Ca2+ release from the envelope of isolated pancreatic acinar nuclei could be activated by nicotinic acid adenine dinucleotide phosphate (NAADP) as well as by inositol 1,4,5-trisphosphate (IP3) and cyclic ADP-ribose (cADPR). Each of these agents reduced the Ca2+ concentration inside the nuclear envelope, and this was associated with a transient rise in the nucleoplasmic Ca2+ concentration. NAADP released Ca2+ from the same thapsigargin-sensitive pool as IP3. The NAADP action was specific because, for example, nicotineamide adenine dinucleotide phosphate was ineffective. The Ca2+ release was unaffected by procedures interfering with acidic organelles (bafilomycin, brefeldin, and nigericin). Ryanodine blocked the Ca2+-releasing effects of NAADP, cADPR, and caffeine, but not IP3. Ruthenium red also blocked the NAADP-elicited Ca2+ release. IP3 receptor blockade did not inhibit the Ca2+ release elicited by NAADP or cADPR. The nuclear envelope contains ryanodine and IP3 receptors that can be activated separately and independently; the ryanodine receptors by either NAADP or cADPR, and the IP3 receptors by IP3.

Introduction

Ca2+ release from intracellular stores plays an important role in cytosolic Ca2+ signal generation in many different cell types (Berridge, 1993; Alvarez et al., 1999; Berridge et al., 2003). The ER is the key organelle (Meldolesi and Pozzan, 1998), possessing two separate types of Ca2+ release channels, namely inositol 1,4,5-trisphosphate (IP3) and ryanodine receptors (Berridge, 1993; Petersen et al., 1994; Pozzan et al., 1994; Ashby and Tepikin, 2002; Bootman et al., 2002). However, several other organelles also have the capacity for storing and releasing Ca2+. The function of the mitochondria and their special role in cellular Ca2+ homeostasis have become increasingly clear in recent years (Pozzan et al., 2000; Gilabert et al., 2001; Collins et al., 2002; Villalobos et al., 2002), whereas the function and importance of Ca2+ release from the nuclear envelope (Malviya et al., 1990; Nicotera et al., 1990; Gerasimenko et al., 1995), the Golgi apparatus (Pinton et al., 1998), the secretory granules (Yoo, 2000), and the endosomes (Gerasimenko et al., 1998) are less clear.

The nucleus sits in an ER socket and the outer nuclear membrane is continuous with the ER membrane. Because the lumen of the nuclear envelope is continuous with the ER lumen, the nuclear Ca2+ store could be regarded as part of the ER Ca2+ store (Petersen et al., 1998). However, the distribution of Ca2+ transport proteins in the ER is very nonuniform. In the polarized pancreatic acinar cells, IP3 receptors are concentrated in the apical secretory pole (Thorn et al., 1993; Lee et al., 1997), Ca2+-induced Ca2+ release can only be initiated in this part of the cell (Ashby et al., 2002), and selective activation of muscarinic receptors on the basal membrane initiates cytosolic Ca2+ signals in the apical pole (Ashby et al., 2003a). To understand nuclear Ca2+ homeostasis, it is therefore not possible simply to extrapolate from the general knowledge of ER properties, but the highly dynamic characteristics of the nucleus (Lyman and Gerace, 2001) have to be investigated directly. Previous work has shown that both IP3 and cyclic ADP-ribose (cADPR) can release Ca2+ from the nuclear envelope into the nucleoplasm, suggesting that the IP3 and ryanodine receptors may be localized predominantly in the inner nuclear membrane (Gerasimenko et al., 1995; Hennager et al., 1995; Humbert et al., 1996; Santella and Kyozuka, 1997; Adebanjo et al., 1999, 2000).

The Ca2+-releasing agent nicotinic acid adenine dinucleotide phosphate (NAADP) was discovered in experiments on sea urchin eggs (Chini et al., 1995; Lee and Aarhus, 1995) and has since been shown to release Ca2+ from internal stores in several cell types (Genazzani and Galione, 1997), including normal pancreatic acinar (Cancela et al., 1999; Petersen and Cancela, 1999; Cancela, 2001) and insulin-secreting β cells (Masgrau et al., 2003; Mitchell et al., 2003). However, the mechanism of action is unclear. In sea urchin eggs, it would appear that NAADP, unlike IP3 or cADPR, mobilizes Ca2+ from a pool that does not have ER characteristics (Genazzani and Galione, 1996). The source may be the so-called reserve granules, the functional equivalent of lysosomes (Churchill et al., 2002). However, in pancreatic acinar cells, the NAADP-elicited Ca2+ spikes occur specifically in the apical granular pole (Cancela et al., 2002), exactly as the spikes elicited by IP3 or cADPR (Thorn et al., 1993, 1994). NAADP does not only elicit cytosolic Ca2+ spiking in the apical granular pole, which is the crucial region for regulation of exocytosis (Nemoto et al., 2001) and fluid secretion (Park et al., 2001), but also plays an important role, together with IP3 and cADPR, in spreading the Ca2+ signal throughout the cell (Cancela et al., 2002). This globalizing action of NAADP must depend on interaction between NAADP and its receptors in the basal part of the acinar cell, which contains the nucleus.

To study specifically the action of NAADP on the nucleus, we have investigated the Ca2+-releasing function of this agent in isolated nuclei from pancreatic acinar cells. We have compared the effects of stimulating with NAADP, cADPR, and IP3, and have also studied interactions between these agents. We find that NAADP is an effective releaser of Ca2+ from the nuclear envelope and, like IP3 and cADPR, causes a reduction in the Ca2+ concentration inside the nuclear envelope as well as increasing the Ca2+ concentration in the nucleoplasm. NAADP releases Ca2+ from the same thapsigargin-sensitive pool that is also the target for IP3 and cADPR. The action of NAADP is specific because β-nicotinamide adenine dinucleotide phosphate (β-NADP) in the same concentration has no effect. The NAADP action is most simply explained by activation of ryanodine receptors because it is abolished by ryanodine and ruthenium red (agents that do not affect the IP3-elicited Ca2+ release), but not by blockade of the IP3 receptors.

Results

Structural and functional characterization of the nuclear envelope

We stained isolated pancreatic acinar nuclei with the low affinity Ca2+-sensitive fluorescent dye Mag-Fura Red™ (Fig. 1 A) or with BODIPY® FL thapsigargin, a fluorescent marker for ER-type Ca2+ pumps (Fig. 1 B). The distributions of these two fluorescent markers were similar, as seen in the overlay picture (Fig. 1 C). The probes were clearly localized in the nuclear envelope, as can be seen by comparison with the transmitted light picture (Fig. 1 D). There was no staining of the nuclei with a mitochondrial marker (MitoTracker® Green) or a marker for acidic organelles (LysoTracker® Red; not depicted). The distribution of fluorescent ryanodine (BODIPY® FL ryanodine), a fluorescent marker for ryanodine receptors (Fig. 1 E), was also similar to the distribution of fluorescent thapsigargin (i.e., most of the staining was localized in the nuclear envelope). BODIPY® FL ryanodine staining could be effectively washed away by 10 μM “cold” nonfluorescent ryanodine, confirming the specificity of the staining (Fig. 1 F).

Isolation of nuclei inevitably involves breaking links with the major part of the ER, which is very widely distributed in the pancreatic acinar cells and is very tightly packed in the basolateral part of the cells surrounding the nucleus (Gerasimenko et al., 2002). As seen in electron microscopical images from intact acinar cells, the ER is indeed very tightly packed around the nucleus, and what appears in transmitted light images as a thin envelope (Fig. 1 D) most likely consists of multiple layers of ER (Fig. 1 G). It can also be seen that the structure of the nucleoplasm is nonuniform, with an apparently patchy coverage of the inner nuclear membrane by chromatin (Fig. 1 G).

Ca2+ release from the nuclear envelope elicited by NAADP, cADPR, or IP3

200 nM NAADP evoked a reduction in Ca2+ concentration inside the nuclear envelope, which was irreversible upon removal of the agent (Fig. 1, H and I; n = 7). The effect of NAADP was specific because two close NAADP analogs, β-NADP and nicotinic acid adenine dinucleotide (NAAD; Chini and De Toledo, 2002), were ineffective. Application of 200 nM β-NADP failed to elicit any Ca2+ release from the envelope (n = 7), and similar results were obtained with 200 nM NAAD (n = 7). 20 μM IP3 (n = 5) and 10 μM cADPR (n = 5) had effects very similar to those elicited by 200 nM NAADP. The dose–response curve for the action of NAADP is shown in Fig. 1 (J and K; n > 6 for each concentration). The lowest NAADP concentration capable of eliciting Ca2+ release was 50 nM, whereas the optimal concentration was in the range of 200 nM to 1 μM. There was no response at a very high NAADP concentration (10 μM). This is in qualitative agreement with work on intact pancreatic acinar cells, where it has been shown that high NAADP concentrations do not evoke any measurable Ca2+ release, presumably due to a very rapid desensitization process (Petersen and Cancela, 1999; Cancela et al., 2000). The maximal stimulus (200 nM NAADP) decreased the nuclear envelope Ca2+ concentration from ∼100–150 μM to ∼30–60 μM (see legend to Fig. 1 for more details).

In the experiments described so far, 1 mM ATP was present and the free Ca2+ concentration in the external solution, representing the cytosol, had been adjusted to 100 nM with a Ca2+-EGTA buffer (low buffer concentration). Under these conditions, Ca2+ reuptake into the envelope store did not occur (Fig. 1, H–K). To carry out experiments with multiple messenger applications, which would make it possible to compare control and test conditions in the same preparation, we searched for a protocol allowing recovery after a stimulation-elicited release of Ca2+ from the nuclear envelope. We found that an increase in the Ca2+ concentration in the solution bathing the isolated nuclei to ∼300 nM made the releasing effects of the various messengers reversible (Fig. 2). The effects of 200 nM NAADP, 10 μM cADPR, 20 μM IP3, or 10 mM caffeine at the elevated external Ca2+ concentration (300 nM) are shown in Fig. 2 (A–E). In all cases, addition of the Ca2+-releasing agent caused a transient reduction in the Ca2+ concentration inside the nuclear envelope (n > 10 for each messenger).

Caffeine is an established activator of ryanodine receptors and can thereby elicit substantial Ca2+ release from both sarcoplasmic reticulum and ER stores (Fabiato, 1985; Solovyova et al., 2002). However, caffeine is also an effective inhibitor of IP3 receptors and can completely block IP3-elicited Ca2+ release (Wakui et al., 1990; Bezprozvanny et al., 1994). In intact pancreatic acinar cells, the effect of caffeine is essentially inhibitory, and caffeine-induced Ca2+ release has only been observed under very special conditions (Osipchuk et al., 1990; Wakui et al., 1990). However, in the isolated nuclei, we found that caffeine itself can induce Ca2+ release (Fig. 2 E; n = 25). As seen in Fig. 2, the response to caffeine stimulation was very similar to that elicited by NAADP, cADPR, or IP3.

In the experiments represented by Fig. 2, the Ca2+ concentration in the solution surrounding the nuclei was ∼300 nM, but the concentration of the Ca2+ buffer (EGTA) was relatively low (100 μM; Fig. 2, all traces except B). This would allow some changes in the Ca2+ concentration near the Ca2+ release channels, and therefore, we also tested the effect of clamping the external Ca2+ concentration to ∼300 nM by using a high concentration of a Ca2+/BAPTA mixture (10 mM). In such experiments, (Fig. 2 B; n = 10), the effect of 200 nM NAADP was not markedly different from that seen in experiments with low Ca2+ buffer concentration.

Fig. 2 (F and G) shows the effects of NAADP in the presence and absence of ATP (n > 3 for each experiment). From these data, it would appear that the transient nature of the NAADP-elicited response is due to ATP-dependent Ca2+ reuptake into the nuclear envelope. This is further supported by the experiment illustrated in Fig. 2 H, in which it is seen that thapsigargin blocks the restoration of the nuclear envelope Ca2+ concentration normally occurring during prolonged stimulation.

The experiments described so far were all performed with a relatively slow stimulation protocol in which the control solution flowing into the bath was simply replaced by one containing the stimulant (e.g., IP3, cADPR, or NAADP). It also seemed desirable to carry out experiments in which more immediate effects of stimulation could be investigated. We used two techniques; local uncaging of caged IP3 and local ionophoretic pipette application of IP3 or cADPR. Fig. 3 (A–E) shows traces representing Ca2+ concentration inside the nuclear envelope obtained in response to local uncaging of caged IP3 at various positions around one isolated nuclear envelope. It is seen that the IP3-elicited reduction in the nuclear envelope Ca2+ concentration occurs much faster with this protocol than in experiments with simple bath exchange (Fig. 2 D). Ca2+ reuptake is also much faster, but this is most likely due to the short-lasting nature of the IP3 stimulus. Ionophoretic IP3 application affords the opportunity to produce short or long stimulation pulses. As seen in Fig. 3 F, Ca2+ reuptake does occur during prolonged IP3 stimulation. This is also the case during cADPR stimulation (Fig. 3 G).

The nuclear envelope Ca2+ store could be one unified space or could consist of several distinct noncommunicating compartments. We attempted to give at least a partial answer to this question by conducting bleaching-recovery experiments. Mag-Fura Red™ in one region of the envelope was bleached, and thereafter substantial recovery, presumably due to diffusion of nonbleached dye from neighboring regions, was observed (Fig. 3 H). This type of experiment demonstrates substantial communication between different parts of the nuclear envelope store, but does not completely rule out a degree of subcompartmentalization.

The recovery of the resting (prestimulation) Ca2+ concentration inside the nuclear envelope during sustained messenger stimulation may seem puzzling because it implies that Ca2+ pump-mediated movement can have an impact on the Ca2+ balance when the Ca2+ release channels might be expected to be open. It would normally be expected that ion movements through channels should be much faster than through pumps. In intact pancreatic acinar cells, Ca2+ reuptake into the ER only occurs after removal of the stimulus producing the Ca2+ release (Mogami et al., 1998). To clarify whether our data were in fact contradicting current model concepts, we attempted to model mathematically the nuclear envelope Ca2+ concentration changes in response to continued IP3 stimulation using values for the IP3 and cytosolic Ca2+ concentrations relevant to our experiments. Fig. 3 (I a) shows the time course of the open-state probability of the IP3 type 2 receptor according to Sneyd's model (Sneyd and Dufour, 2002), whereas Fig. 3 (I b) illustrates the result of a similar model for the probably more relevant IP3 type 3 receptor, based on the data from Swatton and Taylor (2002). Finally, we also took into account the data from Mogami et al. (1998), with regard to the rate of SERCA-mediated Ca2+ uptake into the ER as a function of the Ca2+ concentration in the ER lumen in intact pancreatic acinar cells, to model the time course of the nuclear envelope Ca2+ concentration changes during continuous IP3 application (for further details see supplemental material, available at http://www.jcb.org/cgi/content/full/jcb.200306134/DC1). The result is shown in Fig. 3 J. It is clear that the IP3-elicited reduction in Ca2+ concentration in the ER lumen is largely transient, in agreement with our experimental data (Fig. 2 D and Fig. 3 F).

The two separate Ca2+ release channels can be activated independently

Results of the type shown in Fig. 2 (F and G) indicate that the different Ca2+ channel activators release Ca2+ from a common pool in the nuclear envelope. Thus, IP3 can release Ca2+ from the envelope after NAADP stimulation, but only if Ca2+ reuptake has occurred before the IP3 application. This is different from the situation in sea urchin eggs, where it would appear that NAADP releases Ca2+ from a pool that is separate from the one IP3 acts on (Churchill et al., 2002). In intact pancreatic acinar cells, the local Ca2+-spiking responses in the apical granular pole require interactions between IP3 and ryanodine receptors (Cancela et al., 2000, 2002). Therefore, we tested pharmacologically the different Ca2+ release channels and investigated possible interactions between IP3 and ryanodine receptors in the nuclear envelope.

The borate compound 2-aminoethyldiphenyl borate (2-APB) has been used to inhibit IP3 receptors in different cell types (Ma et al., 2000), but is clearly not a specific IP3 receptor antagonist (Bakowski et al., 2001; Prakriya and Lewis, 2001; Harks et al., 2003). Nevertheless, we tested the ability of 2-APB to influence nuclear Ca2+ release elicited by NAADP (Fig. 4 A), cADPR (Fig. 4 B), and IP3 (Fig. 4 C). 100 μM 2-APB abolished IP3-elicited Ca2+ release (Fig. 4 C; n = 7), but failed to inhibit the responses to cADPR (Fig. 4 B; n = 3) or NAADP (Fig. 4 A; n = 6). During sustained caffeine stimulation in the presence of ATP, there was almost a full recovery of the prestimulation Ca2+ concentration inside the nuclear envelope. Subsequent addition of NAADP (n = 6) or cADPR (n = 5) induced a second Ca2+ release. In contrast, IP3 (n = 6) failed to elicit any response in the presence of 10 mM caffeine, consistent with caffeine's known action as a blocker of IP3 receptors (Wakui et al., 1990; Bezprozvanny et al., 1994; Ehrlich et al., 1994). Heparin, the classical IP3 receptor antagonist (Ehrlich et al., 1994), also blocked IP3-induced Ca2+ release (n = 4), but failed to block responses to NAADP (n = 7) and cADPR (n = 3).

We also used ryanodine which, at high concentrations (>10 μM), is an established inhibitor of ryanodine receptors (Sutko et al., 1997). 100 μM ryanodine did not inhibit the effect of IP3 (Fig. 4 D; n = 7), indicating that the response to IP3 does not, in this preparation, depend on cooperation between IP3 and ryanodine receptors. However, the same concentration of ryanodine (100 μM) abolished the Ca2+ release normally elicited by caffeine (Fig. 4 E; n = 8), NAADP (Fig. 4 F; n = 6), and cADPR (Fig. 4 G; n = 6). 10 μM ruthenium red, an inhibitor of ryanodine receptors (Thorn et al., 1994; Hohenegger et al., 2002), also completely blocked NAADP-induced Ca2+ release from the nuclear envelope (Fig. 4 H; n = 7). These data indicate that both NAADP and cADPR interact functionally with the ryanodine receptors, but most likely via two separate primary receptors (as explained in a later section; see Fig. 6 B), and that the opening of the ryanodine receptors alone can cause Ca2+ release without any need for cooperation with functional IP3 receptor channels.

Ca2+ release into the nucleoplasm elicited by NAADP, cADPR, or IP3

Previously, we have shown that accumulation (in the internal part of isolated nuclei) of Ca2+-sensitive fluorescent indicators labeled with dextrans is a useful way of monitoring Ca2+ concentration changes in the nucleoplasm (Gerasimenko et al., 1995). Using Fluo-4 dextran (MW = 10,000) accumulated inside the isolated nuclei (Fig. 5 A), we found that 200 nM NAADP, 10 μM cADPR, and 20 μM IP3 each elicited a transient Ca2+ concentration rise in the nucleoplasm (Fig. 5, B–D; n > 10 for each messenger). The maximal rise of the nucleoplasmic Ca2+ concentration was ∼0.5 μM.

Ca2+ permeability of the nuclear pores after messenger-elicited Ca2+ release

The transient nature of the nucleoplasmic Ca2+ concentration rise in response to messenger stimulation could be due to Ca2+ reuptake into the nuclear envelope store after the release, or it could be explained by movement of Ca2+ from the nucleoplasm through the nuclear pore complexes into the bathing solution outside the nucleus. Because ATP was not added to the solutions used for the experiments represented by Fig. 5 (E and F), it seems unlikely that the first explanation could apply (see Fig. 2). Because it has been reported that the permeability of the nuclear pore complex could be markedly reduced after depleting the nuclear envelope of Ca2+ (Greber and Gerace, 1995, Lee et al., 1998), we tested the ability of external Ca2+ changes to make an impact on the nucleoplasmic Ca2+ concentration after messenger-induced Ca2+ release. After NAADP stimulation, external application of initially a high Ca2+ concentration (0.5 mM) followed by a Ca2+ chelator (2 mM EGTA) induced a large rise and thereafter a fast decrease in the nucleoplasmic Ca2+ concentration (Fig. 5 E; n = 7). This indicates rapid movement of Ca2+ across the nuclear envelope, most likely through the nuclear pore complexes, in agreement with previous work (Brini et al., 1993; Gerasimenko et al., 1995; Lipp et al., 1997). The same protocol was used after stimulation with 20 μM IP3, and very similar results were obtained (Fig. 5 F; n = 6). These results indicate that the nuclear pore complexes are permeable to Ca2+ even after depletion of the nuclear envelope Ca2+ stores.

Interaction between Ca2+-releasing agents

As already described, a high concentration (10 μM) of NAADP did not evoke any Ca2+ release from the nuclear envelope, most likely due to rapid auto-desensitization (Fig. 1). Subsequent application of IP3 (Fig. 6 A; n = 6) or cADPR (Fig. 6 B; n = 6), during continued exposure to NAADP, elicited normal Ca2+ release responses, indicating that the activation of IP3 or ryanodine receptors does not have an obligatory requirement for operational NAADP receptors. The fact that cADPR can evoke Ca2+ release in the presence of a high desensitizing NAADP concentration (10 μM; Fig. 6 B) indicates that cADPR and NAADP most likely act on two separate receptors, although both agents cause Ca2+ release via opening of ryanodine receptors (Fig. 4).

We investigated the nature of the pool from which Ca2+ could be released by NAADP and the other messengers. When ATP was present, and reaccumulation of lost Ca2+ therefore was possible, thapsigargin was able to elicit renewed Ca2+ release after an NAADP-induced Ca2+ rise in the nucleoplasm (Fig. 6 C; n = 13). This was also the case after application of the triple messenger mixture cADPR + IP3 + NAADP (Fig. 6 D; n = 12).

In the absence of ATP, application of thapsigargin induced a markedly reduced Ca2+ release after exposure to the messenger mixture, suggesting that most of the intranuclear Ca2+ had already been liberated (Fig. 6 E; n = 5). Pretreatment of nuclei with thapsigargin abolished the responses to NAADP (Fig. 6 F; n = 5) or the triple messenger mixture (Fig. 6 G; n = 6), indicating that NAADP and the other messengers release Ca2+ from a thapsigargin-sensitive store and that the whole of the thapsigargin-sensitive Ca2+ store can be released by the messengers.

Does NAADP release Ca2+ from acid compartments?

Recent work on sea urchin eggs indicates that NAADP mobilizes Ca2+ from an acid thapsigargin–insensitive pool, with lysosomal properties, that is separate from those sensitive to IP3 and cADPR (Churchill et al., 2002). Therefore, it seemed important to test whether the NAADP-elicited Ca2+ release from the nuclear envelope is dependent on acidic pools. One way of interfering with organellar acidification is to pretreat with bafilomycin, which is a blocker of the vacuolar type H+ ATPase (Bowman et al., 1988). In the presence of bafilomycin A1, at a near-optimal concentration of 50 nM, we found that NAADP elicited an entirely normal Ca2+ release response (Fig. 7 A; n = 6). We also used 10 μM brefeldin A, a membrane transport blocker that disrupts the Golgi apparatus (Donaldson et al., 1992), and observed that NAADP evoked normal Ca2+ release (Fig. 7 B; n = 6). Finally, we used 7 μM of the protonophore nigericin (Camello-Almaraz et al., 2000), but we failed, also in this case, to find any evidence for a reduction in the magnitude of the NAADP-elicited Ca2+ release (Fig. 7 C; n = 6). These data indicate that the Ca2+ release from pancreatic nuclei elicited by NAADP is unlikely to come from acid compartments.

Discussion

By direct measurements of the Ca2+ concentrations both inside the nuclear envelope store and in the nucleoplasm, we have demonstrated that NAADP has a specific Ca2+-releasing action on isolated pancreatic nuclei (Figs. 1, 2, 4, and 5). Ca2+ is liberated from a thapsigargin-sensitive pool in the nuclear envelope and moves into the nucleoplasm to generate a Ca2+ signal in that compartment. Because the action of NAADP is abolished by ryanodine, but not by blockade of IP3 receptors (Fig. 4), the simplest explanation for its effect is activation of ryanodine receptors. The new experiments described here demonstrate that release of Ca2+ from the nuclear envelope, most likely including adhering ER elements (Fig. 1), is associated with a rise in the nucleoplasmic Ca2+ concentration, confirming our earlier work on liver nuclei (Gerasimenko et al., 1995). We and several other groups (for review see Petersen et al., 1998) have provided evidence indicating that the Ca2+ release channels may at least in part be present in the inner nuclear membrane. It is also clearly possible that Ca2+ released from the ER just outside the outer nuclear membrane can diffuse into the nucleoplasm via open nuclear pore complexes (Fig. 8; Gerasimenko et al., 1995; Lipp et al., 1997).

Ryanodine receptors in the nuclear envelope

In isolated liver nuclei, we have previously demonstrated activation of Ca2+ release by a low concentration of ryanodine and by cADPR (Gerasimenko et al., 1995), indicating the presence of functional ryanodine receptors. In intact pancreatic acinar cells, there is evidence for cADPR-elicited Ca2+ spiking (Thorn et al., 1994), and Ca2+ spiking evoked by the physiological agonists acetylcholine and cholecystokinin, as well as by the Ca2+-releasing agents IP3, cADPR, and NAADP, are all inhibited not only by IP3 receptor antagonists, but also by a high ryanodine concentration (Cancela et al., 2000; Cancela, 2001). A high ryanodine concentration inhibits Ca2+-induced Ca2+ release waves initiated in the apical pole of pancreatic acinar cells (Ashby et al., 2002, 2003b). Here, we have provided fresh evidence for the presence of ryanodine receptors in the nuclear envelope by using specific staining with fluorescent ryanodine (Fig. 1, E and F) and by demonstrating that both NAADP and cADPR elicit Ca2+ release by opening ryanodine-sensitive pathways (Fig. 4). NAADP and cADPR most likely bind to different receptors, which then directly or indirectly activate ryanodine receptors, as cADPR can elicit marked Ca2+ release in the presence of a high desensitizing NAADP concentration (Fig. 6 B).

In view of the evidence for operational ryanodine receptors in the pancreatic cells, it has always been puzzling that caffeine, a well-established activator of ryanodine receptors (Ehrlich et al., 1994; Solovyova et al., 2002), generally fails to evoke Ca2+ release in these cells (Petersen and Cancela, 1999). However, caffeine effectively blocks agonist- and messenger-elicited cytosolic Ca2+ signal generation due to its inhibitory effect on the IP3 receptors (Wakui et al., 1990; Bezprozvanny et al., 1994; Ehrlich et al., 1994; Petersen and Cancela, 1999). In isolated nuclei, we have now obtained the first clear evidence for caffeine-elicited Ca2+ release in pancreatic acinar cells (Fig. 2) which, as expected, is blocked by a high ryanodine concentration (Fig. 4).

The mechanism of action of NAADP

The pioneering work on the Ca2+-releasing effect of NAADP was performed on sea urchin eggs (Chini et al., 1995; Lee and Aarhus, 1995), and in this preparation, the store from which NAADP releases Ca2+ does not appear to have ER properties and is separate from the stores on which IP3 and cADPR act (Churchill et al., 2002). Because NAADP-induced Ca2+ release in the sea urchin eggs does not behave as a Ca2+-induced Ca2+-release system (Chini and Dousa, 1996), it has generally been assumed that NAADP does not act on IP3 or ryanodine receptors, but activates a separate type of Ca2+ release channel that has not yet been characterized (Cancela, 2001). Our new data do not fit this concept. In the pancreatic nucleus, NAADP releases Ca2+ from the same thapsigargin-sensitive store as IP3 (Fig. 2, F–H; Fig. 6; Fig. 8). Furthermore, the Ca2+ release normally elicited by NAADP (but not by IP3) is blocked by a high ryanodine concentration (Fig. 4, D and F) as well as by ruthenium red (Fig. 4 H). Finally, we have shown that interference with acid compartments has no effect on the NAADP-evoked Ca2+ release from the nuclear envelope (Fig. 7). The simplest interpretation of these data suggests that NAADP activates ryanodine receptors. Hohenegger et al. (2002) have recently described NAADP activation of single purified and reconstituted type 1 ryanodine receptors from skeletal muscle. This action, like the one in the pancreatic nuclei, is specific (no effect of NADP), ryanodine sensitive, and maximal at ∼100–300 nM NAADP. Hohenegger et al. (2002) conclude that NAADP most likely directly activates type 1 ryanodine receptors, although an action on a protein tightly coupled to the ryanodine receptor might be regarded as more likely.

Functional NAADP receptors are required specifically for the cytosolic Ca2+ signal generation normally elicited by physiological cholecystokinin concentrations in pancreatic acinar cells (Cancela et al., 2000). In addition to the local Ca2+ spikes in the apical (granular) pole, cholecystokinin also elicits longer lasting global Ca2+ transients that invade the nucleus (Osipchuk et al., 1990; Petersen et al., 1991; Thorn et al., 1993). Ca2+ signal globalization is helped by cooperation between activated IP3, cADPR, and NAADP receptors (Cancela et al., 2002). It seems likely that the NAADP-elicited nuclear Ca2+ release, revealed in this report on isolated nuclei, plays a role in Ca2+ signal globalization. However, we do not yet understand how the CD38/ADP-ribosyl cyclase may be regulated in the pancreatic acinar cells. This enzyme is responsible for the production of both cADPR and NAADP (Cancela, 2001) and also exists in the nucleus, where it has its catalytic site within the nucleoplasm (Adebanjo et al., 1999). There is also evidence for the existence of the polyphosphoinositide cycle inside the nucleus (Divecha et al., 1991). Therefore, the various Ca2+-releasing messengers could be produced inside the nucleus to regulate release of Ca2+ from the nuclear envelope. In the intact cell, the Ca2+ store in the nuclear envelope is part of the unified and lumenally continuous ER store (Petersen et al., 2001), but this report on isolated nuclei reveals that the local control of Ca2+ release can operate in a distinct manner. Although the local Ca2+ spiking in the apical secretory pole region of the pancreatic acinar cells (as well as the global Ca2+-induced Ca2+ waves) depends on cooperative interaction of IP3 and ryanodine receptors (Cancela et al., 2000; Ashby et al., 2002), these receptors can function independently in the nucleus to release Ca2+ into the nucleoplasm.

Materials And Methods

Materials

Mag-Fura Red™ acetoxymethyl ester (AM), Rhod 5N AM, Fluo-4 dextran, BODIPY® FL thapsigargin, MitoTracker® Green, LysoTracker® Red, and caged IP3 were obtained from Molecular Probes, Inc. The protease inhibitor cocktail was obtained from Roche. The rest of the chemicals were purchased from Sigma-Aldrich.

Experimental procedures

Single pancreatic acinar cells or small clusters were acutely isolated from CD1 mouse pancreas as described previously (Thorn et al., 1993). Single nuclei were isolated from pancreatic acinar cells by homogenization and by centrifugation as described in Gerasimenko et al. (1995) with some modifications (Maruyama et al., 1995). The buffer for homogenization contained 140 mM KCl, 10 mM Hepes, 1 mM MgCl2, 100 μM EGTA, 1 mM ATP, and protease cocktail inhibitor (1 tablet per 10 ml of buffer; pH 7.2 adjusted with KOH). The final pellet of nuclei was resuspended in standard buffer (140 mM KCl, 10 mM Hepes, 1 mM MgCl2, 100 μM EGTA [low calcium buffer], 75 μM CaCl2, and 1 mM ATP [pH 7.2 adjusted with KOH]). We have also used the same standard buffer, but with a reduced concentration of MgCl2 (0.1 mM) to check the Mg2+ dependence of the Ca2+ release responses. However, the messenger-induced Ca2+ release from Mag-Fura Red™–loaded nuclear envelopes was not altered by this reduction in the external Mg2+ concentration. In some experiments, we used the standard buffer with the composition given above, but increased the buffering of Ca2+ by using a mixture of 10 mM BAPTA and 7 mM CaCl2.

Isolated nuclei were loaded with 20 μM Mag-Fura Red™ in AM form, with 5 μM Rhod 5N in AM form, or with 20 μM Fluo-4 dextran by incubation for 30–45 min at 4°C. Loaded nuclei were washed by centrifugation. All experiments were performed with single isolated nuclei at RT (20–21°C) in an experimental chamber with a perfusion system that allowed washing of nuclei with standard buffer for several minutes before each experiment. The Ca2+ concentration in the nuclear envelope was assessed by Mag-Fura Red™ fluorescence measurements (excitation 488 nm, emission 550–650 nm) or by Rhod 5N fluorescence measurements (excitation 543 nm, emission 555–630 nm). The nucleoplasmic Ca2+ concentration changes were assessed by Fluo-4 dextran (MW = 10,000) fluorescence measurements (excitation 488 nm, emission 500–550 nm). Nuclear preparations were stained with 0.2 μM BODIPY® FL thapsigargin, 1 μM BODIPY® FL ryanodine, 0.5 μM MitoTracker® Green, or 0.2 μM LysoTracker® Red by incubation with those dyes for 5 min in standard buffer, and were then washed using the perfusion system. EM was performed on a transmission electron microscope (model H-600; Hitachi) as described previously (Johnson et al., 2003).

For flash photolysis experiments, caged IP3 in a concentration of 100 μM was added to the nuclear preparation loaded with Mag-Fura Red™ in AM form. Uncaging was performed using the “regions of interest” option of the Leica confocal software in combination with custom-written macros. Fluorescence of Mag-Fura Red™ was excited at 488 nm, and a second laser (Coherent™) provided light for uncaging in the area of interest at wavelengths of 351 and 364 nm for the duration of the uncaging (74–998 ms). Images were acquired continuously at 74–998-ms intervals before and after uncaging. A similar protocol was used for bleaching-recovery experiments, but 100% of the laser power at 488 nm was used for bleaching Mag-Fura Red™ in a small area of the nuclear envelope.

IP3 and cADPR were also applied by ionophoretic ejection from a microelectrode filled with a solution containing 1 mM IP3 or cADPR (the ejecting current was 50–100 nA, retaining current 5–20 nA). An injection system (HVCS 02; NPI Electronics) was used for these experiments. All experiments were performed using a Leica confocal two-photon system with water immersion objective (63×, NA 1.2).

Online supplemental material

Mathematical model of IP3-induced Ca2+ release and uptake in an isolated nucleus.

Acknowledgments

We thank Dr. C. Vaillant for help with EM and Nina Burdakova for technical assistance.

This work was supported by an MRC Programme Grant (G8801575). O.H. Petersen is an MRC Research Professor. O.V. Gerasimenko is supported by The Wellcome Trust and MRC (G0300076). K. Yano and N.J. Dolman are Wellcome Trust Prize Ph.D. students.

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The online version of this article includes supplemental material.

Abbreviations used in this paper: 2-APB, 2-aminoethyldiphenyl borate; AM, acetoxymethyl ester; β-NADP, β-nicotinamide adenine dinucleotide phosphate; cADPR, cyclic ADP-ribose; IP3, inositol 1,4,5-trisphosphate; NAADP, nicotinic acid adenine dinucleotide phosphate; NADP, nicotinamide adenine dinucleotide phosphate.

Supplementary data