A two-state receptor model for the interaction between angiotensin II type 1 receptors and non-peptide antagonists

doi:10.1016/S0006-2952(00)00546-3

Biochemical Pharmacology

Volume 61, Issue 3, 1 February 2001, Pages 277-284

https://doi.org/10.1016/S0006-2952(00)00546-3 Get rights and content

Abstract

¹The interaction between non-peptide antagonists and the human angiotensin II type 1 (AT₁) receptor in CHO-K1 cells was investigated by incubating the cells with antagonist, followed by a brief exposure to angiotensin II and measurement of the resulting inositol phosphate accumulation. The experimental data, expressed either as angiotensin II concentration–response curves or as antagonist concentration–inhibition curves, were in good agreement with computer-generated data according to a single-state model for the surmountable antagonist losartan and according to a two-step, two-state receptor model for the insurmountable antagonists candesartan, EXP3174, and irbesartan. Experimental and computer-generated data concerning the simultaneous exposure of the receptors to EXP3174 and losartan indicated that losartan produced a concentration-dependent restoration of the maximal response (angiotensin II concentration–response curves) as well as a rightward shift of the insurmountable portion of the EXP3174 inhibition curves, thus counteracting the higher-affinity EXP3174 binding. In conclusion, these findings provide further support for the concept that insurmountable and surmountable AT₁ antagonists are mutually competitive and that insurmountable antagonist–receptor complexes may adopt different states.

Introduction

Several non-peptide AT₁ receptor antagonists have been developed for the clinical treatment of hypertension and congestive heart failure [1]. The interaction between these antagonists and the AT₁ receptor has often been studied by in vitro contraction experiments on rabbit aorta rings/strips and rat portal vein. More recently, relevant information was also obtained by measuring angiotensin II-induced IP production in cell lines expressing transfected AT₁ receptors [2], [3], [4], [5]. Both kinds of studies plead for the existence of two categories of antagonists. Surmountable antagonists, such as losartan, produce parallel rightward shifts of the angiotensin II concentration–response curve and do not affect the maximal response [3], [6]. In contrast, insurmountable antagonists decrease the maximal response to angiotensin II. This decrease may be partial for antagonists such as irbesartan and EXP3174 (the active metabolite of losartan) to almost complete for antagonists such as candesartan [3], [7], [8], [9], [10].

Several theories have been advanced over the past ten years to explain the molecular mechanism of surmountable and insurmountable AT₁ receptor antagonists. They include the presence of allosteric binding sites on the receptor [11], slowly interconverting receptor conformations [12], [13], [14], slow removal of the antagonist from tissue compartments, cells, or matrix surrounding the receptor [15], [16], coexistence of different receptor subpopulations [17], and the ability of the antagonist to modulate the amount of internalized receptors [7] or slow dissociation of the antagonist–receptor complex [18], [19], [20], [21], [22]. This latter possibility has been favored by most authors and is now also strongly supported by the observation that insurmountable AT₁ receptor antagonists only depress the maximal response to angiotensin II when they are pre-exposed to the receptors. Interestingly, the same antagonists are able to produce rightward shifts of the angiotensin II concentration–effect curve without depressing the maximal response when they are added simultaneously [5], [23]. Similarly, antagonists which decreased the maximal binding capacity of labeled angiotensin II when added to the receptors first did not do so under co-incubation conditions [5]. These findings support the proposal that insurmountable AT₁ receptor antagonists inhibit the response to angiotensin II in a competitive fashion and that, when they are pre-exposed to the receptor, their antagonistic action may be so slowly reversible that it cannot be overcome during the ensuing exposure of the receptors to the agonist [5]. Surmountable antagonists are competitive with angiotensin II as well, but their action is readily reversible so that it can be completely overcome by the subsequently added agonist.

It is intriguing why most of the insurmountable antagonists only produce a partial decrease of the maximal response to angiotensin II. As a possible explanation for this phenomenon, it was recently proposed that antagonist–AT₁ receptor complexes may adopt a fast reversible (surmountable) as well as a tight-binding (insurmountable) state and that there is an equilibrium between both states that is dependent on the chemical nature of the bound antagonist [5].

In the present study, we correlated experimental data describing the interaction between insurmountable (candesartan, EXP3174, and irbesartan) AT₁ receptor antagonists and their receptors with computer-generated data according to a two-step, two-state receptor model. Experimental and computer-generated data concerning the simultaneous exposure of the receptors to surmountable and insurmountable AT₁ antagonists also provided further support for the concept that these antagonists are mutually competitive.

Section snippets

Materials

Candesartan [9], EXP3174 [13], losartan [13], and irbesartan [8] were obtained from AstraZeneca. Angiotensin II was obtained from Sigma, myo-[³H]inositol (20 Ci/mmol) was from Pharmacia/Amersham/Biotech. All other chemicals were of the highest grade commercially available.

Cell culture

CHO-K1 cells stably expressing the human angiotensin II AT₁ receptor (CHO-AT₁ cells) were obtained as described by Vanderheyden et al. [3] and were cultured in 75-cm² flasks in DMEM which was supplemented with l-glutamine (2

Results

CHO-AT₁ cells were preincubated for 30 min with increasing concentrations of antagonist, followed by a 5-min challenge with a maximally effective concentration of angiotensin II (10 μM). The resulting experimental IP accumulation data yielded a monophasic inhibition curve for losartan and biphasic curves for the insurmountable antagonists with plateau values at about 5% for candesartan, 25% for EXP3174, and 61% for irbesartan (Fig. 2, Table 2). The complexity of the experiments (involving a

Discussion

AT₁ receptor antagonists only depress the maximal angiotensin II-mediated response when they are allowed to interact with the receptor before the agonist [5], [23]. This depression, or insurmountable antagonism, is traditionally reported by presenting the effect of antagonists on angiotensin II concentration–response curves. It emerges from many reports that when the concentration of an insurmountable antagonist is gradually increased, it will depress the maximal response until a certain level

Acknowledgements

G. V. is Onderzoeksdirecteur of the Fonds voor Wetenschappelijk Onderzoek Vlaanderen, Belgium. We are also most obliged to the Queen Elisabeth Foundation Belgium and the Onderzoeksraad of the Vrije Universiteit Brussel for their kind support. This text presents research results of the Belgian program on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister’s Office, Science Policy Programing. Scientific responsibility is assumed by its authors.

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Molecular and cellular pharmacologyA two-state receptor model for the interaction between angiotensin II type 1 receptors and non-peptide antagonists

Abstract

Introduction

Section snippets

Materials

Cell culture

Results

Discussion

Acknowledgements

J Biol Chem

Eur J Pharmacol

Eur J Pharmacol

Trends Pharmacol Sci

Eur J Pharmacol

Eur J Pharmacol

Biochem Pharmacol

Biochem Biophys Res Commun

Am J Hypertens

Am J Hypertens

, Angiotensin antagonists

Distinction between surmountable and insurmountable selective AT1 receptor antagonists by use of CHO-K1 cells expressing human angiotensin II AT1 receptors

Br J Pharmacol

Quantitative studies on antagonists for 5-hydroxytryptamine

Q J Exp Physiol

Evidence that the apparent complexity of receptor antagonism by angiotensin II analogues is due to a reversible and synoptic action

Br J Pharmacol

Pharmacological characterization of SR 47436, a new non-peptide AT1 subtype angiotensin II receptor antagonist

J Pharmacol Exp Ther

Inhibition of rabbit aortic angiotensin II (AII) receptor by CV-11974, a new neuropeptide AII antagonist

Biochem Pharmacol

Molecular and cellular pharmacology
A two-state receptor model for the interaction between angiotensin II type 1 receptors and non-peptide antagonists

Distinction between surmountable and insurmountable selective AT₁ receptor antagonists by use of CHO-K1 cells expressing human angiotensin II AT₁ receptors

Pharmacological characterization of SR 47436, a new non-peptide AT₁ subtype angiotensin II receptor antagonist