ORGAN BATH EXPERIMENT

Organ Bath Experiment



Organ Bath Experiment

The term antagonist refers to any drug that will block, or partially block, a response. When investigating an antagonist the first thing to check is whether the antagonism is surmountable by increasing the concentration of agonist. The next thing to ask is whether the antagonism is reversible.  After washing away antagonist, does agonist regain response? If an antagonist is surmountable and reversible, it is likely to be competitive (see next paragraph). Investigations of antagonists that are not surmountable or reversible are beyond the scope of this manual.

A competitive antagonist binds reversibly to the same receptor as the agonist. A dose-response curve performed in the presence of a fixed concentration of antagonist will be shifted to the right, with the same maximum response and (generally) the same shape.

Dose

Response: Agonist alone

Response: Agonist & Antagonist X

Response: Agonist & Antagonist Y

1*10-7 M

0.0

0.0

0.0

5*10-7 M

2.6

1.2

0.0

1*10-6 M

18.4

9.3

0.0

5*10-6

104.9

58.5

0.0

1*10-5M

126.2

69.8

0.6

5*10-5 M

129.5

71.9

53.9

1*10-4

129.5

71.9

96.0

5*10-4

129.5

71.9

129.5

1*10-3

129.5

71.9

129.5

Question 1

Gaddum derived the equation that describes receptor occupancy by agonist  in the presence of a competitive antagonist. The agonist is drug A. Its concentration is [A] and its dissociation constant is Ka. The antagonist is called drug B, so its concentration is [B] and dissociation constant is Kb. If the two drugs compete for the same receptors, fractional occupancy by agonist (f) equals:

The presence of antagonist increases the EC50 by a factor equal to 1+[B]/Kb. This is called the dose-ratio. You don't have to know the relationship between agonist occupancy and response for the equation above to be useful in analyzing dose response curves. (Smith 2004 45-71)

You don't have to know what fraction of the receptors is occupied at the EC50 (and it doesn't have to be 50%). Whatever that occupancy, you'll get the same occupancy (and thus the same response) in the presence of antagonist when the agonist concentration is multiplied by the dose-ratio.

The graph below illustrates this point. If concentration A of agonist gives a certain response in the absence of antagonist, but concentration A' is needed to achieve the same response in the presence of a certain concentration of antagonist, then the dose-ratio equals A'/A. You'll get a different dose ratio if you use a different concentration of antagonist.

If the two curves are parallel, you can assess the dose-ratio at any point. However, you'll get the most accurate results by calculating the dose-ratio as the EC50 in the presence of antagonist divided by the EC50 in the absence of antagonist. The figure below shows the calculation of dose ratio.

If the antagonist is competitive, the dose ratio equals one plus the ratio of the concentration of antagonist divided by its Kd for the receptor. (The dissociation constant of the antagonist is sometimes called Kb and sometimes called Kd)

A simple rearrangement gives:

Question 2

If you perform experiments with several concentrations of antagonist, you can create a graph with log(antagonist) on the X-axis and log(dose ratio -1 ) on the Y-axis. If the antagonist is competitive, you expect a slope of ...