Role Of Transient Receptor Potential Channel 6 In Angiotensin â…¡-induced Rat Aortic Smooth Muscle Cell Proliferation

ObjectiveThe cultured rat aortic smooth muscle cells (ASMCs) were proliferated by angiotensin II (AngII) to establish cellular proliferative model. Three kinds of experiments, MTT assay, Western blot analysis and calcium imaging techniques, were used to detect the expression of the Transient receptor potential channel 6 (TRPC6) in ASMC, the proliferation of ASMC and the change of free intracellular Ca2+ concentration ([Ca2+]i). The present study was to analysis the role of TRPC6 in the AngII-induced proliferation in ASMCs.Methods1. Cell culture. Adult male Sprague–Dawley rats (150～200 g and clean stage) were used in the experiments and were killed by cervical dislocation. The thoracic aorta was dissected and rinsed by D-Hank buffer solution to get rid of blood. After removed the tunica externa and endomembrane, the tissue was minced into 1 to 3 mm fragments and planted in petri dishes. The culture medium was made of Dulbecco's modified Eagle's medium supplemented with foetal bovine serum (FBS, 20% v/v), L-glutamine (2 mM), penicillin G (100 units/mL), and streptomycin (100μg/mL). Cells were cultured at 37°C with 5% CO2 in a cell incubator. The medium was changed per three days. When 80% of cells mixing together and appearing"peak and valley", it is the time to sub-culture.2. Morphology and immunofluorescence double staining identification. The 3~5 passages of ASMCs were used to identify the ASMCs with morphology and immunoreactivity. The suitable cells were selected with a phase contrast microscope, digested by enzyme, and cultured on sterile glass covers in a cell incubator. When growing in logarithmic phase, those cells were washed three times for 10 min with 2 ml of PBS, then fixed with 4% paraformaldehyde solution for 20 min at 4°C, and air dry. After blocked the cells for 1 h with 5% BSA at room temperature, the cells were incubated over night with primary antibodies at 4°C (> 14 hrs). Washed three times for 10 min with 2 ml of PBS, the cells were incubated with fluorescent conjugated secondary antibody in PBS for 1 h at 37°C. Washed three times for 10 min with 2 ml of PBS, the cells were counterstained with DAPI (5μg/mL) for 20 minutes at room temperature. Finally, the fluorescence color of the cells were photographed about 15 min with a fluorescence microscope at room temperature in the dark .3. Western blot analysis. The 3~5 passages of ASMCs were used in this experiment. When 80% of cells mixing together, they were rinsed by chilled PBS (0.01 M,pH 7.2～7.3). Repeat the operation twice. Adding 1 ml Single-detergent lysates (50 mmol/L Tris·HCl, pH 8.0,150 mmol/L NaCl,1%TritonX-100,100μg/mL PMSF) on ice to split cells for 5-10 min. After splited, the sample were scraped from dish, and transferred into an EP tube. To reduce viscosity, the sample was sonicated briefly. Following centrifugation (12,000 g, 20 min at 4°C), the protein concentration was determined by the BCA protein assay kits. After sodium laurylsulfate–polyacrylamide gel electrophoresis on a 10% resolving gel, proteins were transferred onto a BioTrace PVDF membrane. The membrane was blocked in freshly prepared PBS containing with 5% nonfat dry milk for 30 min at room temperature, incubated with primary antibodies overnight at 4°C, followed by washed in PBS-0.05% Tween 20 (PBS-T), 3×10 min, and incubated with secondary antibody and HRP-conjuncted antibody for 90 min at room temperature. Immunodetection of proteins by chemiluminescence (ECL kit) was developed by exposure to X-ray film.4. The group and treatment of cells. (i) blank group; (ii) Ang II group: add 0.1μmol/L Ang II; (iii) SKF96365+ Ang II group: add Ang II ( 0.1μmol/L) and different dose of SKF96365 (10,50,100μmol/L); (iv) flufenamic acid group: add different dose of flufenamic acid ( FFA, 10, 20, 40μmol/L ) . 5. MTT assay. Cells at 3~5 passages were used in this experiment. The cells were digested with trypsin for about 1 min at 37 oC, then centrifuged with 1000 r/min for 8 min. After that, the cells were mixed with DMEM solution containing 20% fetal bovine serum to adjust cell density in 2.5×104 /mL. The cells were planted into a 96-well cell culture plate, cultured for 24 hrs in a CO2 incubator at 37 oC, then changed to a serum-free DMEM solution and cultured for another 24 hrs. After cell culture medium was changed, add various kinds of processing factors to medium and continue to culture the cells for 24~72 hrs. Each group has 6 Double-Pores. Four hours before to end the drug action, MTT solution (5 mg/mL, 20μl per well) was added in a96-well cell culture plate. Then discarding the supernatant, adding DMSO 150μl each well, mixing well and measuring absorbance at 490 nm with microplate reader.6. Calcium imaging. Calcium imaging can monitor the change of intracellular free calcium concentration ([Ca2+]i). For imaging experiments, the cells were subcultured at low density in 25 mm Petri dishes in which a 10 mm diameter hole had been cut in the base and replaced by a thin (0.1 mm) glass coverslip. Cells were allowed to grow for at least 24 h in culture medium solution. ASMCs were incubated for 40 min in a bath solution containing 4μmol Fura-2/AM at 37°C. The excitation wavelength of Fura-2/AM which bound to Ca2+ was 340 nm, while the excitation wavelength of Fura-2/AM which was free was 380 nm, the emission wavelength of both was 510 nm. Fluorescent image intensities were expressed as the ratio F340/F380 to allow quantitative estimates of changes in [Ca2+]i. Thus the changes of [Ca2+]i could be evaluated immediately by use of TILLvision software which could display real-time rations.7. Statistical analysis. The data of MTT assay was performed using SPSS software (version 16.0). The data of calcium imaging were analyzed with Igor Pro software (USA). Comparisons between means were performed using Student's t-test. The data among the groups were compared using one way ANOVA. Statistical data are presented as means±SEM. Differences were considered to be significant when p < 0.05.Results1. Identification of ASMC.(i) The cells showed in spindle-shaped, polygonal or irregular shapes. The nucleus showed in ellipsoid or round. The cells grew like a"hill and valley"pattern in a confluent growth. (ii) Immunofluorescence results showed anti-myosin antibody staining positive, indicating that cells were ASMCs, and the purity of cells was of more than 95%.2. Western blot analysis The TRPC6 protein level in wild type rat ASMC was very high compared to the positive control (HEK293 cells which were transfected with TRPC6 gene) and the negative control (HEK293 cells which were transfected with empty vector).3. MTT assay After exposured to different concentration of SKF96365 (10,50,100μmol/L) for 24, 48, 72 hrs, compared with Ang II group, the growth of SMCs was significantly inhibited dose-dependently (p<0.05) . After exposured to different concentration of flufenamic acid (10,20,40μmol/L) for 24 hrs. The proliferation of SMC could be significantly induced in dose-dependent way, compared with Ang II group ( p < 0.05).4. Calcium imaging SKF96365 (50μmol/L), a non-selective TRPC channel blocker, significantly inhibited the Ang II-induced [Ca2+]i rise (n = 10, p < 0.05). Whereas FFA (50μmol/L), a selective TRPC6 channel activators, had no effect on Ang II-induced elevation of [Ca2+]i rise (n = 8, p > 0.05).ConclusionThe expression of TRPC6 channel protein is very high in ASMC. TRPC6 have a critical role in the Ang II -induced ASMC proliferation and the effect of TRPC6 in the cell proliferation may through the change of [Ca2+]i in ASMC.