ABSTRACT
A glutamate-sensitive inward current (Iglu) is described in rat cerebellar granule neurons and related to a glutamate transport mechanism. We examined the features of Iglu using the patch-clamp technique. In steady-state conditions the Iglu measured 8.14 1.9 pA. Iglu was identified as a voltage-dependent inward current showing a strong rectification at positive potentials. L-Glutamate activated the inward current in a dose-dependent manner, with a half-maximal effect at about 18 M and a maximum increase of 51.2 4.4%. The inward current was blocked by the presence of dihydrokainate (0.5 mM), shown by others to readily block the GLT1 isoform. We thus speculate that Iglu could be attributed to the presence of a native glutamate transporter in cerebellar granule neurons.
Subject(s)
Amino Acid Transport System X-AG/physiology , Cerebellum/cytology , Neuroglia/physiology , Animals , Cells, Cultured , Membrane Potentials , Patch-Clamp Techniques , Rats , Rats, WistarABSTRACT
A glutamate-sensitive inward current (Iglu) is described in rat cerebellar granule neurons and related to a glutamate transport mechanism. We examined the features of Iglu using the patch-clamp technique. In steady-state conditions the Iglu measured 8.14 ± 1.9 pA. Iglu was identified as a voltage-dependent inward current showing a strong rectification at positive potentials. L-Glutamate activated the inward current in a dose-dependent manner, with a half-maximal effect at about 18 æM and a maximum increase of 51.2 ± 4.4 percent. The inward current was blocked by the presence of dihydrokainate (0.5 mM), shown by others to readily block the GLT1 isoform. We thus speculate that Iglu could be attributed to the presence of a native glutamate transporter in cerebellar granule neurons
Subject(s)
Animals , Rats , Cerebellum , Neuroglia , Cells, Cultured , Membrane Potentials , Patch-Clamp Techniques , Rats, WistarABSTRACT
To determine whether [Ca(2+)](e) modulates glutamate re-uptake, we studied the uptake mechanism into rat cerebrocortical synaptosomes. The removal of extracellular Ca(2+) caused a negative modulation in the uptake mechanism. The calculated K(50) value was 0.185 +/- 0.019 mM (n = 4). The Michaelis-Menten data analysis indicate that absence of Ca(2+) diminished the V(max) kinetic parameter by about 60% without changing significantly the K(m) suggesting a non-competitive mechanism. We also tested the involvement of intracellular Ca(2+) in this phenomenon by trapping BAPTA into the synaptosomal vesicles to control the Ca(2+) concentration. Our results suggest that intracellular Ca(2+) changes have a less predominant role on the glutamate uptake than do extracellular Ca(2+). These findings argue in favor of an important role of extracellular [Ca(2+)] in maintaining the L-glutamate re-uptake mechanism in the mammalian central nervous system.
Subject(s)
Calcium Signaling/physiology , Calcium/deficiency , Cerebral Cortex/metabolism , Egtazic Acid/analogs & derivatives , Extracellular Space/metabolism , Glutamic Acid/metabolism , Presynaptic Terminals/metabolism , Synaptic Transmission/physiology , Animals , Calcium/pharmacology , Calcium Signaling/drug effects , Cerebral Cortex/drug effects , Chelating Agents , Egtazic Acid/pharmacology , Extracellular Space/drug effects , Female , Glutamic Acid/pharmacokinetics , L-Lactate Dehydrogenase/metabolism , Male , Pharmacokinetics , Presynaptic Terminals/drug effects , Rats , Rats, Wistar , Synaptic Transmission/drug effects , Synaptosomes/drug effects , Synaptosomes/metabolism , TritiumABSTRACT
There is evidence that extracellular glutamate levels are elevated in certain brain regions immediately prior to and during induction and propagation of seizures. There appears to be a correlation between the capacity of removing released glutamate and the genesis of epileptiform activity. Some models make use of metals, such as Co(2+) and Ni(2+), to induce epilepsy. We used patch-clamp recordings to measure the electrogenic glutamate transport in neuronal cells. The present results indicate that Co(2+) (1 mM) and Ni(2+) (5 mM) blocked glutamate transport by 17.6+/-3.9% (n=5, P<0.05) and by 31.8+/-6.2% (n=7, P<0.05), respectively. Ni(2+) inhibited glutamate uptake in a dose-dependent manner. The IC(50) value obtained was 66.6 microM and the maximum inhibition was 40%. We conclude that one mechanism that may explain the seizures induced by exposure to those divalent cations is inhibition of the glutamate transporter.
Subject(s)
Cerebellum/cytology , Cobalt/pharmacology , Convulsants/pharmacology , Glutamic Acid/metabolism , Neurons/metabolism , Amino Acid Transport System X-AG/metabolism , Animals , Biological Transport/drug effects , Biological Transport/physiology , Cells, Cultured , Epilepsy/chemically induced , Epilepsy/metabolism , Nickel/pharmacology , Patch-Clamp Techniques , Rats , Rats, WistarABSTRACT
We report the characterization of a new class of glutamate uptake inhibitors isolated from Phoneutria nigriventer venom. Glutamate transport activity was assayed in rat cerebrocortical synaptosomes by using [(3)H]-L-glutamate. PhTx4 inhibited glutamate uptake in a dose dependent manner. The IC(50) value obtained was 2.35+/-0.9 microg/ml which is in the observed range reported for glutamate uptake blockers. Tx4-7, one of PhTx4 toxins, showed the strongest inhibitory activity (50.3+/-0.69%, n=3).
