Abstract
Intracellular Ca2+ release is mostly mediated by inositol trisphosphate, but intracellular cyclic-ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are important messengers in many systems. Whereas cADPR generally activates type 2 ryanodine receptors (RyR2s), the NAADP-activated Ca2+ release mechanism is less clear. Using knockouts and antibodies against RyRs and Two-Pore Channels (TPCs), we have compared their relative importance for NAADP-induced Ca2+ release from two-photon permeabilized pancreatic acinar cells. In these cells, cholecystokinin-elicited Ca2+ release is mediated by NAADP. TPC2-KO reduced NAADP-induced Ca2+ release by 64%, but the combination of TPC2-KO and an antibody against TPC1, significantly reduced Ca2+ release by 86% (64% vs. 86%, p<0.0002). In RyR3-KO, NAADP-evoked Ca2+ release reduced by ∼50% but, when combined with antibodies against RyR1, responses were 90% inhibited. Antibodies against RyR2 had practically no effect on NAADP-evoked Ca2+ release, but reduced release in response to cADPR by 55%. Antibodies to RyR1 inhibited NAADP-induced Ca2+ liberation by 81%, but only reduced cADPR responses by 30%. We conclude that full NAADP-mediated Ca2+ release requires both TPCs and RyRs. The sequence of relative importance for NAADP-elicited Ca2+ release from the all stores is RyR1>TPC2>RyR3>TPC1>>RyR2. However, when assessing NAADP-induced Ca2+ release solely from the acidic stores (granules/endosomes/lysosomes), antibodies against TPC2 and TPC1 virtually abolished the Ca2+ liberation as did antibodies against RyR1 and RyR3. Our results indicate that the primary, but very small, NAADP-elicited Ca2+ release via TPCs from endosomes/lysosomes triggers the detectable Ca2+-induced Ca2+ release via RyR1 and RyR3 occurring from the granules and the ER.
Highlights
Exocrine gland cells have provided the most important models for elucidating the mechanisms underlying hormone- or neurotransmitter-evoked intracellular Ca2+ release
Our results provide fresh evidence for the importance of both Two-pore channels (TPC) and ryanodine receptor (RyR) in nicotinic acid adenine dinucleotide phosphate (NAADP)-elicited Ca2+ signalling in pancreatic acinar cells
It is well established that repetitive Ca2+ spiking – irrespective of whether it is elicited by ACh or CCK or by intracellular application of inositol trisphosphate (IP3), cADPR or NAADP - is dependent on both operational IP3Rs and RyRs [18] and these findings, together with the direct demonstration that Ca2+ spiking can be elicited by intracellular Ca2+ infusion [40], led to the conclusion that an initial small release of Ca2+ from intracellular stores mediated either by activation of IP3Rs or RyRs caused a much larger release from both IP3Rs and RyRs via the mechanism of Ca2+-induced Ca2+ release (CICR) [18]
Summary
Exocrine gland cells have provided the most important models for elucidating the mechanisms underlying hormone- or neurotransmitter-evoked intracellular Ca2+ release. Studies on pancreatic acinar cells led to many important findings including the discovery of inositol trisphosphate (IP3) as a messenger releasing Ca2+ from intracellular stores [3], localized Ca2+. The acid stores, like the ER, have been shown to respond to all the three intracellular Ca2+ releasing messengers IP3, cADPR and NAADP [18,22]. Our group has emphasized the primary importance of ryanodine receptors for NAADP-induced Ca2+ release in both the secretory granule stores and the ER [22]. We report a comprehensive comparison of the role of different types of RyRs and TPCs in NAADP-induced Ca2+ release in pancreatic acinar cells
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