An efficient cyclic AMP assay for the functional evaluation of beta-adrenergic receptor ligands

beta-Adrenoceptors are G-protein linked receptors that are positively coupl ed to adenylyl cyclase. We wanted to develop an alternative method to the d etection of cAMP using radioimmunoassay for functional analysis of ligands for human beta-adrenoceptors (ARs). Chinese hamster ovary cells that stably expressed the beta(2)-AR were transiently transfected with reporter plasmi ds containing the firefly luciferase gene under the transcriptional control of 6 or 12 cAMP response elements. The transiently transfected cells (elec troporated at 150 volts, single pulse, 70 ms) were grown in 96-well microti ter plates (50,000 cells per well) for 20 hours, exposed to various ligands for 4 hours, and the luciferase activity was assayed. Stimulation of beta- ARs in these transfected cells resulted in a greater than 25-fold induction of luciferase activity. This activity was maximally increased in response to 20 mu M forskolin, 1 mM 3-isobutyl-1-methylxanthine, and 10-30 nM (-)-is oproterenol. Exposure of cells to (-)-isoproterenol elicited a concentratio n dependent luciferase response and these effects of (-)-isoproterenol were blocked by propranolol (K-B = 1.5 nM) and bupranolol (K-B = 1.9 nM). The c oncentration of (-)isoproterenol exhibiting half maximal response was 0.35 nM. This assay offers an excellent alternative to traditional methods of cA MP measurement and has applications to elucidate cAMP mediated signaling pa thways in cells.