Functions of the oxytocin system

Discoveries of OXT-mediated functions have greatly expanded the spectrum of OXT action beyond its classic role as an inducer of uterine contractions and lactation (2). OXT plays a key role in certain kinds of behavioural regulation, such as social recognition, attachment and anxiety (18, 19). Differential pharmacological profiles of the OXTR in the brain and peripheral tissues are associated with the existence of OXTR subtypes (20, 21). Furthermore, the OXTR displays promiscuous coupling to Gq-, Gs- and Gi-protein isoforms forming heterotrimeric complexes, depending on the localization of the receptor within the plasma membrane. The variety of OXTR subtypes, coupling with different G-protein isoforms, results in the activation of multiple signalling pathways. Consequently, this leads to the induction of diverse physiological functions of OXT in different cell types (22, 23) (Figure 2).

Figure 2. Oxytocin receptor signalling pathways.

OXTR activation leads to three different G-protein dependant mechanisms. The major mechanism is mediated by the G^q-PLC-IP³ pathway. When oxytocin binds to the OXTR, it activates Gαq/11 and then phospholipase C (PLC), which induces the cleavage of phosphatidylinositol-4,5-bisphosphate (PIP₂) to inositoltrisphosphate (IP3) and diacylglycerol (DAG). IP3induces Ca²⁺release from Ca²⁺stores. The activation of G_qalso causes membrane depolarization, e. g. in neurones,which facilitates Ca²⁺entry through voltage-gated Ca²⁺channels. Increased cytosolic Ca²⁺ stimulates the Calmodulindependent protein kinase (CaMK) after binding to the Ca²⁺binding protein Calmodulin (CaM). The Ca²⁺/CaM complex then activates CaMK and causes various cellular responses. DAG causes protein kinase C (PKC) activation mediating trophic effects. Additional pathways activated through the OXTR include extracellular-signal regulated kinases (ERK) pathways. Proliferative effects involve ERK-mediated activation of specific gene transcription. Increased transcription of Cyclooxygenase-2 (COX-2) mediates increased production and secretion of prostaglandins, contributing to contractile effects.

OXTR coupling to G_s- and G_i-proteins is linked with the adenylat-cyclase pathway. Anti-proliferative effects observed in certain cell types appear to be mediated via G_i proteins. (Source: 5, modified)

Introduction 16

1.2.1 Oxytocin modulates proliferation

OXT is able to promote, to inhibit or to have no effects on proliferation of various cell types (24, 25, 26). The proliferative effects are altered by cholesterol depletion of membranes or changes in OXTR–caveolae interaction (27), resulting in contrary observations (28, 29) (Figure 3).

Mitogenic effects of OXT are associated with OXTR localization inside caveolae and its coupling to Gαq/11-proteins (30). The first step of the G_q-triggered signalling cascade involves activation of phospholipase C-β (PLC-β) leading to hydrolyzation of phosphatidylinositol-4,5-bisphosphate, producing inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 leads to an increase in intracellular calcium concentrations. This reaction is paralleled by activation of protein kinase C (PKC) through DAG. Finally, PKC induces transient ERK1/2 phosphorylation and thereby promotes proliferation.

Suppression of proliferation is associated with OXTRs located outside caveolae and their coupling to Gi-proteins (30). Upon OXT stimulation, two signalling pathways from this coupling are initiated depending on the cell system. The first cascade results in sustained ERK phosphorylation via PLC-βγ activation and subsequent induction of the cyclin dependant kinase inhibitor p21. The second cascade leads to an increase in cAMP and activation of protein kinase A.

Figure 3. Oxytocin-mediated cell proliferation depends on the cellular localization of its receptor.

(a) Inside caveolae, OXTRs couple to Gq-proteins upon OXT stimulation. Subsequently, Gq-proteins initiate a signalling cascade via phospholipase C-ß (PLCß), phosphatidylinositol-4,5-bisphosphate (PIP2), diacylglycerol (DAG) and protein kinase C (PKC). Finally, PKC activation induces transient ERK1/2 phosphorylation promoting proliferation. (b) Outside caveolae, OXTRs couple to Gi-proteins upon OXT stimulation. Depending on the cell system, two signalling pathways from this coupling are initiated. The first results in sustained ERK phosphorylation via PLCß activation and subsequent induction of the cyclin dependant kinase inhibitor p21. The second pathway leads to an increase in cAMP and activation of protein kinase A (PKA). Triggering of both pathways suppresses proliferation.

(Source: 30, modified)

Introduction 18

1.2.2 Oxytocin modulates neuroendocrine, inflammatory and

oxidative stress responses

A growing body of evidence suggests that oxytocin functions to attenuate stress responsiveness at different levels (31). The OXT-mediated anti-stress effects include inhibition of the hypothalamic-pituitary-adrenal (HPA) axis, as well as alleviation of inflammatory and oxidative stress.

The central response to physical or psychological stressors involves signalling along the HPA axis. Stress exposure leads to the secretion of corticotropin-releasing hormone (CRH) from hypothalamic neurons, targeting the anterior pituitary to release adrenocorticotropin (ACTH). Systemically circulating ACTH, in turn, triggers the release of corticosteroids, e. g.

cortisol, from the adrenal glands. Finally, cortisol participates in an inhibitory feedback loop by blocking the secretion of CRH. Intracerebral OXT has been shown to dampen basal and stress-induced activity of the HPA axis (32, 33). Noteworthy, studies demonstrated that OXT-induced effects on HPA axis activity can be contrarious depending on the length of time of OXT adminstration (34). Acute administration of oxytocin to rats is followed by a short-lasting increased HPA axis activity. In contrast, the long-term effects caused by repeated oxytocin administration are always consistent with a lowered HPA axis activity. However, the inhibitory effect of OXT on the HPA axis is mediated by reducing cortisol/corticosterone levels in response to acute exposure to stressors (35, 33). Human skin is a part of the local, as well as the systemic neuroendocrine network (36). For instance, all key mediators of the HPA axis, including the neuropeptides CRH, proopiomelanocortin-derived ACTH and cortisol/corticosterone, are synthesized and released by skin cells (36-38). In view of possible cross-talks, ACTH and CRH receptors are also located on dermal fibroblasts and keratinocytes (37, 39-41).

Some of the factors that trigger and modulate HPA axis activity are cytokines (42). How closely the OXT system is integrated in neuroendocrine stress responses and immunological networks demonstrates the fact that OXTR expression is also regulated by cytokines.

Treatment with both IL1b and IL6 negatively regulates the transcription of the OXTR gene in cultured human immortalized myometrial cells (43). Furthermore, wound-healing, which involves a well-organized inflammatory phase, has been shown to be facilitated by OXT administration (44, 45). Moreover, in a rat model of dried-latex-induced paw oedema, OXT treatment showed anti-inflammatory and anti-nociceptive activity, thus pointing to a role of OXT in skin inflammation (46).

Inflammation is often accompanied by oxidative stress and vice versa (47). Increased levels of intracellular reactive oxygen species (ROS) can damage macromolecules, leading to lipid peroxidation, oxidation of amino acid side chains, formation of protein-protein cross-links, protein fragmentation, DNA damage and DNA strand breaks (48). Beside ROS generation by environmental stresses, free radicals are naturally generated in the organism. In mitochondria, ROS are produced as a byproduct of cell respiration (49) and play a role in cell signalling (50). In addition, ROS are generated during inflammation by immune cells, such as macrophages, to destroy pathogens. In turn, oxidative stress is able to drive inflammation by activation of transcription factors, such as NF-κB which regulates inflammatory mediator gene expression (51). Small amounts of ROS are usually scavenged by cellular defense systems including nonenzymatic and enzymatic antioxidants. The antioxidant glutathione (GSH) is a tripeptide consisting of glycine, cysteine and glutamate. GSH exists in a reduced (GSH) and an oxidized (GSSG) form. It represents the predominant low molecular weight thiol in mammalian cells, with its intracellular concentrations ranging from 0.2 to 10 mM (52).

85 to 90 % of GSH is freely distributed in the cytosol, but it is also found in mitochondria, peroxisomes, the nuclear matrix and the endoplasmic reticulum.

Beside inflammatory stress, OXT seems to attenuate also oxidative stress. OXT does not only decrease IL6 secretion, but also NADPH-dependent superoxide production in vascular cells and THP-1 macrophages in an in vitro model (53). In addition, subcutaneously administered OXT has been demonstrated to prevent sepsis-induced depletion of GSH contents in colonic and uterine tissues of rats (54). Oxidative stress occurs rapidly after the onset of ischemia. Subcutaneously injected OXT appears to improve renal ischemia-reperfusion induced lipid peroxidation in liver tissue of rats (55). In the same rat model, a reduction of nitrogen monoxide during ischemia which was supposed to be caused by endothelial dysfunction and diminished endothelial nitric oxide synthase activity, was measured. OXT treatment increased nitrogen monoxide levels in the hepatic tissue and thereby enhanced the cytoprotective effects of nitrogen monoxide, like inhibition of oxidative stress, cytokine release, apoptosis, adhesion and aggregation of neutrophil leukocytes. In mice, after induction of experimental stroke, exogenous OXT treatment increased antioxidant activity through elevation of brain GSH peroxidase levels (56). Additionally, OXT itself is an anti-oxidant in aqueous medium (57). OXT is able to scavenge free superoxide and reactive nitrogen species, exhibiting the capacity to directly suppress free radical-mediated damage of cell components (57). The antioxidant activity of OXT is supposed to derive from solvent-exposed tyrosine and tryptophan residues (57). In vitro, significant antioxidant effects can be observed at nanomolar concentrations of OXT. Notably, the potency of this hormone is comparable with classic low molecular weight anti-oxidants (57). Altogether, OXT appears to contribute to the organism’s defense system against oxidative stress.

Introduction 20