Many hormones and neurotransmitters evoke Ca2+ release from intracellular stores, often triggering agonist-specific signatures of intracellular Ca2+ concentration. Inositol trisphosphate (InsP3) and cyclic adenosine 5'-diphosphate-ribose (cADPR) are established Ca2+-mobilizing messengers that activate Ca2+ release through intracellular InsP3 and ryanodine receptors, respectively. However, in pancreatic acinar cells, neither messenger can explain the complex pattern of Ca2+ signals triggered by the secretory hormone cholecystokinin (CCK). We show here that the Ca2+-mobilizing molecule nicotinic acid adenine dinucleotide phosphate (NAADP), an endogenous metabolite of beta-NADP, triggers a Ca2+ response that varies from short-lasting Ca2+ spikes to a complex mixture of short-lasting (1-2s) and long-lasting (0.2-1 min) Ca2+ spikes. Cells were significantly more sensitive to NAADP than to either cADPR or InsP3, whereas higher concentrations of NAADP selectively inactivated CCK-evoked Ca2+ signals in pancreatic acinar cells, indicating that NAADP may function as an intracellular messenger in mammalian cells.

Agonist-evoked cytosolic Ca 2⍣ spikes in mouse pancreatic acinar cells are specifically initiated in the apical secretory pole and are mostly confined to this region. The role played by mitochondria in this process has been investigated. Using the mitochondria-specific fluorescent dyes MitoTracker Green and Rhodamine 123, these organelles appeared as a bright belt concentrated mainly around the secretory granule area. We tested the effects of two different types of mitochondrial inhibitor on the cytosolic Ca 2⍣ concentration using simultaneous imaging of Ca 2⍣ -sensitive fluorescence (Fura 2) and electrophysiology. When carbonyl cyanide m-chlorophenylhydrazone (CCCP) was applied in the presence of the Ca 2⍣ -releasing messenger inositol 1,4,5-trisphosphate (IP 3 ), the local repetitive Ca 2⍣ responses in the granule area were transformed into a global rise in the cellular Ca 2⍣ concentration. In the absence of IP 3 , CCCP had no effect on the cytosolic Ca 2⍣ levels. Antimycin and antimycin ⍣ oligomycin had the same effect as CCCP. Active mitochondria, strategically placed around the secretory pole, block Ca 2⍣ diffusion from the primary Ca 2⍣ release sites in the granule-rich area in the apical pole to the basal part of the cell containing the nucleus. When mitochondrial function is inhibited, this barrier disappears and the Ca 2⍣ signals spread all over the cytosol.

Hormones and neurotransmitters mobilize Ca 2+ from the endoplasmic reticulum via inositol trisphosphate (IP 3 ) receptors, but how a single target cell encodes different extracellular signals to generate speci®c cyto-solic Ca 2+ responses is unknown. In pancreatic acinar cells, acetylcholine evokes local Ca 2+ spiking in the apical granular pole, whereas cholecystokinin elicits a mixture of local and global cytosolic Ca 2+ signals. We show that IP 3 , cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate (NAADP) evoke cytosolic Ca 2+ spiking by activating common oscillator units composed of IP 3 and ryanodine receptors. Acetylcholine activation of these common oscillator units is triggered via IP 3 receptors, whereas cholecystokinin responses are triggered via a different but converging pathway with NAADP and cyclic ADP-ribose receptors. Cholecystokinin potentiates the response to acetylcholine, making it global rather than local, an effect mediated speci®cally by cyclic ADPribose receptors. In the apical pole there is a common early activation site for Ca 2+ release, indicating that the three types of Ca 2+ release channels are clustered together and that the appropriate receptors are selected at the earliest step of signal generation.

In pancreatic acinar cells, low, threshold concentrations of acetylcholine (ACh) or cholecystokinin (CCK) induce repetitive local cytosolic Ca 2+ spikes in the apical pole, while higher concentrations elicit global signals. We have investigated the process that transforms local Ca 2+ spikes to global Ca 2+ transients, focusing on the interactions of multiple intracellular messengers. ACh-elicited local Ca 2+ spikes were transformed into a global sustained Ca 2+ response by cyclic ADP-ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP), whereas inositol 1,4,5-trisphosphate (IP 3 ) had a much weaker effect. In contrast, the response elicited by a low CCK concentration was strongly potentiated by IP 3 , whereas cADPR and NAADP had little effect. Experiments with messenger mixtures revealed a local interaction between IP 3 and NAADP and a stronger global potentiating interaction between cADPR and NAADP. NAADP strongly ampli®ed the local Ca 2+ release evoked by a cADPR/IP 3 mixture eliciting a vigorous global Ca 2+ response. Different combinations of Ca 2+ releasing messengers can shape the spatio-temporal patterns of cytosolic Ca 2+ signals. NAADP and cADPR are emerging as key messengers in the globalization of Ca 2+ signals.

Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic adenosine diphosphate ribose (cADPR) were first demonstrated to mobilize Ca2+ in sea urchin eggs. In the absence of direct measurements of these messengers, pharmacological studies alone have implicated these molecules as intracellular second messengers for specific cell surface receptor agonists. We now report that in mouse pancreatic acinar cells, cholecystokinin, but not acetylcholine, evokes rapid and transient increases in NAADP levels in a concentration-dependent manner. In contrast, both cholecystokinin and acetylcholine-mediated production of cADPR followed a very different time course. The rapid and transient production of NAADP evoked by cholecystokinin precedes the onset of the Ca2+ signal and is consistent with a role for NAADP in the initiation of the Ca2+ response. Continued agonist-evoked Ca2+ spiking is maintained by prolonged elevations of cADPR levels through sensitization of Ca2+ -induced Ca2+ -release channels. This study represents the first direct comparison of NAADP and cADPR measurements, and the profound differences observed in their time courses provide evidence in support of distinct roles of these Ca2+ -mobilizing messengers in shaping specific Ca2+ signals during agonist stimulation.

In order to control cell functions, hormones and neurotransmitters generate an amazing diversity of Ca2+ signals such as local and global Ca2+ elevations and also Ca2+ oscillations. In pancreatic acinar cells, cholecystokinin (CCK) stimulates secretion of digestive enzyme and promotes cell growth, whereas acetylcholine (ACh) essentially triggers enzyme secretion. Pancreatic acinar cells are a classic model for the study of CCK- and ACh-evoked specific Ca2+ signals. In addition to inositol 1,4,5 trisphosphate (IP3), recent studies have shown that cyclic ADPribose (cADPr) and nicotinic acid adenine dinucleotide phosphate (NAADP) release Ca2+ in pancreatic acinar cells. Moreover, it has also been shown that both ACh and CCK trigger Ca2+ spikes by co-activation of IP3 and ryanodine receptors but by different means. ACh uses IP3 and Ca2+, whereas CCK uses cADPr and NAADP. In addition, CCK activates phospholipase A2 and D. The concept emerging from these studies is that agonist-specific Ca2+ signals in a single target cell are generated by combination of different intracellular messengers.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca 2؉-mobilizing intracellular messenger and is linked to a variety of stimuli and cell surface receptors. However, the enzyme responsible for endogenous NAADP synthesis in vivo is unknown, and it has been proposed that another enzyme differing from ADP-ribosyl cyclase family members may exist. The ectoenzyme CD38, involved in many functions as diverse as cell proliferation and social behavior, represents an important alternative. In pancreatic acinar cells, the hormone cholecystokinin (CCK) stimulates NAADP production evoking Ca 2؉ signals by discharging acidic Ca 2؉ stores and leading to digestive enzyme secretion. From cells derived from CD38؊/؊ mice, we provide the first physiological evidence that CD38 is required for endogenous NAADP generation in response to CCK stimulation. Furthermore, CD38 expression in CD38-deficient pancreatic AR42J cells remodels Ca 2؉-signaling pathways in these cells by restoring Ca 2؉ mobilization from lysosomes during CCK-induced Ca 2؉ signaling. In agreement with an intracellular site for messenger synthesis, we found that CD38 is expressed in endosomes. These CD38-containing vesicles, likely of endosomal origin, appear to be proximal to lysosomes but not colocalized with them. We propose that CD38 is an NAADP synthase required for coupling receptor activation to NAADP-mediated Ca 2؉ release from lysosomal stores in pancreatic acinar cells.The Ca 2ϩ -mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) 5 is the most potent of the three established Ca 2ϩ -mobilizing messengers. NAADP was first shown to evoke Ca 2ϩ release from intracellular stores in sea urchin eggs (1). It has been proposed that NAADP initially releases Ca 2ϩ from lysosomal related organelles (2-6), which may then be amplified by Ca 2ϩ -induced Ca 2ϩ release mechanisms in the endoplasmic reticulum (ER) (7). The recent demonstration that NAADP mobilizes Ca 2ϩ from endolysosomal stores by targeting two-pore channels (1, 8, 9) has intensified interest in this messenger and its signaling pathways (10, 11). Two-pore channel-mediated Ca 2ϩ release may be coupled to further Ca 2ϩ release by inositol trisphosphate (IP 3 ) receptors or ryanodine receptors (1,8). To assign NAADP as a second messenger, it was important to show that endogenous levels of NAADP could be controlled by external stimuli, and now NAADP has been shown to be linked to various stimuli and cell surface receptors (12, 13). Dramatic increases in NAADP occur during sea urchin egg fertilization due to production in sperm upon contacting egg jelly (14). Increases in NAADP levels have been also reported in pancreatic ␤-cells after glucose (15) and glucagon-like peptide-1 stimulation (17), in pancreatic acinar cells after CCK stimulation (16), in pulmonary smooth muscle following exposure to endothelin-1 (3), in response to histamine in human myometrial cells (18), and glutamate in neurones (19). However, the enzymes responsible for endogenous NAADP synthesis i...

It remains unclear how different intracellular stores could interact and be recruited by Ca(2+)-releasing messengers to generate agonist-specific Ca(2+) signatures. In addition, refilling of acidic stores such as lysosomes and secretory granules occurs through endocytosis, but this has never been investigated with regard to specific Ca(2+) signatures. In pancreatic acinar cells, acetylcholine (ACh), cholecystokinin (CCK), and the messengers cyclic ADP-ribose (cADPR), nicotinic acid adenine dinucleotide phosphate (NAADP), and inositol 1,4,5-trisphosphate (IP(3)) evoke repetitive local Ca(2+) spikes in the apical pole. Our work reveals that local Ca(2+) spikes evoked by different agonists all require interaction of acid Ca(2+) stores and the endoplasmic reticulum (ER), but in different proportions. CCK and ACh recruit Ca(2+) from lysosomes and from zymogen granules through different mechanisms; CCK uses NAADP and cADPR, respectively, and ACh uses Ca(2+) and IP(3), respectively. Here, we provide pharmacological evidence demonstrating that endocytosis is crucial for the generation of repetitive local Ca(2+) spikes evoked by the agonists and by NAADP and IP(3). We find that cADPR-evoked repetitive local Ca(2+) spikes are particularly dependent on the ER. We propose that multiple Ca(2+)-releasing messengers determine specific agonist-elicited Ca(2+) signatures by controlling the balance among different acidic Ca(2+) stores, endocytosis, and the ER.