Mitochondrial K^ATP Channel And NADPH Oxidase Involved In Angiotensin â…¡-induced Autophagy In VSMCs

Objective:This study was conducted to investigate the role of the mitochondrial ATPsensitive potassium channel and NADPH oxidase in angiotensin â…¡ (Ang â…¡)-inducedautophagy in vascular smooth muscle cells (VSMCs).Methods:Use VSMCs from the male Sprague-Dawley rats as the experiment object.According to different handling, groups were divided into the following: blank group(A), angiotensin â…¡ (Ang â…¡) group (B), Ang â…¡ type1receptor blocker (ARB, Ang â…¡receptor inhibitor)+Ang â…¡ group (C), apocycin (NADPH inhibitor)+Ang â…¡ group(D),5-HD (mitochondrial ATP sensitive potassium channel inhibitor)+Ang â…¡ group(E),3-MA (autophagy inhibitor)+Ang â…¡ group (F), rapamycin (autophagy accelerant)+Ang â…¡ group (G). After experiment in group A, B, C, D and E, fluorescent probenamed dihydroethidium (DHE) was used to detect the level of intracellular reactiveoxygen species (ROS), respectively. MTT was conducted to check up cell survival ratein all groups and western blot was carried out to determine the level changes in theexpression autophagy related protein such as LC3-â…¡, BECN and P62in all groups.Immunofluorescence was used to examine the changes in number of autophagosome inall groups. After knocking out autophagy gene named ATG-5with transfection,Western blot was used to investigate the changes of above-mentioned autophagy relatedprotein expression in group B.Results:1. MTT examination respectively showed no significant effect of Ang â…¡, ARB,apocycin,5-HD,3-MA, or rapamycin on the cell survival rate during cell culture within72h, compared with the blank group, which indicate that treatments or pretreatments inexperiment during cell culture within72h do not influence on cell viability. 2. Western blot displayed that the Ang â…¡ increased the expressions of LC3-â…¡, BECNand ROS but decreased the expression of P62in VSMCs with time and concentrationdependent manner, which suggest that Ang â…¡ promote autophagy of the VSMCs.3. An autophagy inhibitor named3-MA inhibited above-mentioned actions of theAng â…¡ (10-7mol/l,24h) but rapamycin, an autophagy accelerant, enhancedabove-mentioned actions of the Ang â…¡ (10-7mol/l,24h).4. Knocking out autophagy gene named ATG-5with transfection could abolishabove-mentioned actions of the Ang â…¡, which further demonstrate that Ang â…¡contribute to autophagy of the VSMCs.5. ARB such as Olmesartan and Candesartan, Ang â…¡ type1receptor blocker,remarkably inhibited above-mentioned actions of the Ang â…¡ (10-7mol/l,24h), whichsuggest that Ang â…¡ result in autophagy of the VSMCs at least induced by Ang â…¡ type1receptor.6. Apocycin, a NADPH inhibitor, obviously attenuated above-mentioned actions ofthe Ang â…¡ (10-7mol/l,24h), which suggest that Ang â…¡contribute to autophagy of theVSMCs involved in NADPH oxidase action.7.5-HD, a mitochondrial ATP sensitive potassium channel inhibitor, significantlydiminished above-mentioned actions of the Ang â…¡ (10-7mol/l,24h), results of Westernblot being similar to those of immunofluorescence, which suggest that Ang â…¡ induceautophagy of the VSMCs involved in mitochondrial ATP sensitive potassium channelaction.Conclusion:Ang â…¡ increases intracellular ROS expression and promotes autophagy of theVSMCs induced by Ang â…¡ type1receptor, which mechanisms may be involved inactions of mitochondrial ATP sensitive potassium channel and NADPH oxidase.