α2-Adrenergic Agonist-Induced Inhibition of Cyclic Amp Formation in Transfected Cell Lines Using a Microtiter-Based Scintillation Proximity Assay

Abstract

Human embryonic kidney cells (HEK-7) were transfected with the gene for the human alpha2C receptor (alpha2C4). Cells were grown in 96-well microtiter plates and cyclic AMP levels were measured by scintillation proximity assay, a modified radioimmunoassay technique. Radioactivity was quantified using a TopCount Scintillation detector. Cyclic AMP was increased in a dose-dependent manner by the addition of exogenous forskolin. The forskolin-induced enhancement of cyclic AMP was inhibited dose-dependently by the addition of alpha2-adrenergic agonists, and this inhibition was blocked by the addition of adrenergic antagonists. The extent of the inhibitory response caused by alpha2-adrenoceptor agonists was related to the receptor density in clonal cell lines derived from the transfected parental HEK-7 cells. By using cells grown in microtiter format, and employing the technological advantages of scintillation proximity assay and TopCount detection, it was possible to simultaneously evaluate the effects of multiple experimental permutations on cellular production of cyclic AMP with minimal disturbance of the cells and minimal and/or automated manipulation of the cyclic AMP formed. This combination of techniques should allow rapid testing of the actions of adrenergic agonists and antagonists on cells transfected with receptors linked to cyclic AMP formation.