An efficient cyclic AMP assay for the functional evaluation of beta-adrenergic receptor ligands.
beta-Adrenoceptors are G-protein linked receptors that are positively coupled to adenylyl cyclase. We wanted to develop an alternative method to the detection 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 plasmids containing the firefly luciferase gene under the transcriptional control of 6 or 12 cAMP response elements. The transiently transfected cells (electroporated at 150 volts, single pulse, 70 ms) were grown in 96-well microtiter 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 microM forskolin, 1 mM 3-isobutyl-1-methylxanthine, and 10-30 nM (-)-isoproterenol. Exposure of cells to (-)-isoproterenol elicited a concentration 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 concentration of (-)-isoproterenol exhibiting half maximal response was 0.35 nM. This assay offers an excellent alternative to traditional methods of cAMP measurement and has applications to elucidate cAMP mediated signaling pathways in cells.
Vansal
S S
SS
Department of Pharmacology and National Center for the Development of Natural Products, School of Pharmacy, University of Mississippi, University 38677, USA.
Feller
D R
DR
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D000242:Cyclic AMP / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D011863:Radioimmunoassay / Q000379:methods*; D011943:Receptors, Adrenergic, beta / Q000032:analysis* / Q000378:metabolism; D015398:Signal Transduction
D011943:Receptors, Adrenergic, beta; D000242:Cyclic AMP