Department of Neurotoxicology, Mossakowski Medical Research Centre, Polish Academy of Sciences, 02-106 Warsaw, Poland.
Mass Spectrometry Laboratory, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland.
Int J Mol Sci. 2017 Nov 2;18(11):2308. doi: 10.3390/ijms18112308.
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 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.
先前我们已经证明，氨通过增加前体氨基酸精氨酸的摄取来刺激星形胶质细胞中的一氧化氮（NO）合成，这种摄取是通过异源二聚体精氨酸/谷氨酰胺转运体 y+LAT2 完成的。氨还会增加脑内一氧化氮合酶（NOS）内源性抑制剂对称二甲基精氨酸（ADMA）的浓度，但 ADMA 过剩在脑内星形胶质细胞内和细胞外间隙的分布尚未研究。在这里，我们通过与精氨酸竞争 y+LAT2 转运体来检验氨调节 ADMA 及其类似物对称二甲基精氨酸（SDMA）在脑内两个隔室之间分布的假设。在该假设的扩展中，我们分析了形成血脑屏障的内皮细胞中 ADMA/Arg 相互作用。我们通过高效液相色谱（HPLC）和质谱（MS）技术测量了用氨处理的培养皮质星形胶质细胞和大鼠脑内皮细胞系（RBE-4）中精氨酸、ADMA 和 SDMA 的浓度，以及沉默编码 y+LAT2 的基因表达的影响。我们还测试了 ADMA 代谢酶的表达：蛋白精氨酸甲基转移酶（PRMT）和二甲基精氨酸二甲氨基水解酶（DDAH）以及精氨酸摄取到星形胶质细胞。用 5 mM 氨处理 48 小时后，两种细胞类型中的 ADMA 和 SDMA 浓度几乎降低了 50%，而在星形胶质细胞中，沉默基因则显著减弱了这种作用。此外，在培养基中存在 ADMA 的情况下，氨诱导的星形胶质细胞中 y+LAT2 依赖性精氨酸摄取减少。我们的结果表明，上调的 y+LAT2 介导的 ADMA 外排增加可能是氨干扰两种细胞类型内星形胶质细胞内（可能还有内皮细胞内）ADMA 含量，随后干扰 NO 合成的机制。
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