Relationship between subcellular localization of α-adrenoceptor subtypes and function on ischemia

通过稳定表达的活CHO-K1细胞实时成像，同时评估α_1-肾上腺素能受体（α_1-AR）亚型与pH传感器绿色荧光蛋白（α_1-AR/GFpH）偶联的亚细胞定位和邻近pH。α_<1B>-AR/GFpH荧光主要出现在细胞表面，细胞内pH呈均匀分布。去甲肾上腺素（NE,10^<-7>M）刺激α_<1B>-AR，导致细胞内pH升高，促进α_<1B>-AR/GFP快速内化，引起Ca^<2+>振荡。α_<1A>-AR/GFpH荧光不仅在细胞表面检测到，而且在细胞内也检测到，并且在观察期间，这种定位不受NE暴露的影响。NE前，细胞表面的细胞内pH值高于细胞内腔。NE刺激α_<1A>-AR导致细胞内pH降低，仅引起Ca^<2+>短暂性。α_<1D>-AR/GFpH荧光主要在细胞内检测到，这种定位不受NE暴露的影响。α_<1D>-AR/GFpH与α_<1B>-AR/GFpH细胞内pH分布均匀。NE处理α_<1D>-AR/GFpH表达细胞，使细胞pH升高，Ca^<2+>振荡。这些数据表明，转染α_1-AR/GFpH融合蛋白具有功能性，α_1-AR的细胞分布、激动剂介导的内化、Ca^<2+>反应和邻近pH在α_1-AR亚型之间存在差异。这些差异可能与α_1-ARs调控的生理反应的多样性有关。al亚型的亚细胞分布尚不清楚。亚细胞组织的分布将通过共转染亚细胞标记物和抗gfp抗体的免疫电镜检查。

The subcellular localization and adjacent pH of the α_1-adrenoceptor (α_1-AR) subtypes conjugated with pH sensor green fluorescent protein (α_1-AR/GFpH) were simultaneously assessed using real-time imaging of living, stably expressed CHO-K1 cells. The α_<1B>-AR/GFpH fluorescence was detected predominantly on the cell surface and intracellular pH distribution showed homogeneous pattern. Stimulation of the α_<1B>-AR with norepinephrine (NE,10^<-7>M) led to an increase in intracellular pH, promoted rapid α_<1B>-AR/GFP internalization and caused Ca^<2+> oscillations. α_<1A>-AR/GFpH fluorescence was detected not only on the cell surface but also inside he cell and this localization was unaffected by exposure to NE during observation period. Intracellular pH on the cell surface was higher than intracellular compartment before NE. Stimulation of the α_<1A>-AR with NE led to a decrease in intracellular pH and caused only a Ca^<2+> transient. The α_<1D>-AR/GFpH fluorescence was detected mainly intracellularly, and this localization was unaffected by exposure to NE. Intracellular pH distribution of α_<1D>-AR/GFpH was homogeneous as in α_<1B>-AR/GFpH. NE treatment of α_<1D>-AR/GFpH expressing cells increased pH and caused Ca^<2+> oscillations. These data show that transfected α_1-AR/GFpH fusion proteins are functional and that there are differences in the cellular distribution of α_1-AR, agonist-mediated internalization, Ca^<2+> response and neighbor pH among α_1-AR subtypes. These differences could contribute to the diversity in physiologic responses regulated by the α_1-ARs. The subcellular distribution of al-subtypes is still unknown. The distribution of subcellular oragnera will be examined by immunoelectron microscopy using co-transfected subcellar marker and anti-GFP antibody.