Investigating the Effects of Agonists and Their Antagonists on
Receptors in Guinea Pig Ileum
Introduction:
An agonist is a drug that binds and activates a receptor, producing a response. Antagonists combine
with these receptors but do not activate them. In the case of competitive antagonism they reduce
the probability of the agonist combining with the receptor by competing for the same binding site
(Neal, 2009). These molecular interactions; the way in which drugs are absorbed and their
responses, are crucial to our understanding of pharmacology.
This was demonstrated through the smooth longitudinal muscle of guinea pig ileum which contracts
in response to certain agonists acting on its receptors; of which the most relevant to the study are
the muscarinic and histamine receptors on the muscle fibres (Feldburg, 1950).
The main aim was to investigate the action of acetylcholine (ACh) on the muscarinic receptors,
specifically M2 and M3 subtypes present in the ileum (Kardiol, 1991). Not only is this observed alone
but also in the presence of the competitive antagonist atropine (Atr) and how this affects the control
response. Another aim was to show that different agonists, ACh and histamine, can produce the
same contractile response although binding to different receptors. Different receptors explain why
certain drugs act as antagonists to some agonists but not others. Further shown, is how this
selectivity is lost in higher dosages. This was all revealed through observation of the tissue’s
biological response using both a computer simulation and live preparation.
Method:
The apparatus (Appendix 1) consisted of a 2-3cm piece of guinea pig ileum kept in a carbogen
aerated organ bath full of Tyrode solution. The tissue was attached by hook and string, mounted
under tension, to a transducer lever. The dose-response relationship was observed using a
measurement principle based on the ability of typically relaxed ileum tissue to contract in the
presence of ACh. As different doses were added to the bath, the extent of contraction was
monitored as the lever changed its position and recorded into computer program Chart 4.2. The
responses were interpreted using arbitrary units before being converted into percentages using the
maximum response obtained.
The response of the tissue to both ACh alone, the control, and in the presence of Atr was first
observed using a computer simulation program. The ACh concentrations tested ranged from 1x109
M to 1x10-6M. An ACh dosage range from 5x10-9M to 5x10-6M was then tested with three different
dosages of Atr - 5x10-9M, 5x10-8M and 5x10-7M. A dose-response curve was created with the data
after converting it to a percentage using the maximum. This was used to estimate the EC50 for ACh
alone and with 5x10-9M Atr. The amount of histamine required to produce the same response as this
EC50 was found; the two responses used as controls. At these EC50s, ACh and histamine were tested
for response to Atr and mepyramine at concentrations of 6x10-9M and 9x10-9M respectively.
This was followed by the live preparation. Doses increasing in concentration were administered
following a regular dose-wash cycle. ACh was injected, the response monitored for 30 seconds
followed by a wash, then another after 90 seconds (for higher doses), then waiting for the tissue to
return to its initial length by 3 minutes after which the next dose is added. Atr was added to the bath
after ACh alone reached maximal contractile response. Chart 4.2 produced a representative trace
(Appendix 3) and the response peaks were translated to a data pad in arbitrary units (Appendix 4).
The concentration of the bath was calculated (Appendix 2) in order to plot a dose-response curve
and estimate the EC50.
Results:
Table 1: Response of ACh alone and in the presence of three concentrations of Atropine expressed
as percentages of the maximum
[ACh]
Control
Response with 5x10-9 M Response with 5x10-8 M Response with 5x10-7 M
(M)
Response
Atr
Atr
Atr
-9
5x10
0.0
0.0
0.0
0.0
-8
1x10
0.0
0.0
0.0
0.0
-8
2x10
7.0
1.8
0.0
0.0
-8
4x10
17.5
14.0
0.0
0.0
-8
9x10
38.6
28.1
0.0
0.0
7
2x1057.9
52.6
5.3
0.0
-7
6x10
82.5
77.2
19.3
0.0
-6
1x10
94.7
91.2
38.6
0.0
-6
3x10
96.5
101.8
63.2
7.0
-6
5x10
100.0
100.0
75.4
17.5
Table 1 shows an increase in response proportional to the increase in ACh concentration. As the
concentration of Atr increased, a greater amount of ACh was needed in order to achieve a similar
response to the control; the two higher concentrations of Atr not reaching maximum at all. The
highest dosage of ACh produced a 100% response from the control but 17.5% of the maximum
response for the highest concentration of Atr, the lower dosages of ACh producing a response of 0.
Antagonist
Mepyramine (9x10^-9 M)
Atr (6x10^-9 M)
Histamine (2x107M)
Ach (3x10-8M)
Mepyramine (1x10^-5 M)
Atr (1x10^-5 M)
0
10
20
30
40
Response (au)
Figure 1. The Responses of Antagonists Ach and Histamine in the Presence of the Agonists
This graph illustrates the effect of atropine and mepyramine at two different concentrations; one to
abolish the EC50- equivalent response to ACh and histamine and the other to test the effect of using
supramaximal concentrations of antagonists, which in this case was 1x10-5 M ACh and mepyramine.
As shown above, in the presence of 6x10-9 M Atr, ACh response was reduced to 1 au whereas the
response to histamine was normal at 27 au. Increasing the dose to 1x10-5 M, there was no response
from ACh at all, and histamine was still unaffected.
In the presence of low doses of mepyramine (9x10-9M), there was a low response from histamine at
2 au and a usual response from ACh, and increasing the concentration of mepyramine eradicated the
appearance of histamine completely. Increasing the dose also affected ACh as it decreased to 14
from 27 au.
100
90
% Maximum Response to ACh
80
70
60
50
ACh in the absence of
atropine (control)
40
ACh in the presence
of atropine
30
20
10
0
1.00E-10
1.00E-09
1.00E-08
1.00E-07
1.00E-06
[ACh] M
Figure 2. Dose-response curve of Ach alone and in the presence of Atropine.
Figure 2 shows a sigmoidal graph, which in the case of ACh in the presence of Atr has shifted to the
right in a parallel fashion. Both curves reach the maximum. The responses of ACh with Atr are lower
than that of just ACh, the EC50 increased, and the maximum response achieved at a higher dosage.
Discussion:
The response of guinea pig ileum to the agonist ACh was a contraction of the smooth muscle with
the extent of contraction proportional to each increase in dosage as seen in Table 1 and Figure 2.
This was the case until it reached a maximal contractile response of around 75 au. In the presence of
Atr, the same increase in ACh concentrations produced a lower response per dose. This is because
ACh is no longer being taken up in a non-competitive environment. As seen in Table 1, as the
concentration of the antagonist was increased from 5x10-9M to 5x10-7M, higher doses of ACh were
required to reach the maximum response of the control. The EC50s of ACh also increased in the
presence of Atr. This indicates that Atr reduced the potency of ACh by acting as a competitive
antagonist. The mechanism by which this occurs involves Atr competing with ACh for a specific
binding site as it has a high affinity for muscarinic cholinergic receptors like ACh. Atr then occupies
and blocks the site, resulting in less frequent activation of ACh, but it does not actually produce its
own response.
This relationship was confirmed in our results from both Part A and B as expected. When noting
certain characteristics of the dose-response curve (Figure 2), both curves still reach the 100%
maximum even in the presence of Atr. The maximum is surmountable with a high enough
concentration of agonist, which is why the curve with Atr is shifted to the right. This is because
agonist efficacy is not affected by the presence of competitive antagonist. It is known that the
antagonist is competitive as the curves are parallel to each other (Neal, 2009). Table 2 shows that in
the higher range of concentrations of Atr, lower doses of ACh produced no response from the tissue
at all. The log concentration-response curve produced a sigmoidal shape, unlike the rectangular
hyperbola shape obtained from a linear concentration-response curve. The linear curve is rarely
used as it is harder to analyse. Results bunch and overlap (Appendix 4), indicating the scale needs to
be adjusted to estimate values like the EC50 with greater accuracy.
The responses of ACh and histamine to Atr and mepyramine indicated selectivity in their functioning
as antagonists (Figure 1). When both ACh and histamine were used in the presence of Atr it acted as
an antagonist for ACh, but did not affect the response of histamine even at a higher dosage, with
histamine still giving its control EC50 response of approximately 28 au. Similarly, at a low
concentration, mepyramine acted as an antagonist for histamine and did not affect the response of
ACh. However, at a high concentration of mepyramine it resulted in a lower response from ACh.
This ability of drugs to act in this way and give responses as seen in Figure 1 can be explained by
selective antagonism. In such a case a drug acts as an antagonist for specific drugs only and not
others, binding to a certain receptor, though there is an exception to this in the event of overdose.
The results thus imply that Atr and mepyramine work on different receptors. This is as expected for
it is known that histamine does not bind to muscarinic receptors but inhibits histamine by
competition when binding to the H1 receptor. (Kavoussi et al, 2007).
ACh was affected in the 1x10-5M dosage of mepyramine despite it not being antagonist for ACh
because the selectivity of drugs is lost in higher dosages. At the lower and more therapeutic dose,
there was still a high affinity with histamine thus ACh gave its average EC50 response. At higher
concentrations, this affinity is lost as other binding sites are considered for occupation, in this case
the muscarinic receptors. Thus at certain concentrations antagonists can act on more than one
agonist.
As the experiment focused only on the contractile response of the drug - a functional assay, a
measure of affinity expressed as the KD (association constant), could not be obtained due to lack of
information about the amount of drug binding to tissue. The response is concerned with the
activated state whereas the affinity is concerned with the agonist initially binding to the receptor to
form a complex. Also, doses that do not produce an immeasurable response in terms of a
contraction may still have agonist-receptor binding occurring but there is no way of estimating this
reliably with the information given.
The results followed the trends expected; however their quality for the live preparation could have
been made more accurate if time was not a limitation. The purpose of the 3 minute dose-wash cycle
was to bring the tissue back to its relaxed state and to ensure no trace of the previous dose was in
effect. Yet following higher concentrations this cycle was insufficient, seen in the unstable and
sometimes non-uniform baseline of the representative trace. Fluctuations in what should have been
the relaxed state also occurred. This was due to the spontaneous activity of our tissue when it was
not receiving sufficient amount of carbogen and because the tissues had been set up an hour prior
to the experiment. This meant that response peaks would not have all been measured from the
same point. A longer dose-cycle wash would reduce these discrepancies and make the data more
reliable.
Reliability of the results could be improved through repetition of the experiment so that multiple
responses to the each dose could be compared and a more accurate average obtained. Another
source of error, although slight, is the tendency of the tissue to become desensitised after
subsequent doses thus affecting the responses. Tachyphylaxis can occur over a period of time, which
involves the depletion of the neurotransmitter that is involved in the action of the drug. This is not
something that can be improved on however, without changing the tissue several times which would
cause larger inconsistencies, but should be taken into account when analysing results.
Thus it can be seen that in the presence of a competitive antagonist, the response of an agonist is
reduced as the potency is reduced. The maximum response is still surmountable however but
requires a higher dosage of agonist. In the case of a selective antagonist, this affinity to a specific
receptor can be compromised in higher concentrations, as it begins to bind to other receptors.
References:
Ehlert, F.J. (2001), Acetylcholine-Induced Desensitization of Muscarinic Contractile Response in
Guinea Pig Ileum is Inhibited by Pertussis Toxin Treatment, The Journal of Pharmacology and
Experimental Therapeutics, Vol. 299. No. 3 1126-1132
Feldburg, W. (1950), Effects of Ganglion-Blocking Substances on the Small Intestine, National
Institute for Medical Research, 113, pp. 483-50
Kardiol, Z. (1991), Action of acetylcholine on smooth muscle, Department of Pharmacology & Clinical
Pharmacology, pp. 7, 73-77
Kavoussi LR, Novick AC, Partin AW, Peters CA (2007), Wein: Campbell-Walsh Urology, 9th ed. Volume
3. W.B. Saunders.
Neal, M.J. (2009), Medical Pharmacology at a Glance, Blackwell Science, 5th Edition, pp. 8-10
http://www.wisegeek.com/what-is-an-agonist.htm (last accessed 27/3/10)
http://en.wikipedia.org/wiki/Competitive_antagonist (last accessed 29/3/10)
http://en.wikipedia.org/wiki/Tachyphylaxis (last accessed 30/3/10)
Appendices:
Appendix 1: Apparatus used to record contractions of guinea pig ileum. Water circulates through the
equipment as indicated, warming the solution to a physiological temperature. The clamps shown are
opened and closed in a specific order when performing tissue wash.
Appendix 2: Contractile responses to acetylcholine alone and in the presence of atropine
[Ach]
(µmol)
Bath[Ach]
(M)
Response
(au)- Control
% Maximum Response to
ACh (control)
0.01
0.1
0.1
0.1
1
1
1
10
10
10
100
100
100
100
2.00E-10
5.00E-10
1.00E-09
2.00E-09
5.00E-09
1.00E-08
2.00E-08
5.00E-08
1.00E-07
2.00E-07
5.00E-07
1.00E-06
2.00E-06
4.00E-06
1.064
1.3423
0.2319
8.4222
23.3321
58.7586
68.9951
73.8923
76.7242
77.3217
78.7404
79.1633
1.34
1.70
0.29
10.64
29.47
74.22
87.16
93.34
96.92
97.67
99.47
100.00
Response (au)- ACh
& Atropine
% Maximum Response
to ACh and Atropine
0.3192
0.2783
0.8594
0.7939
14.6508
42.6455
56.6524
69.1915
73.1993
74.3916
75.2783
0.42
0.37
1.14
1.05
19.46
56.65
75.26
91.91
97.24
98.82
100.00
25-3-2010
25/03/2010 11:44:43.195 AM
100
Bench 8 (Arb. Unit)
80
60
20
0.1ml of 100um ACh
40
23
2210
2220
24
2230
2240
2250
2260
Appendix 3: Acetylcholine sample representative trace
Appendix 4: Raw data pad
Comment
No.
Comment Text
3
0.4ml of 0.01um ACh
5
0.1ml of 0.1um ACh
7
0.2ml of 0.1um ACh
9
0.4ml of 0.1um ACh
11
0.1ml of 1um ACh
13
0.2ml of 1um ACh
15
0.4ml of 1um ACh
17
0.1ml of 10umol ACh
19
0.2ml of 10um ACh
21
0.4ml of 10um ACh
23
0.1ml of 100um ACh
25
0.2ml of 100um ACh
27
0.4ml of 0.1um ACh
29
0.1ml of 1um ACh
31
0.2ml of 1um ACh
33
0.4ml of 1umol ACh
35
0.1ml of 10umol ACh
37
0.2ml of 10umol ACh
39
0.4ml of 10um ACh
41
0.1ml of 100um ACh
43
0.2ml of 100um ACh
45
0.4ml of 100um ACh
47
0.8ml of 100um ACh
Max - Min
1.064
1.3423
0.2319
8.4222
23.3321
58.7586
68.9951
73.8923
76.7242
77.3217
78.7404
79.1633
0.3192
0.2783
0.8594
0.7939
14.6508
42.6455
56.6524
69.1915
73.1993
74.3916
75.2783
2270
2280
2290
2300
2310
2320
120.00
% Maximum Response
100.00
80.00
60.00
% Maximum Response to Ach
(control)
40.00
% Maximum Response to
Ach+Atropine
20.00
0.00
0.00E+00
-20.00
1.00E-06
2.00E-06
3.00E-06
4.00E-06
Bath [ACh] M
Appendix 4: The linear dose-response relationship
5.00E-06