Introduction

As a consequence of the loss of liver function in chronic liver disease, increased levels of ammonia, manganese, and glutamine have been observed in the brain of hepatic encephalopathy patients.

Objective

In the present study, we explored phosphate activated glutaminase (PAG) activity in mitochondrial enriched fractions under treatment with ammonia and manganese.

Methods

We dissected out the brain cortex, striatum, and cerebellum of male Wistar rats 250−280 g weight; brain sections were pooled to obtain enriched mitochondrial fractions by differential centrifugation. Aliquots equivalent to 200 μg of protein were incubated with semi-log increasing concentrations of ammonia and/or manganese both as chloride salts (from 0 to 10 000 μM) and glutamine (4 mM) for 30 min. Then, the glutamate produced by the reaction was determined by HPLC coupled with fluorescence detection.

Results and discussion

Both manganese and ammonia inhibited PAG in a concentration-dependent manner. Non-linear modeling was used to determine IC50 and IC20 for ammonia (120 μM) and manganese (2 mM). We found that PAG activity under the combination of IC20 of ammonia and manganese was equivalent to the sum of the effects of both substances, being PAG inhibition more pronounced in mitochondrial fractions from cerebellum. The PAG inhibition observed here could potentially explain a pathway for glutamine accumulation, by means of the inhibition of PAG activity as a consequence of increased concentrations of manganese and ammonia in the brain under liver damage conditions.

中文翻译：

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氨和锰对脑磷酸盐激活谷氨酰胺酶的体外抑制作用。

介绍

由于慢性肝病患者肝功能丧失，在肝性脑病患者的大脑中观察到氨、锰和谷氨酰胺水平升高。

客观的

在本研究中，我们研究了氨和锰处理的线粒体富集部分中磷酸盐激活的谷氨酰胺酶 (PAG) 活性。

方法

我们解剖了体重 250-280 g的雄性 Wistar 大鼠的大脑皮层、纹状体和小脑 ；通过差速离心合并脑切片以获得富集的线粒体部分。将相当于 200 μg 蛋白质的等分试样与半对数递增浓度的氨和/或锰作为氯化物盐（从 0 到 10 000 μM）和谷氨酰胺（4 mM）一起温育 30 分钟。然后，通过HPLC结合荧光检测测定反应产生的谷氨酸。

结果和讨论

锰和氨都以浓度依赖的方式抑制 PAG。非线性模型用于确定氨 (120 μM) 和锰 (2 mM) 的 IC50 和 IC20。我们发现在氨和锰的 IC20 组合下的 PAG 活性相当于两种物质的作用总和，PAG 抑制在来自小脑的线粒体部分中更为明显。这里观察到的 PAG 抑制可能解释了谷氨酰胺积累的途径，通过抑制 PAG 活性，这是在肝损伤条件下脑中锰和氨浓度增加的结果。