Synthesis and Properties of Fluorescent .beta.-Adrenoceptor Ligands

We describe the synthesis of bordifluoropyrromethene (BODIPY), fluorescein, and related fluorescent derivatives of the beta-adrenergic ligand CGP 12177. With these probes we screened insect (Sf9) cells stably transformed with the human beta 2-adrenoceptor gene and expressing (2-3.5) x 10(5) human be...

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Bibliographic Details
Published in:Biochemistry (Easton) 1994-08, Vol.33 (31), p.9126-9134
Main Authors: Heithier, Helmut, Hallmann, Dieter, Boege, Fritz, Reilaender, Helmut, Dees, Christian, Jaeggi, Knut A, Arndt-Jovin, Donna, Jovin, Thomas M, Helmreich, Ernst J. M
Format: Article
Language:English
Subjects:
Adrenergic beta-Antagonists - chemical synthesis
Adrenergic beta-Antagonists - metabolism
Animals
Boron Compounds
Cell Line
Fluorescent Dyes - chemical synthesis
Fluorescent Dyes - metabolism
Humans
Imidazoles
Indicators and Reagents
Kinetics
Ligands
Moths
Propanolamines - metabolism
Receptors, Adrenergic, beta - metabolism
Receptors, Adrenergic, beta-2 - metabolism
Recombinant Proteins - metabolism
Structure-Activity Relationship
Transfection
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Summary:We describe the synthesis of bordifluoropyrromethene (BODIPY), fluorescein, and related fluorescent derivatives of the beta-adrenergic ligand CGP 12177. With these probes we screened insect (Sf9) cells stably transformed with the human beta 2-adrenoceptor gene and expressing (2-3.5) x 10(5) human beta 2-adrenoceptors per cell. Among these derivatives only BODIPY-CGP gave a receptor-specific signal sufficiently strong for measuring the on- and off-rate constants and the equilibrium dissociation constant of beta-adrenoceptor-specific binding by spectrofluorometry or photon counting. Similar KD values for BODIPY-CGP binding were obtained by kinetic measurements (approx. 250 pM) and under equilibrium conditions (400 +/- 180 pM), and these were in the same range as those obtained with [3H]CGP 12177 (200 +/- 32 pM). The cell-bound fluorescence could be quenched specifically with nonfluorescent CGP 12177 to near background levels. The disposition of the beta 2-adrenoceptors in BODIPY-CGP-stained Sf9 cells was mainly restricted to the cell surface at 4 and 30 degrees C. Hence, beta-adrenoceptor-expressing cells can be stained specifically with BODIPY-CGP, and beta-adrenoceptors on a single cell can be assessed by photon counting under the fluorescence microscope. Cells can also be scanned by fluorescence-activated flow cytometry.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00197a015