Widespread Distribution of Binding Sites for the Novel Ca2+-mobilizing Messenger, Nicotinic Acid Adenine Dinucleotide Phosphate, in the Brain*

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+-mobilizing agent in invertebrate eggs that has recently been shown to be active in certain mammalian and plant systems. Little, however, is known concerning the properties of putative NAADP receptors. Here, for the first time, we report binding sites for NAADP in brain. In contrast to sea urchin egg homogenates, [32P]NAADP bound reversibly to multiple sites in brain membranes. The rank order of potency of NAADP, 2′,3′-cyclic NAADP and 3′-NAADP in displacing [32P]NAADP was, however, the same in the two systems and in agreement with their ability to mobilize Ca2+ from homogenates. These data indicate that [32P]NAADP likely binds to receptors mediating Ca2+ mobilization. Autoradiography revealed striking heterogeneity in the distribution of [32P]NAADP binding sites throughout the brain. Our data strongly support a role for NAADP-induced Ca2+ signaling in the brain.

Increases in cytosolic [Ca 2ϩ ] can show both temporal and spatial inhomogeneity giving rise to the phenomena of Ca 2ϩ waves and oscillations (1,2). In non-excitable cells, a variety of extracellular stimuli mediate changes in cytosolic [Ca 2ϩ ] through the mobilization of intracellular Ca 2ϩ stores (3). This is achieved through the concerted activation of a family of related intracellular Ca 2ϩ channels/receptor complexes for inositol 1,4,5-trisphosphate (IP 3 ) 1 (4,5) and ryanodine (6). The latter are thought to be activated and/or modulated by cyclic ADP-ribose (cADPR) (7,8). Depletion of intracellular Ca 2ϩ stores activates Ca 2ϩ entry from the extracellular space that serves to refill them, thereby sustaining the Ca 2ϩ signal (9). Conversely, in excitable cells such as neurons, Ca 2ϩ increases are mediated primarily through voltage-and ligand-gated Ca 2ϩ channels located on the plasma membrane. However, accumulating evidence also implicates a role for intracellular Ca 2ϩ stores in neuronal Ca 2ϩ homeostasis (10,11). Thus, in all cells, interplay between mobilization of stored Ca 2ϩ and Ca 2ϩ influx across the plasma membrane is likely important in shaping cytosolic Ca 2ϩ signals.
Recently, a novel Ca 2ϩ -mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP) has been described in sea urchin eggs (7), which, based on cross-desensitization experiments (12) and distinct pharmacology (13), is thought to release Ca 2ϩ independently of IP 3 or ryanodine receptor activation. NAADP-induced Ca 2ϩ mobilization in the sea urchin egg is unique in several respects. First, sub-threshold concentrations of NAADP completely desensitize homogenates to subsequent challenge with maximal concentrations of NAADP that normally evoke full Ca 2ϩ release (13,14). Secondly, NAADP releases Ca 2ϩ from a pool distinct from that mobilized by IP 3 and cADPR (12,15,16), and finally, NAADP-induced Ca 2ϩ mobilization is not regulated by Ca 2ϩ (16,17), a property that underlies regenerative Ca 2ϩ release via IP 3 and ryanodine receptors (1,2). A binding site for NAADP has been previously demonstrated in sea urchin egg microsomes (14); however, little is known concerning the molecular identity or distribution of putative NAADP receptors.
The actions of NAADP have now been extended to mammalian (18,19) and plant (20) preparations. In acinar cells of the pancreas, NAADP is thought to underlie complex Ca 2ϩ signals in response to the brain-gut peptide, cholecystokinin, by providing a "trigger" Ca 2ϩ release that is subsequently propagated by ryanodine and IP 3 receptors (18,21). NAADP can also mobilize Ca 2ϩ from crude brain microsomes (19) raising the possibility that NAADP signaling, like the phosphoinositide pathway (10,11), may be active in the brain. Indeed, NAADP metabolism has been characterized in brain preparations (19,22).
Here we have characterized binding sites for [ 32 P]NAADP in the brain. This is the first report of NAADP binding sites in a mammalian tissue. Our data support a general role for NAADP-mediated Ca 2ϩ signaling in the brain.

EXPERIMENTAL PROCEDURES
Synthesis of Radioligand-[ 32 P]NAADP synthesis described here is a variation of the procedure reported previously (14). [ 32 P]NADP was synthesized from [ 32 P]NAD (2 M; specific activity 1000 Ci/mmol; Amersham Pharmacia Biotech) by incubation with NAD kinase (50 units/ ml; Sigma) and 10 mM MgATP for 2 h at 37°C in a buffer containing 5 mM HEPES (pH 7.5). The remaining ATP and ADP was converted to AMP by incubation with apyrase (2 units/ml; Sigma) for 1 h at 37°C. The reaction was then diluted 5-fold into a medium containing 10 mM MES (pH 5.0), 19 mM nicotinic acid, and ADP-ribosyl cyclase (2 g/ml; Sigma) and incubated for another 2 h to convert [ 32 P]NADP to [ 32 P]NAADP by base exchange. The final mixture was separated by anion exchange high performance liquid chromatography on a 3 ϫ 150-mm column packed with AGMP1 (Bio-Rad). Elution was performed at a flow rate of 1 ml/min using a gradient of trifluoroacetic acid (TFA) that increased linearly from 0 -2% over the first 6 min, to 4% at 11 min, to 8% at 16 min, to 16% at 21 min, to 32% at 26 min, and to 100% (150 mM TFA) at 26.1 min. Fractions were collected every minute and neutralized by the addition of Tris base (final concentration 75 mM), and their radioactivity was determined by Cerenkov counting. A major peak was observed at 25.6 Ϯ 0.4 min (n ϭ 3) that corresponded to authentic * This work was supported in part by the Wellcome Trust (to S. P., G. C. C., and A. G.) and the Medical Research Council (to T. S.). 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.
Tissue Preparation-Whole brains from male CD1 mice or Harlan Sprague-Dawley rats were homogenized at 4°C using an UltraTurax homogenizer in binding medium composed of 20 mM HEPES (pH 7.2) and 1 mM EDTA supplemented with a protease inhibitor mixture (Roche Molecular Biochemicals). Membranes were washed twice by centrifugation (10 min; 20,000 ϫ g) and stored at Ϫ20°C until use. Collection of sea urchin (Lytechinus pictus) eggs and preparation of homogenates were performed as described previously (13).
Binding Assays-Binding of [ 32 P]NAADP (ϳ1 nM) to brain membranes (ϳ500 g/incubation) was performed in binding medium composed of 20 mM HEPES (pH 7.2) and 1 mM EDTA at 37°C. No significant degradation of NAADP (up to 1.5 h) could be detected under these experimental conditions. Binding of [ 32 P]NAADP (ϳ0.2 nM) to sea urchin homogenates (0.5% v/v; ϳ40 g/incubation) was determined at 20°C in a medium composed of 20 mM HEPES (pH 7.2), 250 mM potassium gluconate, 250 mM N-methyl-D-glucamine, and 1 mM MgCl 2 . Bound and free radioligand were separated by rapid filtration. Similar results were obtained with both mouse and rat brain tissue (data not shown).
Autoradiography-Male Harlan Sprague Dawley rats (340 -360 g) were killed by cervical dislocation, and brains were rapidly removed, frozen in isopentane, and stored at Ϫ70°C prior to sectioning. Cryostat sections (12 m) were thaw-mounted on to gelatin-coated slides and returned to Ϫ70°C for storage. Binding of [ 32 P]NAADP (1-2 nM) to the sections was performed for 2 h at 20°C in a medium composed of 20 mM HEPES (pH 7.2) and 1 mM EDTA. Equilibrated samples were then washed with HEPES buffer at 4°C (10 mM; pH 7.2), air dried, and apposed to Hyperfilm (Amersham Pharmacia Biotech) for 12-24 h at Ϫ80°C. Autoradiograms were analyzed using Scion Image (Scion Corporation, Fredrick, MD). Nonspecific binding of [ 32 P]NAADP (determined in the presence of 100 M unlabeled NAADP) to sections cut at the level of the frontal cortex (see Fig. 3A) and thalamus (data not shown) was virtually undetectable.
Other Methods-2Ј,3Ј-Cyclic NAADP and 3Ј-NAADP were synthesized and purified as described (23). Protein was determined using bicinchoninic acid (Sigma). Data are presented as means Ϯ standard error of the mean.

RESULTS AND DISCUSSION
Enzymatically prepared [ 32 P]NAADP bound to crude membrane preparations from brain (Fig. 1). Association of [ 32 P] NAADP followed monophasic kinetics with a first order association rate constant (k ϩ1 ) of 9.4 Ϯ 1 ϫ 10 7 M Ϫ1 min Ϫ1 (n ϭ 3; see Fig. 1A). Binding of [ 32 P]NAADP was specific for NAADP, because a 100,000-fold molar excess of the related nucleotides, NAD, nicotinic acid adenine dinucleotide (NAAD), and cADPR did not displace [ 32 P]NAADP (Fig. 1B). Binding of [ 32 P]NAADP was also insensitive to IP 3 (20 M) (Fig. 1B). These data indicate that [ 32 P]NAADP binds to a unique site distinct from that of known Ca 2ϩ -mobilizing messengers. Apparent (partial) displacement of the radioligand was observed with NADP (100 M). This effect, however, is likely to be due to NAADP contamination in commercial NADP preparations (14).
From competitive equilibrium displacement analysis (Fig. 2), the concentration of NAADP causing half-maximal displacement of the radioligand (IC 50 ) from brain membranes was 200 Ϯ 17 nM (n ϭ 11). In contrast, the IC 50 for sea urchin egg homogenates was 0.47 Ϯ 0.06 nM (n ϭ 12). Differences were also observed in the Hill coefficient (n H ) for binding in the two tissues; n H was 0.76 Ϯ 0.04 (n ϭ 11) and 1.05 Ϯ 0.02 (n ϭ 12) in brain and sea urchin eggs, respectively. Thus, [ 32 P]NAADP likely binds to a single site in the egg preparations (probably the inactivation site; see below), whereas in the brain, multiple binding sites for [ 32 P]NAADP may exist.
[ 32 P]NAADP binding to sea urchin egg microsomes has been reported to be essentially irreversible (14). This was confirmed in the present studies using crude homogenates (data not shown). This unusual property is likely to underlie the remarkable inactivation properties of NAADP-induced Ca 2ϩ release in these cells (13,14). In stark contrast, however, [ 32 P]NAADP binding to brain membranes was completely reversible (Fig.  1C). Thus, an excess of unlabeled NAADP (100 M) initiated dissociation of bound [ 32 P]NAADP with a first order dissociation rate constant (k Ϫ1 ) of 0.07 Ϯ 0.008 min Ϫ1 (n ϭ 3). The clear differences in the reversibility of NAADP binding in the brain and sea urchin egg likely explains the ϳ1000-fold difference in apparent affinity for NAADP (Fig. 2).
Autoradiographic analysis revealed that NAADP binding sites were distributed throughout the brain (Fig. 3). Strikingly, the localization of [ 32 P]NAADP binding sites was far from homogeneous. Binding of [ 32 P]NAADP was particularly high in the medulla (Fig. 3H), midbrain (Fig. 3G), and thalamus (Fig.  3E). Individually, these areas are known to be involved in a diverse range of functions and collectively, contain pathways involved in the processing of somatosensory information. Appreciable levels of binding were also detected in anterior corti-cal regions and globus pallidus (Fig. 3D). In contrast to inositol trisphosphate and ryanodine receptor distribution (24), NAADP binding was low in the striatum (Fig. 3C) and hippocampus (Fig. 3F). Furthermore, [ 32 P]NAADP binding levels in the cerebellum were moderate (Fig. 3H), whereas this region is particularly enriched in the other intracellular Ca 2ϩ release channels (24). Interestingly, [ 32 P]NAADP binding was apparent in both gray and white matter (e.g. corpus callosum; see Fig. 3C), possibly indicating the presence of the binding sites in both neuronal and non-neuronal cells. Whether the observed regional differences in [ 32 P]NAADP binding sites results from differences in expression levels and/or affinity of NAADP receptors remains to be established. Nevertheless, our data point to possible functional heterogeneity in NAADP signaling within the brain.
This study is the first to demonstrate specific binding sites for NAADP in a mammalian tissue. Remarkably, whereas the NAADP analogue specificity was similar in brain and sea urchin eggs, the reversibility of binding between the two systems was in stark contrast. These data indicate differential regulation and/or the existence of multiple NAADP receptor subtypes. Indeed, the apparent affinity for NAADP (and the analogues) were markedly different in brain and egg preparations, and displacement curves in the brain were somewhat shallow. Furthermore, we have demonstrated intriguing heterogeneity in the distribution of NAADP binding sites within the brain and found key differences between the localization of these sites and that of known intracellular Ca 2ϩ release channels. The present data, together with our recent demonstration of NAADP-induced Ca 2ϩ release from brain microsomes (19), and the ability of brain homogenates to both synthesize and degrade NAADP (19,22), strongly support a signaling role for NAADP in the brain.