©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Caged Cyclic ADP-Ribose
SYNTHESIS AND USE (*)

(Received for publication, November 2, 1994; and in revised form, January 31, 1995)

Robert Aarhus (§) Kyle Gee (1) Hon Cheung Lee(§)(¶)

From the Department of Physiology, University of Minnesota, Minneapolis, Minnesota 55455 and Molecular Probes, Inc., Eugene, Oregon 97402

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cyclic ADP-ribose (cADPR) is a recently discovered cyclic nucleotide with Ca mobilizing activity. Caged cADPR was synthesized by reacting cADPR with 2-nitrophenethyldiazoethane. Elemental analyses, ^1H NMR, and extinction coefficient measurements indicate that the product contains only one caging group. Anion exchange high pressure liquid chromatography separated caged cADPR into two forms, which most likely represent isomers. Both forms could be uncaged with equal efficiency by UV exposure to regenerate cADPR. Photolysis of caged cADPR was accomplished effectively with a spectrofluorimeter. The efficiency of uncaging depended on wavelength with UV light shorter than about 320 nm being the most effective. Caged cADPR was biologically inactive and could induce Ca release from sea urchin egg homogenates only after photolysis. Specificity of the Ca release was shown by inhibition by 8-amino-cADPR, a specific antagonist of cADPR. To demonstrate its utility in live cells, caged cADPR was microinjected into sea urchin eggs. Photolysis using a mercury light source effectively regenerated cADPR and resulted in Ca mobilization and activation of cortical exocytosis in the eggs.


INTRODUCTION

Cyclic ADP-ribose (cADPR) (^1)is a recently discovered cyclic nucleotide with signaling activity(1, 2, 3) . Unlike cAMP, cADPR is derived from NAD, and its signaling function is through activation of Ca release from intracellular stores(1) . A wide variety of cells, including invertebrate, amphibian(4) , and mammalian cells, have been shown to be responsive to cADPR (for review, see (5) ). Accumulating evidence indicates that it may be an endogenous regulator of the Ca-induced Ca release process mediated by ryanodine receptors(6, 7, 8) . Its synthetic enzyme, ADP-ribosyl cyclase, is ubiquitous (9, 10, 11) and, in the case of sea urchin eggs, is activated by a cGMP-dependent process(12) . This has led to the recent proposal that cADPR may be involved in the signaling pathway mediated by nitric oxide(13) .

The cyclic structure of cADPR is formed by linking the adenine ring of NAD to the terminal ribose, displacing the nicotinamide group(2) . X-ray crystallography showed that the linkage site is at N-1 of the adenine ring and the cycling glycosidic bond is in the beta-conformation(3) . The structure of cADPR has been also confirmed by chemical synthesis(14) . Detailed knowledge of the structure of the molecule provides rational approaches for synthesizing useful analogs. Indeed, a series of analogs with substitution at the 8-position of the adenine ring has been synthesized and shown to be antagonists of cADPR (15) . Among these, 8-amino-cADPR is the most potent and, at nanomolar concentrations, specifically inhibits the Ca mobilizing effect of cADPR in vitro as well as in intact cells(16) . In this study, we couple a photoactivatable group to one of the phosphates of cADPR. The product, caged cADPR, is biologically inactive until UV activation by photolysis of the caging group. The use of caged cADPR in vitro and in vivo are described.


EXPERIMENTAL PROCEDURES

Synthesis

cADPR was synthesized by incubation of NAD with the Aplysia ADP-ribosyl cyclase and purified by HPLC using an AG MP-1 column as described previously(9) . The purified cADPR (48 mg, 0.092 mmol) was dissolved in 3 ml of ice-cold E-pure water, and the resulting pH of the solution was 2.3. To this stirring solution was added 2-nitrophenethyldiazoethane (0.28 mmol, prepared as described in (17) ) in 3 ml of diethyl ether. The resulting biphasic mixture was vigorously stirred at 0-5 °C in darkness for 3 h, during which the color of the diazoethane solution changed from amber to pale yellow. The ether layer was drawn off, and the diazoethane/ether treatment was repeated 3 more times. The aqueous portion was applied to a Sephadex LH-20 column (2 times 20 cm), and eluted with water; 2-ml fractions were collected. The caged product (silica gel R(F) 0.55, MeOH/CHCl(3)/H(2)O/HAc, 12.5:10:3.5:0.2) was isolated as a fluffy white powder after lyophilization of the combined product fractions (30 mg, 49%). Unreacted cADPR was also recovered (R(F) 0.13, 15 mg, 31%). The caged product could be efficiently photolyzed into free cADPR, even by a hand-held UV lamp as analyzed by thin layer chromatography. As described under ``Results,'' the caged product represented a mixture of two mono-caged isomers, which can be separated by anion exchange HPLC. For the unseparated product, the extinction coefficient at 259 nm was determined to be 17,200 using 0.8 mmol of the product dissolved in water: m.p. 186 °C (decomposition); ^1H NMR (Me(2)SO-d(6)) 9.1-8.6 ppm (m, 2), 8.0-7.5 ppm (m, 4), 6.1-5.8 ppm (m, 4), 5.5 ppm (m, 3), 4.9 ppm (m, 1), 4.5-3.8 ppm (m, 10), 1.65 ppm (m, 2), 1.50 ppm (dd, J = 18.4, 6.5 Hz, 1). Elemental analysis was performed by Desert Analytics (Tucson, AZ) based on the formula of mono-caged cADPR, CHN(6)OP(2)bulletH(2)O.

Ca Release in Homogenates and Intact Eggs

Homogenates of sea urchin egg (Strongylocentrotus purpuratus) were prepared as described previously(1, 7) . Frozen egg homogenates (25%) were thawed at 17 °C 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 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 spectrofluorimetrically in 1.25% homogenates with an excitation wavelength of 490 nm and emission wavelength of 535 nm. The measurements were done in a cuvette maintained at 17 °C, and the homogenates were continuously stirred. The volume of homogenate used was 0.2 ml, and the additions were usually made in 2-µl volumes. All results described are representative of at least three similar experiments.

Lytechinus pictus eggs were used for the microinjection experiments. The procedures for microinjection by pressure were as described previously(16, 18) . Ca changes in the injected eggs were measured using fluo-3. Samples were dissolved in the injection buffer containing 0.5 M KCl, 50 µM EGTA, 10 mM Hepes, pH 6.7. The numbers of eggs injected and the statistics of the results are described in the text.

Photolysis

Activation of caged cADPR in egg homogenates was achieved in a Hitachi spectrofluorimeter (S-2000) by alternating the excitation wavelength every 2 s between 350 nm for photolysis and 490 nm for monitoring fluo-3 fluorescence. Photolysis of caged cADPR in individual eggs was performed by modifying the epifluorescence attachment of a Nikon inverted fluorescence microscope. A second mercury lamp was attached at 90° to the epifluorescence tube. The light of 300-400 nm for photolysis was selected with a UG1 filter (Omega Optical, Brattleboro, VT) and reflected 90°, first with a 400 DCLP dichroic filter and then directed toward the objective with a second BCECF Sp dichroic filter. For monitoring the fluo-3 fluorescence, 490-nm light from the first mercury lamp was selected with a 485DF22 filter. The excitation light passed through the same 400 DCLP dichroic filter and was reflected by the BCECF Sp dichroic filter toward the objective. The fluo-3 fluorescence was selected by a long pass filter with a 500-nm cutoff and monitored by a SIT camera. This optical arrangement allowed simultaneous measurement of fluo-3 fluorescence during photolysis.

HPLC and ^1H NMR 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(2) . Caged cADPR was dissolved in Me(2)SO-d(6) and analyzed with a 400 MHz NMR spectrometer (Bruker AM400).


RESULTS

The novel cyclic structure of cADPR may sometimes convey the false impression of fragility, but, in fact, it is a very stable molecule. The half-time of hydrolysis at pH 2.0 is about 24 h even at 37 °C, and it is several days at room temperature(2, 11) . The stability of cADPR allows the use of the procedure developed by Walker et al.(17) for coupling caging groups to molecules containing phosphate, since it requires incubation at acidic pH for an extended period. The caged cADPR synthesized was a mixture of two forms that could be separated by anion exchange HPLC (Fig. 1). After exposing the caged cADPR mixture to 350-nm UV light in a spectrofluorimeter for 2 h, the peaks corresponding to the two caged forms decreased and a single peak corresponding to cADPR appeared. The time course of the uncaging process is shown in the inset. Since the efficiency of uncaging of the two forms appeared to be equal and both were converted to cADPR, the caged cADPR mixture was used in this study without further purification.


Figure 1: HPLC analyses of caged cADPR. The samples were analyzed by anion exchange HPLC before (dashedcurve) and after (solidcurve) 2 h of exposure to 350-nm UV light in a spectrofluorimeter. The areas of the chromatographic peaks were obtained by integration and plotted in the inset to show the time course of the uncaging process.



In principle, two caging groups could be attached to cADPR, producing bis-caged cADPR. The two forms of caged cADPR separated by HPLC as shown in Fig. 1could represent mono-caged and bis-caged cADPR. However, several lines of evidence indicate clearly that the caged cADPR synthesized contained only a single caging group. First, the measured extinction coefficient of 17,200 M cm at 259 nm agrees well with the 17,500 M cm determined for mono-caged ATP(17, 19) . Bis-caged product would be expected to have extinction coefficient about 5,000 M cm higher at the same wavelength. Second, elemental analyses indicate that the caged cADPR contained 38.42% carbon, which is much closer to the 39.05% calculated for hydrated mono-caged product than the 43.42% calculated for the bis-caged form. Finally, the integration of the ^1H NMR resonances in D(2)O also indicates mono- rather than bis-caged products. Table 1compares the measured ratios of protons in various resonance groups with the calculated values for both mono- and bis-caged products. The number of aryl (Ar) and the adenine (Ad) protons were obtained from integrating the NMR resonances between 9.1 and 7.2 ppm(2, 20) . Similarly, the anomeric (An) and benzylic (CH) protons were obtained from integrating 6.8 to 5.7 ppm, while the methyl (Me) protons of the caging group were from 2.0 to 1.5 ppm. Table 1shows that the integration ratios of all three regions match those calculated for mono- rather than bis-caged product. Therefore, the two forms separated by HPLC as shown in Fig. 1most likely represent isomers of mono-caged cADPR. Fig. 2shows the proposed structure of caged cADPR and illustrates possible mono-caged products that can result from reacting the caging diazoethane with cADPR. Alkylation could occur at either of the two phosphate groups giving rise to constitutional isomers. Also, since each phosphorous is a chiral center and a new chiral center is created at the benzylic position of the caging group upon alkylation of a phosphate, various stereoisomers could be formed. Finally, the orientation of the methyl group in the caging moiety, represented by the wavybond shown in Fig. 2, can also result in stereoisomers. These various isomers are, however, functionally equivalent since, as shown in Fig. 1, the two forms of caged cADPR can be uncaged with similar efficiency and both regenerate cADPR after uncaging; no further attempt was made to characterize the two forms.




Figure 2: The proposed structure of mono-caged cADPR. The chemical structure of cADPR was derived of x-ray crystallography(3) . Possible isomers of mono-caged cADPR include those with the caging group, (2-nitrophenyl)ethyl, on either of the two phosphates and those with a different orientation of the methyl group (denoted by a wavyline).



Fig. 3shows that adding caged cADPR to egg microsomes did not produce any Ca release. Uncaging with UV light resulted in Ca release after a delay. The release was due to cADPR since addition of an antagonist, 8-amino-cADPR(15, 16) , during the Ca release immediately stopped the release process and the Ca was resequestered. In the presence of 8-amino-cADPR, uncaging cADPR with UV light did not produce any Ca release. Also shown in Fig. 3is a control experiment using caged ATP having an identical caging group (Molecular Probes Inc., OR). Caged ATP did not produce any Ca release before or after uncaging with UV light. Therefore, the Ca release observed with caged cADPR was not due to artifacts of exposure to UV or biproducts of the uncaging reaction.


Figure 3: Calcium release from egg homogenates induced by caged cADPR after photolysis. Photolysis of caged cADPR was performed by alternating the excitation wavelength of the spectrofluorimeter between UV (350 nm) and the monitoring wavelength of 490 nm. Ca release from egg homogenates was monitored by fluo-3 fluorescence in the absence ((-)8NH) or presence ((+)8NH) of 2 µM of 8-amino-cADPR, a specific antagonist of cADPR. Caged cADPR and caged ATP were added to the final concentrations indicated.



The uncaging efficiency was assessed in experiments shown in Fig. 4. The response of egg homogenates to cADPR was first calibrated with the addition of increasing concentrations of cADPR as shown in Fig. 4A. Under constant UV illumination, Ca release activity of caged cADPR was similarly tested as shown in Fig. 4B. Comparing the Ca release, it can be seen that uncaging 10 µM of caged cADPR produced as much Ca release as about 100 nM of cADPR, indicating the efficiency of uncaging is about 1%. The low efficiency is most likely due to the low UV intensity of the light source used in the spectrofluorimeter. The fact that relatively low concentrations of caged cADPR were needed and that a readily available instrument like a spectrofluorimeter was sufficient for photolysis illustrates the usefulness of the caged product.


Figure 4: Efficiency of photolysis of caged cADPR. The efficiency was assessed by comparing the Ca release activity of caged cADPR under constant UV illumination with cADPR. Caged cADPR and cADPR were added at the final concentrations indicated. The time scale shown applies to Fig. 4, A and B.



The dependence of the wavelength of the UV light for uncaging is shown in Fig. 5. Shorter UV light was more efficient in uncaging than longer wavelength UV light; not only did the maximal Ca release increase, but the time it took to achieve maximal release was also shorter. The inset of Fig. 5shows that the effects of wavelength on both the maximal Ca release and the time to maximum Ca release appear to level off for light shorter than about 320 nm. This may be because of the reduction in UV light intensity due to absorption by proteins.


Figure 5: The dependence of the photolysis efficiency on wavelength. Photolysis efficiency was assessed by comparing the Ca release activity of caged cADPR under constant UV illumination. Photolysis was performed by alternating the excitation wavelength of the spectrofluorimeter between UV (310-400 nm) and the monitoring wavelength of 490 nm. Ca release from egg homogenates was monitored by fluo-3 fluorescence. The inset shows the dependence of the maximal Ca release and the time to maximum on the photolysis wavelength. Caged cADPR was added to a final concentration of 17.5 µM.



Fig. 6shows that the expected Ca increase can be induced by photolyzing caged cADPR in intact sea urchin eggs. Ca changes in the egg were monitored with the indicator fluo-3, which was co-injected with caged cADPR into the egg. Exposure to UV induced Ca release after a slight delay, which is indicated by a 3-fold increase in fluo-3 fluorescence. The average fluorescence increase was 3.3 ± 1.0-fold (S.E.) measured in 10 eggs injected with 2.3 ± 0.7 µM caged cADPR (Table 2). The time delay between the start of UV exposure and reaching the maximal fluo-3 fluorescence was 44.5 ± 14.1 s (n = 10, S.E.). Also shown in Fig. 6is the absence of Ca change in a control egg injected with the same concentration of caged ATP, indicating the Ca changes observed with caged cADPR were not due to UV exposure or the biproducts of photolysis. In addition to inducing Ca changes, uncaging also induced the cortical reaction, another index of Ca mobilization. The micrographs in Fig. 6show an egg injected with caged cADPR before and after UV exposure. After about 20-30 s of UV exposure, a fertilization membrane is formed surrounding the whole egg. The cortical reaction was induced by photolysis in 11 out of 12 eggs injected with 0.2-5.5 µM caged cADPR. As a control, 10 eggs were injected with 1.9-7.2 µM caged ATP, and none of them underwent a cortical reaction after photolysis. These results show that the caged cADPR can also be effectively uncaged inside live cells.


Figure 6: Calcium release induced by photolysis of caged cADPR in live sea urchin eggs. Ca release in individual eggs was monitored by fluo-3 fluorescence. Caged cADPR (4.9 µM, intracellular) or caged ATP (7.2 µM, intracellular) were co-injected with fluo-3 (0.25 mM, intracellular) into an egg. The injection volumes were about 1.8-2.5% of the egg. All samples were dissolved in the injection buffer described under ``Experimental Procedures.'' Photolysis was induced by UV light at around 360 nm. The micrographs show an egg loaded with caged cADPR before and after UV exposure. A fertilization membrane surrounding the egg was formed after photolysis.





As shown in Table 2, Ca mobilization as indicated by the increase in fluo-3 fluorescence was lower when induced by photolyzing caged cADPR than following fertilization or activation by direct injection of saturating concentrations of cADPR. The time it takes for the fluorescence to reach maximum (t(max)) following uncaging (44 s) was comparable with fertilization (63 s) but was slower than that induced by direct injection of cADPR (9 s). The apparent inefficiency of caged cADPR was mainly due to the barrier filter required for simultaneous measurement of the fluo-3 fluorescence during uncaging, which significantly reduced the UV intensity for uncaging. The cortical reaction following egg activation is another index for Ca mobilization that can be measured without the filter. With the removal of the filter, proper alignment of the light source, and a new mercury light bulb, the time needed to achieve full cortical reaction after turning on the UV excitation for uncaging was reduced to 13 s (Table 2), which is comparable with the 9 s measured by direct injection of saturating concentrations of cADPR. It is expected that the time delay can be further reduced with the use of quartz optics to increase transmittance of UV light. Shown also in Table 2is that the time required to achieve full cortical reaction following fertilization was about 41 s. It is clear that Ca mobilization in sea urchin eggs occurs with a time scale of seconds and that comparable kinetics can be achieved by activation with caged cADPR.


DISCUSSION

Cyclic ADP-ribose is a new cyclic nucleotide whose discovery was not anticipated. The novelty of the discovery necessitates extraordinary measures to establish the authenticity of its Ca-mobilizing activity. The first evidence was that it can induce desensitization, ruling out Ca contamination in the sample as a possible artifact(1) . This was further supported by the fact that its biological effect can be heat-inactivated(18) . We next synthesized a series of antagonists that can block both the specific binding of cADPR to its microsomal receptor as well as its Ca mobilizing activity(15, 16) . In this study, we synthesized caged cADPR and demonstrated, in vitro as well as in vivo, that it is biologically inactive until photolysis. Together, these results definitively establish the authenticity of the Ca-mobilizing activity of cADPR.

Another main advantage of the caged cADPR is its remarkable efficiency. Photolysis can be conveniently induced by readily available instruments such as a spectrofluorimeter or an epi-fluorescence attachment. Because these instruments are not specially optimized for uncaging, the kinetics of activation by photolysis were found to be somewhat slower than direct injection of saturating concentrations of cADPR itself. However, comparable rates can easily be achieved by removal of a barrier filter in the optical setup. Indeed, the uncaging efficiency of caged cADPR appears to be better than, or at least comparable with, other caged compounds. For example, egg activation induced by caged GTPS requires 30 s of UV exposure(21) . It is anticipated that with proper optimization and the use of high UV intensity flash lights, photolysis of caged cADPR can be achieved in a sub-second time scale.

The availability of caged cADPR should eliminate some common problems encountered in investigating Ca mobilization induced by cADPR. For example, Ca leakage during microinjection could be mistaken as Ca release activated by cADPR. With caged cADPR, Ca release is induced by UV photolysis, thus eliminating the possible injection artifact. Similarly, Ca contamination in the sample can easily be recognized since, with caged cADPR, no Ca changes should be observed without photolysis. Also, the use of caged cADPR may provide alternative approaches for introducing cADPR into cells other than by microinjection. For example, cells can be loaded with caged cADPR and an indicator (e.g. fluo-3) by transient permeabilization using procedures such as electroporation or scrape-loading(22) . Viable cells containing the indicator can easily be identified. After resealing and recovery, photolysis can then be used to regenerate cADPR in these cells, and the resultant Ca changes can be monitored by the indicator. Finally, the increase in hydrophobicity due to the caging group could increase permeability of caged cADPR to cells. Once the cells are loaded, cADPR can be generated, at any time, by exposure to UV. With focused laser light, this can even be accomplished at localized regions anywhere inside large cells, such as sea urchin eggs. Therefore, caged cADPR should be a valuable tool for investigating the temporal as well as spatial aspects of Ca mobilization induced by cADPR.


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: cADPR, Cyclic ADP-ribose; HPLC, high pressure liquid chromatography; GTPS, guanosine 5`-3-O-(thio)-triphosphate.


ACKNOWLEDGEMENTS

We thank Robert Swezey for convincing one of us (H. C. L.) of the feasibility of synthesizing caged cADPR 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.