N-acetyl-L-leucine for Niemann-Pick type C: a multinational double-blind randomized placebo-controlled crossover study.

BACKGROUND: Niemann-Pick disease type C (NPC) is a rare autosomal recessive neurodegenerative lysosomal disease characterized by multiple symptoms such as progressive cerebellar ataxia and cognitive decline. The modified amino acid N-acetyl-leucine has been associated with positive symptomatic and neuroprotective, disease-modifying effects in various studies, including animal models of NPC, observational clinical case studies, and a multinational, rater-blinded phase IIb clinical trial. Here, we describe the development of a study protocol (Sponsor Code "IB1001-301") for the chronic treatment of symptoms in adult and pediatric patients with NPC. METHODS: This multinational double-blind randomized placebo-controlled crossover phase III study will enroll patients with a genetically confirmed diagnosis of NPC patients aged 4 years and older across 16 trial sites. Patients are assessed during a baseline period and then randomized (1:1) to one of two treatment sequences: IB1001 followed by placebo or vice versa. Each sequence consists of a 12-week treatment period. The primary efficacy endpoint is based on the Scale for the Assessment and Rating of Ataxia, and secondary outcomes include cerebellar functional rating scales, clinical global impression, and quality of life assessments. DISCUSSION: Pre-clinical as well as observational and phase IIb clinical trials have previously demonstrated that IB1001 rapidly improved symptoms, functioning, and quality of life for pediatric and adult NPC patients and is safe and well tolerated. In this placebo-controlled cross-over trial, the risk/benefit profile of IB1001 for NPC will be evaluated. It will also give information about the applicability of IB1001 as a therapeutic paradigm for other rare and common neurological disorders. TRIAL REGISTRATIONS: The trial (IB1001-301) has been registered at www. CLINICALTRIALS: gov (NCT05163288) and www.clinicaltrialsregister.eu (EudraCT: 2021-005356-10). Registered on 20 December 2021.

A master protocol to investigate a novel therapy acetyl-L-leucine for three ultra-rare neurodegenerative diseases: Niemann-Pick type C, the GM2 gangliosidoses, and ataxia telangiectasia.

BACKGROUND: The lack of approved treatments for the majority of rare diseases is reflective of the unique challenges of orphan drug development. Novel methodologies, including new functionally relevant endpoints, are needed to render the development process more feasible and appropriate for these rare populations and thereby expedite the approval of promising treatments to address patients' high unmet medical need. Here, we describe the development of an innovative master protocol and primary outcome assessment to investigate the modified amino acid N-acetyl-L-leucine (Sponsor Code: IB1001) in three separate, multinational, phase II trials for three ultra-rare, autosomal-recessive, neurodegenerative disorders: Niemann-Pick disease type C (NPC), GM2 gangliosidoses (Tay-Sachs and Sandhoff disease; "GM2"), and ataxia telangiectasia (A-T). METHODS/DESIGN: The innovative IB1001 master protocol and novel CI-CS primary endpoints were developed through a close collaboration between the Industry Sponsor, Key Opinion Leaders, representatives of the Patient Communities, and National Regulatory Authorities. As a result, the open-label, rater-blinded study design is considerate of the practical limitations of recruitment and retention of subjects in these ultra-orphan populations. The novel primary endpoint, the Clinical Impression of Change in Severity© (CI-CS), accommodates the heterogenous clinical presentation of NPC, GM2, and A-T: at screening, the principal investigator appoints for each patient a primary anchor test (either the 8-m walk test (8MWT) or 9-hole peg test of the dominant hand (9HPT-D)) based on his/her unique clinical symptoms. The anchor tests are videoed in a standardized manner at each visit to capture all aspects related to the patient's functional performance. The CI-CS assessment is ultimately performed by independent, blinded raters who compare videos of the primary anchor test from three periods: baseline, the end of treatment, and the end of a post-treatment washout. Blinded to the time point of each video, the raters make an objective comparison scored on a 7-point Likert scale of the change in the severity of the patient's neurological signs and symptoms from video A to video B. To investigate both the symptomatic and disease-modifying effects of treatment, N-acetyl-L-leucine is assessed during two treatment sequences: a 6-week parent study and 1-year extension phase. DISCUSSION: The novel CI-CS assessment, developed through a collaboration of all stakeholders, is advantageous in that it better ensures the primary endpoint is functionally relevant for each patient, is able to capture small but meaningful clinical changes critical to the patients' quality of life (fine-motor skills; gait), and blinds the primary outcome assessment. The results of these three trials will inform whether N-acetyl-L-leucine is an effective treatment for NPC, GM2, and A-T and can also serve as a new therapeutic paradigm for the development of future treatments for other orphan diseases. TRIAL REGISTRATION: The three trials (IB1001-201 for Niemann-Pick disease type C (NPC), IB1001-202 for GM2 gangliosidoses (Tay-Sachs and Sandhoff), IB1001-203 for ataxia telangiectasia (A-T)) have been registered at www.clinicaltrials.gov (NCT03759639; NCT03759665; NCT03759678), www.clinicaltrialsregister.eu (EudraCT: 2018-004331-71; 2018-004406-25; 2018-004407-39), and https://www.germanctr.de (DR KS-ID: DRKS00016567; DRKS00017539; DRKS00020511).

The role of calcium and nicotinic acid adenine dinucleotide phosphate (NAADP) in human osteoclast formation and resorption.

Osteoclasts are specialised bone resorbing cells which form by fusion of circulating mononuclear phagocyte precursors. Bone resorption results in the release of large amounts of calcium into the extracellular fluid (ECF), but it is not certain whether changes in extracellular calcium concentration [Ca(2+)]e influence osteoclast formation and resorption. In this study, we sought to determine the effect of [Ca(2+)]e and NAADP, a potent calcium mobilising messenger that induces calcium uptake, on human osteoclast formation and resorption. CD14+ human monocytes were cultured with M-CSF and RANKL in the presence of different concentrations of calcium and NAADP and the effect on osteoclast formation and resorption evaluated. We found that the number of TRAP+ multinucleated cells and the extent of lacunar resorption were reduced when there was an increase in extracellular calcium and NAADP. This was associated with a decrease in RANK mRNA expression by CD14+ cells. At high concentrations (20 mM) of [Ca(2+)]e mature osteoclast resorption activity remained unaltered relative to control cultures. Our findings indicate that osteoclast formation is inhibited by a rise in [Ca(2+)]e and that RANK expression by mononuclear phagocyte osteoclast precursors is also [Ca(2+)]e dependent. Changes in NAADP also influence osteoclast formation, suggesting a role for this molecule in calcium handling. Osteoclasts remained capable of lacunar resorption, even at high ECF [Ca(2+)]e, in keeping with their role in physiological and pathological bone resorption.

NAADP, a new intracellular messenger that mobilizes Ca2+ from acidic stores.

NAADP (nicotinic acid-adenine dinucleotide phosphate) is a recently described Ca2+-mobilizing molecule. First characterized in the sea urchin egg, it has been shown to mobilize Ca2+ from intracellular stores in a wide range of cells from different organisms. It is a remarkably potent molecule, and recent reports show that its cellular levels change in response to a variety of agonists, confirming its role as a Ca2+-mobilizing messenger. In many cases, NAADP appears to interact with other Ca2+-mobilizing messengers such as IP3 (inositol 1,4,5-trisphosphate) and cADP-ribose in shaping cytosolic Ca2+ signals. What is not clear is the molecular nature of the NAADP-sensitive Ca2+ release mechanism and its subcellular localization. This review focuses on the recent progress made in sea urchin eggs, which indicates that NAADP activates a novel Ca2+ release channel distinct from the relatively well-characterized IP3 and ryanodine receptors. Furthermore, in the sea urchin egg, the NAADP-sensitive store appears to be separate from the endoplasmic reticulum and is most likely an acidic store. These findings have also been reinforced by similar findings in mammalian cells, and a unified model for NAADP-induced Ca2+ signalling is presented.

The NAADP receptor: commentary on Billington et al.

NAADP is a recently described calcium-mobilizing messenger. First discovered as a potent calcium-releasing molecule in sea urchin eggs, its actions have now been reported in several mammalian cell types. In the sea urchin egg, NAADP-sensitive calcium release channels appear distinct from inositol trisphosphate or ryanodine receptors, and are mainly localized to acidic compartments. In this study, Billington et al. extend the pharmacology of the putative NAADP receptor utilizing molecules unrelated to NAADP itself. This work may provide an important step in developing selective NAADP receptor modulators that will help define the role of NAADP in cell signalling.

Interactions between calcium release pathways: multiple messengers and multiple stores.

The discovery of cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) as Ca(2+) releasing messengers has provided additional insight into how complex Ca(2+) signalling patterns are generated. There is mounting evidence that these molecules along with the more established messenger, myo-inositol 1,4,5-trisphosphate (IP(3)), have a widespread messenger role in shaping Ca(2+) signals in many cell types. These molecules have distinct structures and act on specific Ca(2+) release mechanisms. Emerging principles are that cADPR enhances the Ca(2+) sensitivity of ryanodine receptors (RYRs) to produce prolonged Ca(2+) signals through Ca(2+)-induced Ca(2+) release (CICR), while NAADP acts on a novel Ca(2+) release mechanism to produce a local trigger Ca(2+) signal which can be amplified by CICR by recruiting other Ca(2+) release mechanisms. Whilst IP(3) and cADPR mobilise Ca(2+) from the endoplasmic reticulum (ER), recent evidence from the sea urchin egg suggests that the major NAADP-sensitive Ca(2+) stores are reserve granules, acidic lysosomal-related organelles. In this review we summarise the role of multiple Ca(2+) mobilising messengers, Ca(2+) release channels and Ca(2+) stores, and the interplay between them, in the generation of specific Ca(2+) signals. Focusing upon cADPR and NAADP, we discuss how cellular stimuli may draw upon different combinations of these messengers to produce distinct Ca(2+) signalling signatures.

Ca2+ release induced by cyclic ADP ribose in mice lacking type 3 ryanodine receptor.

The action of cyclic-ADP-ribose was studied on calcium release from sarcoplasmic reticulum of skeletal muscles of neonatal and adult wild-type and RyR3-deficient mice. cADPR increased calcium efflux from microsomes, enhanced caffeine-induced calcium release, and, in 20% of the tests, triggered calcium release in single muscle fibers. These responses occurred only in the diaphragm of adult RyR3-deficient mice. cADPR action was abolished by ryanodine, ruthenium red, and 8-brome-cADPR. These results strongly favor a specific action of cADPR on RyR1. The responsiveness of RyR1 appears in adult muscles when RyR3 is lacking.

Pharmacological characterization of the putative cADP-ribose receptor.

cADP-ribose (cADPR), a naturally occurring metabolite of NAD(+), has been shown to be an important regulator of intracellular Ca(2+) release. Considerable evidence suggests that cADPR is the endogenous modulator of the ryanodine receptor (RyR), which mediates Ca(2+)-induced Ca(2+) release (CICR). Indeed, cADPR-mediated Ca(2+) release is subject to functional regulation by other modulators of CICR, including Ca(2+), caffeine and calmodulin. However, the underlying basis behind the effect of such agents on cADPR activity (in particular whether they regulate cADPR binding), as well as the precise nature of the cADPR receptor remains unclear. In the present study, use of (32)P-radiolabelled cADPR has enabled a detailed pharmacological characterization of cADPR-binding sites in sea urchin egg homogenates. We report that cADPR binds specifically to a single class of high affinity receptor. Retainment of binding to membranes after a high-salt wash suggests the involvement of either an integral membrane protein (possibly the RyR itself) or a peripheral protein tightly associated to the membrane. Insensitivity of [(32)P]cADPR binding to either FK506 or rapamycin suggests that this does not concern the FK506-binding protein. Significantly, binding is highly robust, being relatively insensitive to both endogenous and pharmacological modulators of RyR-mediated CICR. In turn, this suggests that such agents modulate cADPR-mediated Ca(2+) release primarily by tuning the 'gain' of the CICR system, upon which cADPR acts, rather than influencing the interaction of cADPR with its target receptor. The exception to this is calmodulin, for which our results indicate an additional role in facilitating cADPR binding.

Coordination of Ca2+ signalling by NAADP.

Nicotinic acid adenine dinucleotide phosphate (NAADP) mobilizes intracellular Ca2+ stores in several cell types. Ample evidence suggests that NAADP activates intracellular Ca2+ channels distinct from those that are sensitive to inositol trisphosphate and ryanodine/cyclic ADP-ribose. Recent studies in intact cells have demonstrated functional coupling ('channel chatter') between Ca2+ release pathways mediated by NAADP, inositol trisphosphate and cyclic ADP-ribose. Thus, NAADP is probably an important determinant in shaping cytosolic Ca2+ signals.

Distinct pharmacology of 2-hydroxycarbazole-induced Ca2+ release in the sea urchin egg.

2-hydroxycarbazole, a compound structurally related to the Ca2+-mobilizing marine toxin 9-methyl-7-bromoeudistomin, has recently been proposed to activate both type 1 and type 2 ryanodine receptors in skeletal and cardiac muscle, respectively. This study was undertaken to evaluate the activity of this compound in the sea urchin egg homogenate, a model system used to characterize intracellular Ca2+ mobilization mechanisms. 2-Hydroxycarbazole was found to potently release Ca2+ in a concentration-dependent manner via a specific mechanism displaying apparent desensitization. Use of selective inhibitors of the Ca2+-mobilizing messengers inositol 1,4,5-trisphosphate, cyclic adenosine diphosphate ribose, and nicotinic acid adenine dinucleotide phosphate, as well as desensitization of homogenates to each of these molecules, failed to inhibit the response to 2-hydroxycarbazole. However, the response to 2-hydroxycarbazole was competitively antagonized by caffeine. Investigation of the Ca2+ stores accessed by 2-hydroxycarbazole revealed Ca2+ release from a thapsigargin-insensitive pool. Finally, 2-hydroxycarbazole failed to enhance [3H]ryanodine binding, suggesting the operation of a nonryanodine receptor mechanism. These results demonstrate that 2-hydroxycarbazole is acting to modulate a Ca2+ release mechanism with distinct pharmacological properties to those previously reported in the sea urchin egg.

NAADP induces Ca2+ oscillations via a two-pool mechanism by priming IP3- and cADPR-sensitive Ca2+ stores.

In sea urchin eggs, Ca2+ mobilization by nicotinic acid adenine dinucleotide phosphate (NAADP) potently self-inactivates but paradoxically induces long-term Ca2+ oscillations. We investigated whether NAADP-induced Ca2+ oscillations arise from the recruitment of other Ca2+ release pathways. NAADP, inositol trisphosphate (IP3) and cyclic ADP-ribose (cADPR) all mobilized Ca2+ from internal stores but only NAADP consistently induced Ca2+ oscillations. NAADP-induced Ca2+ oscillations were partially inhibited by heparin or 8-amino-cADPR alone, but eliminated by the presence of both, indicating a requirement for both IP3- and cADPR-dependent Ca2+ release. Thapsigargin completely blocked IP3 and cADPR responses as well as NAADP-induced Ca2+ oscillations, but only reduced the NAADP-mediated Ca2+ transient. Following NAADP-mediated release from this Ca2+ pool, the amount of Ca2+ in the Ca2+-induced Ca2+ release stores was increased. These results support a mechanism in which Ca2+ oscillations are initiated by Ca2+ release from NAADP-sensitive Ca2+ stores (pool 1) and perpetuated through cycles of Ca2+ uptake into and release from Ca2+-induced Ca2+ release stores (pool 2). These results provide the first direct evidence in support of a two-pool model for Ca2+ oscillations.

Prolonged inactivation of nicotinic acid adenine dinucleotide phosphate-induced Ca2+ release mediates a spatiotemporal Ca2+ memory.

Although numerous extracellular stimuli are coupled to increases in intracellular Ca(2+), different stimuli are thought to achieve specificity by eliciting different spatiotemporal Ca(2+) increases. We investigated the effect of nicotinic acid adenine dinucleotide phosphate (NAADP) inactivation on spatiotemporal Ca(2+) signals in intact sea urchin eggs. The photorelease of NAADP but not inositol 1,4,5-trisphosphate or cyclic ADP-ribose resulted in self-inactivation. When NAADP was released first locally and subsequently globally, the spatial pattern of the first response shaped that of the second. Specifically, the local release of NAADP created a Ca(2+) gradient that was reversed during the subsequent global release of NAADP. Neither cyclic ADP-ribose nor inositol 1,4,5-trisphosphate showed a similar effect. In contrast to homogenates, NAADP inactivation was reversible in intact eggs with resensitization occurring in approximately 20 min. Because initial NAADP responses affect later responses, NAADP can serve as a mechanism for a Ca(2+) memory that has both spatial and temporal components. This NAADP-mediated Ca(2+) memory provides a novel mechanism for cells to control spatiotemporal Ca(2+) increases.

Adp-ribosyl cyclase and cyclic ADP-ribose hydrolase act as a redox sensor. a primary role for cyclic ADP-ribose in hypoxic pulmonary vasoconstriction.

Hypoxic pulmonary vasoconstriction is unique to pulmonary arteries and serves to match lung perfusion to ventilation. However, in disease states this process can promote hypoxic pulmonary hypertension. Hypoxic pulmonary vasoconstriction is associated with increased NADH levels in pulmonary artery smooth muscle and with intracellular Ca(2+) release from ryanodine-sensitive stores. Because cyclic ADP-ribose (cADPR) regulates ryanodine receptors and is synthesized from beta-NAD(+), we investigated the regulation by beta-NADH of cADPR synthesis and metabolism and the role of cADPR in hypoxic pulmonary vasoconstriction. Significantly higher rates of cADPR synthesis occurred in smooth muscle homogenates of pulmonary arteries, compared with homogenates of systemic arteries. When the beta-NAD(+):beta-NADH ratio was reduced, the net amount of cADPR accumulated increased. This was due, at least in part, to the inhibition of cADPR hydrolase by beta-NADH. Furthermore, hypoxia induced a 10-fold increase in cADPR levels in pulmonary artery smooth muscle, and a membrane-permeant cADPR antagonist, 8-bromo-cADPR, abolished hypoxic pulmonary vasoconstriction in pulmonary artery rings. We propose that the cellular redox state may be coupled via an increase in beta-NADH levels to enhanced cADPR synthesis, activation of ryanodine receptors, and sarcoplasmic reticulum Ca(2+) release. This redox-sensing pathway may offer new therapeutic targets for hypoxic pulmonary hypertension.

Unique kinetics of nicotinic acid-adenine dinucleotide phosphate (NAADP) binding enhance the sensitivity of NAADP receptors for their ligand.

Nicotinic acid-adenine dinucleotide phosphate (NAADP) is a novel and potent Ca(2+)-mobilizing agent in sea urchin eggs and other cell types. Little is known, however, concerning the properties of the putative intracellular NAADP receptor. In the present study we have characterized NAADP binding sites in sea urchin egg homogenates. [(32)P]NAADP bound to a single class of high-affinity sites that were reversibly inhibited by NaCl but insensitive to pH and Ca(2+). Binding of [(32)P]NAADP was lost in preparations that did not mobilize Ca(2+) in response to NAADP, indicating that [(32)P]NAADP probably binds to a receptor mediating Ca(2+) mobilization. Addition of excess unlabelled NAADP, at various times after initiation of [(32)P]NAADP binding, did not result in displacement of bound [(32)P]NAADP. These data show that NAADP becomes irreversibly bound to its receptor immediately upon association. Accordingly, incubation of homogenates with low concentrations of NAADP resulted in maximal labelling of NAADP binding sites. This unique property renders NAADP receptors exquisitely sensitive to their ligand, thereby allowing detection of minute changes in NAADP levels.

NAADP-induced calcium release in sea urchin eggs.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent activator of Ca2+ release from intracellular stores described. It acts on a mechanism distinct from inositol trisphosphate and ryanodine receptors, the two major Ca2+ release channels characterised. NAADP-gated Ca2+ release channels do not appear to be regulated by Ca2+ and may be better suited for triggering Ca2+ signals rather than propagating them. They exhibit a remarkable pharmacology for a putative intracellular Ca2+ release channel in that they are selectively blocked by potassium and L-type Ca2+ channel antagonists. Furthermore, in contrast to microsomal Ca2+ stores expressing IP3Rs and RyRs, those sensitive to NAADP are thapsigargin-insensitive, suggesting that they may be expressed on a different part of the endoplasmic reticulum. Perhaps the most unusual feature of the NAADP-gated Ca2+ release mechanisms is its inactivation properties. Unlike the mechanisms regulated by IP3 and cADPR in sea urchin eggs which after induction of Ca2+ release appear to become refractory to subsequent activation, very low concentrations of NAADP are able to inactivate NAADP-induced Ca2+ release fully at concentrations well below those required to activate Ca2+ release. The mechanism and physiological significance of this most unusual desensitisation phenomenon are unclear. More recently, NAADP has been shown to mobilise Ca2+ in ascidian oocytes, brain microsomes and pancreatic acinar cells suggesting a more widespread role in Ca2+ signalling. A possible role for this novel Ca2+ release mechanism in sea urchin egg fertilisation is discussed.

Two neuropeptides recruit different messenger pathways to evoke Ca2+ signals in the same cell.

Bombesin and cholecystokinin (CCK) peptides act as signalling molecules in both the central nervous system and gastrointestinal tract [1-4]. It was reported recently that nicotinic acid adenine dinucleotide phosphate (NAADP) releases Ca2+ from mammalian brain microsomes [5] and triggers Ca2+ signals in pancreatic acinar cells, where it is proposed to mediate CCK-evoked Ca2+ signals [6]. Here, for the first time, we have finely resolved bombesin-induced cytosolic Ca2+ oscillations in single pancreatic acinar cells by whole-cell patch-clamp monitoring of Ca2+-dependent ionic currents [6-8]. Picomolar concentrations of bombesin and CCK evoked similar patterns of cytosolic Ca2+ oscillations, but high, desensitising, NAADP concentrations selectively inhibited CCK, but not bombesin-evoked signals. Inhibiting inositol trisphosphate (IP3) receptors with a high concentration of caffeine blocked both types of oscillations. We further tested whether NAADP is involved in Ca2+ signals triggered by activation of the low-affinity CCK receptor sites. Nanomolar concentrations of CCK evoked non-oscillatory Ca2+ signals, which were not affected by desensitising NAADP receptors. Our results suggest that Ca2+-release channels gated by the novel Ca2+-mobilising molecule NAADP are only essential in specific Ca2+-mobilising pathways, whereas the IP3 receptors are generally required for Ca2+ signals. Thus, the same cell may use different combinations of intracellular Ca2+-releasing messengers to encode different external messages.

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 Ca(2+)-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, [(32)P]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 [(32)P]NAADP was, however, the same in the two systems and in agreement with their ability to mobilize Ca(2+) from homogenates. These data indicate that [(32)P]NAADP likely binds to receptors mediating Ca(2+) mobilization. Autoradiography revealed striking heterogeneity in the distribution of [(32)P]NAADP binding sites throughout the brain. Our data strongly support a role for NAADP-induced Ca(2+) signaling in the brain.

Spatial control of Ca2+ signaling by nicotinic acid adenine dinucleotide phosphate diffusion and gradients.

Intracellular Ca(2+) is able to control numerous cellular responses through complex spatiotemporal organization. Ca(2+) waves mediated by inositol trisphosphate or ryanodine receptors propagate by Ca(2+)-induced Ca(2+) release and therefore do not have an absolute requirement for a gradient in either inositol trisphosphate or cyclic ADP-ribose, respectively. In contrast, we report that although Ca(2+) increases induced by nicotinic acid adenine dinucleotide phosphate (NAADP) are amplified by Ca(2+)-induced Ca(2+) release locally, Ca(2+) waves mediated by NAADP have an absolute requirement for an NAADP gradient. If NAADP is increased such that its concentration is spatially uniform in one region of an egg, the Ca(2+) increase occurs simultaneously throughout this area, and only where there is diffusion out of this area to establish an NAADP gradient is there a Ca(2+) wave. A local increase in NAADP results in a Ca(2+) increase that spreads by NAADP diffusion. NAADP diffusion is restricted at low but not high concentrations of NAADP, indicating that NAADP diffusion is strongly influenced by binding to immobile and saturable sites, probably the NAADP receptor itself. Thus, the range of action of NAADP can be tuned by its concentration from that of a local messenger, like Ca(2+), to that of a global messenger, like IP(3) or cyclic ADP-ribose.

Calcium release from the endoplasmic reticulum of higher plants elicited by the NADP metabolite nicotinic acid adenine dinucleotide phosphate.

Higher plants share with animals a responsiveness to the Ca(2+) mobilizing agents inositol 1,4,5-trisphosphate (InsP(3)) and cyclic ADP-ribose (cADPR). In this study, by using a vesicular (45)Ca(2+) flux assay, we demonstrate that microsomal vesicles from red beet and cauliflower also respond to nicotinic acid adenine dinucleotide phosphate (NAADP), a Ca(2+)-releasing molecule recently described in marine invertebrates. NAADP potently mobilizes Ca(2+) with a K(1/2) = 96 nM from microsomes of nonvacuolar origin in red beet. Analysis of sucrose gradient-separated cauliflower microsomes revealed that the NAADP-sensitive Ca(2+) pool was derived from the endoplasmic reticulum. This exclusively nonvacuolar location of the NAADP-sensitive Ca(2+) pathway distinguishes it from the InsP(3)- and cADPR-gated pathways. Desensitization experiments revealed that homogenates derived from cauliflower tissue contained low levels of NAADP (125 pmol/mg) and were competent in NAADP synthesis when provided with the substrates NADP and nicotinic acid. NAADP-induced Ca(2+) release is insensitive to heparin and 8-NH(2)-cADPR, specific inhibitors of the InsP(3)- and cADPR-controlled mechanisms, respectively. However, NAADP-induced Ca(2+) release could be blocked by pretreatment with a subthreshold dose of NAADP, as previously observed in sea urchin eggs. Furthermore, the NAADP-gated Ca(2+) release pathway is independent of cytosolic free Ca(2+) and therefore incapable of operating Ca(2+)-induced Ca(2+) release. In contrast to the sea urchin system, the NAADP-gated Ca(2+) release pathway in plants is not blocked by L-type channel antagonists. The existence of multiple Ca(2+) mobilization pathways and Ca(2+) release sites might contribute to the generation of stimulus-specific Ca(2+) signals in plant cells.

Cyclic ADP ribose as a calcium-mobilizing messenger.

This Perspective by Galione and Churchill is one in a series on intracellular calcium release mechanisms. The authors review the evidence for cyclic adenosine diphosphate ribose (cADPR) being a second messenger involved in regulating intracellular calcium. In addition, the physiological stimuli and responses mediated by cADPR are discussed. The Perspective is accompanied by a movie showing a calcium wave triggered by cADPR.