RESUMEN
Historically, the rat has been the preferred animal model for behavioral studies. Limitations in genome modification have, however, caused a lag in their use compared to the bevy of available transgenic mice. Here, we have developed several transgenic tools, including viral vectors and transgenic rats, for targeted genome modification in specific adult rat neurons using CRISPR-Cas9 technology. Starting from wild-type rats, knockout of tyrosine hydroxylase was achieved with adeno-associated viral (AAV) vectors expressing Cas9 or guide RNAs (gRNAs). We subsequently created an AAV vector for Cre-dependent gRNA expression as well as three new transgenic rat lines to specifically target CRISPR-Cas9 components to dopaminergic neurons. One rat represents the first knockin rat model made by germline gene targeting in spermatogonial stem cells. The rats described herein serve as a versatile platform for making cell-specific and sequence-specific genome modifications in the adult brain and potentially other Cre-expressing tissues of the rat.
Asunto(s)
Células Madre Germinales Adultas/metabolismo , Encéfalo/metabolismo , Sistemas CRISPR-Cas , Neuronas Dopaminérgicas/metabolismo , Edición Génica/métodos , Marcación de Gen/métodos , Animales , Proteína 9 Asociada a CRISPR/genética , Desoxirribonucleasa I/genética , Dependovirus , Modelos Animales de Enfermedad , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Técnicas de Sustitución del Gen/métodos , Técnicas de Inactivación de Genes , Vectores Genéticos , Integrasas , Proteínas Luminiscentes/genética , Neuronas/metabolismo , Regiones Promotoras Genéticas , ARN Guía de Kinetoplastida , Ratas , Ratas Transgénicas , Tirosina 3-Monooxigenasa/genética , Proteína Fluorescente RojaRESUMEN
Because of unavoidable confounding variables in the direct study of human subjects, it has been difficult to unravel the effects of prenatal cocaine exposure on the human fetal brain, as well as the cellular and biochemical mechanisms involved. Here, we propose a novel approach using a human pluripotent stem cell (hPSC)-based 3D neocortical organoid model. This model retains essential features of human neocortical development by encompassing a single self-organized neocortical structure, without including an animal-derived gelatinous matrix. We reported previously that prenatal cocaine exposure to rats during the most active period of neural progenitor proliferation induces cytoarchitectural changes in the embryonic neocortex. We also identified a role of CYP450 and consequent oxidative ER stress signaling in these effects. However, because of differences between humans and rodents in neocorticogenesis and brain CYP metabolism, translation of the research findings from the rodent model to human brain development is uncertain. Using hPSC 3D neocortical organoids, we demonstrate that the effects of cocaine are mediated through CYP3A5-induced generation of reactive oxygen species, inhibition of neocortical progenitor cell proliferation, induction of premature neuronal differentiation, and interruption of neural tissue development. Furthermore, knockdown of CYP3A5 reversed these cocaine-induced pathological phenotypes, suggesting CYP3A5 as a therapeutic target to mitigate the deleterious neurodevelopmental effects of prenatal cocaine exposure in humans. Moreover, 3D organoid methodology provides an innovative platform for identifying adverse effects of abused psychostimulants and pharmaceutical agents, and can be adapted for use in neurodevelopmental disorders with genetic etiologies.
Asunto(s)
Cocaína/farmacología , Citocromo P-450 CYP3A/metabolismo , Inhibidores de Captación de Dopamina/farmacología , Neocórtex/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Células Madre Pluripotentes/efectos de los fármacos , Línea Celular , HumanosRESUMEN
There has been a marked increase in the availability of synthetic drugs designed to mimic the effects of marijuana. These cannabimimetic drugs, sold illicitly as 'Spice' and related products, are associated with serious medical complications in some users. In vitro studies suggest that synthetic cannabinoids in these preparations are potent agonists at central cannabinoid CB1 receptors (CB1Rs), but few investigations have delineated their cellular effects, particularly in comparison with the psychoactive component of marijuana, Δ9 -tetrahydrocannabinol (Δ9 -THC). We compared the ability of three widely abused synthetic cannabinoids and Δ9 -THC to alter glutamate release and long-term potentiation in the mouse hippocampus. JWH-018 was the most potent inhibitor of hippocampal synaptic transmission (EC50 ~15 nM), whereas its fluoropentyl derivative, AM2201, inhibited synaptic transmission with slightly lower potency (EC50 ~60 nM). The newer synthetic cannabinoid, XLR-11, displayed much lower potency (EC50 ~900 nM) that was similar to Δ9 -THC (EC50 ~700 nM). The effects of all compounds occurred via activation of CB1Rs, as demonstrated by reversal with the selective antagonist/inverse agonist AM251 or the neutral CB1R antagonist PIMSR1. Moreover, AM2201 was without effect in the hippocampus of transgenic mice lacking the CB1R. Hippocampal slices exposed to either synthetic cannabinoids or Δ9 -THC exhibited significantly impaired long-term potentiation (LTP). We find that, compared with Δ9 -THC, the first-generation cannabinoids found in Spice preparations display higher potency, whereas a recent synthetic cannabinoid is roughly equipotent with Δ9 -THC. The disruption of synaptic function by these synthetic cannabinoids is likely to lead to profound impairments in cognitive and behavioral function.
Asunto(s)
Agonistas de Receptores de Cannabinoides/farmacología , Cannabinoides/farmacología , Dronabinol/farmacología , Hipocampo/efectos de los fármacos , Indoles/farmacología , Potenciación a Largo Plazo/efectos de los fármacos , Naftalenos/farmacología , Transmisión Sináptica/efectos de los fármacos , Animales , Ácido Glutámico/efectos de los fármacos , Ácido Glutámico/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor Cannabinoide CB1/genéticaRESUMEN
Fast-scan cyclic voltammetry (FSCV) using carbon fiber electrodes is widely used to rapidly monitor changes in dopamine (DA) levels in vitro and in vivo. Current analytical approaches utilize parameters such as peak oxidation current amplitude and decay times to estimate release and uptake processes, respectively. However, peak amplitude changes are often observed with uptake inhibitors, thereby confounding the interpretation of these parameters. To overcome this limitation, we demonstrate that a simple five-parameter, two-compartment model mathematically describes DA signals as a balance of release (r/ke) and uptake (ku), summed with adsorption (kads and kdes) of DA to the carbon electrode surface. Using nonlinear regression, we demonstrate that our model precisely describes measured DA signals obtained in brain slice recordings. The parameters extracted from these curves were then validated using pharmacological manipulations that selectively alter vesicular release or DA transporter (DAT)-mediated uptake. Manipulation of DA release through altering the Ca(2+)/Mg(2+) ratio or adding tetrodotoxin reduced the release parameter with no effect on the uptake parameter. DAT inhibitors methylenedioxypyrovalerone, cocaine, and nomifensine significantly reduced uptake and increased vesicular DA release. In contrast, a low concentration of amphetamine reduced uptake but had no effect on DA release. Finally, the kappa opioid receptor agonist U50,488 significantly reduced vesicular DA release but had no effect on uptake. Together, these data demonstrate a novel analytical approach to distinguish the effects of manipulations on DA release or uptake that can be used to interpret FSCV data.
Asunto(s)
Cocaína/farmacología , Cuerpo Estriado/efectos de los fármacos , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/efectos de los fármacos , Inhibidores de Captación de Dopamina/farmacología , Dopamina/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Cuerpo Estriado/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Estimulación Eléctrica/métodos , Técnicas Electroquímicas , Masculino , Nomifensina/farmacología , Ratas Sprague-DawleyRESUMEN
Neocortical development involves ordered specification of forebrain cortical progenitors to various neuronal subtypes, ultimately forming the layered cortical structure. Modeling of this process using human pluripotent stem cells (hPSCs) would enable mechanistic studies of human neocortical development, while providing new avenues for exploration of developmental neocortical abnormalities. Here, we show that preserving hPSCs aggregates - allowing embryoid body formation - while adding basic fibroblast growth factor (bFGF) during neuroepithelial development generates neural rosettes showing dorsal forebrain identity, including Mash1(+) dorsal telencephalic GABAergic progenitors. Structures that mirrored the organization of the cerebral cortex formed after rosettes were seeded and cultured for 3 weeks in the presence of FGF18, BDNF and NT3. Neurons migrated along radial glia scaffolding, with deep-layer CTIP2(+) cortical neurons appearing after 1 week and upper-layer SATB2(+) cortical neurons forming during the second and third weeks. At the end of differentiation, these structures contained both glutamatergic and GABAergic neurons, with glutamatergic neurons being most abundant. Thus, this differentiation protocol generated an hPSC-based model that exhibits temporal patterning and a neuronal subtype ratio similar to that of the developing human neocortex. This model was used to examine the effects of cocaine during neocorticogenesis. Cocaine caused premature neuronal differentiation and enhanced neurogenesis of various cortical neuronal subtypes. These cocaine-induced changes were inhibited by the cytochrome P450 inhibitor cimetidine. This in vitro model enables mechanistic studies of neocorticogenesis, and can be used to examine the mechanisms through which cocaine alters the development of the human neocortex.
Asunto(s)
Corteza Cerebral/embriología , Cocaína/química , Células Madre Pluripotentes/citología , Diferenciación Celular , Línea Celular , Inhibidores de Captación de Dopamina/química , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Humanos , Inmunohistoquímica , Neurogénesis , Neuronas/metabolismo , Prosencéfalo/embriología , Especies Reactivas de Oxígeno , Células Madre/metabolismoRESUMEN
The endocannabinoid system has been demonstrated to be active in the pancreatic ß-cell. However the effects of the endocannabinoids (ECs) on insulin secretion are not well defined and may vary depending on the metabolic state of the ß-cell. Specifically it is not known whether the effects of the ECs occur by activation of the cannabinoid receptors or via their direct interaction with the ion channels of the ß-cell. To begin to delineate the effects of ECs on ß-cell function, we examined how the EC, 2-AG influences ß-cell ion channels in the absence of glucose stimulation. The mouse insulinoma cell line R7T1 was used to survey the effects of 2-AG on the high voltage activated (HVA) calcium, the delayed rectifier (K(v)), and the ATP-sensitive K (K(ATP)) channels by whole cell patch clamp recording. At 2mM glucose, 2-AG inhibited the HVA calcium (the majority of which are L-type channels), K(v), and K(ATP) channels. The channel exhibiting the most sensitivity to 2-AG blockade was the K(ATP) channel, where the IC(50) for 2-AG was 1 µM. Pharmacological agents revealed that the blockade of all these channels was independent of cannabinoid receptors. Our results provide a mechanism for the previous observations that CB1R agonists increase insulin secretion at low glucose concentrations through CB1R independent blockade of the K(ATP) channel.
Asunto(s)
Ácidos Araquidónicos/farmacología , Moduladores de Receptores de Cannabinoides/farmacología , Endocannabinoides , Glicéridos/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Insulina/metabolismo , Canales KATP/antagonistas & inhibidores , Animales , Calcio/metabolismo , Línea Celular Tumoral , Glucosa/farmacología , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Ratones , Técnicas de Placa-Clamp , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/metabolismoRESUMEN
BACKGROUND: Stromal-Derived Inducing Activity (SDIA) is one of the most efficient methods of generating dopaminergic (DA) neurons from embryonic stem cells (ESC). DA neuron induction can be achieved by co-culturing ESC with the mouse stromal cell lines PA6 or MS5. The molecular nature of this effect, which has been termed "SDIA" is so far unknown. Recently, we found that factors secreted by PA6 cells provided lineage-specific instructions to induce DA differentiation of human ESC (hESC). METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we compared PA6 cells to various cell lines lacking the SDIA effect, and employed genome expression analysis to identify differentially-expressed signaling molecules. Among the factors highly expressed by PA6 cells, and known to be associated with CNS development, were stromal cell-derived factor 1 (SDF-1/CXCL12), pleiotrophin (PTN), insulin-like growth factor 2 (IGF2), and ephrin B1 (EFNB1). When these four factors, the combination of which was termed SPIE, were applied to hESC, they induced differentiation to TH-positive neurons in vitro. RT-PCR and western blot analysis confirmed the expression of midbrain specific markers, including engrailed 1, Nurr1, Pitx3, and dopamine transporter (DAT) in cultures influenced by these four molecules. Electrophysiological recordings showed that treatment of hESC with SPIE induced differentiation of neurons that were capable of generating action potentials and forming functional synaptic connections. CONCLUSIONS/SIGNIFICANCE: The combination of SDF-1, PTN, IGF2, and EFNB1 mimics the DA phenotype-inducing property of SDIA and was sufficient to promote differentiation of hESC to functional midbrain DA neurons. These findings provide a method for differentiating hESC to form DA neurons, without a requirement for the use of animal-derived cell lines or products.
Asunto(s)
Proteínas Portadoras/fisiología , Diferenciación Celular/fisiología , Quimiocina CXCL12/fisiología , Citocinas/fisiología , Dopamina/fisiología , Células Madre Embrionarias/citología , Efrina-B1/fisiología , Factor II del Crecimiento Similar a la Insulina/fisiología , Neuronas/citología , Animales , Western Blotting , Técnicas de Cocultivo , Células Madre Embrionarias/metabolismo , Humanos , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND AND PURPOSE: Human embryonic stem cells (hESC) are considered a renewable source of dopamine producing neurons, and are of particular interest for their potential clinical use in Parkinson's disease. In this study, we characterized human dopaminergic neurons generated by stromal-derived inducing activity (SDIA) from BG01V2, a strain of human embryonic stem cell line, BG01, characterized by a chromosome 17 trisomy. Similar chromosomal changes have been repeatedly observed in hESC cultures in different laboratories, indicating the importance of chromosome 17 for growth and adaptation of hESC to culture. METHODS: We investigated in vitro proliferation of differentiating cells using a BrDU incorporation assay, and monitored the cell population in long term cultures. Despite the cytogenetic abnormality, TH+ neurons were postmitotic at all stages of differentiation. After 30 days of differentiation, cell division ceased in 91% of the overall population of cells in the culture, indicating intact cell cycle regulation. RESULTS: Expression of midbrain specific marker genes (Otx2, Pax5, Msx-1) showed differentiation of hESC-derived neural progenitor cells into midbrain specific dopamine neurons. These neurons expressed the dopamine transporter (DAT), and displayed functional DAT activity and electrical excitability. CONCLUSIONS: TH+ cells derived from the BG01V2 hESC line using SDIA are postmitotic and have functional characteristics of normal dopaminergic neurons.
Asunto(s)
Diferenciación Celular/fisiología , Dopamina/metabolismo , Células Madre Embrionarias/fisiología , Neuronas/fisiología , Actinas/metabolismo , Bromodesoxiuridina/metabolismo , Línea Celular , Proliferación Celular , Cromosomas Humanos Par 17 , Cocaína/análogos & derivados , Cocaína/farmacocinética , Técnicas de Cocultivo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Humanos , Potenciales de la Membrana/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Factores de Transcripción Otx/metabolismo , Técnicas de Placa-Clamp/métodos , Unión Proteica/efectos de los fármacos , Factores de Tiempo , Tritio/farmacocinética , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Several isomers of 7-methyl-2-exo-([(18)F]fluoropyridinyl-5'-pyridinyl)-7-azabicyclo[2.2.1]heptane have been developed as radioligands with optimized brain kinetics for PET imaging of nAChR. The binding assay demonstrated that all isomers are beta-nAChR selective ligands with Ki = 0.02-0.3 nM. The experimental lipophilicity values of all isomers were in the optimal range for the cerebral radioligands (log D7.4= 0.67-0.99). The isomers with higher binding affinity manifested slow baboon brain kinetics, whereas the isomer with the lowest binding affinity (Ki = 0.3 nM) ((-)-7-methyl-2- exo-[3'-(6-[(18)F]fluoropyridin-2-yl)-5'-pyridinyl]-7-azabicyclo[2.2.1]heptane, [(18)F](-)-6c) and greatest lipophilicity (log D 7.4 = 0.99) exhibited optimal brain kinetics. [(18)F](-)-6c manifests a unique combination of the optimally rapid brain kinetics, high BP and brain uptake, and favorable metabolic profile. Pharmacological studies showed that (-)-6c is an alpha4beta2-nAChR antagonist with low side effects in mice. This combination of imaging properties suggests that [(18)F]-(-)- 6c is a potentially superior replacement for 2-[(18)F]fluoro-A-85380 and 6-[(18)F]fluoro-A-85380, the only available nAChR PET radioligands for humans.
Asunto(s)
2,2'-Dipiridil/análogos & derivados , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Heptanos/síntesis química , Heptanos/farmacología , Antagonistas Nicotínicos/síntesis química , Antagonistas Nicotínicos/farmacología , Receptores Nicotínicos/metabolismo , Tropanos/síntesis química , Tropanos/farmacología , 2,2'-Dipiridil/síntesis química , 2,2'-Dipiridil/química , 2,2'-Dipiridil/farmacología , Animales , Línea Celular , Heptanos/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Lípidos/química , Masculino , Ratones , Estructura Molecular , Antagonistas Nicotínicos/química , Papio , Tomografía de Emisión de Positrones , Ensayo de Unión Radioligante , Ratas , Receptores Nicotínicos/genética , Estereoisomerismo , Relación Estructura-Actividad , Factores de Tiempo , Tropanos/químicaRESUMEN
BACKGROUND: We initiated differentiation of human embryonic stem cells (hESCs) into dopamine neurons, obtained a purified population of neuronal precursor cells by cell sorting, and determined patterns of gene transcription. METHODOLOGY: Dopaminergic differentiation of hESCs was initiated by culturing hESCs with a feeder layer of PA6 cells. Differentiating cells were then sorted to obtain a pure population of PSA-NCAM-expressing neuronal precursors, which were then analyzed for gene expression using Massive Parallel Signature Sequencing (MPSS). Individual genes as well as regions of the genome which were activated were determined. PRINCIPAL FINDINGS: A number of genes known to be involved in the specification of dopaminergic neurons, including MSX1, CDKN1C, Pitx1 and Pitx2, as well as several novel genes not previously associated with dopaminergic differentiation, were expressed. Notably, we found that a specific region of the genome located on chromosome 11p15.5 was highly activated. This region contains several genes which have previously been associated with the function of dopaminergic neurons, including the gene for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, IGF2, and CDKN1C, which cooperates with Nurr1 in directing the differentiation of dopaminergic neurons. Other genes in this region not previously recognized as being involved in the functions of dopaminergic neurons were also activated, including H19, TSSC4, and HBG2. IGF2 and CDKN1C were also found to be highly expressed in mature human TH-positive dopamine neurons isolated from human brain samples by laser capture. CONCLUSIONS: The present data suggest that the H19-IGF2 imprinting region on chromosome 11p15.5 is involved in the process through which undifferentiated cells are specified to become neuronal precursors and/or dopaminergic neurons.
Asunto(s)
Cromosomas Humanos Par 11 , Dopamina/metabolismo , Células Madre Embrionarias/citología , Perfilación de la Expresión Génica , Neuronas/citología , Diferenciación Celular/genética , Línea Celular , Separación Celular , Citometría de Flujo , Humanos , Inmunohistoquímica , Neuronas/metabolismo , Reacción en Cadena de la PolimerasaRESUMEN
The effects of the endocannabinoid anandamide (arachidonylethanolamide, AEA) on the function of alpha4beta2 nicotinic acetylcholine receptors (nAChR) stably expressed in SH-EP1 cells were investigated using the whole-cell patch-clamp technique. In the concentration range of 200 nM to 2 microM, AEA significantly reduced the maximal amplitudes and increased the desensitization of acetylcholine (ACh)-induced currents. The effects of AEA could be neither replicated by the exogenous cannabinoid Delta(9)-tetrahydrocannabinol (1 microM) nor reversed by the selective CB1 receptor antagonist 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide (SR-141716A) (1 microM). The actions of AEA were apparent when applied extracellularly but not during intracellular dialysis. Furthermore, the effects of AEA ACh currents were not altered by the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. The onset and washout of the AEA effects required several minutes (10-30 min), but the latter was significantly decreased in the presence of lipid-free bovine serum albumin (BSA). Moreover, BSA alone increased peak ACh current amplitudes and diminished desensitization rates in naive cells, suggesting a tonic modulation of alpha4beta2 nAChR function by an endogenous AEA-like lipid. Further analysis of AEA effects on alpha4beta2 nAChR-mediated currents, using a two-stage desensitization model, indicated that the first forward rate constant leading to desensitization, k(1), increased nearly 30-fold as a linear function of the AEA concentration. In contrast, the observation that the other three rate constants were unaltered by AEA suggested that AEA raised the energy of the activated state. These results indicate that AEA directly inhibits the function of alpha4beta2 nAChRs in a CB1 receptor-independent manner.
Asunto(s)
Ácidos Araquidónicos/farmacología , Alcamidas Poliinsaturadas/farmacología , Receptores Nicotínicos/efectos de los fármacos , Acetilcolina/farmacología , Línea Celular , Endocannabinoides , Humanos , Cinética , Técnicas de Placa-Clamp , Piperidinas/farmacología , Pirazoles/farmacología , Receptores Nicotínicos/fisiología , Rimonabant , Albúmina Sérica Bovina/metabolismoRESUMEN
Cells grown in monolayer culture offer a convenient system for binding and other experiments under conditions that preserve the complexity of the living state. Kinetics experiments, however, may be distorted by the time course of drug penetration into even so simple a "tissue" as the monolayer. The impediments include unstirred layers both above and between the cells, the congregation of receptors within the confined space between cells, and nonspecific binding to membrane components. The contributions of these factors were investigated in cultures of Chinese hamster ovary (CHO) cells either nontransfected or stably transfected with mu opioid receptors. The dissociation of [3H]naloxone was four times faster under displacement than under infinite dilution conditions, clearly demonstrating the "retention effect" of receptors confined in space. Even the penetration of this ligand between nontransfected cells showed salient delays with respect to diffusion into a slab, indicating that nonspecific, low-affinity binding to membrane components was arresting its progress. The optical sectioning capabilities of confocal microscopy demonstrated that the kinetics of two fluorescent antagonists depended on the vertical plane, providing direct evidence for slowed diffusion down a single cell depth. Modeling shows that kinetic errors increase with receptor density, forward rate constant, and the thickness of the unstirred layer.
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Simulación por Computador , Microscopía Confocal , Modelos Estadísticos , Ensayo de Unión Radioligante , Animales , Colorantes Azulados/metabolismo , Colorantes Azulados/farmacología , Unión Competitiva , Células CHO , Técnicas de Cultivo de Célula , Células Cultivadas , Cricetinae , Difusión , Relación Dosis-Respuesta a Droga , Cinética , Naloxona/metabolismo , Naloxona/farmacología , Antagonistas de Narcóticos/metabolismo , Antagonistas de Narcóticos/farmacología , Octanoles/química , Receptores Opioides mu/análisis , Transfección , Agua/químicaRESUMEN
NTera2, a human embryonal carcinoma (EC) stem cell line, shares many characteristics with human embryonic stem cells (hESCs). To determine whether NTera2 can serve as a useful surrogate for hESCs, we compared global gene expression between undifferentiated NTera2, multiple undifferentiated hESC cell lines, and their differentiated derivatives, and we showed that NTera2 cells share multiple markers with hESCs. Similar to hESCs, NTera2 cells differentiated into TH-positive cells that express dopaminergic markers including AADC, DAT, Nurr1, TrkB, TrkC, and GFRA1 when co-cultured with PA6 cells. Flow cytometry analysis showed that tyrosine hydroxylase (TH) and neural cell adhesion molecule (NCAM) expression increased, whereas SSEA4 expression decreased as cells differentiated. Medium conditioned by PA6 cells stimulated differentiation of NTera2 cells to generate TH-positive cells that expressed dopaminergic markers. Flow cytometry selected polysialylated (PSA-NCAM) cells responded to medium conditioned by PA6 cells by differentiating into TH-positive cells and expressed dopaminergic markers. Sorted cells differentiated for 4 weeks in PA6 cell conditioned media included functional neurons that responded to neurotransmitters and exhibited electronic excitability. Therefore, NTera2 cell dopaminergic neuronal differentiation and PSA-NCAM enrichment provides a useful system for the future study of hESCs.
Asunto(s)
Diferenciación Celular/fisiología , Línea Celular Tumoral , Dopamina/metabolismo , Neuronas/fisiología , Células Madre/fisiología , Animales , Biomarcadores/metabolismo , Forma de la Célula , Técnicas de Cocultivo , Medios de Cultivo Condicionados , Electrofisiología , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Análisis por Micromatrices , Datos de Secuencia Molecular , Moléculas de Adhesión de Célula Nerviosa/química , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuronas/citología , Células Madre/citología , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Because many studies rely upon detergents to solubilize lipophilic agents such as cannabinoid drugs, we examined the effect of commonly employed detergents on the function of the cloned alpha(7) subunit of the nicotinic ACh receptor. Homomeric alpha(7) receptors were expressed in Xenopus oocytes and the two-microelectrode voltage-clamp technique was used to assess their electrophysiological properties. The detergents Tween 80 and Triton X-100 reversibly inhibited ACh (100 microM)-induced inward currents in a concentration-dependent manner, with IC(50) values of 610 nM and 1.4 microM, respectively. The effects of these detergents were independent of membrane potential, and they were not mediated by endogenous Ca(2+)-dependent Cl(-) channels, since they were unaffected by intracellularly injected BAPTA, and recorded in Ca(2+)-free bathing solution containing 2 mM Ba(2+). Both detergents also decreased the maximal effect of ACh, without significantly affecting its EC(50), indicating a non-competitive interaction with the nACh alpha(7) receptors. In contrast to the effects of these detergents, we found that cholic acid (10 microM), DMSO (10 microM) and Tocrisol (0.01% v/v) did not cause a significant effect on nicotinic responses. In conclusion, we demonstrate that the detergents Tween 80 and Triton X-100 are potent inhibitors of neuronal nACh alpha(7) receptors expressed in Xenopus oocytes, and we suggest that studies utilizing these detergents to solubilize lipophilic drugs should be scrutinized for such effects.
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Detergentes/farmacología , Ácido Egtácico/análogos & derivados , Inhibición Neural/efectos de los fármacos , Octoxinol/farmacología , Oocitos/efectos de los fármacos , Polisorbatos/farmacología , Receptores Nicotínicos/fisiología , Acetilcolina/farmacología , Animales , Atropina/farmacología , Bario/farmacología , Quelantes/farmacología , Pollos , Clonación Molecular , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Ácido Egtácico/farmacología , Femenino , Potenciales de la Membrana/efectos de los fármacos , Antagonistas Muscarínicos/farmacología , Técnicas de Placa-Clamp/métodos , ARN Complementario/química , ARN Complementario/metabolismo , Receptores Nicotínicos/efectos de los fármacos , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
The two-stage reaction whereby the antagonist beta-funaltrexamine (beta-FNA) binds covalently to micro opioid receptors makes it a highly discriminating probe into the tertiary structure of the receptor's recognition pocket. To obtain a quantitative measure of how well this pocket is preserved in a mutated form of the receptor, in which His-297 is substituted with glutamine, we employed [3H]-beta-FNA to evaluate the kinetic rate constants for both the reversible as well as the irreversible stages of its binding to wild-type and mutant H297Q micro receptors stably expressed in Chinese hamster ovary cells. The expression levels of the wild-type and mutant H297Q receptors were matched by exploiting the variation in receptor density as a function of plating day and by raising the expression level by pretreatment with naloxone. We found that all of the kinetic rate constants for [3H]-beta-FNA were diminished by about one-half at the mutant H297Q micro receptors with respect to wild-type receptors. By comparison, the association rate constant of [3H]-naloxone likewise decreased by one-half; however, the dissociation rate constant increased 5-fold at the mutant H297Q receptor. We conclude that the mutation has had only minor influence on the recognition site and that the function of position 297 is more likely as a link in the transduction chain.
Asunto(s)
Mutación , Naltrexona/análogos & derivados , Naltrexona/farmacocinética , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Animales , Células CHO , Cricetinae , Regulación de la Expresión Génica/fisiología , Unión Proteica/fisiologíaRESUMEN
The antagonist beta-funaltrexamine (beta-FNA), known to bind covalently to mu-opioid receptors by a two-step, doubly discriminating sequence, was used as a sensitive gauge to compare wildtype to mutant H297Q mu-opioid receptors. We addressed whether this mutation, which enhances the intrinsic activities of alkaloid mu-receptor agents, affects both the reversible and covalent phases of beta-FNA binding. Such altered binding serves as a reporter for the dimensions and topography of the receptor's recognition site. Using the voltage-clamped Xenopus oocyte expression system with coexpressed GIRK potassium channels, we found that beta-FNA blocked the wildtype and mutant H297Q receptors both reversibly and irreversibly, indicating overall conserved tertiary structure in the mutant. The mutant H297Q receptor, however, was more resistant to both phases of blockade, indicating some disturbance of the mutant H297Q receptor recognition site. beta-FNA acted as a partial agonist at the mutant H297Q receptor expressed in both oocytes, as measured by the activation of GIRK channels, and in COS-7 cells assayed by GTPgamma(35)S binding. beta-FNA showed no activity at the wildtype receptor expressed in oocytes, but surprisingly induced binding of GTPgamma(35)S in transfected COS-7 cells. Thus, the topography of the mutant H297Q receptor recognition site is sufficiently conserved to allow the selective binding of beta-FNA, but the decrease in binding affinity and increase in efficacy in oocytes demonstrates clear differences from the wildtype receptor.
Asunto(s)
Naltrexona/análogos & derivados , Naltrexona/farmacología , Antagonistas de Narcóticos/farmacología , Canales de Potasio de Rectificación Interna , Receptores Opioides mu/metabolismo , Animales , Células COS , Relación Dosis-Respuesta a Droga , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Derivados de la Morfina/farmacología , Mutación , Oocitos , Técnicas de Placa-Clamp , Canales de Potasio/metabolismo , Receptores Opioides mu/antagonistas & inhibidores , Receptores Opioides mu/genética , Transfección , XenopusRESUMEN
Wild-type or mutant H297N or H297Q of the mu-opioid receptor were co-expressed with the inwardly rectifying potassium channel GIRK1 in oocytes from Xenopus laevis. Under voltage clamp, pairs of concentration response curves were generated using the agonist normorphine in a bathing medium containing 38.5 mM sodium or an identical medium in which the sodium was replaced by an equimolar concentration of choline. The maximum currents were greater in the presence of sodium by about 30% at wild-type receptors and by about 100% at the mutant receptors. The EC(50) values tended to increase somewhat as well, though these differences reached statistical significance only for the mutant H297Q. Flame photometry detected no change in the intracellular sodium or potassium concentrations of oocytes, suggesting that the effect of sodium was solely extracellular. Thus sodium, long known for its effects on in vitro ligand binding at mu-opioid receptors, also affects overall transduction as revealed in the Xenopus oocyte model of a complete, living cell system.
Asunto(s)
Canales de Potasio de Rectificación Interna , Canales de Potasio/genética , Canales de Potasio/metabolismo , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Sodio/farmacología , Animales , Colina/farmacología , Femenino , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Expresión Génica/fisiología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Mutagénesis/fisiología , Oocitos/fisiología , Técnicas de Placa-Clamp , Potasio/farmacología , Xenopus laevisRESUMEN
5-HT(3) receptors cloned from NCB-20 cells were expressed in Xenopus oocytes, and the effects of forskolin and steroids on the function of the receptors were investigated using the two-electrode voltage-clamp technique. Forskolin, 17-beta-estradiol, and progesterone inhibited the currents activated by 1 microM 5-HT in a reversible and concentration-dependent manner, with IC(50) values of 12, 33, and 89 microM, respectively. The inhibitory effects of forskolin and 17-beta-estradiol were independent of the membrane potential. Forskolin and 17-beta-estradiol significantly reduced the maximal amplitude of the 5-HT concentration-response curve (E(max)) without significantly affecting the EC(50), indicating that these compounds act as noncompetitive inhibitors of the 5-HT(3) receptor. The cAMP analogue, 8-Br-cAMP (0.2 mM), and the protein kinase A activator, Sp-cAMP (0.1 mM), did not affect the amplitude of 5-HT(3) receptor-mediated currents. The membrane-permeable protein kinase A inhibitor Rp-cAMP (0.1 mM) and the estrogen-receptor antagonist tamoxifen (1 microM) did not affect the inhibition of 5-HT-activated current. In addition, 5-HT(3) receptor-mediated currents were inhibited by both 1,9-dideoxy forskolin (30 microM), which does not activate adenylyl cyclase, and wForskolin (30 microM), a charged hydrophilic analogue of forskolin that is membrane impermeable. These results indicate that both forskolin and 17-beta-estradiol inhibit the function of the 5-HT(3) receptor in a noncompetitive manner and that this inhibition is independent of cAMP levels.