Introduction

Majority of drugs structures correlate with specificity to produce pharmacological effects by

interacting with receptor sites with specific three dimensional structures and protein-like
characteristics located on the cell membrane or in the cytoplasm. Receptors bind specifically to
a second molecule which brings about the regulation of a cellular process. A ligand (such as
drugs) may activate or inactivate a specific receptor increasing or decreasing a particular cell
function respectively (Hafner, et al., 1976). These ligands are classified as agonist and
antagonists respectively. An agonist binds and interacts with a receptor producing effects via
mimicking the actions of natural regulators of signal transducing receptors. Whereas antagonist
interacts with the receptor site and blocks or inhibits the normal response for receptor. Most
antagonists which bind reversibly at the receptors binding site compete with agonists, called
competitive agonist .Guinea-pig ileum contains a variety of receptor types including muscarinic
M3 receptors and histamine H1 receptors making this specific ileum an important tool to study the
effects of drugs on specific type of receptors. Preparations of this ileum are also used to
characterize specific receptor or its subtypes, to determine concentration -response curve of an
agonist and to investigate antagonism of drug. Muscarinic and histamine receptors are G-protein
coupled receptors. Five subclasses of muscarinic receptors: M 1, M2, M3, M4, and M5 and three
subclasses of histamine: H1, H2 and H3 are identified.

Acetylcholine is an important

neurotransmitter binds to both muscarinic and nicotinic receptors. Binding of acetylcholine to

muscarinic receptors initiates contraction of smooth muscles of ileum (Caulfield, 1993).
Histamine is an amine produced by decarboxylation of amino acid histidine). It also acts as a
neurotransmitter binding on H1-histamine receptors causing contractions of smooth muscles in
the ileum(Trzeciakowski, 1987).
Aim
Aim of this experiment is to investigate Guineas pig smooth muscle responses to different
concentrations of acetylcholine and histamine. Also to be able identify antagonists X and Y by
observing their antagonistic effects on the concentration -response curve for both acetylcholine
and histamine separately. And to further characterize antagonism of drugs X and Y.
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Materials and methods

A guinea pigs ileum was isolated and maintained in a bath of Holmans solution at 34C under 1
gram resting tension .From Hist stock solution (10-1 M) provided, serial dilutions ranging
from10-1 M to 10-6 M of Hist were prepared. A baseline for about 15 seconds was obtained and at
time 0, 0.2 ml of 10-6 M Hist was added to the bath with ileums contractions (tension g)
recorded for 15-30 seconds. The ileum was washed twice by overflow with 20 mL of Holmans
solution at 15 seconds and 2 minutes. The experiment was repeated with increasing final
concentrations of Hist ranging from 10 -8 M to 10-3 M (table 1). Unknown antagonist Y was
diluted to concentration of 10-7 M in a 500ml Holmans solution. Next, 20ml of 10 -7M antagonist
Y was added to equilibrate ileum in the antagonist solution for 15 minutes. Hist was added
according to table (1) as well, but in the presence of unknown antagonist Y in the final solution
bath as well.

Results were obtained from other groups who used Hist with and without

antagonist Y, and Ach with and without antagonists X and Y.

Table 1: Volume (mL) of Hist drug added to the bath in order to make up to final bath
concentration.
Volume and concentration of Hist added
to bath

Final bath concentration (M)

0.2 mL of 10-6 M

10-8

0.6 mL of 10-6 M

3 10-8

0.2 mL of 10-5 M

10-7

0.6 mL of 10-5 M

310-7

0.2 mL of 10-4 M

10-6

0.6 mL of 10-4 M

310-6

0.2 mL of 10-3 M

10-5

0.6 mL of 10-3 M

310-5

0.2 mL of 10-2 M

10-4

0.6 mL of 10-2 M

310-4
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0.2 mL of 10-1 M

10-3

Calculations:
1) Dilution of 10-1M histamine to 10-2 M:
Hist Stock solution = 1 x 10-1 M, to dilute to 10-2 M in a final volume of 1ml:
C1.V1 = C2.V2

10-1 M xV1 = 10-2 x 1ml

So V1 of Hist to be extracted from stock = (10-2 x 1ml)/10-1 M = 0.1ml

V2= 1 ml; so 1ml-0.1ml = 0.9 ml of Holmans solution should be added up to 1ml of the final
volume.
The same procedure is repeated to obtain serial dilutions from 10-2 M to 10-6 M of Hist solutions.
2) Dilution of antagonist Y to 10-7M:
-Dilution of 10-3M antagonist Y to 10-4M and final volume of 1ml;
C1.V1 = C2.V2

10-3 M xV1 = 10-4 x 1ml

So V1 = (1 x 10-4M x 1ml)/10-3 M = 0.1ml of antagonist Y to be added to 0.9ml of Holmans

solution to make up final solution volume of 1ml.
--Dilution of 10-4M of antagonist Y to 10-7M and final volume of 500ml;
C1.V1 = C2.V2

10-4 M xV1 = 10-7M x 500ml

So V1 = (10-7M x500ml)/10-4 M = 0.5ml of antagonist Y to be added to (500ml-0.5ml= 499.5 ml)

of Holmans solution to make up final solution volume of 500ml.
3) Volumes to be added from His diluted solutions to the final ileum bath:
-To obtain a 10-8M of Hist from diluted 10-6 M Hist solution to make up a final bath volume of
20ml:
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C1.V1 = C2.V2

10-6 M xV1 = 10-8M x 20ml

So V1 = (10-8M x20ml)/10-6 M = 0.2ml of diluted Hist solution to be added to the final ileum bath
solution.
The same procedure is repeated to obtain final bath concentrations in table (1).
Results
Table 2: Final concentrations of Histamine in the solution bath of Holman and magnitude of
contractions (response) of tissues in the guinea-pig isolated ileum bathed in the solution with and
without the presence of antagonist Y.
Tension (g) His

Tension (g)

log (concentration)

without antagonist

Histamine with

10-8

-8.0

Y
2.57

antagonist Y
0

310-8

-7.5

1.26

10-7

-7.0

1.48

310-7

-6.5

1.55

0.02

10-6

-6.0

1.61

0.01

310-6

-5.5

1.47

0.01

10-5

-5.0

1.35

0.37

310-5

-4.5

1.28

0.67

10-4

-4.0

1.12

0.7

310-4

-3.5

1.12

1.5

10-3

-3.0

0.83

4.79

Final Histamine concentration

(M)

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Figure 1: Graph of contraction response (tension in g) of the guinea-pig isolated ileum bathed in
Holmans solution against log (final Histamine concentration M in the bath) for both histamine
with and without the addition of antagonist Y to the bath solution.
Table 3: Final concentrations of Histamine in the solution bath of Holman and magnitude of
contractions (response) of tissues in the guinea-pig isolated ileum bathed in the solution with and
without the presence of antagonist X.
Final Histamine
concentration (M)

log (concentration)

Tension (g) Histamine

without antagonist X

Tension (g)
Histamine with

10-8

-8.0

0.08

antagonist X
0

310-8

-7.5

0.23

-0.05

10-7

-7.0

0.16

0.32

310-7

-6.5

0.47

0.18

10-6

-6.0

0.64

0.58

310-6

-5.5

0.94

1.13

10-5

-5.0

1.13

0.98

310-5

-4.5

1.1

1.3

10-4

-4.0

0.91

0.79

310-4

-3.5

0.72

0.88

10-3

-3.0

0.47

0.69

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Figure 2: Graph of contraction response (tension in g) of the Guinea-pig isolated ileum bathed in
Holmans solution against log (final Histamine concentration M in the bath) for both histamine
with and without the addition of antagonist X to the bath solution.
Table 4: Final concentrations of Acetylcholine in the solution bath of Holman and magnitude of
contractions (response) of tissues in the guinea-pig isolated ileum bathed in the solution with and
without the presence of antagonist X.
Final Acetylcholine
concentration (M)

log ( Final

Tension (g)

Tension (g)

Acetylcholine

Acetylcholine without

Acetylcholine with

concentration M)

antagonist X
0.07

10-8

-8.0

antagonist X
0.6

310-8

-7.5

1.03

0.12

10-7

-7.0

1.26

-0.02

310-7

-6.5

1.61

-0.04

10-6

-6.0

1.32

0.03

310-6

-5.5

1.24

0.1

10-5

-5.0

0.97

0.61

310-5

-4.5

1.12

1.15

10-4

-4.0

1.24

0.22

310-4

-3.5

0.74

0.45

10-3

-3.0

0.72

0.55
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Figure 3: Graph of contraction response (tension in g) of the guinea-pig isolated ileum bathed in
Holmans solution against log (final Acetylcholine concentration M in the bath) for both
histamine with and without the addition of antagonist X to the bath solution.
Table 5: Final concentrations of Acetylcholine in the solution bath of Holman and magnitude of
contractions (response) of tissues in the guinea-pig isolated ileum bathed in the solution with and
without the presence of antagonist Y.
Final Acetylcholine
concentration (M)

log ( Final

Tension (g)

Tension (g)

Acetylcholine

Acetylcholine without

Acetylcholine with

concentration M)

antagonist Y
0.24

10

-8.0

antagonist Y
0.25

310-8

-7.5

1.02

0.13

10-7

-7.0

1.09

0.32

310-7

-6.5

1.36

0.66

10-6

-6.0

2.92

0.73

310-6

-5.5

1.81

0.94

10-5

-5.0

1.17

0.97

310-5

-4.5

0.95

1.07

10-4

-4.0

1.01

1.53

310-4

-3.5

1.08

1.32

10-3

-3.0

1.67

2.47

-8

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Figure 4: Graph of contraction response (tension in g) of the guinea-pig isolated ileum bathed in
Holmans solution against log (final Acetylcholine concentration M in the bath) for both
histamine with and without the addition of antagonist Y to the bath solution.
Discussion
It is noted that increasing the concentrations of both Acetylcholine(Ach) or Histamine(Hist)
neurotransmitters to the ileum of Guinea-pig evoke a bigger response of the tissue via the
increase of magnitude of contractions(g) of the smooth muscles of the ileum as seen in figures
(1) to (4). This is because as the concentration of these drugs increase, more drug-receptor
complexes form which lead in an increase of contraction of smooth muscle tissue (Hafner, et al.,
1976). Thus both Ach and Hist are agonists to the muscarinic M 3 and histamine H1 receptors of
smooth muscles in the ileum respectively. Strength of the agonist-receptor complex efficiency to
produce a maximum functional response is compared relatively to other agonists (Hafner, et al.,
1976). Table (3) shows that concentration-response curve for His (without X) has maximal
responses at 10-5 M of Hist, while maximal responses of Ach (without X) at 310 -7 M of Ach
(table 4). This means that Ach reach its maximum response at lower Ach concentration compared
to Hist. Thus Ach has higher capacity (efficacy) to activate M3 receptor causing a higher
maximal response than Hist at a lower agonist concentration. Moreover, for same agonist (Ach
or Hist), maximal responses values are different among all four ileums even though the
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concentrations of agonists in the bathing solutions are the same. This could be since each ileum
possess different number of muscarinic and histamine receptors than other ileums (Rodriguez, et
al., 2006).Thus ileum with more muscarinic M3 and histamine H1 receptors allow more agonists
to bind to more the receptors, more agonist-receptor complexes are formed and subsequently
greater maximal response compared to other ileums for same agonist. In addition, Figures and
tables (1) to (4) show that as concentrations of agonists (Ach and Hist) increase beyond
maximum concentrations (At maximum response), tension (g) in smooth muscles of the ileum
decrease with it for a quick and short periods. This could be due to desensitization of other
receptors (beside the muscarinic M3 and histamine H1 receptors) via the agonists (Ach and Hist),
thus causing the ileum to undergo quick desensitization, resulting in decrease in contractile
response for a short period of time (few minutes).
The concentration-response curves for Ach and Hist in the absence of antagonists X and Y act as
a control in each graph in order to identify each unknown antagonists X and Y in the presence of
agonists(Hafner, et al., 1976). Concentration-response curve for Hist with antagonist Y in figure
(2) shows decreased tension (g) values as compared to the control. This means that histamine H 1
receptors are inhibited by antagonist Y. Since antagonist Y is occupying some histamine H 1
receptors, Hist cannot bind to these receptors to evoke a tissue response, subsequently decreasing
the contraction (Trzeciakowski, 1987). This means that drug Y is antagonist of histamine H 1
receptors, since Mepyramine is an antagonist at histamine H 1 receptors then drug Y is identified
as Mepyramine. Whereas figure (4) shows the concentration response curve for Ach in the
presence of drug Y is almost similar to the control, with only a slight decreased observed which
means that drug Y has no effect on muscarinic receptors as they are not inhibited by drug
Y(Caulfield, 1993).Ach can still bind to muscarinic receptor to elicit its effect and cause
contraction. Hence, drug Y is not atropine as it is an antagonist of the muscarinic receptors.
However, figure (3) shows that the presence of drug X inhibits the response of muscarinic
receptors as the concentration- response curve shows some negative tension(g) values at the
beginning (table 3). This means that drug X blocks the muscarinic receptors explaining the noresponse (negative tension) observed with few values at the beginning. Therefore, drug X is
antagonist of muscarinic receptors. Since Atropine is an antagonist of muscarinic receptors as
well, then it can be concluded that drug X is atropine
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Figure (1) shows that the concentration-response curve for Hist in the presence of drug Y shifts
parallel to the right of the control curve and reaches almost the same maximal response as the
control but at a higher Hist concentration (table2). This shows that drug Y is a competitive
antagonist of histamine H1 receptors. Since competitive antagonist binds to the receptors via
weak bonds such as hydrophobic bonds for example, the antagonist can then be evacuated from
the receptor, and agonist is still then able to produce the same maximal response observed before
addition of antagonist at a higher concentrations as more agonists are required to compete and
displace the antagonist (Trzeciakowski, 1987).However for figure (3), the concentrationresponse curve for Ach with antagonist X does not show a visible parallel shift to the right of the
control curve due to probably insufficient concentration of agonist administered to able to restore
maximum tissue response with the presence of the antagonist X(Caulfield, 1993). Thus
maximum response of the ileum tissues are lower than that of the curve without the antagonist X
which does not confirm whether drug X is a competitive antagonist or not.
Conclusion
Increasing concentrations of acetylcholine and histamine increases contractions of the guineapigs smooth muscles in the isolated ileum till a maximum response at a maximum concentration
of agonists is reached, which beyond desensitization of the histamine and muscarinic receptors
decreases the tension (response) eventually. Antagonists X and Y are Atropine and Mepyramine
respectively. Based on data and results, drug Y is a competitive antagonists of histamine
receptors, while drug X is an antagonist of acetylcholine muscarinic receptors. Adding sufficient
Ach concentrations with the presence of the antagonist drug X may confirm its competiveness or
not.
References
Caulfield,

M.

P.

(1993).

Muscarinic

receptorscharacterization,

coupling

and

function. Pharmacology & therapeutics, 58(3), 319-379.

Freedman, N. J., & Lefkowitz, R. J. (1995). Desensitization of G protein-coupled
receptors. Recent progress in hormone research, 51, 319-51

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Hafner, D., Heinen, E., & Noack, E. (1976). Mathematical analysis of concentration-response
relationships. Method for the evaluation of the ED50 and the number of binding sites per
receptor molecule using the logit transformation. Arzneimittel-Forschung, 27(10), 1871-1873.
Rodriguez, R., Ventura-Martinez, R., Santiago-Mejia, J., Avila-Costa, M.R & Fortoul, T.I.
(2006). Altered responsiveness of the guinea-pig isolated ileum to smooth muscle stimulants and
to electrical stimulation after in situ ischemia. British Journal of Pharmacology, 4, 371-378.
Trzeciakowski, J. P. (1987). Inhibition of guinea pig ileum contractions mediated by a class of
histamine receptor resembling the H1 subtype. Journal of Pharmacology and Experimental
Therapeutics, 243(3), 874-880.

(Lino & Nojyo, 2006).

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