Discussion

The muscarinic and histamine receptors are among the several receptors which can be

found in the guinea pig ileum. Muscarinic receptors are either ligand-gated G protein coupled

receptors or are metabotropic. These receptors are involved in the parasympathetic nervous

system and it has 7 transmembrane α-helices coupled to intracellular effector system such as

activation or inhibition of protein kinase for a specific response via a G-protein which acts as

a secondary messenger. M1, M2, M3, M4, and M5 are the 5 subtypes of M receptor and different

subtypes of G protein, including Gs, Gq, Go, and Gi protein, are coupled to each M receptor.

These receptors are in charge of controlling the contraction of guinea pig ileum in response to

a drug molecule binding to a receptor. Therefore, the presence of muscarinic receptors is crucial

to determine how a drug affects a particular receptor. Agonists are drugs that bind to receptors

and trigger receptor activation to induce stimulation inside the body. Antagonists will bind to

the same receptor as agonists but do not activate the receptor. As a result, an agonist's ability

to bind to a receptor has been hampered by the blocking of the antagonist, and thus it cannot

exert any pharmacological effects.

Based on the result of experiment 1, the response curve for drug with carbachol only

indicates that when the carbachol concentration increases, the concentration of guinea pig

isolated ileum also increases until it reaches the receptor saturation, which occurs when the

value of log concentration is roughly equal to 6. The point of receptor saturation denotes the

extent to which carbachol molecules have occupied and activated all muscarinic receptors in

the guinea pig ileum to activate the pharmacological reactions in the guinea pig muscle.

Phospholipase C will be activated more and produce more IP3 and DAG molecules as agonist

binding increases. These increases would result in higher IP3 and DAG molecule
concentrations, which would raise the concentration of cystolic Ca2+ ions and promote smooth

muscle contraction. The concentration of carbachol serves as the limiting element in this early

period (before plateau) and directly impacts the strength of muscular contraction. The response

will eventually reach a plateau as the carbachol concentration exceeds the number of receptors

as one receptor only bind to one agonist at one time. Due to the restricted number of accessible

receptor binding sites, even though an excessive amount of carbachol which is a drug is being

injected into the organ solution, the system becomes saturated when all binding sites are

occupied. The excess carbachol could not provide any pharmacological effect as all the binding

side of the receptor is saturated. Thus, despite an increase in drug concentration, the maximum

response is reached and would not increase further. This explains why the log [concentration]

against response graph initially increases before plateauing. In addition, the shape of the log

concentration response curve with carbachol only demonstrates that the strength of guinea pig

isolated ileum contractions is directly proportional to the quantity of drug-receptor complexes

as the higher the quantity of drug receptor complexes in guinea pig ileum, the higher the

magnitude of contractions of guinea pig-isolated ileum. Thus, it demonstrates how the

quantity of available receptors and the concentration of histamine at the receptor site affect the

magnitude of the guinea pig isolated ileum contractions.

In experiment 2, before exposure to the carbachol, atropine is flushed through the ileum

muscle. The results demonstrate that under the same carbachol concentration, the magnitude

of muscle contraction with atropine at the early stage before the plateau is significantly lower

than the magnitude of muscle contraction with carbachol only in experiment 1. It is because

Atropine is an antimuscarinic drug that may reduce and inhibit the response brought on by a

muscarinic agonist. Since atropine is a non-selective muscarinic receptor antagonist, it can bind

to all M receptor subtypes, including the M3 receptor found in the ileum muscles. Competitive

antagonism is the sort of antagonism that the atropine molecule engages in. Due to its
comparable ester and basic groups, atropine has a very similar chemical structure to the agonist

carbachol. Thus, the competitive antagonist Atropine will compete with agonist Carbachol for

the limited binding sites on the same receptor. Atropine competes with the agonist Carbachol

and bind to the receptor sites but it will not induce any pharmacological effect. It is because

Atropine binding does not cause phospholipase C to become activated or for PIP3 to be broken

down into IP3 and DAG. The release of Ca2+ ions from the interior compartments could not

be induced by the low concentration of IP3, which would be low. As a result, the cytoplasmic

Ca2+ ion concentration is low. The binding with calmodulin that is complexed with myosin

light chain kinase reduces due to the absence of Ca2+ ions. As activated myosin light chain

kinase levels drop, so does the amount of regulatory myosin light chain that myosin light chain

kinase can phosphorylate at serine-19. As a result, the ATPase cycle will be less active and the

smooth muscle will have fewer myosin heads that repeatedly bind to, ratchet away from, and

then slide over actin filaments. Thus, the addition of the competitive agonist Atropine at the

early stage (before the plateau) would result in a general decrease in guinea pig ileum muscular

contraction (Kramer, I.M., 2016). Binding of a competitive antagonist to a receptor Atropine

is reversible because competitive antagonists bind to the receptor non-covalently. It can be

overcome by increasing the number of agonists, specifically Carbachol, as agonists displace

antagonists from binding sites. Besides, based on the log concentration-response curve, the

response curve with atropine has shifted to the right compared to the response curve of muscle

concentration with carbachol only. The shifting of the response curve demonstrates that the

affinity for carbachol decreases while atropine is flushed through the ileum muscle at the early

stage before the plateau, and thus leading to an increase in the EC50 of carbachol in the presence

of competitive antagonist Atropine.

Efficacy is the intrinsic ability or ceiling effect which is the ability of a drug to produce

a desired therapeutic effect. Based on the result in the log drug concentration-response curve,
it shows that when EC50 rises, the Emax of the atropine response curve still remains the same.

As a result, it demonstrates that atropine is a competitive antagonist. As Atropine competes

with Carbachol and binds to the same binding sites on the same receptor, atropine reduces the

affinity for carbachol by preventing it from accessing the receptors. As a result, it decreases

the carbachol’s affinity and efficacy while increasing the EC50 of carbachol. As the binding of

Atropine with Carbachol is reversible, the Emax of the log concentration-response curve with

atropine remain unchanged as the non-covalently binding reaction can be overcome by

increasing the number or agonist. However, carbachol will still competes with the antagonist

atropine for the same binding sites when the concentration of carbachol increases. The

magnitude of the guinea pig ileum contractions gradually increases as more atropine molecules

are driven away from the receptor until receptor saturation is reached. Therefore, the maximum

response (Emax) of carbachol is unaffected and will remain the same. The EC50 values of

muscle contraction with and without atropine can be determined from the log concentration-

response curve.
From the result of calculations above, the EC50 of drug with Atropine is higher than the EC50

of drug without Atropine. This indicates that the higher the value of EC50, the less potent a

drug. As a result, when the value of EC50 is high, a larger concentration of the drug is required

to induce 50% of the maximal response. As a result, it demonstrates that carbachol has a higher

affinity and potency to bind to the receptor in the absence of atropine since there is no

competition in the organ bath. Therefore, 50% of the maximal contractile response can be
produced by a lower concentration of carbachol. Besides, the difference in maximal efficacy

(Emax) of drug with Atropine and without Atropine is small which is only 0.18g. As a result,

the efficacy or carbachol with and without the antagonist atropine is regarded as the same.

In experiment 3, the final concentration of 10-8 unknown drug (Drug D) is added into

the reservoir and flushed into the ileum muscles throughout the experiment with carbachol.

Based on the result of experiment 3 in table 2.2, the characteristic of Drug D can be determined

based on the pattern of the dose-response curve graph in figure 1. the initial reading shows that

the muscle contraction after the exposure to Drug D in experiment 3 is lower than the muscle

contraction of pure exposure of carbachol in experiment 1. This indicates that Drug D is a

muscarinic antagonist as the presence of Drug D lowers the response of muscle concentration

due to the presence of Drug D as muscarinic antagonist. Muscarinic antagonist also referred to

as anticholinergics as it blocks the muscarinic cholinergic receptors and will cause mydriasis

and bronchodilation (James Duke,2011). Besides, from the result of response curve graph, the

magnitude of the ileum muscle contraction increases as the concentration of carbachol

increases until reach the saturation point. This indicates that the binding of Drug D with

receptor is reversible. When the binding is reversible, it means that drug D molecules attach to

the receptor non-covalently and can be removed from the receptor by higher concentration

of carbachol to overcome the binding. Based on the result, the combination of Drug D and

carbachol has reached the maximum response similar with the effect of atropine. This shows

that Drug D is a competitive antagonist as it can reach the maximum response of muscle

contraction. The potency and antagonistic effect of Drug D and atropine are similar as the drug-

response curve shows that the Ec50 value of Drug D is similar with atropine. Drug D has
affinity to bind to the receptor as it can produce the Emax which is the maximal response from

the drug-response curve. Drug D have no efficacy as it fails to produce pharmacological effect

and thus the intrinsic activity of unknown drug D is 0.

Conclusion

In a nutshell, by attaching to the corresponding site on a specific receptor, an agonist

will trigger several intracellular reactions. But due to the complementary binding, a

competitive antagonist with a similar chemical structure and conformation may also be able to

bind to the receptor. Competitive antagonists can compete with agonists for receptor binding

because they can bind to the receptor's active site. As there is less chance for the agonists to

bind to the receptor in this competitive situation than when there is no competitive antagonist,

the affinity of the agonist towards the receptor will decrease. As a result, the agonist's EC50

would be higher in the presence of a competitive antagonist, indicating a drop in the agonist's

potency and affinity. As competitive antagonist and receptor binding is reversible, the

antagonistic effects could be lessened and countered by increase the concentration of agonist.

Agonists are more likely to bind to receptors as agonist concentration rises, gradually reducing

the antagonistic effects of competing antagonists.

References
Deranged Physiology. (2015). Potency and efficacy: Deranged physiology. Retrieved April
12, 2021, from _https://derangedphysiology.com/main/cicm-primary-exam/required-
reading/pharmacodynamics/Chapter%2041 5/potency-and-efficacy

Kramer,I,M., 2016, An Introduction to Signal Transduction, Signal Transduction (Third

Edition), Accessed on 13 May 2020 from

https://www.sciencedirect.com/science/article/pii/B9780123948038000024

Tatcher,J,D. , 2010, The Inositol Trisphosphate (IP3) Signal Transduction Pathway, Science

Signaling, Vol. 3, Issue 119, pp. tr3, DOI: 10.1126/ Accessed on 13 May 2020 from

https://stke.sciencemag.org/content/3/119/tr3.full

El-Fakahany, E., & Merkey, B. (2021). 6. characteristics OF Drug-Receptor Interactions.

Retrieved Apnil 12, 2021, from
https://open.lib.umn.edu/pharmacology/chapter/characteristics-of-drug-receptor-
interactions/