Methods

1
The Intrinisic Relative Activity of an Agonist
and its Correlation to Affinity and Efficacy
568.18
John A. Tran and Frederick J. Ehlert Department
of Pharmacology, College of Medicine, University
of California, Irvine, Irvine, California
92697-4625
Figure 2
Figure 1
Abstract We investigated a method developed by
our lab for estimating the receptor-dependent
component of agonist activity called intrinsic
relative activity (RAi). Agonist activity at a
receptor-G protein signaling pathway is usually
described in terms of EC50 and maximal response
(Emax). These parameters can vary tremendously
however, depending upon downstream elements in
the signaling pathway. The RAi is a parameter
estimated from the agonist concentration-response
curve that represents the product of observed
affinity and intrinsic efficacy of the agonist
expressed relative to that of a standard agonist.
Consequently, this parameter should be dependent
mainly on the agonist-receptor-G protein
interaction and unaffected by downstream
signaling elements. To prove the validity of the
RAi analysis, we estimated the affinities and
relative efficacies of a series of agonists using
Furchgotts method of partial receptor
inactivation, and compared the respective product
of these two parameters for each agonist with the
corresponding RAi estimate. In these experiments,
we explored agonistmediated inhibition of
forskolin-stimulated cyclic AMP (cAMP) production
in Chinese hamster ovary cells stably expressing
the human M2 muscarinic receptor (CHO-M2). The
product of affinity and efficacy for the
agonists, oxotremorine-M, oxotremorine,
arecoline, S-aceclidine, pilocarpine, bethanechol
and McN-A-343 expressed relative to carbachol
seemed to correspond with their respective RAi
values. Our results indicate that RAi analysis
can be used as a relative measure of the product
of affinity and efficacy of an agonist-receptor
complex. Supported by NIH grant 69829
Introduction The determination of agonist
activity plays an important role in understanding
the interaction between the agonist and its
receptor, as well as drug design. Generally, in
pharmacological research, agonist activity is
described by EC50 (concentration of drug needed
for half-maximal response) and Emax (maximal
response) parameters which can be extrapolated
from concentration-response curves. However,
these values may vary between the different types
of responses measured and the types of systems
(cell line, isolated tissue) utilized, which can
potentially make the determination of agonist
activity at various receptor subtypes
complex. One method to alleviate this problem is
through the use of Furchgotts method of receptor
inactivation (Furchgott, 1966 Furchgott and
Bursztyn, 1967). This method involves the
comparison of agonist concentration-response
curves before and after receptor inactivation
with an irreversible antagonist. Furchgott
analysis results in the determination of
agonist-receptor dependent values of affinity
(1/Kd) and relative intrinsic efficacy (e),
unlike EC50 and Emax values that include
downstream signaling pathways from the receptor
activation that may vary depending on the
response measured. Affinity and efficacy
measurements can be made because the signaling
pathways before and after receptor alkylation are
the same and, therefore can be canceled out of
the calculation, resulting in agonist-receptor
dependent values. Experimentally, Furchgott
analysis is rarely used because receptor
inactivation is tedious and an irreversible
antagonist may not always be available. Instead,
EC50 and Emax values from concentration-response
curves are used for describing agonist
activity. Recently, our lab developed a method
called the intrinsic relative activity (RAi) of
an agonist (Ehlert et al., 1996 Ehlert et al.,
1999 Ehlert et al., 2001) to alleviate this
problem. This parameter is the product of
affinity and intrinsic efficacy of the
agonist-receptor-G protein signaling pathway of
interest and is probably constant for a given
receptor subtype, irrespective of the functional
response measured. All that is required for the
RAi calculation is the agonist-concentration
response curve. To test if RAi values do indeed
result in the product of affinity and efficacy of
a test agonist relative to a standard agonist,
these values will be determined through Furchgott
analysis in CHO-M2 cells measuring the inhibition
of cAMP production with various muscarinic
agonists (oxotremorine-M, oxotremorine,
arecoline, S-aceclidine, pilocarpine, bethanechol
and McN-A-343) against a standard agonist
(carbachol). Our results suggest that RAi values
are equivalent to the product of affinity and
efficacy.
The RAi analysis (null and operational method) of
the various muscarinic agonist using control
agonist-induced inhibition of cAMP data such as
the control oxotremorine and carbachol data seen
in figure 1a and, the comparison of this
parameter with the product of affinity and
efficacy of those same agonists determined
through the use of Furchgott analysis. Mean
values SEM for three to eight experiments are
shown.
Table 1
Agonist Kd, mM Affinity x Efficacy RAi, Null RAi, Operational
Oxotremorine-M 0.29 (6.54 0.14) 9.39 (0.97 0.08) 4.50 (0.65 0.05) 4.96 (0.70 0.07)
Oxotremorine 0.71 (6.15 0.20) 5.87 (0.77 0.15) 5.60 (0.75 0.17) 5.21 (0.72 0.08)
Carbachol 4.3 (5.36 0.18) 1.0 (0.0) 1.0 (0.0) 1.0 (0.0)
Arecoline 18 (4.76 0.24) 0.32 (-0.50 0.19) 0.53 (-0.28 0.10) 0.51 (-0.29 0.08)
S-Aceclidine 11 (4.95 0.13) 0.36 (-0.44 0.07) 0.66 (-0.18 0.07) 0.23 (-0.64 0.08)
Pilocarpine 11 (4.98 0.07) 0.043 (-1.37 0.15) 0.038 (-1.42 0.10) 0.016 (-1.79 0.09)
Bethanechol 425 (3.37 0.68) 0.018 (-1.74 0.34) 0.024 (-1.63 0.16) 0.03 (-1.52 0.10)
McN-A-343 30 (4.53 0.13) 0.002 (-2.65 0.19) 0.003 (-2.56 0.15) 0.003 (-2.60 0.19)
Methods Furchgott analysis. Pairs of
agonist concentrations yielding the same level of
inhibition of cAMP accumulation before and after
receptor inactivation by 4-DAMP mustard are
plotted on a graph (Fig. 1b) and analyzed by the
following equation 1 The agonist
concentrations before and after receptor
alkylation are denoted by x and x, respectively.
Non-linear regression of equation 1 will result
in values for Kd, which denotes the dissociation
constant of the agonist, and q, which represents
the residual amount of receptors remaining after
4-DAMP mustard treatment. Relative intrinsic
efficacy can be determined by comparing the test
agonist against a standard agonist by estimating
the amount of occupancy needed for each agonist
to elicit the same level of response. A plot of
responses measured (inhibition of cAMP response)
versus occupancy (Fig. 1c) for each agonist can
be made by determining the occupancy that
corresponds to each level of response knowing the
Kd 2 Knowing the affinity and
efficacy values for the test agonist and standard
agonist, the product of the two can be made in
the following manner 3 RAi
analysis. All that is needed for this analysis
is the control curves for the test agonist and
standard agonist. The methods to calculate the
intrinsic relative activity of an agonist in this
type of situation may include the null method
and/or the operational method of RAi calculation
(Griffin et al, 2007). The null method involves
comparing equiactive agonist concentrations by
plotting a graph and fitting the points to the
following equation 4 5
Bi and Ai denote the ith concentrations of the
test agonist (B) and standard agonist (A),
repectively. KA denotes a constant for the
affinity of the standard agonist that was fixed
for nonlinear regression to determine p and q,
where p equals KB/KA while q equals eB/eA. The
operational method of RAi analysis is achieved by
fitting the concentration response curves of each
of the agonists to the following
equation 6 7 R denotes
the agonist response while Xj denotes the
concentration of agonist A or B. Msys and m
denote the maximal response of the system and the
slope factor, respectively. Non-linear
regression, holding KA constant, will yield tj
(tA, tB) and KB estimates. RAi values have been
shown previously (Ehlert et al., 1996 Ehlert et
al., 1999 Ehlert and Griffin, 2001) to be
mathematically equivalent to equation 3.
The Kd, product of affinity and efficacy and RAi
values of the various agonist used in the
inhibition of forskolin-stimulated cAMP
production. The log mean values SEM values are
shown in parenthesis.

• Conclusions
• The RAi values for the agonist-mediated
  inhibition of cAMP production in CHO-M2 cells
  correlate with the product of affinity and
  efficacy of the test agonists (oxotremorine-M,
  oxotremorine, arecoline, S-aceclidine,
  pilocarpine, bethanechol and McN-A-343) relative
  to a standard agonist, carbachol. The affinity
  and efficacy values were determined by Furchgott
  analysis.
• The RAi analysis requires less time because the
  analysis involves only the control agonist
  response curves unlike Furchgott analysis where
  an irreversible antagonist, which here is 4-DAMP
  mustard, is necessary.
• RAi values should not vary between responses
  measured and systems utilized because the
  parameter is the product of affinity and efficacy
  for the agonist-receptor-G protein, properties
  that are inherent to the complex.
• RAi analysis can be used as tool to identify the
  agonist- receptor complexes that are the same
  between different responses measured and/or
  systems utilized even though their EC50 and Emax
  values vary. This analysis also can be used to
  characterize agonist-receptor behavior such as
  agonist-directed signaling or the existence of
  receptor activity modifying proteins by showing
  different RAi values even though the
  agonist-receptor complex is the same.

The inhibition of forskolin-stimulated cAMP
accumulation in CHO-M2 cells (a) by carbachol
(open circles), oxotremorine-M (open triangles)
and 4-DAMP mustard treatment (closed symbols).
Mean values SEM are shown from five to eight
experiments. Furchgott analysis (b, c) using
equation 1 to determine agonist affinity (b Kd
values are shown in Table 1) and equation 2 to
determine the relative efficacy (e c) of
oxotremorine-M compared to carbachol. This
analysis is done using the mean data found in a.
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