The effect of Acetylcholine and Carbachol on the concentration-response curve OF isolated rat ileum

Richard Harris, Rebecka, Jess, 20/11/2015
Aims: To obtain EC50 values for the drugs acetylcholine and carbachol in order to determine the relative potency of each drug with respect to rat ileum. To gain insightful knowledge of common laboratory techniques and procedures for utilisation in future lab sessions.
Introduction:
The ileum is a section of smooth muscle in the gut, specifically the small intestine, after the jejunum and before the caecum. It is involved in the absorption of cobalamin (vitamin B-12), bile salts and anything else that has not been absorbed by the jejunum. The ability of acetylcholine and carbachol to mediate a contractile response in the M2 and M3 muscarinic receptors (MR) was investigated in rat ileum. The M2 MR has been shown to inhibit the relaxant effects of beta-atypical and beta 2-adrenergic receptors. 1 The M3 MRs’ are responsible for the contraction of the ileum. The agonist Atropine inactivates the M3 MRs which leads to the disabling of M2 MRs due to the M3 MRs playing a role in potentiating a response in the M2 MRs. The effect of an antagonist will be simulated in order to assess how much the drugs’ efficacy or affinity will be hindered. The drug acetylcholine is expected to be more potent than carbachol.
Results:
Drug | Dug concentration (mols) | Drug response height (mm) | Avg. drug response height (mm) | Log(Drug concentration) (nmol) | Maximal response height (%) | Avg. Maximal response height (m%) | Ach | A1 (1X10-8) | 20 | 23 | -8.00 | 4.44 | 5.11 | Ach | A2 (1X10-8) | 29 | | -8.00 | 6.17 | | Ach | A3 (1X10-8) | 21 | | -8.00 | 5.53 | | Ach | B1 (3X10-8) | 145 | 133.8 | -7.52 | 32.20 | 29.73 | Ach | B2 (3X10-8) | 122.5 | | -7.52 | 26.06 | | Ach | B3 (3X10-8) | 47.5 | | -7.52 | 12.50 | | Ach | C1 (1X10-7) | 130 | 145.8 | -7.00 | 28.89 | 32.4 | Ach | C2 (1X10-7) | 150 | | -7.00 | 31.91 | | Ach | C3 (1X10-7) | 157.5 | | -7.00 | 41.45 | | Ach | D1 (3X10-7) | 337.5 | 299.2 | -6.52 | 75.00 | 66.49 | Ach | D2 (3X10-7) | 285 | | -6.52 | 60.64 | | Ach | D3 (3X10-7) | 275 | | -6.52 | 72.37 | | Ach | E1 (1X10-6) | 450 | 426.7 | -6.00 | 100.00 | 94.82 | Ach | E2 (1X10-6) | 450 | | -6.00 | 95.74 | | Ach | E3 (1X10-6) | 380 | | -6.00 | 100.00 | | Ach | F1 (3X10-6) | 405 | 450 | -5.52 | 90.00 | 74.30 | Ach | F2 (3X10-6) | 470 | | -5.52 | 100.00 | | Ach | F3 (3X10-6) | 125 | | -5.52 | 32.89 | | Ccl | A1 (1X10-8) | 4.5 | 3.5 | -8.00 | 1.86 | 1.51 | Ccl | A2 (1X10-8) | 3.0 | | -8.00 | 1.20 | | Ccl | A3 (1X10-8) | 3.0 | | -8.00 | 1.46 | | Ccl | B1 (3X10-8) | 11.0 | 10.5 | -7.52 | 4.54 | 4.52 | Ccl | B2 (3X10-8) | 4.0 | | -7.52 | 1.60 | | Ccl | B3 (3X10-8) | 10.0 | | -7.52 | 4.88 | | Ccl | C1 (1X10-7) | 60.0 | 58.0 | -7.00 | 24.74 | 24.95 | Ccl | C2 (1X10-7) | 26.0 | | -7.00 | 10.40 | | Ccl | C3 (1X10-7) | 56.0 | | -7.00 | 27.32 | | Ccl | D1 (3X10-7) | 92.5 | 91.3 | -6.52 | 38.14 | 39.27 | Ccl | D2 (3X10-7) | 56.0 | | -6.52 | 22.40 | | Ccl | D3 (3X10-7) | 90.0 | | -6.52 | 43.90 | | Ccl | E1 (1X10-6) | 180.0 | 167.78 | -6.00 | 74.23 | 72.16 | Ccl | E2 (1X10-6) | 167.5 | | -6.00 | 67.00 | | Ccl | E3 (1X10-6) | 156.0 | | -6.00 | 76.10 | | Ccl | F1 (3X10-6) | 242.5 | 232.5 | -5.52 | 100.00 | 100.00 | Ccl | F2 (3X10-6) | 250.0 | | -5.52 | 100.00 | | Ccl | F3 (3X10-6) | 205.0 | | -5.52 | 100.00 | |

Drug repetitions | EC50 value (mols) | Avg. EC50 value (mols) | Acetylcholine repetition 1 | 1.41X10-7 | 2.50X10-7 | Acetylcholine repetition 2 | 2.24X10-7 | | Acetylcholine repetition 3 | 1.26X10-7 | | Carbachol repetition 1 | 5.01X10-7 | 3.55X10-7 | Carbachol repetition 2 | 8.91X10-7 | | Carbachol repetition 3 | 3.98X10-7 | |

Atropine concentration (mols) | ÁA | Log([Atropine]) | Log([ÁA]-1]) | 1X10-9 | 4 | -9 | 0.47 | 1X10-8 | 18 | -8 | 1.23 | 1X10-7 | 60 | -7 | 1.77 | 1X10-6 | 350 | -6 | 2.54 |

Drug | Concentration (mols) | Maximal response height | % maximal response | EC50 (mols) | Ach | 1X10-8 | 12 | 20 | 3.55X10-8 | Ach | 3X10-8 | 31 | 52 | | Ach | 1X10-7 | 45 | 75 | | Ach | 3X10-7 | 56 | 93 | | Ach | 1X10-6 | 58 | 97 | | Ach | 3X10-6 | 60 | 100 | | Ccl | 1X10-8 | 0 | 0 | 1.2X10-7 | Ccl | 3X10-8 | 9 | 15 | | Ccl | 1X10-7 | 28 | 47 | | Ccl | 3X10-7 | 44 | 73 | | Ccl | 1X10-6 | 55 | 92 | | Ccl | 3X10-6 | 60 | 100 | |
The T-test:
S2= Σ(Y1- Y1)2+Σ(Y2- Y2)2n1+ n2-2= 8.441 = variance t = Y1 - Y2(1n1+ 1n2)S2 = 0.069 df = n1+ n2-2=4 degrees of freedom
Critical value of t Distribution = 2.78, where α=0.05
PA2 = x-intercept = 1.41X10-10 mols
Logged on the graphs are % maximal response vs. log([drug]) (mols). After plotting the points on the graph, the EC50 is calculated by drawing a straight, horizontal line from the 50% mark on the y-axis until it intercepts the curve line, at which point a straight, vertical line is drawn until it intercepts the x-axis. The point it intercepts is marked and the result recorded - this is known as the EC50 value, converted from log([drug]) to mols. The average EC50 value for acetylcholine was 2.50X10-7, lower than the EC50 value for carbachol,3.55X10-7. This effect was also observed in the guinea pig model graphs. The Schild regression graph shows log(DR-1) against log([Carbachol]). The Schild regression allows calculation of the PA2, achieved by extrapolating the trend line, recording the x-intercept and converting it to mols, which in this case came to a value of 1.41X10-9. PA2 is the measure of the affinity of a drug at a particular receptor. The schild regression graph emanates from the plot of the antagonist at varying concentrations with the carbachol agonist, as shown by the graph.

Discussion:
Acetylcholine had a higher potency than the carbachol, as shown by the average EC50’s produced, which stands to reason, as detailed in online reports, whereby acetylcholine is slightly higher in potency than carbachol. The t-test shows that acetylcholine and carbachol are statistically insignificant, which provides evidence for my null hypothesis being false. The average acetylcholine EC50 value was shown to be less than the average carbachol EC50 value, which shows that carbachol is more potent than acetylcholine. The same could be observed in the guinea pig ileum model graphs, which illuminates potential for interspecies activity of the drug. carbachol has a greater efficacy when compared to acetylcholine, probably due to the inactivity of acetylcholinesterase which would invoke hydrolysis. However, the affinity of acetylcholine precedes carbachol for the same reason it has a greater affinity – the acetylcholinesterase. The Schild regression gives rise to a term known as reversible competitive antagonism – this is whereby the agonist, carbachol, can have its effectiveness reduced due to the presence of a competitive antagonist, atropine. The issue arises due to the M3 MRs becoming literally blocked by the atropine, which binds with the active site so the agonist cannot perform its job correctly. If the agonist concentration is too high, the agonist drug can become completely ineffective due to all or most of the active sites being blocked. The effect is reversible, whereby physostigmine or pilocarpine can be injected and will remove, through several mechanisms, the atropine from the active site.
References:
1. Sawyer, G. and Ehlert, F. (1998). Contractile Roles of the M2 and M3 Muscarinic Receptors in the Guinea Pig Colon. [online] aspetjournals.org. Available at: http://jpet.aspetjournals.org/content/284/1/269.long [Accessed 14 Nov. 2015]. 2. GR, CB, PB, FA, AN, CG and CB. (1997). Identification of beta-adrenergic receptor subtypes mediating relaxation in isolated equine ileum. [online] American Journal of veterinary research. Available at: http://www.ncbi.nlm.nih.gov/pubmed/9185969 [Accessed 14 Nov. 2015]. 3. https://en.wikipedia.org/wiki/Muscarinic_acetylcholine_receptor_M3 [Accessed 15 Nov. 2015].S