title: Interactions of full and partial agonists with HT29 cell alpha 2-adrenoceptor: comparative study of [3H]UK-14,304 and [3H]clonidine binding.
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.
authors.affiliation: INSERM U-317, Institut de Physiologie, Université Paul Sabatier, Toulouse, France.
authors.forename: H
authors.initials: H
authors.lastname: Paris
authors.forename: J
authors.initials: J
authors.lastname: Galitzky
authors.forename: J M
authors.initials: JM
authors.lastname: Senard
mesh_terms: D000316:Adrenergic alpha-Agonists / Q000378:metabolism*; D000068438:Brimonidine Tartrate; D003000:Clonidine / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004837:Epinephrine / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007700:Kinetics; D011810:Quinoxalines / Q000378:metabolism*; D011942:Receptors, Adrenergic, alpha / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology; D014316:Tritium; D014407:Tumor Cells, Cultured; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology; D015016:Yohimbine / Q000494:pharmacology
chemical_list: D000316:Adrenergic alpha-Agonists; D011810:Quinoxalines; D011942:Receptors, Adrenergic, alpha; D014316:Tritium; D015016:Yohimbine; D006165:Guanylyl Imidodiphosphate; D014660:Vasoactive Intestinal Peptide; D000068438:Brimonidine Tartrate; D012964:Sodium; D000242:Cyclic AMP; D003000:Clonidine; D004837:Epinephrine