John A Tran Ph.D. Dissertation Defense October 24 th , 2008

1. Intrinsic Relative Activity (RAi) of an Agonist: Understanding Agonist-Receptor Behavior Simply from Agonist Concentration-Response Curves John A Tran Ph.D. Dissertation Defense October 24th, 2008

2. Receptor Theory • Stimulus – Agonist binding and receptor activation • Transducer – downstream signaling mechanisms • Response – result of receptor activation that is measured

3. Characterization of Agonist Activity • EC50 – concentration of agonist that elicits half-maximal response • Emax – maximal response

5. Stimulus • Affinity (1/KX)– ability of the agonist to bind the agonist • Efficacy (e) – ability of the agonist to turn on the receptor Transducer Transducer function unknown Response

6. Furchgott Analysis • Partial inactivation of receptors by irreverisble antagonist • Comparison of equiactive concentrations of the agonist before and after receptor alkylation. q = fraction of receptors remaing after receptor inactivation with irreversible antagonist x’ = concentration of agonist after receptor inactivation that results in the same response as x (Furchgott, 1966; Furchgott and Bursztyn, 1967)

7. Nonlinear regression (Ehlert, 1987)

8. Determination of Affinity Control curve Curve after alkylation Log [x] Log [x’] (Ehlert, 1987)

9. Determine relative efficacy of test agonist relative to standard agonist – e/es Control curve Curve after alkylation 80 % 2 % 4 % (Ehlert, 1987)

10. Furchgott Analysis • Allows the estimation of affinity and efficacy using experimental manipulation of the response

11. What can you get from a concentration-response curve?

12. RAi Analysis • RAi is the product of observed affinity and intrinisic efficacy of the agonist of interest (Y) relative to a standard agonist (X) • RAi is a measure of affinity for the active state of the receptor

13. X Y

15. What is the product of affinity and efficacy? Relative measure of microscopic affinity constant for the active state of the receptor

16. The RAi is a relative measure of the affinity for the active state of the receptor eX XR* eY YR* KX KY

17. Hypothesis • In a biological system, the RAi is equivalent to the product of observed affinity and intrinsic efficacy for the agonist of interest (Y) relative to a standard agonist (X)

18. Aims • Compare RAi with product of affinity and efficacy • Compare RAi value between systems • Determine relative contribution of receptor subtypes for eliciting a response using modified RAi analysis

19. Aim 1 • RAi = product of observed affinity and intrinisic efficacy of test agonist relative standard agonist • Comparison of observed affinity and intrinisic efficacy values procured individually through Furchgott analysis with RAi • Furchgott analysis – irreversible antagonism • RAi analysis – of simple agonist concentration-response curves

20. CHO-M2 cells (Tran et al, submitted)

21. KOxo-M = 0.29 mM KCarb = 4.3 mM

22. RAi = product of affinity and efficacy (Tran et al, submitted)

23. Mouse ileum, “M2 KO” (Tran et al, submitted)

24. Mouse ileum, “M2 KO” (Tran et al, submitted)

25. Aim 1 Summary • RAi values agree with the product of observed affinity and intrinsic efficacy for the test agonist relative to the standard

26. Aim 2 • Compare RAi values between systems • CHO cells vs tissue • The RAi is inherent property of the active state of the receptor that causes it associate with its coupling protein (e.g., G protein). • Therefore RAi values should be consistent between different systems that signal through the same G protein.

27. G1 G1 G2 RAi values are equivalent RAi values not necessarily equivalent

28. M2 receptor Guinea Pig Left Atrium CHO-M2 cells (Tran and Ehlert, in preparation)

29. M2 RAi comparison (Tran and Ehlert, in preparation)

30. (Ehlert et al., 1999) M3 receptor CHO-M3 Mouse ileum, “M2 KO” (Tran et al, submitted)

31. M3 receptor (Tran et al, submitted)

32. Characterization of McN-A-343 Mouse ileum, “M2 KO” (Tran et al, submitted)

33. M2/M3 KO M2 KO M2 KO (Tran et al, submitted)

34. Higher activity of McN-A-343 in M2 KO ileum is a mixed response that may include M1 receptor activation (Tran et al, submitted)

35. b1 receptor CHO-b1 cells Rat Left Atrium (Tran and Ehlert, in preparation)

36. (+) b1 Contraction X (-) b3

37. Unknown dobutamine effect

38. Aim 2 Summary • The RAi is consistent between systems and responses measured signaling from the same G protein pathway • The unique effects of McN-A-343 and dobutamine are likely due to effects in addition to binding the receptor-G protein pathway under study

39. Aim 3 • Determination of the contribution of various receptor subtypes to the response measured in knockout studies using a modified RAi • Relative Contribution (RC) analysis • The contribution of M1, M2 and M3 receptors to oxotremorine-M-induced phosphoinositide hydrolysis in the mouse urinary bladder and ileum • M2 KO • M3 KO • M2/M3 KO

40. R2 R1 • M1, M3 – mediate phosphoinositide hydrolysis through Gq • M2 – may mediate phosphoinositide hydrolysis through Gi • Oxotremorine-M has similar affinities for all the receptors • Assumptions: • Receptors can each mediate the response measured individually • Agonist has same affinity for all the receptors involved

41. RC analysis R2 R1 RC1 = 1 – RC2

42. Mouse Urinary Bladder M3 receptor is solely responsible for response (Tran et al., 2006)

43. Mouse Ileum Complex Result with possible involvement of M2 in addition to M3 as well as another receptor (Tran et al., 2006)

44. M2/M3 KO Antagonist affinities (pKB) AF-DX 116 – 6.42 ± 0.42 Pirenzepine – 8.04 ± 0.47 M1 receptor mediated response Binding affinities (pKd) AF-DX 116 Pirenzepine Esqueda et al, 1996 (Tran et al., 2006)

45. M2/M3 KO M1 receptor exists in the smooth muscle (Tran et al., 2006)

46. M1, RC1 = 15 % (M2/M3 KO) M3, RC3 = 80% (M3 KO) M2, RC2= 1-(RC3 + RC23) = 5% (Tran et al., 2006)

47. Aim 3 Conclusions • The RC analysis was used to estimate the contributions of the M1, M2 and M3 receptors to the oxotremorine-M-induced phosphoinositide hydrolysis • Evidence is shown for a M1 and M2 phosphoinositide response in ileum

48. Summary • The RAi is a relative measure of microscopic affinity constants of the active state of the receptor • The RAi is the product of affinity and efficacy of the test agonist relative to a standard agonist • The RC analysis can be used in knockout studies to determine the contribution of various receptor subtypes to the response measured

49. Frederick Ehlert, Ph.D. Kelvin Gee, Ph.D. Frances Leslie, Ph.D. Ralph Purdy, Ph.D. Qun-Yong Zhou, Ph.D. Alan Goldin, M.D.,Ph.D. Ehlert Lab Michael Griffin, Ph.D. Hinako Suga, Ph.D. Katherine Figueroa, Ph.D. Kirk Pak Alexander Chang Department of Pharmacology Family Acknowledgements