Cyclopiazonic acid cpa

The different experimental rats were sacrificed at 4, 8 and 12 weeks respectively. Bladders were harvested and dipped into 4°C Krebs (containing 119 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄.7H₂O, 25 mM NaHCO₃, 2.5 mM CaCl₂ and 11 mM glucose, pH adjusted to 7.35 with NaOH) solution immediately. The urothelium and submucosa layers were removed by using microinstruments under a microscope (Motic, SMZ-168 Series; China Group Co., Ltd., Xiamen, China; http://www.motic.com/Indu_Stereo/product_432.html) and were longitudinally cut into 3×3×8 mm strips from the ventral bladder. Bladder strips were fixed between electrodes and an automatic organ bath (Panlab; Harvard Apparatus, Holliston, MA, USA) containing 10 ml Krebs solution inflated with 95% O₂ and 5% CO₂ at 37°C, as described previously (16 (link)). Following 30 min, the SOCCs agonist cyclopiazonic acid (CPA, 10 µM) (Sigma-Aldrich; Merck KGaA) was added into the Krebs solution and the inhibitor SKF-96365 (10 µM; Sigma-Aldrich; Merck KGaA) was added into the Krebs solution after 5 min. The spontaneous contractive frequency and amplitude of BSM strips were recorded ~5 min for each intervention after the spontaneous contractions of bladder strips were detected by PowerLab system and the data were analyzed by LabChart version 7.3.7 (AD Instruments, Bella Vista, Australia).

MDA-MB-231 cells plated on glass bottom cell culture dishes were loaded with the calcium-sensitive fluorescent dye Fluo-4/AM (4 mM; Invitrogen) in Hank's Balanced Salt Solution containing 0.02% pluronic acid (Sigma) for 45 min at 37 °C. GABA (Sigma), CGP (Santa Cruz), picrotoxin (Santa Cruz) and cyclopiazonic acid (CPA; Sigma) were applied at concentrations of 2 mM, 10 μM, 10 μM, and 10 μM, respectively. The Ca²⁺-free buffer contained 1 mM EDTA. Fluorescence was measured by an Olympus Confocal Laser Scanning Microscope (DU-897D-CS0) using MetaMorph software. Serial scanning was performed at 488/530 nm excitation/emission wavelengths at 1 s intervals. Fluorescence intensity changes (F%) were shown as the percentage of baseline fluorescence.