ORGAN BATH PRACTICAL SESSION (BIOM2402)

Session 1&2: Organ Bath Practical

Acetylcholine, Scopolamine, Adrenaline, Atropine
The following drugs are available to you.

Acetylcholine (300 mM)

Scopolamine (10 mM)
Adrenaline (1 mM)
Atropine (1 mM)

The main objective of these organ bath practicals are for you to generate cumulative
concentration-response curves for the agonist acetylcholine (Ach), alone (controls), in the
presence of numerous antagonists, and to investigate the mechanisms of pyridostigmine on
rat ileum tissue via analysis of pre-acquired data (video on BlackBoard). This will be done
by;

WEEK ONE (OB1)

a) Preparing serial dilutions of acetylcholine (Dilutions Table 2 from VI session)
b) Synchronising your tissue using a final concentration of 1μM Ach
c) Measuring the contraction of rat ileum TWICE with increasing concentrations of the
drug using the cumulative method of agonist addition (Dilutions Table 2)
d) Measuring the contraction of rat ileum ONCE with cumulative additions of Ach IN
THE PRESENCE OF 10nM Atropine and again in the presence of 100nM Atropine.

WEEK TWO (OB2)

a) Repeat steps A-C from Week One (OB1)
b) Measuring the contraction of rat ileum ONCE with cumulative additions of Ach IN
THE PRESENCE OF 5uM Adrenaline and 3um + 30uM Scopolamine.

AT HOME

c) Access the Pyridostigmine video on Blackboard – watch the experiment performed

with the additions of pyridostigmine and the ‘antagonist’ atropine
d) Generate a written response explaining the MOA of Pyridostigmine; thinking about
how it is functioning without exogenous Ach being added and how the MOA of
atropine works as an ‘antidote’ to these pyridostigmine effects
Introduction
In the virtual Ileum session, you analysed agonist data, assessing both potency and efficacy,
and you assessed how agonist effects are altered in the presence of a competitive antagonist.
For the organ bath practical, you will be generating organ bath data of your own .
The isolated intestine preparation
You will be shown a video of rat ileum dissection and preparation (10 min video). Your
tutors will have already set up in your organ baths. Pieces of intestine from small animals will
give responses for many hours if kept under optimal conditions. For rat ileum, Tyrode’s
buffer aerated with Carbogen gas (95% O2, 5% CO2) can be used, with the tissue maintained
 ORGAN BATH PRACTICAL SESSION (BIOM2402)

at 32°C under a 10 mN tension to establish a reference point against which to compare

tension changes.

General Methods and Materials

Addition of drugs to organ bath
The bath should be filled to the overflow each time. This sets the bath volume to 25 ml.
Add the drugs to the organ bath by means of pipettes fitted with disposable tips. Draw the
required volume of drug solution into the pipette, place the tip just below the surface of the
fluid in the organ bath and inject the solution into the bath where the carbogen is bubbling
(not directly onto the tissue!). Discard pipette tips into the yellow containers located on each
bench. If you are doing a cumulative curve, you need to use the same tip, because you need to
add agonist every 10-15 sec. There is no time for changing pipette tips.

‘Wash-out’ procedure
You don’t want your tissue to be exposed to the agonist for too long, as it will become
fatigued and not work as well towards the end of the practical. You should aim to always
maintain a happy, healthy piece of tissue so that it gives you good concentration-response
data. To remove the agonist, you need to change out the buffer solution in the bath. To do
this, drain the bath, and then allow the Tyrode’s buffer to enter the bath from the coils until
the solution overflows. Repeat this two more times for a total of three washes. This ensures
that all of the agonist is completely washed out of the bath and the tissue. If the previous drug
treatment had produced a sustained contraction, allow the tissue to relax after these washes,
and the tension tracing on the screen starts to return to baseline. When the tissue has relaxed
to its resting length, the recording will have returned to its baseline position. If the tissue
stretches initially, so that tension becomes less than 5 mN, consult with a tutor, who may
increase the tension back to 5-10 mN.

Note: Monitor the level of Tyrode’s buffer in the upper reservoir and re-fill it when needed
from the containers around the lab by the sinks.

Concentrations to be used for the concentration-response curve

As was done during the virtual ileum practical session, you should start your drug additions at
a concentration that is below that which generates a response, and then to increase the agonist
concentration until a maximum contractile response is recorded. In your own experiments, you
will be using the dilution strategy developed in Dilutions 1 and 2 during the virtual ileum
session (page 12).

Now you’re ready to start your prac

Experimental Procedure
1. Prepare your serial drug dilutions of Ach
You will be provided with a 300 mM stock solution of Ach, located in the cool box.
For the experiment, prepare serial dilutions from this highly concentrated stock.
Specifically, prepare serial 10-fold dilutions so that you have working stock
concentrations of 100 mM, 10 mM, 1 mM, 100 μM, 10 μM and 1 μM Ach using your
Dilutions Table 1. Remember to clearly label each of your Eppendorf tubes. If you
have forgotten how to do this, consult your tutor.

2. Synchronise your tissue – check tissue contractility

Before you start acquiring data for your concentration-response curve, you should
‘synchronise’ the smooth muscle in your tissue by adding 25 μl of 1 mM agonist
working stock and immediately washing (for this practical, your agonist is Ach). This
allows you to verify that your tissue is responsive to the agonist. This only needs to be
done the first time that the tissue experiences exogenous drug application…once per
practical session is enough. You should observe a contraction of approx. 10-20 mN
caused by this concentration of Ach. As soon as you observe this contraction,
immediately perform a wash-out step so that the tissue can return to its baseline
state. Please consult a tutor about the magnitude of the contraction measured during
this synchronising step to ensure that your tissue is healthy.

3. Obtain 1-2 cumulative concentration-response curves

You have already calculated the volumes of serial stock solutions required for the
cumulative organ bath experiment (Dilutions Table 2). Make sure that you add the
correct volume of the correct working stock solution to your organ bath to get the
correct response.

Starting at the 3 nM concentration, complete an Ach concentration-response curve

using the cumulative addition method, working through to a concentration that results
in a maximum contractile effect. For this type of experiment, you will need to ensure
that you have made a comment line in labchart immediately prior to adding
acetylcholine. This will allow you to go back and determine which concentration
produced each peak in your data chart. Follow the following timing for this
experiment:

1. When you are ready, click “add comment” on lab chart and then immediately add
the lowest concentration of Ach (to give 3 nM in the organ bath). This time = 0.

2. 10 seconds later, click “add comment” again, and add the next highest
concentration (to give 30 nM in the organ bath).

3. 10 seconds after that, add the next highest concentration and so on until your tissue
cannot contract any further.

4. 10 seconds after the addition of your last concentration, wash out the organ bath (3
times).

5. Show a tutor your cumulative trace on the computer

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6. Next, measure the net contractions for each concentration of drug as above.

7. Begin filling out the experimental data sheet contained at the end of this manual.
Calculate the response to each of the concentrations of agonist as a percentage of
the maximum response.

8. Using the Prism Software, plot the concentration- response curve of Ach using your
data

9. Determine the concentration of Ach that gives approx. 50% of its maximum
response (the EC50)

10. Show a tutor your graph and the EC50 obtained for Ach (don’t forget to include the
units).

4. Obtain 1 cumulative concentration-response curve in the presence of [Antagonist].

11. A stock of 10mM Scopolamine, 1mM Adrenaline and 1mM Atropine will be
available in the ice bucket. Use these to create working stocks or directly add
columns to reach final concentrations suggested above

12. Next, while antagonist is still in the organ bath, construct a new single
cumulative concentration-response curve to Ach. Wash the organ bath and
consider repeating the experiment if you’d like replicates or move on to the
next concentration/antagonist.

13. Obtain the net contraction from agonist at each concentration, and the % Emax
for each concentration, plot this curve ON THE SAME GRAPH as Ach alone
(control curve) and obtain the EC50 value. Make sure the new curve is
properly labelled.

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 EXPERIMENTAL DATA SHEET
Agonist: Acetylcholine

Cumulative agonist data

Test 1 Test 2
[Agonist] Max Max Average
(M) contractile % Emax contractile % Emax % Emax
force (mN) force (mN)

Cumulative agonist data in the presence of Antagonists

Control [Antagonist]
[Agonist] Max Max
(M) contractile % Emax contractile % Emax
force (mN) force (mN)

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OBJECTIVES CHECKLIST FOR OB SESSION 1

 Prepare your Ach serial dilutions (Dilutions Table 1- Last page of VI lab guide)
 Generate a concentration-response curve for Ach using the cumulative approach in
Prism in n≥1 (using Dilutions Table 1) – Control Curve for Atropine
 Generate a concentration-response curve for Ach in the presence of 10nM and 100nM
Atropine using the cumulative approach
 Determine your cumulative Ach EC50 values and potency/efficacy changes for any
cumulative curves from this session (these will be on GraphPad Prism)
 Make sure you show a tutor your results before you leave the practical session.
 Did your results confirm what you already know about the MOA of drugs you have
been given? If not, what experimental or biological factors could have led to these
unexpected results – DISCUSS WITH TUTORS!!!!

OBJECTIVES CHECKLIST FOR OB SESSION 2

 Pull out your Ach serial dilutions (Dilutions Table 1- Last page of VI lab guide)
 Generate a concentration-response curve for Ach using the cumulative approach in
Prism in n≥1 (using Dilutions Table 1) – Control Curve for Scopolamine
 Generate a concentration-response curve for Ach in the presence of 3uM and 30uM
Scopolamine using the cumulative approach
 Generate a concentration-response curve for Ach using the cumulative approach in
Prism in n≥1 (using Dilutions Table 1) – Control Curve for Adrenaline
 Generate a concentration-response curve for Ach in the presence of 5uM Adrenaline
using the cumulative approach.
 Determine your cumulative Ach EC50 values and potency/efficacy changes for any
cumulative curves from this session (these will be on GraphPad Prism)
 Make sure you show a tutor your results before you leave the practical session.
 Did your results confirm what you already know about the MOA of drugs you have
been given? If not, what experimental or biological factors could have led to these
unexpected results – DISCUSS WITH TUTORS!!!!

We are now on the home stretch – please between the end of your OB2 session and Zoom Q&A
sessions, take the time to get all of your Concentration-Response Curves completed (curves, axes,
EC50 values, Emax, Figure legends etc). Additionally, read through the Organ Bath Report
instructions and rubric and bring any questions you have to the Q&A sessions.

Also do NOT forget that there is a pyridostigmine video on Blackboard that you need to watch and
prepare a written response to for your laboratory reports. There is no template for this assignment, but it
is HIGHLY recommended you structure your report to correspond with the criteria sheet (IE:
Introduction, Agonist data, Antagonists and Pyridostigmine sections).