Antagonist preincubation no receptor reserve

Functional studies very often include a pre-incubation step, in which the receptors are pre- equilibrated with the antagonist. Subsequently, the agonist will be added and the response measured. This implies that the antagonist had the opportunity to interact with the receptor for some time without any interference from the agonist. Therefore, one prefers to speak in terms of surmountable (parallel shifts of the dose-response curves) and insurmountable antagonism (depression of the maximal response) rather than in terms of competitive/non-competitive antagonism (Figure 225).

Surmountable antagonists (Figure 227, red curve on right panel only) are competitive antagonists that dissociate sufficiently fast from the receptor. This allows the subsequently added agonist to occupy all receptor sites, at least when its concentration is high enough.

Insurmountable antagonists (Figure 227, green curves) are either:

• Non-competitive antagonists (both allosteric and functional).

• Competitive antagonists, but with such long-lasting action that the subsequently added agonist does not get the opportunity to occupy (stimulate) all the receptor sites at the time the response is measured. In other words: the agonist is not long enough in contact with the receptors to surmount the antagonist's action. This type of antagonist is thus likely to display irreversible (i.e. covalent) or slowly reversible binding characteristics in radioligand binding studies. However, alternative theories have also been elaborated to explain insurmountable antagonism. According to some, the antagonists do not need to remain bound to the receptor to produce a long-lasting effect. They could induce a conformational change in the receptor, dissociate and leave the receptor in a conformation that cannot be recognized by agonist molecules. Alternatively, some antagonists could induce receptor internalization, so that it becomes inaccessible to the agonist molecules.

When functional studies include an antagonist pre-incubation step, surmountable inhibition can only be obtained for competitive antagonists. For insurmountable inhibition, it is not possible to find out whether the antagonist is truly non-competitive or not. However, this distinction can be made based on experiments in which the receptors are co-incubated with agonist and antagonist. The AT1-receptor blocker, candesartan, represents a typical example of an insurmountable, yet competitive antagonist (Figure 228).

Non Competitive Antagonist Agonist

Figure 228 The ATj.receptor blocker, candesartan, is an insurmountable, yet competitive antagonist (response is production of inositol phosphates in CHO-ATj cells). Reprinted with permission from Vanderheyden P.M.L., Fierens F.L.P., De Backer J.-P., Frayman N. and Vauquelin G.(1999) Distinction between surmountable and insurmountable selective ATI receptor antagonists by use of CHO-K1 cells expressing human angiotensin II ATI receptors, British Journal of. Pharmacology, 126, 1057-1065; Reprinted from European Journal of Pharmacology, 372, Fierens, F.L.P., Vanderheyden, P.M.L., De Backer, J.-P. and Vauquelin, G., Insurmountable angiotensin II ATI receptor antagonists: the role of tight antagonist binding, 199-206. Copyright (1999), with permission from Elsevier.

Figure 228 The ATj.receptor blocker, candesartan, is an insurmountable, yet competitive antagonist (response is production of inositol phosphates in CHO-ATj cells). Reprinted with permission from Vanderheyden P.M.L., Fierens F.L.P., De Backer J.-P., Frayman N. and Vauquelin G.(1999) Distinction between surmountable and insurmountable selective ATI receptor antagonists by use of CHO-K1 cells expressing human angiotensin II ATI receptors, British Journal of. Pharmacology, 126, 1057-1065; Reprinted from European Journal of Pharmacology, 372, Fierens, F.L.P., Vanderheyden, P.M.L., De Backer, J.-P. and Vauquelin, G., Insurmountable angiotensin II ATI receptor antagonists: the role of tight antagonist binding, 199-206. Copyright (1999), with permission from Elsevier.

Figure 229 [3H]Candesartan dissociates only slowly from the ATj receptor. Its dissociation coincides with the recovery of functional receptors. Reprinted from European Journal of Pharmacology, 367, Fierens, F., Vanderheyden, P. M., De Backer, J. P. and Vauquelin, G., Binding of the antagonist [3H]candesartan to angiotensin II ATI receptor-transfected Chinese hamster ovary cells, 413-422. Copyright (1999), with permission from Elsevier.

Figure 229 [3H]Candesartan dissociates only slowly from the ATj receptor. Its dissociation coincides with the recovery of functional receptors. Reprinted from European Journal of Pharmacology, 367, Fierens, F., Vanderheyden, P. M., De Backer, J. P. and Vauquelin, G., Binding of the antagonist [3H]candesartan to angiotensin II ATI receptor-transfected Chinese hamster ovary cells, 413-422. Copyright (1999), with permission from Elsevier.

As expected for this situation, [3H]candesartan dissociates only slowly from its receptor (dissociation half-life = 120 min under the same experimental conditions as in the functional assay). The dissociation of this radioligand corresponds to the recovery of functional receptors in washout experiments (i.e. experiments in which the receptors are incubated with antagonist, washed and incubated with fresh medium for various time intervals before adding agonist and measuring the response) (Figure 229). This indicates that the receptor can be activated as soon as the antagonist has dissociated.

Slow dissociation of an antagonist-receptor complex will delay the attainment of a mass action-type competition with the natural agonist/messenger each time the concentration of the latter increases. As recently proposed by Swinney (2004), this constitutes a mechanism to improve the 'biochemical efficiency' of the antagonist: i.e. the relationship between its IC50 to inhibit the agonist's functional response and its binding affinity (Kj). Drugs with high biochemical efficiency are therefore likely to achieve the required clinical effect at low concentrations and therefore to possess a high therapeutic window.

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  • antje
    Is competitive antagonism insurmountable?
    6 years ago

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