Benzoic acid and specific 2-oxo acids activate hepatic efflux of glutamate at OAT2.
Benzoic acid and specific 2-oxo acids activate hepatic efflux of glutamate at OAT2.
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DOI：
10.1016/j.bbamem.2012.08.026
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发表时间：
2013-02
期刊：
影响因子：
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通讯作者：
Till Pfennig;B. Herrmann;T. Bauer;E. Schömig;D. Gründemann
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作者：
Till Pfennig;B. Herrmann;T. Bauer;E. Schömig;D. Gründemann
肝脏是血浆中谷氨酸的主要来源。最近我们发现肝细胞谷氨酸的外流是由转运蛋白OAT2(人类基因符号SLC22A7)催化的。有机阴离子转运蛋白2(OAT2)是一种完整的膜蛋白，在大鼠和人类中主要在肝脏表达，在肾脏中表达较少。许多年前，HäusSinger及其同事在大鼠肝脏的离体灌流实验中证明，苯甲酸或氨基酸的特定2-氧代酸类似物，如2-氧代-4-甲基-戊酸(‘2-氧代-亮氨酸’)强烈刺激谷氨酸的释放(高达7倍)；‘2-氧代-缬氨酸’及相应的氨基酸没有作用。外排刺激的分子机制尚不清楚。在本研究中，人和大鼠OAT2在293细胞中异源表达。外加1 mmol/L苯甲酸可使谷氨酸的OAT_2特异性外流增加20倍，‘2-氧代亮氨酸’也有效，但‘2-氧代-缬氨酸’无效。放射性标记的冬凌草酸的外流也有类似的影响。OAT2的表达没有增加苯甲酸的摄取；因此，苯甲酸不是底物，可以排除反式刺激。相反，进一步的实验表明，谷氨酸外流的增加是由苯甲酸和特定的2-氧代酸与OAT2直接相互作用造成的。我们认为，刺激物结合到不同的细胞外部位，从而加速空底物结合部位到细胞内表面的重新定位。增加OAT2处的谷氨酸外流可能是苯甲酸治疗尿素循环缺陷患者的主要好处。
The liver is the principal source of glutamate in blood plasma. Recently we have discovered that efflux of glutamate from hepatocytes is catalyzed by the transporter OAT2 (human gene symbol SLC22A7). Organic anion transporter 2 (OAT2) is an integral membrane protein of the sinusoidal membrane domain; it is primarily expressed in liver and much less in kidney, both in rats and humans. Many years ago, Häussinger and coworkers have demonstrated in isolated perfused rat liver that benzoic acid or specific 2-oxo acid analogs of amino acids like e.g. 2-oxo-4-methyl-pentanoate (‘2-oxo-leucine’) strongly stimulate release of glutamate (up to 7-fold); ‘2-oxo-valine’ and the corresponding amino acids were without effect. The molecular mechanism of efflux stimulation has remained unclear. In the present study, OAT2 from human and rat were heterologously expressed in 293 cells. Addition of 1mmol/l benzoic acid to the external medium increased OAT2-specific efflux of glutamate up to 20-fold; ‘2-oxo-leucine’ was also effective, but not ‘2-oxo-valine’. Similar effects were seen for efflux of radiolabeled orotic acid. Expression of OAT2 did not increase uptake of benzoic acid; thus, benzoic acid is no substrate, and trans-stimulation can be excluded. Instead, further experiments suggest that increased efflux of glutamate is caused by direct interaction of benzoic acid and specific 2-oxo acids with OAT2. We propose that stimulators bind to a distinct extracellular site and thereby accelerate relocation of the empty substrate binding site to the intracellular face. Increased glutamate efflux at OAT2 could be the main benefit of benzoate treatment in patients with urea cycle defects.