Oxytocin Agonist Research Articles

Possible neural mediation of the central effects of oxytocin on uterine motility

The central nervous system contains the nuclei at the origin of autonomic and neuroendocrine pathways to the uterus. Although the anatomical basis of these pathways is known, the conditions of their recruitment and their interactions in the context of copulation remain to be explored. We tested the hypothesis that some central mechanisms could simultaneously recruit both pathways to the uterus. In this aim, we recorded intrauterine pressure changes in anesthetized female rats at the estrus stage after intracerebroventricular (ICV) administration of oxytocin (OT). Doses of 0.3-300 ng elicited increases of frequency and amplitude of uterine contractions. These effects were partly mimicked by the OT agonist [Thr(4),Gly(7)]OT but not by arginine vasopressin. They were blocked by the OT receptor antagonist atosiban delivered either ICV or intravenously. The latter suggests that ICV OT activated the systemic release of OT. The effects of OT were also blocked by hexamethonium, a ganglionic blocking agent, by atropine, a muscarinic receptor antagonist, and by N(omega)-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthesis. The results reveal that ICV OT recruits autonomic efferent pathways to the uterus. These results support our hypothesis that the activation of central nuclei can promote uterine contractility, and that OT may be a central coordinator of autonomic and neuroendocrine pathways. The hypothalamus, the source of direct OT-ergic projections to the pituitary, the brain stem, and the spinal cord, may be a target of central OT.

Amygdala, Neuropeptides, and Fear Behavior

The neuropeptides vasopressin and oxytocin have opposite effects on fear- and anxiety-related behaviors. At the cellular level, both neuropeptides increase neuronal excitability in different brain regions, including the central amygdala, but the neuronal network underlying these opposite behavioral effects is not yet fully understood. Huber et al. identified discrete, anatomically separate populations of oxytocin and vasopressin receptors within the central amygdala. They used oxytocin and vasopressin agonists and antagonists to elicit electrophysiologic neuronal changes and were able to construct a hypothetical neural network in which oxytocin and vasopressin exerted opposing effects on anxiety and fear. D. Huber, P. Veinante, R. Stoop, Vasopressin and oxytocin excite distinct neuronal populations in the central amygdala. Science 308 , 245-248 (2005). [Abstract] [Full Text]

Non‐Peptide Oxytocin Agonists.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Molecular cloning, pharmacological characterization, and histochemical distribution of frog vasotocin and mesotocin receptors.

The neurohypophysial nonapeptides vasotocin (VT) and mesotocin (MT) are the amphibian counterparts of arginine vasopressin (AVP) and oxytocin (OT). We have here reported the cloning and functional characterization of the receptors for vasotocin (VTR) and mesotocin (MTR) in two species of frog, Rana catesbeiana and Rana esculenta. The frog VTR and MTR cDNAs encode proteins of 419 and 384 amino acids respectively. Frog VTR exhibits a high degree of sequence identity with the mammalian AVP-1a (V1a) receptor while the frog MTR possesses a high degree of sequence identity with the mammalian OT receptor. Activation of VTR induced both c-fos promoter- and cAMP-responsive element (CRE)-driven transcriptional activities, while activation of MTR induced c-fos promoter-driven transcriptional activity but failed to evoke CRE-driven transcriptional activity, suggesting differential G protein coupling between VTR and MTR. The VTR exhibited the highest sensitivity for VT followed by OT>AVP approximately MT, whereas the MTR showed preferential ligand sensitivity for MT>OT>VT>AVP. A V1a agonist but not V2 and OT agonists substantially activated both VTR and MTR with a similar sensitivity. V1a, V2 and OT antagonists inhibited MT-induced MTR activation but not VT-induced VTR activation. In the frog brain, VTR and MTR mRNAs were found to be widely expressed in the telencephalon, diencephalon and mesencephalon, and exhibited very similar regional distribution. In the pituitary, VTR and MTR were expressed in the distal and intermediate lobes but were virtually absent in the neural lobe. Taken together, these data indicated that, although the distribution of VTR and MTR largely overlaps in the frog brain and pituitary, VT and MT may play distinct activities owing to the ligand selectivity and different signaling pathways activated by their receptors.

Non-peptide oxytocin agonists

A library of compounds targeted to the vasopressin/oxytocin family of receptors was screened for activity at a cloned human oxytocin receptor using a reporter gene assay. Potency and selectivity were optimised to afford compound 39, EC 50 = 33 nM. This series of compounds represents the first disclosed, non-peptide, low molecular weight agonists of the hormone oxytocin (OT).

Comparison of Carbetocin and Oxytocin for the Prevention of Postpartum Hemorrhage Following Vaginal Delivery: A Double-Blind Randomized Trial

Objectives: To compare the efficacy of a single 100 µg intramuscular (IM) carbetocin injection, a long-acting oxytocin agonist, to a 2-hour 10 IU oxytocin intravenous (IV) infusion, in reducing the incidence and severity of postpartum hemorrhage (PPH) in women at risk for this condition.Methods: A randomized, double-blind, placebo-controlled study was conducted at 2 hospital centres, including 160 women with at least I risk factor for PPH. Eighty-three women received 100 µg carbetocin IM and an IV placebo immediately after placental delivery, while 77 women received placebo IM and oxytocin IV infusion. Complete blood count was collected at entry and 24 hours postpartum. All outcome measures, including the need for additional uterotonic agents or uterine massage, blood loss, and drop in hemoglobin and hematocrit, were analyzed using chi-square, Fisher exact, and Student t tests.Results: Population profile and risk factors for PPH were similar for each group. No significant difference was observed in the number of women requiring additional uterotonic medication (12 in each group). However, in the carbetocin group, 36 of the 83 women (43.4%) required at least I uterine massage compared to 48 of the 77 women (62.3%) in the oxytocin group (P < .02). Overall, uterotonic intervention was clinically indicated in 37 of the women (44.6%) receiving carbetocin compared to 49 of the women (63.6%) given an IV oxytocin infusion (P < .02). There were no differences in laboratory PPH indicators between the 2 groups.Conclusions: Women receiving a single 100 µg IM carbetocin injection required significantly less uterotonic intervention compared to women receiving a continuous 2-hour IV oxytocin infusion. A single IM carbetocin injection is more likely to prevent PPH in women at risk when compared to a continuous oxytocin IV infusion.

Spinal effects of oxytocin on uterine motility in anesthetized rats.

The rat uterus receives an innervation from the lumbosacral and thoracolumbar segments of the spinal cord. These segments receive descending oxytocinergic projections from the paraventricular nucleus of the hypothalamus. We tested the hypothesis that oxytocin regulates uterine motility through a spinal site of action. Oxytocin was administered in anesthetized female rats either intrathecally at the lumbosacral or thoracolumbar spinal cord levels or intravenously. Uterine activity was revealed by measuring changes of intrauterine pressure using an indwelling balloon placed in one caudal uterine horn. The uterus displayed a spontaneous activity characterized by intrauterine pressure rises, the frequency, amplitude, and duration of which were dependent on the stage of the estrous cycle. Oxytocin delivered at the lumbosacral level affected the frequency (during proestrus, estrus, and diestrus) and amplitude (during proestrus and estrus) of uterine activity. During estrus, oxytocin delivered at the thoracolumbar level affected the frequency, amplitude, and duration of the intrauterine pressure rises. Intravenous oxytocin not only affected intrauterine pressure rises (namely amplitude during proestrus and estrus and frequency and duration during estrus) but also increased the basal tone during estrus. The effects of lumbosacral oxytocin were partly mimicked by the oxytocin agonist [Thr(4),Gly(7)]-oxytocin blocked by the oxytocin receptor antagonist atosiban and by hexamethonium. Arginine vasopressin delivered at the lumbosacral level had no effect. These results support our hypothesis that oxytocin released by descending paraventriculo-spinal pathways and acting on spinal oxytocin receptors modulates the activity of the uterus. This regulation is cycle dependent.

Synthesis and characterization of fluorescent antagonists and agonists for human oxytocin and vasopressin V(1)(a) receptors.

The fluoresceinyl (Flu) group has been linked by an amide bond to the side chain amino group at position 8 of (a) two oxytocin (OT) antagonists, to give d(CH(2))(5)[Tyr(Me)(2),Thr(4),Orn(8)(5/6C-Flu),Tyr-NH(2)(9)]VT (Orn(8)(5/6C-Flu)OTA) (1) and desGly-NH(2),d(CH(2))(5)[D- Tyr(2),Thr(4),Orn(8)(5/6C-Flu)]VT (2), and (b) eight Lys(8) and Orn(8) analogues of potent OT agonists, to give d[Lys(8)(5/6C-Flu)]VT (3), d[Thr(4),Lys(8)(5/6C-Flu)]VT (4), [HO(1)][Lys(8)(5/6C-Flu)]VT (5), [HO(1)][Thr(4),Lys(8)(5/6C-Flu)]VT (6), d[Orn(8)(5/6C-Flu)]VT (7), d[Thr(4),Orn(8)(5/6C-Flu)]VT (8), [HO(1)][Orn(8)(5/6C-Flu)]VT (9), and [HO(1)][Thr(4),Orn(8)(5/6C-Flu)]VT (10). The tetramethylrhodamyl (Rhm) group was attached to the precursor peptide of 9 to give [HO(1)][Orn(8)(5/6C-Rhm)]VT (11). All 11 fluorescent peptides were evaluated in human OT and vasopressin V(1a) (vasoconstrictor), V(1b) (pituitary), and V(2) (antidiuretic) receptor binding and functional assays. With K(d) = 6.24, 217, >10000, and >10000 nM for the OT, V(1a), V(1b), and V(2) receptors, peptide 1 is a potent and selective fluorescent OT antagonist and may be useful for specifically labeling OT receptors while peptide 2 exhibits low affinities for all the receptors. The fluorescent peptides 3-10 are all very potent agonists for the human OT receptor. They exhibit the following K(d) values (nM) for the human OT, V(1a), V(1b), and V(2) receptors, respectively: (3) 0.29, 57, 124, >10000; (4) 1.8, 25.5, 150, >10000; (5) 0.34, 13.7, 66, nd (not determined); (6) 0.32, 17.3, 53, >10000; (7) 0.25, 107, 393, >10000; (8) 0.40, 30, 282, >10000; (9) 0.18, 12.2, 126, nd; (10) 0.17, 11.8, 87, >1000; (11) 0.092, 7.36, nd, nd. Peptide 7 exhibits both a high affinity and a high selectivity for human OT receptors. Peptides 7 and 11 were utilized to study the internalization of the OT receptor-ligand complex. Preliminary studies indicate that this process is similar to that observed for the vasopressin V(1a) receptor and differs from that observed for vasopressin V(2) receptors. Some or all of the fluorescent OT antagonists and agonists reported here are very promising new fluorescent ligands for labeling cells which express the human OT receptor and are also useful tools to follow endocytosis of the receptor-ligand complex.

The social deficits of the oxytocin knockout mouse

Numerous studies have implicated oxytocin (OT) and oxytocin receptors in the central mediation of social cognition and social behavior. Much of our understanding of OT's central effects depends on pharmacological studies with OT agonists and antagonists. Recently, our knowledge of OT's effects has been extended by the development of oxytocin knockout (OTKO) mice. Mice with a null mutation of the OT gene manifest several interesting cognitive and behavioral changes, only some of which were predicted by pharmacological studies. Contrary to studies in rats, mice do not appear to require OT for normal sexual or maternal behavior, though OT is necessary for the milk ejection reflex during lactation. OTKO pups thrive if raised by a lactating female, but OTKO pups emit fewer ultrasonic vocalizations with maternal separation and OTKO adults are more aggressive than WT mice. Remarkably, OTKO mice fail to recognize familiar conspecifics after repeated social encounters, though olfactory and non-social memory functions appear to be intact. Central OT administration into the amygdala restores social recognition. The development of transgenic mice with specific deficits in social memory represents a promising approach to examine the cellular and neural systems of social cognition. These studies may provide valuable new perspectives on diseases characterized by social deficits, such as autism or reactive attachment disorder.

Spinal proerectile effect of oxytocin in anesthetized rats.

The spinal cord contains the neural network that controls penile erection. This network is activated by information from peripheral and supraspinal origin. We tested the hypothesis that oxytocin (OT), released at the lumbosacral spinal cord level by descending projections from the paraventricular nucleus, regulated penile erection. In anesthetized male rats, blood pressure and intracavernous pressure (ICP) were monitored. Intrathecal (it) injection of cumulative doses of OT and the selective OT agonist [Thr(4),Gly(7)]OT at the lumbosacral level elicited ICP rises whose number, amplitude, and area were dose dependent. Thirty nanograms of OT and one-hundred nanograms of the agonist displayed the greatest proerectile effects. Single injections of OT also elicited ICP rises. Preliminary injection of a specific OT-receptor antagonist, hexamethonium, or bilateral pelvic nerve section impaired the effects of OT injected it. NaCl and vasopressin injected it at the lumbosacral level and OT injected it at the thoracolumbar level or intravenously had no effect on ICP. The results demonstrate that OT, acting at the lumbosacral spinal cord, elicits ICP rises in anesthetized rats. They suggest that OT, released on physiological activation of the PVN in a sexually relevant context, is a potent activator of spinal proerectile neurons.

Discovery and design of novel and selective vasopressin and oxytocin agonists and antagonists: the role of bioassays.

Synthetic oxytocin and vasopressin agonists and antagonists have become important tools for research and were instrumental in the identification of the four known receptor subtypes, V1a, V2, V1b (V3) and oxytocin, of these peptide hormones. However, the relative lack of receptor selectivity, particularly of the antagonists, has limited their usefulness as experimental probes and their potential as therapeutic agents. We now present some findings from our continuing studies aimed at the design of more selective oxytocin and vasopressin agonists and antagonists and a structure-activity relationship update on our recently discovered novel hypotensive vasopressin peptides. Bioassays have been, and continue to be, of critical importance in leading to the discovery of the novel agonists, antagonists and hypotensive peptides reported here. This paper highlights three main aspects of these studies. (1) Replacement of the tyrosine2 and/or phenylalanine3 residues in the V2 agonist deamino,[Val4,D-Arg8]arginine-vasopressin (dVDAVP) by thienylalanine resulted in selective V2 agonists with strikingly high potencies. However, the peptide solutions were unstable and lost activity over time. These highly potent V2 agonists, which are devoid of vasopressor activity, are promising leads for improving drugs for treating diabetes insipidus, enuresis and coagulation disorders. (2) Diaminopropionic acid and diaminobutyric acid substitution at position-5 in oxytocin and in V1a antagonists yielded, respectively, the first specific antagonist for the oxytocin receptor, desGly-NH2,d(CH2)5[D-Trp2,Thr4,Dap5]OVT and the first specific antagonist for the vasopressin V1a receptor, d(CH2)5[Tyr(Me)2,Dab5]AVP. The availability of single receptor subtype-specific or selective antagonists will enhance our ability to delineate receptor functions. Utilising these new receptor specific probes, we were able to show that the uterotonic action of vasopressin is mediated principally by oxytocin and not by V1a receptors. (3) Replacement of the phenylalanine3 residue in the V1a/V2/oxytocin antagonist, d(CH2)5[D-Tyr(Et)2,Val4]AVP, with arginine3 yielded the novel, selective, hypotensive vasopressin peptide, d(CH2)5[D-Tyr(Et)2,Arg3,Val4]AVP (Peptide I). Bioassay characterisations of Peptide I show that its vasodepressor action is independent of the peripheral autonomic, bradykinin, nitric oxide and prostaglandin systems and is not mediated by the known classical oxytocin and vasopressin receptors. These findings suggest the existence of a new vasopressin receptor subtype that may be relevant to the vasodilating action of vasopressin in regional vascular beds. Iodinatable hypotensive peptides have been synthesised and could be developed as markers for the putative new receptor. Ongoing structure-activity relationship studies on Peptide I have led to more potent and selective hypotensive peptides for use as new research tools and as leads for the development of a new class of antihypertensive agents.

Historadioautographic Localization, Pharmacology and Ontogeny of V1a Vasopressin Binding Sites in the Rat Kidney

The localization and pharmacological characteristics of vasopressin (VP) binding sites of the V_1a subtype in developing and adult rat kidney were investigated by radioautography on kidney sections incubated in the presence of a radioiodinated selective V_1a antagonist. Their localization after in vivo systemic infusion of the radioligand was also investigated. V_1a binding sites first appear at embryonic day 16 on vascular elements. In the adult, they were localized in the cortex (vascular and tubular structures, juxtaglomerular apparatus), the outer medulla outer stripe (vasa recta) and inner stripe (thin descending limbs of short looped nephrons) and the inner medulla (collecting ducts). Data obtained in vitro were confirmed by in vivo binding at postnatal day 30 (PN30). Whatever their localizations, the V_1a binding sites exhibited full V_1a pharmacological profile in postnatal stages rats and in adult rats: a high affinity (nM range) for VP and for the V_1a agonist, a lower affinity (µM range) for oxytocin and no affinity for the oxytocin agonist. The presence of V_1a binding sites in these different structures raises the question of the putative roles of VP in modulating renal functions. A striking finding is the presence of V_1a binding sites in the outer medullary thin descending limbs of short looped nephrons suggesting their colocalization with urea transporters.

Effect of gonadal steroids on the oxytocin-induced excitation of neurons in the bed nuclei of the stria terminalis at parturition in the rat

Experiments were undertaken to examine the role of ovarian steroids in peripartum programming of oxytocin sensitivity of limbic neurons implicated in oxytocin-induced facilitation of the milk-ejection reflex. In vivo recordings of neurons in the bed nuclei of the stria terminalis and ventrolateral septum of pre-parturient rats which had undergone prior ovariectomy and hysterectomy showed that oestradiol significantly increased the excitatory responses of bed nuclei/ventrolateral septum neurons to intracerebroventricular oxytocin, compared to oil-treated controls. Oestradiol also increased the excitation of bed nuclei neurons to the selective oxytocin agonist, [Thr 4,Gly 7]oxytocin in brain slices from steroid pre-treated ovariectomized hysterectomized rats, so that both the proportion of responsive neurons, and the magnitude of their responses were significantly increased. Parallel autoradiographic studies showed that oxytocin binding in the medial bed nuclei and ventrolateral septum was selectively increased following oestradiol treatment. Progesterone pre-treatment had no effect on either oxytocin sensitivity of bed nuclei/ventrolateral septum neurons recorded in vivo, or on oxytocin binding in the medial bed nuclei and ventrolateral septum, compared to oil-treated controls. Mean responses to [Thr 4,Gly 7]oxytocin in bed nuclei neurons recorded in slices from progesterone-treated rats were larger than controls, but this effect was highly variable. These results demonstrate that oestradiol greatly enhances oxytocin receptor expression and sensitivity of bed nuclei/ventrolateral septum neurons to oxytocin over the peripartum period, consistent with involvement of this steroid in enhancing oxytocin regulation of neuroendocrine and behavioural adaptations required for lactation.

Activin A and follistatin are dynamically regulated during human pregnancy.

Activin A (beta A-beta A) and activin B (beta B-beta B) are related dimeric proteins that regulate numerous cellular activities. Activin activity is bioneutralized by follistatin, a specific and high-affinity binding protein. Recently, our group developed specific and sensitive enzyme-linked immunosorbent activin assays that do not detect either activin isoform when bound to follistatin, therefore, the assays are specific for biologically relevant ligands. Activin A is measurable in the serum of pregnant women (cross-sectional sample collection), while activin B is not detected in maternal serum. However, activin B is measurable in amniotic fluid and cord blood sera. The purpose of this study was to measure serum activin A, activin B, and follistatin prospectively in longitudinally collected samples during pregnancy. This study design offered observations of relative changes in serum hormone concentration with each person serving as an internal reference. Serum samples were collected bimonthly from seven pregnant women beginning within the second month of gestation, and up to, but not including, the onset of labor. Six of the seven women had normal labor and delivery. One patient required pitocin (an oxytocin agonist) for induction of labor which led to delivery. Activin A, activin B, total follistatin, free follistatin, human chorionic gonadotropin, estradiol, progesterone, FSH, and LH were measured in maternal serum samples using specific assays. Serum activin A levels increased in the final month of pregnancy in the six patients who delivered following normal labor (< 0.78 ng/ml (first trimester) to 1-6 ng/ml (term)). Activin B was not detected in any serum sample (< 0.78 pg/ml). Total serum follistatin (free follistatin, follistatin-activin, and follistatin-inhibin) increased 10- to 45-fold in the final month of pregnancy in four of the women undergoing normal labor (10 ng/ml (first trimester) to 100-450 ng/ml (final month)). Total follistatin was high and variable in two women throughout pregnancy. Total follistatin returned to basal serum concentration in three of the patients during the last 2 weeks of pregnancy. Free follistatin was detected throughout pregnancy (range < 2-35 ng/ml). Free follistatin represented a small percentage of the total follistatin throughout the time of pregnancy and did not rise coincident with the rise in total follistatin. Serum activin A and activin B were not detected during the entire course of pregnancy in the one patient who did not have normal labor and total follistatin did not rise in the last trimester of pregnancy. Gonadotropin and steroid hormones were measured in all patients and were within normative ranges for human pregnancy (inclusive of the non-laboring patient). The results suggest that immunodetectable activin A is present in the third trimester of pregnant women who have normal onset labor. The total follistatin assay results suggest that follistatin-activin (or -inhibin) complexes are upregulated during the third trimester of pregnancy. Importantly, activin A production exceeds the binding capacity of circulating follistatin. Because binding protein free activin A is biologically active we conclude that the activin A detected in late pregnancy is biologically relevant. The findings are consistent with our hypothesis that activin A is an endocrine factor during the last trimester of human pregnancy and may be involved in normal labor.

Molecular aspects of monogamy.

Comparative studies of monogamous and nonmonogamous voles demonstrate species differences in the regional expression of oxytocin (OT) receptors in the brain. These species differences have not been observed with other neurotransmitter receptors (except vasopressin). Species differences for OT receptor distribution were also observed in other microtine and murine species selected as monogamous or promiscuous. These chemical neuroanatomic differences appear to be functionally relevant, as treatments with selective OT agonists and antagonists influence those behaviors that appear critical to pair bonding in the monogamous prairie vole. To investigate the mechanism controlling tissue-specific expression of OT receptors, we sequenced the OT receptor gene in both prairie voles and montane voles. The findings are inconclusive. Although both species differ markedly from rat and human in their regulatory (but not their coding) sequences, the species show very subtle differences from each other. Ongoing studies are investigating the consequences of these subtle differences between prairie and montane voles. At the same time, several transactivating factors that might influence OT receptor expression need to be explored. NOTE ADDED IN PROOF: The rat oxytocin receptor gene sequence, cited in FIGURES 4 and 5, was based on an error published in ref. 22. The corrected sequence has now been published (Rosen et al. 1996. Proc. Natl. Acad. Sci USA 93: 12501). The correct sequence shows greater homology with the vole oxytocin receptor gene sequences, but the remaining differences support the argument made herein for species differences in regional receptor expression.

Separate Agonist and Peptide Antagonist Binding Sites of the Oxytocin Receptor Defined by Their Transfer into the V2 Vasopressin Receptor

The neurohypophyseal nonapeptide oxytocin (OT) is the main hormone responsible for the initiation of labor; uterus contraction can be enhanced by application of oxytocin or suppressed by oxytocin antagonists. By transfer of domains from the G protein-coupled OT receptor into the related V2 vasopressin receptor, chimeric "gain in function" V2/OT receptors were produced that were able to bind either OT receptor agonists or a competitive peptide antagonist with high affinity. The binding site for the OT antagonist d(CH2)5[Tyr(Me)2,Thr4,Orn8,Tyr9]vasotocin was found to be formed by transmembrane helices 1, 2, and 7 with a major contribution to binding affinity by the upper part of helix 7. These transmembrane receptor regions could be excluded from participating in OT binding. For agonist binding and selectivity the first three extracellular receptor domains were most important. The interaction of the N-terminal domain and of the first extracellular loop of the OT receptor with the linear C-terminal tripeptidic part of oxytocin was demonstrated. Furthermore, the second extracellular loop of the OT receptor could be identified to interact with the cyclic hormone part. These three domains contribute to OT binding by synergistic interaction with oxytocin but not with the competitive antagonist. Our results provide evidence for the existence of separate domains and different conformations of a peptide hormone receptor involved in binding and selectivity for agonists and peptide antagonists.

Effect of Oxytocin on Testosterone Production by Isolated Rat Leydig Cells is Mediated Via a Specific Oxytocin Receptor1

The effect of the neurohypophysial hormones oxytocin and arginine vasopressin (AVP) on testicular steroidogenesis was reevaluated by use of short-term (< 10 h) cultures of isolated adult rat Leydig cells. Oxytocin at 10(-9), 10(-7), and 10(-5) M concentrations significantly increased basal testosterone production in a dose-dependent manner but had no effect on LH-stimulated testosterone production. The specificity of the effect was determined by use of the specific oxytocin receptor antagonist (OTA). OTA from 10(-9) to 10(-5) M concentrations inhibited the oxytocin-stimulated increase in testosterone production. Furthermore, the oxytocin agonist Thr4 Gly7 oxytocin also induced a dose-dependent increase in basal testosterone production. In contrast, AVP from 10(-9) to 10(-5) M concentrations did not consistently affect basal testosterone production by isolated Leydig cells, but significantly decreased LH-stimulated testosterone production. Inclusion of 10(-7) and 10(-5) M OTA with 10(-7) M AVP did not alter the inhibitory effect of the AVP. These data show that oxytocin and AVP have different effects on testosterone production by Leydig cells in vitro and support the hypothesis that oxytocin acts in the testis through a specific oxytocin receptor.

Oxytocin acts at V 1 receptors to excite sympathetic preganglionic neurones in neonate rat spinal cord in vitro

Intracellular recordings were made from sympathetic preganglionic neurones (SPNs) in transverse slices of thoraco-lumbar spinal cord of young rats (12–20 days old). A small group of SPNs generally having higher membrane potentials ( − 70mV) compared to a remaining group ( − 66mV) showed spontaneous oscillations of their membrane potential. Oxytocin superfused in concentrations of 0.1–30 μM had four effects on SPNs, inducing slow depolarisation, EPSPs, IPSPs and brief rhythmic oscillations. The slow depolarisation was unaffected by TTX whereas this abolished the other changes. The oxytocin-induced depolarisation was associated with a slow inward current and was not reversed at membrane potentials negative to E K , it increased at more positive potentials and was still present in low Ca 2+ and high Mg 2+ solutions. These features of the oxytocin induced current are similar to those of the TTX resistant voltage dependent Na + current described in brainstem autonomic neurones. Vasopressin superfused at concentrations of 0.1 μM to 30 μM had similar effects on SPNs to those of oxytocin. A comparison of the effects of oxytocin and vasopressin on the same neurones revealed that oxytocin was almost 10 times less potent than vasopressin. The effects of oxytocin were not mimicked by a selective oxytocin agonist but were mimicked by a selective vasopressin V 1a agonist and blocked by a selective V 1a antagonist. Therefore it is concluded that the effects of oxytocin on SPNs are mediated by the vasopressin V 1a receptor. It is suggested that oxytocin and vasopressin terminals in the lateral horn are part of a descending system controlling oscillating networks of SPNs in the spinal cord.

Localization and characterization of vasopressin binding sites in the rat brain using an iodinated linear AVP antagonist

The binding characteristics and central distribution of 125I-Linear AVP antagonist, a new ligand for vasopressin binding sites, are described in the following studies. Saturation studies performed on rat brain septal membranes demonstrated that 125I-Linear AVP antagonist binds to a single class of sites with high affinity (55 pM) and limited capacity (88 fmol/mg protein). In autoradiographic studies, 125I-Linear AVP antagonist labeled brain areas known to contain vasopressin receptors without binding to neurophysins. 125I-Linear AVP antagonist also labeled sites in cortex, hypothalamus, ventral tegmental area and substantia nigra. In competition studies, 125I-Linear AVP antagonist binding was most readily blocked by AVP and a selective V 1a agonist. Oxytocin and a selective V 2 ligand were effective only in micromolar concentrations. A selective oxytocin agonist was virtually ineffective in blocking 125I-Linear AVP antagonist binding. In regions that contain a high density of oxytocin binding sites, however, oxytocin-displaceable binding was observed. In agreement with studies on peripheral tissues, the binding profile generated from these studies indicates that 125I-Linear AVP antagonist binds to vasopressin receptors of the V 1a subtype. These results suggest that 125I-Linear AVP antagonist is a valuable ligand for the study of central AVP receptors.

Specific oxytocin agonist stimulates prolactin release but has no effect on inositol phosphate accumulation in isolated rat anterior pituitary cells.

We have previously characterized specific oxytocin receptors in the rat anterior pituitary gland, using a highly selective oxytocin receptor antagonist as radioligand. The aim of the present study was to examine whether occupation of these receptors by oxytocin produces a stimulation of prolactin release and a rise in the accumulation of total inositol phosphates in the rat adenohypophysis. Anterior pituitary cells harvested from randomly cycling and diethylstilboestrol (100 micrograms s.c.)-treated rats were perifused with Dulbecco's minimal essential medium at a rate of 0.3 ml/min. Oxytocin and the specific oxytocin agonist [Thr4-Gly7]-oxytocin (TG-OT) both stimulated a significant prolactin release at concentrations of 10(-6) and 10(-7) M. Oestrogen treatment did not affect the response to oxytocin, indicating that there is no straightforward correlation between receptor number and prolactin secretory response in the rat pituitary gland. The involvement of phosphoinositide hydrolysis was investigated in dispersed anterior pituitary cells and uterine tissue from randomly cycling rats. Oxytocin and arginine-vasopressin stimulated a significant (P < 0.05) and dose-related increase in total inositol phosphates, vasopressin being more potent. The specific oxytocin agonist TG-OT had no effect on total inositol phosphate production in pituitary cells, but when tested in uterine tissue it significantly (P < 0.05) stimulated the accumulation of total inositol phosphate at all concentrations tested (10(-5) to 10(-9) M). In conclusion, the data show that oxytocin has prolactin-releasing activity, acting on specific receptors in the anterior pituitary gland.(ABSTRACT TRUNCATED AT 250 WORDS)