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Previous investigations on pulmonary artery smooth muscle cells have shown that nicotinic acid adenine dinucleotide diphosphate (NAADP) evokes highly localised intracellular Ca²⁺ bursts by mobilising thapsigargin-insensitive Ca²⁺ stores. Such localised Ca²⁺ signals may initiate global Ca²⁺ waves and contraction of the myocytes through the recruitment of ryanodine receptors (RyR) located on the sarcoplasmic reticulum (SR) via Ca²⁺-induced Ca²⁺-release (CICR). In this thesis I have shown that NAADP evokes localised Ca²⁺ signals through the mobilisation of a bafilomycin A1-sensitive, lysosome-related Ca²⁺ store. Lysosomal Ca²⁺ stores facilitate this process by colocalising with a subpopulation of RyRs on the surface of the SR to comprise a highly specialised trigger zone for Ca²⁺ signalling by NAADP. I have also shown that the proposed trigger zone for NAADP-dependent Ca²⁺ signalling may be formed between lysosomes and clusters of RyR subtype 3 (RyR3) located in close proximity to one another in the perinuclear region of cells. Localised Ca²⁺ bursts generated by NAADP-dependent Ca²⁺ release from acidic Ca²⁺ stores and subsequent CICR via RyR3 on the SR may then amplify Ca²⁺ bursts into a propagating Ca²⁺ signal by recruiting clusters of RyR subtype 2 (RyR2) located in the perinuclear and extra-perinuclear regions of the cell. The presence of this trigger zone may explain, in part, why Ca²⁺ bursts by NAADP induce, in an all-or-none manner, global Ca²⁺ signals by CICR via RyRs on the SR. Consistent with a role for NAADP and lysosomes as a discrete and agonist-specific Ca²⁺ signalling pathway utilised by vasoconstrictors, I have shown that endothelin-1 (ET-1), but not phenylephrine or prostaglandin-F2α, mobilises Ca²⁺ stores by NAADP, and that ET-1 initiates Ca²⁺ signalling by NAADP in a receptor subtype-specific manner through the activation of ETB receptors. These findings further advance our understanding of how that spatial organisation of discrete, organellar Ca²⁺ stores underpin the generation of differential Ca²⁺ signalling patterns by different Ca²⁺-mobilising messengers.

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Le calcium est un second messager impliqué dans la plupart des fonctions neuronales et possède un rôle particulièrement important dans la régulation de l'expression génique. Pour activer l'expression de certains gènes, une augmentation de la concentration calcique au sein du noyau est essentielle. L'augmentation du calcium intracellulaire dépend entre autre, de la mobilisation du calcium contenu dans les stocks intracellulaires par des seconds messagers comme l'IP3, le cADPR et le NAADP, plus récemment découvert. Au cours de ma thèse, j'ai montré que dans les neurones d'aplysies, l'Aplysia ADP ribosyl cyclase, l'enzyme de synthèse du cADPR et du NAADP migrait dans le noyau suite à la dépolarisation et à l'entrée de calcium par les canaux voltage dépendants de type L. De plus, grâce à la microscopie confocale et l'utilisation de sondes calciques fluorescentes, nous avons observé sur des noyaux de neurones isolés, que les seconds messagers étaient capables de mobiliser le calcium contenu dans l'enveloppe nucléaire pour générer des oscillations calciques nucléoplasmiques. Nos données pharmacologiques montrent que le NAADP active un récepteur qui lui est propre et que ce dernier coopère avec les RyRs et IP3R pour générer ces signaux. Finalement, afin d'explorer les rôles du NAADP dans la physiologie du neurone entier, j'ai mis au point un modèle de neurones spinaux embryonnaire de souris. Les résultats préliminaires nous permettent de poser de nouvelles hypothèses concernant l'implication du NAADP dans certaines grandes fonctions neuronales régulées par les neurotrophines.

Nicotinic acid-adenine dinucleotide phosphate (NAADP) is a recently described potent intracellular Ca2+-mobilizing messenger active in a wide range of diverse cell types. In the present study, we have investigated the interaction of NAADP with other Ca2+-mobilizing messengers in the release of transmitter at the frog neuromuscular junction. We show, for the first time, that NAADP enhances neurosecretion in response to inositol 1,4,5-trisphosphate (IP3), cADP-ribose (cADPR) and sphingosine 1-phosphate (S1P), but not sphingosylphosphorylcholine. Thapsigargin was without effect on transmitter release in response to NAADP, but blocked the responses to subsequent application of IP3, cADPR and S1P and their potentiation by NAADP. Asynchronous neurotransmitter release may therefore involve functional coupling of endoplasmic reticulum Ca2+ stores with distinct Ca2+ stores targeted by NAADP.