Departamento de Biología Celular y Genética, Facultad de Biología, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain.
Neurochem Int. 2012 Dec;61(8):1314-24. doi: 10.1016/j.neuint.2012.09.008. Epub 2012 Sep 25.
Aquaporin-4 (AQP4) is a water channel protein mainly located in the astroglial plasma membrane, the precise function of which in the brain edema that accompanies hepatic encephalopathy (HE) is unclear. Since ammonia is the main pathogenic agent in HE, its effect on AQP4 expression and distribution in confluent primary astroglial cultures was examined via their exposure to ammonium chloride (1, 3 and 5 mM) for 5 and 10 days. Ammonia induced the general inhibition of AQP4 mRNA synthesis except in the 1 mM/5 day treatment. However, the AQP4 protein content measured was dependent on the method of analysis; an apparent increase was recorded in treated cells in in-cell Western assays, while an apparent reduction was seen with the classic Western blot method, perhaps due to differences in AQP4 aggregation. Ammonia might therefore induce the formation of insoluble AQP4 aggregates in the astroglial plasma membrane. The finding of AQP4 in the pellet of classic Western blot samples, plus data obtained via confocal microscopy, atomic force microscopy (using immunolabeled cells with gold nanoparticles) and scanning electron microscopy, all corroborate this hypothesis. The effect of ammonia on AQP4 seems not to be due to any osmotic effect; identical osmotic stress induced by glutamine and salt had no significant effect on the AQP4 content. AQP4 functional analysis (subjecting astrocytes to a hypo-osmotic medium and using flow cytometry to measure cell size) demonstrated a smaller water influx in ammonia-treated astrocytes suggesting that AQP4 aggregates are representative of an inactive status; however, more confirmatory studies are required to fully understand the functional status of AQP4 aggregates. The present results suggest that ammonia affects AQP4 expression and distribution, and that astrocytes change their expression of AQP4 mRNA as well as the aggregation status of the ensuing protein depending on the ammonia concentration and duration of exposure.
水通道蛋白 4（AQP4）是一种主要位于星形胶质细胞质膜上的水通道蛋白，其在肝性脑病（HE）伴发脑水肿中的精确功能尚不清楚。由于氨是 HE 的主要致病因子，因此通过用氯化铵（1、3 和 5mM）处理共培养的原代星形胶质细胞 5 和 10 天，来检测其对 AQP4 表达和分布的影响。氨诱导除了在 1mM/5 天处理中外的 AQP4mRNA 合成的普遍抑制。然而，所测量的 AQP4 蛋白含量取决于分析方法；在用细胞内 Western 分析测定时，处理细胞中的 AQP4 含量明显增加，而在用经典 Western 印迹法测定时则明显减少，这可能是由于 AQP4 聚集的差异所致。因此，氨可能诱导星形胶质细胞质膜中不溶性 AQP4 聚集的形成。经典 Western 印迹样品沉淀中 AQP4 的发现，以及通过共聚焦显微镜、原子力显微镜（使用金纳米颗粒标记的免疫细胞）和扫描电子显微镜获得的数据，均证实了这一假设。氨对 AQP4 的影响似乎不是由于任何渗透效应引起的；用谷氨酰胺和盐引起的相同渗透压胁迫对 AQP4 含量没有显著影响。AQP4 功能分析（使星形胶质细胞处于低渗介质中，并使用流式细胞术测量细胞大小）表明，在氨处理的星形胶质细胞中，水的内流较小，这表明 AQP4 聚集物代表无活性状态；然而，需要更多的确认性研究来全面了解 AQP4 聚集物的功能状态。目前的结果表明，氨会影响 AQP4 的表达和分布，并且星形胶质细胞会根据氨浓度和暴露时间改变其 AQP4mRNA 的表达以及随之而来的蛋白质的聚集状态。
