[Flow cytometry and laser scanning confocal microscopy analysis of the receptor distribution].
To study the physiological regulation of the receptor protein, a fluorescent probe and detection system for alpha 1B-adrenergic receptors have been developed. By using the anti-peptide antibody developed against the alpha 1B-adrenergic receptor NH2-terminus, we have examined the agonist-regulated alpha 1B-adrenergic receptor redistribution in desensitized cells. Flow cytometry analysis showed that anti-peptide antibody against alpha 1B-adrenergic receptor specifically identifies the receptor in CHO cells, COS-7 cells that were transfected with alpha 1B-adrenergic receptor cDNA and rat hepatocytes. Using a fluoro-labeled receptor ligand, BODIPY FL-prazosin, as a probe, cell surface alpha 1-adrenergic receptor subtypes can be detected by flow cytometry. Laser scanning confocal microscopy visualized the agonist-regulated redistribution process of alpha 1B-adrenergic receptor in living cells; thus, following phenylephrine (10(-6) M) stimulation, receptor antigen at the cell surface rapidly internalized and clustered together in a cell within 30 min. The results showed that the antibody and fluoro-labeled ligand are valuable tools for studying the localization and functional role of the alpha 1-adrenergic receptor subtype.
Department of Molecular Cell Pharmacology, National Childrens' Medical Research Center, Tokyo, Japan.
A
A
Hirasawa
T
T
Awaji
G
G
Tsujimoto
D000818:Animals; D016466:CHO Cells / Q000378:metabolism; D006224:Cricetinae; D005434:Flow Cytometry; D008099:Liver / Q000166:cytology / Q000378:metabolism; D018613:Microscopy, Confocal; D051381:Rats; D018340:Receptors, Adrenergic, alpha-1 / Q000032:analysis*
D018340:Receptors, Adrenergic, alpha-1