Oxytocin may function as a hypothalamic releasing hormone for prolactin and ACTH secretion in the rat. In the present study we have investigated the properties of putative oxytocin receptors in the rat adenohypophysis by radioligand-binding assay.
A novel oxytocin receptor antagonist [1-(β-mercapto-β,β-cyclopentamethylene propionic acid),2-(ortho-methyl)-Tyr2-Thr4-Orn8-Tyr9-NH2]-vasotocin (OTA) was radioiodinated by the iodogen method to a specific activity of 0·6 nCi/fmol. The radioiodinated derivative 125I-labelled OTA (125I-OTA) was reacted with membrane suspensions prepared from the uterus or adenohypophysis of female rats which were (a) ovariectomized for 7 days, (b) ovariectomized and treated with 5 μg oestradiol-17β 48 h before death or (c) implanted with a piece of silicone elastomer tubing containing 50 mg diethylstilboestrol (DES) 5 days before death. In uterine as well as the pituitary membrane suspensions, the radioligand was bound reversibly and with high affinity (dissociation constants 0·2 ± 0·1 and 0·1±0·01 nmol/l respectively; means + s.e.m., n=3) to a single class of sites with limited binding capacity, which varied with the type of pretreatment. Oestradiol-17β increased the binding capacity fivefold in the uterus in ovariectomized rats, but only very low specific radioligand binding was found in pituitary preparations from the same animals. Treatment with DES markedly increased the number of receptors in both the uterus and the adenohypophysis. Studies with several agonist and antagonist analogues revealed no difference in the ligand specificity of the uterine and adenohypophysial sites binding 125I-OTA, indicating that they are the same species of receptor. Furthermore, ligand-binding studies, carried out with tritiated vasopressin as tracer in pituitary membrane preparations, showed that OTA is not bound by pituitary vasopressin receptors at concentrations below 1 μmol/l.
In summary, 125I-OTA is a highly specific radioligand suitable for the analysis of pituitary oxytocin receptors. The low number of oxytocin receptors in the adenohypophysis of rats treated with oestradiol-17β suggests that the prolactin-stimulating action of oxytocin is mediated by highly efficient transmembrane signalling.
Journal of Endocrinology (1989) 122, 465–470