RESUMEN
The antitumor and antimetastatic activity of dopamine D2 receptor antagonists spiperone was studied in C57BL/6 mice in a model of combined pathology (emphysema and lung cancer). Emphysema was induced by administration of LPS and cigarette smoke extract. Lung cancer was induced by injection of Lewis lung carcinoma cells into the lung. It has been shown that under conditions of combined lung pathology, spiperone prevents inflammatory infiltration and emphysematous expansion of the lungs and reduces the size of the primary tumor node, the number of metastases, and the area of the lungs affected by metastases. Spiperone reduces the number of cancer stem cells (CSCs) in the lungs and blood of mice with combined pathology. CSCs isolated from the lungs and blood of mice with combined pathology treated with spiperone had a significantly lower potential to form a tumorosphere in vitro than CSCs from untreated mice with emphysema and lung carcinoma. Thus, blockade of dopamine D2 receptors is a promising approach for correcting combined lung pathology and can be used in the development of a method for treating lung cancer in patients with emphysema.
Asunto(s)
Carcinoma Pulmonar de Lewis , Neoplasias Pulmonares , Ratones Endogámicos C57BL , Células Madre Neoplásicas , Enfisema Pulmonar , Espiperona , Animales , Espiperona/farmacología , Espiperona/uso terapéutico , Ratones , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Carcinoma Pulmonar de Lewis/patología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Enfisema Pulmonar/tratamiento farmacológico , Enfisema Pulmonar/patología , Enfisema Pulmonar/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Antineoplásicos/farmacología , Masculino , Pulmón/efectos de los fármacos , Pulmón/patología , Pulmón/metabolismo , Antagonistas de los Receptores de Dopamina D2/farmacología , Antagonistas de los Receptores de Dopamina D2/uso terapéutico , Receptores de Dopamina D2/metabolismo , Lipopolisacáridos/toxicidadRESUMEN
Early-life viral infections critically influence the brain development and have been variously reported to cause neuropsychiatric diseases such as Schizophrenia, Parkinson's diseases, demyelinating diseases, etc. To investigate the alterations in the dopaminergic system, myelination and associated behavioral impairments following neonatal viral infection, the viral immune activation model was created by an intraperitoneal injection of Poly I:C (5 mg/kg bw/ip) to neonatal rat pups on PND-7. The DA-D2 receptor binding was assessed in corpus striatum by using 3H-Spiperone at 3, 6 and 12 weeks of age. MOG immunolabelling was performed to check myelination stature and myelin integrity, while corpus callosum calibre was assessed by Luxol fast blue staining. Relative behavioral tasks i.e., motor activity, motor coordination and neuromuscular strength were assessed by open field, rotarod and grip strength meter respectively at 3, 6 and 12 weeks of age. Following Poly I:C exposure, a significant decrease in DA-D2 receptor binding, reduction in corpus callosum calibre and MOG immunolabelling indicating demyelination and a significant decrease in locomotor activity, neuromuscular strength and motor coordination signify motor deficits and hypokinetic influence of early life viral infection. Thus, the findings suggest that early life poly I:C exposure may cause demyelination and motor deficits by decreasing DA-D2 receptor binding affinity.
Asunto(s)
Conducta Animal/efectos de los fármacos , Vaina de Mielina/efectos de los fármacos , Neostriado/efectos de los fármacos , Neostriado/metabolismo , Poli I-C/toxicidad , Receptores de Dopamina D2/efectos de los fármacos , Animales , Animales Recién Nacidos , Cuerpo Calloso/citología , Cuerpo Calloso/efectos de los fármacos , Masculino , Actividad Motora/efectos de los fármacos , Fuerza Muscular/efectos de los fármacos , Desempeño Psicomotor/efectos de los fármacos , Ratas , Ratas Wistar , Espiperona/farmacologíaRESUMEN
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.
Asunto(s)
Ácido Hialurónico/metabolismo , Hialuronoglucosaminidasa/farmacología , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Fibrosis Pulmonar Idiopática/metabolismo , Pulmón/efectos de los fármacos , Espiperona/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Colágeno Tipo I/metabolismo , Hialuronoglucosaminidasa/administración & dosificación , Hialuronoglucosaminidasa/farmacocinética , Hidroxiprolina/metabolismo , Fibrosis Pulmonar Idiopática/inducido químicamente , Fibrosis Pulmonar Idiopática/enzimología , Inflamación/metabolismo , Interleucina-1beta/metabolismo , Queratinas/metabolismo , Pulmón/enzimología , Pulmón/metabolismo , Pulmón/patología , Masculino , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Poloxámero/química , Receptores de IgG/metabolismo , Espiperona/administración & dosificación , Factor de Crecimiento Transformador beta/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Endothelial dysfunction and destruction of the pulmonary microcirculation are important pathogenic factors in chronic obstructive pulmonary disease (COPD). In COPD, bronchial obstruction is associated with endothelial dysfunction. Thus, new pharmacological treatment options aimed at restoring the pulmonary endothelium represent a clinical need in COPD therapy. Notch1 has been shown to protect cells against apoptosis, inflammation, and oxidative stress caused by cigarette smoke extract (CSE). Therefore, drug which effect on Notch1 may be a potential therapeutic target for COPD in the future. METHODS: In this study, we assessed the potential of spiperone to mediate regeneration of pulmonary endothelium in model of pulmonary emphysema induced by a CSE and lipopolysaccharide (LPS) in female C57BL/6 mice. RESULTS: Spiperone increased the number of capillaries as well as the expression of the CD31 in the alveolar tissue compared to the controls. Moreover, application of spiperone prevented alveolar wall destruction (DI), and reduced the area of emphysema. Lastly, we demonstrated that spiperone positively influenced mobilization and migration of endothelial progenitor cells (EPC, CD45-CD34+CD31+), CD309+-endothelial cells, and angiogenesis precursors (CD45-CD117+CD309+) into the lung. Spiperone administration significantly reduced the number Notch1 positive CD309+-endothelial cells and Notch1+ EPCs. CONCLUSION: Overall, our results suggest that spiperone mediates endothelial regeneration in an animal model of COPD. Thus, it could represent a novel therapeutic approach for treatment of emphysema associated with COPD.
Asunto(s)
Fumar Cigarrillos , Células Progenitoras Endoteliales , Enfermedad Pulmonar Obstructiva Crónica , Enfisema Pulmonar , Animales , Fumar Cigarrillos/efectos adversos , Células Progenitoras Endoteliales/metabolismo , Endotelio/metabolismo , Endotelio/patología , Femenino , Humanos , Lipopolisacáridos/farmacología , Pulmón , Ratones , Ratones Endogámicos C57BL , Enfermedad Pulmonar Obstructiva Crónica/patología , Enfisema Pulmonar/inducido químicamente , Enfisema Pulmonar/tratamiento farmacológico , Regeneración , Espiperona/metabolismo , Espiperona/farmacología , Espiperona/uso terapéuticoRESUMEN
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.
Asunto(s)
Benzazepinas/administración & dosificación , Levodopa/administración & dosificación , Miopía/tratamiento farmacológico , Espiperona/administración & dosificación , Animales , Benzazepinas/farmacología , Pollos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Inyecciones Intravítreas , Lentes/efectos adversos , Levodopa/farmacología , Masculino , Miopía/etiología , Miopía/metabolismo , Soluciones Oftálmicas , Receptores de Dopamina D2/metabolismo , Espiperona/farmacologíaRESUMEN
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.
Asunto(s)
Antagonistas de Dopamina/farmacología , Células Progenitoras Endoteliales/efectos de los fármacos , Enfisema Pulmonar/tratamiento farmacológico , Receptor Notch1/genética , Receptores de Dopamina D2/genética , Espiperona/farmacología , Deficiencia de alfa 1-Antitripsina/tratamiento farmacológico , Animales , Células Progenitoras Endoteliales/metabolismo , Células Progenitoras Endoteliales/patología , Femenino , Galactosamina/administración & dosificación , Regulación de la Expresión Génica , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Neovascularización Fisiológica/efectos de los fármacos , Elastasa Pancreática/administración & dosificación , Fosforilación/efectos de los fármacos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Enfisema Pulmonar/inducido químicamente , Enfisema Pulmonar/genética , Enfisema Pulmonar/metabolismo , Receptor Notch1/agonistas , Receptor Notch1/metabolismo , Receptores de Dopamina D2/metabolismo , Regeneración/efectos de los fármacos , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Deficiencia de alfa 1-Antitripsina/enzimología , Deficiencia de alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/patologíaRESUMEN
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.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Habénula/metabolismo , Receptores de Dopamina D2/metabolismo , Tejido Adiposo Pardo/efectos de los fármacos , Animales , Benzazepinas/farmacología , Antagonistas de los Receptores de Dopamina D2/farmacología , Masculino , Quinpirol/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Dopamina D1/antagonistas & inhibidores , Espiperona/farmacología , Estrés Psicológico/metabolismo , Temperatura , Termogénesis/efectos de los fármacosRESUMEN
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⯵g/g BW) injection. The present research provides fundamental information about Scyol-VIH and its potential effect in controlling reproduction.
Asunto(s)
Braquiuros/fisiología , Dopamina/farmacología , Hormonas de Invertebrados/metabolismo , Ovario/crecimiento & desarrollo , ARN Mensajero/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Braquiuros/genética , Clonación Molecular , Dopamina/administración & dosificación , Dopaminérgicos/farmacología , Antagonistas de Dopamina/administración & dosificación , Antagonistas de Dopamina/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hormonas de Invertebrados/genética , Ovario/metabolismo , Filogenia , ARN Mensajero/genética , Serotonina/administración & dosificación , Serotonina/farmacología , Serotoninérgicos/administración & dosificación , Serotoninérgicos/farmacología , Maduración Sexual , Espiperona/administración & dosificación , Espiperona/farmacología , Factores de TiempoRESUMEN
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.
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Antagonistas de los Receptores de Dopamina D2/farmacología , Receptores de Dopamina D2/metabolismo , Clozapina/farmacología , Dimerización , Humanos , Unión Proteica , Receptores Acoplados a Proteínas G/metabolismo , Espiperona/farmacologíaRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Canales de Calcio/genética , Regulación Neoplásica de la Expresión Génica , Receptores Purinérgicos P2/genética , Neoplasias Gástricas/tratamiento farmacológico , Uridina Trifosfato/farmacología , beta Catenina/genética , Animales , Calcio/metabolismo , Canales de Calcio/metabolismo , Línea Celular Tumoral , Proliferación Celular , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Humanos , Indoles/farmacología , Metástasis Linfática , Masculino , Ratones , Ratones Desnudos , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y2/genética , Receptores Purinérgicos P2Y2/metabolismo , Transducción de Señal , Espiperona/farmacología , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Análisis de Supervivencia , Carga Tumoral/efectos de los fármacos , Uridina Difosfato/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , beta Catenina/metabolismoRESUMEN
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.
Asunto(s)
Antagonistas de Dopamina/farmacología , Haloperidol/farmacología , Multimerización de Proteína , Receptores de Dopamina D3/química , Espiperona/farmacología , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Unión Proteica , Receptores de Dopamina D3/antagonistas & inhibidores , Receptores de Dopamina D3/metabolismoRESUMEN
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.
Asunto(s)
Aripiprazol/farmacología , Antagonistas de Dopamina/farmacología , Salicilamidas/farmacología , Espiperona/farmacología , Animales , Aripiprazol/administración & dosificación , Conducta Animal/efectos de los fármacos , Condicionamiento Psicológico/efectos de los fármacos , Antagonistas de Dopamina/administración & dosificación , Relación Dosis-Respuesta a Droga , Masculino , Motivación/efectos de los fármacos , Ratas , Ratas Wistar , Receptores Dopaminérgicos/efectos de los fármacos , Receptores Dopaminérgicos/metabolismo , Salicilamidas/administración & dosificación , Espiperona/administración & dosificaciónRESUMEN
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.
Asunto(s)
Encéfalo/efectos de los fármacos , Estimulantes del Sistema Nervioso Central/farmacología , Cognición/efectos de los fármacos , Dopamina/metabolismo , Metanfetamina/farmacología , Desempeño Psicomotor/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Ansiedad/inducido químicamente , Benzazepinas/farmacología , Peso Corporal/efectos de los fármacos , Encéfalo/diagnóstico por imagen , Isótopos de Carbono/farmacocinética , Antagonistas de Dopamina/farmacología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Conducta Exploratoria/efectos de los fármacos , Masculino , Racloprida/farmacocinética , Ratas , Ratas Sprague-Dawley , Receptores Dopaminérgicos/metabolismo , Reconocimiento en Psicología/efectos de los fármacos , Espiperona/farmacologíaRESUMEN
Horizontal cells form the first laterally interacting network of inhibitory interneurons in the retina. Dopamine released onto horizontal cells under photic and circadian control modulates horizontal cell function. Using isolated, identified horizontal cells from a connexin-57-iCre × ROSA26-tdTomato transgenic mouse line, we investigated dopaminergic modulation of calcium channel currents (ICa) with whole cell patch-clamp techniques. Dopamine (10 µM) blocked 27% of steady-state ICa, an action blunted to 9% in the presence of the L-type Ca channel blocker verapamil (50 µM). The dopamine type 1 receptor (D1R) agonist SKF38393 (20 µM) inhibited ICa by 24%. The D1R antagonist SCH23390 (20 µM) reduced dopamine and SKF38393 inhibition. Dopamine slowed ICa activation, blocking ICa by 38% early in a voltage step. Enhanced early inhibition of ICa was eliminated by applying voltage prepulses to +120 mV for 100 ms, increasing ICa by 31% and 11% for early and steady-state currents, respectively. Voltage-dependent facilitation of ICa and block of dopamine inhibition after preincubation with a Gßγ-blocking peptide suggested involvement of Gßγ proteins in the D1R-mediated modulation. When the G protein activator guanosine 5'-O-(3-thiotriphosphate) (GTPγS) was added intracellularly, ICa was smaller and showed the same slowed kinetics seen during D1R activation. With GTPγS in the pipette, additional block of ICa by dopamine was only 6%. Strong depolarizing voltage prepulses restored the GTPγS-reduced early ICa amplitude by 36% and steady-state ICa amplitude by 3%. These results suggest that dopaminergic inhibition of ICa via D1Rs is primarily mediated through the action of Gßγ proteins in horizontal cells.
Asunto(s)
Canales de Calcio/fisiología , Potenciales de la Membrana/fisiología , Receptores de Dopamina D1/metabolismo , Células Horizontales de la Retina/fisiología , 2,3,4,5-Tetrahidro-7,8-dihidroxi-1-fenil-1H-3-benzazepina/farmacología , Animales , Fenómenos Biofísicos/efectos de los fármacos , Fenómenos Biofísicos/genética , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Conexinas/genética , Conexinas/metabolismo , Dopamina/farmacología , Agonistas de Dopamina/farmacología , Antagonistas de Dopamina/farmacología , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Lectinas de Plantas/genética , Lectinas de Plantas/metabolismo , ARN no Traducido/genética , ARN no Traducido/metabolismo , Retina/citología , Células Horizontales de la Retina/efectos de los fármacos , Espiperona/farmacología , omega-Conotoxina GVIA/farmacologíaRESUMEN
BACKGROUND: Cell-based drug screening assays are essential tools for drug discovery and development targeting G protein-coupled receptors, which include dopamine D3 receptors. D3 is notorious for its poor coupling to G protein in most heterologous cell lines, and therefore D3 agonist-stimulated binding of [(35)S]GTPγS to G protein cannot be observed in many "non-functional" D3 expressing cell lines. NEW METHOD: The present work explores the use of an alternate method for assessing agonist activity, consisting of measuring the difference in agonist competition between [(3)H]spiperone bound to low-affinity states of the receptor and that with radioligand bound to high-affinity states (GTP shift assay). COMPARISON WITH EXISTING METHOD: The current study describes the determination of GTP shifts in [(3)H]spiperone binding assays for the assessment of agonists' potencies (at D2 and D3) and efficacies (at D3). Compared with GTPγ(35)S binding assays, the new method removes the cumbersome need of functional D3 cell lines and limited project duration due to short half-life of isotope (35)S. CONCLUSION: The new method allows the estimation of potency (D2 and D3) and efficacy (D3) at the level of receptor and G protein activation in a simple fashion from shifts in monophasic-inhibition curves. Moreover, it does not require [(35)S]GTPγS binding assays with functional D3 cells. This method will have wide applicability for D3-selective agonist screening. It may also be useful for other GPCRs circumventing the need for functional assays and offering the ability to detect agonist activity regardless of the particular signaling pathway.
Asunto(s)
Agonistas de Dopamina/farmacología , Evaluación Preclínica de Medicamentos/métodos , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D3/agonistas , Animales , Células CHO , Cricetulus , Células HEK293 , Humanos , Receptores de Dopamina D2/genética , Receptores de Dopamina D3/genética , Espiperona/farmacología , Radioisótopos de Azufre , Transfección , TritioRESUMEN
The antifibrotic properties of spiperone and its effect on stem and progenitor cells were studied on the model of reversible bleomycin-induced pulmonary fibrosis in C57Bl/6 mice. Spiperone reduced infiltration of the alveolar interstitium and alveolar ducts with inflammatory cells and prevented the growth of the connective tissue in the parenchyma of bleomycin lungs. Apart from anti-inflammatory effect, spiperone suppressed bone marrow hemopoietic cells (CD3, CD45R (B220), Ly6C, Ly6G (Gr1), CD11b (Mac1), TER-119)-, Sca-1+, c-Kit+, CD34- and progenitor hemopoietic cells (granulocyte-erythroid-macrophage-megakaryocytic and granulocyte CFU). Spiperone-induced disturbances of fi brogenesis were paralleled by restoration of endothelial cells in the lung parenchyma, reduction of the number of circulating bone marrow cells and lung mesenchymopoietic cells (mesenchymal multipotent stromal cells (CD31-, CD34-, CD45-, CD44+, CD73+, CD90+, CD106+) and progenitor fi broblast cells), and suppression of multilineage differentiation of multipotent mesenchymal stromal cells (including fi broblast-lineage cells).
Asunto(s)
Antagonistas de Dopamina/farmacología , Fibroblastos/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , Alveolos Pulmonares/efectos de los fármacos , Fibrosis Pulmonar/tratamiento farmacológico , Espiperona/farmacología , Animales , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Bleomicina , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Linaje de la Célula/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Expresión Génica , Granulocitos/citología , Granulocitos/efectos de los fármacos , Granulocitos/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Megacariocitos/citología , Megacariocitos/efectos de los fármacos , Megacariocitos/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Infiltración Neutrófila/efectos de los fármacos , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patologíaRESUMEN
Various D2/3 receptor PET radioligands are sensitive to endogenous dopamine release in vivo. The Occupancy Model is generally used to interpret changes in binding observed in in vivo competition binding studies; an Internalisation Hypothesis may also contribute to these changes in signal. Extension of in vivo competition imaging to other receptor systems has been relatively unsuccessful. A greater understanding of the cellular processes underlying signal changes following endogenous neurotransmitter release may help translate this imaging paradigm to other receptor systems. To investigate the Internalisation Hypothesis we assessed the effects of different cellular environments, representative of those experienced by a receptor following agonist-induced internalisation, on the binding of three D2/3 PET ligands with previously reported sensitivities to endogenous dopamine in vivo, namely [3H]spiperone, [3H]raclopride and [3H]PhNO. Furthermore, we determined the contribution of each cellular compartment to total striatal binding for these D2/3 ligands. These studies suggest that sensitivity to endogenous dopamine release in vivo is related to a decrease in affinity in the endosomal environment compared with those found at the cell surface. In agreement with these findings we also demonstrate that â¼25% of total striatal binding for [3H]spiperone originates from sub-cellular, microsomal receptors, whereas for [3H]raclopride and [3H]PhNO, this fraction is lower, representing â¼14% and 17%, respectively. This pharmacological approach is fully translatable to other receptor systems. Assessment of affinity shifts in different cellular compartments may play a crucial role for understanding if a radioligand is sensitive to endogenous release in vivo, for not just the D2/3, but other receptor systems.
Asunto(s)
Antagonistas de los Receptores de Dopamina D2/metabolismo , Radiofármacos/metabolismo , Receptores de Dopamina D2/metabolismo , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Antagonistas de los Receptores de Dopamina D2/farmacología , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Espacio Extracelular/efectos de los fármacos , Cinética , Masculino , Tomografía de Emisión de Positrones , Racloprida/metabolismo , Racloprida/farmacología , Ensayo de Unión Radioligante , Radiofármacos/farmacología , Ratas Sprague-Dawley , Receptores de Dopamina D3/metabolismo , Espiperona/metabolismo , Espiperona/farmacología , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Tritio/metabolismo , Tritio/farmacologíaRESUMEN
The possibility of boosting antifibrotic activity of testicular hyaluronidase immobilized on polyethylene oxide with spiperone was studied on the bleomycin models of a single (partially reversible pneumofibrosis) and repeated (irreversible pneumofibrosis) injuries to the alveolar epithelium in C57Bl/6 mice. The antifibrotic effect was more pronounced after successive treatment with immobilized hyaluronidase and spiperone than after individual treatment with each of the compounds: no collagen deposition in the parenchyma of bleomycin-damaged lungs was found. The decrease in inflammatory cell (lymphocytes, macrophages, neutrophils, plasma cells) infiltration of the alveoli and alveolar tracts interstitium in mice treated by immobilized hyaluronidase and spiperone did not differ from the anti-inflammatory effect of spiperone monotherapy.
Asunto(s)
Antiinflamatorios/farmacología , Hialuronoglucosaminidasa/farmacología , Fibrosis Pulmonar/tratamiento farmacológico , Espiperona/farmacología , Animales , Antiinflamatorios/uso terapéutico , Bleomicina , Colágeno/metabolismo , Evaluación Preclínica de Medicamentos , Quimioterapia Combinada , Enzimas Inmovilizadas/farmacología , Enzimas Inmovilizadas/uso terapéutico , Hialuronoglucosaminidasa/uso terapéutico , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/prevención & control , Espiperona/uso terapéuticoRESUMEN
Serotonin (5-hydroxytryptamine (5-HT)) is a neurotransmitter that regulates a variety of functions in the nervous, gastrointestinal and cardiovascular systems. Despite such importance, 5-HT signaling pathways are not entirely clear. We demonstrated previously that 4-aminopyridine (4-AP)-sensitive voltage-gated K(+) (Kv) channels determine the resting membrane potential of arterial smooth muscle cells and that the Kv channels are inhibited by 5-HT, which depolarizes the membranes. Therefore, we hypothesized that 5-HT contracts arteries by inhibiting Kv channels. Here we studied 5-HT signaling and the detailed role of Kv currents in rat mesenteric arteries using patch-clamp and isometric tension measurements. Our data showed that inhibiting 4-AP-sensitive Kv channels contracted arterial rings, whereas inhibiting Ca(2+)-activated K(+), inward rectifier K(+) and ATP-sensitive K(+) channels had little effect on arterial contraction, indicating a central role of Kv channels in the regulation of resting arterial tone. 5-HT-induced arterial contraction decreased significantly in the presence of high KCl or the voltage-gated Ca(2+) channel (VGCC) inhibitor nifedipine, indicating that membrane depolarization and the consequent activation of VGCCs mediate the 5-HT-induced vasoconstriction. The effects of 5-HT on Kv currents and arterial contraction were markedly prevented by the 5-HT2A receptor antagonists ketanserin and spiperone. Consistently, α-methyl 5-HT, a 5-HT2 receptor agonist, mimicked the 5-HT action on Kv channels. Pretreatment with a Src tyrosine kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, prevented both the 5-HT-mediated vasoconstriction and Kv current inhibition. Our data suggest that 4-AP-sensitive Kv channels are the primary regulator of the resting tone in rat mesenteric arteries. 5-HT constricts the arteries by inhibiting Kv channels via the 5-HT2A receptor and Src tyrosine kinase pathway.
Asunto(s)
Arterias Mesentéricas/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Receptor de Serotonina 5-HT2A/metabolismo , Serotonina/farmacología , Vasoconstricción , Familia-src Quinasas/metabolismo , 4-Aminopiridina/farmacología , Potenciales de Acción , Animales , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/metabolismo , Células Cultivadas , Ketanserina/farmacología , Masculino , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/fisiología , Contracción Muscular , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/fisiología , Nifedipino/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio con Entrada de Voltaje/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Sprague-Dawley , Antagonistas del Receptor de Serotonina 5-HT2/farmacología , Espiperona/farmacología , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
Pancreatitis was modeled before and after preliminary transplantation of stem cells and serotonin. It was demonstrated that transplantation of mesenchymal stem cells and activation of serotoninergic system prevent the development of pancreatitis.