title: Ammonium-evoked alterations in intracellular sodium and pH reduce glial glutamate transport activity.
abstract: The clearance of extracellular glutamate is mainly mediated by pH- and sodium-dependent transport into astrocytes. During hepatic encephalopathy (HE), however, elevated extracellular glutamate concentrations are observed. The primary candidate responsible for the toxic effects observed during HE is ammonium (NH(4) (+)/NH(3)). Here, we examined the effects of NH(4) (+)/NH(3) on steady-state intracellular pH (pH(i)) and sodium concentration ([Na(+)](i)) in cultured astrocytes in two different age groups. Moreover, we assessed the influence of NH(4) (+)/NH(3) on glutamate transporter activity by measuring D-aspartate-induced pH(i) and [Na(+)](i) transients. In 20-34 days in vitro (DIV) astrocytes, NH(4) (+)/NH(3) decreased steady-state pH(i) by 0.19 pH units and increased [Na(+)](i) by 21 mM. D-Aspartate-induced pH(i) and [Na(+)](i) transients were reduced by 80-90% in the presence of NH(4) (+)/NH(3), indicating a dramatic reduction of glutamate uptake activity. In 9-16 DIV astrocytes, in contrast, pH(i) and [Na(+)](i) were minimally affected by NH(4) (+)/NH(3), and D-aspartate-induced pH(i) and [Na(+)](i) transients were reduced by only 30-40%. Next we determined the contribution of Na(+), K(+), Cl(-)-cotransport (NKCC). Immunocytochemical stainings indicated an increased expression of NKCC1 in 20-34 DIV astrocytes. Moreover, inhibition of NKCC with bumetanide prevented NH(4) (+)/NH(3)-evoked changes in steady-state pH(i) and [Na(+)](i) and attenuated the reduction of D-aspartate-induced pH(i) and [Na(+)](i) transients by NH(4) (+)/NH(3) to 30% in 20-34 DIV astrocytes. Our results suggest that NH(4) (+)/NH(3) decreases steady-state pH(i) and increases steady-state [Na(+)](i) in astrocytes by an age-dependent activation of NKCC. These NH(4) (+)/NH(3)-evoked changes in the transmembrane pH and sodium gradients directly reduce glutamate transport activity, and may, thus, contribute to elevated extracellular glutamate levels observed during HE.
authors.affiliation: Institut für Neurobiologie, Heinrich-Heine-Universität Düsseldorf, Universitätstr. 1, 40225 Düsseldorf, Germany. tony.kelly@uni-duesseldorf.de
authors.forename: Tony
authors.initials: T
authors.lastname: Kelly
authors.forename: Karl W
authors.initials: KW
authors.lastname: Kafitz
authors.forename: Claudia
authors.initials: C
authors.lastname: Roderigo
authors.forename: Christine R
authors.initials: CR
authors.lastname: Rose
mesh_terms: D000375:Aging; D000818:Animals; D001224:Aspartic Acid / Q000494:pharmacology; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D001692:Biological Transport; D002034:Bumetanide / Q000494:pharmacology; D002478:Cells, Cultured; D002491:Central Nervous System Agents / Q000494:pharmacology; D026603:D-Aspartic Acid / Q000494:pharmacology; D018698:Glutamic Acid / Q000378:metabolism*; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D000644:Quaternary Ammonium Compounds / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012964:Sodium / Q000378:metabolism*; D049994:Sodium Potassium Chloride Symporter Inhibitors / Q000494:pharmacology; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism; D064506:Solute Carrier Family 12, Member 2
chemical_list: D002491:Central Nervous System Agents; D000644:Quaternary Ammonium Compounds; C578879:Slc12a2 protein, rat; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; C120673:benzyloxyaspartate; D002034:Bumetanide; D001224:Aspartic Acid; D018698:Glutamic Acid; D026603:D-Aspartic Acid; D012964:Sodium