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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.

Flow cytometry and laser scanning confocal microscopy analysis of the receptor distribution.