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Overview of Receptor Interactions of Agonists and Antagonists
Publication Name:
Current Protocols in Pharmacology
Unit Number:
Unit 4.1
DOI:
10.1002/0471141755.ph0401s42
Online Posting Date:
September, 2008
Abstract
Historically, the earliest methods used to quantitatively measure the fundamental properties of drugs (namely affinity and efficacy) employed isolated tissues, and it is in this realm that the null methods used to define “receptor pharmacology” were described. This unit describes these methods and their use to specifically classify agonists (through potency ratios and determination of relative affinities and efficacies) and antagonists (through analysis of surmountable and insurmountable antagonism) to yield estimates of potency. Different drugs can yield different behaviors in various tissues, so this unit is centered on a flow diagram to indicate the type of analysis appropriate for the behavior observed. For example, some agonists may be full agonists in some tissues and partial agonists in others, while some antagonists may demonstrate surmountable simple competitive antagonism in some tissues and insurmountable non-competitive antagonism in others. Methods exist for determination of affinity and efficacy for all of these behaviors, and these are delineated in this unit. Curr. Protoc. Pharmacol. 42:4.1.1-4.1.24. © 2008 by John Wiley & Sons, Inc.
Keywords: receptor theory; drug classification; in vitro analysis; affinity; efficacy
Figures
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Figure 4.1.1 Components of a dose-response curve. This relationship between the logarithm of the concentration of drug added to the receptor compartment and the resulting biological response is defined by a threshold, a slope, and a maximal response asymptote. The location parameter (position) of the curve along the concentration (x) axis defines the drug potency (alternatively, the system sensitivity), and is most often denoted as the concentration of drug producing half the maximal effective response (ED 50 ). -
Figure 4.1.2 Standard methods to obtain dose-response curves. (A) Cumulative addition of agonist in a preparation producing sustained responses yields a cumulative dose-response curve. (B) If the response is transient, then single concentrations followed by a period of washing with drug-free medium may yield a dose-response curve. (C) If the agonist desensitizes the tissue, then sequential addition of increasing doses may produce a systematic loss in sensitivity. (D) The systematic loss in sensitivity shown in panel C may be reduced by randomized addition of doses in order to distribute the error around the dose-response curve. -
Figure 4.1.3 Two opposing scenarios of equiactive responses to two agonists in isolated tissues. (A) In ideal behavior, two successive dose-response curves are obtained in the tissue and the baseline response of the tissue is unchanged between the procedures. (B) The resting tension (baseline) of the preparation declines after the first dose-response curve. If the dose-response curves are normalized (% maximum of each individual curve), then points of equiactive responses (n) on the normalized curves are not truly equiactive in terms of the real units of responsiveness of the tissue. -
Figure 4.1.4 Graphical depictions of dose-response curves. (A) Data points are joined by straight lines. Under these circumstances, the ED 50 (solid arrow) set by only two data points (in addition to the point determining the maximal asymptote) determines the curve location, which signifies agonist potency. (B) A line utilizing all of the data points is fit to the general logistic function. The position of this curve depends upon the complete data set and a more accurate determination of the ED50 value is obtained (open arrow). -
Figure 4.1.5 Comparison of the relative location parameters of two dose-response curves generated from two agonists (solid and open circles, respectively). (A) If lines joining individual data points are used to define the curve, then the dose ratio (DR) of the agonists depends upon the level of response chosen to make the measurement. (B) If the data points are fit to two logistic functions of common slope, the DR is independent of the level of response. -
Figure 4.1.6 Effect of stimulus-response coupling on the response to an agonist. Low levels of receptor occupancy by a high-efficacy agonist can produce a stimulus that is amplified by a succession of saturable biochemical reactions. Under these conditions, the dose-response curve (solid line) can be shifted to the left of the receptor occupancy curve (dotted line). [A] is the agonist concentration and K A is the equilibrium dissociation constant of the agonist-receptor complex. -
Figure 4.1.7 Flow-chart strategy for the measurement of drug-receptor constants in isolated tissues. Depending upon the responses, there is an array of techniques suitable for assessing drug effects and receptor function. The letters next to the procedures refer to method descriptions in the text. -
Figure 4.1.8 Measurement of potency ratios in isolated tissues. (A) Both agonists A 1 and A2 produce tissue maximal response and have parallel dose-response curves. These agonists most likely possess similar magnitudes of intrinsic efficacy. The potency ratio does not depend upon the level of response at which the measurement is made. (B) Comparison of potencies of a high-efficacy agonist (A1 ) and a low-efficacy agonist (A2 ). The curves tend to be nonparallel and the relative potency depends upon the level of response used to make the measurement. -
Figure 4.1.9 Theory of the method of selective desensitization to identify receptors activated by agonists. In panel (A), two agonists, A 1 and A2 , activate separate receptors, R1 and R2 , respectively. Selective desensitization of R1 eliminates or greatly diminishes the response to agonist A1 , but the response to A2 is unaffected. Panel (B) shows the results if both agonists activate R1 : in this case, both responses are diminished by desensitization. -
Figure 4.1.10 Two patterns of antagonism as described by Gaddum et al. ( -
Figure 4.1.11 The effects of increasing times of equilibration for antagonists producing surmountable (A) and insurmountable (B) antagonism. An initial fast phase of antagonism (represented by the distance between successive curves from left to right) is followed by an approach to a steady state beyond which no further increase in equilibration time produces changes in the antagonism. Arrows indicate increasing equilibration times. -
Figure 4.1.12 The effects of a noncompetitive antagonist on (A) agonist receptor occupancy (amount of response producing [AR] complex) and (B) agonist response to a high-efficacy agonist in a tissue with a receptor reserve for that agonist. Arrows indicate increasing concentration of an irreversible antagonist. -
Figure 4.1.13 Schild analysis of the blockade of responses to an agonist by a competitive antagonist. (A) The effect of a simple competitive antagonist on dose-response curves to an agonist (A). Dose-ratios (DR) 1 to 4 refer to the effects of increasing concentrations of antagonist. (B) The DR produced by the concentrations of antagonist B are plotted as log (DR – 1) values as a function of the respective concentrations of antagonist producing the specific DR (regression according to Equation ( B (–logKB ) on the abscissa. -
Figure 4.1.14 Dose-response curves to an agonist shifted to the right by increasing concentrations of a simple competitive antagonist. The four curves in each part are for different antagonist concentrations, shown increasing from left to right. (A) Data points joined by straight lines. (B) Dose-response lines (general logistic function) fit statistically to the same data, yielding curves of common slope and maxima. -
Figure 4.1.15 Schild analysis of blockade of responses to an agonist by a competitive antagonist. (A) An array of log(DR – 1) values plotted as a function of log[antagonist]. The solid line represents the best fit straight line through the data, and the dotted line is of unit slope through the mean x and y values. (B) A statistical analysis of the error on the slope included unity in the 95% confidence limits. Therefore, the data were refit to a straight line of slope unity (shown as the dotted line). The best-fit straight line of unit slope yields an estimate of the true pK B (intercept). (C) A Schild regression consisting of dose ratios >30. The natural random error on the slope reflects an inordinately large error on the abscissa resulting in a large uncertainty in the estimate of the pKB . (D) Data points obtained near ordinate values of zero (low DRs) allow interpolation of the pKB with increased accuracy. [B] is the concentration of antagonist and KB is the equilibrium dissociation constant for the antagonist-receptor complex. -
Figure 4.1.16 Effect of nonequilibrium steady states on Schild regressions. (A) Effect of saturation of uptake on the agonist. (B) Effect of a saturable uptake process for the antagonist or inadequate time of equilibration of antagonist, where the rate of diffusion is not limiting. The dotted lines represent a slope equal to unity. -
Figure 4.1.17 Detection of heterogeneous receptor populations with Schild analysis. (A) All three agonists (A 1,2,3 ) activate the same receptor (R1 ). The antagonist potency measured by Schild analysis yields the same pKB and the Schild regressions are identical. (B) The agonists differentially activate two receptors for which the antagonist B has differing affinity. The potency of the antagonist varies when measured with the three different agonists, resulting in differing Schild regressions. -
Figure 4.1.18 ”Limitless” (unsaturable) antagonism. Defined as conditions whereby the antagonist, if administered in sufficiently high concentration, will completely eliminate the agonist concentration-response curve either in cases of (A) insurmountable antagonism or (B) surmountable antagonism. Such behavior is indicative of, but not necessarily solely due to, orthosteric antagonistic effects. Shown are curves expressed as a fraction of maximal receptor occupancy or tissue response obtained in the absence of antagonist (curve furthest to left) and presence of increasing concentrations of antagonist. -
Figure 4.1.19 Saturation of antagonistic effects. If the interaction between the agonist and antagonist occurs through each of the two species binding to a separate site on the receptor, then when the allosteric (antagonist) site is saturated, the antagonism will reach a limit. Thus, increasing concentrations of antagonist will fail to produce further dextral displacement of the agonist concentration-response curve for (A) surmountable antagonists and further shift and/or depression of maximal responses for (B) insurmountable antagonists. -
Figure 4.1.20 The measurement of the equilibrium dissociation constant for a noncompetitive antagonist. (A) Dose-response curves to the agonist in the absence (A) and presence (A¢) of a noncompetitive antagonist are obtained and pairs of equiactive concentrations of agonist identified. (B) A double reciprocal plot of the equiactive agonist concentrations yields a linear regression which can be fit to Equation ( B using Equation ( -
Figure 4.1.21 Antagonism with hemi-equilibrium conditions. In some cases the antagonist offset rate from the receptor is so slow as to essentially irreversibly occupy a portion of the receptor population, specifically the portion required by high concentrations of agonist to produce maximal response. Therefore, the concentration-response curves in the presence of the antagonist are shifted to the right as with competitive blockade, but the maxima may be depressed. The depression of maxima may reach a limiting value but, unlike insurmountable allosteric blockade that comes to a limiting value, the dextral displacement continues and does not come to a limiting value. -
Figure 4.1.22 Effect of progressive irreversible reduction of receptor density by chemical alkylation on dose response to a high-efficacy agonist (A). The curves illustrate different levels of alkylation, increasing from left to right. Note that in the region where the maximal response is depressed, the ED 50 value of the dose-response curves (indicated by the arrow) approximates the KA value. -
Figure 4.1.23 The method of partial alkylation of receptors to estimate the K A of an agonist (A). (A) Dose-response curves are obtained before (solid circles) and after (open circles) partial alkylation of the receptors. Maximal response to the agonist is depressed, and equiactive concentrations of agonists are identified (response levels 1 to 3). (B) Reciprocals of equiactive agonist concentrations are plotted, and the resulting straight line evaluated according to Equation (A value from Equation ( -
Figure 4.1.24 Measurement of partial agonist affinity. (A) Equiactive concentrations of a full agonist (A) and a partial agonist (P) are identified (response levels 1 to 4) and compared (B) in a double-reciprocal regression evaluated according to Equation ( P ) according to Equation ( -
Figure 4.1.25 Measurement of the equilibrium dissociation constant of a partial agonist (K P ) by use of dose-ratios to a full agonist produced by a range of concentrations of partial agonist (Kaumann and Marano,P of the partial agonist from all concentrations.
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| Key References | |
|
Furchgott,
1972. See | |
|
Discusses very important concepts in isolated tissue methods with respect to the use of techniques described in this unit. | |
| Kenakin, T.P. 2006. A Pharmacology Primer: Theory, Application and Methods, 2nd ed. Academic Press/Elsevier, Amsterdam. | |
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A survey of methods and procedures for measuring drug activity in isolated tissues. | |
