Aim: To record the dose concentration-response curve (DRC) of acetylcholine and its modification by atropine using rat ileum preparation.
The rat colon is an intestinal smooth muscle. Acetylcholine causes the contractions of the smooth muscle by acting on muscarinic receptors. Atropine blocks muscarinic receptors in the smooth muscle. Therefore, atropine blocks acetylcholine-induced contractions in the rat colon. The concentration-response curve of acetylcholine will be shifted to the right in the presence of atropine. the nature of antagonism is of a competitive type.
The spontaneous contractions of the preparations can be reduced by reducing the calcium content in the physiological solution and maintaining the bath at room temperature (23+ 2 0 C). The muscle (colon) preparation obtained from an unstarved rat gives more stable contractions.
Animal: Rat (150-200g)
Drugs: Acetylcholine stock solution (1mg/ml), Atropine stock solution (1 mg/ml)
Physiological solution: Modified Ringer (contains less calcium)
- Sacrifice the animal by cervical dislocation.
- Cut open the abdomen and identify the colon. the right flexure, i.e. the subhepatic region of the colon where the ascending colon turns to become a transverse colon, is cut out and placed in a shallow dish containing Modified Ringer’s solution.
- The lumen is gently cleaned and a 3 cm long tissue is mounted in the organ bath containing Modified Ringer’s solution (pH 7.4)maintained at 25 0 C and bubbled with carbonated air. The preparation is allowed to equilibrate for 45 min under 500g tension.
- Record the concentration-dependent responses due to acetylcholine using a frontal writing lever. Contact time is 60 sec, and 5 min time cycles are kept for proper recording of the response.
- Add atropine to the reservoir containing Modified Ringer’s solution and irrigate the tissue with atropinised Modified Ringer for 20 min.
- Repeat the concentration-response curve of acetylcholine in presence of atropine.
- Label and fix the tracing, and plot the graph as done in the earlier experiments. Calculate EC 50 values and note the nature of antagonism
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