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Ammonia induces aquaporin-4 rearrangement in the plasma membrane of cultured astrocytes

AuthorsBodega, Guillermo; Köber, Mariana; Penedo García, Marcos ; Luna, Mónica ; Juárez, Silvia; Ciordia, Sergio; Fernández, Benjamín
Hepatic encephalopathy
In-cell Western
Issue DateDec-2012
CitationNeurochemistry International 61(8): 1314-1324 (2012)
AbstractAquaporin-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. © 2012 Elsevier Ltd. All rights reserved.
DescriptionBodega, Guillermo et al.
Publisher version (URL)http://dx.doi.org/10.1016/j.neuint.2012.09.008
Identifiersdoi: 10.1016/j.neuint.2012.09.008
issn: 0197-0186
Appears in Collections:(CNB) Artículos
(IMN-CNM) Artículos
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