Calcium Signaling by Cyclic ADP-ribose, NAADP, and Inositol Trisphosphate Are Involved in Distinct Functions in Ascidian Oocytes*

Mireille Albrieux, Hon Cheung LeeDagger , and Michel Villaz§

From the Laboratoire Canaux Ioniques et Signalisation, DSV/DBMS, 17 rue des Martyrs, F-38054 Grenoble, France and Dagger  The Department of Physiology, University of Minnesota, Minneapolis, Minnesota 55455

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

ADP-ribosyl cyclase catalyzes the synthesis of two structurally and functionally different Ca2+ releasing molecules, cyclic ADP-ribose (cADPR) from beta -NAD and nicotinic acid-adenine dinucleotide phosphate (NAADP) from beta -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, beta -NAD, into unfertilized oocytes. The changes induced by beta -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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 beta -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 beta -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 beta -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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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), beta -NAD (Sigma), beta -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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

cADPR and beta -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."

beta -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 beta -NAD was 170 s, 3-4 times longer than that induced by cADPR (Fig. 2B). The rate of the capacitance increase induced by beta -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 beta -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 beta -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 beta -NAD and its product, cADPR (Fig. 3, A and B).


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Fig. 2.   Effects of beta -NAD on oocyte Ca2+ current and membrane capacitance. A, the current and capacitance changes induced by beta -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 beta -NAD was compared. There was a time lag of about 120 s for beta -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 beta -NAD).


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Fig. 3.   Inhibition of the cADPR and beta -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 beta -NAD (B) were applied through a second patch pipette.

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 beta -NAD to produce cADPR but also an exchange reaction to produce yet another Ca2+ release metabolite from beta -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.

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.

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.

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.

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, beta -NADP, had the same effects, in all respects, as NAADP itself (not shown). This is not surprising since beta -NADP could have been enzymatically converted to NAADP. Also, commercial beta -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.

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.

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
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* This study was supported by the Direction des Sciences du Vivant du CEA, and by INSERM Grant CJF 9709 and Grant HD17484 from National Institutes of Health (to H. C. L.). We acknowledge the support of the Association Française contre les Myopathies.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 33-4 76 88 38 90; Fax: 33-4 76 88 54 87; E-mail: michel.villaz{at}cea.fr.

1 The abbreviations used are: InsP3R, inositol 1,4,5-trisphosphate receptor; NAADP, nicotinic acid-adenine dinucleotide phosphate; cADPR, cyclic ADP-ribose; InsP3, inositol 1,4,5-trisphosphate; ADPR, ADP-ribose; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; RyR, ryanodine receptor; ASW, artificial sea water; pF, picofarad.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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