how do you characterize agonist activity?
DESCRIPTION
How do you characterize agonist activity?. John A Tran Ehlert Lab UCI Department of Pharmacology Research Day June 14, 2008. Introduction. Stimulus – agonist binding and activation Transducer – signaling mechanisms Response – result of receptor activation that is measured. Contraction. - PowerPoint PPT PresentationTRANSCRIPT
How do you characterize agonist activity?
John A TranEhlert Lab
UCI Department of PharmacologyResearch DayJune 14, 2008
Introduction
Stimulus – agonist binding and activation
Transducer – signaling mechanisms
Response – result of receptor activation that is measured
Contraction
http://www.nature.com/gimo/contents/pt1/images/gimo24-f1.jpg
Characterization of Agonist Activity
Contraction
http://www.nature.com/gimo/contents/pt1/images/gimo24-f1.jpg
Response
Agonist Concentration-Response Curves EC50, Emax
Characterization of Agonist Activity
Stimulus Furchgott Analysis
1/Kd – affinity - efficacy
Contraction
http://www.nature.com/gimo/contents/pt1/images/gimo24-f1.jpg
RAi analysis
The RAi is a relative measure of microscopic affinity constants of the active state of the receptor for the agonist.
The parameter is equivalent to the product of affinity and efficacy of the agonist of interest relative to a standard.
Requires only the agonist concentration-response curves already in use.
responsetransducerstimulus
sSx
xi K
KRA
/1
/1
Hypothesis
In biological system, the RAi value is equivalent to the product of affinity and efficacy of the agonist of interest relative to a standard agonist.
sSx
xi K
KRA
/1
/1
Methods
Compare the product of affinity and efficacy with the RAi value
Furchgott analysis – Irreversible antagonism
RAi analysis – agonist concentration-response curves
CHO-M2 Cells
s DetermineDetermine Kd
RAi and the product of affinity and efficacy
CHO-M2 cells
Mouse Ileum, “M2 KO”
Ehlert et al., 1999
Agonist RAi between systems
McN-A-343 Characterization
M2/M3 KO
M2 KO
M2 KO
Higher activity of McN-A-343 in M2 KO ileum is a mixed response that may include M1 receptor activation.
Conclusions
The RAi analysis results in an estimate that is equivalent to the product of affinity and efficacy of the agonist of interest relative to a standard.
The RAi is a relative measure of the microscopic affinity constants of the active state of the receptor.
The RAi analysis can be used as a tool to study unique agonist-receptor related phenomenon.
sSx
xi K
KRA
/1
/1
Acknowledgements
Fred EhlertEhlert Lab
Mike Griffin Hinako Suga Kate Figueroa Kirk Pak Alex Chang
Minoru Matsui