MIT-WPU | School of Pharmacy

WORLD’S FIRST UNIVERSITY

FOR LIFE TRANSFORMATION

5. TO STUDY THE DOSE RESPONSE CURVE (DRC) OF

ACETYLCHOLINE USING FROG RECTUS ABDOMINIS MUSCLE.

Dr. Chinmay Deshmukh

Assistant Professor
Email: chinmay.deshmukh@mitwpu.edu.in
 Reference books
1. A Practical book of Pharmacology II by Dr. Pankaj M. Choudhari, Dr. Dheeraj T. Baviskar and Dr. Prakash

Patil, PV books, S Vikas and company (medical publishers) Jalandhar, 2019.

2. Ghosh M.N., Fundamentals Of Experimental Pharmacology, 3 rd Edition.

3. Kulkarni S.K., Practical Pharmacology And Clinical Pharmacy, Vallabh Publication,.

4. A practical book of Pharmacology-II by Hemant Suryawanshi, Mukesh Patel and Sunil Pawar, Nirali

Prakashan, Second edition, January 2020.

5. X-cology software CD for demonstration

 1. Dose response curve of Acetylcholine
• Requirement: Student kymograph, student
organ bath, aeration tube, aerator, aeration tube
holder, frontal writing lever, lever holder, screw
clip, forceps, rubber tube, tuberculin syringe,
pithing needle, scissor, forceps.
• Drugs: Acetylcholine stock solution (1 mg/ml),
PSS-frog ringer solution bubbled with air
• Animal: Frog
• Physiological and experimental conditions:
• Temperature of organ bath: 37 ± 0.050 C
• Tension on lever: 500 mg
• Aeration: Air bubbled with oxygen and carbon
dioxide
• Magnification value: (should be near to 5)
• Base line: 30 Sec, Response: 60 Sec
• Speed of Drum: 0.25 rpm
• Concentration of standard Ach = 10 µg/ ml
• Stabilization of tissue time: 30 min
 Theory

• Dose response relationship demonstrates hierarchical responses to medication or agonist wherever a rise
of response is recorded with increase of the dose or concentration of drug.

• This relationship is well explained by dose response curve (DRC). The DRC is S shaped curve. The first
part of the curve (25 % graph) has poor discrimination between doses and response whereas central
portion shows bigger sensitivity to concentration and therefore increasing responses [see figure].

• The last part of curve shows the ceiling result where no any additional increase in response is seen even
more increase in dose or concentration.
• Advantages
• Disadvantages
• The method is easy to carry and study.
• The technique is simple and less time consuming. For • Dose–response relationships generally depend
tissues which slowly contract and relax.
on the exposure time and exposure route (e.g.,
• Large dose range can also be plotted and errors are
distributed all through the graph with the help of inhalation, dietary intake); quantifying the
logarithmic transformation of doses in DRC.
• The study of antagonist effect and agonist comparison response after a different exposure time or for a
is easy. different route leads to a different relationship
• Dose-response, which involves the principles of
pharmacokinetics and pharmacodynamics, determines and possibly different conclusions on the effects
the required dose and frequency as well as the
therapeutic index for a drug in a population. of the stressor under consideration
• The therapeutic index (ratio of the minimum toxic • The errors in the method cannot be denied.
concentration to the median effective concentration)
helps determine the efficacy and safety of a drug.
• Increasing the dose of a drug with a small therapeutic
index increases the probability of toxicity or
ineffectiveness of the drug
• The pharmacological profiles of individual drugs can
be compared based on DRC
 Principle

• Frog rectus abdominis muscle contains nicotinic N2 receptors and acetylcholine work as an agonist.
Frog rectus abdominis muscle is voluntary muscle.

• At the neuromuscular junction, nerve impulses liberate Ach from nerve ending into the cleft
between nerve fiber and muscle. This acetylcholine causes a depolarization of the muscle fiber
which in turns sets off a muscle action potential and contraction of the muscle fiber.

• Acetylcholine is cholinergic drug which act on cholinergic receptors namely muscarinic receptors.
Its effect is contraction of smooth muscle so it is employed in pharmacology practical to assess its
effect on smooth muscle (rectus abdominis muscle).
 Acetylcholine
• Acetylcholine is an organic molecule that acts as a neurotransmitter in many organisms, including
humans.

• It is an ester of acetic acid and choline, There are two main classes of acetylcholine receptor (AChR),
nicotinic acetylcholine receptos (nAChR) and muscarinic acetylcholine receptors (mAChR).
Nicotinic AChRs are ionotropic receptors permeable to sodium, potassium, and calcium ions.

• They are stimulated by nicotine and acetylcholine. They are stimulated by Muscatine and
acetylcholine.

• Muscarinic receptors are found in both the central nervous system and in the peripheral nervous
system of the heart, lungs, upper GI tract, and sweat glands. ACh is sometimes used during
cataract surgery to produce rapid constriction of the pupil.
 Effect of Ach on muscle

• Contraction of smooth muscle by acetylcholine is mediated by activation of Muscarinic receptors of

which M2 and M3 subtypes are present in longitudinal muscle of guinea pig intestine.

• In single cells, Muscarinic receptor activation evokes calcium release from stores which raises the
internal free calcium concentration and causes opening of calcium-activated potassium channels.

• The rise in internal calcium suppresses the voltage-dependent inward calcium current.

• A third important effect is the opening of channels which cause depolarization of the membrane
and so increase action potential discharge and contraction in the whole muscle
 Precaution for good responses
• Clean the assembly before experiment
• Balance lever horizontally with load.
• Maintain required temperature of water bath.
• Maintain required and constant speed of drum.
• Maintain slow and smooth aeration to tissue.
• Prepared stock solution, working solution, tyrode solution in distilled water.
• Make PSS for experiment with exact quantities and add calcium chloride at the end in PSS to avoid
turbidity and precipitation. PSS should be clear
• Try to minimize handling of tissue, remove extra tissue cautiously. This is to prevent damage of
receptors.
• Keep less quantity of Tyrode solution in internal tube in such a way that tissue is just dipped into
Tyrode. This will not dilute the drug much and response will be obtained.
• Maintain constant dose response cycle (time cycle) to maintain tissue sensitivity
• Donts
• Do not start button of water bath in absence of water in organ bath
• Do not rotate drum manually when knob is closed.
 Observation & Calculations
Sr. No. Dose of Log Height of % response • Calculations and Graph:
Acetylcholine dose of contraction
• Dose in µg calculations
Ach (response) in
ml µg mm A) Concentration of Ach standard: 10 µg /ml, i.e.
10 µg for 1 ml so for 0.1 ml

1 0.1 1 0 2 13.33
B) % response calculations: (maximum response’s
2 0.2 2 0.3010 4 26.66
dose is considered as 100%)
3 0.4 4 0.6020 5 33.33

4 0.8 8 0.9030 7 46.66

C) Graph:
5 1.6 16 1.2041 10 66.66
• Plot the graph is between percent response on Y
6 3.2 32 1.5051 12 80
axis and log dose on X axis respectively
7 6.4 64 1.8061 13 86.66

8 12.8 128 2.1072 15 100

9 25.6 256 2.4082 15 100

 Interpretation & Result:

• The dose response curve of Acetylcholine using frog rectus abdominis muscle was observed and

ceiling effect was observed at dose of 12.8 and 25.6 ml Ach and height of response is observed as 15

mm (100%)
 Short Questions

1. Define Dose response curve.

2. Write advantages and disadvantages of DRC.

3. Write mechanism of Ach action on contraction of smooth

muscle. 

4. Write principle of determination of dose response relationship

of Ach using frog’s rectus abdominis muscle

THANK YOU
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