RESUMO
Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , DNA Helicases/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Esclerose Tuberosa/metabolismo , Sequência de Aminoácidos , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/química , Evolução Molecular , Feminino , Humanos , Insulina/farmacologia , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fenótipo , Proteínas de Ligação a Poli-ADP-Ribose/química , RNA Helicases/química , Proteínas com Motivo de Reconhecimento de RNA/química , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Peixe-Zebra/metabolismoRESUMO
The propagation of electrical impulses along axons is highly accelerated by the myelin sheath and produces saltating or "jumping" action potentials across internodes, from one node of Ranvier to the next. The underlying electrical circuit, as well as the existence and role of submyelin conduction in saltatory conduction remain, however, elusive. Here, we made patch-clamp and high-speed voltage-calibrated optical recordings of potentials across the nodal and internodal axolemma of myelinated neocortical pyramidal axons combined with electron microscopy and experimentally constrained cable modeling. Our results reveal a nanoscale yet conductive periaxonal space, incompletely sealed at the paranodes, which separates the potentials across the low-capacitance myelin sheath and internodal axolemma. The emerging double-cable model reproduces the recorded evolution of voltage waveforms across nodes and internodes, including rapid nodal potentials traveling in advance of attenuated waves in the internodal axolemma, revealing a mechanism for saltation across time and space.
Assuntos
Potenciais de Ação/fisiologia , Bainha de Mielina/fisiologia , Fibras Nervosas Mielinizadas/fisiologia , Nós Neurofibrosos/fisiologia , Animais , Axônios/metabolismo , Axônios/fisiologia , Masculino , Modelos Neurológicos , Fibras Nervosas Mielinizadas/metabolismo , Técnicas de Patch-Clamp/métodos , Células Piramidais/fisiologia , Ratos , Ratos WistarRESUMO
Muscle spasticity after nervous system injuries and painful low back spasm affect more than 10% of global population. Current medications are of limited efficacy and cause neurological and cardiovascular side effects because they target upstream regulators of muscle contraction. Direct myosin inhibition could provide optimal muscle relaxation; however, targeting skeletal myosin is particularly challenging because of its similarity to the cardiac isoform. We identified a key residue difference between these myosin isoforms, located in the communication center of the functional regions, which allowed us to design a selective inhibitor, MPH-220. Mutagenic analysis and the atomic structure of MPH-220-bound skeletal muscle myosin confirmed the mechanism of specificity. Targeting skeletal muscle myosin by MPH-220 enabled muscle relaxation, in human and model systems, without cardiovascular side effects and improved spastic gait disorders after brain injury in a disease model. MPH-220 provides a potential nervous-system-independent option to treat spasticity and muscle stiffness.
Assuntos
Músculo Esquelético/metabolismo , Miosinas de Músculo Esquelético/efeitos dos fármacos , Miosinas de Músculo Esquelético/genética , Adulto , Animais , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Linhagem Celular , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Masculino , Camundongos , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Espasticidade Muscular/genética , Espasticidade Muscular/fisiopatologia , Músculo Esquelético/fisiologia , Miosinas/efeitos dos fármacos , Miosinas/genética , Miosinas/metabolismo , Isoformas de Proteínas , Ratos , Ratos Wistar , Miosinas de Músculo Esquelético/metabolismoRESUMO
The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global public health. The development of a vaccine is urgently needed for the prevention and control of COVID-19. Here, we report the pilot-scale production of an inactivated SARS-CoV-2 vaccine candidate (BBIBP-CorV) that induces high levels of neutralizing antibodies titers in mice, rats, guinea pigs, rabbits, and nonhuman primates (cynomolgus monkeys and rhesus macaques) to provide protection against SARS-CoV-2. Two-dose immunizations using 2 µg/dose of BBIBP-CorV provided highly efficient protection against SARS-CoV-2 intratracheal challenge in rhesus macaques, without detectable antibody-dependent enhancement of infection. In addition, BBIBP-CorV exhibits efficient productivity and good genetic stability for vaccine manufacture. These results support the further evaluation of BBIBP-CorV in a clinical trial.
Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Avaliação Pré-Clínica de Medicamentos/métodos , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Vacinas de Produtos Inativados/uso terapêutico , Vacinas Virais/uso terapêutico , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/genética , COVID-19 , Vacinas contra COVID-19 , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Modelos Animais de Doenças , Feminino , Cobaias , Imunogenicidade da Vacina , Macaca fascicularis , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Filogenia , Pneumonia Viral/virologia , Coelhos , Ratos , Ratos Wistar , SARS-CoV-2 , Vacinas de Produtos Inativados/efeitos adversos , Células Vero , Vacinas Virais/efeitos adversosRESUMO
The recent discovery that genetically modified α cells can regenerate and convert into ß-like cells in vivo holds great promise for diabetes research. However, to eventually translate these findings to human, it is crucial to discover compounds with similar activities. Herein, we report the identification of GABA as an inducer of α-to-ß-like cell conversion in vivo. This conversion induces α cell replacement mechanisms through the mobilization of duct-lining precursor cells that adopt an α cell identity prior to being converted into ß-like cells, solely upon sustained GABA exposure. Importantly, these neo-generated ß-like cells are functional and can repeatedly reverse chemically induced diabetes in vivo. Similarly, the treatment of transplanted human islets with GABA results in a loss of α cells and a concomitant increase in ß-like cell counts, suggestive of α-to-ß-like cell conversion processes also in humans. This newly discovered GABA-induced α cell-mediated ß-like cell neogenesis could therefore represent an unprecedented hope toward improved therapies for diabetes.
Assuntos
Diabetes Mellitus/tratamento farmacológico , Células Secretoras de Glucagon/citologia , Células Secretoras de Insulina/citologia , Ácido gama-Aminobutírico/administração & dosagem , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Diferenciação Celular/efeitos dos fármacos , Diabetes Mellitus/induzido quimicamente , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Células Secretoras de Glucagon/efeitos dos fármacos , Humanos , Ilhotas Pancreáticas/citologia , Masculino , Camundongos , Proteínas do Tecido Nervoso , Ratos , Ratos Wistar , Ácido gama-Aminobutírico/farmacologiaRESUMO
Arthritis typically involves recurrence and progressive worsening at specific predilection sites, but the checkpoints between remission and persistence remain unknown. Here, we defined the molecular and cellular mechanisms of this inflammation-mediated tissue priming. Re-exposure to inflammatory stimuli caused aggravated arthritis in rodent models. Tissue priming developed locally and independently of adaptive immunity. Repeatedly stimulated primed synovial fibroblasts (SFs) exhibited enhanced metabolic activity inducing functional changes with intensified migration, invasiveness and osteoclastogenesis. Meanwhile, human SF from patients with established arthritis displayed a similar primed phenotype. Transcriptomic and epigenomic analyses as well as genetic and pharmacological targeting demonstrated that inflammatory tissue priming relies on intracellular complement C3- and C3a receptor-activation and downstream mammalian target of rapamycin- and hypoxia-inducible factor 1α-mediated metabolic SF invigoration that prevents activation-induced senescence, enhances NLRP3 inflammasome activity, and in consequence sensitizes tissue for inflammation. Our study suggests possibilities for therapeutic intervention abrogating tissue priming without immunosuppression.
Assuntos
Proteínas do Sistema Complemento/imunologia , Fibroblastos/imunologia , Inflamação/imunologia , Membrana Sinovial/imunologia , Imunidade Adaptativa/imunologia , Animais , Artrite Reumatoide/imunologia , Linhagem Celular , Cães , Humanos , Mediadores da Inflamação/imunologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Ratos Wistar , Transdução de Sinais/imunologiaRESUMO
We present a first-draft digital reconstruction of the microcircuitry of somatosensory cortex of juvenile rat. The reconstruction uses cellular and synaptic organizing principles to algorithmically reconstruct detailed anatomy and physiology from sparse experimental data. An objective anatomical method defines a neocortical volume of 0.29 ± 0.01 mm(3) containing ~31,000 neurons, and patch-clamp studies identify 55 layer-specific morphological and 207 morpho-electrical neuron subtypes. When digitally reconstructed neurons are positioned in the volume and synapse formation is restricted to biological bouton densities and numbers of synapses per connection, their overlapping arbors form ~8 million connections with ~37 million synapses. Simulations reproduce an array of in vitro and in vivo experiments without parameter tuning. Additionally, we find a spectrum of network states with a sharp transition from synchronous to asynchronous activity, modulated by physiological mechanisms. The spectrum of network states, dynamically reconfigured around this transition, supports diverse information processing strategies. PAPERCLIP: VIDEO ABSTRACT.
Assuntos
Simulação por Computador , Modelos Neurológicos , Neocórtex/citologia , Neurônios/classificação , Neurônios/citologia , Córtex Somatossensorial/citologia , Algoritmos , Animais , Membro Posterior/inervação , Masculino , Neocórtex/fisiologia , Rede Nervosa , Neurônios/fisiologia , Ratos , Ratos Wistar , Córtex Somatossensorial/fisiologiaRESUMO
The N-methyl-D-aspartate (NMDA) receptor is a glutamate-activated cation channel that is critical to many processes in the brain. Genome-wide association studies suggest that glutamatergic neurotransmission and NMDA receptor-mediated synaptic plasticity are important for body weight homeostasis1. Here we report the engineering and preclinical development of a bimodal molecule that integrates NMDA receptor antagonism with glucagon-like peptide-1 (GLP-1) receptor agonism to effectively reverse obesity, hyperglycaemia and dyslipidaemia in rodent models of metabolic disease. GLP-1-directed delivery of the NMDA receptor antagonist MK-801 affects neuroplasticity in the hypothalamus and brainstem. Importantly, targeting of MK-801 to GLP-1 receptor-expressing brain regions circumvents adverse physiological and behavioural effects associated with MK-801 monotherapy. In summary, our approach demonstrates the feasibility of using peptide-mediated targeting to achieve cell-specific ionotropic receptor modulation and highlights the therapeutic potential of unimolecular mixed GLP-1 receptor agonism and NMDA receptor antagonism for safe and effective obesity treatment.
Assuntos
Maleato de Dizocilpina , Peptídeo 1 Semelhante ao Glucagon , Receptor do Peptídeo Semelhante ao Glucagon 1 , Obesidade , Receptores de N-Metil-D-Aspartato , Animais , Humanos , Masculino , Camundongos , Ratos , Tronco Encefálico/metabolismo , Tronco Encefálico/efeitos dos fármacos , Modelos Animais de Doenças , Maleato de Dizocilpina/efeitos adversos , Maleato de Dizocilpina/farmacologia , Maleato de Dizocilpina/uso terapêutico , Dislipidemias/tratamento farmacológico , Dislipidemias/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Hiperglicemia/tratamento farmacológico , Hiperglicemia/metabolismo , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/efeitos dos fármacos , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Ratos Sprague-Dawley , Ratos Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidoresRESUMO
Neural stem cells (NSCs) exist in germinal centers of the adult brain and in the carotid body (CB), an oxygen-sensing organ that grows under chronic hypoxemia. How stem cell lineage differentiation into mature glomus cells is coupled with changes in physiological demand is poorly understood. Here, we show that hypoxia does not affect CB NSC proliferation directly. Rather, mature glomus cells expressing endothelin-1, the O2-sensing elements in the CB that secrete neurotransmitters in response to hypoxia, establish abundant synaptic-like contacts with stem cells, which express endothelin receptors, and instruct their growth. Inhibition of glomus cell transmitter release or their selective destruction markedly diminishes CB cell growth during hypoxia, showing that CB NSCs are under the direct "synaptic" control of the mature O2-sensitive cells. Thus, glomus cells not only acutely activate the respiratory center but also induce NSC-dependent CB hypertrophy necessary for acclimatization to chronic hypoxemia.
Assuntos
Corpo Carotídeo/metabolismo , Células-Tronco Neurais/metabolismo , Oxigênio/metabolismo , Centro Respiratório/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Camundongos , Camundongos Transgênicos , Prolil Hidroxilases/metabolismo , Ratos , Ratos WistarRESUMO
N-methyl-d-aspartate receptors (NMDARs) are located in neuronal cell membranes at synaptic and extrasynaptic locations, where they are believed to mediate distinct physiological and pathological processes. Activation of NMDARs requires glutamate and a coagonist whose nature and impact on NMDAR physiology remain elusive. We report that synaptic and extrasynaptic NMDARs are gated by different endogenous coagonists, d-serine and glycine, respectively. The regionalized availability of the coagonists matches the preferential affinity of synaptic NMDARs for d-serine and extrasynaptic NMDARs for glycine. Furthermore, glycine and d-serine inhibit NMDAR surface trafficking in a subunit-dependent manner, which is likely to influence NMDARs subcellular location. Taking advantage of this coagonist segregation, we demonstrate that long-term potentiation and NMDA-induced neurotoxicity rely on synaptic NMDARs only. Conversely, long-term depression requires both synaptic and extrasynaptic receptors. Our observations provide key insights into the operating mode of NMDARs, emphasizing functional distinctions between synaptic and extrasynaptic NMDARs in brain physiology.
Assuntos
Glicina/metabolismo , Plasticidade Neuronal , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/agonistas , Serina/metabolismo , Sinapses , Animais , Membrana Celular , Células Cultivadas , Hipocampo/citologia , Hipocampo/metabolismo , Potenciação de Longa Duração , Depressão Sináptica de Longo Prazo , Neuroglia/metabolismo , Neurônios/citologia , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Excitatory synapses of principal hippocampal neurons are frequently located on dendritic spines. The dynamic strengthening or weakening of individual inputs results in structural and molecular diversity of dendritic spines. Active spines with large calcium ion (Ca2+ ) transients are frequently invaded by a single protrusion from the endoplasmic reticulum (ER), which is dynamically transported into spines via the actin-based motor myosin V. An increase in synaptic strength correlates with stable anchoring of the ER, followed by the formation of an organelle referred to as the spine apparatus. Here, we show that myosin V binds the Ca2+ sensor caldendrin, a brain-specific homolog of the well-known myosin V interactor calmodulin. While calmodulin is an essential activator of myosin V motor function, we found that caldendrin acts as an inhibitor of processive myosin V movement. In mouse and rat hippocampal neurons, caldendrin regulates spine apparatus localization to a subset of dendritic spines through a myosin V-dependent pathway. We propose that caldendrin transforms myosin into a stationary F-actin tether that enables the localization of ER tubules and formation of the spine apparatus in dendritic spines.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Espinhas Dendríticas/metabolismo , Retículo Endoplasmático/metabolismo , Miosina Tipo V/metabolismo , Actinas/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Calmodulina/metabolismo , Retículo Endoplasmático Liso/metabolismo , Células HEK293 , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Espectrometria de Massas , Camundongos Knockout , Miosina Tipo V/genética , Domínios e Motivos de Interação entre Proteínas , Ratos WistarRESUMO
All metazoans depend on the consumption of O2 by the mitochondrial oxidative phosphorylation system (OXPHOS) to produce energy. In addition, the OXPHOS uses O2 to produce reactive oxygen species that can drive cell adaptations1-4, a phenomenon that occurs in hypoxia4-8 and whose precise mechanism remains unknown. Ca2+ is the best known ion that acts as a second messenger9, yet the role ascribed to Na+ is to serve as a mere mediator of membrane potential10. Here we show that Na+ acts as a second messenger that regulates OXPHOS function and the production of reactive oxygen species by modulating the fluidity of the inner mitochondrial membrane. A conformational shift in mitochondrial complex I during acute hypoxia11 drives acidification of the matrix and the release of free Ca2+ from calcium phosphate (CaP) precipitates. The concomitant activation of the mitochondrial Na+/Ca2+ exchanger promotes the import of Na+ into the matrix. Na+ interacts with phospholipids, reducing inner mitochondrial membrane fluidity and the mobility of free ubiquinone between complex II and complex III, but not inside supercomplexes. As a consequence, superoxide is produced at complex III. The inhibition of Na+ import through the Na+/Ca2+ exchanger is sufficient to block this pathway, preventing adaptation to hypoxia. These results reveal that Na+ controls OXPHOS function and redox signalling through an unexpected interaction with phospholipids, with profound consequences for cellular metabolism.
Assuntos
Transporte de Elétrons , Hipóxia/metabolismo , Mitocôndrias/metabolismo , Sistemas do Segundo Mensageiro , Sódio/metabolismo , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Fosfatos de Cálcio/metabolismo , Linhagem Celular Tumoral , Precipitação Química , Humanos , Masculino , Fluidez de Membrana , Camundongos Endogâmicos C57BL , Membranas Mitocondriais/química , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Fosforilação Oxidativa , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Trocador de Sódio e Cálcio/metabolismoRESUMO
Cholesterol is a major constituent of myelin membranes, which insulate axons and allow saltatory conduction. Therefore, Schwann cells, the myelinating glia of the peripheral nervous system, need to produce large amounts of cholesterol. Here, we define a crucial role of the transcription factor Maf in myelination and cholesterol biosynthesis and show that Maf acts downstream from Neuregulin1 (Nrg1). Maf expression is induced when Schwann cells begin myelination. Genetic ablation of Maf resulted in hypomyelination that resembled mice with defective Nrg1 signaling. Importantly, loss of Maf or Nrg1 signaling resulted in a down-regulation of the cholesterol synthesis program, and Maf directly binds to enhancers of cholesterol synthesis genes. Furthermore, we identified the molecular mechanisms by which Nrg1 signaling regulates Maf levels. Transcription of Maf depends on calmodulin-dependent kinases downstream from Nrg1, whereas Nrg1-MAPK signaling stabilizes Maf protein. Our results delineate a novel signaling cascade regulating cholesterol synthesis in myelinating Schwann cells.
Assuntos
Colesterol/biossíntese , Bainha de Mielina/metabolismo , Neuregulina-1/metabolismo , Proteínas Proto-Oncogênicas c-maf/metabolismo , Células de Schwann/metabolismo , Transdução de Sinais , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Colesterol/genética , Regulação da Expressão Gênica , Histona Desacetilases/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-maf/genética , Ratos , Ratos WistarRESUMO
Many neurons including vasopressin (VP) magnocellular neurosecretory cells (MNCs) of the hypothalamic supraoptic nucleus (SON) generate afterhyperpolarizations (AHPs) during spiking to slow firing, a phenomenon known as spike frequency adaptation. The AHP is underlain by Ca2+-activated K+ currents, and while slow component (sAHP) features are well described, its mechanism remains poorly understood. Previous work demonstrated that Ca2+ influx through N-type Ca2+ channels is a primary source of sAHP activation in SON oxytocin neurons, but no obvious channel coupling was described for VP neurons. Given this, we tested the possibility of an intracellular source of sAHP activation, namely, the Ca2+-handling organelles endoplasmic reticulum (ER) and mitochondria in male and female Wistar rats. We demonstrate that ER Ca2+ depletion greatly inhibits sAHPs without a corresponding decrease in Ca2+ signal. Caffeine sensitized AHP activation by Ca2+ In contrast to ER, disabling mitochondria with CCCP or blocking mitochondria Ca2+ uniporters (MCUs) enhanced sAHP amplitude and duration, implicating mitochondria as a vital buffer for sAHP-activating Ca2+ Block of mitochondria Na+-dependent Ca2+ release via triphenylphosphonium (TPP+) failed to affect sAHPs, indicating that mitochondria Ca2+ does not contribute to sAHP activation. Together, our results suggests that ER Ca2+-induced Ca2+ release activates sAHPs and mitochondria shape the spatiotemporal trajectory of the sAHP via Ca2+ buffering in VP neurons. Overall, this implicates organelle Ca2+, and specifically ER-mitochondria-associated membrane contacts, as an important site of Ca2+ microdomain activity that regulates sAHP signaling pathways. Thus, this site plays a major role in influencing VP firing activity and systemic hormonal release.
Assuntos
Cálcio , Retículo Endoplasmático , Mitocôndrias , Neurônios , Ratos Wistar , Núcleo Supraóptico , Vasopressinas , Animais , Ratos , Vasopressinas/metabolismo , Masculino , Feminino , Neurônios/metabolismo , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Cálcio/metabolismo , Núcleo Supraóptico/metabolismo , Potenciais de Ação/fisiologia , Potenciais de Ação/efeitos dos fármacos , Sinalização do Cálcio/fisiologiaRESUMO
Oxytocinergic transmission blocks nociception at the peripheral, spinal, and supraspinal levels through the oxytocin receptor (OTR). Indeed, a neuronal pathway from the hypothalamic paraventricular nucleus (PVN) to the spinal cord and trigeminal nucleus caudalis (Sp5c) has been described. Hence, although the trigeminocervical complex (TCC), an anatomical area spanning the Sp5c, C1, and C2 regions, plays a role in some pain disorders associated with craniofacial structures (e.g., migraine), the role of oxytocinergic transmission in modulating nociception at this level has been poorly explored. Hence, in vivo electrophysiological recordings of TCC wide dynamic range (WDR) cells sensitive to stimulation of the periorbital or meningeal region were performed in male Wistar rats. PVN electrical stimulation diminished the neuronal firing evoked by periorbital or meningeal electrical stimulation; this inhibition was reversed by OTR antagonists administered locally. Accordingly, neuronal projections (using Fluoro-Ruby) from the PVN to the WDR cells filled with Neurobiotin were observed. Moreover, colocalization between OTR and calcitonin gene-related peptide (CGRP) or OTR and GABA was found near Neurobiotin-filled WDR cells. Retrograde neuronal tracers deposited at the meningeal (True-Blue, TB) and infraorbital nerves (Fluoro-Gold, FG) showed that at the trigeminal ganglion (TG), some cells were immunopositive to both fluorophores, suggesting that some TG cells send projections via the V1 and V2 trigeminal branches. Together, these data may imply that endogenous oxytocinergic transmission inhibits the nociceptive activity of second-order neurons via OTR activation in CGRPergic (primary afferent fibers) and GABAergic cells.
Assuntos
Estimulação Elétrica , Ocitocina , Núcleo Hipotalâmico Paraventricular , Ratos Wistar , Receptores de Ocitocina , Transmissão Sináptica , Animais , Masculino , Núcleo Hipotalâmico Paraventricular/fisiologia , Núcleo Hipotalâmico Paraventricular/metabolismo , Ocitocina/metabolismo , Ocitocina/análogos & derivados , Ratos , Receptores de Ocitocina/metabolismo , Receptores de Ocitocina/antagonistas & inibidores , Transmissão Sináptica/fisiologia , Nociceptores/fisiologia , Nociceptores/metabolismo , Nociceptividade/fisiologia , Potenciais de Ação/fisiologia , Potenciais de Ação/efeitos dos fármacos , Meninges/fisiologia , Inibição Neural/fisiologiaRESUMO
We recently established a method for the isolation of serum-free oligosaccharides, and characterized various features of their structures. However, the precise mechanism for how these glycans are formed still remains unclarified. To further investigate the mechanism responsible for these serum glycans, here, we utilized rat primary hepatocytes to examine whether they are able to secrete free glycans. Our findings indicated that a diverse array of free oligosaccharides such as sialyl/neutral free N-glycans (FNGs), as well as sialyl lactose/LacNAc-type glycans, were secreted into the culture medium by primary hepatocytes. The structural features of these free glycans in the medium were similar to those isolated from the sera of the same rat. Further evidence suggested that an oligosaccharyltransferase is involved in the release of the serum-free N-glycans. Our results indicate that the liver is indeed secreting various types of free glycans directly into the serum.
Assuntos
Hepatócitos , Oligossacarídeos , Animais , Ratos , Hepatócitos/metabolismo , Oligossacarídeos/sangue , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Células Hep G2 , Humanos , Masculino , Ratos WistarRESUMO
G-protein-gated inward rectifier K+ (GIRK) channels play a critical role in the regulation of the excitability of cardiomyocytes and neurons and include GIRK1, GIRK2, GIRK3 and GIRK4 subfamily members. BD1047 dihydrobromide (BD1047) is one of the representative antagonists of the multifunctional Sigma-1 receptor (S1R). In the analysis of the effect of BD1047 on the regulation of Gi-coupled receptors by S1R using GIRK channel as an effector, we observed that BD1047, as well as BD1063, directly inhibited GIRK currents even in the absence of S1R and in a voltage-independent manner. Thus, we aimed to clarify the effect of BD1047 on GIRK channels and identify the structural determinants. By electrophysiological recordings in Xenopus oocytes, we observed that BD1047 directly inhibited GIRK channel currents, producing a much stronger inhibition of GIRK4 compared to GIRK2. It also inhibited ACh-induced native GIRK current in isolated rat atrial myocytes. Chimeric and mutagenesis studies of GIRK2 and GIRK4 combined with molecular docking analysis demonstrated the importance of Leu77 and Leu84 within the cytoplasmic, proximal N-terminal region and Glu147 within the pore-forming region of GIRK4 for inhibition by BD1047. The activator of GIRK channels, ivermectin, competed with BD1047 at Leu77 on GIRK4. This study provides us with a novel inhibitor of GIRK channels and information for developing pharmacological treatments for GIRK4-associated diseases.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Receptores sigma , Receptor Sigma-1 , Animais , Ratos , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/química , Simulação de Acoplamento Molecular , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Oócitos/metabolismo , Receptores sigma/metabolismo , Receptores sigma/antagonistas & inibidores , Receptores sigma/genética , Receptores sigma/química , Xenopus laevis , Ratos WistarRESUMO
Acetaminophen (ACE) is a widely used analgesic and antipyretic drug with various applications, from pain relief to fever reduction. Recent studies have reported equivocal effects of habitual ACE intake on exercise performance, muscle growth, and risks to bone health. Thus, this study aimed to assess the impact of a 6-week, low-dose ACE regimen on muscle and bone adaptations in exercising and non-exercising rats. Nine-week-old Wistar rats (n = 40) were randomized to an exercise or control (no exercise) condition with ACE or without (placebo). For the exercise condition, rats ran 5 days per week for 6 weeks at a 5% incline for 2 min at 15 cm/s, 2 min at 20 cm/s, and 26 min at 25 cm/s. A human equivalent dose of ACE was administered (379 mg/kg body weight) in drinking water and adjusted each week based on body weight. Food, water intake, and body weight were measured daily. At the beginning of week 6, animals in the exercise group completed a maximal treadmill test. At the end of week 6, rats were euthanized, and muscle cross-sectional area (CSA), fiber type, and signaling pathways were measured. Additionally, three-point bending and microcomputer tomography were measured in the femur. Follow-up experiments in human primary muscle cells were used to explore supra-physiological effects of ACE. Data were analyzed using a two-way ANOVA for treatment (ACE or placebo) and condition (exercise or non-exercise) for all animal outcomes. Data for cell culture experiments were analyzed via ANOVA. If omnibus significance was found in either ANOVA, a post hoc analysis was completed, and a Tukey's adjustment was used. ACE did not alter body weight, water intake, food intake, or treadmill performance (p > .05). There was a treatment-by-condition effect for Young's Modulus where placebo exercise was significantly lower than placebo control (p < .05). There was no treatment by condition effects for microCT measures, muscle CSA, fiber type, or mRNA expression. Phosphorylated-AMPK was significantly increased with exercise (p < .05) and this was attenuated with ACE treatment. Furthermore, phospho-4EBP1 was depressed in the exercise group compared to the control (p < .05) and increased in the ACE control and ACE exercise group compared to placebo exercise (p < .05). A low dose of ACE did not influence chronic musculoskeletal adaptations in exercising rodents but acutely attenuated AMPK phosphorylation and 4EBP1 dephosphorylation post-exercise.
Assuntos
Acetaminofen , Condicionamento Físico Animal , Animais , Humanos , Ratos , Acetaminofen/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Peso Corporal , Carboidratos , Músculo Esquelético/metabolismo , Condicionamento Físico Animal/fisiologia , Ratos WistarRESUMO
Cisplatin (Cis) is among the most powerful antineoplastic medications, nevertheless, its serious side effects; particularly nephrotoxicity designates a major concern. Previous studies reported that ezetimibe (Eze), a well-known antihyperlipidemic drug, exerts additional trivial pharmacological effects. In this work, we displayed Eze as an intriguing protective candidate in a cisplatin-induced nephrotoxicity rat model through AMPK activation. Eze (10 mg/kg, p.o.) was administered for two weeks and Cis (10 mg/kg, i.p.) was administered on the 10th day to induce nephrotoxicity in male Wistar rats. Treatment with Eze greatly augmented the phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK) and the antioxidant regulator; nuclear factor erythroid 2-related factor 2 (Nrf2), thus, mitigating oxidative injury through induction of the antioxidant enzymes, such as heme oxygenase-1 (HO-1) and glutathione reductase (GR). As well, Eze relieved inflammation by reducing protein expression of thioredoxin-interacting protein (TXNIP) and nucleotide-binding domain-like receptor protein 3 (NLRP3), which led to a decrease in the release of caspase-1, in addition to, the inflammatory markers IL-18 and IL-1 ß. Besides, Eze ameliorated apoptosis in the renal cells through inhibiting the phosphorylated Apoptosis signal-regulating kinase-1(p-ASK1), caspase-3 and reducing Bax/Bcl2ratio. Correspondingly, histopathological examination corroborated the previous biochemical findings. Collectively, Eze exerts significant renal protection against Cis-induced nephrotoxicity via antioxidant, anti-inflammatory and anti-apoptotic pathways that are probably mediated, at least partly, via activating AMPK/Nrf2/HO-1 pathway and conquering both TXNIP/NLRP3 inflammasome and TXNIP/ASK1 signaling pathways. To confirm the protective effect of Eze via AMPK-activation, an AMPK-inhibitor, dorsomorphin (Dors), when co-administered with Eze abolished its protective effect.
Assuntos
Cisplatino , Proteína 3 que Contém Domínio de Pirina da Família NLR , Ratos , Masculino , Animais , Cisplatino/toxicidade , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Antioxidantes/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Ezetimiba/farmacologia , Ratos Wistar , Estresse Oxidativo , Proteínas de Ciclo Celular/metabolismoRESUMO
Mesenchymal stem cells (MSCs) reveal multifaceted immunoregulatory properties, which can be applied for diverse refractory and recurrent disease treatment including acute graft-versus-host disease (aGVHD). Distinguishing from MSCs with considerable challenges before clinical application, MSCs-derived exosomes (MSC-Exos) are cell-free microvesicles with therapeutic ingredients and serve as advantageous alternatives for ameliorating the outcomes of aGVHD. MSC-Exos were enriched and identified by western blotting analysis, NanoSight, and transmission electron microscopy (TEM). Bone marrow-derived MSCs (denoted as MSCs) and exosomes (denoted as MSC-Exos) were infused into the aGVHD SD-Wister rat model via tail vein, and variations in general growth and survival of rats were observed. The level of inflammatory factors in serum was quantized by enzyme-linked immunosorbent assay (ELISA). The pathological conditions of the liver and intestine of rats were observed by frozen sectioning. The ratios of CD4+/CD8+ and Treg cell proportions in peripheral blood, together with the autophagy in the spleen and thymus, were analyzed by flow cytometry. After treatment with MSC-Exos, the survival time of aGVHD rats was prolonged, the clinical manifestations of aGVHD in rats were improved, whereas the pathological damage of aGVHD in the liver and intestine was reduced. According to ELISA, we found that MSC-Exos revealed ameliorative effect upon aGVHD inflammation (e.g., TNF-α, IL-2, INF-γ, IL-4, and TGF-ß) compared to the MSC group. After MSC-Exo treatment, the ratio of Treg cells in peripheral blood was increased, whereas the ratio of CD4+/CD8+ in peripheral blood and the autophagy in the spleen and thymus was decreased. MSC-Exos effectively suppressed the activation of immune cells and the manifestation of the inflammatory response in the aGVHD rat model. Our data would supply new references for MSC-Exo-based "cell-free" biotherapy for aGVHD in future.