Biol Res 37: 559-563, 2004 R

ARTICLE

To be or not to be… Nicotinic acid adenine dinucleotide phosphate (NAADP): A new intracellular second messenger?

EDUARDO NUNES CHINI

Department of Anesthesiology, Mayo Clinic and Foundation, Rochester, Minnesota, 55905 USA

Dirección para Correspondencia

ABSTRACT

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent activator of intracellular Ca²⁺ release in several vertebrate and invertebrate systems. The role of the NAADP system in physiological processes is being extensively investigated at the present time. The NAADP receptor and its associated Ca²⁺ pool have been hypothesized to be important in several physiological processes including fertilization, T cell activation, and pancreatic secretion. However, whether NAADP is a new second messenger or a tool for the discovery of a new Ca²⁺ channel is still an unanswered question. Research developed over the last two years has provided some important clues to whether NAADP is or not a physiological cellular messenger. In this short review, I will discuss some of these new findings that are helping us to find an answer to the important question: Is NAADP a second messenger or not?

Key words: NAADP, Ca²⁺, channels, cADPR, intracellular, second messenger

INTRODUCTION

The discovery of intracellular second messengers represented a major step in understanding how extracellular signals are capable of regulating cellular behavior (5). In this regard, the release of intracellular calcium ions (Ca²⁺) plays a fundamental role in cell signaling (5; see also Carafoli, this issue, reference 7). Release of Ca²⁺ from intracellular stores, such as the endoplasmic and sarcoplasmic reticulum, is a key component in several intracellular signaling pathways (5).

Recently, we and others have found that the nucleotide nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent activator of intracellular Ca²⁺ release (10, 24). This nucleotide activates an intracellular Ca²⁺ release mechanism that differs in many ways from that modulated by both IP₃ and cADPR (9-10, 12, 14-16, 18, 20-25). In contrast to IP₃ and cADPR, the research on NAADP is only in its infancy, and further experimentation is needed to determine the precise role of this Ca²⁺-releasing pathway in cell signaling. In this review, I discuss several aspects of NAADP research and analyze some of the recent data that indicates that NAAADP may be an intracellular second messenger.

THE NAADP MOLECULE

NAADP has a molecular mass only 1 Da larger than b_NADP (Fig. 1) (10, 12, 24). The difference between the molecule of NAADP and NADP is the change of an NH₂ of the amide in NADP to OH of the carboxyl group in NAADP (10, 12, 24). With this substitution NADP is transformed into one of the most potent activators of intracellular Ca²⁺ release known to mankind (10, 12, 24) (Fig. 1).

Figure 1. Base-exchange reaction: The nicotinamide group of nicotinamide adenine dinucleotide phosphate (NADP+) is exchanged for nicotinic acid, resulting in nicotinic acid adenine dinucleotide phosphate (NAADP).

NAADP-INDUCED Ca²⁺ RELEASE

The mechanism of Ca²⁺ release elicited by NAADP was initially characterized in sea urchin eggs (5-12). In initial studies, the most striking feature of NAADP was its ability to induce Ca²⁺ release even after IP₃ and ryanodine channels had been desensitized previously (10, 24). This behavior suggests that another Ca²⁺ release mechanism, possibly a new Ca²⁺ channel, is involved in NAADP-mediated Ca²⁺ release. Several lines of evidence support this notion, including: 1) antagonists of ryanodine and IP₃ channels were ineffective in blocking NAADP-mediated Ca²⁺ release (10); 2) known modulators of ryanodine and IP₃ channels—such as Ca²⁺, Mg²⁺, caffeine, ryanodine, ruthenium red, and procaine—as well as pH did not influence NAADP-mediated Ca²⁺ release (10, 12); and 3) L-type Ca²⁺ channel antagonists could inhibit NAADP-induced Ca²⁺ release but not IP₃-induced Ca²⁺ release or CICR (21-22). Together, these findings revealed a distinct pharmacological behavior of the NAADP Ca²⁺ release system of sea urchin eggs, further strengthening the hypothesis that NAADP is an activator of a novel Ca²⁺ release system.

In recent years, we and others have observed that several cells from vertebrates and invertebrates are responsive to NAADP (2-4, 8-10, 12, 14-16, 18, 20-25, 28-32). Furthermore, NAADP has been implicated in several physiological processes, including among other processes, fertilization, pancreatic secretion and T-lymphocyte activation (8, 19, 31, 4).

The molecular identity of the NAADP receptor is still obscure. Nevertheless, specific binding of radioactive NAADP has been described in sea urchin eggs (29) and in rat brain with autoradiographic techniques (30). A single and saturable binding site was more thoroughly characterized in sea urchin egg (23, 29). Ca²⁺ and pH did not affect NAADP binding, which probably explains why Ca²⁺ and pH do not influence NAADP-induced Ca²⁺ release in sea urchin eggs. More importantly, NAADP binding seemed to be irreversible (23, 29), suggesting an explanation for the molecular basis of the inactivation phenomenon observed with subthreshold concentrations of NAADP. Ongoing work from several laboratories will surely very soon provide us with the anxiously anticipated structure of the NAADP receptor.

IS CD38 THE ENZYME RESPONSIBLE FOR THE SYNTHESIS OF NAADP?

This is one of the most important questions that we face at this time on this new field of research. Synthesis of NAADP has been previously described in several tissues including brain, liver, spleen, heart, and kidney glomeruli. Synthesis of NAADP can be catalyzed in vitro by a NAD(P)ase, analogous to the lymphocyte antigen CD38 (1, 11, 13, 26), in a reaction called the base-exchange reaction (Fig.1). The enzyme catalyzes the exchange of nicotinamide for nicotinic acid on the molecule of NADP⁺, generating NAADP (Fig.1, 1, 11, 13, 26). Whether NAADP can be generated via the base-exchange reaction in vivo is still an open question. Under the present experimental conditions used for synthesis of NAADP, the concentrations of substrate needed, namely nicotinic acid, are several times higher than would be expected to be present in intact cells (1, 11, 13, 26). Furthermore, the optimal pH for this reaction is out of the physiological range (1, 11, 13, 26). However, compartmentalization of nicotinic acid and NADP into an acid environment could theoretically provide a possible milieu for the synthesis of NAADP in vivo. Another theoretical problem is the fact that in mammalian cells, the base-exchange reaction seems to be catalyzed by CD38, which is an ectoenzyme. This feature, therefore, raises the question of how substrates would be available to the CD38 catalytic site and, once NAADP is generated, how it would be made available in the cytosol to induce Ca²⁺ release.

Despite the limitations discussed, the base-exchange reaction is the only pathway currently described for the synthesis of NAADP in biological systems (1, 11, 13, 26). However, as discussed above, whether the base-exchange reaction occurs under physiological conditions is still an open question. Using CD38 knockout mice, we determined that CD38 is the major enzyme responsible for the base-exchange reaction in mouse tissue (11). In our experiments, however, we performed in vitro synthesis of NAADP, and we did not measure intracellular levels of NAADP. Recent work from our laboratory using a method designed to detect in vivo levels of NAADP indicates that CD38 may not be the enzyme responsible for the synthesis of NAADP in vivo. Furthermore, our recent data also indicate that the base-exchange reaction may not be the physiological pathway for the synthesis of NAADP.

IS NAADP A NEW SECOND MESSENGER?

In a recent review we have proposed that several requirements had to be fulfilled before NAADP could be considered an intracellular messenger (12). These include:

1) NAADP levels must be determined in cells.
2) The physiological pathways for the synthesis of NAADP must be defined.
3) The concentration of intracellular NAADP must be regulated by external or internal stimuli.
4) A correlation between stimulated intracellular NAADP levels and Ca²⁺ release must be established.

We then concluded our review describing that at that time none of these requirements had been demonstrated for NAADP (12). Very recent data, however, has indicated that NAADP may be a second messenger. In 2003 an NAADP-like molecule present in both invertebrate and mammalian cells was described for the first time (19, 27). Furthermore, it was also found that the intracellular levels of the NAADP-like molecule were modulated by extracellular stimulus (19, 27), indicating that NAADP is present in cells and that its synthesis or mobilization is regulated. These data strongly indicate that NAADP is indeed a second messenger. However, several other questions are still unanswered. In fact, as discussed above, the pathway for the synthesis of NAADP has not been elucidated. Whether intracellular generated NAADP can regulate cellular functions has not been demonstrated either, and finally, the structure of the NAADP receptor has not been reported. However, several laboratories are working as "we speak" on these important questions, and I believe that the missing pieces of the NAADP puzzle are very close to clarification. Certainly, the future holds new and exciting discoveries in this field.

ACKNOWLEDGEMENTS

We acknowledge the excellent secretarial assistance provided by Lea Dacy, and for the excellent technical assistance of Mr. Michael Thompson. The Mayo Foundation and the Federation for Anesthesia Research (FAER) supported my research.

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Corresponding author: E. N. Chini, Dept. of Anesthesiology Mayo Clinic and Foundation 200 First St., Rochester, MN, 55905 USA. Phone: (1-507) 255-0992, Fax: (1-507) 284-8566, E-mail: chini.eduardo@mayo.edu

Received: January 16, 2004. Accepted: March 8, 2004.