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.
Highlights
Hyperammonemia (HA) is defined as a clinical condition in which the ammonium ion concentration in the blood and brain exceeds 130 and 500 μM respectively [1,2]
The present study extended our knowledge of described our previous study demonstrated that ammonia-induced increase of nitric oxide (NO) synthesis is associated with increased [3H]arginine uptake via upregulated in y+LAT2 [15]
Our present work showed that in primary astrocytes, y+LAT2 activity may modulate intra/extracellular asymmetric dimethylarginine (ADMA) distribution independently from activation of ADMA metabolism. This may be concluded from both unchanged expression and protein level of protein arginine methyltransferase (PRMT)-1 and dimethylarginine dimethyl aminohydrolase (DDAH)-1 in ammonia treated astrocytes and reduction of intracellular symmetric dimethylarginine (SDMA), a dimethylarginine, which is not metabolized via the same enzymatic pathway as ADMA
Summary
Hyperammonemia (HA) is defined as a clinical condition in which the ammonium ion concentration in the blood and brain exceeds 130 and 500 μM respectively [1,2]. One of the principle mechanisms by which ammonia damages the brain is oxidative/nitrosative stress (ONS) [4]. Activation of NO synthesis by ammonia contributes to ONS in astrocytes [6,7,8], neurons [9], and esAnpcdetciovietahstei(oRlinOalSo)cfefNolllOlso(wseye.gdn.t,bhryeasrtiasbpbridayirnaeamecnmtidoonontwihaietchloiaNnltOrciebtloultpleirsnoedtou(RcOeBNhEiSg-4hin)lycaerseltlarcloitcnivyeet)ensailt[ri6ko–ge8e][n,1n0sp]e.eucWrioenshs(iRle[N9i]Sn, )an[n5ed]u. Onia may eInlictihtisADstuMdAy tehxips ohrytpfroothmestihs ewcaesllstebstyedy+iLnAcTul2t,uarepdroacsetrsoscylitkeesl.yRtaot bberafiancielintdaotethdebliyalucperllesgwulearteion of the cacrorniseirdaenreddcaosmonpeeotitthioernbbryaininccormeapsaerdtmaergnitnininwe huipchtatkhe ombescehravneidsmuonpdeerratehse.se conditions [13]. In this study this hypothesis was tested in cultured astrocytes. Rat brain endothelial cells were considered as one other brain compartment in which the mechanism operates
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