Oxytocin is a neurohypophyseal hormone containing nine amino acids. It is synthesized by neuron cell bodies in the paraventricular nuclei of the hypothalamus and then transported through the axons to the posterior pituitary were it is stored in the axon's terminal. It is then released in response to various physiological stimuli. Oxytocin for many years has been regarded as the hormone for pregnancy because of its involvement in parturition and lactation, however, it has various roles outside pregnancy especially in the central nervous system where it is involved in the control of human behavior (Lee et al, 2009). The only clinically available oxytocin antagonist is atosiban and is a mixed oxytocin/vasopressin v1a antagonist and is not suitable for long-term maintenance treatment as it is not orally bioavailable (Tsatsaris et al 2004). For this reason, a lot of research is dedicated to finding an oxytocin antagonist which is highly selective to OTRs and is orally biovailable in order to find a replacement for atosiban on the market. The aim of this experiment was to investigate the selectivity of the oxytocin antagonist [β-Mercapto-β, β-cyclopentamethylenepropionyl1,O-Me-Tyr2,Orn8] - oxytocin (OTA) on other receptors in rat tissue. The effects of acetylcholine (ACh), Serotonine (5-HT), angiotensin II (Ang II), angiotensin IV (Ang IV) and vasopressin were determined in the presence and absence of OTA. In order to determine the selectivity of OTA, its antagonistic effects were assessed on contractile responses induced by ACh, 5-HT, vasopressin and Ang IV in rat isolated colon and 5-HT, vasopressin and Ang II in rat uterus. The research showed that OTA did not have a significant effect on the contractions induced by Ang IV, Ang II, acetylcholine, 5-HT, and vasopressin receptors.
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Key words: oxytocin; oxytocin antagonism; β-Mercapto-β, β-cyclopentamethylenepropionyl1,O-Me-Tyr2,Orn8 - oxytocin.
Oxytocin(OT) is a neurohypophyseal hormone containing nine amino acids. Its sequence is Cysteine-Tyrosine-Isoleucine-Glutamine-Asparagine-Cysteine-Proline-Leucine-Glycine amide of which the cysteine residues form a sulphur bridge. The neuron cell bodies in the paraventricular nuclei of the hypothalamus are responsible for the synthesis of oxytocin. Once oxytocin is synthesised, it is transported through the axons to the posterior pituitary, where it is stored in the axons terminals (Seeley et al, 2003). It is then released from posterior pituitary in response to various physiological stimuli.
OT receptors are coupled to Gq/11α class GTP binding proteins, which together with GBγ stimulate the activity of phospholipase C-β isoforms. This then leads to the generation of inositol triphosphate and 1,2-diacyl-glycerol. The release of Ca2+ from the stores can be triggered by inositol triphosphate and diacylglycerol stimulates protein kinase C, which in turn phosphorylates unidentified target proteins.(Gimpl and Fahrenholz, 2001). Furthermore, a number of events are initiated in response to the increase in intracellular Ca2+ for example, the activation of neuronal and endothelial isoforms of nitric oxide (NO) synthase are triggered by the formation of Ca2+ calmodulin complexes. In turn, NO stimulates the soluble guanylate cyclase to produce cGMP. (Gimpl and Fahrenholz, 2001). According to Sanborn et al, 1998., in the smooth muscle cells, the Ca2+ calmodulin complexes have the ability to trigger the activation of myosin light-chain kinase activity which initiates smooth muscle contraction, e.g. in the myometrial or mammary myoepithelial cells.(Sanborn et al, 1998). Furthermore, the rising Ca2+ levels control cellular excitability, modulate their firing patterns, and lead to transmitter release in neurosecretory cells (Gimpl and Fahrenholz, 2001).
It has being suggested in recent years that oxytocin is expressed throughout the gastrointestinal tract and that it contributes to the control of the gastrointestinal tract. An experiment by Ohlson et al, 2006 set out to investigate this hypothesis. Oxytocin immunoreactivity was demonstrated throughout the gastrointestinal tract and was shown by the expression of mRNA. It was evident in the experiment that oxytocin was expressed, however, the experiment failed to show the presence of oxytocin receptors. Moisten et al 2004 gave an explanation which suggested that there may have been expressions at levels below the detection limit with the antibodies and methods used in the study by Ohlson et al.
Oxytocin receptors are also expressed in the brain. According to Febo et al 2009 and Li et al 1997, areas in the brain which contain OTRs include the ventralmedial nucleus of the hypothalamus, the amygdala, the lateral septum, the bed nucleus of the atria temnalis, the anterior olfactory nucleus, the preoptic and ventral tegmental areas and the hippocampus. Oxytocin receptors are also expressed in the stomach and this was shown by Qin et al, 2009. According to Gimpl and Fahrenholz, 2001, oxytocin shows many physiological roles including mammary and uterine smooth muscle contractions, neurotransmission in the central nervous system as well as autocrine and /or paracrine functions in the ovaries and testes.
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For many years, oxytocin has been regarded as the hormone for pregnancy. This is because it is involved in the stimulation, onset and progression of labour and also the stimulation of milk ejection. Furthermore, it is recognised as having a variety of roles outside pregnancy especially in the central nervous system where it is involved in the control of human behaviour (Lee et al,2009).
Clinically, oxytocin has being proven to induce labour in pregnant women. This is because when oxytocin binds to the oxytocin receptors (OTRs), which are more abundant during pregnancy, and are expressed in the myometrial cells in the mammalian uterus, it functions as a potent stimulant of the uterine contractions. The agonist oxytocin binds to the extracellular region, and the transmembrane domain of the receptor, which enables the intracellular part to couple to G-proteins and initiate a cascade of events, liberating Ca2+. This then causes smooth muscle contractions. (Zingg and Lapotte, 2003).
The initial findings of du Vigneaud in 1966 found that modification to position 1 (fig.1) of oxytocin by gedimethyl and consequently, by cyclic spiro substituent by Nestor et al, 1975 gave oxytocin antagonism. This raised an interest in OTR antagonist and since then, a lot of research has being dedicated to this field. Similar simple modifications of the cyclic peptide oxytocin have been done. Researchers such as Williams et al, 1996, Allan et al, 2006, Manning et al, 2008 and also Gimpl et al, 2008 made similar simple modifications such as introducing an ornithine at position 8 and capping of the 2-tyrosine hydroxyl group as a methyl or ethyl ether. Conformationally constrained bicyclic analogues were exploited as well as the introduction of non-natural and D-amino acids. The effort of the researchers led to the discovery of the most prominent oxytocin antagonist, atosiba
Figure 1: Structures of oxytocin and atosiban
Fig.1.Structure of agonist oxytocin (1) and antagonist atosiban (2).
Diagram from Journal of Medicinal Chemistry, 2010, vol.53.No.18
Atosiban is a synthesised cyclic nonapeptide that behaves as a competitive antagonist for oxytocin receptors. The oxytocin molecule has been modified in position 1, 2, 4, and 8, and as a consequence can inhibit the uterotonic action of oxytocin completely, competitively and dose dependently. It has also been shown to inhibit uterine contractions and delay preterm delivery (Tsatsaris et al. 2004). For this reason, it is intravenously administered clinically for the acute treatment of preterm labour.
Atosiban is a mixed oxytocin/vasopressin V1a antagonist and is not suitable for long-term maintenance treatment as it is not orally bioavailable (Tsatsaris et al. 2004). For this reason, a lot of research is now dedicated to finding an oxytocin antagonist which is highly selective to OTRs and is orally bioavailable in order to find a replacement for atosiban on the market.
The research is focusing on the selectivity of the antagonism of the oxytocin antagonist [β-Mercapto-β, β-cyclopentamethylenepropionyl1,O-Me-Tyr2,Orn8 ]- Oxytocin (OTA) on rat tissue. OTA is a potent oxytocin antagonist and will be used in conjunction with different receptor agonists such as oxytocin, vasopressin, ACh, 5-HT, histamine, Ang II and Ang IV, on rat tissue in both the presence and absence of OTA. In previous research conducted at the University of Brighton, the selectivity of OTA was tested by determining its antagonistic effects against the contractile effects of oxytocin, ang IV and acetylcholine in the rat isolated uterus. The research showed significant reduction in contractions induced by oxytocin 10-7 M in the presence of OTA 10-7M (p=0.002) however contractions induced by Ang IV and ACh at 10-6M were not significantly affected by the OTA 10-8 M nor 10-7M (Gard et al unpublished).
The research is looking at the selectivity of OTA in rat colon against the contractile responses of oxytocin, vasopressin, ACh, 5-HT, histamine, and Ang IV, and in isolated rat uterus against 5-HT, Ang II, histamine and vasopressin. The structures of oxytocin and vasopressin are very similar and there is often cross reactivity on their respective receptors. OTA is expected to have an effect on the contractile responses of vasopressin. However, with regards to the other agonist, OTA is not expected to have an effect.
Figure 2 : Below is the structure of the OTA used in the experiment.
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Fig.2. structure of β-Mercapto-β, β-cyclopentamethylenepropionyl1,O-Me-Tyr2,Orn8 -oxytocin
The effects of acetylcholine, 5-HT, histamine, ang II, ang IV and vasopressin were determined in the presence and absence of the selective oxytocin receptor antagonist [β-Mercapto-β,β-cyclopentamethylenepropionyl1,O-Me-Tyr2,Orn8 ]- oxytocin (OTA). To determine the selectivity of OTA, its antagonistic effects were accessed on contractile responses of acetylcholine, 5-HT, histamine, vasopressin, Ang II and ang IV in the rat isolated colon and 5-HT, histamine, vasopressin and Ang II in the rat isolated uterus.
2.2 Isolated smooth muscle contractility
The male and female rats were killed by cervical dislocation and their colon and uterus removed respectively post-mortem. The colon and uterus were then suspended in Krebs solution (KS) and De Jalon's solution respectively, normally at 32°C, gassed with 95% O2 and 5% CO2 under a resting tension of 1g. The isotonic contractile responses of ang II and ang IV, all at 10-9M to 10-5M, were recorded using a five minute dose cycle and 30 seconds contact time. With the rest of the agonist, an approximate of a two minute dose cycle was used with 30 second contact time and the tissue washed before waiting until it achieved its baseline and then adding another concentration. Three replications were made for each drug concentration in different tissues. The effects of OTA were determined by adding it to the
tissue a minute before the agonist was added.
The rat colon was divided into three separate tissues namely ascending, descending and transverse colon. The uterus was freed from its fat and cut horizontally so that it became a single piece of tissue rather than it being hollow tissue.
2.3 Animal husbandry
Male and female Sprague-Dawley rats with a mean weight of 150g were obtained from Harlan. They were fed a diet of standard laboratory chow. A 12 hour light-dark cycle was applied and lights came on at 07:00 hours.
2.4 Drugs and chemicals
Oxytocin, [β-Mercapto-β, β-cyclopentamethylenepropionyl1,O-Me-Tyr2,Orn8 ]-oxytocin were obtained from Sigma-Aldrich; ang IV, ang II and acetylcholine were obtained from Bachem.
2.5 Data analysis
The contractile responses of tissues were measured by an isometric transducer. The software used to record the measurements was PowerlabTM. The data was analysed in Graphpad PrismTM. The effects of the agonists on rat isolated tissue and uterine smooth muscle contractility were plotted as group means plus and minus the standard error of the means. The graph a single concentration of the agonist in the presence of OTA was plotted as the percentage of the initial contraction of the agonist in the presence of the lowest of the lowest dose of OTA
Effects of various agonists on rat isolated tissue and rat uterus in the presence and absence of OTA.
Fig.3 shows the dose response curve of ACh in rat colon. ACh caused contractions in rat colon as expected. Sigmoid curves were formed with logEC50 values of the line of best fit being -6.229 (95% CI -6.742 to -5.716), -6.887(95% CI -7.803 to -5.971) and -6.983 (95% CI -7.951 to -6.015). The curve of the descending colon is steeper than the transverse and ascending colon.
Fig.4 shows a dose response curve of Ang IV in three separate rat tissues. Ang IV caused contraction in all the tissues. LogEC50 values of line of best fit is -5.873 (95% CI -6.056 to -5.690) ascending colon, -5.924 in transverse colon and -5.902 (95% CI -6.686 to -5.119) descending colon. There is little difference in the shape of the graphs.
Fig. 5 shows a dose response curves of 5-HT in three rat tissues. 5-HT caused contraction in all three tissues. A sigmoid curve is formed with LogEC50 values of the line of best fit being -7.315 (95% CI -7.315 to -6.634), -6.018 (95% CI -6.576 to -5.460), and -7.413 (95% CI -8.385 to -6.440) respectively.
Fig.6 shows the dose response curve of angiotensin II in rat uterus. Ang II caused contractions in the rat uterus. A sigmoid curve is formed with Log EC50 values of line of best fit -7.434 (95% CI -10.75 to -4.118).
Fig.7 shows a dose response curve of 5-HT in rat uterus.5-HT caused contractions which formed a sigmoid curve with Log EC50 value of line of best fit -7.108 (95% CI -7.536 to -6.679).
Fig. 8 shows a dose response curve of vasopressin in rat uterus. Vasopressin caused contractions in the rat uterus. A sigmoid curve is formed with Log EC50 value of line of best fit been -7.115 (95% CI -7.568 to -6.663).
Fig.9 shows the effect acetylcholine 10-6M in the presence of increasing concentration of OTA. OTA did not have a significant effect (p>0.05) on the contractile effects caused by acetylcholine in both ascending and descending colon.
Fig.10 shows the contractile effects of 5-HT 10-6M, Vasopressin 10-6M and Ang II 10-7M in the presence of increasing concentration of OTA. OTA did not have a significant effect (p> 0.05) on the contractile effects of the agonists in the rat uterus.
Fig.11 shows the contractile effects of Ang IV 10-6M in ascending and descending colon in the presence of increasing concentration of OTA. OTA did not have a significant effect (p>0.05) in both ascending and descending colon.
Fig.12 shows the contractile effects of 5-HT 10-6M in increasing concentration of OTA. OTA did not have a significant effect (p>0.05) on the contractions caused by 5-HT 10-6M in both ascending and descending colon.
The aim of the experiment was to investigate how selective OTA is with respect to other receptors. Previous research conducted at the University of Brighton has shown OTA having antagonistic effects on the oxytocin receptor in the rat uterus (Gard et al, unpublished). By using a single concentration of the agonists of various receptors in the presence of increasing concentration of OTA, the research was able to determine the effects of OTA on the contractile responses caused by 5-HT, Ach, Ang IV in rat isolated colon and 5-HT, vasopressin and Ang II in the rat uterus. The results of the current study have shown that OTA does not have antagonising effects on receptors of ACh, Vasopressin, 5-HT, Ang II and Ang IV. In the previous research conducted at the University of Brighton, OTA was shown not to have antagonising effects on contractions induced by Ang II and Ang IV on the rat uterus (Murray, unpublished) and also not to have antagonising effects on contractions induced by ACh (Gard et al, unpublished) on the rat uterus. The current research further investigated the effects of OTA on contractions induced by ACh (figure 9) and Ang IV (figure 11) in the rat isolated colon. OTA was found not have antagonising effects on the contractions induced by ACh and Ang IV in the rat colon. The results are in inline with the previous research conducted. Interestingly, OTA seemed to potentiate the contractions induced by ang IV 10-6M in the ascending rat colon (figure 11). In one of the tissues, effect of contraction induced by Ang IV 10-6M more than doubled in the presence of OTA 10-6M. Further research can be done with an increased tissue sample to further investigate the effects of OTA on the ascending rat colon.
The initial stage of the experiment was to determine the effects of oxytocin, vasopressin, 5-HT, ACh, Ang IV and histamine in rat isolated colon and vasopressin, 5-HT, histamine and angiotensin II in the rat uterus. There is no significant difference in the distribution of the receptors of acetylcholine, 5-HT and angiotensin IV in the rat isolated colon. This is indicated by the 95% CI of the results in the ascending, descending and transverse colon as they overlap in the tissues. 5-HT was the most potent of the three agonists in the rat isolated uterus and Ang II was the most potent of the three agonists (Ang II, vasopressin and 5-HT) in the rat uterus.
There was a lot of variance in the data of the contractile response of acetylcholine, 5-HT and angiotensin IV in the rat isolated colon. The size/length of the tissue affects how much that tissue pulls in respect to its agonist. During the course of the experiment, the division of the rat colon into ascending, descending and transverse was hard to keep constant. It would be ideal in future to try and keep the length of the tissue constant. This would help reduce the error bars.
The research showed that OTA did not have antagonising effect on contractions induced by 5-HT, Ang II, Ang IV, vasopressin and ACh. Interestingly, OTA increased contractions induced by Ang IV 10-6M. Further research should be considered with a higher tissue sample on the effects of Ang IV 10-6M with increasing concentration of OTA.