Oxytocin Binding Sites Research Articles

Relationship Between Oxytocin and Pain Modulation and Inflammation.

Oxytocin (OXT), which is a well-known neurohypophysial hormone that is synthesized in the paraventricular (PVN) and supraoptic nuclei (SON) of the hypothalamus, is secreted from the posterior pituitary (PP) into the systemic circulation, where it plays an essential role in reproduction, especially during and after childbirth. Many recent studies have shown that OXT contributes to the modulation of several functions, such as social recognition, trust building, anti-nociception, anti-inflammation, stress relief and suppression of feeding. However, little is known about the neuronal networks responsible for OXT effects. Endogenious OXT has two regulations: the 1st regulation is humoral regulation, in which OXT is delivered to target organs from PP via the bloodstream; the 2nd regulation is nerve regulations, in which OXT from parvocellular neurosecretory neurons in the PVN directly project to the central nerve system (CNS). OXT binding sites, as well as OXT receptor expression, are located in various regions of the CNS, including the dorsal horn of spinal cord in rats, where it plays an important role in nociception. We examined the response to acute and chronic nociception/-inflammation in rat models using OXT-monomeric red fluorescent protein 1 (mRFP1) transgenic rats. We used formalin test as acute nociceptive/-inflammatory rat models and adjuvant arthritis as chronic nociceptive/-inflammatory rat models. We studied the effects of acute and chronic nociception/-inflammation on OXT-mRFP1 expression in the hypothalamus, posterior pituitary and spinal cord, and examined the role that OXT plays in acute and chronic nociceptive responses in rats. This review focuses on pain modulation and anti-inflammation by OXT according to previous clinical and animal research.

Oxytocin-Induced Analgesia and Scratching Are Mediated by the Vasopressin-1A Receptor in the Mouse

The neuropeptides oxytocin (OXT) and arginine vasopressin (AVP) contribute to the regulation of diverse cognitive and physiological functions including nociception. Indeed, OXT has been reported to be analgesic when administered directly into the brain, the spinal cord, or systemically. Here, we characterized the phenotype of oxytocin receptor (OTR) and vasopressin-1A receptor (V1AR) null mutant mice in a battery of pain assays. Surprisingly, OTR knock-out mice displayed a pain phenotype identical to their wild-type littermates. Moreover, systemic administration of OXT dose-dependently produced analgesia in both wild-type and OTR knock-out mice in three different assays, the radiant-heat paw withdrawal test, the von Frey test of mechanical sensitivity, and the formalin test of inflammatory nociception. In contrast, OXT-induced analgesia was completely absent in V1AR knock-out mice. In wild-type mice, OXT-induced analgesia could be fully prevented by pretreatment with a V1AR but not an OTR antagonist. Receptor binding studies demonstrated that the distribution of OXT and AVP binding sites in mouse lumbar spinal cord resembles the pattern observed in rat. AVP binding sites diffusely label the lumbar spinal cord, whereas OXT binding sites cluster in the substantia gelatinosa of the dorsal horn. In contrast, quantitative real-time reverse transcription (RT)-PCR revealed that V1AR but not OTR mRNA is abundantly expressed in mouse dorsal root ganglia, where it localizes to small- and medium-diameter cells as shown by single-cell RT-PCR. Hence, V1ARs expressed in dorsal root ganglia might represent a previously unrecognized target for the analgesic action of OXT and AVP.

Telencephalic binding sites for oxytocin and social organization: A comparative study of eusocial naked mole‐rats and solitary cape mole‐rats

African mole-rats provide a unique taxonomic group for investigating the evolution and neurobiology of sociality. The two species investigated here display extreme differences in social organization and reproductive strategy. Naked mole-rats (NMRs) live in colonies, dominated by a queen and her consorts; most members remain nonreproductive throughout life but cooperate in burrowing, foraging, and caring for pups, for which they are not biological parents (alloparenting). In contrast, Cape mole-rats (CMRs) are solitary and intolerant of conspecifics, except during fleeting seasonal copulation or minimal maternal behavior. Research on other mammals suggests that oxytocin receptors at various telencephalic sites regulate social recognition, monogamous pair bonding, and maternal/allomaternal behavior. Current paradigms in this field derive from monogamous and polygamous species of New World voles, which are evolutionarily remote from Old World mole-rats. The present findings indicate that NMRs exhibit a considerably greater level of oxytocin receptor (OTR) binding than CMRs in the: nucleus accumbens; indusium griseum; central, medial, and cortical amygdaloid nuclei; bed nucleus of the stria terminalis; and CA1 hippocampal subfield. In contrast, OTR binding in the piriform cortex is intense in CMRs but undetectable in NMRs. We speculate that the abundance of OTR binding and oxytocin-neurophysin-immunoreactive processes in the nucleus accumbens of NMRs reflects their sociality, alloparenting behavior, and potential for reproductive attachments. In contrast, the paucity of oxytocin and its receptors at this site in CMRs may reflect a paucity of prosocial behaviors. Whether similarities in OTR expression between eusocial mole-rats and monogamous voles are due to gene conservation or convergent evolution remains to be determined.

Distribution of vasopressin and oxytocin binding sites in the brain and upper spinal cord of the common marmoset

The aim of this study was to label selectively and to map central vasopressin (AVP) and oxytocin (OT) binding sites in the common marmoset. [ 125I]VPA, a compound selective in rodents and human for the AVP V 1a receptor, yielded the same labeling pattern as [ 3H]AVP, thus suggesting that most AVP receptors present in the marmoset brain are of the V 1a subtype. Numerous areas exhibited AVP binding sites, among which the olfactory bulb, the accumbens nucleus, the bed nucleus of the stria terminalis, the hypothalamic suprachiasmatic, arcuate and ventromedial nuclei, the medial amygdaloid nucleus, the nucleus of the solitary tract and the cerebral cortex. Binding sites for [ 125I]OTA, a selective OT receptor antagonist in rat and human, were markedly less abundant than [ 125I]VPA ones, and, to a few exceptions, expressed in different areas. Neither AVP, nor OT binding sites were detected in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei identified by neurophysin immunoreactivity. Marked species-related differences were observed in the distribution of both AVP and OT binding sites. Altogether, our data provide a morphological basis to investigate the function of central AVP and OT in the marmoset.

Receptors and neural effects of oxytocin in the rodent hypothalamus and preoptic region.

Vasopressin and oxytocin are produced in and secreted from not only hypothalamo-hypophysial neurons which shed their products into the circulation to act as hormones or releasing factors, but also from neurons whose axons form tracts which remain within the central nervous system. Using tritiated or radioiodinated ligands, binding sites for vasopressin and for oxytocin have been detected by in vitro autoradiography. In the rat hypothalamus binding sites for vasopressin are present in the suprachiasmatic, sigmoid and arcuate nuclei, and oxytocin receptors in the area of the ventromedial nucleus. Electrophysiological evidence obtained using single cell recordings in slices suggests that oxytocin-binding sites present in the ventromedial hypothalamus and in the bed nucleus of the stria terminalis mostly represent functional, neuronal receptors. The expression of these receptors (but not of the vasopressin receptors) depends on gonadal steroid hormones, as does that of uterine and mammary gland oxytocin receptors. Modifications of the hormonal status associated with, for example, puberty and lactation cause 'up-regulation' of central and peripheral oxytocin receptors. The central administration of oxytocin facilitates (and the administration of oxytocin agonists inhibits) maternal behaviour and the milk ejection reflex, therefore the hormonal and neural actions of oxytocin appear to be complementary in ensuring the birth and development of the offspring.

A neuromodulatory role for oxytocin within the supramammillary nucleus

Oxytocin functions as both a neurohypophysial hormone and central neuromodulatory peptide, and has been implicated in reproductive behaviours, anxiety and reward, as well as facilitation of the neuroendocrine milk-ejection reflex. A potential substrate for oxytocin is the supramammillary nucleus (SuM), a structure that contains oxytocin binding sites and serves as an important relay within the limbic system. Hence, this study investigated the neuromodulatory role of oxytocin within the SuM. Firstly, the effect of oxytocin on neuronal firing within the SuM was studied, using in vitro brain slices from virgin female rats. Oxytocin (10(-6)M) excited approximately 50% of SuM neurones, and similar results were obtained with the selective oxytocin agonist, Thr(4) Gly(7) oxytocin (TGOT) (10(-6) and 10(-7)M). The remaining neurones were unaffected. The TGOT response was blocked by application of the oxytocin antagonist, [d(CH(2))51,Tyr(Me)(2),Thr(4),Orn(8),Tyr-NH29]-vasotocin. Repeat doses of TGOT caused diminution of the response, indicative of desensitisation. In the second series of experiments, immunocytochemical techniques were used to study the oxytocinergic innervation of the SuM. The supramammillary decussation was found to contain numerous oxytocinergic fibres, and some could be seen coursing ventrally to enter the SuM. Whereas, some were clearly "en passant" fibres innervating the neurohypophysis, others followed a more convoluted and branching course, and appeared to terminate within the nucleus. Finally, in vivo microinfusion studies investigated whether oxytocin injected into the SuM facilitated the milk-ejection reflex, a well known action of central oxytocin. Oxytocin microinfusion in the region of the SuM caused a pronounced facilitation of the reflex, contrasting with the much smaller effects of microinfusions made rostral or caudal to the nucleus. Collectively, these results strongly support a neuromodulatory role for oxytocin within the SuM. This could have important implications for understanding the diverse neuroendocrine and behavioural functions of central oxytocin, including its role in reward.

Oxytocin and electrical stimulation of the paraventricular hypothalamic nucleus produce antinociceptive effects that are reversed by an oxytocin antagonist

In recent years, oxytocin has been implicated in a wide diversity of functions. The role of oxytocin in analgesia and pain modulation represents an important new function of an endogenous system controlling sensorial information. The paraventricular (PV) nucleus of the hypothalamus is one of the most important sources of oxytocin, and it has a very well-defined projection to the spinal cord. The location of this PV spinal cord projection correlates well with oxytocin binding sites at the dorsal horn of the spinal cord. In this work, we used rats with a chronic (46 days) sciatic loose ligature, an electrical stimulating electrode, and an intrathecal cannula, which reached the L4–L5 levels of the spinal cord. We compared the oxytocin effects with electrical stimulation of the PV and observed a significant reduction of the withdrawal responses to mechanical and cold stimulation applied to the ipsilateral and contralateral hind paws. An oxytocin antagonist administered intrathecally blocked the PV effects. Naloxone was also intrathecally injected 2 min before the PV stimulation, and we also observed a significant reduction of the withdrawal responses; however, this reduction was less pronounced. Our results support the hypothesis that oxytocin is part of the descending inhibitory control mechanisms having an important antinociceptive action. We cannot exclude a minor opiate participation in the OT action.

Sexually dimorphic expression of oxytocin binding sites in forebrain and spinal cord of the rat

The central actions of oxytocin on reproduction-related functions and behaviors are strongly steroid-dependent and gender specific. This study characterizes sexual differences in the oxytocin binding site expression in forebrain and spinal cord of the rat. Using film autoradiography, we quantified the density of oxytocin binding sites in the ventromedial hypothalamic nucleus, the medial and central nuclei of the amygdala, the medial bed nucleus of the stria terminalis and the spinal cord dorsal horns both in adult male and female rats, and during development. In addition, neonatal castrated males and intact neonatal females treated with a single injection of testosterone (1 mg) were examined. Data showed a sexual dimorphism in the expression of oxytocin binding sites in the spinal cord dorsal horns and in restricted areas of the forebrain that are sensitive to gonadal steroids such as the ventromedial hypothalamic nucleus, but not in gonadal steroid insensitive sites such as the central nucleus of the amygdala. Adult males had higher oxytocin binding site densities in the ventromedial hypothalamic nucleus and dorsal horns than females. In the forebrain, but not in the dorsal horn, this sexual difference required a perinatal exposure to testosterone. Neonatal castration only abolished the sexual difference in the ventromedial hypothalamic nucleus of adults, but not in the dorsal horn. Furthermore, females that received a single injection of testosterone 1 day after birth showed significant increases in the density of oxytocin binding sites in the ventromedial hypothalamic nucleus, medial nucleus of the amygdala and medial bed nucleus of the stria terminalis. In addition, the findings suggest that the sexual difference in the ventromedial hypothalamic nucleus also requires gonadal hormones in adulthood. Our data support the hypothesis that sexually dimorphic oxytocin binding sites may contribute to the regulatory central actions of oxytocin in gender specific functions and behaviors such as nociception and reproduction.

Oxytocin-induced excitation of neurones in the rat central and medial amygdaloid nuclei

Central oxytocin plays an important role in regulating emotionality. The amygdala expresses gonadal steroid-sensitive oxytocin binding sites in both the central and medial sub-nuclei, although the densities markedly differ between these nuclei. These studies examined the in vitro electrophysiological effects of oxytocin in the two amygdaloid nuclei and compared responses in female rats in different reproductive states (virgin, pregnant and lactating). Oxytocin (10 −9–10 −6M) caused a concentration-dependent increase in the firing rate of 20–36% of the neurones in both nuclei. Although autoradiographic studies using the oxytocin receptor antagonist [ 125I] d(CH 2) 5[Tyr(Me) 2,Thr 4,Orn 8,Tyr-NH 2 9]-vasotocin showed a higher density of binding in the central nucleus of the amygdala than medial nucleus of the amygdala, neurones in the central nucleus of the amygdala had a much lower sensitivity to oxytocin: equivalent responses obtained with 10 −6M in the central nucleus of the amygdala and 10 −8M in the medial nucleus of the amygdala, and neurones in the central nucleus of the amygdala were insensitive to concentrations below 10 −6M. Furthermore, repeated applications of oxytocin induced homologous desensitization in the central nucleus of the amygdala, but not medial nucleus of the amygdala—a single application of oxytocin producing long duration suppression of responses. This indicates that oxytocin has contrasting modes of action in the amygdala. Studies made across the reproductive cycle showed that lactating animals exhibited a larger proportion of oxytocin-responsive neurones in the medial nucleus of the amygdala and a smaller proportion in the central nucleus of the amygdala, compared with virgin or pregnant animals, indicating a peripartum shift in relative activation within the amygdala. However, changes in responses were not accompanied by changes in the density of oxytocin binding sites. These data show that oxytocin has a markedly different efficacy on neuronal activation in the central and medial sub-nuclei of the amygdala. The relative shift in excitatory responses between these two nuclei may underlie some of the neuroendocrine, behavioral and anxiolytic effects which have been ascribed to oxytocin in the periparturient rat.

Presence of functional vasopressin receptors in spinal ventral horn neurons of young rats: a morphological and electrophysiological study.

The objective of the present work was double. (i) Light microscopic autoradiography was used to determine the distribution of vasopressin and oxytocin binding sites in the spinal cord of rats. (ii) Whole-cell recordings were performed in lumbar spinal cord slices in order to assess whether these receptors are functional, whether they are located pre- or postsynaptically and whether they are present in motoneurons. In newborns, vasopressin binding sites of the V1a type were present in all laminae of the central gray at all segmental levels, whereas oxytocin binding sites were found only in the superficial layers of the dorsal horn. In adults, binding sites for both neuropeptides were also present, but were less dense. The dissociation constants for vasopressin were similar in newborns and adults. Whole-cell recordings showed that in identified motoneurons vasopressin exerted a direct effect, by inducing a membrane depolarization or by generating a sustained inward current, and an indirect effect, by enhancing glycinergic and GABAergic inhibitory transmission. Vasopressin-induced facilitation of inhibitory transmission could also be demonstrated in unidentified ventral horn neurons. All these effects were mediated by V1a but not V1b receptors. In some neurons, glycinergic transmission was also facilitated by a selective oxytocin receptor agonist. Our data, together with data obtained previously in brainstem motor nuclei, suggest that vasopressin of hypothalamic origin could play a role in motricity. The neuropeptide could act as a neuromodulator, because it would not directly activate motoneurons, but rather render them more responsive to incoming excitatory inputs. Vasopressin may thus act as a regulator of muscular force.

Actions of oxytocin and interactions with glutamate on spontaneous and evoked dorsal spinal cord neuronal activities

Among the numerous pain control mechanisms that have been proposed, those acting at the spinal cord have been broadly studied, but little is known about how neuropeptides originating in supraspinal structures may relate to pain and analgesic mechanisms. Oxytocin (OT), in addition to its well known hormonal action, produces neuronal effects in various regions of the central nervous system. Indeed, some parvocellular neurons in the hypothalamic paraventricular nucleus (PVN) are oxytocinergic and project to the caudal part of the brain and the spinal cord. Moreover, the rat spinal cord shows a good overlap between the oxytocinergic hypothalamo-spinal neuron projections and the distribution of OT binding sites. However, the physiological significance of these binding sites is largely unknown. Extracellular unit activity of spinal cord neurons was recorded at the T13–L1 levels in male rats anesthetized with halotane. Somatic stimulation was applied to the inner and outer thigh of the ipsilateral hindpaw, and glutamate (GLU) and OT were locally delivered by pressure using pipettes coupled to recording electrodes. Our results show that spinal cord neurons, mainly located in the dorsal horn, in the intermediolateral cell column (IML) and in the intermediomedial gray matter (IMM), respond to the application of OT (71.5%) with activation (48%) or inhibition (52%). In some cases, opposite OT effects were observed during simultaneous recordings of two cells, suggesting OT activation of an inhibitory interneuron followed by the inhibition of the second recorded neuron. Increases in neuronal firing rate produced by GLU could be blocked by prior OT application. Finally, OT could reduce or partially block the responses to tactile and nociceptive somatic stimulation. We found that spinal cord neurons are sensitive to OT indicating that OT binding sites are functionally active. OT effects suggest the activation of inhibitory interneurons acting on a second order projecting cells to modulate afferent tactile and nociceptive information.

Changes in oxytocin receptor in bovine preovulatory follicles between the gonadotropin surge and ovulation

In cattle, production of oxytocin by granulosa cells of preovulatory follicles is induced by the LH/FSH surge and intrafollicular oxytocin increases dramatically toward the end of the interval between the surge and ovulation. We reported previously that oxytocin modulates steroid production by both theca and granulosa cells obtained from bovine preovulatory follicles, implying actions of oxytocin on both cell types of preovulatory follicles. The objective of the present study was to examine the temporal expression of oxytocin receptor mRNA and protein in both theca and granulosa cells of bovine periovulatory follicles. To induce luteal regression and initiate a follicular phase, heifers were injected with prostaglandin F 2α on Day 6 or 7 of the estrous cycle and 36 h later, a GnRH analogue was administered to induce the LH/FSH surge. The periovulatory follicle was isolated at 0, 3.5, 12, or 24 h after GnRH injection. A significant increase in the levels of mRNA for oxytocin was detected in granulosa, but not theca, cells of periovulatory follicles at 12 and 24 h after GnRH injection, relative to time 0. In contrast, the levels of oxytocin receptor mRNA and specific binding sites for oxytocin in granulosa cells had decreased significantly at 12 and 24 h post-GnRH. In theca cells, the levels of oxytocin receptor mRNA were significantly lower at 12 and 24 h compared with values at 3.5 h, but specific binding of oxytocin to thecal cell membranes was not different at any time point. Immunopositive staining for oxytocin receptor was localized to both the theca and granulosa cell layer of periovulatory follicles at all four times of follicle isolation. These results suggest the direct action of oxytocin on both theca and granulosa cells of bovine periovulatory follicles through binding to its receptor, supporting the hypothesis that follicular oxytocin plays an important role(s) in the regulation of the final stage of follicular development. Down-regulation of oxytocin receptor mRNA and oxytocin binding may serve to temporally limit the actions of oxytocin on the preovulatory follicle.

Historadioautographic Localization of Oxytocin and V1a Vasopressin Binding Sites in the Kidney of Developing and Adult Rabbit, Mouse and Merione and of Adult Human

The localization of oxytocin (OT) binding sites and vasopressin (VP) binding sites of the V1a subtype was investigated by radioautography in kidneys of rabbits, mice and meriones during postnatal development and in the adult, and in the human kidney. Kidney sections were incubated in the presence of selective radioiodinated OT and V1a antagonists, respectively. The localizations were compared with those previously described in the rat. The main finding of the study was the almost constant presence in the cortex of V1a binding sites in the connecting tubule, the cortical collecting duct and in the juxtaglomerular apparatus (on the intra- and extraglomerular mesangium and the afferent arteriole). This distribution suggests an interaction of VP via V1a receptors and the kallikrein-kinin system in the kidney. OT binding sites, in comparison with V1a binding sites, were fewer and less constantly detectable in the kidney of the different species. In the mouse, their presence on the limbs of Henle’s loop in the medulla points to the possibility of their involvement in the medullary concentrating process. In the kidneys of the various species, OT and V1a binding sites occurred always in differential structures. In contrast, in the human kidney cortex, a colocalization of OT and V1a binding sites was almost constantly observed. This raises the question as to the specificity of the neurohypophysial hormone receptors in the human kidney.

Oxytocin receptor gene expression in rat mammary gland: structural characterization and regulation.

The differential, tissue-specific regulation of oxytocin (OT) binding sites allows the neurohypophysial nonapeptide OT to fulfill a dual role: to induce uterine contractions at parturition and to mediate milk ejection during lactation. Whereas uterine OT binding sites are up-regulated prior to parturition and are rapidly down-regulated thereafter, mammary gland OT binding sites gradually increase throughout gestation and remain up-regulated during the ensuing lactation period. Here, we structurally characterized OT receptor (OTR) mRNA in mammary gland and analyzed its expression during gestation and lactation and in response to steroid treatment. In mammary gland tissues, we found a 6.7 and a 5.4 kb OTR mRNA species, and both species were further analyzed by RACE (rapid amplification of cDNA ends). The 6.7 kb mRNA was found to be common to mammary gland and uterus and to extend 618 nucleotides beyond the published sequence of the rat OTR gene. The 5.4 kb mRNA species is unique to the mammary gland and terminates at a mammary gland-specific polyadenylation site that is not preceded by a classical polyadenylation signal. RT-PCR analysis did not provide any evidence for differences in the coding regions, suggesting that both uterine and mammary gland OTR mRNAs encode the same receptor protein. Furthermore, primer extension experiments showed that no differences exist in the specific transcriptional initiation sites of the OTR gene in the two tissues. During pregnancy, OTR mRNA per mammary gland increased approximately 150-fold and remained high during lactation, consistent with the previously identified regulation of OT binding sites and the role of OT during lactation. Whereas estrogen administration strongly induced the uterine OTR mRNA levels (>5-fold), mammary gland remained unaffected by steroid treatment. Moreover, tamoxifen had no effect on the mammary gland OTR mRNA level. In summary, our data demonstrate a differential control of OTR expression in uterus versus mammary gland and a mammary gland-specific OTR mRNA polyadenylation site. However, this differential control apparently does not involve the expression of different receptor genes nor the utilization of tissue-specific transcriptional initiation sites.

Oxytocin receptor agonists enhance inhibitory synaptic transmission in the rat hippocampus by activating interneurons in stratum pyramidale.

Oxytocin probably plays a role as a neurotransmitter/neuromodulator in the hippocampus of the rat. Oxytocin binding sites are present in the subiculum and CA1 region and oxytocin can excite a class of CA1 nonpyramidal neurons. In the present work we characterized the effect of oxytocin on hippocampal synaptic transmission. Whole-cell recordings were obtained from pyramidal neurons, in conditions of nearly symmetrical chloride concentrations. The selective oxytocin receptor agonist, [Thr4,Gly7]-oxytocin (TGOT), caused an increase in the frequency and amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) in virtually all neurons. These peptide-enhanced IPSCs were blocked by bicuculline, but not by strychnine, and reversed near 0 mV, indicating that they were mediated by gamma-aminobutyric acid (GABA)A receptors. On average, TGOT caused a nearly threefold increase in the frequency and almost a doubling in the amplitude of spontaneous IPSCs. TGOT did not influence the frequency and the amplitude of miniature IPSCs or spontaneous excitatory postsynaptic currents (EPSCs), and had no effect on evoked IPSCs. The peptide did not affect the basic membrane properties of pyramidal neurons or their GABA sensitivity. Thus, TGOT facilitated inhibitory transmission by exerting an excitatory action on the soma and/or dendrites of GABAergic interneurons. Extracellular recordings were performed in interneurons located in various hippocampal strata. Their sensitivity to TGOT was compared to that of substance P (SP). Interneurons in stratum pyramidale were excited both by TGOT and by SP. By contrast, stratum radiatum interneurons responded to SP but not to TGOT. In stratum oriens, half of the interneurons responded to SP, but only a minority to TGOT. Thus, oxytocin-responsive interneurons appear to be preferentially located in close vicinity of pyramidal neurons.

Pharmacologic characterization of the oxytocin receptor in human uterine smooth muscle cells.

[(3)H]-oxytocin was used to characterize the oxytocin receptor found in human uterine smooth muscle cells (USMC). Specific binding of [(3)H]-oxytocin to USMC plasma membranes was dependent upon time, temperature and membrane protein concentration. Scatchard plot analysis of equilibrium binding data revealed the existence of a single class of high-affinity binding sites with an apparent equilibrium dissociation constant (K(d)) of 0.76 nM and a maximum receptor density (B(max)) of 153 fmol mg(-1) protein. The Hill coefficient (n(H)) did not differ significantly from unity, suggesting binding to homogenous, non-interacting receptor populations. Competitive inhibition of [(3)H]-oxytocin binding showed that oxytocin and vasopressin (AVP) receptor agonists and antagonists displaced [(3)H]-oxytocin in a concentration-dependent manner. The order of potencies for peptide agonists and antagonists was: oxytocin>[Asu(1,6)]-oxytocin>AVP= atosiban>d(CH(2))(5)Tyr(Me)AVP>[Thr(4),Gly(7)]-oxytocin>dDAVP, and for nonpeptide antagonists was: L-371257>YM087>SR 49059>OPC-21268>SR 121463A>OPC-31260. Oxytocin significantly induced concentration-dependent increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) and hyperplasia in USMC. The oxytocin receptor antagonists, atosiban and L-371257, potently and concentration-dependently inhibited oxytocin-induced [Ca(2+)](i) increase and hyperplasia. In contrast, the V(1A) receptor selective antagonist, SR 49059, and the V(2) receptor selective antagonist, SR 121463A, did not potently inhibit oxytocin-induced [Ca(2+)](i) increase and hyperplasia. The potency order of antagonists in inhibiting oxytocin-induced [Ca(2+)](i) increase and hyperplasia was similar to that observed in radioligand binding assays. In conclusion, these data provide evidence that the high-affinity [(3)H]-oxytocin binding site found in human USMC is a functional oxytocin receptor coupled to [Ca(2+)](i) increase and cell growth. Thus human USMC may prove to be a valuable tool in further investigation of the physiologic and pathophysiologic roles of oxytocin in the uterus. British Journal of Pharmacology (2000) 129, 131 - 139

Oxytocinergic innervation of autonomic nuclei controlling penile erection in the rat

In the rat, spinal autonomic neurons controlling penile erection receive descending pathways that modulate their activity. The paraventricular nucleus of the hypothalamus contributes oxytocinergic fibers to the dorsal horn and preganglionic sympathetic and parasympathetic cell columns. We used retrograde tracing techniques with pseudorabies virus combined with immunohistochemistry against oxytocin and radioligand binding detection of oxytocinergic receptors to evidence the oxytocinergic innervation of thoracolumbar and lumbosacral spinal neurons controlling penile erection. Spinal neurons labelled with pseudorabies virus transsynaptically transported from the corpus cavernosum were present in the intermediolateral cell column and the dorsal gray commissure of the thoracolumbar and lumbosacral spinal cord. Confocal laser scanning microscopic observation of the same preparations revealed close appositions between oxytocinergic varicosities and pseudorabies virus-infected neurons, suggesting strongly the presence of synaptic contacts. Electron microscopy confirmed this hypothesis. Oxytocin binding sites were present in the superficial layers of the dorsal horn, the dorsal gray commissure and the intermediolateral cell column in both the thoracolumbar and lumbosacral segments. In rats, stimulation of the paraventricular nucleus induces penile erection, but the link between the nucleus and penile innervation remains unknown. Our findings support the hypothesis that oxytocin, released by descending paraventriculo-spinal pathways, activates proerectile spinal neurons.

Oxytocin gene expression and action in goat testis

Gene expression, immunohistochemical localization, binding and effects of oxytocin (OT) on androgen production in the testis of adult goats were studied. Using polymerase chain reaction (PCR) analysis we were able to detect OT gene transcripts in the goat testis. Immunohistochemistry revealed that both OT and neurophysin epitopes were expressed together in the intratubular regions, especially in the Sertoli cells, suggesting the production of OT in these cells. However, enzyme immunoassay found no difference in OT concentration between testicular arterial and venous plasma. Saturable, specific [ 3H]-OT binding sites were present in membrane fractions of the goat testis. Scatchard analysis indicated an apparent affinity of 42 ± 7 L/nmol and binding capacity of 24 ± 4 fmol OT bound/mg DNA. In vitro treatment of goat testes for 6 h with 100 nM OT led to a 3.5-fold increase (P<0.001) in 5α-dihydrotestosterone (DHT) production in spite of the induced decrease in testosterone, suggesting that OT not only affects testosterone production but also modulates its conversion to DHT. These results indicate that the goat testis produces OT, which may be involved in the local control of androgen biosynthesis.

Role of oxytocin in the initiation of term and preterm labor in rats: Changes in oxytocin receptor density and plasma oxytocin concentration and the effect of an oxytocin antagonist, L-366,509

Objective: Our purpose was to compare the functional roles of oxytocin in term and preterm labor in rats by both biochemical and pharmacologic means. Study Design: We determined the myometrial oxytocin receptor density and the maternal plasma concentrations of oxytocin and progesterone on gestational days 18, 20, and 22 (morning) and at the onset of delivery (day 22 afternoon) in rats with labor at term and at the onset of delivery (day 20 afternoon) in rats in preterm labor induced by the combined use of bilateral ovariectomy and estradiol injection. We also evaluated the effects of an oxytocin antagonist, L-366,509, on the initiation of both term and preterm labor. Results: The number of tritiated oxytocin binding sites in myometrial membranes rapidly increased on gestational day 22 (morning) in rats with term labor. Plasma progesterone level decreased in an inverse fashion. A rapid increase in circulating oxytocin concentration was observed at the onset of delivery in rats in labor at term. Both the plasma oxytocin concentration and the receptor density had the same values in rats with preterm labor as in rats with term labor. L-366,509 delayed the initiation of labor in rats with term and preterm labor in a dose-dependent manner. Conclusion: It is confirmed biochemically and pharmacologically that oxytocin plays an important role in the initiation of both term and preterm labor in rats. The oxytocin antagonist examined was able to delay term and preterm labor, so it might prove useful in clinical practice for the treatment of preterm labor. (Am J Obstet Gynecol 1999;180:621-7.)

Historadioautographic localisation of oxytocin and vasopressin binding sites in the central nervous system of the merione ( Meriones shawi)

The distribution of vasopressin and oxytocin binding sites in the central nervous system of the merione ( Meriones shawi), a rodent adapted to desert life, was studied by means of conventional film radioautography at macroscopic scale and historadioautography at cellular level using radioiodinated ligands highly selective for either oxytocin or type V1a vasopressin receptors. Both types of binding sites exhibited the same selectivity for endogenous peptides as in the rat. Distribution of oxytocin binding sites was similar in some structures (limbic system, spinal cord) to that described in the rat and in other rodents. Vasopressin binding sites were much more widely distributed in the merione than in the rat brain. In addition to locations common to most rodents (lateral septum and suprachiasmatic nucleus), in merione vasopressin binding sites occurred in several areas known to express oxytocin binding sites in the rat (olfactory system, hypothalamus). Colocalisation of vasopressin and oxytocin binding sites, which occurred in the CA1 and CA2 fields of Ammon’s horns of the hippocampus, the caudate-putamen and the fundus striati of the merione, has so far not been reported in any other rodent.