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Title: Excitatory amino acid-stimulated uptake of /sup 22/Na+ in primary astrocyte cultures

Abstract

In this study we have found that L-glutamic acid, as well as being taken up by a Na+-dependent mechanism, will stimulate the uptake of 22Na+ by primary astrocyte cultures from rat brain in the presence of ouabain. By simultaneously measuring the uptake of 22Na+ and L-3H-glutamate a stoichiometry of 2-3 Na+ per glutamate was measured, implying electrogenic uptake. Increasing the medium K+ concentration to depolarize the cells inhibited L-3H-glutamate uptake, while calculations of the energetics of the observed L-3H-glutamate accumulation also supported an electrogenic mechanism of at least 2 Na+:1 glutamate. In contrast, kinetic analysis of the Na+ dependence of L-3H-glutamate uptake indicated a stoichiometry of Na+ to glutamate of 1:1, but further analysis showed that the stoichiometry cannot be resolved by purely kinetic studies. Studies with glutamate analogs, however, showed that kainic acid was a very effective stimulant of 22Na+ uptake, but 3H-kainic acid showed no Na+ -dependent uptake. Furthermore, while L-3H-glutamate uptake was very sensitive to lowered temperatures, glutamate-stimulated 22Na+ uptake was relatively insensitive. These results indicate that glutamate-stimulated uptake of 22Na+ in primary astrocytes cultures cannot be explained solely by cotransport of Na+ with glutamate, and they suggest that direct kainic acid-type receptor induced stimulation of Na+more » uptake also occurs. Since both receptor and uptake effects involve transport of Na+, accurate measurements of the Na+ :glutamate stoichiometry for uptake can only be done using completely specific inhibitors of these 2 systems.« less

Authors:
; ;
Publication Date:
Research Org.:
Albany Medical College, Albany, NY (USA)
OSTI Identifier:
6258041
Resource Type:
Journal Article
Resource Relation:
Journal Name: J. Neurosci.; (United States); Journal Volume: 9:4
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; AMINO ACIDS; BIOCHEMICAL REACTION KINETICS; SODIUM COMPOUNDS; UPTAKE; ANIMAL CELLS; BRAIN; CATIONS; CELL CULTURES; GLUTAMIC ACID; MEMBRANE TRANSPORT; NERVE CELLS; OUABAIN; POTASSIUM; RATS; SODIUM 22; STOICHIOMETRY; TRACER TECHNIQUES; TRITIUM COMPOUNDS; ALKALI METAL COMPOUNDS; ALKALI METAL ISOTOPES; ALKALI METALS; ANIMALS; BETA DECAY RADIOISOTOPES; BETA-PLUS DECAY RADIOISOTOPES; BODY; CARBOHYDRATES; CARBOXYLIC ACIDS; CARDIAC GLYCOSIDES; CARDIOTONICS; CARDIOVASCULAR AGENTS; CENTRAL NERVOUS SYSTEM; CHARGED PARTICLES; DRUGS; ELEMENTS; GLYCOSIDES; IONS; ISOTOPE APPLICATIONS; ISOTOPES; KINETICS; LABELLED COMPOUNDS; LIGHT NUCLEI; MAMMALS; METALS; NERVOUS SYSTEM; NUCLEI; ODD-ODD NUCLEI; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANS; RADIOISOTOPES; REACTION KINETICS; RODENTS; SODIUM ISOTOPES; SOMATIC CELLS; STROPHANTHINS; VERTEBRATES; YEARS LIVING RADIOISOTOPES 550201* -- Biochemistry-- Tracer Techniques

Citation Formats

Kimelberg, H.K., Pang, S., and Treble, D.H. Excitatory amino acid-stimulated uptake of /sup 22/Na+ in primary astrocyte cultures. United States: N. p., 1989. Web.
Kimelberg, H.K., Pang, S., & Treble, D.H. Excitatory amino acid-stimulated uptake of /sup 22/Na+ in primary astrocyte cultures. United States.
Kimelberg, H.K., Pang, S., and Treble, D.H. 1989. "Excitatory amino acid-stimulated uptake of /sup 22/Na+ in primary astrocyte cultures". United States. doi:.
@article{osti_6258041,
title = {Excitatory amino acid-stimulated uptake of /sup 22/Na+ in primary astrocyte cultures},
author = {Kimelberg, H.K. and Pang, S. and Treble, D.H.},
abstractNote = {In this study we have found that L-glutamic acid, as well as being taken up by a Na+-dependent mechanism, will stimulate the uptake of 22Na+ by primary astrocyte cultures from rat brain in the presence of ouabain. By simultaneously measuring the uptake of 22Na+ and L-3H-glutamate a stoichiometry of 2-3 Na+ per glutamate was measured, implying electrogenic uptake. Increasing the medium K+ concentration to depolarize the cells inhibited L-3H-glutamate uptake, while calculations of the energetics of the observed L-3H-glutamate accumulation also supported an electrogenic mechanism of at least 2 Na+:1 glutamate. In contrast, kinetic analysis of the Na+ dependence of L-3H-glutamate uptake indicated a stoichiometry of Na+ to glutamate of 1:1, but further analysis showed that the stoichiometry cannot be resolved by purely kinetic studies. Studies with glutamate analogs, however, showed that kainic acid was a very effective stimulant of 22Na+ uptake, but 3H-kainic acid showed no Na+ -dependent uptake. Furthermore, while L-3H-glutamate uptake was very sensitive to lowered temperatures, glutamate-stimulated 22Na+ uptake was relatively insensitive. These results indicate that glutamate-stimulated uptake of 22Na+ in primary astrocytes cultures cannot be explained solely by cotransport of Na+ with glutamate, and they suggest that direct kainic acid-type receptor induced stimulation of Na+ uptake also occurs. Since both receptor and uptake effects involve transport of Na+, accurate measurements of the Na+ :glutamate stoichiometry for uptake can only be done using completely specific inhibitors of these 2 systems.},
doi = {},
journal = {J. Neurosci.; (United States)},
number = ,
volume = 9:4,
place = {United States},
year = 1989,
month = 4
}
  • Primary astrocyte cultures prepared from the cerebral cortices of neonatal rats showed significant accumulation of serotonin (5-hydroxytryptamine; (/sup 3/H)-5-HT). At concentrations in the range of 0.01 to 0.7 microM (/sup 3/H)-5-HT, this uptake was 50 to 85% Na+ dependent and gave a Km of 0.40 +/- 0.11 microM (/sup 3/H)-5-HT and a Vmax of 6.42 +/- 0.85 (+/- SEM) pmol of (/sup 3/H)-5-HT/mg of protein/4 min for the Na+-dependent component. In the absence of Na+ the uptake was nonsaturable. Omission of the monoamine oxidase inhibitor pargyline markedly reduced the Na+-dependent component of (/sup 3/H)-5-HT uptake but had a negligible effectmore » on the Na+-independent component. This suggest significant oxidative deamination of serotonin after it has been taken up by the high affinity system, followed by release of its metabolite. The authors estimated that this system enabled the cells to concentrate (/sup 3/H)-5-HT up to 44-fold at an external (/sup 3/H)-5-HT concentration of 10(-7) M. Inhibition of (/sup 3/H)-5-HT uptake by a number of clinically effective antidepressants was also consistent with a specific high affinity uptake mechanism for 5-HT, the order of effectiveness of inhibition being chlorimipramine greater than fluoxetine greater than imipramine = amitriptyline greater than desmethylimipramine greater than iprindole greater than mianserin. Uptake of (/sup 3/H)-5-HT was dependent on the presence of Cl- as well as Na+ in the medium, and the effect of omission of both ions was nonadditive. Varying the concentration of K+ in the media from 1 to 50 mM had a limited effect on (/sup 3/H)-5-HT uptake.« less
  • A functional N-methyl-D-aspartate (NMDA) receptor has been identified on HT-4 cells, a clonal neural cell line, in which glutamate activates the receptor to elicit neurotransmitter secretion. Specific inhibitors of the NMDA receptor block-glutamate-mediate secretion, and the characteristics of NMDA-mediated secretion parallel the reported properties of the NMDA receptor. Excitatory amino acid secretion can be elicited by potassium-evoked depolarization and is not the simple reversal of the uptake system. 2-Amino-4-phosphonobutyrate (APB) inhibits depolarization-induced secretion of excitatory amino acids but has no effect on excitatory amino acid uptake, suggesting that the APB binding protein in the brain represents a component involved inmore » the secretion of excitatory amino acids.« less
  • The isoxazole amino acid 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionic acid (AMPA) (1), which is a highly selective agonist at the AMPA subtype of excitatory amino acid (EAA) receptors, has been used as a lead for the development of two novel EAA receptor antagonists. One of the compounds, 2-amino-3-(3-(carboxymethoxy)-5-methylisoxazol-4-yl)propionic acid (AMOA, 7), was synthesized via O-alkylation by ethyl chloroacetate of the amino acid protected AMPA derivative 4. The other compound, 2-amino-3-(2-(3-hydroxy-5-methylisoxazol-4-yl)-methyl-5-methyl-3-+ ++oxoisoxazolin -4-yl)propionic acid (AMNH, 14) was synthesized with use of 4-(chloromethyl)-3-methoxy-5-methylisoxazole (8) as the starting material. The intermediate 4-(chloromethyl)-2-(3-methoxy-5-methylisoxazol-4-yl)methyl-5-me thylisoxazolin- 3-one (11) was converted into the acetamidomalonate (12), which was stepwise deprotected tomore » give 14. Compounds 7 and 14 were stable in aqueous solution at pH values close to physiological pH. Neither 7 nor 14 showed detectable affinities for the receptor, ion channel, or modulatory sites of the N-methyl-D-aspartic acid (NMDA) receptor complex. Quantitative receptor autoradiographic and conventional binding techniques were used to study the affinities of 7 and 14 for non-NMDA receptor sites. Both compounds were inhibitors of the binding of (3H)AMPA (IC50 = 90 and 29 microM, respectively). Compounds 14 and 7 were both very weak inhibitors of the high-affinity binding of radioactive kainic acid ((3H)KAIN). Compound 14, but not 7, was, however, shown to be an inhibitor of low-affinity (3H)KAIN binding as determined in the presence of 100 mM calcium chloride. In the rat cortical slice preparation, 7 was shown to antagonize excitation induced by 1 with some selectivity, whereas 14 proved to be a rather selective antagonist of KAIN-induced excitation.« less
  • The effect of ATP and other purines on /sup 45/Ca uptake was studied in primary cultures of rat astrocytes. Treatment of the cells with ATP for 1 to 30 min brought about an increase in cellular /sup 45/Ca. Stimulation of calcium influx by ATP was investigated using a 90 sec exposure to /sup 45/Ca and over a concentration range of 0.1 nM to 3 mM; a biphasic dose-response curve was obtained with EC50 values of 0.3 nM and 9 uM, indicating the presence of low and high affinity purinergic binding sites. Similar levels of /sup 45/Ca influx at 90 secmore » were observed with ATP, ADP and adenosine (all at 100 uM). Prior treatment of the cultures with LaCl3 blocked the purine-induced /sup 45/Ca influx. These findings indicate that one pathway for calcium entry in astrocytes involves purinergic receptor-operated, calcium channels.« less