Identification and Analysis of Unknown Agonists G and H

Group 6: Alecco Philippi, Alex Kim, Alyssa Fryer, Kyra Chin, and Mark A. Pistea

UNKNOWN G UNKNOWN H
Increasing concentrations of unknown G were administered Figure 2. Plot representing Increasing concentrations of drug H were administered to Figure 5. Plot representing
muscle responses to unknown rabbit ileum tissue, which resulted in a contractile response. muscle responses to drug H
to rabbit ileum tissue, resulting in a decrease in tension–and drug G at increasing and acetylcholine (Ach) at
by extension–muscular relaxation. This relaxation was concentrations. A sigmoidal, The effect of drug H rapidly increased at a threshold increasing concentrations.
observed to have a gradual onset and prolonged action
non-linear regression was concentration of 10-7 M. EC50 was determined to be ~2.74×10-7 Sigmoidal, non-linear regressions
performed and fit to the plots to were performed and fit to the plots
(Figure 3A). A near-maximal response induced by drug G illustrate the dose-response M. to illustrate the
occurred at a concentration of ~10-5 M, which corresponds curve. The EC50 of drug G is Drug H demonstrated rapid onset with transient action. A concentration-response curves.
marked at a concentration of 1.98 The EC50 values of drug H and
to a ~78.4% decrease from baseline tension (1.0147 g to ×10-7 M, corresponding to a
near-maximal response was observed at a concentration of
Ach are marked at concentrations
0.2196 g). EC50 for drug G was observed to be tension of approximately 0.62 g. ~1.00x10-6 M–representative of a ~401.1% increase from of 2.74×10-7 M and 5.21×10-7 M,
The Emax of drug G is baseline tension (0.4986 g to 2.4987 g) (Figure 5). respectively. Muscle tension was
approximately 1.98×10-7 M (Figure 2). approximately 0.16 g. approximately 1.85 g at EC50 for
Acetylcholine (Ach) is known to elicit effects similar to both drug H and Ach. Emax
those observed in our results. Ach binds to muscarinic and values were determined to be
approximately 2.70 g and 2.77 g
Antagonist - Phentolamine nicotinic receptors to elicit parasympathomimetic effects on for drug H and Ach, respectively.
Phentolamine is an α-adrenoceptor antagonist, which is involved in preventing sympathetic activation of gut smooth the rabbit ileum used in the experiment4.
muscle.1 A 33.66% decrease was observed from baseline muscle tension to the phentolamine trial containing unknown G EXPERIMENTAL DESIGN
(Figure 4). Given that the action of unknown G is inhibited in the presence of an α-adrenoceptor antagonist, it is likely an Antagonist - Atropine
α-agonist. Atropine is a competitive antagonist of muscarinic agonists such as Ach5. Atropine competes for a common binding site on all
muscarinic receptors. When drug H was added to the organ bath in the presence of atropine, a reduced contractile response was
Antagonist - Propranolol observed (Figure 6) A ~94.8% increase from baseline was observed in contractile response in the atropine-treated bath with drug
Propranolol is a β-adrenoceptor antagonist2. When unknown G was added in the presence of propranolol, a relaxation H, which is relatively small compared to the contractile response observed with only drug H (~401.1%). Our results demonstrate
response was observed, albeit not as strong as the trial with drug G alone. A 39.75% decrease from baseline tension was that drug H exerts an effect on muscarinic receptors and is thus a muscarinic agonist.
observed in the propranolol trial; a 78.36% decrease was observed with drug G alone (Figure 4). Our results suggest that
propranolol is antagonistic towards the action of drug G. By extension, our results also suggest that drug G is a β-agonist. Antagonist - Hexamethonium
Hexamethonium is a nicotinic receptor antagonist that functions non-competitively through binding to the allosteric binding
2A site6. A smaller contractile response compared to that of the Drug H (~142.7% increase versus ~401.1%) trial was observed in
2B the rabbit ileum in the presence of hexamethonium (Figure 6), indicating that drug H is a nicotinic agonist.

6A 6B 6C

Figure 1: Flow chart outlining the logical method that was followed based on
receptor activation to determine the action of unknown agonist G and H.
Figure 6A (left): An increase in tension after adding drug H to the organ bath. 6B (middle): The contractile response after the
addition of atropine and drug H. 6C (right): The contractile response following the addition of hexamethonium and drug H. Yellow
circles highlighting response shortly after addition of unknown H.
Conclusion - Drug G
2C Figure 7. A comparison of
Drug G appears to be an α and β adrenergic muscle responses to unknown
agonist, as its effects on muscle contractility are Conclusion drug H under various
conditions. 0.25 mL of a 1:10
inhibited by the presence of both α and β The unknown drug H was identified to be a muscarinic and nicotinic dilution of stock drug H solution
adrenoreceptor antagonists1,2. Relaxation responses agonist. This was confirmed by the ~94.8% and ~142.7% increases in was added to the organ bath for
contractile responses observed in the presence of muscarinic and nicotinic all trials using drug H. 0.25 mL of
decreased by 33.66% and 39.75% from baseline 10-1 M hexamethonium solution
Figure 3A (top left): A decrease in tension after tension, whereas the maximum effect of drug G antagonists from baseline values, respectively. was added to the organ bath
Figure 4. A comparison of muscle responses adding drug G to the organ bath. 3B (top right): (10-3 M final bath concentration)
The relaxation response following the addition of responded to a 78.36% decrease from baseline
to unknown drug G before and after Based on the results, drug H may likely be Ach. To validate this hypothesis, for the hexamethonium trial. 0.25
phentolamine and propranolol exposure. phentolamine. 3C (bottom): The relaxation tension. mL of 10-3 M atropine solution
0.75 mL of 10-3 M phentolamine was added to a response following the addition of propranolol. Blue tissue responses to the drug may be assessed in conjunction with (10-5 M final bath concentration)
25 mL organ bath to achieve a final bath circles highlighting response shortly after addition of physostigmine–an acetylcholinesterase inhibitor. Prolonged action of the drug was added to the atropine trial.
concentration of 3x10-5 M phentolamine, with unknown H. with physostigmine would prove that drug H’s identity as Ach7. No effectors were added to the
the same preparations for propranolol, resulting baseline trial. The rabbit ileum
in a final bath concentration of 3x10-5 M. 0.75
Unknown G is likely norepinephrine or epinephrine. To confirm this, these drugs may be
was washed prior to each trial.
mL of a 1:10 dilution of stock drug G solution injected in an animal, and blood pressure tracings may be collected. Since epinephrine
was added to the bath for all drug conditions. No
effectors were added to the baseline trial. The
works on β2-receptors in the heart (norepinephrine does not), a slight decrease in blood
rabbit ileum was washed prior to each trial. pressure, before an increase, would point towards the drug being epinephrine3.
REFERENCES CONTRIBUTIONS
(1) Kim, N.; Goldstein, I.; Moreland, R.; Traish, A. Alpha-Adrenergic Receptor Blockade by Phentolamine Increases the Efficacy of Vasodilators in Penile Corpus Cavernosum. Int J Impot Res 2000, 12 (S1), S26–S36. https://doi.org/10.1038/sj.ijir.3900504.
(2) Srinivasan, A. Propranolol: A 50-Year Historical Perspective. Ann Indian Acad Neurol 2019, 22 (1), 21. https://doi.org/10.4103/aian.AIAN_201_18. Kyra: Intro and conclusion for drug G, part of analysis Mark: Formatting, Antagonists (group H) discussions, References
(3) Colangelo, C.; Shichkova, P.; Keller, D.; Markram, H.; Ramaswamy, S. Cellular, Synaptic and Network Effects of Acetylcholine in the Neocortex. Front. Neural Circuits 2019, 13, 24. https://doi.org/10.3389/fncir.2019.00024. paragraph, formatting, Figure 2 Alecco: Made some concentration-response curves, wrote information regarding unknown H.
(4) Charkoudian N, Rabbitts JA. Sympathetic neural mechanisms in human cardiovascular health and disease. Mayo Clin Proc. 2009;84(9):822-830. doi:10.1016/S0025-6196(11)60492-8 Alyssa: Analysis paragraph for Drug G, Experimental Alex: Title, Figures 1. - 4. and their captions, other calculations with data (percent change).
(5) Lochner, M.; Thompson, A. J. The Muscarinic Antagonists Scopolamine and Atropine Are Competitive Antagonists at 5-HT 3 Receptors. Neuropharmacology 2016, 108, 220–228. https://doi.org/10.1016/j.neuropharm.2016.04.027.
design, and Formatting
(6) Papke, R. L.; Wecker, L.; Stitzel, J. A. Activation and Inhibition of Mouse Muscle and Neuronal Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes. J Pharmacol Exp Ther 2010, 333 (2), 501–518. https://doi.org/10.1124/jpet.109.164566.
(7) Hoover, D. Physostigmine. In xPharm: The Comprehensive Pharmacology Reference; Elsevier, 2007; pp 1–6. https://doi.org/10.1016/B978-008055232-3.61705-2.