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1.
Small ; 18(18): e2200205, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35355419

RESUMEN

Optical interrogation of cellular electrical activity has proven itself essential for understanding cellular function and communication in complex networks. Voltage-sensitive dyes are important tools for assessing excitability but these highly lipophilic sensors may affect cellular function. Label-free techniques offer a major advantage as they eliminate the need for these external probes. In this work, it is shown that endogenous second-harmonic generation (SHG) from live cells is highly sensitive to changes in transmembrane potential (TMP). Simultaneous electrophysiological control of a living human embryonic kidney (HEK293T) cell, through a whole-cell voltage-clamp reveals a linear relation between the SHG intensity and membrane voltage. The results suggest that due to the high ionic strengths and fast optical response of biofluids, membrane hydration is not the main contributor to the observed field sensitivity. A conceptual framework is further provided that indicates that the SHG voltage sensitivity reflects the electric field within the biological asymmetric lipid bilayer owing to a nonzero χeff(2) tensor. Changing the TMP without surface modifications such as electrolyte screening offers high optical sensitivity to membrane voltage (≈40% per 100 mV), indicating the power of SHG for label-free read-out. These results hold promise for the design of a non-invasive label-free read-out tool for electrogenic cells.


Asunto(s)
Microscopía de Generación del Segundo Armónico , Colorantes , Células HEK293 , Humanos , Potenciales de la Membrana
2.
Int J Mol Sci ; 22(11)2021 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-34071169

RESUMEN

Polygodial is a "hot" peppery-tasting sesquiterpenoid that was first described for its anti-feedant activity against African armyworms. Using the haploid deletion mutant library of Saccharomyces cerevisiae, a genome-wide mutant screen was performed to shed more light on polygodial's antifungal mechanism of action. We identified 66 deletion strains that were hypersensitive and 47 that were highly resistant to polygodial treatment. Among the hypersensitive strains, an enrichment was found for genes required for vacuolar acidification, amino acid biosynthesis, nucleosome mobilization, the transcription mediator complex, autophagy and vesicular trafficking, while the resistant strains were enriched for genes encoding cytoskeleton-binding proteins, ribosomal proteins, mitochondrial matrix proteins, components of the heme activator protein (HAP) complex, and known regulators of the target of rapamycin complex 1 (TORC1) signaling. WE confirm that polygodial triggers a dose-dependent vacuolar alkalinization and that it increases Ca2+ influx and inhibits glucose-induced Ca2+ signaling. Moreover, we provide evidence suggesting that TORC1 signaling and its protective agent ubiquitin play a central role in polygodial resistance, suggesting that they can be targeted by polygodial either directly or via altered Ca2+ homeostasis.


Asunto(s)
Antifúngicos/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Antifúngicos/química , Calcio , Farmacorresistencia Fúngica/genética , Homeostasis/efectos de los fármacos , Concentración de Iones de Hidrógeno , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Pruebas de Sensibilidad Microbiana , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Nucleosomas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sesquiterpenos/química , Sesquiterpenos/farmacología , Transducción de Señal , ATPasas de Translocación de Protón Vacuolares
3.
BMC Neurosci ; 16: 41, 2015 Jul 14.
Artículo en Inglés | MEDLINE | ID: mdl-26169500

RESUMEN

BACKGROUND: GABAA receptor-mediated neurotransmission is greatly influenced by cation-chloride cotransporter activity during developmental stages. In embryonic neurons Na-K-2Cl (NKCC1) cotransporters mediate active chloride uptake, thus increasing the intracellular chloride concentration associated with GABA-induced depolarization. At fetal stages near term, oxytocin-induced NKCC1 downregulation has been implicated in the developmental shift from depolarizing to hyperpolarizing GABA action. Mature dorsal root ganglion neurons (DRGN), however, express high NKCC1 levels and maintain high intracellular chloride levels with consequent GABA-induced depolarization. RESULTS: Gramicidin-perforated patch-clamp recordings were used to assess the developmental change in chloride homeostasis in rat cultured small DRGN at the embryonic day 16 (E16) and 19 (E19). The results were compared to data previously obtained in fetal DRGN at E14 and in mature cells. A significant NKCC1 downregulation, leading to reduction in excitatory GABAergic transmission, was observed at E16 and E19. CONCLUSION: These results indicate that NKCC1 activity transiently decreases in DRGN at fetal stages near term. This developmental shift in GABAergic transmission may contribute to fetal analgesia and neuroprotection at birth.


Asunto(s)
Ganglios Espinales/embriología , Ganglios Espinales/fisiología , Potenciales de la Membrana/fisiología , Neuronas/fisiología , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Bumetanida/farmacología , Células Cultivadas , Cloruros/metabolismo , Regulación hacia Abajo , Ganglios Espinales/efectos de los fármacos , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Ratas Wistar , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacología
4.
EMBO J ; 29(19): 3408-20, 2010 Oct 06.
Artículo en Inglés | MEDLINE | ID: mdl-20818335

RESUMEN

The amyloid peptides Aß(40) and Aß(42) of Alzheimer's disease are thought to contribute differentially to the disease process. Although Aß(42) seems more pathogenic than Aß(40), the reason for this is not well understood. We show here that small alterations in the Aß(42):Aß(40) ratio dramatically affect the biophysical and biological properties of the Aß mixtures reflected in their aggregation kinetics, the morphology of the resulting amyloid fibrils and synaptic function tested in vitro and in vivo. A minor increase in the Aß(42):Aß(40) ratio stabilizes toxic oligomeric species with intermediate conformations. The initial toxic impact of these Aß species is synaptic in nature, but this can spread into the cells leading to neuronal cell death. The fact that the relative ratio of Aß peptides is more crucial than the absolute amounts of peptides for the induction of neurotoxic conformations has important implications for anti-amyloid therapy. Our work also suggests the dynamic nature of the equilibrium between toxic and non-toxic intermediates.


Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/toxicidad , Neuronas/metabolismo , Fragmentos de Péptidos/toxicidad , Placa Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/ultraestructura , Análisis de Varianza , Animales , Benzotiazoles , Biofisica , Colorantes Fluorescentes , Humanos , Cinética , Ratones , Microelectrodos , Microscopía Electrónica de Transmisión , Técnicas de Placa-Clamp , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/ultraestructura , Unión Proteica , Espectroscopía Infrarroja por Transformada de Fourier , Tiazoles
5.
J Cell Sci ; 124(Pt 7): 1115-25, 2011 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-21385841

RESUMEN

Loss-of-function mutations in the gene encoding the mitochondrial PTEN-induced putative kinase 1 (PINK1) are a major cause of early-onset familial Parkinson's disease (PD). Recent studies have highlighted an important function for PINK1 in clearing depolarized mitochondria by mitophagy. However, the role of PINK1 in mitochondrial and cellular functioning in physiological conditions is still incompletely understood. Here, we investigate mitochondrial and cellular calcium (Ca(2+)) homeostasis in PINK1-knockdown and PINK1-knockout mouse cells, both in basal metabolic conditions and after physiological stimulation, using unbiased automated live single-cell imaging in combination with organelle-specific fluorescent probes. Our data reveal that depletion of PINK1 induces moderate fragmentation of the mitochondrial network, mitochondrial membrane depolarization and increased production of reactive oxygen species. This results in reduced uptake of Ca(2+) by mitochondria after physiological stimulation. As a consequence, cells with knockdown or knockout of PINK1 display impaired mitochondrial ATP synthesis, which is exacerbated under conditions of increased ATP demand, thereby affecting cytosolic Ca(2+) extrusion. The impairment in energy maintenance was confirmed in the brain of PINK1-knockout mice by in vivo bioluminescence imaging. Our findings demonstrate a key role for PINK1 in the regulation of mitochondrial homeostasis and energy metabolism under physiological conditions.


Asunto(s)
Calcio/metabolismo , Metabolismo Energético , Mitocondrias/metabolismo , Enfermedad de Parkinson/enzimología , Proteínas Quinasas/deficiencia , Adenosina Trifosfato/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Técnicas de Silenciamiento del Gen , Homeostasis , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/enzimología , Mitocondrias/genética , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Proteínas Quinasas/genética , Especies Reactivas de Oxígeno/metabolismo
6.
Neurobiol Dis ; 44(3): 284-91, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21807097

RESUMEN

APP.V717I and Tau.P301L transgenic mice develop Alzheimer's disease pathology comprising important aspects of human disease including increased levels of amyloid peptides, cognitive and motor impairment, amyloid plaques and neurofibrillary tangles. The combined model, APP.V717I×Tau.P301L bigenic mice (biAT mice) exhibit aggravated amyloid and tau pathology with severe cognitive and behavioral defects. In the present study, we investigated early changes in synaptic function in the CA1 and CA3 regions of acute hippocampal slices of young APP.V717I, Tau.P301L and biAT transgenic animals. We have used planar multi-electrode arrays (MEA) and improved methods for simultaneous multi-site recordings from two hippocampal sub-regions. In the CA1 region, long-term potentiation (LTP) was severely impaired in all transgenic animals when compared with age-matched wild-type controls, while basal synaptic transmission and paired-pulse facilitation were minimally affected. In the CA3 region, LTP was normal in Tau.P301L and APP.V717I but clearly impaired in biAT mice. Surprisingly, frequency facilitation in CA3 was significantly enhanced in Tau.P301L mice, while not affected in APP.V717I mice and depressed in biAT mice. The findings demonstrate important synaptic changes that differ considerably in the hippocampal sub-regions already at young age, well before the typical amyloid or tau pathology is evident.


Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Electrodos , Hipocampo/patología , Hipocampo/fisiopatología , Sinapsis/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Biofisica , Modelos Animales de Enfermedad , Estimulación Eléctrica/métodos , Potenciales Postsinápticos Excitadores/genética , Potenciales Postsinápticos Excitadores/fisiología , Humanos , Técnicas In Vitro , Isoleucina/genética , Potenciación a Largo Plazo/genética , Ratones , Ratones Transgénicos , Mutación/genética , Sinapsis/fisiología , Factores de Tiempo , Valina/genética , Proteínas tau/genética
7.
J Bioenerg Biomembr ; 43(6): 587-92, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22072073

RESUMEN

A growing body of evidence suggests that mitochondrial dysfunctions play a crucial role in the pathogenesis of various neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting both upper and lower motor neurons. Although ALS is predominantly a sporadic disease, approximately 10% of cases are familial. The most frequent familial form is caused by mutations in the gene encoding Cu/Zn superoxide dismutase 1 (SOD1). A dominant toxic gain of function of mutant SOD1 has been considered as the cause of the disease and mitochondria are thought to be key players in the pathogenesis. However, the exact nature of the link between mutant SOD1 and mitochondrial dysfunctions remains to be established. Here, we briefly review the evidence for mitochondrial dysfunctions in familial ALS and discuss a possible link between mutant SOD1 and mitochondrial dysfunction.


Asunto(s)
Esclerosis Amiotrófica Lateral/enzimología , Enfermedades Genéticas Congénitas/enzimología , Mitocondrias/enzimología , Superóxido Dismutasa/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/patología , Humanos , Mitocondrias/genética , Mitocondrias/patología , Mutación , Superóxido Dismutasa/genética , Superóxido Dismutasa-1
8.
Cell Calcium ; 100: 102479, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34610487

RESUMEN

Ca2+ and pH homeostasis are closely intertwined and this interrelationship is crucial in the cells' ability to adapt to varying environmental conditions. To further understand this Ca2+-pH link, cytosolic Ca2+ was monitored using the aequorin-based bioluminescent assay in parallel with fluorescence reporter-based assays to monitor plasma membrane potentials and intracellular (cytosolic and vacuolar) pH in yeast Saccharomyces cerevisiae. At external pH 5, starved yeast cells displayed depolarized membrane potentials and responded to glucose re-addition with small Ca2+ transients accompanied by cytosolic alkalinization and profound vacuolar acidification. In contrast, starved cells at external pH 7 were hyperpolarized and glucose re-addition induced large Ca2+ transients and vacuolar alkalinization. In external Ca2+-free medium, glucose-induced pH responses were not affected but Ca2+ transients were abolished, indicating that the intracellular [Ca2+] increase was not prerequisite for activation of the two primary proton pumps, being Pma1 at the plasma membrane and the vacuolar and Golgi localized V-ATPases. A reduction in Pma1 expression resulted in membrane depolarization and reduced Ca2+ transients, indicating that the membrane hyperpolarization generated by Pma1 activation governed the Ca2+ influx that is associated with glucose-induced Ca2+ transients. Loss of V-ATPase activity through concanamycin A inhibition did not alter glucose-induced cytosolic pH responses but affected vacuolar pH changes and Ca2+ transients, indicating that the V-ATPase established vacuolar proton gradient is substantial for organelle H+/Ca2+ exchange. Finally, a systematic analysis of yeast deletion strains allowed us to reveal an essential role for both the vacuolar H+/Ca2+ exchanger Vcx1 and the Golgi exchanger Gdt1 in the dissipation of intracellular Ca2+.


Asunto(s)
Proteínas de Saccharomyces cerevisiae , ATPasas de Translocación de Protón Vacuolares , Glucosa , Concentración de Iones de Hidrógeno , ATPasas de Translocación de Protón/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo
9.
Front Genet ; 11: 266, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32457789

RESUMEN

The yeast Saccharomyces cerevisiae is a powerful model to study the molecular mechanisms underlying α-synuclein (α-syn) cytotoxicity. This is due to the high degree of conservation of cellular processes with higher eukaryotes and the fact that yeast does not endogenously express α-synuclein. In this work, we focused specifically on the interplay between α-syn and intracellular Ca2+ homeostasis. Using temperature-sensitive SEC4 mutants and deletion strains for the vacuolar Ca2+ transporters Pmc1 and Vcx1, together with aequorin-based Ca2+ recordings, we show that overexpression of α-syn shifts the predominant temporal pattern of organellar Ca2+ release from a biphasic to a quasi-monophasic response. Fragmentation and vesiculation of vacuolar membranes in α-syn expressing cells can account for the faster release of vacuolar Ca2+. α-Syn further significantly reduced Ca2+ storage resulting in increased resting cytosolic Ca2+ levels. Overexpression of the vacuolar Ca2+ ATPase Pmc1 in wild-type cells prevented the α-syn-induced increase in resting Ca2+ and was able to restore growth. We propose that α-syn-induced disruptions in Ca2+ signaling might be an important step in initiating cell death.

10.
Biol Cell ; 100(1): 39-49, 2008 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17868032

RESUMEN

BACKGROUND INFORMATION: The IP(3)R (inositol 1,4,5-trisphosphate receptor) is a tetrameric channel that accounts for a large part of the intracellular Ca(2+) release in virtually all cell types. We have previously demonstrated that caspase-3-mediated cleavage of IP(3)R1 during cell death generates a C-terminal fragment of 95 kDa comprising the complete channel domain. Expression of this truncated IP(3)R increases the cellular sensitivity to apoptotic stimuli, and it was postulated to be a constitutively active channel. RESULTS: In the present study, we demonstrate that expression of the caspase-3-cleaved C-terminus of IP(3)R1 increased the rate of thapsigargin-mediated Ca(2+) leak and decreased the rate of Ca(2+) uptake into the ER (endoplasmic reticulum), although it was not sufficient by itself to deplete intracellular Ca(2+) stores. We detected the truncated IP(3)R1 in different cell types after a challenge with apoptotic stimuli, as well as in aged mouse oocytes. Injection of mRNA corresponding to the truncated IP(3)R1 blocked sperm factor-induced Ca(2+) oscillations and induced an apoptotic phenotype. CONCLUSIONS: In the present study, we show that caspase-3-mediated truncation of IP(3)R1 enhanced the Ca(2+) leak from the ER. We suggest a model in which, in normal conditions, the increased Ca(2+) leak is largely compensated by enhanced Ca(2+)-uptake activity, whereas in situations where the cellular metabolism is compromised, as occurring in aging oocytes, the Ca(2+) leak acts as a feed-forward mechanism to divert the cell into apoptosis.


Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Caspasa 3/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Animales , Apoptosis/fisiología , Línea Celular , Inhibidores Enzimáticos/metabolismo , Femenino , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Meiosis/fisiología , Ratones , Ratones Noqueados , Oocitos/citología , Oocitos/fisiología , Estaurosporina/metabolismo , Tapsigargina/metabolismo
11.
Adv Sci (Weinh) ; 5(4): 1700731, 2018 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-29721420

RESUMEN

Microelectrode arrays (MEAs) have proved to be useful tools for characterizing electrically active cells such as cardiomyocytes and neurons. While there exist a number of integrated electronic chips for recording from small populations or even single cells, they rely primarily on the interface between the cells and 2D flat electrodes. Here, an approach that utilizes residual stress-based self-folding to create individually addressable multielectrode interfaces that wrap around the cell in 3D and function as an electrical shell-like recording device is described. These devices are optically transparent, allowing for simultaneous fluorescence imaging. Cell viability is maintained during and after electrode wrapping around the cel and chemicals can diffuse into and out of the self-folding devices. It is further shown that 3D spatiotemporal recordings are possible and that the action potentials recorded from cultured neonatal rat ventricular cardiomyocytes display significantly higher signal-to-noise ratios in comparison with signals recorded with planar extracellular electrodes. It is anticipated that this device can provide the foundation for the development of new-generation MEAs where dynamic electrode-cell interfacing and recording substitutes the traditional method using static electrodes.

12.
Cell Calcium ; 42(1): 59-69, 2007 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-17241659

RESUMEN

Motor neuron death in amyotrophic lateral sclerosis (ALS) has been linked to selective vulnerability towards AMPA receptor-mediated excitotoxicity. We investigated intracellular mechanisms leading to impairment of motor neuron Ca2+ homeostasis with near physiological AMPA receptor activation. Using fast solution exchange on patch-clamped cultured neurons, kainate (KA) was applied for 2s. This induced a transient increase in the cytosolic Ca2+ concentration ([Ca2+]c) for seconds. Inhibition of the mitochondrial uniporter by RU-360 abolished the decay of the Ca2+ transient and caused immediate [Ca2+]c overload. Repetitive short KA stimulation caused a slowing of the decay of the Ca2+ transient and a gradual increase in peak and baseline [Ca2+]c in motor neurons, but not in other neurons, indicating saturation of the mitochondrial buffer. Furthermore, mitochondrial density was lower in motor neurons and, in a network of neurons with physiological synaptic AMPA receptor input, RU-360 acutely induced an increase in Ca2+ transients. We conclude that motor neurons have an insufficient mitochondrial capacity to buffer large Ca2+ elevations which is partly due to a reduced mitochondrial density per volume compared to non-motor neurons. This may exert deleterious effects in motor neuron disease where mitochondrial function is thought to be compromised.


Asunto(s)
Calcio/fisiología , Ácido Kaínico/farmacología , Mitocondrias/fisiología , Neuronas Motoras/fisiología , Receptores AMPA/fisiología , Animales , Células Cultivadas , Microscopía Confocal , Modelos Neurológicos , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/ultraestructura , Técnicas de Placa-Clamp , Ratas , Compuestos de Rutenio/farmacología
13.
FASEB J ; 20(6): 756-8, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16455754

RESUMEN

Both the commitment event and the modality of cell death in photodynamic therapy (PDT) remain poorly defined. We report that PDT with endoplasmic reticulum (ER)-associating hypericin leads to an immediate loss of SERCA2 protein levels, causing disruption of Ca2+ homeostasis and cell death. Protection of SERCA2 protein rescues ER-Ca2+ levels and prevents cell death, suggesting that SERCA2 photodestruction with consequent incapability of the ER to maintain intracellular Ca2+ homeostasis is causal to cell killing. Apoptosis is rapidly initiated after ER-Ca2+ depletion and strictly requires the BAX/BAK gateway at the mitochondria. Bax-/-Bak-/- double-knockout (DKO) cells are protected from apoptosis but undergo autophagy-associated cell death as revealed by electron microscopy and biochemical analysis. Autophagy inhibitors, but not caspase antagonists, significantly reduce death of DKO cells, suggesting that sustained autophagy is lethal. Thus, following ER photodamage and consequent disruption of Ca2+ homeostasis, BAX and BAK proteins model PDT-mediated cell killing, which is executed through apoptosis in their presence or via an autophagic pathway in their absence.


Asunto(s)
Apoptosis/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Perileno/análogos & derivados , Fotoquimioterapia , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , Antracenos , Autofagia/efectos de los fármacos , Autofagia/efectos de la radiación , Calcio/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , Línea Celular , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/efectos de la radiación , Fibroblastos/metabolismo , Eliminación de Gen , Humanos , Ratones , Perileno/farmacología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína X Asociada a bcl-2/genética
14.
J Pharmacol Toxicol Methods ; 87: 48-52, 2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28549786

RESUMEN

Drug-induced cardiotoxicity poses a negative impact on public health and drug development. Cardiac safety pharmacology issues urged for the preclinical assessment of drug-induced ventricular arrhythmia leading to the design of several in vitro electrophysiological screening assays. In general, patch clamp systems allow for intracellular recordings, while multi-electrode array (MEA) technology detect extracellular activity. Here, we demonstrate a complementary metal oxide semiconductor (CMOS)-based MEA system as a reliable platform for non-invasive, long-term intracellular recording of cardiac action potentials at high resolution. Quinidine (8 concentrations from 10-7 to 2.10-5M) and verapamil (7 concentrations from 10-11 to 10-5M) were tested for dose-dependent responses in a network of cardiomyocytes. Electrophysiological parameters, such as the action potential duration (APD), rates of depolarization and repolarization and beating frequency were assessed. In hiPSC, quinidine prolonged APD with EC50 of 2.2·10-6M. Further analysis indicated a multifactorial action potential prolongation by quinidine: (1) decreasing fast repolarization with IC50 of 1.1·10-6M; (2) reducing maximum upstroke velocity with IC50 of 2.6·10-6M; and (3) suppressing spontaneous activity with EC50 of 3.8·10-6M. In rat neonatal cardiomyocytes, verapamil blocked spontaneous activity with EC50 of 5.3·10-8M and prolonged the APD with EC50 of 2.5·10-8M. Verapamil reduced rates of fast depolarization and repolarization with IC50s of 1.8 and 2.2·10-7M, respectively. In conclusion, the proposed action potential-based MEA platform offers high quality and stable long-term recordings with high information content allowing to characterize multi-ion channel blocking drugs. We anticipate application of the system as a screening platform to efficiently and cost-effectively test drugs for cardiac safety.


Asunto(s)
Potenciales de Acción/fisiología , Antiarrítmicos/farmacología , Cardiotoxinas/farmacología , Células Madre Pluripotentes Inducidas/fisiología , Miocitos Cardíacos/fisiología , Semiconductores , Potenciales de Acción/efectos de los fármacos , Animales , Animales Recién Nacidos , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Microelectrodos , Miocitos Cardíacos/efectos de los fármacos , Quinidina/farmacología , Ratas , Ratas Wistar
15.
Sci Rep ; 7(1): 7195, 2017 08 03.
Artículo en Inglés | MEDLINE | ID: mdl-28775328

RESUMEN

Epilepsy is a chronic brain disorder characterized by recurrent seizures due to abnormal, excessive and synchronous neuronal activities in the brain. It affects approximately 65 million people worldwide, one third of which are still estimated to suffer from refractory seizures. Glutamic acid decarboxylase (GAD) that converts glutamate into GABA is a key enzyme in the dynamic regulation of neural network excitability. Importantly, clinical evidence shows that lowered GAD activity is associated with several forms of epilepsy which are often treatment resistant. In the present study, we synthetized and explored the possibility of using ethyl ketopentenoate (EKP), a lipid-permeable GAD-inhibitor, to induce refractory seizures in zebrafish larvae. Our results demonstrate that EKP evoked robust convulsive locomotor activities, excessive epileptiform discharges and upregulated c-fos expression in zebrafish. Moreover, transgenic animals in which neuronal cells express apoaequorin, a Ca2+-sensitive bioluminescent photoprotein, displayed large luminescence signals indicating strong EKP-induced neuronal activation. Molecular docking data indicated that this proconvulsant activity resulted from the direct inhibition of both gad67 and gad65. Limited protective efficacy of tested anti-seizure drugs (ASDs) demonstrated a high level of treatment resistance of EKP-induced seizures. We conclude that the EKP zebrafish model can serve as a high-throughput platform for novel ASDs discovery.


Asunto(s)
Inhibidores Enzimáticos/farmacología , Glutamato Descarboxilasa/antagonistas & inhibidores , Convulsiones/metabolismo , Convulsiones/fisiopatología , Animales , Conducta Animal , Biomarcadores , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/química , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Glutamato Descarboxilasa/química , Glutamato Descarboxilasa/genética , Modelos Moleculares , Conformación Molecular , Actividad Motora , Convulsiones/tratamiento farmacológico , Convulsiones/genética , Relación Estructura-Actividad , Pez Cebra
16.
Cell Calcium ; 39(4): 325-36, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16458354

RESUMEN

The N-terminal 1-225 amino acids (aa) of the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) function as a suppressor/coupling domain. In this study we used IP(3)R-deficient B-lymphocytes to investigate the effects of modifications in this domain on IP(3) binding and Ca(2+)-release activity. Although the N-terminal 1-225 aa of IP(3)R3 had the same role as in IP(3)R1, the suppression of IP(3) binding for IP(3)R1 was lost when the suppressor/coupling domains were exchanged between the two isoforms. Resulting chimeric receptors showed a higher sensitivity to IP(3)-induced activation (IICR). Deletion of 11 aa in IP(3)R1 ([Delta76-86]-IP(3)R1) or replacing aa 76-86 of the IP(3)R1 in the suppressor/coupling domain by 13 aa of IP(3)R3 ([75-87 T3]-IP(3)R1) also resulted in increased IP(3) binding and sensitivity of IICR. These residues constitute the only part of the suppressor/coupling domain that is strikingly different between the two isoforms. Expression of [Delta76-86]-IP(3)R1 and of [75-87 T3]-IP(3)R1 increased the propensity of cells to undergo staurosporine-induced apoptosis, but had no effect on the Ca(2+) content in the endoplasmic reticulum. In the cell model used, our observations suggest that the sensitivity of the Ca(2+)-release activity of IP(3)R1 to IP(3) influences the sensitivity of the cells to apoptotic stimuli and that the suppressor/coupling domain may have an anti-apoptotic function by attenuating the sensitivity of IICR.


Asunto(s)
Apoptosis/fisiología , Canales de Calcio/fisiología , Calcio/fisiología , Inositol 1,4,5-Trifosfato/metabolismo , Receptores Citoplasmáticos y Nucleares/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión/fisiología , Calcio/metabolismo , Canales de Calcio/genética , Caspasa 3 , Caspasas/metabolismo , Línea Celular , Pollos , Secuencia Conservada , Genes Supresores/fisiología , Humanos , Receptores de Inositol 1,4,5-Trifosfato , Datos de Secuencia Molecular , Estructura Terciaria de Proteína/fisiología , Receptores Citoplasmáticos y Nucleares/genética , Homología de Secuencia de Aminoácido
17.
Cell Calcium ; 40(1): 41-51, 2006 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-16675011

RESUMEN

Presenilins (PS) are proteins involved in the pathogenesis of autosomal-dominant familial cases of Alzheimer's disease. Mutations in PS are known to induce specific alterations in cellular Ca2+ signaling which might be involved in the pathogenesis of neurodegenerative diseases. Mouse embryonic fibroblasts (MEF) deficient in PS1 and PS2 (PS DKO) as well as the latter rescued with PS1 (Rescue), were used to investigate the underlying mechanism of these alterations in Ca2+ signaling. PS DKO cells were characterized by a decrease in the [Ca2+]ER as measured by ER-targeted aequorin luminescence and an increased level of type 1 inositol 1,4,5-trisphosphate receptor (IP3R1). The lower [Ca2+]ER was associated with an increase in a Ca2+ leak from the ER. The increased IP3R1 expression and the concomitant changes in ER Ca2+ handling were reversed in the Rescue cells. Moreover using RNA-interference mediated reduction of IP3R1 we could demonstrate that the up-regulation of this isoform was responsible for the increased Ca2+ leak and the lowered [Ca2+]ER PS DKO cells. Finally, we show that the decreased [Ca2+]ER in PS DKO cells was protective against apoptosis.


Asunto(s)
Canales de Calcio/biosíntesis , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/genética , Receptores Citoplasmáticos y Nucleares/biosíntesis , Regulación hacia Arriba/fisiología , Animales , Canales de Calcio/fisiología , Células Cultivadas , Receptores de Inositol 1,4,5-Trifosfato , Proteínas de la Membrana/deficiencia , Ratones , Ratones Noqueados , Presenilina-1 , Presenilina-2 , Receptores Citoplasmáticos y Nucleares/fisiología
18.
Biochim Biophys Acta ; 1742(1-3): 103-12, 2004 Dec 06.
Artículo en Inglés | MEDLINE | ID: mdl-15590060

RESUMEN

Recent evidence highlights the functional importance of the Golgi apparatus as an agonist-sensitive intracellular Ca(2+) store. Besides Ca(2+)-release channels and Ca(2+)-binding proteins, the Golgi complex contains Ca(2+)-uptake mechanisms consisting of the well-known sarco/endoplasmic reticulum Ca(2+)-transport ATPases (SERCA) and the much less characterized secretory-pathway Ca(2+)-transport ATPases (SPCA). SPCA supplies the Golgi compartments and, possibly, the more distal compartments of the secretory pathway with both Ca(2+) and Mn(2+) and, therefore, plays an important role in the cytosolic and intra-Golgi Ca(2+) and Mn(2+) homeostasis. Mutations in the human gene encoding the SPCA1 pump (ATP2C1) resulting in Hailey-Hailey disease, an autosomal dominant skin disorder, are discussed.


Asunto(s)
ATPasas Transportadoras de Calcio/metabolismo , Aparato de Golgi/metabolismo , Manganeso/metabolismo , Proteínas de Transporte de Membrana/metabolismo , ATPasas Transportadoras de Calcio/química , Humanos , Manganeso/química , Proteínas de Transporte de Membrana/química , Modelos Moleculares , Pénfigo Familiar Benigno/metabolismo , Conformación Proteica
19.
Biochim Biophys Acta ; 1600(1-2): 19-31, 2002 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-12445455

RESUMEN

Intracellular calcium release is a fundamental signaling mechanism in all eukaryotic cells. The ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate receptor (IP(3)R) are intracellular calcium release channels. Both channels can be regulated by calcium and calmodulin (CaM). In this review we will first discuss the role of calcium as an activator and inactivator of the IP(3)R, concluding that calcium is the most important regulator of the IP(3)R. In the second part we will further focus on the role of CaM as modulator of the IP(3)R, using results of the voltage-dependent Ca(2+) channels and the RyR as reference material. Here we conclude that despite the fact that different CaM-binding sites have been characterized, their function for the IP(3)R remains elusive. In the third part we will discuss the possible functional role of CaM in IP(3)-induced Ca(2+) release (IICR) by direct and indirect mechanisms. Special attention will be given to the Ca(2+)-binding proteins (CaBPs) that were shown to activate the IP(3)R in the absence of IP(3).


Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Calmodulina/fisiología , Inositol 1,4,5-Trifosfato/fisiología , Animales , Sitios de Unión , Calcineurina/fisiología , Canales de Calcio/metabolismo , Proteínas de Unión al Calcio/fisiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Inositol 1,4,5-Trifosfato/química , Inositol Polifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Estructura Terciaria de Proteína , Canal Liberador de Calcio Receptor de Rianodina/química , Canal Liberador de Calcio Receptor de Rianodina/metabolismo
20.
J Neuropathol Exp Neurol ; 64(7): 605-12, 2005 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-16042312

RESUMEN

AMPA receptor-mediated excitotoxicity has been implicated in the selective degeneration of motor neurons in amyotrophic lateral sclerosis (ALS). Motor neurons in vitro are particularly vulnerable to excessive AMPA receptor stimulation and one of the factors underlying this selective vulnerability is the presence of a large proportion of Ca2+ -permeable (i.e. GluR2-lacking) AMPA receptors. However, the precise role of GluR2-lacking AMPA receptors in motor neuron degeneration remains to be defined. We therefore studied the impact of GluR2 deficiency on motor neuron death in vitro and in vivo. Cultured motor neurons from GluR2-deficient embryos displayed an increased Ca2+ influx through AMPA receptors and an increased vulnerability to AMPA receptor-mediated excitotoxicity. We deleted the GluR2 gene in mutant SOD1G93A mice by crossbreeding them with GluR2 knockout mice. GluR2 deficiency clearly accelerated the motor neuron degeneration and shortened the life span of mutant SOD1G93A mice. These findings indicate that GluR2 plays a pivotal role in the vulnerability of motor neurons in vitro and in vivo, and that therapies that limit Ca2+ entry through AMPA receptors might be beneficial in ALS patients.


Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Neuronas Motoras/patología , Degeneración Nerviosa/patología , Receptores AMPA/deficiencia , Médula Espinal/patología , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Técnicas In Vitro , Ratones , Ratones Noqueados , Neuronas Motoras/metabolismo , Técnicas de Placa-Clamp , Receptores AMPA/genética , Receptores AMPA/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa-1
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