From the Laboratoire Canaux Ioniques et Signalisation, DSV/DBMS, 17 rue des Martyrs, F-38054 Grenoble, France and
The
Department of Physiology, University of Minnesota, Minneapolis,
Minnesota 55455
ADP-ribosyl cyclase catalyzes the synthesis of
two structurally and functionally different Ca2+
releasing molecules, cyclic ADP-ribose (cADPR) from
-NAD and nicotinic acid-adenine dinucleotide phosphate (NAADP) from
-NADP. Their Ca2+-mobilizing effects in ascidian oocytes were
characterized in connection with that induced by inositol
1,4,5-trisphosphate (InsP3). Fertilization of the oocyte is
accompanied by a decrease in the oocyte Ca2+ current and an
increase in membrane capacitance due to the addition of membrane to the
cell surface. Both of these electrical changes could be induced by
perfusion, through a patch pipette, of nanomolar concentrations of
cADPR or its precursor,
-NAD, into unfertilized oocytes. The changes
induced by
-NAD showed a distinctive delay consistent with its
enzymatic conversion to cADPR. The cADPR-induced changes were inhibited
by preloading the oocytes with a Ca2+ chelator, indicating
the effects were due to Ca2+ release induced by cADPR.
Consistently, ryanodine (at high concentration) or 8-amino-cADPR, a
specific antagonist of cADPR, but not heparin, inhibited the
cADPR-induced changes. Both inhibitors likewise blocked the membrane
insertion that normally occurred at fertilization consistent with it
being mediated by a ryanodine receptor. The effects of NAADP were
different from those of cADPR. Although NAADP induced a similar
decrease in the Ca2+ current, no membrane insertion
occurred. Moreover, pretreatment of the oocytes with NAADP inhibited
the post-fertilization Ca2+ oscillation while cADPR did
not. A similar Ca2+ oscillation could be artificially
induced by perfusing into the oocytes a high concentration of
InsP3 and NAADP could likewise inhibit such an
InsP3-induced oscillation. This work shows that three
independent Ca2+ signaling pathways are present in the
oocytes and that each is involved in mediating distinct changes
associated with fertilization. The results are consistent with a
hierarchical organization of Ca2+ stores in the oocyte.
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INTRODUCTION |
Ca2+ signaling in cells generally involves both
its influx from the extracellular medium and its release from
intracellular stores. Two families of intracellular Ca2+
release channels have been characterized, namely, the inositol 1,4,5-trisphosphate receptors
(InsP3R)1 and the
ryanodine receptors (RyR). Unlike the InsP3R, the
physiological agonist for RyR has not been identified although it is
known that its Ca2+ channel property can be modulated by a
plant alkaloid, ryanodine. The skeletal muscle RyR isoform is known to
be activated directly by conformational coupling with the voltage
sensors at the cell surface and does not seem to require an agonist (1,
2). On the other hand, other RyR isoforms may require specific ligand for activation, and cyclic ADP-ribose (cADPR) has been proposed to be
such an agonist for RyR (3). Cyclic ADP-ribose was discovered during an
investigation of Ca2+ signaling mechanisms in sea urchin
eggs (4-6). Subsequent work on a variety of cells indicates cADPR is
likely an endogenous regulator of the RyR (7-10). Cyclic ADP-ribose is
synthesized from
-NAD by the enzyme ADP-ribosyl cyclase, which was
first described in sea urchin eggs (4, 6) and has since been shown to
be ubiquitous (Ref. 11, and reviewed in Ref. 12). A soluble form of the
cyclase has been purified from Aplysia ovotestis, and its
crystalline structure has been solved recently (13-15). A
membrane-bound homolog of the Aplysia cyclase is CD38, a
lymphocyte antigen (16). CD38 is catalytically different from the
Aplysia cyclase in that it not only can cyclize
-NAD into
cADPR but can also catalyze the hydrolysis of cADPR to ADP-ribose (17).
Both CD38 and the soluble cyclase can, additionally, catalyze the
exchange of the nicotinamide group of
-NADP with nicotinic acid to
produce nicotinic acid-adenine dinucleotide phosphate (NAADP) (18), a
metabolite that can release Ca2+ from intracellular stores
different from that mobilized by cADPR (19).
Ca2+ is involved in a complex series of changes
accompanying fertilization of ascidian oocytes (20-23). An immediate
decrease in depolarization-activated Ca2+ current is
followed by a progressive increase in membrane capacitance (24). The
latter is indicative of membrane insertion into the cell surface.
Subsequently, a prolonged Ca2+ oscillation occurs, lasting
until meiosis is complete. Perfusion of high concentrations of inositol
1,4,5-trisphosphate (InsP3) into unfertilized oocytes
through a whole-cell patch pipette can elicit repetitive
Ca2+ transients similar to that observed during
fertilization (24). However, neither the rapid decrease of
Ca2+ current nor membrane insertion is induced by
InsP3, suggesting other Ca2+ messengers may be
involved in mediating these early changes at fertilization (24). In
this study, we used the whole-cell patch-clamp technique to introduce
three independent Ca2+ messengers, cADPR, NAADP, and
InsP3 into the oocytes (25). The resulting Ca2+
mobilization was monitored by two independent methods, by electrical measurements to sense the localized Ca2+ changes near the
plasma membrane and by fluorescent indicator to sample the cytoplasmic
Ca2+ changes. Results show that all three Ca2+
signaling mechanisms are present in the oocytes and are involved not
only in mediating the early electrical changes at the cell surface
associated with fertilization but also in modulating the subsequent
Ca2+ oscillation.
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EXPERIMENTAL PROCEDURES |
Ascidian Eggs--
As described previously (24), specimens of
the hermaphroditic ascidian Phallusia mammillata were
collected near Sète on the French Mediterranean coast. Mature
oocytes were extracted from the oviduct and kept in artificial sea
water (ASW). Sperm was drawn directly from the spermiduct. Chorions and
follicle cells surrounding the oocyte were removed either manually,
using fine sharpened tungsten needles, or enzymatically (26).
Fertilization was induced by inseminating dechorionated eggs with a
dilute suspension of sperm.
Solutions--
Similar media were described in a previous study
(24). They included artificial sea water (ASW) containing NaCl, 400 mM; KCl, 10 mM; MgCl2, 50 mM; CaCl2, 10 mM; Hepes, 10 mM, pH 8.0. The pipette solution was composed of sucrose,
400 mM; KCl, 200 mM; NaCl, 10 mM;
MgCl2, 1 mM; EGTA, 1 mM; Hepes, 20 mM, pH 7.2. Ryanodine (Calbiochem) is prepared as a stock
solution at 13.5 mM in ethanol. Stock solutions of Fura-2
dextran 10,000 (Molecular Probes), InsP3 (Calbiochem),
cADPR (Amersham Pharmacia Biotech),
-NAD (Sigma),
-NADP (Sigma),
ADPR (Sigma), NAADP (Molecular Probes), 8-amino-cADPR (Molecular
Probes), BAPTA (Sigma), and heparin (Sigma) were each prepared in
pipette solution. The concentration values of the compounds used in
this study refer to their concentrations in the pipettes. The oocytes
(~130 µm diameter) were filled by diffusion through the whole-cell
patch pipette within minutes. The concentrations of the stock solution
of cADPR and NAADP were verified by absorbance measurements at 260 nm
and by using the published values for extinction coefficients (6,
19).
Electrophysiological Recordings--
Dechorionated eggs in ASW
were patch-clamped in the whole-cell configuration using pipettes
pulled to resistances of 1.5-3 megohms with a Mecanex BB-CH puller
(Geneva, Switzerland). Currents were recorded under voltage-clamp
conditions with an RK300 amplifier (BioLogic, Claix, France) and pCLAMP
software (Axon Instruments, Foster City, CA). Series resistance was
compensated electronically (0.5-1.5 megohms). Eggs were successively
patched with different pipettes, allowing for changes in pipette
solutions as described previously (24, 25). Eggs were patch-clamped in
the whole-cell configuration and recorded for hours, with the same
pipette or with different pipettes, without disturbing the cell
integrity (24, 25, 27, 28). Capacitance of the oocyte membrane was evaluated from the steps of current induced under triangle-wave voltage
command, as previously explained (29, 30).
Calcium Imaging--
Most of the procedure was performed as
described previously (24). Dechorionated eggs were loaded with 1 mM Fura-2 dextran applied during a 10-min period by
diffusion from the patch pipette. Eggs were observed using an
epifluorescence Nikon Diaphot 300 microscope through a CF-Fluor ×20
objective (numerical aperture 0.75). Fluorescence was collected from
the entire cell. Eggs were illuminated at 340 and 380 nm successively
using a Lambda 10 optical filter changer (Sutter Instrument Company)
and a Technical-Video epifluorescence DX-5 device (Woods Hole, MA).
Fluorescence emission was recorded at 510 nm using an Extended Isis CCD
camera (Photonic Science, Robertsbridge, UK). Data acquisition and
calcium measurement were performed with the Starwise Fluo 220 system
(Imstar, Paris). During the acquisition process, at each sampling time,
the two source intensity images (excited at 340 and 380 nm) were stored on a hard disk. The calculations of calcium concentration were done
according to the formula of Grynkiewicz (31) with predefined values of
calibration parameters to provide a pixel by pixel ratiometric image of
[Ca2+]i as in ref. (24). In the
results that are shown below, [Ca2+]i were averaged over the
apparent whole egg diameter. Data were sampled at 4-s intervals.
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RESULTS |
cADPR and
-NAD Activate Calcium Release and Induce Changes in
the Oocyte Calcium Current and Membrane Capacitance--
Perfusion of
cADPR into an unfertilized ascidian egg through a whole-cell patch
pipette elicited two electrophysiological effects, a rapid decrease in
the depolarization-activated Ca2+ current and a concomitant
but slower increase in membrane capacitance (Fig.
1A). The latter change is
indicative of membrane insertion into the cell surface (24). The
magnitude of these changes were highly significant, amounting to about
60% decrease in the Ca2+ current and 20% increase in
capacitance. In control experiments, perfusion with standard pipette
solution without cADPR produced no observable electrical changes (not
shown). The measured amplitude of the Ca2+ current remained
constant even when the same oocyte was repeatedly patched by different
pipettes (24, 25, 27, 28). If the oocyte was preloaded by perfusion
with a high concentration of ryanodine (100 µM), a RyR
blocker, application of cADPR subsequently was ineffective in eliciting
any changes (24). In contrast, an inhibitor of the InsP3R,
heparin, at up to 3 mg/ml, did not prevent the cADPR effects (not
shown). These results indicate that cADPR-induced changes may be
mediated by a RyR-like Ca2+ release channel. That
Ca2+ release indeed was involved was further demonstrated
by preloading the oocytes with a Ca2+ chelator, BAPTA. As
illustrated in Fig. 1B, subsequent application of cADPR
produced no change in either the Ca2+ current or the
membrane capacitance. These electrical measurements thus provide a very
sensitive assay for monitoring Ca2+ release especially in
the localized regions adjacent to the plasma membrane. A
concentration-response curve summarizing 37 recordings of the
Ca2+ current is shown in Fig. 1C. The
half-maximal effective concentration of cADPR was about 0.1 nM, among the lowest values ever reported (32).

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Fig. 1.
Calcium release activated by cADPR in the
ascidian oocyte. A, perfusion of cADPR (10 nM) into an oocyte induced a rapid decrease in
Ca2+ current and an increase in oocyte capacitance.
B, these effects were abolished when 5 mM BAPTA
was perfused into the oocyte prior to cADPR application, indicating
that the observed electrical changes were due to a Ca2+
release activated by cADPR. C, concentration-response curve
for the current change induced by cADPR. The Ca2+ current
was measured 2 min after the perfusion of cADPR into the oocyte
(means ± S.D., 37 oocytes measured). Compounds were perfused into
the oocyte through a patch pipette in the whole-cell configuration. The
oocyte Ca2+ current was measured subsequently by
depolarization pulses between -60 and +30 mV in steps of +10 mV from a
holding potential of -80 mV. The peak current intensity measured after
a depolarization to -20 mV was used in the plot. The oocytes were
incubated in ASW solutions at room temperature. The details of the
current measurement were as described previously (24) and are outlined
under "Experimental Procedures."
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-NAD, the precursor of cADPR, was similarly effective in inducing
the two electrical changes as shown in Fig.
2A. However, the kinetics were
significantly slower, especially the decrease of the Ca2+
current. The half-time for the current decrease induced by
-NAD was
170 s, 3-4 times longer than that induced by cADPR (Fig.
2B). The rate of the capacitance increase induced by
-NAD
was similarly slower, with an initial rate of about 20 pF/min
(cf. Fig. 2A) as compared with about 50 pF/min in
the case of cADPR (cf. Fig. 1A). This time lag is
consistent with the presence of an ADP-ribosyl cyclase in the oocytes
converting
-NAD to cADPR, which is then responsible for inducing the
observed electrical changes. It would thus be expected that
8-amino-cADPR, a specific antagonist of cADPR (33), should block the
effects of
-NAD. This was found to be the case as shown in Fig.
3. Perfusion of a high concentration (1 µM) of 8-amino-cADPR by itself did not elicit any current
or capacitance changes but totally blocked the activating effects of
both
-NAD and its product, cADPR (Fig. 3, A and
B).

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Fig. 2.
Effects of -NAD on oocyte Ca2+
current and membrane capacitance. A, the current and
capacitance changes induced by -NAD were qualitatively similar to
those triggered by cADPR except with slower kinetics. The initial
velocity of the capacitance change shown was about 20 pF/min. It was
significantly slower than that activated by cADPR (about 50 pF/min) as
shown in Fig. 1. B, the time required to reach the
half-maximal current change induced by cADPR and -NAD was compared.
There was a time lag of about 120 s for -NAD compared with
cADPR at various concentrations, from 1 nM to 10 µM for each of the two compounds. Values are means ± S.D. (27 oocytes measured with cADPR, 9 oocytes with -NAD).
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Fig. 3.
Inhibition of the cADPR and -NAD-induced
changes by 8-amino-cADPR. 8-amino-cADPR (1 µM
pipette concentration) was first perfused into the oocyte for 6 min
before the indicated concentrations of cADPR (A) or -NAD
(B) were applied through a second patch pipette.
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Fertilization of ascidian oocytes is accompanied by a large
increase in membrane capacitance similar to that induced by cADPR (24,
27, 30). Evidence suggests that the fertilization-induced membrane
insertion is mediated by Ca2+ released via the RyR (24). As
will be shown later (cf. Fig. 9B), this membrane
insertion was totally abolished by 8-amino-cADPR.
NAADP Activates Calcium Release and Decreases the Oocyte Calcium
Current without Affecting Membrane Capacitance--
Results above
suggest that an ADP-ribosyl cyclase is present and operative in
ascidian oocytes. The cyclase is known to be a multifunctional enzyme
capable of catalyzing not only the cyclization of
-NAD to produce
cADPR but also an exchange reaction to produce yet another
Ca2+ release metabolite from
-NADP, NAADP (Ref. 18, and
reviewed in Ref. 12). The Ca2+ stores sensitive to NAADP in
sea urchin eggs homogenates can be separated from those sensitive to
cADPR and InsP3 by density centrifugation (19) and appear
to possess a Ca2+ transport system that is insensitive to
thapsigargin (34). The effects of NAADP in ascidian oocytes are
likewise very different from those of cADPR. Although perfusion of
nanomolar concentrations of NAADP into the oocytes induced a decrease
in Ca2+ current, the change was much slower than that
induced by cADPR (compare Figs. 1A and 4A). The
concentration-response curve of NAADP is shown Fig.
4B, summarizing 24 assays with
NAADP concentrations ranging from nanomolar to micromolar. The
half-maximal effective concentration was about 3 nM,
30-fold higher than that of cADPR (Fig. 1C).

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Fig. 4.
Effects of NAADP on the oocyte current and
capacitance. A, perfusion of NAADP (10 nM
pipette concentration) into the oocyte decreased the oocyte current but
had no effect on the membrane capacitance. B, the
concentration-response curve for the current change induced by NAADP
(means ± S.D.). The Ca2+ current was measured 2 min
after perfusion of NAADP into the oocyte and was evaluated at the peak
intensity of the depolarization-induced current as described in the
legend of Fig. 1.
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The most dramatic difference between the effects of NAADP and cADPR
was, however, in the capacitance measurements. Contrary to that
observed with cADPR, NAADP never elicited any capacitance changes as
shown in Figs. 4A and 5A. This is further
emphasized in Fig. 5A. Of the
19 oocytes perfused with NAADP, none of them showed any increase in
membrane capacitance. In contrast, all 31 oocytes responded to cADPR
with a capacitance increase averaging to about 40 pF after 5 min. As a
control, ADP-ribose (ADPR), the hydrolysis product of cADPR, was also
tested, and it too did not induce any capacitance changes (Fig.
5A).

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Fig. 5.
Characterization of the cADPR, NAADP, and
ADPR effects on the oocyte current and capacitance. A,
the capacitance change induced by cADPR after 5 min (from nanomolar to
micromolar concentrations, 31 oocytes), NAADP (from nanomolar to
micromolar concentrations, 19 oocytes), or ADP-ribose (ADPR) (from
nanomolar to micromolar concentrations, 7 oocytes) are compared. Values
are means ± S.D. Only cADPR could induce an increase in the
oocyte capacitance. B, oocytes were first preloaded with
either 5 mM BAPTA, 1 µM 8-amino-cADPR, or
standard pipette solution (control) and subsequently
perfused with an agonist, either cADPR, NAADP, or ADPR. The resultant
decrease in the oocyte Ca2+ current was evaluated 2 min
after the agonist application. Values are means ± S.D.
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Of the 19 oocytes perfused with NAADP and not showing a capacitance
change, all responded with an average of about 60% decrease in
Ca2+ current after 2 min of perfusion, a magnitude similar
to that induced by cADPR (Fig. 5B). The current decrease
induced by either NAADP or cADPR could be blocked by preloading the
oocytes with BAPTA, indicating in both cases that the effect was due to
Ca2+ release activated by the agonists. There was, however,
a major difference between these agonists with respect to the action of the antagonist, 8-amino-cADPR, which inhibited only the current decrease induced by cADPR but not that induced by NAADP (Fig. 5B). This latter result indicates that the Ca2+
release mechanism activated by NAADP was distinct from that activated by cADPR, consistent with that reported in sea urchin eggs (19). This
difference was also supported by an experiment in which application of
a high concentration of ryanodine to oocytes (100 µM)
blocked the cADPR-sensitive decrease in Ca2+ current (24)
but, similar to 8-amino-cADPR, did not inhibit the NAADP-induced
changes (data not shown).
It has previously been reported that ADPR can interfere with channels
and Ca2+ events in ascidian oocytes (35). This is confirmed
in Fig. 5B. The effect of ADPR on the current, however, was
not mediated by Ca2+ release since preloading the oocytes
with BAPTA, which effectively eliminated the actions of both cADPR and
NAADP, did not inhibit the effect of ADPR. The effect of ADPR appeared
to be specific to that molecule since other similar compounds such as 1 mM ADP (not shown) or 8-amino-cADPR (Fig. 3A)
did not cause a decrease in the current. An unexpected feature of the
ADPR effect was its sensitivity to inhibition by 8-amino-cADPR (Fig.
5B). It is possible that the action of ADPR is related to
its ability to covalently react with amino groups of proteins (36).
Irrespective of the exact mechanism, it is clear that ADPR did not
mobilize Ca2+ since its action was not blocked by BAPTA
(Fig. 5B) and thus was not investigated further.
The NAADP Signaling Is Independent of RyR, but Is Related to
InsP3R--
The effects of NAADP and cADPR on the current
are independent (Fig. 6A). As
usual, perfusion of NAADP into the oocyte decreased its
Ca2+ current. Subsequent perfusion of cADPR into the same
oocyte induced a further decrease in its Ca2+ current and
an increase in membrane capacitance. As described above, both
cADPR-induced changes can be attributed to the involvement of RyR. The
magnitude of the subsequent current change was typical of that induced
by cADPR alone, indicating that cADPR and NAADP act independently and
that their effects on the current were essentially additive.

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Fig. 6.
The interrelationship of the effects induced
by NAADP, cADPR, and InsP3. A, the effects
of NAADP and cADPR are independent. An oocyte was first perfused with
10 nM NAADP. After the decrease in the oocyte current
stabilized, 10 nM cADPR was perfused into the same oocyte
through a different patch pipette. B, an oocyte was first
perfused with InsP3. After the decrease in the oocyte
current stabilized, NAADP was perfused into the same oocyte through a
different patch pipette. Further decrease in oocyte current was
recorded. The panel on the right shows the mean of the
current decrease induced by NAADP following the pretreatment of either
standard pipette solution (Standard) (n = 19, mean ± S.D.), InsP3 (n = 2), or 3 mg/ml heparin (n = 2). C, conditions are
similar to panel B except NAADP was applied first, followed
by InsP3. The panel on the right
shows the mean of the current decrease induced by 100 nM
InsP3 following the pretreatment of either buffer
(Standard) (n = 13), NAADP
(n = 4), or 3 mg/ml heparin (n = 6).
Values are mean ± S.D. The results of this figure are obtained
with concentrations of cADPR or NAADP ranging from nanomolar to
micromolar. The peak Ca2+ current decrease was evaluated 2 min after InsP3 application.
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The inactivating effect of NAADP on the oocyte Ca2+ current
is, in some respects, similar to that observed with InsP3
(24). Fig. 6B shows that perfusion with InsP3
induced a slow decrease in the current very much like that seen with
NAADP (Fig. 4A). Also, neither NAADP nor InsP3
altered the membrane capacitance. However, the receptor for NAADP is
distinct from that of InsP3 since neither heparin, an
InsP3 receptor antagonist, nor pretreatment of the oocytes
with InsP3 inhibited the action of NAADP (Fig. 6B, right panel). Nevertheless, the
InsP3 and the NAADP-sensitive Ca2+ stores
appear to be able to interact functionally. Thus, as shown in Fig.
6C, pretreatment of the oocytes with NAADP could render InsP3 incapable of causing a decrease in the
Ca2+ current. Indeed, pretreatment with NAADP
(n = 4, Fig. 6C, right panel) was found to
be as effective as heparin in blocking the subsequent action of
InsP3. It thus appears that the order of addition is
important. Prior activation by NAADP inhibits InsP3 but the
converse is not true; no effect was seen on the action of NAADP by
pretreatment with InsP3. These intriguing and apparently paradoxical observations were investigated further.
Inhibitory Effect of NAADP on Calcium Oscillation--
It was
found that the action of InsP3 was complex (24). As shown
in Fig. 7A, it was capable of
inducing not only a single event of Ca2+ release but
prolonged Ca2+ oscillation lasting as long as
InsP3 was applied through the patch pipette. Simultaneous
perfusion of both NAADP and InsP3 did not block the first
Ca2+ release induced by InsP3 but effectively
and reproducibly eliminated the subsequent Ca2+ oscillation
(n = 11), as illustrated in Fig. 7B. All the
InsP3-induced Ca2+ oscillations, including the
first one, could be inhibited if the oocytes were first preincubated
for minutes with NAADP before InsP3 application (Fig.
7C). This inhibitory effect is reversible, since subsequent
simultaneous application of InsP3 and NAADP 30 min later
induces a pattern of Ca2+ signal identical to that depicted
in Fig. 7B (not shown). It thus appears that, when
InsP3 and NAADP were simultaneously applied, there was
insufficient time for NAADP to effect total inhibition of the
InsP3-induced oscillation (Fig. 7B). The results
obtained with NAADP preincubation (Fig. 7C) are consistent
with those shown in Fig. 6C.

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Fig. 7.
Inhibition of the InsP3-induced
Ca2+ oscillation by NAADP. A, perfusion of
a high concentration of InsP3 into an oocyte triggered a
Ca2+ oscillation. The intracellular Ca2+
concentration was measured fluorimetrically using Fura-2 dextran. The
graph shown is representative of 16 experiments. B,
perfusion of a combination of NAADP and InsP3, each at 10 µM, activated only a single Ca2+ transient,
and the prolonged oscillation was inhibited. Lower concentrations of
NAADP, from nanomolar to micromolar, had similar inhibitory effects on
the Ca2+ oscillation (not shown). The graph shown is
representative of eleven experiments. C, after a
preapplication of 10 µM NAADP for 5 min, subsequent
simultaneous perfusion of NAADP and InsP3 was unable to
elicit any Ca2+ signal on the tested unfertilized
oocyte.
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The InsP3-induced Ca2+ oscillation was very
similar to that which occurs after fertilization (24). As shown in Fig.
8A, fertilization was
accompanied by an initial Ca2+ transient lasting more than
5 min, which was then followed by several smaller transients with a
periodicity of about 2-3 min and each lasting for about 1 min.
Pretreatment of the oocyte with NAADP for several minutes reproducibly
eliminated these oscillations (Fig. 8B). The first
Ca2+ transient was not inhibited by NAADP but did appear to
be significantly shortened by the NAADP treatment (n = 3). This first Ca2+ transient has previously been shown to
be contributed by both Ca2+ release through
InsP3R and RyR as well as by Ca2+ influx (24).
Although NAADP did not inhibit the first Ca2+ transient
after fertilization, it was effective in blocking the post-fertilization Ca2+ oscillation, which is likely to be
mediated mainly by InsP3. The inhibitory effect of NAADP on
the Ca2+ oscillation was specific since neither
pretreatment with cADPR nor 8-amino-cADPR was capable of blocking the
post-fertilization Ca2+ oscillation (data not shown). On
the other hand, the precursor of NAADP,
-NADP, had the same effects,
in all respects, as NAADP itself (not shown). This is not surprising
since
-NADP could have been enzymatically converted to NAADP. Also,
commercial
-NADP preparations are known to be contaminated with
significant amounts of NAADP. Indeed, it was the contamination that had
led to the discovery of the Ca2+ releasing effect of NAADP
(19).

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Fig. 8.
Inhibition of the fertilization-induced
Ca2+ oscillation by NAADP. A, normal
Ca2+ oscillation following insemination. The time of
fertilization is monitored by the initial change in the oocyte current
recorded electrophysiologically and is indicated by the
arrow labeled F. The graph shown is
representative of nineteen experiments. B, perfusion of
NAADP (100 nM) before and during fertilization inhibited
the oscillation. The graph shown is representative of three
experiments.
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Further evidence that the inhibitory effect of NAADP was restricted
only to the Ca2+ oscillation is shown in Fig.
9A. Pretreatment of oocytes
with NAADP did not abolish the fertilization-associated membrane
insertion, as indicated by a normal increase in membrane capacitance,
nor did it totally inhibit the first Ca2+ transient, which
is consistent with that shown in Fig. 8B. In contrast,
8-amino-cADPR totally eliminated the capacitance increase as shown in
Fig. 9B, whereas the Ca2+ oscillation pattern
following the first transient was not affected by this cADPR antagonist
(not shown).

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Fig. 9.
Effects of NAADP and 8-amino-cADPR on the
fertilization-induced changes. Intracellular Ca2+
concentration (line curve) was monitored simultaneously with
measurements of the oocyte membrane capacitance (black
triangles). The time of fertilization (F) was
determined as described in the legend of Fig. 8. A,
perfusion of NAADP (1 nM) through the patch pipette in the
whole-cell configuration did not inhibit the capacitance increase
normally associated with fertilization nor did it inhibit the
post-fertilization Ca2+ first transient. B,
perfusion of 8-amino-cADPR (1 µM) inhibited the
capacitance increase normally associated with fertilization without
affecting the post-fertilization Ca2+ transient.
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These results show that three independent Ca2+ signaling
mechanisms are present and operative in ascidian oocytes. Each
mechanism is activated by a different Ca2+ messenger,
cADPR, NAADP and InsP3, and each appears to be involved in
mediating specific changes occurring during fertilization and early
development of the oocyte.
 |
DISCUSSION |
It is generally believed that cells possess multiple types of
Ca2+ stores (reviewed in Ref. 37). The recent discoveries
of cADPR and NAADP (4-6, 19) in addition to InsP3 provide
credence to such a belief. Indeed, it has been shown that the
NAADP-sensitive stores can be physically separated from those sensitive
to the other two Ca2+ agonists (32, 38). Of the three, the
InsP3-dependent mechanism is ubiquitous in
cells (39). The cADPR-dependent mechanism is also quite
widespread, being present in a variety of cells from plant to mammalian
tissues (32). In contrast, the distribution of the NAADP-mechanism in
cells is just beginning to be explored. This study is of notable
importance since it is the first to demonstrate that the mechanism is
present in a cell other than sea urchin eggs. Although both sea urchins
and ascidians are marine animals, they diverged in evolution hundreds
of millions of years ago. The fact that NAADP-dependent
Ca2+ signaling is present in these two widely different
species suggests that the mechanism may prove to be as widespread as
those mediated by cADPR or InsP3. In this study,
pharmacological evidence is obtained, indicating all three mechanisms
may be mediated by its own independent receptor, consistent with that
shown in sea urchin eggs (32). Thus, 8-amino-cADPR inhibited the effect
of cADPR while heparin blocked the effect of InsP3, and
neither antagonist had any effect on the action of NAADP (Figs. 5 and
6).
The intriguing question of why cells possess three different
Ca2+ signaling mechanisms was explored in this study. Our
results show that each of the three mechanisms has its own special
function. Evidence is presented indicating the cADPR mechanism is
involved in mediating the insertion of membranes associated with
fertilization. Thus, neither NAADP nor InsP3 can induce an
increase in the membrane capacitance (Figs. 4 and 6). Perfusion of
cADPR into oocytes mimicked the capacitance increase seen at
fertilization while preapplication of 8-amino-cADPR blocked the
increase naturally occurring (Figs. 1 and 9). It is likely that cADPR
is also responsible for mediating the decrease in membrane
Ca2+ current during fertilization. Although all three
Ca2+ agonists can induce a decrease in membrane
Ca2+ current in the oocyte, only cADPR can do so with fast
enough kinetics (Figs. 1, 4, and 6) as compared with those of
fertilization (24). The fast changes induced by cADPR or fertilization
are consistent with the cortical localization of the Ca2+
stores (25).
That this effect of cADPR on the current is due to Ca2+
release activated by the agonist is shown by the fact that preloading the oocytes with BAPTA totally inhibited the change (Fig.
5B). However, measurements of the cytosolic Ca2+
using Fura-2 dextran detected no Ca2+ changes induced by
either cADPR (24) or NAADP (Fig. 7C). This could be due to
the relatively low temporal and spatial resolution of our classical
fura-2 measurement. Future experiments using other probes, in
conjunction with confocal microscopy may allow direct measurement of
the local Ca2+ in the cortical region. In any case, the
experiments using BAPTA presently serve to validate the current
measurement as a method for monitoring localized Ca2+
release close to the plasma membrane. This technique has been widely
used and is generally accepted (24, 25, 35). The use of the
Ca2+ chelator to distinguish between
Ca2+-dependent and -independent effects is
important since, in principle, factors other than Ca2+ may
be able to induce a similar change in Ca2+ current. This is
the case for ADPR, whose inactivating effect on the Ca2+
current is not inhibitable by BAPTA, indicating it is not mediated by
Ca2+ mobilization (Fig. 5B). The ADPR-effect is
likely to be due to a direct interaction of the metabolite with ion
channels. Indeed, it has been reported that ADPR can directly activate
a K+-channel in arterial smooth muscles (40).
In contrast to cADPR and NAADP, perfusion with InsP3
induces a cytoplasmic Ca2+ oscillation in the oocytes
similar to that observed after fertilization. Preloading the oocytes
with heparin blocks the oscillation (41). Together, these results
indicate that post-fertilization Ca2+ oscillation is
mediated by InsP3. The exact mechanism of how these
Ca2+ oscillations are generated is not known, but our
results point to the critical involvement of the NAADP-sensitive
Ca2+ stores. It has previously been shown in sea urchin
eggs that NAADP itself is an inactivator of the
NAADP-dependent Ca2+ release mechanism and can
totally desensitize the release mechanism even at non-activating
concentrations (38, 42). In the ascidian oocytes, our results show that
pretreatment with NAADP, which presumably would discharge the stores
and inactivate the release mechanism, can effectively inhibit the
Ca2+ oscillation. It is possible that the oscillation
requires the functional interaction between the InsP3- and
the NAADP-sensitive stores. Inactivating the NAADP-mechanism by the
pretreatment could disrupt the critical interaction and thus block the
oscillation, even if the InsP3-sensitive stores are fully
functional. Indeed, it has previously been proposed that the
interaction between the NAADP- and the
cADPR/InsP3-sensitive Ca2+ stores may be
responsible for mediating the Ca2+ oscillation seen in sea
urchin eggs (32, 42). Irrespective of the exact mechanism, the
inhibition by pretreatment with NAADP does suggest the
involvement of NAADP, together with InsP3, in mediating the post-fertilization Ca2+ oscillation.
Moreover, the known pH regulation of the NAADP production could
allow the natural alkalinization of the cytoplasmic pH that occurs after fertilization to influence the pattern of
post-fertilization Ca2+ oscillation (43).
The results presented in this study are consistent with a model where
the three independent Ca2+ stores are arranged in a
hierarchical manner. It is proposed that the cADPR-sensitive stores are
localized next to the plasma membrane. Their mobilization by cADPR
would rapidly raise the cortical Ca2+ concentration,
resulting in activation of a fast decrease in oocyte current and
membrane insertion. Since the action of cADPR can be blocked by
ryanodine (24), it is suggested that a RyR-like release channel is
involved. Such a channel has indeed been immunolocalized to the
cortical region of the oocytes (25). The next level of the hierarchical
organization is suggested to be the NAADP-sensitive stores. The farther
distance of these stores from the plasma membrane accounts for the
NAADP-induced current decrease being slower than that of cADPR. The
attenuation of the Ca2+ release from these stores by local
buffering could also contribute to the slow change and could account
for the ineffectiveness of NAADP to activate the capacitance increase.
The cortical region where the cADPR- and NAADP- sensitive stores are
localized may represent a diffusion barrier for either large molecules,
such as Fura-2 dextran, or for Ca2+ ions. The latter could
be due to rapid resequestration of Ca2+ and/or buffering by
Ca2+ binding proteins. Thus the cortical Ca2+
changes can be detected readily by electrical measurements but not by
the cytoplasmic Fura-2 dextran. In contrast, the Ca2+
release induced by InsP3 can be detected by the cytoplasmic
probe, indicating that the stores are distributed in the cytoplasm. It is proposed that the InsP3-sensitive stores interact
specifically with the NAADP-sensitive stores to generate the observed
Ca2+ oscillation. The exact nature of the interaction
remains to be determined. One possibility is that the Ca2+
released from one type of store is sequestered by the other, resulting
in overloading and activating spontaneous release. A similar proposal
has been advanced to account for the effect of NAADP on
Ca2+ oscillations seen in sea urchin eggs (32, 42).
Self-desensitization of the NAADP mechanism by pretreatment with the
agonist could disrupt the crucial interaction between the
Ca2+ stores and thus terminate the oscillation. This
hierarchical model was designed to account for results presented in
this study and will serve as our working model for future
investigations of the Ca2+ signaling pathways in the
oocyte.
We are grateful to Michel Cantou and Laurent
Libicz for scubadiving and collecting animals. We thank Isabelle
Marty, Marie-Jo Moutin, and Christophe Arnoult for helpful comments on
earlier drafts of the manuscript. We thank Gérard Baux and
colleagues for communication of results before publication. We thank
Philippe Deterre for providing the opportunity for fruitful discussions at the "3rd CD38 Workshop" in Paris. We also thank Richard Graeff for critical reading and editing of the manuscript.