©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Derivative of NADP Mobilizes Calcium Stores Insensitive to Inositol Trisphosphate and Cyclic ADP-ribose (*)

(Received for publication, October 18, 1994; and in revised form, November 22, 1994)

Hon Cheung Lee (§) Robert Aarhus

From the Department of Physiology, University of Minnesota, Minneapolis, Minnesota 55455

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have previously shown that alkaline treatment of NADP generates a derivative which can mobilize Ca from sea urchin egg homogenates (Clapper, D. L., Walseth, T. F., Dargie, P. J., and Lee, H. C.(1987) J. Biol. Chem. 262, 9561-9568). In this study, the active derivative was purified and shown by high pressure liquid chromatography to be distinct from NADP and NADPH. However, its proton NMR spectrum was virtually identical to that of NADP. The mass of its molecular ion was measured by high resolution mass spectrometry to be 743.0510, one mass unit larger than the corresponding ion of NADP. These results are consistent with the active derivative being nicotinic acid adenine dinucleotide phosphate (NAADP). Ca release induced by NAADP was saturable with a half-maximal concentration of about 30 nM. The release was specific since NADP and nicotinic acid adenine dinucleotide were ineffective even at 10-40-fold higher concentrations. The NAADP-dependent Ca release showed desensitization and was insensitive to heparin and a specific antagonist of cyclic ADP-ribose (cADPR), 8-amino-cADPR. The release mechanism did not require calmodulin. This is similar to the inositol trisphosphate-sensitive release but distinct from that of cADPR. That the NAADP-sensitive Ca stores were different from those sensitive to inositol trisphosphate- or cADPR was further indicated by their differences in distribution on Percoll density gradients. Microinjection of NAADP into live sea urchin eggs induced transient elevation of intracellular Ca and triggered the cortical reaction, indicating the NAADP-dependent mechanism is operative in intact cells.


INTRODUCTION

It is generally accepted that inositol trisphosphate (IP(3)) (^1)is a second messenger for mobilizing internal Ca stores (reviewed in (1) ). However, most cells also contain IP(3)-insensitive stores. We have developed a bioassay for Ca release activators using sea urchin eggs(2) . The eggs have an extensive network of endoplasmic reticulum that can be easily isolated by homogenization. The homogenates, when used without further fractionation, are a good representation of various intracellular Ca stores in the cell. In addition to IP(3), NAD and NADP were also found to be effective in releasing Ca from the homogenates(2) .

The Ca release induced by NAD exhibited a prominent initial delay, suggesting enzymatic conversion of NAD to an active metabolite(2) . This was shown to be the case, and subsequent characterizations led to the discovery of cyclic ADP-ribose (cADPR)(3) , the structure of which has been unambiguously determined by x-ray crystallography(4) . In addition to sea urchin eggs, an invertebrate cell, amphibian sympathetic neurons(5) , and a variety of mammalian cells (reviewed in (6) ) have been shown to be responsive to cADPR. Accumulating evidence indicates it may be an endogenous regulator of the Ca-induced Ca release process mediated by the ryanodine receptors(7, 8, 9) . ADP-ribosyl cyclase(10) , the synthetic enzyme of cADPR, is ubiquitous(11, 12) and, in the case of sea urchin eggs, has been shown to be regulated by a cGMP-dependent process(13) . This has led to the recent proposal that cADPR may be involved in the signaling pathway mediated by nitric oxide (14) .

Unlike the Ca release induced by NAD, NADP released Ca without a delay(2) . Alkaline treatment of NADP greatly increased its Ca release activity suggesting it is a derivative of NADP which is responsible for the activity(2) . In this study, we present evidence that the active derivative is nicotinic acid adenine dinucleotide phosphate (NAADP) and characterize its Ca release activity in fractionated microsomes as well as in live sea urchin eggs.


EXPERIMENTAL PROCEDURES

Alkaline Activation of NADP

NADP (about 3 µmol) obtained from Sigma was purified by anion-exchange HPLC (described later) and dissolved in a buffer containing 80 mM K(2)CO(3), 20 mM KHCO(3), pH 10.5. The mixture was incubated at 57 °C for 10-20 min. The increase in Ca releasing activity was assayed periodically by adding small aliquots of the mixture to egg homogenates (described later). At the time when the Ca release activity was maximal, the incubation was terminated by dilution with a cold buffer containing 5 mM Tris and the pH was titrated to 7.5 with HCl. The active derivative produced was purified by HPLC as described later.

The Ca Release Assay

Homogenates of sea urchin egg (Strongylocentrotus purpuratus and Lytechinus pictus) were prepared as described previously(2, 8, 9) . Frozen egg homogenates (25%) were thawed at 17 ° for 20 min and diluted to 5% with a medium containing 250 mMN-methylglucamine, 250 mM potassium gluconate, 20 mM HEPES, 1 mM MgCl(2), 2 units/ml of creatine kinase, 8 mM phosphocreatine, 0.5 mM ATP, and 3 µM fluo-3, pH 7.2 adjusted with acetic acid (GluIM). The homogenates were diluted to 2.5% and finally 1.25% with the medium described and were incubated at 17 °C for 1 h between dilutions. Ca release was measured using Fluo 3 in 0.2 ml of homogenates at 17 °C.

Fractionation of Egg Microsomes and Microinjection of Intact Eggs

2 ml of 25% egg homogenate of S. purpuratus were layered on 10 ml of 25% Percoll prepared as described previously(8, 9) . After centrifugation for 30 min at 25,000 revolutions/min in a Beckman Ti50 rotor at 10 °C, the microsomes were collected by inserting a syringe needle directly into the band of vesicles on the upper part of the gradient. Alternatively, the bottom of the centrifugation tube was punctured, and 1-ml fractions were collected.

L. pictus eggs were used for the microinjection experiments. The procedures for microinjection by pressure and for measuring the Ca changes in the injected eggs using Indo 1 were as described previously(15, 16) . Samples were dissolved in the injection buffer containing 0.5 M KCl, 50 µM EGTA, 10 mM HEPES, pH 6.7. Possible Ca contamination was removed by passing the NAADP samples through a 2-ml Chelex column. To heat-inactivate NAADP, the samples were put into a stoppered tube and heated in a microwave oven for 30 min at full power. The concentration of NAADP was determined by absorbance using the extinction coefficient of 17,440 at 254 nm obtained from NADP standards.

HPLC, ^1H NMR, and Mass Spectroscopy Analyses

HPLC separation was done with columns packed with the AG MP-1 resin (Bio-Rad) and eluted with a nonlinear gradient of trifluoroacetic acid similar to that described previously(3) . Purified NADP and NAADP were dissolved in D(2)O and analyzed with a 500 MHz NMR spectrometer in a facility of the Department of Chemistry, University of Minnesota, Minneapolis. All mass spectrometry measurements were done at the Midwest Center for Mass Spectrometry (Lincoln, NE) using a Kratos Analytical Instruments MS-50 triple analyzer equipped with a fast atom bombardment source. Samples were dissolved in water at 1 µg/µl, and a 1-µl aliquot was added to a matrix of triethanolamine for negative ion spectra. Fast atom bombardment by 6-kV argon atoms was used to desorb the preformed ions from the matrix which was supported on a copper probe held at -8 kV.


RESULTS

Structural Determination

In a previous study, we have shown that there are three different Ca release systems in egg homogenates(2) . In addition to the IP(3)- and the cADPR-sensitive systems there is another Ca release mechanism which is sensitive to NADP. Homogenates desensitized to high concentrations of IP(3) and cADPR could still respond to NADP. Alkaline treatment of NADP greatly increased its potency suggesting a derivative and not NADP itself was responsible for stimulating the Ca release. Unlike NAD, NADP released Ca without delay, indicating the active derivative of NADP was already present in the NADP samples and did not require enzymatic conversion. This was confirmed by analyzing the samples using anion exchange HPLC. Fig. 1shows a typical chromatograph. Fractions were collected and assayed for Ca release activity using the egg homogenates. The major peak corresponding to NADP eluted at about 21.3 min, and it did not have Ca release activity. All the activity was found in a small UV absorbing peak (indicated by an asterisk) eluted at about 27.5 min, 6.2 min later than NADP. These results show that the Ca release activity came from a contaminant in the NADP sample and not from NADP itself. The active impurity was found in at least three to four batches of NADP from Sigma. Also shown in Fig. 1is a chromatograph of NADP activated by the alkaline treatment. The increase of the active component after the alkaline treatment of NADP correlated with the increase in Ca release activity.


Figure 1: HPLC analyses of NADP and alkaline-activated NADP. Top panel, commercial NADP (about 3 µmol) was chromatographed on an AG MP-1 column. Elution was monitored at 254 nm. Aliquots (2 µl) from each fractions were tested for Ca release activity using sea urchin egg homogenates as a bioassay. Bottom panel, the fractions corresponding to the NADP peak were lyophilized and treated under alkaline conditions for 20 min as described under ``Experimental Procedures.'' The alkaline-activated NADP was similarly analyzed by HPLC. Both panels, the UV peaks that exhibited Ca release activity are indicated by an asterisk.



Fig. 2compares the HPLC chromatograph of the active component purified from alkaline-activated NADP (A-NADP) with NADP and NADPH. The elution times of the three were different. When a mixture of the three were analyzed, the chromatograph showed three distinct peaks indicating A-NADP was definitely different from NADP and NADPH. The proton NMR spectra of A-NADP and NADP were, however, virtually identical as shown in Fig. 3. Therefore, all the non-exchangable protons on the NADP molecule were unchanged.


Figure 2: HPLC chromatographs of NADP, NADPH, and alkaline-treated NADP. NADP (30 nmol), NADPH (16 nmol), and A-NADP (14 nmol) were analyzed individually and in combination by anion-exchange HPLC. An analytic HPLC column was used for the analysis, giving slightly different retention times than the preparative column used in Fig. 1.




Figure 3: ^1H NMR spectra of NADP and alkaline-treated NADP. About 4 µmol of HPLC purified NADP and 2 µmol of A-NADP were dissolved in D(2)O (99.996%), and the spectra were obtained using a 500 MHz NMR spectrometer.



Fig. 4shows the mass spectra of A-NADP and NADP. The two main peaks in the A-NADP spectrum represented the molecular ions, [M-2H] and [M-3H+Na], and they had m/z values of 743 and 765, respectively. The corresponding ions of NADP had m/z values of 742 and 764, respectively. High resolution mass spectrometry of [M-2H] of NAADP gave an exact mass of 743.0510, which was one atomic mass unit higher than the corresponding molecular ion of NADP. Analyses by HPLC shown in Fig. 2definitively ruled out the possibility that the derivative was NADPH since the elution time of the derivative was 6.35 min earlier. A possible structure that is consistent with these results is that of NAADP shown in Fig. 5. This structure is consistent with both A-NADP and NADP having the same proton NMR spectrum since the different protons, those on the carboxyl and the amide groups, are exchangeable with D(2)O and, therefore, do not show up in the NMR spectra. Conversion of -NH(2) (16 atomic mass units) to -OH (17 atomic mass units) resulted in the molecule gaining one mass unit, which is consistent with the mass spectrometry measurements (Fig. 4). In fact, the calculated mass of this structure is identical to the measured mass of A-NADP to within 0.8 parts/million. The structure is also consistent with the HPLC data. Conversion of the amide to a carboxyl group would make the molecule more negatively charged and therefore would have a longer retention time on the anion exchange column as observed (Fig. 2).


Figure 4: Mass spectra of NADP and alkaline-treated NADP. HPLC purified NADP and A-NADP were analyzed by fast atom bombardment mass spectrometry.




Figure 5: Structure of NADP and its active derivative. The active derivative NADP is proposed to be NAADP.



Characteristics of the Ca Release

NAADP was very effective in releasing Ca from egg homogenates. As shown in Fig. 6, the half-maximal concentration of NAADP measured in L. pictus homogenates was about 30 nM, which is as potent as cADPR and both are more effective than IP(3)(16) . The response showed saturation at about 100 nM. Similar to IP(3) and cADPR, high concentrations of NAADP could also desensitize the egg microsomes as shown in Fig. 7a. The Ca release elicited by 102 nM of NAADP was very fast, and the microsomes failed to respond to another addition of NAADP even after the released Ca was totally resequestered. The desensitized microsomes could still respond normally to cADPR, indicating the two release mechanisms were independent of each other. This is further shown in Fig. 7b. The antagonist, 8-amino-cADPR(15, 17) , specifically blocked the cADPR-, but not the NAADPdependent Ca release. Similarly, Fig. 7c shows the NAADP-dependent release was not inhibited by heparin even at 2 mg/ml, which totally blocked the IP(3)-sensitive release. Therefore, the three Ca release mechanisms are completely different. Two compounds with structures similar to NAADP, NADP at 1.1 µM (Fig. 7d) and nicotinic acid adenine dinucleotide (NAAD) at 4 µM (Fig. 7e), did not release any Ca from the homogenates, demonstrating the high degree of specificity of the NAADP-sensitive Ca release system.


Figure 6: The concentration dependence of the Ca release induced by NAADP. Ca release was measured in 1.25% (v/v) L. pictus egg homogenates using Fluo 3 as a Ca indicator. The fluorescence changes were calibrated with known amounts of CaCl(2).




Figure 7: Characteristics of the Ca release induced by NAADP. Ca release was measured in S. purpuratus egg homogenates. a, each addition gave final concentrations of 102 nM of NAADP and 100 nM of cADPR. b, each addition gave final concentrations of 170 nM of 8-amino-cADPR (8-NH(2)), 100 nM cADPR, or 102 nM of NAADP. c, each addition gave final concentrations of 2 mg/ml heparin (Hep), 1.2 µM of IP(3) or 102 nM NAADP. d, NADP was added to a final concentration of 1.1 µM. e, each addition gave final concentrations of 4 µM of NAAD or 102 nM NAADP.



It has previously been shown that egg microsomes can be cleanly separated from soluble proteins and other organelles by Percoll density gradient centrifugation. The purified microsomes lose their sensitivity to cADPR but can be restored by the addition of calmodulin(9) . No such dependence on calmodulin was seen with the NAADP-sensitive release. As shown in Fig. 8A, purified microsomes responded to NAADP equally well in the presence or absence of calmodulin. Fig. 8B shows the same preparation of microsomes exhibited nearly absolute requirement of calmodulin for the cADPR sensitivity. In the presence of calmodulin, 150 nM of cADPR produced a saturating response, while the same concentration of cADPR induced no Ca release in the absence of calmodulin. The independence of the NAADP response to calmodulin is similar to that of IP(3). As shown in Fig. 8C, the same preparation of microsomes responded to IP(3) equally well in the presence and absence of calmodulin.


Figure 8: The NAADP-sensitive Ca release does not require calmodulin. S. purpuratus microsomes were purified by Percoll gradient centrifugation. The top microsomal band (cf.Fig. 9) was incubated without or with 7 µg/ml of calmodulin (CaM). NAADP, cADPR, and IP(3) were added to the final concentrations indicated. Ca release was measured with 1.5 µM Fluo 3.




Figure 9: Fractionation of egg homogenates with Percoll density centrifugation. Egg (S. purpuratus) microsomes were fractionated by Percoll gradient centrifugation. Fractions were collected by puncturing the bottom of the centrifuge tube (Fraction 1). Each 1 ml fraction was diluted 5-fold with the homogenization buffer (GluIM) containing 5 µg/ml of calmodulin. Ca release was assayed for each diluted fraction by the addition of 120 nM cADPR, 136 nM NAADP, or 5 µM IP(3) and calibrated with known amounts of Ca.



The results described above indicate NAADP activates Ca release by a mechanism different from that of IP(3) and cADPR. To determine if the Ca stores sensitive to NAADP are also distinct from that of IP(3) and cADPR, Percoll density fractionation of the homogenates was performed, and the results are shown in Fig. 9. The fractions were incubated with calmodulin and challenged with a maximal concentration of each of the three Ca agonists. Consistent with previous results(2, 8) , the IP(3)- and cADPR-sensitive stores comigrate and were concentrated in two fractions (6 and 7) from the upper part of the gradient. These fractions also contain most of the glucose-6-phosphatase activity, an endoplasmic reticulum marker(2, 8) . In contrast, the NAADP-sensitive stores had a much broader distribution. Fraction 2 and 3 from the bottom of the gradient contained the majority of the mitochondria and yolk granules(2, 8) . A small contamination of the cADPR-sensitive stores was present in fraction 2 but no detectable IP(3)-sensitive stores. It is clear from these results that the NAADP-sensitive stores are distinct from the IP(3)- and cADPR-sensitive stores.

Fig. 10shows microinjection of NAADP into an live sea urchin egg induced a large transient increase of intracellular Ca as monitored by the fluorescence ratio of Indo 1 (Fig. 10). Similar to what was shown in egg homogenates, the injected egg became desensitized to NAADP afterward and failed to respond to another injection of NAADP. Heating NAADP for 30 min in a microwave oven destroyed its ability to release Ca from egg homogenates (data not shown). As a control, the same amount of heat-inactivated NAADP was injected, and no Ca change was seen. In addition to the observed Ca changes, the injected eggs also underwent a massive cortical exocytotic reaction, another index for Ca mobilization. Microinjection of 10 picoliter (1.5% of egg volume) of 23 µM NAADP into intact eggs (intracellular concentration about 0.35 µM) activated the cortical reaction in seven out of seven eggs. As a control, heat-inactivated NAADP was microinjected into the same number of eggs, and none had a cortical reaction.


Figure 10: Intracellular Ca changes induced by microinjection of NAADP into L. pictus eggs. Intracellular Ca changes were monitored by the fluorescence intensity ratio (405 nm/485 nm) of Indo 1. At the time indicated, heat-inactivated NAADP or NAADP was microinjected (0.5% of egg volume) into the eggs. Each injection delivered about 115 nM into the egg, assuming uniform distribution. The concentration of NAADP or heat-inactivated NAADP was 23 µM in the micropipette.




DISCUSSION

We have previously demonstrated that both NAD and NADP mobilize Ca through mechanisms distinct from that of IP(3), a well known and accepted pathway(2) . The Ca mobilizing activities were not due to the pyridine nucleotides themselves but were attributed to their derivatives (or metabolites)(2) . We had chosen to focus our investigations on the derivative of NAD because the kinetics of the NAD-induced Ca release clearly indicates the conversion is catalyzed by an enzyme. Our investigations eventually led to the discovery of cADPR (3, 4) and its synthetic enzyme, ADP-ribosyl cyclase(10, 11, 12) . We were expecting that the active derivative of NADP may turn out to be also cyclic, perhaps a phosphorylated form of cADPR. It came as a surprise when the NMR results showed that all the non-exchangeable protons of the nicotinamide group were present in NAADP (Fig. 3). Indeed, the spectrum was virtually identical to that of NADP. Since all non-exchangable protons were unchanged, we next focused on exchangeable protons. The obvious choice was the amide group. Mass spectrometry measurements indicated the derivative was one mass unit higher than NADP, which suggests the modification was the conversion of the nicotinamide group to nicotinic acid. This was confirmed by high resolution mass measurements showing the calculated mass of NAADP agreed with the measured mass to within 0.8 parts/million.

The conversion of NADP to NAADP involves a rather simple deamidation of the amide group. It is likely the reaction could be catalyzed by a cellular enzyme. Deamidation of nicotinamide to nicotinic acid is a key pathway in NAD metabolism and is catalyzed by nicotinamide deamidase (EC 3.5.1.19). This enzyme has been purified from yeast(18) . It is not known whether it can deamidate NADP. So far, we have not been able to demonstrate consistent conversion of NADP to NAADP in sea urchin egg homogenates. Although we use NADP as the starting material in our chemical synthesis of NAADP, it may not be the actual substrate of the synthesizing enzyme. For example, the synthesizing enzyme could be a kinase which phosphorylates NAAD to NAADP. Indeed, the NAD kinase is particularly abundant in sea urchin eggs (19) . Whether it can phosphorylate NAAD has not been determined. The NAD kinase is of particular interest because it is a Ca- and calmodulin-sensitive enzyme that has been shown to be transiently activated after fertilization with a time course corresponding to the Ca changes(19) . Another possibility is that the enzymatic synthesis of NAADP may be inhibited by a tightly associated regulatory component. Judging from the potency of NAADP in mobilizing Ca, it is very likely that the enzyme is tightly regulated. Finally, the synthesis enzyme may be particularly labile. We simply may not have arrived at the right conditions to preserve its activity in a cell-free system.

As shown in this study, NAADP is as active as cADPR and both are more effective than IP(3) when tested with the same preparations of egg microsomes. The kinetics of the NAADP-induced Ca release is also significantly faster than the cADPR-sensitive release (cf.Fig. 7), making it the most effective Ca release activator known in sea urchin egg. NAADP is not a nonspecific ionophore since it can produce desensitization. It also does not function as an inhibitor of the Ca sequestration mechanism since inhibition of the pump by either thapsigargine (8) or removal of ATP (16) can only effect a slow leakage and not fast release as seen with NAADP. Also, as shown in Fig. 7, the Ca released by NAADP was effectively resequestered even in the presence of NAADP. The fact that it is active at nanomolar concentrations suggests a specific receptor may be involved. This putative receptor is likely to be different from the IP(3) and the cADPR receptors since microsomes desensitized to the other two agonists still respond to NAADP, and specific blockers of the other two receptors had no effect on the NAADP-sensitive release. Fractionation using Percoll gradient centrifugation shows that the NAADP receptor is present in Ca stores distinct from those sensitive to either IP(3) or cADPR. That the distribution of the NAADP-sensitive stores in the gradient did not resemble that of glucose-6-phosphatase suggests the stores may not be components of the endoplasmic reticulum, the most accepted site for Ca storage.

The most intriguing question raised by this and previous studies on cADPR is whether there are more second messengers for Ca mobilization than generally believed. It is accepted that IP(3) is a second messenger for Ca. The evidence for cADPR is not yet definitive but certainly is more than suggestive. The discovery of NAADP brings forth a third candidate. This proliferation of candidates reminds one of the analogous situation with neurotransmitters, which began with the discovery of acetylcholine but soon blossomed to a myriad of exotic possibilities.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HD17484 and HD32040 (to H. C. L.). 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.

§
To whom correspondence should be addressed: 6-182 Lyon Lab., Dept. of Physiology, University of Minnesota, Minneapolis, MN 55455. Tel.: 612-625-7120; Fax: 612-625-0991.

(^1)
The abbreviations used are: IP(3), inositol 1,4,5-trisphosphate; cADPR, cyclic ADP-ribose; NAADP, nicotinic acid adenine dinucleotide phosphate; A-NADP, alkaline-activated NADP; NAAD, nicotinic acid adenine dinucleotide; HPLC, high pressure liquid chromatography.


ACKNOWLEDGEMENTS

We thank Roger Hayes and the Mid West Mass Spectrometry Center at the University of Nebraska, Lincoln, NE, for the mass spectroscopic measurements, the Chemistry department of the University of Minnesota, Minneapolis, MN, for the proton NMR measurements, and Richard Graeff and Timothy Walseth for critical reading of the manuscript.


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