Platelets play a vital role in maintaining haemostasis. Human platelet activation depends on Ca 2+ release, leading to cell activation, granule secretion and aggregation. NAADP (nicotinic acid–adenine dinucleotide phosphate) is a Ca 2+ -releasing second messenger that acts on acidic Ca 2+ stores and is used by a number of mammalian systems. In human platelets, NAADP has been shown to release Ca 2+ in permeabilized human platelets and contribute to thrombin-mediated platelet activation. In the present study, we have further characterized NAADP-mediated Ca 2+ release in human platelets in response to both thrombin and the GPVI (glycoprotein VI)-specific agonist CRP (collagen-related peptide). Using a radioligand-binding assay, we reveal an NAADP-binding site in human platelets, indicative of a platelet NAADP receptor. We also found that NAADP releases loaded 45 Ca 2+ from intracellular stores and that total platelet Ca 2+ release is inhibited by the proton ionophore nigericin. Ned-19, a novel cell-permeant NAADP receptor antagonist, competes for the NAADP-binding site in platelets and can inhibit both thrombin- and CRP-induced Ca 2+ release in human platelets. Ned-19 has an inhibitory effect on platelet aggregation, secretion and spreading. In addition, Ned-19 extends the clotting time in whole-blood samples. We conclude that NAADP plays an important role in human platelet function. Furthermore, the development of Ned-19 as an NAADP receptor antagonist provides a potential avenue for platelet-targeted therapy and the regulation of thrombosis.

NAADP (nicotinic acid–adenine dinucleotide phosphate) is an unusual second messenger thought to mobilize acidic Ca 2+ stores, such as lysosomes or lysosome-like organelles, that are functionally coupled to the ER (endoplasmic reticulum). Although NAADP-sensitive Ca 2+ stores have been described in neurons, the physiological cues that recruit them are not known. Here we show that in both hippocampal neurons and glia, extracellular application of glutamate, in the absence of external Ca 2+ , evoked cytosolic Ca 2+ signals that were inhibited by preventing organelle acidification or following osmotic bursting of lysosomes. The sensitivity of both cell types to glutamate correlated well with lysosomal Ca 2+ content. However, interfering with acidic compartments was largely without effect on the Ca 2+ content of the ER or Ca 2+ signals in response to ATP. Glutamate but not ATP elevated cellular NAADP levels. Our results provide evidence for the agonist-specific recruitment of NAADP-sensitive Ca 2+ stores by glutamate. This links the actions of NAADP to a major neurotransmitter in the brain.

NAADP (nicotinic acid–adenine dinucleotide phosphate) is a potent Ca 2+ -mobilizing messenger that stimulates Ca 2+ release in a variety of cells. NAADP-sensitive Ca 2+ channels are thought to reside on acidic Ca 2+ stores and to be functionally coupled to IP 3 (inositol 1,4,5-trisphosphate) and/or ryanodine receptors located on the endoplasmic reticulum. Whether NAADP-sensitive Ca 2+ channels ‘chatter’ to other channels, however, is not clear. In the present study, we have used a cell-permeant NAADP analogue to probe NAADP-mediated responses in rat medulla oblongata neurons. NAADP-AM (NAADP-acetoxymethyl ester) evoked global cytosolic Ca 2+ signals in isolated neurons that were reduced in amplitude by removal of external Ca 2+ , abolished by disruption of acidic compartments and substantially inhibited by blockade of ryanodine receptors. In rat medullary slices, NAADP-AM depolarized neurons from the nucleus ambiguus in the presence of intracellular EGTA, but not of the faster Ca 2+ chelator BAPTA [1,2-bis-( o -aminophenoxy)ethane- N , N , N ′, N ′-tetra-acetic acid]. Depolarization was also dependent upon extracellular Ca 2+ , acidic stores and ryanodine receptors. In voltage-clamp mode, NAADP-AM induced an inward current with a reversal potential of approx. 0 mV. The results of the present study reveal the presence of acidic NAADP-sensitive Ca 2+ stores in medulla neurons, the mobilization of which results not only in global Ca 2+ signals but also in local signals that activate non-selective cation channels on the cell surface resulting in depolarization. Thus NAADP is capable of co-ordinating channels both within the cell interior and at the cell membrane representing a novel mechanism for excitation of central neurons.

NAADP (nicotinic acid–adenine dinucleotide phosphate), the most potent Ca 2+ -mobilizing second messenger, is active in a wide range of organisms and cell types. Until now, all NAADP-producing enzymes have been thought to be members of the ADP-ribosyl cyclase family. ADP-ribosyl cyclases exhibit promiscuous substrate selectivity, synthesize a variety of products and are regulated in a limited manner, which may be non-physiological. In the present paper, we report the presence of an enzyme on the surface of sea urchin sperm that exhibits bell-shaped regulation by Ca 2+ over a range (EC 50 of 10 nM and IC 50 of 50 μM) that is physiologically relevant. Uniquely, this surface enzyme possesses complete selectivity for nucleotides with a 2′-phosphate group and exhibits only base-exchange activity without any detectable cyclase activity. Taken together, these findings indicate that this novel enzyme should be considered as the first true NAADP synthase.

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.

Nicotinic acidŐadenine 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.