QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online January 12, 2006
doi: 10.1242/10.1242/jcs.02721

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gerasimenko, J. V. Articles by Gerasimenko, O. V. Search for Related Content PubMed PubMed Citation Articles by Gerasimenko, J. V. Articles by Gerasimenko, O. V.

NAADP, cADPR and IP₃ all release Ca²⁺ from the endoplasmic reticulum and an acidic store in the secretory granule area

MRC Secretory Control Research Group, The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool, L69 3BX, UK

View larger version (39K): [in a new window]   Fig. 1. Two-photon permeabilization of pancreatic acinar cells and the effects of Ca2+ releasing messengers. (A) A pancreatic acinar cell doublet loaded with Fluo-5N AM before permeabilization. Blue dot shows the position of two-photon light application. (B) Same cell doublet after permeabilization and perfusion with Texas Red dextran (Mr 3x103). Only the lower cell has been permeabilized and is therefore bright due to diffusion of Texas Red dextran into the cytoplasm. (C) Same cell doublet after washing out of Texas Red dextran. Note reduced fluorescence of Fluo-5N in the lower permeabilized cell. (D) Transmitted light picture of the doublet (after permeabilization) shown in A-C. (E) IP3 (10 µM), applied to the doublet shown in A-D, elicited a reduction in [Ca2+] in the intracellular stores in the lower (permeabilized) cell (red trace), whereas there was no response in the upper (intact) cell (blue trace). (F) Typical reduction of [Ca2+] in the intracellular stores induced by NAADP (100 nM) in permeabilized cell. (G) Typical cADPR (10 µM)-induced Ca2+ release in the permeabilized cell. (H) IP3 (10 µM) induces additional Ca2+ release after the NAADP-elicited reduction in the intra-organellar [Ca2+]. (I) NAADP (100 nM) induces additional Ca2+ release after the IP3-elicited response.

View larger version (36K): [in a new window]   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.

View larger version (26K): [in a new window]   Fig. 3. Bafilomycin A1 and nigericin abolish Ca2+ release responses from the thapsigargin-insensitive Ca2+ store. (A) Nigericin (7 µM) blocks NAADP (100 nM)-elicited Ca2+ release in the presence of thapsigargin (10 µM). (B) Nigericin does not block NAADP-elicited Ca2+ release in the absence of thapsigargin. (C) Nigericin blocks IP3 (10 µM)-evoked Ca2+ release in the presence of thapsigargin. (D) Nigericin does not block IP3-elicited Ca2+ release in the absence of thapsigargin. (E) Bafilomycin A1 (1 µM) abolishes NAADP-evoked Ca2+ release in the presence of thapsigargin. (F) Bafilomycin A1 fails to inhibit NAADP response in the absence of thapsigargin. (G) Bafilomycin A1 abolishes IP3-evoked Ca2+ release in the presence of thapsigargin. (H) Bafilomycin A1 fails to inhibit IP3 response in the absence of thapsigargin. Cells were loaded with Fluo-5N AM. All traces represent whole-cell regions of interest of permeabilized cells.

View larger version (40K): [in a new window]   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.

View larger version (26K): [in a new window]   Fig. 5. NAADP-induced Ca2+ release from both the ER and the store in the granular pole is blocked by ruthenium red and ryanodine. (A) Control trace showing that both first and second additions of NAADP trigger similar Ca2+ releases from the internal stores of permeabilized cells. (B) NAADP induces typical control Ca2+ release, but cannot release Ca2+ in the presence of ruthenium red. (C) Ruthenium red (10 µM) abolishes both cADPR-elicited (10 µM) and NAADP-elicited (100 nM) Ca2+ release. (D) IP3 (10 µM) induces typical Ca2+ release response in the presence of ruthenium red. (E) NAADP induces typical control Ca2+ release, but in the presence of ryanodine the NAADP response is blocked. (F) cADPR induces typical control Ca2+ release response, but in the presence of ryanodine (100 µM) both the cADPR and NAADP responses are blocked. (G) Control trace showing that both first and second additions of cADPR induce similar Ca2+ releases from the internal stores of permeabilized cells. (H) IP3 induces typical Ca2+ release response in the presence of ryanodine. Cells were loaded with Fluo-5N AM. All traces represent experiments on permeabilized cells. The region of interest is the whole cell in all cases.

View larger version (14K): [in a new window]   Fig. 6. Clamping [Ca2+] at 100 nM does not block Ca2+ release from ER, but blocks NAADP response from thapsigargin-insensitive Ca2+ store. (A) Ca2+ concentration was clamped (100 nM) by using a Ca2+/BAPTA mixture with a high concentration of BAPTA (10 mM). Both NAADP (100 nM) and IP3 (10 µM) induced Ca2+ release. (B) NAADP failed to induce Ca2+ release after application of thapsigargin (10 µM) at clamped [Ca2+]. (C) IP3 induced Ca2+ release after treatment with thapsigargin at clamped [Ca2+]. (D) cADPR (10 µM) induced Ca2+ release after treatment with thapsigargin at clamped [Ca2+]. All traces represent whole-cell regions of interest. Cells were loaded with Fluo-5N AM.

View larger version (28K): [in a new window]   Fig. 7. (A) Ionomycin releases Ca2+ from the ER but not from acidic stores. (a) NAADP-induced additional 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 ionomycin (n=6, P<0.005, asterisk shows time point when the amplitudes at the granular and basal areas have been compared using t-test). Bars represent standard errors. (c) No response is seen in the basal area. (B) IP3-induced additional Ca2+ response in the presence of 10 µM ionomycin from the secretory granule area of a permeabilized cell. (C) Nigericin-induced additional Ca2+ response in the presence of 10 µM ionomycin from the secretory granule area of a permeabilized cell. In A, B and C cells were loaded with Fluo-5N AM. (D-E) Thapsigargin does not discharge acidic Ca2+ store in intact cells. (D) Subsequent applications of 10 nM and then 10 µM thapsigargin in the absence of external Ca2+ did not prevent an addition small CCK-induced (1 nM CCK) Ca2+ release. Cells were loaded with Fluo-4 AM. (E) Averaged traces from 100 seconds of the experiment shown in D (dotted blue box) shows in detail the response to a subsequent application of 1 nM CCK in the absence of external Ca2+ (n=8, P<0.0005; asterisks show time points at which relative fluorescence was compared using a t-test). Bars represent standard errors.

View larger version (38K): [in a new window]   Fig. 8. Schematic model of Ca2+ signalling in pancreatic acinar cells highlighting the two Ca2+ stores in the apical pole. Any of the three Ca2+ releasing messengers tested, IP3, cADPR or NAADP, can induce Ca2+ release from both the ER and the acidic Ca2+ store. IP3 activates IP3Rs in both stores, whereas cADPR and NAADP activate RyRs in both stores, but via separate binding sites and/or activation mechanisms. The reason for the preferential apical localization of the physiological cytosolic Ca2+ signals is most likely interaction between the two stores (which can of course only occur in the secretory granular area) as well as interaction between RyRs and IP3Rs (most likely to occur in the secretory granule area, which has by far the highest concentration of IP3Rs). CICR, Ca2+-induced Ca2+ release.