IDENTIFICATION OF ALPHA-2-ADRENOCEPTORS AND NONADRENERGIC IDAZOXAN BINDING-SITES IN RABBIT COLON EPITHELIAL-CELLS

α2-Adrenoceptors are possibly involved in the regulation of the hydroelectrolytic flux across the digestive mucosa. As no data are available concerning the existence of these receptors in colon epithelial cells, we aimed to investigate the existence of α2-adrenoceptors in this tissue using tritiated antagonists. [3H]Yohimbine and [3H]rauwolscine were not usable to label colonic α2-adrenoceptors because of their very high level of non-specific binding. In contrast, the methoxy derivative of idazoxan, [3H]RX821002, appeared a convenient radioligand for the purpose. [3H]RX821002 bound with high affinity (kd = 6.2 ± 0.8 nM) to a single population of non-interacting sites (Bmax = 193 ± 17 fmol/mg protein). The rank order of potency of catecholanaine enantiomers and adrenergic drugs to inhibit [3H]RX821002 binding demonstrated that the labelled sites are α2-adrenoceptors and that 53% of the receptors are in a high-affinity state sensitive to GTP + NaCl [3H]Idazoxan also bound to colocyte membranes, but inhibition by (-)-adrenaline and various imidazoline compounds indicated that this radioligands labels α2-adrenoceptors and non-adrenergic sites. When experiments were performed under binding conditions impeding the interaction of [3H]idazoxan with the α2-adrenoceptors (i.e. in presence of 10-4 M (-)-adrenaline), the Bmax of non-adrenergic idazoxan binding sites was 97 ± 8 fmol/mg protein and the KD was 3.5 + 0.5 nM. The sites were pharmacologically characterized with various imidazoline and non-imidazoune drugs. In order to study the putative relationship between α2-adrenoceptors and non-adrenergic idazoxan binding sites, the expression of both kinds of sites was investigated along the crypt-to-surface axis. Crypt cells had a higher number of α2-adrenoceptors than surface cells, whereas the number of non-adrenergic idazoxan binding sites remained constant. The results show that (i) α2-adrenoceptors coexist with non-adrenergic idazoxan binding sites in rabbit colocytes; (ii) the number of ayadrenoceptors is higher in crypt cells than in surface cells and (iii) α2-adrenoceptors and non-adrenergic binding sites are different and unrelated. © 1990.