Nicotinic Acid Adenine Dinucleotide Phosphate Triggers Ca2+ Release from Brain Microsomes Judit Bak*, Peter White†, György Timár*, Ludwig Missiaen‡, Armando A
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Brief Communication 751 Nicotinic acid adenine dinucleotide phosphate triggers Ca2+ release from brain microsomes Judit Bak*, Peter White†, György Timár*, Ludwig Missiaen‡, Armando A. Genazzani†§ and Antony Galione† Mobilization of Ca2+ from intracellular stores is an To test whether brain microsomes also possessed an important mechanism for generating cytoplasmic Ca2+ NAADP-sensitive Ca2+-release mechanism, they were signals [1]. Two families of intracellular Ca2+-release prepared from the cerebrum and passively loaded with 45 2+ µ 2+ channels — the inositol-1,4,5-trisphosphate (IP3) Ca . NAADP (3 M) induced Ca release from micro- receptors and the ryanodine receptors (RyRs) — have somes in the presence of 100 nM free Ca2+ in the extra- been described in mammalian tissues [2]. Recently, microsomal buffer (Figure 1a). Under the same conditions, µ µ nicotinic acid adenine dinucleotide phosphate (NAADP), maximal concentrations of IP3 (5 M) or cADPR (2 M) a molecule derived from NADP+, has been shown to also enhanced 45Ca2+ release from the vesicles (Figure 1a), trigger Ca2+ release from intracellular stores in as did Ca2+ itself. Microsomes were unable to release Ca2+ invertebrate eggs [3–6] and pancreatic acinar cells [7]. in the presence of the quench solution (see Materials and The nature of NAADP-induced Ca2+ release is unknown methods), demonstrating the specificity of the release. 2+ but it is clearly distinct from the IP3- and cyclic ADP Although the amount of Ca release induced by NAADP ribose (cADPR)-sensitive mechanisms in eggs (reviewed was significantly smaller than that triggered by IP3 or in [8,9]). Furthermore, mammalian cells can synthesize cADPR, release appeared to have similar kinetics, thus and degrade NAADP, suggesting that NAADP-induced indicating that an NAADP-sensitive Ca2+-mobilizing Ca2+ release may be widespread and thus contribute to mechanism is present in brain intracellular organelles. the complexity of Ca2+ signalling [10,11]. Here, we show for the first time that NAADP evokes Ca2+ release from NAADP released Ca2+ with a half-maximal effective con- µ rat brain microsomes by a mechanism that is distinct centration (EC50) of approximately 1 M (Figure 1b), from those sensitive to IP3 or cADPR, and has a which is similar to the EC50 values reported for IP3 and remarkably similar pharmacology to the action of cADPR in this preparation [13–14]. This is, however, signif- NAADP in sea urchin eggs [12]. Membranes prepared icantly higher than the nanomolar concentrations required from the same rat brain tissues are able to support the for NAADP’s effects in pancreatic acinar cells [7]. To deter- synthesis and degradation of NAADP. We therefore mine whether NAADP acts on a Ca2+-release mechanism suggest that NAADP-mediated Ca2+ signalling could play an important role in neuronal Ca2+ signalling. Figure 1 Addresses: *Department of Medical Biochemistry, Semmelweis University of Medicine, PO Box 262, Budapest 1444, Hungary. (a) †Department of Pharmacology, Oxford University, Mansfield Road, 8 (b) 6 Oxford OX1 3QT, UK. ‡Laboratorium voor Fysiologie, Katholieke 6 Universiteit, Leuven B-3000, Belgium. 5 § Present address: Department of Pharmacology, University of release 4 release Cambridge, Tennis Court Road, Cambridge CB2 1QJ, UK. 2+ 2+ 4 Ca 2 Ca Correspondence: Armando Genazzani (nmol/mg protein) E-mail: [email protected] 0 (nmol/mg protein/5 sec) 3 0102030 0.1 1 10 Received: 15 April 1999 Time (sec) NAADP (µM) Revised: 1 June 1999 Current Biology Accepted: 9 June 1999 NAADP releases Ca2+ in rat brain microsomes (a) Comparison of Published: 5 July 1999 Ca2+-mobilizing characteristics of 2 µM cADPR (green circles), 3 µM µ NAADP (pink circles) and 5 M IP3 (blue circles) in the presence of Current Biology 1999, 9:751–754 0.1 µM Ca2+. On its own, 0.1 µM Ca2+ elicited a significant Ca2+ http://biomednet.com/elecref/0960982200900751 release (black circles). Squares represent Ca2+-efflux triggered by Ca2+ (black) or NAADP (pink) in the presence of the quench medium. © Elsevier Science Ltd ISSN 0960-9822 NAADP-induced Ca2+ release was observed in all four separate preparations of brain microsomes tested. (b) Concentration-response Results and discussion curve of NAADP-induced Ca2+ efflux. Experiments were peformed in µ 2+ Brain microsomes were one of the first mammalian cell the presence of 0.1 M Ca and therefore the basal release observed can be attributed to Ca2+-induced Ca2+ release. Values represent preparations in which cADPR and IP3 were shown to mean ± standard error. trigger Ca2+ release independently of each other [13–14]. 752 Current Biology, Vol 9 No 14 Figure 2 (a) 120 (b) (c) 100 6 75 80 4 50 release release release 2+ 2+ 40 2+ 2 Ca Ca 25 Ca (% ionomycin release) (% of respective control) 0 0 0 (nmol/mg protein/5 sec) 9 8 7 6 5 4 3 3 pCa IP -cADPR Heparin cADPR 2 DiltiazemVerapamil NAADP + NAADP 3 8-NH IP Current Biology cADPR + NAADP Characterization of NAADP-induced Ca2+ release. (a) Effect of of 45Ca2+ efflux was determined 5 sec after dilution into Ca2+-release 2+ 2+ inhibitors of Ca release on NAADP-induced (open bars) and medium. (c) Ca release triggered by a maximal concentration of IP3 cADPR-induced (grey bars) Ca2+ release. NAADP was used at a (5 µM), cADPR (2 µM) or NAADP (3 µM), or combinations of these concentration of 3 µM, and cADPR at 2 µM. The rate of 45Ca2+ efflux agents, in brain microsomes. Values are expressed as a percentage of was determined 5 sec after dilution into Ca2+-release medium. the release elicited by the Ca2+ ionophore ionomycin. The open parts (b) Effect of free Ca2+ concentration on NAADP-induced Ca2+ release of the bars represent the additional Ca2+ efflux that would be expected (circles) and Ca2+-induced Ca2+ release (squares). Free Ca2+ if the actions of the agents were fully additive. concentration was adjusted with EGTA as described in [14]. The rate 2+ distinct from those activated by IP3 and cADPR, we tested sensitivity of these mechanisms to modulators of Ca - the sensitivity of NAADP-induced Ca2+ release to various release channels [14]. In the absence of exogenous modula- drugs known to modify Ca2+ release from intracellular tors, raising Ca2+ in the incubation medium triggered an stores. Concentrations of heparin (600 µg/ml) that can increase in Ca2+ efflux as a result of CICR (Figure 2b), with 2+ µ 2+ inhibit IP3-induced Ca efflux (data not shown) did not maximal release occurring at around 1 M Ca . The acti- 2+ inhibit either cADPR-induced or NAADP-triggered Ca vation of both IP3 receptors and RyRs often shows a similar µ 2+ release (Figure 2a). Similarly, 8-NH2-cADPR (5 M), a bell-shaped dependence on the Ca concentration in the competitive antagonist of cADPR [15], reduced cADPR- vicinity of the cytoplasmic face of the release channels [16]. induced Ca2+ release but did not affect Ca2+ release At submicromolar free Ca2+ concentrations, the rate of Ca2+ induced by either IP3 (data not shown) or NAADP. High efflux from vesicles is augmented by cADPR, but above concentrations of L-type Ca2+-channel blockers selectively 1 µM free Ca2+ the effect of cADPR is less pronounced suppress NAADP-evoked Ca2+ release in sea urchin egg [14]. In contrast, we found NAADP-induced Ca2+ release homogenates, while having little effect on release induced to be largely independent of Ca2+ concentration in the µ by either cADPR or IP3 [12]. In brain microsomes, 100 M extravesicular medium (Figure 2b). This finding agrees verapamil or diltiazem — both potent inhibitors of L-type with results obtained in sea urchin eggs, where NAADP- Ca2+ channels — significantly reduced NAADP-induced induced Ca2+ release is neither potentiated by Ca2+ or Sr2+ Ca2+ efflux without affecting that evoked by cADPR nor inhibited by Mg2+ [17,18], in contrast to the effects of (Figure 2a). In the presence of permissive Ca2+ concentra- these ions on cADPR-induced Ca2+ release [19,20]. Our tions (for example 100 nM, as in this set of experiments), data also suggest that NAADP draws part of the Ca2+ that it part of the response seen on addition of NAADP, cADPR releases from the same stores as used in CICR, because the 2+ 2+ or IP3 can be attributed to Ca -induced Ca release difference in the magnitude of release between these two (CICR), as shown in Figure 1a. Therefore, the residual mechanisms is inversely proportional to the efficiency of response observed when the cADPR- and NAADP-sensi- CICR. Therefore, significant functional or structural tive mechanisms are blocked is likely to represent CICR. overlap exists between the Ca2+ pools released by Ca2+ and NAADP. This finding, together with a study on NAADP- Passive loading of microsomes has the advantage over induced Ca2+ release in pancreatic acinar cells, has led to ATP-driven loading that the concentration of free extrami- the proposal that NAADP-induced Ca2+ release may act as crosomal Ca2+ can be readily set by altering the composi- a trigger to evoke CICR via other Ca2+-release channels [7]. tion of the extravesicular solutions. This enables investigation of the effect of cytoplasmic Ca2+ concentra- It has been proposed that in sea urchin egg homo- tion on the Ca2+-release mechanisms themselves and the genates and in microsomes, NAADP releases Ca2+ from a Brief Communication 753 substantially different Ca2+-storage compartment from Figure 3 that which is sensitive to IP3 and cADPR [18,21]. To investigate the extent of overlap of different Ca2+ stores (a) (b) 120 200 in brain microsomes, we investigated the effects of com- 100 Synthesis binations of different Ca2+-mobilizing agents on Ca2+ 150 80 Degradation release.