**2. Oxytocin synthesis and function**

Oxytocin (OT) is a nine amino acid neuropeptide synthesized by the magnocellular neurons of the supraoptic and paraventricular nuclei of the hypothalamus. It is released into the circulation by exocytosis from the posterior pituitary and nerve terminals in response to various stimuli. The amino acids sequence in the OT molecule is: Cysteine-Tyrosine-Isoleukine-Glutamine-Asparagine-Cysteine-Proline-Leukine-Glycinamide, and with a sulfur bridge between the two cysteines. The structure of OT is very similar to that of the nonapeptide vasopressin, which differs from oxytocin by two amino acids. Oxytocin is also

Oxytocin and Myometrial Contractility in Labor 245

In humans, circulating oxytocin is not necessary for the initiation and completion of parturition, since normal labor can be achieved in cases of pituitary dysfunction (Phelan et al., 1978). Additionally, oxytocin circulation levels do not increase significantly in pregnancy or at the beginning of labor but are increased at the expulsive stage, while oxytocin pulsatile changes occur in pregnant women at term. Apart from in the pituitary, OT is also produced locally, and, in fact, placental OT acting in a paracrine fashion may be more important than circulating OT for the mechanism of labor. OTR is also up-regulated at the end of gestation and sensitivity to oxytocin-induced contractions is greatly increased compared to the nonpregnant uterus. A significant increase in the number of oxytocin receptors in the myometrium and decidua is observed in women with both term and preterm labor (Petraglia et al., 1996). Although steroid hormones are also thought to influence the number of OTR, the mechanisms of regulation are complex and not yet fully elucidated (Mirando et al., 1990, Wathes et al., 1996; Zingg et al., 1995). After parturition the binding sites of OT in uterus decline rapidly, while OTR expression in mammary glands remains high during the

However, continuous exposure to high doses of oxytocin leads to desensitization and downregulation of OTR (Plested and Bernal, 2001). Desensitization is a phenomenon that prevents overstimulation of cells after prolonged agonist stimulation. This phenomenon is observed in GPCR receptors and is brought about by means of different mechanisms at many levels, such as phosphorylation, internalization or changes at the receptor mRNA levels. Rapid desensitization of GPCR receptors, taking place within seconds or minutes, occurs in two steps: 1. phosphorylation of the receptor, causing inhibition of G-protein activation; 2. binding of proteins, called arrestins, preventing G-protein activation and promoting receptor internalization. Internalization of the receptor after continuous OT stimulation is yet another mechanism of desensitization. Though it has been suggested that once internalized, the receptor does not return to the cell surface, recent data suggest that intracellular trafficking and recycling of the OTR to the cell surface does indeed take place (Conti et al., 2009). OTR desensitization is a phenomenon that occurs after prolonged agonist stimulation, i.e. lasting for several hours (Terzidou, 2007). Continuous OT treatment reduces the mRNA of the OTR, this possibly due to suppression of OTR transcription or destabilization of the mRNA molecule. In cultured human myometrial cells, treatment with OT for up to 20 hours causes OTR desensitization which effects in a reduction of the OT binding sites from 210 x 103 sites/cell to only 20.1 x 103 sites/cell, without receptor internalization. However, while the total amount of OTR protein is not diminished, treatment reduces OTR mRNA levels (Phaneuf et al., 1998). In vivo, in women with oxytocin-induced or oxytocin-augmented labor there is also a reduction in myometrial oxytocin binding sites and in OTR mRNA levels. Compared to women not in labor, in cases of oxytocin-augmented or oxytocin-induced labor, the median number of binding sites was reduced from to 477 fmol/mg-1 protein to 140 fmol/mg-1 protein and 118 fmol/mg-1 protein, respectively, both differences being statistically significant. Compared to women not in labor, in cases of labor augmentation and induction OTR mRNA levels were reduced by 60 and 300-fold, respectively (Phaneuf et al., 2000). Oxytocin receptor down-regulation has great significance in clinical practice. Long-term oxytocin infusion may fail to augment labor or may lead to postpartum uterine atony which cannot be managed with additional

**4. Changes in circulating oxytocin and oxytocin receptor levels in labor** 

period of lactation (Petraglia et al., 2010).

synthesized in such peripheral tissues as the uterus, corpus luteum, placenta, amnion, and testis (Gimpl and Fahrenholz, 2001).

Oxytocin, involved in numerous physiological and pathological processes, exerts a variety of actions, including the regulation of the hypothalamo-pituitary-adrenal axis in response to stress, cell proliferation, pregnancy, luteal function, maternal behavior, erectile function, and ejaculation (Viero et al., 2010).

#### **3. The oxytocin receptor signaling**

Oxytocin has only one receptor, which belongs to the rhodopsin-type class I G-protein coupled receptor (GPCR) superfamily. The gene of the oxytocin receptor is present in a single copy on chromosome 3p25 and contains 3 introns and 4 exons. Oxytocin and other molecules of similar structure, such as arginine vasopressin (AVP) and oxytocin agonists or antagonists, can bind to the receptor. The affinity for OT is about 10-fold higher than for AVP. The cell surface transmembrane OTR is activated after binding of OT molecule, and the receptor subsequently causes activation of the various intracellular signal pathways, this finally resulting in the numerous effects of the hormone, including contraction. OTR is coupled to the Gq/11 a- class guanosine triphosphate (GTP) binding proteins. Binding of OT activates, via Gαq/11, phospholipase C (PLC) which hydrolyzes phosphatidylinositol 4,5 bisphosphate (PIP2) to inositol 1,4,5- triphosphate (InsP3) and diacylglycerol (DAG). InsP3 results in the release of Ca2+ ions from intracellular stores, while DAG activates protein kinases type C (PKC), which further phosphorylates other proteins, thus bringing about a trophic effect on myometrial cells via the eukaryotic translation elongation factor 2 (eEF2). Release of Ca2+ ions initiates smooth muscle contractions as Ca2+ binds to calmodulin and the Ca2+-calmodulin system activates myosin light-chain kinase. This mechanism causes myometrial contractions, as well as contraction of mammary myoepithelial cells leading to milk ejection (Gimpl and Fahrenholz, 2001). The major pathway that mediates the signal of OTR after binding of OT is the Gq/PLC/InsP3 pathway. The OTR is, however, also coupled with other G proteins, Gs and Gi, which give rise to various other cellular effects, e.g. inhibition of cellular growth (Viero et al., 2010).

OTR additionally acts on voltage-gated or receptor coupled channels; this activation, which leads to membrane depolarization and the entry of extracellular Ca2+ into the cells, eventually triggers various cellular responses and further promotes smooth muscle contractility.

OTR also activates the mitogen-activated protein kinase (MAPK) and the Rho kinase pathways. Rho associated protein kinases are involved in many cellular phenomena, among them cell migration, cell cycle control and cell contractility. Activation of OTR and MAPK results, in both cases, in elevated cytosolic phospholipase A2 (cPLA2) activity. cPLA2 hydrolyzes phospholipids while liberating arachidonic acid, that results in increased production of prostaglandins via cyclooxygenase-2 (COX-2), an enzyme up-regulated by MAPK (Molnar and Hertelendy, 1995; Soloff et al., 2000). RhoA kinase increases phoshporylated myosin light chains. The increase in intracellular Ca2+ ions, the activation of the Rho and MAP kinase pathways, and the increased production and secretion of prostaglandins all together result in the contractile effects of OT-OTR activation.

synthesized in such peripheral tissues as the uterus, corpus luteum, placenta, amnion, and

Oxytocin, involved in numerous physiological and pathological processes, exerts a variety of actions, including the regulation of the hypothalamo-pituitary-adrenal axis in response to stress, cell proliferation, pregnancy, luteal function, maternal behavior, erectile function, and

Oxytocin has only one receptor, which belongs to the rhodopsin-type class I G-protein coupled receptor (GPCR) superfamily. The gene of the oxytocin receptor is present in a single copy on chromosome 3p25 and contains 3 introns and 4 exons. Oxytocin and other molecules of similar structure, such as arginine vasopressin (AVP) and oxytocin agonists or antagonists, can bind to the receptor. The affinity for OT is about 10-fold higher than for AVP. The cell surface transmembrane OTR is activated after binding of OT molecule, and the receptor subsequently causes activation of the various intracellular signal pathways, this finally resulting in the numerous effects of the hormone, including contraction. OTR is coupled to the Gq/11 a- class guanosine triphosphate (GTP) binding proteins. Binding of OT activates, via Gαq/11, phospholipase C (PLC) which hydrolyzes phosphatidylinositol 4,5 bisphosphate (PIP2) to inositol 1,4,5- triphosphate (InsP3) and diacylglycerol (DAG). InsP3 results in the release of Ca2+ ions from intracellular stores, while DAG activates protein kinases type C (PKC), which further phosphorylates other proteins, thus bringing about a trophic effect on myometrial cells via the eukaryotic translation elongation factor 2 (eEF2). Release of Ca2+ ions initiates smooth muscle contractions as Ca2+ binds to calmodulin and the Ca2+-calmodulin system activates myosin light-chain kinase. This mechanism causes myometrial contractions, as well as contraction of mammary myoepithelial cells leading to milk ejection (Gimpl and Fahrenholz, 2001). The major pathway that mediates the signal of OTR after binding of OT is the Gq/PLC/InsP3 pathway. The OTR is, however, also coupled with other G proteins, Gs and Gi, which give rise to various other cellular effects, e.g.

OTR additionally acts on voltage-gated or receptor coupled channels; this activation, which leads to membrane depolarization and the entry of extracellular Ca2+ into the cells, eventually triggers various cellular responses and further promotes smooth muscle

OTR also activates the mitogen-activated protein kinase (MAPK) and the Rho kinase pathways. Rho associated protein kinases are involved in many cellular phenomena, among them cell migration, cell cycle control and cell contractility. Activation of OTR and MAPK results, in both cases, in elevated cytosolic phospholipase A2 (cPLA2) activity. cPLA2 hydrolyzes phospholipids while liberating arachidonic acid, that results in increased production of prostaglandins via cyclooxygenase-2 (COX-2), an enzyme up-regulated by MAPK (Molnar and Hertelendy, 1995; Soloff et al., 2000). RhoA kinase increases phoshporylated myosin light chains. The increase in intracellular Ca2+ ions, the activation of the Rho and MAP kinase pathways, and the increased production and secretion of

prostaglandins all together result in the contractile effects of OT-OTR activation.

testis (Gimpl and Fahrenholz, 2001).

**3. The oxytocin receptor signaling** 

inhibition of cellular growth (Viero et al., 2010).

contractility.

ejaculation (Viero et al., 2010).

#### **4. Changes in circulating oxytocin and oxytocin receptor levels in labor**

In humans, circulating oxytocin is not necessary for the initiation and completion of parturition, since normal labor can be achieved in cases of pituitary dysfunction (Phelan et al., 1978). Additionally, oxytocin circulation levels do not increase significantly in pregnancy or at the beginning of labor but are increased at the expulsive stage, while oxytocin pulsatile changes occur in pregnant women at term. Apart from in the pituitary, OT is also produced locally, and, in fact, placental OT acting in a paracrine fashion may be more important than circulating OT for the mechanism of labor. OTR is also up-regulated at the end of gestation and sensitivity to oxytocin-induced contractions is greatly increased compared to the nonpregnant uterus. A significant increase in the number of oxytocin receptors in the myometrium and decidua is observed in women with both term and preterm labor (Petraglia et al., 1996). Although steroid hormones are also thought to influence the number of OTR, the mechanisms of regulation are complex and not yet fully elucidated (Mirando et al., 1990, Wathes et al., 1996; Zingg et al., 1995). After parturition the binding sites of OT in uterus decline rapidly, while OTR expression in mammary glands remains high during the period of lactation (Petraglia et al., 2010).

However, continuous exposure to high doses of oxytocin leads to desensitization and downregulation of OTR (Plested and Bernal, 2001). Desensitization is a phenomenon that prevents overstimulation of cells after prolonged agonist stimulation. This phenomenon is observed in GPCR receptors and is brought about by means of different mechanisms at many levels, such as phosphorylation, internalization or changes at the receptor mRNA levels. Rapid desensitization of GPCR receptors, taking place within seconds or minutes, occurs in two steps: 1. phosphorylation of the receptor, causing inhibition of G-protein activation; 2. binding of proteins, called arrestins, preventing G-protein activation and promoting receptor internalization. Internalization of the receptor after continuous OT stimulation is yet another mechanism of desensitization. Though it has been suggested that once internalized, the receptor does not return to the cell surface, recent data suggest that intracellular trafficking and recycling of the OTR to the cell surface does indeed take place (Conti et al., 2009). OTR desensitization is a phenomenon that occurs after prolonged agonist stimulation, i.e. lasting for several hours (Terzidou, 2007). Continuous OT treatment reduces the mRNA of the OTR, this possibly due to suppression of OTR transcription or destabilization of the mRNA molecule. In cultured human myometrial cells, treatment with OT for up to 20 hours causes OTR desensitization which effects in a reduction of the OT binding sites from 210 x 103 sites/cell to only 20.1 x 103 sites/cell, without receptor internalization. However, while the total amount of OTR protein is not diminished, treatment reduces OTR mRNA levels (Phaneuf et al., 1998). In vivo, in women with oxytocin-induced or oxytocin-augmented labor there is also a reduction in myometrial oxytocin binding sites and in OTR mRNA levels. Compared to women not in labor, in cases of oxytocin-augmented or oxytocin-induced labor, the median number of binding sites was reduced from to 477 fmol/mg-1 protein to 140 fmol/mg-1 protein and 118 fmol/mg-1 protein, respectively, both differences being statistically significant. Compared to women not in labor, in cases of labor augmentation and induction OTR mRNA levels were reduced by 60 and 300-fold, respectively (Phaneuf et al., 2000). Oxytocin receptor down-regulation has great significance in clinical practice. Long-term oxytocin infusion may fail to augment labor or may lead to postpartum uterine atony which cannot be managed with additional

Oxytocin and Myometrial Contractility in Labor 247

**Dose and administration** 

Clinically, atosiban, which requires continuous intravenous administration, is as effective as β2-adrenergic agonists, but without producing their adverse effects. Subcutaneously administered after a period of preterm labor, atosiban given as maintenance therapy was not shown to be associated with a reduction of the incidence of preterm birth nor with any improvement of neonatal outcome. In a study in which a total of 513 women were randomized to receive either atosiban or placebo administered with a subcutaneous infusion pump in order to prevent recurrence of preterm birth, atosiban compared to placebo did not reduce the incidence of preterm birth before 37 weeks (RR 0.89; 95% CI 0.71 to 1.12), 32 weeks (RR 0.85; 95% CI 0.47 to 1.55), or 28 weeks (RR 0.75; 95% CI 0.28 to 2.01). Outcomes were also similar for both groups with respect to birth weight, respiratory distress syndrome, patent ductus arteriosus, necrotizing enterocolitis, and intraventricular

In Europe and other countries atosiban is the only oxytocin/vasopressin antagonist used today for preterm delivery. However, this does not apply to the USA where the Food and Drug Administration has not granted approval of the drug as a tocolytic because of

Clinical studies have determined that atosiban is safer than beta-receptor agonists. A large study (Worldwide Atosiban versus Beta-agonists Study Group, 2001) demonstrated that atosiban was comparable in clinical effectiveness to conventional beta-agonist therapy (ritodrine, salbutamol or terbutaline), but was better tolerated and was associated with fewer maternal cardiovascular side effects (ClinicalTrials.gov, 2001). Atosiban is also safer than calcium channel blockers. Meanwhile, clinical studies have shown nifedipine to be equally effective as atosiban, although the maternal side effects were significantly more common among women allocated to nifedipine rather than atosiban (Al-Omari et al., 2006). Cyclooxygenase inhibitors act as tocolytics by inhibiting prostaglandins production but also present significant side effects (King et al., 2005). Conversely, evidence is as yet not strong enough for recommendation of the use of nitric oxide donors as inhibitors of preterm delivery (Duckitt and Thornton, 2002). Nevertheless, atosiban has not been proven to be superior in terms of neonatal

sufficient lack of evidence as to its efficacy and improvement of neonatal outcomes.

outcome, concerns having been expressed in other studies (Papatsonis et al., 2005).

Atosiban's limited bioavailability—which necessitates parenteral administration and hospitalization—together with its low affinity for OTR and the binding to V1a receptors that causes side effects, have led to endeavors for the identification of new peptide and nonpeptide oxytocin antagonists for the management of preterm labor. While many such substances have been discovered, these drugs are still being evaluated at the experimental level and clinical studies in most cases have ceased or have been completed unsuccessfully (Manning et al., 2008). These compounds are either peptide or non-peptide molecules.

Step 1 6.75 mg i.v. bolus, in one minute Step 2 18 mg/h i.v. i.v. infusion for 3 hours Step 3 6 mg/h i.v. i.v. infusion for up to 45 hours **Side effects:** Nausea, vomiting, hyperglycemia, headaches, dizziness, tachycardia, hot flushes,

hypotension, injection site reactions, pruritis, rush, pyrexia, insomnia

hemorrhage (Papatsonis et al., 2009).

Table 1. Dose, method of administration, and side effects of atosiban.

oxytocin infusion. However, oxytocin is normally secreted in pulses, this pulsatile secretion likely being a mechanism that prevents desensitization from occurring. This might explain why in women in labor, induction of labor requires significantly lower doses of oxytocin when oxytocin is administered in pulses, compared with continuous oxytocin infusion (Dawood, 1995).
