SYNTHESIS AND PROPERTIES OF FLUORESCENT BETA-ADRENOCEPTOR LIGANDS

We describe the synthesis of bordifluoropyrromethene (BODIPY), fluores cein, and related fluorescent derivatives of the beta-adrenergic ligan d CGP 12177. With these probes we screened insect (Sf9) cells stably t ransformed with the human beta(2)-adrenoceptor gene and expressing (2- 3.5) x 10(5) human beta(2)-adrenoceptors per cell. Among these derivat ives only BODIPY-CGP gave a receptor-specific signal sufficiently stro ng for measuring the on- and off-rate constants and the equilibrium di ssociation constant of beta-adrenoceptor-specific binding by spectrofl uorometry or photon counting. Similar K-D values for BODIPY-CGP bindin g were obtained by kinetic measurements (approx. 250 pM) and under equ ilibrium conditions (400 +/- 180 pM), and these were in the same range as those obtained with [H-3]CGP 12177 (200 +/- 32 pM). The cell-bound fluorescence could be quenched specifically with nonfluorescent CGP 1 2177 to near background levels. The disposition of the beta(2)-adrenoc eptors in BODIPY-CGP-stained Sf9 cells was mainly restricted to the ce ll surface at 4 and 30 degrees C. Hence, beta-adrenoceptor-expressing cells can be stained specifically with BODIPY-CGP, and beta-adrenocept ors on a single cell can be assessed by photon counting under the fluo rescence microscope. Cells can also be scanned by fluorescence-activat ed flow cytometry.