α₂-Adrenoceptors in the HT 29 human colon adenocarcinoma cell line: Characterization with [³H]clonidine; Effects on cyclic AMP accumulation

Abstract

In the present work, [³H]clonidine was used to characterize α₂-adrenoceptors on the human adenocarcinoma cell line HT 29. The effects of α₂-adrenergic stimulation on cellular cyclic AMP levels were also investigated. The binding of [³H]clonidine on HT 29 cell membrane preparations was rapid and reversible. Scatchard analysis of the saturation curves indicated the existence of a single class of non-interacting sites with a K_D of 1.29 ± 0.07 nM and a B_max of 114 ± 7 fmol/mg of cell membrane protein. The binding sites for [³H]clonidine showed the required specificity for α₂-adrenoceptors. The potencies of α-adrenergic compounds to displace [³H]clonidine binding ranked as follows: yohimbine > phentolamine ⪢ prazosin for antagonists and clonidine > epinephrine > norepinephrine > phenylephrine ⪢ amidephrine for agonists. When tested on intact cells, epinephrine, norepinephrine and clonidine were found to counteract, in a dose-dependent manner, the increase of cyclic AMP triggered by vasoactive intestinal peptide (VIP). Such inhibitory effects were abolished by the addition of yohimbine but not of prazosin. The physiological amines were the most efficient agonists: both epinephrine and norepinephrine inhibited VIP-induced cyclic AMP accumulation by 50–55% with K_D values of 50 nM and 300 nM respectively. Clonidine was a partial agonist only, provoking a weak (25–30%) inhibition of VIP-induced cyclic AMP accumulation even at high concentrations. These results indicate that, like normal colocytes, human colon adenocarcinoma cells HT 29 possess α₂-adrenoceptors, the stimulation of which is associated with an inhibition of cyclic AMP production. The presence of functionally active α₂-adrenoceptors on this cultured cell line makes HT 29 a good model for further studies of the physiology of α₂-adrenoceptors.