Identification and coupling to adenylate cyclase of three different [3H]CGP 12177 binding sites in Caco-2 cell membranes

Abstract

In the present investigation the identification of β -adrenoceptor (β -ARs) subtypes in the Caco-2 cell line was performed using radiometric assays. β -ARs were measured using increasing concentrations of the highly specific β -AR antagonist ( −)[3H]CGP 12177 (0.06–4 nM), whereas the β1- and β2-AR subtypes discriminated through selective binding assays using the highly selective unlabelled antagonists CGP 20712A and ICI 118551. Atypicalβ -ARs were measured using an incubation system formed by higher concentrations (0.6–20 nM) of ( −)[3H]CGP 12177. β - Atypical binding site concentrations ( 69 ± 5 fmol mg ml−1of membrane protein) were higher than β1-ARs ( 7 ± 1) andβ2 -ARs ( 24 ± 2), respectively. The different β -AR subtype affinities were characterized by binding inhibition experiments and the adrenergic agonists displaced the radioligand from its specific binding sites in the following order of potency: isoproterenol > clenbuterol > dobutamine > SR 58611A; for antagonists the order of potency was: propranolol ∼= ICI118551 ∼= CGP20712A. For atypical β -ARs the order was: SR 58611A > clenbuterol > dobutamine > isoproterenol for agonists and propranolol > CGP 20712A > ICI 118551 for antagonists. As far as in vitro functional studies are concerned, β -AR subtypes were shown to be coupled to adenylyl cyclase as their stimulation produced cAMP in an amount significantly higher than basal values. cAMP production after stimulation with dobutamine, clenbuterol, isoproterenol, and SR 58611A was measured using a cAMP radioassay kit. The order of efficacy suggested that the stimulation of β2-ARs was the most effective in inducing the activation of cell signalling mechanisms. The identification of functional β -ARs in a cancer cell line represents the first step in the study of the possible adrenergic control of cellular activities (e.g. proliferation and/or differentiation), which could suggest the use of this cancer cell line as a model for the study of cell activity or possibly new therapeutic strategies.