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1.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070506

RESUMO

Concentration of hyaluronic acid (HA) in the lungs increases in idiopathic pulmonary fibrosis (IPF). HA is involved in the organization of fibrin, fibronectin, and collagen. HA has been proposed to be a biomarker of fibrosis and a potential target for antifibrotic therapy. Hyaluronidase (HD) breaks down HA into fragments, but is a subject of rapid hydrolysis. A conjugate of poloxamer hyaluronidase (pHD) was prepared using protein immobilization with ionizing radiation. In a model of bleomycin-induced pulmonary fibrosis, pHD decreased the level of tissue IL-1ß and TGF-ß, prevented the infiltration of the lung parenchyma by CD16+ cells, and reduced perivascular and peribronchial inflammation. Simultaneously, a decrease in the concentrations of HA, hydroxyproline, collagen 1, total soluble collagen, and the area of connective tissue in the lungs was observed. The effects of pHD were significantly stronger compared to native HD which can be attributed to the higher stability of pHD. Additional spiperone administration increased the anti-inflammatory and antifibrotic effects of pHD and accelerated the regeneration of the damaged lung. The potentiating effects of spiperone can be explained by the disruption of the dopamine-induced mobilization and migration of fibroblast progenitor cells into the lungs and differentiation of lung mesenchymal stem cells (MSC) into cells of stromal lines. Thus, a combination of pHD and spiperone may represent a promising approach for the treatment of IPF and lung regeneration.


Assuntos
Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/farmacologia , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/efeitos dos fármacos , Espiperona/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Hialuronoglucosaminidase/administração & dosagem , Hialuronoglucosaminidase/farmacocinética , Hidroxiprolina/metabolismo , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/enzimologia , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Queratinas/metabolismo , Pulmão/enzimologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Poloxâmero/química , Receptores de IgG/metabolismo , Espiperona/administração & dosagem , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
2.
FEBS J ; 288(5): 1514-1532, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32783364

RESUMO

Dopamine receptors are G protein-coupled receptors that have several essential functions in the central nervous system. A better understanding of the regulatory mechanisms of ligand binding to the receptor may open new possibilities to affect the downstream signal transduction pathways. The majority of the available ligand binding assays use either membrane preparations, cell suspensions, or genetically modified receptors, which may give at least partially incorrect understanding of ligand binding. In this study, we implemented an assay combining fluorescence and bright-field microscopy to measure ligand binding to dopamine D3 receptors in live mammalian cells. For membrane fluorescence intensity quantification from microscopy images, we developed a machine learning-based user-friendly software membrane tools and incorporated it into a data management software aparecium that has been previously developed in our workgroup. For the experiments, a fluorescent ligand NAPS-Cy3B was synthesized by conjugating a dopaminergic antagonist N-(p-aminophenethyl)spiperone with a fluorophore Cy3B. The subnanomolar affinity of NAPS-Cy3B makes it a suitable ligand for the characterization of D3 receptors in live HEK293 cells. Using a microplate compatible automated widefield fluorescence microscope, together with the membrane tools software, enables the detection and quantification of ligand binding with a high-throughput. The live cell assay is suitable for the characterization of fluorescent ligand binding and also in the competition experiments for the screening of novel unlabeled dopaminergic ligands. We propose that this simple yet more native-like approach is feasible in GPCR research, as it enables the detection of ligand binding in an environment containing more components involved in the signal transduction cascade.


Assuntos
Bioensaio , Carbocianinas/química , Antagonistas de Dopamina/farmacologia , Receptores Dopaminérgicos/metabolismo , Software , Espiperona/análogos & derivados , Dopamina/metabolismo , Dopamina/farmacologia , Antagonistas de Dopamina/síntese química , Células HEK293 , Humanos , Processamento de Imagem Assistida por Computador/métodos , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Cinética , Ligantes , Aprendizado de Máquina , Microscopia de Fluorescência/métodos , Microscopia de Fluorescência/estatística & dados numéricos , Ligação Proteica , Espiperona/química
3.
Nat Commun ; 11(1): 6442, 2020 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-33353947

RESUMO

In addition to the serotonin 5-HT2A receptor (5-HT2AR), the dopamine D2 receptor (D2R) is a key therapeutic target of antipsychotics for the treatment of schizophrenia. The inactive state structures of D2R have been described in complex with the inverse agonists risperidone (D2Rris) and haloperidol (D2Rhal). Here we describe the structure of human D2R in complex with spiperone (D2Rspi). In D2Rspi, the conformation of the extracellular loop (ECL) 2, which composes the ligand-binding pocket, was substantially different from those in D2Rris and D2Rhal, demonstrating that ECL2 in D2R is highly dynamic. Moreover, D2Rspi exhibited an extended binding pocket to accommodate spiperone's phenyl ring, which probably contributes to the selectivity of spiperone to D2R and 5-HT2AR. Together with D2Rris and D2Rhal, the structural information of D2Rspi should be of value for designing novel antipsychotics with improved safety and efficacy.


Assuntos
Antipsicóticos/química , Receptores de Dopamina D2/química , Espiperona/química , Animais , Sítios de Ligação , Células HEK293 , Humanos , Ligantes , Camundongos , Modelos Moleculares , Ligação Proteica
4.
Sci Rep ; 10(1): 13242, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764736

RESUMO

Animal models have demonstrated a link between dysregulation of the retinal dopamine system and the development of myopia (short-sightedness). We have previously demonstrated that topical application of levodopa in chicks can inhibit the development of form-deprivation myopia (FDM) in a dose-dependent manner. Here, we examine whether this same protection is observed in lens-induced myopia (LIM), and whether levodopa's protection against FDM and LIM occurs through a dopamine D1- or D2-like receptor mechanism. To do this, levodopa was first administered daily as an intravitreal injection or topical eye drop, at one of four ascending doses, to chicks developing LIM. Levodopa's mechanism of action was then examined by co-administration of levodopa injections with D1-like (SCH-23390) or D2-like (spiperone) dopamine antagonists in chicks developing FDM or LIM. For both experiments, levodopa's effectiveness was examined by measuring axial length and refraction after 4 days of treatment. Levodopa inhibited the development of LIM in a dose-dependent manner similar to its inhibition of FDM when administered via intravitreal injections or topical eye drops. In both FDM and LIM, levodopa injections remained protective against myopia when co-administered with SCH-23390, but not spiperone, indicating that levodopa elicits its protection through a dopamine D2-like receptor mechanism in both paradigms.


Assuntos
Benzazepinas/administração & dosagem , Levodopa/administração & dosagem , Miopia/tratamento farmacológico , Espiperona/administração & dosagem , Animais , Benzazepinas/farmacologia , Galinhas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Injeções Intravítreas , Lentes/efeitos adversos , Levodopa/farmacologia , Masculino , Miopia/etiologia , Miopia/metabolismo , Soluções Oftálmicas , Receptores de Dopamina D2/metabolismo , Espiperona/farmacologia
5.
Bull Exp Biol Med ; 168(6): 718-723, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32328949

RESUMO

We studied the effects of spiperone, a selective blocker of dopamine D2 receptors, on the model of pulmonary emphysema provoked by administration of elastase and D-galactosamine hydrochloride to female C57BL/6 mice and characterized by activation of proteases in the lungs and systemic deficiency of its inhibitor α1-antitrypsin. In this model, spiperone prevented the development of inflammatory reaction and reduced the area of emphysematous expanded alveolar tissue. The expression of angiogenic marker CD31 in the lungs increased under these conditions. Regeneration of the damaged microvascular bed under the action of spiperone resulted from recruiting of Notch1+ endothelial progenitor cells (CD45-CD31+CD34+) into the lungs and blockade of the inhibitory effect of dopamine on phosphorylation of VEGF-2 receptors in endothelial cells of different maturity. In addition, spiperone produced a protective effect on hepatocytes and restored the production and secretion of α1-antitrypsin by these cells.


Assuntos
Antagonistas de Dopamina/farmacologia , Células Progenitoras Endoteliais/efeitos dos fármacos , Enfisema Pulmonar/tratamento farmacológico , Receptor Notch1/genética , Receptores de Dopamina D2/genética , Espiperona/farmacologia , Deficiência de alfa 1-Antitripsina/tratamento farmacológico , Animais , Células Progenitoras Endoteliais/metabolismo , Células Progenitoras Endoteliais/patologia , Feminino , Galactosamina/administração & dosagem , Regulação da Expressão Gênica , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/efeitos dos fármacos , Elastase Pancreática/administração & dosagem , Fosforilação/efeitos dos fármacos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Enfisema Pulmonar/induzido quimicamente , Enfisema Pulmonar/genética , Enfisema Pulmonar/metabolismo , Receptor Notch1/agonistas , Receptor Notch1/metabolismo , Receptores de Dopamina D2/metabolismo , Regeneração/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Deficiência de alfa 1-Antitripsina/enzimologia , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/patologia
6.
Sci Rep ; 9(1): 19512, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31862967

RESUMO

Emotional hyperthermia is the increase in body temperature that occurs as a response to an animal detecting a salient, survival-relevant stimulus. Brown adipose tissue (BAT) thermogenesis, controlled via its sympathetic innervation, contributes to this temperature increase. Here, we have used an intruder rat experimental model to determine whether quinpirole-mediated activation of dopamine D2 receptors attenuates emotional hyperthermia in conscious rats. In anesthetized rats, we determined whether systemic quinpirole reduces BAT nerve discharge induced by activation of the medullary raphé and the lateral habenula (LHb). We measured BAT and body temperature with chronically implanted thermistors in conscious, freely moving, individually housed, male rats (resident rats). Either vehicle or quinpirole was administered, intraperitoneally, to the resident rat 30 min before introduction of a caged intruder rat. Quinpirole, in a dose-dependent manner, reduced intruder-elicited increases in BAT and body temperature. Pre-treatment with the D2 antagonist spiperone, but not the selective D1 antagonist SCH-23390, prevented this quinpirole-elicited decrease. In anesthetized rats, quinpirole abolished BAT sympathetic nerve discharge elicited by bicuculline-mediated activation of the LHb, but not the medullary raphé. Thus, activation of dopamine D2 receptors reduces the BAT thermogenesis that contributes to emotional hyperthermia. We provide evidence that these dopamine D2 receptors are located in the thermogenic pathway between the LHb and the lower brainstem pre-sympathetic control centre in the medullary raphé.


Assuntos
Tecido Adiposo Marrom/metabolismo , Habenula/metabolismo , Receptores de Dopamina D2/metabolismo , Tecido Adiposo Marrom/efeitos dos fármacos , Animais , Benzazepinas/farmacologia , Antagonistas dos Receptores de Dopamina D2/farmacologia , Masculino , Quimpirol/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D1/antagonistas & inibidores , Espiperona/farmacologia , Estresse Psicológico/metabolismo , Temperatura , Termogênese/efeitos dos fármacos
7.
Anim Reprod Sci ; 208: 106122, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31405473

RESUMO

The vitellogenesis-inhibiting hormone (VIH), also known as gonad-inhibiting hormone, is a neuropeptide hormone in crustaceans that belongs to the crustacean hyperglycemic hormone (CHH)-family peptide. There is regulation vitellogenesis by VIH during gonad maturation in crustaceans. A full-length Scylla olivacea VIH (Scyol-VIH) was identified through reverse transcription polymerase chain reaction and rapid amplification of cDNA ends. The open reading frame consists of 378 nucleotides, which encodes a 126-amino acid precursor protein, including a 22-residue signal peptide and a 103-amino acid mature peptide in which 6 highly conserved cysteine residues are present. There was expression of the Scyol-VIH gene in immature female Scylla olivacea in the eyestalk, brain and ventral nerve cord. The Scyol-VIH gene expression was localized to the eyestalk X-organ, brain neuronal clusters 6 and 11, and in multiple neuronal clusters of the ventral nerve cord. The relative abundance of Scyol-VIH mRNA transcript in the eyestalk was relatively greater in immature stage females, then decreased as ovarian maturation progressed. Furthermore, eyestalk Scyol-VIH increased after dopamine (5 µg/g BW) injection. The present research provides fundamental information about Scyol-VIH and its potential effect in controlling reproduction.


Assuntos
Braquiúros/fisiologia , Dopamina/farmacologia , Hormônios de Invertebrado/metabolismo , Ovário/crescimento & desenvolvimento , RNA Mensageiro/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Braquiúros/genética , Clonagem Molecular , Dopamina/administração & dosagem , Dopaminérgicos/farmacologia , Antagonistas de Dopamina/administração & dosagem , Antagonistas de Dopamina/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônios de Invertebrado/genética , Ovário/metabolismo , Filogenia , RNA Mensageiro/genética , Serotonina/administração & dosagem , Serotonina/farmacologia , Serotoninérgicos/administração & dosagem , Serotoninérgicos/farmacologia , Maturidade Sexual , Espiperona/administração & dosagem , Espiperona/farmacologia , Fatores de Tempo
8.
Int J Mol Sci ; 20(7)2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30987329

RESUMO

Dopamine D2 receptors (D2R) are known to form transient homodimer complexes, of which the increased formation has already been associated with development of schizophrenia. Pharmacological targeting and modulation of the equilibrium of these receptor homodimers might lead to a better understanding of the critical role played by these complexes in physiological and pathological conditions. Whereas agonist addition has shown to prolong the D2R dimer lifetime and increase the level of dimer formation, the possible influence of D2R antagonists on dimerization has remained rather unexplored. Here, using a live-cell reporter assay based on the functional complementation of a split Nanoluciferase, a panel of six D2R antagonists were screened for their ability to modulate the level of D2LR dimer formation. Incubation with the D2R antagonist spiperone decreased the level of D2LR dimer formation significantly by 40-60% in real-time and after long-term (≥16 h) incubations. The fact that dimer formation of the well-studied A2a-D2LR dimer was not altered following incubation with spiperone supports the specificity of this observation. Other D2R antagonists, such as clozapine, risperidone, and droperidol did not significantly evoke this dissociation event. Furthermore, molecular modeling reveals that spiperone presents specific Tyr1995.48 and Phe3906.52 conformations, compared to clozapine, which may determine D2R homodimerization.


Assuntos
Antagonistas dos Receptores de Dopamina D2/farmacologia , Receptores de Dopamina D2/metabolismo , Clozapina/farmacologia , Dimerização , Humanos , Ligação Proteica , Receptores Acoplados a Proteínas G/metabolismo , Espiperona/farmacologia
9.
10.
Bioorg Med Chem Lett ; 29(8): 959-964, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30808590

RESUMO

We have synthesized 3 analogs of the dopamine D2 receptor (D2 DR) antagonist spiperone that can be conjugated to streptavidin-coated quantum dots via a pegylated biotin derivative. Using fluorescent imaging we demonstrate that substitution on the spiro position is tolerated, whilst the length and rigidity of a spacer arm attached to spiperone is important in controlling specific labeling as well as minimizing nonspecific labeling to cells and the surface of cell culture dishes. The ligand with the most rigid linker IDT772 (4) had the best binding profile and had high specific binding to D2 DR expressing HEK-293T cells with low nonspecific binding to plates and HEK-293T cells that lacked the D2 DR.


Assuntos
Biotina/química , Pontos Quânticos/química , Receptores de Dopamina D2/metabolismo , Espiperona/química , Antagonistas dos Receptores de Dopamina D2/química , Células HEK293 , Humanos , Ligantes , Microscopia de Fluorescência , Receptores de Dopamina D2/química , Receptores de Dopamina D2/genética , Estreptavidina/química
11.
Pain ; 160(2): 334-344, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30325872

RESUMO

Neuropathic pain represents a challenge to clinicians because it is resistant to commonly prescribed analgesics due to its largely unknown mechanisms. Here, we investigated a descending dopaminergic pathway-mediated modulation of trigeminal neuropathic pain. We performed chronic constriction injury of the infraorbital nerve from the maxillary branch of trigeminal nerve to induce trigeminal neuropathic pain in mice. Our retrograde tracing showed that the descending dopaminergic projection from hypothalamic A11 nucleus to spinal trigeminal nucleus caudalis is bilateral. Optogenetic/chemogenetic manipulation of dopamine receptors D1 and D2 in the spinal trigeminal nucleus caudalis produced opposite effects on the nerve injury-induced trigeminal neuropathic pain. Specific excitation of dopaminergic neurons in the A11 nucleus attenuated the trigeminal neuropathic pain through the activation of D2 receptors in the spinal trigeminal nucleus caudalis. Conversely, specific ablation of the A11 dopaminergic neurons exacerbated such pain. Our results suggest that the descending A11-spinal trigeminal nucleus caudalis dopaminergic projection is critical for the modulation of trigeminal neuropathic pain and could be manipulated to treat such pain.


Assuntos
Encéfalo/patologia , Antagonistas de Dopamina/uso terapêutico , Neurônios Dopaminérgicos/patologia , Receptores de Dopamina D2/metabolismo , Espiperona/uso terapêutico , Doenças do Nervo Trigêmeo/terapia , Animais , Benzazepinas/uso terapêutico , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Condicionamento Operante/fisiologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Neurônios Dopaminérgicos/fisiologia , Lateralidade Funcional , Hiperalgesia/fisiopatologia , Hipotálamo/efeitos dos fármacos , Hipotálamo/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Limiar da Dor/fisiologia , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Doenças do Nervo Trigêmeo/fisiopatologia
12.
Sci Rep ; 7(1): 2459, 2017 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-28550303

RESUMO

Although purinegic signaling is important in regulating gastric physiological functions, it is currently unknown for its role in gastric cancer (GC). We demonstrate for the first time that the expression of P2Y6 receptors was markedly down-regulated in human GC cells and primary GC tissues compared to normal tissues, while the expression of P2Y2 and P2Y4 receptors was up-regulated in GC cells. Moreover, the expression levels of P2Y6 receptors in GC tissues were correlated to tumor size, differentiation, metastasis to lymph nodes, and the survival rate of the patients with GC. Ncleotides activated P2Y6 receptors to raise cytosolic Ca2+ concentrations in GC cells through store-operated calcium entry (SOCE), and then mediated Ca2+-dependent inhibition of ß-catenin and proliferation, eventually leading to GC suppression. Furthermore, UTP particularly blocked the G1/S transition of GC cells but did not induce apoptosis. Collectively, we conclude that nucleotides activate P2Y6 receptors to suppress GC growth through a novel SOCE/Ca2+/ß-catenin-mediated anti-proliferation of GC cells, which is different from the canonical SOCE/Ca2+-induced apoptosis in other tumors.


Assuntos
Antineoplásicos/farmacologia , Canais de Cálcio/genética , Regulação Neoplásica da Expressão Gênica , Receptores Purinérgicos P2/genética , Neoplasias Gástricas/tratamento farmacológico , Uridina Trifosfato/farmacologia , beta Catenina/genética , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Humanos , Indóis/farmacologia , Metástase Linfática , Masculino , Camundongos , Camundongos Nus , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y2/genética , Receptores Purinérgicos P2Y2/metabolismo , Transdução de Sinais , Espiperona/farmacologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Análise de Sobrevida , Carga Tumoral/efeitos dos fármacos , Difosfato de Uridina/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/metabolismo
13.
Chembiochem ; 18(16): 1639-1649, 2017 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-28557180

RESUMO

Unbiased chemoproteomic profiling of small-molecule interactions with endogenous proteins is important for drug discovery. For meaningful results, all protein classes have to be tractable, including G protein-coupled receptors (GPCRs). These receptors are hardly tractable by affinity pulldown from lysates. We report a capture compound (CC)-based strategy to target and identify GPCRs directly from living cells. We synthesized CCs with sertindole attached to the CC scaffold in different orientations to target the dopamine D2 receptor (DRD2) heterologously expressed in HEK 293 cells. The structure-activity relationship of sertindole for DRD2 binding was reflected in the activities of the sertindole CCs in radioligand displacement, cell-based assays, and capture compound mass spectrometry (CCMS). The activity pattern was rationalized by molecular modelling. The most-active CC showed activities very similar to that of unmodified sertindole. A concentration of DRD2 in living cells well below 100 fmol used as an experimental input was sufficient for unambiguous identification of captured DRD2 by mass spectrometry. Our new CCMS workflow broadens the arsenal of chemoproteomic technologies to close a critical gap for the comprehensive characterization of drug-protein interactions.


Assuntos
Antagonistas dos Receptores de Dopamina D2/química , Imidazóis/química , Indóis/química , Receptores de Dopamina D2/análise , Animais , Antagonistas dos Receptores de Dopamina D2/síntese química , Antagonistas dos Receptores de Dopamina D2/efeitos da radiação , Células HEK293 , Humanos , Imidazóis/síntese química , Imidazóis/efeitos da radiação , Indóis/síntese química , Indóis/efeitos da radiação , Ligantes , Simulação de Acoplamento Molecular , Ensaio Radioligante , Ratos , Receptores de Dopamina D2/efeitos da radiação , Espiperona/química , Relação Estrutura-Atividade , Suínos , Espectrometria de Massas em Tandem , Raios Ultravioleta
14.
Sci Rep ; 7(1): 2134, 2017 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-28522847

RESUMO

The dopamine D3 receptor (D3R) is a molecular target for both first-generation and several recently-developed antipsychotic agents. Following stable expression of this mEGFP-tagged receptor, Spatial Intensity Distribution Analysis indicated that a substantial proportion of the receptor was present within dimeric/oligomeric complexes and that increased expression levels of the receptor favored a greater dimer to monomer ratio. Addition of the antipsychotics, spiperone or haloperidol, resulted in re-organization of D3R quaternary structure to promote monomerization. This action was dependent on ligand concentration and reversed upon drug washout. By contrast, a number of other antagonists with high affinity at the D3R, did not alter the dimer/monomer ratio. Molecular dynamics simulations following docking of each of the ligands into a model of the D3R derived from the available atomic level structure, and comparisons to the receptor in the absence of ligand, were undertaken. They showed that, in contrast to the other antagonists, spiperone and haloperidol respectively increased the atomic distance between reference α carbon atoms of transmembrane domains IV and V and I and II, both of which provide key interfaces for D3R dimerization. These results offer a molecular explanation for the distinctive ability of spiperone and haloperidol to disrupt D3R dimerization.


Assuntos
Antagonistas de Dopamina/farmacologia , Haloperidol/farmacologia , Multimerização Proteica , Receptores de Dopamina D3/química , Espiperona/farmacologia , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Receptores de Dopamina D3/antagonistas & inibidores , Receptores de Dopamina D3/metabolismo
15.
Eur J Nucl Med Mol Imaging ; 44(6): 1033-1041, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28083689

RESUMO

PURPOSE: Music can induce different emotions. However, its neural mechanism remains unknown. The aim of this study was to use functional magnetic resonance imaging (fMRI) and position emission tomography (PET) imaging for mapping of neural changes under the most popular music in healthy volunteers. METHODS: Blood-oxygen-level-dependent (BOLD) fMRI and monoamine receptor PET imaging with 11C-N-methylspiperone (11C-NMSP) were conducted under the popular music Gangnam Style and light music A Comme Amour in healthy subjects. PET and fMRI images were analyzed by using the Statistical Parametric Mapping software (SPM). RESULTS: Significantly increased fMRI BOLD signals were found in the bilateral superior temporal cortices, left cerebellum, left putamen and right thalamus cortex. Monoamine receptor availability was increased significantly in the left superior temporal gyrus and left putamen, but decreased in the bilateral superior occipital cortices under the Gangnam Style compared with the light music condition. Significant positive correlation was found between 11C-NMSP binding and fMRI BOLD signals in the left temporal cortex. Furthermore, increased 11C-NMSP binding in the left putamen was positively correlated with the mood arousal level score under the Gangnam Style condition. CONCLUSION: Popular music Gangnam Style can arouse pleasure experience and strong emotional response. The left putamen is positively correlated with the mood arousal level score under the Gangnam Style condition. Our results revealed characteristic patterns of brain activity associated with Gangnam Style, and may also provide more general insights into the music-induced emotional processing.


Assuntos
Mapeamento Encefálico , Imageamento por Ressonância Magnética , Imagem Multimodal , Música , Tomografia Computadorizada por Raios X , Adulto , Radioisótopos de Carbono , Emoções , Voluntários Saudáveis , Humanos , Masculino , Espiperona/análogos & derivados , Adulto Jovem
16.
J Neural Transm (Vienna) ; 124(5): 655-667, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28116523

RESUMO

Previously, we created an 8-h limited-access dual bottle drinking paradigm to deliver methylphenidate (MP) to rats at two dosages that result in a pharmacokinetic profile similar to patients treated for attention deficit hyperactivity disorder. Chronic treatment resulted in altered behavior, with some effects persisting beyond treatment. In the current study, adolescent male Sprague-Dawley rats were split into three groups at four weeks of age: control (water), low-dose MP (LD), and high-dose MP (HD). Briefly, 4 mg/kg (low dose; LD) or 30 mg/kg (high dose; HD) MP was consumed during the first hour, and 10 mg/kg (LD) or 60 mg/kg (HD) MP during hours two through eight. Following three months of treatment, half of the rats in each group (n = 8-9/group) were euthanized, and remaining rats went through a 1-month abstinence period, then euthanized. In vitro receptor autoradiography was performed to quantify binding levels of dopamine transporter (DAT), dopamine type 1 (D1R)-like receptors, and dopamine type 2 (D2R)-like receptors using [3H] WIN35,428, [3H] SCH23390, and [3H] Spiperone, respectively. Immediately following treatment, HD MP-treated rats had increased DAT and D1R-like binding in several subregions of the basal ganglia, particularly more caudal portions of the caudate putamen, which correlated with some previously reported behavioral changes. There were no differences between treatment groups in any measure following abstinence. These findings suggest that chronic treatment with a clinically relevant high dose of MP results in reversible changes in dopamine neurochemistry, which may underlie some effects on behavior.


Assuntos
Estimulantes do Sistema Nervoso Central/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Metilfenidato/farmacologia , Receptores de Dopamina D1/metabolismo , Administração Oral , Animais , Autorradiografia , Benzazepinas , Cocaína/análogos & derivados , Corpo Estriado/citologia , Dopaminérgicos , Relação Dose-Resposta a Droga , Masculino , Compostos Radiofarmacêuticos , Ratos Endogâmicos SHR , Receptores de Dopamina D2/metabolismo , Espiperona , Trítio
17.
Behav Brain Res ; 320: 282-290, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-27993694

RESUMO

Methamphetamine (MA) studies in animals usually involve acute, binge, or short-term exposure to the drug. However, addicts take substantial amounts of MA for extended periods of time. Here we wished to study the effects of MA exposure on brain and behavior, using an animal model analogous to this pattern of MA intake. MA doses, 4 and 8mg/kg/day, were based on previously reported average daily freely available MA self-administration levels. We examined the effects of 16 week MA treatment on psychomotor and cognitive function in the rat using open field and novel object recognition tests and we studied the adaptations of the dopaminergic system, using in vitro and in vivo receptor imaging. We show that chronic MA treatment, at doses that correspond to the average daily freely available self-administration levels in the rat, disorganizes open field activity, impairs alert exploratory behavior and anxiety-like state, and downregulates dopamine transporter in the striatum. Under these treatment conditions, dopamine terminal functional integrity in the nucleus accumbens is also affected. In addition, lower dopamine D1 receptor binding density, and, to a smaller degree, lower dopamine D2 receptor binding density were observed. Potential mechanisms related to these alterations are discussed.


Assuntos
Encéfalo/efeitos dos fármacos , Estimulantes do Sistema Nervoso Central/farmacologia , Cognição/efeitos dos fármacos , Dopamina/metabolismo , Metanfetamina/farmacologia , Desempenho Psicomotor/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Ansiedade/induzido quimicamente , Benzazepinas/farmacologia , Peso Corporal/efeitos dos fármacos , Encéfalo/diagnóstico por imagem , Isótopos de Carbono/farmacocinética , Antagonistas de Dopamina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Comportamento Exploratório/efeitos dos fármacos , Masculino , Racloprida/farmacocinética , Ratos , Ratos Sprague-Dawley , Receptores Dopaminérgicos/metabolismo , Reconhecimento Psicológico/efeitos dos fármacos , Espiperona/farmacologia
18.
Behav Pharmacol ; 28(1): 30-36, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27748674

RESUMO

Rats repeatedly exposed to the bar test following injections with a dopamine D2-like receptor antagonist such as haloperidol show increased descent latencies, suggesting that contextual stimuli may lose their ability to elicit approach and other responses. Here, we showed that rats took progressively longer to initiate descent from a horizontal bar across sessions following daily intraperitoneal treatment (paired group) with the D2-like receptor antagonist, spiroperidol (0.125 and 0.25 mg/kg), but not in the control group that received 0.25 mg/kg in their home cage and testing following saline. When both groups were tested following an injection of spiroperidol or following saline, a sensitized and a conditioned increase in descent latency, respectively, were observed in the paired but not in the unpaired group. No evidence of sensitization or conditioning was found with the substituted benzamide compound, eticlopride (0.15-0.5 mg/kg), or the D2-like receptor partial agonist, aripiprazole (0.25-0.5 mg/kg). The different effects of these agents on learning may be related to different region-specific affinities for dopamine receptors or differences in receptor dissociation profiles. We suggest that the behavioural changes observed in spiroperidol-treated rats may reflect inverse incentive learning.


Assuntos
Aripiprazol/farmacologia , Antagonistas de Dopamina/farmacologia , Salicilamidas/farmacologia , Espiperona/farmacologia , Animais , Aripiprazol/administração & dosagem , Comportamento Animal/efeitos dos fármacos , Condicionamento Psicológico/efeitos dos fármacos , Antagonistas de Dopamina/administração & dosagem , Relação Dose-Resposta a Droga , Masculino , Motivação/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores Dopaminérgicos/efeitos dos fármacos , Receptores Dopaminérgicos/metabolismo , Salicilamidas/administração & dosagem , Espiperona/administração & dosagem
19.
Sci Rep ; 6: 33233, 2016 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-27615810

RESUMO

G protein-coupled receptors (GPCRs), including dopamine receptors, represent a group of important pharmacological targets. An increased formation of dopamine receptor D2 homodimers has been suggested to be associated with the pathophysiology of schizophrenia. Selective labeling and ligand-induced modulation of dimerization may therefore allow the investigation of the pathophysiological role of these dimers. Using TIRF microscopy at the single molecule level, transient formation of homodimers of dopamine receptors in the membrane of stably transfected CHO cells has been observed. The equilibrium between dimers and monomers was modulated by the binding of ligands; whereas antagonists showed a ratio that was identical to that of unliganded receptors, agonist-bound D2 receptor-ligand complexes resulted in an increase in dimerization. Addition of bivalent D2 receptor ligands also resulted in a large increase in D2 receptor dimers. A physical interaction between the protomers was confirmed using high resolution cryogenic localization microscopy, with ca. 9 nm between the centers of mass.


Assuntos
Receptores de Dopamina D2/metabolismo , Espiperona/metabolismo , Animais , Células CHO , Cricetulus , Antagonistas de Dopamina/metabolismo , Humanos , Cinética , Ligantes , Microscopia de Fluorescência , Ligação Proteica , Multimerização Proteica , Transporte Proteico , Análise de Célula Única
20.
Microb Pathog ; 97: 231-5, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27265677

RESUMO

Helicobacter pylori is a major human pathogen related to gastric adenocarcinoma and gastroduodenal diseases. Treatment of H. pylori infections is complicated by the rise of antibiotic resistance, necessitating investigation of alternative therapies. One such alternative is passive immunization by oral administration of antibacterial immunoglobulin. In the present study, chicken immunoglobulin (IgY) was used for passive immunotherapy against a major virulence factor of H. pylori, namely recombinant HP-Nap protein. Recombinant HP-Nap was prepared and used to immunize hens. IgY was purified from the eggs by polyethylene glycol precipitation method with a total IgY-HP-NAP yield of 30 mg per egg. The inhibitory effect of specific IgY on H. pylori attachment was investigated in AGS cell line infected by the bacteria. The results demonstrate the potent effect of IgY- HP-NAP in inhibition of H. pylori attachment to the AGS cells.


Assuntos
Aderência Bacteriana/efeitos dos fármacos , Células Epiteliais/microbiologia , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/imunologia , Imunoglobulinas/metabolismo , Fatores Imunológicos/metabolismo , Fatores de Virulência/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Galinhas , Humanos , Imunoglobulinas/isolamento & purificação , Fatores Imunológicos/isolamento & purificação , Espiperona/análogos & derivados , Fatores de Virulência/imunologia
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