Dans les cellules acineuses du pancréas exocrine, les agonistes acétylcholine (ACh) et Cholécystokinine (CCK) déclenchent des oscillations calciques via les messagers libérant du calcium IP3 et cADPR pour l'ACh, et NAADP et cADPR pour la CCK. L'existence de plusieurs messagers et de plusieurs réserves intracellulaires de Ca2+ pose la question d'une possible coopération de ces messagers, et du recrutement sélectif des différentes réserves de Ca2+.<br />Notre travail montre que lors de l'initiation des oscillations calciques induites par la CCK, le NAADP recrute le Ca2+ du lysosome et de l'endosome, tandis que dans le cas de l'ACh, l'IP3 recrute le Ca2+ des granules de zymogène. Cette réponse calcique est ensuite maintenue et amplifiée par le réticulum endoplasmique (RE) sous contrôle du cADPR. <br />Nous proposons que les messagers déterminent les signatures calciques des agonistes en contrôlant la contribution de chaque réserve acide et du RE.<br />Notre travail montre que la principale enzyme de synthèse de NAADP chez la souris est l'antigène de surface CD38, bien qu'une autre enzyme de synthèse existe dans le cerveau. Nos résultats montrent aussi pour la première fois que dans les muscles squelettiques, le monoxyde d'azote inhibe la synthèse de NAADP.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca²⁺ mobilising messenger in mammalian and non-mammalian cells. Studies on a variety of cell types suggest that NAADP evokes Ca²⁺ release from a lysosome-related store and via activation of a receptor distinct from either ryanodine receptors (RyR) or inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R). However, the identity of the NAADP receptor has, until now, remained elusive. In this thesis I have shown that NAADP-evoked Ca²⁺ release from lysosomes is underpinned by two-pore channels (TPCs), of which there are 3 subtypes, TPC1, TPC2 and TPC3. When stably over-expressed in HEK293 cells, TPC2 was found to be specifically targeted to lysosomes, while TPC1 and TPC3 were targeted to endosomes. Initial Ca²⁺ signals via TPC2, but not those via TPC1, were amplified into global Ca²⁺ waves by Ca²⁺-induced Ca²⁺ release (CICR) from the endoplasmic reticulum (ER) via IP₃Rs. I have shown that, consistent with a role for TPCs in NAADP-mediated Ca²⁺ release, TPC2 is expressed in pulmonary arterial smooth muscle cells (PASMCs), is likely targeted to lysosomal membranes, and that TPCs also underpin NAADP-evoked Ca²⁺ signalling in this cell type. However, and in contrast to HEK293 cells, in PASMCs NAADP evokes spatially restricted Ca²⁺ bursts that are amplified into global Ca²⁺ waves by CICR from the sarcoplasmic reticulum (SR) via a subpopulation of RyRs, but not via IP₃Rs. I have demonstrated that lysosomes preferentially co-localise with RyR subtype 3 (RyR3) in the perinuclear region of PASMCs to comprise a “trigger zone” for Ca²⁺ signalling by NAADP, away from which a propagating Ca²⁺ wave may be carried by subsequent recruitment of RyR2. The identification of TPCs as a family of NAADP receptors may further our understanding of the mechanisms that confer the versatility of Ca²⁺ signalling which is required to regulate such diverse cellular functions as gene expression, fertilization, cell growth, and ultimately cell death.