CDB15:0001193 OXT — OXTR

ABSTRACT: Oxytocin is a nonapeptide hormone (CYIQNCPLG-NH2, OT), controlling labor and lactation in mammalian females, via interactions with specific cellular membrane receptors (OTRs). The native hormone is cyclized via a 1-6 disulfide and its receptor belongs to the GTP-binding (G) protein-coupled receptor (GPCR) family, also known as heptahelical transmembrane (7TM) or serpentine receptors. Using a technique combining multiple sequence alignments with available experimental constraints, a reliable OTR model was built. Subsequently, the OTR complexes with a selective agonist [Thr4,Gly7]OT, a selective cyclohexapeptide antagonist L-366,948 and oxytocin itself were modeled and relaxed using a constrained simulated annealing (CSA) protocol. All three ligands seem to prefer similar modes of binding to the receptor, manifested by repeating receptor residues which directly interact with the ligands. Those involved in the three complexes are putative helices: TM3: R113, K116, Q119, M123; TM4: Q171, and TM5: I201 and T205. Most of them are the equivalent residues/positions to those found in our earlier studies, regarding related vasopressin V2 receptor/bioligand interactions.

ABSTRACT: We have discovered a new, potent, selective, and orally active oxytocin receptor antagonist, (2S,4Z)-N-[(2S)-2-hydroxy-2-phenylethyl]-4-(methoxyimino)-1-[(2'-methyl[1,1'-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxamide (compound 1). We report the biochemical, pharmacological, and pharmacokinetic characterization in vitro and in vivo of this compound. Compound 1 competitively inhibits binding of [3H]oxytocin and the peptide antagonist 125I-ornithine vasotocin analog to human and rat oxytocin receptor expressed in human embryonic kidney 293-EBNA or Chinese hamster ovary cells with nanomolar potency. Selectivity against vasopressin receptor subtypes is >6-fold for V1a and >350-fold for V2 and V1b. Compound 1 inhibits oxytocin-evoked intracellular Ca2+ mobilization (IC50 = 8 nM). Compound 1 has no intrinsic agonist activity at the oxytocin receptor. Oxytocininduced contraction of isolated rat uterine strips is blocked by compound 1 (pA2 = 7.82). In anesthetized nonpregnant rats, single administration of compound 1 by i.v. or oral routes causes dose-dependent inhibition of contractions elicited by repeated injections of oxytocin with ED50 = 3.5 mg/kg i.v. and 89 mg/kg p.o., respectively. Compound 1 significantly inhibits spontaneous uterine contractions in pregnant rats near term when administered intravenously or orally. We conclude that compound 1 is a potent, selective, and orally active nonpeptide oxytocin receptor antagonist, which is a suitable candidate for evaluation as a potential tocolytic agent for the management of preterm labor.

ABSTRACT: Just before the onset of labour, uterine myometrium becomes extremely sensitive to oxytocin, for which it is a primary target tissue, because of a dramatic increase in the number of oxytocin receptors. We report here the structure and expression of the human oxytocin receptor complementary DNA isolated by expression cloning. The encoded receptor is a 388-amino-acid polypeptide with 7 transmembrane domains typical of G protein-coupled receptors. The oxytocin receptor, expressed in Xenopus oocytes, specifically responds to oxytocin and induces an inward membrane current. Messenger RNAs for the receptor are of two sizes, 3.6 kilobases in breast, and 4.4 kilobases in ovary, uterine endometrium and myometrium. The mRNA level in the myometrium is very high at term. We conclude that the increase in receptor number in the myometrium at labour is, at least in part, due to the increase in mRNA.

ABSTRACT: The human oxytocin (OT) receptor was stably expressed in 293 embryonic kidney cells (293/OTR), characterized pharmacologically and compared to human uterine myometrial receptors. The cloned receptor is expressed at a reasonably high density (0.82 fmole/microgram protein) and exhibits high affinity for [3H]OT (Kd = 0.32nM), similar to the value found in human myometrial tissue. The rank-order of potency for various antagonist and agonist ligands from several structural classes is also similar between the cloned and native receptor, as seen in a comparison of their inhibitory constants for [3H]OT binding. Agonist affinity at the cloned OT receptor is decreased by guanine nucleotide analogs, demonstrating functional G-protein-coupling. The OT receptor in 293 cells, like in human myometrium, is also coupled to the inositol phosphate pathway. In 293/OTR cells, OT stimulates inositol phosphate accumulation with an EC50 = 4.1 nM, an effect blocked by a potent and selective OT antagonist, L-366,948. Additionally, the cloned receptor in 293 cells desensitizes to high concentrations of OT, similar to the desensitization in myometrial tissue and also described for several other G-protein-coupled receptors. These results illustrate the utility of the 293 cell line for expressing human OT receptors in an environment quite comparable to the native myometrial tissue.