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Vasopressin (AVP) and oxytocin (OT) receptors (nomenclature as recommended by NC-IUPHAR [94]) are activated by the endogenous cyclic nonapeptides vasopressin and oxytocin. These peptides are derived from precursors which also produce neurophysins (neurophysin I for oxytocin; neurophysin II for vasopressin). Vasopressin and oxytocin differ at only 2 amino acids (positions 3 and 8). There are metabolites of these neuropeptides that may be biologically active [69].

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15538. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Oxytocin is a master regulator of the social brain. In some animal models of autism, notably inMagel2tm1.1Mus-deficient mice, peripheral administration of oxytocin in infancy improves social behaviors until adulthood. However, neither the mechanisms responsible for social deficits nor the mechanisms by which such oxytocin administration has long-term effects are known. Here, we aimed to clarify these oxytocin-dependent mechanisms focusing on social memory performance.We showed thatMagel2tm1.1Mus-deficient mice present a deficit in social memory and studied the hippocampal circuits underlying this memory. We showed a co-expression ofMagel2andoxytocin-receptorin the dentate gyrus and CA2/CA3 hippocampal regions. Then, we demonstrated: an increase of the GABAergic activity of CA3-pyramidal cells associated with an increase in the quantity of oxytocin-receptors and of somatostatin interneurons. We also revealed a delay in the GABAergic development sequence inMagel2tm1.1Mus-deficient pups, linked to phosphorylation modifications of KCC2. Above all, we demonstrated the positive effects of subcutaneous administration of oxytocin in the mutant neonates, restoring neuronal alterations and social memory.Although clinical trials are debated, this study highlights the mechanisms by which peripheral oxytocin-administration in neonates impacts the brain and demonstrates the therapeutic value of oxytocin to treat infants with autism spectrum disorders.

Oxytocin is currently being considered as a novel therapeutic for anxiety disorders due to its ability to promote affiliative behaviors. In the nucleus accumbens (NAc) activation of oxytocin receptors (OTR) promotes social approach (time spent near an unfamiliar individual). Here, we show that stressful social experiences reduce the expression of NAc OTR mRNA, coinciding with decreases in social approach. Social stressors also increase social vigilance, characterized as orienting to an unfamiliar individual without approaching. Vigilance is a key component of behavioral inhibition, a personality trait that is a risk factor for anxiety disorders. To understand whether NAc OTR can modulate both social approach and vigilance, we use pharmacological approaches to assess the impact of activation or inhibition of NAc OTR downstream pathways on these behaviors. First, we show that in unstressed male and female California mice, inhibition of OTR by an unbiased antagonist (L-368,899) reduces social approach but does not induce social vigilance. Next, we show that infusion of Atosiban, an OTR-Gq antagonist/OTR-Gi agonist, has the same effect in unstressed females. Finally, we show that Carbetocin, a biased OTR-Gq agonist, increases social approach in stressed females while simultaneously inhibiting social vigilance. Taken together these data suggest that OTR in the NAc differentially modulate social approach and social vigilance, primarily through an OTR-Gq mechanism. Importantly, pharmacological inhibition of OTR alone is insufficient to induce vigilance in unstressed mice, suggesting that mechanisms modulating social approach may be distinct from mechanisms modulating social vigilance.

The neuropeptides oxytocin (OXT) and vasopressin (AVP) have been identified as modulators of emotional social behaviors and associated with neuropsychiatric disorders characterized by social dysfunction. Experimental and therapeutic use of OXT and AVP via the intranasal route is the subject of extensive clinical research. However, the large-scale functional substrates directly engaged by these peptides and their functional dynamics remain elusive. By using cerebral blood volume (CBV) weighted fMRI in the mouse, we show that intranasal administration of OXT rapidly elicits the transient activation of cortical regions and a sustained activation of hippocampal and forebrain areas characterized by high oxytocin receptor density. By contrast, intranasal administration of AVP produced a robust and sustained deactivation in cortico-parietal, thalamic and mesolimbic regions. Importantly, intravenous administration of OXT and AVP did not recapitulate the patterns of modulation produced by intranasal dosing, supporting a central origin of the observed functional changes. In keeping with this notion, hippocampal local field potential recordings revealed multi-band power increases upon intranasal OXT administration. We also show that the selective OXT-derivative TGOT reproduced the pattern of activation elicited by OXT and that the deletion of OXT receptors does not affect AVP-mediated deactivation. Collectively, our data document divergent modulation of brainwide neural systems by intranasal administration of OXT and AVP, an effect that involves key substrates of social and emotional behavior. The observed divergence calls for a deeper investigation of the systems-level mechanisms by which exogenous OXT and AVP modulate brain function and exert their putative therapeutic effects.

Vasopressin (AVP) and oxytocin (OT) receptors (nomenclature as recommended by NC-IUPHAR [92]) are activated by the endogenous cyclic nonapeptides vasopressin and oxytocin. These peptides are derived from precursors which also produce neurophysins (neurophysin I for oxytocin; neurophysin II for vasopressin). Vasopressin and oxytocin differ at only 2 amino acids (positions 3 and 8). There are metabolites of these neuropeptides that may be biologically active [67].

Dimeric/oligomeric states of G-protein coupled receptors have been difficult to target. We report here bivalent ligands consisting of two identical oxytocin-mimetics that induce a three order magnitude boost in G-protein signaling of oxytocin receptors (OTRs) in vitro and a 100- and 40-fold gain in potency in vivo in the social behavior of mice and zebrafish. Through receptor mutagenesis and interference experiments with synthetic peptides mimicking transmembrane helices (TMH), we show that such superpotent behavior follows from the binding of the bivalent ligands to dimeric receptors based on a TMH1-TMH2 interface. Moreover, in this arrangement, only the analogues with a well-defined spacer length (∼25 Å) precisely fit inside a channel-like passage between the two protomers of the dimer. The newly discovered oxytocin bivalent ligands represent a powerful tool for targeting dimeric OTR in neurodevelopmental and psychiatric disorders and, in general, provide a framework to untangle specific arrangements of G-protein coupled receptor dimers.

International audience; Oxytocin (OT) is a neuropeptide elaborated by the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. Magnocellular OT neurons of these nuclei innervate numerous forebrain regions and release OT into the blood from the posterior pituitary. The PVN also harbors parvocellular OT cells that project to the brainstem and spinal cord, but their function has not been directly assessed. Here, we identified a subset of approximately 30 parvocellular OT neurons, with collateral projections onto magnocellular OT neurons and neurons of deep layers of the spinal cord. Evoked OT release from these OT neurons suppresses nociception and promotes analgesia in an animal model of inflammatory pain. Our findings identify a new population of OT neurons that modulates nociception in a two tier process: (1) directly by release of OT from axons onto sensory spinal cord neurons and inhibiting their activity and (2) indirectly by stimulating OT release from SON neurons into the periphery.

Oxytocin (OT) is a neuropeptide, which can be seen to be one of the molecules of the decade due to its profound prosocial effects in nonvertebrate and vertebrate species, including humans. Although OT can be detected in various physiological fluids (blood, saliva, urine, cerebrospinal fluid) and brain tissue, it is unclear whether peripheral and central OT releases match and synergize. Moreover, the pathways of OT delivery to brain regions involved in specific behaviors are far from clear. Here, we discuss the evolutionarily and ontogenetically determined pathways of OT delivery and OT signaling, which orchestrate activity of the mesolimbic social decision-making network. Furthermore, we speculate that both the alteration in OT delivery and OT receptor expression may cause behavioral abnormalities in patients afflicted with psychosocial diseases.

BackgroundThe neuropeptide oxytocin (OT) is a key regulator of social and emotional behaviors. The effects of OT are context dependent, and it has been proposed that OT increases the salience of both positive and negative social cues. Here we tested whether the bed nucleus of the stria terminalis (BNST) mediates anxiogenic effects of OT.MethodsFirst, we studied the effects of systemic administration of an OT receptor (OTR) antagonist L-368,899 on social behavior in male and female California mice exposed to social defeat. We examined the effect of L-368,899 on G protein activation and used early growth response factor 1 immunohistochemistry to identify potential sites of OTR action. Finally, we examined the effects of L-368,899 infused in the BNST on behavior.ResultsA single dose of systemic L-368,899 increased social approach in stressed female mice and decreased social approach in male mice naïve to defeat. L-368,899 prevented OT activation of G proteins and did not activate G proteins in the absence of OT. Intranasal OT, which reduces social approach in female mice but not male mice, increased early growth response factor 1 immunoreactivity in the nucleus accumbens core and anteromedial BNST in female mice but not in male mice. Stressed female mice that received an infusion of L-368,899 into the anteromedial BNST but not the nucleus accumbens core increased social approach and decreased social vigilance responses.ConclusionsOur results suggest that OTR activation in anteromedial BNST induces a vigilance response in which individuals avoid, yet attend to, unfamiliar social contexts. Our results suggest that OTR antagonists may have unappreciated therapeutic potential for stress-induced psychiatric disorders.

Nanoparticles (NPs) have received much attention in recent years for their diverse potential biomedical applications. However, the synthesis of NPs with desired biodistribution and pharmacokinetics is still a major challenge, with NP size and surface chemistry being the main factors determining the behavior of NPs in vivo. Here we report on the surface chemistry and in vitro cellular uptake of magnetic iron oxide NPs coated with zwitterionic dopamine sulfonate (ZDS). ZDS-coated NPs were compared to similar iron oxide NPs coated with PEG-like 2-[2-(2-methoxyethoxy)ethoxy]acetic acid (MEEA) to investigate how surface chemistry affects their in vitro behavior. ZDS-coated NPs had a very dense coating, guaranteeing high colloidal stability in several aqueous media and negligible interaction with proteins. Treatment of HepG2 cells with increasing doses (2.5-100 mug Fe/mL) of ZDS-coated iron oxide NPs had no effect on cell viability and resulted in a low, dose-dependent NP uptake, inferior than most reported data for the internalization of iron oxide NPs by HepG2 cells. MEEA-coated NPs were scarcely stable and formed micrometer-sized aggregates in aqueous media. They decreased cell viability for dose >=50 mug Fe/mL, and were more efficiently internalized than ZDS-coated NPs. In conclusion, our data indicate that the ZDS layer prevented both aggregation and sedimentation of iron oxide NPs and formed a biocompatible coating that did not display any biocorona effect. The very low cellular uptake of ZDS-coated iron NPs can be useful to achieve highly selective targeting upon specific functionalization.

Despite the accumulating evidence suggesting that the neuropeptide oxytocin (OT) plays a role in neuropsychiatric disorders characterized by social dysfunction, the influence of OT on the nonsocial aspects of learning and memory have been less investigated. To foster research in this area, we review the effects of OT on learning and memory in animal models and humans. In healthy animal models, OT improves memory consolidation and extinction, but only if given at a low dose immediately after the acquisition phase. On the contrary, OT effects in healthy humans have been inconsistent; although, in this case, OT was always given before the acquisition phase and no dose-response curves have ever been drawn up. Interestingly, a specific impairment in the reversal of learning has been found in mice devoid of OT receptors and OT has been demonstrated to enhance fear extinction in rodents. All together, these data suggest that OT plays a role in elementary forms of behavioral flexibility and adaptive responses and support its therapeutic potential in neuropsychiatric disorders characterized by cognitive inflexibility and/or impairment (autism, schizophrenia, Alzheimer's disease, Parkinson disease, stroke, posttraumatic stress disorder). Accordingly, OT has been shown to improve cognitive flexibility in OT receptor-deficient mice, and scattered findings indicate that intranasal OT has positive effects on the memory of patients with schizophrenia or posttraumatic stress disorders. Further studies of the therapeutic potential of OT as an enhancer of learning and memory are warranted.

Recognition of other's emotions influences the way social animals interact and adapt to the environment. The neuropeptide oxytocin (OXT) has been implicated in different aspects of emotion processing. However, the role of endogenous OXT brain pathways in the social response to different emotional states in conspecifics remains elusive. Here, using a combination of anatomical, genetic, and chemogenetic approaches, we investigated the contribution of endogenous OXT signaling in the ability of mice to discriminate unfamiliar conspecifics based on their emotional states. We found that OXTergic projections from the paraventricular nucleus of the hypothalamus (PVN) to the central amygdala (CeA) are crucial for the discrimination of both positively and negatively valenced emotional states. In contrast, blocking PVN OXT release into the nucleus accumbens, prefrontal cortex, and hippocampal CA2 did not alter this emotion discrimination. Furthermore, silencing each of these PVN OXT pathways did not influence basic social interaction. These findings were further supported by the demonstration that virally mediated enhancement of OXT signaling within the CeA was sufficient to rescue emotion discrimination deficits in a genetic mouse model of cognitive liability. Our results indicate that CeA OXT signaling plays a key role in emotion discrimination both in physiological and pathological conditions.

Carbetocin, a long‐acting oxytocin analogue, has been reported to elicit interesting and peculiar behavioural effects. The present study investigated the molecular pharmacology of carbetocin, aiming to better understand the molecular basis of its action in the brain. Using bioluminescence resonance energy transfer biosensors, we characterised the effects of carbetocin on the three human oxytocin/vasopressin receptors expressed in the nervous system: the oxytocin receptor (OXTR) and the vasopressin V1a (V1aR) and V1b (V1bR) receptors. Our results indicate that (i) carbetocin activates the OXTR but not the V1aR and V1bR at which it may act as an antagonist; (ii) carbetocin selectively activates only the OXTR/Gq pathway displaying a strong functional selectivity; (iii) carbetocin is a partial agonist at the OXTR/Gq coupling; (iv) carbetocin promotes OXTR internalisation via a previously unreported β‐arrestin‐independent pathway; and (v) carbetocin does not induce OXTR recycling to the plasma membrane. Altogether, these molecular pharmacology features identify carbetocin as a substantially different analogue compared to the endogenous oxytocin and, consequently, carbetocin is not expected to mimic oxytocin in the brain. Whether these unique features of carbetocin could be exploited therapeutically remains to be established.

We used a bioluminescence resonance energy transfer biosensor to screen for functional selective ligands of the human oxytocin (OT) receptor. We demonstrated that OT promoted the direct engagement and activation of G(q) and all the G(i/o) subtypes at the OT receptor. Other peptidic analogues, chosen because of specific substitutions in key OT structural/functional residues, all showed biased activation of G protein subtypes. No ligand, except OT, activated G(oA) or G(oB), and, with only one exception, all of the peptides that activated G(q) also activated G(i2) and G(i3) but not G(i1), G(oA), or G(oB), indicating a strong bias toward these subunits. Two peptides (DNalOVT and atosiban) activated only G(i1) or G(i3), failed to recruit β-arrestins, and did not induce receptor internalization, providing the first clear examples of ligands differentiating individual G(i/o) family members. Both analogs inhibited cell proliferation, showing that a single G(i) subtype-mediated pathway is sufficient to prompt this physiological response. These analogs represent unique tools for examining the contribution of G(i/o) members in complex biological responses and open the way to the development of drugs with peculiar selectivity profiles. This is of particular relevance because OT has been shown to improve symptoms in neurodevelopmental and psychiatric disorders characterized by abnormal social behaviors, such as autism. Functional selective ligands, activating a specific G protein signaling pathway, may possess a higher efficacy and specificity on OT-based therapeutics.

In this article, we discuss the mathematical and computational framework required to develop a general-purpose simulation tool for bio-electronic applications. Electrochemical and fluid-mechanical transport of substances, chemical reactions and electrical transduction of biological signals are described through the coupled use of systems of partial and ordinary differential equations (PDEs and ODEs). Functional iteration techniques for system decoupling and mixed-hybridized finite element discretization methods are proposed and validated in the simulation of realistic problems in Electrophysiology and Biochemistry.

Oxytocin (OT), the main neuropeptide of sociality, is expressed in neurons exclusively localized in the hypothalamus. During the last decade, a plethora of neuroendocrine, metabolic, autonomic and behavioral effects of OT has been reported. In the urgency to find treatments to syndromes as invalidating as autism, many clinical trials have been launched in which OT is administered to patients, including adolescents and children. However, the impact of OT on the developing brain and in particular on the embryonic and early postnatal maturation of OT neurons, has been only poorly investigated. In the present review we summarize available (although limited) literature on general features of ontogenetic transformation of the OT system, including determination, migration and differentiation of OT neurons. Next, we discuss trajectories of OT receptors (OTR) in perinatal period. Furthermore, we provide evidence that early alterations, from birth, in the central OT system lead to severe neurodevelopmental diseases such as feeding deficit in infancy and severe defects in social behavior in adulthood, as described in Prader-Willi syndrome. Our review intends to propose a hypothesis about developmental dynamics of central OT pathways, which are essential for survival right after birth and for the acquisition of social skills later on. A better understanding of the embryonic and early postnatal maturation of the OT system may lead to better OT-based treatments in Prader-Willi syndrome and autism.

In this study, we investigated the functional role of the localization of human OTR in caveolin-1 enriched membrane domains. Biochemical fractionation of MDCK cells stably expressing the WT OTR-GFP indicated that only minor quantities of receptor are partitioned in caveolin-1 enriched domains. However, when fused to caveolin-2, the OTR protein proved to be exclusively localized in caveolin-1 enriched fractions, where it bound the agonist with increased affinity and efficiently coupled to Galpha(q/11). Interestingly, the chimeric protein was unable to undergo agonist-induced internalization and remained confined to the plasma membrane even after prolonged agonist exposure (120 min). A striking difference in receptor stimulation was observed when the OT-induced effect on cell proliferation was analysed: stimulation of the human WT OTR inhibited cell growth, whereas the chimeric protein had a proliferative effect. These data indicate that the localization of human OTR in caveolin-1 enriched microdomains radically alters its regulatory effects on cell growth; the fraction of OTR residing in caveolar structures may therefore play a crucial role in regulating cell proliferation.

Autism spectrum disorders (ASDs) are characterized by impaired communication, social impairments, and restricted and repetitive behaviors and interests. Recently, altered motivation and reward processes have been suggested to participate in the physiopathology of ASDs, and μ-opioid receptors (MORs) have been investigated in relation to social reward due to their involvement in the neural circuitry of reward. Mice lacking a functional MOR gene (Oprm1 (-/-) mice) display abnormal social behavior and major autistic-like core symptoms, making them an animal model of autism. The oxytocin (OXT) system is a key regulator of social behavior and co-operates with the opioidergic system in the modulation of social behavior. To better understand the opioid-OXT interplay in the central nervous system, we first determined the expression of the oxytocin receptor (OXTR) in the brain of WT C57BL6/J mice by quantitative autoradiography; we then evaluated OXTR regional alterations in Oprm1 (-/-) mice. Moreover, we tested these mice in a paradigm of social behavior, the male-female social interaction test, and analyzed the effects of acute intranasal OXT treatment on their performance. In autoradiography, Oprm1 (-/-) mice selectively displayed increased OXTR expression in the Medial Anterior Olfactory Nucleus, the Central and Medial Amygdaloid nuclei, and the Nucleus Accumbens. Our behavioral results confirmed that Oprm1 (-/-) male mice displayed social impairments, as indicated by reduced ultrasonic calls, and that these were rescued by a single intranasal administration of OXT. Taken together, our results provide evidence of an interaction between OXT and opioids in socially relevant brain areas and in the modulation of social behavior. Moreover, they suggest that the oxytocinergic system may act as a compensative mechanism to bypass and/or restore alterations in circuits linked to impaired social behavior.

Despite the continuous advancements in bio-molecular detection and fluidic systems integration, the realization of portable and high performance devices for diagnostic applications still presents major difficulties, mostly because of the need to combine adequate sensitivity with low cost of production and operational simplicity and speed. In this context, we propose a compact device composed of a smartphone and a custom-designed cradle, containing only a disposable sensing cartridge, a tiny magnetic stirrer and a few passive optical components. The detection principle is the previously proposed Reflective Phantom Interface that is based on measuring the intensity of light reflected by the surface of an amorphous fluoropolymer substrate, which has a refractive index very close to that of the aqueous sample solution and hosts various antibodies immobilized within spots. The reflectivity of dozens of spots is monitored in real time by the phone[U+05F3]s camera using the embedded flash LED as the illumination source. We test the performance of the combined device targeting heterologous immunoglobulins and antigens commonly used as markers for diagnoses of hepatitis B and HIV. Target concentrations as low as a few ng/ml can be rapidly and robustly determined by comparing the rate of increase of the signal after the addition of the sample with that measured after the subsequent addition of a standard solution with known concentration. The features of the proposed system enable the realization of novel handheld biosensing devices suitable for those applications where multiple targets have to be rapidly detected even without the presence of trained personnel. © 2014 Elsevier B.V.

Oxytocin (OT) inhibits the proliferation of breast-cancer cells in vitro via a specific G-coupled receptor. To elucidate the intracellular mechanism involved in this biological effect, different G-coupled receptor mediators have been investigated in untreated and OT-treated MDA-MB231 breast-carcinoma cells. In these cells, after OT treatment, a significant cAMP increase was observed using a radioimmunoassay procedure, whereas the Ca2+ (determined with the fluorescent probe fura-2) and the inositol phosphate (determined after cell labeling with myo(2-(3)H)-inositol) concentrations were not modified, contrary to what has been observed in myometrial and myo-epithelial cells. The PKA inhibitor PKI (6-22) amide reverted the effect of OT, indicating that the anti-proliferative effect of the peptide is strictly related to the cAMP-PKA pathway. OT treatment did not modify tyrosine phosphorylation either. Our results indicate that in breast epithelial cells devoid of contractile activity, cAMP is the intracellular mediator of OT action, whereas the Ca2+-phosphoinositide system is not involved.

The neuropeptides oxytocin (OT) and vasopressin (AVP) have been shown to play a central role in social behaviors; as a consequence, they have been recognized as potential drugs to treat neurodevelopmental and psychiatric disorders characterized by impaired social interactions. However, despite the basic and preclinical relevance of mouse strains carrying genetic alterations in the OT/AVP systems to basic and preclinical translational neuroscience, the pharmacological profile of mouse OT/AVP receptor subtypes has not been fully characterized. To fill in this gap, we have characterized a number of OT and AVP agonists and antagonists at three murine OT/AVP receptors expressed in the nervous system as follows: the oxytocin (mOTR) and vasopressin V1a (mV1aR) and V1b (mV1bR) subtypes. These three receptors were transiently expressed in vitro for binding and intracellular signaling assays, and then a homology model of the mOTR structure was constructed to investigate how its molecular features compare with human and rat OTR orthologs. Our data indicate that the selectivity profile of the natural ligands, OT and AVP, is conserved in humans, rats, and mice. Furthermore, we found that the synthetic peptide [Thr(4)Gly(7)]OT (TGOT) is remarkably selective for the mOTR and, like the endogenous OT ligand, activates Gq and Gi and recruits β-arrestins. Finally, we report three antagonists that exhibit remarkably high affinities and selectivities at mOTRs. These highly selective pharmacological tools will contribute to the investigation of the specific physiologic and pathologic roles of mOTR for the development of selective OT-based therapeutics.

Intranasal administration of oxytocin (OXT) might be a promising new adjunctive therapy for mental disorders characterized by social behavioral alterations such as autism and schizophrenia. Despite promising initial studies in humans, it is not yet clear the specificity of the behavioral effects induced by chronic intranasal OXT and if chronic intranasal OXT could have different effects compared with single administration. This is critical for the aforementioned chronic mental disorders that might potentially involve life-long treatments. As a first step to address these issues, here we report that chronic intranasal OXT treatment in wild-type C57BL/6J adult mice produced a selective reduction of social behaviors concomitant to a reduction of the OXT receptors throughout the brain. Conversely, acute intranasal OXT treatment produced partial increases in social behaviors towards opposite-sex novel-stimulus female mice, while on the other hand, it decreased social exploration of same-sex novel stimulus male mice, without affecting social behavior towards familiar stimulus male mice. Finally, prolonged exposure to intranasal OXT treatments did not alter, in wild-type animals, parameters of general health such as body weight, locomotor activity, olfactory and auditory functions, nor parameters of memory and sensorimotor gating abilities. These results indicate that a prolonged over-stimulation of a 'healthy' oxytocinergic brain system, with no inherent deficits in social interaction and normal endogenous levels of OXT, results in specific detrimental effects in social behaviors.

The intrahelical salt bridge between \(E/D^{3.49}\) and \(R^{3.50}\) within the E/DRY motif on helix 3 (H3) and the interhelical hydrogen bonding between the E/DRY and residues on H6 are thought to be critical in stabilizing the class A G protein-coupled receptors in their inactive state. Removal of these interactions is expected to generate constitutively active receptors. This study examines how neutralization of \(E^{3.49/6.30}\) in the thromboxane prostanoid (TP) receptor alters ligand binding, basal, and agonist-induced activity and investigates the molecular mechanisms of G protein activation. We demonstrate here that a panel of full and partial agonists showed an increase in affinity and potency for E129V and E240V mutants. Yet, even augmenting the sensitivity to detect constitutive activity (CA) with overexpression of the receptor or the G protein revealed resistance to an increase in basal activity, while retaining fully the ability to cause agonist-induced signaling. However, direct G protein activation measured through bioluminescence resonance energy transfer (BRET) indicates that these mutants more efficiently communicate and/or activate their cognate G proteins. These results suggest the existence of additional constrains governing the shift of TP receptor to its active state, together with an increase propensity of these mutants to agonist-induced signaling, corroborating their definition as superactive mutants. The particular nature of the TP receptor as somehow "resistant" to CA should be examined in the context of its pathophysiological role in the cardiovascular system. Evolutionary forces may have favored regulation mechanisms leading to low basal activity and selected against more highly active phenotypes.

Recognizing and quantifying specific biomolecules in aqueous samples are constantly needed in research and diagnostic laboratories. As the typical detection procedures are rather lengthy and involve the use of labeled secondary antibodies or other agents to provide a signal, efforts have been made over the last 10 y to develop alternative label-free methods that enable direct detection. We propose and demonstrate an extremely simple, low-cost, label-free biodetector based on measuring the intensity of light reflected by the interface between a fluid sample and an amorphous fluoropolymer substrate having a refractive index very close to that of water and hosting various antibodies immobilized in spots. Under these index-matching conditions, the amount of light reflected by the interface allows straightforward quantification of the amount of antigen binding to each spot. Using antibodies targeting heterologous immunoglobulins and antigens commonly used as markers for diagnoses of hepatitis B and HIV, we demonstrate the limit of detection of a few picograms per square millimeter of surface-bound molecules. We also show that direct and real-time access to the amount of binding molecules allows the precise extrapolation of adhesion rates, from which the concentrations of antigens in solution can be estimated down to fractions of nanograms per milliliter.

Summary Despite intensive investigation over the past 20 years, the specific role played by individual Gi protein family members in mediating complex cellular effects is still largely unclear. Therefore, we investigated the role of specific Gi proteins in mediating somatostatin (SS) effects in somatotroph cells. Because our previous data showed that SS receptor type 5 (SST5) carrying a spontaneous R240W mutation in the third intracellular loop had a similar ability to inhibit intracellular cAMP levels to the wild-type protein but failed to mediate inhibition of growth hormone (GH) release and cell proliferation, we used this model to check specific receptor–G-protein coupling by a bioluminescent resonance energy transfer analysis. In HEK293 cells, wild-type SST5 stimulated the activation of Gαi1–3 and GαoA, B, whereas R240W SST5 maintained the ability to activate Gαi1–3 and GαoB, but failed to activate the splicing variant GαoA. To investigate the role of the selective deficit in GαoA coupling, we co-transfected human adenomatous somatotrophs with SST5 and a pertussis toxin (PTX)-resistant GαoA (GαoA(PTX-r)) protein. In PTX-treated cells, GαoA(PTX-r) rescued the ability of the selective SST5 analog BIM23206 to inhibit extracellular signal-related kinase 1/2 (ERK1/2) phosphorylation, GH secretion and intracellular cAMP levels. Moreover, we demonstrated that silencing of GαoA completely abolished SST5-mediated inhibitory effects on GH secretion and ERK1/2 phosphorylation, but not on cAMP levels. In conclusion, by analysing the coupling specificity of human SST5 to individual Gαi and Gαo subunits, we identified a crucial role for GαoA signalling in human pituitary cells.

We recently reviewed the status of peptide and nonpeptide agonists and antagonists for the V(1a), V(1b) and V(2) receptors for arginine vasopressin (AVP) and the oxytocin receptor for oxytocin (OT). In the present review, we update the status of peptides and nonpeptides as: (i) research tools and (ii) therapeutic agents. We also present our recent findings on the design of fluorescent ligands for V(1b) receptor localisation and for OT receptor dimerisation. We note the exciting discoveries regarding two novel naturally occurring analogues of OT. Recent reports of a selective VP V(1a) agonist and a selective OT agonist point to the continued therapeutic potential of peptides in this field. To date, only two nonpeptides, the V(2) /V(1a) antagonist, conivaptan and the V(2) antagonist tolvaptan have received Food and Drug Administration approval for clinical use. The development of nonpeptide AVP V(1a), V(1b) and V(2) antagonists and OT agonists and antagonists has recently been abandoned by Merck, Sanofi and Pfizer. A promising OT antagonist, Retosiban, developed at Glaxo SmithKline is currently in a Phase II clinical trial for the prevention of premature labour. A number of the nonpeptide ligands that were not successful in clinical trials are proving to be valuable as research tools. Peptide agonists and antagonists continue to be very widely used as research tools in this field. In this regard, we present receptor data on some of the most widely used peptide and nonpeptide ligands, as a guide for their use, especially with regard to receptor selectivity and species differences.

Objective To assess the expression of the oxytocin receptor (OTR) and the role of oxytocin (OT) in the proliferation of myometrial and leiomyoma cells. Design Prospective laboratory study. Setting Research laboratory at the Italian National Research Council. Patient(s) Twenty-two women who underwent therapeutic myomectomy for fibroids. Intervention(s) Primary cultures of leiomyoma and myometrium cells were established from eutopic and ectopic myometrial tissues. An immortalized myometrial cell line (h-TERTmyo) and a leiomyosarcoma cell line (SK-UT-1) were also characterized. Main Outcome Measure(s) Expression of OTR and desmin mRNA was determined by quantitative real-time polymerase chain reaction. Cell growth was determined by 3-[4,5-dimethylthiazol-2-yl]5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H tetrazolium assay. Apoptosis was determined by annexin V cell staining and flow cytometry analysis. Result(s) Oxytocin stimulated proliferation of primary myometrial and leiomyoma cells but inhibited the proliferation of h-TERTmyo and SK-UT-1, indicating a change in phenotype during immortalization. A progressive and rapid decrease in desmin and OTR mRNA was observed in primary cultures, indicating that myometrial cells dedifferentiate very rapidly in culture. The relative expression of OTR mRNA varied widely in both myometrial and leiomyoma smooth muscle cells, but there was no significant difference. Conclusion(s) These results indicate that OT stimulates the proliferation of both myometrial and leiomyoma cells, demonstrating that the OT/OTR system plays an important role in regulating uterine cell growth and providing a rationale for evaluating the use of OTR antagonists in managing uterine myomas.

As in the case of most G protein-coupled receptors, agonist stimulation of human oxytocin receptors (OTRs) leads to desensitization and internalization; however, little is known about the subsequent intracellular OTR trafficking, which is crucial for reestablishing agonist responsiveness. We examined receptor resensitization by first using HEK293T cells stably expressing human OTRs. Upon agonist activation, the receptors were almost completely sequestered inside intracellular compartments that were not labeled by lysosomal markers, thus indicating that the internalized receptors were not sorted to these degrading organelles. Binding and fluorescence assays showed that almost 85% of the receptors had returned to the cell surface after 4 h, by which time cell responsiveness to the agonist was also completely restored, as shown by measuring phospholipase C activation. Similar results were also obtained in the presence of cycloheximide, thus indicating that receptor recycling and not de novo receptor synthesis was responsible for the resensitization. Notably, very similar internalization and recycling kinetics were observed in endogenous OTRs expressed on myometrial cells. We also investigated the role of β-arrestin2 in OTR recycling as these receptors have been previously classified as slowly or nonrecycling receptors on the basis of their stable association with this interacting protein. Our data suggest that the stable OTR/β-arrestin2 interaction plays an important role in determining the rate of recycling of human OTRs, but does not determine the fate of endocytosed receptors. Subsequent investigations of receptor recycling pathways showed that OTRs localize in vesicles containing the Rab5 and Rab4 small GTPases (markers of the “short cycle”), whereas there was no colocalization with Rab11 (a marker of the “long cycle”) or Rab7 (a marker of vesicles directed to endosomal/lysosomal compartments). Taken together, these data indicate that OTRs are capable of very efficient and complete resensitization due to receptor recycling via the short cycle.

G-protein-coupled receptors (GPCRs) are integral membrane proteins, hence it is not surprising that a number of their structural and functional features are modulated by both proteins and lipids. The impact of interacting proteins and lipids on the assembly and signalling of GPCRs has been extensively investigated over the last 20-30 years, and a further impetus has been given by the proposal that GPCRs and/or their immediate signalling partners (G proteins) can partition within plasma membrane domains, termed rafts and caveolae, enriched in glycosphingolipids and cholesterol. The high content of these specific lipids, in particular of cholesterol, in the vicinity of GPCR transmembranes can affect GPCR structure and/or function. In addition, most GPCRs are post-translationally modified with one or more palmitic acid(s), a 16-carbon saturated fatty acid, covalently bound to cysteine(s) localised in the carboxyl-terminal cytoplasmic tail. The insertion of palmitate into the cytoplasmic leaflet of the plasma membrane can create a fourth loop, thus profoundly affecting GPCR structure and hence the interactions with intracellular partner proteins. This review briefly highlights how lipids of the membrane and the receptor themselves can influence GPCR organisation and functioning. © 2009 Society for Endocrinology.

We have recently shown that oxytocin inhibits cell growth when the vast majority of oxytocin receptors (OTRs) are excluded from detergent-resistant membranes (DRMs; the biochemical counterpart of lipid rafts), but has a strong mitogenic effect when the receptors are targeted to these plasma membrane domains upon fusion with caveolin-2, a resident raft protein. The aim of this study was to investigate whether the manipulation of total cell cholesterol can influence OTR localization and signaling. Our data indicate that cholesterol depletion in HEK-293 cells does not affect the signaling events mediated by the OTRs located outside DRMs. When treated with 2 mM methyl-β-cyclodextrin (MβCD), the receptors remained outside and continued to inhibit cell growth. On the contrary, the MβCD treatment of cells expressing receptors fused to caveolin-2 led to their redistribution outside DRMs, and converted the receptor-mediated proliferative effect into cell growth inhibition. These data indicate that 1) once released from DRMs, the receptors fused to caveolin-2 signal exactly as wild-type OTRs and 2) their DRM location is responsible for the specific OTR signaling leading to cell proliferation. Finally, we evaluated whether cholesterol loading could force the OTRs into lipid rafts and change their signaling, but, after cell treatment with an MβCD/cholesterol complex, receptor stimulation continued to lead to cell growth inhibition, thus indicating that increasing cell cholesterol levels is not sufficient per se to affect OTR signaling.

In human myometrial cells, the promiscuous coupling of the oxytocin receptors (OTRs) to G(q) and G(i) leads to contraction. However, the activation of OTRs coupled to different G protein pathways can also trigger opposite cellular responses, e.g. OTR coupling to G(i) inhibits, whereas its coupling to G(q) stimulates, cell proliferation. Drug analogues capable of promoting a selective receptor-G protein coupling may be of great pharmacological and clinical importance because they may target only one specific signal transduction pathway. Here, we report that atosiban, an oxytocin derivative that acts as a competitive antagonist on OTR/G(q) coupling, displays agonistic properties on OTR/G(i) coupling, as shown by specific (35)S-labeled guanosine 5'-3-O-(thio) trisphosphate ([(35)S]GTPgammaS) binding. Moreover, atosiban, by acting on a G(i)-mediated pathway(,) inhibits cell growth of HEK293 and Madin-Darby canine kidney cells stably transfected with OTRs and of DU145 prostate cancer cells expressing endogenous OTRs. Notably, atosiban leads to persistent ERK1/2 activation and p21(WAF1/CIP1) induction, the same signaling events leading to oxytocin-mediated cell growth inhibition via a G(i) pathway. Finally, atosiban exposure did not cause OTR internalization and led to only a modest decrease (20%) in the number of high affinity cell membrane OTRs, two observations consistent with the finding that atosiban did not lead to any desensitization of the oxytocin-induced activation of the G(q)-phospholipase C pathway. Taken together, these observations indicate that atosiban acts as a "biased agonist" of the human OTRs and thus belongs to the class of compounds capable of selectively discriminating only one among the multiple possible active conformations of a single G protein-coupled receptor, thereby leading to the selective activation of a unique intracellular signal cascade.

This review describes the advances in our understanding of the role of G-protein coupled receptor (GPCR) localisation in membrane microdomains known as lipid rafts and caveolae. The growing interest in these specialised regions is due to the recognition that they are involved in the regulation of a number of cell functions, including the fine-tuning of various signalling molecules. As a number of GPCRs have been found to be enriched in lipid rafts and/or caveolae by means of different experimental approaches, we first discuss the pitfalls and uncertainties related to the use of these different procedures. We then analyse the addressing signals that drive and/or stabilise GPCRs in lipid rafts and caveolae, and explore the role of rafts/caveolae in regulating GPCR trafficking, particularly in receptor exo- and endocytosis. Finally, we review the growing evidence that lipid rafts and caveolae participate in the regulation of GPCR signalling by affecting both signalling selectivity and coupling efficacy.

We have recently shown that oxytocin inhibits cell proliferation when the vast majority of oxytocin receptors are excluded from caveolin-1-enriched microdomains, and that, on the contrary, it has a mitogenic effect when the receptors are targeted to these plasma membrane domains. In this study, we investigated whether the receptors located inside and outside caveolar microdomains initiate different signalling pathways and how this may lead to opposite effects on cell proliferation. Our data indicate that, depending on their localization, oxytocin receptors transactivate EGFR and activate ERK1/2 using different signalling intermediates. The final outcome is a different temporal pattern of EGFR and ERK1/2 phosphorylation, which is more persistent when the receptors are located outside caveolar microdomains and inhibit cell growth, and very transient when they are located in caveolar microdomains and stimulate cell growth. Finally, only the activation of receptors located outside caveolar microdomains correlates with the activation of the cell cycle inhibitor p21(WAF1/CIP1), thus suggesting that the antiproliferative OTR effects may, in this case, be achieved by a sustained activation of EGFR and MAPK leading to the induction of this cell cycle regulator.

Despite oxytocin receptors (OTR) being present in human chorio-decidual tissues, their expression and role in placental trophoblast cells in the context of tumor growth or physiological functions related to cell proliferation have never been examined. In the present study we demonstrate the presence and functionality of OTR in normal human trophoblast cell lines (ED77 and ED27) and a choriocarcinoma cell line (BeWo). RT-PCR and immunofluorescence analysis revealed the presence of OTR messenger RNA and protein in these cells. Binding studies using [(125)I]oxytocin ([(125)I]OT) antagonist confirmed the presence of specific binding sites in ED27, ED77, and BeWo cells. OTR functionality was demonstrated by measuring the OT-induced increase in the intracellular calcium concentrations. This effect was dose dependent and was blocked by the selective OT antagonist d(CH(2))(5)[Tyr(Me)(2),Thr(4), Tyr-NH(2)(9)]OVT (OT antagonist). Furthermore, two proteins with apparent molecular masses of 125 and 60 kDa became tyrosine phosphorylated in all of the cell lines after OT stimulation (and an additional protein of 45 kDa in BeWo choriocarcinoma cells), suggesting that this peptide can stimulate tyrosine kinase activity. Finally, we observed a dose-dependent OT stimulation of cell proliferation associated with OTR activation that was completely abolished by the selective OT antagonist. These findings provide the first evidence of the presence of functional OTR in normal trophoblast cell lines as well as in choriocarcinoma cells and show that a specific effect of OT on normal and neoplastic trophoblast is to promote cellular proliferation.

The aim of this study was to investigate the molecular changes associated with the transition of the human oxytocin receptor from its inactive to its active states. Mutation of the conserved arginine of the glutamate/aspartate-arginine-tyrosine motif located in the second intracellular domain gave rise to the first known constitutively active oxytocin receptor (R137A), whereas mutation of the aspartic acid located in the second transmembrane domain led to an inactive receptor (D85A). The structural features of the constitutively active and inactive receptor mutants were compared with those of the wild type in its free and agonist-bound states. The results suggest that, although differently triggered, the activation process induced by the agonist and the activating mutation are characterized by the opening of a solvent exposed site formed by the 2nd intracellular loop, the cytosolic extension of helix 5, and the 3rd intracellular loop; on the contrary, the D85A mutation prevents oxytocin from triggering the opening of a cytosolic site. On the basis of these findings, we hypothesize that this cytosolic crevice plays an important role in G protein recognition. Finally, comparative analysis of the free- and agonist-bound forms of the wild-type oxytocin receptor and alpha1B adrenergic receptor suggests that the highly conserved polar amino acids and the seven helices play similar mechanistic roles in the different G protein-coupled receptors.

Oxytocin (OT) and vasopressin (AVP) receptors belong to the G-protein-coupled receptor family; in these receptors, the peptide agonists bind to a region delimited by the upper third of the transmembrane part of the molecule (1) and the molecular interactions responsible for agonist selectivity have been extensively investigated (2–5). However, nothing is known on the intramolecular changes that characterize the active conformation(s) of these receptors. In order to get a better understanding of the process of receptor activation, we started a project aimed at the identification of the key residues involved in stabilizing the active conformation(s) of the human oxytocin receptor.

We investigated the mechanisms that regulate the efficacy of agonists in the arginine-vasopressin (AVP)/oxytocin (OT) receptor system. In this paper, we present evidence that AVP, a full agonist of the vasopressin receptors, acts as a partial agonist on the oxytocin receptor. We also found that AVP becomes a full agonist when two aromatic residues of the oxytocin receptor are replaced by the residues present at equivalent positions in the vasopressin receptor subtypes. Our results indicate that these two residues modulate the response of the oxytocin receptor to the partial agonist AVP.

We have isolated cDNA and genomic clones coding for the human alpha 7 neuronal nicotinic receptor subunit, the major component of brain nicotinic receptors that are blocked by alpha-bungarotoxin. The human alpha 7 neuronal nicotinic cDNA encodes a mature protein of 479 amino acids that is highly homologous to the rat alpha 7 neuronal nicotinic subunit (90%). We have mapped the human alpha 7-nicotinic receptor subunit gene to chromosome 15, band q14, a region frequently rearranged in patients carrying a bisatellite 15 chromosome, large inv dup (15), whose clinical features include mental retardation and seizures.

A beta subunit of the neuronal nicotinic receptor, sharing 88% homology with the rat beta 4 subunit, has been cloned from a human neuroblastoma cell line. The gene encoding the human beta 4 subunit is expressed in association with the alpha 3 gene in neuroblastoma and small-cell lung carcinoma cell lines. Patch-clamp experiments and radioligand binding assays confirm that these neuroendocrine tumor cell lines express functional neuronal nicotinic receptors. We suggest that these receptors might play a crucial role in the control of neurotransmitter and hormone secretion from neurosecretory human tumors.

alpha-Bungarotoxin (alpha Bgtx) is a toxin known to interact with muscle nicotinic receptors and with some neuronal nicotinic receptors. We show that alpha Bgtx binding sites are also expressed in nonmuscle and nonneuronal human cells, including small cell lung carcinoma and several epithelial cell lines. These receptors are immunologically related to the alpha Bgtx receptors of unknown function described in the nervous system and in the IMR32 neuroblastoma cell line and are distinct from muscle nicotinic receptors. We have also cloned from IMR32 cells the human alpha 5-nicotinic receptor subunit, which is supposed to participate in the formation of alpha Bgtx receptors. Transcripts corresponding to the alpha 5-subunit gene were found not only in neuroblastoma cells but also in all the cell lines expressing alpha Bgtx receptors, with the exception of the TE671 cell line, whose nicotinic receptor subunits are of the muscle type. We conclude that both alpha Bgtx receptors and the alpha 5-nicotinic subunit gene are not neuron-specific, as previously thought, but are expressed in a number of human cell lines of various origin.

Using indirect immunofluorescence techniques, we have found that calcitonin gene-related peptide-like immunoreactivity is present in the neuromuscular junctions of somatic muscles as well as in almost all motor neurons of the lumbar enlargement of 1-week-old rats. It gradually decreases in both motor neurons and motor nerve endings as the animal grows up and completely disappears from the neuromuscular junctions in adult rats, persisting only in the motor nerve endings on the intrafusal fibers. In situ hybridization experiments have shown that the down-regulation of calcitonin gene-related peptide-like immunoreactivity is strictly related to a reduction in CGRP mRNA levels in the spinal motor neurons. These results indicate that the expression of CGRP is developmentally regulated in spinal cord alpha motor neurons. They also suggest that the peptide may play an important role at the immature neuromuscular junction.