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The ileum is the longest segment of the small intestine and it contains a circular smooth muscular layer. The smooth muscle contains muscarinic acetylcholine receptors which should respond to the presence of acetylcholine (ACh) and initiate a contraction in the muscle (Widmaier et al., 2016). The aim of this practical was to examine the effect of increasing ACh concentrations on a segment of guinea pig ileum. This was done through calculating the EC50 which is the concertation of the agonist that produces 50% of the maximal response and the Emax which is the maximal response produced by the agonist (Rang et al., 2016). It is expected that there will be a positive correlation between the concentration of ACh and the strength of the tissues contractions
A guinea pig was humanely killed by cervical dislocation and the ileum was removed. A short segment of ileum (1cm – 1.5cm) was threaded at each end and mounted in a 38ml organ bath. The organ bath containing aerated Tyrode’s solution and it was maintained at a temperature of 36°C. The segment of ileum was attached to one end of a lever connected to an isotonic transducer so that the voltage of the contractions could be recorded in the Powerlab data collection apparatus. A 1g weight was attached to the other end of the lever so that the tissue would be under tension. Addition of Acetylcholine
A range of serial dilutions of Acetylcholine (ACh) were made to produce final organ bath concentrations between 1*10-9M to 8.192*10-6 M. The ACh was added in a 3-minute dose cycle. The lowest concentration of ACh was added to the organ bath and the tissue’s response was observed for 30 seconds. The solution was washed out by overflow to remove the ACh. In order for the temperature to equilibrate the tissue was allowed to rest for two minutes. Increasing concentrations of ACh were added to the organ bath in twofold increments and the tissue’s response recorded until a maximal response was reached.
A data trace was produced in LabChart. This was used to calculate the value of Emax and to calculate the percentage maximal response from the voltages recorded. A graph of percentage maximal response against log[ACh (M)] was produced in Microsoft Excel. From the graph the value of EC50 was calculated.
Figure 1: Graph showing the response of a short segment of guinea pig ileum to increasing concentrations of acetylcholine. The data is expressed as a percentage of the maximal response recorded in millivolts. The graph shows an EC50 value of 3.98*10-8 M (dashed line).
Figure 2: Data trace showing the effects of differing ACh concentrations on a segment of guinea pig ileum. The data is expressed as the strength of the tissue’s contraction in millivolts.
As shown in Figure 1 there is a positive correlation between the concentration of ACh and the strength of the contractions recorded. ACh produced little contraction in the tissue until a threshold concentration of 4*10-6M was reached. From 4*10-9M to 1.6*10-8M there was little difference in the strength of the contractions. However, there was sharp increase in the strength of the contractions of the tissue between 1.6*10-8M and 6.4*10-8M. A maximal response was recorded at an ACh concentration of 2.56*10-7M. At higher concentrations of ACh figure 1 shows a decrease in the strength of the contractions. The ACh produced an pEC50 value of 7.4 and Figure 2 shows that it produced an Emax value of 353mv.
The are some flaws with the experimental design. There is a small sample size, so the data is susceptible to outlying values. However, this can be solved by repeating the experiment with a larger number of guinea pigs. The results are similar to results obtained in previous studies. According to a study by Ringdahl (1986) the pEC50 of ACh acting on a guinea pig ileum is 7.44.
The results agree with what was expected as there is a positive correlation between the concentration of ACh and the strength of the muscle contractions recorded. The ACh may not have initially generated a response as there was not a high enough concentration to bind to the muscarinic acetylcholine receptor and generate an action potential. The small increase in response may have resulted from receptors on some of the smooth muscle cells binding with enough ACh to generate an action potential causing the muscle to contract. At high concentrations of ACh the ACh can bind to receptors on a majority of the smooth muscle cell which results in a large response. The fall in maximal response could be the result of the receptors becoming desensitised, but it could also be due to experimental error.
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