RESUMO
Insulin-regulated aminopeptidase (IRAP, cystinyl aminopeptidase, CysAP) and aminopeptidase M (alanyl aminopeptidase, AlaAP) are closely related enzymes involved in cognitive, metabolic, and cardiovascular functions. These functions may be modulated by the type of fat used in the diet. In order to analyze a possible coordinated response of both enzymes we determined simultaneously their activities in frontal cortex, liver, and plasma of adult male rats fed diets enriched with fats differing in their percentages of saturated, mono or polyunsaturated fatty acids such as sesame, sunflower, fish, olive, Iberian lard, and coconut. The systolic blood pressure, food intake, body and liver weight as well as glucose and total cholesterol levels in plasma were measured. The type of fat in the diet influences the enzymatic activities depending on the enzyme and its location. These results suggest cognitive improvement properties for diets with predominance of polyunsaturated fatty acids. Physiological parameters such as systolic blood pressure, food intake, and biochemical factors such as cholesterol and glucose in plasma were also modified depending on the type of diet, supporting beneficial properties for diets rich in mono and polyunsaturated fatty acids. Inter-tissue correlations between the analyzed parameters were also modified depending on the type of diet. If the type of fat used in the diet modifies the behavior and relationship between CysAP and AlaAP in and between frontal cortex, liver and plasma, the functions in which they are involved could also be modified.
RESUMO
While historically 'in vitro' binding data were obtained by analyzing equilibrium experiments, kinetic data are increasingly appreciated to provide information on the time a particular compound remains bound to its target. This information is of biological importance to understand the molecular mechanism of a drug not only to evaluate the time a particular receptor/enzyme is blocked in the case of antagonists/inhibitors but also to investigate its contribution to the efficacy to mediate signaling in the case of agonists. There is accumulating evidence that many drugs binding to either membrane-bound receptors or enzymes are found to display long duration of action which can be ascribed to slow dissociation from their target proteins. In the present review three such examples are discussed which encompass ligands that bind to membrane-bound proteins and from which it appears that the tight binding kinetics is influenced by the cellular/membrane environment of the target protein.
Assuntos
Ligantes , Receptores de Superfície Celular/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/química , Bloqueadores do Receptor Tipo 1 de Angiotensina II/metabolismo , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/química , Inibidores da Dipeptidil Peptidase IV/metabolismo , Humanos , Cinética , Ligação Proteica , Receptor Tipo 1 de Angiotensina/química , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores de Superfície Celular/agonistas , Receptores de Superfície Celular/antagonistas & inibidores , Transdução de SinaisRESUMO
Derivatization of biologically active peptides by conjugation with fluorophores or radionuclide-bearing moieties is an effective and commonly used approach to prepare molecular tools and diagnostic agents. Whereas lysine, cysteine, and N-terminal amino acids have been mostly used for peptide conjugation, we describe a new, widely applicable approach to peptide conjugation based on the nonclassical bioisosteric replacement of the guanidine group in arginine by a functionalized carbamoylguanidine moiety. Four arginine-containing peptide receptor ligands (angiotensin II, neurotensin(8-13), an analogue of the C-terminal pentapeptide of neuropeptide Y, and a neuropeptide FF analogue) were subject of this proof-of-concept study. The N(ω)-carbamoylated arginines, bearing spacers with a terminal amino group, were incorporated into the peptides by standard Fmoc solid phase peptide synthesis. The synthesized chemically stable peptide derivatives showed high receptor affinities with Ki values in the low nanomolar range, even when bulky fluorophores had been attached. Two new tritiated tracers for angiotensin and neurotensin receptors are described.
Assuntos
Angiotensina II/farmacologia , Arginina/análogos & derivados , Neuropeptídeo Y/farmacologia , Neurotensina/farmacologia , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Receptores de Angiotensina/metabolismo , Receptores de Neuropeptídeos/metabolismo , Receptores de Neurotensina/metabolismo , Angiotensina II/química , Arginina/química , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Ligantes , Estrutura Molecular , Neuropeptídeo Y/química , Neurotensina/química , Oligopeptídeos/química , Fragmentos de Peptídeos/química , Receptores de Neuropeptídeos/agonistas , Receptores de Neurotensina/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
Within the hippocampus, the major somatostatin (SRIF) receptor subtype, the sst2A receptor, is localized at postsynaptic sites of the principal neurons where it modulates neuronal activity. Following agonist exposure, this receptor rapidly internalizes and recycles slowly through the trans-Golgi network. In epilepsy, a high and chronic release of somatostatin occurs, which provokes, in both rat and human tissue, a decrease in the density of this inhibitory receptor at the cell surface. The insulin-regulated aminopeptidase (IRAP) is involved in vesicular trafficking and shares common regional distribution with the sst2A receptor. In addition, IRAP ligands display anticonvulsive properties. We therefore sought to assess by in vitro and in vivo experiments in hippocampal rat tissue whether IRAP ligands could regulate the trafficking of the sst2A receptor and, consequently, modulate limbic seizures. Using pharmacological and cell biological approaches, we demonstrate that IRAP ligands accelerate the recycling of the sst2A receptor that has internalized in neurons in vitro or in vivo. Most importantly, because IRAP ligands increase the density of this inhibitory receptor at the plasma membrane, they also potentiate the neuropeptide SRIF inhibitory effects on seizure activity. Our results further demonstrate that IRAP is a therapeutic target for the treatment of limbic seizures and possibly for other neurological conditions in which downregulation of G-protein-coupled receptors occurs. SIGNIFICANCE STATEMENT: The somatostatin type 2A receptor (sst2A) is localized on principal hippocampal neurons and displays anticonvulsant properties. Following agonist exposure, however, this receptor rapidly internalizes and recycles slowly. The insulin-regulated aminopeptidase (IRAP) is involved in vesicular trafficking and shares common regional distribution with the sst2A receptor. We therefore assessed by in vitro and in vivo experiments whether IRAP could regulate the trafficking of this receptor. We demonstrate that IRAP ligands accelerate sst2A recycling in hippocampal neurons. Because IRAP ligands increase the density of sst2A receptors at the plasma membrane, they also potentiate the effects of this inhibitory receptor on seizure activity. Our results further demonstrate that IRAP is a therapeutic target for the treatment of limbic seizures.
Assuntos
Cistinil Aminopeptidase/metabolismo , Hipocampo/metabolismo , Receptores de Somatostatina/metabolismo , Convulsões/metabolismo , Convulsões/prevenção & controle , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Sistema Límbico/metabolismo , Masculino , Camundongos , Transporte Proteico/fisiologia , Ratos , Ratos WistarRESUMO
AIMS: To better understand the functional role of soluble (Sol) and membrane-bound (MB) cystinyl-aminopeptidase (CysAP) activities, we studied differentially their organ distribution in adult male Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR)with or without treatment with captopril.We searched for a possible tissue-specific association of CysAP with water balance and blood pressure. MAIN METHODS: We used twenty WKY rats distributed in ten controls and ten captopril-treated, and sixteen SHR divided in eight controls and eight captopril-treated. Captopril (100 mg/kg/day) was administered in drinking water for 4 weeks. Systolic blood pressure, water intake and diuresis were measured individually. CysAP was assayed fluorometrically using L-cystine-di-ß-naphthylamide as substrate. KEY FINDINGS: Sol or MB activities were generally higher in SHR compared to WKY notably in hypothalamus and kidney than in the other tissues. Captopril mainly decreased CysAP in SHR whereas it increased in WKY. The distribution of Sol CysAP was more homogeneous among tissues ofWKY than SHR. In contrast, the distribution of MB CysAP was more heterogeneous than Sol CysAP in both WKY and SHR. This suggests that MB CysAP activity acts in a more tissue-specific manner than Sol CysAP. The majority of the significant correlations between tissue activities and the measured physiological parameters were observed mostly in renal medulla and hypothalamus. SIGNIFICANCE: Sol and MB CysAP activities, acting separately or in concert and mainly in renal medulla, regulate the function of their susceptible endogenous substrates, and may participate meaningfully in the control of blood pressure and fluid balance.
Assuntos
Pressão Sanguínea/fisiologia , Cistinil Aminopeptidase/metabolismo , Hipocampo/enzimologia , Medula Renal/enzimologia , Equilíbrio Hidroeletrolítico/fisiologia , Animais , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Captopril/farmacologia , Masculino , Especificidade de Órgãos/efeitos dos fármacos , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Equilíbrio Hidroeletrolítico/efeitos dos fármacosRESUMO
INTRODUCTION: The insulin-regulated aminopeptidase (IRAP) is expressed in several cell types, where it is mainly located in specialized secretory endosomes that are quickly recruited to the cell surface upon cell type-specific activation. Here we describe for the first time the expression and subcellular distribution of IRAP in macrophages. METHODS: IRAP mRNA expression, protein expression and presence at the cell surface was investigated by real-time polymerase chain reaction (PCR), Western blot and [(3)H]IVDE77 binding, respectively. RESULTS: IRAP mRNA expression was increased by interferon-γ (IFN-γ) and lipopolysaccharide (LPS), but not by anti-inflammatory cytokines (interleukin (IL)-4, IL-10, transforming growth factor ß (TGF-ß)). IFN-γ increased [(3)H]IVDE77 binding steadily over time, while LPS quickly and transiently recruited IRAP to the cell surface. Combined stimulations with IFN-γ and LPS showed the same pattern as LPS alone. Latex particles also induced a transient recruitment of IRAP to the cell surface, but no difference was observed in phagocytic uptake between wild-type and IRAP(-/-) macrophages, suggesting that the enzymatic activity of IRAP is not required for the ingestion of particles. CONCLUSION: IRAP is more highly expressed in pro-inflammatory M1-activated macrophages and its presence at the cell surface is modulated upon exposure to IFN-γ, LPS or exogenous particles.
Assuntos
Cistinil Aminopeptidase/metabolismo , Macrófagos Peritoneais/enzimologia , Células 3T3-L1 , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cistinil Aminopeptidase/deficiência , Cistinil Aminopeptidase/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Inflamação/patologia , Interferon gama/farmacologia , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Lectinas Tipo C/metabolismo , Ligantes , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Fagocitose/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Superfície Celular/metabolismoRESUMO
The hexapeptide angiotensin IV (Ang IV) induces diverse biological effects such as memory enhancement and protection against ischemic stroke. Studies on the mechanism of Ang IV however are hampered by its instability and its lack of selectivity. The high-affinity binding site for Ang IV is the insulin-regulated aminopeptidase (IRAP, EC 3.4.11.3), but Ang IV also acts as a weak agonist for the Ang II-receptor (AT1), implying the need for stable and highly selective Ang IV-analogues. Here we present the screening of novel Ang IV-analogues, selected on basis of high affinity for IRAP, high selectivity (compared to aminopeptidase N and the AT1 receptor) and resistance against proteases. The selected compound IVDE77 possesses a number of advantages compared to Ang IV: (i) it has a 40 times higher affinity for IRAP (Ki 1.71 nM), (ii) it does not activate the AT1 receptor, (iii) it is easily radiolabeled with tritium and (iv) it is resistant to proteolysis, even in human plasma. In addition, pre-treatment of intact CHO-K1 cells with IVDE77 led to a virtually complete inhibition of subsequent intracellular accumulation of [(3)H]IVDE77-IRAP complexes. IVDE77 thus represents the first Ang IV-analogue able to abolish IRAP-availability completely at the cell surface in vitro. In summary, IVDE77 is a useful tool for the detection of IRAP under physiological conditions, and may contribute to elucidating the mechanism of Ang IV to ascertain which functions are IRAP-dependent.
Assuntos
Angiotensina II/análogos & derivados , Azepinas/farmacologia , Cistinil Aminopeptidase/metabolismo , Oligopeptídeos/farmacologia , Angiotensina II/farmacologia , Animais , Ligação Competitiva , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , LigantesRESUMO
Oxytocin is a neuromodulator with antidepressant-like effects. In vitro, oxytocin is rapidly cleaved by insulin-regulated aminopeptidase (IRAP). Oxytocin metabolites are known to exert strong central activities that are different from the effects of the parent molecule. Our goal is to investigate in vivo whether IRAP deletion modifies the antidepressant-like effects of oxytocin. Male and female C57Bl/6 mice, IRAP wild-type (IRAP(+/+)) and knock-out (IRAP(-/-)) mice were injected subcutaneously with saline, oxytocin or oxytocin combined with angiotensin IV. One hour after injection, immobility was timed during a 5 min forced swim that was preceded by an open field to study locomotor behaviour. Oxytocin induced antidepressant-like effects in male (0.25 mg/kg oxytocin) and female (0.15 mg/kg oxytocin) C57Bl/6 mice subjected to the forced swim test. Oxytocin did not influence locomotor behaviour in mice, as shown with the open field. These findings were reproduced in transgenic male (aged 3-6 months) and female (aged 12-18 months) IRAP(+/+) mice. However, the major findings of our study were that the antidepressant-like effect was reversed in angiotensin IV treated IRAP(+/+) mice and was completely absent in age- and gender-matched IRAP(-/-) mice. The lack of an antidepressant-like effect of oxytocin in young male and middle-aged female IRAP(-/-) mice attributes an important role to IRAP in mediating this effect.
Assuntos
Aminopeptidases/metabolismo , Antidepressivos/uso terapêutico , Depressão/tratamento farmacológico , Ocitocina/uso terapêutico , Fatores Etários , Angiotensina II/análogos & derivados , Angiotensina II/farmacologia , Animais , Antidepressivos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Cistinil Aminopeptidase/deficiência , Cistinil Aminopeptidase/genética , Depressão/sangue , Depressão/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Comportamento Exploratório/efeitos dos fármacos , Feminino , Imipramina/uso terapêutico , Resposta de Imobilidade Tônica/efeitos dos fármacos , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Ocitocina/sangue , Ocitocina/farmacologiaRESUMO
The histidine residue in angiotensin IV was replaced by various conformationally constrained amino acids. The substitution of the His(4)-Pro(5) dipeptide sequence by the constrained Trp analogue Aia-Gly, in combination with beta(2)hVal substitution at the N-terminus, provided a new stable analogue H-(R)-beta(2)hVal-Tyr-Ile-Aia-Gly-Phe-OH (AL-40) that is a potent ligand for the Ang IV receptor IRAP and selective versus AP-N and the AT1 receptor.
Assuntos
Angiotensina II/análogos & derivados , Histidina , Sequência de Aminoácidos , Aminopeptidases/metabolismo , Angiotensina II/química , Angiotensina II/metabolismo , Animais , Azepinas/química , Biomimética , Células CHO , Ácidos Carboxílicos/química , Cricetinae , Cricetulus , Cistinil Aminopeptidase/metabolismo , Humanos , Fenilalanina/química , Conformação Proteica , Receptor Tipo 1 de Angiotensina/metabolismo , Especificidade por SubstratoRESUMO
OBJECTIVES: Angiotensin (Ang) IV was reported to increase renal cortical blood flow (CBF) via putative angiotensin IV receptor (AT4) stimulation but reduce total renal blood flow (RBF) via angiotensin II type 1 (AT1) receptors. We investigated the effect of Ang IV on simultaneously measured mean arterial pressure (MAP), RBF, and CBF. The possible involvement of AT1 or AT4 receptors, the possible natriuretic effect, and responses to central administration were also explored. METHODS AND RESULTS: Intravenous injections of Ang IV dose dependently increased MAP and decreased CBF and RBF; these effects were abolished by AT1 receptor blockade. These reductions in CBF and RBF highly correlated as did renal vascular responses to Ang II and fenoldopam. Ang IV did not induce renal vasodilation even following AT1 receptor blockade. Intrarenal Ang IV infusion reduced CBF and RBF but had no natriuretic effect. Central Ang IV administration induced an AT1-mediated immediate increase in MAP and renal vascular resistance and a secondary increase in RBF. AT4 selective ligands, LVV-hemorphin-7 and AT4-16 (intravenous, intrarenal or intracerebroventricular), had no effects on MAP, RBF or urinary sodium excretion. Additional in-vitro experiments indicated that the majority of the Ang IV-sensitive aminopeptidase activity in kidney membranes is attributed to aminopeptidase-N. CONCLUSION: Insulin-regulated aminopeptidase (IRAP)/AT4 receptors are involved in neither the regulation of RBF or CBF nor in the handling of renal sodium. Ang IV increases MAP and induces renal vasoconstriction via stimulation of brain and peripheral AT1 receptors and may be involved in the regulation of renal blood flow and blood pressure.
Assuntos
Angiotensina II/análogos & derivados , Pressão Sanguínea/fisiologia , Receptor Tipo 1 de Angiotensina/fisiologia , Vasoconstrição/fisiologia , Angiotensina II/fisiologia , Animais , Cistinil Aminopeptidase/metabolismo , Rim/irrigação sanguínea , Masculino , Ratos , Receptores de Angiotensina/metabolismo , Fluxo Sanguíneo Regional , Sistema Renina-AngiotensinaRESUMO
For decades, angiotensin (Ang) II was considered as the end product and the only bioactive peptide of the renin-angiotensin system (RAS). However, later studies revealed biological activity for other Ang fragments. Amongst those, Ang IV has drawn a lot of attention since it exerts a wide range of central and peripheral effects including the ability to enhance learning and memory recall, anticonvulsant and anti-epileptogenic properties, protection against cerebral ischemia, activity at the vascular level and an involvement in atherogenesis. Some of these effects are AT(1) receptor dependent but others most likely result from the binding of Ang IV to insulin-regulated aminopeptidase (IRAP) although the exact mechanism(s) of action that mediate the Ang IV-induced effects following this binding are until now not fully known. Nevertheless, three hypotheses have been put forward: since Ang IV is an inhibitor of the catalytic activity of IRAP, its in vivo effects might result from a build-up of IRAP's neuropeptide substrates. Second, IRAP is co-localized with the glucose transporter GLUT4 in several tissue types and therefore, Ang IV might interact with the uptake of glucose. A final and more intriguing hypothesis ascribes a receptor function to IRAP and hence an agonist role to Ang IV. Taken together, it is clear that further work is required to clarify the mechanism of action of Ang IV. On the other hand, a wide range of studies have made it clear that IRAP might become an important target for drug development against different pathologies such as Alzheimer's disease, epilepsy and ischemia.
Assuntos
Angiotensina II/análogos & derivados , Cistinil Aminopeptidase/metabolismo , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Animais , Catálise/efeitos dos fármacos , Humanos , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 1 de Angiotensina/fisiologia , Sistema Renina-Angiotensina/efeitos dos fármacos , Sistema Renina-Angiotensina/fisiologiaRESUMO
Locally administered angiotensin IV causes a dose-dependent increase of the dopamine levels in the striatum of the rat. The aminopeptidases insulin-regulated aminopeptidase (IRAP) and/or aminopeptidase N (AP-N) are proposed to be involved in this effect since both enzymes are inhibited by angiotensin IV. In agreement with this hypothesis we demonstrate that by using the AP-N selective inhibitor 7B, about 60% of the aminopeptidase activity in striatal membranes could be attributed to AP-N (pK(i)=9.20). Higher concentrations of 7B are capable of inhibiting IRAP as well (pK(i)=7.26). Interestingly, in vivo, inhibition of IRAP or AP-N activity does not appear to be involved in the angiotensin IV-mediated effect in the striatum since 7B itself is not capable to induce dopamine release such as observed with angiotensin IV. However, 7B at a concentration selective for inhibition of AP-N (100 nM) potentiates the angiotensin IV-mediated increase of dopamine, suggesting that inhibition of AP-N lengthens the half-life of angiotensin IV. On the other hand, inhibition of both AP-N and IRAP by perfusion of 500 nM 7B completely abolishes the effect of angiotensin IV. We therefore hypothesize that the effect of angiotensin IV on dopamine release in the striatum is mediated via activation of IRAP and/or AP-N, possibly acting as receptors for angiotensin IV.
Assuntos
Angiotensina II/análogos & derivados , Antígenos CD13/metabolismo , Corpo Estriado/metabolismo , Cistinil Aminopeptidase/metabolismo , Dopamina/metabolismo , Terminações Pré-Sinápticas/metabolismo , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Animais , Antígenos CD13/antagonistas & inibidores , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Corpo Estriado/efeitos dos fármacos , Cistinil Aminopeptidase/antagonistas & inibidores , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , Terminações Pré-Sinápticas/efeitos dos fármacos , Ratos , Receptores de Angiotensina/efeitos dos fármacos , Receptores de Angiotensina/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
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.
Assuntos
Antígenos CD13/antagonistas & inibidores , Cátions Bivalentes/metabolismo , Quelantes/farmacologia , Animais , Antígenos CD13/metabolismo , Cátions Bivalentes/farmacologia , Células Cultivadas , Quelantes/metabolismo , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Ácido Edético/farmacologia , Humanos , Fenantrolinas/farmacologia , Proteínas Recombinantes/metabolismo , Suínos , Zinco/farmacologiaRESUMO
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.
Assuntos
Angiotensina II/análogos & derivados , Antígenos CD13/metabolismo , Cistinil Aminopeptidase/metabolismo , Inibidores Enzimáticos/metabolismo , Receptores de Angiotensina/metabolismo , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Antagonistas de Receptores de Angiotensina , Animais , Ligação Competitiva , Antígenos CD13/antagonistas & inibidores , Antígenos CD13/genética , Células CHO , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Cricetinae , Cricetulus , Cistinil Aminopeptidase/antagonistas & inibidores , Cistinil Aminopeptidase/genética , Cães , Inibidores Enzimáticos/farmacologia , Humanos , Radioisótopos do Iodo , Cinética , Ligantes , Ligação Proteica , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
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.
Assuntos
Cálcio/química , Cistinil Aminopeptidase/química , Magnésio/química , Metais/química , Zinco/química , 2,2'-Dipiridil/química , Animais , Sítios de Ligação , Células CHO , Cátions Bivalentes/química , Membrana Celular/química , Quelantes/química , Cricetinae , Cistinil Aminopeptidase/metabolismo , Ácido Edético/química , Ácido Egtázico/química , Ácido Pentético/química , Fenantrolinas/química , Ligação ProteicaRESUMO
BACKGROUND: We previously identified the G-protein-coupled receptor Mas, encoded by the Mas proto-oncogene, as an endogenous receptor for the heptapeptide angiotensin-(1-7); however, the receptor is also suggested to be involved in actions of angiotensin II. We therefore tested whether this could be mediated indirectly through an interaction with the angiotensin II type 1 receptor, AT1. METHODS AND RESULTS: In transfected mammalian cells, Mas was not activated by angiotensin II; however, AT1 receptor-mediated, angiotensin II-induced production of inositol phosphates and mobilization of intracellular Ca2+ was diminished by 50% after coexpression of Mas, despite a concomitant increase in angiotensin II binding capacity. Mas and the AT1 receptor formed a constitutive hetero-oligomeric complex that was unaffected by the presence of agonists or antagonists of the 2 receptors. In vivo, Mas acts as an antagonist of the AT1 receptor; mice lacking the Mas gene show enhanced angiotensin II-mediated vasoconstriction in mesenteric microvessels. CONCLUSIONS: These results demonstrate that Mas can hetero-oligomerize with the AT1 receptor and by so doing inhibit the actions of angiotensin II. This is a novel demonstration that a G-protein-coupled receptor acts as a physiological antagonist of a previously characterized receptor. Consequently, the AT1-Mas complex could be of great importance as a target for pharmacological intervention in cardiovascular diseases.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Angiotensina II/antagonistas & inibidores , Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Células CHO , Cálcio/metabolismo , Cricetinae , Técnicas In Vitro , Fosfatos de Inositol/metabolismo , Artérias Mesentéricas/efeitos dos fármacos , Camundongos , Camundongos Knockout , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/genética , Transfecção , Vasoconstrição/efeitos dos fármacosRESUMO
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.
Assuntos
Angiotensina II/análogos & derivados , Cátions Bivalentes/farmacologia , Cistinil Aminopeptidase/metabolismo , Angiotensina II/metabolismo , Animais , Células CHO , Quelantes , Cricetinae , Ácido Edético/farmacologia , Ácido Egtázico/farmacologia , Feminino , Radioisótopos do Iodo , Fenantrolinas/farmacologia , Ensaio RadioliganteRESUMO
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.
Assuntos
Angiotensina II/análogos & derivados , Angiotensina II/metabolismo , Células CHO/enzimologia , Cistinil Aminopeptidase/metabolismo , Receptores de Angiotensina/metabolismo , Animais , Sítios de Ligação , Catálise , Membrana Celular/metabolismo , Cricetinae , Feminino , Radioisótopos do Iodo , LigantesRESUMO
A bioassay-guided fractionation of the 80 % ethanolic extract from Bocconia frutescens L. roots, showing a dose-dependent inhibitory effect towards both [(3)H]-angiotensin II and [(3)H]-BQ-123 binding to the human angiotensin II AT 1 and endothelin 1 ET(A) receptors, led to an alkaloidal subfraction as the only responsible fraction for the activity of the whole extract. Among the alkaloids present in this fraction sanguinarine and chelerythrine were significant inhibitors of [(3)H]-angiotensin II binding (hAT 1 receptor), with IC(50) values within the micromolar range. On the contrary, the [(3)H]-BQ-123 binding (ET(A) receptor) was only weakly inhibited. Moreover, other members of the isoquinoline alkaloid family such as chelidonine and some protoberberine alkaloids exhibited no affinity for the two receptors. The present work shows the possible structure-activity relationship for these benzophenanthridine alkaloids on a screening bioassay using both stably transfected Chinese hamster ovary (CHO) and the human neuroblastoma SK-N-MC cells. Furthermore, the ability of these compounds to block AT(1) and/or ET(A) receptors may provide some justification for the traditional use of Bocconia frutescens L. to control hypertension.
Assuntos
Alcaloides/farmacologia , Papaveraceae , Extratos Vegetais/farmacologia , Raízes de Plantas/química , Receptores de Angiotensina/metabolismo , Receptores de Endotelina/metabolismo , Alcaloides/química , Angiotensina II/metabolismo , Animais , Benzofenantridinas , Ligação Competitiva/efeitos dos fármacos , Células CHO , Cricetinae , Relação Dose-Resposta a Droga , Humanos , Concentração Inibidora 50 , Isoquinolinas , Ligantes , Estrutura Molecular , Peptídeos Cíclicos/metabolismo , Fenantridinas/química , Fenantridinas/farmacologia , Extratos Vegetais/química , Relação Quantitativa Estrutura-Atividade , Receptor Tipo 1 de Angiotensina , Receptor de Endotelina A , Trítio/metabolismo , Células Tumorais CultivadasRESUMO
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.