ABSTRACT
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.
Subject(s)
Amino Acid Transport System A/metabolism , Auditory Pathways/metabolism , Glutamine/metabolism , Neurons/metabolism , Rhombencephalon/metabolism , Animals , Auditory Pathways/cytology , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Immunohistochemistry , Neurons/drug effects , Organ Culture Techniques , Patch-Clamp Techniques , Proline/metabolism , Proline/pharmacology , Protein Isoforms/metabolism , Rats , Rats, Wistar , Rhombencephalon/cytology , Signal Transduction/drug effects , Signal Transduction/physiology , Sodium Channel Blockers/pharmacology , beta-Alanine/analogs & derivatives , beta-Alanine/pharmacologyABSTRACT
GABA(C) receptors contain rho subunits and mediate feedback inhibition from retinal amacrine cells to bipolar cells. We previously identified the cytoskeletal protein MAP1B as a rho1 subunit anchoring protein. Here, we analyze the structural basis and functional significance of the MAP1B-rho1 interaction. Twelve amino acids at the C terminus of the large intracellular loop of rho1 (and also rho2) are sufficient for interaction with MAP1B. Disruption of the MAP1B-rho interaction in bipolar cells in retinal slices decreased the EC(50) of their GABA(C) receptors, doubling the receptors' current at low GABA concentrations without affecting their maximum current at high concentrations. Thus, anchoring to the cytoskeleton lowers the sensitivity of GABA(C) receptors and provides a likely site for functional modulation of GABA(C) receptor-mediated inhibition.
Subject(s)
Amino Acid Transport Systems, Neutral , Microtubule-Associated Proteins/metabolism , Receptors, GABA-B , Receptors, GABA/metabolism , Animals , Binding Sites/genetics , Binding, Competitive/drug effects , Binding, Competitive/genetics , Blotting, Western , COS Cells , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cells, Cultured , Dose-Response Relationship, Drug , GABA Antagonists/pharmacology , Glutathione Transferase/genetics , Glycine Agents/pharmacology , Glycine Plasma Membrane Transport Proteins , In Vitro Techniques , Microtubule-Associated Proteins/genetics , Mutagenesis, Site-Directed , Patch-Clamp Techniques , Peptides/genetics , Peptides/pharmacology , Phosphinic Acids/pharmacology , Protein Structure, Tertiary/genetics , Pyridines/pharmacology , Receptors, GABA/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Retina/drug effects , Retina/metabolism , Transfection , gamma-Aminobutyric Acid/metabolism , gamma-Aminobutyric Acid/pharmacokineticsABSTRACT
Transporters are thought to assist in the termination of synaptic transmission at some synapses by removing neurotransmitter from the synapse. To investigate the role of glutamate transport in shaping the time course of excitatory transmission at the mossy fiber-granule cell synapse, the effects of transport impairment were studied using whole-cell voltage- and current-clamp recordings in slices of rat cerebellum. Impairment of transport by L-trans-pyrrolidine-2,4-dicarboxylate (PDC) produced a prolongation of the decay of the AMPA receptor-mediated current after a repetitive stimulus, as well as prolongation of single stimulus-evoked EPSCs when AMPA receptor desensitization was blocked. PDC also produced a prolongation of both single and repetitive-evoked NMDA receptor-mediated EPSCs. Enzymatic degradation of extracellular glutamate did not reverse the PDC-induced prolongation of AMPA receptor-mediated current after a repetitive stimulus, suggesting that transporter binding sites participate in limiting glutamate spillover. In current-clamp recordings, PDC dramatically increased the total area of the EPSP and the burst duration evoked by single and repetitive stimuli. These data indicate that glutamate transporters play a significant role in sculpting the time course of synaptic transmission at granule cell synapses, most likely by limiting the extent of glutamate spillover. The contribution of transporters is particularly striking during repetitive stimulus trains at physiologically relevant frequencies. Hence, the structural arrangement of the glomerulus may enhance the contribution of transporters to information processing by limiting the extent of glutamate spillover between adjacent synapses.
Subject(s)
Cerebellum/physiology , Evoked Potentials/physiology , Neurons/physiology , Synaptic Transmission/physiology , Aging , Animals , Aspartic Acid/pharmacology , Benzothiadiazines/pharmacology , Dicarboxylic Acids/pharmacology , Electric Stimulation , Evoked Potentials/drug effects , Female , In Vitro Techniques , Male , Neurotransmitter Uptake Inhibitors/pharmacology , Patch-Clamp Techniques , Pyrrolidines/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, AMPA/physiology , Synaptic Transmission/drug effects , Time FactorsABSTRACT
A physician or athletic trainer will often be faced with an athlete complaining of chest pain during or after an event. Chest pain in children and adolescents is usually of a noncardiac origin; only 5% of cases are due to cardiac problems. With a properly documented history and physical evaluation, one can usually identify the etiology of the chest discomfort or at least rule out any serious difficulties. The various diagnostic possibilities include cardiac, musculoskeletal, pulmonary, gastrointestinal, and psychiatric causes of pain. We discuss several specific conditions, as well as the signs, symptoms, and basic management.
