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 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.
Heithier
H
H
Medizinische Universitätsklinik, Abteilung für Klinische Biochemie und Pathobiochemie, Würzburg, Germany.
Hallmann
D
D
Boege
F
F
Reiländer
H
H
Dees
C
C
Jaeggi
K A
KA
Arndt-Jovin
D
D
Jovin
T M
TM
Helmreich
E J
EJ
D000319:Adrenergic beta-Antagonists / Q000138:chemical synthesis* / Q000378:metabolism; D000818:Animals; D001896:Boron Compounds; D002460:Cell Line; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000378:metabolism; D006801:Humans; D007093:Imidazoles; D007202:Indicators and Reagents; D007700:Kinetics; D008024:Ligands; D009036:Moths; D011412:Propanolamines / Q000378:metabolism*; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection
D000319:Adrenergic beta-Antagonists; D001896:Boron Compounds; D005456:Fluorescent Dyes; D007093:Imidazoles; D007202:Indicators and Reagents; D008024:Ligands; D011412:Propanolamines; D011943:Receptors, Adrenergic, beta; D018343:Receptors, Adrenergic, beta-2; D011994:Recombinant Proteins; C088725:BODIPY-CGP 12177; C033394:CGP 12177