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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.


(A) Schematic distribution of RyRs type-dependent responses to 100 nM NAADP in pancreatic acinar cells from wt (a) or RyR3-KO (b) (based on knockouts and antibodies data shown in Fig. 4). (B) Schematic distribution of RyRs type-dependent responses to cADPR in pancreatic acinar cells from wt (a) or from RyR3-KO (b) (based on knockouts and antibodies data shown in Fig. 4). (C) Comparison of the relative importance of the RyRs and TPCs for the NAADP-induced Ca2+ responses in permeabilized pancreatic acinar cells. Ryanodine (100 μM) practically abolished responses to NAADP (0.71 ± 0.30%, n = 7) as compared to control (27.3 ± 1.8%, n = 12). Ruthenium Red (10 μM) also abolished responses to NAADP (0.33 ± 0.11, n = 6; not shown). Mixture of antibodies to all types of RyRs (RyR1 + RyR2 + RyR3) also dramatically reduced responses (1.27 ± 0.15%, n = 3). Specific inhibitor of NAADP-induced signalling Ned-19 has significantly reduced responses (8.6 ± 1.12%, n = 5). Mixture of antibodies to TPC1 and TPC2 reduced responses (5.3 ± 0.24%, n = 7). Antibodies to TPC1 applied to the cells isolated from TPC2 knockouts reduced responses (3.9 ± 0.27%, n = 7). Error bars show ± SEM. Cells were loaded with Fluo-5N in AM form before permeabilization.
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fig0025: (A) Schematic distribution of RyRs type-dependent responses to 100 nM NAADP in pancreatic acinar cells from wt (a) or RyR3-KO (b) (based on knockouts and antibodies data shown in Fig. 4). (B) Schematic distribution of RyRs type-dependent responses to cADPR in pancreatic acinar cells from wt (a) or from RyR3-KO (b) (based on knockouts and antibodies data shown in Fig. 4). (C) Comparison of the relative importance of the RyRs and TPCs for the NAADP-induced Ca2+ responses in permeabilized pancreatic acinar cells. Ryanodine (100 μM) practically abolished responses to NAADP (0.71 ± 0.30%, n = 7) as compared to control (27.3 ± 1.8%, n = 12). Ruthenium Red (10 μM) also abolished responses to NAADP (0.33 ± 0.11, n = 6; not shown). Mixture of antibodies to all types of RyRs (RyR1 + RyR2 + RyR3) also dramatically reduced responses (1.27 ± 0.15%, n = 3). Specific inhibitor of NAADP-induced signalling Ned-19 has significantly reduced responses (8.6 ± 1.12%, n = 5). Mixture of antibodies to TPC1 and TPC2 reduced responses (5.3 ± 0.24%, n = 7). Antibodies to TPC1 applied to the cells isolated from TPC2 knockouts reduced responses (3.9 ± 0.27%, n = 7). Error bars show ± SEM. Cells were loaded with Fluo-5N in AM form before permeabilization.

Mentions: Fig. 5 A and B presents schematically the relative importance of RyR sub-types for NAADP- and cADPR-induced Ca2+ release. By analysing the distribution of RyR type-dependent responses to 100 nM NAADP or cADPR in cells from wt and RyR3 KO mice in the presence or in the absence of RyR type specific antibodies (Fig. 4F and G) we have shown that RyR types 1 and 3 are most important for NAADP-elicited Ca2+ release, whereas RyR2 is mainly responsible for the cADPR-induced responses.


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)

(A) Schematic distribution of RyRs type-dependent responses to 100 nM NAADP in pancreatic acinar cells from wt (a) or RyR3-KO (b) (based on knockouts and antibodies data shown in Fig. 4). (B) Schematic distribution of RyRs type-dependent responses to cADPR in pancreatic acinar cells from wt (a) or from RyR3-KO (b) (based on knockouts and antibodies data shown in Fig. 4). (C) Comparison of the relative importance of the RyRs and TPCs for the NAADP-induced Ca2+ responses in permeabilized pancreatic acinar cells. Ryanodine (100 μM) practically abolished responses to NAADP (0.71 ± 0.30%, n = 7) as compared to control (27.3 ± 1.8%, n = 12). Ruthenium Red (10 μM) also abolished responses to NAADP (0.33 ± 0.11, n = 6; not shown). Mixture of antibodies to all types of RyRs (RyR1 + RyR2 + RyR3) also dramatically reduced responses (1.27 ± 0.15%, n = 3). Specific inhibitor of NAADP-induced signalling Ned-19 has significantly reduced responses (8.6 ± 1.12%, n = 5). Mixture of antibodies to TPC1 and TPC2 reduced responses (5.3 ± 0.24%, n = 7). Antibodies to TPC1 applied to the cells isolated from TPC2 knockouts reduced responses (3.9 ± 0.27%, n = 7). Error bars show ± SEM. Cells were loaded with Fluo-5N in AM form before permeabilization.
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fig0025: (A) Schematic distribution of RyRs type-dependent responses to 100 nM NAADP in pancreatic acinar cells from wt (a) or RyR3-KO (b) (based on knockouts and antibodies data shown in Fig. 4). (B) Schematic distribution of RyRs type-dependent responses to cADPR in pancreatic acinar cells from wt (a) or from RyR3-KO (b) (based on knockouts and antibodies data shown in Fig. 4). (C) Comparison of the relative importance of the RyRs and TPCs for the NAADP-induced Ca2+ responses in permeabilized pancreatic acinar cells. Ryanodine (100 μM) practically abolished responses to NAADP (0.71 ± 0.30%, n = 7) as compared to control (27.3 ± 1.8%, n = 12). Ruthenium Red (10 μM) also abolished responses to NAADP (0.33 ± 0.11, n = 6; not shown). Mixture of antibodies to all types of RyRs (RyR1 + RyR2 + RyR3) also dramatically reduced responses (1.27 ± 0.15%, n = 3). Specific inhibitor of NAADP-induced signalling Ned-19 has significantly reduced responses (8.6 ± 1.12%, n = 5). Mixture of antibodies to TPC1 and TPC2 reduced responses (5.3 ± 0.24%, n = 7). Antibodies to TPC1 applied to the cells isolated from TPC2 knockouts reduced responses (3.9 ± 0.27%, n = 7). Error bars show ± SEM. Cells were loaded with Fluo-5N in AM form before permeabilization.
Mentions: Fig. 5 A and B presents schematically the relative importance of RyR sub-types for NAADP- and cADPR-induced Ca2+ release. By analysing the distribution of RyR type-dependent responses to 100 nM NAADP or cADPR in cells from wt and RyR3 KO mice in the presence or in the absence of RyR type specific antibodies (Fig. 4F and G) we have shown that RyR types 1 and 3 are most important for NAADP-elicited Ca2+ release, whereas RyR2 is mainly responsible for the cADPR-induced responses.

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.