Abstract
Arachidonic acid has been proposed to be a messenger molecule released following synaptic activation of glutamate receptors and during ischemia. Here we demonstrate that micromolar levels of arachidonic acid inhibit glutamate uptake mediated by EAAT1, a human excitatory amino acid transporter widely expressed in brain and cerebellum, by reducing the maximal transport rate approximately 30%. In contrast, arachidonic acid increased transport mediated by EAAT2, a subtype abundantly expressed in forebrain and midbrain, by causing the apparent affinity for glutamate to increase more than 2-fold. The results demonstrate that the response of different glutamate transporter subtypes to arachidonic acid could influence synaptic transmission and modulate excitotoxicity via positive or negative feedback according to the transporter(s) present in a particular region.
Highlights
Arachidonic acid has been proposed to be a messenger molecule released following synaptic activation of glutamate receptors and during ischemia
We demonstrate that micromolar levels of arachidonic acid inhibit glutamate uptake mediated by EAATI, a human excitatory amino acid transporter widely expressed in brain and cerebellum, by reducing tbe maximal transport rate approximately 30%
Arachidonic acid has been proposed to be a messenger molecule that influences synaptic transmission released by synaptic activation of ionotropic and metabotropic glutamate receptors [8, 9]
Summary
Expression of EAAT Subtypes in Oocytes and HEK293 Cells---Capped RNA was transcribed from linearized pOTV plasmids containing the coding region of EAATl-3 [6] using T7 polymerase (Boehringer Mannheim). 50 ng of RNA was injected into stage V oocytes, and experiments were performed 2-6 days later. Expression of EAAT Subtypes in Oocytes and HEK293 Cells---Capped RNA was transcribed from linearized pOTV plasmids containing the coding region of EAATl-3 [6] using T7 polymerase (Boehringer Mannheim). HEK-293 cells stably transformed with the pCEP4/lE-EAAT plasmids were selected using hygromycin (Sigma) at a concentration of 250 /Lg/ml. Oocytes were voltage-clamped at -60 mV and continuously superfused with ND-96 recording solution or solution containing test compounds at various concentrations. The percentage decrease (EAAT1) or increase (EAAT2) in the transport current was calculated by comparing the current amplitudes induced by 30 p.M L-glutamate co-applied with varying concentrations of arachidonic acid to the control transport current amplitudes in the same oocytes.
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