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

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.

Keywords: ammonia; astrocytes; asymmetric dimethylarginine (ADMA); nitric oxide; rat brain endothelial cell line (RBE-4) cells; symmetric dimethylarginine (SDMA); y+LAT2.

Scheme 1

Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are formed by the methylation of arginine residues in a variety of proteins catalyzed by protein arginine methyltransferases (PRMT). After proteolysis, arginine and its methylated forms are released as free forms. ADMA elimination occurs primarily and specifically by its degradation by dimethylarginine dimethyl aminohydrolases (DDAH-1 and -2), wherein only DDAH-1 is widely expressed in the brain [20,21]. In turn, SDMA is extracted in urine.

Figure 1

The effect of silencing of the Slc7a6 gene on y⁺LAT2 protein level in astrocytes in relation to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein level. Upper panel shows representative Western blots. Results are mean ± SD; n = 4, * p < 0.05 vs. control; ^# p < 0.05 vs. ammonia.

Figure 2

Expression of mRNAs coding for enzymes of ADMA metabolism, PRMT1 and DDAH1 genes in control and ammonia-treated astrocytes.

Figure 3

PRMT-1 and DDAH-1 protein level in primary astrocytes treated with 5 mM ammonia for 48 h. Upper panel shows representative Western blots.

Figure 4

The effect of ammonia and Slc7a6 gene silencing on nitrite+nitrate (NOx) concentration in the medium derived from cultured astrocytes. Result are mean ± SD; n = 5, * p < 0.05 vs control, ^# p < 0.05 vs. ammonia.

Figure 5

The effect of ADMA on [³H]arginine uptake in control and 5 mM ammonia treated primary astrocytes cultures. Result are mean ± SD; n = 6, * p < 0.01vs control, ^# p < 0.01 vs. ammonia.