Internalization and distribution of three α 1 ‐adrenoceptor subtypes in HEK293A cells before and after agonist stimulation 4

Aim: To examine the subcellular distribution of the 3 α 1 ‐adrenoceptor (α 1 ‐AR) subtypes and their internalization and trafficking upon agonist stimulation in human embryonic kidney 293 A cells. Methods: Confocal real‐time imaging, enzyme linked immunosorbent assay (ELISA) and whole cell [ 3 H]‐prazosin binding assay were applied to detect the distribution and localization of the 3 α 1 ‐AR subtypes. Results: α 1A ‐AR was found both on the cell surface and in the cytoplasm; α 1B ‐AR, however, was predominantly detected on the cell surface, while α 1D ‐AR was detected mainly in the intracellular compartments. After stimulation with phenylephrine, localization changes were detected by confocal microscopy for α 1A ‐ and α 1B ‐AR, but the localization of α 1D ‐AR were unaffected. Phenylephrine stimulation promoted a more rapid internalization of α 1B ‐AR than α 1A ‐AR. α 1D ‐AR internalization was detected only by ELISA. Whole cell [ 3 H]‐prazosin binding assay showed that α 1A ‐AR functional receptors were detected both on the cell surface and in the cytoplasm; α 1B ‐AR, however, were detected predominantly on the cell surface, while α 1D ‐AR were detected mainly in intracellular compartments. Phenylephrine stimulation promoted internalization of α 1A ‐ and α 1B ‐AR. Conclusion: Phenylephrine stimulation induced changes in the localization of the 3 α 1 ‐AR.