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

用活的、稳定表达的CHO-K1细胞实时成像检测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)结合的亚细胞定位和邻近pH。α_&lt；1B&gt；-AR/GFpH荧光主要分布在细胞表面，胞内pH分布均匀。去甲肾上腺素(NE，10^&lt；-7&gt；M)刺激α_1B&gt；-AR可引起细胞内pH升高，促进α_&lt；1B&gt；-AR/GFP内化，引起细胞内钙振荡。α_&lt；1a&gt；-AR/GFpH荧光不仅存在于细胞表面，而且存在于细胞内，这种定位不受观察期间NE的影响。去甲肾上腺素前细胞表面的胞内pH高于胞内室。去甲肾上腺素刺激α_&lt；1a&gt；-AR导致细胞内pH降低，仅引起钙瞬变。α_&lt；1D&gt；-AR/GFpH荧光主要在细胞内检测到，这种定位不受NE的影响。与α_&lt；1B&gt；-AR/GFpH一样，α_&lt；1D&gt；-AR/GFpH细胞内pH分布均匀。去甲肾上腺素处理α_&lt；1d&gt；-AR/GFpH表达细胞的pH升高，引起Ca~(2+)振荡。结果表明，α_1-AR/GFpH融合蛋白具有功能，不同亚型α_1-AR的细胞分布、激动剂介导的内化、Ca~(2+)反应和邻近pH存在差异。这些差异可能导致了α_1-ARs调控的生理反应的多样性。Al-亚型的亚细胞分布尚不清楚。使用共转染的亚细胞标志和抗GFP抗体，用免疫电子显微镜观察亚细胞的分布。

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.