Rapid changes in the cytosolic levels of free Ca, termed Ca signaling, are involved in the regulation of diverse cellular events including fertilization, muscle contraction, secretion, and proliferation(1, 2) . Often, Ca signaling is initiated by the release of internal Ca stores into the cytosolic compartment when endogenous second messengers activate membrane Ca channels. Two distinct families of Ca channels have been recognized to play key roles in intracellular Ca mobilization in many cell types. One family consists of channels sensitive to IP, ()a second messenger generated from cellular phospholipids in response to numerous hormones and neurotransmitters(1, 3, 4) . A second family is composed of the ryanodine-sensitive Ca channels(5, 6) . One possible second messenger of ryanodine-sensitive channels is Ca itself, in a process termed Ca-induced Ca release(6) . Recently, cADPR, a naturally occurring metabolite of NAD (7, 8) , has been implicated as a second messenger of ryanodine-sensitive channels. In sea urchin egg microsomes, cADPR is a potent mediator of Ca release, and its action is cross-potentiated by agents known to affect ryanodine-sensitive Ca channels(9, 10, 11, 12, 13) . In mammals, cADPR has been shown to elicit Ca release from microsomes isolated from several different tissues (14, 15, 16, 17, 18) and permeabilized cells(19, 20) .

While cADPR appears to be a link between NAD metabolism and Ca homeostasis, the metabolic signals that regulate cADPR synthesis and/or degradation are unknown, and our knowledge of cADPR metabolic enzymes is limited. An ADPR cyclase that catalyzes the stoichiometric conversion of NAD to cADPR has been isolated from the marine mollusk Aplysia californica(21, 22) . In mammals, cADPR appears to be metabolized by multifunctional enzymes that contain ADPR cyclase, cADPR hydrolase, and NAD glycohydrolase activities (23, 24, 25, 26) . While it is not yet clear if all mammalian NAD glycohydrolases are involved in cADPR metabolism, many of these enzymes effectively use both NAD and NADP as substrates(27, 28, 29, 30, 31, 32, 33) , raising the possibility that they may catalyze the formation of a cyclic nucleotide from NADP. We report here that the Aplysia ADPR cyclase, an enzyme with strong sequence homology to several mammalian NAD glycohydrolases, efficiently catalyzes the conversion of NADP to 2`-P-cADPR. In addition, a multifunctional canine spleen NAD glycohydrolase (23) containing ADPR cyclase and cADPR hydrolase activities displays a kinetic preference for NADP over NAD and utilizes 2`-P-cADPR as a substrate, indicating that 2`-P-cADPR is a likely metabolite in mammalian cells. We also report that 2`-P-cADPR is as active as cADPR in eliciting Ca release from rat brain microsomes. Together with recent reports that another possible metabolite of NADP causes Ca mobilization(34, 35) , the results described here raise the possibility that NADP metabolism may be linked to Ca signaling.

MATERIALS AND METHODS

Activity of Aplysia ADPR Cyclase and Canine Spleen NAD Glycohydrolase on NAD and NADP

Activity of Canine Spleen NAD Glycohydrolase on 2`-P-cADPR

Enzymatic Synthesis and Purification of 2`-P-cADPR

For preparation of 2`-P-cADPR, 1 ml of 10 mM NADP (Calbiochem) in buffer A was passed through a 0.3-ml Aplysia ADPR cyclase column at 4 °C. The eluate was collected, and an aliquot was withdrawn for HPLC analysis. HPLC analysis was carried out on a 3.9 300-mm µBondapak C column (Waters) with isocratic elution with 100 mM potassium phosphate buffer, pH 6.0, at a flow rate of 1 ml/min. The 2`-P-cADPR was purified by anion-exchange chromatography followed by preparative HPLC. In brief, the eluate from the Aplysia ADPR cyclase column was applied to a Poly-Prep column containing 0.5 ml of Bio-Rad AG 1-X2 anion-exchange resin previously equilibrated with buffer A. The column was then washed with 20 ml of buffer A followed by 5 ml of deionized water. Elution was performed using 5 ml of 100 mM ammonium formate buffer, pH 4.0. The eluate was applied to a 10 270-mm Dynamax preparative reversed-phase HPLC column (Rainin Instrument Co. Inc.) with isocratic elution with 0.05% trifluoroacetic acid at a flow rate of 2 ml/min. The 2`-P-cADPR peak was collected and lyophilized overnight. To eliminate residual trifluoroacetic acid, the preparation was redissolved in deionized water and subjected to two additional cycles of lyophilization. The final sample was reconstituted in deionized water and stored at -20 °C. The concentration of 2`-P-cADPR in stock solutions was determined by absorbance at 254 nm using an extinction coefficient of 14,300(36) .

Characterizations of 2`-P-cADPR

H NMR analysis was done using a Varian VXR-400 NMR spectrometer. Three µmol of 2`-P-cADPR was lyophilized three times in 99.9% D0 prior to NMR analysis: H NMR (D0) 8.94 (s, 1H), 8.31 (s, 1H), 6.13 (d, 1H, J = 4 Hz), 6.07 (d, 1H, J = 4 Hz), 5.43 (br s, 1H), 4.80 (br s, 1H), 4.46-4.54 (m, 1H), 4.39 (d, 1H, J = 5 Hz), 4.26-4.34 (m, 2H), and 3.96-4.08 (m, 2H). Assignment of the anomeric ribose protons and the 2`-proton was based on spectra of cADPR ()previously published(36, 37, 38) .

Ultraviolet absorption spectra were obtained on a Hitachi U2000 spectrophotometer. The spectral properties of 2`-P-cADPR were determined as a function of pH using a number of different buffers at a final concentration of 50 mM, which was also used as a reference. The buffers used at the indicated pH values were sodium acetate, pH 5.0; MES, pH 6.0; HEPES, pH 7.0 and 8.0; TAPS, pH 8.5 and 9.0; and CAPS, pH 10 and 11.

Preparation of Rat Brain Microsomes

Calcium Release Assays

RESULTS

Action of A. californica ADPR Cyclase on NADP

To identify the product generated from NADP, an immobilized enzyme preparation was prepared by coupling the purified enzyme to agarose. This preparation has been previously used to convert micromole amounts of NAD to cADPR by a single passage at 4 °C through a 0.3-ml column of the immobilized enzyme. ()The result obtained when a 1-ml solution of 10 mM NADP was passed through a column of immobilized enzyme is shown in Fig. 1B. Passage through the column resulted in the disappearance of 90% of NADP and the appearance of nicotinamide and unidentified material eluting at 4 min. The material eluting at 4 min was purified by anion-exchange chromatography followed by preparative reversed-phase HPLC. The final preparation showed a single peak on reversed-phase HPLC (Fig. 1C). The NADP preparation used contained a small amount of contaminating material eluting at 6 min (Fig. 1A). A recent report has shown that a commercial preparation of NADP (Sigma) contained contaminating NAADP, which is active in mobilizing Ca from sea urchin egg microsomes (35) . Therefore, the possibility that the contaminant in our NADP preparation (Calbiochem) was NAADP was examined. When the NADP preparation was subjected to anion-exchange HPLC, the contaminating material eluted at 7 min compared with an elution time of 60 min for NAADP, demonstrating that the material did not correspond to NAADP.

Figure 1: Utilization of NADP by Aplysia ADPR cyclase. A sample of NADP was passed through a column of immobilized A. californica ADPR cyclase, and aliquots of the reaction mixture were analyzed by reversed-phase HPLC as described under ``Materials and Methods.'' The HPLC chromatograms of the NADP solution before and after passage through the ADPR cyclase column are shown in A and B, respectively. The material eluting at 4 min was subsequently purified as described under ``Materials and Methods,'' and an aliquot of the purified material was analyzed by reversed-phase HPLC as shown in C.

Characterization of Material Produced from NADP

Figure 2: Treatment of material produced from NADP with alkaline phosphatase (phosphomonoesterase). Material derived from NADP by passage through an ADPR cyclase column was purified and incubated in the absence (A) or presence (B) of bacterial alkaline phosphatase. As a control, an equal amount of the alkaline phosphatase was incubated alone (C). The reaction mixtures were analyzed by anion-exchange HPLC.

Further information was obtained from H NMR spectroscopy (Fig. 3). The H NMR spectrum was very similar to that of cADPR(36, 37, 38) . The spectrum also indicated that the material was primarily a single compound. A unique similarity to the spectrum of cADPR was the presence of two sets of chemical shifts between 6.0 and 6.2 ppm, which correspond to the anomeric protons of both ribose moieties. The chemical shifts at this frequency indicate that both ribose anomeric carbon atoms are bound to nitrogen, i.e. that the nucleotide is cyclic. A second unique similarity was an isolated signal with a chemical shift between 5.4 and 5.5 ppm. In cADPR, this signal is due to the ribose 2`-proton, which is shifted far downfield relative to the other ribose protons(36, 37, 38) . This signal appears as a well resolved triplet, while a broad singlet was seen for the cyclic form of 2`-P-ADPR. This difference can be attributed to the presence of a phosphate esterified to the 2`-carbon, with a resulting phosphorus-proton coupling.

Figure 3: H NMR spectrum of material produced from NADP. Material derived from NADP by passage through an ADPR cyclase column was purified and prepared for NMR analysis, and an NMR spectrum was obtained as described under ``Materials and Methods.'' The proposed structure is shown in Fig. 5.

Figure 5: Proposed structure of 2`-P-cADPR.

The position of cyclization was studied by obtaining UV absorption spectra as a function of pH. Fig. 4shows spectra obtained at pH values of 5.0, 9.0, and 11.0. With increasing pH, the compound displayed a hyperchromic effect at 260 nm, a more pronounced shoulder at 267 nm, and a markedly increased absorbance in the region at 280-310 nm. These pH-dependent spectral changes are unique to cADPR and other adenine nucleotides substituted at N-1 of the adenine ring(41) , indicating that N-1 was the position of cyclization in 2`-P-cADPR. The spectral changes in the region at 280-310 nm are due to dissociation of an adenine ring proton(41) . Fig. 4(inset) shows a plot of the absorbance at 300 nm as a function of pH. From these data, a pK of 9.0 was determined for 2`-P-cADPR. The corresponding pK value for cADPR is 8.2(41) . The higher pK value of 9.0 can be attributed to the presence of the additional phosphate group present in the molecule. Taken together, the data from both enzymatic and spectral characterizations demonstrate that the compound generated from NADP by the action of immobilized A. californica ADPR cyclase is 2`-P-cADPR, cyclized at N-1 of the adenine moiety (Fig. 5).

Figure 4: Ultraviolet absorption spectra of material produced from NADP as a function of pH. The spectra were obtained at pH 11 (-), pH 9.0 (), and pH 5.0(- - -). Inset, the absorbance at 300 nm is plotted as a function of pH.

Utilization of NADP and 2`-P-cADPR by Canine Spleen NAD Glycohydrolase

Figure 6: Activity of canine spleen NAD glyohdrolase with NADP and 2`-P-cADPR as substrates. The activity of canine spleen NAD glycohydrolase (23) with NADP (A) or 2`-P-cADPR (B) as substrate was determined as described under ``Materials and Methods.'' Representative Lineweaver-Burk plots are shown. From three separate experiments, apparent K values of 1.6 ± 0.2 µM for NADP and 140 ± 14 µM for 2`-P-cADPR were determined.

Ca Mobilizing Activity of 2`-P-cADPR

Figure 7: Comparison of Ca mobilizing characteristics of 2`-P-cADPR, cADPR, IP, 2`-P-ADPR, and ADPR from rat brain microsomes. A, C, and E show the microsomal response to additions of 10 µM 2`-P-cADPR, cADPR, and IP, respectively. Additions of 50 µM 2`-P-ADPR and ADPR are shown in B and D, respectively.

The Ca release elicited by both 2`-P-cADPR and cADPR was dose-dependent (Fig. 8). EC values of 1.3 µM for 2`-P-cADPR and 1.5 µM for cADPR were determined. The Ca release was specific for the cyclic nucleotides as addition of 50 µM 2`-P-ADPR (Fig. 7B) and ADPR (Fig. 7D) did not result in detectable Ca release. Also shown is Ca release following addition of 10 µM IP (Fig. 7E). In contrast to the patterns observed with 2`-P-cADPR and cADPR, the IP response showed a rapid phase of Ca release followed by rapid re-uptake of Ca into the microsomes.

Figure 8: Ca release from rat brain microsomes elicited by 2`-P-cADPR and cADPR. Isolated rat brain microsomes were treated with different concentrations of either 2`-P-cADPR or cADPR, and Ca release was monitored fluorometrically using the Ca indicator Fura-2 as described under ``Materials and Methods.'' Measurements were made at 100 s following addition of cADPR or 2`-P-cADPR. Approximately 30 nM Ca was released by saturating concentrations of 2`-P-cADPR and cADPR, and this value was normalized to 100%. All other measurements are expressed relative to the maximal value.

Current evidence indicates that cADPR and IP elicit Ca release through distinct mechanisms(10) . To determine if the mechanism of Ca release induced by 2`-P-cADPR was also distinct from the IP system, cross-desensitization and inhibition studies were done. Fig. 9A shows that a second addition of IP resulted in Ca release that was only 4% of the first addition, consistent with desensitization of the IP-sensitive release mechanism. However, a subsequent addition of 2`-P-cADPR elicited approximately the same amount of Ca release observed without prior additions of IP. Fig. 9B shows that a second addition of 2`-P-cADPR resulted in Ca release that was 29% of the first addition, indicating a partial desensitization of the 2`-P-cADPR release mechanism. In this case, a subsequent addition of IP still resulted in Ca release that was approximately the same as observed without prior additions of 2`-P-cADPR (Fig. 9B). The relationship between 2`-P-cADPR and IP was examined further by the use of heparin, a potent inhibitor of IP-sensitive Ca channels(1) . Pretreatment with 600 µg/ml heparin completely abolished the action of IP, but the Ca release activity of 2`-P-cADPR was unaffected (data not shown). In total, these data provide evidence that the mechanism of 2`-P-cADPR-induced Ca release is distinct from the IP pathway.

Figure 9: Interaction of IP- and 2`-P-cADPR-induced Ca release from rat brain microsomes. Rat brain microsomes were treated with two successive additions of IP followed by 2`-P-cADPR (A) and conversely, two successive additions of 2`-P-cADPR followed by IP (B). The final concentrations of each Ca-mobilizing agent added are indicated.

Evidence that 2`-P-cADPR and cADPR were acting via a similar mechanism was obtained by examination of the effects of 2`-P-cADPR on cADPR-induced Ca release and vice versa. In the experiment shown in Fig. 10A, a second addition of 20 µM 2`-P-cADPR resulted in Ca release that was 24% of the first addition. The subsequent addition of cADPR resulted in Ca release that was 32% of a control without 2`-P-cADPR pretreatment, indicating that the 2`-P-cADPR release mechanism was cross-desensitized to that of cADPR, even though the release mechanism was only partially desensitized. A similar result was observed when successive additions of cADPR were followed by addition of 2`-P-cADPR (Fig. 10B).

Figure 10: Cross-desensitization of Ca release by 2`-P-cADPR and cADPR. The effects of two successive additions of saturating concentrations of 2`-P-cADPR on subsequent Ca release by cADPR and vice versa are shown in A and B, respectively. The final concentration (20 µM) of the nucleotides represents the concentration that induced maximal Ca release from the rat brain microsomes (see Fig. 8).

Partial desensitization of Ca release was also observed following addition of subsaturating amounts of 2`-P-cADPR. For example, when the microsomes were treated with successive additions of 1 µM 2`-P-cADPR, the response to the second, third, and fourth additions was reduced to 25-35% of the initial addition (data not shown). In the same experiment, when 1 µM cADPR was added following the four additions of 2`-P-cADPR, Ca release was 25% of a control without 2`-P-cADPR pretreatment, again indicating cross-desensitization between the 2`-P-cADPR and cADPR mechanisms.

Evidence that both 2`-P-cADPR and cADPR were operating through a ryanodine-sensitive Ca channel was obtained by examining the effect of procaine, a partial antagonist of ryanodine-sensitive channels(10) . When the microsomes were pretreated with 5 mM procaine, the Ca release induced by 1 µM cADPR or 2`-P-cADPR was inhibited by 45 and 50%, respectively, relative to the controls (Fig. 11).

Figure 11: Effect of procaine on Ca mobilizing activity of cADPR and 2`-P-cADPR. The microsomes were pretreated with 5 mM procaine for 1 h. Either cADPR or 2`-P-cADPR was then added, and the Ca release was monitored fluorometrically. The results are expressed as percentage of the controls in which procaine was omitted from the incubation mixture.

DISCUSSION

Studies with cADPR have provided a link between NAD metabolism and regulation of Ca signaling(10) . The results described here, together with recent reports that another possible NADP metabolite, NAADP, causes Ca release in sea urchin egg microsomes(34, 35) , suggest a possible link between NADP metabolism and Ca signaling. As shown here, the Aplysia ADPR cyclase utilizes NAD and NADP with very similar efficiency to generate cyclic nucleotides (Fig. 1). In mammalian cells, the only enzymes that have been demonstrated to metabolize cADPR are NAD glycohydrolases that catalyze both the synthesis of cADPR from NAD and the hydrolysis of cADPR to ADPR(23, 24, 25, 26) . Our results with the canine spleen enzyme using NADP as a substrate indicate that the conversion of NADP to 2`-P-cADPR in vivo is likely. The K value of the enzyme for NADP is well below estimated cellular concentrations of NADP(42) , and the enzyme displays a kinetic preference for NADP over NAD as reflected by a significantly higher ratio of k/K for NADP compared with NAD. Evidence that the multifunctional canine spleen enzyme uses the same mechanism (23) with NADP as with NAD is supported by the observation that NAD and NADP are competitive substrates and that the enzyme also uses both cADPR and 2`-P-cADPR as substrates. While it remains to be determined if NADP is converted to 2`-P-cADPR in mammalian cells, the efficiency with which the canine spleen enzyme uses NADP warrants a search for 2`-P-cADPR in vivo.

The maintenance of Ca homeostasis requires the precise regulation of cytosolic free Ca levels. These levels are in turn controlled by membrane Ca channels through which Ca moves between different intracellular compartments and between intracellular and extracellular compartments. The elevation of cytosolic free Ca levels is often initiated by activation of members of one or both of two different families of intracellular Ca channels, the IP-sensitive channels (1, 3, 4) and the ryanodine-sensitive channels(5, 6) . Studies of sea urchin egg microsomes indicate that cADPR modulates ryanodine-sensitive Ca channels(9, 10, 11, 12, 13) . Although less well characterized, cADPR also appears to act on ryanodine-sensitive Ca channels in mammalian cell microsomes(14, 15) . The results presented here indicate that 2`-P-cADPR, similar to cADPR, causes Ca release by a mechanism distinct from IP. Microsomes desensitized to further addition of IP could still release Ca in response to 2`-P-cADPR and vice versa (Fig. 9). Also, levels of heparin that completely blocked release by IP did not affect Ca release by 2`-P-cADPR.

Our results also suggest that 2`-P-cADPR elicits Ca release by a mechanism similar to that of cADPR based on the similar kinetics of Ca release elicited by the two nucleotides (Fig. 7), the similar dose-response curves (Fig. 8), cross-desensitization (Fig. 10), and the partial inhibition of Ca release by procaine (Fig. 11). The possibility that 2`-P-cADPR was being converted to cADPR by the brain microsomes and that the observed Ca release was due to cADPR can be ruled out by the observations that there was no detectable conversion of 2`-P-cADPR to cADPR under the conditions used and that very similar dose-response curves of cADPR and 2`-P-cADPR were observed. The concentrations of 2`-P-cADPR and cADPR that gave half-maximal Ca release in our study, 1.3-1.5 µM (Fig. 8), are somewhat higher than those reported by other workers who observed maximal release at 0.25 µM cADPR for rat brain microsomes(14) . Whether this reflects differences in the amount of Ca loaded into the microsomes or some other difference will require further study.

It is interesting that the kinetics of Ca release of 2`-P-cADPR and cADPR differ from those of IP in the brain microsomes. Addition of IP to the microsomes resulted in a rapid release followed by a rapid re-uptake of Ca, while 2`-P-cADPR and cADPR caused an initial rapid release followed by a slower but prolonged increase in Ca (Fig. 7). Previous studies also have observed a prolonged Ca release by cADPR in rat pituitary cells (20) and rat brain microsomes(14) . The difference in kinetics suggests that the mode of Ca channel activation caused by cADPR and 2`-P-cADPR may be fundamentally different from that of IP. The rate of Ca release during the prolonged phase that occurs following 2`-P-cADPR and cADPR is probably underestimated as it presumably reflects the actual rate of Ca release minus the rate of Ca re-uptake.

Even if cADPR and 2`-P-cADPR act on the same Ca channels, the linkage of both NAD and NADP metabolism to Ca signaling raises interesting metabolic possibilities as the NAD(H) and NADP(H) pools are functionally distinct. The NAD(H) pool is maintained in a highly oxidized state (42) as NAD serves as a hydride acceptor in multiple catabolic reactions and as a source of ADP-ribose for cellular ADP-ribose transfer enzymes(43) . In contrast, the NADP(H) pool is normally maintained in a highly reduced state to provide NADPH as a source of reducing equivalents for anabolic pathways(42) . The highly reduced state of the NADP(H) pool under normal metabolic conditions makes it a potentially sensitive indicator of metabolic conditions that cause oxidative stress. In that vein, it is of interest that the studies of Richter and Kass (44) have closely linked agents that oxidize the NADP(H) pool to the activation of a mitochondrial NAD glycohydrolase and to rapid Ca efflux from mitochondria.

FOOTNOTES

*

This work was supported in part by National Institutes of Health Grant CA43894. 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. Tel.: 606-257-5283; Fax: 606-257-7585.

()

The abbreviations used are: IP, 1-D-myo-inositol 1,4,5-trisphosphate; cADPR, cyclic adenosine diphosphoribose; ADPR, adenosine diphosphoribose; 2`-P-cADPR, 2`-phospho-cyclic adenosine diphosphoribose; 2`-P-ADPR, 2`-phosphoadenosine diphosphoribose; HPLC, high performance liquid chromatography; MES, 2-(N-morpholino)ethanesulfonic acid; TAPS, 3-[tris(hydroxymethyl)methyl]aminopropanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid; NAADP, nicotinic acid-adenine dinucleotide phosphate.

()

D. Cervantes-Laurean and M. K. Jacobson, unpublished data.

()

H. Kim and M. K. Jacobson, unpublished data.

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