(Received for publication, March 7, 1996)
From the
Ca mobilization from intracellular stores
constitutes an important mechanism for generating cytoplasmic
Ca
signals. Inositol trisphosphate (InsP
)
and ryanodine receptors are the two families of intracellular
Ca
release channels that have been identified, which
may be regulated by separate intracellular messengers, InsP
and cyclic adenosine 5`-diphosphate ribose, respectively. A third
molecule, nicotinic acid adenine dinucleotide phosphate (NAADP), has
recently been recognized as a potent Ca
releasing
agent in sea urchin eggs and microsomes. We now report that
non-releasing concentrations of NAADP fully and irreversibly inactivate
the NAADP-sensitive Ca
release mechanism. This
phenomenon occurred both in intact sea urchin eggs and in homogenates
and is not shared by either InsP
or cyclic adenosine
5`-diphosphate ribose. The novel properties of this Ca
release mechanism, giving a one-shot Ca
release, may be suited to irreversible cellular events.
Calcium stores in sea urchin eggs provide an excellent model to
study Ca release
mechanisms(1, 2, 3, 4) . The sea
urchin egg Ca
stores contain inositol trisphosphate
(InsP
)(
)-sensitive Ca
channels (1) and also ryanodine-sensitive release channels, activated by
the pyridine nucleotide metabolite, cyclic adenosine 5`-diphosphate
ribose (cADPR)(2) . Recently, a third distinct Ca
release mechanism has been identified which is potently and
selectively activated by a novel pyridine nucleotide, nicotinic acid
adenine dinucleotide phosphate (NAADP)(5, 6) . NAADP
releases Ca
from egg homogenates and microsomal
fractions in the presence of mitochondrial inhibitors and also when
microinjected into the egg(6, 7) . Neither heparin, an
established InsP
receptor antagonist, nor the ryanodine
receptor inhibitors, procaine and ruthenium red(8) , or the
selective cADPR antagonist 8-NH
-cADPR (9) block
NAADP-induced Ca
release(5, 6) ,
indicating that NAADP acts at an alternative site. In addition,
depletion of endoplasmic reticulum (ER) stores by pretreatment with
thapsigargin (10 µM) does not significantly affect NAADP
Ca
release, although it reduces release by both
InsP
and cADPR(10) . This result is in accordance
with the suggestion that the NAADP-sensitive Ca
store
may reside in an intracellular compartment distinct from the
InsP
- and cADPR-sensitive stores (6) .
We now
report that non-releasing concentrations of NAADP fully and
irreversibly inactivate the NAADP-sensitive Ca release mechanism both in intact sea urchin eggs and in
homogenates and that this property is not shared by either InsP
or cADPR. Moreover, NAADP mobilizes Ca
by
activating a Ca
release channel in the microsomal
membrane, which is selectively blocked by classical inhibitors of
L-type voltage-sensitive Ca
channels. These
properties suggest that this release mechanism might contribute to
complex patterns of Ca
signals widely observed in
intracellular signaling.
In the present study, NAADP potently released Ca in a dose-dependent manner from sea urchin homogenates (EC
approximately 32 nM) and triggered a Ca
wave in the intact sea urchin egg ( Fig. 1and Fig. 2A). Surprisingly, very low concentrations of
NAADP, which evoked little or no Ca
release, were
found to fully inactivate the NAADP-sensitive Ca
release mechanism both in the homogenate and the intact egg (Fig. 1, A and C, and Fig. 2, A and B).
Figure 1:
A, representative fluorimetric traces
of Ca release by 500 nM NAADP (top
trace) and of homologous desensitization by sub-threshold
concentrations of NAADP in 2.5% sea urchin homogenate. Homogenates were
pretreated for 3 min with the illustrated concentrations of NAADP and
then challenged with 500 nM NAADP. Ca
release is expressed as relative fluorescence units (R.F.U.). B, representative fluorimetric trace
showing the absence of cross desensitization by NAADP on cADPR and
InsP
-induced Ca
release. Concentration of
NAADP (N) and cADPR (cA) was 500 nM unless
stated. InsP
was 1 µM. C-E,
dose-response curves to NAADP, cADPR, and InsP
in
homogenate (filled squares) and residual release by maximal
concentrations of agonists (500 nM NAADP; 500 nM cADPR; 1 mM InsP
) after 3 min preincubation
with stated concentrations (open circles). Values are mean
± standard deviation of 6-9
determinations.
Figure 2:
Pseudocolor images of Ca
levels, measured using Fura-2, in sea urchin eggs. A,
injection of NAADP into sea urchin eggs resulted in a Ca
release that spread in a wave form across the egg followed by a
slower, smaller amplitude Ca
release. Subsequent
injection of a higher concentration of NAADP could not elicit
substantial Ca
release in the egg. B, lower
concentrations (5 nM) of NAADP resulted in less Ca
release, but these too prevented Ca
release by
a subsequent NAADP injection. C, InsP3 injections released
Ca
but this did not desensitise a subsequent
Ca
release response to higher concentrations of
InsP
applied some 1500 s later. The time course of the
average Ca
change within the egg (measured with
Fura-2, Molecular Probes) and the accompanying ratios of 340 and 380 nm
excitation fluorescence images are shown. Open symbols on the
time course represent the average Ca
levels within
the egg determined from the numbered images shown below the
time course.
Within the intact egg NAADP (approximate
cytoplasmic concentrations of 50 and 500 nM) released
Ca in a biphasic manner (Fig. 2A).
The pattern of the Ca
release was an initial
Ca
peak, followed by a partial recovery, and a later,
slower and smaller amplitude release as previously observed by others (6) (peak [Ca
] was 1897 ±
268 nM, n = 5). We were able to demonstrate
the inactivation phenomenon of NAADP-induced Ca
release in the whole egg by first injecting into the egg as
little as 5 nM NAADP, which then prevented any further
response of the egg to larger injections of NAADP. 5 nM NAADP,
in itself produced only a small rise in Ca
, as shown
in Fig. 2B, but significantly reduced Ca
release by a subsequent injection of 500 nM NAADP even
after waiting at least 1500 s (267 ± 29 nM, n = 4). This persistent inactivation contrasted with the
inactivation of InsP
and cADPR receptors, which underwent a
transient desensitization (data not shown), but recovered within 30 min (Fig. 2C). In fact, InsP
(2
µM) injections released Ca
in the whole
egg in a monophasic manner (1102 ± 61 nM, n = 3) but in contrast to NAADP, a higher concentration of
InsP
(20 µM) was still able to release maximal
Ca
at least 1500 s later (1982 ± 163
nM, n = 3). Like InsP
,
preapplication of 1 µM cADPR to the egg (giving 1094
± 307 nM Ca
release, n = 3) did not block the Ca
response to 10
µM cADPR at least 1500 s later (mean Ca
1845 ± 78 nM, n = 3; data not
shown).
In the homogenate, the extent of the inactivation was both
concentration (Fig. 1, A and C) and
time-dependent (Fig. 3). Cross-desensitization to InsP and cADPR by NAADP (20 nM to 1 µM) did not
occur (Fig. 1B). Instead full inactivation occurred by
pretreatment with 1-2 nM NAADP, but substantial
inactivation still occurred with concentrations as low as 100 pM (Fig. 1, A and C). Within 60 s full
inactivation occurred with a non-stimulating concentration of 5 nM NAADP, but took longer and was less extensive by pretreatment with
lower concentrations (Fig. 3). Inactivation was also apparently
irreversible, since the receptor did not resensitize after 12 h of
exposure to 2 nM NAADP (data not shown), and after
reconstituting purified microsomes prepared from desensitized
homogenate in fresh buffer not containing NAADP (data not shown). In
the homogenate, Ca
release by InsP
and
cADPR desensitized only after treatment with stimulating concentrations
and in a manner paralleling the extent of channel activation (Fig. 1, D and E; see also (13) ).
Figure 3:
Time course of the inactivation by various
concentrations of NAADP in 2.5% sea urchin homogenate. The homogenate
was challenged with NAADP (500 nM) after addition at time zero
of sub-threshold concentrations (as indicated) of NAADP. Values are
median of 3-6 replicates from two experiments. Ca release by NAADP (500 nM) in the absence of any NAADP
pretreatment was 10.5 ± 0.65 nmol.
The graded release of Ca induced by NAADP (Fig. 1C) suggests that release by this agent is
quantal(14) . Changes in luminal or cytosolic Ca
concentrations or pool depletion have been proposed to explain
this phenomenon for both InsP
R and RyRs (see (14) for a review). However, it seems unlikely that
NAADP-induced Ca
release relies on these mechanisms
since it is terminated by desensitization and can occur at
concentrations that are non-stimulating. Instead, it appears to be an
intrinsic property of the receptor mechanism as has also been proposed
for the InsP
R(13) .
Various experiments suggest
that the inactivation mechanism is independent of enzymatic activity.
First, in Percoll gradient-purified
microsomes(1, 15) , full desensitization at either
non-stimulating or stimulating NAADP concentrations was identical to
that seen in whole egg homogenates (data not shown), ruling out a
requirement for cytosolic components. Second, the general kinase
inhibitor staurosporine (10 µM) had no effect on the
extent of inactivation (data not shown) in the homogenate. Third, the
release and inactivation were unaltered by performing the experiments
at 4 °C, consistent with NAADP regulating intracellular
Ca fluxes via a channel mechanism.
The
inactivation event was independent of small electrochemical or pH
gradient changes across the membrane since pretreatment for 1 h with
gramicidin, valinomycin, or nigericin (all at 1 µM) did
not significantly alter Ca release by NAADP or
InsP
(data not shown; see also (16) ).
The
nature of the NAADP Ca release mechanism is unknown
but is clearly separate from the InsP
and cADPR mechanisms.
Diltiazem, nifedipine, BAY K8644, and verapamil, classical modulators
of L-type voltage-gated Ca
channels(17) ,
fully blocked maximal Ca
release by NAADP (Fig. 4) but not that by cADPR or InsP
(Fig. 4) and did not alter the NAADP-induced inactivation
phenomenon (data not shown). The potent N-type voltage-gated
Ca
channel blocker
-conotoxin (17) was
without any effect (Fig. 4).
Figure 4:
A, representative fluorimetric traces of
the inhibition by diltiazem on NAADP-induced Ca release. B, effect of classical L-channel
Ca
-modulators and
-conotoxin on Ca
mobilization induced by NAADP, cADPR, or InsP
.
Antagonists were added 20 s prior to agonists. Values are mean ±
standard deviation of 3-9 determinations from 1-3 separate
experiments. Diltiazem, nifedipine, verapamil, and BAY K8644 were
diluted in dimethyl sulfoxide (Me
SO), and 5 µl of
Me
SO were added to the control.
The desensitization of the NAADP receptor shares similarities with neuronal nicotinic receptors(18) . However, nicotinic receptor desensitization by non-stimulating concentrations of agonists is never complete; it is also reversible and may involve phosphorylation. None of these features are shown by the NAADP-sensitive mechanism.
Recent reports have
indicated that mammalian cells possess the metabolic machinery to use
NAADP as an intracellular messenger. First, NAADP is generated and
degraded in various rat tissues, including brain, liver, and
spleen(19) . Second, it has been shown that ADP-ribosyl
cyclase, the enzyme responsible for the cyclization of NAD to cADPR, and CD38, a lymphocyte differentiation antigen, can
also synthesize NAADP(20) . Ca
mobilizing
effects of NAADP have not yet been reported for other cell types, but
an agonist-stimulated Ca
release pathway that is
blocked by nifedipine and diltiazem has been described in neutrophils (21) and low affinity binding sites for L-type
Ca
-channel blockers are present in cardiac
sarcoplasmic reticulum(22) .
Multiple Ca release mechanisms may contribute to complex patterns of
Ca
signals widely observed during intracellular
signaling(23) . The characteristics of NAADP Ca
release described here suggest that the receptor may function as
an irreversible biochemical switch activated in a one-off manner by a
rapid surge in intracellular NAADP concentrations. Such a response may
be suited to irreversible events such as fertilization, as recently
suggested(7) , cell division or cell death.