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How do you characterize agonist activity?

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.

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How do you characterize agonist activity?

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  1. How do you characterize agonist activity? John A Tran Ehlert Lab UCI Department of Pharmacology Research Day June 14, 2008

  2. 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

  3. Characterization of Agonist Activity • Response • Agonist Concentration-Response Curves • EC50, Emax Contraction http://www.nature.com/gimo/contents/pt1/images/gimo24-f1.jpg

  4. Characterization of Agonist Activity • Stimulus • Furchgott Analysis • 1/Kd – affinity • e - efficacy Contraction http://www.nature.com/gimo/contents/pt1/images/gimo24-f1.jpg

  5. RAianalysis • 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.

  6. 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.

  7. Methods • Compare the product of affinity and efficacy with the RAi value • Furchgott analysis – Irreversible antagonism • RAi analysis – agonist concentration-response curves

  8. CHO-M2 Cells Determine Kd

  9. RAi and the product of affinity and efficacy CHO-M2 cells

  10. Mouse Ileum, “M2 KO”

  11. Agonist RAi between systems Ehlert et al., 1999

  12. McN-A-343 Characterization

  13. M2/M3 KO M2 KO M2 KO

  14. Higher activity of McN-A-343 in M2 KO ileum is a mixed response that may include M1 receptor activation.

  15. 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.

  16. Acknowledgements • Fred Ehlert • Ehlert Lab • Mike Griffin • Hinako Suga • Kate Figueroa • Kirk Pak • Alex Chang • Minoru Matsui

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