Specific Oxytocin Receptor Antagonist Research Articles

Oxytocin treatment rescues irritability-like behavior in Cc2d1a conditional knockout mice.

Irritability, a state of excessive reactivity to negative emotional stimuli, is common in individuals with autism spectrum disorder (ASD). Although it has a significant negative impact of patients' disease severity and quality of life, the neural mechanisms underlying irritability in ASD remain largely unclear. We have previously demonstrated that male mice lacking the Coiled-coil and C2 domain containing 1a (Cc2d1a) in forebrain excitatory neurons recapitulate numerous ASD-like behavioral phenotypes, including impaired social behaviors and pronounced repetitive behaviors. Here, using the bottle-brush test (BBT) to trigger and evaluate aggressive and defensive responses, we show that Cc2d1a deletion increases irritability-like behavior in male but not female mice, which is correlated with reduced number of oxytocin (OXT)-expressing neurons in the paraventricular nucleus (PVN) of the hypothalamus. Intranasal OXT administration or chemogenetic activation of OXT neurons in the PVN rescues irritability-like behavior in Cc2d1a conditional knockout (cKO) mice. Administration of a selective melanocortin receptor 4 agonist, RO27-3225, which potentiates endogenous OXT release, also alleviates irritability-like behavior in Cc2d1a cKO mice, an effect blocked by a specific OXT receptor antagonist, L-368,899. We additionally identify a projection connecting the posterior ventral segment of the medial amygdala (MeApv) and ventromedial nucleus of the ventromedial hypothalamus (VMHvl) for governing irritability-like behavior during the BBT. Chemogenetic suppression of the MeApv-VMHvl pathway alleviates irritability-like behavior in Cc2d1a cKO mice. Together, our study uncovers dysregulation of OXT system in irritability-like behavior in Cc2d1a cKO mice during the BBT and provide translatable insights into the development of OXT-based therapeutics for clinical interventions.

Oxytocin alters the morphology of hypothalamic neurons via the transcription factor myocyte enhancer factor 2A (MEF-2A).

Oxytocin (OT) has gained attention not only as anxiolytic drug and as potential treatment option for autistic children; it also acts as a growth and differentiation factor in neuronal cells. While behavioral effects of OT have been studied in detail, knowledge about the cellular effects of OT is relatively sparse. In this study, we present evidence for three hypotheses: 1) OT leads to neurite retraction in hypothalamic neurons via the OT receptor (OTR) 2) The transcription factor MEF-2A is a central regulator of OT-induced neurite retraction, and 3) The MAPK pathway is critical for OT-induced MEF-2A activation. Incubation of rat hypothalamic H32 cells with 10 nM to 1 μM OT, vasopressin, and the specific OTR agonist TGOT, over the course of 12 h resulted in a time-dependent, significant retraction of neurites. In addition, the size of the nuclear compartment increased, whereas the overall cell size remained unchanged. OT treatment for 10 h increased the cellular viability significantly, and this effect could be blocked by a specific OTR antagonist, providing evidence for a specific and pro-active effect of OT on neurite retraction, and not as an unspecific side effect of apoptosis. The molecular mechanism that controls OT-induced neurite retraction includes a reduced phosphorylation of the transcription factor MEF-2A at Serine 408 (S408). This dephosphorylation is under the control of the OTR-coupled MAPK pathway, as blocking MEK1/2 by U0126 inhibited MEF-2A activation and subsequent neurite retraction. The siRNA-mediated knockdown of MEF-2A prevented the OT-induced neurite retraction, providing direct evidence for a role of MEF-2A in morphological alterations induced by OT treatment. In summary, the present study reveals a previously unknown OTR-coupled MAPK-MEF-2A pathway, which is responsible for OT-induced neurite retraction of hypothalamic neurons.

11C]PF-3274167 as a PET radiotracer of oxytocin receptors: Radiosynthesis and evaluation in rat brain

IntroductionOxytocin plays a major role in the regulation of social interactions in mammals by interacting with the oxytocin receptor (OTR) expressed in the brain. Furthermore, the oxytocin system appears as a possible therapeutic target in autism spectrum disorders and other psychiatric troubles, justifying current pharmacological researches. Since no specific PET radioligand is currently available to image OTR in the brain, the aim of this study was to radiolabel the specific OTR antagonist PF-3274167 and to evaluate [11C]PF-3274167 as a potential PET tracer for OTR in rat brains. Methods[11C]PF-3274167 was prepared via the O-methylation of its desmethyl precursor with [11C]methyl iodide. The lipophilicity of the radioactive compound was evaluated by measuring the n-octanol-buffer partition coefficient (logD). Autoradiography experiments were performed on rat brain tissue to evaluate the in vitro distribution of the [11C]PF-3274167. MicroPET experiments were conducted with and without pre-injection of ciclosporin in order to evaluate the influence of the P-glycoprotein (P-gp) on the brain uptake. Results[11C]PF-3274167 was synthesized with high radiochemical and chemical purities (>95%) and good specific activity. The measured logD was 1.93. In vitro, [11C]PF-3274167 did not show any evidence of specific binding to OTR. PET imaging showed that [11C]PF-3274167 uptake in rat brain was very low in basal conditions but increased significantly after the administration of ciclosporin, suggesting that it is a substrate of the P-gp. In the ciclosporin-pre-injected rat, however, [11C]PF-3274167 distribution did not match with the known distribution of OTR in rats. Conclusion[11C]PF-3274167 is not a suitable tracer for imaging of OTR in rat brain, probably because of a too low affinity for this receptor in addition to a poor brain penetration.

Involvement of the oxytocin system in the nucleus accumbens in the regulation of juvenile social novelty-seeking behavior

Exploration of novel environments, stimuli, and conspecifics is highly adaptive during the juvenile period, as individuals transition from immaturity to adulthood. We recently showed that juvenile rats prefer to interact with a novel individual over a familiar cage mate. However, the neural mechanisms underlying this juvenile social novelty-seeking behavior remain largely unknown. One potential candidate is the oxytocin (OXT) system, given its involvement in various motivated social behaviors. Here, we show that administration of the specific oxytocin receptor antagonist desGly-NH2,d(CH2)5-[Tyr(Me)2,Thr4]OVT reduces social novelty seeking-behavior in juvenile male rats when injected into the nucleus accumbens (10ng/0.5μl/side). The same drug dose was ineffective at altering social novelty-seeking behavior when administered into the lateral septum or basolateral amygdala. These results are the first to suggest the involvement of the OXT system in the nucleus accumbens in the regulation of juvenile social novelty-seeking behavior.

Oxytocin receptor neurotransmission in the dorsolateral bed nucleus of the stria terminalis facilitates the acquisition of cued fear in the fear-potentiated startle paradigm in rats

Oxytocin (OT) is a hypothalamic neuropeptide that modulates fear and anxiety-like behaviors. Dorsolateral bed nucleus of the stria terminalis (BNSTdl) plays a critical role in the regulation of fear and anxiety, and expresses high levels of OT receptor (OTR). However, the role of OTR neurotransmission within the BNSTdl in mediating these behaviors is unknown. Here, we used adult male Sprague-Dawley rats to investigate the role of OTR neurotransmission in the BNSTdl in the modulation of the acoustic startle response, as well as in the acquisition and consolidation of conditioned fear using fear potentiated startle (FPS) paradigm. Bilateral intra-BNSTdl administration of OT (100 ng) did not affect the acquisition of conditioned fear response. However, intra-BNSTdl administration of specific OTR antagonist (OTA), (d(CH2)51, Tyr(Me)2, Thr4, Orn8, des-Gly-NH29)-vasotocin, (200 ng), prior to the fear conditioning session, impaired the acquisition of cued fear, without affecting a non-cued fear component of FPS. Neither OTA, nor OT affected baseline startle or shock reactivity during fear conditioning. Therefore, the observed impairment of cued fear after OTA infusion resulted from the specific effect on the formation of cued fear. In contrast to the acquisition, neither OTA nor OT affected the consolidation of FPS, when administered after the completion of fear conditioning session. Taken together, these results reveal the important role of OTR neurotransmission in the BNSTdl in the formation of conditioned fear to a discrete cue. This study also highlights the role of the BNSTdl in learning to discriminate between threatening and safe stimuli.

Septal oxytocin administration impairs peer affiliation via V1a receptors in female meadow voles

The peptide hormone oxytocin (OT) plays an important role in social behaviors, including social bond formation. In different contexts, however, OT is also associated with aggression, social selectivity, and reduced affiliation. Female meadow voles form social preferences for familiar same-sex peers under short, winter-like day lengths in the laboratory, and provide a means of studying affiliation outside the context of reproductive pair bonds. Multiple lines of evidence suggest that the actions of OT in the lateral septum (LS) may decrease affiliative behavior, including greater density of OT receptors in the LS of meadow voles that huddle less. We infused OT into the LS of female meadow voles immediately prior to cohabitation with a social partner to determine its effects on partner preference formation. OT prevented the formation of preferences for the partner female. Co-administration of OT with a specific OT receptor antagonist did not reverse the effect, but co-administration of OT with a specific vasopressin 1a receptor (V1aR) antagonist did, indicating that OT in the LS likely acted through V1aRs to decrease partner preference. Receptor autoradiography revealed dense V1aR binding in the LS of female meadow voles. These results suggest that the LS is a brain region that may be responsible for inhibitory effects of OT administration on affiliation, which will be important to consider in therapeutic administrations of OT.

Oxytocin enhances the expression of morphine-induced conditioned place preference in rats

Drug addiction is characterized by drug-seeking and drug-taking and has devastating consequences on addicts as well as on society. Environmental contexts previously associated with drug use can elicit continued drug use and facilitate relapse. Accumulating evidence suggests that the neuropeptide oxytocin might be a potential treatment for behavioral disorders, including drug addiction. Here, we investigated the effects of central oxytocin administration on the acquisition and expression of morphine-induced conditioned place preference (CPP), a model for measuring the rewarding effects of drugs of abuse, in male Wistar rats. Intracerebroventricular (ICV) administration of oxytocin (0.2μg) or the specific oxytocin receptor antagonist (OTA), desGly-NH2, d(CH2)5[Tyr(Me)2, Thr4] OVT, (0.75μg), on the conditioning days did not affect the acquisition of morphine-induced CPP. By contrast, ICV oxytocin, but not OTA, administration immediately prior to the post-conditioning session enhanced the expression of morphine-induced CPP, possibly by activation of oxytocin receptors in the nucleus accumbens shell (NAcSh). The oxytocin enhancement of morphine-induced CPP was not associated with any changes in the locomotor activity of morphine-conditioned rats. Together, these data suggest that central administration of exogenous oxytocin enhances the expression of morphine-induced CPP, at least in part, via activation of oxytocin receptors within the NAcSh.

Oxytocin in the medial prefrontal cortex regulates maternal care, maternal aggression and anxiety during the postpartum period.

The neuropeptide oxytocin (OT) acts on a widespread network of brain regions to regulate numerous behavioral adaptations during the postpartum period including maternal care, maternal aggression, and anxiety. In the present study, we examined whether this network also includes the medial prefrontal cortex (mPFC). We found that bilateral infusion of a highly specific oxytocin receptor antagonist (OTR-A) into the prelimbic (PL) region of the mPFC increased anxiety-like behavior in postpartum, but not virgin, females. In addition, OTR blockade in the postpartum mPFC impaired maternal care behaviors and enhanced maternal aggression. Overall, these results suggest that OT in the mPFC modulates maternal care and aggression, as well as anxiety-like behavior, during the postpartum period. Although the relationship among these behaviors is complicated and further investigation is required to refine our understanding of OT actions in the maternal mPFC, these data nonetheless provide new insights into neural circuitry of OT-mediated postpartum behaviors.

Social learning reverses conditioned aversion to nicotine via oxytocin receptor in the infralimbic cortex in rats (839.4)

The initial aversive response to cigarettes in most smokers is thought to be ameliorated by the presence of peers who also smoke. We previously reported that social learning (i.e. detecting an odor cue associated with nicotine from peer rats) reversed the development of conditioned taste aversion (CTA) between self‐administered nicotine and a contingent olfactogustatory cue in adolescent rats (PMID:21796102). Herein, we focused on the role of oxytocin in infralimbic cortex (IL) as a mechanism for the effect of social learning. Bilateral lesions of IL before (F1,13=18.1, p=0.001) but not after (F1,16=0.481, p>0.05) self‐administration reduced the effect of social learning on tests of CTA. We also found that carbon disulfide contained in rats’ breath was the signal mediating social learning and its effect also required intact IL. We then infused a specific oxytocin receptor antagonist, desGly‐NH2‐d(CH2)5[D‐Tyr]OVT, (0.028 ~ 2.8 μg/ml, a gift from Dr. Mannings) bilaterally into the IL, which dose‐dependently reduced the effect of carbon disulfide on nicotine CTA (F4,30=3.9, p=0.01). The highest dose of the antagonist had no effect on i.v. saline control rats. These results suggested that the release of oxytocin in the IL is a critical signal that reversed the conditioned aversion between nicotine and its olfactogustatory cue, which in turn enabled voluntary nicotine intake in adolescent rats.Key words: nicotine; aversion; social learning; infralimbic cortex; oxytocin

Sex-specific modulation of juvenile social play by vasopressin.

Social play activities among juveniles are thought to contribute to the development of social and emotional skills in humans and animals. Conversely, social play deficits are observed in developmental neuropsychiatric disorders. Importantly, many of these disorders show sex differences in incidence, course of the disease, and severity of symptoms. We hypothesized that sex differences in the neural systems controlling social behavior can contribute to these differences. We therefore studied the involvement of the sexually dimorphic vasopressin and oxytocin systems, which have been implicated in these disorders, in juvenile social play behavior. Single-housed 5-week-old juvenile male and female rats were exposed in their home cage to an age-and sex-matched novel conspecific for 10 min, and social play behaviors were recorded. We found no consistent sex differences in duration or elements of social play in vehicle-treated rats. However, intracerebroventricular injection of the specific vasopressin 1a receptor (V1aR) antagonist (CH2)5Tyr(Me(2))AVP significantly reduced social play behaviors in males while increasing them in females. Intracerebroventricular injection of the specific oxytocin receptor antagonist des-Gly-NH2,d(CH2)5[Tyr(Me)(2),Thr(4)]OVT did not alter social play in either sex. To locate the effects of V1aR blockade on social play, we targeted the lateral septum, a sexually dimorphic brain region showing denser vasopressin fibers in males than in females and an abundant expression of V1aR in both sexes. Surprisingly, blockade of V1aR in the lateral septum increased social play behaviors in males, but decreased them in females. These findings suggest sex- and brain region-specific roles for vasopressin in the regulation of social play behavior in juvenile rats.

PI3K/Akt responses to oxytocin stimulation in Caco2BB gut cells

Recently, we discovered oxytocin receptor (OTR) expression in the developing gut villus epithelium that emerges in villus-crypt junctions after weaning. Oxytocin (OT) and OTR regulate many physiological functions in various tissues; however, their function in gut epithelium is unknown. We explored responses of PI3K and Akt phosphoisoforms to OT stimuli in the Caco2BB human gut cell line. In Caco2BB cells, PI3K and pAkt levels peaked at 62.5 nM OT. At higher concentrations, PI3K decreased more gradually than pAkt(S473) suggesting that the pAkt(S473) response is separate from PI3K. At ≤7.8 nM OT, pAkt(T308) increased while pAkt(S473) decreased. Using a specific OTR antagonist, we demonstrated that responses of pAkt(T308) to OT depend on OTR in contrast to the partial OTR-dependence of the pAkt(S473) response. Differential pAkt phosphoisoform responses included pAkt phosphoserine 473 persistently free of phosphothreonine 308. The reduction in PI3K after 62.5 nM OT for 30 min coincided with OTR internalization. The PI3K/Akt activation profile was somewhat different in other cell lines (MCF-7 breast cancer cells, HT29 gut cells), which have PI3K activating mutations, that were examined to establish experimental parameters. In Caco2BB cells, the divergent effects of OT upon pAkt phosphoisoforms suggests separate sub-pathways; pAkt (T308) activation depends on OTR via the PI3K pathway and pAkt(S473) presumably results from its specific kinase mTORC2 (mammalian target of rapamycin complex 2). Thus, OT may modulate gut cell functions downstream of mTOR complexes (e.g., translation control as suggested by others in uterine cells). We will next explore OT-stimulated kinase activities downstream of mTOR related to pAkt phosphoisoforms.

A single postnatal injection of oxytocin rescues the lethal feeding behaviour in mouse newborns deficient for the imprinted Magel2 gene

The onset of feeding at birth is a vital step for the adaptation of the neonate to extra uterine life. Prader-Willi syndrome (PWS) is a complex neurogenetic disorder caused by the alteration of several imprinted contiguous genes including MAGEL2. PWS presents with various clinical manifestations, including poor suckling behaviour and feeding problems in neonates. Hypothalamic defects have been proposed, but the pathophysiological mechanisms remain poorly understood. Here, we report that a Magel2-deficient mouse with 50% neonatal mortality had an altered onset of suckling activity and subsequent impaired feeding, suggesting a role of MAGEL2 in the suckling deficit seen in PW newborns. The hypothalamus of Magel2 mutant neonates showed a significant reduction in oxytocin (OT). Furthermore, injection of a specific OT receptor antagonist in wild-type neonates recapitulated the feeding deficiency seen in Magel2 mutants, and a single injection of OT, 3-5 h after birth, rescued the phenotype of Magel2 mutant pups, allowing all of them to survive. Our study illustrates the crucial role of feeding onset behaviour after birth. We propose that OT supply might constitute a promising avenue for the treatment of feeding difficulties in PW neonates and potentially of other newborns with impaired feeding onset.

Blocking oxytocin receptors inhibits vaginal marking to male odors in female Syrian hamsters

In Syrian hamsters ( Mesocricetus auratus), precopulatory behaviors such as vaginal scent marking are essential for attracting a suitable mate. Vaginal marking is dependent on forebrain areas implicated in the neural regulation of reproductive behaviors in rodents, including the medial preoptic/anterior hypothalamus (MPOA-AH). Within MPOA-AH, the neuropeptide oxytocin (OT) acts to facilitate copulation (lordosis), as well as ultrasonic vocalizations towards males. It is not known, however, if OT in this area also facilitates vaginal marking. In the present study, a specific oxytocin receptor antagonist (OTA) was injected into MPOA-AH of intact female Syrian hamsters to determine if oxytocin receptor-dependent signaling is critical for the normal expression of vaginal marking elicited by male, female, and clean odors. OTA injections significantly inhibited vaginal marking in response to male odors compared with vehicle injections. There was no effect of OTA on marking in response to either female or clean odors. When injected into the bed nucleus of the stria terminalis (BNST), a nearby region to MPOA-AH, OTA was equally effective in decreasing marking. Finally, the effects of OTA appear to be specific to vaginal marking, as OTA injections in MPOA-AH or BNST did not alter general locomotor activity, flank marking, or social odor investigation. Considered together, these results suggest that OT in MPOA-AH and/or BNST normally facilitates male odor-induced vaginal marking, providing further evidence that OT generally supports prosocial interactions among conspecifics.

Estradiol upregulates the expression of oxytocin receptor in colon in rats

The study was designed to investigate the effect of estradiol on the excitatory effect of oxytocin (OT) on colon motility. Female Wistar rats were used, and some of them were ovariectomized (OVX) and treated with vehicle or estradiol (E(2)). A plastic balloon made of condom was inserted into colon to monitor the change of colonic pressure in vivo. Longitudinal muscle strips of distal colon were prepared to monitor the spontaneous contraction of colon in vitro. Expression of OT receptor (OTR) was investigated by Western blot analysis. Expression of OTR mRNA was detected by RT-PCR. Immunohistochemistry was used to locate OTR. In OVX rats, pretreatment of E(2) (4-100 microg/kg sc) dose-dependently increased the excitatory effect of OT on colon motility both in vivo and in vitro and increased the expression of OTR and OTR mRNA in colon. Systemic administration of OT excited the colon motility in vivo in rats at perioda of proestrus and estrus but did not influence it at diestrus period, when the concentration of plasma E(2) was lowest in the estrous cycle. Pretreatment of atosiban, the specific OTR antagonist, and TTX, the blocker of voltage-dependent sodium channel on nerve fiber, attenuated the excitatory effect of OT on colon motility. OTR was located in myenteric plexus of colon. These results suggested that E(2) increased the excitatory effect of OT on colon motility by upregulating the expression of OTR in myenteric plexus.

Oxytocin receptor activity in the ventrocaudal periaqueductal gray modulates anxiety-related behavior in postpartum rats.

Postpartum rats are less anxious than diestrous virgin females, a phenomenon requiring that mothers have recent contact with their infants. Oxytocin (OT) is one of many neurochemicals released intracerebrally while mothers interact with infants, and we investigated whether OT receptor activity in the ventrocaudal periaqueductal gray (cPAGv) contributes to mothers' reduced anxiety. Infusion of the highly specific OT receptor antagonist, desGly-NH2,d(CH2)5[D-Tyr2,Thr-sup-4]OVT, into the cPAGv reduced the percentage of time dams spent in the open arms of an elevated plus-maze, but had no effect on the open-arm behavior of diestrous virgins. Conversely, after separating dams from their litters for 4 hr to increase anxiety, a lower (2 ng) but not higher (5 ng) dose of OT infused into each hemisphere of the cPAGv doubled the percentage of time dams spent in open arms, but did not do so in virgins. OTergic manipulations inconsistently affected risk-assessment behaviors (stretch-attend postures, head dips) in both virgins and dams. Therefore, OT receptor activation in the cPAGv is an important consequence of contact with infants that reduces some anxiety-related behaviors in mother rats.

Mutual regulation of vasopressin- and oxytocin-induced glucagon secretion in V1b vasopressin receptor knockout mice

[Arg8]-vasopressin (AVP) and oxytocin (OT) are neurohypophysial hormones which exert various actions, including the control of blood glucose, in some peripheral tissues. To investigate the type of receptors involved in AVP- and OT-induced glucagon secretion, we investigated the effect of these peptides on glucagon secretion in islets of wild-type (V1bR+/+) and vasopressin V1b receptor knockout (V1bR-/-) mice. AVP-induced glucagon secretion was significantly inhibited by the selective V1b receptor antagonist, SSR149415 (30%), and OT-induced glucagon secretion by the specific OT receptor antagonist, d(CH2)5[Tyr(Me)2, Thr4, Tyr-NH(2)(9)]OVT (CL-14-26) (45%), in islets of V1bR+/+mice. AVP- and OT-induced glucagon secretions were not by the antagonist of each, but co-incubation with both 10(-6) M SSR149415 and 10(-6) M CL-14-26 further inhibited AVP- and OT-induced glucagon secretions in islets of V1bR+/+ mice (57 and 69% of the stimulation values respectively). In addition, both AVP and OT stimulated glucagon secretion with the same efficacy in V1bR-/- mice as in V1bR+/+ mice. AVP- and OT-induced glucagon secretion in V1bR-/- mice was significantly inhibited by CL-14-26. These results demonstrate that V1b receptors can mediate OT-induced glucagon secretion and OT receptors can mediate AVP-induced glucagon secretion in islets from V1bR+/+mice in the presence of a heterologous antagonist, while AVP and OT can stimulate glucagon secretion through the OT receptors in V1bR-/-mice, suggesting that the other receptor can compensate when one receptor is absent.

Oxytocin microinjected into dorsal motor nucleus of the vagus excites gallbladder motility via NMDA receptor–NO-cGMP pathway

Our recent study indicated that, in the dorsal motor nucleus of the vagus (DMV), the N-methyl- d-aspartic acid (NMDA) receptor–nitric oxide (NO)–cGMP pathway participated in the regulation of gallbladder motility in rabbits. Oxytocin (OT) is involved as a neurotransmitter in autonomic regulation. The aim of the present experiments is to investigate the effect of OT microinjected into DMV on the gallbladder motility and the involvement of NMDA receptor–NO–cGMP pathway. A frog bladder connected with transducer was inserted into the gallbladder to record the gallbladder pressure. Microinjection of OT (10–50 nmol/L, 100 nl) dose dependently increased the strength of gallbladder phasic contraction. The excitatory effect of OT (10 nmol/L, 100 nl) was completely abolished by atosiban (10 mmol/L, 100 nl), the specific OT receptor antagonist, but was not influenced by [deamino-Pen 1, O-Me-Tyr 2,Arg 8]-vasopressin (10 mmol/L, 100 nl), the V 1 receptor antagonist. Pretreatment of ketamine (10 mmol/L, 100 nl), the NMDA receptor antagonist, suppressed the gallbladder motor response to OT; but pretreatment of 6-Cyaon-7-Nitroquinoxaline-2,3-(1H,4H)-Dione (CNQX; 10 mmol/L, 100 nl), the non-NMDA receptor antagonist, did not affect it. Pretreatment of l-NAME (10 mmol/L, 100 nl), the nitric oxide synthase (NOS) inhibitor, or methyl blue (10 mmol/L, 100 nl), the guanylyl cyclase inhibitor, inhibited the excitatory effect of OT on gallbladder motility. Hence, we deduced that the microinjection of OT into the DMV enhanced the gallbladder motility through binding specific OT receptors and activating the NMDA receptor–NO–cGMP pathway.

Effect of an oxytocin receptor antagonist and rho kinase inhibitor on the [Ca ++] i sensitivity of human myometrium

Objective The purpose of this study was to assess the possible mechanism and effect of oxytocin receptor on the sensitization of the contractile proteins to [Ca ++] i. Study design Myometrial strips were prepared from biopsy specimens that had been taken at term (37–40 weeks of gestation) cesarean delivery from 22 women, before the onset of labor. Simultaneous measurements of [Ca ++] i and tension were performed on spontaneously contracting strips. The effects of a specific oxytocin receptor antagonist, L371,257, and a rho kinase inhibitor, Y-27632, on the [Ca ++] i and tension transients were determined. Results Application of L371,257 (90 nmol/L) to spontaneously contracting muscle strips reduced peak tension to 43%±12% of its original value without affecting peak [Ca ++] i (105%±15%). More tension is developed at each [Ca ++] i during the falling phase, relative to the rising phase of each spontaneous contraction; this asymmetric [Ca ++] i-tension relationship was abolished by L371,257. The subsequent application of oxytocin (3 μmol/L) reversed the effects of the antagonist on tension. An inhibitor of rho kinase (Y-27632) caused a concentration-dependent inhibition of spontaneous contractions without affecting the underlying Ca ++ signals. The addition of oxytocin (1 nmol/L) to spontaneously active myometrium increased both the duration of the Ca ++ signal and the sensitivity of the contractile machinery to [Ca ++] i; only the latter effect was prevented by Y-27632. Conclusion Oxytocin independently regulates the duration of the periodic Ca ++ signals and the sensitivity of the contractile machinery to Ca ++. The latter is likely to be mediated by rho kinase, which is essential for the effective coupling of increases in [Ca ++] i to tension.

Modulation by oxytocin of ATP-activated currents in rat dorsal root ganglion neurons

The modulatory effect of oxytocin (OT) on ATP-activated currents (I ATP) was studied in freshly isolated dorsal root ganglion (DRG) neurons of rats using whole cell clamp technique. In most of the neurons examined (50/70, 71.4%) extracellular application of OT (10 −9–10 −5 mol/L) suppressed I ATP while in the rest (20/70, 28.6%) no modulatory effect was observed. OT shifted the ATP concentration-response curve downwards with a decrease of 39.8±4.2% in the maximal current response and with no significant change of Kd value. This OT-induced inhibition of I ATP showed no voltage dependence, and could be blocked by [d(CH 2) 5,Tyr(Me) 2,Thr 4,Tyr-NH 2 9]-OVT (d(CH 2) 5-OVT) (10 −8 mol/L), a specific OT receptor antagonist. Intracellular application of H-9 (4×10 −5 mol/L, an inhibitor of protein kinase A) ( n=12), BAPTA (10 −2 mol/L, a chelator of calcium ions) ( n=4) could reverse the inhibitory effect of extracellular OT (10 −7 mol), while inclusion of H-7 (2×10 −5 mol/L, a protein kinase C inhibitior) ( n=8) and KN-93 (10 −5 mol/L, an inhibitor of CaMKII) ( n=9) in the recording pipette did not affect this effect. The results suggested that OT inhibition on ATP-activated currents was mediated by OT receptors in the membrane of DRG neurons; and this inhibitory effect involved the transduction of intracellular cAMP-PKA and Ca 2+.

Oxytocin in the NTS

The role of brain stem oxytocinergic projections in the modulation of heart rate control during exercise is discussed on the basis of both changes in endogenous peptide content and heart rate changes observed during exercise. Running on a treadmill caused an increase in oxytocin content in dorsal/ventral brain stem areas and spinal cord, specifically in trained rats. Trained rats pretreated with a specific oxytocin receptor antagonist into the dorsal brain stem area (corresponding to the nucleus tractus solitarii and dorsal motor nucleus of the vagus, or NTS/DMV) showed a significant potentiation of exercise tachycardia with no change in the blood pressure response. The same treatment in sedentary rats was without effect. On the other hand, administration of exogenous oxytocin into this area caused significant blunting of exercise tachycardia in both groups, with no change in the pressure response. It is proposed that long-descending oxytocinergic pathways from the hypothalamus to the NTS/DMV area serve as a link between the two main neural controllers of the circulation--that is, the central command and feedback control mechanisms driven by the peripheral receptor signals. Our results strongly suggest that oxytocinergic input to NTS/DMV, by restraining the tachycardic response of trained individuals, contributes to the smaller response observed after training, without compromising cardiac output adjustment and the circulatory demand during exercise.