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Fig. 2. NAADP, IP₃ or cADPR elicit Ca²⁺ release from both thapsigargin-sensitive and thapsigargin-insensitive intracellular stores. (A) NAADP induces large Ca²⁺ release before and small Ca²⁺ release after thapsigargin. (a) Representative trace (from whole permeabilized cell) shows that a short application of IP₃ (10 µM) induces a typical Ca²⁺ release, which is similar to that subsequently obtained in response to NAADP (100 nM). Thereafter, thapsigargin (10 µM) induces a more substantial liberation of Ca²⁺. NAADP (100 nM) added on the plateau of the thapsigargin response induces a further small Ca²⁺ release. Cells were loaded with Fluo-5N AM. (b) Same experiment as shown in a with the region of interest (ROI) in the granular area (blue trace). NAADP (100 nM) induces Ca²⁺ release from the store in the secretory granule area in the presence of thapsigargin (10 µM). (c) Averaged traces from the last 100 seconds of the experiments shown in b and d, (dotted boxes) with application of 100 nM NAADP in the continuous presence of 10 µM thapsigargin. Blue trace, granular area; red trace basal area (n=20, P<0.001, asterisk shows the time point at which the amplitudes in the granular and basal areas were compared using a t-test; bars represent standard errors). (d) Same experiment as in a, but now the ROI is in the basal area (red trace). NAADP (100 nM) does not induce any noticeable Ca²⁺ release in the presence of thapsigargin (10 µM). (B) IP₃ induces small Ca²⁺ response after application of thapsigargin. (a) In a separate experiment, IP₃ (10 µM) induces a small Ca²⁺ release in the granular area of the permeabilized cell in the presence of thapsigargin (10 µM). Cells were loaded with Fluo-5N AM. (b) Averaged traces of the last 100 seconds (dotted boxes) of the experiments shown in a and c (from granular (blue) and basal (red) area, respectively) with application of 10 µM IP₃ in the continuous presence of 10 µM thapsigargin (n=10, P<0.001, asterisk shows the time point at which the amplitudes in the granular and basal areas were compared using a t-test; bars represent standard errors) (c) In the basal area of the same cell, IP₃ (10 µM) fails to elicit further Ca²⁺ release in the presence of thapsigargin (10 µM). (C) cADPR induces a small Ca²⁺ release after thapsigargin. (a) cADPR (10 µM) releases Ca²⁺ in the presence of thapsigargin (10 µM) in the granular area of the permeabilized cell. Cells were loaded with Fluo-5N AM. (b) Averaged traces the last 100 seconds (dotted boxes) of the experiments shown in a and c (from granular (blue) and basal (red) area, respectively), with application of 10 µM cADPR in the continuous presence of 10 µM thapsigargin (n=8, P<0.002, asterisk shows the time point at which the amplitudes were compared using a t-test; bars represent standard errors). (c) Same experiment as shown in a, but with the ROI in the basal area. Whereas cADPR (10 µM) releases Ca²⁺ in the absence of thapsigargin, there is no effect of the messenger in the presence of the SERCA pump inhibitor.

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