Pharmacology Adrianos Boutsias Abstract word number: 247

March 26, 2019 General Studies

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

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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)*1/(volume 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).

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)*1/(volume 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.

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

x

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