The neurohypophyseal nonapeptides OT (oxytocin) and AVP (arginine vasopressin) play an important role in many physiological functions through GPCR (G-protein-coupled-receptor) signal transduction. In humans and other mammalian species, OT and AVP target the OTR (oxytocin receptor) and the three vasopressin receptors V1aR, V1bR and V2R. Centrally, it functions as a neurotransmitter where it is involved in complex social behaviour, maternal care, stress and anxiety. Peripherally, OT acts as a hormone that triggers uterine smooth muscle, mammary glands, testis, prostate glands, kidney, thymus, pancreas and adipocytes. The heart, especially the right atrium is also one of the main sources of OT.
(Insights into the molecular evolution of oxytocin receptor ligand binding, 2013 )
OT is a hormone acting on classical endocrine, paracrine and autocrine ways. It regulates basic cardiovascular functions, for instance, cardiomyocytes survival, tissue regeneration, stem cells differentiation, hypertrophy, glucose uptake potentiation, negative inotrophic and chronotrophic effects, anti-inflammatory and pro-inflammatory cytokines balance, hydromineral balance by ANP release, vasodilatation and endothelial cell growth via NO pathway, electrical activity control and vascular reactivity.
Ischemic heart disease (IHD) is among the most important and top ranked causes of death in the world, and its prevention and management are actively being investigated. Based on the researches in the recent decade, reperfusion or re-establishing blood flow into the ischemic myocardium can limit the infarction size and reduce adverse sequels. However, reperfusion itself causes striking damages, due to oxygen radicals production, calcium ions flux e neutrophils infiltration. Preconditioning and postconditioning represent potent and reproducible methods to render the myocardium more resistant against irreversible structural and functional damage but still related injuries remain the head of mortality and morbidity. Thus, numerous studies focused on OT’s protective effects against ischemia-reperfusion induced tissue damage, proposing it as a novel cardioprotective agent.
(Is oxytocin a therapeutic factor for ischemic heart disease?, 2013 )
The encoded receptor is a 389-amino acid polypeptide with 7 transmembrane domains and belongs to the class I G protein-coupled receptor (GPCR) family.
The human OT receptor mRNAs were found to be of two sizes, 3.6 kb in breast and 4.4 kb in ovary, endometrium, and myometrium. The OT receptor gene is present in single copy in the human genome and was mapped to the gene locus 3p25–3p26.2. The gene spans 17 kb and contains 3 introns and 4 exons:
* Exons 1 and 2 correspond to the 5′-prime noncoding region
* Exons 3 and 4 encode the amino acids of the OT receptor
* Intron 3, which is the largest at 12 kb, separates the coding region immediately after the putative transmembrane domain 6
* Exon 4 contains the sequence encoding the seventh transmembrane domain, the COOH terminus, and the entire 3′-noncoding region, including the polyadenylation signals
OT receptors are functionally coupled to Gq/11α class GTP binding proteins that stimulate together with Gβγ the activity of phospholipase C-β isoforms. This leads to the generation of inositol trisphosphate and 1,2-diacylglycerol. Inositol trisphosphate triggers Ca2+release from intracellular stores, whereas diacylglycerol stimulates protein kinase C, which phosphorylates unidentified target proteins. Finally, in response to an increase of intracellular [Ca2+], a variety of cellular events are initiated. For example, the forming Ca2+-calmodulin complexes trigger activation of neuronal and endothelial isoforms of nitric oxide (NO) synthase. NO in turn stimulates the soluble guanylate cyclase to produce cGMP. In smooth muscle cells, the Ca2+-calmodulin system triggers the activation of myosin light-chain kinase activity which initiates smooth muscle contraction, e.g., in myometrial or mammary myoepithelial cells. In neurosecretory cells, rising Ca2+ levels control cellular excitability, modulate their firing patterns, and lead to transmitter release. Further Ca2+-promoted processes include gene transcription and protein synthesis.
In the heart, the activation of OT receptors and subsequent elevation of intracellular [Ca2+], stimulate exocytosis and atrial natriuretic peptide (ANP) secretion. ANP then exerts a negative chrono- and inotropic effect via activation of guanylyl cyclase and release of cGMP. A rapid reduction in the effective circulating blood volume is produced by an acute reduction in cardiac output, coupled with ANP's peripheral vasodilating actions. The ANP released would also act on the kidneys to cause natriuresis, and ANP acts within the brain to inhibit water and salt intake, leading to a gradual recovery of circulating blood volume to normal.
(The oxytocin receptor system: structure, function, and regulation, 2001 )
The residues Arg34,Phe103, Tyr209 and Phe284 were important for ligand binding and selectivity at the human OTR. Additionally, Asp85 and Lys270 are involved in receptor signalling. Interestingly, all OT and OT-related receptors share the Asp85 residue in the TM (transmembrane) domain 2 and the Lys270 residue in ICL (intracellular loop) 3, and these residues were reported to be important for receptor activation. Particularly, the Asp85 is also conserved in other class A GPCRs and is thought to play a more general role in receptor–G-protein signalling. The N-terminal Arg34 is highly conserved, indicating its importance for ligand binding. Phe103 was demonstrated to be important for ligand selectivity in the OTR and is thought to interact with the residue at position 8 of the
peptide ligand. OT receptors with a phenylalanine in that position generally bind native ligands that have either a leucine or an isoleucine residue at position 8. Tyr209 and Phe284, located in the TM region are two other important residues for ligand–receptor binding. In the human receptor, Tyr209 and Phe284 interact with residues at positions 2 and 3 of OT. Tyr209 is highly conserved among all OT-like receptors, being a tyrosine in most sequences or another aromatic residue. All native peptide ligands contain an aromatic residue (tyrosine or phenylalanine) in position 2 and a hydrophobic or aromatic residue in position 3 (isoleucine, phenylalanine, valine or tryptophan) indicating that this ligand–receptor interaction may indeed be conserved throughout evolution. The importance of N-terminal hydrophobicity of the ligands is further supported by the identification of the superagonist desamino-OT, where the deletion of the N-terminal amine led to a more hydrophobic and potent OT analogue.
Sequence logos of short sections of aligned receptors in key positions that are involved in ligand recognition, binding and receptor functionality for the human OTR as well as residues found in contact with the ligand, obtained from the GPCR ligand co-crystal after structural super-positioning, are shown. The sections (solid boxes) include the N-terminal residue Arg34 (a), Asp85 in TM1 (b) in TM2 ©, Phe103 in the ECL (extracellular loop) 1 (d), TM3 (e), ECL2 (f), Tyr209 in TM5 (g), Lys270 in ICL3 (h), Phe284 in TM6 (i) and TM7 (j). Numbering of residues is based on the human OTR sequence. Residue positions of importance are highlighted by grey boxes and OT receptor residues are highlighted in red. Common GPCR–ligand interacting residues are labelled with an asterisk and residues that correlate with the respective ligand sequence are coloured in blue.
Ligands are recognized by the extracellular region of the receptor and interact with residues in the three-dimensional environment of the receptor TM domain. To identify whether and which residues are oriented towards the binding pocket and interact with the ligand, an homology model of the human OTR on the template of the mouse μ-opioid receptor crystal structure was developed by Koehbach et al. With this approach, they define a common binding site, deep within the vestibule of the receptor structure that is shared by all GPCR ligands (agonists and antagonists). The positions with the highest frequency of direct contact with ligands include Gln119 and Met123 in helix 3, Ile204 and Val208 in helix 5, Trp288, Phe291, Phe292 and Gln295 in helix 6 and residue Met315 in helix 7. The α-carbon atoms of all these residues are within 16 A° (1 A°=0.1 nm) from each other. The side chains are all orientated towards the ligand-binding vestibule and many of them are in direct contact with each other. Residues at the rim of the vestibule displayed lower interaction frequency and it appears that this strongly depends on the size and orientation of the ligand.
(Insights into the molecular evolution of oxytocin receptor ligand binding )
Oxytocin has antioxidant and anti-inflammatory effects on vascular cells and macrophages. OT receptors were identified in this cell types, and OT decreased NADPH-dependent superoxide production and IL-6 secretion in these cells. A possible explanation for the decreased NADPH oxidase activity was that OT was acting as a superoxide scavenger. Different studies have shown that OT
scavenged free radicals and prevented lipid peroxidation. The putative anti-inflammatory effects of OT was assessed examinating IL-6 secretion in oxytocin-treated macrophages and endothelial cells. OT decreased lipopolysaccharides stimulated IL-6 secretion from macrophages by 56% and endothelial cells by 26%, suggesting an attenuation of inflammatory processes in these cells.
(Oxytocin attenuates NADPH-dependent superoxide activity and IL-6 secretion in macrophages and vascular cells, 2008 )
Cardiac OT is involved in the regulation of myocardial glucose uptake by hypoxia in physiological conditions such as stress, acting via phosphoinositide-3-kinase(PI3K) pathways. Calcium-calmodulin kinase and AMP-activated protein kinase (AMPK) pathways are also involved in OT-mediated glucose uptake in cardiomyocytes. This can improve cell survival and consequently heart performance.
OT is a direct and an indirect negative chronotropic agent, with protective functions on I/R-induced myocardial injury. As indirect agent, stimulates atrial natriuretic peptide (ANP) release, that acts via OT receptor to activate guanylyl cyclase and cGMP release. cGMP, in turn, induces negative chronotropic and inotropic effects. Direct effects of exogenous OT can led to natriuresis and drop mean arterial pressure. Besides, in the vascular bed, OT stimulates ANP and subsequent NO release followed by vasorelaxation. Transient opening of mPTP (a multiprotein complex comprised of voltage-dependent anion channel whose opening cause inner membrane potential reduction), mitochondrial
depolarization, ATP loss and massive production of ROS precede apoptosis and necrosis phenomena during cardiac I/R. OT-induced hemodynamic stability and anti-stunning effects may mediate through ROS inhibition and mPTP closure.Also intrinsic OT may play an important physiological role in regulating
vascular tone, as well as cardiac function. It is synthesized and released by heart as well as large vessels, with the peak concentration in the right atrium. After its release, OT acts on its cardiac receptors and decreases heart rate and contractility. It also has a potent well known anti-stress and
subsequent supportive cardiovascular effects. Acute stress has recently been shown to cause myocardial I/R damage which is reversed by cardiac OT receptors (Fig. 1).
(The Oxytocin Receptor System: Structure, Function, and Regulation, 2001 )
NITRIC OXIDE (NO) PROTECTION
Myocardial cells are one important sources of NO production. NO has direct and indirect regulatory cardiac functions: it directly acts on excitation–contraction coupling, autonomic signaling modulation of myocytes and mitochondrial respiration. The indirect effects include coronary vessel tone regulation, thrombogenicity, angiogenesis, proliferative, and inflammatory properties. OT is known to stimulate NO release during I/R in rats (Fig. 1).
OXYTOCIN AND SOCIAL SUPPORT
OT plays a central role in the regulation of stress-related processes. For example, central OT administration reduces stress-induced corticosterone release and anxiety behavior in rats. OT treatment decreases anxiety and the neuroendocrine response to stress in social interactions, whereas treatment with an OT receptor antagonist heightens stress and anxiety related behaviors.
AVP, instead, seems to play an anxiogenic role and is essential for coordinating the behavioral and metabolic responses to stress.Experiments of chronic isolation on animal models showed decreased OTR mRNAs level in heart and elevated plasma OT level. This results suggest that exposure to a chronic stressor may alter OT production or possibly gene transcription, specially in female, which have a more sensitive response to effects of isolation. This mechanism may protect females somewhat from the negative consequences of isolation.The down-regulation of OTR mRNA expression in the hypothalamus and the heart following chronic isolation may be a response to prolonged elevation of circulating OT.
(Exposure to chronic isolation modulates receptors mRNAs for oxytocin and vasopressin in the hypothalamus and heart, 2013 )
Though the amino acid sequence is rather short, its first complete synthesis more than 55 years ago was a struggle of many years because of difficulties, in the first place, in elucidating the structure, in degradation and in obtaining a sufficient amount of purified material. Oxytocin has a narrow therapeutic window (i.e., the drug dosage which is effective is restricted) and is eliminated from the circulatory system within minutes. Therefore, the most precise and reliable mode of delivering OT is through infusing it directly into the blood. Administered orally, the nonapeptide can be destroyed by proteolytic enzymes in the gastrointestinal tract. The most common therapeutic application is the stimulation of maternal labor. Possible administration routes include intraveinous infusion or intranasal/sublingual administration (Oxytocin spray ).
In terms of therapeutic consequences, the diversity of OTR signaling is extremely challenging. The coupling of OTR to different G proteins exhibiting opposite effects renders the definition of “agonist” and “antagonist” rather questionable. All OTR ligands have putatively the potential to stimulate dual signaling responses. Therefore, an “agonist” or “antagonist” can only be defined relative to the cellular context (e.g., cell type, stage of development, phosphorylation level, receptor subtype expression/trafficking level). The ability to design compounds that can discriminate between many diverse pathways and predominantly activate a specific intracellular reaction will be at the heart of future OTR-based drug strategies.
(Oxytocin: Crossing the Bridge between Basic Science and Pharmacotherapy, 2013 )
Knowledge of the effects of isolation, especially chronic isolation, on endogenous OT and AVP systems, including receptors, may aid in understanding the mechanisms through which isolation is detrimental, or conversely social support is protective, against disorders neural and cardiovascular systems, that may be characterized by excessive or inappropriate emotional and autonomic reactivity. Different studies demonstrated that stable social environment, characterized by increased affiliative social behavior,slowed the progression of cardiovascular injuries in animal models. In addition, social environment differentially modulated inflammatory and oxidative stress mechanisms associated with atherosclerosis progression. Given the extensive literature that relates social behavior to oxytocin, emerge that elevations in peripheral or local oxytocin level as a function of social environment could work directly on vascular and cardiac cells to slow or prevent the progression of cardiovascular damages.
Image 1 from Is oxytocin a therapeutic factor for ischemic heart disease?, 2013