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1.
Mov Disord ; 35(2): 245-255, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31840869

RESUMO

BACKGROUND: Motor symptoms of Parkinson's disease (PD) are caused by degeneration and progressive loss of nigrostriatal dopamine neurons. Currently, no cure for this disease is available. Existing drugs alleviate PD symptoms but fail to halt neurodegeneration. Glial cell line-derived neurotrophic factor (GDNF) is able to protect and repair dopamine neurons in vitro and in animal models of PD, but the clinical use of GDNF is complicated by its pharmacokinetic properties. The present study aimed to evaluate the neuronal effects of a blood-brain-barrier penetrating small molecule GDNF receptor Rearranged in Transfection agonist, BT13, in the dopamine system. METHODS: We characterized the ability of BT13 to activate RET in immortalized cells, to support the survival of cultured dopamine neurons, to protect cultured dopamine neurons against neurotoxin-induced cell death, to activate intracellular signaling pathways both in vitro and in vivo, and to regulate dopamine release in the mouse striatum as well as BT13's distribution in the brain. RESULTS: BT13 potently activates RET and downstream signaling cascades such as Extracellular Signal Regulated Kinase and AKT in immortalized cells. It supports the survival of cultured dopamine neurons from wild-type but not from RET-knockout mice. BT13 protects cultured dopamine neurons from 6-Hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP+ )-induced cell death only if they express RET. In addition, BT13 is absorbed in the brain, activates intracellular signaling cascades in dopamine neurons both in vitro and in vivo, and also stimulates the release of dopamine in the mouse striatum. CONCLUSION: The GDNF receptor RET agonist BT13 demonstrates the potential for further development of novel disease-modifying treatments against PD. © 2019 International Parkinson and Movement Disorder Society.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Doença de Parkinson/metabolismo , Substância Negra/metabolismo , Animais , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Dopamina/metabolismo , Dopamina/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Camundongos , Oxidopamina/farmacologia , Doença de Parkinson Secundária/induzido quimicamente , Substância Negra/efeitos dos fármacos
2.
J Neuroinflammation ; 9: 133, 2012 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-22715976

RESUMO

BACKGROUND: The sphingosine 1-phosphate (S1P) receptor modulator FTY720P (Gilenya®) potently reduces relapse rate and lesion activity in the neuroinflammatory disorder multiple sclerosis. Although most of its efficacy has been shown to be related to immunosuppression through the induction of lymphopenia, it has been suggested that a number of its beneficial effects are related to altered endothelial and blood-brain barrier (BBB) functionality. However, to date it remains unknown whether brain endothelial S1P receptors are involved in the maintenance of the function of the BBB thereby mediating immune quiescence of the brain. Here we demonstrate that the brain endothelial receptor S1P5 largely contributes to the maintenance of brain endothelial barrier function. METHODS: We analyzed the expression of S1P5 in human post-mortem tissues using immunohistochemistry. The function of S1P5 at the BBB was assessed in cultured human brain endothelial cells (ECs) using agonists and lentivirus-mediated knockdown of S1P5. Subsequent analyses of different aspects of the brain EC barrier included the formation of a tight barrier, the expression of BBB proteins and markers of inflammation and monocyte transmigration. RESULTS: We show that activation of S1P5 on cultured human brain ECs by a selective agonist elicits enhanced barrier integrity and reduced transendothelial migration of monocytes in vitro. These results were corroborated by genetically silencing S1P5 in brain ECs. Interestingly, functional studies with these cells revealed that S1P5 strongly contributes to brain EC barrier function and underlies the expression of specific BBB endothelial characteristics such as tight junctions and permeability. In addition, S1P5 maintains the immunoquiescent state of brain ECs with low expression levels of leukocyte adhesion molecules and inflammatory chemokines and cytokines through lowering the activation of the transcription factor NFκB. CONCLUSION: Our findings demonstrate that S1P5 in brain ECs contributes to optimal barrier formation and maintenance of immune quiescence of the barrier endothelium.


Assuntos
Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/imunologia , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Imunidade Celular , Receptores de Lisoesfingolipídeo/fisiologia , Idoso de 80 Anos ou mais , Barreira Hematoencefálica/metabolismo , Linhagem Celular , Células Cultivadas , Endotélio Vascular/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Imunidade Celular/genética , Lentivirus/genética , Masculino , Receptores de Lisoesfingolipídeo/deficiência , Receptores de Lisoesfingolipídeo/genética
3.
Glia ; 58(12): 1465-76, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20648639

RESUMO

Sphingolipids are a class of biologically active lipids that have a role in multiple biological processes including inflammation. Sphingolipids exert their functions by direct signaling or through signaling by their specific receptors. Phosphorylated FTY720 (FTY720P) is a sphingosine 1-phosphate (S1P) analogue that is currently in trial for treatment of multiple sclerosis (MS), which targets all S1P receptors but S1P(2). To date, however, it remains unknown whether FTY720P may exert direct anti-inflammatory effects within the central nervous system (CNS), because data concerning S1P receptor expression and regulation under pathological conditions in the human brain are lacking. To investigate potential regulation of S1P receptors in the human brain during MS, we performed immunohistochemical analysis of S1P receptor 1 and 3 expression in well-characterized MS lesions. A strong increase in S1P receptor 1 and 3 expression on reactive astrocytes was detected in active and chronic inactive MS lesions. In addition, we treated primary cultures of human astrocytes with the proinflammatory cytokine tumor necrosis factor-alpha to identify the regulation of S1P(1/3) on astrocytes under pathological conditions. Importantly, we demonstrate that FTY720P exerts an anti-inflammatory action on human astrocytes by limiting secretion of proinflammatory cytokines. Our data demonstrate that reactive astrocytes in MS lesions and cultured under proinflammatory conditions strongly enhance expression of S1P receptors 1 and 3. Results from this study indicate that astrocytes may act as a yet-unknown target within the CNS for the anti-inflammatory effects observed after FTY720P administration in the treatment of MS.


Assuntos
Esclerose Múltipla/fisiopatologia , Receptores de Lisoesfingolipídeo/metabolismo , Regulação para Cima/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Astrócitos/metabolismo , Encéfalo/citologia , Células Cultivadas , Quimiocinas/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Cloridrato de Fingolimode , Humanos , Imunossupressores/farmacologia , Masculino , Pessoa de Meia-Idade , Propilenoglicóis/farmacologia , Receptores de Lisoesfingolipídeo/genética , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Receptores de Esfingosina-1-Fosfato , Linfócitos T/metabolismo , Regulação para Cima/efeitos dos fármacos
4.
Free Radic Biol Med ; 45(3): 256-64, 2008 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-18452719

RESUMO

Reactive oxygen species (ROS) play a pivotal role in the development of neuroinflammatory disorders, such as multiple sclerosis (MS). Here, we studied the effect of ROS on protein expression in brain endothelial cells (BECs) using proteomic techniques and show that long-term exposure to ROS induces adaptive responses in BECs to counteract an oxidative attack. ROS induce differential protein expression in BECs, among which is peroxiredoxin-1 (Prx1). To further study the role of Prx1 we established a BEC line overexpressing Prx1. Our data indicate that Prx-1 overexpression protects BECs from ROS-induced cell death, reduces adhesion and subsequent transendothelial migration of monocytes by decreasing intercellular adhesion molecule-1 expression, and enhances the integrity of the BEC layer. Interestingly, vascular Prx1 immunoreactivity was markedly upregulated in inflammatory lesions of experimental autoimmune encephalomyelitis (EAE) animals and active demyelinating MS lesions. These findings indicate that enhanced vascular Prx1 expression may reflect the occurrence of vascular oxidative stress in EAE and MS. On the other hand, it may function as an endogenous defense mechanism to inhibit leukocyte infiltration and counteract ROS-induced cellular injury.


Assuntos
Barreira Hematoencefálica/metabolismo , Encéfalo/irrigação sanguínea , Células Endoteliais/metabolismo , Peroxirredoxinas/metabolismo , Espécies Reativas de Oxigênio/toxicidade , Animais , Barreira Hematoencefálica/patologia , Western Blotting , Encéfalo/metabolismo , Encéfalo/patologia , Adesão Celular/fisiologia , Linhagem Celular , Movimento Celular/fisiologia , Encefalomielite Autoimune Experimental/metabolismo , Citometria de Fluxo , Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Espectrometria de Massas , Esclerose Múltipla/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Análise Serial de Proteínas , Ratos , Ratos Endogâmicos Lew , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa
5.
Psychopharmacology (Berl) ; 200(3): 367-80, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18581099

RESUMO

RATIONALE: While individual differences in vulnerability to psychostimulants have been largely attributed to dopaminergic neurotransmission, the role of serotonin is not fully understood. OBJECTIVES: To study the rewarding and motivational properties of cocaine in the serotonin transporter knockout (SERT-/-) rat and the involvement of compensatory changes in 5-HT1A receptor function are the objectives of the study. MATERIALS AND METHODS: The SERT-/- rat was tested for cocaine-induced locomotor activity, cocaine-induced conditioned place preference, and intravenous cocaine self-administration. In addition, the function and expression of 5-HT1A receptors was assessed using telemetry and autoradiography, respectively, and the effect of 5-HT1A receptor ligands on cocaine's psychomotor effects were studied. RESULTS: Cocaine-induced hyperactivity and conditioned place preference, as well as intravenous cocaine self-administration were enhanced in SERT-/- rats. Furthermore, SERT-/- rats displayed a reduced hypothermic response to the 5-HT1A receptor agonist 8-OHDPAT. S-15535, a selective somatodendritic 5-HT1A receptor agonist, reduced stress-induced hyperthermia (SIH) in wild-type controls (SERT+/+), while it increased SIH in SERT-/- rats. As 5-HT1A receptor binding was reduced in selective brain regions, these thermal responses may be indicative for desensitized 5-HT1A receptors. We further found that both 8-OHDPAT and S-15535 pretreatment increased low-dose cocaine-induced locomotor activity in SERT-/- rats, but not SERT+/+ rats. At a high cocaine dose, only SERT+/+ animals responded to 8-OHDPAT and S-15535. CONCLUSION: These data indicate that SERT-/- -associated 5-HT1A receptor adaptations facilitate low-dose cocaine effects and attenuate high-dose cocaine effects in cocaine supersensitive animals. The role of postsynaptic and somatodendritic 5-HT1A receptors is discussed.


Assuntos
Transtornos Relacionados ao Uso de Cocaína/genética , Técnicas de Inativação de Genes , Motivação , Receptor 5-HT1A de Serotonina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Animais , Autorradiografia , Comportamento de Escolha/efeitos dos fármacos , Cocaína/administração & dosagem , Cocaína/farmacologia , Transtornos Relacionados ao Uso de Cocaína/psicologia , Condicionamento Operante/efeitos dos fármacos , Genótipo , Infusões Intravenosas , Atividade Motora/efeitos dos fármacos , Ensaio Radioligante , Ratos , Ratos Wistar , Autoadministração , Meio Social
6.
FASEB J ; 21(13): 3666-76, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17586731

RESUMO

The blood-brain barrier (BBB) prevents the entrance of circulating molecules and immune cells into the central nervous system. The barrier is formed by specialized brain endothelial cells that are interconnected by tight junctions (TJ). A defective function of the BBB has been described for a variety of neuroinflammatory diseases, indicating that proper regulation is essential for maintaining brain homeostasis. Under pathological conditions, reactive oxygen species (ROS) significantly contribute to BBB dysfunction and inflammation in the brain by enhancing cellular migration. However, a detailed study about the molecular mechanism by which ROS alter BBB integrity has been lacking. Here we demonstrate that ROS alter BBB integrity, which is paralleled by cytoskeleton rearrangements and redistribution and disappearance of TJ proteins claudin-5 and occludin. Specific signaling pathways, including RhoA and PI3 kinase, mediated observed processes and specific inhibitors of these pathways prevented ROS-induced monocyte migration across an in vitro model of the BBB. Interestingly, these processes were also mediated by protein kinase B (PKB/Akt), a previously unknown player in cytoskeleton and TJ dynamics that acted downstream of RhoA and PI3 kinase. Our study reveals new insights into molecular mechanisms underlying BBB regulation and provides novel opportunities for the treatment of neuroinflammatory diseases.


Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Junções Íntimas/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Linhagem Celular Transformada , Humanos , Ratos
7.
Oligonucleotides ; 17(1): 122-33, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17461769

RESUMO

Brain tissue has become a challenging therapeutic target, in part because of failure of conventional treatments of brain tumors and a gradually increasing number of neurodegenerative diseases. Because antisense oligonucleotides are readily internalized by neuronal cells in culture, these compounds could possibly serve as novel therapeutic agents to meet such a challenge. In previous in vitro work using cell culture systems, we have demonstrated that intracellular delivery requires a vector such as cationic liposomes since free oligonucleotides remain largely trapped in the endocytic pathway following cellular uptake. Here we studied the cellular uptake properties of oligonucleotides by explants of rat brain (brain slices), and by in vivo brain tissue after administration of oligonucleotides by bolus injection. In contrast to in vitro uptake, we show that in brain slices oligonucleotides were taken up by neuronal and nonneuronal cells, irrespective of their assembly with cationic liposomes. In either case, a diffuse distribution of oligonucleotides was seen in the cytosol and/or nucleus. Uptake of oligonucleotides by brain slices as a result of membrane damage, potentially arising from the isolation procedure, could be excluded. Interestingly, internalization was inhibited following treatment of the tissue with antibody GN-2640, directed against a nucleic acid channel, present in rat kidney cells. Our data support the view that an analogous channel is present in brain tissue, allowing entry of free oligonucleotides but not plasmids. Indeed, for delivery of the latter and accomplishment of effective transfection, cationic lipids were needed for gene translocation into both brain slices and brain tissue in vivo. These data imply that for antisense therapy to become effective in brain, cationic lipid-mediated delivery will only be needed for specific cell targeting but not necessarily for delivery per se to accomplish nuclear deposition of oligonucleotides into brain cells and subsequent down-regulation of disease-related targets.


Assuntos
Encéfalo/metabolismo , Núcleo Celular/metabolismo , Ácidos Nucleicos/metabolismo , Oligonucleotídeos/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Anticorpos/farmacologia , Cátions/química , Rim/citologia , Rim/metabolismo , Lipídeos/química , Ácidos Nucleicos/antagonistas & inibidores , Oligonucleotídeos/antagonistas & inibidores , Oligonucleotídeos/química , Plasmídeos/química , Plasmídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual
8.
J Med Chem ; 47(3): 627-43, 2004 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-14736243

RESUMO

A series of novel 3,4-diarylpyrazolines was synthesized and evaluated in cannabinoid (hCB(1) and hCB(2)) receptor assays. The 3,4-diarylpyrazolines elicited potent in vitro CB(1) antagonistic activities and in general exhibited high CB(1) vs CB(2) receptor subtype selectivities. Some key representatives showed potent pharmacological in vivo activities after oral dosing in both a CB agonist-induced blood pressure model and a CB agonist-induced hypothermia model. Chiral separation of racemic 67, followed by crystallization and an X-ray diffraction study, elucidated the absolute configuration of the eutomer 80 (SLV319) at its C(4) position as 4S. Bioanalytical studies revealed a high CNS-plasma ratio for the development candidate 80. Molecular modeling studies showed a relatively close three-dimensional structural overlap between 80 and the known CB(1) receptor antagonist rimonabant (SR141716A). Further analysis of the X-ray diffraction data of 80 revealed the presence of an intramolecular hydrogen bond that was confirmed by computational methods. Computational models and X-ray diffraction data indicated a different intramolecular hydrogen bonding pattern in the in vivo inactive compound 6. In addition, X-ray diffraction studies of 6 revealed a tighter intermolecular packing than 80, which also may contribute to its poorer absorption in vivo. Replacement of the amidine -NH(2) moiety with a -NHCH(3) group proved to be the key change for gaining oral biovailability in this series of compounds leading to the identification of 80.


Assuntos
Pirazóis/síntese química , Receptor CB1 de Canabinoide/antagonistas & inibidores , Sulfonamidas/síntese química , Administração Oral , Animais , Ácido Araquidônico/metabolismo , Ligação Competitiva , Disponibilidade Biológica , Células CHO , Cricetinae , Cristalografia por Raios X , Febre/induzido quimicamente , Febre/fisiopatologia , Humanos , Hipotensão/induzido quimicamente , Hipotensão/fisiopatologia , Masculino , Camundongos , Modelos Moleculares , Conformação Molecular , Pirazóis/química , Pirazóis/farmacologia , Ensaio Radioligante , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologia
9.
Eur J Neurosci ; 26(7): 2066-73, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17897403

RESUMO

Impulsivity and aggression have been suggested to inversely correlate with central serotonin (5-HT) levels in a trait-like manner. However, this relationship is far from straightforward. In the present study we addressed the effect of lifelong reduced or absent serotonin transporter (SERT) function, which is associated with constitutively increased extracellular 5-HT levels, on impulsivity and aggression. We used unique SERT knockout rats in a resident-intruder test, five-choice serial reaction time task and serial reversal learning task to assay aggression, inhibitory control and behavioural flexibility, respectively. Homozygous SERT knockout rats (SERT( -/-)) displayed reduced aggression and improved inhibitory control, but unchanged behavioural flexibility. The behavioural phenotype of heterozygous SERT knockout rats (SERT( +/-)) was not different from that of wild-type controls in any of the behavioural paradigms. We determined monoamine (metabolite) tissue levels in the medial prefrontal cortex, orbitofrontal cortex, lateral hypothalamus, raphe nuclei and cerebrospinal fluid, and found that the 5-HT levels, but not other monoamine tissue levels, were reduced in SERT( -/-) rats. In addition, the 5-hydroxyindoleacetic acid (5-HIAA)/5-HT ratio in cerebrospinal fluid was increased in these rats. In conclusion, our data show that the absence of the SERT affects aggression and inhibitory control, but not behavioural flexibility, characteristics that may reflect the trait-like consequences of constitutive changes in central 5-HT levels.


Assuntos
Comportamento Animal/fisiologia , Inibição Psicológica , Proteínas da Membrana Plasmática de Transporte de Serotonina/deficiência , Agressão/fisiologia , Análise de Variância , Animais , Animais Geneticamente Modificados , Aprendizagem da Esquiva/fisiologia , Córtex Cerebral/anatomia & histologia , Córtex Cerebral/metabolismo , Comportamento de Escolha/fisiologia , Ácido Hidroxi-Indolacético/líquido cefalorraquidiano , Ratos , Ratos Wistar , Tempo de Reação/genética , Serotonina/metabolismo
10.
Int J Neuropsychopharmacol ; 10(6): 727-39, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17076936

RESUMO

There is evidence for neurodevelopment disturbances in schizophrenia. In rats, a neonatal basolateral amygdala lesion induces behavioural features in adults reminiscent of the symptomatology of schizophrenia. Dopamine plays a key role in the pathogenesis of schizophrenia, and cannabis use has been implicated in the risk for developing schizophrenia. The effects of an excitotoxic, bilateral basolateral amygdala lesion on postnatal days 7 or 21 were compared when the rats were adult. The behavioural response to a novelty challenge and the level of dopamine receptors and cannabinoid receptors in the brain using in-vitro autoradiography was determined. In brain tissue punches concentrations of monoamines and metabolites were determined by high-performance liquid chromatography. The neonatal lesion, but not the later lesion induced behavioural hyperactivity and biochemical effects. The neonatal lesion reduced the density of dopamine D2-like, but not D3-, and less markely D1-like receptors and increased dopamine turnover. These effects were observed in the mesolimbic, but not in the striatal regions. In contrast, density of cannabinoid receptors was increased in the striatal, but not the mesolimbic regions of these animals. Noradrenergic neurotransmission was reduced in both regions. The present findings contribute to the idea that the neonatal basolateral amygdala lesion induces features in adults reminiscent of the neurodevelopmental disturbances in schizophrenia, with a focus on the amygdala-prefrontal cortex-nucleus accumbens circuit.


Assuntos
Tonsila do Cerebelo/fisiologia , Animais Recém-Nascidos/fisiologia , Monoaminas Biogênicas/fisiologia , Moduladores de Receptores de Canabinoides/fisiologia , Tonsila do Cerebelo/patologia , Animais , Autorradiografia , Comportamento Animal/efeitos dos fármacos , Química Encefálica/efeitos dos fármacos , Dopamina/metabolismo , Agonistas de Aminoácidos Excitatórios/toxicidade , Ácido Hidroxi-Indolacético/metabolismo , Ácido Ibotênico/toxicidade , Processamento de Imagem Assistida por Computador , Masculino , Norepinefrina/metabolismo , Ratos , Ratos Wistar , Receptor CB1 de Canabinoide/efeitos dos fármacos , Receptor CB1 de Canabinoide/metabolismo , Receptores de Dopamina D1/efeitos dos fármacos , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/efeitos dos fármacos , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/efeitos dos fármacos , Serotonina/metabolismo
11.
Neurobiol Dis ; 22(2): 312-22, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16472549

RESUMO

Chemokine receptors represent promising targets to attenuate inflammatory responses and subsequent secondary damage after brain injury. We studied the response of the chemokines CXCL1/CINC-1 and CXCL2/MIP-2 and their receptors CXCR1 and CXCR2 after controlled cortical impact injury in adult rats. Rapid upregulation of CXCL1/CINC-1 and CXCL2/MIP-2, followed by CXCR2 (but not CXCR1), was observed after injury. Constitutive neuronal CXCR2 immunoreactivity was detected in several brain areas, which rapidly but transiently downregulated upon trauma. A second CXCR2-positive compartment, mainly colocalized with the activated microglia/macrophage marker ED1, was detected rapidly after injury in the ipsilateral cortex, progressively emerging into deeper areas of the brain later in time. It is proposed that CXCR2 has a dual role after brain injury: (i) homologous neuronal CXCR2 downregulation would render neurons more vulnerable to injury, whereas (ii) chemotaxis and subsequent differentiation of blood-borne cells into a microglial-like phenotype would be promoted by the same receptor.


Assuntos
Dano Encefálico Crônico/imunologia , Lesões Encefálicas/complicações , Quimiocinas/imunologia , Encefalite/imunologia , Degeneração Neural/imunologia , Receptores de Interleucina-8B/imunologia , Animais , Biomarcadores/metabolismo , Dano Encefálico Crônico/metabolismo , Dano Encefálico Crônico/fisiopatologia , Diferenciação Celular/imunologia , Movimento Celular/imunologia , Sobrevivência Celular/imunologia , Quimiocina CXCL1 , Quimiocina CXCL2 , Quimiocinas CXC/imunologia , Quimiocinas CXC/metabolismo , Modelos Animais de Doenças , Regulação para Baixo/imunologia , Ectodisplasinas , Encefalite/metabolismo , Encefalite/fisiopatologia , Gliose/imunologia , Gliose/metabolismo , Gliose/fisiopatologia , Masculino , Proteínas de Membrana/imunologia , Microglia/imunologia , Degeneração Neural/metabolismo , Degeneração Neural/fisiopatologia , Neuroimunomodulação/imunologia , Ratos , Ratos Wistar , Receptores de Interleucina-8B/metabolismo , Fatores de Necrose Tumoral/imunologia , Regulação para Cima/imunologia
12.
Synapse ; 60(8): 599-608, 2006 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-17001660

RESUMO

Present Parkinson's disease treatment strategies are far from ideal for a variety of reasons; it has therefore been suggested that partial dopamine receptor agonism might be a potential therapeutic approach with potentially fewer side effects. In the present study, we describe the in vitro characterization of the nonergot ligand SLV308 (7-[4-methyl-1-piperazinyl]-2(3H)-benzoxazolonemonohydrochloride). SLV308 binds to dopamine D(2), D(3), and D(4) receptors and 5-HT(1) (A) receptors and is a partial agonist at dopamine D(2) and D(3) receptors and a full agonist at serotonin 5-HT(1) (A) receptors. At cloned human dopamine D(2,L) receptors, SLV308 acted as a potent but partial D(2) receptor agonist (pEC(50) = 8.0 and pA(2) = 8.4) with an efficacy of 50% on forskolin stimulated cAMP accumulation. At human recombinant dopamine D(3) receptors, SLV308 acted as a partial agonist in the induction of [(35)S]GTPgammaS binding (intrinsic activity of 67%; pEC(50) = 9.2) and antagonized the dopamine induction of [(35)S]GTPgammaS binding (pA(2) = 9.0). SLV308 acted as a full 5-HT(1) (A) receptor agonist on forskolin induced cAMP accumulation at cloned human 5-HT(1) (A) receptors but with low potency (pEC(50) = 6.3). In rat striatal slices SLV308 concentration-dependently attenuated forskolin stimulated accumulation of cAMP, as expected for a dopamine D(2) and D(3) receptor agonist. SLV308 antagonized the inhibitory effect of quinpirole on K(+)-stimulated [(3)H]-dopamine release from rat striatal slices (pA(2) = 8.5). In the same paradigm, SLV308 had antagonist properties in the presence of quinpirole (pA(2) = 8.5), but the partial D(2) agonist terguride had much stronger antagonistic properties. In conclusion, SLV308 combines high potency partial agonism at dopamine D(2) and D(3) receptors with full efficacy low potency serotonin 5-HT(1) (A) receptor agonism and is worthy of profiling in in vivo models of Parkinson's disease.


Assuntos
Benzoxazóis/química , Benzoxazóis/farmacologia , Agonistas de Dopamina/farmacologia , Doença de Parkinson/tratamento farmacológico , Piperazinas/farmacologia , Receptores de Dopamina D2/agonistas , Agonistas do Receptor 5-HT1 de Serotonina , Agonistas do Receptor de Serotonina/farmacologia , Animais , Benzoxazóis/isolamento & purificação , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/fisiologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Células CHO , Colforsina/farmacologia , Cricetinae , AMP Cíclico/metabolismo , Agonistas de Dopamina/química , Agonistas de Dopamina/isolamento & purificação , Relação Dose-Resposta a Droga , Interações Medicamentosas/fisiologia , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Lisurida/análogos & derivados , Lisurida/farmacologia , Masculino , Estrutura Molecular , Doença de Parkinson/metabolismo , Doença de Parkinson/fisiopatologia , Piperazinas/química , Piperazinas/isolamento & purificação , Quimpirol/farmacologia , Ensaio Radioligante , Ratos , Ratos Wistar , Receptor 5-HT1A de Serotonina/genética , Receptor 5-HT1A de Serotonina/metabolismo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/agonistas , Receptores de Dopamina D3/genética , Receptores de Dopamina D3/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Agonistas do Receptor de Serotonina/química , Agonistas do Receptor de Serotonina/isolamento & purificação
13.
Eur J Neurosci ; 17(6): 1319-22, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12670322

RESUMO

Neurotensin has been implicated in the pathophysiology of schizophrenia. The neonatal amygdala lesion in rat has been proposed to be a neurodevelopmental model for some aspects of schizophrenia. [125I] Neurotensin binding was assessed in adult rats using in vitro autoradiography following a lesion of the basolateral amygdala at postnatal day 7 (Pd 7) or postnatal day 21 (Pd 21). The Pd 7 and Pd 21 lesions differentially affected neurotensin receptor densities in the hippocampal complex and (less pronounced) in the dopaminergic cell regions, implying a neurodevelopmental cause. These results may be of relevance for the involvement of neurotensin in the pathogenesis of schizophrenia.


Assuntos
Tonsila do Cerebelo/patologia , Encéfalo/metabolismo , Neurotensina/metabolismo , Receptores de Neurotensina/metabolismo , Animais , Animais Recém-Nascidos , Autorradiografia , Hipocampo/metabolismo , Radioisótopos do Iodo , Ratos , Ratos Wistar , Esquizofrenia/metabolismo
14.
Exp Cell Res ; 291(2): 313-25, 2003 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-14644154

RESUMO

Successful application of antisense oligonucleotides (ODNs) in cell biology and therapy will depend on the ease of design, efficiency of (intra)cellular delivery, ODN stability, and target specificity. Equally essential is a detailed understanding of the mechanism of antisense action. To address these issues, we employed phosphorothioate ODNs directed against specific regions of the mRNA of the serotonin 5HT1A receptor, governed by sequence and structure. We demonstrate that rather than various intracellular factors, the gene sequence per se primarily determines the antisense effect, since 5HT1a autoreceptors expressed in RN46A cells, postsynaptic receptors expressed in SN48 cells, and receptors overexpressed in LLP-K1 cells are all efficiently downregulated following ODN delivery via a cationic lipid delivery system. The data also reveal that the delivery system as such is a relevant parameter in ODN delivery. Antisense ODNs bound extensively to the RNA matrix in the cell nuclei, thereby interacting with target mRNA and causing its subsequent degradation. Antisense delivery effectively diminished the mRNA pool, thus resulting in downregulation of newly synthesized 5HT1A proteins, without the appearance of truncated protein fragments. In conjunction with the selected mRNA target sequences of the ODNs, the latter data indicated that effective degradation rather than a steric blockage of the mRNA impedes protein expression. The specificity of the antisense approach, as described in this study, is reflected by the effective functional downregulation of the 5-HT1A receptor.


Assuntos
Regulação para Baixo , Matriz Nuclear/metabolismo , Oligorribonucleotídeos Antissenso/farmacologia , RNA Mensageiro/metabolismo , Receptor 5-HT1A de Serotonina/biossíntese , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Microscopia Eletrônica , Oligorribonucleotídeos Antissenso/metabolismo , Ratos , Receptor 5-HT1A de Serotonina/genética , Antagonistas do Receptor 5-HT1 de Serotonina , Transfecção/métodos
15.
Chembiochem ; 5(3): 340-8, 2004 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-14997526

RESUMO

Corticotropin Releasing Factor (CRF) antagonists are considered promising for treatment of stress-related illnesses such as major depression and anxiety-related disorders. We report here the design, synthesis and biological evaluation of 91 truncated astressin analogues in order to deduce the pharmacophoric amino acid residues. Such truncated peptides may serve as valuable lead structures for the development of new small, non-peptide-based CRF antagonists. N-Terminal truncation of astressin led to active CRF antagonists that are substantially reduced in size and are selectively active at the human CRF receptor type 1 in vitro and in vivo. Subsequently, an alanine scan in combination with further truncated derivatives led to the proposal of a new pharmacophoric model of peptide-based CRF antagonists. It was found that the astressin(27-41)C sequence is the shortest active CRF antagonist. The first eight N-terminal amino acid residues were found to be an important structural determinant and were replaceable by alanine residues, thus enhancing the alpha-helical propensity. A covalent structural constraint is of utmost importance for the preorganization of the C-terminal amino acid residues. The C-terminal heptapeptide sequence, however, was found to be crucial for the antagonistic activity, since substitution or deletion of any residue led to inactive compounds.


Assuntos
Hormônio Liberador da Corticotropina/antagonistas & inibidores , Hormônio Liberador da Corticotropina/química , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Hormônio Liberador da Corticotropina/farmacologia , Desenho de Fármacos , Humanos , Fragmentos de Peptídeos/farmacologia , Estrutura Secundária de Proteína , Estresse Psicológico/tratamento farmacológico , Relação Estrutura-Atividade
16.
Chem Pharm Bull (Tokyo) ; 50(8): 1109-13, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12192147

RESUMO

The design, synthesis and biological activities of potent pyrazole-based tricyclic CB1 receptor antagonists (2) are described. The key synthetic step involves the ring closure of the lithiated alpha, gamma-keto ester adduct (4). The optimal nitroderivative (28) in this series exhibits a high CB1 receptor affinity (pKi=7.2) as well as very potent antagonistic activity (pA2=8.8) in vitro. The regioselectivity of the pyrazole ring closure is shown to depend strongly on the aromatic substitution pattern of the applied arylhydrazine.


Assuntos
Canabinoides/síntese química , Canabinoides/farmacologia , Desenho de Fármacos , Receptores de Droga/antagonistas & inibidores , Animais , Canabinoides/química , Ratos , Receptores de Canabinoides
17.
Eur J Neurosci ; 16(9): 1751-60, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12431228

RESUMO

To gain a greater insight into the relationship between hyperactivity of the corticotropin-releasing hormone (CRH) system and autonomic and physiological changes associated with chronic stress, we developed a transgenic mouse model of central CRH overproduction. The extent of central and peripheral CRH overexpression, and the amount of bioactive CRH in the hypothalamus were determined in two lines of CRH-overexpressing (CRH-OE) mice. Furthermore, 24 h patterns of body temperature, heart rate, and activity were assessed using radiotelemetry, as well as cumulative water and food consumption and body weight gain over a 7-day period. CRH-OE mice showed increased amounts of CRH peptide and mRNA only in the central nervous system. Despite the presence of the same CRH transgene in their genome, only in one of the two established lines of CRH-OE mice (line 2122, but not 2123) was overexpression of CRH associated with increased levels of bioactive CRH in the hypothalamus, increased body temperature and heart rate (predominantly during the light (inactive) phase of the diurnal cycle), decreased heart rate variability during the dark (active) phase, and increased food and water consumption, when compared with littermate wildtype mice. Because line 2122 of the CRH transgenic mice showed chronic stress-like neuroendocrine and autonomic changes, these mice appear to represent a valid animal model for chronic stress and might be valuable in the research on the consequences of CRH excess in situations of chronic stress.


Assuntos
Sistema Nervoso Autônomo/fisiologia , Hormônio Liberador da Corticotropina/metabolismo , Hipotálamo/metabolismo , Animais , Temperatura Corporal/fisiologia , Sistema Nervoso Central/química , Sistema Nervoso Central/fisiologia , Hormônio Liberador da Corticotropina/análise , Frequência Cardíaca/fisiologia , Hipotálamo/química , Masculino , Camundongos , Camundongos Transgênicos/imunologia , Camundongos Transgênicos/fisiologia , Estresse Fisiológico , Regulação para Cima/fisiologia
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