Methods

1
A Simple Method for Estimation of Agonist
Activity at GPCRs Characterization of Responses
Elicited by M2 and M3 Muscarinic Receptors
724.10
John A. Tran1, Alex Chang1, Minoru Matsui2 and
Frederick J. Ehlert1 1Department of Pharmacology,
School of Medicine, University of California,
Irvine, Irvine, California 92697-4625 2Department
of Physiology, Dartmouth Medical School, Lebanon,
New Hampshire
Figure 5
Figure 3
Figure 1
Introduction Agonist activity is usually
characterized by estimation of the EC50 and Emax
values for eliciting a particular response. It
well known that these parameters can vary for the
same agonist receptor complex, depending upon the
response being measured. A more invariant
approach would be to estimate the observed
affinity and relative efficacy of the agonist
using the method of partial receptor inactivation
(Furchgott, 1966 Furchgott and Bursztyn, 1967).
However this technique is more tedious and rarely
used. Recently, we developed a method for
estimating the product of affinity and intrinsic
efficacy of an agonist expressed relative to that
of another agonist, simply through the analysis
of the agonist concentration-response curve
(Ehlert et al., 1996 Ehlert et al., 1999 Ehlert
and Griffin, 2001 Griffin et al., 2007). This
parameter is termed the intrinsic relative
activity (RAi), and it is mainly dependent on the
agonist-receptor-G protein signaling pathway and
independent of other system parameters.
Consequently, the RAi estimate is useful for
comparing agonist activity across a variety of
systems and for quantifying ligand directed
signaling. In the present the present report,
we have estimated the RAi values of muscarinic
agonists for eliciting M2 and M3 muscarinic
responses, and compared these estimates with
those calculated from the product of observed
affinity and intrinsic efficacy estimated by
Furchgotts method of partial receptor
inactivation. We found good agreement between
the two estimates, which illustrates that our
simple RAi estimate is an accurate measure of the
product of affinity and efficacy of the agonist.
We also found evidence that part of the
contractile response to the novel muscarinic
agonist McN-A-343 in the mouse ileum may be
mediated through the M1 muscarinic receptor.
Methods Furchgott Analysis Equiactive
concentrations of agonist before (X) and after
(X) partial receptor alkylation by 4-DAMP
mustard were estimated, and the following
equation was fitted to the data by nonlinear
regression analysis to estimate the observed
dissociation constant (Kd) and proportion of
residual receptors (q) 1 Knowing the
Kd value of the agonist, it is possible to plot
response against receptor occupancy (Fig 1d, 2d).
The relative efficacy of the agonist was
estimated from this plot using the following
equation occupancy 2 The
relative intrinsic efficacy estimation is made by
comparing the level of occupancy of the various
agonists need to elicit the same amount of
response measured as compared to the standard
agonist, carbachol. The product of affinity and
efficacy of the agonist of interest relative to
the standard can then be made in the following
manner 3 RAi analysis The only
requirements necessary for the RAi estimate are
the concentration-curves of the test and standard
agonists. There are two methods to calculate the
RAi value based on (1) a null equation and (2)
the operational model (Griffin et al., 2007).
The null method involves a fitting the following
null equation to equiactive concentrations of the
test and standard agonist 4 5
Bi and Ai denote the ith concentrations of the
test agonist (B) and standard agonist (A),
respectively. KA denotes the dissociation
constant of the standard agonist, and p and q
represent the ratios of KB/KA and eB/eA. The
operational model was used to estimate RAi by
fitting the concentration-response curves of the
agonists simultaneously to following
equation 6 7 The
agonist response is denoted by R, Xj denotes the
concentration of agonist A or B, Msys denotes the
maximal response of the system, and m denotes the
transducer slope factor. Non-linear regression
analysis yields estimates for tj/Kj for each
agonist (tA/KA, tB/KB). When the Emax of the
test agonist B is less than that of A, it is also
possible to estimate KB.
M2 muscarinic receptor-mediated inhibition of
forskolin-stimulated cAMP in CHO cells. a
Concentration-response curves of selected
muscarinic agonists for inhibition of
forskolin-stimulated cAMP accumulation in CHO M2
cells. The data represent the mean values SEM
of 3 to 8 experiments. b Examples of the effect
of 4-DAMP mustard, an irreversible antagonist, on
carbachol- and oxotremorine-M-mediated inhibition
of forskolin-stimulated cAMP accumulation. c
Relationship between equiactive agonist
concentrations before and after 4-DAMP mustard
treatment. d The normalized response of
selected agonists is plotted against receptor
occupancy.
The estimate of RAi from agonist
concentration-response curves yields a parameter
equivalent to the product of affinity and
efficacy. a Comparison between the product of
affinity and efficacy and the RAi values
estimated from the cAMP response in CHO-M2 cells.
b Comparison between the product of affinity
and efficacy with RAi values estimated from the
contraction response in the M2 KO ileum as well
as RAi values for the phosphoinositide response
in CHO-M3 cells previously reported by Ehlert et
al., 1999.
Effect of tetrodotoxin (TTX) on contraction in
the M2/M3 KO (a) and M2KO (b, c) ileum by
McN-A-343 (a, c) and carbachol (b). Mean values
SEM of 3 to 9 experiments are shown.
Figure 2
Figure 4

• Conclusions
• The RAi value estimated from the agonist
  concentration-response curve for M2 and M3
  muscarinic responses is equivalent to the product
  of affinity and efficacy estimated by the method
  of partial inactivation.
• The RAi estimate is mainly a function of the
  agonist-receptor-G protein interaction and is
  therefore independent of downstream elements in
  the signaling cascade. Consequently, the RAi
  estimate is useful for comparing agonist activity
  across different systems and for detecting
  ligand-directed signaling.
• RAi analysis can be used to identify novel
  agonist behavior, such as the behavior of
  McN-A-343 in gastrointestinal smooth muscle,
  which is generally assumed to be an assay for M3
  function. The anomalously high RAi estimate of
  McN-A-343 in mouse ileum suggests a role for the
  M1 receptor in this response.

M3 muscarinic receptor-stimulated contractions in
the ileum from M2 muscarinic receptor knockout
(M2 KO) mice. a Concentration-response curves
of selected muscarinic agonists for eliciting
contraction in the ileum from M2 KO mice are
shown. The data represent the mean values SEM
of 6 to 13 experiments. b Examples of the
effect of 4-DAMP mustard on carbachol- and
S-aceclidine-stimulated contractions. Mean
values SEM from 5 - 8 experiments are shown.
c Relationship between equiactive agonist
concentrations before and after 4-DAMP mustard
treatment. d The normalized response of
selected agonists is plotted against receptor
occupancy.
Effect of pirenzepine (a, b) and atropine (c, d)
on the contractile action of carbachol (a, c) and
McN-A-343 (b, d) in the isolated ileum from the
M2KO mouse. Mean values SEM from 3 experiments
are shown.
Ehlert FJ and Griffin MT (2001). Annalytical
Pharmacology 234-47. Ehlert FJ, Griffin MT and
Glidden PF (1996). J Pharmacol Exp Ther
2791335-1344. Ehlert FJ, Griffin MT, Sawyer GW
and Bailon R (1999). J Pharmacol Exp Ther
289981-992. Furchgott RF (1966). Adv. Drug Res.
321-55. Furchgott RF and Bursztyn P (1967). Ann.
New York Acad. Sci. 144882-899. Griffin MT,
Figueroa KW, Liller S and Ehlert FJ (2007). J
Pharmacol Exp Ther 3211193-1207.