Abstract

The HT29 cell line expresses alpha 2-adrenoceptors that are negatively coupled to the adenylate cyclase system and is, in this respect, a valuable model for in vitro study of alpha 2-adrenergic receptivity in a tissue from human origin. In these cancerous cells, UK-14,304 is a full agonist of the alpha 2-adrenergic-mediated inhibition of the vasoactive intestinal peptide-induced cyclic AMP accumulation, whereas clonidine acts only as a partial agonist. In the present report, we used [3H]UK-14,304 as radioligand and compared its binding characteristics with those of [3H]clonidine in order to better understand the difference between full and partial agonism on the basis of agonist/receptor interactions. [3H]UK-14,304 labeled with high affinity (KD = 0.39 +/- 0.05 nM) a single class of sites having the pharmacological specificity of an alpha 2-adrenoceptor. Comparison of [3H]UK-14,304, [3H]clonidine, and [3H]yohimbine Bmax proved that both 3H-agonists labeled the same number of sites (172 +/- 14 versus 179 +/- 21 fmol/mg of protein), whereas the 3H-antagonist recognized more sites (246 +/- 22 fmol/mg of protein). Inhibition of [3H]yohimbine by the two agonists was consistent with the existence of an heterogeneous population of receptors and analysis of the data according a two-site inhibition model showed 1) that the KiL/KiH ratio was higher for UK-14,304 than for clonidine and 2) that the percentages of high affinity state receptor recognized by both agonists were identical (56 +/- 4% with UK-14,304 and 59 +/- 5% with clonidine). Kinetics of [3H]UK-14,304 and [3H]clonidine binding indicated more complex agonist-receptor interactions than equilibrium data did. Association as well as dissociation of both radioligands appeared to be biphasic, suggesting a relative heterogeneity of 3H-agonist binding sites. Kinetic behavior of [3H]UK-14,304 only differed from that of [3H]clonidine by a much slower dissociation rate and by its ability to induce the formation of a tightly bound component, which corresponded to the formation of a very stable full agonist/receptor/Gi complex. Effects of guanosine-5'-(imido)-triphosphate and GTP on [3H]UK-14,304 and [3H]clonidine binding also proved that the agonists were not similarly sensitive to guanine nucleotides.