Endosomes, lysosomes and lysosome-related organelles are emerging as important Ca 2+ storage cellular compartments with a central role in intracellular Ca 2+ signalling. Endocytosis at the plasma membrane forms endosomal vesicles which mature to late endosomes and culminate in lysosomal biogenesis. During this process, acquisition of different ion channels and transporters progressively changes the endolysosomal luminal ionic environment (e.g. pH and Ca 2+ ) to regulate enzyme activities, membrane fusion/fission and organellar ion fluxes, and defects in these can result in disease. In the present review we focus on the physiology of the inter-related transport mechanisms of Ca 2+ and H + across endolysosomal membranes. In particular, we discuss the role of the Ca 2+ -mobilizing messenger NAADP (nicotinic acid adenine dinucleotide phosphate) as a major regulator of Ca 2+ release from endolysosomes, and the recent discovery of an endolysosomal channel family, the TPCs (two-pore channels), as its principal intracellular targets. Recent molecular studies of endolysosomal Ca 2+ physiology and its regulation by NAADP-gated TPCs are providing exciting new insights into the mechanisms of Ca 2+ -signal initiation that control a wide range of cellular processes and play a role in disease. These developments underscore a new central role for the endolysosomal system in cellular Ca 2+ regulation and signalling.

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.

NAADP (nicotinic acid–adenine dinucleotide phosphate)-induced Ca 2+ release has been proposed to occur selectively from acidic stores in several cell types, including sea urchin eggs. Using fluorescence measurements, we have investigated whether NAADP-induced Ca 2+ release alters the pH L (luminal pH) within these acidic stores in egg homogenates and observed their prompt, concentration-dependent alkalinization by NAADP (but not β-NAD + or NADP). Like Ca 2+ release, the pH L change was desensitized by low concentrations of NAADP suggesting it was secondary to NAADP receptor activation. Moreover, this was a direct effect of NAADP upon the acidic stores and not secondary to increases in cytosolic Ca 2+ as it was not mimicked by IP 3 (inositol 1,4,5-trisphosphate), cADPR (cyclic adenine diphosphoribose), ionomycin, thapsigargin or by direct addition of Ca 2+ , and was not blocked by EGTA. The results of the present study further support acidic stores as targets for NAADP and for the first time reveal an adjunct role for NAADP in regulating the pH L of intracellular organelles.

Nicotinic acid—adenine dinucleotide phosphate (NAADP) is a newly described Ca 2+ -mobilizing nucleotide that appears to target intracellular Ca 2+ -release channels distinct from those sensitive to inositol trisphosphate or ryanodine/cyclic ADP-ribose. Little, however, is known concerning the regulation of cellular NAADP levels. In the present study, we have characterized the metabolism of NAADP by brain membranes. From HPLC and MS analyses we show that loss of NAADP was associated with the appearance of a major product that is likely to be nicotinic acid—adenine dinucleotide (NAAD), the dephosphorylated form of NAADP. Dephosphorylation of NAADP, but not 3′-NAADP, was dramatically attenuated by Ca 2+ chelators and stimulated by Ca 2+ over a physiological range in a calmodulin-insensitive manner. In contrast, NADP was metabolized predominantly to ADP-ribose phosphate via glycohydrolase activity, although slower Ca 2+ -dependent dephosphorylation of both NADP and 2′-AMP could also be demonstrated. This is the first report describing a Ca 2+ -regulated 2′-specific phosphatase which is probably the major pathway for the inactivation of NAADP in brain. Our data provide a potential feedback mechanism for limiting NAADP-induced Ca 2+ release within cells through stimulation of NAADP metabolism by Ca 2+ and strongly support a signalling role for this novel nucleotide in the brain.

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.

Actions of photoreleased cADP-ribose (cADPR), a novel regulator of calcium-induced calcium release (CICR) from ryanodine-sensitive stores, were investigated in cardiac myocytes. Photoreleased cADPR caused an increase in the magnitude of whole-cell calcium transients studied in mammalian cardiac ventricular myocytes (both guinea-pig and rat) using confocal microscopy). Approx. 15 s was required following photorelease of cADPR for the development of its maximal effect. Photoreleased cADPR also increased the frequency of calcium ‘sparks’, which are thought to be elementary events which make up the whole-cell calcium transient, and were studied in rat myocytes, but had little or no effect on spark characteristics (amplitude, rise time, decay time and distance to half amplitude). The potentiating effects of photoreleased cADPR on both whole-cell transients and the frequency of calcium sparks were prevented by cytosolic application of the antagonist 8-amino-cADPR (5 μ M). These experiments, therefore, provide the first evidence in any cell type for an effect of cADPR on calcium sparks, and are the first to show the actions of photoreleased cADPR on whole-cell calcium transients in mammalian cells. The observations are consistent with the effects of cADPR in enhancing the calcium sensitivity of CICR from the sarcoplasmic reticulum in cardiac ventricular myocytes, leading to an increase in the probability of occurrence of calcium sparks and to an increase in whole-cell calcium transients. The slow time-course for development of the full effect on whole-cell calcium transients might be taken to indicate that the influence of cADPR on CICR may involve complex molecular interactions rather than a simple direct action of cADPR on the ryanodine-receptor channels.

The sea urchin egg has been used as a system to study calcium-release mechanisms induced by inositol 1,4,5-trisphosphate (IP 3 ), cADP-ribose (cADPR), and more recently, nicotinic acid–adenine dinucleotide phosphate (NAADP). In order that cADPR and NAADP may be established as endogenous messengers for calcium release, the existence of intracellular enzymes capable of metabolizing these molecules must be demonstrated. In addition, intracellular levels of cADPR and NAADP should be under the control of extracellular stimuli. It has been shown that cGMP stimulates the synthesis of cADPR in the sea urchin egg. The present study shows that the sea urchin egg is capable of synthesizing and degrading NAADP. cADPR and NAADP synthetic activities appear to be separate, with different cellular localizations, pH and temperature optima. We suggest that in the sea urchin egg, cADPR and NAADP production may be differentially regulated by receptor-coupled second messengers, with cADPR production being regulated by cGMP and NAADP production modulated by cAMP.

Cyclic ADP ribose (cADPR) is a potent Ca 2+ -releasing agent, and putative second messenger, the endogenous levels of which are tightly regulated by synthetic (ADP-ribosyl cyclases) and degradative (cADPR hydrolase) enzymes. These enzymes have been characterized in a number of mammalian and invertebrate tissues and their activities are often found on a single polypeptide. β-NAD + , cGMP and nitric oxide (NO) have been reported to mobilize Ca 2+ in the sea urchin egg via the cADPR-mediated pathway. We now report that in sea urchin egg homogenates, nicotinamide inhibits the Ca 2+ -mobilizing action of β-NAD + , cGMP and NO, but has no effect on cADPR-induced Ca 2+ release. Moreover, nicotinamide inhibits cGMP-induced regenerative Ca 2+ waves in the intact sea urchin egg. By successfully separating the cADPR-metabolizing machinery from that which releases Ca 2+ , we have shown that nicotinamide inhibits cADPR-mediated Ca 2+ signalling at the level of cADPR generation. Importantly, nicotinamide had no effect upon the hydrolysis of cADPR, and its selective action on cyclase activity was supported by its inhibition of purified Aplysia ADP-ribosyl cyclase, which does not exhibit detectable hydrolytic activity. The action of nicotinamide in blocking Ca 2+ release by β-NAD + , cGMP and NO strongly suggests that these agents act as modulators of cADPR synthesis rather than to sensitize calcium release channels to cADPR.

Nicotinic acid–adenine dinucleotide phosphate (NAADP) is a novel intracellular Ca 2+ releasing agent recently described in sea-urchin eggs and egg homogenates. Ca 2+ release by NAADP is independent of that induced by either inositol trisphosphate (Ins P 3 ) or cyclic adenosine dinucleotide phosphate (cADPR). We now report that in sea urchin egg homogenates, NAADP releases Ca 2+ from a Ca 2+ pool that is distinct from those that are sensitive to Ins P 3 and cADPR. This organelle has distinct Ca 2+ uptake characteristics: it is insensitive to thapsigargin and cyclopiazoic acid, but maintenance of the pool shows some requirement for ATP. Although the different Ca 2+ pools have different characteristics, there appears to be some degree of overlap or cross-talk between the NAADP- and cADPR/Ins P 3 -sensitive Ca 2+ pools. Ca 2+ -induced Ca 2+ release is unlikely to account for the apparent overlap between stores, since NAADP-induced Ca 2+ release, in contrast with that stimulated by cADPR, is not potentiated by bivalent cations.