Jean-Paul De Backer

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.

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.

Tight binding of the angiotensin AT1 receptor antagonist [3H]candesartan is independent of receptor internalization

Abstract Angiotensin II induces angiotensin AT1 receptor internalization via Clathrin coated pits formation. We investigated whether insurmountable inhibition by the non-peptide antagonist 2-ethoxy-1-[(2′-(1H-tetrazol-5-yl) biphenyl-4-yl) methyl]-1H-benzimidazoline-7-carboxylic acid (candesartan) was related to receptor internalization. Mild acid treatment can discriminate between internalized and cell surface bound [3H]angiotensin II. In contrast, it provides no information about the subcellular localization of bound [3H]candesartan since this binding is acid resistant. The internalization of [3H]angiotensin II is rapidly inhibited in the presence of 0.4 M sucrose. Yet, no such rapid effect was noticed for [3H]candesartan. [3H]candesartan displays insurmountable/long lasting binding to the vast majority of both wild type and L314 truncated rat angiotensin AT1A receptors with impaired receptor internalization. In agreement with previously published AT1 angiotensin receptor visualization experiments, the present data suggest that non-peptide antagonist-angiotensin AT1 receptor complexes remain at the cell surface. Insurmountable antagonism of candesartan is therefore independent from receptor internalization via clathrin-coated pits.

Antagonist-radioligand binding to D2L-receptors in intact cells.

D(2)-dopamine receptors mediate most of the physiological actions of dopamine and are important recognition sites for antipsychotic drugs. Earlier binding studies were predominantly done with broken cell preparations with the tritiated D(2)-receptor antagonists [(3)H]-raclopride, a hydrophilic benzamide, and [(3)H]-spiperone, a highly hydrophobic butyrophenone. Here we compared [(3)H]-raclopride and [(3)H]-spiperone binding properties in intact Chinese Hamster Ovary cells stably expressing recombinant human D(2L)-receptors. Specific binding of both radioligands occurred to a comparable number of sites. In contrast to the rapid dissociation of [(3)H]-raclopride in both medium only and in the presence of an excess of unlabelled ligand [(3)H]-spiperone dissociation was only observed in the latter condition, and it was still slower than in broken cell preparations. However, this could not explain the pronounced difference in the potency of some unlabelled ligands to compete with both radioligands. To integrate these new findings, a model is proposed in which raclopride approaches the receptor from the aqueous phase, while spiperone approaches the receptor by lateral diffusion within the membrane.

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.

Antagonist interaction with endogenous AT1 receptors in human cell lines

Using Chinese Hamster Ovary cells expressing human AT(1) receptors cells (CHO-hAT(1)), it was previously shown that insurmountable inhibition of the angiotensin II response by non-peptide antagonists is related to the duration of their receptor occupancy. In the present study it was shown that these antagonists displayed similar binding characteristics to endogenously expressed AT(1) receptors in human adrenal cortex cells (NCI-h295) and renal vascular smooth muscle cells (HVSMC). Competition binding studies with [(3)H]candesartan for NCI-h295 cells, with [(125)I]Sar(1)-Ile(8) angiotensin II for HVSMC and with both radioligands for CHO-hAT(1) cells displayed the same potency order for unlabelled antagonists: candesartan>EXP3174>irbesartan>losartan. The AT(2) receptor antagonist PD123319 displayed low potency in all instances. The apparent half-lives of the antagonist-AT(1) receptor complexes in NCI-h295 cells and HVSMC were comparable to those obtained under identical conditions with CHO-hAT(1) cells. Angiotensin II increased the inositol phosphate accumulation dose dependently with half-maximal response at 17.4+/-1.6nM for NCI-h295 cells and 4.5+/-0.8nM for HVSMC. Pre-incubation of the cells with losartan only produced concentration-dependent rightward shifts of the angiotensin II concentration-response curve. The maximal response was decreased by 85-92% with candesartan, 70-88% with EXP3174 and 60% with irbesartan. The similar binding and inhibitory properties of these antagonists among the investigated cell types validates the use of CHO-hAT(1) cells for investigating pharmacological properties of human AT(1) receptors.

Human neuropeptide YY1 receptors exert unequal control of the extracellular acidification rate in different cell lines

The ability of the human neuropeptide YY1 receptor subtype to increase the extracellular acidification rate in different cell lines was investigated by using the Cytosensor Microphysiometer. In CHO-Y1 cells (Chinese Hamster Ovary cells expressing the cloned human neuropeptide YY1 receptor), neuropeptide Y increased the acidification rate by up to 15% of the basal level with a -Log(EC50) of 7.42. As expected for neuropeptide YY1 receptors, this response was potently inhibited by the neuropeptide YY1-selective non-peptide antagonist BIBP3226 ((R)-N2-(diphenylacetyl)-N-[(4-hydroxy-phenyl)methyl]-D-arginine amide). Its enantiomer BIBP3435 ((S)-N2-(diphenylacetyl)-N-[(4-hydroxy-phenyl)methyl]-D-arginin amide) was less potent. The antagonists themselves did not affect the extracellular acidification rate at concentrations up to 10 microM. In SK-N-MC cells (a neuroblastoma cell line of human origin that expresses the neuropeptide YY1 receptor) no change of the acidification rate could be observed in the presence of neuropeptide Y at concentrations up to 1 microM. For control, the neuropeptide YY1 receptors were also investigated by assessing whole cell radioligand binding and, at the functional level, by assessing their ability to decrease the forskolin-induced accumulation of cAMP. The specific (i.e., neuropeptide Y-displaceable) binding of [3H]neuropeptide Y was to a homogeneous class of high-affinity sites in both SK-N-MC and CHO-Y1 cells. The equilibrium dissociation constants for [3H]neuropeptide Y, the total number of binding sites and the kinetic constants for association and for dissociation were similar. Neuropeptide Y produced a dose-dependent inhibition of forskolin-induced cAMP accumulation in SK-N-MC cells (-log(EC50) = 9.40) but it did not affect cAMP accumulation in CHO-Y1 cells. Non-transfected CHO-K1 cells were used as negative control throughout the study. No binding or response could be observed in these cells. Our data suggest that the signalling mechanisms of neuropeptide YY1 receptors are closely related to the cell type in which they are expressed.

Antagonist-D2S-dopamine receptors interactions in intact Chinese recombinant ovary cells

D2‐type dopamine receptors are major recognition sites for antipsychotic drugs. There are two splice variants: D2S and D2L with an additional 29 amino acid sequence in the third intracellular loop. Only little comparative information is hitherto available about their pharmacological properties and none of these studies dealt with intact cell systems. This prompted us to investigate the binding properties of [3H]‐raclopride, a hydrophilic benzamide, and [3H]‐spiperone, a highly hydrophobic butyrophenone, to intact CHO cells expressing recombinant human D2L‐receptors. Presently, we have repeated and extended this experimental approach to the human D2S‐receptors in the same cell system. Except for a slower dissociation of [3H]‐spiperone from D2S, the binding properties of these and other antagonists were not significantly different for both isoforms (P > 0.05). The very slow dissociation of the atypical antipsychotic clozapine was surprising in light of its low affinity. Two experiments pointed out the existence of non‐competitive interactions between raclopride and spiperone for D2S as well as D2L (A. Packeu, J. P. De Backer & G. Vauquelin, in preparation). Alongside the different physicochemical properties of these ligands, this finding fits with a model wherein the hydrophilic raclopride approaches the D2L‐receptor from the aqueous phase, while the hydrophobic spiperone approaches the receptor by lateral diffusion between the membrane lipids. These different modes of approach could imply the existence of topologically distinct ligand binding sites at D2‐receptors.D(2)-type dopamine receptors are major recognition sites for antipsychotic drugs. There are two splice variants: D(2S) and D(2L) with an additional 29 amino acid sequence in the third intracellular loop. Only little comparative information is hitherto available about their pharmacological properties and none of these studies dealt with intact cell systems. This prompted us to investigate the binding properties of [(3)H]-raclopride, a hydrophilic benzamide, and [(3)H]-spiperone, a highly hydrophobic butyrophenone, to intact CHO cells expressing recombinant human D(2L)-receptors. Presently, we have repeated and extended this experimental approach to the human D(2S)-receptors in the same cell system. Except for a slower dissociation of [(3)H]-spiperone from D(2S), the binding properties of these and other antagonists were not significantly different for both isoforms (P > 0.05). The very slow dissociation of the atypical antipsychotic clozapine was surprising in light of its low affinity. Two experiments pointed out the existence of non-competitive interactions between raclopride and spiperone for D(2S) as well as D(2L) (A. Packeu, J. P. De Backer & G. Vauquelin, in preparation). Alongside the different physicochemical properties of these ligands, this finding fits with a model wherein the hydrophilic raclopride approaches the D(2L)-receptor from the aqueous phase, while the hydrophobic spiperone approaches the receptor by lateral diffusion between the membrane lipids. These different modes of approach could imply the existence of topologically distinct ligand binding sites at D(2)-receptors.

A two-state model of antagonist-AT1 receptor interaction: further support by binding studies at low temperature

The molecular mechanism of insurmountable antagonism was investigated to a large extent in Chinese hamster ovary cells transfected with the human angiotensin II receptor type 1 (AT(1)) receptor. It was proposed that AT(1) receptor antagonists interact with their receptor according to a two-state receptor model. Briefly, this theoretical model reveals that antagonist bound AT(1) receptor can adopt a fast and a slow reversible state. The first, fast reversible state is similar for all antagonists, while the slow reversible state displays the characteristics of each antagonist. In the present study, we performed competition experiments with the AT(1) receptor antagonists candesartan, EXP3174, irbesartan, losartan and ligand [3H]-angiotensin II at 0-4 degrees. This gave the opportunity to verify the two-state model for the first time with experimental data.