1. Volume 140, Issue 7, Pages 2107-2115.e4 (June 2011)
Genetic and Pharmacologic Inhibition of the Ca2+ Influx Channel TRPC3 Protects Secretory Epithelia From Ca2+-Dependent Toxicity 
Min Seuk Kim, Kyu Pil Lee, Dongki Yang, Dong Min Shin, Joel Abramowitz, Shigeki Kiyonaka, Lutz Birnbaumer, Yasuo Mori, Shmuel Muallem 
Gastroenterology 
Volume 140, Issue 7, Pages e4 (June 2011)
DOI: /j.gastro
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2. Figure 1
Deletion of Trpc3 and Pyr3 similarly inhibit receptor-evoked Ca2+ oscillations and Ca2+ influx. Fura2-loaded pancreatic acini from (A and C) wild-type or (B and D) Trpc3−/− mice were used to measure [Ca2+]i in response to stimulation with 10 pmol/L CCK-8 (A and B) to induce Ca2+ oscillations or 10 nmol/L CCK-8 (C and D) to evoke a sustained response. The upper traces in A and B are the control, and the lower traces are from acini that were treated with 3 μmol/L Pyr3. In C and D, the dark traces are the controls and the gray traces are acini treated with Pyr3. Panels E and F show the mean ± SEM of the frequency and plateau, respectively, of the Ca2+ signals. Panel G shows example traces of the time course and I/V of the CRAC-like current in the absence and presence of Pyr3 and the mean ± SEM of 5 experiments. *P < .05 or better with n = 6–8 experiments with acini from 3 mice of each phenotype.
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3. Figure 2
Deletion of Trpc3 and Pyr3 similarly inhibit SOC-mediated Ca2+ influx. Fura2-loaded (A and B) pancreatic or (C and D) SMG acini from (A and C) wild-type or (B and D) Trpc3−/− mice were used to measure Ca2+ release in response to inhibition of the sarcoplasmic/endoplasmic Ca2+ adenosine triphosphatase pumps with 25 μmol/L cyclopiazonic acid (CPA) in Ca2+-free solution and Ca2+ influx in response to readdition of Ca2+. Dark traces are controls, and gray traces are cells treated with 3 μmol/L Pyr3. *P < .05 or better with n = 6–8 experiments with acini from 3 mice of each phenotype. NS, not significant. In panel E, GMS acini were stimulated with 0.3 (upper traces) or 100 μmol/L carbachol (lower traces) and treated with or without 3 μmol/L Pyr3, as indicated (gray lower trace). The upper traces show examples of individual cells. The lower traces are averages of 4–5 experiments in cells stimulated in Ca2+-free solution to measure Ca2+ release and then Ca2+ was re-added to measure influx.
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4. Figure 3
Inhibition of Trpc3 by Pyr3 ameliorates CCK-8–induced acute pancreatitis. Mice were treated with or without 0.1 μg/g body wt Pyr3 and injected with saline (controls) or cerulein to induce acute pancreatitis. Panel A shows the mean ± SEM of plasma amylase content in 3–6 mice under each condition. Panel B shows typical images of damaged tissue under the various treatments, and the columns are the mean ± SEM of at least 5 images from each of the 3–6 mice used in each condition. In Panel C, isolated pancreatic acini were stimulated for 1 hour with 10 nmol/L CCK-8 in the presence or absence of 3 μmol/L Pyr3 and incubated for 20 minutes with fluorescence trypsin substrate. The figure shows example images and the mean ± SEM of 6 experiments.
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5. Figure 4
Effect of Trpc3 deletion and inhibition of Trpc3 activity by Pyr3 on granules mistargeting and induction of autophagy. In panel A, tissue sections were obtained from mice treated with or without Pyr3 and with and without cerulein to induce acute pancreatitis. The sections were costained with Lamp2 and the secretory granules marker amylase. Overlap of Lamp2 with amylase was determined by MetaMorph (Universal Imaging Corp, Marlow, Buckinghamshire, United Kingdom), and the columns show the mean ± SEM. In panel B, tissue sections from mice treated with or without Pyr3 and with and without cerulein were stained with the autophagy marker LC3. The number of autophagosomes was determined by MetaMorph and the columns show the mean ± SEM of the indicated number of experiments. In panel C, isolated SMG and pancreatic acini from wild-type (upper panel) or Trpc3−/− mice (lower panel) treated with or without 3 μmol/L Pyr3 were stimulated with 1 mmol/L carbachol or 10 nmol/L CCK-8, respectively, for 30 minutes, and extracts were used to analyze accumulation of LC3-II. The columns are the mean ± SEM (n = 3) for acini from wild-type mice. In acini from 3 Trpc3−/− mice, LC3 did not change.
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6. Figure 5
Effect of Trpc3 deletion and inhibition of Trpc3 activity by Pyr3 on salivary secretion in vivo. Anesthetized wild-type (squares and triangles) injected with saline (squares) or 0.1 μg/g body wt Pyr3 (triangles) and Trpc3−/− mice (circles) were used to measure cumulative salivary secretion in response to injection of pilocarpine, as detailed in Materials and Methods. The results are the mean ± SEM of 4–7 mice under each condition. *P < .05 or better relative to secretion by untreated wild-type mice.
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7. Figure 6
Deletion of Trpc3 and inhibition of Trpc3 activity by Pyr3 inhibit induction of autophagy and granules mistargeting in SMG acini. Acini obtained from (A and D) wild-type or (B and E) Trpc3−/− mice and treated with phosphate-buffered saline (control) or 3 μmol/L Pyr3 were left unstimulated (upper images) or were stimulated with the supramaximal concentration of 1 mmol/L carbachol (Car). The acini were fixed and stained for the autophagy marker for LC3 (A and B) or costained for the late endosomal/lysosomal marker LAMP2 and the secretory granules marker amylase (D and E). The number of LC3 particles was counted with MetaMorph, and the mean ± SEM of the fold increase measured in 8 experiments with acini from wild-type mice and 5 experiments with acini from Trpc3−/− mice is shown in panel C. The percentage of LAMP2/amylase overlap was determined with MetaMorph, and the mean ± SEM of the overlap measured in 7 experiments with acini from wild-type mice and 5 experiments with acini from Trpc3−/− mice is shown in panel F. The P values are listed in the figures.
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8. Figure 7
Deletion of Trpc3 and inhibition of Trpc3 activity by Pyr3 inhibit PERK phosphorylation and cell damage. SMG and pancreatic acini obtained from (A) wild-type or (B) Trpc3−/− mice were treated with phosphate-buffered saline (vehicle) or 3 μmol/L Pyr3, as indicated. Part of the SMG acini were stimulated with 1 mmol/L carbachol (Car) for 30 minutes, and part of the pancreatic acini were stimulated with 10 nmol/L CCK-8 (CCK) for 30 minutes. Lysates prepared from the acini were analyzed for phosphor-PERK (p-PERK). Analysis of β-actin provided the loading controls. In panel B, the first and last lanes are positive controls obtained with acini from wild-type mice. In panel C, the supernatants of SMG and pancreatic acini treated with or without 3 μmol/L Pyr3 and stimulated with 1 mmol/L carbachol or 10 nmol/L CCK-8 for 30 minutes, respectively, were collected and used to measure released G6PD. G6PD release is expressed as percent of total G6PD and is given as the mean ± SEM of 3 and 4 experiments with SMG and pancreatic acini, respectively. The P values are listed in the figure.
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9. Supplementary Figure 1
Inhibition of Trpc3 activity with Pyr3 inhibits pathologic vacuoles formation. (A) Pancreatic and (B) SMG acini obtained from wild-type mice were treated with phosphate-buffered saline (control) or 3 μmol/L Pyr3, as indicated. The acini were maintained in media containing Texas Red dextran and were stimulated with (A) 10 nmol/L CCK-8 or (B) 1 mmol/L carbachol for 30 minutes, as detailed in Materials and Methods. Images of the acini were then obtained, and the number of vacuoles was counted in the indicated number of cells. Example images are shown for stimulated acini with and without Pyr3, and the respective columns show the mean ± SEM of the number of cells analyzed that are listed in the columns.
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10. Supplementary Figure 2
Deletion of Trpc3 and inhibition of TRPC3 activity with Pyr3 inhibit accumulation of ceramide. (A) Pancreatic and (B) SMG acini obtained from wild-type mice were treated with phosphate-buffered saline (control) or 3 μmol/L Pyr3, as indicated. Part of the pancreatic acini was stimulated with 10 nmol/L CCK-8 (CCK) for 30 minutes, and part of the SMG acini was stimulated with 1 mmol/L carbachol (Car) for 30 minutes, as indicated. The acini were then fixed and probed with anti-ceramide monoclonal antibodies. Fluorescence intensity was analyzed with MetaMorph and is given relative to that in control cells. The respective columns show the mean ± SEM of 5 experiments with pancreatic acini (red) and 6–9 experiments with SMG acini (yellow).
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