title: Induction of inducible nitric oxide synthase expression in ammonia-exposed cultured astrocytes is coupled to increased arginine transport by upregulated y(+)LAT2 transporter.
abstract: One of the aspects of ammonia toxicity to brain cells is increased production of nitric oxide (NO) by NO synthases (NOSs). Previously we showed that ammonia increases arginine (Arg) uptake in cultured rat cortical astrocytes specifically via y(+)L amino acid transport system, by activation of its member, a heteromeric y(+)LAT2 transporter. Here, we tested the hypothesis that up-regulation of y(+)LAT2 underlies ammonia-dependent increase of NO production via inducible NOS (iNOS) induction, and protein nitration. Treatment of rat cortical astrocytes for 48 with 5 mM ammonium chloride ('ammonia') (i) increased the y(+)L-mediated Arg uptake, (ii) raised the expression of iNOS and endothelial NOS (eNOS), (iii) stimulated NO production, as manifested by increased nitrite+nitrate (Griess) and/or nitrite alone (chemiluminescence), and consequently, (iv) evoked nitration of tyrosine residues of proteins in astrocytes. Except for the increase of eNOS, all the above described effects of ammonia were abrogated by pre-treatment of astrocytes with either siRNA silencing of the Slc7a6 gene coding for y(+)LAT2 protein, or antibody to y(+)LAT2, indicating their strict coupling to y(+)LAT2 activity. Moreover, induction of y(+)LAT2 expression by ammonia was sensitive to Nf-κB inhibitor, BAY 11-7085, linking y(+)LAT2 upregulation to the Nf-κB activation in this experimental setting as reported earlier and here confirmed. Importantly, ammonia did not affect y(+)LAT2 expression nor y(+)L-mediated Arg uptake activity in the cultured cerebellar neurons, suggesting astroglia-specificity of the above described mechanism. The described coupling of up-regulation of y(+)LAT2 transporter with iNOS in ammonia-exposed astrocytes may be considered as a mechanism to ensure NO supply for protein nitration. Ammonia (NH4(+)) increases the expression and activity of the L-arginine (Arg) transporter (Arg/neutral amino acids [NAA] exchanger) y(+)LAT2 in cultured rat cortical astrocytes by a mechanism involving activation (nuclear translocation) of the transcription factor nuclear factor-Nuclear factor-κB (Nf-κB-p65). Up-regulation of y(+)LAT2 transporter is coupled with increased inducible nitric oxide synthase (iNOS) expression, which leads to increase nitric oxide (NO) synthesis and protein nitration.
authors.affiliation: Department of Neurotoxicology, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Zielińska
authors.affiliation: Department of Neurotoxicology, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Milewski
authors.affiliation: Department of Neurotoxicology, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Marta
authors.initials: M
authors.lastname: Skowrońska
authors.affiliation: Medical Center of Postgraduate Education, Warsaw, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Gajos
authors.affiliation: Department of Neurochemistry, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Elżbieta
authors.initials: E
authors.lastname: Ziemińska
authors.affiliation: Medical Center of Postgraduate Education, Warsaw, Poland.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Beręsewicz
authors.affiliation: Department of Neurotoxicology, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Jan
authors.initials: J
authors.lastname: Albrecht
mesh_terms: D027182:Amino Acid Transport System y+ / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001253:Astrocytes / Q000166:cytology*; D002478:Cells, Cultured; D027301:Fusion Regulatory Protein 1, Light Chains / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology*; D026901:Membrane Transport Proteins / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D017208:Rats, Wistar; D015533:Transcriptional Activation / Q000502:physiology*; D015854:Up-Regulation
chemical_list: D027182:Amino Acid Transport System y+; D027301:Fusion Regulatory Protein 1, Light Chains; D026901:Membrane Transport Proteins; D016328:NF-kappa B; C486582:Slc7a8 protein, rat; D009569:Nitric Oxide; D001120:Arginine; D052247:Nitric Oxide Synthase Type II
keywords: ammonia; arginine; astrocytes; nitration; nitric oxide synthase; y+LAT2 transporter