c

FAR 161/2

Basic Pharmacology and

Immunopharmacology
PRACTICAL 1

ISOLATED GUINEA-PIG ILEUM:

AGONIST DOSE-RESPONSE CURVE

NAME : ZULKIFLI BIN KHAIRUDDIN

MATRIC NO. : 107225
I/C NO. : 910304-08-6409
GROUP :B
OBJECTIVE:
To study contractile responses of ileal segments isolated from a guinea-pig with increasing doses
of acetylcholine (ACh).

INTRODUCTION:
Guinea-pig isolated ileum

One of the basic principles of pharmacology holds that, within certain limits, the response
of a tissue organ to a drug is related in magnitude to the concentration of the drug in region of the
receptor with which it combines. To test and examine further this principle, a preparation which
is amenable to the recording of a range of magnitudes of response is required. Thus, the isolated
ileum of the guinea-pig is the most suitable preparation for this purpose, since it lacks
spontaneous activity (unlike ileal segments isolated from rabbits) when put under favourable
conditions (temperature approximately 32oC or room temperature, 1 gram (10mN) tension, in
Tyrode solution which is aerated with 95% O2 & 5% CO2). These factors are vital to ensure that
the effect of increasing drug concentration on contraction of the isolated guinea-pig ileum is
measured accurately. In addition, guinea-pig ileal segments are robust and therefore able to
respond to drugs for a few hours ›
›

Agonist dose-response curves

Dose and response are related and can be represented by a dose-response curve. Data
from toxicology testing can be represented by a dose-response curve, from which scientists can
describe the threshold and potency of chemicals.
The response to a drug may be:

1.c Graded ± these responses can be measured, eg: tissue contractile responses
2.c Quantal ± this type of response is either present or not at all (³all or none´) eg: death,
recovery, sleep

The responses in this experiment is graded. Graded dose-response relationship can be used to
determine maximum efficacy of a drug. Dose-response curves can be used to plot the results of
many kinds of experiments. The X-axis plots concentration of a drug or hormone. The Y-axis
plots response, which could be almost anything. For example, the response might be enzyme
activity, accumulation of an intracellular second messenger, membrane potential, secretion of a
hormone, heart rate or contraction of a muscle.

The term "dose" is often used loosely. The term "dose" strictly only applies to experiments
performed with animals or people, where you administer various doses of drug. You don't know
the actual concentration of drug -- you know the dose you administered. However, the term
"dose-response curve" is also used more loosely to describe ›
› experiments where you
apply known concentrations of drugs. The term "concentration-response curve" is a more precise
label for the results of these experiments.
METHOD:
Refer the Pharmacology Practical Manual (page 3-7)

RESULTS:
i)c Investigation of the relationship between the dose of ACh and its contractile response
in isolated segments of guinea-pig ileum

Dose of Ach (µg) Log dose ACh Contractile response(cm) % of Emax

1.0 0 0.00 0.00
2.0 0.3 0.30 15.00
4.0 0.6 0.50 25.00
8.0 0.9 1.30 65.00
16.0 1.2 1.70 85.00
24.0 1.4 2.00 100.00
32.0 1.5 2.00 100.00

Threshold dose = 2.0 ug

Maximum dose = 24.0 ug

 ii)c Investigation of the variation in contractile responses observed in segments of
isolated guinea-pig ileum, with repetition of a single dose of ACh.
c
Our group did not have time to do this experiment. Thus, we took our friends¶ result
for this experiment.

The concentration that they took is as the concentration ACh will give response about
50% Emax.

Readings Responses (cm)

1 1.4
2 1.2
3 1.1
4 1.4
5 1.3
6 1.5
7 1.7
8 1.6
9 2.0
10 2.1

Mean magnitude of contractile response

= 1.4 + 1.2 + 1.1 + 1.4 + 1.5 + 1.3+ 1.7 + 1.6 + 2.0 + 2.1

10

= 1.33

Smallest magnitude = 1.1 cm

Largest magnitude = 2.1 cm

 iii)c Responses and percentage of maximum responses to increasing doses of
acetylcholine in the presence of atropine (1.00x10l M)

Acetylcholine concentration Response(cm) % of Emax

2.00x10 0.00 0.00
4.00x10 0.00 0.00
8.00x10 0.00 0.00
1.6x10 0.00 0.00
3.20x10 0.50 3.33
6.4X10 1.20 8.00
1.28X10 3.50 23.33
2.56X10 6.00 40.00
5.12X10 8.30 53.33
1.02X10 12.10 80.67
2.05X10 12.90 86.00
4.10X10 14.10 94.00
8.20X10 15.00 100.00
c

iv)c Responses and percentage of maximum responses to increasing doses of

acetylcholine in the presence of papaverine (1.00x10l
M)

Acetylcholine concentration Response(cm) % of Emax

2.00x10 0.00 0.00
4.00x10 0.00 0.00
8.00x10 1.00 6.67
1.6x10 2.20 14.67
3.20x10 4.30 28.67
6.4X10 5.80 38.67
1.28X10 7.00 46.67
2.56X10 8.60 57.33
5.12X10 9.80 65.33
1.02X10 10.10 67.33
2.05X10 10.10 67.33
4.10X10 10.10 67.33
8.20X10 10.10 67.33
XUESTIONS:

1.c Investigation of the relationship between the dose of ACh and its contractile
response in isolated segments of guinea-pig ileum.

m c

 


›

m
  ›
m


m


m› 
 



› 
m› 

From the experiment, increasing the dose of the drug will increase the contractile
response in guinea-pig ileum. The drug use in this experiment is Acetylcholine (ACh). The
threshold dose is 2.0 µg and the maximum dose is 24.0 µg. As a conclusion, increasing the dose
will increase the contractile response of the guinea-pig ileum. The contractile response will reach
the maximum response. So, increasing the dose of the drug will make the response is constant.
This is regarding to the limited receptor.

2.c Investigation of the variation in the contractile response observed in segments of

isolated guinea-pig ileum, with repetition of a single dose of ACh.

m c 
m


m›m›
›


m


 



m 



hen the same concentration of the drug is injected repeatedly and continually, we found
that the magnitude of contractile response is increasing and then decreasing. This is because
tachyphylaxis occurs. Tachyphylaxis is the phenomenon that explains about the less sensitivity
of the receptor towards the drug because the drug is repeatedly and continually be given in the
long period of time. In this experiment, when we repeatedly inject the drug into the Tyrode
solution, the magnitude of the contraction response will decrease.

 c 

m


 ›


m








›









 
m›
›
 




 
m
m





›
 



m




›



 
m›


›
›

 

› 



›m

 ›
m›
›


mm› 










 Two methods can be applied in order to determine the unknown concentration of ACh,
by using isolated guinea-pig ileum preparation. There are :

i ) Graph method

The same method as the experiment before is applied to the guinea-pig ileum and the
dose use is the unknown concentration of ACh. Drug is injected and the contractile response of
guinea-pig ileum is measured by using the kymograph trace. From the previous experiment,
known that the maximum response is 2.0cm. Let say the response obtained from the unknown
concentration of ACh is x cm. So, the percentage of the maximum response for unknown
concentration of ACh can be calculated with the following formula.

% maximum response = E X 100%

Emax

% maximum response = x X 100%

2.0

From the graph percentage maximum response against log-dose of ACh, extrapolate the
x-axis that give the percentage response. The value from the x-axis is the value of log dose
for unknown concentration of ACh. hen the value is antilog, the unknown concentration of
ACh can be known in µg.

ii ) Estimation method

This method is not very accurate because the result obtained is only an estimation and not
specific. This method can be done only on qualitative not quantitative. Drug with concentration
0.5 µg is assume as known concentration of ACh. It is use in the organ bath which contains
isolated guinea-pig ileum. Kymograph pen is trace on the graph paper as responds to the
contractile response of isolated guinea-pig ileum. The kymograph trace for the contractile
response is measured and jotted down on the paper. Rinse the organ bath and ileum twice using
the Tyrode solution in 2 minutes. Then, the unknown concentration of ACh is injected in the
organ bath. The kymograph trace for the contractile response of unknown concentration of ACh
is measure and compare with the 0.5 µg concentration of ACh. If the response obtained from the
unknown concentration of ACh is higher than known concentration of ACh, it can be conclude
its concentration is more than 0.5 µg and vice versa. This method is not encouraged to be use
because the percentage of error to occur is high.
DISCUSSION:
Agonists are drugs or ligands that bind to the same active site of receptor with
endogenous ligand that caused the same signals. The magnitude signals of agonists either the
same or less than the magnitude signals of endogenous ligand.

The linear response when the log concentration of agonists formed when agonists added
to the biological system. Acetylcholine is agonists that have high affinity towards cholinergic
receptor. The isolated guinea-pig ileum can contract faster when presence of acetylcholine.

Atropine is competitive antagonists. hen the solution that contain isolated guinea-pig
ileum and acetylcholine added with atropine, the response obtained is below than the solution
isolated guinea-pig ileum and acetylcholine without atropine. It is because agonist will competed
with antagonist to bind at the same site of receptor. Antagonists will prevent the agonists to bind
and also will prevent the contraction of isolated guinea-pig ileum. But, when we replace the
atropine with papaverine, the effect of response is below than the presence of atropine. It is
because papaverine is irreversible antagonist. Irreversible antagonist will bind to the active site
of receptor, it will not detach from the receptor. It is contrast than atropine because atropine will
detach from the receptor, thus, agonists can bind to the receptor after that. The differences
between atropine and papaverine can be shown at the graph.

|c  
›m›




m› ›









m 
›
 m›


 


›
›m





 ›
m›
›



c c

In this experiment, it is shown that the concentration of drug is parallel to the response of
isolated guinea-pig. Its mean that when the concentration of the drug is increase, the contractile
response in isolated segment of guinea-pig ileum also increase. This is because the empty
receptor will bind with the drug continuously. The more concentration of drug, more the drug
can bind with the receptor and contribute high response.

?c  
›m›



m›m›
›
 m›


 




›




›m


 ›
m›
›

›


››

m
› 






c

Drug at the same concentration that inject repeatedly and continually will cause the
receptor less response towards the drug. This is known as tachyphylaxis. Therefore when a dose
of ACh which give a response of 50% Emax is repeatedly injected in the organ bath, the
magnitude of the response is decrease. It is because its receptor less response toward the ACh.

c
PRECAUTIONS:
2c Make sure hand is washed thoroughly before handling the ileum.
2c Ensure that both thread and ileal segment are never in contact with the walls of the organ
bath chamber.
2c Make sure accurate volume of solution is measured while preparing the serial dilution.
2c Make sure that the ileal segment is always completely immersed in Tyrode solution
2c Hold the ileum by using the hands, not using the forceps. It is because to prevent the
ileum to damage.
2c Make sure the speed rate of kymograph is around 2 or 3.
2c Make sure the tyrode solution in the organ bath will changed every 3 minutes.

CONCLUSION:
c From the experiment, we had study the contractile responses of ileal segments isolated
from a guinea-pig ileum to increasing doses of ACh. The threshold dose is 2.0 µg and the
maximum dose is 24.0 µg. Increase the dose of ACh will increase the magnitude of contractile
response. The magnitude of contractile response will increase until reach the maximum response.
hen the same dose of ACh is repeatedly and continually injected to the Tyrode solution the
magnitude of contractile response of ileal is decrease with time. This is because phenomenon
known as tachyphylaxis occurs.

REFERENCES:
Vc Pharmacology Practical Manual
Vc Notes lectures pharmacology. (Introduction to Pharmacology & Mechanisms Drug
Action)
Vc Dorland¶s Pocket Medical Dictionary 27th edition