Seminar assignments - The effects of antagonistic and agonistic substances on guinea pigileum PDF

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The effects of antagonistic and agonistic substances on Guinea Pig
Ileum
...

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Pharmacology 300884

Date: 11th October 2013

The effects of antagonistic and agonistic substances on Guinea Pig Ileum

Abstract:

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The experiment focused on drug administration to determine the effects of different antagonists on the contraction of a piece of guinea pig ileum using a range of agonistic substances. An understanding of the substances used were obtained through research before undertaking the simulations. From the information obtained through the research it was known that there are multiple receptor types within the ileum and that this would cause different antagonists to have varied effects on the contractility of the ileum based on the agonistic substance used. In this study the program “Pharmocological experiments on guinea pig ileum” was used to create the simulations of the actual experiment. Antagonistic substances were tested against agonistic substances to determine how they react with each other and to gain knowledge to be able to characterise the type of antagonist it is and the receptor types it associates with, if any at all. The simulation allowed for assumptions to be made that the procedure was carried out correctly, it also allowed for increases and decreases in drug dosage to observe the effects. This simulation gave an insight into the properties of the antagonists and the results were in line with what was expected of the substances from research findings.

Introduction:

Ex-vivo tissue preparation can be a very useful way to assess the effects of chemical and physical agents on specific parts of the anatomy. Because of its contractile properties, smooth muscle is used, it allows a straightforward measurement of the force it produces as an indication of effect. The ileum is a portion of the intestine that lies between the pyloric sphincter and colon. It consists of a tube of muscle and epithelial layers, innervated and vascularised by bundles of fibers (Vohora 2009). The contractility of the muscle layers is modulated by the nervous inputs in the myenteric plexus and within the myenteric plexus are ganglia linking pre and post ganglionic neurons, acetylcholine acts to transmit between them (Ehlert and Ansari 2001). There are three different types of receptors present in the guinea pig ileum, they are cholinergic receptors, serotonergic receptors and histaminergic receptors (Giraldo,Vigano and Hammer 2005). The different agonistic substances act on one or more of these receptors to produce a response. It is up to the antagonistic substance to inhibit the response caused by the agonist but they vary in their mechanisms of action. This study aims to investigate the pharmalogical effect and mechanism of action of antagonistic substances atropine, hexamethonium, eserine, mepyramine and papverine on the contractility of the guinea pig ileum using the agonistic substances acetylcholine, carbachol, metacholine, histamine and barium chloride.

Methods: 2

Materials The software “Pharmalogical Experiments on Isolated Guinea Pig Ileum” was used to simulate an experimental setup on an approximately two centimetre piece of guinea pig ileum. The guinea pig ileum was suspended in Tyrode’s solution in an organ bath. The experiment also required an isotonic lever connected to a transducer to record results as well as a water jacket to maintain the temperature of the organ bath. Antagonistic substances atropine, hexamethonium, eserine, mepyramine and papverine are used as tests opposed to agonistic substances acetylcholine, carbachol, metacholine, histamine and barium chloride. Setup The ileum was carefully suspended in an organ bath with the bottom end of the tissue fixed and the top end attached to the isotonic lever. As the isotonic lever is connected to a transducer, whatever movements in terms of contractility made by the tissue is converted to an electric signal. The signal then gets sent to a chart recorder which displays the signal as a trace. The organ bath needs to be maintained at 37 degrees Celsius which is achieved by the water jacket circulating water around it at this temperature. Procedure Tests were first carried out with the agonist without the presence of an antagonist to determine the ‘ceiling response’ of the agonistic substance. This was done by testing a minimal quantity of the agonist with the ileum and then increasing the dose until it reached its plateau. Once the ceiling dose became known the graph was reproduced using the submaximal dose of the agonist, antagonistic substances was then added to determine any change in response of the ileum contractions. The antagonist needed to be added to the organ bath and allowed to equilibrate for five minutes, the simulation allowed us to assume that this was done. The agonist was added using a three minute cycle which included thirty seconds exposure to the ileum then a wash out of the organ bath, it could also be assumed that the organ bath was adequately washed out.

Results: Atropine

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Hexamethonium

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Eserine

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Mepyramine

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Papaverine

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Discussion:

The goal was to determine the pharmacological effects and mechanism of action of a given set of antagonistic test substances on the guinea pig ileum. To cause a contraction in the ileum a range of agonistic substances were used. These substances included acetylcholine, a nicotinic and muscarinic agonist, carbachol, a muscarinic agonist, metacholine, a muscarinic agonist, histamine, a neurotransmitter which acts on H1 receptors in the gut to cause contraction and barium chloride, barium ions induce contraction of smooth muscle and cause some release of acetylcholine (Blackwood and Bolton 2003). The antagonistic substances tested were atropine, a muscarinic antagonist, hexamethonium, a ganglionic nicotinic agonist, eserine, a competitive acetyl cholinesterase inhibitor, mepyramine, a histamine H1 receptor antagonist and papverine, which acts via intracellular second messenger to cause muscle relaxation and non-competitively inhibit actions of stimulants (Vogel 2008). Atropine can be seen to limit the muscarinic actions of acetylcholine, carbachol and metacholine however it has no effect as an antagonist on histamine. It can also be seen that a higher dosage of atropine causes barium chloride to be antagonised. These findings allow for atropine to be classified as a competitive antagonist for muscarinic receptors and acetylcholine however it needs to be in high concentration to act on H1 receptors therefore showing no decrease in contraction in histamine (Angelo and Marcello 2008). Hexamethonium is known to be a ganglionic nicotinic antagonist that works by blocking nicotinic receptors (Eglen, Michel and Cornett 2001). In the tests performed it shows that hexamethonium played no role in antagonising any of the agonists. This allows hexamethonium to be classified as a non-competitive antagonist as it does not compete for any of the tested agonistic receptors. Eserine functions as a cholinesterase inhibitor (Unno, Kwon and Okamoto 2003). Its mechanism is to prevent the hydrolysis of acetylcholine which in turn enhances its effect. It is for this reason it can be classified that eserine has no antagonistic properties. The results show that there was no effect on any of the agonistic substances in terms of contractility decreased. Mepyramine is a histamine H1 receptor antagonist meaning it competes with a histamine receptor agonist for a binding site (Horio, Nakamura and Ishida 2000). It can be seen that mepyramine is most effective as an antagonist when it is paired with histamine however in larger doses it can also decrease the response of the muscarinic receptor agonists. This can be explained because although mepyramine does not have any effect on the acetylcholine molecules themselves it interferes with the binding of the agonists to the muscarinic receptors. Papverine is a known muscle relaxant. A high dosage was required to have an antagonistic effect on the contracting of the ileum caused by the agonists. This shows that it is not competing to bind at receptor sites but rather works as a non-competitive antagonist. It 17

primarily inhibits muscle contraction by inhibition of mitochondrial respiration (Shimizu, Yoshihara and Takahashi 2000).

In conclusion the results of the antagonistic substances effects on the guinea pig ileum using the agonistic substances have correlated directly with what was expected to occur based on the findings from the research carried out prior to the experiment.

References:

Angelo A, Marcello C 2008, The Role of Histamine H1, H2 and H3Receptors on Enteric Ascending Synaptic Transmission in the Guinea Pig Ileum, The Journal of Pharmacology, vol. 287 no. 3 pp. 952-957 Blackwood AM, Bolton TB 2003, Mechanism of carbachol-evoked contractions of guineapig ileal smooth muscle close to freezing point, Journal of Pharmacology and Chemotherapy, vol. 109 no. 4 pp. 1029-1037

Eglen RM, Michel AD, Cornett CM 2001, ‘The interaction of hexamethonium with muscarinic receptor subtypes in vitro’, British Journal of Pharmacology, vol. 98 pp. 499-506 Ehlert FJ, Ansari KZ 2001, Acetylcholine-induced desensitization of muscarinic contractile response in Guinea pig ileum is inhibited by pertussis toxin treatment, The Journal of Pharmacology and Experimental Therapeutics, vol. 299 no.3 pp. 1126-1132

Giraldo E, Vigano MA, Hammer R 2005, ‘Characterization of muscarinic receptors in guinea pig ileum longitudinal smooth muscle’, Molecular Pharmacology, vol. 33 pp. 617-625

Horio S, Nakamura S, Ishida Y 2000, Alterations in histamine receptors of guinea-pig ileal smooth muscle produced during agonist-induced desensitization, British Journal of Pharmacology’, vol. 101 pp. 587-590 Shimizu K, Yoshihara E, Takahashi M 2000, ‘Mechanism of relaxant response to papaverine on the smooth muscle of non-pregnant rat uterus’, Journal of smooth muscle research, vol. 36 pp. 83-91

Unno T, Kwon SC, Okamoto H 2003, ‘Receptor signaling mechanisms underlying muscarinic agonist-evoked contraction in guinea-pig ileal longitudinal smooth muscle’, British Journal of Pharmacology, vol. 139 no.2 pp. 337-350 18

Vogel HG 2008, Drug discovery and evaluation: Pharmacological Assays, Springer-Verlag, New York Vohora D 2009, The third Histamine Receptor, CRC Press, Florida

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