Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

OTR (Oxytocin Receptor)

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_101738

Synonyms

Historical Background

The biological activities of the pituitary gland have been recognized for over 120 years. In 1895, Oliver and Schäfer discovered that an element of pituitary extract could elevate blood pressure in mammals – the so-called pressor effect. A few years later, Howell identified that this activity resided from the posterior lobe of the pituitary. In 1906, Sir Henry Dale, while studying the vasopressor action of the extract, found that it also possessed a powerful stimulating action on the uterus of a pregnant cat. He termed the agent responsible, “oxytocin” from the Greek words ωχνξ, τoχoxξ meaning “quick birth.” Shortly afterwards the same extract was shown to cause milk secretion from mammary tissue. Around 50 years later, the American biochemist, Vincent du Vigneaud determined the chemical structure of oxytocin (OT). He characterized and sequenced a peptide of 9 amino acids which formed a 6 amino-acid cyclic ring structure via a disulphide bond between two cysteine residues and 3 residue tail. This was followed shortly by its synthesis. Oxytocin was in fact the first peptide hormone to be synthesized and for his work; du Vigneaud was awarded the Nobel Prize for Chemistry in 1955.

As with most endocrine systems, however, the physiological actions of hormones are mediated through binding to specific receptors on target organs. Despite some of the biological activities of OT and its structure being known for some time, it wasn’t until 20 years after its synthesis that the receptor responsible for eliciting these effects – the oxytocin receptor (OTR) – was identified. In a 1973 study, Soloff and Swartz demonstrated using a pharmacological ligand binding assay, the specific and high affinity binding of radiolabeled oxytocin to the mammary gland of the rat. They proposed that the mammary gland possessed “oxytocin binding substances which may be the hormone recognition sites of an oxytocin-receptor system.” In addition they determined that the binding part of the receptor was a protein because it was susceptible to inhibition by trypsin. Similar studies in the uterus of the rat and sow and human soon followed.

It was not until 1992 however that the cDNA encoding the receptor for oxytocin was isolated and identified by Kimura. In their study they injected defolliculated oocytes from Xenopus with mRNA prepared from the muscle of the uterus (myometrium). Subsequent application of OT under voltage-clamped conditions caused a large inward membrane current. The receptor mRNA responsible was determined to be between 4 and 5 kb in size. Using a size-fractionated sublibrary of cDNA clones, followed by screening of progressively smaller sublibraries, they isolated a single cDNA clone which encoded the human OTR. When transcribed in vitro and the RNA injected into oocytes, this clone enabled them to be responsive to OT (Kimura et al. 1992). The molecular cloning of the OTR and determination of its amino acid structure paved the way for studies investigating the regulation of the gene’s expression including the development of total and conditional OTR and OT gene knockout mice (Nishimori et al. 1996; Takayanagi et al. 2005; Lee et al. 2008).

The OXTR gene is present in single copy in the human genome and is mapped to chromosome 3 at locus 3p25-26.2 (Inoue et al. 1994) and spans approximately 17 kilobases (kb). In most mammals the gene consists of 3 exons; however, in the human and mouse a third intron interrupts the first exon. Therefore in these species the OXTR gene consists of 4 exons and 3 introns (Gimpl and Fahrenholz 2001). Introns 1 and 2 are relatively short (639 and 166 bp). Intron 3 is the largest intron at approximately 12 kb and separates the coding region found immediately after the sixth transmembrane spanning domain. Exons 1 and 2 correspond to the 5′ noncoding region, followed by exons 3 and 4 which contain the amino acids that encode the receptor: Exon 3 encodes from 142 bp upstream of the translation start site (ATG initiation codon) and spans 922 bp downstream which includes transmembrane domains 1-6 and beyond. Exon 4 contains the sequences encoding the seventh transmembrane domain and the C-terminus as well as the 3′ noncoding region (Fig. 1) (Inoue et al. 1994).
OTR (Oxytocin Receptor), Fig. 1

Organization of the human oxytocin receptor gene. The OTR is located on chromosome 3 at position 3p25.3. It is comprised of 4 exons (boxes labeled 1-4). Only exons 3 and 4 encode the amino acid sequence for the receptor (areas highlighted in blue), flanked by the start (ATG) and stop codons (TGA). The regions encoding the 7 transmembrane-spanning domains are represented by dark blue boxes and are numbered by roman numerals I-VII (Adapted from Inoue et al. 1994)

Primer extension analysis identified that the transcription start sites are located 618 and 621 bp upstream of the initiation codon. A variety of nucleotide consensus sequences that are typically involved in transcriptional regulation in many genes were found in close proximity to this, including a TATA-like motif and a potential binding site for SP-1. A number of other transcription factor binding sites are also found in the 5′ flanking region including those for AP-1, AP-2 c-Myb, and four inverted GATA-1 motifs. The rat OXTR was also shown to contain a potential estrogen response element (ERE), but a functional ERE has not been found in other species, including human (Kimura et al. 2003).

Receptor Structure, Ligand Binding, and Signaling

The encoded receptor contains 389 amino acids in its polypeptide chain and belongs to the rhodopsin-type class I G protein-coupled receptor (GPCR) superfamily, typically characterized by their highly conserved 7 transmembrane (TM) alpha helical domains (Fig. 2). Data from a number of species including pig, rat, sheep, bovine, mouse, and rhesus monkey have also been identified and show essentially the same basic structure. Two different-sized messenger RNAs for the receptor were found: 3.6 kb in mammary gland and a larger 4.4 kb in ovary, endometrium, and myometrium.
OTR (Oxytocin Receptor), Fig. 2

Schematic structure of the human oxytocin receptor. Membrane topology of the human OTR is shown where each amino acid is denoted by a blue circle, and residues involved in ligand binding and signal transduction discussed in the text are highlighted. Residues in red indicate those identified as being involved in binding of oxytocin to the receptor while residues in purple denote those sites shown to confer altered receptor activity when mutated. Areas of proposed functional importance in the cytoplasmic C-terminal tail, including clusters of serine residues whish are thought to be involved in GRK or β-arrestin binding and receptor internalization and desensitization are highlighted in green. Putative N-glycosylation and palmitoylation sites are also highlighted and a putative disulphide bond between the first and second extracellular loops is also shown (Adapted from Gimpl and Fahrenholz 2001)

The structure of the receptor, modeling of the ligand docking at the molecular level and subsequent activation, as well as the post-oxytocin signaling mechanisms have been well described. In general, when activated, conserved sequence residues in GPCRs lead to signal transduction via the recruitment of G proteins. The switching from the inactive to active form of the OTR is assumed to follow that of other GPCR models with a change in the relative orientation of the transmembrane domains revealing a G protein binding site. Oxytocin is suggested to bind to the extracellular loops and the NH2 terminal domain, as well as to residues within the transmembrane domains themselves (Gimpl and Fahrenholz 2001). Mutagenesis and molecular modeling has suggested that the binding site of OT is located in in a narrow cleft buried in the TM core of the receptor, formed by the ring-like arrangement of the TM domains. The cyclic portion of the oxytocin peptide interacts with TM 3, 4, and 6 while the linear C-terminal tail region interacts with TM 2 and 3 and the first intracellular loop.

Biochemical studies with the human OTR and OT have identified four key residues that are important for ligand binding and agonist selectivity (see Fig. 2): Arg34 positioned within an important 12 residue sequence within the N-terminus, Phe103 positioned in the first extracellular loop and two aromatic residues, Tyr209 and Phe284 in TM 5 and 6 respectively, which are thought to bind to residues 2 and 3 of OT (Zingg and Laporte 2003). In terms of OTR signaling, site-directed mutagenesis has also identified key residues in the OTR sequence that are responsible for conformational changes in the receptor, intracellular activation of G proteins, and coupling to downstream effectors (see Fig. 2). A point mutation in the amino acid aspartic acid at position 85 to alanine in TM2 in the human OTR results in receptor inactivity. Changing a lysine to alanine residue at position 270 within the C-terminal part of the third intracellular loop of OTR decreases phosphatidyl turnover. In contrast an arginine to alanine transition at position 137 which forms part of a conserved glutamate (or aspartate)–arginine–tyrosine motif increases basal OTR activity.

The carboxy terminus contains two well-conserved cysteine residues, thought to be sites of palmitoylation and therefore conferring anchorage of the receptor’s cytoplasmic tail into the membrane lipid bilayer. Additionally the NH2-terminal domain of the receptor also contains sites for possible glycosylation (Fig. 2). These differences in glycosylation patterns may be responsible for differences in the molecular masses of the myometrial versus mammary OTRs reported. However, glycosylation does not appear to be important for the proper expression of the OTR or its function since receptor binding characteristics in deglycosylation mutants involving exchange of Asp for Asn in three positions remained unchanged (Gimpl and Fahrenholz 2001).

Homology with Other GPCRs

The OTR shares high sequence homology with the vasopressin receptors of which there are three subtypes: V1aR, V1bR, and V2R. The highest homology between the vasopressin (AVP)/OT receptor types is found in the extracellular loops (∼80%) and the transmembrane helices. This high sequence similarity especially in the extracellular domains can give rise to agonist cross-reactivity. In regards to AVP/OT agonist binding, the OTR is relatively unselective, having only a tenfold higher affinity for OT over AVP. AVP can act as a partial agonist at OTR but requires 100-fold greater concentration to induce the same response as OT. In fact much of our understanding about the functional structure of OTRs including the determination of the important binding domains which confer different ligand affinities comes from chimeric “gain of function mutants” in which different domains or residues of the OTR have been exchanged for the equivalent regions on the different AVPRs.

Oxytocin receptors are functionally coupled to the heterotrimeric G-proteins, comprised of three subunits Gα, Gβ, and Gγ which cycle between active and inactive signaling states in response to guanine nucleotides. Together they can stimulate a number of signaling pathways that regulate a diverse number of cellular processes. Oxytocin receptors have been shown to recruit and activate the Gq and Gi subfamily (Gi1, Gi2, and Gi3) as well as the two members of the Go family (GoA and GoB) of G proteins (Busnelli et al. 2012). There is less evidence, however, to suggest that OTR activation results in the recruitment or activation of Gαs.

Classically, the activation of Gαq/11 results in the activation of phospholipase C-β (PLC-β) which, in turn, controls the hydrolysis of phosphatidylinositol 4,5-bispohosphate (PIP2) into inositol-tris-phosphate (IP3) and diacylglycerol (DAG). Inositol-tris-phosphate controls the mobilization of Ca2+ from intracellular stores such as the sarcoplasmic reticulum (SR) thereby raising intracellular Ca2+ and promoting cell contraction, while DAG leads to activation of protein kinase type C (PKC). OTR activation linked to Gq signaling also leads to the stimulation of phospholipase A2 production and an increase in cyclooxygenase 2 (COX-2) levels, both resulting in increased prostaglandin production. Other signaling pathways activated include the MAP-kinase (MAPK) cascade and induction of c-fos and c-jun which are linked to the proliferative effects of OT. Signaling through Gαi/o results in the inhibition of adenylate cyclase activity and a reduction in levels of cAMP. In addition, inhibition of cell growth is reported to be Gi-mediated.

In in vitro assays using HEK293 cell lines stably transfected with human OTR cDNA, the Gαi and Gαo isoforms were found to be activated by OT with an EC50 tenfold greater than that required to activate Gαq, suggesting that the Gi and Go-mediated pathways are activated at much higher concentrations of oxytocin than the Gq pathway (Busnelli et al. 2012). How this is translated into a physiological response in vivo, however, will depend on the local concentration of OT as well as the relative expression of the individual G protein subunits. In addition, that OTRs can activate multiple G proteins can give rise to heterogeneity in the overall cellular response following their activation. The signaling pathways may therefore act synergistically or may have opposite effects on the same cell function.

Many GPCRs, including the OTR, have also been shown to associate and form dimers which can be homo- or heterodimers in nature. OTR heterodimerization reported includes in vitro dimerization with the highly related V1aR and V1bRs and heterodimerization with the dopamine D2 and adrenergic β2 receptors in vivo.

Expression, Localization, and Regulation

Oxytocin has both central and peripheral actions, with roles in many physiological and pathological processes including reproduction, for example, parturition and lactation, maternal behavior, erectile dysfunction, and ejaculation. Oxytocin can also modulate social behavior via increasing empathy, trust, and pair bonding. Not surprisingly therefore, the OTR has been found to be expressed in humans throughout the body: in reproductive structures including myometrium, endometrium, gestational tissues (amnion and decidua), ovary, testes, and breast, as well as other organs including kidney, heart, adrenal gland, and in neural regions of the brain, such as the frontal cortex, amygdala, hypothalamus, and olfactory nucleus.

The localization of receptors to the plasma membrane was determined by investigating the distribution of [3H] oxytocin binding sites among various subcellular fractions of rat myometrium obtained by differential centrifugation. More specifically, OTRs have been found to be localized to the cholesterol-rich and caveolin-containing membrane domains of the plasma membranes known as caveolae which is Latin for “little caves” (Gimpl and Fahrenholz 2001). These form small omega-shaped cell surface invaginations. The localization of the receptors to these domains may be conveyed by a cholesterol-binding motif within the extracellular domains of the receptor. In contrast to most other GPCRs, the OTR undergoes quite large and cell-specific up- and downregulation of expression. A number of factors have been shown to regulate OTR expression.

Sex steroids: In uterus, brain, and kidney, estrogens are a major stimulant of OTR expression. However, OTR mRNA levels in the mammary gland remain unaffected by estrogen administration. Studies of the rat and human OXTR promoter identified a potential but nonclassical estrogen responsive element (ERE), but there are doubts over its functionality (Kimura et al. 2003). Estrogen’s effects may not follow the classical ER- ERalpha-ERE- target gene pathway. Instead, the mechanism through which estrogen regulates expression is likely to involve multiple factors acting indirectly or via other promoter elements with the OTR. Progesterone (P4) on the other hand is inhibitory towards OTR expression. Pregnant rats treated with P4 failed to show the same upregulation of OTR mRNA at parturition as controls. Ovariectomy of pregnant rats in mid-gestation results in a significant increase in myometrial OTR mRNA levels compared to sham-operated controls and induces preterm labor. The elevation in OTR mRNA levels associated with ovariectomy-induced preterm labor was completely blocked by the administration of progesterone (Ou et al. 1998).

Cholesterol: Cholesterol is one of the most abundant lipids within the lipid bilayer and therefore its concentration can regulate the function and organization of many membrane proteins including GPCRs. The OTR is known to favor residing within cholesterol-rich portions of the membrane (Gimpl and Fahrenholz 2001). It is within these cholesterol-rich domains that they confer a higher affinity for agonist binding. OTRs are said to be sensitive to levels of membrane cholesterol, and disruption of cholesterol content affects OTR signaling.

Stretch: Mechanical stretch, e.g., of the myometrium (smooth muscle of the uterus) during pregnancy has also shown to be involved in the upregulation of OTR expression, and it suggested that this may contribute to the higher rates of preterm birth in multiple pregnancy. In studies of unilaterally pregnant rats in which the nongravid uterine horn was mechanically stretched, the nongravid stretched horn shows equivalent OTR upregulation as the gravid horn at parturition (Ou et al. 1998). The nongravid, nonstretched uterus, however, shows low expression suggesting that both endocrine signals and stretch are a contributing factor to OTR expression in pregnancy and labor.

Internalization and desensitization: A further dimension to the regulation of OTR signaling is via its capacity, like other GPCRs, to desensitize and internalize. In the setting of persistent agonist binding, desensitization is initiated by phosphorylation of the receptor by G-protein-coupled receptor kinases (GRKs). These phosphorylate GPCRs which increases their affinity for β- arrestins. β-arrestins contain motifs which allow them to function as scaffold proteins linking the receptor to components of the clathrin-dependent endocytic machinery and hence prompts receptor endocytosis. The OTR has been shown to recruit β-arrestin following OTR stimulation and has been shown to co-localize with β-arrestins in endocytic vesicles. Removal of the receptor from the membrane therefore uncouples it from further G protein signaling. The recruitment of β-arrestin to the OTR is dependent upon a highly conserved region within its carboxy terminal containing a series of serine clusters. Mutations within this region lead to unstable OTR-β-arrestin interactions and prevent agonist-mediated receptor internalization (Smith et al. 2006). There is also evidence to suggest that OTR can undergo receptor desensitization via a non-β-arrestin pathway involving PKC.

Epigenetics and OXTR methylation: The OXTR gene contains a CpG island that stretches through exon 1 to exon 3 from about 20 to 2350 bp downstream of the transcription start site. Luciferase reporter gene assays showed that this CpG region has significant promoter activity (Kusui et al. 2001). A specific region of this CpG island (termed MT2) appears to be responsible for the majority of DNA methylation-induced silencing of OTR.

Physiological Functions in Labor and Lactation

Oxytocin is most widely known for its ability to contract the uterus and for milk ejection during lactation. In the uterus, oxytocin stimulates and maintains uterine contractions by elevating intracellular Ca levels. Binding of oxytocin to OTRs primarily leads to the activation of Gαq/11 resulting in the PLC-mediated hydrolysis of PIP2, increased IP3 formation, and hence increased intracellular Ca via release of Ca2+ from the SR (Fig. 3). In smooth muscle cells such as myometrium, elevated Ca brings about contraction via stimulation of Ca2+-dependent calmodulin which, in turn, activates myosin light chain kinase (MLCK). Subsequent phosphorylation of the regulatory myosin light chains by MLCK brings about cross-bridge cycling and generation of force (Arrowsmith and Wray 2014). Relaxation is brought about by dephosphorylation of myosin via MLC phosphatase (MLCP) and restoring normal intracellular Ca levels.
OTR (Oxytocin Receptor), Fig. 3

Proposed signaling pathways activated by oxytocin binding to the OTR in myometrium and other uterine tissues including decidua and amnion (from Arrowsmith and Wray 2014). Binding of oxytocin to its receptor activates Gαq/11, which activates phospholipase C-β, which in turn hydrolyses phosphoinositide-bis-phoshate (PIP2) into inositol-tris-phosphate (IP3) and diacylglycerol (DAG). IP3 causes release of Ca from the sarcoplasmic reticulum (SR) and DAG activates protein kinases type C (PKC). Activation of Gαq/11 is also suggested to cause the opening of voltage-operated Ca2+ channels (VOCCs) and Ca2+ entry. This may be as a result of direct activation or indirect activation of channel opening, e.g., via receptor-operated channel (ROC) opening. Inhibition of the Ca exit from the cell by inhibition of Ca2+−ATPase also promotes increased [Ca2+]i. The reduction in lumenal SR [Ca2+] is considered to trigger store-operated Ca2+ entry (SOCE). The elevation in [Ca2+]i leads to formation of the Ca2+-calmodulin complex which then activates myosin light-chain kinase (MLCK), resulting in acto-myosin cross-bridge cycling and myometrial contraction. In addition, DAG activation of PKC activates the mitogen-activated protein kinase (MAPK) cascade resulting in increased phospholipase A2 (PLA2) activity and prostaglandin E2 (PGE2) production, which also contributes to contraction (mechanism not shown). DAG-activated PKC also signals for phosphorylation of C-kinase-activated protein phosphatase-1 inhibitor 17 kDa (CPI-17), whereas oxytocin binding to OTR also activates Rho-A which in turn activates RhoA-associated protein kinase (ROCK). Both phosphorylated CPI-17 and ROCK inhibit myosin light chain phosphatase (MLCP), leading to increased MLC phosphorylation and is the proposed mechanism of Ca2+ sensitization in the myometrium. Oxytocin receptor signaling in other uterine tissues (e.g., decidua and amnion) also signals for production of prostaglandins and pro-inflammatory cytokines (not shown), which may mediate local paracrine signaling with the myometrium. Red pathways indicate signaling pathways with direct influences on [Ca]i, whereas purple and turquoise lines indicate Ca2+-independent pathways to contraction, including Ca2+ sensitization (purple lines) and the production of prostaglandins (turquoise pathways). Dotted lines indicate where mechanisms are not yet fully determined

There is also evidence to suggest that Ca2+ from extracellular sources also contributes to the oxytocin-induced rise in intracellular Ca2+. Oxytocin-induced intracellular Ca2+ increase is greater in the presence of extracellular Ca2+ than that in its absence, suggesting that activation of OTR also affects Ca influx mechanisms. One mechanism is via voltage gated, i.e., L-type channels or receptor-operated channels. However, this Ca2+ influx was found to be insensitive to the L-type channel blocker nifedipine. It is therefore suggested that oxytocin augments Ca2+ entry via a process known as capacitative Ca2+ entry or store-operated Ca2+ entry (SOCE) – a process which is independent of voltage-operated Ca entry. Instead the lowering of SR Ca concentration triggers Ca entry. It is likely that oxytocin affects L-type channel activity indirectly such as via the opening of other cation channels (e.g., Ca2+-activated Cl- channels) or SOCE, which would then lead to membrane depolarization and subsequent opening of voltage-operated channels. The evidence to suggest that oxytocin can affect Ca extrusion mechanisms, e.g., by inhibition of Ca2+-ATPases and Ca efflux from cells, thus prolonging the elevation of Ca2+ is limited.

Oxytocin binding to its receptor can also lead to activation of Rho proteins (likely via Gα12/13 proteins) which can regulate the interaction between actin and myosin independently of Ca2+. Rho activation leads to activation of Rho kinase and subsequent phosphorylation and inhibition of MLC phosphatase (MLCP). Inhibition of MLCP therefore removes the inhibitory break on contraction and allows for modulation of force production without the need for a change in intracellular Ca concentration. This process is known as Ca sensitization (Somlyo et al. 1999). In rats, pretreatment with a specific inhibitor of Rho-kinase decreased the level of oxytocin-induced MLC phosphorylation, suggesting that the RhoA/Rho kinase cascade is involved in the contraction response mediated by oxytocin. When examined in human myometrium, however, the inhibition of RhoA-Rho kinase pathway produced only moderate effects. Additionally, OTR activation leading PKC-mediated production of DAG can also contribute to sensitization via inhibition of MLCP directly, or via a smooth muscle specific inhibitor of MLCP known as C-kinase-activated protein phosphatase-1 inhibitor 17 kDa (CPI-17) (see Fig. 3).

OTR signaling in cultured myometrial and amnion cells (cells of the fetal membranes) also activates NFκB and MAPK signaling and stimulates the production of pro-inflammatory cytokines such as IL-6 and IL-8 and prostaglandin production via upregulation of cyclooxygenase type 2 (COX-2) (Fig. 3). This inflammatory signaling and upregulation of prostaglandin synthesis is known to play roles in parturition including fetal membrane remodeling, cervical ripening, and myometrial activation. Additionally OT is also implicated in the achievement of coordinated, synchronous myometrium contractions required for labor. This is via its role in promoting upregulation of connexin proteins, including connexin 43 which is a major protein involved in the formation of gap junctions providing communication between myometrial cells. Activation of MAPK signaling pathways in myometrium, which is thought to be mediated by Gαq-11 and βγ release, may also lead to MAPK mediated induced myometrial cell proliferation.

In late pregnancy and preterm labor, the expression of myometrial OTRs significantly increases shifting the myometrium towards increased sensitivity to OT. After parturition, OTR expression in myometrium rapidly declines, whereas mammary gland OTR expression remains raised throughout lactation. This tissue-specific regulation of OTR expression is suggested to be the one mechanism which enables circulating OT to switch its target organ and therefore its physiological function, i.e., from to inducing and augmenting uterine contractions during parturition to milk ejection during lactation.

The milk-ejection reflex describes a process whereby oxytocin is released in response to neonatal sucking. During this process, pulsatile waves of OT are released from the posterior pituitary causing the myoepithelial cells surrounding mammary gland alveoli to contract, allowing the collected milk to be released into ducts and open into nipple pores. The signaling pathway leading to contraction of the myoepithelial cells is largely attributed to receptor coupling to Gαq-11 and the activation of the PLC/Ca2+ signaling pathways. Unlike myometrium, however, the increased Ca2+ in myoepithelial cells induced by OT is only transient and not sustained, which is indicative of Ca release from intracellular stores and not Ca influx mechanisms. The release of OT also occurs in most women before the tactile stimulus of suckling. A second release of OT follows in response to the suckling stimulus itself, highlighting other OTR signaling roles such as on maternal behavior.

Physiological Functions Within the Brain

The widespread distribution of OTRs within the brain has firmly established OT as a central neurotransmitter. However, while the peripheral role of oxytocin in labor and lactation is well known and the signaling pathways activated by OTR are well established, much remains to be understood regarding the behavioral and cellular mechanisms of OT’s functions in the brain.

In rodents, receptors for OTR are abundantly expressed throughout the brain while in humans expression is more restricted. Receptor autoradiography studies and immunostaining for OTR found it to be highly expressed in in the basal nucleus of Meynert, diagonal band of Broca and lateral septal nucleus as well as the hypothalamus, anterior cingulate cortex, olfactory nucleus, and amygdala (Boccia et al. 2013). Many of these regions are associated with the central control of stress, anxiety, and social behavior including pair bonding, parental care, aggression, and social memory. Therefore, in the brain, OT is suggested play a key role in the regulation of social cognition and behavior including roles in attachment, social exploration, and social recognition as well as having roles in the fear and stress response.

Following release from dendrites within the hypothalamic neurons, OT is suggested to reach other regions of the brain by diffusion. The precise signaling pathways elicited by OTR activation in these neuronal cells, however, is not fully understood. In immortalized neuronal cells OTR coupling to Gq or to Gi/o resulted in opposite effects on cell excitability. Gq activation inhibits inwardly rectifying potassium channel conductance while Gi/Go coupling was shown to promote inwardly rectifying currents (Gravati et al. 2010). The balance of signaling between the different G-proteins will therefore determine cell function and hence affect behavioral outcomes.

Pathophysiological Implications

The pathophysiological functions of OT and OTR in reproduction have been demonstrated in mouse gene ablation studies (Nishimori et al. 1996; Young et al. 1996; Lee et al. 2008). Interestingly however, neither OT nor its receptor appear necessary for labor. Oxytocin-deficient females are fertile, display normal mating behavior, become pregnant, and deliver their offspring on time without complications and show normal maternal behavior. However, their pups die within 24 hours due to the mothers’ inability to eject milk and nurse them. The mammary glands of the OT-deficient mice were shown to contain milk, and readministration of exogenous OT was able to restore milk ejection, confirming that OT is required for milk ejection but not its production. Similarly, normal parturition has been noted in the absence of OT in cases of women with clinical pituitary gland dysfunction.

OTR-null mice are also viable and show no obvious defects in fertility, sexual behavior, or parturition. However, they show defects in lactation due to lack of milk ejection and also reduced maternal nurturing. OTR- and OT-knockout females postpartum were shown to retrieve fewer pups when scattered compared to their wild types and those that do retrieve pups gather fewer pups. OTR-knockout female mice also groom themselves and their pups less than their wild-type counterparts. Additionally, OTR-null male pups show increased aggressive behaviors compared to their wild-type littermates, further confirming that the OTR is also important in the regulation of development of social behavior.

Labor Dystocia, Postpartum Hemorrhage, and Preterm Birth

Oxytocin is one of the most frequently used drugs in obstetrics, for promoting uterine contractions for labor induction and augmentation and to prevent postpartum hemorrhage. OTR antagonists have also been developed to inhibit preterm labor contractions and treating dysmenorrhea. The goal of labor augmentation is to enhance inadequate contractions to achieve vaginal delivery. Oxytocin is given intravenously and the rate of administration is tailored to the rate of contraction. Paradoxically, prolonged OT infusion results in decreased uterine contractility due to receptor desensitization and internalization. Radioligand studies in cultured cells have shown consistently that OT exposure results in decreased OT -OTR binding and leads to receptor internalization. Clinical studies have shown a loss of uterine activity, measured by intrauterine pressure catheter recordings, with OT infusion. Desensitization of the receptor can increase the risk of cesarean section delivery due to dysfunctional labor and also poses the risk of uterine atony leading to postpartum hemorrhage as forceful uterine contractions are also required to clamp the uterine blood vessels to stem bleeding after delivery. Therefore prolonged OT administration may be counterproductive to the augmentation of uterine contractions. In fact, OT is released from the pituitary in a pulsatile manner. The pulse frequency increases during labor reaching its maximum during the second stage. This pulsatile release may in fact be a mechanism to prevent agonist-mediated desensitization. In contrast, uterine hyperstimulation (or hypertony) in which contractions become too strong or too frequent following OT administration can also occur which can be detrimental to the fetus.

Preterm birth (<37 weeks gestation) accounts for the majority of neonatal morbidity and mortality. As premature uterine contractions are one of the most recognized signs and causes of spontaneous preterm labor, there has been much effort in developing anticontraction medications (known as tocolytics) to inhibit preterm labor contractions to prolong pregnancy. This includes the development of OTR antagonists. The OTR antagonist, atosiban, is widely used clinically to reduce uterine activity in threatened preterm labor. Atosiban is a peptide analogue of oxytocin which competes with oxytocin for binding at the OTR, thus preventing oxytocin-induced rises in intracellular calcium and promoting relaxation of the myometrium. Atosiban is however a biased ligand that antagonises Gαq/11 signaling but acts as a Gαi agonist in a number of cell lines. Its coupling to Gαi signaling may in fact contribute to a pro-labor effect by activation of inflammatory pathways.

Additionally, owing to its similarity in structure to oxytocin and vasopressin, atosiban is also an antagonist at the vasopressin 1a receptor (V1aR) and has higher efficacy at the V1aR over OTRs. The role of vasopressin receptors in human myometrium is less clear – the uterus is responsive to AVP and it expresses V1aR. Interestingly, a more selective OTR antagonist barusiban was no better than placebo in inhibiting in vivo preterm labor contractions. There is much research interest in developing more effective OTR antagonists with greater selectivity and efficacy for inhibiting preterm labor contractions.

Behavioral Disorders

Research across species has shown that OT plays key roles modulating social perception, social cognition, and social behavior, thereby promoting social approach, affiliation, and promoting the maintenance of social relationships. Not surprisingly then, a dysfunction of OT or impairment of OTR signaling has been associated with a number of mental disorders including autism spectrum disorders and schizophrenia, as well as involvement in a collection of mood and anxiety disorders (Cochran et al. 2013).

Variations in the OTR gene may partly explain individual differences in OT-related social behavior. Two single nucleotide polymorphisms (SNPs) in the third intron have been suggested to be particularly promising candidates to explain differences in oxytocinergic functioning (Meyer-Lindenberg et al. 2011): Genetic association studies have revealed reproducible and significant links of some OTR gene polymorphisms to specific social traits and behaviors. Rs225498 (G-A transition) has been linked to emotional deficits and females heterozygous for this polymorphism with a familial history of depression were found to have the highest levels of depression and anxiety. While those with rs53576 (G-A transition) polymorphisms showed a deficit in social behaviors including empathy and mother’s sensitivity towards her child’s behavior. However, the effects of these SNPs are thought to be small and do not change the amino acid sequence of the receptor. Polymorphisms in the OXT and OXTR genes have also been associated with schizophrenia and autism. Downregulation of OTR mRNA and binding sites for OT have also been detected in a number of brain regions involved in social cognition in schizophrenic patients. OXTR methylation may be associated with autism, high callous–unemotional traits, and differential activation of brain regions involved in social perception. Therefore, both genetic and epigenetic factors may have a large impact on defining social personalities and traits.

Exogenous OT administration has been reported to rescue social deficits in preclinical animal models characterized by autistic-like symptoms in which OTR expression was completely or partially absent. Some human studies have evaluated the therapeutic value of endogenously applied neuropeptides such as OT, typically administered via a nasal spray, in ameliorating social dysfunction in patients affected by many different psychiatric or neurodevelopmental disorders. But the evidence for consistency in their therapeutic potential is limiting. One factor being that only a small fraction of these peptides can pass through the blood brain barrier.

Summary

Activation of OTRs results in signaling via a number of pathways; the main pathway being the Gq/PLC/IP3 pathway which results in increased intracellular Ca2+. In myometrium this leads to contraction, and in mammary cells this contributes to milk ejection. Additionally, OTR signaling in decidua and fetal membranes leads to increased prostaglandin synthesis and production of proinflammatory cytokines which can potentiate OT’s actions and aid labor onset. A number of agonists and antagonists to the OTR have been developed for their therapeutic potential, including the antagonist atosiban which has tocolytic qualities and is used for inhibiting preterm labor contractions.

Oxytocin also plays a central role in social behavior, and impairment of OTR signaling is indicated in a number of behavioral pathologies. Oxytocin analogues and OTR antagonists are therefore also useful research tools in furthering our understanding of OTR signaling and OT functions in systems such as the CNS. The therapeutic potential of OT analogues in the treatment of pathophysiological behaviors is still under examination, but targeting the OTR brings hope for alleviating these social disorders.

References

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© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Harris-Wellbeing Preterm Birth Research Centre, Department of Cellular and Molecular Physiology, Institute of Translational MedicineUniversity of LiverpoolLiverpoolUK