Angiotensin IV displays only low affinity for native insulin-regulated aminopeptidase (IRAP).

Radioligand binding studies revealed that Ang IV binds to insulin-regulated aminopeptidase (IRAP)/'AT(4) receptors' with high affinity. Yet, as these experiments were routinely carried out in the presence of chelators, only the catalytic zinc-depleted apo-form of IRAP was labelled. While the chelators remove the catalytic zinc from IRAP and protect Ang IV from proteolytic degradation, the aminopeptidase N selective inhibitor '7B' only exerts the latter effect. By using 7B along with the new stable Ang IV-analog [(3) H]AL-11, we here show that the native enzyme is only a low-affinity target for Ang IV.

Binding of "AT4 receptor" ligands to insulin regulated aminopeptidase (IRAP) in intact Chinese hamster ovary cells.

Insulin regulated aminopeptidase (IRAP) recognises "AT(4)-receptor" ligands like angiotensin IV (Ang IV) and peptidomimetics like AL-11. The metabolic stability and high affinity of [(3)H]AL-11 for catalytically active IRAP allowed its detection in Chinese hamster ovary (CHO-K1) cell membranes in the absence of chelators (Demaegdt et al., 2009). Here, we show that, contrary to [(3)H]Ang IV, [(3)H]AL-11 displays high affinity and specificity for IRAP in intact CHO-K1 cells as well. After binding to IRAP at the surface, [(3)H]AL-11 is effectively internalized by an endocytotic process. Unexpectedly, surface binding and internalization of [(3)H]AL-11 was not affected by pretreating the cells with Ang IV but declined with AL-11. In the latter case surface expression of IRAP even increased. After elimination of simpler explanations, it is proposed that metabolically stable "AT(4)-receptor" ligands undergo semi-continuous cycling between the cell surface and endosomal compartments. The in vivo efficacy of stable and unstable "AT(4)-receptor" ligands could therefore differ.

Antagonist-D2S-dopamine receptor interactions in intact recombinant Chinese hamster ovary cells [corrected].

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.

Non-competitive interaction between raclopride and spiperone on human D-receptors in intact Chinese hamster ovary cells.

We recently investigated the binding properties of the antagonists [(3)H]-raclopride and [(3)H]-spiperone to intact Chinese hamster ovary cells expressing recombinant human D(2long)-dopamine receptors (CHO-D(2L) cells). Compared with saturation binding with [(3)H]-raclopride, raclopride reduced [(3)H]-spiperone binding with to low potency in competition binding experiments. The present findings illustrate the ability of spiperone to inhibit [(3)H]-raclopride binding non-competitively. While raclopride only decreases the apparent K(D) of [(3)H]-raclopride in saturation binding experiments, spiperone only decreases the number of sites to which [(3)H]-raclopride binds with high affinity. Also, while the IC(50) of raclopride depends on the concentration of [(3)H]-raclopride in competition experiments, this is not the case for spiperone. Kinetic studies reveal that the binding of raclopride at its high affinity sites does not affect the association of subsequently added [(3)H]-spiperone nor the rebinding of freshly dissociated [(3)H]-spiperone to the same or surrounding receptors. Yet, spiperone does not affect the dissociation rate of [(3)H]-raclopride and raclopride does not affect the (genuine) dissociation rate of [(3)H]-spiperone. The easiest way to interpret the present findings in molecular terms is to assume that D(2L)-receptors or their dimeric complexes possess two distinct binding sites: one with high affinity/accessibility for [(3)H]-raclopride and the other one with high affinity/accessibility for [(3)H]-spiperone. The ability of bound spiperone to inhibit high affinity raclopride binding while the reverse is not the case suggests for the occurrence of non-reciprocal allosteric interactions. These new findings could point at the occurrence of allosteric interactions between different classes of D(2)-receptor antagonists.

Selective labeling of IRAP by the tritiated AT(4) receptor ligand [3H]Angiotensin IV and its stable analog [3H]AL-11.

'AT(4) receptors' through which Angiotensin IV (Ang IV) improves memory acquisition, were recently identified as insulin regulated aminopeptidase (IRAP). Radioligand binding studies have hitherto been performed with iodinated Ang IV in the presence of divalent cation chelators EDTA and 1,10-phenanthrolin. Hence, they referred to the apo-form of IRAP. Presently, binding of [(3)H]Ang IV and [(3)H]AL-11, a stable Ang IV analog, was compared on Chinese hamster ovary (CHO-K1) and mouse hippocampal (P40H1) cell membranes. With chelators, their high affinity sites showed the same pharmacological profile as for [(125)I]Ang IV binding. Without chelators, only high affinity binding was perceived for [(3)H]AL-11. The same pharmacological profile was recorded in both membrane preparations; it was different from the one in the presence of chelators and corresponded to catalytically active IRAP (despite the concurrent presence of aminopeptidase N (APN) in P40H1 cell membranes). This confirms that the active and apo-forms of IRAP have a distinct pharmacological profile.

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.

Synergistic inhibition of the enzymatic activity of aminopeptidase N by divalent metal ion chelators.

Membranes of HEK293 cells that were transfected with human aminopeptidase N (AP-N, CD13, EC 3.4.11.2) and purified soluble porcine kidney AP-N were used to study inhibition of its enzyme activity by divalent cation chelators. Whereas pre-incubation for 10 min with ethylenediaminetetraacetic acid (EDTA), did not or only weakly affected the enzyme activity, the bidentate chelator 1,10-phenanthroline produced a complete and concentration-dependent inhibition of AP-N. The corresponding curves had Hill slopes of 2.50 +/- 0.23 and 2.73 +/- 0.01 for soluble and recombinant AP-N respectively. EDTA increased the potency of 1,10-phenanthroline till a limit, at which Hill slopes became close to unity. In the absence of EDTA, the inhibition by 1,10-phenanthroline was only weakly affected by the substrate concentration. On the other hand, competition between 1,10-phenanthroline and the substrate took place in the presence of EDTA. Similar findings were reported for the related metallopeptidase cystinyl aminopeptidase and point towards a model in which 1,10-phenanthroline inhibit enzyme activity by decreasing the free Zn2+ concentration. Moreover, EDTA is capable of removing a modulatory ion from an allosteric site at the enzyme, facilitating the direct interaction between 1,10-phenanthroline and the catalytic Zn2+. Compatible with this model, Ca2+ may bind to this allosteric site resulting in the potentiation of Zn2+-mediated re-activation of the enzyme activity in the presence of EDTA and 1,10-phenanthroline.

Angiotensin AT4 receptor ligand interaction with cystinyl aminopeptidase and aminopeptidase N: [125I]Angiotensin IV only binds to the cystinyl aminopeptidase apo-enzyme.

Due to its high affinity for [(125)I]Angiotensin IV, cystinyl aminopeptidase (CAP) has recently been assigned as the 'angiotensin AT(4) receptor'. Since the aminopeptidase N (AP-N) activity is also susceptible to inhibition by Angiotensin IV, it might represent an additional target for this peptide. Based on [(125)I]Angiotensin IV binding and catalytic activity measurements, we compared the ligand interaction properties of recombinant human CAP and human AP-N. Both enzymes displayed distinct pharmacological profiles. Although their activity is inhibited by Angiotensin IV and LVV-hemorphin 7, both peptides are more potent CAP-inhibitors. On the other hand, substance P and l-methionine have a higher potency for AP-N. High affinity binding of [(125)I]Angiotensin IV to CAP occurs in the presence of chelators but not to AP-N in either the absence or presence of chelators. These differences were exploited to determine whether CAP and/or AP-N are present in different cell lines (CHO-K1, COS-7, HEK293, SK-N-MC and MDBK). We provide evidence that CAP predominates in these cell lines and that, comparatively, CHO-K1 cells display the highest level of this enzyme.

Ligand binding and functional properties of human angiotensin AT1 receptors in transiently and stably expressed CHO-K1 cells.

Chinese Hamster Ovary Cells (CHO-K1) were transiently and stably transfected to express the human angiotensin AT(1) receptor. Cell surface receptor expression was maximal 2 days after transient transfection. Their pharmacological and signalling properties differed from stably expressed receptors. Receptor reserve was significant in the transient cells but not in stable cells, explaining the higher potency of angiotensin II and the lower degree of insurmountable inhibition by candesartan in the transient cells. [Sar(1)Ile(8)]angiotensin II (sarile) is a potent angiotensin AT(1) receptor antagonist for the stable cells but is a partial agonist, producing 19% of the maximal response by angiotensin II, in transient cells. Internalization of [(3)H]angiotensin II and [(125)I]sarile (i.e., acid-resistant binding) was more pronounced in stable cells. CHO-K1 cells were also transiently transfected with the enhanced green fluorescence-AT(1) receptor gene. Confocal microscopy revealed rapid internalization induced by angiotensin II and sarile but not by candesartan. The above disparities may result from differences in receptor maturation and/or cellular surrounding.

Metal ion modulation of cystinyl aminopeptidase.

Cystinyl aminopeptidase has one Zn2+-binding motif and is a member of the M1 aminopeptidase family. Ion modulation of its catalytic activity was studied in membranes of CHO-K1 cells (Chinese-hamster ovary K1 cells) using L-leucine-p-nitroanilide as substrate. The planar bidentate chelators 1,10-phenanthroline and 2,2'-bipyridine inhibited the activity in a concentration-dependent manner with Hill slopes of 3.32+/-1.78 and 2.10+/-0.26 respectively. The acetic acid-containing chelators EDTA, EGTA and DTPA (diethylenetriamine-N,N,N',N'',N''-penta-acetic acid) weakly affected the activity, but they increased the potency of the planar chelators up to a limit, at which Hill slopes became close to unity. Moreover, competition between 1,10-phenanthroline and the substrate only took place in the presence of EDTA. These findings are compatible with a model in which the bidentate chelators inhibit enzyme activity by decreasing the free Zn2+ concentration. By removing a modulatory ion from an allosteric site at the enzyme, the acetic acid-containing chelators facilitate the direct interaction between the bidentate chelators and the catalytic Zn2+. The inhibitory effect of EDTA plus 1,10-phenanthroline could be completely reversed by Zn2+. Ca2+ and Mg2+ increased the potency of Zn2+ for this process. This is expected if they interact with the modulatory site to decrease the sensitivity of the enzyme towards 1,10-phenanthroline. Conversely, the bidendate chelators increased the high-affinity [125I]angiotensin IV binding to the membranes and this was potentiated by the acetic acid-containing chelators. These findings support the concept that high-affinity [125I]angiotensin IV binding, previously referred to as 'AT4 receptor binding', only occurs for the cystinyl aminopeptidase apoenzyme.

Synergistic modulation of cystinyl aminopeptidase by divalent cation chelators.

Membranes of Chinese hamster ovary (CHO-K1) cells were used to study the opposite modulation of enzyme activity and [125I]Ang IV binding to cystinyl aminopeptidase (EC 3.4.11.3) by divalent cation chelators. Whereas ethylene diamine tetraacetic acid (EDTA) or ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) alone only slightly affected the enzyme activity, 1,10-phenanthrolin (1,10-PHE) produced a complete and concentration-dependent inhibition. Interestingly EDTA (> or =0.05 mM) or EGTA (> or =0.15 mM) enhanced the inhibitory effect of 1,10-PHE. Two-site analysis of the corresponding inhibition curves revealed that EDTA and EGTA converted enzymes with low sensitivity towards 1,10-PHE into enzymes with high sensitivity. The combined inhibition by EDTA (0.1 mM) and 1,10-PHE (0.1 mM) could be prevented and reversed by addition of Zn2+ (at about 0.04-0.1 mM). In contrast, specific binding of [125I]Ang IV was enhanced in the presence of 1,10-PHE. Binding was only slightly affected by EDTA or EGTA alone. Furthermore, the stimulatory effect of 1,10-PHE was potentiated by EDTA (> or =0.05 mM) as well as EGTA (> or =0.15 mM). In the presence of EDTA (0.1 mM) and 1,10-PHE (0.1 mM), specific [125I]Ang IV binding was completely inhibited by Zn2+ (IC50= 39.7 +/- 6.2 microM). The present data show that divalent cations such as Zn2+ are essential for the enzyme activity of cystinyl aminopeptidase and inhibitory for [125I]Ang IV binding. Modulation of the effects of 1,10-PHE by other chelators such as EDTA or EGTA, suggests that, in addition to the binding site for zinc in the catalytic site, cystinyl aminopeptidase also bears a regulatory divalent cation binding site.

Endogenous cystinyl aminopeptidase in Chinese hamster ovary cells: characterization by [125I]Ang IV binding and catalytic activity.

The angiotensin II C-terminal hexapeptide fragment angiotensin IV (Ang IV) exerts central and cardiovascular effects. Cystinyl aminopeptidase (EC 3.4.11.3), a membrane-associated zinc-dependent metallopeptidase of the M1 family, has recently been found to display high affinity for Ang IV and it was proposed to represent the AT4 receptor. We present evidence for the presence of endogenous cystinyl aminopeptidase in membranes from Chinese hamster ovary (CHO-K1) cells by binding studies with [125I]Ang IV and by measuring the cleavage of L-leucine-p-nitroanilide. The equilibrium dissociation constant of [125I]Ang IV in saturation binding studies (KD= 0.90 nM) was similar to the value (KD= 0.70 nM) calculated from the association and dissociation rates. Binding was displaced with high potency by the "AT4 receptor" ligands (Ang IV > divalinal1-Ang IV approximately LVV-hemorphin-7 approximately LVV-hemorphin-6 > Ang (3-7) > Ang III > Ang (4-8)) but not by AT1/AT2 receptor antagonists. Enzymatic activity in CHO-K1 cell membranes was competitively inhibited upto 94% by Ang IV and other "AT4 receptor" ligands (Ang IV > Ang III approximately divalinal1-Ang IV approximately Ang (3-7) approximately LVV-hemorphin-7 > Ang (4-8) approximately LVV-hemorphin-6). High affinity binding of [125I]Ang IV required the presence of metal chelators and the ligands such as Ang IV and LVV-hemorphin-7 displayed higher potency in the binding studies as in the enzyme assay. This difference in potency varied from one peptide to another. These pharmacological properties match those previously reported for the recombinantly-expressed human cystinyl aminopeptidase in embryonal kidney cells.

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.

Antagonist interaction with endogenous AT(1) 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.

Distinct binding properties of the AT(1) receptor antagonist [(3)H]candesartan to intact cells and membrane preparations.

[(3)H]-2-Ethoxy-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]-1H-benzimidazoline-7-carboxylic acid ([(3)H]candesartan), a non-peptide angiotensin II type 1 receptor (AT(1) receptor) antagonist bound with high affinity and specificity to intact adherent human AT(1) receptor transfected Chinese hamster ovary cells. The binding characteristics were preserved when cells were suspended, but the dissociation was 3-4-fold faster and the affinity 2-fold lower, while examining [(3)H]candesartan binding to cell membranes. These data suggested the role of the intracellular organisation of living CHO-hAT(1) cells in antagonist-AT(1) receptor interactions. Yet, a specific role of microtubule or actin filaments of the cytoskeleton, receptor phosphorylation by Protein Kinase C, membrane polarity, cytoplasmic components like ATP and the need of an intact cell membrane could be excluded. The potential effect of protease degradation or receptor oxidation during the membrane preparation was also unlikely. The dissociation rate and the equilibrium dissociation constant of [(3)H]candesartan increased with the temperature for both intact cells and membranes. Thermodynamic studies suggested that the bonds between candesartan and the hAT(1) receptor may be of different nature in intact CHO-hAT(1) cells and membranes thereof. Whereas the binding was almost completely enthalpy-driven on intact cells, there was a mixed contribution of both enthalpy and entropy on membranes.

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.