(Received for publication, July 10, 1996, and in revised form, October 30, 1996)
From the Department of Pharmacology, Three endogenous molecules have now been shown to
release Ca2+ in the sea urchin egg: inositol
trisphosphate (InsP3), cyclic adenosine 5 Intracellular Ca2+ stores in the sea urchin egg
express both inositol trisphosphate
(InsP3)1-sensitive
Ca2+ channels (1) and ryanodine-sensitive Ca2+
channels (RyRs). The latter are activated by a pyridine nucleotide metabolite, cyclic adenosine 5 A unique feature of the NAADP-induced Ca2+ release is that
it can be selectively inactivated by subthreshold concentrations of
this molecule, which per se do not cause Ca2+
release (6, 8). The mechanism of this unusual form of desensitization is unclear. However, the binding of NAADP cannot be displaced when
microsomes are pretreated with [32P]NAADP and then
challenged with a high concentration of NAADP after 3 min (8), perhaps
indicating that treatment with nonstimulating concentrations of NAADP
results in a conformational change of the receptor occluding further
association and dissociation of NAADP binding.
Recent reports have indicated that mammalian cells possess the
metabolic machinery to regulate intracellular levels of NAADP and
therefore possibly utilize it as an intracellular messenger. NAADP can
be synthesized in cell extracts from In the present study we have investigated the transient kinetics of the
NAADP-induced Ca2+ release mechanism in the sea urchin egg
homogenates and compared it with the transient kinetics of the
InsP3-sensitive and cADPR-sensitive Ca2+
release. Such approach can lead to further understanding of the similarities and differences between these release mechanisms and to
provide information on the activation and inactivation mechanisms of
NAADP-induced Ca2+ release. NAADP-induced Ca2+
release is composed of a fast and a slow phase, which can be described
by two rate constants. Similar biexponential equations can also fit
InsP3-induced and cADPR-induced Ca2+ release.
NAADP triggers Ca2+ release after a pronounced latency
inversely dependent on the concentration of NAADP, while no latency is
present before InsP3- and cADPR-induced Ca2+
release. Desensitization by subthreshold concentrations of NAADP does
not affect either of the rate constants, but affects the magnitude of
release. This finding suggests that NAADP desensitization proceeds in
an all-or-none manner, with inactivation affecting a subpopulation of
receptors while others are unaffected.
Eggs were obtained by
stimulating ovulation of female Lytechinus pictus (Marinus,
Inc., Long Beach, CA) with a intracoelomic injection of KCl. These were
then washed twice in artificial sea water (NaCl, 435 mM;
MgCl2, 40 mM; MgSO4, 15 mM; CaCl2, 11 mM; KCl, 10 mM; NaHCO3, 2.5 mM; EDTA, 1.0 mM at pH 8.0) and jelly removed by filtration through
90-µm nylon mesh.
Homogenates (2.5%) of
unfertilized Lytechinus pictus eggs were prepared as
described previously (11), and Ca2+ loading was achieved by
incubation at room temperature for 3 h in an intracellular medium
(IM) consisting of potassium gluconate, 250 mM;
N-methylglucamine, 250 mM; Hepes, 20 mM (pH 7.2); MgCl2, 1 mM; ATP, 0.5 mM; phosphocreatine, 10 mM; creatine
phosphokinase, 10 units/ml; oligomycin, 1 mg/ml; antimycin, 1 mg/ml;
sodium azide, 1 mM; fluo-3, 3 mM. Free
Ca2+ concentration was measured by monitoring fluorescence
intensity at excitation and emission wavelengths of 490 and 535 nm,
respectively. Fluorimetry was performed at 17 °C using 500 µl of
homogenate in a Perkin-Elmer LS-50B fluorimeter. Additions were made in
5-µl volume, and all chemicals were added in IM containing 10 µM EGTA. Basal concentrations of Ca2+ were
typically between 100 and 150 nM. Sequestered
Ca2+ was determined by monitoring decrease in fluo-3
fluorescence during microsomal loading and by measuring
Ca2+ release in response to ionomycin (5 µM)
and was constant between experiments.
Microsomes were purified from 50% egg homogenates by
the Percoll density centrifugation method described previously (11). Briefly, a fractionating buffer was prepared by diluting Percoll stock
to 25% in a modified intracellular medium (333 mM
N-methylglucamine, 333 mM potassium acetate, 27 mM Hepes, 1.3 mM MgCl2, pH titrated to 7.2 with acetic acid). This was then supplemented with 0.5 mM ATP, 2 units/ml creatine phosphokinase, 4 mM
phosphocreatine, 10 µM EGTA. 1 ml of 50% egg homogenate
was layered on 9 ml of this solution and centrifuged at 27,000 × g for 30 min at 15 °C. The top fraction (1 ml) was
collected and represented the supernatant fraction. The
Ca2+-storing, cADPR- and InsP3-sensitive
microsomes formed a distinct tight band half way down the tube. 1 ml of
this was collected using a disposable syringe to puncture the vessel
wall. The fractions were aliquoted and stored at Rapid kinetics of
Ca2+ release by InsP3, cADPR, and NAADP were
carried out on 2.5% homogenate prepared as described above.
Stopped-flow measurements were performed as described elsewhere
(12-14). Ca2+-loaded homogenate was introduced in a 2.5-ml
syringe of a stopped-flow fluorimeter (Applied Photophysics, model SX17
MV), while a 250-µl syringe was filled with either IM,
InsP3, or NAADP (diluted in IM) at a concentration 10 times
the concentration desired in the mixing chamber, as the mixing ratios
of the two syringes were 10:1. Temperature of homogenate in either the
syringes or mixing compartment was maintained at 17 °C by a
circulating water bath. Fluorescence changes of fluo-3 were monitored
with an exciting the sample at 490 nm and measuring the emission above
515 nm. Fluo-3 fluorescence was captured over 55 s in a split-time
base mode, with 200 recordings taken in the first 5 s and 200 recordings taken in the remaining 50 s. Each experiment represents
the average of at least six acquisitions.
The averaged traces were then analyzed using nonlinear regression
analysis programs supplied by Applied photophysics and Biosoft. The
progress of Ca2+ release from at least three different
homogenate preparations used in this study were shown to be biphasic
and could be best fitted to a biexponential profile using the following
equation,
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgment
REFERENCES
-diphosphate
ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate
(NAADP), a derivative of NADP. While the mechanism through which the
first two molecules are able to release Ca2+ is established
and well characterized with InsP3 and cADPR-activating InsP3 and ryanodine receptors, respectively, the newly
described NAADP has been shown to release Ca2+ via an
entirely different mechanism. The most striking feature of this novel
Ca2+ release mechanism is its inactivation, since
subthreshold concentrations of NAADP are able to fully and irreversibly
desensitize the channel. In the present study we have investigated the
fast kinetics of activation and inactivation of NAADP-induced
Ca2+ release. NAADP was found to release Ca2+
in a biphasic manner, and such release was preceded by a pronounced latent period, which was inversely dependent on concentration. Moreover, the kinetic features of NAADP-induced Ca2+
release were not altered by pretreatment with low concentrations of
NAADP, although the extent of Ca2+ release was greatly
affected. Our data suggest that the inactivation of NAADP-induced
Ca2+ release is an all-or-none phenomenon, and while some
receptors have been fully inactivated, those that remain sensitive to
NAADP do so without any change in kinetic features.
-diphosphate ribose (cADPR) (2). Recently, a third distinct Ca2+ release mechanism has been
identified that is potently and selectively activated by a different
pyridine nucleotide, nicotinic acid adenine dinucleotide phosphate
(NAADP) (3, 4). NAADP-induced Ca2+ release can be induced
both in the intact egg by microinjection and from microsomes of egg
homogenates (4-6). Ca2+ release induced by NAADP is not
blocked by heparin, a selective inhibitor of InsP3
receptors, nor by 8-amino-cADPR, a selective inhibitor of the
cADPR-sensitive Ca2+ release (3, 4). Moreover, NAADP does
not cross-desensitize with either InsP3 nor cADPR, showing
that it induces Ca2+ release via a mechanism independent of
InsP3Rs and RyRs (3, 4, 6). This is also supported by the
distinct pharmacological properties of the NAADP-sensitive mechanism.
For example, NAADP-induced Ca2+ release is blocked by
L-type Ca2+ channel blockers such as nifedipine and
diltiazem, which have no effect on either of the other two
Ca2+ release mechanisms in the sea urchin egg homogenates
(6). The NAADP-sensitive Ca2+ store, unlike cADPR- and
InsP3-sensitive stores, is insensitive to thapsigargin and
cyclopiazoic acid, suggesting that the NAADP-sensitive Ca2+
release mechanism resides on a distinct intracellular organelle (7).
-NADP in the presence of
nicotinic acid in various rat tissues, including brain, liver, and
spleen (9). Furthermore, 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 (10). The regulation of this class of enzymes is still
unclear, but it appears that NAADP is synthesized in the presence of
-NADP and nicotinic acid at more acidic pH, while cADPR is produced
in the presence of
-NAD at more neutral pH values (10).
Collection of Sea Urchin Eggs
70 °C until
use.
where A1, A2,
k1, and k2 are the
amplitudes and rate constants of Ca2+ release for the fast
and slow phases, respectively, and t is the time (seconds).
The amplitudes A1 and A2
are expressed in arbitrary units and relate to the fluorescence
intensity changes of fluo-3. Since the fluo-3 signal in these
experiments was never saturating, these units are related to
Ca2+ changes. In experiments where NAADP was used,
Ca2+ release was preceded by a latency or lag phase. This
latency was quantified and then subtracted prior to further analysis of the Ca2+ release process. In a control experiment the
effect of the Ca2+ pumps responsible for the re-uptake of
Ca2+ in these preparations were also quantified, and the
rate constants of the uptake were essentially negligible compared with
Ca2+ release by either InsP3, cADPR, or NAADP.
In fact, the rate constant for Ca2+ was 0.0008 ± 0.000004 s
(Eq. 1)
1, while the rate constants observed for
Ca2+ release were at least 30 times faster.
NAADP was either purchased from Research Biochemicals International (St. Albans, United Kingdom (UK)) or was a kind gift of Prof. T. F. Walseth (University of Minnesota), fluo-3 was from Molecular Probes (Cambridge, UK). All other chemicals were from Sigma (Poole, UK).
NAADP released Ca2+ dose-dependently with
an EC50 of 25 ± 5 nM (Fig.
1, A and B). Furthermore, NAADP, 3 nM, which per se induced only a negligible
release, was able to desensitize the mechanism to a subsequent addition
of a maximal concentration of NAADP (200 nM) (Fig.
1C; see also Refs. 6 and 8). Performing experiments at
7 °C did not alter significantly the extent of Ca2+
release nor the extent of inactivation (Fig. 1).
If quantal release, first described for the InsP3-induced Ca2+ release, is defined by the observation that submaximal concentrations of mobilizing agent can rapidly release Ca2+ from the Ca2+ stores without affecting their ability to respond to further maximal concentrations of the same agent (15-17) (incremental detection), NAADP does not release Ca2+ in a quantal manner. It therefore appears either quantal Ca2+ release, which has been also described for the ryanodine receptors (18), is not central in the operation of all intracellular Ca2+ channels or that incremental detection is not necessarily associated with quantal release.
To investigate the fast kinetics of the Ca2+ fluxes
activated by NAADP, InsP3, or cADPR, we have employed
stopped-flow analysis, with a temporal resolution of 25 ms. In the
present experiments Ca2+ pump inhibitors have not been used
for two reasons: (i) the rate of Ca2+ uptake in our
preparations was at least 30 times slower than the slower
Ca2+ release parameter examined and therefore negligible
(see also "Materials and Methods"); (ii) NAADP-induced
Ca2+ release is known to be affected differently from cADPR
and InsP3 by Ca2+ uptake inhibitors
(e.g. it is insensitive to thapsigargin and cyclopiazoic
acid) (7). When NAADP, InsP3, or cADPR were added to the
homogenate, it resulted in an increased fluorescence signal representing Ca2+ release (Figs.
2, 3, 4). Supramaximal
concentrations of NAADP (1 µM) (6) released
Ca2+ with a t1/2 of 11.2 ± 1.0 s. Supramaximal concentrations of cADPR (3 µM)
(6) released Ca2+ with a t1/2 of
6.5 ± 0.9 s. In comparison, addition of maximal InsP3 concentrations (10 µM) (6) resulted in
a more rapid Ca2+ release, with a t1/2
of 2.7 ± 0.8 s. InsP3-induced Ca2+
release appears slower to that observed in cerebellar microsomes, where
maximal InsP3 concentrations release Ca2+ with
a t1/2 of less than a second (14). This difference
may well reflect the relative abundance of InsP3-sensitive
channels in the two systems.
An important and consistent feature of the NAADP-induced
Ca2+ release was that low concentrations of NAADP did not
release Ca2+ instantly, but rather a pronounced latency
period was observed prior to release (Figs. 2 and 5).
This latent period had a duration of up to several seconds and was
found to be inversely dependent on the concentration of NAADP used
(Fig. 5). However, at higher NAADP concentrations (3 and 10 µM), Ca2+ release was not preceded by a
detectable latency (Figs. 2 and 5). In contrast, no lag phase was
observed with any concentration of InsP3 (Fig. 3) This is
in agreement with most reports on the fast kinetics of the
InsP3 receptor (14) and consistent with direct gating of
InsP3Rs by InsP3, although a lag phase has been reported for InsP3-induced Ca2+ release in rat
basophilic leukemia cells (13). cADPR was also not preceded by a lag
phase. To investigate whether this lag phase was due to the conversion
of NAADP to an active metabolite, stopped-flow experiments were
performed at 7 °C. If an enzymatic conversion was responsible for
the latency, then lowering the temperature should prolong the lag
phase. The lag phases at both 7 and 17 °C did not markedly differ at
all concentrations tested (Fig. 4). Moreover, NAADP was able to release
Ca2+ to the same extent in both conditions (Fig. 1). It
therefore seems unlikely that an enzymatic conversion is responsible
for the observed latency and that Ca2+ pumps affect release
in this system.
The marked lag phase observed could be dependent on the requirement of a modulatory protein to bind the NAADP receptor. To investigate this possibility, fluorimetric determinations of NAADP-induced Ca2+ release under various conditions were performed. In Percoll-purified microsomes, which should lack soluble cytosolic proteins, NAADP still released Ca2+, although to a minor extent in respect to the 2.5% homogenate. Unlike cADPR-sensitive Ca2+ stores, NAADP-sensitive Ca2+ stores are not all contained in this microsomal fraction (4), and the minor extent of release observed is most likely due to this observation. Addition of calmodulin (1 µM) did not affect NAADP-induced Ca2+ release in microsomes, in contrast to the potentiating effect that it has on cADPR-induced Ca2+ release (19). Addition of FK506 (10 µM) and rapamycin (10 µM), which are known to affect Ca2+-releasing mechanisms by inhibiting modulatory proteins associated with RyRs and InsP3Rs (20), did not affect NAADP-induced Ca2+ release significantly. Moreover, the reconstitution of the preparation with 2.5% supernatant, only slightly enhanced the total amount of Ca2+ released by NAADP (120 ± 12% compared with controls). Ca2+ release by NAADP is also unaffected by preincubation of 2.5% homogenate with cholesteryl hemisuccinate (5 mM), an agent which rigidifies membranes and slows down protein movement in the membrane (21, 22). Taken together, these observations suggest that modulatory proteins are unlikely to be involved in the opening of the channel, although the possibility of a closely associated uncharacterized modulatory protein cannot presently be ruled out. The observed lag phase could also be due to the presence of one or more slow temperature-insensitive limiting steps prior to channel opening.
NAADP-induced Ca2+ release was best fitted to a
biexponential function (see Fig. 6 for an example) as
defined by the equation given above as were InsP3- and
cADPR-induced Ca2+ release. A monoexponential process
proved to be completely unsatisfactory in all three cases cases. On the
other hand, the Ca2+ release process that followed addition
of low concentrations of cADPR (below 50 nM) could not be
fitted to any standard mathematical fit routinely employed. The pattern
of Ca2+ release induced by low concentrations of cADPR
appeared sigmoidal, with the rate of release slowly increasing
throughout the first few seconds of release. This can be explained by
the regulatory role that cADPR plays on Ca2+-induced
Ca2+ release (2). It is possible that at low concentrations
of cADPR, the early events of Ca2+ release potentiate
further Ca2+ release. Rate constants for both the fast and
slow phases of Ca2+ release induced by NAADP,
InsP3, and cADPR were comparable in value at saturating
concentrations of agonists used (for 10 µM NAADP the fast
rate constant was 0.42 s1 and the slow rate constant was
0.04 s
1; for 10 µM InsP3 the
fast and slow rate constants were 0.40 and 0.08 s
1,
respectively, and for 5 µM cADPR the fast and slow rate
constants were 0.45 and 0.05 s
1, respectively; Figs.
2, 3, 4). The InsP3- and cADPR-induced Ca2+
release rate constants increased with concentration reaching a maximum
at around 1 µM (Figs. 3 and 4). The increases in the rate
constants appear to closely follow the trend in increase in amplitude.
Unlike in cerebellar microsomes (14), the slow rate constant for
InsP3 was responsible for most Ca2+ release.
This was also observed for NAADP-induced Ca2+ release (Fig.
2). However, for NAADP-induced Ca2+ release, the slow rate
constant was concentration-insensitive, although the extent of
Ca2+ release by this phase increased steadily with
increasing concentrations of NAADP. This finding is difficult to
rationalize. The most likely explanation is that a fine balance exists
between the activation and inactivation of this receptor. Since the
EC50 for inactivation is ~100-fold lower than for
activation, at low concentrations a high proportion of receptors will
be desensitized without being activated. Increasing concentrations of
NAADP will increase the probability that the receptors will activate
before inactivating and increase the amplitude of release. As shown in
Fig. 7 (for text see below), partial inactivation
modifies the amplitude but not the kinetic parameters and therefore
this model is plausible. In contrast, the fast rate constant increased
with increasing concentrations of NAADP, but the extent of
Ca2+ release appeared to plateau at 100 nM and
contributed no more than 25% of the overall release. Experiments
conducted at 7 °C did not substantially alter NAADP-induced
Ca2+ release, although the slow rate constant decreased by
approximately 30% (data not shown). The presence of two kinetic
components can suggest that NAADP releases Ca2+ from two
populations of Ca2+ stores with different
Ca2+-accumulating capacities, which would account for the
two phases. Another hypothesis is that in our homogenate preparation
the majority of vescicles sensitive to NAADP have a low proportion of
NAADP channels, which account for the slow phase of Ca2+
release, but a few vescicles contain a high proportion of NAADP receptors. With increasing concentrations of NAADP a higher proportion of receptors on this latter population of vescicles is activated and
therefore Ca2+ fluxes are faster.
The cooperativity of NAADP-induced Ca2+ release was assessed for the fast rate constants and found to have a Hill coefficient of 1.1, suggesting that either only one binding site is involved in Ca2+ release, of that if multiple sites are present they act independently of each other in influencing Ca2+ channel activity. In comparison, it has been demonstrated previously that cADPR-induced Ca2+ release in sea urchin eggs is a cooperative process with a Hill coefficient of approximately 1.8 (23).
We then examined the kinetic properties of NAADP-induced Ca2+ release after partial desensitization with lower concentrations of NAADP. When this was performed, the magnitude of Ca2+ release by maximal concentrations of NAADP was reduced in a concentration-dependent manner (Fig. 7). Interestingly, both the fast and slow rate constants were unaffected by the pretreatment with low desensitizing concentrations of NAADP (Fig. 7). One possibility is that inactivation occurs in an all-or-none manner, with submaximal inactivating concentrations of NAADP causing the full inactivation of a proportion of receptors. Increasing the magnitude of the pretreatment, this proportion of inactivated receptors would progressively increase. In such a way, while a proportion of the receptors has been shifted to the desensitized form by the pretreatment, the active ones preserved the kinetic characteristics expressed by the control.
In conclusion, NAADP-induced Ca2+ release, although distinct, shows some common kinetic features with the established Ca2+-releasing molecule InsP3. Both release Ca2+ in a biphasic manner and with similar rate constants. In both cases the slow phase of release is responsible for the majority of Ca2+ release. NAADP, unlike InsP3, releases Ca2+ after a latency. Furthermore, NAADP-induced Ca2+ release appears not to be cooperative. Moreover, NAADP inactivation is most likely an intrinsic all-or-none property of the receptor. In fact, partial inactivation does not alter the kinetic properties of the release, although it alters the amplitude, which suggests that the number of receptors responding has diminished but the active receptors have not been altered by pretreatment.
We acknowledge Jaswinder Sethi for fruitful discussions.