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Int. J. Mol. Sci. 2017, 18(11), 2308; https://doi.org/10.3390/ijms18112308

Ammonia Reduces Intracellular Asymmetric Dimethylarginine in Cultured Astrocytes Stimulating Its y+LAT2 Carrier-Mediated Loss

1
Department of Neurotoxicology, Mossakowski Medical Research Centre, Polish Academy of Sciences, 02-106 Warsaw, Poland
2
Mass Spectrometry Laboratory, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland
*
Author to whom correspondence should be addressed.
Received: 25 September 2017 / Revised: 19 October 2017 / Accepted: 27 October 2017 / Published: 2 November 2017
(This article belongs to the Special Issue Amino Acids Transport and Metabolism)
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Abstract

Previously we had shown that ammonia stimulates nitric oxide (NO) synthesis in astrocytes by increasing the uptake of the precursor amino acid, arginine via the heteromeric arginine/glutamine transporter y+LAT2. Ammonia also increases the concentration in the brain of the endogenous inhibitor of nitric oxide synthases (NOS), asymmetric dimethylarginine (ADMA), but distribution of ADMA surplus between the intraastrocytic and extracellular compartments of the brain has not been studied. Here we tested the hypothesis that ammonia modulates the distribution of ADMA and its analog symmetric dimethylarginine (SDMA) between the two compartments of the brain by competition with arginine for the y+LAT2 transporter. In extension of the hypothesis we analyzed the ADMA/Arg interaction in endothelial cells forming the blood-brain barrier. We measured by high-performance liquid chromatography (HPLC) and mass spectrometry (MS) technique the concentration of arginine, ADMA and SDMA in cultured cortical astrocytes and in a rat brain endothelial cell line (RBE-4) treated with ammonia and the effect of silencing the expression of a gene coding y+LAT2. We also tested the expression of ADMA metabolism enzymes: protein arginine methyltransferase (PRMT) and dimethylarginine dimethyl aminohydrolase (DDAH) and arginine uptake to astrocytes. Treatment for 48 h with 5 mM ammonia led to an almost 50% reduction of ADMA and SDMA concentration in both cell types, and the effect in astrocytes was substantially attenuated by silencing of the Slc7a6 gene. Moreover, the y+LAT2-dependent component of ammonia-evoked arginine uptake in astrocytes was reduced in the presence of ADMA in the medium. Our results suggest that increased ADMA efflux mediated by upregulated y+LAT2 may be a mechanism by which ammonia interferes with intra-astrocytic (and possibly intra-endothelial cell) ADMA content and subsequently, NO synthesis in both cell types. View Full-Text
Keywords: astrocytes; rat brain endothelial cell line (RBE-4) cells; ammonia; nitric oxide; asymmetric dimethylarginine (ADMA); symmetric dimethylarginine (SDMA); y+LAT2 astrocytes; rat brain endothelial cell line (RBE-4) cells; ammonia; nitric oxide; asymmetric dimethylarginine (ADMA); symmetric dimethylarginine (SDMA); y+LAT2
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).
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Milewski, K.; Bogacińska-Karaś, M.; Fręśko, I.; Hilgier, W.; Jaźwiec, R.; Albrecht, J.; Zielińska, M. Ammonia Reduces Intracellular Asymmetric Dimethylarginine in Cultured Astrocytes Stimulating Its y+LAT2 Carrier-Mediated Loss. Int. J. Mol. Sci. 2017, 18, 2308.

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