Regulation of endogenous human NPFF2 receptor by neuropeptide FF in SK-N-MC neuroblastoma cell line

Neuropeptide FF has many functions both in the CNS and periphery. Two G protein-coupled receptors (NPFF1 and NPFF2 receptors) have been identified for neuropeptide FF. The expression analysis of the peptide and receptors, together with pharmacological and physiological data, imply that NPFF2 receptor would be the primary receptor for neuropeptide FF. Here, we report for the first time a cell line endogenously expressing hNPFF2 receptor. These SK-N-MC neuroblastoma cells also express neuropeptide FF. We used the cells to investigate the hNPFF2 receptor function. The pertussis toxin-sensitive inhibition of adenylate cyclase activity upon receptor activation indicated coupling to Gi/o proteins. Upon agonist exposure, the receptors were internalized and the mitogen-activated protein kinase cascade was activated. Upon neuropeptide FF treatment, the actin cytoskeleton was reorganized in the cells. The expression of hNPFF2 receptor mRNA was up-regulated by neuropeptide FF. Concomitant with the receptor mRNA, the receptor protein expression was increased. The homologous regulation of hNPFF2 receptor correlates with our previous results in vivo showing that during inflammation, the up-regulation of neuropeptide FF mRNA precedes that of NPFF2 receptor. The regulation of hNPFF2 receptor by NPFF could also be important in the periphery where neuropeptide FF has been suggested to function as a hormone.     

Angiotensin AT4 ligands are potent,competitive inhibitors of insulin regulated aminopeptidase (IRAP)

Angiotensin IV (Ang IV) exerts profound effects on memory and learning, a phenomenon ascribed to its binding to a specific AT4 receptor. However the AT4 receptor has recently been identified as the insulin-regulated aminopeptidase (IRAP). In this study, we demonstrate that AT4 receptor ligands, including Ang IV, Nle1-Ang IV, divalinal-Ang IV, and the structurally unrelated LVV-hemorphin-7, are all potent inhibitors of IRAP catalytic activity, as assessed by cleavage of leu-beta-naphthylamide by recombinant human IRAP. Both Ang IV and divalinal-Ang IV display competitive kinetics, indicating that AT4 ligands mediate their effects by binding to the catalytic site of IRAP. The AT4 ligands also displaced [125I]-Nle1-Ang IV or [125I]-divalinal1-Ang IV from IRAP-HEK293T membranes with high affinity, which was up to 200-fold greater than in the catalytic assay; this difference was not consistent among the peptides, and could not be ascribed to ligand degradation. Although some AT4 ligands were subject to minor cleavage by HEK293T membranes, none were substrates for IRAP. Of a range of peptides tested, only vasopressin, oxytocin, and met-enkephalin were rapidly cleaved by IRAP. We propose that the physiological effects of AT4 ligands result, in part, from inhibition of IRAP cleavage of neuropeptides involved in memory processing.     

Human AT1 receptor is a single copy gene: Characterization in a stable cell line

To address conflicting reports concerning the number of angiotensin II (AII) receptor type 1 (AT₁) coding loci in vertebrates, Southern blot analysis was used to determine the genomic representation of AT₁ receptor genes in animals comprising a divergent evolutionary spectrum. The data demonstrate that the AT₁ receptor gene is present as a single genomic copy in a broad spectrum of animals including human, monkey, dog, cow, rabbit, and chicken. In contrast, members of the rodent taxonomic order contain two genes in their genomes. These two genes may have arisen in rodents as a consequence of a gene duplication event that occurred during evolution following the branching of rodents from the mammalian phylogenetic tree. In order to investigate the properties of the human AT₁ receptor in a pure cell system, the recombinant human AT₁ receptor was stably expressed in mouse L cells. An isolated cell line, designated LhAT₁-D6, was found to express abundant levels of recombinant receptor [430±15 fmol/mg] exhibiting high affinity [K_D=0.15±0.02 nM] for [¹²⁵I][SAR¹, IIe⁸] angiotensin II (SIA). The pharmacological profile of ligands competing for [¹²⁵I] SIA binding to the expressed recèptor was in accordance with that of the natural receptor. Radioligand binding of the expressed receptor was decreased in the presence of the non-hydrolyzable analog of GTP, guanosine 5-(-thio) triphosphate [GTPS]. Angiotensin II evoked a rapid efflux of⁴⁵Ca²⁺ from LhAT₁-D6 cells that was blocked by AT₁ receptor specific antagonists. In addition, AII inhibited forskolin-stimulated cAMP accumulation in these cells which was blocked by the AT-1 antagonist. Thus, the LhAT₁-D6 cell line provides a powerful tool to explore the human AT₁ receptor regulation.     

Muscarinic binding sites in a catecholaminergic human neuroblastoma cell line

Tyrosine hydroxylase (TH) a characteristic enzyme activity for the catecholaminergic clonal cell line LA-N-1 and choline acetyltransferase (ChAT) a characteristic enzyme activity for the cholinergic clonal cell line LA-N-2 were previously shown to be increased in these cells exposed to 10^–5 M retinoic acid (RA) as differentiating agent. An investigation of the receptor characteristics suggests a complementarity between the two cell lines. The binding of QNB, a muscarinic ligand, was undetectable with the LA-N-2 cells but was present in the LA-N-1 cells and possessed a kD of 1.8 nM and 2.2 nM and a B_max of 0.56 and 0.68 for control and RA grown cells respectively. There was a gradual increase in QNB binding to LA-N-1 cells from 2 days in vitro (DIV) until 6 DIV in both control and RA grown cells. An IC₅₀ of 2.5×10^–8 M and 0.9×10^–8 M for atropine inhibition was obtained for the control and RA grown cells respectively. The corresponding values for carbachol inhibition were 7×10^–2 M and 3×10^–2 M respectively. The inhibition by the agonist oxotremorine is comparable to that of carbachol and 1 mM pilocarpine inhibited the binding by 21%. QNB binding showed a low affinity for pirenzepine and for AF-DX-116 but was inhibited with a rather high affinity by 4-DAMP (IC₅₀:110 M) thus suggesting the presence of an M₃ receptor. Acetylcholine (100 M) plus eserine (50 M) and BW284c55 (1 M), an acetylcholinesterase inhibitor, reduced the binding of QNB by approximately 25%. Nicotine (1 mM) caused a 36% reduction of binding and hemicholinium-3 (HC-3) (1 M), an inhibitor of choline uptake, inhibited the binding by 53%. There was a down regulation of QNB binding observed with cells grown for 24 hours with either the antagonist atropine or the agonists carbachol or oxotremorine. Low amounts of -bungarotoxin (-BgTx) binding sites were barely detectable in both LA-N-1 and LA-N-2 cells. The LA-N-1 muscarinic receptor is coupled to polyphosphoinositide hydrolysis without increased cyclic AMP formation further suggesting its being an M₃ receptor.     

Angiotensin IV protects against angiotensin II-induced cardiac injury via AT4 receptor

Angiotensin II (Ang II) is an important regulator of cardiac function and injury in hypertension. The novel Ang IV peptide/AT4 receptor system has been implicated in several physiological functions and has some effects opposite to those of Ang II. However, little is known about the role of this system in Ang II-induced cardiac injury. Here we studied the effect of Ang IV on Ang II-induced cardiac dysfunction and injury using isolated rat hearts, neonatal cardiomyocytes and cardiac fibroblasts. We found that Ang IV significantly improved Ang II-induced cardiac dysfunction and injury in the isolated heart in response to ischemia/reperfusion (I/R). Moreover, Ang IV inhibited Ang II-induced cardiac cell apoptosis, cardiomyocyte hypertrophy, and proliferation and collagen synthesis of cardiac fibroblasts; these effects were mediated through the AT4 receptor as confirmed by siRNA knockdown. These findings suggest that Ang IV may have a protective effect on Ang II-induced cardiac injury and dysfunction and may be a novel therapeutic target for hypertensive heart disease.     

Differential toll-like receptor 3 (TLR3) expression and apoptotic response to TLR3 agonist in human neuroblastoma cells

Background  

Toll-like receptor-3 (TLR-3) is a critical component of innate immune system against dsRNA viruses and is expressed in the central nervous system. However, it remains unknown whether TLR3 may serve as a therapeutic target in human neuroblastoma (NB).     

Sub-cellular localization of insulin-regulated membrane aminopeptidase, IRAP to vesicles in neurons

Angiotensin IV and LVV-hemorphin 7 promote robust enhancing effects on learning and memory. These peptides are also competitive inhibitors of the insulin-regulated membrane aminopeptidase, suggesting that the biological actions of these peptides may result from inhibition of IRAP activity. However, the normal function of IRAP in the brain is yet to be determined. The present study investigated the sub-cellular distribution of IRAP in four neuronal cell lines and in the mouse brain. Using sub-cellular fractionation, IRAP was found to be enriched in low density microsomes, while lower levels of IRAP were also present in high density microsomes, plasma membrane and mitochondrial fractions. Dual-label immunohistochemistry confirmed the presence of IRAP in vesicles co-localized with the vesicular maker VAMP2, in the trans Golgi network co-localized with TGN 38 and in endosomes co-localized with EEA1. Finally using electron microscopy, IRAP specific immunoreactivity was predominantly associated with large 100-200 nm vesicles in hippocampal neurons. The location, appearance and size of these vesicles are consistent with neurosecretory vesicles. IRAP precipitate was also detected in intracellular structures including the rough endoplasmic reticulum, Golgi stack and mitochondrial membranes. The sub-cellular localization of IRAP in neurons demonstrated in the present study bears striking parallels with distribution of IRAP in insulin responsive cells, where the enzyme plays a role in insulin-regulated glucose uptake. Therefore, we propose that the function of IRAP in neurons may be similar to that in insulin responsive cells.     

Interaction of angiotensin II with the C-terminal 300-320 fragment of the rat angiotensin II receptor AT1a monitored by NMR

Interaction between angiotensin II (Ang II) and the fragment peptide 300-320 (fCT300-320) of the rat angiotensin II receptor AT1a was demonstrated by relaxation measurements, NOE effects, chemical shift variations, and CD measurements. The correlation times modulating dipolar interactions for the bound and free forms of Ang II were estimated by the ratio of the nonselective and single-selective longitudinal relaxation rates. The intermolecular NOEs observed in NOESY spectra between HN protons of 9Lys(fCT) and 6His(ang), 10Phe(fCT) and 8Phe(ang), HN proton of 3Tyr(fCT) and Halpha of 4Tyr(ang), 5Phe(fCT)Hdelta and Halpha of 4Tyr(ang) indicated that Ang II aromatic residues are directly involved in the interaction, as also verified by relaxation data. Some fCT300-320 backbone features were inferred by the CSI method and CD experiments revealing that the presence of Ang II enhances the existential probability of helical conformations in the fCT fragment. Restrained molecular dynamics using the simulated annealing protocol was performed with intermolecular NOEs as constraints, imposing an alpha-helix backbone structure to fCT300-320 fragment. In the built model, one strongly preferred interaction was found that allows intermolecular stacking between aromatic rings and forces the peptide to wrap around the 6Leu side chain of the receptor fragment.     

The fine structure of continuous human neuroblastoma lines SK-N-SH,SK-N-BE(2), and SK-N-MC

Summary Cell cultures of the continuous human neuroblastoma lines SK-N-SH, SK-N-BE(2), and SK-N-MC at exponential and stationary growth phase have been examined by electron microscopy. At the level of fine structure these cells did not show typical neuronal differentiation such as extensive granular endoplasmic reticulum or neurites with microtubules and neurofilaments. Instead they were characterized by abundant free ribosomes, moderate Golgi complexes, and usually scant granular endoplasmic reticulum, features similar to the fine structure of early normal embryonic autonomic neurons. However, in several respects appearance of differentiated features of the neuroblastoma cells did not follow the pattern observed for normal neurons, suggesting noncoordinate, expression of neuronal phenotypic properties. First, an occasional neuroblastoma cell had as extensive granular endoplasmic reticulum as would be found at later stages in normal developing neurons. Second, the cellular processes of these neuroblastoma cells did not have the fine structure of developing or mature axons in vivo. Third, few dense core vesicles were found in SK-N-SH and SK-N-BE(2), though these organelles are numerous in early normal adrenergic neurons and the adrenergic character of these two lines is apparent from other studies that have demonstrated expression of neurotransmitter synthesizing enzymes (SK-N-MC is cholinergic). The fine structural characterization of these continuous human neuroblastoma cell lines will allow this parameter to be utilized with other approaches in future experimental studies. This work was supported by PSC-BHE Research Award 11612 from the City University of New York and in part by the National Cancer Institute Core Grant CA-08748 to the Sloan-Kettering Institute. E. N. B. was the recipient of a predoctoral fellowship under USPHS Training Grant GM02050.     

A Globin Fragment, LVV-Hemorphin-7, Induces [3H]Thymidine Incorporation in a Neuronal Cell Line via the AT4 Receptor

The AT4 receptor was characterized initially as a specific binding site for angiotensin IV, a C-terminal fragment of the vasoactive peptide angiotensin II. Recently, we found that LVV-hemorphin-7, a fragment of beta globin, is an abundant peptide in the brain and binds to the AT4 receptor with high affinity and specificity. In the neuroblastoma/glioma hybrid cell line, NG108-15, LVV-hemorphin-7 and angiotensin IV competed for 125I-angiotensin IV binding in a biphasic fashion with IC50 values of 1.2 x 10(-10) and 1.1 x 10(-9) M for the high-affinity site, respectively, and 6.7 x 10(-8) and 1.5 x 10(-8) M for the low-affinity site, respectively. Both peptides were internalized rapidly by the cells. However, LVV-hemorphin-7, but not angiotensin IV, elicited a 1.8-fold increase in DNA synthesis in a dose-dependent manner. Furthermore, co-incubation of the cells with an excess of angiotensin IV (10(-6) M) inhibited LVV-hemorphin-7-stimulated DNA synthesis. Therefore, whereas LVV-hemorphin-7 and angiotensin IV were capable of binding to the AT4 receptor, only LVV-hemorphin-7 elicited [3H]thymidine incorporation in NG108-15 cells. In contrast, angiotensin IV behaved as an antagonist. The current finding suggests that LVV-hemorphin-7 is a functional peptide in the central nervous system and in view of its abundance in neural tissue, compared with angiotensin IV, may be of significant physiological importance.     

Different kinases desensitize the human delta-opioid receptor (hDOP-R) in the neuroblastoma cell line SK-N-BE upon peptidic and alkaloid agonists

In a previous work, we described a differential desensitization of the human δ-opioid receptor (hDOP-R) by etorphine (a non-selective and alkaloid agonist) and δ-selective and peptidic agonists (DPDPE ([d-Pen^2,5]enkephalin) and deltorphin I (Tyr-d-Ala-Phe-Asp-Val-Val-Gly-NH₂)) in the neuroblastoma cell line SK-N-BE (Allouche et al., Eur. J. Pharmacol., 371, 235, 1999). In the present study, we explored the putative role of different kinases in this differential regulation.

First, selective chemical inhibitors of PKA, PKC and tyrosine kinases were used and we showed a significant reduction of etorphine-induced opioid receptor desensitization by the bisindolylmaleimide I (PKC inhibitor) while genistein (tyrosine kinase inhibitor) was potent to impair desensitization induced by the different agonists. When the PKA was inhibited by H89 pretreatment, no modification of opioid receptor desensitization was observed whatever the agonist used.

Second, we further studied the role of G protein-coupled receptor kinases (GRKs) and by using western-blot experiments we observed that only the GRK2 isoform was expressed in the SK-N-BE cells. Next, the neuroblastoma cells were transfected with the wild type GRK2 or its dominant negative mutant GRK2-K220R and the inhibition on cAMP level was determined in naïve and agonist-pretreated cells. We showed that over-expression of GRK2-K220R totally abolished etorphine-induced receptor desensitization while no effect was observed with peptidic agonists and over-expression of GRK2 selectively impaired cAMP inhibition promoted by etorphine suggesting that this kinase was involved in the regulation of hDOP-R activated only by etorphine.

Third, correlation between functional experiments and phosphorylation of the hDOP-R after agonist activation was assessed by western-blot using the specific anti-phospho-DOP-R Ser³⁶³ antibody. While all agonists were potent to increase phosphorylation of opioid receptor, we showed no impairment of receptor phosphorylation level after PKC inhibitor pretreatment. Upon agonist activation, no enhancement of receptor phosphorylation was observed when the GRK2 was over-expressed while the GRK2-K220R partially reduced the hDOP-R Ser³⁶³ phosphorylation only after peptidic agonists pretreatment.

In conclusion, hDOP-R desensitization upon etorphine exposure relies on the GRK2, PKC and tyrosine kinases while DPDPE and deltorphin I mediate desensitization at least via tyrosine kinases. Although the Ser³⁶³ was described as the primary phosphorylation site of the mouse DOP-R, we observed no correlation between desensitization and phosphorylation of this amino acid.     

Characterization of an N-terminal secreted domain of the type-1 human metabotropic glutamate receptor produced by a mammalian cell line

A Chinese hamster ovary cell line has been established which secretes the N-terminal domain of human mGlu1 receptor. The secreted protein has been modified to contain a C-terminal hexa-histidine tag and can be purified by metal-chelate chromatography to yield a protein with an apparent molecular weight of 130 kDa. Following treatment with dithiothreitol the apparent molecular weight is reduced to 75 kDa showing that the protein is a disulphide-bonded dimer. N-terminal protein sequencing of both the reduced and unreduced forms of the protein yielded identical sequences, confirming that they were derived from the same protein, and identifying the site of signal-peptide cleavage of the receptor as residue 32 in the predicted amino acid sequence. Endoglycosidase treatment of the secreted and intracellular forms of the protein showed that the latter was present as an endoglycosidase H-sensitive dimer, indicating that dimerization is taking place in the endoplasmic reticulum. Characterization of the binding of [3H]quisqualic acid showed that the protein was secreted at levels of up to 2.4 pmol/mL and the secreted protein has a Kd of 5.6 +/- 1.8 nm compared with 10 +/- 1 nm for baby hamster kidney (BHK)-mGlu1alpha receptor-expressing cell membranes. The secreted protein maintained a pharmacological profile similar to that of the native receptor and the binding of glutamate and quisqualate were unaffected by changes in Ca2+ concentration.     

Increased epidermal growth factor receptor in multidrug-resistant human neuroblastoma cells

Multidrug-resistant human neuroblastoma cell lines obtained by selection with vincristine or actinomycin D from two independent clonal lines, SH-SY5Y and MC-IXC, have 3- to 30-fold more cell surface epidermal growth factor (EGF) receptors than the drug-sensitive parental cells as indicated by EGF binding assays and immunoprecipitation, affinity-labeling, and phosphorylation studies. Reversion to drug sensitivity in one line was accompanied by a return to the parental level of EGF receptor. SH-EP cells, a clone derived from the same neuroblastoma cell line as SH-SY5Y but which displays melanocyte rather than neuronal lineage markers, also express significantly more EGF receptor than SH-SY5Y cells. By nucleic acid hybridization analysis with a molecularly cloned probe, increased receptor level in multidrug-resistant cells was shown to be the result of higher levels of EGF receptor mRNA in drug-resistant than in drug-sensitive cells. The increased steady state amount of specific RNA did not result from amplification of receptor-encoding genes. A small difference was observed in the electrophoretic mobility under denaturing conditions of EGF receptor immunoprecipitated from drug-resistant and drug-sensitive cells. Quantitative and qualitative modulation of the EGF receptor might reflect alterations in the transformation and/or differentiation phenotype of the resistant cells or might result from unknown selective pressures associated with the development of multidrug resistance.     

AT4 receptor structure-binding relationship: N-terminal-modified angiotensin IV analogues

The effect of structural changes in the N-terminal amino acid of AIV, with respect to AT₄ receptor binding, was examined by competition with [¹²⁵I]AIV in bovine adrenal membranes. Analogues with modifications of the first residue -amino group possessed lower affinities than the primary amine-containing parent compound. Peptides with a residue 1 -carbon in the conformation exhibited poor affinity for the AT₄ receptor. Modifications of the residue 1 R-group demonstrate that a straight chain aliphatic moiety containing four carbons is optimal for receptor-ligand binding, as evidenced by the extremely high affinity of [Nle¹]AIV (K_i = 3.59±0.51 pM). Replacement of the 1–2 peptide bond of AIV with the methylene bond isostere Ψ (CH₂-NH), increased the K_i approximately fivefold, indicating that the peptide bond may be replaced wihle maintaining relatively high-affinity receptor binding.     

Small potent ligands to the insulin-regulated aminopeptidase (IRAP)/AT(4) receptor.

Angiotensin IV analogs encompassing aromatic scaffolds replacing parts of the backbone of angiotensin IV have been synthesized and evaluated in biological assays. Several of the ligands displayed high affinities to the insulin-regulated aminopeptidase (IRAP)/AT(4) receptor. Displacement of the C-terminal of angiotensin IV with an o-substituted aryl acetic acid derivative delivered the ligand 4, which exhibited the highest binding affinity (K(i) = 1.9 nM). The high affinity of this ligand provides support to the hypothesis that angiotensin IV adopts a gamma-turn in the C-terminal of its bioactive conformation.Ligand (4) inhibits both human IRAP and aminopeptidase N-activity and induces proliferation of adult neural stem cells at low concentrations. Furthermore, ligand 4 is degraded considerably more slowly in membrane preparations than angiotensin IV. Hence, it might constitute a suitable research tool for biological studies of the (IRAP)/AT(4) receptor.     

Conformational flexibility of three cytoplasmic segments of the angiotensin II AT1A receptor: a circular dichroism and fluorescence spectroscopy study.

The conformation of three synthetic peptides encompassing the proximal and distal half of the third intracellular loop (Ni3 and Ci3) and a portion of the cytoplasmic tail (fCT) of the angiotensin II AT1A receptor has been studied using circular dischroism and fluorescence spectroscopies. The results show that the conformation of the peptides is modulated in various ways by the environmental conditions (pH, ionic strength and dielectric constant). Indeed, Ni3 and fCT fold into helical structures that possess distinct stability and polarity due to the diverse forces involved: mainly polar interactions in the first case and a combination of polar and hydrophobic interactions in the second. The presence of these various features also produce distinct intermolecular interactions. Ci3, instead, exists as an ensemble of partially folded states in equilibrium. Since the corresponding regions of the angiotensin II AT1A receptor are known to play an important role in the receptor function, due to their ability to undergo conformational changes, these data provide some new clues about their different conformational plasticity.     

P2X7 receptor activation leads to increased cell death in a radiosensitive human glioma cell line

Gliomas are the most lethal tumors of central nervous system. ATP is an important signaling molecule in CNS and it is a selective P2X7 purinergic receptor ligand at high concentrations. Herein, we investigated whether the activation of P2X7R might be implicated in death of a radiosensitive human glioma lineage. The effects of P2X7R agonists (ATP and BzATP) and irradiation (2 Gy) on glioma cells were analyzed by MTT assay and annexin-V/PI determination, whereas mRNA and protein P2X7R expression was assessed by qRT-PCR and flow cytometry, respectively. P2X7R pore formation was functionality examined by analyzing ethidium bromide uptake. The human glioma cells U-138 MG and U-251 MG were resistant to death when treated with either ATP (5 mM) or BzATP (100 μM), but the radiosensitive M059J glioma cells displayed a significant decrease of cell viability (32.4 ± 4.1 % and 25.6 ± 3.3 %, respectively). The M059J lineage expresses significantly higher mRNA P2X7R levels when compared to the U-138 MG and U-251 cell lines (0.40 ± 0.00; 0.28 ± 0.01, and 0.31 ± 0.01, respectively), and irradiation upregulated P2X7R expression (0.55 ± 0.08) in this lineage. Noteworthy, P2X7R protein doubled after irradiation on M059J lineage, and increased in 50 % and 42.6 % when comparing M059J-irradiated to irradiated U-138 MG and U-251 MG cells, respectively. Ethidium bromide uptake was significantly increased in 104 % and 77.8 % when comparing M059J to U-138 MG and U-251MG, respectively. Finally, the selective P2X7R antagonist A740003 significantly decreased the cell death caused by irradiation. We provide novel evidence indicating that M059J human glioma cell line is ATP-P2X7R sensitive, pointing out the relevance of the purinergic P2X7R on glioma radiosensitivity.     

Conformational constraints in angiotensin IV to probe the role of Tyr2, Pro5 and Phe6

The aromatic amino acids Tyr and Phe in angiotensin IV (Ang IV) were conformationally constrained by the use of β‐Me substituted analogs, or cyclic constrained analogs. None of these modifications was allowed for Tyr¹, while only e‐β‐MePhe⁶ substitution resulted in an AngIV analog with high IRAP potency and selectivity versus AP‐N or the AT₁ receptor. This indicates an important role of the orientation of the Phe⁶ for inducing selectivity. Pro⁵ replacement with 2‐aminocyclopentanecarboxylic acid maintained IRAP potency and abolished AT₁ affinity. These results confirm the importance of conformational constrained amino acids to generate selectivity in bioactive peptides. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.