Identification of alpha 2-adrenoceptors and non-adrenergic idazoxan binding sites in rabbit colon epithelial cells.
alpha 2-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 alpha 2-adrenoceptors in this tissue using tritiated antagonists. [3H]Yohimbine and [3H]rauwolscine were not usable to label colonic alpha 2-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 catecholamine enantiomers and adrenergic drugs to inhibit [3H]RX821002 binding demonstrated that the labelled sites are alpha 2-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 alpha 2-adrenoceptors and non-adrenergic sites. When experiments were performed under binding conditions impeding the interaction of [3H]idazoxan with the alpha 2-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-imidazoline drugs. In order to study the putative relationship between alpha 2-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 alpha 2-adrenoceptors than surface cells, whereas the number of non-adrenergic idazoxan binding sites remained constant. The results show that (i) alpha 2-adrenoceptors coexist with non-adrenergic idazoxan binding sites in rabbit colocytes; (ii) the number of alpha 2-adrenoceptors is higher in crypt cells than in surface cells and (iii) alpha 2-adrenoceptors and non-adrenergic binding sites are different and unrelated.
Senard
J M
JM
Laboratoire de Pharmacologie Médicale et Clinique, Faculté de Médecine, INSERM U317, Toulouse, France.
Langin
D
D
Estan
L
L
Paris
H
H
D000317:Adrenergic alpha-Antagonists / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D003106:Colon / Q000166:cytology / Q000378:metabolism*; D004146:Dioxanes / Q000378:metabolism*; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D019329:Idazoxan / Q000031:analogs & derivatives; D066298:In Vitro Techniques; D007700:Kinetics; D011817:Rabbits; D011942:Receptors, Adrenergic, alpha / Q000145:classification / Q000378:metabolism*
D000317:Adrenergic alpha-Antagonists; D004146:Dioxanes; D011942:Receptors, Adrenergic, alpha; C058525:2-methoxyidazoxan; D019329:Idazoxan