INVITED REVIEW
Nicotinic acid adenine dinucleotide phosphate: a new intracellular second messenger?

Eduardo N. Chini1 and Frederico G. S. De Toledo2

1 Department of Anesthesiology, Mayo Clinic and Foundation, Rochester, Minnesota 55905; and 2 Department of Internal Medicine, University of Miami, Jackson Memorial Medical Center, Miami, Florida 33136


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
STRUCTURE AND DISCOVERY OF...
UNIQUE MECHANISM OF...
CELLS RESPONSIVE TO NAADP
NAADP RECEPTOR
SYNTHESIS AND DEGRADATION OF...
REGULATION OF NAADP SYNTHESIS
ROLE OF NAADP IN...
NAADP CA2+ POOL
CONCLUSION---NAADP: A NEW...
REFERENCES

Nicotinic acid adenine dinucleotide phosphate (NAADP) is one of the most potent stimulators of intracellular Ca2+ release known to date. The role of the NAADP system in physiological processes is being extensively investigated at the present time. Exciting new discoveries in the last 5 years suggest that the NAADP-regulated system may have a significant role in intracellular Ca2+ signaling. 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.

calcium; endoplasmic reticulum; fertilization; sea urchin eggs; cyclic adenosine 5'-diphosphate-ribose


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
STRUCTURE AND DISCOVERY OF...
UNIQUE MECHANISM OF...
CELLS RESPONSIVE TO NAADP
NAADP RECEPTOR
SYNTHESIS AND DEGRADATION OF...
REGULATION OF NAADP SYNTHESIS
ROLE OF NAADP IN...
NAADP CA2+ POOL
CONCLUSION---NAADP: A NEW...
REFERENCES

THE DISCOVERY of intracellular second messengers represented a major step in understanding how extracellular signals are capable of regulating cellular behavior. In this regard, the release of intracellular calcium ions (Ca2+) plays a fundamental role in cell signaling. Release of Ca2+ from intracellular stores, such as the endoplasmic and sarcoplasmic reticulum, is a key component in several intracellular signaling pathways. Ca2+ fluxes display complex spatial and temporal signatures, enabling more information to be encoded by Ca2+ signals. To meet the demands of this complexity, cells rely on precise regulation of Ca2+ channel activity (7). Understanding of the regulation of intracellular Ca2+ release and its relationship to extracellular stimuli was greatly enhanced by the discovery of the inositol 1,4,5-trisphosphate (IP3) signaling pathway (7). In addition to IP3-induced Ca2+ release, cells contain other mechanisms for intracellular Ca2+ release (7, 23, 25, 27, 33, 35).

One of these Ca2+-releasing pathways is regulated by the newly discovered nucleotide cyclic ADP-ribose (cADPR). cADPR was discovered in 1987 by H. C. Lee and collaborators (23), who observed that incubation of sea urchin egg homogenates with nicotinamide adenine dinucleotide (NAD+) resulted in Ca2+ release from microsomal stores (23, 33). Subsequent studies revealed that the Ca2+ release activity of NAD+ was actually due to conversion of NAD+ to an active metabolite, later identified as a cyclic compound derived from the ADP-ribose moiety of NAD+ and named cADPR (39). In 1991, it was concluded that cADPR mobilizes Ca2+ by activation or sensitization of the so-called ryanodine receptor/channel (RyR) (26).

The Ca2+-releasing properties of cADPR suggested a signaling role for this molecule. Since the discovery of cADPR, much interest has been raised about the possible role of other nucleotides as second messengers involved in control of intracellular Ca2+. In fact, it was discovered that another nucleotide, nicotinic acid adenine dinucleotide phosphate (NAADP), is a potent activator of intracellular Ca2+ release (13, 36). This nucleotide activates an intracellular Ca2+ release mechanism that differs in many ways from that modulated by both IP3 and cADPR (1, 7-13, 16-18, 20-23, 25-31, 33-38). 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 we discuss several aspects of NAADP research and the potential role of NAADP in cellular signal transduction. NAADP has been the subject of recent descriptive reviews by other authors (27, 37, 39, 44). While briefly describing the major findings in the field, this review is devoted to a critical appraisal of several key issues that need to be resolved to determine whether NAADP is a new second messenger or a tool for the discovery of a new class of Ca2+ channels. In any case, studies of the NAADP Ca2+ release system will provide exciting new information about the complex mechanism of intracellular Ca2+ mobilization.


    STRUCTURE AND DISCOVERY OF NAADP
TOP
ABSTRACT
INTRODUCTION
STRUCTURE AND DISCOVERY OF...
UNIQUE MECHANISM OF...
CELLS RESPONSIVE TO NAADP
NAADP RECEPTOR
SYNTHESIS AND DEGRADATION OF...
REGULATION OF NAADP SYNTHESIS
ROLE OF NAADP IN...
NAADP CA2+ POOL
CONCLUSION---NAADP: A NEW...
REFERENCES

In 1987 it was discovered that incubation of NADP in alkaline pH generated a Ca2+-releasing metabolite (23). However, it was not until 1995 that it was described for the first time that a nicotinic acid derivative of NADP was a potent mobilizer of intracellular Ca2+ in sea urchin egg homogenates, an experimental system in which IP3-induced Ca2+ release and Ca2+-induced Ca2+ release (CICR) can be measured easily in real time (13, 36). Our laboratory analyzed the products of alkali-treated beta -NADP, and, using several physicochemical methods, we (13) found that the Ca2+ releasing activity was mediated by a nucleotide with a molecular mass only 1 Da larger than beta -NADP (Fig. 1). We concluded that the substance with Ca2+-releasing properties was a NADP-related compound that has a nicotinic acid instead of a nicotinamide in the molecule. We (13) then described this molecule as NAADP. In fact, the only difference between NAADP and NADP is the change of an NH2 of the amide in NADP to OH of the carboxyl group in NAADP. This substitution accounts for a difference of 1 Da between the compounds (Fig. 1).


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 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).

The structural requirements of NAADP-induced Ca2+ release system appear to be very stringent, because several structural analogs of NAADP have no effect on intracellular Ca2+ release (13, 35). Of particular interest, the phosphate in position 2' is crucial for the biological activity of NAADP, because NAAD has no Ca2+-mobilizing property (13, 35). However, changing the position of the third phosphate from 2' to 3' has no effect on the Ca2+-releasing properties of the molecule (13, 35). In fact, whether the third phosphate is in position 2' or 3' or whether it is cyclic on positions 2' and 3' does not change the Ca2+-mobilizing properties of this nucleotide (13, 35). Recently, a fluorescent analog of NAADP, 1,N6-etheno-NAADP, with Ca2+-mobilizing properties was synthesized (37). This compound may be a useful tool for the identification of the NAADP receptor.


    UNIQUE MECHANISM OF INTRACELLULAR CA2+ RELEASE
TOP
ABSTRACT
INTRODUCTION
STRUCTURE AND DISCOVERY OF...
UNIQUE MECHANISM OF...
CELLS RESPONSIVE TO NAADP
NAADP RECEPTOR
SYNTHESIS AND DEGRADATION OF...
REGULATION OF NAADP SYNTHESIS
ROLE OF NAADP IN...
NAADP CA2+ POOL
CONCLUSION---NAADP: A NEW...
REFERENCES

The mechanism of Ca2+ release elicited by NAADP was initially characterized in sea urchin eggs (13, 26-31, 36, 46). In initial studies, the most striking feature of NAADP was its ability to induce Ca2+ release even after IP3 and ryanodine channels had previously been desensitized (13, 36). This behavior suggested that another Ca2+ release mechanism, possibly a new Ca2+ channel, was involved in NAADP-mediated Ca2+ release. Other lines of evidence supported this notion, including findings that 1) antagonists of ryanodine and IP3 channels were ineffective in blocking NAADP-mediated Ca2+ release (13); 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 (Table 1; Refs. 11-13, 16-18, 25, 27-31, 33, 35, 36); and 3) L-type Ca2+ channel antagonists could inhibit NAADP-induced Ca2+ release but not IP3-induced Ca2+ release or CICR (28-31). 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.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Unique properties of NAADP-induced Ca2+ release system

A remarkable distinct property of the NAADP Ca2+ release system is the self-inactivation mechanism elicited by "low" doses of NAADP: sea urchin egg homogenates preexposed to a subthreshold concentration of NAADP that does not elicit Ca2+ release per se become unresponsive to further challenges of maximal doses of NAADP (1, 28, 33). This self-inactivation mechanism is time and dose dependent, suggesting that a specific NAADP binding site is required. This behavior is also suggestive of irreversible binding of NAADP to the receptor, possibly locking the Ca2+ channel in a closed state, but this remains to be demonstrated. The inactivation mechanism might permit the NAADP Ca2+ release system to be activated in cells only once, or not at all if a low concentration of NAADP inactivates the receptor first. These characteristics suggest that the mechanism of Ca2+ release induced by NAADP may be highly tuned to detect sudden increases in NAADP concentration. Moreover, if the self-inactivation mechanism of the NAADP Ca2+ release system indeed occurs in vivo, it raises the intriguing question of whether it could represent a simple form of cell memory (22), such as that required in one-time events like egg fertilization or lymphocyte activation. All these characteristics make NAADP a rather unique trigger of intracellular Ca2+ release.


    CELLS RESPONSIVE TO NAADP
TOP
ABSTRACT
INTRODUCTION
STRUCTURE AND DISCOVERY OF...
UNIQUE MECHANISM OF...
CELLS RESPONSIVE TO NAADP
NAADP RECEPTOR
SYNTHESIS AND DEGRADATION OF...
REGULATION OF NAADP SYNTHESIS
ROLE OF NAADP IN...
NAADP CA2+ POOL
CONCLUSION---NAADP: A NEW...
REFERENCES

Invertebrate cells. Ca2+ release induced by NAADP was first described in sea urchin egg homogenates and in intact sea urchin eggs (13, 36, 46). These preparations provide an easy system to measure Ca2+ release from microsomal stores with fluorescent Ca2+-sensitive molecular probes. In addition, both IP3-responsive channels and RyR are present in the same preparation. In this experimental system, NAADP induced a robust Ca2+ release from both thapsigargin (tg)-sensitive and -insensitive stores (29). As discussed previously, NAADP-induced Ca2+ release remarkably differs in many ways from the Ca2+ release elicited by IP3 and cADPR (13, 36): 1) Ca2+ release induced by NAADP is not cross-desensitized by cADPR or IP3; 2) NAADP-induced Ca2+ release is not inhibited by antagonists of RyR and IP3 channels; and 3) NAADP-induced Ca2+ release is inhibited by L-type Ca2+ channel blockers whereas the cADPR and IP3 channels are not.

The precise role of NAADP-mediated Ca2+ release in sea urchin egg fertilization is not known. However, preliminary evidence indicates that NAADP-sensitive Ca2+ stores are activated during fertilization (46). In fact, we (46) showed that fertilization of the sea urchin egg leads to a complete inactivation of the NAADP-induced Ca2+ release. These data indicate that the Ca2+ pool regulated by NAADP may have an important role during sea urchin egg fertilization. NAADP-induced Ca2+ release has also been demonstrated in intact starfish and ascidian oocytes (47).

Plant cells. Recently, NAADP-induced Ca2+ release was described in cauliflower and red beet microsomes (42). However, the physiological role of this compound in plants remains unknown.

Mammalian cells. Until recently, research on NAADP-induced Ca2+ release was largely limited to invertebrate cells, in part because of the advantages of measuring Ca2+ fluxes in sea urchin preparations. More recently, NAADP-induced Ca2+ release has been shown to be widespread in mammalian cells and tissues (Table 2), including rat brain, T lymphocytes, vascular smooth muscle cells, cardiac myocytes, fibroblasts, and HL-60 cells (3-5, 20, 50).

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Tissues and cells in which NAADP-induced Ca2+ release has been described

It is important to note that although recent evidence indicates that mammalian cells appear to have the NAADP-responsive Ca2+ channel, the majority of studies have been carried out in microsomal vesicles passively loaded with Ca2+. In the future it will be important to determine whether NAADP can activate intracellular Ca2+ release in more physiological conditions. A few studies have been conducted in intact cells, however. The extensive work of Cancela and collaborators (9, 10) demonstrated that intact pancreatic acinar cells have a NAADP-responsive Ca2+ release system. However, the effect of NAADP on intracellular Ca2+ has been measured indirectly with whole cell patch clamp of Ca2+-dependent currents through Cl- and nonselective cation channels (9, 10). Moreover, the direct effect of NAADP on these currents is not known, and it is presumed that the effects of NAADP on the whole cell patch-clamp system are completely due to the effect of NAADP on intracellular Ca2+. Another important work in intact cells was conducted in human Jurkat T lymphocytes (5). Microinjection of NAADP in these cells was shown to induce a dose-dependent mobilization of intracellular Ca2+, which was studied with imaging using an intracellular Ca2+-sensitive fluorescent dye. Akin to sea urchin eggs, NAADP-induced Ca2+ release in T cells displayed characteristics dissimilar to the cADPR and IP3 Ca2+ release systems, suggesting that it may activate a unique Ca2+ channel (5). In addition to Ca2+ release properties, NAADP was implicated in T cell activation, because self-inactivation of the NAADP system abolished subsequent stimulation of Ca2+ signaling via the T cell receptor-CD3 complex (5).

An important point to be considered in evaluating the role of NAADP-induced Ca2+ release in any cell type is the validation of specificity of the NAADP effect. For instance, it is of utmost importance to document negative controls with the NAADP analogs NAAD and NADP. However, these controls appear to be lacking in many reports. Without these appropriate controls, claims of finding specific NAADP-induced Ca2+ release should be viewed with caution.

In summary, Ca2+ mobilization induced by NAADP has been found in both intact cells and cell-free preparations of various vertebrates and invertebrates (Table 2). Despite this progress, it remains to be established whether a physiological rather than a pharmacological NAADP signaling system is present in a widespread mode in cells. In contrast to the IP3- and Ca2+-induced Ca2+ release systems, characterization of a ubiquitous functional NAADP signaling system is still in a very early stage.


    NAADP RECEPTOR
TOP
ABSTRACT
INTRODUCTION
STRUCTURE AND DISCOVERY OF...
UNIQUE MECHANISM OF...
CELLS RESPONSIVE TO NAADP
NAADP RECEPTOR
SYNTHESIS AND DEGRADATION OF...
REGULATION OF NAADP SYNTHESIS
ROLE OF NAADP IN...
NAADP CA2+ POOL
CONCLUSION---NAADP: A NEW...
REFERENCES

The molecular identity of the NAADP receptor is still obscure. Nevertheless, specific binding of radioactive NAADP has been described in sea urchin eggs (8, 43) and in rat brain with autoradiographic techniques (45). A single and saturable binding site was more thoroughly characterized in sea urchin eggs (8, 43). In microsomes, the dissociation constant (Kd) was 280 pM, consistent with a high-affinity binding receptor (8). NADP and NAADPH appear to compete for binding (8, 43), but it is unclear whether this is due to NAADP present as a contaminant in NADP and NAADPH solutions used in these studies. 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 (8, 44), suggesting an explanation for the molecular basis of the inactivation phenomenon observed with subthreshold concentrations of NAADP.

Furthermore, NAADP binding has also been described in brain microsomes (45), and with autoradiographic techniques those authors described that NAADP binding sites are diffusely distributed in rat brain tissues.


    SYNTHESIS AND DEGRADATION OF NAADP
TOP
ABSTRACT
INTRODUCTION
STRUCTURE AND DISCOVERY OF...
UNIQUE MECHANISM OF...
CELLS RESPONSIVE TO NAADP
NAADP RECEPTOR
SYNTHESIS AND DEGRADATION OF...
REGULATION OF NAADP SYNTHESIS
ROLE OF NAADP IN...
NAADP CA2+ POOL
CONCLUSION---NAADP: A NEW...
REFERENCES

We (15, 19, 20, 41, 50) previously described synthesis of NAADP in several tissues including brain, liver, spleen, heart, and kidney glomeruli. Synthesis of NAADP can be catalyzed in vitro by a NAD(P)ase, analog to the lymphocyte antigen CD38 (2, 19), in a reaction called the base-exchange reaction (Fig. 2; Ref. 6). The enzyme catalyzes the exchange of nicotinamide for nicotinic acid on the molecule of NADP+, generating NAADP (Fig. 2; Refs. 6, 13, 16, 19). 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 (6, 13, 19). Furthermore, the optimal pH for this reaction is out of the physiological range (2, 20). 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 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.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 2.   Potential alternative routes for synthesis of NAADP. NAADP could conceivably be generated from NADP by replacing the NH2 group of nicotinamide for a OH group. Another possible route would be phosphorylation of nicotinic acid adenine dinucleotide (NAAD) by a putative NAAD kinase.

For these reasons, it is important to consider other theoretical pathways for the synthesis of NAADP in vivo (Fig. 2). Conceivably, NAADP might be generated by deamination of NADP+ (Fig. 2) or phosphorylation of NAAD+. The latter is a particularly attractive hypothetical route because NAAD+ is a compound present in cells and NAADP might be then catalyzed by NAD+ kinase with ATP as a 2'-phosphate donor (Fig. 2). These alternative synthetic pathways ought to be explored in future studies. Notably, Lerner et al. (40) characterized a human NAD+ kinase in vitro and found no evidence that it could synthesize NAADP by phosphorylation of NAAD+. Nevertheless, these data do not completely exclude the possibility that NAAD+ phosphorylation might perhaps occur in vivo, if intracellular cofactors or other putative physiological conditions are required to modify the enzyme and enable the reaction. They also do not exclude the possibility that other isoforms might catalyze the reaction. Therefore, the postulated NAAD+ phosphorylation pathway seems unlikely at this point but cannot be completely discarded yet.

Despite the limitations discussed, the base-exchange reaction is the only pathway currently described for the synthesis of NAADP in biological systems (2, 4, 6, 13, 19, 20, 42, 50). In this regard, an important observation is that enzymes with ADP-ribosyl cyclase activity (capacity for synthesis of cyclic ADP-ribose) are also able to catalyze the synthesis of NAADP through the base-exchange reaction (2, 14, 19, 24). In fact, the mammalian version of the ADP-ribosyl cyclase (CD38) is capable of generating both NAADP and cADPR (2, 14). This observation led to the proposal of a cross talk between these two possible signaling pathways (34). However, as discussed above, whether the base-exchange reaction occurs under physiological conditions is still an open question. Using CD38 knockout mice, we (13a) determined that CD38 is the major enzyme responsible for the base-exchange reaction in mouse tissues. However, in one study (50) the capacity for synthesis of NAADP by the base-exchange reaction in cells did not correlate with the presence of NAADP-induced Ca2+ release in the same cells. As result, this discrepancy raises doubts about the role of the base-exchange reaction as the physiological route for the synthesis of NAADP.

Far less is known about the pathways of NAADP degradation. NAADP hydrolysis has been described in several mammalian tissues including kidney, heart, spleen, liver, and brain (15). This activity appears to be mediated by the tissue alkaline phosphatase, and, in fact, isolated alkaline phosphatase is capable of NAADP hydrolysis (13).


    REGULATION OF NAADP SYNTHESIS
TOP
ABSTRACT
INTRODUCTION
STRUCTURE AND DISCOVERY OF...
UNIQUE MECHANISM OF...
CELLS RESPONSIVE TO NAADP
NAADP RECEPTOR
SYNTHESIS AND DEGRADATION OF...
REGULATION OF NAADP SYNTHESIS
ROLE OF NAADP IN...
NAADP CA2+ POOL
CONCLUSION---NAADP: A NEW...
REFERENCES

To be considered a second messenger, the intracellular concentration of NAADP would be expected to change in response to physiological stimuli. In fact, because low concentrations of NAADP inactivate the NAADP receptor, one would expect that NAADP levels have to rise rapidly in the cytosol to activate Ca2+ release. Such a rapid rise in NAADP synthesis would demand a fast activation of enzymes involved in NAADP synthesis. Very little is known, however, about how enzymes involved in NAADP metabolism are regulated. One study in sea urchin eggs demonstrated that both cAMP and cGMP could enhance synthesis of NAADP by a membrane-bound enzyme (49). However, cGMP did not affect NAADP synthesis in another study (32). These important points need to be addressed in future studies.

Regulation of NAADP synthesis might theoretically also be upregulated by hormones or agents that increase expression of enzymes capable of catalyzing the base-exchange reaction, such as ADP-ribosyl cyclases and CD38 (14, 24, 48). For example, we (20, 24) demonstrated that retinoic acid enhances the activity of ADP-ribosyl cyclase in rat smooth muscle and mesangial cells. Likewise, when cultured rat mesangial cells were incubated with 9-cis-retinoic acid, increased synthesis of NAADP was observed (20). The exact role of this and other examples of convergence of the cADPR and NAADP synthetic pathways still remains largely unexplored.


    ROLE OF NAADP IN INTRACELLULAR CA2+ HOMEOSTASIS
TOP
ABSTRACT
INTRODUCTION
STRUCTURE AND DISCOVERY OF...
UNIQUE MECHANISM OF...
CELLS RESPONSIVE TO NAADP
NAADP RECEPTOR
SYNTHESIS AND DEGRADATION OF...
REGULATION OF NAADP SYNTHESIS
ROLE OF NAADP IN...
NAADP CA2+ POOL
CONCLUSION---NAADP: A NEW...
REFERENCES

The unique Ca2+-releasing properties of NAADP make it an exciting candidate for an intracellular messenger. The data obtained in sea urchin egg homogenates, in which the normal architecture of intracellular Ca2+ stores is lost, indicate that NAADP-induced Ca2+ release is completely distinct and independent of the other intracellular Ca2+-releasing systems regulated by cADPR and IP3 (13, 36). As discussed above, in sea urchin egg homogenates the NAADP-regulated Ca2+ system is not inhibited by inhibitors of the cADPR and IP3 systems. However, in intact cells the NAADP system appears to interact actively with other intracellular Ca2+ systems (9, 10, 21, 47). In fact, it has been proposed, for example, that in pancreatic acinar cells NAADP would be the trigger of Ca2+ oscillations induced by cholecystokinin (CCK) (9, 10) and that Ca2+ released by NAADP, in response to CCK, would activate the CICR mediated by cADPR and IP3. These interactions between Ca2+ systems will lead to amplification of the Ca2+ signaling and generation of the Ca2+ oscillation (9, 10). In fact, self-inactivation of the NAADP receptor in intact pancreatic acinar cells attenuates the Ca2+ signal in response to CCK (9, 10).

A similar role for NAADP has been proposed for the mobilization of Ca2+ in intact starfish oocyte and sea urchin eggs (21, 47). In these cells, microinjection or release of caged NAADP leads to a robust Ca2+ release followed by oscillations (21, 47). It appears that in these intact invertebrate cells NAADP-induced Ca2+ release can further promote Ca2+ mobilization by activation or sensitization of the ryanodine and IP3 receptors (21, 47). In fact, in both intact sea urchin eggs and starfish oocytes the NAADP-induced Ca2+ oscillations can be inhibited by dual block of the cADPR and IP3 systems with 8-amino-cADPR (a cADPR antagonist) and heparin (an IP3 inhibitor). In contrast, NAADP-induced Ca2+ oscillations in these intact cells are insensitive to either heparin or 8-amino-cADPR alone (21, 47).

In fact, the Ca2+ released from the NAADP pool can modulate the intracellular Ca2+ release by at least two different mechanisms: 1) a mode of priming the intracellular Ca2+ pools as described by Churchill and Galione (21); and 2) direct sensitization of the CICR (Fig. 3). In the first case, it was demonstrated that the Ca2+ released from the NAADP pool will increase the amount of Ca2+ in tg-dependent stores (21). These stores correspond to the cADPR- and IP3-regulated pools (21). In this model, NAADP promotes Ca2+ oscillation by releasing Ca2+ from its tg-independent store and then the Ca2+ released is taken by CICR stores. The Ca2+ priming of the CICR stores leads to a cycle of Ca2+ overload, release, and reuptake that corresponds to the Ca2+ oscillations (21).


View larger version (50K):
[in this window]
[in a new window]
 
Fig. 3.   Interaction between NAADP and other Ca2+-release systems. Cross talk between the NAADP Ca2+-release system and the Ca2+-induced Ca2+-release (CICR) system may occur in 2 distinct modes. A: NAADP opens a distinct Ca2+-channel resulting in Ca2+ release from specific intracellular stores. Released Ca2+ is transported by Ca2+-ATPases into cyclic ADP-ribose (cADPR)-sensitive intracellular stores. These stores are then "primed" for Ca2+ release on activation of the ryanodine channel (RyR) by agonists such as cADPR or Ca2+ (CICR). B: Ca2+ released by NAADP directly activates RyR, resulting in CICR, which can be amplified by further CICR.

In the second case, the Ca2+ released by NAADP could affect the apparent affinity of the RyR (9, 10). In this case, Ca2+ released by NAADP sensitizes the RyR to its agonists by a mechanism similar to the so-called CICR (Fig. 3B).


    NAADP CA2+ POOL
TOP
ABSTRACT
INTRODUCTION
STRUCTURE AND DISCOVERY OF...
UNIQUE MECHANISM OF...
CELLS RESPONSIVE TO NAADP
NAADP RECEPTOR
SYNTHESIS AND DEGRADATION OF...
REGULATION OF NAADP SYNTHESIS
ROLE OF NAADP IN...
NAADP CA2+ POOL
CONCLUSION---NAADP: A NEW...
REFERENCES

The NAADP-regulated Ca 2+ store in sea urchin eggs is physically distinct from the cADPR and IP3 pools. In fact, two different mechanisms of intracellular Ca2+ uptake are observed in sea urchin egg homogenates. Sea urchin egg homogenates have both tg-sensitive and -insensitive Ca2+ uptake systems. These data indicate that egg homogenates have both a sarco(endo) plasmic reticulum Ca2+-ATPase (SERCA)-like pool and also a second different mechanism of Ca2+ uptake that is not mediated by a SERCA-like enzyme. Genazzani and Galione (29) demonstrated that cADPR and IP3 promoted Ca2+ release only through the tg-sensitive pools. In contrast, NAADP is able to induce Ca2+ release from both tg-sensitive and -insensitive pools, indicating that, in sea urchin egg homogenates, the NAADP and cADPR Ca2+ pools are at least to some extent independent (29). More recently, it was demonstrated that the NAADP and cADPR pools can be segregated to opposite poles of intact sea urchin eggs by centrifugation (38). In addition, it was demonstrated that the NAADP-regulated Ca2+ pool in sea urchin eggs is distinct from the endoplasmic reticulum and mitochondria. This new, yet unidentified, Ca2+ pool provides exciting possibilities in NAADP research, and its identification may lead to the discovery of a new type of intracellular organelle involved in Ca2+ homeostasis.


    CONCLUSION---NAADP: A NEW INTRACELLULAR MESSENGER OR A PHARMACOLOGICAL TOOL FOR DISCOVERY OF A UNIQUE CA2+ CHANNEL?
TOP
ABSTRACT
INTRODUCTION
STRUCTURE AND DISCOVERY OF...
UNIQUE MECHANISM OF...
CELLS RESPONSIVE TO NAADP
NAADP RECEPTOR
SYNTHESIS AND DEGRADATION OF...
REGULATION OF NAADP SYNTHESIS
ROLE OF NAADP IN...
NAADP CA2+ POOL
CONCLUSION---NAADP: A NEW...
REFERENCES

Over the last decade, intensive research on mechanisms of intracellular calcium regulation has led to the discovery of potential new second messengers and a novel Ca2+ release system. The role of the NAADP system in physiological processes is being extensively investigated at the present time, and the title of this review may imply that we can provide an answer for this question. However, although NAADP displays many characteristics of a signal transduction molecule, in our opinion, we are far from answering whether NAADP is indeed an intracellular messenger.

Several requirements must be fulfilled before NAADP can be considered an intracellular messenger. 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.

To date, none of these requirements has been completely fulfilled, and it would be premature to promote NAADP to the status of second messenger at this point. In fact, it is possible that NAADP may not be an intracellular messenger, and, in analogy to ryanodine, NAADP may be a pharmacological, rather than physiological, agonist of a new intracellular Ca2+ channel. However, even if NAADP turns out not to be a physiological agonist, it will lead to the discovery of a new class of intracellular Ca2+ channels with unique properties relevant to cell physiology. As discussed in this review, several pieces of the NAADP puzzle await clarification through more research investigation. Certainly, the future holds new and exciting discoveries in this field.


    ACKNOWLEDGEMENTS

We acknowledge the excellent secretarial assistance provided by Lea Dacy.


    FOOTNOTES

Mayo Foundation and the American Heart Association supported this research.

Address for reprint requests and other correspondence: E. N. Chini, Dept. of Anesthesiology, Mayo Clinic and Foundation, 200 First St., Rochester, MN 55905 (E-mail: chini.eduardo{at}mayo.edu).

10.1152/ajpcell.00475.2001


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
STRUCTURE AND DISCOVERY OF...
UNIQUE MECHANISM OF...
CELLS RESPONSIVE TO NAADP
NAADP RECEPTOR
SYNTHESIS AND DEGRADATION OF...
REGULATION OF NAADP SYNTHESIS
ROLE OF NAADP IN...
NAADP CA2+ POOL
CONCLUSION---NAADP: A NEW...
REFERENCES

1.   Aarhus, R, Dickey DM, Graeff RM, Gee KR, Walseth TF, and Lee HC. Activation and inactivation of Ca2+ release by NAADP+. J Biol Chem 271: 8513-8516, 1996[Abstract/Free Full Text].

2.   Aarhus, R, Graeff RM, Dickey DM, Walseth TF, and Lee HC. ADP-ribosyl cyclase and CD38 catalyze the synthesis of a calcium-mobilizing metabolite from NADP. J Biol Chem 270: 30327-30333, 1995[Abstract/Free Full Text].

3.   Bak, J, Billington RA, Timar G, Dutton AC, and Genazzani AA. NAADP receptors are present and functional in the heart. Curr Biol 11: 987-990, 2001[ISI][Medline].

4.   Bak, J, White P, Timar G, Missiaen L, Genazzani AA, and Galione A. Nicotinic acid adenine dinucleotide phosphate triggers Ca2+ release from brain microsomes. Curr Biol 9: 751-754, 1999[ISI][Medline].

5.   Berg, I, Potter BVL, Mayr GW, and Guse A. Nicotinic acid adenine dinucleotide phosphate (NAADP+) is an essential regulator of T-lymphocyte Ca2+-signaling. J Cell Biol 150: 581-588, 2000[Abstract/Free Full Text].

6.   Bernofsky, C. Nicotinic acid adenine dinucleotide phosphate (NAADP+). Methods Enzymol 66: 105-112, 1980[Medline].

7.   Berridge, MJ, Lipp P, and Bootman MD. The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1: 11-21, 2000[ISI][Medline].

8.   Billington, RA, and Genazzani AA. Characterization of NAADP+ binding in sea urchin eggs. Biochem Biophys Res Commun 276: 112-116, 2000[ISI][Medline].

9.   Cancela, JM, Churchill GC, and Galione A. Coordination of agonist-induced Ca2+-signalling patterns by NAADP in pancreatic acinar cells. Nature 398: 74-76, 1999[ISI][Medline].

10.   Cancela, JM, Gerasimenko OV, Gerasimenko JV, Tepikin AV, and Petersen OH. Two different but converging messenger pathways to intracellular Ca2+ release: the roles of nicotinic acid adenine dinucleotide phosphate, cyclic ADP-ribose and inositol trisphosphate. EMBO J 19: 2549-2557, 2000[Abstract/Free Full Text].

11.   Chini, EN. Effect of volatile anesthetics on cADP-ribose-induced Ca2+ release system. J Appl Physiol 91: 516-521, 2001[Abstract/Free Full Text].

12.   Chini, EN, Beers KW, Chini CC, and Dousa TP. Specific modulation of cyclic-ADP ribose-induced Ca2+ release by polyamines. Am J Physiol Cell Physiol 269: C1042-C1047, 1995[Abstract/Free Full Text].

13.   Chini, EN, Beers KW, and Dousa TP. Nicotinate adenine dinucleotide phosphate (NAADP) triggers a specific calcium release system in sea urchin eggs. J Biol Chem 270: 3216-3223, 1995[Abstract/Free Full Text].

13a.   Chini, EN, Chini CS, Kato I, Takasawa S, and Okamoto H. CD38 is the major enzyme responsible for the synthesis of NAADP in mammalian tissues. Biochem J 362: 125-130, 2002[ISI][Medline].

14.   Chini, EN, de Toledo FG, Thompson MA, and Dousa TP. Effect of estrogen upon cyclic ADP-ribose metabolism: beta -estradiol stimulates ADP-ribosyl cyclase in rat uterus. Proc Natl Acad Sci USA 94: 5872-5876, 1997[Abstract/Free Full Text].

15.   Chini, EN, and Dousa TP. Enzymatic synthesis and degradation of nicotinate adenine dinucleotide phosphate (NAADP), a Ca2+-releasing agonist, in rat tissues. Biochem Biophys Res Commun 205: 167-174, 1995.

16.   Chini, EN, and Dousa TP. Nicotinate-adenine dinucleotide phosphate-induced Ca2+-release does not behave as a Ca2+-induced Ca2+-release system. Biochem J 316: 709-711, 1996[ISI][Medline].

17.   Chini, EN, and Dousa TP. Palmitoyl-CoA potentiates the Ca2+ release elicited by cyclic ADP-ribose. Am J Physiol Cell Physiol 270: C530-C537, 1996[Abstract/Free Full Text].

18.   Chini, EN, Liang MY, and Dousa TP. Differential effect of pH upon cyclic-ADP-ribose and nicotinate-adenine dinucleotide phosphate-induced Ca2+ release systems. Biochem J 335: 499-504, 1998[ISI][Medline].

19.   Chini, EN, Thompson MA, and Dousa TP. Enzymatic synthesis of NAADP by ADP-ribosyl cyclases (Abstract). FASEB J 10: A143, 1996.

20.   Cheng, JF, Yusufi ANK, Thompson MA, Chini EN, and Grande JP. Nicotinic acid adenine dinucleotide phosphate: a new Ca2+ releasing agent in kidney. J Am Soc Nephrol 12: 54-60, 2001[Abstract/Free Full Text].

21.   Churchill, GC, and Galione A. NAADP induces Ca2+ oscillation via a two-pool mechanism by priming IP3- and cADPR-sensitive Ca2+ stores. EMBO J 20: 2666-2671, 2001[Abstract/Free Full Text].

22.   Churchill, GC, and Galione A. Prolonged inactivation of NAADP-induced Ca2+ release mediates a spatial temporal Ca2+ memory. J Biol Chem 276: 11223-11224, 2001[Abstract/Free Full Text].

23.   Clapper, DL, Walseth TF, Dargie PJ, and Lee HC. Pyridine nucleotide metabolites stimulate calcium release from sea urchin egg microsomes desensitized to inositol trisphosphate. J Biol Chem 262: 9561-9568, 1987[Abstract/Free Full Text].

24.   De Toledo, FGS, Cheng J, Liang M, Chini EN, and Dousa TP. ADP-ribosyl cyclase in rat vascular smooth muscle cells: properties and regulation. Circ Res 86: 1153-1159, 2000[Abstract/Free Full Text].

25.   Dousa, TP, Chini EN, and Beers KW. Adenine nucleotide diphosphates: emerging second messengers acting via intracellular Ca2+ release. Am J Physiol Cell Physiol 271: C1007-C1024, 1996[Abstract/Free Full Text].

26.   Galione, A, Lee HC, and Busa WB. Ca2+-induced Ca2+ release in sea urchin egg homogenates: modulation by cyclic ADP-ribose. Science 261: 1143-1146, 1991.

27.   Galione, A, Patel S, and Churchill GC. NAADP-induced calcium release in sea urchin eggs. Biol Cell 92: 197-204, 2000[ISI][Medline].

28.   Genazzani, AA, Empson RM, and Galione A. Unique inactivation properties of NAADP-sensitive Ca2+ release. J Biol Chem 271: 11599-11602, 1996[Abstract/Free Full Text].

29.   Genazzani, AA, and Galione A. Nicotinic acid-adenine dinucleotide phosphate mobilizes Ca2+ from a thapsigargin-insensitive pool. Biochem J 315: 721-725, 1996[ISI][Medline].

30.   Genazzani, AA, and Galione A. A Ca2+ release mechanism gated by the novel pyridine nucleotide, NAADP. Trends Pharmacol Sci 18: 108-110, 1997[ISI][Medline].

31.   Genazzani, AA, Mezna M, Dickey DM, Michelangeli F, Walseth TF, and Galione A. Pharmacological properties of the Ca2+-release mechanism sensitive to NAADP in the sea urchin egg. Br J Pharmacol 121: 1489-1495, 1997[Abstract].

32.   Graeff, RM, Franco L, De Flora A, and Lee HC. Cyclic GMP-dependent and -independent effects on the synthesis of the calcium messengers cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate. J Biol Chem 273: 118-125, 1998[Abstract/Free Full Text].

33.   Lee, HC. Mechanisms of calcium signaling by cyclic ADP-ribose and NAADP. Physiol Rev 77: 1133-1164, 1997[Abstract/Free Full Text].

34.   Lee, HC. A unified mechanism of enzymatic synthesis of two calcium messengers: cyclic ADP-ribose and NAADP. J Biol Chem 380: 785-793, 1999.

35.   Lee, HC. Physiological functions of cyclic ADP-ribose and NAADP as calcium messengers. Annu Rev Pharmacol Toxicol 41: 317-345, 2001[ISI][Medline].

36.   Lee, HC, and Aarhus R. A derivative of NADP mobilizes calcium stores insensitive to inositol trisphosphate and cyclic ADP-ribose. J Biol Chem 270: 2152-2157, 1995[Abstract/Free Full Text].

37.   Lee, HC, and Aarhus R. Fluorescent analogs of NAADP with calcium mobilizing activity. Biochim Biophys Acta 1425: 263-271, 1998[ISI][Medline].

38.   Lee, HC, and Aarhus R. Functional visualization of the separate but interacting calcium stores sensitive to NAADP and cyclic ADP-ribose. J Cell Sci 113: 4413-4420, 2000[Abstract/Free Full Text].

39.   Lee, HC, Walseth TF, Bratt GT, Hayes RN, and Clapper DL. Structural determination of a cyclic metabolite of NAD+ with intracellular Ca2+-mobilizing activity. J Biol Chem 264: 1608-1615, 1989[Abstract/Free Full Text].

40.   Lerner, F, Niere M, Ludwig A, and Ziegler M. Structural and functional characterization of human NAD kinase. Biochem Biophys Res Commun 288: 69-74, 2001[ISI][Medline].

41.   Liang, M, Chini EN, Cheng J, and Dousa TP. Synthesis of NAADP and cADPR in mitochondria. Arch Biochem Biophys 371: 317-325, 1999[ISI][Medline].

42.   Navazio, L, Bewell MA, Siddiqua A, Dickinson GD, Galione A, and Sanders D. Calcium release from the endoplasmic reticulum of higher plants elicited by the NADP metabolite nicotinic acid adenine dinucleotide phosphate. Proc Natl Acad Sci USA 97: 8693-8698, 2000[Abstract/Free Full Text].

43.   Patel, S, Churchill GC, and Galione A. Unique kinetics of nicotinic acid-adenine dinucleotide phosphate (NAADP) binding enhance the sensitivity of NAADP receptors for their ligand. Biochem J 352: 725-729, 2000[ISI][Medline].

44.   Patel, S, Churchill GC, and Galione A. Coordination of Ca2+ signaling by NAADP. Trends Biochem Sci 26: 482-489, 2001[ISI][Medline].

45.   Patel, S, Churchill GC, Sharp T, and Galione A. Widespread distribution of binding sites for the novel Ca2+-mobilizing messenger, nicotinic acid adenine dinucleotide phosphate, in the brain. J Biol Chem 275: 36495-36497, 2000[Abstract/Free Full Text].

46.   Perez-Terzic, CM, Chini EN, Shen SS, Dousa TP, and Clapham DE. Ca2+ release triggered by nicotinate adenine dinucleotide phosphate in intact sea urchin eggs. Biochem J 312: 955-959, 1995[ISI][Medline].

47.   Santella, L, Kyozuka K, Genazzani AA, De Riso L, and Carafoli E. Nicotinic acid adenine dinucleotide phosphate-induced Ca2+ release. J Biol Chem 275: 8301-8306, 2000[Abstract/Free Full Text].

48.   Takahashi, K, Kukimoto I, Tokita K, Inageda K, Inoue S, Kontani K, Hoshino S, Nishina H, Kanaho Y, and Katada T. Accumulation of cyclic ADP-ribose measured by a specific radioimmunoassay in differentiated human leukemic HL-60 cells with all-trans-retinoic acid. FEBS Lett 371: 204-208, 1995[ISI][Medline].

49.   Wilson, HL, and Galione A. Differential regulation of nicotinic acid-adenine dinucleotide phosphate and cADP-ribose production by cAMP and cGMP. Biochem J 331: 837-843, 1998[ISI][Medline].

50.   Yusufi, ANK, Cheng JF, Thompson MA, Chini EN, and Grande JP. Nicotinic acid-adenine dinucleotide phosphate (NAADP) elicits specific microsomal Ca2+ release from mammalian cells. Biochem J 353: 531-536, 2001[ISI][Medline].


Am J Physiol Cell Physiol 282(6):C1191-C1198
0363-6143/02 $5.00 Copyright © 2002 the American Physiological Society