NMJ recap/workshop

Question 1

Concentration response relationship - linear plot

Answer 1

The relationship between concentration and response is hyperbolic
EC50 is the concentration of agonist that elicits a half maximal response

Question 2

Concentration response relationship to carbachol - linear plot

Answer 2

a linear plot is inconvenient when plotting a wide range of concentrations as it greatly compresses data at the lower end of the scale and the EC50 is ambiguous
It is easier to plot this with micromolar units

Question 3

Concentration response relationship - semi-logarithmic plot

Answer 3

the relationship between concentration and response is sigmoidal

Question 4

Finding an EC50

Answer 4

the EC50 is the concentration of agonist that elicits a half maximal response
Draw a line from the 50% maximal percentage on the Y-axis until it reaches the plot

Question 5

Concentration response relationships to carbachol in the absence and presence of (+)-tubocurarine - semi-logarithmic plot

Answer 5

in the presence of the antagonist tubocurarine the plot shifts to the right but shows no changes in maximan response

Question 6

Reversible competitive antagonism

Answer 6

the antagonist competes with the agonist for the same binding site on the receptor
The interaction between the antagonist and the receptor is reversible
The antagonism is surmountable over a wide range of agonist concentrations
In the presence of an antagonist, the agonist concentration-response curve is shifted to the right without a change in maximal response of change in slope (i.e. the shape of the concentration response curve remains unchanged)
No change in maximal response - still reaches 100% - no change in the shape of the curve
Shift to the right

Question 7

Concentration ratio

Answer 7

the ‘shift to the right’ is best expressed as a concentration ratio. This is the factor by which the agonist concentration must be increased to, to restore a given response in the presence of an antagonist (e.g. the EC50)

Question 8

concentration ratio calculation

Answer 8

CR = Xa’ / Xa
Where Xa’ is the EC50 of the agonist and antagonist
Where Xa is the EC50 of the agonist alone
> This gives you the concentration of agonist that must be present to achieve the same response with the antagonist present.

Question 9

Calculating apparent pA2 from concentration rations

Answer 9

apparent pA2 = log10((CR-1)/[B])
Where [B] is the antagonist concentration

Question 10

Competitive antagonism: setting up the Schild plot

Answer 10

agonist concentration-response curve over increasing concentration of antagonist.
An increase in concentration causes a shift to the right on the plot
The antagonism is still surmountable with increased concentrations of agonist.

Question 11

Competitive antagonism: the Schild plot: calculate EC50

Answer 11

Compare EC50 for agonist and (agonist + antagonist) concentration

Question 12

Competitive antagonism: the Schild plot: calculate the Log10 concentration ratios -1

Answer 12

log10 concentration ratio - 1 = log 10((Xa’/Xa) -1)

Question 13

Competitive antagonism: the Schild Plot

Answer 13

if the antagonist is competitive, the slope of the regression should = 1.0
X intercept = KB - the equilibrium constant for the antagonist

Question 14

Competitive antagonism: the Schild Plot: Calculate pA2

Answer 14

pA2 = -log KB

Question 15

pA2

Answer 15

an indication of antagonist potency
pA2 is the negative log of the molar concentration of an antagonist that makes it necessary to double the concentration of the agonist needed to elicit the original response obtained in the absence of antagonist

Question 16

Competitive antagonism

Answer 16

competitive antagonist cause a parallel rightward shift of the agonist concentration response curve but maximum response is unchanged

Question 17

Non-competitive antagonism

Answer 17

non-competitive antagonists depress the slope and maximum concentration response curve, but do not cause a rightward shift

Question 18

Effects of increasing the concentration of a competitive antagonist

Answer 18

Progressive rightward shift of agonist concentration-response curve on concentration axis but no depression of the slope - a maximal response

Question 19

Effects of increasing the concentration of a non-competitive antagonist

Answer 19

progressive depression of maximum response and slope, but no shift of agonist concentration response curve on the concentration axis.

Question 20

Calculating a concentration ratio (CR)

Answer 20

CR = EC50 for agonist in the presence of antagonist / EC50 for agonist in the absence of antagonist

Question 21

Calculating the apparent pA2

Answer 21

• pA2 = -log KB

Question 22

Constructing a concentration-inhibition plot

Answer 22

make measurements of twitch amplitude (representing muscle force)
% control response is given by:
> ([amplitude in presence of antagonist/amplitude in absence of antagonist] x100)

Question 23

IC50

Answer 23

The concentration of an antagonist where the agonist-evoked response (or ligand-binding, in the case of binding experiments) is reduced by 50%
Useful in estimating potency of irreversible competitive antagonists and non-competitive antagonists