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Fig. 4. The NAADP-sensitive acidic Ca²⁺ store is substantial and appears not to be located in lysosomes or the Golgi. (A) Comparison of the relative content and relative responses to Ca²⁺ releasing messengers from the ER (blue, on the left) Ca²⁺ store (response to 10 µM thapsigargin or Ca²⁺ releasing messenger in the presence of bafilomycin A1 or nigericin) and the acidic Ca²⁺ store (pink, on the right; subsequent application of 10 µM ionomycin/7 µM nigericin and 2 mM of EGTA or Ca²⁺ releasing messenger after 10 µM thapsigargin). Bars represent standard errors. Asterisks show the amplitudes compared with control using t-test, P>0.005. (B) GPN (50 µM) induces cytosolic Ca²⁺ signals on its own and inhibits the responses to both CCK (2 pM) and ACh (10 nM); intact cells were loaded with Fluo-4 AM. (C) GPN (50 µM) in the presence of the protease inhibitor CI-1 does not inhibit the cytosolic responses to CCK (2 pM) or ACh (10 nM); intact cells were loaded with Fluo-4 AM. (D) Treatment of cells with 10 µM CI-1 did not affect GPN-induced permeabilization of lysosomes measured with either LysoTracker Red (red, on the left) or BODIPY FL-pepstatin A (green, on the right). All columns represent relative amplitudes of GPN-induced decrease of LysoTracker Red and BODIPY FL-pepstatin A fluorescence intensity in pancreatic acinar cells in the presence or absence of CI-1. Bars indicate s.e.m. (E) GPN (50 µM) alone blocks NAADP-induced responses from the acidic store of permeabilized cells. (F) NAADP-induced Ca²⁺ release in the presence of GPN and Cl-1 after application of thapsigargin. (a) GPN (50 µM) with CI-1 (10 µM) does not block NAADP-induced Ca²⁺ response from the secretory granule area of the permeabilized cell. (b) Averaged traces from the last 100 seconds (dotted boxes) of the experiments shown in a and c (from the granular (blue) and basal (red) areas, respectively) with application of 100 nM NAADP in the continuous presence of 10 µM thapsigargin, and GPN with CI-1 (n=10, P<0.002, asterisk shows the time point at which the amplitudes were compared using t-test; bars represent standard errors. (c) No response is seen in the basal area. (G) Pre-treatment with brefeldin A does not block response to NAADP. (a) Brefeldin A does not block NAADP-induced Ca²⁺ responses in the secretory granular area of the permeabilized cell. (b) Averaged traces from the last 100 seconds (dotted boxes) of the experiments shown in a and c (from the granular (blue) and basal (red) areas, respectively) with application of 100 nM NAADP in the continuous presence of 10 µM thapsigargin and 10 µM brefeldin A (n=7, P<0.0007, asterisk shows time point at which the amplitudes were compared using a t-test). Bars represent standard errors. (c) No response is seen in the basal area. Cells were loaded with Fluo-5N AM.

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