Abstract
The reperfusion of ischemic brain tissue following a cerebral stroke causes oxidative stress, and leads to the generation of reactive oxygen species (ROS). Apart from inflicting oxidative damage, the latter may also trigger the upregulation of aquaporin 4 (AQP4), a water-permeable channel expressed by astroglial cells of the blood-brain barrier (BBB), and contribute to edema formation, the severity of which is known to be the primary determinant of mortality and morbidity. The mechanism through which this occurs remains unknown. In the present study, we have attempted to address this question using primary astrocyte cultures treated with hydrogen peroxide (H2O2) as a model system. First, we showed that H2O2 induces a significant increase in AQP4 protein levels and that this is inhibited by the antioxidant N-acetylcysteine (NAC). Second, we demonstrated using cell surface biotinylation that H2O2 increases AQP4 cell-surface expression independently of it’s increased synthesis. In parallel, we found that caveolin-1 (Cav1) is phosphorylated in response to H2O2 and that this is reversed by the Src kinase inhibitor 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). PP2 also abrogated the H2O2-induced increase in AQP4 surface levels, suggesting that the phosphorylation of tyrosine-14 of Cav1 regulates this process. We further showed that dominant-negative Y14F and phosphomimetic Y14D mutants caused a decrease and increase in AQP4 membrane expression respectively, and that the knockdown of Cav1 inhibits the increase in AQP4 cell-surface, expression following H2O2 treatment. Together, these findings suggest that oxidative stress-induced Cav1 phosphorylation modulates AQP4 subcellular distribution and therefore may indirectly regulate AQP4-mediated water transport.
Highlights
Aquaporin 4 (AQP4) is the most abundant water-permeable channel found in the mammalian brain, where it is expressed within the astrocytes that form part of the blood-brain barrier (BBB) and is detected in especially high quantities at the astrocytic endfeet that encapsulate the blood vessels (Nielsen et al, 1997)
In order to assess the effect of oxidative stress on AQP4 expression, we analyzed primary astrocyte cultures treated for 1 h with increasing concentrations of H2O2 via immunoblotting, from which it was seen that H2O2 induces an upregulation of AQP4 protein levels (Figures 1A,B)
Cav1 is phosphorylated by the tyrosine-protein kinase Src in a wide variety of cell types (Glenney and Zokas, 1989; Li et al, 1996; Gottlieb-Abraham et al, 2013), and we found that the H2O2-induced phosphorylation of Cav1 decreases when astrocytes were pre-treated with the Src inhibitor PP2 for
Summary
Aquaporin 4 (AQP4) is the most abundant water-permeable channel found in the mammalian brain, where it is expressed within the astrocytes that form part of the blood-brain barrier (BBB) and is detected in especially high quantities at the astrocytic endfeet that encapsulate the blood vessels (Nielsen et al, 1997) It is currently uncertain whether AQP4 is. The initial phase of this swelling, termed ‘‘cytotoxic edema’’ develops when the anoxic conditions brought on by the cessation of blood flow cause astrocytes to enter a state of metabolic derangement This results in the influx and accumulation of extracellular Na+, followed soon after by Cl− and water, driven by the electrochemical gradient at the perivascular endfeet (reviewed in Liang et al, 2007). These findings are the first to show that Cav phosphorylation plays a key role in the regulation of AQP4 cell surface expression in oxidative stress, possibly by altering AQP4 internalization and trafficking, resulting in its redistribution to specific compartments of the cell
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