Frederik Lp Fierens

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 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. [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 (0.1 μM) 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. 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 μM 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.

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.

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

Abstract— 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°C. It was saturable and occurred to a homogeneous class of sites with a KD 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 ≥ EXP3I74 > 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.

Long-lasting angiotensin type 1 receptor binding and protection by candesartan: comparison with other biphenyl-tetrazole sartans.

Background The ability of biphenyl-tetrazole angiotensin type 1 (AT1) receptor antagonists (BTsartans) to block angiotensin II (Ang II)-mediated responses has been extensively investigated in vascular tissues and, more recently, in cell lines expressing the human AT1-receptor. When pre-incubated, BTsartans acted surmountably (shifting the Ang II concentration–response curve to the right) or insurmountably (also decreasing the maximal response). It was shown that their insurmountable behaviour is due to the formation of tight, long-lasting complexes with the receptor. Partial insurmountable antagonism is due to the co-existence of tight and loose complexes. The proportion of insurmountable antagonism, the potency and the dissociation rate of the BTsartans decreases in the order: candesartan > EXP3174 (losartans active metabolite) > valsartan > irbesartan ≫ losartan. Objective It is of interest to explore how tight AT1-receptor binding of BTsartans such as candesartan might contribute to their long-lasting clinical effect. Methods Computer-assisted simulations (COPASI program) were performed to follow the receptor-occupation and protection by different antagonists as a function of time. Free antagonist concentrations were allowed to decrease exponentially with time. Results The simulations suggest that slow dissociation does not tangibly prolong receptor occupancy if the free antagonist is eliminated at a slower pace (as is the case for BTsartans). Yet when surmountable and insurmountable antagonists occupy the same amount of receptors, insurmountable antagonists offer appreciably better protection against fluctuations in natural messenger concentration. Conclusion Slow receptor dissociation and slow antagonist elimination are likely to act in synergy to produce long-lasting receptor protection.

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 [3H]-irbesartan to human recombinant angiotensin type 1 receptors

The aim of the present work was to investigate the binding properties of the selective AT1-receptor antagonist irbesartan to human AT1-receptors by direct radioligand binding. For this purpose the specific binding of [3H]-irbesartan to intact Chinese Hamster Ovary (CHO) cells expressing human recombinant AT1-receptors was determined. Specific binding of [3H]-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 AT1 antagonists (AIIAs) with the same potency order as previous results from [3H]-angiotensin II and [3H]-candesartan binding to human AT1-receptors. Whereas the dissociation rate of [3H]-irbesartan was essentially independent of the radioligand concentration, it was much slower at 12°C when compared with 37°C. Moreover, the dissociation rate was similar, as determined in washout experiments in the absence or presence of unlabelled AT1 antagonists. At 37°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.

Role of basic amino acids of the human angiotensin type 1 receptor in the binding of the non-peptide antagonist candesartan

To explain the insurmountable/long-lasting binding of biphenyltetrazole-containing AT1-receptor antagonists such as candesartan, to the human angiotensin II type 1-receptor, a model is proposed in which the basic amino acids Lys199 and Arg 167 of the receptor interact respectively with the carboxylate and the tetrazole group of the antagonists. To validate this model, we have investigated the impact of substitution of Lys199 by Ala or Gln and of Arg167 by Ala on the binding properties of [3H]candesartan and on competition binding by candesartan, EXP3174, irbesartan, losartan, angiotensin II (Ang II) and [Sar1-Ile8]angiotensin. Our results indicate that both amino acids play an important role in the AT1-receptor ligand binding. Whereas the negative charge of Lys 199 is involved in an ionic bond with the end-standing carboxylate group of the peptide ligands, its polarity also contributes to the non-peptide antagonist binding. Substitution of Arg167 by Ala completely abolished [3H]Ang II, as well as [3H] candesartan, binding. Whereas these results are in line with the proposed model, it cannot be excluded that both amino acid residues are important for the structural integrity of the AT1-receptor with respect to its ligand binding properties.

Molecular characterization of the high-affinity [3H]neuropeptide Y-binding component from the venom of Conus anemone

The venom of the marine snail Conus anemone contains the ‘ANPY toxin’ which binds neuropeptide Y (NPY) and related insect peptides with nanomolar affinity. This toxin has initially been proposed to be a major 18.5 kDa component of the venom. Here we demonstrate that the 18.5 kDa proteins of venom produce at least five different bands in native electrophoresis and that none of them binds [3H]NPY. Instead, the ANPY toxin migrates as a distinct band on native electrophoresis and is only present as a minor component in the venom. Its approximate molecular weight is 17.5 kDa and its [3H]NPY binding activity is extremely stable below 37 °C, even in the absence of protease inhibitors.