Acta Pharmacologica Sinica 2007 March; 28 (3): 359-366; doi: 10.1111/j.1745-7254.2007.00509.x

 
Original Article
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Internalization and distribution of three α1-adrenoceptor subtypes in HEK293A cells before and after agonist stimulation1
 

Shu-yi WANG2, Yao SONG2, Ming XU2, Qi-hua HE3, Qi-de HAN2, You-yi ZHANG2,4

2Institute of Vascular Medicine, Peking University Third Hospital and Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Beijing 100083, China; 3Center of Medical Analysis, Peking University, Health Science Center, Beijing 100083, China

 

Aim: To examine the subcellular distribution of the 3 α1-adrenoceptor (α1-AR) subtypes and their internalization and trafficking upon agonist stimulation in human embryonic kidney 293A cells.

 

Methods: Confocal real-time imaging, enzyme linked immunosorbent assay (ELISA) and whole cell [3H]-prazosin binding assay were applied to detect the distribution and localization of the 3 α1-AR subtypes.

 

Results: α1A-AR was found both on the cell surface and in the cytoplasm; α1B-AR, however, was predominantly detected on the cell surface, while α1D-AR was detected mainly in the intracellular compartments. After stimulation with phenylephrine, localization changes were detected by confocal microscopy for α1A- and α1B-AR, but the localization of α1D-AR were unaffected. Phenylephrine stimulation promoted a more rapid internalization of α1B-AR than α1A-AR. α1D-AR internalization was detected only by ELISA. Whole cell [3H]-prazosin binding assay showed that α1A-AR functional receptors were detected both on the cell surface and in the cytoplasm; α1B-AR, however, were detected predominantly on the cell surface, while α1D-AR were detected mainly in intracellular compartments. Phenylephrine stimulation promoted internalization of α1A- and α1B-AR.


Conclusion:
Phenylephrine stimulation induced changes in the localization of the 3 α1-AR.

 

Keywords: adrenoceptor; agonist stimulation; confocal image; enzyme-linked immunosorbent assay; [3H]-prazosin binding assay; internalization; subcellular distribution; subtype

 
1 Project supported by the National Key Basic Research Program of the People's Republic of China (No G2000056906) and the National Natural Science Foundation of China (No 30490172, 30171083).

4 Correspondence to Prof You-yi ZHANG.
Phn/Fax 86-10-8280-2306.
E-mail zhangyy@bjmu.edu.cn
Received 2006-08-11     Accepted 2006-10-13

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