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Acute insult of ammonia leads to calcium-dependent glutamate release from cultured astrocytes: An effect of pH

Item Type:Article
Title:Acute insult of ammonia leads to calcium-dependent glutamate release from cultured astrocytes: An effect of pH
Creators Name:Rose, C. and Kresse, W. and Kettenmann, H.
Abstract:Hyperammonemia is a key factor in the pathogenesis of hepatic encephalopathy (HE) as well as other metabolic encephalopathies, such as those associated with inherited disorders of urea cycle enzymes and in Reye's syndrome. Acute HE results in increased brain ammonia (up to 5 mM), astrocytic swelling, and altered glutamatergic function. In the present study, using fluorescence imaging techniques, acute exposure (10 min) of ammonia (NH 4+/NH3) to cultured astrocytes resulted in a concentration-dependent, transient increase in [Ca2+]i. This calcium transient was due to release from intracellular calcium stores, since the response was thapsigargin-sensitive and was still observed in calcium-free buffer. Using an enzyme-linked fluorescence assay, glutamate release was measured indirectly via the production of NADH (a naturally fluorescent product when excited with UV light). NH4+/NH3 (5 mM) stimulated a calcium-dependent glutamate release from cultured astrocytes, which was inhibited after preincubation with 1,2-bis(2-aminophenoxy)ethane-N,N,N,′N′-tetraacetic acid acetoxymethyl ester but unaffected after preincubation with glutamate transport inhibitors dihydrokainate and DL-threo-β-benzyloxyaspartate. NH 4+/NH3 (5 mM) also induced a transient intracellular alkaline shift. To investigate whether the effects of NH 4+/ NH3 were mediated by an increase in pH i, we applied trimethylamine (TMA+/TMA) as another weak base. TMA+/TMA (5 IMM) induced a similar transient increase in both pHi and [Ca2+]i (mobilization from intracellular calcium stores) and resulted in calcium-dependent release of glutamate. These results indicate that an acute exposure to ammonia, resulting in cytosolic alkalinization, leads to calcium-dependent glutamate release from astrocytes. A deregulation of glutamate release from astrocytes by ammonia could contribute to glutamate dysfunction consistently observed in acute HE.
Keywords:Adenosine Triphosphate, Amino Acid Transport System X-AG, Ammonia, Aspartic Acid, Astrocytes, Calcium, Cultured Cells, Drug Dose-Response Relationship, Egtazic Acid, Endoplasmic Reticulum, Fluoresceins, Glutamic Acid, Hydrogen-Ion Concentration, Kainic Acid, Methylamines, Fluorescence Microscopy, Fluorescence Spectrometry, Thapsigargin, Ultraviolet Rays, Animals, Mice
Source:Journal of Biological Chemistry
ISSN:0021-9258
Publisher:American Society for Biochemistry and Molecular Biology (U.S.A.)
Volume:280
Number:22
Page Range:20937-20944
Date:1 January 2005
Official Publication:https://doi.org/10.1074/jbc.M412448200
PubMed:View item in PubMed

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