Secretagogues cause ubiquitination and down-regulation of inositol 1,4,5-trisphosphate receptors in rat pancreatic acinar cells  Richard J.H. Wojcikiewicz*, Stephen A. Ernst‡, David I. Yule§  Gastroenterology  Volume 116, Issue 5, Pages 1194-1201 (May 1999) DOI: 10.1016/S0016-5085(99)70023-5 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 IP3 receptor down-regulation in vitro. Isolated acinar cells were incubated as shown. Then, Triton X-100 extracts were prepared, electrophoresed, and probed for type III IP3 receptor content with anti-typeIIIR. Data are chemiluminescence images that correspond to the ~230–300-kilodalton regions of gels (n = at least 3 independent experiments). Arrows mark the migration position of the type III receptor (250 ± 5 kilodaltons; n = 3). (A) Cells were incubated for a range of times without stimulus (lanes 1–4) or with 10 nmol/L CCK (lanes 5–11). (B) Cells were incubated for 120 minutes with a range of CCK concentrations. (C) Cells were incubated for 120 minutes without stimulus (lanes 1 and 2) or with 10 nmol/L BBS (lanes 3 and 4), 100 μmol/L CCh (lanes 5 and 6), 100 nmol/L VIP (lanes 7 and 8), or 10 nmol/L CCK (lanes 9 and 10). Gastroenterology 1999 116, 1194-1201DOI: (10.1016/S0016-5085(99)70023-5) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Specificity of CCK-induced IP3 receptor down-regulation in vitro. Isolated acinar cells were incubated for 60 minutes with a range of CCK concentrations without thapsigargin (lanes 1–7) or with 1 μmol/L thapsigargin (lanes 8–10). Triton X-100 extracts were then prepared, electrophoresed, and probed with the antibodies indicated. Data are chemiluminescence images that correspond to regions of gels in which the target proteins migrated (n = 3 independent experiments). Arrows indicate migration positions of the type III receptor, PKCϵ, PLCβ1, HSP90, and HSP70 (~250, ~90, ~150, ~90, and ~70 kilodaltons, respectively). Gastroenterology 1999 116, 1194-1201DOI: (10.1016/S0016-5085(99)70023-5) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Ubiquitination of IP3 receptors in vitro. Isolated acinar cells were preincubated for 60 minutes without or with inhibitors (20 μg/mL ALLN or 6 μmol/L lactacystin), then incubated with or without CCK for 45 minutes. Triton X-100 extracts were prepared, and type III receptors were immunoprecipitated with anti-typeIIIR. Samples of the extracts (A, top panel) and immunoprecipitates (all other panels in A and B) were electrophoresed and probed with the indicated antibodies. Data are chemiluminescence images (n = 2 independent experiments). Arrows, square brackets, and arrowheads mark the migration positions of the type III receptor (~250 kilodaltons), ubiquitin immunoreactivity (~260–320 kilodaltons), and type III receptor fragments (~200 and 220 kilodaltons), respectively. Gastroenterology 1999 116, 1194-1201DOI: (10.1016/S0016-5085(99)70023-5) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Redistribution and down-regulation of IP3 receptors in vivo. Rats were injected with (A and E) saline or (B–D and F) 50 μg/kg cerulein, pancreata were removed, and type III receptor immunoreactivity was determined in 5–10-μm-thick cryostat sections by using anti-typeIIIR. In control animals, type III receptors are in close association with the luminal plasma membrane (A and E; arrows indicate juxtaluminal staining). One hour after cerulein injection, the overall level of fluorescence is reduced slightly and the staining around lumens, which are now dilated, is more diffuse (B and F; arrowheads indicate diffuse staining). These changes were more pronounced 2 and 3 hours after cerulein injection (C and D, respectively). A–D, low magnification; E and F, high magnification (n = 4 independent analyses). Gastroenterology 1999 116, 1194-1201DOI: (10.1016/S0016-5085(99)70023-5) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 Down-regulation and ubiquitination of IP3 receptors in vivo. Rats were injected with saline (lane 1), 50 μg/kg cerulein (lanes 2 and 3), or 0.5 μg/kg cerulein (lane 4). After 15 minutes (lanes 1, 2, and 4) or 60 minutes (lane 3), pancreata were removed, Triton X-100 extracts were prepared, and IP3 receptors were immunoprecipitated with anti-typeIIIR. Samples of the (A) extracts and (B) immunoprecipitates were then electrophoresed and probed with the antibodies indicated. Data are chemiluminescence images corresponding to the regions of gels in which the target proteins migrated (n = 2 independent experiments). Arrows and square bracket indicate migration positions of target proteins and ubiquitin immunoreactivity, respectively. Molecular masses are the same as cited in Figures 2 and 3. Gastroenterology 1999 116, 1194-1201DOI: (10.1016/S0016-5085(99)70023-5) Copyright © 1999 American Gastroenterological Association Terms and Conditions