title: Depolarization-induced release of [(3)H]D-aspartate from GABAergic neurons caused by reversal of glutamate transporters.
abstract: Cultured neocortical neurons, which predominantly consist of GABAergic neurons exhibit a pronounced stimulus-coupled GABA release. Since the cultures may contain a small population of glutamatergic neurons and the GABAergic neurons have a high content of glutamate it was of interest to examine if glutamate in addition to gamma-aminobutyric acid (GABA) could be released from these cultures. The neurons were preloaded with [(3)H]D-aspartate and subsequently its release was followed during depolarization induced by a high potassium concentration or the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor agonists, AMPA and kainate. Depolarization of the neurons with 55 mM potassium increased the release of [(3)H]D-aspartate by more than 10-fold. When the non-specific calcium-channel blockers cobalt or lanthanum were included in the stimulation buffer with potassium, the release of [(3)H]D-aspartate was decreased by about 40%. These results indicated that some of the released [(3)H]D-aspartate might originate from a vesicular pool. When AMPA was applied to the neurons, the release of [(3)H]D-aspartate was increased 2-fold and could not be prevented or decreased by addition of cobalt. Since AMPA has a rapid desensitizing effect on AMPA receptors, it was examined whether AMPA under non-desensitizing conditions was able to induce an increased release of [(3)H]D-aspartate as compared to the conditions of applying AMPA alone. The desensitization of AMPA receptors was blocked by 6-chloro-3,4-dihydro-3-(2-norbornen-5-yl)-2H-1,2, 4-benzothiadiazine-7-sulphonamide-1,1-dioxide (cyclothiazide). Under the non-desensitizing conditions, the AMPA-induced release of [(3)H]D-aspartate was highly enhanced showing about a 10-fold increase over basal release. Addition of cobalt or lanthanum did not decrease the amount of [(3)H]D-aspartate released, indicating that the release originated from a cytoplasmic pool. Kainate, which induces an almost non-desensitizing effect on AMPA receptors, showed similar results as observed for AMPA under non-desensitizing conditions. The NMDA receptor antagonist (5R,10 S)-(+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) had only minor effects on the [(3)H]D-aspartate release induced by AMPA and kainate. Thus, the depolarization-induced release of [(3)H]D-aspartate from cultured GABAergic neurons appears to be caused mainly by reversal of the glutamate transporters.
authors.affiliation: The Royal Danish School of Pharmacy, PharmaBiotec Research Center, Department of Pharmacology, Universitetsparken 2, DK-2100, Copenhagen, Denmark.
authors.forename: J B
authors.initials: JB
authors.lastname: Jensen
authors.forename: D S
authors.initials: DS
authors.lastname: Pickering
authors.forename: A
authors.initials: A
authors.lastname: Schousboe
mesh_terms: D000818:Animals; D001224:Aspartic Acid / Q000493:pharmacokinetics*; D001581:Benzothiadiazines / Q000494:pharmacology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D004232:Diuretics; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D005260:Female; D018698:Glutamic Acid / Q000378:metabolism; D007608:Kainic Acid / Q000494:pharmacology; D051379:Mice; D008815:Mice, Inbred Strains; D009474:Neurons / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D011247:Pregnancy; D018091:Receptors, AMPA / Q000378:metabolism*; D018092:Kainic Acid Receptors / Q000378:metabolism*; D049993:Sodium Chloride Symporter Inhibitors / Q000494:pharmacology; D014316:Tritium; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000502:physiology*
chemical_list: D001224:Aspartic Acid; D001581:Benzothiadiazines; D004232:Diuretics; D018690:Excitatory Amino Acid Agonists; D018698:Glutamic Acid; D007608:Kainic Acid; D011188:Potassium; D018091:Receptors, AMPA; D018092:Kainic Acid Receptors; D049993:Sodium Chloride Symporter Inhibitors; D014316:Tritium; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D005680:gamma-Aminobutyric Acid; C004639:cyclothiazide