# Effect of physostigmine on DRC of acetylcholine using frog rectus abdominis muscle and rat ileum respectively

Aim: To record the effect of Physostigmine (eserine ) on the concentration-response curve of acetylcholine using rat ileum preparation.

Principle:

The rat colon is an intestinal smooth muscle. Ach causes the contraction of the muscle by acting on the muscarinic receptor. The spontaneous contraction of muscle preparation can be reduced by the calcium level of the physiological salt solution and by maintaining the water bath at room temperature. The muscle preparation obtained from a starved rat gives a stable preparation. Physostigmine increased the levels of Ach.

Physostigmine is an anticholinesterase substance and it inhibits the metabolic breakdown of acetylcholine. As a result, the action of acetylcholine is potentiated. The concentration-response curve of acetylcholine will be shifted to the left in the presence of physostigmine.

Requirements:

Animal –frog

Drugs –Acetlycholine stock solution (1 mg/ml),

Physostigmine stock solution (1mg/ml)

Physiological solution – modified ringer with less calcium

Procedure:

1. albino rats (150-200 g) are fasted (deprived of food and water) overnight and saline(25ml/kg) is administered orally with the help of an oral feeding cannula.
2. Those animals are divided into four groups containing three rats in each as follows: (i) Ist group- only normal saline (N.Saline) (ii) IInd group- saline + Urea (900 mg/kg; oral) (iii) IIIrd group-saline +hydroflumethiazide(1mg/kg; oral) (iv) IVth group-saline +frusemide(5mg/kg; oral)
3. After the administration of drugs, animals are placed in four different metabolic cages.
4. Urine is collected in a measuring cylinder.
5. The time, when the first drop of urine is collected in a cylinder for each group is noted and the volume is recorded at intervals of 15 min for 3-4 hrs.
6. The difference in the volume collected at different time intervals and total volume can be compared with various diuretics.
7. Record a concentration-response curve of acetylcholine using at least four doses.
8. Add physostigmine (2 µg/ml) to the reservoir containing the frog ringer and irrigate the tissue with a sterilized ringer for 30 min.
9. Repeat the concentration-response curves of acetylcholine in the presence of physostigmine.
10. Label and fix both the concentration–response curves.
11. Plot both the concentration–response curves of acetylcholine, ie. one in the absence and the other in the presence of physostigmine. note the potentiation in the response of acetylcholine.

Observation:

Report:

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