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Both RyRs and TPCs are required for NAADP-induced intracellular Ca²⁺ release.

Gerasimenko JV, Charlesworth RM, Sherwood MW, Ferdek PE, Mikoshiba K, Parrington J, Petersen OH, Gerasimenko OV - Cell Calcium (2015)

Bottom Line: Antibodies against RyR2 had practically no effect on NAADP-evoked Ca(2+) release, but reduced release in response to cADPR by 55%.We conclude that full NAADP-mediated Ca(2+) release requires both TPCs and RyRs.Our results indicate that the primary, but very small, NAADP-elicited Ca(2+) release via TPCs from endosomes/lysosomes triggers the detectable Ca(2+)-induced Ca(2+) release via RyR1 and RyR3 occurring from the granules and the ER.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Group, Cardiff School of Biosciences, Cardiff University, Cardiff, Wales, UK.

No MeSH data available.


Ned-19 at high concentration (100 μM) considerably inhibited CCK responses in pancreatic acinar cells. A–B. Representative traces of responses to 5 pM CCK (A) or 20 nM ACh (B) followed by application of 100 μM Ned-19 in the continuous presence of CCK (A, n = 9) or ACh (B, n = 14). C–D. Quantification of the area under the curve (during 400 s from the addition point of 5 pM CCK or 100 μM Ned-19) in the experiments shown in A and B (41.3 ± 4.2 a.u. for 5 pM CCK and 0.5 ± 0.6 a.u. for 5 pM CCK and 100 μM Ned-19). Quantification of the area under the curve for ACh and Ned-19 is shown in D: 35.7 ± 12 a.u. for 20 nM ACh and 37.9 ± 11.6 a.u. for 20 nM ACh and 100 μM Ned-19 (D)). Error bars show ± SEM. *p < 4.9*10−8; n/s-non significant p > 0.78. Cells were loaded with Fluo-4 in AM form.
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fig0005: Ned-19 at high concentration (100 μM) considerably inhibited CCK responses in pancreatic acinar cells. A–B. Representative traces of responses to 5 pM CCK (A) or 20 nM ACh (B) followed by application of 100 μM Ned-19 in the continuous presence of CCK (A, n = 9) or ACh (B, n = 14). C–D. Quantification of the area under the curve (during 400 s from the addition point of 5 pM CCK or 100 μM Ned-19) in the experiments shown in A and B (41.3 ± 4.2 a.u. for 5 pM CCK and 0.5 ± 0.6 a.u. for 5 pM CCK and 100 μM Ned-19). Quantification of the area under the curve for ACh and Ned-19 is shown in D: 35.7 ± 12 a.u. for 20 nM ACh and 37.9 ± 11.6 a.u. for 20 nM ACh and 100 μM Ned-19 (D)). Error bars show ± SEM. *p < 4.9*10−8; n/s-non significant p > 0.78. Cells were loaded with Fluo-4 in AM form.

Mentions: Previously it has been shown that Ca2+ responses elicited by physiological concentrations of CCK can be specifically blocked by inactivation of the NAADP receptor, while responses to ACh were unaffected [18,23]. The relationship between CCK action and NAADP signalling was shown by measurements of the intracellular level of NAADP that were increased in a dose-dependent manner upon stimulation with physiological concentrations of CCK. In contrast, ACh did not change the intracellular NAADP concentrations [17]. We employed the cell-permeable NAADP analogue and selective antagonist, Ned-19 [32] in order to explore differences between the mechanisms of ACh- and CCK-evoked Ca2+ signal generation. In a previous study, Ned-19, at a high concentration (100 μM), was shown to block NAADP-mediated Ca2+ release and binding to the NAADP receptor [33]. In our experiments on intact acinar cells, exposure of the cells to 100 μM Ned-19 was able to block completely cytosolic Ca2+ responses induced by 5 pM CCK (Fig. 1A and C), but was unable to inhibit Ca2+ oscillations produced by 20 nM ACh (Fig. 1B and D).


Both RyRs and TPCs are required for NAADP-induced intracellular Ca²⁺ release.

Gerasimenko JV, Charlesworth RM, Sherwood MW, Ferdek PE, Mikoshiba K, Parrington J, Petersen OH, Gerasimenko OV - Cell Calcium (2015)

Ned-19 at high concentration (100 μM) considerably inhibited CCK responses in pancreatic acinar cells. A–B. Representative traces of responses to 5 pM CCK (A) or 20 nM ACh (B) followed by application of 100 μM Ned-19 in the continuous presence of CCK (A, n = 9) or ACh (B, n = 14). C–D. Quantification of the area under the curve (during 400 s from the addition point of 5 pM CCK or 100 μM Ned-19) in the experiments shown in A and B (41.3 ± 4.2 a.u. for 5 pM CCK and 0.5 ± 0.6 a.u. for 5 pM CCK and 100 μM Ned-19). Quantification of the area under the curve for ACh and Ned-19 is shown in D: 35.7 ± 12 a.u. for 20 nM ACh and 37.9 ± 11.6 a.u. for 20 nM ACh and 100 μM Ned-19 (D)). Error bars show ± SEM. *p < 4.9*10−8; n/s-non significant p > 0.78. Cells were loaded with Fluo-4 in AM form.
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fig0005: Ned-19 at high concentration (100 μM) considerably inhibited CCK responses in pancreatic acinar cells. A–B. Representative traces of responses to 5 pM CCK (A) or 20 nM ACh (B) followed by application of 100 μM Ned-19 in the continuous presence of CCK (A, n = 9) or ACh (B, n = 14). C–D. Quantification of the area under the curve (during 400 s from the addition point of 5 pM CCK or 100 μM Ned-19) in the experiments shown in A and B (41.3 ± 4.2 a.u. for 5 pM CCK and 0.5 ± 0.6 a.u. for 5 pM CCK and 100 μM Ned-19). Quantification of the area under the curve for ACh and Ned-19 is shown in D: 35.7 ± 12 a.u. for 20 nM ACh and 37.9 ± 11.6 a.u. for 20 nM ACh and 100 μM Ned-19 (D)). Error bars show ± SEM. *p < 4.9*10−8; n/s-non significant p > 0.78. Cells were loaded with Fluo-4 in AM form.
Mentions: Previously it has been shown that Ca2+ responses elicited by physiological concentrations of CCK can be specifically blocked by inactivation of the NAADP receptor, while responses to ACh were unaffected [18,23]. The relationship between CCK action and NAADP signalling was shown by measurements of the intracellular level of NAADP that were increased in a dose-dependent manner upon stimulation with physiological concentrations of CCK. In contrast, ACh did not change the intracellular NAADP concentrations [17]. We employed the cell-permeable NAADP analogue and selective antagonist, Ned-19 [32] in order to explore differences between the mechanisms of ACh- and CCK-evoked Ca2+ signal generation. In a previous study, Ned-19, at a high concentration (100 μM), was shown to block NAADP-mediated Ca2+ release and binding to the NAADP receptor [33]. In our experiments on intact acinar cells, exposure of the cells to 100 μM Ned-19 was able to block completely cytosolic Ca2+ responses induced by 5 pM CCK (Fig. 1A and C), but was unable to inhibit Ca2+ oscillations produced by 20 nM ACh (Fig. 1B and D).

Bottom Line: Antibodies against RyR2 had practically no effect on NAADP-evoked Ca(2+) release, but reduced release in response to cADPR by 55%.We conclude that full NAADP-mediated Ca(2+) release requires both TPCs and RyRs.Our results indicate that the primary, but very small, NAADP-elicited Ca(2+) release via TPCs from endosomes/lysosomes triggers the detectable Ca(2+)-induced Ca(2+) release via RyR1 and RyR3 occurring from the granules and the ER.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Group, Cardiff School of Biosciences, Cardiff University, Cardiff, Wales, UK.

No MeSH data available.