Lecture 2

Question 1

Define Hill langmuir equation ?

Answer 1

predict relationship between receptor occupancy
PAR =[A]/Ka+[A]

Question 2

The rate of association

Answer 2

K+1

Question 3

The rate of dissociation ?

Answer 3

K-1

Question 4

The overall dissociation rate constant ?

Answer 4

Ka= k-1/k+1

gives the measure of affinity of agonist for a receptor

how well agonist is able to bind to receptor

Question 5

How does receptor occupancy relate to tissue response ?

Answer 5

once activated this triggers a response which can be seen and measured by
CONC REPONSE CURVES

Question 6

Define Agonist ?

Answer 6

A substance that binds to receptors that activates it and causes a biological response

Question 7

What are concentration response curves ?

Answer 7

Force of contraction by different concc of agonists

Question 8

Hill equation ?

Answer 8

memorise

Question 9

What is EC50 ?

Answer 9

the maximum response at 50 %

Question 10

What does the Hill equation measure ?

Answer 10

Relationship between tissue concentration VS agonist concentration

Question 11

What does the Hill-langmuir equation measure ?

Answer 11

Meaures the relationship between the AGONIST -RECEPTOR occupancy VS agonist concentration

Question 12

Why do we get dissconect between tissue response & agonist occupancy ?

Answer 12

additional response –> agonist efficacy
Maybe because it causes changes in receptor and then activates receptors to induce tissue response

Question 13

What is efficacy ?

Answer 13

the strength to invoke a response and produce maximal reponse

Question 14

Define Antagonist ?

Answer 14

bind to receptor & do nothing they prevent agonist from binding

only governed by affinity
only association/dissocation

Question 15

Agonist VS antagonist

Answer 15

Both compete for the binding site

Question 16

How does competative antagonism work ?

Answer 16

its surmountable so if there is enough agonists there able to overcome by increasing the conc
causes a shift to the right but NO CHANGE to dose ratio

Question 17

How to determine affinity for competative antagonist ?

Answer 17

1.Schild analysis :dose response curves
2.Radiologand binding assay :directly measuring affinity using radiolablled antagonist

Question 18

What does parallel shift mean ?

Answer 18

i think it assumes the response in absence &presence of antagonist =SAME then n
the no of receptors occupied by agonist must be same
the if increase conc of agonist in order to overcome antagonism thats present caused by antagonist

Question 19

Schilds equation ?

Answer 19

predicts parallel shift in conc response curve that is constant r for any given antagonist concentration [B] or dissociation constant Kg

r-1 = [B]/Kb

Question 20

What does the dissociation constant (Kb) tell us ?

Answer 20

Kb=physiochemical constant
Kb=same for a given receptor & drug combo in any tissue ,any species as long as the receptor is the same

Question 21

What can we use Kb for when considering the definiton ?

Answer 21

Used to identify receptors within a tissue
varies with receptor types
Quantitaviley compare the affinity of diff drugs on same receptor (varies with diff drugs)

Question 22

What does a radioligand binding assay do ?

Answer 22

2nd technique to identify the affinity for competative antagonist

to compae drugs

Question 23

What can the radioligand bining studies tell us ?

Answer 23

1.measuring conc of bound receptors (RECEPTOR DENSITY)
2.measure dissociation constant (AFFINITY) of radiolabelled antagonists

Question 24

What is needed for a radioligand assay ?

Answer 24

1. source of receptor ligand a highly specific radiolabeeled ligand
   2.Source of receptors -potential target tissues

Question 25

What is radiolabelling ?

Answer 25

chemical reaction introducing radioactive isotopes to compound these radioligands should have increased specificity activity so small quanitities can me measured

Question 26

How is the tissue used in it ?

Answer 26

homogenized and seperated in aaliquots containing some amounts of the sample Aliquots=increased conc of radiolabelled so increased binding Reach saturation as limited no of receptors

Question 27

The incubation stepof radioligand binidng ?

Answer 27

adding radioligand -crucial step IN-VITRO (in test tubes) Conditions need to be optimised to ensure eqm reached between receptor & radioligand

Question 28

What are external factors of radio ligand binding assay ?

Answer 28

* PH,temp,salt conc * enzymes released as they may degrade * Duration of incubation of tissues with the radio ligand

Question 29

The FILTRATION part of radio ligand binding assay ?

Answer 29

add excess so all is covered some radioligand must be removed as its not bound before measuring do 3 types -millipore filtration -column chromatograohy -EQM dialysis

Question 30

Then measure radioactivity ? | of radio ligand binding assay

Answer 30

corrrelate with the amount of receptors increase levels of radio ligand =increased amount of receptors =UNTIL PLATEAU

Question 31

How does Millipore filtration work ?

Answer 31

-very small pores only small molecules pass through (RL free complexes) not big (RL receptor complex) sample is loaded onto filter and ligand washed away after few succesive centrifugations

Question 32

How does column chromatography work ?

Answer 32

-sample lloaded on column and traps small molecules (RL free comples) instead of big -bigger molecules can pass through freelu centrfugation can be used to pellet

Question 33

How deos filttration centrifugation work ?

Answer 33

a process that is used to separate or concentrate contaminants suspended in a liquid medium. A centrifuge will spin the oil at high rotational speeds and separate the particulates from the liquid.

Question 34

What is equilibrium dialysis ?

Answer 34

-based on osmosis -sample in membrane (pores)allows exchange of smallmolecules between smaple and buffer surrounding -free RL diffuses to surrounding buffer leaving the bound ligand inside

Question 35

How are the results of radio ligand binding assay interpreted ?

Answer 35

only those who bind to specific receptors CRITERIA 1.Specificity 2.Saturability 3.Reversibility 4.Bio relevance

Question 36

Define Specificity ?

Answer 36

Ligand must bind to receptor only unlabelled will eplace labelle in all receptro sites if not replaced 1.interaction not reversible 2.non specific binding can be saturated if interaction is meaured = decreased radioactivity or 0

Question 37

Define Saturbility ?

Answer 37

finite no receptors Increased ocn of RL receptors to fixed conc of tissue sample = icreased receptor binding once all binded to receptors =additon of RL means no binding

Question 38

Define Reversibility ?

Answer 38

binding is reversible allows competition for receptor binding sites labelled and not labelled ligands

Question 39

What is competative binding assay ?

Answer 39

binding of radiolabelled ligand (fixed conc) decreases as non-labelled ligand conc increases

Question 40

What is KL ?

Answer 40

measure of dissociation constant of radiolabelled ligand (affinity)

Question 41

How do we measure bio availability ?

Answer 41

in vitro only 1 part of process 1.not represent physiological environment 2.dont know stability -injested 3.Drug metabolism 4.Drug absorption 5.Stability diff in phys environment

Question 42

What are some risks ?

Answer 42

when homogenised other proteins are exposed prod non-specific binding conditions some ligans bind to glass tube causes curve to never saturate due to extra binding sites

Question 43

How to overcome risks ?

Answer 43

Must consider 2 properties 1.binding interaction of ligand receptor =DYNAMIC ligand-receptor complex =REVERSIBLE 2.limite amount of receptor in our prep