Effect Of Carbachol And Atropine On Rat Ileum

Abstract

Previous research has found that cholinergic receptors cause contraction of smooth muscle in the ileum, upon stimulation from acetylcholine and its agonists. This response has also been shown to be partially blocked by antagonists such as atropine. In this study a dose response curve was constructed to determine the potency of carbochol (agonist). Carbachols contractile effect on isolated rat ileum, when atropine was introduced (antagonist) was also investigated. An isolated tissue bath was constructed and the tension along a longitudinal section of rat ileum was measured to determine the contractile effect of carbachol and atropine. The potency of carbachol varied slightly from previous studies with a value of 2.78x10-6, however atropine was shown to partially block cholinergic receptors causing a 40.8% reduction in response, which was also shown to be recoverable. Therefore, atropine reduces carbachols effect to contract the smooth muscle in ileum tissue.

Introduction

Contraction of smooth muscle present in the gastrointestinal tract is mediated by acetylcholine and its derivatives, which act on M2 and M3 muscarinic receptors, of which can be found in many species including the Ileum of a rat (Bolton, T.B. & Lim, S.P. 1991). Contraction of the tissue arises as the M3 receptor is coupled to the GQ protein, upon stimulation it activates phospholipase c, initiating the production of inositol triphosphate (IP3) and diacylglycerol (DAG). This leads to the release of Ca2+ into the cell via intracellular stores and the latter uses protein kinase C to act on Ca2+ sensitive ion channels. M2 receptors result in the inhibition of adenylate cyclase ultimately also leading to a rise in Ca2+ within the cell, triggering a contraction (Unno, T., et al. 2005)

Muscarinic receptors can be activated or inhibited using a variety of synthetic drug molecules. Carbochol is an acetylcholine agonist, it is classed as a parasympathomimetic that stimulates both muscarinic and nicotinic receptors. Whilst, Atropine is a synthetic form an alkaloid derived from the plant Atropa Belladonna and acts as an anti-parasympathetic, competitive antagonist on muscarinic receptors (PubChem. Accessed 16,2020).

In this study an agonist was used to simulate the effects of the natural ligand acetylcholine. An agonist is a molecule that binds to a receptor and stimulates a response, the extent of this interaction can be defined as the drugs affinity. However, to elicit a response the ligand must also have efficacy, this can be defined as the “response produced when a specified number of receptors are occupied”. Efficacy can also be described in terms of the potency of the drug, this is the “activity of a drug at a particular concentration”, often this is defined further as the EC50 which is “the concentration at which 50% of the receptors are occupied by the ligand, producing 50% of the maximum response” (Neubig, R.R., et al. 2003).Therefore, the aim of this study was to establish the potency of the agonist carbochol on an isolated section of rat ileum. Further to this, the experiment also aimed to investigate the ability of carbochol to contract rat ileum that had been exposed to the antagonist atropine.

Method

Lab chart and data pad were used to record the results. The latter was set at 4mm/sec with a voltage of 1mV. An organ bath was set up using 50 ml of Krebs-Henseleit physiological saline with a constant stream of bubbles supplied. A 2cm section of rat ileum was suspended in the bath connected to an isometric transducer, calibrated using a 0.5g weight. 0.1ml of the carbochol dilutions were administered to the bath consecutively once the response plateaued; until there was no further increase in response. The bath concentrations used were as follows 1x10-8, 3x10-8, 5x10-8 this was repeated, by increasing each concentration by a power of ten each time. The last concentration was 3x10-4. Once the maximum response was obtained the tissue was rinsed several times to allow recovery. 3 separate doses of the EC70 for carbachol were applied, washing the tissue in between each repeat to find the standard concentration. 0.1mL of 1x10-8 atropine was added to the bath and left for approximately 10 minutes, after, the standard concentration of carbachol was added without washing. Recovery was tested by rinsing then applying the standard concentration using the same method that was used to determine this concentration. Final bath concentrations were converted into log values and a %maximum response was obtained for each concentration.

Results

The effect of an acetylcholine agonist (carbachol) and antagonist (atropine) on the ability of a longitudinal section of rat ileum to contract.

Log dose response curve for carbochol showing a graded response in isolated rat ileum.

The contractile effect of carbochol on isolated rat ileum is demonstrated via a concentration response curve. Percentage maximum response is plotted against the Log of each carbochol concentration. Each point is a percentage of the maximum response produced by the agonist. Increasing concentrations of carbachol resulted in a greater response in the tissue (greater tension generated) until a maximum was reached, this is reflected in the sigmoidal shape of the graph. The derived EC50 of carbochol was 2.78x10-6 and an EC70 value of 8.2x10-6 was also obtained.

Atropine supresses isolated rat ileums response to carbachol. Values for the first and third bar are recorded as mean ± standard error (SEM) (Standard = 0.74±0.07, Recovery=0.85±0.01). The first bar shows the contractility produced by the standard concentration of carbachol before the introduction of atropine. The second bar represents the response of the tissue after the antagonist was introduced and the third bar shows the recovery of the tissues after the removal of the antagonist.

In the presence of the antagonist atropine, the response to the standard concentration of carbochol showed a decline. The percentage reduction in response was 40.84%. The removal of the antagonist showed a recovery of the tissue, this was signified by an increase in grams of tension generated. The average percentage recovery for the tissue was 115%.

Discussion

This investigation explored the contractile effect of carbachol and atropine on rat ileum. The acetylcholine agonist carbochol elicited a contractile response in the isolated rat ileum, the calibration curve suggested that the EC50 (potency) of the agonist was 2.87x10-6. This value is considerably higher than that found in previous studies with (Morales, M.A. et al., 2013) reporting a value of 1.15x10-9 and 2.44x10-7 was found by (Glaza, I. et al. 2011). However, this could be due to age of the rat used, as there is evidence to suggest that the sensitivity of muscarinic acetylcholine receptors may decline with age (Lopes, G.S., et al. 2007). The introduction of atropine (antagonist) caused a 40.84% decline in the response of the tissue compared to the maximum response produced by the standard concentration, suggesting atropine disrupts and partially blocks the contractile effect carbochol has on the smooth muscle of rat ileum. The near 50% decline in the contractile response of the rat ileum suggest that atropine is a competitive, reversible antagonist. This can be concluded because the average response in the presence of the standard concentration of carbochol after the removal of the antagonist was 115% suggesting that there was a full recovery of the muscarinic receptors in the tissue. This is to be expected as the effect of a reversible antagonists can be counteracted by increasing the agonist concentration or in the case of this study washing the tissue, which results in the removal of the antagonist from the binding site.

Upon reproduction of the study, a number of improvements could be implemented as fig. 2. showed the results did vary somewhat. The first one being to repeat the administration of each drug concentration several times, as the response of the rat ileum is likely to vary due to biological variation. Furthermore, despite undertaking repeats for the response to the standard concentration and the recovery of the tissue after the antagonist was removed, each individual experiment could be reproduced independently using fresh stock solutions, to ensure the drug is at its optimum condition. The drug’s effectiveness will decline with time which may impact its effect on the tissue. Furthermore, the experiment could be reproduced with other types/species/ages of animal to examine the response of cholinergic receptors in these organisms. Combining these alterations in the method would help to achieve more reliable results that better account for variation. Furthermore, the construction of a Schild plot would be beneficial for this investigation as it would all the pA2 of atropine to be determined. This is the “concentration of the antagonist (atropine) that is needed to create a 2-fold increase in the agonist (carbochol) which would abolish the effect of the antagonist” and recover the response produced before the introduction of it (atropine) (Neubig, R.R., et al. 2003).

In conclusion, the experiment aimed to determine the potency of the cholinergic agonist carbachol and explore the contractile effect that introducing the antagonist atropine would have on a longitudinal section of isolated rat ileum. Carbochol was shown to produce a contractile response in isolated rat ileum, with an EC50 value being obtained. Atropine was shown to disrupt the contractile effect of carbochol, by partially blocking the muscarinic receptors, therefore decreasing its efficacy and preventing it from producing a maximal response in the ileum tissue. However, it should be noted that these results may lack reliability due to the time constraints of the study.

Bibliography

1. Bolton, T.B. and Lim, S.P., 1991. Action of acetylcholine on smooth muscle. Zeitschrift fur Kardiologie, 80, pp.73-77.
2. GlazaBCDE, I., Szadujkis-SzadurskiAC, L., Szadujkis-SzadurskiC, R., GajdusBD, M. and OlkowskaF, J., 2011. Modulating activity of M. Postepy Hig Med Dosw (Online), 65, pp.478-481.
3. Gokhale, S.D., Gulati, O.D. and Panchal, D.I., 1967. An analysis of the sympathomimetic effects of acetylcholine of the rat ileum. British journal of pharmacology and chemotherapy, 30(1), p.35.
4. Lopes, G.S., Smaili, S.S., Neto, A.C., Vladimirova, I., Jurkiewicz, A. and Jurkiewicz, N.H., 2007. Aging-induced decrease of cholinergic response and calcium sensitivity on rat jejunum contractions. The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, 62(3), pp.264-270.
5. Morales, M.A., Ahumada, F., Castillo, E., Burgos, R., Christen, P., Bustos, V. and Muñoz, O., 2013. Inhibition of cholinergic contractions of rat ileum by tropane-type alkaloids present in Schizanthus hookeri. Zeitschrift für Naturforschung C, 68(5-6), pp.203-209.
6. National Center for Biotechnology Information. PubChem Database. Carbachol, CID=5831, https://pubchem.ncbi.nlm.nih.gov/compound/Carbachol (accessed on Apr. 16, 2020)
7. National Center for Biotechnology Information. PubChem Database. Atropine, CID=174174, https://pubchem.ncbi.nlm.nih.gov/compound/Atropine (accessed on Apr. 28, 2020)
8. Neubig, R.R., Spedding, M., Kenakin, T. and Christopoulos, A., 2003. International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. XXXVIII. Update on terms and symbols in quantitative pharmacology. Pharmacological reviews, 55(4), pp.597-606.
9. Unno, T., Matsuyama, H., Sakamoto, T., Uchiyama, M., Izumi, Y., Okamoto, H., Yamada, M., Wess, J. and Komori, S., 2005. M2 and M3 muscarinic receptor‐mediated contractions in longitudinal smooth muscle of the ileum studied with receptor knockout mice. British journal of pharmacology, 146(1), pp.98-108.