Jose M. Cancela

Coordination of agonist-induced Ca2+-signalling patterns by NAADP in pancreatic acinar cells

Many hormones and neurotransmitters evoke Ca2+ release fromintracellular stores, often triggering agonist-specific signatures of intracellular Ca2+ concentration. Inositol trisphosphate (InsP3)1 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 β-NADP, triggers a Ca2+ response that varies from short-lasting Ca2+ spikes to a complex mixture of short-lasting (1–2 s) 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.

Active mitochondria surrounding the pancreatic acinar granule region prevent spreading of inositol trisphosphate‐evoked local cytosolic Ca2+ signals

Agonist‐evoked cytosolic Ca2+ 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 Ca2+ concentration using simultaneous imaging of Ca2+‐sensitive fluorescence (Fura 2) and electrophysiology. When carbonyl cyanide m‐chlorophenylhydrazone (CCCP) was applied in the presence of the Ca2+‐releasing messenger inositol 1,4,5‐trisphosphate (IP3), the local repetitive Ca2+ responses in the granule area were transformed into a global rise in the cellular Ca2+ concentration. In the absence of IP3, CCCP had no effect on the cytosolic Ca2+ levels. Antimycin and antimycin + oligomycin had the same effect as CCCP. Active mitochondria, strategically placed around the secretory pole, block Ca2+ diffusion from the primary Ca2+ 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 Ca2+ signals spread all over the cytosol.

Two different but converging messenger pathways to intracellular Ca2+ release: the roles of nicotinic acid adenine dinucleotide phosphate, cyclic ADP-ribose and inositol trisphosphate

Hormones and neurotransmitters mobilize Ca2+ from the endoplasmic reticulum via inositol trisphosphate (IP3) receptors, but how a single target cell encodes different extracellular signals to generate specific cytosolic Ca2+ responses is unknown. In pancreatic acinar cells, acetylcholine evokes local Ca2+ spiking in the apical granular pole, whereas cholecystokinin elicits a mixture of local and global cytosolic Ca2+ signals. We show that IP3, cyclic ADP‐ribose and nicotinic acid adenine dinucleotide phosphate (NAADP) evoke cytosolic Ca2+ spiking by activating common oscillator units composed of IP3 and ryanodine receptors. Acetylcholine activation of these common oscillator units is triggered via IP3 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 specifically by cyclic ADP‐ribose receptors. In the apical pole there is a common early activation site for Ca2+ release, indicating that the three types of Ca2+ release channels are clustered together and that the appropriate receptors are selected at the earliest step of signal generation.

Transformation of local Ca2+ spikes to global Ca2+transients: the combinatorial roles of multiple Ca2+ releasing messengers

In pancreatic acinar cells, low, threshold concentrations of acetylcholine (ACh) or cholecystokinin (CCK) induce repetitive local cytosolic Ca2+ spikes in the apical pole, while higher concentrations elicit global signals. We have investigated the process that transforms local Ca2+ spikes to global Ca2+ transients, focusing on the interactions of multiple intracellular messengers. ACh‐elicited local Ca2+ spikes were transformed into a global sustained Ca2+ response by cyclic ADP‐ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP), whereas inositol 1,4,5‐trisphosphate (IP3) had a much weaker effect. In contrast, the response elicited by a low CCK concentration was strongly potentiated by IP3, whereas cADPR and NAADP had little effect. Experiments with messenger mixtures revealed a local interaction between IP3 and NAADP and a stronger global potentiating interaction between cADPR and NAADP. NAADP strongly amplified the local Ca2+ release evoked by a cADPR/IP3 mixture eliciting a vigorous global Ca2+ response. Different combinations of Ca2+ releasing messengers can shape the spatio‐temporal patterns of cytosolic Ca2+ signals. NAADP and cADPR are emerging as key messengers in the globalization of Ca2+ signals.

New Ca2+-releasing messengers: are they important in the nervous system?

In the nervous system, Ca2+ signalling is determined primarily by voltage-gated Ca2+-selective channels in the plasma membrane, but there is increasing evidence for involvement of intracellular Ca2+ stores in such signalling. It is generally assumed that neurotransmitter-elicited release of Ca2+ from internal stores is primarily mediated by Ins(1,4,5)P3, as originally discovered in pancreatic acinar cells. The more-recently discovered Ca2+-releasing messenger, cyclic ADP-ribose (cADPR), which activates ryanodine receptors, has so far only been implicated in a few cases, and the possible importance of another Ca2+-releasing molecule, nicotinic acid adenine dinucleotide phosphate (NAADP), has been ignored. Recent investigations of the action of the brain-gut peptide cholecystokinin on pancreatic acinar cells have indicated that NAADP and cADPR receptors are essential for Ca2+ release. Tools are available for testing the possible involvement of NAADP and cADPR in neurotransmitter-elicited intracellular Ca2+ release, and such studies could reveal complex mechanisms that control this release in the nervous system.

Brain mitochondrial defects amplify intracellular [Ca2+] rise and neurodegeneration but not Ca2+ entry during NMDA receptor activation

According to the “indirect” excitotoxicity hypothesis, mitochondrial defects increase Ca2+ entry into neurons by rendering NMDA‐R hypersensitive to glutamate. We tested this hypothesis by investigating in the rat striatum and cultured striatal cells how partial mitochondrial complex II inhibition produced by 3‐nitropropionic acid (3NP) modifies the toxicity of the NMDA‐R agonist quinolinate (QA). We showed that nontoxic 3NP treatment, leading to partial inhibition of complex II activity, greatly exacerbated striatal degeneration produced by slightly toxic QA treatment through an “all‐or‐nothing” process. The potentiation of QA‐induced cell death by 3NP was associated with increased calpain activity and massive calpain‐mediated cleavage of several postsynaptic proteins, suggesting major neuronal Ca2+ deregulation in the striatum. However, Ca2+ anomalies probably do not result from NMDA‐R hypersensitivity. Indeed, brain imaging experiments using [18F]fluorodeoxyglucose indirectly showed that 3NP did not increase QA‐induced ionic perturbations at the striatal glutamatergic synapses in vivo. Consistent with this, the exacerbation of QA toxicity by 3NP was not related to an increase in the QA‐induced entry of 45Ca2+ into striatal neurons. The present results demonstrate that the potentiation of NMDA‐R‐mediated excitotoxicity by mitochondrial defects involves primarily intracellular Ca2+ deregulation, in the absence of NMDA‐R hypersensitivity.—Jacquard, C., Trioulier, Y., Cosker, F., Escartin, C., Bizat, N., Hantraye, P., Cancela*, J. M., Bonvento, G., Brouillet, E. Brain mitochondrial defects amplify intracellular [Ca2+] rise and neurodegeneration but not Ca2+ entry during NMDA receptor activation. FASEB J. 20, E245–E259 (2006)

Co-ordination of Ca(2+) signalling in mammalian cells by the new Ca(2+)-releasing messenger NAADP.

Ca2+ signalling is one of the most important means in mammalian cells of relaying the action of hormones and neurotransmitters. The great diversity of agonist-induced Ca2+ signatures, visualized by optical imaging techniques, can be explained by the production of intracellular messengers triggering Ca2+ release from internal stores and/or by different coupling of Ca2+ release to Ca2+ entry. Several messengers, such as inositol trisphosphate and cyclic ADP-ribose, have been identified to date. More recent studies have reported the important role of a newly discovered Ca2+ releasing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). These studies have shown important interactions of these messengers in the generation of specific Ca2+ signals. NAADP acts at a very low concentration and seems to have a key role in sensitising cyclic ADP-ribose and inositol trisphosphate receptors. These points will be discussed in the present review.

Intracellular glucose switches between cyclic ADP-ribose and inositol trisphosphate triggering of cytosolic Ca2+ spiking

Cyclic ADP-ribose (cADPR) is a potentially important intracellular Ca2+ releasing messenger [1-5]. In pancreatic acinar cells where intracellular infusion of both inositol trisphosphate (IP3) and cADPR evoke repetitive Ca2+ spiking [6], the cADPR antagonist 8-NH2-cADPR [7], which blocks cADPR-evoked but not IP3-evoked Ca2+ spiking, can abolish Ca2+ spiking induced by physiological levels of the peptide hormone cholecystokinin (CCK) [8]. We have tested the effect of intracellular glucose on the ability of IP3, cADPR and CCK to induce cytosolic Ca2+ spikes in pancreatic acinar cells. In order to gain access to the intracellular cytosol, we used the whole-cell configuration of the patch-clamp technique [9] and monitored cytosolic Ca2+ concentration changes by measuring the Ca(2+)-dependent ionic current [10-13]. Glucose (300 microM to 10 mM) in the patch pipette/intracellular solution prevented cADPR from evoking Ca2+ spiking. The same effect was observed with 2-deoxy-glucose, but not L-glucose. In contrast, glucose potentiated IP3-evoked Ca2+ spiking. CCK evoked Ca2+ spiking irrespective of the presence or absence of intracellular glucose, but the cADPR antagonist 8-NH2-cADPR blocked CCK-evoked Ca2+ spiking only in the absence of intracellular glucose. This suggests that the hormone can evoke Ca2+ spiking via either the IP3 or the cADPR pathway. The intracellular glucose level may control a switch between these two pathways.

Nerve guidance: Attraction or repulsion by local Ca2+ signals

Recent studies have shown that cytosolic Ca2+ signals, generated on one side of a nerve growth cone, can induce turning either towards or away from the side of the Ca2+ signal, depending on the global Ca2+ level. The results indicate that local Ca2+ signals may provide important directional cues for axon guidance.

0346 : Characterization of the calcium deregulation in cardiomyocytes from mdx mice, the main rodent model of the Duchenne muscular dystrophy

The Duchenne muscular dystrophy (DMD) is caused by an absence or mutation of the protein dystrophin, which leads to smooth, skeletal and cardiac muscles degeneration. Intracellular calcium deregulation, notably abnormally high cytosolic calcium concentration, is thought to contribute to muscle necrosis and to cardiomyopathy development. Despite its importance in cell necrosis, the mechanisms involved in this calcium deregulation are not fully understood. In our study, we have investigated several parameters of the calcium homeostasis in cardiomyocytes from mdx mice, the main rodent model of the DMD. All the parameters have been measured by using confocal microscopy calcium imaging experiments in cardiomyocytes loaded with the calcium indicator Fluo-3AM. Our results show an increase of the occurrence of spontaneous calcium events such as a higher frequency of calcium sparks and waves in the mdx cardiomyocytes compared with wild type mice. We also found that the calcium sensitivity of the ryanodine receptors (RyRs) is increased in cardiomyocytes from mdx mice. Finally, the sarcoplasmic reticulum (SR) calcium content has been evaluated by caffeine-induced calcium release. We found reduced calcium content of the SR in the mdx mice, which could be explained, at least in part, by an excessive spontaneous calcium activity due to the enhanced RyRs sensitivity. Our data suggest that targeting this SR calcium “leak” with pharmacological tools could be beneficial for the DMD patients. The author hereby declares no conflict of interest