnormal protein degradation. It is thought that in the case of Parkinson's disease, the de-regulation of LRRK2 pathways results in over-activation of NAADP and two-pore channels, which in turn over-amplifies calcium signaling in response to external stimuli. NAADP appears to be linked to LRRK2 pathways as the addition of NED-19, an NAADP antagonist, mitigates the effects of LRRK2 de-regulation and the blocking of LRRK2 pathways simultaneously blocks NAADP-dependent autophagy overresponse.

Further Readings

1. Bak, Judit, et al. "NAADP receptors are present and functional in the heart." Current Biology 11.12 (2001): 987-990.
2. Brailoiu, Eugen, et al. "Essential requirement for two-pore channel 1 in NAADP-mediated calcium signaling." The Journal of cell biology 186.2 (2009): 201-209.
3. Calcraft, Peter J., et al. "NAADP mobilizes calcium from acidic organelles through two-pore channels." Nature 459.7246 (2009): 596-600.
4. Churchill, Grant C., et al. "NAADP mobilizes Ca²⁺ from reserve granules, lysosome-related organelles, in sea urchin eggs." Cell 111.5 (2002): 703-708.
5. Gerasimenko, Julia V., et al. "NAADP, cADPR and IP₃ all release Ca²⁺ from the endoplasmic reticulum and an acidic store in the secretory granule area." Journal of Cell Science 119.2 (2006): 226-238.
6. Gerasimenko, Julia V., et al. "NAADP mobilizes Ca²⁺ from a thapsigargin-sensitive store in the nuclear envelope by activating ryanodine receptors." J Cell Biol 163.2 (2003): 271-282.
7. Gòmez-Suaga, Patricia, et al. "Leucine-rich repeat kinase 2 regulates autophagy through a calcium-dependent pathway involving NAADP." Human molecular genetics (2011): ddr481.
8. Gul, Rukhsana, et al. "Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Cyclic ADP-Ribose (cADPR) Mediate Ca²⁺ Signaling in Cardiac Hypertrophy Induced by β-Adrenergic Stimulation." PloS one 11.3 (2016): e0149125.
9. Lee, Hon Cheung, and Robert Aarhus. "A derivative of NADP mobilizes calcium stores insensitive to inositol trisphosphate and cyclic ADP-ribose." Journal of Biological Chemistry 270.5 (1995): 2152-2157.
10. Lee, Hon Cheung. "Cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate (NAADP) as messengers for calcium mobilization." Journal of Biological Chemistry 287.38 (2012): 31633-31640.
11. Masgrau, Roser, et al. "NAADP: a new second messenger for glucose-induced Ca²⁺ responses in clonal pancreatic β cells." Current biology 13.3 (2003): 247-251.
12. Naylor, Edmund, et al. "Identification of a chemical probe for NAADP by virtual screening." Nature chemical biology 5.4 (2009): 220-226.
13. Patel, Sandip, and Roberto Docampo. "Acidic calcium stores open for business: expanding the potential for intracellular Ca²⁺ signaling." Trends in cell biology 20.5 (2010): 277-286.
14. Pitt, Samantha J., et al. "TPC2 is a novel NAADP-sensitive Ca²⁺ release channel, operating as a dual sensor of luminal pH and Ca²⁺." Journal of Biological Chemistry 285.45 (2010): 35039-35046.
15. Rosen, Daniel, et al. "Analogues of the nicotinic acid adenine dinucleotide phosphate (NAADP) antagonist Ned-19 indicate two binding sites on the NAADP receptor." Journal of Biological Chemistry 284.50 (2009): 34930-34934.
16. Yamasaki, Michiko, et al. "Role of NAADP and cADPR in the induction and maintenance of agonist-evoked Ca²⁺spiking in mouse pancreatic acinar cells." Current biology 15.9 (2005): 874-878.