Bidirectional Ca²⁺ signaling occurs between the endoplasmic reticulum and acidic organelles

A universal signal transduction mechanism for extracellular stimuli is the release of Ca²⁺ from intracellular stores (Berridge et al., 2003) with stimulus-specific Ca²⁺ patterns fine-tuned by appropriate combinations of three Ca²⁺-mobilizing intracellular messengers: D-myo-inositol 1,4,5-trisphosphate (IP₃), cyclic ADP-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP; Cancela et al., 2002; Morgan and Galione, 2008). Thus, multiple messengers entrain Ca²⁺ oscillations and waves, e.g., at fertilization (Churchill and Galione, 2001; Santella et al., 2004; Moccia et al., 2006; Whitaker, 2006; Davis et al., 2008), the activation of T cells (Steen et al., 2007; Davis et al., 2012) or pancreatic acinar cells (Cancela et al., 2002; Yamasaki et al., 2004).

While IP₃ and cADPR target their cognate receptors on the neutral sarcoplasmic or endoplasmic reticulum (SR/ER), NAADP evokes Ca²⁺ release from acidic Ca²⁺ stores (Churchill et al., 2002; Morgan et al., 2011), probably by activating complexes of the two-pore channel (TPC) family (E. Brailoiu et al., 2009, 2010a; Calcraft et al., 2009; Zong et al., 2009; Ruas et al., 2010; Morgan et al., 2011; Davis et al., 2012).

To date, the cross talk (“channel chatter”; Patel et al., 2001) between the NAADP and IP₃/cADPR pathways has centered upon “anterograde” signaling from the acidic stores to the ER in the “trigger” hypothesis (or two-pool model; Churchill and Galione, 2001): NAADP activates TPCs on acidic stores to provide the critical “pacemaker” trigger Ca²⁺ that is subsequently amplified by IP₃ receptors (IP₃Rs) and/or ryanodine receptors (RyRs) on the neutral ER/SR (Morgan et al., 2011), either via Ca²⁺-induced Ca²⁺ release (CICR; Patel et al., 2001; Kinnear et al., 2004; Zong et al., 2009; Brailoiu et al., 2010b; Ruas et al., 2010; Davis et al., 2012) or by luminal priming (Churchill and Galione, 2001; Macgregor et al., 2007).

It is unknown whether Ca²⁺ signals travel in the reverse direction from ER to the acidic Ca²⁺ stores to make channel chatter a two-way conversation that might be important for regenerative cycles of Ca²⁺ oscillations and waves. Consequently, we have investigated whether ER (IP₃/cADPR) signals communicate with acidic Ca²⁺ stores (NAADP) by using a novel single-cell approach for monitoring acidic store activation. The activation of acidic Ca²⁺ stores is difficult to extract from cytosolic Ca²⁺ recordings that are the net result of multiple processes. This issue can be offset by monitoring the organelle lumen itself using optical reporters, e.g., targeted to ER, mitochondria, and secretory granules (Arnaudeau et al., 2001; Pinton et al., 2007; Santodomingo et al., 2008). By analogy, we have monitored the luminal pH (pH_L) of acidic Ca²⁺ stores as a readout of activation because a prompt alkalinization accompanies NAADP-induced Ca²⁺ release in sea urchin eggs (Morgan and Galione, 2007a,b; Morgan, 2011), pancreatic acinar cells (Cosker et al., 2010), and atrial myocytes (Collins et al., 2011).

Hence, we have investigated sea urchin eggs where the sperm stimulus couples to NAADP, cADPR, and IP₃ (Morgan, 2011). Given that, of these three, NAADP is unique in changing acidic store pH_L in egg homogenate and that these stores are well distributed throughout the sea urchin egg (Lee and Epel, 1983; Churchill et al., 2002; Morgan and Galione, 2007a,b; Ramos et al., 2010), we have imaged Ca²⁺ store alkalinization to “map” where and when acidic stores are activated during a physiological stimulus. Our results suggest that the ER and acidic vesicles are in close apposition and that Ca²⁺ released from the ER by IP₃/cADPR stimulates the NAADP pathway in an unexpected retrograde manner, thereby amplifying acidic Ca²⁺ store signaling. This may have profound implications for Ca²⁺ oscillations and waves in all systems.

Ca²⁺ release evoked by NAADP or fertilization is accompanied by a rapid increase in the pH_L of acidic Ca²⁺ stores (Morgan and Galione, 2007a,b). In the intact sea urchin egg, this predominantly cortical pH_L response was termed a pH_Lash (pronounced “flash”) that was independent of cytosolic pH changes and exocytosis (Morgan and Galione, 2007a). Because NAADP is unique in evoking this pH_L change (Morgan and Galione, 2007b), we exploited this as a “reporter” of acidic store activation (i.e., NAADP-induced Ca²⁺ release), first testing its sensitivity and spatiotemporal fidelity.

We photo-released NAADP from its microinjected caged precursor using a UV laser. No pH_L (or Ca²⁺) responses to UV light were observed in the absence of caged NAADP (Δratio: 0.03 ± 0.01; n = 10, P > 0.05). While measuring pH_L, the uniform uncaging of NAADP (Fig. 1 A) replicated the injection of free NAADP (Morgan and Galione, 2007a), i.e., a prompt increase in the pH_L of acidic vesicles with the largest response in the cell periphery and a small but detectable response in the egg center. In contrast, when measuring Ca²⁺, the uniform uncaging of NAADP evoked a uniform Ca²⁺ response (Fig. 1 B). However, rapid Ca²⁺ diffusion and the contribution of ER Ca²⁺ stores (Churchill and Galione, 2000) render it unsuitable for mapping acidic store activation, so we focused on pH_L as a more reliable readout.

By varying the UV laser power, the pH_L response of both the periphery and the center increased as a function of the NAADP concentration (Fig. 1 C). Indeed, when the magnitude of the responses was normalized to the maximum response, there was no difference in the sensitivity of the two regions, only in their dynamic range (Fig. 1 D). Consequently, the ratio of the responses in the periphery and center is almost invariant with NAADP concentration (the center being ∼30% of the periphery; Fig. 1 E). Kinetically, the maximal pH_Lash response (at 70% UV) occurred with a time to peak of 6.4 ± 0.4 s and a lag of 3.2 ± 0.3 s (n = 14). Such response times are congruent with other second messenger reporters (Nikolaev et al., 2004).

To address spatial fidelity, we focally uncaged NAADP at one pole of the egg (Fig. 1, F–H). In the majority of eggs (n = 22), the pH_L response precisely overlapped with the site of exposure to UV and remained at the site until it waned (Fig. 1, F–H; Fig. S1). This indicated that neither the diffusion of NAADP nor of the target vesicles themselves were confounding factors over this period. Thus, pH_L faithfully mapped experimental increases in cytosolic NAADP and acidic store activation. However, in a minority of eggs (n = 6) the initial, polarized response did eventually propagate to the antipode after remaining stationary at the UV site for 19 ± 3 s (Fig. S1 F). This regenerative phase clearly required positive feedback and probably reflects the secondary Ca²⁺ oscillations and waves that can be entrained by uncaging NAADP (Lee et al., 1997; Churchill and Galione, 2000, 2001).

If pH_L is a faithful reporter of the NAADP/TPC pathway, then responses should be inhibited by NAADP antagonists. Therefore, we tested several antagonists with the fertilization-induced pH_Lash. Note that at fertilization, the pH_L of acidic vesicles changes differently in different regions of the egg: the periphery shows a prompt alkalinization (the pH_Lash) that accompanies the main Ca²⁺ wave and can be mimicked by NAADP (Morgan and Galione, 2007a), whereas the center shows a slow acidification that is driven by an unrelated mechanism dependent upon extracellular Na⁺ (Lee and Epel, 1983; Morgan and Galione, 2007a).

We first tested the fluorescent NAADP receptor antagonist, Ned-19 (Fig. S2; Naylor et al., 2009; Barceló-Torns et al., 2011). Preincubation of eggs with Ned-19 resulted in a concentration-dependent increase in Ned-19 loading, as assessed by its intrinsic fluorescence (Fig. 2 C), but which did not affect the resting pH_L (% control, 80 µM, 95 ± 2; 160 µM, 105 ± 3; P > 0.05, n = 47–83). In keeping with a role for NAADP, Ned-19 inhibited the sperm-induced pH_Lash (Fig. 2, A–C). This was not due to a general interference with fertilization because it had little effect upon the slower acidification of the central acidic granules (Fig. 2, A–C; Lee and Epel, 1983; Morgan and Galione, 2007a) or upon fertilization envelope lifting driven by the other remaining fertilization messengers, cADPR and IP₃ (Fig. 2 B, inset). Furthermore, we confirmed that Ned-19 inhibited the pH_Lash evoked by injection of NAADP itself (Fig. 2 D).

Unlike RyRs and IP₃Rs, NAADP receptors are inhibited by high concentrations of L-type Ca²⁺ channel blockers in sea urchin egg and other cell types (Genazzani et al., 1997; Mándi et al., 2006; Zhang and Li, 2007). Conveniently, both the main fertilization-induced Ca²⁺ wave (Shen and Buck, 1993) and the pH_Lash itself (Fig. S2 E) are independent of Ca²⁺ influx. As a selective NAADP antagonist (Fig. S2, A and B), the phenylalkylamine, diltiazem, selectively inhibited the sperm-induced pH_Lash compared with the central acidification (Fig. 2 F) and, like Ned-19, did not interfere with fertilization envelope lifting (unpublished data). Diltiazem also inhibited the responses to NAADP injection in terms of the pH_Lash and fertilization envelope lifting (consistent with a block of Ca²⁺ release, Fig. 2 G).

Similarly, another L-type Ca²⁺ channel blocker, nifedipine (a dihydropyridine), also inhibited the sperm-induced pH_Lash (but not the central acidification, Fig. 2 E). This partial inhibition by nifedipine was at its limit of solubility and probably reflects its lower membrane permeability compared with diltiazem (XlogP3 of 2.2 and 3.1, respectively). Finally, the nucleotide mimetic, PPADS, inhibited NAADP responses (Fig. S2; Billington and Genazzani, 2007) and selectively inhibited the pH_Lash when microinjected into the egg, precluding an extracellular site of action (Fig. 2 E). Together, the data affirm NAADP as the main pH_L messenger at fertilization and strengthen pH_L as a physiologically relevant reporter of the NAADP pathway.

With a means of mapping acidic store activation, we asked whether Ca²⁺ released from the ER can signal to acidic vesicles. First, we elevated Ca²⁺ independently of sperm using ionomycin, a Ca²⁺ ionophore that mobilizes Ca²⁺ from neutral (and not acidic) Ca²⁺ stores (Fasolato et al., 1991; see Fig. 5 A) and which acts only weakly at the plasma membrane (Morgan and Jacob, 1994). Ionomycin evoked a prompt Ca²⁺ rise in eggs (Fig. 3, Eii), as well as a pH_Lash (Fig. 3, Ei). However, the pH_L response was slower, mimicking the sperm response in its magnitude (Fig. 3, A–C) and kinetics (time to peak (s) ionomycin: 69 ± 2; sperm: 60 ± 2; n = 31–39). Because ionomycin has no direct effect upon the pH_L of sea urchin egg acidic vesicles (Morgan and Galione, 2007b) it follows that it is the released Ca²⁺ that affects acidic vesicles.

Their relative kinetics imply that the pH_Lash is downstream of Ca²⁺ and this is consistent with the one mirroring the other in space: the pH_L (Fig. 3, Di) and Ca²⁺ (Fig. 3, Dii) responses to sperm each occurred as waves (that propagate away from the sperm entry point; Morgan and Galione, 2007a), whereas ionomycin induced a synchronous cortical elevation of either pH_L or Ca²⁺ (Fig. 3, Ei and ii). Taken together, the kinetic and spatial interrelationship of the two parameters is consistent with Ca²⁺ driving pH_L changes.

How could the Ca²⁺ released by ionomycin be stimulating acidic vesicles? As argued previously for sperm (Morgan and Galione, 2007a), the pH_Lash is not limited by the time of the acidic stores to respond to NAADP (an ∼10-times faster process), so the kinetics may reflect the time taken to generate NAADP. We therefore tested whether ionomycin could elevate NAADP levels, as measured in populations of eggs with a radioreceptor assay (Lewis et al., 2007). As observed previously, fertilization increased NAADP in two phases: the first phase represents an increase in NAADP in the sperm as they contact the egg jelly, the second phase is due to de novo synthesis of NAADP inside the egg (Fig. 3 F; Churchill et al., 2003). Remarkably, ionomycin increased NAADP in eggs with kinetics that not only overlapped with the second “egg” phase but also that mirrored the slow time to peak of the pH_L response (each peaked at ∼70 s; Fig. 3 F). The maximum magnitude of the ionomycin-evoked NAADP increase was variable between preparations, being 30 ± 18% (n = 4) of that evoked by sperm.

If ionomycin increases NAADP, then its pH_Lash should be sensitive to NAADP inhibitors. We also used SKF96365 to block NAADP (Moccia et al., 2004; Bezin et al., 2008) after first confirming its >50-fold selectivity for NAADP over cADPR and IP₃ (Fig. S2, C and D). Although SKF96365 could not be used with sperm because it blocks sperm chemotaxis and the acrosome reaction (Hirohashi and Vacquier, 2003; Yoshida et al., 2003; Treviño et al., 2006), it was an effective inhibitor of the ionomycin-induced pH_Lash (Fig. 3 I). Similarly, Ned-19 and diltiazem also inhibited the ionomycin pH_Lash (Fig. 3, G and H) with a similar potency (and selectivity over the slow central acidification) to that seen against the sperm-induced response (Fig. 2, C and F). The data suggest that Ca²⁺ release from the ER by ionomycin can stimulate a pH_Lash via NAADP generation and action.

We next investigated if more physiological routes of Ca²⁺ release from the ER could support acidic store activation. Sea urchin eggs are sensitive to both ER-targeting messengers, IP₃ and cADPR (Churchill and Galione, 2001; Morgan and Galione, 2007b), and we first confirmed that microinjection of these messengers elicited robust Ca²⁺ responses similar to those with NAADP itself (Fig. 4, A and B). When subsequently measuring pH_L changes, not only did NAADP evoke a pH_Lash but so did cADPR and IP₃ (Fig. 4, C and D). Although the examples shown are among the best responses seen with cADPR and IP₃, overall they were weaker stimuli of the pH_Lash than was NAADP (Fig. 4 E), with IP₃ being the least efficacious. This differential messenger profile for the pH_Lash contrasted with the similar global Ca²⁺ responses (Fig. 4, A and B). The fact that IP₃ and cADPR do not directly affect pH_L in egg homogenate (Morgan and Galione, 2007b) implies that these receptors do not reside on acidic vesicles themselves, and that the mechanism that normally couples Ca²⁺ to pH_L is lost upon homogenization.

Such differential coupling of second messengers to the pH_Lash might be explained by local Ca²⁺ domains; i.e., a high local Ca²⁺ required for a pH_Lash might be readily attained by NAADP but less so by cADPR and IP₃. We therefore tested whether other Ca²⁺ signals differentially couple to the pH_Lash. First, release of Ca²⁺ from the ER was evoked by inhibiting SERCA (sarco-endoplasmic reticulum Ca²⁺ ATPase) with cyclopiazonic acid (CPA). In most cells, CPA stimulated a small, slow Ca²⁺ release (Fig. 5, A–C) that failed to evoke a pH_Lash (Fig. 5, D–F), even though a subsequent addition of ionomycin was successful (Fig. 5, D–F). This is consistent with the CPA-induced Ca²⁺ rise failing to reach a local Ca²⁺ threshold. Nonetheless, in ∼28% of cells, CPA produced a secondary peak of Ca²⁺ release after a long delay (285 ± 24 s; Fig. 5, A–C) that translated into a pH_Lash after 361 ± 15 s. Overall, CPA coupled weakly to a pH_Lash.

In contrast to CPA, photolysis of caged Ca²⁺ elicited a very rapid increase in Ca²⁺ whose amplitude was indistinguishable (P > 0.05) from that produced by sperm (Fig. 5, G–I). However, in spite of the globally similar Ca²⁺ signals, the photolysis of caged Ca²⁺ failed to produce a pH_Lash in 90% of eggs (only two eggs gave a pH_Lash; Fig. 5, J–L). This weak coupling provides strong evidence that it is the local and not global Ca²⁺ that is an important determinant of the coupling efficiency.

We then asked what mechanism couples IP₃/cADPR to a pH_Lash, with potential pathways depicted in Fig. 6 A. We first tested whether ER Ca²⁺ recruits the NAADP pathway by using three NAADP receptor antagonists. At concentrations that block NAADP itself, Ned-19, diltiazem, and SKF96365 also inhibit the pH_Lash response to both cADPR and IP₃ (Fig. 6, B and C). This places NAADP action downstream of ER Ca²⁺ because the inhibitors do not affect Ca²⁺ release evoked by IP₃ or cADPR (Fig. S2; Genazzani et al., 1997; Naylor et al., 2009), as confirmed by the weak effect of the inhibitors upon Ca²⁺-dependent exocytosis (fertilization envelope lifting; Fig. 6 D). We therefore exclude pathway 2 (Fig. 6 A).

Because IP₃Rs and RyRs are on the ER, Ca²⁺ must diffuse from the ER to target sites that affect acidic stores, and this should be blocked by EGTA. Accordingly, EGTA ablated the pH_Lash response to either cADPR or IP₃ (Fig. 6, B and C). This contrasted with the robust pH_L response to NAADP in the presence of EGTA (Fig. 6, B and C; Morgan and Galione, 2007b). The data suggest that IP₃ and cADPR stimulate acidic stores after Ca²⁺ diffusion from the ER to target domains (Fig. 6 A, pathway 1 or 3).

Interestingly, EGTA did slightly modify the response to NAADP, reducing the amplitude (Fig. 5, B and C) and slowing the upstroke kinetics (time to peak [s]: Ctrl, 6 ± 0; EGTA, 28 ± 3; P < 0.001). The fact that EGTA (a slow Ca²⁺ buffer) strongly inhibited the IP₃/cADPR pH_Lash but was weaker toward the NAADP pH_Lash was further evidence of local Ca²⁺ domains around acidic vesicles facilitating the pH_Lash. As a final confirmation, we tested the fast Ca²⁺ buffer, BAPTA, which is able to dissipate Ca²⁺ gradients in microdomains (Kidd et al., 1999; G. Brailoiu et al., 2009). The NAADP-induced pH_Lash was almost completely abolished by BAPTA (Fig. 6, B and C). We conclude that Ca²⁺ release by NAADP evokes locally high Ca²⁺ concentrations around the acidic vesicles that are required for acidic vesicle activation (alkalinization). Together the data support the main highlighted pathway 3 in Fig. 6 A in which IP₃- or cADPR-induced Ca²⁺ release from the ER activates acidic vesicles via the Ca²⁺-sensitive NAADP pathway.

To complement NAADP inhibition, we examined the effect of NAADP receptor desensitization. Microinjection of NAADP into the egg demonstrably desensitized both the Ca²⁺ and pH_Lash responses to a second NAADP injection (Fig. 7, Ai and Ci); crucially, NAADP desensitization inhibited the IP₃-induced pH_Lash (Fig. 7, Ciii) without affecting IP₃-induced Ca²⁺ release (Fig. 7, Aiii). Thus, desensitization uncoupled IP₃-induced Ca²⁺ release from the pH_Lash and mimicked the action of NAADP antagonism.

Conversely, if IP₃ recruits NAADP receptors, then injection of IP₃ first should cross-desensitize them. We found that both the Ca²⁺ (Fig. 7, Aiv) and pH_Lash (Fig. 7, Civ) responses to NAADP were reduced by prior IP₃ injection, consistent with heterologous desensitization of the NAADP receptors.

Unfortunately, the analogous cross-desensitization of the cADPR pH_Lash response was not technically possible because when NAADP was injected first, the subsequent cADPR-induced Ca²⁺ release was profoundly cross-desensitized (Fig. S3), probably reflecting the secondary recruitment of the cADPR pathway by NAADP (Churchill and Galione, 2000) and its consequent, persistent desensitization (Thomas et al., 2002). Nonetheless, the data overall support our model of bidirectional communication.

The corollary of such cross talk is that ER Ca²⁺ is important physiologically for facilitating acidic store activation via NAADP. We first tested whether cytosolic Ca²⁺ was important for the pH_Lash by microinjecting eggs with EGTA. After verifying that EGTA blocked the Ca²⁺ response at fertilization (Fig. 8 A), we showed that it did indeed inhibit the pH_Lash (when measured throughout the entire periphery; Fig. 8 B). However, when analyzed more closely, some eggs injected with EGTA showed a highly localized, nonpropagating pH_L increase (Fig. 8, C and D). Moreover, this “hot spot” coincided with the point of sperm contact (Fig. 8 C, inset) and may correspond to the bolus of NAADP delivered to the egg by sperm (see Discussion; Churchill et al., 2003). An additional hot spot of pH_L was observed in another part of the egg periphery (presumably due to polyspermy when fertilization envelope lifting is inhibited by EGTA). This suggests that Ca²⁺ is physiologically important for propagating acidic store activation, apparently by amplifying the initial sperm-induced trigger.

We then tested whether this facilitating Ca²⁺ was released from the ER by IP₃/cADPR. Consequently, fertilization was effected in eggs that had been microinjected with a cocktail of IP₃ and cADPR antagonists (heparin and 8-NH₂-cADPR, respectively; Churchill and Galione, 2000) or with an injection marker alone. In the absence of inhibitors, the pH_Lash proceeded as a wave away from the point of sperm entry (Morgan and Galione, 2007a) to be subsequently mirrored in the antipode (Fig. 3, Di; Morgan and Galione, 2007a). One would predict that the effect of blocking ER Ca²⁺ release would be similar to microinjecting EGTA and, qualitatively, this is what we observed: a nonpropagating, local pH_L response remained at one pole of the cell (Fig. 8, E and F) that was smaller in amplitude (Fig. 8 G) and slower (Fig. 8 H) than the initiation site response in control eggs. These data are consistent with ER Ca²⁺ release amplifying acidic store activation during fertilization.

These functional data imply a close physical apposition of acidic Ca²⁺ stores and the ER that was borne out by examining the cellular architecture. In the cortex where the pH_Lash was observed, acidic vesicles and ER were densely packed and, irrespective of the slice depth (1–22 µm), were consistently closely apposed (Fig. 9). Indeed, at this spatial resolution, the vast majority of vesicles were juxtaposed to ER cisternae (Fig. 9, inset), possibly reflecting vesicle tethering in order to maintain acidic vesicle–ER junctions (Kilpatrick et al., 2012; Patel and Brailoiu, 2012).

The current anterograde model of NAADP signaling describes acidic Ca²⁺ stores as the providers of local, trigger Ca²⁺, which is amplified by Ca²⁺-sensitive ER Ca²⁺ channels, IP₃Rs, or RyRs (channel chatter; Patel et al., 2001; G. Brailoiu et al., 2009; Morgan et al., 2011). Indeed, in the sea urchin egg, NAADP couples to either channel family (Churchill and Galione, 2000, 2001), hinting at a close apposition of all three channels that is made possible by the structural intimacy of the ER and acidic vesicles observed by either light (Davis et al., 2008; Morgan, 2011) or electron microscopy (Sardet, 1984; Poenie and Epel, 1987; Henson et al., 1989; Fishkind et al., 1990; McPherson et al., 1992). With such a functional triad, we wondered whether channel chatter is a two-way conversation with the ER signaling in a retrograde manner back to the acidic vesicle.

Therefore, we used pH_L as a marker of acidic store activation by NAADP. By uncaging NAADP, we verified that pH_L responds over an appropriate, quasi-linear concentration range and is rapid, spatially sensitive and is not distorted by diffusion. Importantly, NAADP is unique in directly activating acidic vesicles because the alkalinization in egg homogenate cannot be recapitulated by ER Ca²⁺ release agents (IP₃, cADPR, ionomycin, or SERCA inhibition; Morgan and Galione, 2007b). Finally, we verify that fertilization-induced pH_L changes are primarily dependent upon the NAADP pathway (Morgan and Galione, 2007a) judging by the effect of four NAADP inhibitors. pH_L therefore bears all the requisite hallmarks of an NAADP/acidic store reporter.

Our primary hypothesis was that Ca²⁺ release from the ER resulted in acidic vesicle activation, and it was supported by several lines of evidence. First, the Ca²⁺ and pH_L responses mirror each other in space (see the focal photolysis of caged NAADP, sperm-induced waves, or uniform ionomycin-induced responses). Second, Ca²⁺ precedes pH_L with sperm (Morgan and Galione, 2007a) or ionomycin. Third, releasing Ca²⁺ from the ER (with ionomycin, CPA, IP₃, or cADPR) can drive a pH_Lash. Fourth, inhibiting a rise of Ca²⁺ (with Ca²⁺ buffers or intracellular channel antagonists) abrogates the pH_Lash.

In terms of the underlying pathway, we conclude that NAADP is an obligate component because the pH_Lash was blocked by NAADP antagonism or desensitization, irrespective of the ER stimulus (ionophore, second messenger, fertilization). We are confident that the antagonist pharmacology is reliable because: (1) five structurally unrelated inhibitors inhibit the pH_Lash; (2) these inhibitors are selective for the pH_Lash over the NAADP-independent central acidification; (3) they are weak inhibitors of Ca²⁺ release evoked by IP₃ or cADPR (Genazzani et al., 1997; Mándi et al., 2006; Zhang and Li, 2007; Naylor et al., 2009) or Ca²⁺-dependent exocytosis of the fertilization envelope; (4) NAADP desensitization mimicked NAADP antagonism; and (5) pH_Lash blockade is independent of effects upon plasma membrane Ca²⁺ channels or basal pH_L. But how does ER Ca²⁺ activate acidic stores? We propose that there are two Ca²⁺-dependent processes that can contribute to the pH_Lash—one drives NAADP synthesis, the second facilitates NAADP receptor (TPC) activation—and we shall discuss the evidence.

The most direct evidence for Ca²⁺-dependent NAADP synthesis was that ionomycin stimulated NAADP levels in egg populations and, correspondingly, the ionomycin-evoked pH_Lash was kinetically sluggish (consistent with the time to generate NAADP) and sensitive to NAADP antagonism. Unfortunately, we are prevented from directly testing the importance of Ca²⁺ during fertilization because L. pictus eggs possess low esterase activity, which precludes EGTA/AM-loading in the NAADP radioreceptor assay (Morgan and Galione, 2008). Ca²⁺-dependent messenger production has precedents in IP₃ (Thaler et al., 2004), cADPR (Kim et al., 2008; Shawl et al., 2009), and cAMP (Cooper, 2003) and is an emerging theme for NAADP too, having been postulated in sperm (Vasudevan et al., 2010). At present we cannot say whether Ca²⁺ is the sole stimulus of NAADP synthesis at fertilization, but it suggests that Ca²⁺ can be a component.

Nevertheless, NAADP synthesis cannot readily explain all aspects of the Ca²⁺-dependent activation of acidic vesicles. For instance, the slow kinetics (≤70 s) of the ionomycin-induced pH_Lash are credibly consistent with synthesis, but the more rapid effects of IP₃ or cADPR (≤4 s) are more difficult to rationalize in such terms (unless exquisitely coupled). We therefore invoke a direct action of Ca²⁺ upon the acidic stores themselves.

First, for acidic vesicle activation, NAADP exhibited a co-requirement for Ca²⁺ because the pH_Lash was sensitive to Ca²⁺ chelators, EGTA and BAPTA. Moreover, the degree of inhibition was commensurate with the kinetics of Ca²⁺ buffering (slow EGTA was weaker than was fast BAPTA). With ∼2.5 mM chelator in the cytosol, one can calculate (Stern, 1992; Dargan and Parker, 2003 [assuming a 200 µm²/s Ca²⁺ diffusion coefficient appropriate for high concentrations of Ca²⁺ around a channel; Allbritton et al., 1992; Naraghi and Neher, 1997]) that the maximum range of Ca²⁺ action after release from a channel would be ∼300 nm in the presence of EGTA but ∼10 nm in the presence of BAPTA. In other words, the Ca²⁺ released by NAADP must be acting locally in microdomains 10–300 nm from the TPC to facilitate the pH_Lash (compare microdomains in other systems; G. Brailoiu et al., 2009). Given that Ca²⁺ channel complexes span 15–28 nm in diameter (Samsó and Wagenknecht, 1998; Wolf et al., 2003; Taylor et al., 2004), we propose that Ca²⁺ feedback reinforces TPC activation at the intra- as well as intermolecular level.

It is this Ca²⁺ sensitivity of the NAADP system that may partly explain the trans-stimulation of acidic stores by Ca²⁺ from the ER. The NAADP-induced pH_Lash was robust in the presence of EGTA, indicating that TPC activation remained detectable. Nonetheless, the pH_Lash evoked by IP₃ or cADPR was completely blocked by EGTA pointing to the coupling mechanism being affected. The effect of EGTA is unlikely to be due to simply blocking Ca²⁺ release at IP₃Rs/RyRs because local CICR and ER channel opening persist in the presence of the slow chelator (Dargan and Parker, 2003; Dargan et al., 2004; Shuai and Parker, 2005; Smith and Parker, 2009). We conclude that EGTA prevents a pH_Lash by buffering the ER Ca²⁺ within the junction before it reaches target sites on adjacent vesicles.

Our staining of the ER and acidic vesicles revealed a close proximity, and electron micrographs of the sea urchin egg cortex confirm the close (≤300 nm) apposition of acidic vesicles and ER elements (Sardet, 1984; Poenie and Epel, 1987; Henson et al., 1989; Fishkind et al., 1990; McPherson et al., 1992). Nevertheless, our functional data suggest that the resident IP₃Rs and RyRs, though close, must be farther than 300 nm from the Ca²⁺-sensitive targets in order to be EGTA sensitive. This can be rationalized if the channels are slightly along the ER branches and RyR immunogold labeling is consistent with such distances (McPherson et al., 1992), but comparable data for IP₃Rs are not available. A preferential closeness of RyRs and TPCs is feasible because RyRs (McPherson et al., 1992) and TPCs (Ruas et al., 2010) are relatively denser in the cortical (i.e., pH_Lash) region compared with IP₃Rs (Parys et al., 1994). In addition, the larger Ca²⁺ conductance of RyRs compared with IP₃Rs (Pitt et al., 2010) might also contribute to preferential coupling. Such communication between ER and acidic vesicles is lost upon homogenization (Morgan and Galione, 2007b), conceivably because it destroys the native apposition of the two organelles.

Given the inhibition by NAADP antagonism/desensitization, ER Ca²⁺ appears to stimulate TPCs on the acidic vesicles. Considering that cytosolic Ca²⁺ effects at the NAADP receptor have hitherto been discounted (Chini and Dousa, 1996; Genazzani and Galione, 1996; Bak et al., 1999; Mándi et al., 2006), such a Ca²⁺ stimulation may instead occur at the luminal face of NAADP-regulated channels (after Ca²⁺ uptake): high luminal [Ca²⁺] appears to enhance the conductance of sea urchin NAADP receptors (Morgan et al., 2012), TPC1 (Rybalchenko et al., 2012) and TPC2 (Pitt et al., 2010), analogous to the luminal regulation of IP₃Rs and RyRs (Burdakov et al., 2005). Ca²⁺ transfer from ER to acidic vesicles would be reminiscent of bidirectional communication between other inter-organelle partnerships such as ER/mitochondria (Arnaudeau et al., 2001; Rizzuto and Pozzan, 2006).

Is retrograde signaling from ER to acidic vesicle physiological? NAADP antagonism confirmed that the fertilization-induced pH_Lash is driven primarily by NAADP (Morgan and Galione, 2007a), but the fact that Ca²⁺ chelation and ER channel blockers also inhibit the pH_Lash at fertilization points to an additional role of cADPR/IP₃. In particular, this blockade of the ER “balkanizes” the pH_Lash around the sperm entry site and is consistent with the retrograde signal amplifying acidic store activation.

What might be the order of events at fertilization? When a sperm contacts the egg jelly, NAADP first rapidly increases within the sperm head (Churchill et al., 2003) to drive the acrosome reaction (Vasudevan et al., 2010) and, incidentally, to provide a preformed bolus of NAADP that can be delivered to the egg upon fusion (Churchill et al., 2003). In the absence of egg amplification (i.e., plus EGTA or ER channel blockade), the early focal pH_L change around the sperm entry point is certainly consistent with a visualization of this NAADP delivery.

Because the volume of the sperm is ∼6 orders of magnitude smaller than the egg’s, dilution of the bolus in the vast egg cytosol necessitates further NAADP production within the egg. We therefore propose that the observed slow second phase of NAADP synthesis (Churchill et al., 2003) can be driven by an increase in Ca²⁺ that is dependent upon cADPR/IP₃; this is analogous to waves of IP₃ synthesis stimulated by Ca²⁺ in sea urchin eggs (Kuroda et al., 2001; Thaler et al., 2004).

If cADPR and IP₃ provide ER Ca²⁺ to facilitate the NAADP pathway, then these ER messengers should precede the second (egg) phase of NAADP synthesis (Churchill et al., 2003); their involvement in the initiation and propagation of the fertilization Ca²⁺ wave (Galione et al., 1993; Lee et al., 1993; Davis et al., 2008) and the kinetics of cADPR or IP₃ production (Kuroda et al., 2001; Leckie et al., 2003; Thaler et al., 2004) favor this idea.

In addition to this novel retrograde signaling mode, conventional anterograde signals from the acidic vesicles to the ER do occur in the egg: NAADP-induced Ca²⁺ release recruits cADPR and IP₃, as expected (Churchill and Galione, 2000). Hence, we envisage mutually supportive Ca²⁺ feedback between acidic vesicles and ER as the Ca²⁺ wave front propagates across the egg.

Our current work expands the channel chatter model into a two-way conversation between ER and acidic Ca²⁺ stores. Reports of NAADP acting downstream of IP₃ or cADPR are few and have not detailed a mechanism (e.g., in ascidian oocytes [Albrieux et al., 1998] and T-lymphocytes [Berg et al., 2000]), but the implications may be far reaching: we do not understand how NAADP is choreographed during Ca²⁺ oscillations and waves in mammalian cells, in spite of its importance (Cancela et al., 1999). Our model provides a reinforcement loop whereby consecutive rounds of acidic vesicle-to-ER and ER-to-acidic vesicle Ca²⁺ feedback occur during oscillations, e.g., during the pacemaker rise of the next spike or at the Ca²⁺ wave front (Fig. S4). Local ER/acidic vesicle communication has also been highlighted recently in mammalian cells (Kilpatrick et al., 2012; Sanjurjo et al., 2012). That the ER can signal to acidic vesicles may also have far-reaching implications for endo-lysosomal functions such as resident enzyme activity, autophagy, and the pathology of diseases that affect lysosomal Ca²⁺ fluxes (Lloyd-Evans et al., 2008; Morgan et al., 2011; Coen et al., 2012).

Sea urchin eggs from Lytechinus pictus were harvested by intracoelomic injection of 0.5 M KCl and collected in artificial sea water (ASW [mM]: 435 NaCl, 40 MgCl₂, 15 MgSO₄, 11 CaCl₂, 10 KCl, 2.5 NaHCO₃, and 20 Tris, pH 8.0), and de-jellied by passage through 100-µm nylon mesh (EMD Millipore). Sperm, on the other hand, were collected “dry” and maintained at 4°C until use.

Eggs were maintained at room temperature in ASW and imaged on glass poly-d-lysine–coated coverslips mounted on a confocal laser scanning microscope (LSM 510 Meta; Carl Zeiss); an Axiovert 200M (Carl Zeiss) equipped with Zeiss objectives (10× Neofluar, NA 0.3; 40× Fluar, NA 1.3) was controlled by LSM software (Carl Zeiss). Excitation/emission (nm) wavelengths per channel were 351/>385 (UV), 488/505–530 (green), 543/>560 (red), 633/645–719 (far-red). When inhibitors were tested at fertilization, eggs were preincubated with the inhibitors (or vehicle) but sperm were preactivated in ASW without inhibitors for 20–30 s before their addition to eggs in order to minimize drug effects upon sperm. Images were analyzed using custom-written Magipix software (R. Jacob, King’s College London, London, England, UK).

pH_L was usually monitored ratiometrically in eggs co-loaded with 10 µM Acridine orange and 1 µM LysoTracker red DND-99 for 15–20 min at room temperature and imaged using green/red channels, respectively, as described previously (Morgan and Galione, 2007a). Data are expressed as the ratio of the Acridine orange/LysoTracker red signals with an increase in the ratio reflecting an increase in pH_L.

In pH_L experiments where different second messengers were consecutively injected, eggs were labeled with 10 µM Acridine orange only (10–30 min) and its fluorescence monitored simultaneously with that of the injection marker (see following section). Because of the slight change of cell shape elicited by the first injection (due to fertilization envelope lifting), the peripheral region of interest was always redefined to monitor the fluorescence response to the second injection.

Cytosolic Ca²⁺ was measured in two modes: ratiometric recording involved coinjecting eggs with 10 kD dextran conjugates of fluo-4 (Ca²⁺-sensitive) and Alexa Fluor 647 (Ca²⁺-insensitive) at pipette concentrations of 1 mM and 250 µM, respectively (data are expressed as the green/far-red ratio); alternatively, single wavelength recording used either fluo-4 dextran (green channel) or rhod-dextran (high affinity form, red channel) only.

Caged NAADP was synthesized in-house using sequential reactions (Lee et al., 1997; Morgan et al., 2006). In brief, 2-nitroacetophenone hydrazone was synthesized from 2-nitroacetophenone and hydrazine monohydrate under acidic conditions; the chloroform-extracted hydrazone was then converted to 1(2-nitrophenyl)diazoethane using MnO₂ and finally incubated with NAADP under acidic conditions to cage the phosphate groups. The caged product was purified by HPLC and stored at −80°C. After treatment with alkaline phosphatase beads (to remove contaminating free NAADP), caged NAADP was then microinjected into eggs (50-µM pipette concentration) together with 1 mM fluo-4 dextran to measure Ca²⁺ or with 50–200 µM Alexa Fluor 647 dextran as an injection marker for pH_L recordings. Photolysis was effected with a Coherent Enterprise UV laser (351 nm) and exposure was either global (70% power, 5 frames at 1 Hz) or focal (10–70% power, 2 iterations) as controlled by an acousto-optical tunable filter.

Caged Ca²⁺ (NP-EGTA) at 25 mM in 10 mM Hepes (pH 7) was coinjected with either fluo-4 dextran or Alexa Fluor 647 dextran. Focal photolysis was effected by UV laser (351- and 364-nm lines, 20 iterations at 50–75% power).

We exploited the intrinsic fluorescence of Ned-19 to monitor its loading into intact eggs (excitation at 364 nm; emission >385 nm). To minimize photobleaching, images were captured discontinuously after known times of Ned-19 preincubation and all with identical acquisition settings. We also confirmed that the fluorescence of Ned-19 did not interfere with Acridine orange fluorescence in vitro: 10 µM Acridine orange fluorescence was recorded in a fluorimeter (excitation 488 nm, emission 526 nm; model LS-50B, PerkinElmer) in a medium containing (mM): 250 potassium gluconate, 250 N-methylglucamine, 20 Hepes, and 1 MgCl₂, pH 5.5. In the presence of 100 and 200 µM Ned-19, the Acridine orange signal was 106 ± 2 and 109 ± 3% of that in the absence of Ned-19, respectively (n = 3, P > 0.1).

Labeling of ER and acidic vesicles was performed as described previously (Terasaki and Jaffe, 1991; Davis et al., 2008). In summary, a saturated solution of DiI (DiIC₁₈(3)) was prepared by vortexing a few grains of DiI in 200 µl of soybean oil and was microinjected into the egg center. The DiI diffused from the central oil droplet into the contiguous membrane system of the ER in 15–30 min, during which time acidic vesicles were labeled by addition of 1 µM LysoTracker green DND-26 (Invitrogen). Confocal 1-µm optical sections were collected using the standard green and red channel settings.

For the NAADP assay, NAADP and [³²P]NAADP were enzymatically synthesized in-house using the base-exchange reaction of Aplysia ADP-ribosyl cyclase (ARC; Lee et al., 1997; Morgan et al., 2006; Vasudevan et al., 2010). For NAADP, 13 mM NADP and 100 mM nicotinic acid were incubated at pH 4.5 with ARC for 1 h at room temperature and NAADP purified by HPLC. Two stages were required for [³²P]NAADP: first, [³²P]NAD was phosphorylated to [³²P]NADP using human NAD kinase and 10 mM ATP; second, the product was converted to [³²P]NAADP by incubating with 100 mM nicotinic acid and ARC at pH 4.5 for 1 h at room temperature, and purified by HPLC.

The time course of NAADP changes in populations of eggs stimulated with sperm or ionomycin was determined biochemically. For a given experiment, 1 ml of eggs was diluted into 20 ml of ASW. A 2-ml aliquot was taken from this solution at each time point and centrifuged at 9,000 g. Centrifugation was then stopped as swiftly as possible, and the supernatant discarded. 100 µl HClO₄ was then added. To disrupt the cells, sonication was performed (Jencons Vibra-Cell at amplitude 60) for three bursts of 5 s. The time point was taken as the start of sonication and samples were then placed on ice. The denatured protein was pelleted by centrifugation at 9,000 g for 10 min and stored at −80°C for later analysis. The supernatant was neutralized with an equal volume of 2 M KHCO₃ and vortexed. Centrifugation at 9,000 g for 10 min was again used to remove the KClO₄ precipitate. The resulting supernatant was stored at −80°C for NAADP analysis. The protein concentration in the precipitated pellet was determined using the BCA reagent.

As reported in detail previously (Lewis et al., 2007), NAADP levels were determined using the NAADP-binding protein from sea urchin (L. pictus) egg homogenate, which is highly selective for NAADP (Churamani et al., 2004; Lewis et al., 2007). First, we added 25 µl of test sample to each tube and then added 125 µl of 1% (vol/vol) sea urchin egg homogenate in intracellular medium and incubated the reaction for 10 min at 25°C. To each tube we then added 0.2 nM of [³²P]NAADP (∼50,000 cpm) diluted in 100 µl of intracellular medium (250 mM N-methyl-d-glucamine, 250 mM potassium gluconate, 1 mM MgCl₂, and 20 mM Hepes, pH 7.2) and incubated the reaction for 10 min at 25°C. Bound NAADP was then trapped onto Whatman GF/B filter papers using a Brandel cell harvester. We washed the filters three times with 1 ml of a buffer containing 20 mM Hepes and 500 mM potassium acetate, pH 7.4, and the bound radioactivity was estimated by phosphorimaging. The amount of NAADP in each test sample was determined by comparison with a standard curve containing known amounts of NAADP. Results are normalized to the protein content (pmol NAADP/mg protein).

Fluorescence traces in all figures are from single cells representative of n eggs from ≥3 preparations unless indicated otherwise. Data are expressed as the mean ± SEM. Two datasets were compared using Student’s t test, whereas multiple groups were analyzed using ANOVA and a Tukey-Kramer or Dunnett’s post-test. A nonparametric ANOVA (Kruskal-Wallis and Dunn post-test) was applied when required. Data were paired where appropriate and significance assumed at P < 0.05.

Caged NAADP, free NAADP, and [³²P]NAADP were synthesized in-house. [³²P]β-NAD⁺ was obtained from GE Healthcare. IP₃ was from LC Laboratories. Cyclic ADP-ribose, NAADP, IP₃, PPADS (pyridoxalphosphate-6-azophenyl-2′4′-disulfonic acid), EGTA, BAPTA, heparin (low MW), 8-NH₂-cADPR, diltiazem, nifedipine, cyclopiazonic acid, ADP-ribosyl cyclase, and soybean oil were obtained from Sigma-Aldrich. Ned-19 (mixed isomers) was from IBScreen (Moscow, Russia). Acridine orange, LysoTracker red DND-99, rhod-dextran (10 kD, high affinity form), Fluo-4 dextran (10 kD), Alexa Fluor 647 dextran (10 kD), NP-EGTA (potassium salt), and DiIC₁₈(3) were from Invitrogen. Ionomycin and SKF96365 were from EMD Millipore. NAD kinase was a kind gift from M. Ziegler (University of Bergen, Bergen, Norway). All other reagents were of analytical grade.

Fig. S1 shows the regenerative propagation of the pH_L response away from the focal photolysis region in a minority of eggs and compares such real responses with theoretical models. Fig. S2 compares the effect of NAADP antagonists upon Ca²⁺ release in egg homogenate evoked by NAADP, cADPR, and IP₃; the effect of removing extracellular Ca²⁺ on the pH_Lash in intact eggs is also shown. Fig. S3 shows that cADPR-induced Ca²⁺ release in intact eggs is desensitized by prior injection of NAADP. Fig. S4 is a scheme summarizing how Ca²⁺ from the ER may stimulate the NAADP pathway in eggs and mammalian cells.

We thank Clive Garnham for technical assistance, Margarida Ruas for invaluable suggestions, and the Wellcome Trust for financial support.

There are no conflicts of interest.