RESUMEN
Tumor necrosis factor receptor 2 (TNFR2), a membrane-bound tumor necrosis factor receptor expressed by regulatory T cells (Tregs), participates in Treg proliferation. Although a specific TNFR2 pathway has been reported, the signaling mechanism has not been completely elucidated. This study sought to clarify TNFR2 signaling in human Tregs using amplicon sequencing and single-cell RNA sequencing to assess Tregs treated with a TNFR2 agonist antibody. Pathway enrichment analysis based on differentially expressed genes highlighted tumor necrosis factor α signaling via nuclear factor kappa B, interleukin-2 signal transducer and activator of transcription 5 signaling, interferon-γ response, and cell proliferation-related pathways in Tregs after TNFR2 activation. TNFR2-high Treg-focused analysis found that these pathways were fully activated in cancer Tregs, showing high TNFR2 expression. Collectively, these findings suggest that TNFR2 orchestrates multiple pathways in cancer Tregs, which could help cancer cells escape immune surveillance, making TNFR2 signaling a potential anticancer therapy target.
Asunto(s)
Receptores Tipo II del Factor de Necrosis TumoralRESUMEN
Autoimmune responses to aquaporin 4 (AQP4) cause neuromyelitis optica (NMO); thus, specific immunotolerance to this self-antigen could represent a new NMO treatment. We generated the liposome-encapsulated AQP4 peptide 201-220 (p201-220) to induce immunotolerance. Liposomes were generated using phosphatidylserine and the polyglycidol species PG8MG. The in vivo tissue distribution of the liposomes was tested using an ex vivo imaging system. To confirm the antigen presentation capacity of PG8MG liposomes, dendritic cells were treated with PG8MG liposome-encapsulated AQP4 p201-220 (AQP4-PG8MG liposomes). Immunotolerance induction by AQP4-PG8MG liposomes was evaluated using the ex vivo cell proliferation of lymph node cells isolated from AQP4 p201-220-immunized AQP4-deficient mice. Fluorescent dye-labeled PG8MG liposomes were distributed to the lymph nodes. AQP4 p201-220 was presented on dendritic cells. AQP4-PG8MG liposomes were tended to suppress immune responses to AQP4 p201-220. Thus, the encapsulation of AQP4 peptides in PG8MG liposomes represents a new strategy for suppressing autoimmune responses to AQP4.
Asunto(s)
Antígenos/inmunología , Acuaporina 4/inmunología , Proliferación Celular , Antígenos de Histocompatibilidad Clase II/inmunología , Liposomas , Ganglios Linfáticos/citología , Péptidos/inmunología , Animales , Femenino , Ratones , Ratones Endogámicos C57BLRESUMEN
Pulmonary hypertension (PH) is a life-threatening lung disease. PH with concomitant lung diseases, e.g., idiopathic pulmonary fibrosis, is associated with poor prognosis. Development of novel therapeutic vasodilators for treatment of these patients is a key imperative. We evaluated the efficacy of dual activation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) using an active, small-molecule phosphodiesterase (PDE4)/PDE5 dual inhibitor (Compound A). Compound A increased both cAMP and cGMP levels in WI-38 lung fibroblasts and suppressed the expressions of type-1 collagen α1 chain and fibronectin. Additionally, compound A reduced right ventricular weight/left ventricular weight+septal weight ratio, brain natriuretic peptide expression levels in right ventricle, CâC motif chemokine ligand 2 expression levels in lung, and plasma surfactant protein D. Our data indicate that dual activation of cAMP/cGMP pathways may be a novel treatment strategy for PH.
Asunto(s)
AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Hipertensión Pulmonar/inducido químicamente , Hipertensión Pulmonar/tratamiento farmacológico , Inflamación/terapia , Pulmón/efectos de los fármacos , Monocrotalina/toxicidad , Inhibidores de Fosfodiesterasa 5/uso terapéutico , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Células Cultivadas , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Modelos Animales de Enfermedad , Epitelio/lesiones , Fibronectinas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Humanos , Pulmón/metabolismo , Pulmón/patología , Inhibidores de Fosfodiesterasa 5/farmacología , Ratas Wistar , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
Fibrosis of the lung constitutes a major clinical challenge and novel therapies are required to alleviate the associated morbidity and mortality. Investigating the antifibrotic efficacy of drugs that are already in clinical practice offers an efficient strategy to identify new therapies. The phosphodiesterase 4 (PDE4) inhibitors, approved for the treatment of chronic obstructive pulmonary disease, harbor therapeutic potential for pulmonary fibrosis by augmenting the activity of endogenous antifibrotic mediators that signal through cyclic AMP. In this study, we tested the efficacy of several PDE4 inhibitors including a novel compound (Compound 1) in a murine model of lung fibrosis that results from a targeted type II alveolar epithelial cell injury. We also compared the antifibrotic activity of PDE4 inhibition to the two therapies that are FDA-approved for idiopathic pulmonary fibrosis (pirfenidone and nintedanib). We found that both preventative (day 0-21) and therapeutic (day 11-21) dosing regimens of the PDE4 inhibitors significantly ameliorated the weight loss and lung collagen accumulation that are the sequelae of targeted epithelial cell damage. In a therapeutic protocol, the reduction in lung fibrosis with PDE4 inhibitor administration was equivalent to pirfenidone and nintedanib. Treatment with this class of drugs also resulted in a decrease in plasma surfactant protein D concentration, a reduction in the plasma levels of several chemokines implicated in lung fibrosis, and an in vitro inhibition of fibroblast profibrotic gene expression. These results motivate further investigation of PDE4 inhibition as a treatment for patients with fibrotic lung disease.
Asunto(s)
Células Epiteliales Alveolares/patología , Benzamidas/uso terapéutico , Isoquinolinas/uso terapéutico , Inhibidores de Fosfodiesterasa 4/uso terapéutico , Fibrosis Pulmonar/tratamiento farmacológico , Aminopiridinas/uso terapéutico , Animales , Benzamidas/administración & dosificación , Benzamidas/sangre , Células Cultivadas , Quimiocinas/sangre , AMP Cíclico/metabolismo , Ciclopropanos/uso terapéutico , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Fibroblastos/metabolismo , Humanos , Isoquinolinas/administración & dosificación , Isoquinolinas/sangre , Ratones Endogámicos C57BL , Ratones Transgénicos , Inhibidores de Fosfodiesterasa 4/administración & dosificación , Inhibidores de Fosfodiesterasa 4/sangre , Fibrosis Pulmonar/sangre , Fibrosis Pulmonar/prevención & control , Proteína D Asociada a Surfactante Pulmonar/sangre , Piridinas/uso terapéutico , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Pulmonary hypertension (PH) is a life-threatening lung disease. Despite the availability of several approved drugs, the development of a new treatment method is needed because of poor prognosis. Tissue selective drug delivery systems can avoid the adverse effects of current therapy and enhance efficacy. We evaluated the possibility of delivering drugs to the lungs of a PH rat model using fluorescence dye-labeled nanosized liposomes. To evaluate the tissue distribution following systemic exposure, fluorescent dye-labeled, 40-180 nm liposomes with and without polyethylene glycol (PEG) were intravenously administered to a monocrotaline-induced PH (MCT) rat model and tissue fluorescence was measured. Fluorescent dye-containing liposomes were intratracheally administered to the MCT model to evaluate the distribution of the liposome-encapsulated compound following local administration to reduce systemic exposure. The lung vascular permeability, plasma concentration of surfactant protein (SP)-D, lung reactive oxygen species (ROS) production, and macrophage marker gene cluster of differentiation (CD68) expression were measured. PEG and 80-nm liposome accumulation in the lung was elevated in the MCT model compared to that in normal rats. The intratracheally administered liposomes were delivered selectively to the lungs of the MCT model. The lung vascular permeability, plasma SP-D concentration, and CD68 expression were significantly elevated in the lungs of the MCT model, and were all significantly and positively correlated to liposome lung accumulation. Liposomes can accumulate in the lungs of an MCT model by enhancing vascular permeability by the inflammatory response. Therefore, drug encapsulation in liposomes could be an effective method of drug delivery in patients with PH.
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Colorantes Fluorescentes/metabolismo , Hipertensión Pulmonar/metabolismo , Liposomas/metabolismo , Monocrotalina , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Liberación de Fármacos , Colorantes Fluorescentes/administración & dosificación , Colorantes Fluorescentes/química , Hipertensión Pulmonar/inducido químicamente , Liposomas/química , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Masculino , Imagen Óptica , Tamaño de la Partícula , Permeabilidad , Polietilenglicoles/química , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Propiedades de SuperficieRESUMEN
Drug delivery systems maximize the efficacy of drugs by improving their pharmacokinetic profiles, pharmacodynamic effects, or both and reducing their adverse effects. One of the most advanced, clinically available formulations are liposome-encapsulated drugs. In this study, we aimed to determine if liposomes can selectively deliver compounds in gastrointestinal diseases. Initially, we evaluated the correlation between the diarrhea score and accumulation of fluorescence (FL)-labeled liposome using in vivo imaging systems in various disease states of an inflammatory bowel disease mouse model. The result showed that FL-labeled liposome accumulation and colon tissue weight, which reflect the disease state were highly and positively correlated. Then, to confirm the accumulation of liposomes at injured sites of the colon, we administered both FL-labeled liposomes and luminescence probes for detecting reactive oxygen species (ROS) to the mouse model. The imaging data showed that liposome accumulation tended to coincide with ROS detected sites and the correlation coefficient indicated a significantly positive correlation between liposome accumulation and ROS detection levels. Finally, we evaluated the involvement of macrophages in the uptake mechanism of the liposomes by analyzing the relationship between FL-labeled liposome accumulation and macrophage marker gene expression levels. The result showed that the expression of each macrophage marker gene and liposome accumulation showed a significant positive correlation. Therefore, the macrophages considerably contributed to the uptake mechanism of the liposomes. These data suggest that liposomes could be an attractive delivery tool for enhancing the accumulation of drug candidates through macrophages in injured colonic tissues. This approach is expected to provide new treatment options for patients with colitis.
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Colon/metabolismo , Sistemas de Liberación de Medicamentos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Traslado Adoptivo , Animales , Antígenos CD/genética , Antígenos de Diferenciación/genética , Antígenos de Diferenciación Mielomonocítica/genética , Colon/lesiones , Modelos Animales de Enfermedad , Femenino , Colorantes Fluorescentes/administración & dosificación , Colorantes Fluorescentes/farmacocinética , Marcadores Genéticos , Enfermedades Inflamatorias del Intestino/etiología , Enfermedades Inflamatorias del Intestino/metabolismo , Liposomas , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Nanomedicina , Especies Reactivas de Oxígeno/metabolismo , Receptores Depuradores de Clase A/genética , Linfocitos T/inmunologíaRESUMEN
Duchenne muscular dystrophy (DMD) is the most common inherited muscular dystrophy. Patients experience DMD in their 20s from cardiac or respiratory failure related to progressive muscle wasting. Currently, the only treatments for the symptoms of DMD are available. Muscle fibrosis, a DMD feature, leads to reduced muscle function and muscle mass, and hampers pharmaceutical therapeutic efficacy. Although antifibrotic agents may be useful, none is currently approved. Phosphodiesterase 4 (PDE4) inhibitors have exhibited antifibrotic effects in human and animal models. In this study, we showed beneficial effects of the PDE4 inhibitor piclamilast in the DMD mdx mouse. Piclamilast reduced the mRNA level of profibrotic genes, including collagen 1A1, in the gastrocnemius and diaphragm, in the mdx mouse, and significantly reduced the Sirius red staining area. The PDE5 inhibitors sildenafil and tadalafil ameliorated functional muscle ischemia in boys with DMD, and sildenafil reversed cardiac dysfunction in the mdx mouse. Single-treatment piclamilast or sildenafil showed similar antifibrotic effects on the gastrocnemius; combination therapy showed a potent antifibrotic effect, and piclamilast and combination therapy increased peroxisome proliferator-activated receptor γ coactivator-1α mRNA in mouse gastrocnemius. In summary, we confirmed that piclamilast has significant antifibrotic effects in mdx mouse muscle and is a potential treatment for muscle fibrosis in DMD.-Nio, Y., Tanaka, M., Hirozane, Y., Muraki, Y., Okawara, M., Hazama, M., Matsuo, T. Phosphodiesterase 4 inhibitor and phosphodiesterase 5 inhibitor combination therapy has antifibrotic and anti-inflammatory effects in mdx mice with Duchenne muscular dystrophy.
Asunto(s)
Distrofia Muscular Animal/tratamiento farmacológico , Distrofia Muscular de Duchenne/tratamiento farmacológico , Inhibidores de Fosfodiesterasa 4/uso terapéutico , Inhibidores de Fosfodiesterasa 5/uso terapéutico , Animales , Antiinflamatorios/uso terapéutico , Benzamidas/uso terapéutico , Fibrosis/tratamiento farmacológico , Fibrosis/enzimología , Fibrosis/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Contracción Muscular/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/enzimología , Distrofia Muscular Animal/metabolismo , Distrofia Muscular de Duchenne/enzimología , Distrofia Muscular de Duchenne/metabolismo , PPAR gamma/genética , Piridinas/uso terapéutico , ARN Mensajero/genética , Citrato de Sildenafil/uso terapéuticoRESUMEN
Somatostatin receptor subtype 5 (SSTR5) has emerged as a novel attractive drug target for type 2 diabetes mellitus. Starting from N-benzyl azetidine derivatives 1 and 2 as in-house hit compounds, we explored the introduction of a carboxyl group into the terminal benzene of 1 to enhance SSTR5 antagonistic activity by the combination of the substituents at the 3-position of the isoxazoline. Incorporation of a carboxyl group at the 4-position of the benzene ring resulted in a significant enhancement in potency, however, the 4-benzoic acid derivative 10c exhibited moderate human ether-a-go-go related gene (hERG) inhibitory activity. A subsequent optimization study revealed that replacement of the 4-benzoic acid with an isonipecotic acid dramatically reduced hERG inhibition (5.6% inhibition at 30µM) by eliminating π-related interaction with hERG K+ channel, which resulted in the identification of 1-(2-((2,6-diethoxy-4'-fluorobiphenyl-4-yl)methyl)-5-oxa-2,6-diazaspiro[3.4]oct-6-en-7-yl)piperidin-4-carboxylic acid 25a (hSSTR5/mSSTR5 IC50=9.6/57nM). Oral administration of 25a in high-fat diet fed C57BL/6J mice augmented insulin secretion in a glucose-dependent manner and lowered blood glucose concentration.
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/farmacología , Receptores de Somatostatina/antagonistas & inhibidores , Animales , Células CHO , Espectroscopía de Resonancia Magnética con Carbono-13 , Cricetulus , Descubrimiento de Drogas , Hipoglucemiantes/química , Hipoglucemiantes/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Espectroscopía de Protones por Resonancia MagnéticaRESUMEN
Endoplasmic reticulum (ER) stress caused by accumulation of misfolded proteins is observed in several kinds of diseases. Since ER stress is reported to be involved in the progression of non-alcoholic steatohepatitis (NASH), highly sensitive and simple measurement methods are required for research into developing novel therapy for NASH. To investigate the involvement of ER stress in NASH pathogenesis in a mouse model, an assay for liver ER stress was developed using ER stress activated indicator-luciferase (ERAI-Luc) mice. To establish the assay method for detection of ER stress in the liver, tunicamycin (TM) (0.3 mg/kg i. p.) was administered to ERAI-Luc mice, and the luciferase activity was measured in ex vivo and in vivo. To evaluate ER stress in the NASH model, ERAI-Luc mice were fed a modified choline-deficient l-amino acid-defined (mCDAA) diet for 14 weeks. After measurement of ER stress by luminescence imaging, levels of liver lipids and pro-fibrotic and pro-inflammatory gene expression were measured as NASH-related indexes. In non-invasive whole-body imaging, TM elevated luciferase activity in the liver, induced by activation of ER stress. The highest luminescence in the liver was confirmed by ex vivo imaging of isolated tissues. In parallel with progression of NASH, elevated luminescence induced by ER stress in liver was observed in mCDAA diet-fed ERAI-Luc mice. Luciferase activity was significantly and positively correlated to levels of triglyceride and free cholesterol in the liver, as well as to the mRNA expression of type 1 collagen α1 chain and tumor necrosis factor α. These data indicated that the use of ERAI-Luc mice was effective in the detection of ER stress in the liver. Moreover, the NASH model using ERAI-Luc mice can be a useful tool to clarify the role of ER stress in pathogenesis of NASH and to evaluate effects of drugs targeted against ER stress.
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Deficiencia de Colina/genética , Colágeno Tipo I/genética , Estrés del Retículo Endoplásmico/genética , Alimentos Formulados/efectos adversos , Hígado/metabolismo , Enfermedad del Hígado Graso no Alcohólico/genética , Factor de Necrosis Tumoral alfa/genética , Animales , Bioensayo , Colesterol/metabolismo , Deficiencia de Colina/etiología , Deficiencia de Colina/metabolismo , Deficiencia de Colina/patología , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Modelos Animales de Enfermedad , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Regulación de la Expresión Génica , Genes Reporteros , Hepatocitos/metabolismo , Hepatocitos/patología , Hígado/patología , Luciferasas/genética , Luciferasas/metabolismo , Mediciones Luminiscentes , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Transducción de Señal , Triglicéridos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Tunicamicina/farmacologíaRESUMEN
We reported elsewhere that orexin neurons are directly hyperpolarized by noradrenaline (NA) and dopamine. In the present study, we show that NA, dopamine, and adrenaline all directly hyperpolarized orexin neurons. This response was inhibited by the alpha2 adrenergic receptor (alpha2-AR) antagonist, idazoxan or BRL44408, and was mimicked by the alpha2-AR-selective agonist, UK14304. A low concentration of Ba2+ inhibited NA-induced hyperpolarization, which suggests that activation of G protein coupled inward rectifier potassium channels is involved in the response. In the presence of a high concentration of idazoxan, NA induced depolarization or inward current. This response was inhibited by alpha1-AR antagonist, prazosin, which suggests the existence of alpha1-ARs on the orexin neurons along with alpha2-AR. We also examined the effects of NA on glutamatergic and GABAergic synaptic transmission. NA application dramatically increased the frequency and amplitude of spontaneous inhibitory synaptic currents (sIPSCs) and inhibited excitatory synaptic currents (sEPSCs) in orexin neurons; however, NA decreased the frequency of miniature EPSCs (mEPSCs) and IPSCs and the amplitude of evoked EPSCs and IPSCs through the alpha2-AR, because the NA response on mPSCs was inhibited by idazoxan. These results suggest that the NA-induced increase in sIPSC frequency and amplitude is mediated via alpha1-ARs on the somata of GABAergic neurons that innervate the orexin neurons. Calcium imaging using orexin/YC2.1 transgenic mouse brain revealed that NA-induced inhibition of orexin neurons is not altered by sleep deprivation or circadian time in mice. The evidence presented here revealed that orexin neurons are regulated by catecholamines in a complex manner.
Asunto(s)
Catecolaminas/fisiología , Péptidos y Proteínas de Señalización Intracelular/análisis , Péptidos y Proteínas de Señalización Intracelular/fisiología , Neuronas/química , Neuronas/fisiología , Neuropéptidos/análisis , Neuropéptidos/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Agonistas alfa-Adrenérgicos/farmacología , Antagonistas Adrenérgicos alfa/farmacología , Animales , Calcio/fisiología , Femenino , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/fisiología , Idazoxan/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Neuronas/efectos de los fármacos , Norepinefrina/fisiología , Receptores de Orexina , Orexinas , Receptores Adrenérgicos alfa 1/análisis , Receptores Adrenérgicos alfa 1/fisiología , Receptores Adrenérgicos alfa 2/análisis , Receptores Adrenérgicos alfa 2/fisiología , Receptores Acoplados a Proteínas G , Receptores de Neuropéptido , Privación de Sueño/fisiopatología , Transmisión Sináptica/fisiología , Tetrodotoxina/farmacología , Tirosina 3-Monooxigenasa/análisis , Tirosina 3-Monooxigenasa/fisiologíaRESUMEN
Orexin A and B are neuropeptides implicated in the regulation of sleep/wakefulness and energy homeostasis. The regulatory mechanism of the activity of orexin neurons is not precisely understood. Using transgenic mice in which orexin neurons specifically express yellow cameleon 2.1, we screened for factors that affect the activity of orexin neurons (a total of 21 peptides and six other factors were examined) and found that a sulfated octapeptide form of cholecystokinin (CCK-8S), neurotensin, oxytocin, and vasopressin activate orexin neurons. The mechanisms that underlie CCK-8S-induced activation of orexin neurons were studied by both calcium imaging and slice patch-clamp recording. CCK-8S induced inward current in the orexin neurons. The CCKA receptor antagonist lorglumide inhibited CCK-8S-induced activation of orexin neurons, whereas the CCKB receptor agonists CCK-4 (a tetrapeptide form of cholecystokinin) and nonsulfated CCK-8 had little effect. The CCK-8S-induced increase in intracellular calcium concentration was eliminated by removing extracellular calcium but not by an addition of thapsigargin. Nifedipine, omega-conotoxin, omega-agatoxin, 4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride, and SNX-482 had little effect, but La3+, Gd3+, and 2-aminoethoxydiphenylborate inhibited CCK-8S-induced calcium influx. Additionally, the CCK-8S-induced inward current was dramatically enhanced in the calcium-free solution and was inhibited by the cation channel blocker SKF96365, suggesting an involvement of extracellular calcium-sensitive cation channels. CCK-8S did not induce an increase in intracellular calcium concentration when membrane potential was clamped at -60 mV, suggesting that the calcium increase is induced by depolarization. The evidence presented here expands our understanding of the regulation of orexin neurons and the physiological role of CCK in the CNS.
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Colecistoquinina/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neuronas/fisiología , Neuropéptidos/metabolismo , Receptor de Colecistoquinina A/fisiología , Animales , Calcio/metabolismo , Conductividad Eléctrica , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Líquido Extracelular/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Técnicas In Vitro , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Ratones , Ratones Transgénicos , Inhibición Neural/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Receptores de Orexina , Orexinas , Técnicas de Placa-Clamp , Receptor de Colecistoquinina A/metabolismo , Receptores Acoplados a Proteínas G , Receptores de Neuropéptido , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sincalida/análogos & derivados , Sincalida/farmacología , Transmisión Sináptica/efectos de los fármacosRESUMEN
The finding of orexin/hypocretin deficiency in narcolepsy patients suggests that this hypothalamic neuropeptide plays a crucial role in regulating sleep/wakefulness states. However, very little is known about the synaptic input of orexin/hypocretin-producing neurons (orexin neurons). We applied a transgenic method to map upstream neuronal populations that have synaptic connections to orexin neurons and revealed that orexin neurons receive input from several brain areas. These include the amygdala, basal forebrain cholinergic neurons, GABAergic neurons in the preoptic area, and serotonergic neurons in the median/paramedian raphe nuclei. Monoamine-containing groups that are innervated by orexin neurons do not receive reciprocal connections, while cholinergic neurons in the basal forebrain have reciprocal connections, which might be important for consolidating wakefulness. Electrophysiological study showed that carbachol excites almost one-third of orexin neurons and inhibits a small population of orexin neurons. These neuroanatomical findings provide important insights into the neural pathways that regulate sleep/wakefulness states.
Asunto(s)
Hipotálamo/anatomía & histología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Vías Nerviosas/anatomía & histología , Neuronas/citología , Neuropéptidos/metabolismo , Animales , Tronco Encefálico/anatomía & histología , Tronco Encefálico/efectos de los fármacos , Tronco Encefálico/ultraestructura , Carbacol/farmacología , Agonistas Colinérgicos/farmacología , Proteínas Fluorescentes Verdes/genética , Humanos , Hipotálamo/efectos de los fármacos , Hipotálamo/ultraestructura , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Transgénicos , Vías Nerviosas/efectos de los fármacos , Neuronas/fisiología , Orexinas , Técnicas de Placa-Clamp , Proteínas Recombinantes de Fusión/genética , Tetrodotoxina/genética , Vigilia/fisiologíaRESUMEN
Both orexin and serotonin (5-HT) have important roles in the regulation of sleep-wakefulness, as well as in feeding behavior. We examined the effects of 5-HT on orexin/hypocretin neurons, using hypothalamic slices prepared from orexin/enhanced green fluorescent protein (EGFP) transgenic mice in which EGFP is expressed exclusively in orexin neurons. Patch-clamp recording from EGFP-expressing cells showed that 5-HT hyperpolarized all orexin neurons in a concentration-dependent manner. The response was inhibited by the 5-HT1A receptor antagonist WAY100635. A 5-HT1A receptor agonist, 8-hydroxy-2-(dl-N-propyl-amino)tetralin, also evoked hyperpolarization on orexin neurons with potency comparable with 5-HT. A low concentration of Ba2+ (30 microM) inhibited 5-HT-induced hyperpolarization. Single-channel recording revealed that the conductance of 5-HT-induced channel activity was 33.8 pS, which is in good agreement with that of the G-protein-coupled inward rectifier potassium channel (GIRK). Moreover, 5-HT1A receptor-like immunoreactivity was observed on orexin neurons, and 5-HT transporter immunoreactive nerve endings are in close apposition to orexin neurons. Intracerebroventricular injection of the 5-HT1A receptor-selective antagonist WAY100635 (100 ng) increased locomotor activity during the latter half of dark phase in wild-type mice but not in orexin/ataxin-3 mice in which orexin neurons are specifically ablated, suggesting that activation of orexin neurons is necessary for the WAY100635-induced increase in locomotor activity. These results indicate that 5-HT hyperpolarizes orexin neurons through the 5-HT1A receptor and subsequent activation of the GIRK and that this inhibitory serotonergic input to the orexin neurons is likely to be important for the physiological regulation of this neuropeptide system.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neuronas/fisiología , Neuropéptidos/metabolismo , Receptor de Serotonina 5-HT1A/fisiología , Animales , Ataxina-3 , Femenino , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Proteínas Fluorescentes Verdes/genética , Hipotálamo/citología , Hipotálamo/metabolismo , Inmunohistoquímica , Técnicas In Vitro , Inyecciones Intraventriculares , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Actividad Motora/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Neuropéptidos/genética , Proteínas Nucleares , Receptores de Orexina , Orexinas , Técnicas de Placa-Clamp , Canales de Potasio de Rectificación Interna/agonistas , Receptor de Serotonina 5-HT1A/biosíntesis , Receptores Acoplados a Proteínas G , Receptores de Neuropéptido , Proteínas Represoras , Serotonina/farmacología , Antagonistas de la Serotonina/administración & dosificación , Antagonistas de la Serotonina/farmacología , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Agonistas de Receptores de Serotonina/farmacología , Tetrodotoxina/farmacología , Factores de TranscripciónRESUMEN
Orexins are a pair of neuropeptides implicated in energy homeostasis and arousal. Here we characterize the electrophysiological properties of orexin neurons using slice preparations from transgenic mice in which orexin neurons specifically express green fluorescent protein. Orexin neurons showed high frequency firing with little adaptation by injecting a positive current. The hyperpolarization-activated current was observed in orexin neurons by a negative current injection. The neurotransmitters, which were implicated in sleep/wake regulation, affected the activity of orexin neurons; noradrenaline and serotonin hyperpolarized, while carbachol depolarized orexin neurons in either the presence or absence of tetrodotoxin. It has been reported that orexins directly or indirectly activate the nuclei that are the origin of the neurons containing these neurotransmitters. Our data suggest that orexin neurons have reciprocal neural circuitries between these nuclei for either a positive or negative feedback loop and orchestrate the activity of these neurons to regulate the vigilance states.