ANTAGONISM OF ACETYLCHOLINE-DEPENDENT CONTRACTIONS

IN GUINEA-PIG ILEUM

Determination of “apparent pA₂”for telenzepine using the Schild-Gaddum

 dose response curves

Introduction:

In determining the potency of antagonist drugs on blockade of a particular receptor, the antagonist has to be tested in the presence of a selective agonist drug for the receptor of interest. However, the antagonist’s potency can only be accurately determined when the agonist concentration-response curves are constructed in the absence and presence of increasing concentration of the antagonist. This is the method devised by Schild (Arunlakshana & Schild, 1959), which is routinely used in pharmacological research to determine the apparent pA2 or potency of antagonist drugs.

Aim:

Now that the pA2 for atropine has been determined, the aim behind this practical is to determine the pA2 for another muscarinic receptor antagonist, telenzepine, using the same experimental set-up.

Learning objectives:

1. Construct concentration-response curves for acetylcholine in the absence and presence of increasing concentrations of telenzepine
2. Using the data generated, construct agonist-dose response curves and determine the dose-shift ratio.
3. Construct a Schild plot and calculate the pA2 value for telenzepine

Method:

1. The guinea-pig ileum will be set up as in previous experiments.

• Using 10µg/ml stock acetylcholine, construct a full concentration response curve for acetylcholine by initially adding 10µl of acetylcholine and then doubling the volume there after. Follow the same time cycles as in previous experiments (30 sec contact followed by 60 sec wash).

• After washing the tissue, add 10nM (final concentration) of telenzepine to a 1 Litre Tyrode in the reservoir from the 10µM stock telenzepine solution.

• Flush the tissue with the new Tyrode solution containing 10nM telenzepine up to 6 times to ensure that the tissue is exposed to telenzepine.

• Wait 5 minutes, then repeat the acetylcholine concentration response as detailed in step 2.

• Wash the tissue several times with the telenzepine free Tyrode several times to ensure complete removal of telenzepine.

• Then repeat 3-6, four more times, each by introducing an increasing concentration of telenzepine. Use the following final telenzepine concentrations, 20, 40 and 80nM.

Data analysis:

1. On the same graph paper plot the log concentration-responses to acetylcholine alone and in the presence of increasing concentrations of telenzepine in molar concentration.

• On a linear part of the acetylcholine concentration-response curves (preferably the EC50), read off the concentration of agonist giving the same response by reading from the y-axis into the x-axis (See figure 1).

• Calculate the dose ratio for each of the concentration responses in the presence of telenzepine by dividing the value read off from the concentration response in the presence of telenzepine (A’) over the value read off in the absence of telenzepine (A) = (A’/A) (See figure 2).

• Repeat A’/A for all concentration response curves in the presence of antagonist.

• Subtract the dose-ratio from 1 and then calculate the log = log((A’/A)-1)

• Calculate –log of atropine in molar concentrations

• To produce a Schild plot on graph paper plot the –log antagonist concentration (Molar) on the x-axis against the dose-ratio (log((A’/A-1))) on the y-axis (See figure 3).

• Extrapolate the line back to the x-axis.

• The value generated from that is pA2.

Figure 1: Concentration-response for agonist alone and in the presence of increasing concentration of antagonist.

Discussion

Compare your results to those published in peer reviewed papers. Your pA₂ result should compare favourably with published data, relate why this is. Also compare the calculated pA2 value for telenzepine from this practical to the pA2 value for atropine detremined from the previous practical. Comment on the significance of these results in the light of what is known about the molecular mode of actions of atropine and telenzepine. Relate this to the scenario, and its clinical use.

References

• Arunlakshana, O.  &  Schild, H.O. (1959) Brit. J. Pharmacol. 14, 48.
• Rang & Dale (All editions). Check Calcium blockers in the index.