Experimental Pharmacology for Undergraduates & Postgraduates J Sujatha Devi
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EXPERIMENTS FOR THE UNDERGRADUATES

Effect of Drugs on the Isolated Skeletal Muscle (Frog Rectus Muscle)1

 
EFFECT OF ACETYL CHOLINE
 
Aim
To study the effect of Acetyl choline.
 
Apparatus Required
  1. Rudolf Magnus organ bath
  2. Kymograph
  3. Stop watch.
 
Nutrient Solution
Constituents of frog ringer solution—For 5 liters
1. Sodium chloride
32.5 gm
2. Potassium chloride (15%)
4.65 cc
3. Calcium chloride (16%)
3.75 cc
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Fig. 1.1: Rudolf Magnus organ bath
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4. Sodium bicarbonate
1 gm
5. Dextrose
10 gm
Mix all the above mentioned chemicals in 5 liters of distilled water. Calcium chloride should be added at the end of the preparation of nutrient solution, to avoid the formation of white precipitate.
 
Drugs Used
Acetyl choline.
 
Arrangement of the Bath
The main instrument used in this experiment is the Rudolf Magnus organ bath. It consists of an outer bath and an inner bath.
The outer bath—Water is filled in this bath and can be heated by an electric heating element. It has got a thermostat, so that the temperature can be adjusted according to the experiment.
The inner bath—The capacity should be kept constant at 20 ml [put a small red mark on the inner bath]. This inner bath is connected to a reservoir containing the nutrient solution. The flow of the nutrient solution in and out of the inner bath is regulated by stop cocks.
Tissue holder [‘J'tube] is kept inside the inner bath. The isolated tissue is mounted to the tissue holder and the other end of the tissue is tied with a thread to the simple lever of the 1st order.
The lever writes on the kymograph. The speed of the kymograph is one revolution in every 64 minutes.
The magnification of the response obtained should be adjusted between 10 to 15 times of the actual response.
The upper bent end of the tissue holder is connected to an aerator that provides oxygen to the tissue by supplying air.
 
Dissection and Mounting of the Tissue
Dissection: A pithed frog is laid on its back in a tray. The skin on the abdominal wall is removed. The two rectimuscles in the midline extending from xiphisternum to the symphysis pubis is identified. The borders of the muscle is dissected out from the adjacent abdominal wall muscles. The two rectimuscles also separated with a scissors. With the help of a needle and thread, put a tight ligature round the muscle, just above the symphysis pubis and just below the xiphisternum with a long thread attached. Then cut and remove the muscle strip from the symphysis pubis and xiphisternum.
Mounting: The lower end of the isolated rectus muscle is tied closely to the bent portion of the (lower end) tissue holder at its centre. The upper end of the tissue with the long thread is tied to the simple lever placed above, so that when the muscle contracts, the writing point goes up.
During the dissection and attaching muscle to the tissue holder, the muscle must be kept moist with the frog ringer solution.
 
Procedure
  1. Allow the tissue to relax for 30 minutes and wash the tissue, for 2 times by allowing the nutrient solution in and out of the inner bath.
  2. Switch on the kymograph and see whether the steady base line is drawn on the drum.
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Fig. 1.2: Recording on the drum
 
Effect of Acetyl Choline
About 0.05 ml of 100 μg/ml concentration of Acetyl choline is added to the inner bath. The muscle contraction is noted. The drum is stopped and the tissue is washed. The experiment is repeated after an interval of 5 minutes every time, with 0.1 ml, 0.2 ml, 0.4 ml, 0.8 ml of Acetyl choline.
 
Inference
  1. The ACh is an agonist and produces contraction of skeletal muscle.
  2. Increase in dose of ACh produces increase in response. This is known as dose response curve.
  3. 0.4 ml produces the maximum effect. So in this curve 0.4 ml is the maximum dose, and the response is called as maximum response. It can also be called as Ceiling dose and Ceiling effect, respectively.
  4. 0.8 ml is called as supramaximal dose and the response produced by it is called as supra—maximal response.
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POTENTIATING EFFECT OF PHYSOSTIGMINE
The drum is started and 0.4 ml of ACh of 100 μg/ml concentration is added to the organ bath. The muscle contraction is noted on the drum. The drum is stopped and the tissue is washed. The experiment is repeated after an interval of 5 minutes.
Then 0.4 ml of Physostigmine [eserine] of 1 mg/ml concentration is added to the inner bath. After 5 minutes the drum is started and 0.4 ml of ACh is added and the response is noted.
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Inference
Physostigmine is an anticholinesterase drug. So it potentiates the effect of ACh.
 
BLOCKING EFFECT OF D-TUBOCURARINE
The drum is started and 0.4 ml of ACh of 100 μg/ml is added to the inner bath. The contraction is noted on the drum. The procedure is repeated again after washing the tissue.
Then 0.4 ml of 200 μg/ml of D-Tubocurarine is added to the inner bath. After 2 minutes the drum is started and 0.4 ml of 100 μg/ml of ACh is added to the bath. The effect is recorded on the drum.
 
Inference
D-Tubocurarine is a skeletal muscle relaxant and it is a blocker [Antagonist]. So it blocks the neuromuscular receptors [NM] and blocks the effect of the agonist—ACh.
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How to Proceed in the Practical Examination?
Example—
Find out the nature of the given test drug by using isolated skeletal muscle preparation.
Mount the (Frog rectus) isolated skeletal muscle tissue preparation. Draw the contractions of 0.2 ml, and 0.4 ml of ACh solution. Add 0.1 ml of the given test solution to the inner bath and draw the contraction on the drum. Again draw the contraction of 0.4 ml of ACh solution. As per the observations of the graph, detect the test drug, and write the inference accordingly.
Example—1
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Inference
  1. The test drug is a cholinergic drug, since it produces contraction of the isolated skeletal muscle.
  2. It produces no change in the effect of known ACh.
Example—2
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Inference
The test drug is a blocker (antagonist of Nm receptor). Probably D-Tubocurarine, since it is not producing any contraction of skeletal muscle and also it blocks the effect of ACh.
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Example—3
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Inference
The test drug is indirectly acting cholinergic drug like Physostigmine, since it is an anticholinesterase drug. It potentiates the effect of ACh.
Probable oral questions—
  1. What is the use of outer bath?
    The temperature of the water in the outer bath can be adjusted (using autostat) so that the temperature will be conveyed to the nutrient solution and the isolated tissue inside the inner bath.
  2. What temperature should be maintained for Isolated frog tissue?
    Atmospheric temperature will be sufficient, since the frog is an amphibian.
  3. What is the use of aerator?
    It supplies air and oxygen to the tissue. Air bubbles produced by the aerator mix the drug in the nutrient solution of inner bath evenly.
  4. What is the magnification of the graph and how will you increase?
    The actual contraction produced by the isolated tissue is magnified for about 10 to15 times and drawn on the drum by the lever.
    By adjusting the distance between the fulcrum to the writing point, and fulcrum to the (thread tied) tissue (i.e. effort arm and load arm) the magnification can be adjusted.
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  5. What type and order of lever is used here?
    Simple lever is used. It is of first order. Fulcrum is in middle, load on one end and effort on other end.
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  6. What is the mechanism of action of ACh on isolated frog rectus?
    Acetyl choline (ACh) is an agonist and acts on the NM receptor situated in the neuro-muscular junction of the (skeletal muscle) frog rectus.
  7. What is the mechanism of action of physostigmine?
    It inhibits the cholinesterase enzyme thereby potentiates ACh effect. So it is an indirectly acting cholinergic drug.
  8. What is the mechanism of action of D-tubocurarine?
    It is a reversible (competitive) antagonist of NM receptors. It blocks the NM receptors thereby prevents the effect of the agonist, i.e. ACh.
  9. Why is there an increase in quantity of dextrose in nutrient solution?
    Skeletal muscle needs more dextrose for its contraction.
  10. How will you increase contraction of skeletal muscle?
    By increasing the quantity of dextrose and by adding eserine (physostigmine) in nutrient solution, skeletal muscle contraction can be increased in case of low response.