Lab 2: Potency

Question 0

What are the main components of an organ bath?

Answer 0

Reservoir
Heating coil
Transducer
Tissue
Organ bath 
Tissue/oxygen hook 
Tap

Question 1

What is an organ bath?

Answer 1

a pharmacology screening tool designed for studying isolated tissue. This is used to determine the concentration-response relationship in a contractile tissue

Question 2

What is the purpose of the reservoir?

Answer 2

Holds the krebs solution before it goes into the organ bath

Question 3

What does the heating coil/water jacket do?

Answer 3

Warms the solution to 37°C which is the body temperature of the guineapig ileum

Question 4

What does the transducer do?

Answer 4

Detects the contractile force exerted by the tissue

Question 5

What does the tissue do?

Answer 5

This is suspended in the organ bath and studied while it is immersed in differing concentrations of agonists/antagonists

Question 6

What does tissue/oxygen hook do?

Answer 6

Holds the tissue in place, providing a baseline tension force.
Also delivers carbogen to the organ bath
(The Oxygen tube allows constant aeration of the krebs solution so tissue has an oxygen supply.)

Question 7

What does the tap do?

Answer 7

Removes water from the bath. It is used to wash the tissue upon each addition of drug to ensure a fresh supply of krebs solution

Question 8

What is the capacity of the organ bath?

Answer 8

20ml

Question 9

What is krebs solution?

Answer 9

A mixture of 
glucose 
sodium 
potassium 
calcium 
magnesium and 
chloride

This nourishes the tissue and keeps it alive

Question 10

What is carbogen?

Answer 10

95% oxygen and 5% carbon dioxide

Question 11

What is the link between the organ bath and the computer?

Answer 11

The tissue hooks and the force transducer.
The transducer detects movement from the tissue and translates it into a signal as an electrical impulse to the computer.

Question 12

What is the software used to quantitate the signal translated by the force transducer?

Answer 12

Chart. This shows each contraction as a blip in the trace.

Question 13

What are Gilson pipettes?

Answer 13

Automatic pipettes used in the pharmacology lab.
P1000 is used to measure 100μl - 1000μl
P500 is used to measure 50μl - 500μl
P20 is used to measure 2μl to 20μl

The top number is the maximum place holder

Question 14

What are agonists?

Answer 14

Drugs that have the ability to bind to receptors for endogenous substances as the first step in their mechanism of action.
These have affinity and efficacy at receptors because they elicit a response

Question 15

What are antagonists?

Answer 15

Drugs that bind to receptors but do not elicit a response.

Thus they have affinity but no efficacy

Question 16

What are the different types of antagonists?

Answer 16

Competitive reversible antagonist (most common)
Competitive irreversible antagonist
Non competitive antagonist
Physiological antagonist

Question 17

What is a competitive reversible antagonist?

Answer 17

Antagonist which competes with the endogenous ligand or agonist for the receptor.
The binding is weaker than an irreversible antagonists, therefore its inhibition can be reversed.
Produce parallel rightward shifts of the concentration response curves to endogenous compounds/agonists without altering the maximal response

Question 18

What is a non competitive antagonist?

Answer 18

An antagonist which decreases the response to the agonist by binding to a separate site from the agonist.
The binding of the agonist is therefore unaffected but this will prevent the cascade of events that an agonist binding would normally initiate

Question 19

What is the potency of an agonist?

Answer 19

EC50, the concentration that produces 50% of the maximum response.

It can also be expressed as pD2, the negative log of EC50

Question 20

What is the potency of a competitive reversible antagonist?

Answer 20

Expressed as pA2,
This is the negative log of the molar concentration of antagonist causing a 2x shift of the concentration response curve for the endogenous compound or agonist,

Question 21

What is the potency if a non competitive antagonist or a competitive irreversible antagonist?

Answer 21

Expressed as pD2’
This is the negative log of the molar concentration of antagonist that causes a 50% reduction in the maximal response to the endogenous compound or agonist,

Question 22

What is the tissue used in the potency lab?

Answer 22

Guinea pig ileum

Question 23

What are the agonists used in the potency lab?

Answer 23

Carbachol which is an acetylcholine analogue

Serotonin (5HT)

Question 24

What is the competitive reversible antagonist used in the potency lab?

Answer 24

Atropine

Question 25

What is the non competitive antagonist used in the potency lab?

Answer 25

Verapamil

Question 26

How was the response of the tissue to the drugs investigated?

Answer 26

Before each experiment the tissue is equilibrated with the krebs solution for 30 minutes.

Part A of the experiment investigated the tissue responses to two different agonists, carbachol and serotonin.
Carbachol was the control.

Part B of the experiment investigated the tissue responses to carbachol, and then carbachol in the presence of atropine.

Part C of the experiment investigated the tissue responses to carbachol and then carbachol in the presence of verapamil

These parts were then analysed into concentration response curves to determine the potencies of the drugs and what type (agonist, competitive antag, non competitive antag, etc.)

Question 27

How were the 10 fold dilution series undertaken?

Answer 27

We know the volume of the organ bath, the desired final concentration of drug, and the initial concentration of drugs we have.
We used C1V1=C2V2 to work out the quantity of agonist which needed to be added to the organ bath.
This was 20μl
This was pipetted from the second highest concentration.
The next dilution was made by pipetteing 20μL of the third highest concentration and etc.

The very first dilution was just 200μL of the highest concentration of drug

Question 28

What is the purpose of the 5 minute cycle?

Answer 28

We conducted the experiment on a 5 minute cycle which consisted of addition of the agonist, observing any tissue response, washing the tissue with fresh krebs solution, adjusting the resting tension and re-washing the tissue.
This eliminated any effects of accumulating drug.

Question 29

How was chart used to measure the contractions ?

Answer 29

A baseline tension of 500 mg was recorded. Any blip in this was measured as a contraction.
The force is the difference between the baseline and the highest peak of the contraction

Question 30

What is the mechanism of carbachol?

Answer 30

Carbachol is an analogue of acetylcholine and acts on the muscarinic receptors of the PNS. The key isoform found in smooth muscle is the M3 receptor.
It uses the same inositol phosphate signalling pathway as serotonin, where it binds to the G linked receptor, increasing the IP3 concentration in the cells. this releases stores of Ca in the sarcoplasmic reticulum. This Ca interacts with calmodulin, activating myosin-light chain kinase which in turn activates myosin to generate muscle contraction

Question 31

What is the mechanism of Serotonin?

Answer 31

Serotonin is an endogenous molecule and has affinity for many receptors, including the 5HT-2A receptor, involved in causing contraction in the smooth muscle of the ileum.
It also binds to a G linked receptor, increasing the IP3 concentration in the cells. this releases stores of Ca in the sarcoplasmic reticulum. This Ca interacts with calmodulin, activating myosin-light chain kinase which in turn activates myosin to generate muscle contraction

Question 32

What is the mechanism of atropine?

Answer 32

Atropine is a competitive antagonist and competes for the same binding site as carbachol on the muscarinic receptor in the smooth muscle. This causes less of the agonist to be able to bind, resulting in a smaller contractile effect at the same concentration of agonist.

Question 33

What is the mechanism of action of verapamil ?

Answer 33

Verapamil is a calcium channel blocker, and inhibits muscle contraction by blocking the release of calcium into the cell. This prevents the activation of myosin light chain kinase and the activation of myosin, so no contractile force can be generated.

Question 34

How was a concentration response curve generated?

Answer 34

The data collected from each group underwent primary and secondary exclusions to obtain a consistent data set where each value was within 2SDs from the mean. The mean response of the tissue to the concentrations of each drug was graphed as concentration response curves and the trendlines were drawn on by hand.