Background & Aims: Excessive Ca2+ influx mediates many cytotoxic processes, including those associated with autoimmune inflammatory diseases such as acute pancreatitis and Sjgren syndrome. Transient receptor potential (canonical) channel (TRPC) 3 is a major Ca2+ influx channel in pancreatic and salivary gland cells. We investigated whether genetic or pharmacologic inhibition of TRPC3 protects pancreas and salivary glands from Ca2+-dependent damage. Methods: We developed a Ca2+- dependent model of cell damage for salivary gland acini. Acute pancreatitis was induced by injection of cerulein into wild-type and Trpc3-/- mice. Mice were also given the Trpc3-selective inhibitor pyrazole 3 (Pyr3). Results: Salivary glands and pancreas of Trpc3-/- mice were protected from Ca2+-mediated cell toxicity. Analysis of Ca2+ signaling in wild-type and Trpc3-/- acini showed that Pyr3 is a highly specific inhibitor of Tprc3; it protected salivary glands and pancreas cells from Ca 2+-mediated toxicity by inhibiting the Trpc3-mediated component of Ca2+ influx. Conclusions: TRPC3-mediated Ca2+ influx mediates damage to pancreas and salivary glands. Pharmacologic inhibition of TRPC3 with the highly selective TRPC3 inhibitor Pyr3 might be developed for treatment of patients with acute pancreatitis and Sjgren syndrome.
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The sources of the antibodies used are as follows: anti-LC3B, α-amylase, and β-actin were purchased from Cell Signaling (Danvers, MA), and anti-ceramide, LAMP2, and phosphor-PERK were obtained from Alexis Biochemicals (San Diego, CA), Sigma-Aldrich (St Louis, MO), and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. All experiments with animals were approved by the Animal Use and Care Committees of UT Southwestern Medical Center and the National Institutes of Health. Preparation of acini from each tissue was as described previously. 1 Briefly, mice were killed by cervical dislocation and the pancreas and salivary glands were collected. After mincing, each tissue was digested for 10 minutes with collagenase P (0.25 mg/mL; Roche Diagnostics, Basel, Schweiz, Switzerland) in standard solution A that contained 0.1% bovine serum albumin, 0.1% pyruvic acid, 0.02% soybean trypsin inhibitor, 140 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl 2 , 10 mmol/L HEPES (pH 7.4), 10 mmol/L glucose, and 1 mmol/L CaCl 2 (310 mOsm). The digests were washed in enzyme-free solution A, and the acini were maintained in solution A for 1 hour to stabilize the cells before use.
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