Unique inactivation properties of NAADP-sensitive Ca2+ release.

Ca mobilization from intracellular stores constitutes an important mechanism for generating cytoplasmic Ca signals. Inositol trisphosphate (InsP) and ryanodine receptors are the two families of intracellular Ca release channels that have been identified, which may be regulated by separate intracellular messengers, InsP and cyclic adenosine 5′-diphosphate ribose, respectively. A third molecule, nicotinic acid adenine dinucleotide phosphate (NAADP), has recently been recognized as a potent Ca releasing agent in sea urchin eggs and microsomes. We now report that non-releasing concentrations of NAADP fully and irreversibly inactivate the NAADP-sensitive Ca release mechanism. This phenomenon occurred both in intact sea urchin eggs and in homogenates and is not shared by either InsP or cyclic adenosine 5′-diphosphate ribose. The novel properties of this Ca release mechanism, giving a one-shot Ca release, may be suited to irreversible cellular events.

ance with the suggestion that the NAADP-sensitive Ca 2ϩ store may reside in an intracellular compartment distinct from the InsP 3 -and cADPR-sensitive stores (6).
We now report that non-releasing concentrations of NAADP fully and irreversibly inactivate the NAADP-sensitive Ca 2ϩ release mechanism both in intact sea urchin eggs and in homogenates and that this property is not shared by either InsP 3 or cADPR. Moreover, NAADP mobilizes Ca 2ϩ by activating a Ca 2ϩ release channel in the microsomal membrane, which is selectively blocked by classical inhibitors of L-type voltagesensitive Ca 2ϩ channels. These properties suggest that this release mechanism might contribute to complex patterns of Ca 2ϩ signals widely observed in intracellular signaling.

MATERIALS AND METHODS
Collection of Sea Urchin Eggs-Eggs were obtained by stimulating ovulation of female Lytechinus pictus (Marinus, Inc., Long Beach, CA) with a intracoelomic injection of KCl. These were then washed twice in artificial sea water (435 mM NaCl, 40 mM MgCl 2 , 15 mM MgSO 4 , 11 mM CaCl 2 , 10 mM KCl, 2.5 mM NaHCO 3 , 1.0 mM EDTA, pH 8.0), and jelly removed by filtration through 90-m nylon mesh.
Ca 2ϩ Release Assays-Homogenates (2.5%) of unfertilized Lytechinus pictus eggs were prepared as described previously (11) and Ca 2ϩ loading was achieved by incubation at room temperature for 3 h in an intracellular medium consisting of 250 mM potassium gluconate, 250 mM Nmethylglucamine, 20 mM Hepes, pH 7.2,1 mM MgCl 2 , 0.5 mM ATP, 10 mM phosphocreatine, 10 units/ml creatine phosphokinase, 1 mg/ml oligomycin, 1 mg/ml antimycin, 1 mM sodium azide, 3 mM fluo-3. Free Ca 2ϩ concentration was measured by monitoring fluorescence intensity at excitation and emission wavelengths of 490 and 535 nm, respectively. Fluorimetry was performed at 17°C using 500 l of homogenate in a Perkin-Elmer LS-50B fluorimeter. Additions were made in a 5-l volume, and all chemicals were added in intracellular medium containing 10 M EGTA. Basal concentrations of Ca 2ϩ were typically between 100 and 150 nM. Sequestered Ca 2ϩ was determined by monitoring decrease in fluo-3 fluorescence during microsomal loading and by measuring Ca 2ϩ release in response to ionomycin (5 M) and was constant between experiments.
Imaging of Intracellular [Ca 2ϩ ] i in Eggs-Eggs were transferred to polylysine-coated glass coverslips and microinjected with fura-2, pentapotassium salt (2 mM in the pipette) in buffer consisting of 0.5 M KCl, 20 mM Pipes, pH 6.7, to a final cellular concentration of approximately 20 M. Injection volumes were estimated as approximately 1% of the egg volume. All experiments were performed at 22°C. Free cytosolic Ca 2ϩ concentration was determined by ratioing fluorescence intensities at excitation wavelengths of 340 and 380 nm, using an emission wavelength of 510 nm. Ratio images were obtained using a fluorimetric imaging system and IONVISION software supplied by Improvision Ltd, University of Warwick Science Park, Coventry, United Kingdom (UK). Standard CaCl 2 solutions were used to calibrate the system, and viscosity corrections were made (12). Materials-NAADP was from RBI (St. Albans, UK), fluo-3 and fura-2 were from Molecular Probes (Cambridge, UK), and BAY K8644 was from Calbiochem (Nottingham, UK). All other chemicals were from Sigma (Poole, UK).

RESULTS AND DISCUSSION
In the present study, NAADP potently released Ca 2ϩ in a dose-dependent manner from sea urchin homogenates (EC 50 approximately 32 nM) and triggered a Ca 2ϩ wave in the intact sea urchin egg ( Figs. 1 and 2A). Surprisingly, very low concentrations of NAADP, which evoked little or no Ca 2ϩ release, were found to fully inactivate the NAADP-sensitive Ca 2ϩ release mechanism both in the homogenate and the intact egg ( Fig. 1, A and C, and Fig. 2, A and B).
Within the intact egg NAADP (approximate cytoplasmic concentrations of 50 and 500 nM) released Ca 2ϩ in a biphasic manner ( Fig. 2A). The pattern of the Ca 2ϩ release was an * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed. FIG. 2. Pseudocolor images of Ca 2؉ levels, measured using Fura-2, in sea urchin eggs. A, injection of NAADP into sea urchin eggs resulted in a Ca 2ϩ release that spread in a wave form across the egg followed by a slower, smaller amplitude Ca 2ϩ release. Subsequent injection of a higher concentration of NAADP could not elicit substantial Ca 2ϩ release in the egg. B, lower concentrations (5 nM) of NAADP resulted in less Ca 2ϩ release, but these too prevented Ca 2ϩ release by a subsequent NAADP injection. C, InsP3 injections released Ca 2ϩ but this did not desensitise a subsequent Ca 2ϩ release response to higher concentrations of initial Ca 2ϩ peak, followed by a partial recovery, and a later, slower and smaller amplitude release as previously observed by others (6) (peak [Ca 2ϩ ] was 1897 Ϯ 268 nM, n ϭ 5). We were able to demonstrate the inactivation phenomenon of NAADPinduced Ca 2ϩ release in the whole egg by first injecting into the egg as little as 5 nM NAADP, which then prevented any further response of the egg to larger injections of NAADP. 5 nM NAADP, in itself produced only a small rise in Ca 2ϩ , as shown in Fig. 2B, but significantly reduced Ca 2ϩ release by a subsequent injection of 500 nM NAADP even after waiting at least 1500 s (267 Ϯ 29 nM, n ϭ 4). This persistent inactivation contrasted with the inactivation of InsP 3 and cADPR receptors, which underwent a transient desensitization (data not shown), but recovered within 30 min (Fig. 2C). In fact, InsP 3 (2 M) injections released Ca 2ϩ in the whole egg in a monophasic manner (1102 Ϯ 61 nM, n ϭ 3) but in contrast to NAADP, a higher concentration of InsP 3 (20 M) was still able to release maximal Ca 2ϩ at least 1500 s later (1982 Ϯ 163 nM, n ϭ 3). Like InsP 3 , preapplication of 1 M cADPR to the egg (giving 1094 Ϯ 307 nM Ca 2ϩ release, n ϭ 3) did not block the Ca 2ϩ response to 10 M cADPR at least 1500 s later (mean Ca 2ϩ 1845 Ϯ 78 nM, n ϭ 3; data not shown).
In the homogenate, the extent of the inactivation was both concentration (Fig. 1, A and C) and time-dependent (Fig. 3).
Cross-desensitization to InsP 3 and cADPR by NAADP (20 nM to 1 M) did not occur (Fig. 1B). Instead full inactivation occurred by pretreatment with 1-2 nM NAADP, but substantial inactivation still occurred with concentrations as low as 100 pM (Fig.  1, A and C). Within 60 s full inactivation occurred with a non-stimulating concentration of 5 nM NAADP, but took longer and was less extensive by pretreatment with lower concentrations (Fig. 3). Inactivation was also apparently irreversible, since the receptor did not resensitize after 12 h of exposure to 2 nM NAADP (data not shown), and after reconstituting purified microsomes prepared from desensitized homogenate in fresh buffer not containing NAADP (data not shown). In the homogenate, Ca 2ϩ release by InsP 3 and cADPR desensitized only after treatment with stimulating concentrations and in a manner paralleling the extent of channel activation (Fig. 1, D and E; see also Ref. 13).
The graded release of Ca 2ϩ induced by NAADP (Fig. 1C) suggests that release by this agent is quantal (14). Changes in luminal or cytosolic Ca 2ϩ concentrations or pool depletion have been proposed to explain this phenomenon for both InsP 3 R and RyRs (see Ref. 14 for a review). However, it seems unlikely that NAADP-induced Ca 2ϩ release relies on these mechanisms since it is terminated by desensitization and can occur at concentrations that are non-stimulating. Instead, it appears to be an intrinsic property of the receptor mechanism as has also been proposed for the InsP 3 R (13).
Various experiments suggest that the inactivation mechanism is independent of enzymatic activity. First, in Percoll gradient-purified microsomes (1,15), full desensitization at either non-stimulating or stimulating NAADP concentrations was identical to that seen in whole egg homogenates (data not shown), ruling out a requirement for cytosolic components. Second, the general kinase inhibitor staurosporine (10 M) had no effect on the extent of inactivation (data not shown) in the homogenate. Third, the release and inactivation were unaltered by performing the experiments at 4°C, consistent with NAADP regulating intracellular Ca 2ϩ fluxes via a channel mechanism.
The inactivation event was independent of small electrochemical or pH gradient changes across the membrane since pretreatment for 1 h with gramicidin, valinomycin, or nigericin (all at 1 M) did not significantly alter Ca 2ϩ release by NAADP or InsP 3 (data not shown; see also Ref. 16).
The nature of the NAADP Ca 2ϩ release mechanism is unknown but is clearly separate from the InsP 3 and cADPR mechanisms. Diltiazem, nifedipine, BAY K8644, and verapamil, classical modulators of L-type voltage-gated Ca 2ϩ channels (17), fully blocked maximal Ca 2ϩ release by NAADP (Fig. 4) but not that by cADPR or InsP 3 (Fig. 4) and did not alter the NAADP-induced inactivation phenomenon (data not shown). The potent N-type voltage-gated Ca 2ϩ channel blocker -cono- toxin (17) was without any effect (Fig. 4).
The desensitization of the NAADP receptor shares similarities with neuronal nicotinic receptors (18). However, nicotinic receptor desensitization by non-stimulating concentrations of agonists is never complete; it is also reversible and may involve phosphorylation. None of these features are shown by the NAADP-sensitive mechanism.
Recent reports have indicated that mammalian cells possess the metabolic machinery to use NAADP as an intracellular messenger. First, NAADP is generated and degraded in various rat tissues, including brain, liver, and spleen (19). Second, it has been shown that ADP-ribosyl cyclase, the enzyme responsible for the cyclization of NAD ϩ to cADPR, and CD38, a lymphocyte differentiation antigen, can also synthesize NAADP (20). Ca 2ϩ mobilizing effects of NAADP have not yet been reported for other cell types, but an agonist-stimulated Ca 2ϩ release pathway that is blocked by nifedipine and diltiazem has been described in neutrophils (21) and low affinity binding sites for L-type Ca 2ϩ -channel blockers are present in cardiac sarcoplasmic reticulum (22).
Multiple Ca 2ϩ release mechanisms may contribute to complex patterns of Ca 2ϩ signals widely observed during intracellular signaling (23). The characteristics of NAADP Ca 2ϩ release described here suggest that the receptor may function as an irreversible biochemical switch activated in a one-off manner by a rapid surge in intracellular NAADP concentrations. Such a response may be suited to irreversible events such as fertilization, as recently suggested (7), cell division or cell death.