U1P4 EXPERIMENTS ON GUINEA PIG ILEUM

This computer simulation and wet-lab supports material covered in the BMED2401 lectures: Drug
targets and drug-receptor binding, Responses to agonists and Responses to antagonists.

Things to do before the session:

Read through these notes and re-read your lecture notes (given above)
Complete the pre-work online

What you will need for this session:

These notes
Your lecture notes (given above)
Lab coat, protective eyewear and closed in shoes
Motivation and patience J

This experiment will be carried out in parts.

PRE-WORK is to be completed using Lt online in your own time PRIOR TO YOUR

PRACTICAL. Access the pre-work via Kuracloud. If you have any problems accessing the
pre-work please contact Tina Hinton (tina.hinton@sydney.edu.au).

PART 1 is a simulation of the guinea pig ileum experiment you will complete on the
computer in threes. See notes below.

PART 2 is the organ bath experiment you will complete in threes in Lt. If you have any
problems accessing the experiment please ask your demonstrator.

PART 3 is additional exercises that you need to carry out in class or in your own time. See
exercises below.
A NOTE ON ANIMAL ETHICS

For the use of animals in research or teaching an ethics application needs to be approved. The
application needs to be obtained every three to five years and an annual report needs to be
returned. In addition while animals are under our care they are monitored daily and are provided
with environmental enrichment, which addresses having a balanced/varied diet, socialization and
activities.

TASK: Visit the Sydney site: http://sydney.edu.au/research_support/ethics/animal/ and write down

the Code of Practice 3 Rs that need to be addressed when writing an application.

PART 1: EXPERIMENTS ON GUINEA PIG ILEUM SIMULATION

Time required: Part 1 should take about 45 min to complete.

PART A. Response to ACh and the effect of an antagonist on the agonist response:

You will be using a computer simulation to generate the data for this part of the experiment.

To access the programs, you need to login to the computer using your unikey login and password.
Citrix Profile Management will load. The green Citrix Receiver screen should load automatically on
your desktop.
Type the keyword Ileum into the search function at the top right hand side of the Citrix Receiver
screen. PCCAL Guinea Pig Ileum 4.0 should come up as an option. Click on this option to load the
PCCAL Guinea Pig Ileum program on to your desktop.
Click on the icon for the program to start it up. A screen looking like this below should eventually
come up. You are ready to start.

Click on
Receptors
here

You will see that there are several modules available. Today you will just be looking at the last part
of the module "Receptors". We strongly recommend, however, that you take a look at the
following modules in your own time:
 Experimental Set-Up
Agonists
Antagonists
Receptors (in full)

Click on page
14 here

Click on page 14 of Receptors for the simulation and data generation

You will come up with a screen that looks like this below. You can add an agonist by selecting the
agonist from the AGONIST box.

Click here to
select an
agonist
concentration

Select an
agonist by
clicking on it

You need to enter an agonist concentration by clicking on the blue volume and concentration panel
next to the agonist box.

Choose an
agonist
volume and
concentration
to add to the
tissue

Once agonist volume and concentration is selected, click OK, then click on Show Results to
observe the contraction of the ileum at the dose chosen. Notice if you click Show Results again
you might get a slightly different amplitude of contraction. This is due to inherent biological
variation.

Click here to
observe the
ileum
response to
the chosen
dose of
agonist

To add an antagonist you need to repeat the steps above, but using the red ANTAGONIST box.
 Choose an
antagonist
volume and
concentration
to add to the
tissue

Once antagonist volume and concentration is selected, click OK, then click on Show Results to
observe the contraction of the ileum at the dose chosen.

Aims: The first aim of this activity is to demonstrate the action of an agonist on smooth muscle
contraction, and how this response is affected by competitive antagonism. Another purpose of this
activity is to demonstrate that different drugs can produce the same response (contraction of
smooth muscle) through different mechanisms (i.e. receptors) and that it is possible to differentiate
between receptors by showing that certain antagonists block some agonists but not others.
Objectives: After finishing this activity you should be able to:
say whether a drug is an agonist or an antagonist
determine what type of antagonism is occurring
suggest sites of action for some different agonists and antagonists.

Procedure

Control Responses to ACh

To add a concentration of Agonist,
1. Find a concentration of ACh that produces the maximal contractile response (note that a
maximal response for ACh is around 60 arbitrary units).
2. Add the doses of ACh provided in Table 1, starting with a concentration of 1 x 10-9 M. It is a
good idea to repeat each dose to see what sort of variability you get (remember, this is a
simulation of the live preparation).
3. Record your results in arbitrary units in Table 1 below.
4. Convert your response in arbitrary units to a % of the maximal ACh response and record in
Table 1.

Table 1. Control Responses

[ACh] (M) Response (au) Response (%max ACh)

1 x 10-9
3 x 10-9
5 x 10-9
1 x 10-8
2 x 10-8
4 x 10-8
9 x 10-8
2 x 10-7
6 x 10-7
1 x 10-6

What do you notice about the amplitude of contractile response to the last two or three
concentrations of agonist?

Responses in the presence of an antagonist

1. Investigate the effects of an antagonist on the ACh response by "adding" 5 x 10-9 M
atropine (Atr) to the bath and re-testing the responses to ACh. Note that you will start with
5 x 10-9 M ACh and will be testing additional concentrations (keep in mind that you will need
a higher concentration of ACh to achieve a maximal response in the presence of atropine).
2. Record your results in arbitrary units in Table 2 below.
3. Convert your response in arbitrary units to a % of the maximal ACh response and record in
Table 2.
4. Repeat steps 1-3, instead testing 5 x 10-8 and 5 x 10-7 atropine
NOTE You will need to test additional ACh concentrations to reach the maximum
response with higher concentrations of Atr present - include them in the table.
 Table 2. Response to ACh in the presence of the atropine

[ACh] (M) Atr 5x10-9 M Atr 5x10-8 M Atr 5x10-7 M

Response Response Response Response Response Response
(au) (%max (au) (%max (au) (%max
ACh) ACh) ACh)
5 x 10-9
1 x 10-8
2 x 10-8
4 x 10-8
9 x 10-8
2 x 10-7
6 x 10-7
1 x 10-6
3 x 10-6
5 x 10-6

What do you notice about the amplitude of response to 4 x 10-8 M ACh alone (Table 1) compared
to that in the presence of 5 x 10-9 M atropine (Table 2)?

What dose of ACh is required to give a maximal contractile response when administered alone and
in the presence of atropine? Explain.

Plot your results from the simulated experiment on the semi-logarithmic graph below, being sure to
plot both control ACh data and data for ACh in the presence of the three concentrations of
antagonist (if you wish in your own time you can create a Schild plot and determine the pA2; the
slope will tell you what type of antagonist you used).
 100

90
0
80
% Maximum Response to ACh

0
70
0
60
0
50
0
40
0
30
0
20
0
10
0
0 1x10-9 1x10-8 1x10-7 1x10-6 1x10-5 1x10-4

[ACh] M

Figure 3. Write your own legend:

What do you notice about the curve for ACh in the presence of Atr compared to that of ACh alone?

What is the approximate EC50 (effective concentration that produces 50% of the maximal response)
for ACh alone? (just estimate from the curve note that the X axis is logarithmic, not linear)

What is the approximate EC50 for ACh in the presence of 5 x 10-9 M Atr? Why is this value greater
than that for ACh alone?

What sort of antagonism is occurring with Atr? What features of the dose-response curve show
this?
PART B. Differentiating the receptors

Testing the effect of histamine

1. Re-test the approximate EC50 of ACh determined above. Write this value and response (in
arbitrary units) in table 3 below. Repeat the EC50 response twice (why?).
2. Find a dose of histamine that produces a response similar in magnitude to the EC50 of the
ACh concentration chosen above. Repeat this response. Start with a concentration of
1x10-8 M histamine.
3. Record your results in Table 3 below. These responses to the two agonists are the control
responses.

Table 3. Control Responses

Agonist Bath [Agonist] (M) Response (au)

ACh
ACh repeat
Histamine
Histamine repeat

What do you notice about the EC50 of histamine compared to ACh? What does this suggest about
drug potency?

Differentiating the receptors

1. Choose concentrations of antagonist (atropine and mepyramine) that just abolish the EC50-
equivalent response to ACh and histamine. Record your results in table 4 below.
2. Test the effect when using supramaximal concentrations of antagonists. For example test
the effect using 1 x10-5M atropine and then 1 x10-5M mepyramine on EC50-equivalent
responses to ACh and histamine.

Table 4. Responses in the presence of the antagonists

Antagonist Agonist Bath [Agonist] (M) Response (au)

Atropine ACh
(_________M) Histamine
Mepyramine ACh
(_________M) Histamine
Atropine (high) ACh
(1 x10-5M) Histamine
Mepyramine (high) ACh
(1 x10-5M) Histamine
What effect does addition of atropine to the organ bath have on the response of the ileum to
histamine? What about the addition of mepyramine? What can you say about the site of action of
atropine and mepyramine given these results?

Selectivity vs specificity

What happened when you used supramaximal concentrations of atropine or mepyramine with
respect to the responses to histamine and ACh, respectively (be careful here) and why?

Check your answers with your demonstrator and LOG OUT OF THE COMPUTER YOU HAVE
BEEN USING FOR THE SIMULATION before moving on to the live tissue experiment.

PART 2: EXPERIMENTS ON GUINEA PIG ILEUM LIVE PREPARATION

For the live preparation practical you are working in threes. Login to the one computer that
is tethered to the organ bath.

Login to Kuracloud using the Kuracloud shortcut. All three group members can login to the
one BMED2401 Guinea Pig Ileum practical. Make sure you use Google Chrome as the
browser!

Follow the instructions in Lt to undertake your practical Experiments on Guinea Pig Ileum.
PART 3: SCHILD ANALYSIS AND MEDICINAL CHEMISTRY EXERCISES

PART A. Schild Analysis

The Schild plot is traditionally used to determine the apparent binding constant (KB) and the type
of antagonism (slope = ~1). The plot is created as linear-linear with the abscissa (x) axis log
transformed). For this exercise you may need to use the internet, lecture material or your textbook
(Rang et al.) for Pharmacology. The function that describes the plot is shown below: where rA is the
dose ratio, i.e the EC50 in the presence of antagonist over the EC50 in the absence of an
antagonist; XB is the concentration of the antagonist.

The Schild equation is thus:

log(rA-1) = logXB - logKB

rA = XA/XA
where XA = EC50 agonist in presence of antagonist; XA = EC50 agonist alone

ACh alone
ACh + 5 nM Atr
ACh + 50 nM Atr
ACh + 500 nM Atr

Figure 4: Dose-response curves for ACh alone and in the presence of increasing
concentrations of atropine

Table 6: Calculations for Schild Analysis

Atropine (M) (XB) Atropine (log(M)) ACh EC50 (M) rA Log(rA-1)

(logXB)
0 4 x 10-8
5 x 10-9 2 x 10-7
5 x 10-8 2 x 10-6
5 x 10-7 1.5 x 10-5

Use the above data and figure to generate a Schild plot and determine the approximate KB value
(hint value from graph will need to be transformed) for atropine.
 3-

Log(rA-1) 2 -

1-

0-
-9 - -7 -6 -5 -4
8 LogXB

1. From your plot can you suggest the type of antagonism? What aspect of the plot permits you to
determine the type of antagonism? (Note: this question has a higher level of difficulty)

2. Assuming the slope is one for your experiment what was the pA2 for atropine?

3. If you used another cholinomimetic, such as methacholine, would the rA be the same given that
you used the same concentration of atropine?

PART B: Structure-Activity Relationships

You may want to refer to the list of different types of binding forces given in U1L13 Receptor-
Ligand Binding and in the associated information sheet on Blackboard to answer these questions.

1. Write down the form of acetylcholine (structure given below) that you would expect to
predominate at physiological pH.
2. Identify two functional groups of acetylcholine that could give reasonably strong binding to the
relevant receptor.

3. What sort of bonds would occur between these functional groups and the receptor?

4. Compare the structures of acetylcholine and nicotine and identify two functional groups of each
agonist that could give reasonably strong binding to the relevant receptor.

5. Explain how structural differences between acetylcholine and histamine could explain their
selectivity at their respective receptors. (Answer this very briefly and in simple terms.
Consider receptor affinity resulting from different binding forces from the various functional
groups, and different molecular shape and size).

6. Identify an extra structural feature in atropine that might be responsible for the intrinsic activity
of the antagonist. What type of bonding force would be expected between this extra structural
feature and the receptor?

Acetylcholine Atropine
CH3 H CH3
N N
O CH3
CH3
N +H
CH3 O CH3 HOCH2 HOCH2
O O
-H
O O

Nicotine Hexamethonium
CH3
H H
+H + CH3
N N CH3 + N
H CH3
N CH3
CH3 CH3
N -H N CH3

Histamine Mepyramine
CH3 CH2
H H H
+H N +H N
N NH2 N NH3 N N CH3 N N CH3

N -H N -H
CH3O CH3O

Check your answers with your demonstrator before leaving.

ASSESSMENT

This practical will be assessed in U1A1. See your course guide and Blackboard site for further
details.