(Received for publication, November 2, 1994; and in revised form, January 31, 1995)
From the
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,
H 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.
Cyclic ADP-ribose (cADPR) ()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
-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.
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.
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
H NMR resonances in
D
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
) 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.
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.