Ammonia induces aquaporin-4 rearrangement in the plasma membrane of cultured astrocytes |
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Authors: | Guillermo Bodega,Isabel Suá rez,Luis A. Ló pez-Ferná ndez,Marí a I. Garcí a,Mariana Kö ber,Marcos Penedo,Mó nica Luna,Silvia Juá rez,Sergio Ciordia,Marc Oria,Joan Có rdoba,Benjamí n Ferná ndez |
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Affiliation: | 1. Departamento de Biología Celular y Genética, Facultad de Biología, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain;2. Laboratorio de Farmacogenética y Farmacogenómica, Servicio de Farmacia, Hospital General Universitario Gregorio Marañón, 28007 Madrid, Spain;3. Instituto de Microelectrónica de Madrid/CSIC, Tres Cantos, 28760 Madrid, Spain;4. Proteomics, Centro Nacional de Biotecnología/CSIC, Campus de Cantoblanco, 28049 Madrid, Spain;5. Servei de Medicina Interna-Hepatología, Hospital Universitari Vall d’Hebron, 08035 Barcelona, Spain;6. Departamento de Biología Celular, Facultad de Biología, Universidad Complutense, 28040 Madrid, Spain |
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Abstract: | 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. |
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Keywords: | AFM, atomic force microscope ALF, acute liver failure AQP, aquaporin FSC, forward scattered light HE, hepatic encephalopathy ICW, in-cell Western SEM, scanning electron microscope WB, Western blotting |
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