1. To record DRC of Acetylcholine using rat ileum
Lab 6
To record DRC of Acetylcholine using rat ileum

2. Drug Receptor Interaction
A drug combines with the receptor and may initiate a sequence of effects.
The ability of drug to combine with the receptor to form the drug-receptor complex is defined as affinity.
The ability of drug to initiate the effect is termed as intrinsic activity.
Agonist is the drug which possess both affinity as well as intrinsic activity.
Antagonist possess affinity but no intrinsic activity.

3. Dose Response Relationship
Graded dose response curve
Cumulative dose response curve: simpler, less time, and less drug required
The affinity of a drug can be determined by finding out the pD2 value.
pD2 is defind as -ve log of molar concentration of agonist which produces half (50%) of the maximum response or pD2 = -log [EC50]

4. Dose – Response Curve
It is a relationship between Dose and (Response %)
From this curve we can see :
Potency: A measure of the activity of a drug in a biological system.
Efficacy: The ability of a drug to produce a desired or intended effect.

5. Dose – Response Curve
Therapeutic index (the ratio LD50:ED50): A ratio that compares the blood concentration at which a drug becomes toxic and the concentration at which the drug is effective. The larger the therapeutic index (TI), the safer the drug is.
ED50: The amount of drug that produces a therapeutic response in 50% of the people taking it

6. OBJECTIVE
To record a dose response curve (D.R.C.) of acetylcholine using rat intestinal smooth muscle (ileum) with graded doses.

7. PRINCIPLE
Dose (concentration) response curves demonstrate graded responses to drugs or agonists where an increase in response is recorded with a subsequent increase in the dose or the concentration of the drug.
A cumulative dose response curve is obtained by increasing the concentration of the drug in the organ bath step by step without washing the preceding doses.
This technique is simple and less time consuming. It is generally employed in those preparations where the tissue is slowly contracting and slowly relaxing
e.g., Frog rectus abdominus, Rabbit aortic strip, Rat seminal vesicle and Rat vas deferens etc.

8. REQUIREMENTS Animals: Rat Tissue: Intestine (ILEUM)
Apparatus: EmkaBath2 Organ bath
Drugs: Acetylcholine (Ach) in different concentrations (10 µg/ml; 100 µg/ml; 1000 µg/ml; 10 mg/ml)
Physiological salt solution: Tyrode solution

9. PROCEDURE Calibrate the instruments emkaBath2 with weight 0 & 10 grams
Anesthetize the rat with Ether and sacrifice by bleeding to death and lay it on its back on the dissecting board.
Open the abdomen, expose the caecum and identify the ileum.
Cut and prepare centimeters length of tissue and fix the tissue between upper & lower tissue holder.
Mount the preparation in upright position in the organ bath containing Tyrode solution (just enough to submerge the tissue)
Start recording in a lab tutor experiment and adjust the tissue tension by using the micro positioner.

10. PROCEDURE
Maintain the bath temperature at 37˚C and bubble the organ bath with carbogen gas (95% oxygen + 5 % carbodioxide) or aeration, since it is a mammalian tissue.
Relax the tissue for min, during relax, wash the tissue with fresh Tyrode solution, for at least four times, this is called Equilibration or Stabilization.
Once the tissue is stabilized, start the experiment.
Start recording with 30 seconds baseline, then add the graded doses of acetylcholine to obtain contractile response.
Add 0.1 ml Ach to the bath and take the response for 30 sec. Give 2-3 washing.
Repeat the recording with another dose. For e.g. 0.1 ml, 0.2 ml, 0.4 ml, 0.8 ml, 1.6 ml, 3.2 ml and so on, till the maximum response. Its called ceiling dose.
Measure the height of contraction at different doses of Ach.
Tabulate the observations into three columns as: (1) dose of Ach, (2) height of contraction (in mm) and (3) % response.
Plot the dose response curve on semi-log graph paper, taking log dose of Ach on X-axis and % response on Y-axis.
Determine the dose of Ach which produces 50% response. Convert to its molar concentration.
Calculate pD2 of Ach as follows: pD2 = -log [EC50]

11. Dose – Response Curve D + R DR complex Response
wash cm
0.1ml ml ml ml ml

12. Dose – Response Curve Response % Response (cm) Dose 16.6 1 0.1 50 3
(x/ max )*100
Response (cm)
Dose
(ml)
16.6 1
0.1 50 3
0.2 66 4
0.4
100 6
0.8
1.6

13. Dose – Response Curve
ED 50 = 2.75 ml

15. Schematic illustration of the dose-response curves for a series of agonists (A, B, C and D) that have the same efficacy, but differ in terms of their potency. The most potent drug (Drug A) has the lowest EC50 value, and is approximately fold more potent than Drug D.

16. Dose-response relationships for four agonists that vary in efficacy
Dose-response relationships for four agonists that vary in efficacy. Each drug has essentially the same EC50 value (equi-potent), but differ in terms of the maximum response they can produce at high concentrations that saturate all available receptor sites. Drug A has a relative efficacy that is 2 times than Drug C, and ~100 times more than Drug D.

17. pD2 value
1. To determine the concentration of Ach in the organ tube which produces 50% response is determined as: Qty (ml) to produce 50% response X Conc of Ach used Y = Bath capacity (ml) 2. For the calculation of molar concentration: Molecular weight of Ach = g/l = 1M 182 mg/ml = 1M 182 µg/ml = 10-3 M i.e. Y µg/ml = ? 3. To determine pD2 value: pD2 = - log [EC50]