(Received for publication, September 1, 1995; and in revised form, December 11, 1995)
From the
Cyclic adenosine diphosphoribose (cADPR), a metabolite of NAD,
appears to modulate changes in intracellular free Ca levels by activation of ryanodine-sensitive Ca
channels. We report here that an ADPR cyclase purified from Aplysia californica readily catalyzes the conversion of NADP
to 2`-phospho-cyclic adenosine diphosphoribose (2`-P-cADPR), cyclized
at N-1 of the adenine moiety. An enzyme from canine spleen previously
shown to contain NAD glycohydrolase, ADPR cyclase, and cADPR hydrolase
activities also utilized NADP and 2`-P-cADPR as substrates. The
apparent K
value for NADP was 1.6
µM compared with 9.9 µM for NAD, and the V
with NADP was twice that with NAD, indicating
that 2`-P-cADPR is a likely metabolite in mammalian cells. 2`-P-cADPR
was as active as cADPR in eliciting Ca
release from
rat brain microsomes, but was unable to elicit Ca
release following conversion to 2`-P-ADPR by the action of canine
spleen NAD glycohydrolase. 2`-P-cADPR and
1-D-myo-inositol 1,4,5-trisphosphate (IP
)
appear to act by distinct mechanisms as microsomes desensitized to
IP
still released Ca
in response to
2`-P-cADPR and vice versa. Also, inhibition of IP
-induced
Ca
release by heparin had no effect on release by
2`-P-cADPR. Both 2`-P-cADPR and cADPR appear to act by a similar
mechanism based on similar kinetics of Ca
release,
similar dose-response curves, cross-desensitization, and partial
inhibition of release by procaine. The results of this study suggest
that 2`-P-cADPR may function as a new component of Ca
signaling and a possible link between NADP metabolism and
Ca
homeostasis.
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.
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) .
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% D
0 prior to NMR analysis:
H NMR
(D
0)
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.
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.
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.
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.
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.
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.