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.

Transport of L-Arginine Related Cardiovascular Risk Markers

“…A number of proinflammatory cytokines, notably IFN-γ, have been described to exerts significant effects on the expression of y+LAT1 [ 163 ] but not y+LAT2, indicating that SLC7A7 is sensitive to IFN-γ. Furthermore, recent in vitro studies using astrocytes and rat brain endothelial cells demonstrated that y + LAT2 may have protective effects against hyperammonemic conditions in the brain [ 164 ]. In this study, ammonia-mediated up-regulation of y + LAT2 diminished NO synthesis by increasing intracellular ADMA and SDMA depletion without any significant changes in L-arginine concentrations.…”

Disturbances in Nitric Oxide Cycle and Related Molecular Pathways in Clear Cell Renal Cell Carcinoma

“…Ammonia activates NO production by stimulating arginine uptake and regulating the distribution of ADMA and SDMA. The intracellular L-Arg/ADMA ratio modulates iNOS activity and NO levels [89]. These findings warrant further investigations into the role of ammonia as a modulator of NO production.…”

“…ADMA and NMMA compete with L-arginine for binding to the active site of NOS, acting as competitive inhibitors for all isoforms. SDMA competes with CAT transporters for L-arginine, indirectly exerting inhibitory effects on NO synthesis [3,4,24,26,89].…”

Tumor cell-derived asymmetric dimethylarginine regulates macrophage functions and polarization

“…The Slc7a2A has about a 2-fold greater efficiency in transporting l-arginine than ADMA, while the Slc7a2B does not have such preference [ 42 ]. Furthermore, it has been shown that ADMA fails to compete for arginine uptake unless mM concentrations are used [ 37 ]. The ADMA concentration for treatment was therefore set at 5 mM (1 mg/mL) to ensure the uptake of ADMA into cells.…”