NAADP induces Ca²⁺ oscillations via a two-pool mechanism by priming IP₃- and cADPR-sensitive Ca²⁺ stores

Abstract

In sea urchin eggs, Ca²⁺ mobilization by nicotinic acid adenine dinucleotide phosphate (NAADP) potently self-inactivates but paradoxically induces long-term Ca²⁺ oscillations. We investigated whether NAADP-induced Ca²⁺ oscillations arise from the recruitment of other Ca²⁺ release pathways. NAADP, inositol trisphosphate (IP₃) and cyclic ADP-ribose (cADPR) all mobilized Ca²⁺ from internal stores but only NAADP consistently induced Ca²⁺ oscillations. NAADP-induced Ca²⁺ oscillations were partially inhibited by heparin or 8-amino-cADPR alone, but eliminated by the presence of both, indicating a requirement for both IP₃- and cADPR-dependent Ca²⁺ release. Thapsigargin completely blocked IP₃ and cADPR responses as well as NAADP-induced Ca²⁺ oscillations, but only reduced the NAADP-mediated Ca²⁺ transient. Following NAADP-mediated release from this Ca²⁺ pool, the amount of Ca²⁺ in the Ca²⁺-induced Ca²⁺ release stores was increased. These results support a mechanism in which Ca²⁺ oscillations are initiated by Ca²⁺ release from NAADP-sensitive Ca²⁺ stores (pool 1) and perpetuated through cycles of Ca²⁺ uptake into and release from Ca²⁺-induced Ca²⁺ release stores (pool 2). These results provide the first direct evidence in support of a two-pool model for Ca²⁺ oscillations.

Introduction

Nicotinic acid adenine dinucleotide phosphate (NAADP) potently mobilizes Ca²⁺ in several cell types and species including sea urchin eggs (Lee and Aarhus, 1995), ascidian oocytes (Albrieux et al., 1998), starfish oocytes (Santella et al., 2000), mouse pancreatic acinar cells (Cancela et al., 1999), rat brain microsomes (Bak et al., 1999), plant microsomes (Navazio et al., 2000) and human T-lymphocytes (Berg et al., 2000). NAADP operates through a pathway that, although as yet undefined at the molecular level, is pharmacologically and physically distinct from the inositol 1,4,5-trisphosphate (IP₃)- and cyclic ADP-ribose (cADPR)-sensitive Ca²⁺ release pathway (Lee, 1997). In the sea urchin egg, NAADP is unique among the Ca²⁺-releasing messengers in at least four respects (Genazzani and Galione, 1997; Lee, 1997; Galione et al., 2000). First, NAADP does not exhibit positive feedback by Ca²⁺, termed Ca²⁺-induced Ca²⁺ release (CICR) (Chini and Dousa, 1996). Secondly, NAADP potently self-inactivates even when present at concentrations less than those required to activate detectable Ca²⁺ release (Aarhus et al., 1996; Genazzani et al., 1996; Lee et al., 1997). Thirdly, once bound to its receptor, radiolabelled NAADP is occluded and cannot be displaced by non-labelled NAADP, which is thought to be related to its self-inactivation mechanism (Aarhus et al., 1996; Billington and Genazzani, 2000; Patel et al., 2000). Fourthly, NAADP mobilizes Ca²⁺ from a store that is distinct pharmacologically and physically from the IP₃- and cADPR-sensitive Ca²⁺ stores (Lee and Aarhus, 1995, 2000; Genazzani and Galione, 1996).

NAADP stimulates Ca²⁺ oscillations in both pancreatic acinar cells (Cancela et al., 1999) and sea urchin eggs (Aarhus et al., 1996; Genazzani et al., 1996; Lee et al., 1997). In pancreatic acinar cells, NAADP releases Ca²⁺, which is then amplified by CICR through IP₃ and ryanodine receptors (Cancela et al., 1999). Amplification of NAADP-induced Ca²⁺ release through CICR pathways also occurs in starfish eggs (Santella et al., 2000) and sea urchin eggs (Churchill and Galione, 2000). In sea urchin eggs, however, a ‘trigger’ mechanism is unlikely to underlie the long-term Ca²⁺ oscillations induced by NAADP because of self-inactivation (Aarhus et al., 1996; Billington and Genazzani, 2000; Patel et al., 2000). Based on this, Lee and coworkers (Aarhus et al., 1996; Lee, 1997; Lee et al., 1997) suggested that these Ca²⁺ oscillations are more likely to involve overloading and spontaneous release from the IP₃- and/or cADPR-sensitive pathways. However, we demonstrated recently that NAADP self-inactivation is reversible in intact sea urchin eggs when NAADP release is submaximal and spatially localized (Churchill and Galione, 2001), raising the possibility that NAADP-induced Ca²⁺ oscillations are due to cycles of desensitization and re-sensitization.

Several theoretical models have been proposed to explain Ca²⁺ oscillations, and can be classified by the number of Ca²⁺ stores required (one or two) and whether the level of IP₃ must also oscillate (Berridge and Galione, 1988; Tsien and Tsien, 1990; Berridge, 1993). Depending on the cell type and experimental conditions, evidence exists for mechanisms requiring oscillating levels of IP₃ (Harootunian et al., 1991; Hirose et al., 1999) and control by positive and/or negative feedback of Ca²⁺ on Ca²⁺ release channels with a steady level of IP₃ (Wakui et al., 1989; Hajnoczky and Thomas, 1997). Direct evidence does not exist for the two-pool mechanism proposed by Berridge and colleagues (Berridge, 1988, 1991; Berridge and Galione, 1988), which envisages transfer of Ca²⁺ from IP₃-sensitive stores into CICR stores, which overload and spontaneously release Ca²⁺.

The sea urchin egg is highly responsive to IP₃, cADPR and NAADP (Clapper and Lee, 1985; Clapper et al., 1987; Lee and Aarhus, 1995) and thus provides an excellent system in which to study the interaction between Ca²⁺ release pathways. Here we show that NAADP induces long-term Ca²⁺ oscillations by releasing Ca²⁺ from a separate store (pool 1) and is then taken up by CICR stores (pool 2). The shuttling of Ca²⁺ primes the CICR stores and results in cycles of Ca²⁺ overloading, release and re-uptake.

Results and discussion

NAADP but not IP₃ or cADPR induces long-term Ca²⁺ oscillations

Lee and coworkers (Aarhus et al., 1996; Lee, 1997; Lee et al., 1997) reported that NAADP but not IP₃ or cADPR resulted in Ca²⁺ oscillations in sea urchin eggs, and photorelease was much more effective than microinjection of NAADP (Aarhus et al., 1996). However, Swann and Whitaker (1986) reported that microinjection of IP₃ also resulted in a second Ca²⁺ increase that occurred several minutes after the primary Ca²⁺ increase. Therefore, to investigate the mechanism by which NAADP gives rise to Ca²⁺ oscillations, we first compared the Ca²⁺ responses of sea urchin eggs to NAADP, IP₃ and cADPR.

The photorelease of supramaximal amounts of messenger revealed that NAADP released more Ca²⁺ than cADPR, which in turn released more than IP₃ (Figures 1 and 6A). That NAADP evokes a larger increase than either IP₃ or cADPR supports the notion that NAADP is releasing Ca²⁺ from both a common store and a store responsive to only NAADP, as suggested by the data from sea urchin homogenates (Lee and Aarhus, 1995; Genazzani and Galione, 1996). NAADP induced long-lasting (at least 80 min) Ca²⁺ oscillations in 26 of 29 experiments (Figure 1), as demonstrated previously (Aarhus et al., 1996; Genazzani et al., 1996; Lee, 1997). NAADP-induced Ca²⁺ oscillations persisted in the absence of extracellular Ca²⁺ (data not shown), indicating that the oscillations involved mobilization from intracellular Ca²⁺ stores, as reported previously (Aarhus et al., 1996; Lee et al., 1997). In contrast to the high proportion of eggs exhibiting Ca²⁺ oscillations after photorelease of NAADP, Ca²⁺ oscillations were induced less frequently after photorelease of cADPR (eight of 17 experiments, p = 0.004) and IP₃ (one of nine experiments, p <0.0001; Figure 1). Additionally, the magnitude of the Ca²⁺ oscillations was much larger after NAADP-mediated Ca²⁺ release than after either cADPR- or IP₃-mediated Ca²⁺ release (Figure 1). Taken together, it is clear that Ca²⁺ oscillations are more robust and frequent after a Ca²⁺ increase mediated by NAADP than after either cADPR or IP₃.

Fig. 1. Induction of Ca²⁺ oscillations by photorelease of NAADP, cADPR and IP₃. The number of similar responses (numerator) relative to the total number of experiments (denominator) is shown next to each trace. The final intracellular concentrations were: Oregon Green 488 BAPTA Dextran, 10 µM; caged NAADP, 0.5 µM; caged cADPR, 5 µM and caged IP₃, 5 µM.

Fig. 6. Histograms of the maximal amplitude and duration of the Ca²⁺ transients elicited by the designated treatments. Maximal amplitude (A) was calculated as F/F_o and duration (B) was calculated as the time during which F/F_o remained above half the maximal amplitude. All data are from the Ca²⁺ transient elicited by photorelease of the messenger indicated. Histograms are the mean ± standard error of the mean for n = 3–29. The inset bar represents the least significant difference (LSD), which was calculated from the pooled error.

The Ca²⁺ increase that occurs 1–3 min after the NAADP-mediated Ca²⁺ increase occurs before Ca²⁺ recovers to baseline and appears as either a spike clearly separated from the primary increase or as only a shoulder during recovery from the primary increase (Figure 1) (Aarhus et al., 1996; Genazzani et al., 1996; Lee, 1997). To enable quantitative comparisons of data with and without a discernible Ca²⁺ spike, we determined the duration that Ca²⁺ was elevated above its half-maximal concentration (Figure 6B). This measure validly described all treatments except for the effect of removing extracellular Ca²⁺ (Figure 6B), in which case it is an underestimate because the first oscillation is separated from the NAADP-induced Ca²⁺ increase to the extent that Ca²⁺ decreases below its half-maximal concentration before the first oscillation occurs (Aarhus et al., 1996; Lee et al., 1997). Duration data show that the initial Ca²⁺ increase was more prolonged when initiated by NAADP than when initiated by either IP₃ or cADPR (Figures 1 and 6B).

The later Ca²⁺ increases that occur 5–15 min after the NAADP-induced Ca²⁺ increase exhibit irregular patterns (Figure 1) (Aarhus et al., 1996; Genazzani et al., 1996; Lee, 1997). The time of onset, duration, amplitude and frequency of the Ca²⁺ increases is decidedly variable (Figure 1, NAADP). Often there are small Ca²⁺ transients of short duration (∼1 min) superimposed on larger prolonged Ca²⁺ transients (∼5–15 min). This suggests that there are at least two independent oscillatory mechanisms operating simultaneously. Ca²⁺ oscillations with a similar ‘signature’ occur after fertilization of sea urchin eggs with increases occurring before nuclear envelope breakdown, metaphase–anaphase transition and cytokinesis (Poenie et al., 1985; Whitaker and Larman, 2001).

In view of our recent data showing that NAADP desensitization is reversible in intact eggs (Churchill and Galione, 2001), we determined whether re-sensitization could explain NAADP-induced Ca²⁺ oscillations. Under the conditions used in the current study the eggs remained insensitive to NAADP for at least 2 h (data not shown), probably due to the use of supramaximal concentrations of NAADP. Thus, Lee and colleagues’ original argument that the long-term Ca²⁺ oscillations cannot be due to NAADP because of desensitization (Aarhus et al., 1996; Lee, 1997; Lee et al., 1997) remains valid.

NAADP-induced Ca²⁺ oscillations require both IP₃- and cADPR-dependent Ca²⁺ release pathways

To determine the potential roles of IP₃- and cADPR-dependent Ca²⁺ release pathways in the later oscillations (≥5 min after NAADP photorelease), the effects of IP₃ and cADPR antagonists on NAADP-induced Ca²⁺ oscillations were investigated. The involvement of IP₃ was probed with heparin, which inhibits IP₃ binding to its receptor (Taylor and Broad, 1998). NAADP induced oscillations in four of six experiments (p = 0.20) in the presence of 2.5 mg/ml heparin and in three of seven experiments (p = 0.07) in the presence of 5 mg/ml heparin (Figure 2). Heparin, however, inhibited the first Ca²⁺ oscillation after the NAADP-induced Ca²⁺ increase and shortened the duration of this Ca²⁺ transient (Figures 2 and 6B). The involvement of cADPR was probed with 8-amino-cADPR (8NH₂, Figure 6), which inhibits cADPR binding to its receptor (Walseth and Lee, 1993). The presence of 8-amino-cADPR (5 µM) did not block Ca²⁺ oscillations in six of six experiments (Figure 2, p >0.99). Nevertheless, 8-amino-cADPR, as did heparin, decreased the amplitude and frequency of the Ca²⁺ oscillations (Figure 2), indicating a partially inhibitory effect. In contrast to the variable and partially inhibitory effects of either heparin or 8-amino-cADPR alone, the combination of both compounds eliminated NAADP-induced later Ca²⁺ oscillations in seven of seven experiments (Figure 2, p <0.0001). In the presence of both inhibitors, NAADP released more Ca²⁺ than it did in the presence of either inhibitor alone (Figures 2 and 6A), possibly due to prevention of a basal leak of Ca²⁺ from a common store, which would lead to greater loading of this common store. These data demonstrate that both IP₃- and cADPR-mediated pathways for Ca²⁺ release are required for NAADP-induced Ca²⁺ oscillations.

Fig. 2. Requirement of NAADP-induced Ca²⁺ oscillations on Ca²⁺ release pathways mediated by IP₃ and cADPR. The final intracellular concentrations were: Oregon Green 488 BAPTA Dextran, 10 µM; caged NAADP, 0.5 µM; heparin, 2.5 or 5 mg/ml; 8-amino-cADPR, 5 µM; and heparin and 8-amino-cADPR combined, 2.5 mg/ml and 5 µM, respectively. The number of similar responses relative to the total number of experiments is shown next to each trace. The positive control for NAADP-induced Ca²⁺ oscillations was from the same batch of eggs.

That NAADP-induced Ca²⁺ oscillations could only be blocked completely by inhibition of both CICR pathways is similar to the findings reported for inhibition of fertilization-induced Ca²⁺ waves in sea urchin eggs (Galione et al., 1993; Lee et al., 1993), NAADP-induced Ca²⁺ release in starfish eggs (Santella et al., 2000) and for amplification of NAADP-mediated Ca²⁺ waves in sea urchin eggs (Churchill and Galione, 2000). The situation is different from that in pancreatic acinar cells, where NAADP-induced Ca²⁺ oscillations can be blocked by inhibiting either the IP₃ or cADPR pathway (Cancela et al., 1999). In the case of pancreatic acinar cells, the interaction was suggested to occur at the level of CICR, with Ca²⁺ released via the NAADP-mediated pathway serving as ‘trigger Ca²⁺’, which feeds forward on to either IP₃ or ryanodine receptors thereby amplifying the response (Cancela et al., 1999). Such a mechanism cannot explain the Ca²⁺ oscillations that occur after NAADP-induced Ca²⁺ mobilization because the NAADP receptors are completely desensitized to NAADP for at least 2 h after the initial release using this particular protocol (data not shown). Indeed, the first Ca²⁺ oscillation, which is sometimes distinct and at other times appears as a shoulder after the initial NAADP-mediated Ca²⁺ increase (Figure 1) (Aarhus et al., 1996; Genazzani et al., 1996; Lee, 1997), occurs during recovery from the NAADP-induced Ca²⁺ transient when Ca²⁺ is decreasing. This is at odds with a cytosol-based CICR mechanism, which would provide amplification during the increase in Ca²⁺.

A Ca²⁺ store that is thapsigargin insensitive and NAADP sensitive is present in intact eggs

If NAADP-induced Ca²⁺ oscillations are due to overloading and spontaneous Ca²⁺ release from CICR stores, then Ca²⁺ would have to be moved into the CICR stores from an NAADP-sensitive Ca²⁺ store that is functionally and physically separate. The NAADP-sensitive Ca²⁺ store is pharmacologically and physically distinct in sea urchin egg homogenates (Lee and Aarhus, 1995; Genazzani and Galione, 1996). The presence of such a store in intact sea urchin eggs, however, was unknown until a recent report that demonstrated spatial separation of the NAADP-sensitive Ca²⁺ stores from the IP₃- and cADPR-sensitive Ca²⁺ stores in intact sea urchin eggs whose organelles were stratified by centrifugation (Lee and Aarhus, 2000). We used a pharmacological approach to determine whether NAADP-sensitive Ca²⁺ stores in intact sea urchin eggs were functionally distinct from IP₃- and cADPR-sensitive Ca²⁺ stores. Treatment of intact eggs with the Ca²⁺ pump inhibitor thapsigargin (2 µM) for 30 min or more completely eliminated the response to both IP₃ and cADPR (Figures 3A and 6A), as reported previously for homogenates (Genazzani and Galione, 1996). In contrast, thapsigargin reduced but did not eliminate the initial response to NAADP (Figures 3A and 6A). This result demonstrates that in the intact egg NAADP targets Ca²⁺ stores that are functionally separate from the CICR stores. This conclusion is consistent with that of Lee and Aarhus (2000), who demonstrated recently that the NAADP-sensitive store was separable from the endoplasmic reticulum and segregated with mitochondria. However, it is unlikely that mitochondria are the target for NAADP because NAADP-sensitive stores are distinct from cytochrome C oxidase, a mitochondrial marker, in fractionated sea urchin egg homogenates (Lee and Aarhus, 1995), and mitochondria are sinks not sources for Ca²⁺ in non-fertilized eggs (Eisen and Reynolds, 1985). Therefore, NAADP probably targets an organelle that remains to be characterized.

Fig. 3. NAADP-induced Ca²⁺ oscillations require thapsigargin-sensitive Ca²⁺ stores. (A) Effect of thapsigargin on the initial response to photorelease of an IP₃–cADPR mixture and NAADP. (B) Effect of thapsigargin on long-term NAADP-induced Ca²⁺ oscillations shown on a different time scale. Eggs were treated with thapsigargin (2 µM) for ≥30 min and then exposed to UV. The final intracellular concentrations were: Oregon Green 488 BAPTA Dextran, 10 µM; caged NAADP, 0.5 µM, and both caged cADPR, 5 µM and caged IP₃, 5 µM.

NAADP-induced Ca²⁺ oscillations require thapsigargin-sensitive Ca²⁺ stores

Pretreatment with thapsigargin eliminated NAADP- induced Ca²⁺ oscillations in eight of eight experiments (Figure 3B; p <0.001 compared with the NAADP control), demonstrating a requirement for IP₃- and cADPR-sensitive Ca²⁺ stores. These data are consistent with the elimination of NAADP-induced Ca²⁺ oscillations obtained with heparin and 8-amino-cADPR (Figure 2), and consistent with a mechanism in which the CICR stores (IP₃- and cADPR-sensitive) play a role in the NAADP-induced Ca²⁺ oscillations.

CICR stores contain more Ca²⁺ after an NAADP-mediated Ca²⁺ transient

Next we directly assessed the effect of an NAADP-mediated Ca²⁺ release on the amount of Ca²⁺ in the CICR stores. As only the CICR stores are sensitive to thapsigargin (Genazzani and Galione, 1996 and Figure 3), the size of the Ca²⁺ transient induced by thapsigargin was used as a measure of CICR store loading. Figure 4 shows that thapsigargin (5 µM) induced a significantly (p = 0.01) larger Ca²⁺ transient after an NAADP-induced Ca²⁺ transient (maximum amplitude of 2.0 ± 0.27 ratio units, n = 10) than in control eggs (maximum amplitude of 1.3 ± 0.07 ratio units, n = 6). These data indicate that a substantial amount of the Ca²⁺ released by NAADP is taken up by the CICR stores. In contrast, the amplitude of Ca²⁺ release upon photorelease of NAADP was not increased after Ca²⁺ mobilization by either IP₃ (maximum amplitude of 2.2 ± 0.14 ratio units, t-test, p = 0.53) or both IP₃ and cADPR (maximum amplitude of 2.2 ± 0.13 ratio units, t-test, p = 0.62). The mechanism for the preferential accumulation of NAADP-released Ca²⁺ into the CICR stores is unknown. A possible mechanism could be faster removal of Ca²⁺ by thapsigargin-sensitive Ca²⁺ pumps than by thapsigargin-insensitive Ca²⁺ pumps.

Fig. 4. Overloading of CICR stores in response to an NAADP-mediated Ca²⁺ release. The extracellular solution was changed from artificial sea water containing 10 mM Ca²⁺ to one containing no added Ca²⁺ and 2 mM BAPTA. Thapsigargin (5 µM) was added 2 min later. The final intracellular concentrations were: Oregon Green 488 BAPTA Dextran, 10 µM and caged NAADP, 0.5 µM.

Ca²⁺ oscillations can be induced by photorelease of Ca²⁺

With a Ca²⁺-overloading mechanism, any means of overloading Ca²⁺ stores should induce long-term Ca²⁺ oscillations (Berridge and Galione, 1988; Berridge, 1991). Photoreleasing Ca²⁺ (NP-EGTA) (Ellis-Davies and Kaplan, 1994) resulted in an increase equivalent to that induced by NAADP (Figures 5 and 6A), and induced long-term Ca²⁺ oscillations in four of 10 experiments (Figure 5). The pattern of the oscillations generated by photoreleasing Ca²⁺ (Figure 5) was similar but not identical to that generated by NAADP (Figure 1). The amount of caged Ca²⁺ remaining after the first period of photorelease was determined by two additional periods of photorelease, one immediately after recovery to basal levels and one 70 min after the first photorelease (Figure 5). These additional UV illuminations failed to increase Ca²⁺, demonstrating that the first period of photorelease destroyed most of the NP-EGTA. The amount of Ca²⁺ can be approximated from this information. Given that NP-EGTA was at a concentration of 500 µM and that the resting Ca²⁺ (∼100 nM) is approximately at the K_d (80 nM) for NP-EGTA (Ellis-Davies and Kaplan, 1994), there would be ∼250 µM caged Ca²⁺ (and 250 µM free NP-EGTA). Complete photorelease of the caged Ca²⁺ would liberate ∼250 µM Ca²⁺. Although an increase in Ca²⁺ is sufficient for inducing Ca²⁺ oscillations in sea urchin eggs about half the time, it does not induce Ca²⁺ oscillations as often as NAADP, and fails to induce the higher frequency component. Therefore, Ca²⁺ alone can partially, but not completely, mimic NAADP-induced Ca²⁺ oscillations.

Fig. 5. Ca²⁺ oscillations induced by photorelease of Ca²⁺. The final intracellular concentrations were: Oregon Green 488 BAPTA Dextran, 10 µM and NP-EGTA, 500 µM. The number of similar responses relative to the total number of experiments is shown next to each trace.

Mechanism for NAADP-induced Ca²⁺ oscillations

The mechanism by which NAADP induces Ca²⁺ oscillations and a summary of the evidence for this mechanism are presented in Figure 7. The initial Ca²⁺ release mediated by NAADP is largely independent of any additional processes (pathway 1, Figure 7), except for an amplification by CICR pathways, as described previously (Churchill and Galione, 2000). A portion (approximately one-third) of the Ca²⁺ mobilized by NAADP comes from a thapsigargin-resistant store that is distinct from the CICR stores. This released Ca²⁺ is then taken up into CICR stores. Ca²⁺ uptake continues until the stores overload and spontaneously release Ca²⁺ as demonstrated previously (Galione et al., 1991; Missiaen et al., 1991). A necessary feature of this mechanism is that the released Ca²⁺ is taken up into thapsigargin-sensitive stores more quickly than into thapsigargin-insensitive (NAADP-sensitive) stores, but how this is accomplished is unknown. The first Ca²⁺ oscillation after the NAADP-induced transient requires IP₃-dependent Ca²⁺ release (pathway 2, Figure 7). Both IP₃- and cADPR-sensitive Ca²⁺ stores participate in the later cycles of Ca²⁺ uptake, overload and release (pathway 3, Figure 7). This Ca²⁺-overloading mechanism can also explain the occasional induction of long-term Ca²⁺ oscillations by IP₃ (Figure 1 and Swann and Whitaker, 1986) and cADPR (Figure 1), in which overloading would occur via Ca²⁺ influx. This mechanism meets the criteria of a two-pool model for Ca²⁺ oscillations (Berridge, 1988; Berridge and Galione, 1988; Tsien and Tsien, 1990) because one Ca²⁺ store is the source of priming Ca²⁺ (NAADP sensitive) and a separate Ca²⁺ store mediates the oscillations (CICR), and oscillations can be triggered by any increase in Ca²⁺ that primes the CICR stores. Therefore, this mechanism represents the first direct demonstration of the two-pool model for Ca²⁺ oscillations.

Fig. 7. NAADP induces Ca²⁺ oscillations by priming CICR stores. (A) The effect of each treatment is designated as having no effect (open circle), partial inhibition (grey circle) and complete inhibition (black circle). (B) Cartoon of the proposed mechanism. NAADP releases Ca²⁺ from a functionally separate Ca²⁺ store (designated 1). This Ca²⁺ is taken up into the CICR stores, thereby priming them. This results in overloading and spontaneous Ca²⁺ release initially from IP₃-sensitive Ca²⁺ stores (designated 2). Subsequently, Ca²⁺ is taken up into and released from both IP₃- and cADPR-sensitive Ca²⁺ stores (designated 3).

Materials and methods

Microinjection and Ca²⁺ imaging were performed as described previously (Churchill and Galione, 2000). Briefly, sea urchin (Lytechinus pictus) eggs (Marinus, Long Beach, CA) were obtained by intracoelomic injection of 0.5 M KCl, shed into artificial sea water (in mM, NaCl 435, MgCl₂ 40, MgSO₄ 15, CaCl₂ 11, KCl 10, NaHCO₃ 2.5, EDTA 1), dejellied by passing through 90 µm nylon mesh, and then washed twice by centrifugation. Eggs were transferred to polylysine-coated glass coverslips, pressure microinjected (Picospritzer, World Precision Instruments) with Oregon Green 488 BAPTA Dextran (Molecular Probes) and caged compounds and/or inhibitors. The Ca²⁺-sensitive dye was imaged by laser-scanning confocal microscopy (TCS NT, Leica) and caged compounds were photolysed with an ultraviolet laser. Images were processed with the software NIH Image to create a self-ratio by dividing each image by an image acquired before stimulation. Where appropriate, parametric data were subjected to analysis of variance with means separated by Fisher’s least significant difference test with significance taken as p ≤0.05. Proportions (e.g. number of eggs exhibiting oscillations versus number not) were analysed by creating a 2 × 2 contingency table, with NAADP compared with another treatment and then subjected to Fisher’s exact probability test.