Receptor classification and antagonist affinity

Adrenergic receptors were initially classified in 1948 by Ahlquist into the a- and P-subtypes on basis of differences in the order of potencies of agonists (Figure 61):

• The a-adrenergic receptor is associated with most of the excitatory functions (e.g. vasoconstriction and contraction of the smooth muscle of the uterus). The potency of catecholamines to trigger these responses decreases in the order: adrenaline > noradrenaline >> isoproterenol.

• The P-adrenergic receptor is associated with most of the inhibitory functions (e.g. vasodilatation and relaxation of the uterine and bronchial smooth muscle) and an important excitatory function (myocardial stimulation). The potency of catecholamines to trigger these responses decreases in the order: isoproterenol >> adrenaline > noradrenaline.

However, because the EC50 values of agonist dose-response curves do not necessarily reflect their KD, such classification could be hazardous in certain circumstances. This is well illustrated in the following example (Figure 62). Consider that agonists A and B have the same affinity, but that the intrinsic efficacy (e) of B is well above than that of A. Whereas the competition binding curves with these two unlabelled agonists will overlap (because of their equal KD), functional assays in systems with an outspoken 'receptor reserve' are likely to reveal a higher potency for B when compared to A.

NORADRENALINE (NOREPINEPHRINE)

ADRENALINE (EPINEPHRINE)

-^_jp-üHUH-UH2-NM-UH(UH3>2 ISOPROTERENOL

«-adrenergic receptor p-adrenergic receptor

concentration (Log) concentration (Log)

Figure 61 Distinction between a- and P-adrenergic receptors.

concentration (Log) concentration (Log)

Figure 61 Distinction between a- and P-adrenergic receptors.

LJ UJ

e(B)>e<A) f y

: EC50(B)

/ EC50(A) /

f receptor J occupancy

/

KD(A) = KD(B)

Figure 62 Different potency ratios for agonists may be obtained by binding studies and physiological experiments in systems with a large 'receptor reserve' (same KD for A and B, but the intrinsic efficacy (e) of B is well above that of A).

The distinct potency patterns might eventually lead to the conclusion that the labelled receptors (binding studies) are different from those which produce the agonist-mediated response.

An axiom of receptor pharmacology is that agonist potency ratios represent a unique identifier of receptors: i.e. the rank order of agonist potencies is dependent on the molecular properties of affinity and efficacy and, hence, a constant that should be independent of the experimental system. However, agonist EC50 values and intrinsic activities (a) constitute weak arguments for classifying receptors.

Antagonist Kj values could be calculated from their IC50 values from competition binding experiments by the Cheng and Prusoff (Cheng and Prusoff, 1973) formula (Section 2.3) provided that the radioligand's KD value was known. However, because of the potential (but a priori unknown) involvement of cellular amplification phenomena in functional assays, agonist EC50 values from agonist dose- response curves could also reflect tissue-dependent factors besides the actual agonist-receptor interaction and, accordingly, they do not necessarily reflect the true KD for the receptor. This handicap prevents the calculatation of antagonist Ki values from their IC50 values from inhibition experiments. Yet, IC50 values may be compared to one another when they are obtained under strictly identical conditions. This provides information about the rank order of antagonist affinities and about antagonist affinity ratios (Figure 63).

Fortunately, antagonist Ki values can be obtained with functional studies by the method developed by Schild and coworkers (Arunlakshana and Schild, 1959). This method is based on the fact that competitive antagonists produce parallel rightward

-11 -10 -9 -8 -7 -6 -5 antagonist concentration (log M)

Figure 63 Ability of different antagonists to inhibit angiotensin II (0.1 ^M) induced inositol phosphate accumulation in CHO cells expressing the human AT1 receptor. Reprinted from British Journal of Pharmacology, 126, Vanderheyden, P.M.L., Fierens, F.L.P., De Backer, J.-P., Frayman, N. and Vauquelin, G., Distinction between surmountable and insurmountable selective ATI receptor antagonists by use of CHO-K1 cells expressing human angiotensin II ATI receptors, 1057-1065, © (1999).

-11 -10 -9 -8 -7 -6 -5 antagonist concentration (log M)

Figure 63 Ability of different antagonists to inhibit angiotensin II (0.1 ^M) induced inositol phosphate accumulation in CHO cells expressing the human AT1 receptor. Reprinted from British Journal of Pharmacology, 126, Vanderheyden, P.M.L., Fierens, F.L.P., De Backer, J.-P., Frayman, N. and Vauquelin, G., Distinction between surmountable and insurmountable selective ATI receptor antagonists by use of CHO-K1 cells expressing human angiotensin II ATI receptors, 1057-1065, © (1999).

shifts of the agonist dose-response curve (Figure 64). In other words, an agonist may produce a certain response at concentration [LJ when present alone and in the presence of a competitive antagonist (at concentration [I]), the agonist concentration must be increased to [L2] to obtain the same response. The ratio of these equi-active agonist concentrations ([L2]/[LJ) is often referred to as 'dose ratio, DR' or 'concentration ratio, CR'.

Figure 64 Agonist dose-response curve: effect of a fixed concentration of a competitive antagonist.

CGP12177 concentration {Log M)

Figure 65 CGP12177 (P3-selective agonist) dose response (LipoLysis in rat fat cells) curve: effect of 0.04, 0.2 and 1 mM metoprolol ^-selective antagonist). Reproduced from Van Liefde, I., Van Witzenburg, A. and Vauquelin, G. (1992) Multiple beta adrenergic receptor subclasses mediate the l-isoproterenol-induced lipolytic response in rat adipocytes. Journal of Pharmacology and Experimental Therapeutics, 262, 552-558, with permission from the American Society for Pharmacology and Experimental Theraputics.

CGP12177 concentration {Log M)

Figure 65 CGP12177 (P3-selective agonist) dose response (LipoLysis in rat fat cells) curve: effect of 0.04, 0.2 and 1 mM metoprolol ^-selective antagonist). Reproduced from Van Liefde, I., Van Witzenburg, A. and Vauquelin, G. (1992) Multiple beta adrenergic receptor subclasses mediate the l-isoproterenol-induced lipolytic response in rat adipocytes. Journal of Pharmacology and Experimental Therapeutics, 262, 552-558, with permission from the American Society for Pharmacology and Experimental Theraputics.

When [I] equals the inhibitor's KD, it will be twice as difficult for the agonist to produce the same response: i.e. [L2] = 2 X [Li]. The method presented below allows us to calculate this particular value of [I] by linear regression analysis of dose-response data obtained in the presence of different antagonist concentrations.

As an example, Figure 65 shows the dose-response curve of CGP12177 to stimulate lipolysis in rat adipocytes, and the ability of increasing concentrations of the P-adrenergic antagonist metoprolol to produce rightward shifts of this curve. Figure 65 clearly shows that the dose ratio will be more pronounced when the antagonist concentration increases. The [L1] value for the control curve (i.e. without agonist) corresponds to the agonist concentration producing an arbitrarily chosen response. [L2] values of the agonist are then measured for the curves obtained in the presence of the different [I]. The next step is to plot Log([L2]/[L1] -1) versus Log([I]). This plot is referred to as a Schild Plot (Arunlakshana and Schild, 1959) and reflects the following equation:

Log([L2]/[L1] -1) = 0 corresponds to the intercept of the plot with the abscissa and, in this situation, Log(Kj) is equal to Log([I]). Hence, the antagonist's Log(Kj) (often referred to in the literature as pA2') can easily be calculated by linear regression of the Schild Plot (Figure 66). Schild regressions represent the most useful physiological tool

Schild Regression

Figure 66 SchiLd plot of the shifted dose-response curves of metoprolol (and for similar experiments with the antagonists propranolol and atenolol). Reproduced from Van Liefde, I., Van Witzenburg, A. and Vauquelin, G. (1992) Multiple beta adrenergic receptor subclasses mediate the l-isoproterenol-induced lipolytic response in rat adipocytes. Journal of Pharmacology and Experimental Therapeutics, 262, 552-558, with permission from the American Society for Pharmacology and Experimental Theraputics.

for pharmacological receptor classification. It is, for example, on basis of such studies that the P-adrenergic receptors in rat adipocytes were discovered to constitute a new subclass, possessing unusually low affinity for antagonists. Indeed, the K value of metoprolol (9.3 ^M) is well above the values typical for pr and p2-adrenergic receptors.

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