RESUMEN
Glutamine plays multiple roles in the CNS, including metabolic functions and production of the neurotransmitters glutamate and GABA. It has been proposed to be taken up into neurons via a variety of membrane transport systems, including system A, which is a sodium-dependent electrogenic amino acid transporter system. In this study, we investigate glutamine transport by application of amino acids to individual principal neurons of the medial nucleus of the trapezoid body (MNTB) in acutely isolated rat brain slices. A glutamine transport current was studied in patch-clamped neurons, which had the electrical and pharmacological properties of system A: it was sodium-dependent, had a non-reversing current-voltage relationship, was activated by proline, occluded by N-(methylamino)isobutyric acid (MeAIB), and was unaffected by 2-aminobicyclo-[2.2.1]-heptane-2-carboxylic acid (BCH). Additionally, we examined the expression of different system A transporter isoforms using immunocytochemical staining with antibodies raised against system A transporter 1 and 2 (SAT1 and SAT2). Our results indicate that both isoforms are expressed in MNTB principal neurons, and demonstrate that functional system A transporters are present in the plasma membrane of neurons. Since system A transport is highly regulated by a number of cellular signaling mechanisms and glutamine then goes on to activate other pathways, the study of these transporters in situ gives an indication of the mechanisms of neuronal glutamine supply as well as points of regulation of neurotransmitter production, cellular signaling and metabolism in the native neuronal environment.
Asunto(s)
Sistema de Transporte de Aminoácidos A/metabolismo , Vías Auditivas/metabolismo , Glutamina/metabolismo , Neuronas/metabolismo , Rombencéfalo/metabolismo , Animales , Vías Auditivas/citología , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Inmunohistoquímica , Neuronas/efectos de los fármacos , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Prolina/metabolismo , Prolina/farmacología , Isoformas de Proteínas/metabolismo , Ratas , Ratas Wistar , Rombencéfalo/citología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Bloqueadores de los Canales de Sodio/farmacología , beta-Alanina/análogos & derivados , beta-Alanina/farmacologíaRESUMEN
The study of synaptic transmission in brain slices generally entails the patch-clamping of postsynaptic neurones and stimulation of identified presynaptic axons using a remote electrical stimulating electrode. Although patch recording from postsynaptic neurones is routine, many presynaptic axons take tortuous turns and are severed in the slicing procedure, blocking propagation of the action potential to the synaptic terminal and preventing synaptic stimulation. Here we demonstrate a method of using calcium imaging to select postsynaptic cells with functional synaptic inputs prior to patch-clamp recording. We have used this method for exploring transmission in the auditory brainstem at the medial nucleus of the trapezoid body neurones, which are innervated by axons from the contralateral cochlear nucleus. Brainstem slices were briefly loaded with the calcium indicator fura-2 AM and stimulated with an electrode placed on the midline. Electrical stimulation caused a rise in intracellular calcium concentration in those postsynaptic neurones with active synaptic connections. Since <10% of the medial nucleus of the trapezoid body neurones retain viable synaptic inputs following the slicing procedure, preselecting those cells with active synapses dramatically increased our recording success. This detection method will greatly ease the study of synaptic responses in brain areas where suprathreshold synaptic inputs occur but connectivity is sparse.
Asunto(s)
Calcio/metabolismo , Núcleo Coclear/citología , Núcleo Coclear/fisiología , Sinapsis/fisiología , Animales , Vías Auditivas/citología , Vías Auditivas/fisiología , Estimulación Eléctrica , Colorantes Fluorescentes , Fura-2 , Ratas , Ratas EndogámicasRESUMEN
1. The K(ATP) channel opener diazoxide has been proposed to protect cardiac muscle against ischaemia by opening mitochondrial K(ATP) channels to depolarize the mitochondrial membrane potential, DeltaPsi(m). We have used the fluorescent dye TMRE to measure DeltaPsi(m) in adult rat freshly isolated cardiac myocytes exposed to diazoxide and metabolic inhibition. 2. Diazoxide, at concentrations that are highly cardioprotective (100 or 200 microM), caused no detectable increase in TMRE fluorescence (n=27 cells). However, subsequent application of the protonophore FCCP, which should collapse DeltaPsi(m), led to large increases in TMRE fluorescence (>300%). 3. Metabolic inhibition (MI: 2 mM NaCN+1 mM iodoacetic acid (IAA) led to an immediate partial depolarization of DeltaPsi(m), followed after a few minutes delay by complete depolarization which was correlated with rigor contracture. Removal of metabolic inhibition led to abrupt mitochondrial repolarization followed in many cells by hypercontracture, indicated by cell rounding and loss of striated appearance. 4. Prior application of diazoxide (100 microM) reduced the number of cells that hypercontracted after metabolic inhibition from 63.7+/-4.7% to 24.2+/-1.8% (P< 0.0001). 5-hydroxydeanoate (100 microM) reduced the protection of diazoxide (46.8+/-2.7% cells hypercontracted, P< 0.0001 vs diazoxide alone). 5. Diazoxide caused no detectable change in flavoprotein autofluorescence (n=26 cells). 6. Our results suggest that mitochondrial depolarization and flavoprotein oxidation are not inevitable consequences of diazoxide application in intact cardiac myocytes, and that they are also not essential components of the mechanism by which it causes protection.
Asunto(s)
Diazóxido/farmacología , Flavoproteínas/metabolismo , Mitocondrias Cardíacas/efectos de los fármacos , Miocardio/citología , Miocardio/metabolismo , Canales de Potasio/metabolismo , Vasodilatadores/farmacología , Animales , Separación Celular , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Mitocondrias Cardíacas/metabolismo , Oxidación-Reducción , Ratas , Ratas WistarRESUMEN
Manipulating the expression of a protein can provide a powerful tool for understanding its function, provided that the protein is expressed at physiologically-significant concentrations. We have developed a simple method to measure (1) the concentration of an overexpressed protein in single cells and (2) the covariation of particular physiological properties with a protein's expression. As an example of how this method can be used, teratocarcinoma cells were transfected with the neuron-specific calcium binding protein calretinin (CR) tagged with green fluorescent protein (GFP). By measuring GFP fluorescence in microcapillaries, we created a standard curve for GFP fluorescence that permitted quantification of CR concentrations in individual cells. Fura-2 measurements in the same cells showed a strong positive correlation between CR-GFP fusion protein expression levels and calcium clearance capacity. This method should allow reliable quantitative analysis of GFP fusion protein expression.
Asunto(s)
Indicadores y Reactivos , Proteínas Luminiscentes , Proteína G de Unión al Calcio S100/metabolismo , Animales , Calbindina 2 , Calcio/metabolismo , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes , Humanos , Masculino , Proteína G de Unión al Calcio S100/análisis , Teratocarcinoma , Neoplasias Testiculares , Células Tumorales CultivadasRESUMEN
Membrane dynamics within the chick ciliary neuronal growth cone were investigated by using the membrane-impermeant dye FM1-43. A depolarization-evoked endocytosis was observed that shared many properties with the synaptic vesicle recycling previously described at the presynaptic terminal. In addition, in the absence of depolarization a basal level of constitutive endocytotic activity was observed at approximately 30% of the rate of evoked endocytosis. This constitutive endocytosis accounted for large amounts of membrane retrieval: the equivalent of the entire growth cone surface area could be internalized within a 30 min period. Endosomes generated via constitutive and evoked processes were highly mobile and could move considerable distances both within the growth cone and out to the neurite. In addition to their different requirements for formation, evoked and constitutive endosomes displayed a significant difference in release properties. After a subsequent depolarization of labeled growth cones, evoked endosomes were released although constitutive endosomes were not released. Furthermore, treatment with latrotoxin released evoked endosomes, but not constitutive endosomes. Although the properties of evoked endosomes are highly reminiscent of synaptic vesicles, constitutive endosomes appear to be a separate pool resulting from a distinct and highly active process within the neuronal growth cone.
Asunto(s)
Membrana Celular/fisiología , Endocitosis/fisiología , Endosomas/fisiología , Neuronas/fisiología , Compuestos de Piridinio , Compuestos de Amonio Cuaternario , Animales , Membrana Celular/ultraestructura , Células Cultivadas , Embrión de Pollo , Cobalto/farmacología , Endocitosis/efectos de los fármacos , Endosomas/ultraestructura , Colorantes Fluorescentes , Ganglios Parasimpáticos/citología , Ganglios Parasimpáticos/fisiología , Cinética , Neuronas/citología , Cloruro de Potasio/farmacología , Vesículas Sinápticas/fisiología , Vesículas Sinápticas/ultraestructuraRESUMEN
We have described how a combination of electrical, ion-sensing, and glutamate-sensing techniques has advanced our understanding of glutamate uptake into isolated salamander retinal glial cells. The next steps in understanding glutamate transport will inevitably depend strongly on molecular biological methods, as described elsewhere in this book, but will also require more detailed study of transporters in their normal environment, perhaps by using patch-clamping or imaging techniques to study cells in situ.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Cerebelo/fisiología , Ácido Glutámico/metabolismo , Neuroglía/fisiología , Células de Purkinje/fisiología , Retina/citología , Sistema de Transporte de Aminoácidos X-AG , Animales , Transporte Biológico , Estimulación Eléctrica , Fluoresceínas , Colorantes Fluorescentes , Ácido Glutámico/análogos & derivados , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Cinética , Neuroglía/metabolismo , Técnicas de Placa-Clamp , Ratas , UrodelosRESUMEN
1. Zinc may be released from some presynaptic glutamatergic neurons, including hippocampal mossy fibres and retinal photoreceptors. We whole-cell-clamped glial (Müller) cells isolated from the salamander retina to investigate the effect of zinc on glutamate transporters in these cells. Glutamate-evoked currents in these cells are generated largely by carriers homologous to the mammalian GLAST/EAAT1 transporter. 2. Zinc inhibited both glutamate uptake into the cells, and glutamate release by reversal of the uptake process. The IC50 for inhibition of uptake (< 1 microM) was similar to or below the values for zinc modulating NMDA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and GABA receptors, and 100-fold less than the calculated value for the rise in extracellular zinc concentration evoked by depolarization with potassium in area CA3 of the hippocampus. 3. Although zinc altered the apparent affinity of the transporter for glutamate and Na+, it did not act simply by binding competitively to the glutamate-, Na(+)-, K(+)- or H(+)-binding sites on the transporter. Zinc inhibited both forward and reversed glutamate transport from the outside of the cell membrane, but not from the inside. The inhibitory action of zinc on uptake was voltage independent, indicating a zinc-binding site outside the membrane field. 4. As well as inhibiting glutamate transport, zinc potentiated activation of the anion conductance in the Müller cell glutamate transporter. However, zinc reduced the current mediated by the anion conductance in the cone synaptic terminal glutamate transporter (homologous to the mammalian EAAT5), indicating that zinc has different actions on different glutamate transporter subtypes. 5. By acting on glutamate transporters, zinc may have a neuromodulatory role during synaptic transmission and a neuroprotective role during transient ischaemia.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/efectos de los fármacos , Neuroglía/efectos de los fármacos , Retina/citología , Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Zinc/farmacología , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Potenciales de Acción/efectos de los fármacos , Sistema de Transporte de Aminoácidos X-AG , Animales , Aniones/metabolismo , Proteínas de la Membrana Bacteriana Externa/efectos de los fármacos , Sitios de Unión , Unión Competitiva/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Sinergismo Farmacológico , Conductividad Eléctrica , Espacio Extracelular/química , Ácido Glutámico/farmacocinética , Concentración de Iones de Hidrógeno , Canales Iónicos/efectos de los fármacos , Canales Iónicos/fisiología , Potenciales de la Membrana/efectos de los fármacos , Potasio/metabolismo , Retina/efectos de los fármacos , Retina/metabolismo , Células Fotorreceptoras Retinianas Conos/citología , Sodio/metabolismo , Urodelos , Zinc/metabolismoRESUMEN
Glutamate transporters in neurones and glia, four of which have been cloned from mammals, play a crucial role in controlling the extracellular glutamate concentration in the brain. In normal conditions, they remove glutamate from the extracellular space and thereby help to terminate glutamatergic synaptic transmission and to prevent the extracellular glutamate concentration from rising to neurotoxic values. Glutamate transport on these carriers is thought to be driven by the cotransport of Na+, the counter-transport of K+, and either the cotransport of H+ or the counter-transport of OH-. Activating the transporters also activates an anion conductance in their structure, the anion flux through which is not coupled to glutamate movement and varies widely for the different transporters. During hypoxia or ischaemia, glutamate transporters can run backwards, releasing glutamate into the extracellular space, triggering the death of neurones and thus causing mental and physical handicap. The rate of glutamate release by this process is slowed by the acid pH occurring in hypoxia/ischaemia, which may help protect the brain during transient, but not sustained, ischaemia.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Encéfalo/metabolismo , Ácido Glutámico/metabolismo , Proteínas del Tejido Nervioso/fisiología , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfato/metabolismo , Sistema de Transporte de Aminoácidos X-AG , Animales , Isquemia Encefálica/metabolismo , Canales de Cloruro/metabolismo , Clonación Molecular , Epilepsia/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Hipoxia Encefálica/metabolismo , Proteínas del Tejido Nervioso/genética , Potasio/metabolismo , Transducción de Señal , Sodio/metabolismo , Transmisión SinápticaRESUMEN
Glutamate uptake is driven by the cotransport of Na+ ions, the countertransport of K+ ions, and either the countertransport of OH- or the cotransport of H+ ions. In addition, activating glutamate uptake carriers has been shown to lead to activation of an anion conductance present in the carrier structure. Here we characterize the ion selectivity and gating of this anion conductance. The conductance is small with Cl- as the permeant anion, but it is large with NO3- or ClO4- present, undermining the earlier use of NO3- and ClO4- to suggest that OH- countertransport rather than H+ cotransport helps drive uptake. Activation of the anion conductance can be evoked by extra- or intracellular glutamate and can occur even when glutamate transport is inhibited. By running the carrier backward and detecting glutamate release with AMPA receptors in neurons placed near the glial cells, we show that anion flux is not coupled thermodynamically to glutamate movement, but OH-/H+ transport is. The possibility that cell excitability is modulated by the anion conductance associated with glutamate uptake suggests a target for therapeutic drugs to reduce glutamate release in conditions like epilepsy.
Asunto(s)
Sistema de Transporte de Aminoácidos X-AG , Proteínas Portadoras/metabolismo , Glutamatos/farmacología , Neuroglía/metabolismo , Retina/citología , Simportadores , Animales , Aniones/metabolismo , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Cloruros/metabolismo , Conductividad Eléctrica , Agonistas de Aminoácidos Excitadores/farmacología , Proteínas de Transporte de Glutamato en la Membrana Plasmática , Glutamatos/metabolismo , Concentración de Iones de Hidrógeno , Activación del Canal Iónico/fisiología , Neuroglía/efectos de los fármacos , Nitratos/metabolismo , Técnicas de Placa-Clamp , Potasio/metabolismo , Sodio/metabolismo , Sodio/farmacología , UrodelosRESUMEN
Glutamate uptake into glial cells helps to keep the brain extracellular glutamate concentration, [glu]o, below levels that kill neurons. Uptake is powered by the transmembrane gradients of Na+, K+ and pH. When the extracellular [K+] rises in brain ischaemia, uptake reverses, releasing glutamate into the extracellular space. Here we show, by monitoring glutamate transport electrically and detecting released glutamate with ion channels in neurons placed outside glial cells, that a raised [H+] inhibits both forward and reversed glutamate uptake. No electroneutral reversed uptake was detected, contradicting the idea that forward and reversed uptake differ fundamentally. Suppression of reversed uptake by the low pH occurring in ischaemia will slow the Ca(2+)-independent release of glutamate with can raise [glu]o to a neurotoxic level, and will thus protect the brain during a transient loss of blood supply.