Pharmacology an antagonist disallows a receptor’s wild-type

Pharmacology Laboratory Report on:
The muscular effect of acetylcholine, carbachol and butyrylcholine on isolated rat ileum tissue.
Module number: BMS242
Student Registration number: 170158804
Name: Adrianos Boutsias
Abstract word number: 247
Remainder report word number: 1,439
Abstract
Neurotransmitters released by postganglionic neurones regulate smooth muscle activity in the gastrointestinal (GI) tract. An agonist is a substance, that, when bound to a receptor, initiates a physiological response, while an antagonist disallows a receptor’s wild-type function. The objectives of this study were to answer two questions: first, how does smooth muscle respond under influence from several neurotransmitters? and second, how quickly does the tissue contract under exposure to said neurotransmitters? In order to tackle these questions, acetylcholine, butyrylcholine and carbachol stocks were retrieved, diluted serially and pipetted into an organ bath, where their total bath concentrations were calculated: dose(g)molecular weight*1volume of bath. In the bath, isolated rat ileum, immersed in 20ml Krebs, was connected to a force transducer-micropositioner apparatus via a metal hook, allowing for a response (g) to be measured when varying concentrations of drugs were added. By this, conclusions on which drug was a full or partial agonist/antagonist were derived, once a drug’s maximal plateau was observed. Thus, a logarithmic graph was drawn, where the EC50 for every drug was calculated. The EC50 is the concentration at which half-maximal response is witnessed; here, acetylcholine which displayed the least drastic increase in response compared to increases in drug concentration, had the largest EC50 value, followed by carbachol and finally butyrylcholine. Acetylcholine, which produced the largest response force (0.23g) was concluded as a full agonist, butyrylcholine was a partial agonist, while carbachol was an antagonist, due to all % responses above 10 µg/µl carbachol being negative.

Introduction
The field of research into the enteric nervous system (ENS) is an ever evolving and multidisciplinary one. Given that a single neuron may possess the ability to synthesise and release multiple neurotransmitters (Boron, W., F. and Boulpaep, E., L. 2012), this gives rise to the intricacy and complexity behind this fascinating biological system. However, within the ENS there is further complexity: besides simply the ability for the ENS to produce and metabolise neurotransmitters such as acetylcholine, non-neuronal acetylcholine machinery exists in epithelial cells found in both the large and small intestine (Takahashi, T., et al. 2018). As a result, an abundance of disorders are linked to the ENS, with the most well-researched congenital disease being Hirschsprung disease (Lake, J., L. and Heuckeroth R., O. 2013).
The aims of this experiment are to derive whether acetylcholine, butyrylcholine and carbachol are either partial or full agonists or otherwise via eliciting a response (g) in isolated rat ileum and to calculate the EC50 of each. The EC50 is the concentration (M) of a drug that causes a half-maximal response (Jiang, X. and Kopp-Schneider, A. 2014). In this case, the response was in the form of tension felt on a force-transducer connected to a micropositioner due to the contraction reflex of the rat ileum. The hypothesis was that acetylcholine would be a full agonist, while butyrylcholine and carbachol would be partial agonists and that the EC50 of acetylcholine would be highest, followed by carbachol and then butyrylcholine.

This work seeks to understand more on the mechanism behind the interaction between the enteric nervous system and the effector intestinal smooth muscle. By this, a standard on how all neurotransmitters affect the GI may be produced. Comparing this standard to a case by control comparison, disorders may be more easily identified in people throughout the world.

Methodology
Preparing the organ bath
Prior to setting up the ileum, 20ml of Kreb’s solution was placed into the organ bath. For aeration, 95% O2: 5% CO2 was bubbled into the bath at a steady rate.

Setting up the tissue sample
Rat ileum tissue was retrieved whilst submerged in Kreb’s on a petri dish. A 60cm long piece of thread was passed through one end of the tissue using a needle. Multiple knots were tied and one end was trimmed. This procedure was then repeated on the other end of the tissue, but prior to trimming, a metal hook was placed next to the tissue and the excess thread was knotted around the hook. The hook was tied as closely to the ileum as possible. The hook end was fixed to a holder close to the chamber wall. The remaining untrimmed thread was tied onto a force transducer, connected to a micropositioner set within the middle of its range. The thread was not to be in contact with either the chamber or the hook and the thread was taut. The ileum was to not be stretched.
For calibration of base-line tension and equilibration of tissue
Using Labchart, the program was started and the base-line tension was observed and set between 0.5-1.5g. For calibration, the tissue was left in Kreb’s for 5 minutes until stabilisation.
Dilution of the drugs
50mg/ml acetylcholine (Ach), 50mg/ml carbachol and 10mg/ml butyrylcholine stocks were used and all underwent serial dilutions. For Ach, a 1:2 dilution was performed to produce a 25mg/ml sample, as well as a 1:5 dilution from the stock to produce 10mg/ml Ach which was followed by 4 consecutive 1:10 dilutions. A 20mg/ml sample was also made. The Ach procedure was repeated for carbachol. Butyrylcholine was diluted 10-fold 4 times.

Producing a dose-response curve
The organ bath volume was set to 20ml and the baseline was observed for 30 seconds. The first drug was then added and left for 45 seconds. The bath/tissue were then washed and drained with Kreb’s multiple times and 90 seconds after the third refilling the next drug would be added. Increasing doses of drug were added until a maximal response tension was recorded.

Plotting of results and statistics
Semi-log graph paper was used. Response (% of max) was on the y-axis and concentration of drugs (mM) was on the x-axis (log scale). The EC50 (µM) of each drug was calculated. Full and partial agonists were also derived. Class data was used and saw standard deviation calculations to identify any outliers and subsequently any truemean values were calculated for each drug’s EC50 (µM).

Results
In general, acetylcholine and butyrylcholine induced an increased positive response percentage of contraction in the rat ileum. Whereas carbachol displayed the reversed effect, where negative values of % response were witnessed. All drugs showed a linear relationship between an increasing bath concentration (µM) and % response. Further, all showed no identifiable response when the pipetted diluted concentration of the drug was both 1 µg/µl and 10 µg/µl, except for carbachol which illustrated a -8.69 % response. A dosage of 100 µg/µl displayed a swift rise in % response for butyrylcholine (0-30.4%) while acetylcholine and carbachol showed less dramatic changes of 8.7% and -13.0% respectably. Acetylcholine (25,000 µg/µl) reached the highest positive contraction response (0.23g) while butyrylcholine (10,000 µg/µl) reached 0.16g and carbachol (25,000 µg/µl) gave a -0.20 g final measurement (Table 1).

0000
Dose Acetylcholine (µg/µl) 1 10 100 1,000 10,000 20,000 25,000
Response (g) 0.00 0.00 0.02 0.04 0.07 0.20 0.23
% Response 0.0 0.0 8.7 17.4 30.4 87.0 100.0
Bath Concentration (µM) 0.28 2.80 28.00 280.00 2,800.00 5,950.00 6,880.00
Dose Carbachol (µg/µl) 1 10 100 1,000 10,000 20,000 25,000
Response (g) 0.00 -0.02 -0.05 -0.18 -0.20 -0.20 -0.20
% Response 0 -8.69 -21.7 -78.3 -87.0 -87.0 -87.0
Bath Concentration (µM) 0.28 2.80 28.00 280.00 2,800.00 5,950.00 6,880.00
Dose Butyrylcholine (µg/µl) 1 10 100 1,000 10,000
Response (g) 0.00 0.00 0.07 0.12 0.16
% Response 0.0 0.0 30.4 52.2 69.6
Bath Concentration (µM) 0.24 2.40 24.00 240.00 2,400.00
Table 1. Table showing dose responses for acetylcholine, carbachol and butyrylcholine. Bath concentration (µM) was calculated: dose(g)molecular weight*1volume of bath . Percentage responses of carbachol and butyrylcholine were compared to the maximal response of acetylcholine. 20,000 µg/µl and 25,000 µg/µl doses for Butyrylcholine were not successfully included in this study.

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Fig.1 Semi-logarithmic plot for the dose response curves for acetylcholine, carbachol and butyrylcholine when pipetted into an organ bath filled with 20ml Kreb’s. % response is the proportion of contraction displayed by isolated rat ileum. The EC50 was denoted by the dotted lines intercepting each curve. Concentrations of x<1 µM were omitted from this graph.

The EC50 was measured for every drug; acetylcholine was concluded as 4,032.6µM, butyrylcholine was 46.7 µM and carbachol was 70.1 µM with the use of EC50-measuring programs (fig.1).

Acetylcholine Carbachol Butyrylcholine
EC50(µM) 0.7 23.0 0.0
0.1 0.0 1.0
150.0 0.0 0.0
0.0 95.0 0.0
8.0 22.0 0.0
1.2 0.0 2.3
0.0 1.3 0.3 3.7 12.0 11,800.0? 1,900.0 400.0 0.0 0.0 7,230.0 ? 0.3 Table 2. Class data depicting EC50 (µM) calculations for acetylcholine, carbachol and butyrylcholine drugs. ? = outlier EC50 value.

Mean values prior to removal of values as outliers for all drugs were as follows: acetylcholine = 646.8 µM, butyrylcholine = 0 µM and carbachol = 1,327.2 µM. Standard deviations calculations for all drugs yielded a 1,885.8 µM value for acetylcholine, 0.9 µM for butyrylcholine and 3,927.4 µM for carbachol. This led to the EC50 value of 7,230.0 µM for acetylcholine to be omitted, as well as the 11,800.0 µM value for carbachol. Butyrylcholine displayed no anomalous values. As a result, truemean values were calculated as 176.6 µM, 0 µM and 18.1 µM for acetylcholine, butyrylcholine and carbachol respectively (Table 2).

Discussion
Based on the previous works by Dryn et al (2018), acetylcholine is the most abundant and commonly observed neurotransmitter in the ENS and furthermore Kamila and Slawomir (2018) mention that acetylcholine (along with vasoactive intestinal polypeptide (VIP), substance P and galanin) is the most important substance found in enteric neurones for full smooth muscle contraction. Therefore, the data depicting acetylcholine as a full agonist with a maximal response of 0.23g agrees with this (Table 1).
Carbachol and butyrylcholine have been previously termed as choline ester analogues and were seen to induce responses in acetylcholine receptors in Deroceras buccal muscle tissue, however, they were noted to not produce as strong a contraction as acetylcholine (Wright, T. and Huddart, H. 2002).
Despite this research, the class data suggests otherwise to the data at hand: butyrylcholine had no effect on ileum smooth muscle contraction, as the truemean value of EC50 was 0 µM (Table 2). This may be due to the fact that the butyrylcholine experiment was completed last and the lack of renewing the ileum tissue meant that either the used tissue was exhausted, or no longer alive. The class data also illustrates that acetylcholine reached maximal contraction at a much higher concentration than carbachol, at a difference of 158.8 µM truemean EC50. According to Boron and Boulpaep (2012), action potentials of smooth muscles could be either continuous or brief; the results may be explained with the notion that acetylcholine-induced muscle contractions are more prolonged and have less of a rapid effect on the muscle tissue than carbachol.
Furthermore, in addition to acetylcholine being labelled as a full agonist, the evidence that carbachol produced a less-than-full response while simultaneously causing negative contractions for all concentrations above 10 µg/µl suggests that carbachol acted as an antagonistic, partial neurotransmitter. This disagrees with the both the aforementioned literature by Wright and Huddart (2002) and the class data.

Overall, the data when compared to the hypothesis agrees with how acetylcholine produced maximal contraction and the highest EC50 and also agrees with how carbachol would produce the second-highest EC50, followed by butyrylcholine. However, while the hypothesis agrees with butyrylcholine being a partial agonist, the negative % response results from carbachol went against to what was expected.

This disagreement for carbachol in the data as well as some irregularities in the class data could be due to many sources of error. For one, no repeats were done for any drug due to lack of time and so no SEM was calculated. Furthermore, the same ileum tissue was used throughout the entire experiment as previously mentioned and as such later deviating results may have been affected by this. An inaccurate syringe was used to transport 20ml Kreb’s every time to the organ bath, so little consistency was achieved between rinses. The ileum tissue was stretched when sewn to the metal hook, so underestimates in response (g) followed inevitably.
References
Boron, W. and Boulpaep, E. (2012). Medical physiology. 2nd ed. Philadelphia: Saunders, pp.887-888.
Takahashi, T., Shiraishi, A. and Murata, J. (2018). The Coordinated Activities of nAChR and Wnt Signaling Regulate Intestinal Stem Cell Function in Mice. International Journal of Molecular Sciences, online 19(3), p.738. Available at: https://www.ncbi.nlm.nih.gov/pubmed/29510587 Accessed 3 Oct. 2018.

Lake, J. and Heuckeroth, R. (2013). Enteric nervous system development: migration, differentiation, and disease. American Journal of Physiology-Gastrointestinal and Liver Physiology, online 305(1), pp.G1-G24. Available at: https://www.ncbi.nlm.nih.gov/pubmed/23639815 Accessed 3 Oct. 2018.

Jiang, X. and Kopp-Schneider, A. (2014). Summarizing EC50 estimates from multiple dose-response experiments: A comparison of a meta-analysis strategy to a mixed-effects model approach. Biometrical Journal, online 56(3), pp.493-512. Available at: https://www.ncbi.nlm.nih.gov/pubmed/24478144 Accessed 3 Oct. 2018.

Dryn, D., Luo, J., Melnyk, M., Zholos, A. and Hu, H. (2018). Inhalation anaesthetic isoflurane inhibits the muscarinic cation current and carbachol-induced gastrointestinal smooth muscle contractions. European Journal of Pharmacology, online 820, pp.39-44. Available at: https://www.ncbi.nlm.nih.gov/pubmed/29198958 Accessed 4 Oct. 2018.

Szymanska, K. and Gonkowski, S. (2018). Bisphenol A—Induced changes in the enteric nervous system of the porcine duodenum. NeuroToxicology, online 66, pp.78-86. Available at: https://www.sciencedirect.com/science/article/pii/S0161813X18300871 Accessed 4 Oct. 2018.

T., W. and H., H. (2002). The nature of the acetylcholine and 5-hydroxytryptamine receptors in buccal smooth muscle of the pest slug Deroceras reticulatum. Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology, online 172(3), pp.237-249. Available at: https://link.springer.com/article/10.1007%2Fs00360-001-0248-6 Accessed 4 Oct. 2018.