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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.


Related in: MedlinePlus

NAADP-elicited Ca2+ release from intracellular stores was reduced in pancreatic acinar cells isolated from RyR3 KO mice. (A) Control application of NAADP (100 nM) to permeabilized pancreatic acinar cells isolated from wt mice (n = 12). (B) Application of NAADP (100 nM) to permeabilized pancreatic acinar cells isolated from RyR3 KO mice (n = 8). (C) Permeabilized cells from RyR3 KO were incubated with RyR1 antibodies for 20 min (1:100) followed by subsequent applications of 100 nM NAADP and 10 μM thapsigargin (n = 10). (D) Permeabilized cells from RyR3 KO were incubated with RyR1 antibodies for 20 min (1:100) followed by subsequent applications of 10 μM cADPR and 10 μM thapsigargin (10.6 ± 0.9%, n = 9 as compared to control cADPR responses from wt 21.6 ± 0.7, n = 11). (E) Comparison of amplitudes of Ca2+ responses to NAADP (100 nM), IP3 (10 μM) and thapsigargin (10 μM) obtained using permeabilized cells from wt mice and RyR3 KO mice in the presence or absence of treatment with antibody against RyR1 (n > 4 for each group). Data represent mean values ± SEM. Cells were loaded with Fluo-5N AM.
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fig0020: NAADP-elicited Ca2+ release from intracellular stores was reduced in pancreatic acinar cells isolated from RyR3 KO mice. (A) Control application of NAADP (100 nM) to permeabilized pancreatic acinar cells isolated from wt mice (n = 12). (B) Application of NAADP (100 nM) to permeabilized pancreatic acinar cells isolated from RyR3 KO mice (n = 8). (C) Permeabilized cells from RyR3 KO were incubated with RyR1 antibodies for 20 min (1:100) followed by subsequent applications of 100 nM NAADP and 10 μM thapsigargin (n = 10). (D) Permeabilized cells from RyR3 KO were incubated with RyR1 antibodies for 20 min (1:100) followed by subsequent applications of 10 μM cADPR and 10 μM thapsigargin (10.6 ± 0.9%, n = 9 as compared to control cADPR responses from wt 21.6 ± 0.7, n = 11). (E) Comparison of amplitudes of Ca2+ responses to NAADP (100 nM), IP3 (10 μM) and thapsigargin (10 μM) obtained using permeabilized cells from wt mice and RyR3 KO mice in the presence or absence of treatment with antibody against RyR1 (n > 4 for each group). Data represent mean values ± SEM. Cells were loaded with Fluo-5N AM.

Mentions: To test for a role of RyRs in NAADP-induced Ca2+ signalling, we have employed permeabilized pancreatic acinar cells isolated from RyR3 KO mice. The NAADP (100 nM) elicited Ca2+ release from the internal stores of RyR3 KO cells was reduced to about 48% of the control value obtained from wt cells (Fig. 4A, B and E). These data strongly suggest that RyR3 is involved in NAADP-induced Ca2+ signalling. Pre-incubation of permeabilized cells isolated from RyR3−/− cells with a RyR1 antibody almost abolished the NAADP responses (∼90% inhibition) (Fig. 4C and E). A similar protocol using RyR3 KO cells in the presence of RyR1 antibody was able to block cADPR-mediated Ca2+ release by only 49% (Fig. 4D and E). Responses to IP3 (10 μM) or thapsigargin (10 μM) in cells from RyR3 KO animals were not significantly different from responses in wt cells (Fig. 4E).


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)

NAADP-elicited Ca2+ release from intracellular stores was reduced in pancreatic acinar cells isolated from RyR3 KO mice. (A) Control application of NAADP (100 nM) to permeabilized pancreatic acinar cells isolated from wt mice (n = 12). (B) Application of NAADP (100 nM) to permeabilized pancreatic acinar cells isolated from RyR3 KO mice (n = 8). (C) Permeabilized cells from RyR3 KO were incubated with RyR1 antibodies for 20 min (1:100) followed by subsequent applications of 100 nM NAADP and 10 μM thapsigargin (n = 10). (D) Permeabilized cells from RyR3 KO were incubated with RyR1 antibodies for 20 min (1:100) followed by subsequent applications of 10 μM cADPR and 10 μM thapsigargin (10.6 ± 0.9%, n = 9 as compared to control cADPR responses from wt 21.6 ± 0.7, n = 11). (E) Comparison of amplitudes of Ca2+ responses to NAADP (100 nM), IP3 (10 μM) and thapsigargin (10 μM) obtained using permeabilized cells from wt mice and RyR3 KO mice in the presence or absence of treatment with antibody against RyR1 (n > 4 for each group). Data represent mean values ± SEM. Cells were loaded with Fluo-5N AM.
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fig0020: NAADP-elicited Ca2+ release from intracellular stores was reduced in pancreatic acinar cells isolated from RyR3 KO mice. (A) Control application of NAADP (100 nM) to permeabilized pancreatic acinar cells isolated from wt mice (n = 12). (B) Application of NAADP (100 nM) to permeabilized pancreatic acinar cells isolated from RyR3 KO mice (n = 8). (C) Permeabilized cells from RyR3 KO were incubated with RyR1 antibodies for 20 min (1:100) followed by subsequent applications of 100 nM NAADP and 10 μM thapsigargin (n = 10). (D) Permeabilized cells from RyR3 KO were incubated with RyR1 antibodies for 20 min (1:100) followed by subsequent applications of 10 μM cADPR and 10 μM thapsigargin (10.6 ± 0.9%, n = 9 as compared to control cADPR responses from wt 21.6 ± 0.7, n = 11). (E) Comparison of amplitudes of Ca2+ responses to NAADP (100 nM), IP3 (10 μM) and thapsigargin (10 μM) obtained using permeabilized cells from wt mice and RyR3 KO mice in the presence or absence of treatment with antibody against RyR1 (n > 4 for each group). Data represent mean values ± SEM. Cells were loaded with Fluo-5N AM.
Mentions: To test for a role of RyRs in NAADP-induced Ca2+ signalling, we have employed permeabilized pancreatic acinar cells isolated from RyR3 KO mice. The NAADP (100 nM) elicited Ca2+ release from the internal stores of RyR3 KO cells was reduced to about 48% of the control value obtained from wt cells (Fig. 4A, B and E). These data strongly suggest that RyR3 is involved in NAADP-induced Ca2+ signalling. Pre-incubation of permeabilized cells isolated from RyR3−/− cells with a RyR1 antibody almost abolished the NAADP responses (∼90% inhibition) (Fig. 4C and E). A similar protocol using RyR3 KO cells in the presence of RyR1 antibody was able to block cADPR-mediated Ca2+ release by only 49% (Fig. 4D and E). Responses to IP3 (10 μM) or thapsigargin (10 μM) in cells from RyR3 KO animals were not significantly different from responses in wt cells (Fig. 4E).

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.


Related in: MedlinePlus