Patrick M.L Vanderheyden

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

CHO‐K1 cells that were stably transfected with the gene for the human AT1 receptor (CHO‐AT1 cells) were used for pharmacological studies of non‐peptide AT1 receptor antagonists. In the presence of 10u2003mM LiCl, angiotensin II caused a concentration‐dependent and long‐lasting increase of inositol phosphates accumulation with an EC50 of 3.4u2003nM. No angiotensin II responses are seen in wild‐type CHO‐K1 cells. [3H]‐Angiotensin II bound to cell surface AT1 receptors (dissociates under mild acidic conditions) and is subject to rapid internalization. Non‐peptide selective AT1 antagonists inhibited the angiotensin II (0.1u2003μM) induced IP accumulation and the binding of [3H]‐angiotensin II (1u2003nM) with the potency order: candesartan > EXP3174 > irbesartan > losartan. Their potencies are lower in the presence of bovine serum albumin. Preincubation with the insurmountable antagonist candesartan decreased the maximal angiotensin II induced inositol phosphate accumulation up to 94% and, concomitantly, decreased the maximal binding capacity of the cell surface receptors. These inhibitory effects were half‐maximal for 0.6u2003nM candesartan and were attenuated by simultaneous preincubation with 1u2003μM losartan indicating a syntopic action of both antagonists. Losartan caused a parallel rightward shift of the angiotensin II concentration‐response curves and did not affect the maximal binding capacity. EXP3174 (the active metabolite of losartan) and irbesartan showed a mixed‐type behavior in both functional and binding studies. Reversal of the inhibitory effect was slower for candesartan as compared with EXP3174 and irbesartan and it was almost instantaneous for losartan, suggesting that the insurmountable nature of selective AT1 receptor antagonists in functional studies was related to their long‐lasting inhibition.

Binding of the antagonist [3H]candesartan to angiotensin II AT1 receptor-tranfected Chinese hamster ovary cells

Abstract Binding of the non-peptide angiotensin II AT1 antagonist [ 3 H ](2-ethoxy-1-[(2′-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]-1H-benzimidazoline-7-carboxylic acid ([ 3 H ]candesartan) to human angiotensin II AT1 receptor-transfected Chinese hamster ovary (CHO-AT1) cells was inhibited to the same extent by angiotensin II and non-peptide angiotensin II AT1 antagonists. No binding was observed in control CHO-K1 cells. Dissociation was slow (k−1=0.0010±0.0001 min−1) after removal of the free [ 3 H ]candesartan but increased 5-fold upon addition of supramaximal concentrations of angiotensin II AT1 antagonists. Angiotensin II responses recovered equally slow from candesartan-pretreatment. When washed and further incubated, these angiotensin II responses also recovered more rapidly in the presence of 2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2′-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole (losartan), indicating that unlabelled ligands prevented reassociation. [ 3 H ]candesartan saturation binding experiments required a long time to reach equilibrium. Therefore, the equilibrium dissociation constant (Kd=51±8 pM) was calculated from the association and dissociation rate constants. Our findings indicate that the insurmountable nature of candesartan in functional studies is related to its slow dissociation from the receptor.

Insurmountable angiotensin AT1 receptor antagonists: the role of tight antagonist binding.

Angiotensin II increased the inositol phosphates production (EC50 = 3.4+/-0.7 nM) in Chinese hamster ovary (CHO) cells expressing the cloned human angiotensin AT1 receptor (CHO-AT1 cells). Coincubation with angiotensin AT1 receptor antagonists produced parallel rightward shifts of the concentration-response curve without affecting the maximal response. The potency order is 2-ethoxy-1-[(2-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]-1H-benz imidazoline-7-carboxylic acid (candesartan) > 2-n-butyl-4-chloro-1-[(2-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]i midazole-5-carboxylic acid (EXP3174) > 2-n-butyl-4-spirocyclopentane-1-[(2-(1H-tetrazol-5-yl)biphe nyl-4-yl)methyl]2-imidazolin-5-one (irbesartan)> of 2-n-butyl-4-chloro-5-hydroxymethyl-1-(2-(1H-tetrazol-5-yl)bipheny l-4-yl)methyl]imidazole (losartan). Additionally, preincubation with these antagonists depressed the maximal response, i.e., 95%, 70%, 30% of the control response for candesartan, EXP3174 and irbesartan and not detectable for losartan. Increasing the antagonist concentration or prolonging the preincubation time did not affect this depression. Furthermore, these values remained constant for candesartan and EXP3174, when the angiotensin II incubation time varied between 1 and 5 min. Our data indicate that antagonist-receptor complexes are divided into a fast reversible/surmountable population and a tight binding/insurmountable population at the very onset of the incubation with angiotensin II.

Monoclonal antibodies against the native or denatured forms of muscarinic acetylcholine receptors.

BALB/c mice were immunized with affinity‐purified muscarinic acetylcholine receptors from calf brain and their splenocytes fused with NS1 myeloma cells. Hybrid cultures were grown and selected for production of antibodies on the basis of enzyme immunoassays on calf and rat forebrain membrane preparations. Thirty‐four clones were retained and six of them further subcloned. Two of these subclones produced antibodies that selectively recognized muscarinic acetylcholine receptor‐bearing membranes. The M‐35b antibodies interacted only with native digitonin‐solubilized receptors, and not with denatured receptors. The M‐23c antibodies did not react with active digitonin‐solubilized receptors but recognized the denatured form. The M‐23c antibodies should thus be useful in the purification of the receptor and its precursor translation products, while the M‐35b antibodies could be used for the immunocytochemical localization of the receptor in cells and tissues of different species.

Purification of muscarinic acetylcholine receptors by affinity chromatography.

Calf forebrain homogenates contain 2.8 pM muscarinic acetylcholine receptors per mg of protein. [3H]Antagonist saturation binding experiments under equilibrium conditions revealed a single class of sites with equilibrium dissociation constants of 0.82 nM for [3H]dexetimide and 0.095 nM for [3H]quinuclidinyl benzilate. Displacement binding studies with agonists revealed the presence of low and high affinity sites. Here we describe the solubilization of muscarinic acetylcholine receptors with digitonin and their purification by affinity chromatography using an affinity gel which consisted of dexetimide coupled to Affi‐Gel 10 (i.e., carboxy N‐hydroxysuccinimide esters linked via a 1 nm spacer arm to agarose beads). Purified proteins were obtained by specific elution with muscarinic drugs, i.e., the antagonist atropine and the irreversible ligand propylbenzilylcholine mustard. SDS‐polyacrylamide gel electrophoresis of the radioiodinated purified preparations revealed a major 70‐K protein.

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

The 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.

Models and methods for studying insurmountable antagonism.

Insurmountable antagonists depress the concentration-response curves of subsequently added agonists. The longevity of the antagonist-receptor complex and the existence of allosteric binding sites are the most frequent explanations for this phenomenon. Yet, observed antagonist behaviour often depends on the tissue, the animal species, the duration of the measured response and the study design. Intact cell studies allow greater flexibility and tighter control of the experimental conditions and therefore have the potential to offer a better insight into the molecular basis of insurmountable antagonism.

New insights in insurmountable antagonism

Antagonists that produce parallel rightward shifts of agonist dose–response curves with no alteration of the maximal response are traditionally classified as surmountable, while insurmountable antagonists also depress the maximal response. Although the longevity of the antagonist–receptor complex is quoted in many studies to explain insurmountable antagonism, slowly interconverting receptor conformations, allosteric binding sites, and receptor internalization have been evoked as alternative explanations. To complicate matters even further, insurmountable antagonism is not only drug‐related; it may also depend on the tissue, species and experimental design. For the sake of drug development, it is important to elucidate the molecular mechanisms of insurmountable antagonism. New experimental approaches, such as intact cell studies and the use of computer‐assisted simulations based on dynamic receptor models, herald the advent of better insight in the future.

Reversible and syntopic interaction between angiotensin receptor antagonists on Chinese hamster ovary cells expressing human angiotensin II type 1 receptors.

Evidence for a competitive type of interaction between angiotensin II type 1 (AT(1)) antagonists on Chinese hamster ovary cells expressing the human AT(1) receptor (CHO-AT(1)) was obtained by analyzing the binding of [(3)H]-2-ethoxy-1-[(2-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]-1H- ben zimidazoline-7-carboxylic acid ([(3)H]candesartan) and by measuring the AT-induced production of inositol phosphates. The AT(1) antagonists candesartan, 2-n-butyl-4-chloro-1-[(2-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]+ ++imid azole-5-carboxylic acid (EXP3174), or 2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2-(1H-tetrazol-5-yl)bip hen yl- 4-yl)methyl]imidazole (losartan) produced a concentration-dependent increase in the apparent K(d) values of [(3)H]candesartan in saturation binding experiments, while the B(max) values were unchanged. Furthermore, the dissociation rate of the radioligand initiated by 1 microM unlabelled candesartan was not changed in the presence of 10 microM losartan, 10 microM EXP3174, or 10 microM irbesartan (2-n-butyl-4-spirocyclopentane-1-[(2-(1H-tetrazol-5-yl)b iph enyl-4-yl) methyl]2-imidazolin-5-one)). Preincubation of the CHO-AT(1) cells with candesartan, EXP3174, and irbesartan caused a reduction in the maximal AT-induced inositol mono-, bis-, and trisphosphate production. This insurmountable effect was reversed in the presence of 1 microM losartan. In line with this finding, the insurmountable antagonist concentration-inhibition curves at 10 microM AT were shifted to the right in the presence of losartan. For candesartan this effect was concentration-dependent, yielding a pK(B) value for losartan of 7.7, which is similar to the pK(B) from previously obtained AT concentration-response curves. Finally, the dissociation rate of candesartan, EXP3174, irbesartan, and losartan was determined by measuring the recovery of AT responses after antagonist pretreatment and washing of the cells with medium containing 1 microM losartan to prevent re-association of the insurmountable antagonists. In addition, similar kinetic data were obtained from the slowing of the [(3)H]candesartan association rate to antagonist preincubated cells.

Angiotensin II type 1 receptor antagonists. Why do some of them produce insurmountable inhibition

Chinese hamster ovary (CHO) cells expressing human recombinant angiotensin II type 1 (AT(1)) receptors offer a useful experimental system in which antagonist binding and inhibition of AT-induced inositol mono-, bis-, and trisphosphate accumulation can be measured under identical experimental conditions. The major conclusions of the current work are: All investigated AT(1) antagonists are competitive with respect to AT. They bind to a common or overlapping binding site on the receptor in a mutually exclusive way. Reduction of the maximal angiotensin II response, i.e. insurmountable inhibition, is observed only when the cells are preincubated with candesartan, EXP3174, or irbesartan and is strictly related to the dissociation rate of the antagonist-receptor complex. On the other hand, inhibition by losartan is fully surmountable by AT, and its dissociation is very rapid. With respect to the binding kinetics, the antagonist-receptor complex can adopt a fast and a slow reversible state. The equilibrium between both states, which is dependent upon the nature of the antagonists, determines the extent of insurmountable inhibition. Consequently, the dissociation rate of the different antagonists correlates with the amount of insurmountable inhibition. In addition to the relatively slow dissociation of candesartan, reassociation to the receptor, which is measurable in CHO-AT(1) cells, likely contributes to its long-lasting blood pressure lowering effect in vivo.