Objective Principle Requirements Experimental specifications (conditions) Preparation of ACh and Atropine stock and std. solutions Preparation of Tyrode solution (PSS) Procedure Kymograph recording of contractions Observation table Calculation Graphical presentation of CRC/ DRC Result and interpretation

1. Experiment No. 9
Effect of atropine on DRC of acetylcholine
using rat ileum
Mr. Vishal Balakrushna Jadhav
Assistant Professor (Pharmacology)
GES’s Sir Dr. M. S. Gosavi COPER, Nashik-5

2. Overview of Discussion
• Objective
• Principle
• Requirements
• Experimental specifications (conditions)
• Preparation of ACh and Atropine stock and std. solutions
• Preparation of Tyrode solution (PSS)
• Procedure
• Kymograph recording of contractions
• Observation table
• Calculation
• Graphical presentation of CRC/ DRC
• Result and interpretation
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3. Objective
To study antagonistic effect of atropine on the CRC/
DRC of acetyl choline (ACh) using isolated rat ileum
muscle preparation.
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4. Combined effect of drugs
4
When two or more drugs are given simultaneously or in quick succession
they may be either indifferent to each other or exhibit synergism or
antagonism. The interaction may take place at pharmacokinetic level or
at pharmacodynamic level.
Antagonism When one drug decreases or abolishes the action of another,
they are said to be antagonistic-
Effect of drugs A + B < Effect of drug A + Effect of drug B
Usually in an antagonistic pair one drug is inactive as such but decreases
the effect of the other.
Receptor antagonism One drug (antagonist) blocks the receptor action of
the other (agonist). This is a very important mechanism of drug action,
because physiological signal molecules act through their receptors,
blockade of which can produce specific and often profound
pharmacological effects.
Principle

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Receptor antagonists are selective (relatively), i.e. an anticholinergic will
oppose contraction of intestinal smooth muscle induced by cholinergic
agonists, but not that induced by histamine or 5-HT (they act through a
different set of receptors).
Receptor antagonism can be of competitive or noncompetitive type.
1) Competitive antagonism (equilibrium type)
The competitive antagonist is chemically similar to the agonist, competes
with it and binds to the same site to the exclusion of the agonist
molecules. Because the antagonist has affinity but no intrinsic activity, no
response is produced and the log DRC of the agonist is shifted to the right.
Since antagonist binding is reversible and depends on the relative
concentration of the agonist and antagonist molecules, higher
concentration of the agonist progressively overcomes the block- a parallel
shift of the agonist DRC with no suppression of maximal response is
obtained. The extent of shift is dependent on the affinity and
concentration of the antagonist.
A partial agonist having affinity for the same receptor, also competes with
and antagonizes a full agonist, while producing a submaximal response of
its own.

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2) Non-competitive antagonism
The antagonist is chemically unrelated to the agonist, binds to a different
allosteric site altering the receptor in such a way that it is unable to
combine with the agonist or is unable to transduce the response, so that
the downstream chain of events are uncoupled. This is also called as
allosteric antagonism.
Because the agonist and the antagonist are combining with different sites,
there is no competition between them-even high agonist concentration is
unable to reverse the block completely. Increasing concentrations of the
antagonist progressively flatten the agonist DRC. Noncompetitive
antagonists have been produced experimentally, but are not in clinical use.
3) Non-equilibrium (non-competitive) antagonism
Certain antagonists bind to the receptor with strong (covalent) bonds or
dissociate from it slowly so that agonist molecules are unable to reduce
receptor occupancy of the antagonist molecules law of mass action
cannot apply-an irreversible or non-equilibrium antagonism is produced.
The agonist DRC is shifted to the right and the maximal response is
lowered (if spare receptors are few).

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Since flattening of agonist DRC is a feature of noncompetitive antagonism;
non-equilibrium antagonism has also been called 'a type of noncompetitive
antagonism'. This appears inappropriate because the antagonist is binding
to the same site as the agonist, e.g. phenoxybenzamine is a non-equilibrium
antagonist of adrenaline at α- adrenergic receptors.
Features of competitive and noncompetitive antagonism are compared below-

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Since flattening of agonist DRC is a feature of noncompetitive antagonism;
non-equilibrium antagonism has also been called 'a type of noncompetitive
antagonism'. This appears inappropriate because the antagonist is binding
to the same site as the agonist, e.g. phenoxybenzamine is a non-equilibrium
antagonist of adrenaline at α- adrenergic receptors.
Features of competitive and noncompetitive antagonism are compared below-

9. Rat ileum is the smooth muscles which is sensitive to ACh and shows
contraction. ACh acts on muscarinic M3- subtype of receptor which
function through Gq protein and activate membrane bound
phospholipase C (PLc) → generating inositol trisphosphate (IP3) and
diacylglycerol (DAG), the secondary messengers release Ca2+
intracellularly → cause depolarization and thereby smooth muscle
contraction.
Atropine acts as competitive antagonist of ACh at muscarinic receptors
and produces competitive, reversible and surmountable type of
antagonism characterized by parallel shift of CRC/ DRC of ACh towards
the right side in the presence of atropine without suppression of
maxima. Such an antagonism is reversible as increased concentration of
ACh abolishes the access of atropine onto the muscarinic receptors.
Atropine or other anticholinergics neither react chemically with ACh nor
do they interfere with the hydrolysis of ACh.
Atropine shows some structural similarities with ACh which induces
antagonism- the critical distance between the nitrogen (-N) and the
carbonyl oxygen (-C=O) is 7A°.
Overnight fasted rats are used to record better response of ACh on
intestinal smooth muscles such as ileum.

10. Requirements
Animal: Albino rats (150-200 g, overnight fasted)
Physiological solution: Tyrode solution.
Drug- ACh (Stock solution: 1 mg/ml), Atropine (Stock
solution: 0.1 mg/ml)
Chemical- Fixing solution.
Instruments: Sherrington recording drum , Student organ
bath, Aerator, Insulin or tuberculin syringe to inject drugs
in small fractions, Dissecting board and various dissecting
instruments. Frontal writing lever and stand, Pipette,
Stop watch etc.
Miscellaneous: Kymograph paper, plasticin, clips, and
thread.
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11. Experimental specifications (conditions)
 Isolated tissue- Isolated rat ileum preparation
 Drug- ACh (Stock solution: 1 mg/ml), ACh std. (1/ 10/ 100
μg/ml), Atropine (Stock solution: 0.1 mg/ml), Atropine std. (2
μg/ml)
 Physiological salt solution (PSS)- Tyrode solution.
 Time cycle- Total- 5 minutes, Base line- 30 seconds, Contact
time- 90 seconds, Washing period- 3 minutes
 Applied load/ tension- 0.5 g
 Bath capacity- 40 ml
 Bath temperature- 32- 35°C
 Speed of rotation of drum- 0.25 mm/ second
 Magnification value (Mf) = d (F-W)/ d (F-T)
 Aeration- Normal air (1- 2 bubbles/ second) 11

12. Preparation of ACh stock and standard solutions
Stock solution- 1 mg/ml or 1000 μg/ml
 Dissolve 1 mg of ACh in 1 ml of distilled water → 1000 μg/ml
Standard solutions
 Dilute 1 ml of stock solution up to 10 ml with distilled water → 100 μg/ml
 Dilute 1 ml of 100 μg/ml solution up to 10 ml with distilled water → 10 μg/ml
 Dilute 1 ml of 10 μg/ml solution up to 10 ml with distilled water → 1 μg/ml
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Preparation of Atropine stock and standard solutions
Stock solution- 0.1 mg/ml or 100 μg/ml
 Dissolve 1 mg of atropine sulphate in 10 ml of Tyrode solution → 100 μg/ml
Standard solutions
 Dilute 0.2 ml of stock solution up to 10 ml with Tyrode solution → 2 μg/ml

13. Preparation of Tyrode solution (PSS)
 Prepare 1 litre of Tyrode solution by dissolving NaCl (8.0
g), KCl (0.2 g), MgCl2 (0.1 g), NaHCO3 (1.0 g), NaH2PO4
(0.05 g) and glucose (2.0 g) in distilled water.
 MgCl2 should be added at last.
 CaCl2 (0.2 g) should be dissolved separately in distilled
water to avoid chances of precipitation of salt.
 Mix CaCl2 solution to the higher volume of PSS.
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14. Procedure
 Albino rat is sacrificed by a blow on the head and carotid
bleeding.
 Cut open the abdomen and lift the caecum to trace the ileocecal
junction. Cut and remove a few centimeter long of the ileal
portion and immediately place it in the watch glass containing
Tyrode solution. Trim the mesentery and with gentle care clean
the contents of the ileum by pushing the Tyrode solution into
the lumen of the ileum. Utmost care should be taken to avoid
any damage to the gut muscle. Cut the ileum into small
segments of 2-3 cm long.
 Take a piece of ileum of 2-3 cm long and tie the thread to the top
and the bottom ends without closing the lumen, and mount the
tissue in the organ bath containing Tyrode solution maintained
at 32- 35°C and bubbled with O2 or air. A tension of 0.5 g is
applied and the tissue is allowed to equilibrate for 30 minutes
before adding the drugs to the organ bath.
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15.  Record the CRC/ DRC of ACh std. using at least five gradual doses.
 Add atropine (2 μg/ml) to the reservoir containing Tyrode solution
and irrigate the tissue with atropinized Tyrode for 30 minutes.
 Repeat the CRC/ DRC of ACh std. (same conc.) in presence of
atropine.
 Properly label and fix both the CRCs/ DRCs of ACh std. in absence
and in presence of atropine.
 Measure the height of the response (cm/ mm) and plot both the
CRCs/ DRCs graphically.
 Note the reduction (antagonism) in the response of ACh. Read the
relative EC50/ ED50 values and calculate the dose ratio using the
formula-
 Describe the shift of CRC/ DRC of ACh std. in presence of atropine
and interpret the result.
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16. Kymograph recording of contractions
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Fig. Dose Response Curve (DRC) of ACh Std. in absence and
in presence of atropine.

17. Observation table
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18. Graphical presentation
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19. Calculations
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Magnification value (Mf) = d (F-W)/ d (F-T)
Where-
d (F-W) → distance between fulcrum and stylus (writing tip)
d (F-T) → distance between fulcrum and point of attachment of
tissue
Dose Ratio (DR)
DR= Antilog (A)/ Antilog (B)

20. Result and interpretation
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The antagonistic effect of atropine on CRC/ DRC of ACh std. using
isolated rat ileum muscle preparation was interpreted as the shift of
CRC/ DRC of ACh std. towards the right side in the presence of
atropine without suppression of maxima, which is an indicative of
competitive type of receptor antagonism.
The dose ratio by the graph % Response Vs log (dose) was found to
be-...............
The magnification value was found to be- ...............

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Thank You...