Elevated ammonium levels: differential acute effects on three glutamate transporter isoforms

TY - JOUR

T1 - Elevated ammonium levels

T2 - differential acute effects on three glutamate transporter isoforms

AU - Søgaard, Rikke

AU - Novak, Ivana

AU - MacAulay, Nanna

PY - 2012/3

Y1 - 2012/3

N2 - Increased ammonium (NH(4)(+)/NH(3)) in the brain is a significant factor in the pathophysiology of hepatic encephalopathy, which involves altered glutamatergic neurotransmission. In glial cell cultures and brain slices, glutamate uptake either decreases or increases following acute ammonium exposure but the factors responsible for the opposing effects are unknown. Excitatory amino acid transporter isoforms EAAT1, EAAT2, and EAAT3 were expressed in Xenopus oocytes to study effects of ammonium exposure on their individual function. Ammonium increased EAAT1- and EAAT3-mediated [(3)H]glutamate uptake and glutamate transport currents but had no effect on EAAT2. The maximal EAAT3-mediated glutamate transport current was increased but the apparent affinities for glutamate and Na(+) were unaltered. Ammonium did not affect EAAT3-mediated transient currents, indicating that EAAT3 surface expression was not enhanced. The ammonium-induced stimulation of EAAT3 increased with increasing extracellular pH, suggesting that the gaseous form NH(3) mediates the effect. An ammonium-induced intracellular alkalinization was excluded as the cause of the enhanced EAAT3 activity because 1) ammonium acidified the oocyte cytoplasm, 2) intracellular pH buffering with MOPS did not reduce the stimulation, and 3) ammonium enhanced pH-independent cysteine transport. Our data suggest that the ammonium-elicited uptake stimulation is not caused by intracellular alkalinization or changes in the concentrations of cotransported ions but may be due to a direct effect on EAAT1/EAAT3. We predict that EAAT isoform-specific effects of ammonium combined with cell-specific differences in EAAT isoform expression may explain the conflicting reports on ammonium-induced changes in glial glutamate uptake.

AB - Increased ammonium (NH(4)(+)/NH(3)) in the brain is a significant factor in the pathophysiology of hepatic encephalopathy, which involves altered glutamatergic neurotransmission. In glial cell cultures and brain slices, glutamate uptake either decreases or increases following acute ammonium exposure but the factors responsible for the opposing effects are unknown. Excitatory amino acid transporter isoforms EAAT1, EAAT2, and EAAT3 were expressed in Xenopus oocytes to study effects of ammonium exposure on their individual function. Ammonium increased EAAT1- and EAAT3-mediated [(3)H]glutamate uptake and glutamate transport currents but had no effect on EAAT2. The maximal EAAT3-mediated glutamate transport current was increased but the apparent affinities for glutamate and Na(+) were unaltered. Ammonium did not affect EAAT3-mediated transient currents, indicating that EAAT3 surface expression was not enhanced. The ammonium-induced stimulation of EAAT3 increased with increasing extracellular pH, suggesting that the gaseous form NH(3) mediates the effect. An ammonium-induced intracellular alkalinization was excluded as the cause of the enhanced EAAT3 activity because 1) ammonium acidified the oocyte cytoplasm, 2) intracellular pH buffering with MOPS did not reduce the stimulation, and 3) ammonium enhanced pH-independent cysteine transport. Our data suggest that the ammonium-elicited uptake stimulation is not caused by intracellular alkalinization or changes in the concentrations of cotransported ions but may be due to a direct effect on EAAT1/EAAT3. We predict that EAAT isoform-specific effects of ammonium combined with cell-specific differences in EAAT isoform expression may explain the conflicting reports on ammonium-induced changes in glial glutamate uptake.

KW - Ammonium Chloride

KW - Animals

KW - Biological Transport

KW - Carbon Radioisotopes

KW - Cloning, Molecular

KW - Excitatory Amino Acid Transporter 1

KW - Excitatory Amino Acid Transporter 2

KW - Excitatory Amino Acid Transporter 3

KW - Female

KW - Glutamic Acid

KW - Humans

KW - Hydrogen-Ion Concentration

KW - Methylamines

KW - Oocytes

KW - Patch-Clamp Techniques

KW - Protein Isoforms

KW - Quaternary Ammonium Compounds

KW - Scintillation Counting

KW - Synaptic Transmission

KW - Tritium

KW - Xenopus laevis

U2 - 10.1152/ajpcell.00238.2011

DO - 10.1152/ajpcell.00238.2011

M3 - Journal article

C2 - 22159086

SN - 0363-6143

VL - 302

SP - C880-C891

JO - American Journal of Physiology: Cell Physiology

JF - American Journal of Physiology: Cell Physiology

IS - 6

ER -