(Received for publication, October 6, 1994; and in revised form, December 5, 1994)
From the
Transient fluxes of intracellular ionized calcium
(Ca) from intracellular stores are integral
components of regulatory signaling pathways operating in numerous
biological regulations, including in early stages of egg fertilization.
Therefore, we explored whether NADP, which is rapidly generated by
phosphorylation of NAD upon fertilization may, directly or indirectly,
exert a regulatory role as a trigger of Ca
release
from intracellular stores in sea urchin eggs. NADP had no effect, but
we found that the deamidated derivative of NADP, nicotinate adenine
dinucleotide phosphate (
-NAADP), is a potent and specific stimulus
(ED
16 nM) for Ca
release in
sea urchin egg homogenates. NAADP triggers the Ca
release via a mechanism which is distinct from the well-known
Ca
release systems triggered either by
inositol-1,4,5-triphosphate (IP
) or by cyclic adenosine
diphospho-ribose (cADPR). The NAADP-induced release of Ca
is not blocked by heparin, an antagonist of IP
, or by
procaine or ruthenium red, antagonists of cADPR. However, it is
selectively blocked by thionicotinamide-NADP which does not inhibit the
actions of IP
or cADPR. NAADP produced by heating of NADP
in alkaline (pH = 12) medium or synthetized enzymatically by
nicotinic acid-NADP reaction catalyzed by NAD glycohydrolase have
identical properties. The results presented herein thus describe a
novel endocellular Ca
-releasing system controlled by
NAADP as a specific stimulus. The NAADP-controlled Ca
release system may be an integral component of multiple
intracellular regulations occurring in fertilized sea urchin eggs,
which are mediated by intracellular Ca
release, and
may also have similar role(s) in other tissues.
One of the earliest and essential events following egg
fertilization is a transient release of calcium (Ca)
from intracellular stores(1, 5) . It is firmly
established that inositol-1,4,5 trisphosphate (IP
) (
)is one of the intracellular agents that triggers
Ca
release from intracellular stores via a specific
receptor channel(2) . More recently, Lee and associates (3) discovered that cyclic ADP-ribose (cADPR), a metabolite of
-NAD, also triggers Ca
release in both sea
urchin egg homogenates and intact eggs(3) , but via a
biochemical mechanism which is entirely different from that for
IP
(4) . In contrast to IP
, cADPR
triggers Ca
release by a ryanodine channel in a
calmodulin-dependent
mechanism(5, 6, 7, 8) . According to
recent reports by Lee et al.(10) and Galione et
al.(9) , both IP
and cADPR systems are
involved in Ca
release during egg fertilization.
However, Epel (11) pointed out that the Ca
which is released following egg fertilization acts as a modulator
with multiple targets within the cell and suggested possible
hierarchies in the regulation by Ca
(12) .
This notion raises the possibility that intracellular Ca
[Ca
]
release
following egg fertilization, and perhaps
[Ca
]
release in other
cells and tissues, may be stimulated by biomodulators other than
IP
and cADPR which control distinct Ca
channels.
It is of particular interest that one of the early
biochemical events in fertilization of the sea urchin egg is a marked
increase in activity of NAD kinase, which catalyzes rapid conversion of
NAD to NADP(13, 14) . Importantly, the surge in NADP
concentration precedes an increase in the cell respiratory rate, and
this feature may suggest, according to Epel and
coworkers(13, 14) , that the increased NADP has a
regulatory rather than a metabolic role(13, 14) . In
view of findings by Clapper et al.(3) that -NAD
is converted to the potent Ca
-releasing compound
cADPR, we considered that
-NADP may also be converted to a
metabolite which acts as a distinct
[Ca
]
releasing factor
that may also contribute to the regulation of
[Ca
]
release during
fertilization of the sea urchin egg. We found that NADP alone does not
trigger [Ca
]
release.
In search of new
[Ca
]
-releasing factors
derived from NADP, we focused on an early observation by Lee and
associates (3) who in the course of their study of cADPR
synthesis from
-NAD noted that exposure of NADP to strong alkaline
pH resulted in generation of a Ca
releasing activity
that was apparently distinct from activity of IP
or cADPR.
In this report we provide evidence that a derivative of NADP with a
deamidated nicotinamide moiety, NAADP is a potent
[Ca
]
-releasing agent,
and its action is blocked specifically by thionicotinamide-NADP.
Figure 1:
[Ca2+]-mobilizing
activity of ALK-NADP in the sea urchin egg homogenate bioassay. ALK-NADP was produced by alkaline treatment, i.e. incubation of HPLC-purified NADP in NaOH buffer (pH = 12)
at 60 °C for 30 min then neutralized to pH = 7, and the
resulting mixture of compounds was separated by HPLC (see Fig. 2). The concentration of ALK-NADP was then estimated
assuming that the extinction coefficient at 254 nm is the same for both
NADP and ALK-NADP. The time course of
[Ca
]
release from a
1.25% sea urchin egg homogenate was measured fluorometrically using
Fluo-3 as described previously(7) . Changes in fluorescence
were calibrated by the addition of known amounts of Ca
to the sea urchin egg homogenate. Inset, dependence of
[Ca
]
release on
ALK-NADP concentration. The ED
of ALK-NADP for induction
of Ca
release was
16
nM.
Figure 2:
HPLC purification of ALK-NADP (NAADP).
HPLC profile of NADP before (A) and after (B)
alkaline treatment (see Fig. 1). Samples were analyzed by
anion-exchange HPLC using an AG MP-1 column, with a non-linear gradient
ordinate, right side (panel A) of 150 mM trifluoracetic acid (TFA); (- -
-) and water at a flow rate 4 ml/min (UV
absorbance
(-) ordinate, left side). The
[Ca
]
release activity
in the eluates was determined in fractions using the sea urchin egg
homogenate [Ca
]
release bioassay. Alkaline treatment of NADP (see text)
generated several peaks that absorb at UV
nm (panel
B). The [Ca
]
releasing activity (- -
) in the eluate coincided with
a single UV peak that eluted at 17 min. This peak was rechromatographed
on an AG MP-1 column. The second chromatogram showed only the single
peak that was eluted at 17 min (not shown). These samples were also
analyzed by a TLC polyethyleneimine-cellulose system using 0.2 M NH
HCO
as developing buffer for 2 h. The
compound ALK-NADP appeared as a single UV spot on the TLC plate (R
=
0.25).
Binding of [H]IP
was performed as described previously(14) , with minor
modifications. Sea urchin egg homogenates (2 mg/ml) were incubated with
10 nM [
H]IP
(17 Ci/mmol) at
4 °C in IM for 3 min. The filters were then washed and
radioactivity counted essentially as described for
[
H]cADPR binding.
NAADP was biosynthetized
via the base-exchange reaction catalyzed by NAD-glycohydrolase as
described by Bernovsky(16) . Nicotinic acid (500 mM)
and NADP (10 mM) were incubated in 20 mM triethanolamine (pH 7.6) with 0.26 g/ml calf spleen
NAD-glycohydrolase (NAD(P)ase; Sigma) at 37 °C for 90 min. The
reaction was stopped by addition of an equal volume of acetone. The
mixture was centrifuged at 2,000
g for 2 min and after
acetone was evaporated under a stream of N
gas supernatant
was used for HPLC analysis, as described above. The HPLC-purified
NAADP
was assayed by the quantitative conversion to
NAAD
(nicotinic acid adenine dinucleotide reduced
form) following incubation with calf intestine alkaline phosphatase
(Sigma) as described by Bernovsky (16) (not shown).
Sea
urchins L. pictus were obtained from Marines, Inc.; Long
Beach, CA. Fluo-3 was from Molecular Probes Inc., Eugene, OR, and
IP and ryanodine were purchased from Calbiochem. Cyclic
ADP-ribose and [
H]cADPR were from Amersham Corp,
[
H]IP
was from DuPont NEN. All other
reagents, including nucleotides listed in Table 1, of the highest
purity grade available were supplied from Sigma.
HPLC-purified NADP does not trigger release of intracellular
Ca from sea urchin eggs homogenates. However,
following an ``alkaline treatment,'' i.e. after
incubation of NADP with NaOH (pH = 12) at 60 °C for 30 min
and adjustment to neutral pH, a product of NADP was generated (denoted
further as ``ALK-NADP'') that triggers the release of
[Ca
]
from sea urchin egg
homogenates (Fig. 1). The product of alkaline-treated NADP was
then purified by anion-exchange HPLC using a gradient of TFA (Fig. 2); the fractions were monitored for UV absorption at 254
nm and analyzed for [Ca
]
releasing activity in sea urchin egg homogenate bioassay (see
``Materials and Methods''). As shown in Fig. 2,
alkaline treatment results in the decrease of the area peak of NADP,
and several distinct UV peaks appeared. Of these peaks, the
[Ca
]
releasing activity was
found in eluate of a single UV peak that was eluted by higher ionic
strength trifluoroacetic acid solvent (at 18 min), thus indicating that
the bioactive product is more electronegative than NADP. In all
subsequent experiments we used the HPLC-purified ALK-NADP, the product
of the alkaline treatment of NADP. The
[Ca
]
release elicited by
ALK-NADP was dose dependent, and the maximum effect that was comparable
to the Ca
release responses achieved by maximally
effective concentrations of cADPR or IP
(Fig. 1).
The concentration of ALK-NADP needed for half-maximal Ca
release in sea urchin homogenates is about 16 nM,
suggesting that the potency of ALK-NADP is comparable to cADPR and is
more potent than IP
(3) . ALK-NADP-triggered
[Ca
]
release was temperature
dependent in the range of 10-30 °C; however, increasing the
temperature of sea urchin eggs homogenate to 42 °C completely
abolished release in response to ALK-NADP. To determine whether the
Ca
release mechanism activated by ALK-NADP is clearly
distinct from the Ca
release triggered by cADPR or
IP
, we conducted a series of experimental maneuvers,
including the use of pharmacological tools as described below.
Figure 3:
Homologous desensitization of the sea
urchin egg homogenate. Experimental conditions were as described under
``Materials and Methods.'' Arrows indicate the
sequential addition of the
[Ca]
-mobilizing
compounds; 160 nM cADPR, 160 nM ALK-NADP, 2
µM IP
, 1.4 mM caffeine and 100
µM ryanodine.
It was described previously that sea urchin
egg homogenates treated with ryanodine and/or caffeine were
desensitized not only to subsequent addition of these agents but also
to cADPR, while IP could still trigger
[Ca
]
release(4, 7) . Addition of ryanodine or
caffeine to the sea urchin egg homogenate causes relatively slow
[Ca
]
release and, after
resequestration of Ca
subsequent addition of cADPR
fails to trigger [Ca
]
release (Fig. 3). In contrast, the same sea urchin egg homogenate
responds promptly to the addition of ALK-NADP by a marked release of
[Ca
]
(Fig. 3E).
The reverse was also true: desensitization of the sea urchin egg
homogenate to subsequent additions of ALK-NADP does not alter
Ca
release triggered by ryanodine or caffeine (data
not shown).
Figure 4:
Specificity of
[Ca2+]-releasing activity
of NAADP)determined by inhibitors.
[Ca
]
release was
triggered by addition (
) of 160 nM ALK-NADP; with
control, no additions (A); with 1 mM procaine (B); with 320 µg/ml heparin (C); 36 µM ruthenium red (D); or 40 µM thio-NADP (E).
While evaluating the effects of NADP derivatives (Table 1) we found that thionicotinamide-NADP (thio-NADP)
strongly blocked [Ca]
release
induced by ALK-NADP ( Fig. 4and Fig. 5), but had no
inhibitory effect upon [Ca
]
release triggered by cADPR (Fig. 5) or by IP
(not shown). The inhibitory effect of thio-NADP was dose
dependent, with a half-maximal inhibitory concentration of
approximately 3 µM (Fig. 5, inset).
Figure 5:
Inhibition of ALK-NADP-induced
Ca release by thio-NADP
.
[Ca
]
release from sea
urchin egg homogenates (1.25%) was monitored using Fluo-3 as
Ca
indicator. 160 nM ALK-NADP, 24 µM thio-NADP, and 160 nM cADPR were added as indicated
(
) on the abscissa in the figure. The inset of
the figure shows the dose-dependent inhibition of
[Ca
]
release induced
by 160 nM ALK-NADP by pretreatment (60 s) of sea urchin egg
homogenates with increasing concentrations of thio-NADP
(ID
3 µM).
Figure 6:
Binding of
[H]-cADPR, upper panel, and
[
H]-IP
, lower panel, to sea
urchin egg homogenates; for details see ``Materials and
Methods.'' Effect of addition of unlabeled ligands in 1000
higher concentration. Upper panel (a): A, 10 nM [
H]cADPR alone; B, with 10
µM cADPR; C, with 10 µM ADPR; D, with 10 µM ALK-NADP. Lower panel (b): A, 10 nM [
H]IP
alone; B, with 10 µM IP
; C, with 10 µM cADPR; D, with 10
µM ALK-NADP. Each bar denotes mean ± S.E.
of six experiments.
Of particular interest and importance is the
finding that thio-NADP, which after alkaline treatment did not exhibit
[Ca]
releasing activity, is
effective as a specific and dose-dependent inhibitor of the
[Ca
]
releasing activity of
ALK-NADP, but does not interfere with
[Ca
]
release elicited by
IP
or cADPR ( Fig. 4and Fig. 5).
Incubation of ALK-NADP with alkaline phosphatase or with snake venom
phosphodiesterase completely abolished the
[Ca]
releasing activity. This
indicates that the 2`-phosphate on the adenosine moiety and an intact
diester moiety are both essential for biologic activity of ALK-NADP. At
neutral pH, the [Ca
]
releasing
activity of ALK-NADP in aqueous solution remained intact even when
heated to 100 °C for 2 h, whereas boiling in strong acid (pH
= 2) or strong alkali (pH = 12) abolished the
Ca
releasing activity of ALK-NADP. The ALK-NADP
compound is absorbed on anion-exchange resin (Dowex
1) or on
charcoal, but is not extracted from aqueous solution by ether (data not
shown).
The NMR analysis of ALK-NADP confirmed the presence of all
non-exchangeable proton peaks associated with the nicotinamide group
H,-4,-5,-6, although they were shifted; the two adenine
protons HA
and -8 were still present, and the two anomeric
protons of the ribose units of ALK-NADP were in the same spectral
region as in NADP (not shown). The mass spectrum of
HPLC-purified ALK-NADP (Fig. 7) revealed that ALK-NADP differs
from NADP by only 1 Da, and NADP presented a molecular ion species (in
negative mode) at m/z 742 and satiated species at 764
(+Na) and 786 (+2Na). Analysis of ALK-NADP under the same
conditions (Fig. 7) revealed a molecular species at m/z 743 and satiated species at 765 (+Na).
Figure 7: Mass spectrometry of NADP and ALK-NADP. Upper panel, NADP. Lower panel: ALK-NADP. The spectra were obtained as described under ``Materials and Methods.'' ALK-NADP shows molecular mass 1 Da higher than NADP.
Based on results of all analytical procedures and other experimental evidence we propose that ALK-NADP, a product of alkaline treatment of NADP, is NADP derivative with deamidated nicotinamide moiety, and hence identical to nicotinate (nicotinic acid) NAADP.
In view of these conclusions, we
explored whether non-enzymatically produced ALK-NADP has properties
that are identical to NAADP generated by an enzymatic reaction. NAADP
was prepared by an enzymatic exchange reaction between NADP and
nicotinic acid catalyzed by NAD-glycohydrolase as described by
Brenofsky(14) . Incubation of NADP with nicotinic acid in the
presence of calf spleen NAD-glycohydrolase indeed resulted in the
production of 2:2:1 of NADP/NAADP/2`P-ADPR. Importantly, NAADP prepared
in this manner co-elutes by HPLC with ALK-NADP (Fig. 8) and
shows the same [Ca]
releasing
activity (ED
) as non-enzymatically prepared ALK-NADP,
including homologous desensitization (Fig. 9) and identical
specific inhibition by thionicotinamide-NADP. Furthermore, repeat
additions of enzymatically prepared NAADP does not produce
desensitization of the [Ca
]
release induced by cADPR (Fig. 9) or IP
(data
not shown). Finally, incubation of ALK-NADP with alkaline phosphatase
results in a compound which is inactive (see above) and which coelutes
by HPLC with authentic nicotinate adenine dinucleotide (NAAD) (Fig. 10). Thus, all evidence presented here leads to the
conclusion that ALK-NADP is identical with NAADP.
Figure 8:
Enzymatic biosynthesis of
NAADPcatalyzed by NAD(P)-glycohydralase. The
nicotinate analog of NADP
(NAADP) was formed
by the base exchange reaction as described under ``Materials and Methods.'' The reaction mixture was diluted 50-fold,
and 0.5 ml of the mixture was subjected to anion-exchange HPLC as
described in Fig. 2. The figure shows HPLC analysis of time 0
min(- - - ), and 90 min (-) of incubation. Both NAADP and
ALK-NADP UV peaks (A
nm) were coeluted at 18 min
(compare Fig. 2).
Figure 9:
[Ca]
release activity of biosynthetic NAADP.
[Ca
]
release in 1.25%
sea urchin egg homogenate was monitored using Fluo-3 as the
Ca
indicator. Arrows on the abscissa indicate addition of 160 nM NAADP produced by the
NAD(P)-glycohydrolase-catalyzed exchange reaction as described in Fig. 8, 160 nM ALK-NADP (produced by alkaline treatment
of NADP), and 160 nM cADPR.
Figure 10:
Hydrolysis of ALK-NADP by alkaline
phosphatase. ALK-NADP (80 µM) produced and purified as
described under ``Materials and Methods'' was incubated in a
medium containing 40 mM Tris-HCl buffer (pH 8.2) at 35 °C
for 10 min in the presence (-) or absence(- - - ) of 10
units/ml alkaline phosphatase, and the change in
[Ca]
release activity
was monitored by the addition of an aliquot of the mixture to the sea
urchin egg homogenate assay. Results show inactivation of the
[Ca
]
-releasing
compound by alkaline phosphatase. The reaction was stopped by addition
of an equal volume of acetone. The mixtures were centrifuged at 2,000
g for 2 min, and after acetone evaporation the
supernatant (80 nM) was used for anion-exchange HPLC analysis,
as described under ``Materials and Methods.'' (NA denotes nicotinic acid.)
It was previously shown that after treatment with alkali the
NADP promotes Ca release from intracellular stores of
sea urchin egg homogenates by a mechanism that is apparently distinct
from cADPR and IP
-induced
[Ca
]
release (3) .
Experimental evidence presented herein leads to the conclusion that one
of the products of alkaline treatment of NADP is a derivative of NADP
with a deamidated nicotinamide moiety, NAADP, and that this nucleotide
has distinct and specific biologic properties. NAADP has the capacity
to trigger, in nanomolar concentrations, the release of
[Ca
]
via a mechanism which is
clearly different from those of the well-known
[Ca
]
-releasing agents IP
and cADPR. The evidence indicating that the action of NAADP upon
[Ca
]
release is different from
that of IP
or cADPR includes the finding of homologous
desensitization and the absence of cross-desensitization with IP
or cADPR (Fig. 3). The lack of inhibition of
NAADP-triggered [Ca
]
release by
known antagonists of cADPR and IP
(Fig. 4) and its
specific inhibition by thio-NADP (Fig. 5), which has no blocking
effect upon other [Ca
]
releasing systems, constitute pharmacologic evidence for the
specificity of NAADP action ( Fig. 4and 5). All of these
observations are consistent with the results of binding-displacement
experiments with [
H]cADPR and
[
H]IP
(Fig. 6) which show that
the site of interaction of NAADP is distinct from those of cADPR and
IP
. Taken together, the evidence thus supports the
hypothesis that the [Ca
]
release mechanisms triggered by NAADP, cADPR, and IP
are different.
The results of various analytical procedures
employed in our study strongly support our conclusion that the
[Ca]
-releasing compound
resulting from alkaline treatment of
-NADP is
-NAADP.
Evidence includes UV spectral analysis, mass spectrometry, NMR
analyses, sensitivity to specific enzymes, as well as electrostatic
properties deduced from an ion-exchange HPLC and Dowex absorption
studies. The identical properties of the non-enzymatically prepared
ALK-NADP with the compound prepared by enzymatically catalyzed exchange
reaction according to Bernovsky(16) , in chemical and
[Ca
]
releasing studies, further
affirms that the newly identified
[Ca
]
-releasing substance is
indeed
-NAADP.
Furthermore, the results allow the possibility that NAD-glycohydrolase can catalyze exchange of nicotinate on NADP in vivo and thus constitute a possible route for NAADP biosynthesis. NAAD is known to be an abundant and essential compound that is an intermediary metabolite in the biosynthetic pathways of NAD and NADP(18) . Thus, besides the nicotinate-NADP exchange reaction catalyzed by NAD-glycohydrolase(16) , another route of biosynthesis of NAADP could be via phosphorylation of NAAD to NAADP by NAD kinase with ATP as the phosphate donor(13) . Possibly, the NAD kinase may also accept NAAD as a substrate or, alternatively, analogous kinase(s) may exist with a substrate specificity for NAAD. Finally, NADP could be enzymatically converted to NAADP by a one-step enzymatic reaction catalyzing deamidation of nicotinamide to nicotinate. However, while some of our observations suggest that NAADP can be generated enzymatically, the biosynthetic and catabolic pathways for NAADP in various tissues and cells remain to be determined.
Sea
urchin eggs are a relatively simple system that can serve as a model
for the study of the
[Ca]
-mediated biochemical
mechanisms that occur during fertilization in which the cADPR signaling
pathway(3) , as well as another novel NAADP-triggered
[Ca
]
-releasing system, has been
identified in the present study. Consequently, the release of
[Ca
]
in sea urchin eggs after
fertilization may be controlled not only by IP
and/or by
cADPR, but also by NAADP. The significance of the complex control of
[Ca
]
release in the course of
fertilization is not yet apparent. Perhaps multiple mechanisms for
[Ca
]
release may be needed for
the redundant control(9) , or alternatively the regulatory
functions may be hierarchial in nature(11) , i.e. aimed to control multiple targets(9) . Finally, our
discovery of a new specific NAADP-controlled
[Ca
]
release system in sea
urchin eggs opens the possibility that this signaling pathway may be
present in various other cell types and tissues.