©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Nicotinate Adenine Dinucleotide Phosphate (NAADP) Triggers a Specific Calcium Release System in Sea Urchin Eggs (*)

(Received for publication, October 6, 1994; and in revised form, December 5, 1994)

Eduardo N. Chini (§) Kelly W. Beers (¶) Thomas P. Dousa (**)

From the Nephrology Research Unit, Division of Nephrology and Internal Medicine, Departments of Medicine and Physiology, Mayo Clinic and Foundation, Mayo Medical School, Rochester, Minnesota 55905

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Transient fluxes of intracellular ionized calcium (Ca) from intracellular stores are integral components of regulatory signaling pathways operating in numerous biological regulations, including in early stages of egg fertilization. Therefore, we explored whether NADP, which is rapidly generated by phosphorylation of NAD upon fertilization may, directly or indirectly, exert a regulatory role as a trigger of Ca release from intracellular stores in sea urchin eggs. NADP had no effect, but we found that the deamidated derivative of NADP, nicotinate adenine dinucleotide phosphate (beta-NAADP), is a potent and specific stimulus (ED 16 nM) for Ca release in sea urchin egg homogenates. NAADP triggers the Ca release via a mechanism which is distinct from the well-known Ca release systems triggered either by inositol-1,4,5-triphosphate (IP(3)) or by cyclic adenosine diphospho-ribose (cADPR). The NAADP-induced release of Ca is not blocked by heparin, an antagonist of IP(3), or by procaine or ruthenium red, antagonists of cADPR. However, it is selectively blocked by thionicotinamide-NADP which does not inhibit the actions of IP(3) or cADPR. NAADP produced by heating of NADP in alkaline (pH = 12) medium or synthetized enzymatically by nicotinic acid-NADP reaction catalyzed by NAD glycohydrolase have identical properties. The results presented herein thus describe a novel endocellular Ca-releasing system controlled by NAADP as a specific stimulus. The NAADP-controlled Ca release system may be an integral component of multiple intracellular regulations occurring in fertilized sea urchin eggs, which are mediated by intracellular Ca release, and may also have similar role(s) in other tissues.


INTRODUCTION

One of the earliest and essential events following egg fertilization is a transient release of calcium (Ca) from intracellular stores(1, 5) . It is firmly established that inositol-1,4,5 trisphosphate (IP(3)) (^1)is one of the intracellular agents that triggers Ca release from intracellular stores via a specific receptor channel(2) . More recently, Lee and associates (3) discovered that cyclic ADP-ribose (cADPR), a metabolite of beta-NAD, also triggers Ca release in both sea urchin egg homogenates and intact eggs(3) , but via a biochemical mechanism which is entirely different from that for IP(3)(4) . In contrast to IP(3), cADPR triggers Ca release by a ryanodine channel in a calmodulin-dependent mechanism(5, 6, 7, 8) . According to recent reports by Lee et al.(10) and Galione et al.(9) , both IP(3) and cADPR systems are involved in Ca release during egg fertilization. However, Epel (11) pointed out that the Ca which is released following egg fertilization acts as a modulator with multiple targets within the cell and suggested possible hierarchies in the regulation by Ca(12) . This notion raises the possibility that intracellular Ca [Ca] release following egg fertilization, and perhaps [Ca] release in other cells and tissues, may be stimulated by biomodulators other than IP(3) and cADPR which control distinct Ca channels.

It is of particular interest that one of the early biochemical events in fertilization of the sea urchin egg is a marked increase in activity of NAD kinase, which catalyzes rapid conversion of NAD to NADP(13, 14) . Importantly, the surge in NADP concentration precedes an increase in the cell respiratory rate, and this feature may suggest, according to Epel and coworkers(13, 14) , that the increased NADP has a regulatory rather than a metabolic role(13, 14) . In view of findings by Clapper et al.(3) that beta-NAD is converted to the potent Ca-releasing compound cADPR, we considered that beta-NADP may also be converted to a metabolite which acts as a distinct [Ca] releasing factor that may also contribute to the regulation of [Ca] release during fertilization of the sea urchin egg. We found that NADP alone does not trigger [Ca] release. In search of new [Ca]-releasing factors derived from NADP, we focused on an early observation by Lee and associates (3) who in the course of their study of cADPR synthesis from beta-NAD noted that exposure of NADP to strong alkaline pH resulted in generation of a Ca releasing activity that was apparently distinct from activity of IP(3) or cADPR. In this report we provide evidence that a derivative of NADP with a deamidated nicotinamide moiety, NAADP is a potent [Ca]-releasing agent, and its action is blocked specifically by thionicotinamide-NADP.


MATERIALS AND METHODS

[Ca](i) Release Bioassay

Homogenates from Lytechinus pectus eggs were prepared as described previously(3) . Eggs were washed once in artificial sea water, twice in Ca-free sea water containing 1 mM EGTA, twice in Ca-free sea water without EGTA, and homogenized in an intracellular medium (IM) containing an ATP-regenerating system and protease inhibitors as described previously(3) . Frozen homogenates 25% were thawed in a 17 °C water bath and then diluted to 1.25% with IM containing 250 N-methylglutamine, 250 mM K-gluconate, 20 mM HEPES buffer (pH 7.2), 1 mM MgCl(2), 2 units/ml creatine kinase, 4 mM phosphocreatine, and 1 mM ATP. After incubation at 17 °C for 3 h, 3 µM Fluo-3 were added. Fluorescence was measured in a 250-µl cuvette at 17 °C with a circulating water bath and continuously mixed with a magnetic stirring bar, with 490 nm excitation and 535 nm emission using an Hitachi (F-2000) spectrofluorimeter (Fig. 1).


Figure 1: [Ca2+]-mobilizing activity of ALK-NADP in the sea urchin egg homogenate bioassay. ALK-NADP was produced by alkaline treatment, i.e. incubation of HPLC-purified NADP in NaOH buffer (pH = 12) at 60 °C for 30 min then neutralized to pH = 7, and the resulting mixture of compounds was separated by HPLC (see Fig. 2). The concentration of ALK-NADP was then estimated assuming that the extinction coefficient at 254 nm is the same for both NADP and ALK-NADP. The time course of [Ca] release from a 1.25% sea urchin egg homogenate was measured fluorometrically using Fluo-3 as described previously(7) . Changes in fluorescence were calibrated by the addition of known amounts of Ca to the sea urchin egg homogenate. Inset, dependence of [Ca] release on ALK-NADP concentration. The ED of ALK-NADP for induction of Ca release was 16 nM.




Figure 2: HPLC purification of ALK-NADP (NAADP). HPLC profile of NADP before (A) and after (B) alkaline treatment (see Fig. 1). Samples were analyzed by anion-exchange HPLC using an AG MP-1 column, with a non-linear gradient ordinate, right side (panel A) of 150 mM trifluoracetic acid (TFA); (- bullet - bullet -) and water at a flow rate 4 ml/min (UV absorbance (-) ordinate, left side). The [Ca] release activity in the eluates was determined in fractions using the sea urchin egg homogenate [Ca] release bioassay. Alkaline treatment of NADP (see text) generated several peaks that absorb at UV nm (panel B). The [Ca] releasing activity (- -bullet) in the eluate coincided with a single UV peak that eluted at 17 min. This peak was rechromatographed on an AG MP-1 column. The second chromatogram showed only the single peak that was eluted at 17 min (not shown). These samples were also analyzed by a TLC polyethyleneimine-cellulose system using 0.2 M NH(4)HCO(3) as developing buffer for 2 h. The compound ALK-NADP appeared as a single UV spot on the TLC plate (R = 0.25).



HPLC Purification of the Nucleotides

HPLC was performed by anion-exchange chromatography using AG MP-1 (Bio-Rad) resin packed into a column (1 times 10 cm) as described previously(3) , with a non-linear gradient of 150 mM trifluoracetic acid and water as described in Fig. 1; nucleotides were monitored by UV absorption at 254 nm. ALK-NADP was purified as shown in (Fig. 2). The ALK-NADP peak was rechromatographed by a second run on an AG MP-1 column, which showed a single peak that was eluted at 17 min. The ALK-NADP peak was collected and evaporated to dryness on a SpeedVac concentrator. The ALK-NADP used in all experiments was at least 97% pure as determined by HPLC. About 4-7% of the total NADP was converted to ALK-NADP by alkaline treatment. Samples of ALK-NADP were also analyzed by a TLC polyethyleneimine-cellulose system using 0.2 M NH(4)HC0(3) (pH = 7) as developing buffer; ALK-NADP appeared as a single UV spot on TLC plate (R(f) = 0.25).

Alkaline Treatment

NADP and NADP analogs were incubated in a buffer containing (final concentrations) 10 mM NaOH (pH = 12) at 60 °C for 10 min and, after neutralizing with HCl back to pH 7, the release activity was tested in sea urchin egg homogenate assay. Neither NADP nor any of other examined compounds (Table 1) triggered release activity before the alkaline treatment. The release activity was determined by addition of 4-10 µl of the compound to sea urchin egg homogenate assay system (Fig. 1).



Binding of cADPR

Binding to its receptor was determined by using [^3H]cADPR. Sea urchin egg homogenates (2 mg/ml) were diluted in IM with the addition of 1 mM EGTA and incubated with 20 nM [^3H]cADPR (100 dpm/fmol) for 10 min on ice. After incubation, the mixture (100 µg of protein) was filtered through prewashed fiberglass GF/B filters (Whatman Co.) under vacuum and rapidly washed twice with 2 ml of ice-cold IM. Radioactivity retained on the filters was determined using standard scintillation counting techniques.

Binding of [^3H]IP(3) was performed as described previously(14) , with minor modifications. Sea urchin egg homogenates (2 mg/ml) were incubated with 10 nM [^3H]IP(3) (17 Ci/mmol) at 4 °C in IM for 3 min. The filters were then washed and radioactivity counted essentially as described for [^3H]cADPR binding.

Mass Spectroscopy Analysis

Analysis was performed using electrospray ionization in negative and positive modes on an MAT 9000 system with ESI 2. The results of ALK-NADP analysis were compared with those of standard solutions of beta-NADP and cADPR. The sheath liquid used in the negative mode was MeOH/H(2)O/NH(4)OH (90:10:0.5%, v/v) flowing at 5 µl/min. The sheath liquid used in the positive mode was IPA/H(2)O/A(C)OH (90:10:1) flowing at 10 µl/min. The samples were dissolved in H(2)O and diluted 1/100 for a final concentration of 50 pmol/µl with either sheath liquid for negative mode or MeoH/H(2)O/formic acid (50:50:0.5%, v:v) for positive mode. The spectra were obtained over a mass range of 50-900 amv at the rate of 10 s/decade.

NMR Analyses

Analyses were conducted at the NMR facility at the Mayo Clinic, Rochester, MN. NMR results were obtained at 500 mHz on a Brucker AMX-500 spectrometer. Digital resolution of one-dimensional spectra was 0.339 H2. The spectra were obtained from 1 µmol of beta-NADP and 1 µmol of ALK-NADP purified as described above. Experiments were performed in D(2)O-99.996% or 10% D(2)0, 90% water. The partial alignments of the peaks in the NADP spectra were based on previously published data(15) .

NAADP was biosynthetized via the base-exchange reaction catalyzed by NAD-glycohydrolase as described by Bernovsky(16) . Nicotinic acid (500 mM) and NADP (10 mM) were incubated in 20 mM triethanolamine (pH 7.6) with 0.26 g/ml calf spleen NAD-glycohydrolase (NAD(P)ase; Sigma) at 37 °C for 90 min. The reaction was stopped by addition of an equal volume of acetone. The mixture was centrifuged at 2,000 times g for 2 min and after acetone was evaporated under a stream of N(2) gas supernatant was used for HPLC analysis, as described above. The HPLC-purified NAADP was assayed by the quantitative conversion to NAAD (nicotinic acid adenine dinucleotide reduced form) following incubation with calf intestine alkaline phosphatase (Sigma) as described by Bernovsky (16) (not shown).

Sea urchins L. pictus were obtained from Marines, Inc.; Long Beach, CA. Fluo-3 was from Molecular Probes Inc., Eugene, OR, and IP(3) and ryanodine were purchased from Calbiochem. Cyclic ADP-ribose and [^3H]cADPR were from Amersham Corp, [^3H]IP(3) was from DuPont NEN. All other reagents, including nucleotides listed in Table 1, of the highest purity grade available were supplied from Sigma.


RESULTS

HPLC-purified NADP does not trigger release of intracellular Ca from sea urchin eggs homogenates. However, following an ``alkaline treatment,'' i.e. after incubation of NADP with NaOH (pH = 12) at 60 °C for 30 min and adjustment to neutral pH, a product of NADP was generated (denoted further as ``ALK-NADP'') that triggers the release of [Ca](i) from sea urchin egg homogenates (Fig. 1). The product of alkaline-treated NADP was then purified by anion-exchange HPLC using a gradient of TFA (Fig. 2); the fractions were monitored for UV absorption at 254 nm and analyzed for [Ca](i) releasing activity in sea urchin egg homogenate bioassay (see ``Materials and Methods''). As shown in Fig. 2, alkaline treatment results in the decrease of the area peak of NADP, and several distinct UV peaks appeared. Of these peaks, the [Ca](i) releasing activity was found in eluate of a single UV peak that was eluted by higher ionic strength trifluoroacetic acid solvent (at 18 min), thus indicating that the bioactive product is more electronegative than NADP. In all subsequent experiments we used the HPLC-purified ALK-NADP, the product of the alkaline treatment of NADP. The [Ca](i) release elicited by ALK-NADP was dose dependent, and the maximum effect that was comparable to the Ca release responses achieved by maximally effective concentrations of cADPR or IP(3) (Fig. 1). The concentration of ALK-NADP needed for half-maximal Ca release in sea urchin homogenates is about 16 nM, suggesting that the potency of ALK-NADP is comparable to cADPR and is more potent than IP(3)(3) . ALK-NADP-triggered [Ca](i) release was temperature dependent in the range of 10-30 °C; however, increasing the temperature of sea urchin eggs homogenate to 42 °C completely abolished release in response to ALK-NADP. To determine whether the Ca release mechanism activated by ALK-NADP is clearly distinct from the Ca release triggered by cADPR or IP(3), we conducted a series of experimental maneuvers, including the use of pharmacological tools as described below.

Desensitization of [Ca](i) Release

The sea urchin egg homogenate [Ca](i) release system displays homologous desensitization to sequential additions of saturating concentrations of the same [Ca](i)-releasing agent, but neither IP(3) nor cADPR cross-desensitizes the response of the assay system to other compounds(3, 4) . To determine whether or not the release mechanism induced by ALK-NADP is distinct from both cADPR and IP(3) systems, we conducted the following desensitization experiments (Fig. 3). As shown in Fig. 3, A and B, after homologous desensitization of the sea urchin homogenate to either cADPR (Fig. 3A) or IP(3) (Fig. 3B) subsequent [Ca](i) release response induced by ALK-NADP is not diminished. Conversely, subsequent additions of ALK-NADP causes homologous desensitization of the sea urchin egg homogenate to ALK-NADP (Fig. 3, C and D) while the [Ca](i) release responses to IP(3) and cADPR are not affected (Fig. 3, C and D).


Figure 3: Homologous desensitization of the sea urchin egg homogenate. Experimental conditions were as described under ``Materials and Methods.'' Arrows indicate the sequential addition of the [Ca]-mobilizing compounds; 160 nM cADPR, 160 nM ALK-NADP, 2 µM IP(3), 1.4 mM caffeine and 100 µM ryanodine.



It was described previously that sea urchin egg homogenates treated with ryanodine and/or caffeine were desensitized not only to subsequent addition of these agents but also to cADPR, while IP(3) could still trigger [Ca](i) release(4, 7) . Addition of ryanodine or caffeine to the sea urchin egg homogenate causes relatively slow [Ca](i) release and, after resequestration of Ca subsequent addition of cADPR fails to trigger [Ca](i) release (Fig. 3). In contrast, the same sea urchin egg homogenate responds promptly to the addition of ALK-NADP by a marked release of [Ca](i) (Fig. 3E). The reverse was also true: desensitization of the sea urchin egg homogenate to subsequent additions of ALK-NADP does not alter Ca release triggered by ryanodine or caffeine (data not shown).

Effect of Specific [Ca](i) Release Antagonists

We further characterized the [Ca](i) release induced by ALK-NADP with the use of various antagonists. As is the case for all other known [Ca](i)-releasing agents, the effect of ALK-NADP was inhibited by 3,4,5-trimethoxibenzoic acid 8-(diethylemine)octal ether) (TMB-8), a nonspecific blocker of [Ca](i) release from intracellular stores. ALK-NADP-triggered [Ca](i) release was neither inhibited by heparin, an antagonist of IP(3) (Fig. 4), nor by procaine or ruthenium red, blockers of [Ca](i) release by cADPR (Fig. 4). In these experiments we used antagonists at concentrations that were previously found to cause maximum inhibition of the cADPR-induced or IP(3)-induced [Ca](i) release in our preparation.


Figure 4: Specificity of [Ca2+]-releasing activity of NAADP)determined by inhibitors. [Ca] release was triggered by addition () of 160 nM ALK-NADP; with control, no additions (A); with 1 mM procaine (B); with 320 µg/ml heparin (C); 36 µM ruthenium red (D); or 40 µM thio-NADP (E).



While evaluating the effects of NADP derivatives (Table 1) we found that thionicotinamide-NADP (thio-NADP) strongly blocked [Ca](i) release induced by ALK-NADP ( Fig. 4and Fig. 5), but had no inhibitory effect upon [Ca](i) release triggered by cADPR (Fig. 5) or by IP(3) (not shown). The inhibitory effect of thio-NADP was dose dependent, with a half-maximal inhibitory concentration of approximately 3 µM (Fig. 5, inset).


Figure 5: Inhibition of ALK-NADP-induced Ca release by thio-NADP. [Ca] release from sea urchin egg homogenates (1.25%) was monitored using Fluo-3 as Ca indicator. 160 nM ALK-NADP, 24 µM thio-NADP, and 160 nM cADPR were added as indicated () on the abscissa in the figure. The inset of the figure shows the dose-dependent inhibition of [Ca]release induced by 160 nM ALK-NADP by pretreatment (60 s) of sea urchin egg homogenates with increasing concentrations of thio-NADP (ID 3 µM).



Ligand Binding and Displacement Studies

A low capacity high affinity specific binding site for [^3H]cADPR was described in sea urchin egg microsomes, and analogous binding sites are detectable in whole sea urchin egg homogenates,(17) . To test whether ALK-NADP could interact with the cADPR-binding sites, aliquots of sea urchin egg homogenates were incubated with [^3H]cADPR, as described under ``Materials and Methods.'' As shown in Fig. 6, the binding of [^3H]cADPR to the sea urchin egg homogenates was inhibited (ID 20 nM) by addition of authentic unlabeled cADPR; in contrast neither ALK-NADP nor ADPR (non-cyclic adenosine diphospho-ribose) competed for the cADPR-binding sites even at the highest concentrations tested (Fig. 6a). Similarly, [^3H]IP(3) bound to the same sea urchin egg homogenate preparation was displaced by unlabeled IP(3), whereas neither ALK-NADP nor cADPR competed appreciably for IP(3) binding (Fig. 6b).


Figure 6: Binding of [^3H]-cADPR, upper panel, and [^3H]-IP(3), lower panel, to sea urchin egg homogenates; for details see ``Materials and Methods.'' Effect of addition of unlabeled ligands in 1000 times higher concentration. Upper panel (a): A, 10 nM [^3H]cADPR alone; B, with 10 µM cADPR; C, with 10 µM ADPR; D, with 10 µM ALK-NADP. Lower panel (b): A, 10 nM [^3H]IP(3) alone; B, with 10 µM IP(3); C, with 10 µM cADPR; D, with 10 µM ALK-NADP. Each bar denotes mean ± S.E. of six experiments.



Determination of ALK-NADP Structure

After clearly establishing distinct functional characteristics of the [Ca](i) releasing activity of ALK-NADP, we set out to determine its chemical structure. The elution profile of NADP and ALK-NADP by anion-exchange HPLC suggests that the ALK-NADP is more electronegative than NADP (Fig. 2). Further information concerning the structural nature of ALK-NADP was obtained in experiments when several NADP-related nucleotides were subjected to similar alkaline treatment as NADP (``Materials and Methods'') and tested in [Ca](i) release assay. Comparison of ALK beta-NADP to ALK alpha-NADP suggests that the active compound retained the beta-glycosidic bond, which is essential for [Ca](i) releasing activity since alkaline treatment of alpha-NADP did not produce an active compound (Table 1). Interestingly, alkaline-treated beta-NADP analogs with 3`-phosphate or 2`,3`-cyclic monophosphate generated full [Ca](i) releasing activity (Table 1). On the other hand, modifications of NADP on the adenine moiety caused loss of activity. For example, alkaline treatment of either nicotinamide hypoxanthine dinucleotide phosphate (NADP analog with deaminated adenine) or 1,N^6-etheno NADP did not produce an active [Ca](i)-releasing product, thereby suggesting that preservation of the unmodified adenine moiety is essential for the biologic activity of ALK-NADP. The UV spectrum of ALK-NADP was not different from NADP at wavelengths ranging from 200 to 400 nm also indicating that after the alkaline chemical reaction, the integrity of adenine moiety of NADP is preserved. Neither the reduced form of NADP (NADPH), nor any of the NADP derivatives (Table 1) in reduced form, could be converted to compounds with [Ca](i) releasing activity by the alkaline treatment.

Of particular interest and importance is the finding that thio-NADP, which after alkaline treatment did not exhibit [Ca](i) releasing activity, is effective as a specific and dose-dependent inhibitor of the [Ca](i) releasing activity of ALK-NADP, but does not interfere with [Ca](i) release elicited by IP(3) or cADPR ( Fig. 4and Fig. 5).

Incubation of ALK-NADP with alkaline phosphatase or with snake venom phosphodiesterase completely abolished the [Ca](i) releasing activity. This indicates that the 2`-phosphate on the adenosine moiety and an intact diester moiety are both essential for biologic activity of ALK-NADP. At neutral pH, the [Ca](i) releasing activity of ALK-NADP in aqueous solution remained intact even when heated to 100 °C for 2 h, whereas boiling in strong acid (pH = 2) or strong alkali (pH = 12) abolished the Ca releasing activity of ALK-NADP. The ALK-NADP compound is absorbed on anion-exchange resin (Dowex times 1) or on charcoal, but is not extracted from aqueous solution by ether (data not shown).

The NMR analysis of ALK-NADP confirmed the presence of all non-exchangeable proton peaks associated with the nicotinamide group H,-4,-5,-6, although they were shifted; the two adenine protons HA(2) and -8 were still present, and the two anomeric protons of the ribose units of ALK-NADP were in the same spectral region as in NADP (not shown). The mass spectrum of HPLC-purified ALK-NADP (Fig. 7) revealed that ALK-NADP differs from NADP by only 1 Da, and NADP presented a molecular ion species (in negative mode) at m/z 742 and satiated species at 764 (+Na) and 786 (+2Na). Analysis of ALK-NADP under the same conditions (Fig. 7) revealed a molecular species at m/z 743 and satiated species at 765 (+Na).


Figure 7: Mass spectrometry of NADP and ALK-NADP. Upper panel, NADP. Lower panel: ALK-NADP. The spectra were obtained as described under ``Materials and Methods.'' ALK-NADP shows molecular mass 1 Da higher than NADP.



Based on results of all analytical procedures and other experimental evidence we propose that ALK-NADP, a product of alkaline treatment of NADP, is NADP derivative with deamidated nicotinamide moiety, and hence identical to nicotinate (nicotinic acid) NAADP.

In view of these conclusions, we explored whether non-enzymatically produced ALK-NADP has properties that are identical to NAADP generated by an enzymatic reaction. NAADP was prepared by an enzymatic exchange reaction between NADP and nicotinic acid catalyzed by NAD-glycohydrolase as described by Brenofsky(14) . Incubation of NADP with nicotinic acid in the presence of calf spleen NAD-glycohydrolase indeed resulted in the production of 2:2:1 of NADP/NAADP/2`P-ADPR. Importantly, NAADP prepared in this manner co-elutes by HPLC with ALK-NADP (Fig. 8) and shows the same [Ca](i) releasing activity (ED) as non-enzymatically prepared ALK-NADP, including homologous desensitization (Fig. 9) and identical specific inhibition by thionicotinamide-NADP. Furthermore, repeat additions of enzymatically prepared NAADP does not produce desensitization of the [Ca](i) release induced by cADPR (Fig. 9) or IP(3) (data not shown). Finally, incubation of ALK-NADP with alkaline phosphatase results in a compound which is inactive (see above) and which coelutes by HPLC with authentic nicotinate adenine dinucleotide (NAAD) (Fig. 10). Thus, all evidence presented here leads to the conclusion that ALK-NADP is identical with NAADP.


Figure 8: Enzymatic biosynthesis of NAADPcatalyzed by NAD(P)-glycohydralase. The nicotinate analog of NADP (NAADP) was formed by the base exchange reaction as described under ``Materials and Methods.'' The reaction mixture was diluted 50-fold, and 0.5 ml of the mixture was subjected to anion-exchange HPLC as described in Fig. 2. The figure shows HPLC analysis of time 0 min(- - - ), and 90 min (-) of incubation. Both NAADP and ALK-NADP UV peaks (A nm) were coeluted at 18 min (compare Fig. 2).




Figure 9: [Ca] release activity of biosynthetic NAADP. [Ca]release in 1.25% sea urchin egg homogenate was monitored using Fluo-3 as the Ca indicator. Arrows on the abscissa indicate addition of 160 nM NAADP produced by the NAD(P)-glycohydrolase-catalyzed exchange reaction as described in Fig. 8, 160 nM ALK-NADP (produced by alkaline treatment of NADP), and 160 nM cADPR.




Figure 10: Hydrolysis of ALK-NADP by alkaline phosphatase. ALK-NADP (80 µM) produced and purified as described under ``Materials and Methods'' was incubated in a medium containing 40 mM Tris-HCl buffer (pH 8.2) at 35 °C for 10 min in the presence (-) or absence(- - - ) of 10 units/ml alkaline phosphatase, and the change in [Ca] release activity was monitored by the addition of an aliquot of the mixture to the sea urchin egg homogenate assay. Results show inactivation of the [Ca]-releasing compound by alkaline phosphatase. The reaction was stopped by addition of an equal volume of acetone. The mixtures were centrifuged at 2,000 times g for 2 min, and after acetone evaporation the supernatant (80 nM) was used for anion-exchange HPLC analysis, as described under ``Materials and Methods.'' (NA denotes nicotinic acid.)




DISCUSSION

It was previously shown that after treatment with alkali the NADP promotes Ca release from intracellular stores of sea urchin egg homogenates by a mechanism that is apparently distinct from cADPR and IP(3)-induced [Ca](i) release (3) . Experimental evidence presented herein leads to the conclusion that one of the products of alkaline treatment of NADP is a derivative of NADP with a deamidated nicotinamide moiety, NAADP, and that this nucleotide has distinct and specific biologic properties. NAADP has the capacity to trigger, in nanomolar concentrations, the release of [Ca](i) via a mechanism which is clearly different from those of the well-known [Ca](i)-releasing agents IP(3) and cADPR. The evidence indicating that the action of NAADP upon [Ca](i) release is different from that of IP(3) or cADPR includes the finding of homologous desensitization and the absence of cross-desensitization with IP(3) or cADPR (Fig. 3). The lack of inhibition of NAADP-triggered [Ca](i) release by known antagonists of cADPR and IP(3) (Fig. 4) and its specific inhibition by thio-NADP (Fig. 5), which has no blocking effect upon other [Ca](i) releasing systems, constitute pharmacologic evidence for the specificity of NAADP action ( Fig. 4and 5). All of these observations are consistent with the results of binding-displacement experiments with [^3H]cADPR and [^3H]IP(3) (Fig. 6) which show that the site of interaction of NAADP is distinct from those of cADPR and IP(3). Taken together, the evidence thus supports the hypothesis that the [Ca](i) release mechanisms triggered by NAADP, cADPR, and IP(3) are different.

The results of various analytical procedures employed in our study strongly support our conclusion that the [Ca](i)-releasing compound resulting from alkaline treatment of beta-NADP is beta-NAADP. Evidence includes UV spectral analysis, mass spectrometry, NMR analyses, sensitivity to specific enzymes, as well as electrostatic properties deduced from an ion-exchange HPLC and Dowex absorption studies. The identical properties of the non-enzymatically prepared ALK-NADP with the compound prepared by enzymatically catalyzed exchange reaction according to Bernovsky(16) , in chemical and [Ca](i) releasing studies, further affirms that the newly identified [Ca](i)-releasing substance is indeed beta-NAADP.

Furthermore, the results allow the possibility that NAD-glycohydrolase can catalyze exchange of nicotinate on NADP in vivo and thus constitute a possible route for NAADP biosynthesis. NAAD is known to be an abundant and essential compound that is an intermediary metabolite in the biosynthetic pathways of NAD and NADP(18) . Thus, besides the nicotinate-NADP exchange reaction catalyzed by NAD-glycohydrolase(16) , another route of biosynthesis of NAADP could be via phosphorylation of NAAD to NAADP by NAD kinase with ATP as the phosphate donor(13) . Possibly, the NAD kinase may also accept NAAD as a substrate or, alternatively, analogous kinase(s) may exist with a substrate specificity for NAAD. Finally, NADP could be enzymatically converted to NAADP by a one-step enzymatic reaction catalyzing deamidation of nicotinamide to nicotinate. However, while some of our observations suggest that NAADP can be generated enzymatically, the biosynthetic and catabolic pathways for NAADP in various tissues and cells remain to be determined.

Sea urchin eggs are a relatively simple system that can serve as a model for the study of the [Ca](i)-mediated biochemical mechanisms that occur during fertilization in which the cADPR signaling pathway(3) , as well as another novel NAADP-triggered [Ca](i)-releasing system, has been identified in the present study. Consequently, the release of [Ca](i) in sea urchin eggs after fertilization may be controlled not only by IP(3) and/or by cADPR, but also by NAADP. The significance of the complex control of [Ca](i) release in the course of fertilization is not yet apparent. Perhaps multiple mechanisms for [Ca](i) release may be needed for the redundant control(9) , or alternatively the regulatory functions may be hierarchial in nature(11) , i.e. aimed to control multiple targets(9) . Finally, our discovery of a new specific NAADP-controlled [Ca](i) release system in sea urchin eggs opens the possibility that this signaling pathway may be present in various other cell types and tissues.


FOOTNOTES

*
This research was supported by United States Public Health Service Grant DK-30579 from the National Institute of Diabetes and Digestive and Kidney Disease and by the Mayo Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Research Fellow of the Mayo Foundation.

Recipient of a research training fellowship from NIH Renal Diseases Research Training Grant DK-07013.

**
To whom correspondence and reprint requests should be addressed: 921B Guggenheim Bldg., Mayo Clinic, Rochester, MN 55905. Tel.: 507-284-4343; Fax: 507-284-8566.

(^1)
The abbreviations used are: IP(3), inositol-1,4,5-trisphosphate; cADPR, cyclic ADP-ribose; TMB-8, 3,4,5-trimethoxibenzoic acid 8-(diethylemine)octal ether); thio-NADP, thionicotinamide-NADP; HPLC, high performance liquid chromatography; TLC, thin layer chromatography; 2`P-ADPR, adenosine diphosphoribose 2` phosphate.


ACKNOWLEDGEMENTS

We thank Dr. S. Naylor and L. Benson for mass spectrometry analysis and helpful discussion and M. E. Bennett for excellent secretarial assistance. We gratefully acknowledge the support of the Granger Analytical NMR Laboratory and the Granger Foundation.


REFERENCES

  1. Shapiro, B. M., Schackmann, R. W., and Gabel, C. A. (1981) Annu. Rev. Biochem. 50, 815-843 [CrossRef][Medline] [Order article via Infotrieve]
  2. Berridge, M. J., and Irvine, R. F. (1989) Nature 341, 197-205 [CrossRef][Medline] [Order article via Infotrieve]
  3. Clapper, D. L., Walseth, R. F., Dargie, P. J., and Lee, H. C. (1987) J. Biol. Chem. 262, 9561-9568 [Abstract/Free Full Text]
  4. Dargie, P. J., Agre, M. C., and Lee, H. C. (1990) Cell Regul. 1, 279-290 [Medline] [Order article via Infotrieve]
  5. Galione, A., Lee, H. C., and Busa, W. B. (1991) Science 253, 1143-1146 [Medline] [Order article via Infotrieve]
  6. Galione, A. (1994) Mol. Cell. Endocrinol. 98, 125-131
  7. Lee, H. C. (1993) J. Biol. Chem. 268, 293-299 [Abstract/Free Full Text]
  8. Lee, H. C., Aarhus, R., Graeff, R., Gurnack, M. E., and Walseth, T. F. (1994) Nature 370, 307-309 [CrossRef][Medline] [Order article via Infotrieve]
  9. Galione, A., McDougall, A., Busa, W. B., Willmott, N., Gillot, J., and Whitaker, M. J. (1993) Science 261, 348-352 [Medline] [Order article via Infotrieve]
  10. Lee, H. C., Aaurhus, R., and Walseth, T. F. (1993) Science 261, 352-355 [Medline] [Order article via Infotrieve]
  11. Epel, D. (1980) in The Cell Surface: Mediator of Development Processes (Subtelny, S., and Wessels, N., eds) pp. 169-185, Academic Press, Orlando, FL
  12. Epel, D. (1964) Biochem. Biophys. Res. Commun. 17, 62-68
  13. Epel, D., Patton, C., Wallace, R. W., and Cheung, W. Y. (1981) Cell 23, 543-549 [Medline] [Order article via Infotrieve]
  14. Shosham-Barmatz, V., Zhang, G. H., Garretson, L., and Kraus-Friedmann, N. (1990) Biochem. J. 268, 699-705 [Medline] [Order article via Infotrieve]
  15. Lee, H. C., Walseth, T. F., Bratt, G. T., Hayes, R. N., and Clapper D. L. (1989) J. Biol. Chem. 264, 1608-1615 [Abstract/Free Full Text]
  16. Bernovsky, C. (1982) Methods Enzymol. 66, 105-112
  17. Lee, H. C. (1991) J. Biol. Chem. 266, 2276-2281 [Abstract/Free Full Text]
  18. Preiss, J., and Hanaller, P. (1958) J. Biol. Chem. 233, 493-496 [Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.