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1.
Eur J Med Chem ; 165: 142-159, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30665144

RESUMO

Purine nucleotides such as ATP and ADP are important extracellular signaling molecules in almost all tissues activating various subtypes of purinoreceptors. In the brain, the P2Y1 receptor (P2Y1R) subtype mediates trophic functions like differentiation and proliferation, and modulates fast synaptic transmission, both suggested to be affected in diseases of the central nervous system. Research on P2Y1R is limited because suitable brain-penetrating P2Y1R-selective tracers are not yet available. Here, we describe the first efforts to develop an 18F-labeled PET tracer based on the structure of the highly affine and selective, non-nucleotidic P2Y1R allosteric modulator 1-(2-[2-(tert-butyl)phenoxy]pyridin-3-yl)-3-[4-(trifluoromethoxy)phenyl]urea (7). A small series of fluorinated compounds was developed by systematic modification of the p-(trifluoromethoxy)phenyl, the urea and the 2-pyridyl subunits of the lead compound 7. Additionally, the p-(trifluoromethoxy)phenyl subunit was substituted by carborane, a boron-rich cluster with potential applicability in boron neutron capture therapy (BNCT). By functional assays, the new fluorinated derivative 1-{2-[2-(tert-butyl)phenoxy]pyridin-3-yl}-3-[4-(2-fluoroethyl)phenyl]urea (18) was identified with a high P2Y1R antagonistic potency (IC50 = 10 nM). Compound [18F]18 was radiosynthesized by using tetra-n-butyl ammonium [18F]fluoride with high radiochemical purity, radiochemical yield and molar activities. Investigation of brain homogenates using hydrophilic interaction chromatography (HILIC) revealed [18F]fluoride as major radiometabolite. Although [18F]18 showed fast in vivo metabolization, the high potency and unique allosteric binding mode makes this class of compounds interesting for further optimizations and investigation of the theranostic potential as PET tracer and BNCT agent.


Assuntos
Encéfalo/diagnóstico por imagem , Compostos de Fenilureia/farmacologia , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/síntese química , Receptores Purinérgicos P2Y1/análise , Sítio Alostérico , Terapia por Captura de Nêutron de Boro/métodos , Radioisótopos de Flúor , Compostos de Fenilureia/síntese química , Compostos de Fenilureia/química
2.
J Cereb Blood Flow Metab ; 39(11): 2144-2156, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-30334687

RESUMO

Extracellular ATP, which is released from damaged cells after ischemia, activates P2 receptors. P2Y1 receptors (P2Y1R) have received considerable attention, especially in astrocytes, because their activation plays a central role in the regulation of neuron-to-glia communication. However, the functions or even existence of P2Y1R in microglia remain unknown, despite the fact that many microglial P2 receptors are involved in several brain diseases. Herein, we demonstrate the presence and functional capability of microglial P2Y1R to provide neuroprotective effects following ischemic stress. Cerebral ischemia resulted in increased microglial P2Y1R expression. The number of injured hippocampal neurons was significantly higher in P2Y1 R knockout (KO) mice than wildtype mice after forebrain ischemia. Propidium iodide (PI) uptake, a marker for dying cells, was significantly higher in P2Y1R KO hippocampal slices compared with wildtype hippocampal slices at 48 h after 40-min oxygen-glucose deprivation (OGD). Furthermore, increased PI uptake following OGD was rescued by ectopic overexpression of P2Y1R in microglia. In summary, these data suggest that microglial P2Y1R mediate neuroprotective effects against ischemic stress and OGD insult.


Assuntos
Microglia/química , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Receptores Purinérgicos P2Y1/fisiologia , Animais , Isquemia Encefálica , Morte Celular/efeitos dos fármacos , Glucose/deficiência , Hipocampo/metabolismo , Hipocampo/patologia , Hipóxia , Camundongos , Camundongos Knockout , Neurônios/efeitos dos fármacos , Receptores Purinérgicos P2Y1/análise
3.
PLoS One ; 11(6): e0157587, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27301021

RESUMO

Endocytic sorting and lysosomal degradation are integral to the regulation of G protein-coupled receptor (GPCR) function. Upon ligand binding, classical GPCRs are activated, internalized and recycled or sorted to lysosomes for degradation, a process that requires receptor ubiquitination. However, recent studies have demonstrated that numerous GPCRs are sorted to lysosomes independent of receptor ubiquitination. Here, we describe an ubiquitin-independent lysosomal sorting pathway for the purinergic GPCR P2Y1. After activation, P2Y1 sorts to lysosomes for degradation independent of direct ubiquitination that is mediated by a YPX3L motif within the second intracellular loop that serves as a binding site for the adaptor protein ALIX. Depletion of ALIX or site-directed mutation of the YPX3L motif inhibits P2Y1 sorting into the lumen of multivesicular endosomes/lysosomes and degradation. These findings confirm the function of YPX3L motifs as lysosomal targeting sequences for GPCRs and demonstrate that ALIX mediates the ubiquitin-independent degradation of certain GPCRs.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Lisossomos/metabolismo , Desnaturação Proteica , Receptores Purinérgicos P2Y1/metabolismo , Ubiquitina/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Motivos de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/análise , Proteínas de Ciclo Celular/análise , Complexos Endossomais de Distribuição Requeridos para Transporte/análise , Células HeLa , Humanos , Receptores Purinérgicos P2Y1/análise , Ubiquitinação , ATPases Vacuolares Próton-Translocadoras/análise , ATPases Vacuolares Próton-Translocadoras/metabolismo
4.
An. R. Acad. Farm ; 81(3): 247-257, jul.-sept. 2015. ilus, graf
Artigo em Inglês | IBECS | ID: ibc-146511

RESUMO

Nucleotides are important signalling molecules in both the peripheral and central nervous system. However, the in vitro study of their receptors can be hampered by the heterogeneity of primary neuronal cultures. The use of clonal neuroblastoma cell lines allows to circumvent this difficulty, so these lines are often used as a model to analyze the properties, regulation and physiological role of nucleotide receptors in neural tissues. Expression studies indicated the presence of P2Y1, P2Y6, P2Y11, P2Y13, P2X1, P2X4, P2X5, P2X6 and P2X7 proteins in SK-N-MC cells. Functional analyses showed transient [Ca2+]i increases upon application of ADP, 2-MeSADP or ADPβS. Responses to these agonists seem to be mediated by a P2Y1 receptor, as demonstrated by the almost complete blockade exerted by the P2Y1-selective antagonist MRS2179. ATP was also able to induce [Ca2+]i increases in SK-N-MC cells. Responses to ATP were partially blocked by MRS2179 and the P2X antagonist TNP-ATP, thus suggesting that ATP can interact with two different P2 receptors: a P2Y1 receptor, inhibited by MRS2179, and a TNP-ATP sensitive P2X receptor. To characterize the P2X receptor responsible for the MRS2179-resistant component of the ATP response, we analyze the effect of several P2X agonists on [Ca2+]i. Cells did not show responses to either α,β-meATP or BzATP, although [Ca2+]i increases could be observed when cells were challenged with CTP. Both the response to CTP and the MRS2179-resistant component of ATP response were potentiated by ivermectin. Such pharmacological profile is consistent with the presence of a functional P2X4 receptor in SK-N-MC cell line


Los nucleótidos son importantes moléculas señalizadoras en el sistema nervioso. El estudio in vitro de sus receptores puede verse obstaculizado por la heterogeneidad de los cultivos neuronales. El uso de líneas celulares de neuroblastoma permite eludir esta dificultad y dichas líneas se utilizan frecuentemente como un modelo con el que analizar las propiedades, regulación y función de los receptores de nucleótidos en tejidos neurales. Estudios de expresión indicaron la presencia de proteínas P2Y1, P2Y6, P2Y11, P2Y13, P2X1, P2X4, P2X5, P2X6 y P2X7 en las células SK-N-MC. Análisis funcionales mostraron incrementos transitorios de [Ca2+]i tras la aplicación de ADP, 2- MeSADP o ADPβS, respuestas que parecen estar mediadas a través un receptor P2Y1, como se pone de manifiesto por el bloqueo casi total ejercido por el antagonista selectivo P2Y1, MRS2179. El ATP también indujo incrementos de [Ca2+]i en las células SK-N-MC, siendo su respuesta parcialmente bloqueada por MRS2179 y por el antagonista P2X TNP-ATP, lo que sugiere que el ATP puede interactuar con dos receptores P2 diferentes: un receptor P2Y1, inhibido por MRS2179, y un receptor P2X sensible a TNP-ATP. Se caracterizó el receptor P2X analizando el efecto de varios agonistas en la [Ca2+]i. Ninguna célula mostró respuestas a α,β- meATP o BzATP, aunque se observaron incrementos de [Ca2+]i cuando las células fueron estimuladas con CTP. Tanto la respuesta a CTP como el componente de la respuesta a ATP resistente a MRS2179, se potenciaron en presencia de ivermectina. Todos estos datos sugieren la presencia de un receptor P2X4 funcional en las células SK-N-MC


Assuntos
Nucleotídeos/análise , Nucleotídeos/farmacologia , Neuroblastoma/tratamento farmacológico , Receptores Purinérgicos P2Y1/análise , Receptores Purinérgicos P2Y1/química , Receptores Purinérgicos/química , Receptores Purinérgicos P2X7/análise , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X5/análise , Receptores Purinérgicos P2X5/química , Western Blotting/métodos , Western Blotting , Imuno-Histoquímica/métodos , Imuno-Histoquímica
5.
Hypertension ; 62(2): 263-73, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23753413

RESUMO

Catecholaminergic C1 cells of the rostral ventrolateral medulla (RVLM) are key determinants of the sympathoexcitatory response to peripheral chemoreceptor activation. Overactivation of this reflex is thought to contribute to increased sympathetic activity and hypertension; however, molecular mechanisms linking peripheral chemoreceptor drive to hypertension remain poorly understood. We have recently determined that activation of P2Y1 receptors in the RVLM mimicked effects of peripheral chemoreceptor activation. Therefore, we hypothesize that P2Y1 receptors regulate peripheral chemoreceptor drive in this region. Here, we determine whether P2Y1 receptors are expressed by C1 neurons in the RVLM and contribute to peripheral chemoreceptor control of breathing, sympathetic activity, and blood pressure. We found that injection of a specific P2Y1 receptor agonist (MRS2365) into the RVLM of anesthetized adult rats increased phrenic nerve activity (≈55%), sympathetic nerve activity (38 ± 6%), and blood pressure (23 ± 1 mm Hg), whereas application of a specific P2Y1 receptor antagonist (MRS2179) decreased peripheral chemoreceptor-mediated activation of phrenic nerve activity, sympathetic nerve activity, and blood pressure. To establish that P2Y1 receptors are expressed by C1 cells, we determine in the brain slice preparation using cell-attached recording techniques that cells responsive to MRS2365 are immunoreactive for tyrosine hydroxylase (a marker of C1 cells), and we determine in vivo that C1-lesioned animals do not respond to RVLM injection of MRS2365. These data identify P2Y1 receptors as key determinants of peripheral chemoreceptor regulation of breathing, sympathetic nerve activity, and blood pressure.


Assuntos
Neurônios Adrenérgicos/fisiologia , Pressão Sanguínea/fisiologia , Células Quimiorreceptoras/fisiologia , Bulbo/fisiologia , Receptores Purinérgicos P2Y1/fisiologia , Sistema Nervoso Simpático/fisiologia , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Animais , Nervo Frênico/fisiologia , Ratos , Receptores Purinérgicos P2Y1/análise , Respiração
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