title: Effects of ammonia on high affinity glutamate uptake and glutamate transporter EAAT3 expression in cultured rat cerebellar granule cells.
abstract: Increased levels of extracellular glutamate are a consistent feature of hepatic encephalopathy (HE) associated with liver failure and other hyperammonemic pathologies. Reduction of glutamate uptake has been described in ammonia-exposed cultured astrocytes, synaptosomes, and in animal models of hyperammonemia. In the present study, we examine the effects of pathophysiological concentrations of ammonia on D-aspartate (a non-metabolizable analog of glutamate) uptake by cultured rat cerebellar granule neurons. Exposure of these cells to ammonia resulted in time-dependent (24% reduction at 24h and 60% reduction at 5 days, P<0.001) and dose-dependent (21, 37, and 57% reduction at 1, 2.5, and 5mM for 5 days, P<0.01) suppression of D-aspartate uptake. Kinetic analyses revealed significant decreases in the velocity of uptake (V(max)) (37% decrease at 2.5mM NH(4)Cl, P<0.05 and 52% decrease at 5mM NH(4)Cl, P<0.001) as well as significant reductions in K(m) values (25% reduction at 2.5mM NH(4)Cl, P<0.05 and 45% reduction at 5mM NH(4)Cl, P<0.001). Western blotting, on the other hand, showed no significant changes in the neuronal glutamate transporter EAAC1/EAAT3 protein, the only glutamate transporter currently known to be expressed by these cells. In addition, 1H combined with 13C-NMR spectroscopy studies using the stable isotope [1-13C]-glucose demonstrated a significant increase in intracellular glutamate levels derived from the oxidative metabolism of glucose, rather than from the deamidation of exogenous glutamine in cultured granule neurons exposed to ammonia. The present study provides evidence that the effects of ammonia on glutamate uptake are not solely an astrocytic phenomenon and that unlike the astrocytic glutamate transporter counterpart, EAAT3 protein expression in cultured cerebellar granule cells is not down-regulated when exposed to ammonia. Decrease of glutamate uptake in these cellular preparations may afford an additional regulatory mechanism aimed at controlling intracellular levels of glutamate and ultimately the releasable pool of glutamate in neurons.
authors.affiliation: Neuroscience Research Unit, Centre Hospitalier de l'Université de Montréal, Campus Saint-Luc, 1058 St-Denis St., Que., H2X 3J4, Montreal, Canada. helen.chan@sympatico.ca
authors.forename: Helen
authors.initials: H
authors.lastname: Chan
authors.forename: Claudia
authors.initials: C
authors.lastname: Zwingmann
authors.forename: Marc
authors.initials: M
authors.lastname: Pannunzio
authors.forename: Roger F
authors.initials: RF
authors.lastname: Butterworth
mesh_terms: D027322:Amino Acid Transport System X-AG / Q000378:metabolism*; D000643:Ammonium Chloride / Q000494:pharmacology*; D000704:Analysis of Variance; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D002247:Carbon Isotopes; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D050580:Excitatory Amino Acid Transporter 3; D005947:Glucose / Q000378:metabolism; D050491:Glutamate Plasma Membrane Transport Proteins; D018698:Glutamic Acid / Q000378:metabolism*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000378:metabolism; D027981:Symporters / Q000378:metabolism*
chemical_list: D027322:Amino Acid Transport System X-AG; D002247:Carbon Isotopes; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; C493398:Slc1a1 protein, rat; D027981:Symporters; D000643:Ammonium Chloride; D001224:Aspartic Acid; D018698:Glutamic Acid; D012964:Sodium; D007770:L-Lactate Dehydrogenase; D005947:Glucose