Experimental rats were euthanized by cervical dislocation followed by exsanguination. The ileum was then removed, cleared mucosa, connective tissue and fat and then submerged in physiological solution (Tyrode’s solution) at room temperature and bubbled with oxygen. A piece of ileum (2–5 cm) was immersed in an organ bath under a resting tension of 1.0 g at 37 °C. The tissue was allowed to stabilize for 30 min before contractile responses were recorded. The physiological solution was made up with the following composition (g): NaH2PO4 (1.0), MgSO4 (2.0), KCl (4.0), NaHCO3 (20), NaCl (160.0), glucose (20.0), MgCl2 (55.6) and CaCl2 (110.0). Concentrations are given as final concentrations in the bath solution. To record isometric contractions, organs were suspended by metal rods in organ baths (25ml), connected to a force transducer attached to an amplifier and connected to an A/D converter into a PC running LabChart software. The ileum was stimulated electrically with a sampling range of 20mV at a rate of 1 k/sec. A sequence of responses was obtained to drugs in doses chosen to give similar contractions, mainly 80% of the maximal. The drugs were injected into the bath in 0.1 to 0.3 ml volumes with 0.9% saline.
Drugs. The drugs used and their molecular weights (g/mol): acetylcholine chloride (181.7), histamine phosphate (111.1), Carbachol chloride (182.7) and Pilocarpine (244.7). All substances were dissolved and diluted in 0.9% saline solution. All the drugs were then dissolved in Krebs solution, the lower concentrations made from concentrated stock solutions in 0.9% saline solution. (Write about why you chose these drugs)
Drug injection cycle. A standardised approach of half-log doses was used to construct a dose – response curve. After sensitisation of the ileum tissue to each drug, a small dose of 0.1mL of 2.5×10-6M of a drug was added to the organ bath to produce a final organ bath concentration of 1×10-8M and the response was noted. After thirty seconds the organ bath was emptied and refilled twice with 25mL of fresh Tyrode’s solution. After three minutes a larger dose of 0.3mL was then added to the organ bath and the response was recorded. This procedure was repeated with larger doses of agonist every three minutes until a maximum response was observed (no further increase in response after addition of larger concentrations).