4. Drug Receptor Interaction

Question 1

what was the aim of this practical to examine?

Answer 1

how the size of the response that may be recorded from a piece of isolated tissue varies when a drug is added.

Question 2

what is a receptor

Answer 2

Highly specialized proteins embedded in cell membranes that possess structurally defined binding sites which specifically interact with particular complementary sites on the drug molecule

Question 3

agonist vs an antagonist

Answer 3

Agonists - Drugs that bind to receptors and ELICIT a response
Antagonists - Drugs bind to receptors with high affinity but do NOT activate receptors (i.e. do not initiate a signalling mechanism).
They prevent an agonist from binding to the receptor, if both bind to the same site.

Question 4

what is the ileum

Answer 4

the final and longest part of the small intestine. This is the part of the GI tract through which most drugs are absorbed.

Question 5

what does the ileum consist of

Answer 5

smooth muscle

Question 6

Smooth muscle contraction is dependent upon what

Answer 6

influx of calcium ions

Question 7

what is the major neurotransmitter released in the GI tract

Answer 7

acetylcholine

Question 8

what does acetylincholine cause

Answer 8

the contraction of the ileum

Question 9

The effects of acetylcholine on ileum smooth muscle can be measured experimentally in vitro - what does this mean

Answer 9

in a lab setting using components of the organism isolated from its usual biological surrounded

Question 10

The effects of acetylcholine on ileum smooth muscle can be measured experimentally in vitro - how?

Answer 10

using small sections of rat or guinea-pig ileum maintained in aerated physiological saline solution in an organ bath (~25 ml). Different concentrations of acetylcholine can then be added to the organ bath and contractile responses measured using a transducer/amplifier/chart recorder system

Question 11

go over the transducer etc experimental set up

Answer 11

https://ucl-eu-west-2-moodle-sitedata.s3.eu-west-2.amazonaws.com/f9/4b/f94b349a33286d5cea7a9a01fa54e761cbcf10c1?response-content-disposition=inline%3B%20filename%3D%22CR%20Practical%20document%202023-2024r.pdf%22&response-content-type=application%2Fpdf&X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIA47YHZF637GKGWUJC%2F20250512%2Feu-west-2%2Fs3%2Faws4_request&X-Amz-Date=20250512T162716Z&X-Amz-SignedHeaders=host&X-Amz-Expires=21584&X-Amz-Signature=eb761dd8792c63a7fc58c9bfe46378c54c96a5292b7a7b85851d2e4c6cf44288

Question 12

Depending on the responses of the tissue when the drug is added, this method can be used to determine whether drugs are agonists or antagonists: what effects do agonists have

Answer 12

Agonists will produce a response when applied to a tissue and this will vary depending on the concentration applied.

Question 13

how do we know the max response of the agonist has been produced - what indicates it

Answer 13

we start by adding very low concentrations of the drug and increasing the concentration until at least two concentrations produce a similar response.

Question 14

how do we set up the ileum in the organ bath

Answer 14

place ileum piece in the petri dish provided together with a volume of Tyrode solution
- Using the needle and cotton provided, attach a short length (~ 5 cm) of cotton to one end and tie it to form a loop then attach a longer length (~ 25 cm) of cotton to the other end.
- then remove the tissue holder and attach ileum to the organ bath

Question 15

what do we do after we set up the ileum

Answer 15

1. add increasing concentrations of acetylcholine until three acetylcholine concentrations produce responses that are recorded using the online LT practical.
2. Add the appropriate volume of stock atropine solution to the organ bath so that the final concentration of atropine is 10-8 M. 3. Allow to equilibrate (5 minutes.) Add the concentration of ACh that produced a first response in the presence of atropine.

• repeat with higher ACh concentrations

Question 16

what type of drug is acetylcholine

Answer 16

an agonist

Question 17

how do we know acetylcholine is an agonist

Answer 17

When applied to the isolated ileum tissue, acetylcholine produced contraction of the smooth muscle.

The magnitude of contraction increased with increasing concentrations of acetylcholine, showing a concentration-dependent response.

This is characteristic of an agonist, as it binds to muscarinic receptors on smooth muscle and activates them, mimicking the action of endogenous acetylcholine released by parasympathetic nerves.

The response eventually plateaus, indicating a maximum (saturating) effect—another hallmark of an agonist.

Question 18

where / what receptors do atropine an acetylcholine bind to

Answer 18

muscarinic (M3) receptors

Question 19

how is atropine a competitive antagonist of muscarinic acetylcholine receptors

Answer 19

It blocks muscarinic receptors (especially the M3 subtype) found on smooth muscle in the ileum.

These receptors are normally activated by acetylcholine, which causes smooth muscle contraction.

By competing with acetylcholine for the same binding site on the M3 receptor, atropine prevents this contraction from occurring.

Question 20

is the antagonism of atropine reversible or not

Answer 20

it’s reversible, if you add enough acetylcholine, it can displace atropine and restore the response (which is why the ACh response can return at high concentrations, though reduced)

Question 21

What do you predict will be the response of the ileum to acetylcholine?

Answer 21

The ileum will contract — and this contraction will be:
Concentration-dependent (greater contractions with higher concentrations of ACh),
Rapid, since ACh binds directly to M3 receptors and activates them,
Reproducible, if you wash and repeat the exposure properly.
- we measure the contracting as tension on the transducer

Question 22

why will the ileum react to the acetylcholine

Answer 22

Acetylcholine is a muscarinic receptor agonist, and the ileum (part of the GI tract) contains M3 muscarinic receptors on smooth muscle cells.

Question 23

What do you predict will be the response of the ileum to atropine by itself

Answer 23

No response/significant contraction:

Question 24

why does just atropine cause no reaction

Answer 24

Atropine doesn’t activate the muscarinic receptors — it just blocks them.
Since it’s an antagonist, it has no intrinsic activity — it only prevents acetylcholine (or other agonists) from activating the receptor.
So, unless there’s some baseline cholinergic tone in the tissue (unlikely in isolated tissue unless nerve stimulation is ongoing), atropine alone won’t cause a contraction.

Question 25

Predicted response when both Atropine and Acetylcholine are present?

Answer 25

The response of the ileum to acetylcholine will be reduced or completely blocked, depending on: The concentration of atropine, and The concentration of acetylcholine.

Question 26

What were the observed responses of the ileum to increasing doses of acetylcholine?

Answer 26

As you increased the dose of acetylcholine in the organ bath, you would have seen: Gradually stronger contractions of the ileum tissue. A dose-dependent response — meaning that as the concentration of acetylcholine increased, the amplitude (strength) of the contraction increased. Eventually, you reached a maximum response, beyond which adding more ACh did not increase the contraction — this is known as a plateau.

Question 27

Mechanism Behind This Observation: of ACh causing contractions

Answer 27

Acetylcholine binds to M3 muscarinic receptors on the smooth muscle of the ileum. These receptors are Gq-coupled GPCRs → activation leads to: Stimulation of phospholipase C (PLC) Production of inositol triphosphate (IP₃) Release of Ca²⁺ from intracellular stores Increased intracellular calcium leads to: Activation of calmodulin and myosin light chain kinase (MLCK) Contraction of the smooth muscle As more ACh is added: More receptors are activated → stronger contraction Once all receptors are occupied (saturated), further ACh doesn't increase contraction — this is the maximum (Emax) response.

Question 28

what happens if you add histamine to the ileum

Answer 28

When histamine binds to these receptors, it can cause contraction

Question 29

why does adding histamines cause a contraction too?

Answer 29

The ileum also has histamine H₁ receptors on its smooth muscle. When histamine binds to these receptors, it can cause contraction — just like acetylcholine, but via a different receptor type. So, if you add histamine: You’ll often observe a contraction of the ileum tissue, independent of acetylcholine. The contraction may differ in magnitude, speed, or duration, depending on tissue sensitivity and receptor density.

Question 30

what receptors do histamine work on

Answer 30

H1 receptors, which are also Gq coupled, and so lead to an increase in intracellular Ca2+, causing the smooth muscle contraction

Question 31

how do we know that histamines act on a diff receptor to ACh

Answer 31

Receptor Selectivity: If you block muscarinic receptors with atropine, acetylcholine no longer works — but histamine still causes contraction. This confirms that histamine acts via a different receptor.

Question 32

describe atropine

Answer 32

muscarinic cholinergic receptor antagonist

Question 33

The smooth muscle of the ileum is innervated by what nervous system

Answer 33

the parasympathetic nervous system

Question 34

The neurotransmitter release from parasympathetic nerve endings is acetylcholine (ACh) and the receptors present on the muscle are what receptors?

Answer 34

cholinergic receptors, specifically of the muscarinic subtype.

Question 35

competitive antagonists bind to receptors at agonist binding site, but unlike agonists, are unable to do what?

Answer 35

elicit a response.

Question 36

why is the effect of the antagonist surmountable

Answer 36

because increasing the agonist concentration should overcome the effect of the antagonist - this is as the antagonist is present, the agonist is limited in its access to the active binding site, therefore its activity is decreased.

Question 37

Tissue mounting steps:

Answer 37

Take a piece of ileum and place it in the petri dish provided together with a volume of Tyrode solution (see video). Using the needle and cotton provided, attach a short length (~ 5 cm) of cotton to one end and tie it to form a loop then attach a longer length (~ 25 cm) of cotton to the other end. Remove the stainless steel pipe from the organ bath and slip the loop of cotton over the hook situated at the base of the pipe then return the pipe with the ileum attached to the organ bath. Place a small piece of plasticine on the arm of the transducer situated above the organ bath and a larger piece (approximately twice the size) to the other arm. Apply some tension to the longer length of cotton then attach it to the small piece of plasticine on the transducer arm.

Question 38

Experimental Method

Answer 38

After the preparation had been set up, add increasing concentrations of ACh were added until the tissue contracted. Estimate the concentrations of ACh that give approximately 30% and 70% of the maximum response. Test these concentrations twice to ensure that you achieve consistent responses. Disconnect the container on the stand above the organ bath and replace it with the ATROPINE container provided. This should be filled to the 1 L mark with Tyrode solution together with the appropriate volume of stock atropine solution so that the final concentration of atropine is 10-8 M. Flush the organ bath three times with this solution and allow to equllibrate for at least 5 minutes. The tissue will now be bathed in Tyrode solution containing the required concentration of atropine. Add increasing concentrations of ACh until the heights of contraction corresponding to the 30% and 70% responses found in the absence of atropine are achieved. Repeat the above with atropine solutions at 10-7 M and then 10-6 M

Question 39

Which receptor is the agonist (ACh) acting on and what type of receptor (e.g. inotropic/metabotropic) is it?

Answer 39

ACh acts on the M2 and M3 muscarinic receptors, which are metabotropic receptors, as they transmit signals within a cell by activating G proteins

Question 40

name TWO other clinically-used agonists acting on this receptor.

Answer 40

1. Pilocarpine: this is used to treat dryness of the mouth and throat 2. Bethanechol chloride: this is used to treat certain disorders of the urinary tract or bladder, by helping cause urination/emptying of the bladder.

Question 41

What is the intracellular signalling mechanism activated by that M3 receptor?

Answer 41

ACh will bind to the M3 muscarinic receptors, which causes Gq/11 protein activation. Then, phospholipase C is activated, leading to the hydrolysis of PIP2 to produce second messengers (DAG and IP3 - for G protein activation). This leads to more Ca2+ ions being released, which would stimulate more contraction (of the smooth muscle in the rat ileum's).

Question 42

how can we tell graphically if something is a full agonist

Answer 42

- level of high efficiency, - able to elicit 100% of the maximum response.

Question 43

how can we tell graphically if something is a partial agonist

Answer 43

lower efficiency compared to the other ones - does not elicit the maximum response,

Question 44

The control graph (no atropine) should show what/look like what

Answer 44

a sigmoidal curve with an approximately linear region in the middle of the ACh concentration range between approximately 30% and 70% of the maximum response.

Question 45

what is the EC 50 value on the graph

Answer 45

point at where 50% of response (on y axis) was reached

Question 46

Competitive shift of log concentration response relation expected in the presence of fixed antagonist concentration [A]: shifts the graph where?

Answer 46

shifts it to the right, looks the same, just more agonist needed to overcome it

Question 47

how do we work out the dose ratio for the schild plot after

Answer 47

just divide the dose at a specific same point for each line D2, or D3, etc all by D1 to work out the ratio : if ur confused just double check ur lab report

Question 48

Schild analysis is then based on this simple shift factor relation: what equation

Answer 48

DR = 1+ A/KA

Question 49

what do we plot in a schild plot on each axis

Answer 49

y = Log (DR-1) x = log [antagonist conc]

Question 50

The Schild plot should (if true competitive antagonism applies) yield what

Answer 50

a straight line with a fixed slope of +1.

Question 51

Log(DR - 1) =

Answer 51

Log[A] - Log[KA]

Question 52

how do we derive the KA for atropine from the schild plot

Answer 52

KA: From the original Schild equation: Log(DR - 1) = Log[A] - Log[KA] When DR=2, Log (DR-1)=0, then: Log [A] = Log [KA]. Therefore taking Antilogs: KA= Antilog {Log[A]} And: KA=Antilog{ x axis intercept, when DR=2}

Question 53

how do we derive pA2 =

Answer 53

= −log KA

Question 54

why is there is no further increase in the contractions produced by ACh despite the addition of higher concentrations of drug to the tissue

Answer 54

Despite the addition of higher concentrations, there is no further increase in the contractions, because all of the binding sites of the muscarinic receptors would have been saturated by ACh. There are no more available binding sites for the ACh to bind to bind to, so adding a higher concentration won't have any effect. We can say that Max / the maximum concentration rate has already been reached.

Question 55

a lower dose = what potency

Answer 55

higher potency