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Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus that is linked directly to the development of Kaposi's sarcoma. KSHV establishes a latent infection in B cells, which can be reactivated to initiate lytic replication, producing infectious virions. Using pharmacological and genetic silencing approaches, we showed that the voltage-gated K+ channel Kv1.3 in B cells enhanced KSHV lytic replication. The KSHV replication and transcription activator (RTA) protein increased the abundance of Kv1.3 and led to enhanced K+ channel activity and hyperpolarization of the B cell membrane. Enhanced Kv1.3 activity promoted intracellular Ca2+ influx, leading to the Ca2+-driven nuclear localization of KSHV RTA and host nuclear factor of activated T cells (NFAT) proteins and subsequently increased the expression of NFAT1 target genes. KSHV lytic replication and infectious virion production were inhibited by Kv1.3 blockers or silencing. These findings highlight Kv1.3 as a druggable host factor that is key to the successful completion of KSHV lytic replication.
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Herpesvirus Humano 8 , Canal de Potasio Kv1.3 , Factores de Transcripción NFATC , Replicación Viral , Herpesvirus Humano 8/fisiología , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Humanos , Canal de Potasio Kv1.3/metabolismo , Canal de Potasio Kv1.3/genética , Canal de Potasio Kv1.3/antagonistas & inhibidores , Factores de Transcripción NFATC/metabolismo , Factores de Transcripción NFATC/genética , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Inmediatas-Precoces/genética , Transactivadores/metabolismo , Transactivadores/genética , Linfocitos B/virología , Linfocitos B/metabolismo , Calcio/metabolismo , Sarcoma de Kaposi/virología , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/genéticaRESUMEN
Increasing the selectivity of chemotherapies by converting them into prodrugs that can be activated at the tumour site decreases their side effects and allows discrimination between cancerous and non-cancerous cells. Herein, the use of metabolic glycoengineering (MGE) to selectively label MCF-7 breast cancer cells with tetrazine (Tz) activators for subsequent activation of prodrugs containing the trans-cyclooctene (TCO) moiety by a bioorthogonal reaction is demonstrated. Three novel Tz-modified monosaccharides, Ac4ManNTz 7, Ac4GalNTz 8, and Ac4SiaTz 16, were used for expression of the Tz activator within sialic-acid rich breast cancer cells' surface glycans through MGE. Tz expression on breast cancer cells (MCF-7) was evaluated versus the non-cancerous L929 fibroblasts showing a concentration-dependant effect and excellent selectivity with ≥35-fold Tz expression on the MCF-7 cells versus the non-cancerous L929 fibroblasts. Next, a novel TCO-N-mustard prodrug and a TCO-doxorubicin prodrug were analyzed in vitro on the Tz-bioengineered cells to probe our hypothesis that these could be activated via a bioorthogonal reaction. Selective prodrug activation and restoration of cytotoxicity were demonstrated for the MCF-7 breast cancer cells versus the non-cancerous L929 cells. Restoration of the parent drug's cytotoxicity was shown to be dependent on the level of Tz expression where the Ac4ManNTz 7 and Ac4GalNTz 8 derivatives (20 µM) lead to the highest Tz expression and full restoration of the parent drug's cytotoxicity. This work suggests the feasibility of combining MGE and tetrazine ligation for selective prodrug activation in breast cancer.
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Antineoplásicos , Neoplasias de la Mama , Profármacos , Profármacos/química , Profármacos/farmacología , Profármacos/síntesis química , Humanos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/síntesis química , Femenino , Estructura Molecular , Ensayos de Selección de Medicamentos Antitumorales , Relación Estructura-Actividad , Células MCF-7 , Relación Dosis-Respuesta a Droga , Proliferación Celular/efectos de los fármacos , Ingeniería Metabólica , Supervivencia Celular/efectos de los fármacosRESUMEN
The motility of microglia involves intracellular signaling pathways that are predominantly controlled by changes in cytosolic Ca2+ and activation of PI3K/Akt (phosphoinositide-3-kinase/protein kinase B). In this letter, we develop a novel biophysical model for cytosolic Ca2+ activation of the PI3K/Akt pathway in microglia where Ca2+ influx is mediated by both P2Y purinergic receptors (P2YR) and P2X purinergic receptors (P2XR). The model parameters are estimated by employing optimization techniques to fit the model to phosphorylated Akt (pAkt) experimental modeling/in vitro data. The integrated model supports the hypothesis that Ca2+ influx via P2YR and P2XR can explain the experimentally reported biphasic transient responses in measuring pAkt levels. Our predictions reveal new quantitative insights into P2Rs on how they regulate Ca2+ and Akt in terms of physiological interactions and transient responses. It is shown that the upregulation of P2X receptors through a repetitive application of agonist results in a continual increase in the baseline [Ca2+], which causes the biphasic response to become a monophasic response which prolongs elevated levels of pAkt.
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Microglía , Proteínas Proto-Oncogénicas c-akt , Microglía/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Receptores Purinérgicos/metabolismoRESUMEN
Obesity is driven by an imbalance between caloric intake and energy expenditure, causing excessive storage of triglycerides in adipose tissue at different sites around the body. Increased visceral adipose tissue (VAT) is associated with diabetes, while pericardial adipose tissue (PAT) is associated with cardiac pathology. Adipose tissue can expand either through cellular hypertrophy or hyperplasia, with the former correlating with decreased metabolic health in obesity. The aim of this study was to determine how VAT and PAT remodel in response to obesity, stress, and exercise. Here we have used the male obese Zucker rats, which carries two recessive fa alleles that result in the development of hyperphagia with reduced energy expenditure, resulting in morbid obesity and leptin resistance. At 9 weeks of age, a group of lean (Fa/Fa or Fa/fa) Zucker rats (LZR) and obese (fa/fa) Zucker rats (OZR) were treated with unpredictable chronic mild stress or exercise for 8 weeks. To determine the phenotype for PAT and VAT, tissue cellularity and gene expression were analyzed. Finally, leptin signaling was investigated further using cultured 3T3-derived adipocytes. Tissue cellularity was determined following hematoxylin and eosin (H&E) staining, while qPCR was used to examine gene expression. PAT adipocytes were significantly smaller than those from VAT and had a more beige-like appearance in both LZR and OZR. In the OZR group, VAT adipocyte cell size increased significantly compared with LZR, while PAT showed no difference. Exercise and stress resulted in a significant reduction in VAT cellularity in OZR, while PAT showed no change. This suggests that PAT cellularity does not remodel significantly compared with VAT. These data indicate that the extracellular matrix of PAT is able to remodel more readily than in VAT. In the LZR group, exercise increased insulin receptor substrate 1 (IRS1) in PAT but was decreased in the OZR group. In VAT, exercise decreased IRS1 in LZR, while increasing it in OZR. This suggests that in obesity, VAT is more responsive to exercise and subsequently becomes less insulin resistant compared with PAT. Stress increased PPAR-γ expression in the VAT but decreased it in the PAT in the OZR group. This suggests that in obesity, stress increases adipogenesis more significantly in the VAT compared with PAT. To understand the role of leptin signaling in adipose tissue remodeling mechanistically, JAK2 autophosphorylation was inhibited using 5 µM 1,2,3,4,5,6-hexabromocyclohexane (Hex) in cultured 3T3-derived adipocytes. Palmitate treatment was used to induce cellular hypertrophy. Hex blocked adipocyte hypertrophy in response to palmitate treatment but not the increase in lipid droplet size. These data suggest that leptin signaling is necessary for adipocyte cell remodeling, and its absence induces whitening. Taken together, our data suggest that leptin signaling is necessary for adipocyte remodeling in response to obesity, exercise, and psychosocial stress.
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Tejido Adiposo , Leptina , Masculino , Ratas , Animales , Ratas Zucker , Pericardio , Palmitatos , Estrés Psicológico , Hipertrofia , ObesidadRESUMEN
INTRODUCTION: Automated Patch Clamp (APC) technology has become an integral element in ion channel research, drug discovery and development pipelines to overcome the use of the highly time-consuming manual patch clamp (MPC) procedures. This automated technology offers increased throughput and promises a new model in obtaining ion channel recordings, which has significant relevance to the development of novel therapies and safety profiling of candidate therapeutic compounds. AREAS COVERED: This article reviews the recent innovations in APC technology, including platforms, and highlights how they have facilitated usage in both industry and academia. The review also provides an overview of the ion channel research endeavors and how APC platforms have contributed to the understanding of ion channel research, pharmacological tools and therapeutics. Furthermore, the authors provide their opinion on the challenges and goals for APC technology going forward to accelerate academic research and drug discovery across a host of therapeutic areas. EXPERT OPINION: It is clear that APC technology has progressed drug discovery programs, specifically in the field of neuroscience and cardiovascular research. The challenge for the future is to keep pace with fundamental research and improve translation of the large datasets obtained.
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Ensayos Analíticos de Alto Rendimiento , Canales Iónicos , Humanos , Ensayos Analíticos de Alto Rendimiento/métodos , Descubrimiento de Drogas/métodos , Tecnología , Técnicas de Placa-ClampRESUMEN
The voltage-gated K+ channel plays a key role in atrial excitability, conducting the ultra-rapid rectifier K+ current (IKur) and contributing to the repolarization of the atrial action potential. In this study, we examine its regulation by hydrogen sulfide (H2S) in HL-1 cardiomyocytes and in HEK293 cells expressing human Kv1.5. Pacing induced remodeling resulted in shorting action potential duration, enhanced both Kv1.5 channel and H2S producing enzymes protein expression in HL-1 cardiomyocytes. H2S supplementation reduced these remodeling changes and restored action potential duration through inhibition of Kv1.5 channel. H2S also inhibited recombinant hKv1.5, lead to nitric oxide (NO) mediated S-nitrosylation and activated endothelial nitric oxide synthase (eNOS) by increased phosphorylation of Ser1177, prevention of NO formation precluded these effects. Regulation of Ikur by H2S has important cardiovascular implications and represents a novel and potential therapeutic target.
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Fibrilación Atrial , Sulfuro de Hidrógeno , Canales de Potasio con Entrada de Voltaje , Humanos , Sulfuro de Hidrógeno/farmacología , Sulfuro de Hidrógeno/metabolismo , Fibrilación Atrial/metabolismo , Células HEK293 , Canal de Potasio Kv1.5/genética , Canal de Potasio Kv1.5/metabolismo , Miocitos Cardíacos/metabolismoRESUMEN
The conformation and self-assembly of four lipopeptides, peptide amphiphiles comprising peptides conjugated to lipid chains, in aqueous solution have been examined. The peptide sequence in all four lipopeptides contains the integrin cell adhesion RGDS motif, and the cytocompatibility of the lipopeptides is also analyzed. Lipopeptides have either tetradecyl (C14, myristyl) or hexadecyl (C16, palmitoyl) lipid chains and peptide sequence WGGRGDS or GGGRGDS, that is, with either a tryptophan-containing WGG or triglycine GGG tripeptide spacer between the bioactive peptide motif and the alkyl chain. All four lipopeptides self-assemble above a critical aggregation concentration (CAC), determined through several comparative methods using circular dichroism (CD) and fluorescence. Spectroscopic methods [CD and Fourier transform infrared (FTIR) spectroscopy] show the presence of ß-sheet structures, consistent with the extended nanotape, helical ribbon, and nanotube structures observed by cryogenic transmission electron microscopy (cryo-TEM). The high-quality cryo-TEM images clearly show the coexistence of helically twisted ribbon and nanotube structures for C14-WGGRGDS, which highlight the mechanism of nanotube formation by the closure of the ribbons. Small-angle X-ray scattering shows that the nanotapes comprise highly interdigitated peptide bilayers, which are also present in the walls of the nanotubes. Hydrogel formation was observed at sufficiently high concentrations or could be induced by a heat/cool protocol at lower concentrations. Birefringence due to nematic phase formation was observed for several of the lipopeptides, along with spontaneous flow alignment of the lyotropic liquid crystal structure in capillaries. Cell viability assays were performed using both L929 fibroblasts and C2C12 myoblasts to examine the potential uses of the lipopeptides in tissue engineering, with a specific focus on application to cultured (lab-grown) meat, based on myoblast cytocompatibility. Indeed, significantly higher cytocompatibility of myoblasts was observed for all four lipopeptides compared to that for fibroblasts, in particular at a lipopeptide concentration below the CAC. Cytocompatibility could also be improved using hydrogels as cell supports for fibroblasts or myoblasts. Our work highlights that precision control of peptide sequences using bulky aromatic residues within "linker sequences" along with alkyl chain selection can be used to tune the self-assembled nanostructure. In addition, the RGDS-based lipopeptides show promise as materials for tissue engineering, especially those of muscle precursor cells.
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Lipopéptidos , Nanoestructuras , Lipopéptidos/farmacología , Lipopéptidos/química , Adhesión Celular , Secuencia de Aminoácidos , Mioblastos , Dicroismo CircularRESUMEN
Cav2.2 channels are key regulators of presynaptic Ca2+ influx and their dysfunction and/or aberrant regulation has been implicated in many disease states; however, the nature of their involvement in Alzheimer's disease (AD) is less clear. In this short communication, we show that recombinant hCav2.2/b1b/a2d1 channels are modulated by human synthetic AD-related protofibrillar amyloid beta Ab1-42 peptides. Structural studies revealed a time-dependent increase in protofibril length, with the majority of protofibrils less than 100 nm at 24 h, while at 48 h, the majority were longer than 100 nm. Cav2.2 modulation by Ab1-42 was different between a 'low' (100 nM) and 'high' (1 µM) concentration in terms of distinct effects on individual biophysical parameters. A concentration of 100 nM Ab1-42 caused no significant changes in the measured biophysical properties of Cav2.2 currents. In contrast, 1 µM Ab1-42 caused an inhibitory decrease in the current density (pA/pF) and maximum conductance (Gmax), and a depolarizing shift in the slope factor (k). These data highlight a differential modulation of Cav2.2 channels by the Ab1-42 peptide. Discrete changes in the presynaptic Ca2+ flux have been reported to occur at an early stage of AD; therefore, this study reveals a potential mechanistic link between amyloid accumulation and Cav2.2 channel modulation.
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The hypoxic ventilatory response (HVR) is critical to breathing and thus oxygen supply to the body and is primarily mediated by the carotid bodies. Here we reveal that carotid body afferent discharge during hypoxia and hypercapnia is determined by the expression of Liver Kinase B1 (LKB1), the principal kinase that activates the AMP-activated protein kinase (AMPK) during metabolic stresses. Conversely, conditional deletion in catecholaminergic cells of AMPK had no effect on carotid body responses to hypoxia or hypercapnia. By contrast, the HVR was attenuated by LKB1 and AMPK deletion. However, in LKB1 knockouts hypoxia evoked hypoventilation, apnoea and Cheyne-Stokes-like breathing, while only hypoventilation and apnoea were observed after AMPK deletion. We therefore identify LKB1 as an essential regulator of carotid body chemosensing and uncover a divergence in dependency on LKB1 and AMPK between the carotid body on one hand and the HVR on the other.
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Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Quinasas Activadas por AMP , Cuerpo Carotídeo , Hipoxia , Quinasas de la Proteína-Quinasa Activada por el AMP/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Apnea , Cuerpo Carotídeo/metabolismo , Humanos , Hipercapnia/metabolismo , Hipoventilación/metabolismo , Hipoxia/metabolismoRESUMEN
Hydrogen sulfide (H2S) is gaining interest as a mammalian signalling molecule with wide ranging effects. S-sulfhydration is one mechanism that is emerging as a key post translational modification through which H2S acts. Ion channels and neuronal receptors are key target proteins for S-sulfhydration and this can influence a range of neuronal functions. Voltage-gated K+ channels, including Kv2.1, are fundamental components of neuronal excitability. Here, we show that both recombinant and native rat Kv2.1 channels are inhibited by the H2S donors, NaHS and GYY4137. Biochemical investigations revealed that NaHS treatment leads to S-sulfhydration of the full length wild type Kv2.1 protein which was absent (as was functional regulation by H2S) in the C73A mutant form of the channel. Functional experiments utilising primary rat hippocampal neurons indicated that NaHS augments action potential firing and thereby increases neuronal excitability. These studies highlight an important role for H2S in shaping cellular excitability through S-sulfhydration of Kv2.1 at C73 within the central nervous system.
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Hipocampo/citología , Sulfuro de Hidrógeno/farmacología , Canales de Potasio Shab/genética , Canales de Potasio Shab/metabolismo , Potenciales de Acción , Animales , Células Cultivadas , Regulación hacia Abajo , Células HEK293 , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Morfolinas/farmacología , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Compuestos Organotiofosforados/farmacología , Fosforilación , Cultivo Primario de Células , RatasRESUMEN
Since its development on the cusp of the new millennium, automated patch clamp (APC) technology has matured over the last two decades. The increased throughput it afforded promised a new paradigm in ion channel recordings: It offered the potential to overcome the time-consuming, low-throughput bottleneck arising from manual patch clamp (MPC) investigations. This chapter highlights the advances in technology, showing how APC platforms have 'democratised' ion channel recordings, lowering the technical bar whilst substantially raising throughput. It will describe the background of the seminal first-generation and updates on advances in second-generation platforms. Furthermore, the chapter summarises the advances APC has made in ion channel studies, including finding new tool compounds and medicines. New functionality and applications on APC platforms give ion channel researchers flexible tools to study ion channels with high quality and high throughput.
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Fenómenos Electrofisiológicos , Canales Iónicos , Electrofisiología , Técnicas de Placa-ClampRESUMEN
Microglia, the major endogenous immune cells of the central nervous system, mediate critical degenerative and regenerative responses in ischaemic stroke. Microglia become "activated", proliferating, and undergoing changes in morphology, gene and protein expression over days and weeks post-ischaemia, with deleterious and beneficial effects. Pro-inflammatory microglia (commonly referred to as M1) exacerbate secondary neuronal injury through the release of reactive oxygen species, cytokines and proteases. In contrast, microglia may facilitate neuronal recovery via tissue and vascular remodelling, through the secretion of anti-inflammatory cytokines and growth factors (a profile often termed M2). This M1/M2 nomenclature does not fully account for the microglial heterogeneity in the ischaemic brain, with some simultaneous expression of both M1 and M2 markers at the single-cell level. Understanding and regulating microglial activation status, reducing detrimental and promoting repair behaviours, present the potential for therapeutic intervention, and open a longer window of opportunity than offered by acute neuroprotective strategies. Pharmacological modulation of microglial activation status to promote anti-inflammatory gene expression can increase neurogenesis and improve functional recovery post-stroke, based on promising preclinical data. Cell-based therapies, using preconditioned microglia, are of interest as a method of therapeutic modulation of the post-ischaemic inflammatory response. Currently, there are no clinically-approved pharmacological options targeting post-ischaemic inflammation. A major developmental challenge for clinical translation will be the selective suppression of the deleterious effects of microglial activity after stroke whilst retaining (or enhancing) the neurovascular repair and remodelling responses of microglia.
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The self-assembly of amyloid peptides (Aß), in particular Aß1-42, into oligomers and fibrils is one of the main pathological events related to Alzheimer's disease. Recent studies have demonstrated the ability of carbon monoxide-releasing molecules (CORMs) to protect neurons and astrocytes from Aß1-42 toxicity. In fact, CORMs are able to carry and release controlled levels of CO and are known to exert a wide range of anti-inflammatory and anti-apoptotic activities at physiologically relevant concentrations. In order to investigate the direct effects of CORMs on Aß1-42, we studied the reactivity of CORM-2 and CORM-3 with Aß1-42 in vitro and the potential inhibition of its aggregation by mass spectrometry (MS), as well as fluorescence and circular dichroism spectroscopies. The application of an electrospray ionization-MS (ESI-MS) method allowed the detection of stable Aß1-42/CORMs adducts, involving the addition of the Ru(CO)2 portion of CORMs at histidine residues on the Aß1-42 skeleton. Moreover, CORMs showed anti-aggregating properties through formation of stable adducts with Aß1-42 as demonstrated by a thioflavin T fluorescence assay and MS analysis. As further proof, comparison of the CD spectra of Aß1-42 recorded in the absence and in the presence of CORM-3 at a 1:1 molar ratio showed the ability of CORM-3 to stabilize the peptide in its soluble, unordered conformation, thereby preventing its misfolding and aggregation. This multi-methodological investigation revealed novel interactions between Aß1-42 and CORMs, contributing new insights into the proposed neuroprotective mechanisms mediated by CORMs and disclosing a new strategy to divert amyloid aggregation and toxicity.
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There can be a fine line between therapeutic intervention and substance abuse, and this point is clearly exemplified in herbal cannabis and its products. Therapies involving cannabis have been the treatment of last resort for some cases of refractory epilepsy, and this has been among the strongest medical justifications for legalization of marijuana. In order to circumvent the narcotic effects of Δ9-tetrahydrocannabinol (THC), many studies have concentrated on its less intoxicating isomer cannabidiol (CBD). However, CBD, like all natural cannabinoids, is a controlled substance in most countries, and its conversion into THC can be easily performed using common chemicals. We describe here the anticonvulsant properties of 8,9-dihydrocannibidiol (H2CBD), a fully synthetic analogue of CBD that is prepared from inexpensive, non-cannabis derived precursors. H2CBD was found to have effectiveness comparable to CBD both for decreasing the number and reducing the severity of pentylenetetrazole-induced seizures in rats. Finally, H2CBD cannot be converted by any reasonable synthetic route into THC, and thus has the potential to act as a safe, noncontroversial drug for seizure mitigation.
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Anticonvulsivantes/uso terapéutico , Cannabidiol/uso terapéutico , Cannabinoides/uso terapéutico , Convulsiones/tratamiento farmacológico , Animales , Anticonvulsivantes/farmacología , Cannabidiol/farmacología , Cannabinoides/farmacología , Modelos Animales de Enfermedad , Masculino , Pentilenotetrazol , Ratas , Ratas Wistar , Convulsiones/inducido químicamente , Resultado del TratamientoRESUMEN
Tuberous sclerosis complex (TSC) is a rare disease caused by mutations in the TSC1 or TSC2 genes and is characterized by widespread tumour growth, intractable epilepsy, cognitive deficits and autistic behaviour. CBD has been reported to decrease seizures and inhibit tumour cell progression, therefore we sought to determine the influence of CBD on TSC pathology in zebrafish carrying a nonsense mutation in the tsc2 gene. CBD treatment from 6 to 7 days post-fertilization (dpf) induced significant anxiolytic actions without causing sedation. Furthermore, CBD treatment from 3 dpf had no impact on tsc2-/- larvae motility nor their survival. CBD treatment did, however, reduce the number of phosphorylated rpS6 positive cells, and their cross-sectional cell size. This suggests a CBD mediated suppression of mechanistic target of rapamycin (mTOR) activity in the tsc2-/- larval brain. Taken together, these data suggest that CBD selectively modulates levels of phosphorylated rpS6 in the brain and additionally provides an anxiolytic effect. This is pertinent given the alterations in mTOR signalling in experimental models of TSC. Additional work is necessary to identify upstream signal modulation and to further justify the use of CBD as a possible therapeutic strategy to manage TSC.
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Cannabidiol/farmacología , Proteína S6 Ribosómica/efectos de los fármacos , Esclerosis Tuberosa/tratamiento farmacológico , Animales , Encéfalo/metabolismo , Cannabidiol/metabolismo , Cannabinoides/metabolismo , Cannabinoides/farmacología , Modelos Animales de Enfermedad , Péptidos y Proteínas de Señalización Intracelular/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/genética , Locomoción/efectos de los fármacos , Fosforilación/efectos de los fármacos , Convulsiones/patología , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/efectos de los fármacos , Esclerosis Tuberosa/fisiopatología , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa/metabolismo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/efectos de los fármacos , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Alzheimer's disease is the most common form of dementia, it is estimated to affect over 40 million people worldwide. Classically, the disease has been characterized by the neuropathological hallmarks of aggregated extracellular amyloid-ß and intracellular paired helical filaments of hyperphosphorylated tau. A wealth of evidence indicates a pivotal role for the innate immune system, such as microglia, and inflammation in the pathology of Alzheimer's disease. The over production and aggregation of Alzheimer's associated proteins results in chronic inflammation and disrupts microglial clearance of these depositions. Despite being non-excitable, microglia express a diverse array of ion channels which shape their physiological functions. In support of this, there is a growing body of evidence pointing to the involvement of microglial ion channels contributing to neurodegenerative diseases such as Alzheimer's disease. In this review, we discuss the evidence for an array of microglia ion channels and their importance in modulating microglial homeostasis and how this process could be disrupted in Alzheimer's disease. One promising avenue for assessing the role that microglia play in the initiation and progression of Alzheimer's disease is through using induced pluripotent stem cell derived microglia. Here, we examine what is already understood in terms of the molecular underpinnings of inflammation in Alzheimer's disease, and the utility that inducible pluripotent stem cell derived microglia may have to advance this knowledge. We outline the variability that occurs between the use of animal and human models with regards to the importance of microglial ion channels in generating a relevant functional model of brain inflammation. Overcoming these hurdles will be pivotal in order to develop new drug targets and progress our understanding of the pathological mechanisms involved in Alzheimer's disease.
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Automated patch clamp (APC) technology was first developed at the turn of the millennium. The increased throughput it afforded promised a new paradigm in ion channel recordings, offering the potential to overcome the time-consuming, low-throughput bottleneck, arising from manual patch clamp investigations. This has relevance to the fast-paced development of novel therapies for chronic pain. This review highlights the advances in technology, using select examples that have facilitated APC usage in both industry and academia. It covers both first generation and the latest developments in second-generation platforms. In addition, it also provides an overview of the pain research field and how APC platforms have furthered our understanding of ion channel research and the development of pharmacological tools and therapeutics. APC platforms have much to offer to the ion channel research community, and this review highlights areas of best practice for both academia and industry. The impact of APC platforms and the prospects of ion channel research and improved therapeutics for chronic pain will be evaluated. LINKED ARTICLES: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.
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Automatización , Investigación Biomédica , Fenómenos Electrofisiológicos , Canales Iónicos/metabolismo , Dolor/metabolismo , Técnicas de Placa-Clamp , Animales , Humanos , Manejo del DolorRESUMEN
The voltage-gated K+ channel has key roles in the vasculature and in atrial excitability and contributes to apoptosis in various tissues. In this study, we have explored its regulation by carbon monoxide (CO), a product of the cytoprotective heme oxygenase enzymes, and a recognized toxin. CO inhibited recombinant Kv1.5 expressed in HEK293 cells in a concentration-dependent manner that involved multiple signalling pathways. CO inhibition was partially reversed by superoxide dismutase mimetics and by suppression of mitochondrial reactive oxygen species. CO also elevated intracellular nitric oxide (NO) levels. Prevention of NO formation also partially reversed CO inhibition of Kv1.5, as did inhibition of soluble guanylyl cyclase. CO also elevated intracellular peroxynitrite levels, and a peroxynitrite scavenger markedly attenuated the ability of CO to inhibit Kv1.5. CO caused nitrosylation of Kv1.5, an effect that was also observed in C331A and C346A mutant forms of the channel, which had previously been suggested as nitrosylation sites within Kv1.5. Augmentation of Kv1.5 via exposure to hydrogen peroxide was fully reversed by CO. Native Kv1.5 recorded in HL-1 murine atrial cells was also inhibited by CO. Action potentials recorded in HL-1 cells were increased in amplitude and duration by CO, an effect mimicked and occluded by pharmacological inhibition of Kv1.5. Our data indicate that Kv1.5 is a target for modulation by CO via multiple mechanisms. This regulation has important implications for diverse cellular functions, including excitability, contractility and apoptosis.