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First published online 13 December 2005
doi: 10.1242/jcs.02731

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ER stress disrupts Ca²⁺-signaling complexes and Ca²⁺ regulation in secretory and muscle cells from PERK-knockout mice

Guojin Huang^1,*, Jian Yao^1,*, Weizhong Zeng¹, Yusuke Mizuno¹, Kristine E. Kamm¹, James T. Stull¹, Heather P. Harding², David Ron² and Shmuel Muallem^1,

¹ Department of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA
² Skirball Institute of Biomolecular Medicine and the Departments of Cell Biology, Medicine and Pharmacology, New York University School of Medicine, 530 First Avenue, New York, NY 10016, USA

View larger version (26K): [in a new window]   Fig. 1. Fragmentation of the ER in PERK-/- cells does not affect reloading of the internal Ca2+ stores. WT (A) and PERK-/- pancreatic acini (B) were stimulated with 1 mM carbachol (Carb) to discharge the agonist-mobilized intracellular Ca2+ pool. Reloading was initiated by termination of the stimulated state with 10 µM atropine (Atr). At different times after initiation of reloading the cells were re-stimulated with 10 nM CCK to estimate the extent of reloading, which was calculated as the percentage of the maximal response measured with cells stimulated only with CCK. The upper and lower traces in A and B show the reloading after a 30 second and 10 minute treatment with atropine and the time courses of reloading are plotted in panel (C) for WT () and PERK-/- cells (). The traces in A,B and the summary in C are the mean (black trace) ± s.e.m. (gray lines) of at least three experiments. All experiments were performed in the presence of 1 mM Ca2+o to enable measurement of the reloading.

View larger version (21K): [in a new window]   Fig. 2. Fragmentation of the ER reduces the rate of [Ca2+]i increase. (A) Examples of the Ca2+ increase in individual acini obtained from WT (solid traces) and PERK-/- mice (dashed traces) stimulated with 5 or 25 µM carbachol, as indicated by the bars. WT () and PERK-/- () pancreatic acinar cells were stimulated with different concentrations of carbachol and the extent (B) and rate (C) of the [Ca2+]i increase were measured. The results are the mean ± s.e.m. of three acinar preparations with at least four experiments with every cell preparation. Similar findings were observed in the presence (1 mM) and absence (no Ca2+ and 0.1 EGTA) of Ca2+o and therefore results obtained in the presence and absence of Ca2+o were averaged in B and C.

View larger version (61K): [in a new window]   Fig. 3. Ca2+ waves in PERK-/- pancreatic acinar cells. PERK-/- pancreatic acinar cells were stimulated with the indicated concentrations of carbachol. (A,B) Selective images of cells stimulated with 5 µM (A) or 100 µM carbachol (B). (C) Changes in [Ca2+]i at the apical (green regions of interest and traces) and basal poles (red regions of interest and traces). (D) The rate of the Ca2+ waves recorded in cells stimulated with 0.1 and 1 mM carbachol. The results are the mean ± s.e.m. of 5/11 cells stimulated with 100 µM carbachol that showed a Ca2+ wave and of 8/8 cells stimulated with 1 mM carbachol (Carb). All results in A and B were obtained in the absence of Ca2+o and similar results were obtained in the presence of 1 mM Ca2+o. The averages are from cells stimulated in the presence or absence of Ca2+o.

View larger version (38K): [in a new window]   Fig. 4. Deletion of PERK does not affect IP3 production, localization of IP3Rs and IP3-mediated Ca2+ release in pancreatic acinar cells. (A,B) WT (filled symbols and columns) and PERK-/- pancreatic acini (open symbols and columns) were stimulated with the indicated concentrations of carbachol (A), bombesin (BS) or CCK (B) for 2-10 seconds and the mass of 1,4,5 IP3 was measured. (C) Pancreatic acini were fixed and used for immunolocalization of IP3R1 (upper images), IP3R2 (middle images) or IP3R3 (bottom images) in WT and PERK-/- cells. (D) Pancreatic acini from WT and PERK-/- mice were permeabilized with streptolysin O and after stabilization of medium Ca2+ at about 55 nM, increasing concentrations of IP3 were added to the incubation medium to measure the ability of IP3 to release Ca2+ from the ER ( indicates addition of 0.15 µM IP3; arrow indicates addition of 5 µM IP3). The results of three experiments are summarized in E and are given as the mean ± s.e.m.

View larger version (29K): [in a new window]   Fig. 5. Ca2+-induced Ca2+ release in WT and PERK-/- parotid acini. WT (A,C) and PERK-/- parotid acini (B,D) were incubated in Ca2+-free medium and either stimulated with 1 mM carbachol (A,B) or treated with 10 µM CPA for 2 minutes. Then the acini were alternately exposed to medium containing 7.5 or 0 mM Ca2+ to induce CICR. (E) The averaged peak increases in [Ca2+]i. (F) Ratios between the first and second peaks (carbachol stimulation) and first and third peak (CPA treatment) evoked by addition of 7.5 mM Ca2+ to WT (dotted lines) and PERK-/- cells (dashed lines) in A-D. Summaries are from at least four experiments each and are given as the mean ± s.e.m.

View larger version (27K): [in a new window]   Fig. 6. Ca2+-induced Ca2+ release in WT and PERK-/- urinary bladder smooth muscle strips. Changes in [Ca2+]i (A) and force (C) in UBSM strips from WT (upper traces in each set) and PERK-/- mice (lower traces in each set) depolarized with 20-80 mM K+. The average [Ca2+]i and force increases in three WT () and three PERK-/- () mice are summarized in B and D, respectively and are given as the mean ± s.e.m. The responses were calculated as a percentage of those induced by 80 mM KCl in each strip.

View larger version (24K): [in a new window]   Fig. 7. Cav1.2 and caffeine-induced Ca2+ release in WT and PERK-/- urinary bladder smooth muscle cells. (A) WT (black traces and column) and PERK-/- (gray traces and column) cells were used to measure Cav1.2 current. (B) The voltage dependence of Cav1.2 in WT () and PERK-/- cells (). WT (C) and PERK-/- UBSM strips (D) were exposed to caffeine concentrations between 5-40 mM and then depolarized with 80 mM K+ to measure the activity of the RyRs in the UBSM. (E) The average response of four strips from two WT and two PERK-/- mice given as the mean ± s.e.m.

View larger version (53K): [in a new window]   Fig. 8. Co-immunoprecipitation of plasma membrane- and ER-resident Ca2+-signaling proteins in WT and PERK-/- cells. (A) Extracts from pancreatic acini of three WT and three PERK-/- mice were used to measure expression (input) of the indicated proteins and for immunoprecipitation of PMCA. The immunoprecipitates were analyzed for co-immunoprecipitation of PMCA, IP3R3 and SERCA2b. (B) Extracts from UBSM of three WT and three PERK-/- mice were used to measure expression of the indicated proteins and for immunoprecipitation of Cav1.2. The immunoprecipitates were analyzed for co-immunoprecipitation of Cav1.2 and RyR2.

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