(Received for publication, October 18, 1994; and in revised form, November 22, 1994)
From the
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.
It is generally accepted that inositol trisphosphate
(IP) (
)is a second messenger for mobilizing
internal Ca
stores (reviewed in (1) ).
However, most cells also contain IP
-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
, 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.
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.
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:
H 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
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
O and, therefore, do not show up in the
NMR spectra. Conversion of -NH
(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.
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
.
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
), 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
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
. As
shown in Fig. 8C, the same preparation of microsomes
responded to IP
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
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
and calibrated with known amounts of
Ca
.
The results described above indicate NAADP activates
Ca release by a mechanism different from that of
IP
and cADPR. To determine if the Ca
stores sensitive to NAADP are also distinct from that of IP
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
- 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
-sensitive stores. It is clear from these results that
the NAADP-sensitive stores are distinct from the IP
- 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.
We have previously demonstrated that both NAD and NADP mobilize Ca
through mechanisms
distinct from that of IP
, 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 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
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
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
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.