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S. E. Chadio and F. A. Antoni


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

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V. J. Ayad, S. A. McGoff and D. C. Wathes


The presence of oxytocin receptors in ovine oviduct has been investigated. High-affinity binding sites for [3H]oxytocin were detected in crude membrane fractions prepared from the oviducts of ewes killed during the oestrous period. The dissociation constant calculated for these sites in competition studies was 1·7 nmol/l. Similar dissociation constants were calculated for [Arg8]-vasopressin and the oxytocin-specific agonists [Gly7]-oxytocin and [Thr4, Gly7]-oxytocin, indicating that these sites represent oxytocin receptors. At least one additional site of lower affinity and undetermined identity was present.

The relative concentration of oxytocin-binding sites in preparations of oviduct membranes were estimated in ewes killed at different stages of the oestrous cycle using a single concentration of [3H]oxytocin. Binding was low during the luteal phase of the cycle but increased to a maximum at oestrus (77·7 fmol/mg protein). Binding fell after ovulation, reaching what appeared to be basal concentrations by the early luteal stage of the cycle. Binding to oviductal membranes from prepubertal, anoestrous and pregnant ewes was also low, but in anoestrous animals which had been treated with progesterone and oestrogen it was similar to values measured in ewes at oestrus. These results are consistent with the existence of oviductal oxytocin receptors which are regulated by ovarian steroids.

We conclude that oxytocin receptors are present in the oviduct of the ewe around the time of ovulation. The significance of oxytocin to events taking place in the oviduct at this time remains to be determined.

Journal of Endocrinology (1990) 124, 353–359

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A Kanda, K Takuwa-Kuroda, E Iwakoshi-Ukena, Y Furukawa, O Matsushima and H Minakata

We reported that the common octopus, Octopus vulgaris, in common with vertebrates, possesses two members of the oxytocin/vasopressin superfamily: octopressin (OP) and cephalotocin (CT). This was the first observation of its kind in invertebrates. As OP and CT have different biological activities, the presence of specific receptors has been proposed. We cloned the cDNA of an orphan receptor from Octopus brain and found it to encode a polypeptide of 397 amino acids that displays sequences characteristic of G-protein coupled receptors. The orphan receptor showed high homology to receptors of the oxytocin/vasopressin superfamily and seemed to conserve the agonist-binding pocket common to the oxytocin and vasopressin receptors. Xenopus oocytes that express the orphan receptor responded to the application of CT by an induction of membrane Cl(-) currents coupled to the inositol phosphate/Ca(2+) pathway. OP and the other members of the oxytocin/vasopressin superfamily did not activate this receptor. HPLC fractionation of the Octopus brain extract combined with an oocyte assay yielded a single substance that was identical to CT. On the basis of these results, we conclude that the cloned receptor is the CT receptor (CTR). Expression of CTR mRNA in Octopus was detected in the central and the peripheral nervous systems, the pancreas, the oviduct and the ovary. This receptor may mediate physiological functions of CT in Octopus such as neurotransmission, reproduction and metabolism.

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T. Engstrom, A. Atke and H. Vilhardt


Binding of [3H]oxytocin to purified myometrial plasma membranes was unaffected by continuous infusion of bradykinin over 5 days in rats pretreated with oestradiol 2 days before collection of tissue. In contrast, oxytocin treatment resulted in a 76% decrease in maximal binding of [3H]oxytocin and thereby in oxytocin receptor concentration without affecting the dissociation constant. The K M value (molar concentration giving half maximal contraction) of isolated uterine strips stimulated with oxytocin was increased and maximal contractile responses were reduced following oxytocin infusions.

The binding of [3H]bradykinin to purified plasma membranes was influenced by treatment with both oxytocin and bradykinin. Bradykinin infusions down-regulated the bradykinin receptor concentration by 19%, while the receptor affinity remained unchanged. Maximal contraction (Emax) values of isolated strips stimulated with bradykinin exhibited a slightly attenuated response and K M values were significantly enhanced. Long-term treatment with oxytocin down-regulated myometrial bradykinin receptors by 31%. In addition, oxytocin infusions caused Emax to decrease and K M to increase in experiments with isolated uterine strips stimulated with bradykinin.

It is concluded that the down-regulation of oxytocin and bradykinin receptors following prolonged exposure to oxytocin may result from changes in a common pathway for intracellular peptide receptor processing. Likewise, the increased K M values of isolated myometrial strips (despite unchanged dissociation constants) suggest that prolonged oxytocin treatment affects the coupling between receptor activation and contractile response.

J. Endocr. (1988) 118, 81–85

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V. J. Ayad, T. J. Parkinson, E. L. Matthews and M. L. Wild


Previous autoradiographic studies have suggested that the regulation of oxytocin receptors differs between endometrial cell types during the ovine oestrous cycle, and that those present on luminal epithelial cells are of particular importance to the regulation of prostaglandin F release during luteal regression. The present autoradiographic study compares the distribution of the endometrial oxytocin receptor in day-15 non-pregnant and pregnant ewes. The distribution of the endometrial oxytocin receptor in day-15 non-pregnant ewes infused with systemic or intrauterine oxytocin has also been investigated.

Continuous, s.c. infusion of oxytocin (150 mmol/24 h) into ewes (n = 6) between days 10 and 15 of the oestrous cycle significantly increased plasma oxytocin concentrations (to approximately 100 pmol/l). There was no similar increase in systemic oxytocin concentrations in ewes receiving intrauterine (i.u.) oxytocin infusions (10 nmol/24 h) between days 10 and 15 of the oestrous cycle (n = 6). Luteolysis was inhibited in all six animals infused with oxytocin (s.c.) and endometrial oxytocin receptor concentrations were significantly lower on day 15 in these animals (12·8 ± 6·5 (s.e.m.) fmol/mg protein; P<0·001) and in pregnant ewes (18·4 ± 15·4 fmol/mg protein; P <0·001; n = 8) than in ewes infused with saline (248·6±67·1 fmol/mg protein; n = 6). While the 125I-labelled oxytocin receptor antagonist, [1-(β-mercapto-β,β-cyclopentamethylene propionic acid), 2-(ortho-methyl)-Tyr2, Thr4, Orn8, Tyr9-NH2]-vasotocin (125I-labelled OTA) clearly labelled glandular epithelia, luminal epithelium and caruncular stromal cells specifically on day 15 in saline (s.c.)-infused ewes, such specific labelling appeared to be reduced or absent from pregnant animals and those infused with oxytocin (s.c.). A significant reduction in the density of labelling of caruncular stroma (P < 0·05) and luminal epithelium (P < 0·001) was confirmed using quantitative densitometric analysis. The reduction in the labelling of endometrium in oxytocin-infused ewes was not caused by the binding of exogenous oxytocin to endometrial binding sites. Oxytocin infusion (i.u.) did not inhibit luteolysis, nor was there any significant difference in the endometrial oxytocin receptor concentration in this group of ewes on day 15 compared with those infused with saline (i.u.). There was also clear specific labelling of luminal epithelial cells with 125I-labelled OTA in ewes receiving oxytocin infused i.u. and quantification of autoradiograms failed to differentiate between these animals and those infused with saline (i.u.).

It was concluded that systemic oxytocin infusion and the early establishment of pregnancy led to a clear reduction in the endometrial oxytocin receptor concentration on luminal epithelial cells, glandular epithelial cells and caruncular stromal cells, but that i.u. oxytocin infusions did not affect any of these receptor populations and notably not the luminal epithelial oxytocin receptor. The results support the contention that the luminal epithelial oxytocin receptor is involved in the luteolytic process.

Journal of Endocrinology (1993) 137, 423–431

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P R Riley, D R E Abayasekara, H J Stewart and A P F Flint


The entire coding region of an ovine endometrial oxytocin receptor (OTR) cDNA was generated by PCR, subcloned into the SV40 major late promoter expression vector pSVLJ and transiently expressed in Cos-7 cells. A specific OTR antagonist, 125I-labelled d(CH2)5 [Tyr(Me)2,Thr4,Tyr-NH2 9]-vasotocin (OTA), was used to describe the binding kinetics of the expressed receptor which had a K d of 4·5 nm and Bmax of 2·4 nm/mg protein (6·8 × 105 receptor molecules/transfected cell). The functional properties of the expressed OTR were determined by measuring oxytocin-induced phosphoinositide (PI) hydrolysis. Oxytocin increased PI turnover in OTR transfected cells fourfold in excess of residual endogenous activity, and stimulated phospholipase C (PLC) activity in a dose- and time-dependent manner, confirming that the expressed OTR cDNA was functional. Arginine vasopressin also stimulated PI turnover in a dose-dependent manner; thresholds of responses to oxytocin and arginine vasopressin were 10−9 m and 10−7 m respectively. OTA did not increase PI turnover and competitively inhibited the oxytocin-induced response. Direct activation of the pathway by aluminium fluoride and guanosine (3′-Othio)-triphosphate (GTPγS) confirmed that the OTR was G-protein linked. Co-incubation of GTPγS with oxytocin shifted the PI-response threshold from 10−7 m to 10−9 m and significantly increased the level of response, suggesting that maximum PI turnover was agonist-dependent. The G-protein involved in mediating the signal transduction pathway was pertussis toxin-insensitive and, therefore, probably a member of the Gq subfamily. The PLC inhibitor, U73122, had no effect on oxytocin-induced PI turnover, consistent with the response in endometrial tissue. These data suggest that the signalling pathway mediated by expressed OTR is similar to that attributed to OTR occupancy in ovine endometrium.

Journal of Endocrinology (1996) 149, 389–396

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A. Atke, H. Vilhardt and P. Melin


Purified myometrial plasma membrane fractions were prepared from rats treated with oestradiol to induce oestrus. The binding affinities of 11 antagonistic oxytocin analogues to the oxytocin receptor of the plasma membranes were measured. Furthermore, lipophilicity of the peptides was assessed by reversed-phase high pressure liquid chromatography. No significant correlation was found between lipophilicity of the analogues and values for antagonistic potencies or binding affinities. Also, receptor-binding affinity did not correlate with in-vitro antagonistic activity whereas a significant correlation was obtained between binding affinities and in-vivo antagonistic potency for analogues void of partial agonist properties. It is concluded that neither receptor affinity nor lipophilicity in the analogues can predict the potency of the antagonists in vitro. However, receptor affinity was found to be a relatively good predictor of the in-vivo potency, while the usefulness of measuring antagonistic potency in vitro is questioned.

J. Endocr. (1988) 118, 187–192

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E L Matthews and V J Ayad


The purpose of the present study was to investigate the presence of high-affinity oxytocin-binding sites (putative oxytocin receptors) in the cervix of the non-pregnant ewe. [3H]Oxytocin binding to the peripheral layers of cervical tissue (comprising the serosal layer and the least dense collagenous and muscular layers) and the remaining dense collagenous cervical tissue were studied separately. [3H] Oxytocin-binding sites were detected in membrane fractions prepared from both of these regions, but binding to the peripheral cervix was variable and binding site concentrations were low, hence these were not characterized further. A high-affinity oxytocin-binding site, having a dissociation constant of 1·8 nmol/l, was characterized in the dense collagenous regions of the cervix of ewes killed during the oestrous period. Similar dissociation constants were determined for [Arg8]-vasopressin and the oxytocin-specific agonist [Thr4, Gly7]-oxytocin in competition studies.

[3H] Oxytocin binding to peripheral cervical tissue and to the dense collagenous cervix was generally low or undetectable during the luteal phase, but increased in both tissues around the time of luteolysis. Although specific binding to both tissues was variable during the oestrous period, it was higher at this time than during the luteal phase. [3H] Oxytocin-binding site concentrations were also found to be higher within the inner dense collagenous cervix of oestrous ewes than of pregnant, ovariectomized or anoestrous animals. During the oestrous cycle, oxytocin-binding site concentrations reached a maximum in the dense collagenous cervical tissue on the day of oestrus (141·8 ±44 (s.e.m.) fmol/mg protein), showing a general decline during the following days back to luteal phase values. This compared with concentrations of 513·3 ±132·1 and 216·1 ± 13·9 fmol/mg protein, measured for comparative purposes in endometrial and myometrial membrane preparations, respectively, on the day of oestrus in the same group of ewes. However, in membrane preparations of peripheral cervical tissue higher concentrations were measured on day 14 than on the following 2 days and maximal concentrations were not reached until the day after oestrus (52·7 ± 4·2 fmol/mg protein). Concentrations were maintained at similar values during the subsequent 2 days and significant specific binding was still measurable in both regions of the cervix on day 5.

The localization of oxytocin-binding sites within dense collagenous cervical tissue was investigated autoradiographically using the 125I-labelled specific oxytocin receptor antagonist [1(β-mercapto-β,β-cyclopentamethylene propionic acid), 2-(ortho-methyl)-Tyr2, Thr4, Orn8, Tyr9 -NH2]-vasotocin. The only clear specific labelling was localized to the luminal epithelium of the uterine section of the cervix of oestrous ewes, with labelling in ewes in the luteal phase clearly reduced or absent.

The results demonstrate the presence of a high-affinity oxytocin-binding site within the cervix of the oestrous ewe which is associated with secretory cells and which undergoes similar changes in concentration during the oestrous cycle to uterine oxytocin receptor sites. The significance of this novel putative site of oxytocin action remains to be established.

Journal of Endocrinology (1994) 142, 397–405

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V. J. Ayad, E. L. Matthews, D. C. Wathes, T. J. Parkinson and M. L. Wild


The present study was designed to determine the localization of the endometrial oxytocin receptor during the ovine oestrous cycle, particularly on day 14, the time of initiation of luteal regression in the ewe. Samples were obtained from 29 ewes at different stages of the oestrous cycle (several during the luteal phase and on every day between day 14 (− 2) and day + 3 of the oestrous period).

Oxytocin receptors were localized autoradiographically in sections of uterine tissue, using the 125I-labelled oxytocin receptor antagonist [1-(β-mercapto-β,β-cyclopentamethylene propionic acid), 2-(ortho-methyl)-Tyr2,Thr4,Orn8,Tyr9-NH2]-vasotocin (125I-labelled OTA). There was some variation in the pattern of 125I-labelled OTA labelling between different uterine tissue samples from the same ewe and also between samples obtained from different ewes thought to be at the same stage of the oestrous cycle. A clear overall pattern did, however, emerge with 125I-labelled OTA-binding sites distributed between luminal epithelial cells, glandular epithelial cells and caruncular stromal cells to varying extents on different days of the cycle.

During the luteal phase (days 5–12) clear specific labelling of endometrial tissue was generally absent. On day 14 labelling was evident on the luminal epithelium, but only in nine tissue samples out of a total of 18 studied, indicating that the entire luminal surface did not contain oxytocin receptors at this time. Between the day before oestrus and day 3 of the oestrous cycle the luminal epithelium was consistently labelled. The most extensive labelling of the remaining endometrial tissue was observed on the day of oestrus, with 125I-labelled OTA-binding sites clearly present on the stromal cells within caruncles and on a large proportion of secretory epithelia. This contrasted with the day before and the day after oestrus when labelling of glandular tissue was confined to the superficial endometrium, and labelling of caruncular stromal cells, although sometimes evident, was never as intense as on day 0. On days 2 and 3 labelling varied between being similar to that found on day 1 and being confined to the luminal epithelium and very few superficial secretory glands.

The results of this study lead us to conclude that the oxytocin receptor shows a differential distribution between stromal cells, epithelial cells lining secretory glands and luminal epithelial cells during the oestrous cycle; that the steroidal regulation of the oxytocin receptor differs between endometrial cell types; and that control of the luminal epithelial oxytocin receptors is probably of particular importance to the regulation of prostaglandin F release at luteal regression and during the maternal recognition of pregnancy.

Journal of Endocrinology (1991) 130, 199–206

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T Engstrom, P Bratholm, NJ Christensen and H Vilhardt

The objective of the present study was to further elucidate our previous observation that beta2-adrenoceptor activation induces oxytocin receptor (OTR) expression in rat myometrium. We wanted to investigate whether the mechanism behind this effect was under the influence of gonadal steroids. Ovariectomized non-pregnant rats were treated with estrogen, progesterone or a combination of both for 3 days. Some rats were concomitantly treated with isoproterenol. Estrogen treatment increased both OTR mRNA production and maximal binding of [3H]-oxytocin to isolated myometrial plasma membranes, but it did not affect contractility of isolated uterine strips challenged with oxytocin. When the estrogen regimen was combined with isoproterenol treatment, an augmented maximal contractile response (Emax) to oxytocin was observed although no further increase in OTR mRNA and binding was seen. Progesterone treatment did not in itself alter OTR mRNA, OTR binding or Emax. However, OTRs were induced at the level of gene expression when progesterone was supplemented with isoproterenol infusion. Finally, progesterone suppressed the effect of estrogen on OTR mRNA production and binding when the two compounds were administered together. However, when isoproterenol treatment was added this effect was abolished and Emax was enhanced more than that seen following treatment with estrogen alone. These data suggest that beta2-adrenoceptor activation represents an important regulator of OTR expression/function in estrogen- and progesterone-dominated rat myometrium.