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Objective To evaluate the effect of adenosine receptor antagonist SCH442416 on the expression of Kir2.1,Kir4.1 and TASK-1 in rat Müller cell at an elevated hydrostatic pressure in vitro.Methods Thirty SPF Sprague Dawley rats were purchased from Shanghai Slack Laboratory Animals Ltd.Cultured Müller cells were divided into normal control group (n =6),40 mmHg/24 hours (1 mmHg =0.133 kPa) group (n =6) and adenosine + SCH442416 intervention group (n =6).Müller cells were treated with 40 mmHg pressure for 24 hours in 40 mmHg/24 hours group,and Müller cells were treated with 40 mmHg pressure for 24 hours + 10 μ mol/L adenosine + 100 nmol/L SCH442416 in adenosine + SCH442416 intervention group.The real-time PCR,Western blot,whole-cell patch-clamp recordings and immunohistochemistry were used to detect Kir2.1,Kir4.1 and TASK-1 expression and Müller cells Kir currents.The experimental procedures were in accordance with the National Institutes of Health (NIH) guidelines for the Care and Use of Laboratory,and follow the 3R principle.Results Western blot assay showed that,following 40 mmHg pressure cultured for 24 hours,the expression of Kir4.1 and TASK-1 protein in Müller cell were significantly decreased by 38.6％ and 52.6％ compared with the normal control group,with significant differences between the two groups (both at P =0.000);Kir2.1 protein expression decreased by 14.7％,with insignificant difference between the two groups (P =0.082).Kir4.1 and TASK protein expression in adenosine + SCH442416 intervention group was increased by 60.7％ and 61.4％ compared with the 40 mmHg/24 hours group,with significant differences between the two groups (both at P =0.000);Kir2.1 protein expression in adenosine + SCH442416 intervention group was increased by 8.8％ compared with the 40 mmHg/24 hours group,with insignificant difference between them (P =0.354).Real-time PCR assay showed that,following 40 mmHg pressure cultured for 24 hours,Kir2.1,Kir4.1 and TASK-1 mRNA expression in Müller cells were significantly decreased compared with the normal control group,with significant differences between the two groups (P =0.047,0.001,0.000);Kir4.1 and TASK-1 mRNA expression in Müller cells in the adenosine + SCH442416 intervention group was significantly increased compared with the 40 mmHg/24 hours group,with significant differences between the two groups (P =0.038,0.030);however,there is no significant change in Kir2.1 mRNA expression (P =0.612).Conclusions SCH442416 upregulates the expression of Kir4.1 and TASK-1 mRNA and protein,but weakly affects the expression of Kir2.1.
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<p><b>OBJECTIVE</b>To investigate the changes of the two- pore K channel TASK-1 in diabetic rats with myocardial injury.</p><p><b>METHODS</b>Thirty-six SD rats were divided into normal group (N), diabetes at 4 weeks (DM 4W) group, and diabetes at 8 weeks (DM 8W) group. The cardiac functions of the rats were determined using cardiac ultrasonography, and the body weight and heart weight of the rats at different time points were measured to calculate the heart/body weight ratio (HW/BW). Myocardial fibrosis in the rats was assessed using Masson's staining. The protein expression of TASK-1 in the myocardium was detected using Western blotting. Whole- cell patch clamp technique was used to record the action potential duration (APD) and twopore domain potassium channel TASK- 1 current in acutely isolated rat ventricular myocytes. meanwhile, The inhibition of TASK-1 current was observed by the TASK-1 specific inhibitor ML-365.</p><p><b>RESULTS</b>Compared with the normal group, the diabetic rats showed significantly increased HW/BW ( < 0.05), end- diastole left ventricular diameter (LVIDd), end- systolic left ventricular diameter (LVIDs), and TASK-1 protein expression, with obviously decreased left ventricular diameter shortening rate (FS) and ejection fraction (EF) ( < 0.01). Masson staining showed that in diabetic rats, the collagen fibers were thickened, interwoven into a network with uneven arrangement and increased deposition. Compared with DM 4W group, the rats in DM 8W group exhibited progressive increases in LVIDd, LVIDs, HW/BW, and TASK-1 expression ( < 0.01 or 0.05); FS and EF were further decreased ( < 0.01). Masson staining showed worsened morphological changes of the myocardium with increased deposition. Compared with that in the normal group, the current of TASK- 1 in diabetic rats at 8 weeks was significantly reduced ( < 0.01) and the duration of action potential was extended ( < 0.05). The TASK-1 current was successfully inhibited by ML-365.</p><p><b>CONCLUSIONS</b>Diabetes can induce myocardial fibrosis and aggravate myocardial injury possibly in relation to changes in the protein expression and current of the two-port potassium channel TASK-1.</p>
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Hydrogen peroxide (H2O2) causes oxidative stress and is considered as an inducer of cell death in various tissues. Two-pore domain K+ (K2p) channels may mediate K+ efflux during apoptotic volume decreases (AVD) in zygotes and in mouse embryos. In the present study, we sought to elucidate linkage between K2p channels and cell death by H2O2. Thus K2p channels (TASK-1, TASK-3, TREK-1, TREK-2) were stably transfected in HEK-293 cells, and cytotoxicity assay was preformed using cell counting kit-8 (CCK-8). Cell survival rates were calculated using the cytotoxicity assay data and dose-response curve was fitted to the H2O2 concentration. Ionic currents were recorded in cell-attached mode. The bath solution was the normal Ringer solution and the pipette solution was high K+ solution. In HEK-293 cells expressing TREK-1, TREK-2, TASK-3, H2O2 induced cell death did not change in comparison to non-transfected HEK-293. In HEK-293 cells expressing TASK-1, however, dose-response curve was significantly shifted to the left. It means that H2O2 induced cell death was increased. In cell attached-mode recording, application of H2O2 (300micrometer) increased activity of all K2P channels. However, a low concentration of H2O2 (50micrometer) increased only TASK-1 channel activity. These results indicate that TASK-1 might participate in K+ efflux by H2O2 at low concentration, thereby inducing AVD.