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.

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.

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.

Lys(199) mutation of the human angiotensin type 1 receptor differentially affects the binding of surmountable and insurmountable non-peptide antagonists.

Many slow dissociating (insurmountable) non-peptide angiotensin type 1 receptor (AT1) antagonists contain,besides the acidic biphenyltetrazole substructure of losartan, a second acidic group to stabilise antagonist-receptor complexes. To investigate the involved basic amino-acids of the human AT1-receptor, wild-type and mutant receptors were transiently transfected in CHO-K1 cells and characterised by [3H]candesartan binding. Lys199-->Gln substitution decreased the affinity 45-fold for candesartan (95% insurmountable),18-fold for EXP3174 (70% insurmountable), 10-fold for irbesartan (40% insurmountable) and 5-fold for losartan (surmountable). His256 -->Ala substitution had only minor effects. This suggests that Lys199 is important for the tight binding of non-peptide antagonists.

Binding characteristics of [(3)H]-irbesartan to human recombinant angiotensin type 1 receptors.

The aim of the present work was to investigate the binding properties of the selective AT(1)-receptor antagonist irbesartan to human AT(1)-receptors by direct radioligand binding. For this purpose the specific binding of [(3)H]-irbesartan to intact Chinese Hamster Ovary (CHO) cells expressing human recombinant AT(1)-receptors was determined. Specific binding of [(3)H]-irbesartan rapidly reached equilibrium and was saturable with a KD of 1.94 +/- 0.12 to a homogeneous class of binding sites. Its binding was inhibited by other AT(1) antagonists (AIIAs) with the same potency order as previous results from [(3)H]-angiotensin II and [(3)H]-candesartan binding to human AT(1)-receptors. Whereas the dissociation rate of [(3)H]-irbesartan was essentially independent of the radioligand concentration, it was much slower at 12 degrees C when compared with 37 degrees C. Moreover, the dissociation rate was similar, as determined in washout experiments in the absence or presence of unlabelled AT(1) antagonists. At 37 degrees C the dissociation rate constant corresponded to a half-life of approximately seven minutes, which is sufficient to explain the partially insurmountable inhibition by irbesartan in previous studies. In contrast, other phenomena such as the plasma half life and tissue-related factors are necessary to explain its sustained in vivo antihypertensive effect.

Interaction between the partially insurmountable antagonist valsartan and human recombinant angiotensin II type 1 receptors.

The interaction between the AT1 receptor-selective antagonist valsartan, and its human receptor, was investigated by direct radioligand binding as well as by its inhibition of angiotensin II induced inositol phosphate accumulation in CHO cells expressing human recombinant AT1 receptors. Specific binding of [3H]-valsartan rapidly reached equilibrium at 37 degrees C. It was saturable and occurred to a homogeneous class of sites with a K(D) of 0.88+/-0.076. It was inhibited by other AT1 receptor antagonists with the same potency order as previously described for the binding of [3H]-angiotensin II and [3H]-candesartan to human AT1 receptors (i.e. candesartan > or = EXP3174 > valsartan = irbesartan = angiotensin II > losartan). When valsartan and angiotensin II were applied simultaneously to the CHO-AT1 cells. the antagonist caused a rightward shift of the angiotensin II concentration response curve. Hence, valsartan interacts with the AT1 receptor in a manner that is competitive with angiotensin II. Pre-incubation of the cells with 0.5, 5 and 50 nM valsartan caused an additional, concentration-dependent, up to 55% decline of the maximal response. The partial nature of this insurmountable inhibition by valsartan was confirmed by biphasic antagonist concentration-inhibition curves. These data reflect the co-existence of a fast reversible/surmountable as well as a tight binding/insurmountable valsartan receptor complex. In agreement, pre-incubation of the CHO-AT1 cells with 5 and 50 nM valsartan produced a partial inhibition of the angiotensin II induced increase of the free intracellular calcium concentration. [3H]-Valsartan dissociated from its receptors with a half-life of 17 min. In functional recovery experiments with valsartan-pre-treated cells, the angiotensin II-mediated response was half-maximally restored within approximately 30 min. These kinetic data suggest that the insurmountable inhibition by valsartan is related to its relatively slow dissociation from the human AT1 receptors.

Inhibition of angiotensin II-induced inositol phosphate production by triacid nonpeptide antagonists in CHO cells expressing human AT1 receptors.

PURPOSE: The aim of the present work is to describe the inhibitory properties of LY301875 and LY303336, two polysubstituted 4-aminoimidazole AT1 receptor antagonists, on CHO cells expressing human recombinant AT1 receptors. METHODS: The binding of [3H]-angiotensin II to intact cells as well as to angiotensin II induced inositol phosphate accumulation is measured. RESULTS: Both antagonists inhibit specific [3H]-angiotensin II binding to AT1 receptors in these cells, with IC50 values of 5.9 and 5.2 nM, respectively. Preincubation of the cells with LY301875 results in a decline of up to 80% of the maximal angiotensin II-stimulated inositol phosphate (IP) production. A near complete decline of the maximal response is observed for LY303336. This insurmountable inhibition is attenuated for both antagonists when losartan is included during the preincubation of the cells. CONCLUSIONS: Functional recovery experiments, in which antagonist-preincubated cells are washed and exposed to fresh media, suggest that the insurmountable inhibition by LY301875 and LY303336 is related to their relatively slow dissociation from the AT1 receptors. As already described for losartan and the derived insurmountable AT1 antagonists candesartan. EXP3174, and irbesartan, coincubation experiments reveal that LY301875 and LY303336 interact with the AT1 receptor in a manner that is competitive with angiotensin II.

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.

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

1. 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. 2. In the presence of 10 mM LiCl, angiotensin II caused a concentration-dependent and long-lasting increase of inositol phosphates accumulation with an EC50 of 3.4 nM. No angiotensin II responses are seen in wild-type CHO-K1 cells. 3. [3H]-Angiotensin II bound to cell surface AT1 receptors (dissociates under mild acidic conditions) and is subject to rapid internalization. 4. Non-peptide selective AT1 antagonists inhibited the angiotensin II (0.1 microM) induced IP accumulation and the binding of [3H]-angiotensin II (1 nM) with the potency order: candesartan > EXP3174 > irbesartan > losartan. Their potencies are lower in the presence of bovine serum albumin. 5. 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 0.6 nM candesartan and were attenuated by simultaneous preincubation with 1 microM losartan indicating a syntopic action of both antagonists. 6. 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. 7. 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.

The effects of candesartan on human AT1 receptor-expressing Chinese hamster ovary cells.

Chinese hamster ovary cells expressing the cloned human angiotensin II receptor of the AT1 subtype (CHO-AT1 cells) were used as an 'in vitro" model system to investigate the action mechanism of the nonpeptide AT1 receptor blocker candesartan. In the presence of 10 mM LiCl, angiotensin II causes a long-lasting increase in the production of inositol phosphates in these cells. This effect is dose-dependent with half-maximal stimulation (EC50) at 3 nM angiotensin II. Pre-incubation of the cells for 30 min at 37 degrees C with candesartan decreases the maximal response to angiotensin II by up to 90%, with a half-maximal decrease at 0.5 nM candesartan. At this concentration, candesartan only produces a slight rightward shift of the angiotensin II dose-response curve. Recovery experiments on CHO-AT1 cells reveal that the inhibitory effect of candesartan is only slowly reversed after removal of the blocker. The insurmountable effect of candesartan can therefore be ascribed to its long-lasting inhibition of the AT1 receptor.