Regulation of mitochondrial glutamine/glutamate metabolism by glutamate transport: studies with (15)N.
Regulation of mitochondrial glutamine/glutamate metabolism by glutamate transport: studies with (15)N.
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DOI：
10.1152/ajpcell.2001.280.5.c1151
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发表时间：
2001-05
期刊：
影响因子：
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通讯作者：
T. Welbourne;I. Nissim
中科院分区：
文献类型：
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作者：
T. Welbourne;I. Nissim
我们主要关注质膜谷氨酸摄取在调节细胞内谷氨酸脱氢酶（GA）和谷氨酸脱氢酶（GDH）通量以及确定近端小管样LLC-PK（1）-F（+）细胞系中细胞内谷氨酸的命运中的作用。我们使用高亲和力谷氨酸转运抑制剂D-天冬氨酸（D-Asp）和DL-苏型-β-羟基天冬氨酸（THA）来阻断细胞外摄取，然后使用[（15）N]谷氨酸或[2-（15）N]谷氨酰胺来跟踪谷氨酰胺和谷氨酸的代谢命运和分布。在与[2-（15）N]谷氨酰胺（99原子%过量）孵育的单层细胞中，谷氨酰胺和谷氨酸在整个细胞内和细胞外隔室中达到平衡。在5 mM D-Asp和0.5 mM THA存在下，谷氨酰胺分布保持不变，但细胞内谷氨酸富集量下降33%（P < 0.05），细胞外谷氨酸富集量增加39%（P < 0.005）。随着谷氨酸摄取的阻断，细胞内谷氨酸浓度降低了37%（P < 0.0001），而细胞内谷氨酰胺浓度保持不变。谷氨酰胺从媒体和估计的细胞内GA通量的消失增加与细胞内谷氨酸浓度的下降。从[2-（15）N]谷氨酰胺形成并在培养基中回收的标记的谷氨酸和NH分别增加12倍和3倍，与加速的GA和GDH通量一致。然而，标记的丙氨酸形成减少了37%，表明抑制转氨作用。虽然D-Asp和THA单独促进GA和GDH通量，但只有THA抑制转氨作用。这些结果是一致的谷氨酸转运调节和被调节的谷氨酰胺和谷氨酸代谢在上皮细胞。
We focused on the role of plasma membrane glutamate uptake in modulating the intracellular glutaminase (GA) and glutamate dehydrogenase (GDH) flux and in determining the fate of the intracellular glutamate in the proximal tubule-like LLC-PK(1)-F(+) cell line. We used high-affinity glutamate transport inhibitors D-aspartate (D-Asp) and DL-threo-beta-hydroxyaspartate (THA) to block extracellular uptake and then used [(15)N]glutamate or [2-(15)N]glutamine to follow the metabolic fate and distribution of glutamine and glutamate. In monolayers incubated with [2-(15)N]glutamine (99 atom %excess), glutamine and glutamate equilibrated throughout the intra- and extracellular compartments. In the presence of 5 mM D-Asp and 0.5 mM THA, glutamine distribution remained unchanged, but the intracellular glutamate enrichment decreased by 33% (P < 0.05) as the extracellular enrichment increased by 39% (P < 0.005). With glutamate uptake blocked, intracellular glutamate concentration decreased by 37% (P < 0.0001), in contrast to intracellular glutamine concentration, which remained unchanged. Both glutamine disappearance from the media and the estimated intracellular GA flux increased with the fall in the intracellular glutamate concentration. The labeled glutamate and NH formed from [2-(15)N]glutamine and recovered in the media increased 12- and 3-fold, respectively, consistent with accelerated GA and GDH flux. However, labeled alanine formation was reduced by 37%, indicating inhibition of transamination. Although both D-Asp and THA alone accelerated the GA and GDH flux, only THA inhibited transamination. These results are consistent with glutamate transport both regulating and being regulated by glutamine and glutamate metabolism in epithelial cells.