Hyperosmolar mannitol simulates expression of aquaporins 4 and 9 through a p38 mitogen-activated protein kinase-dependent pathway in rat astrocytes

The membrane pore proteins, aquaporins (AQPs), facilitate the osmotically driven passage of water and, in some instances, small solutes. Under hyperosmotic conditions, the expression of some AQPs changes, and some studies have shown that the expression of AQP1 and AQP5 is regulated by MAPKs. However, the mechanisms regulating the expression of AQP4 and AQP9 induced by hyperosmotic stress are poorly understood. In this study, we observed that hyperosmotic stress induced by mannitol increased the expression of AQP4 and AQP9 in cultured rat astrocytes, and intraperitoneal infusion of mannitol increased AQP4 and AQP9 in the rat brain cortex. In addition, a p38 MAPK inhibitor, but not ERK and JNK inhibitors, suppressed their expression in cultured astrocytes. AQPs play important roles in maintaining brain homeostasis. The expression of AQP4 and AQP9 in astrocytes changes after brain ischemia or traumatic injury, and some studies have shown that p38 MAPK in astrocytes is activated under similar conditions. Since mannitol is commonly used to reduce brain edema, understanding the regulation of AQPs and p38 MAPK in astrocytes under hyperosmotic conditions induced with mannitol may lead to a control of water movements and a new treatment for brain edema.     

Aquaporin-4 as a New Target against Methamphetamine-Induced Brain Alterations: Focus on the Neurogliovascular Unit and Motivational Behavior

Methamphetamine (METH) abuse/misuse is a worldwide problem, and despite extensive characterization of its neurotoxicity over the last years, many questions remain unanswered. Recently, it was shown that METH compromises the blood-brain barrier (BBB) and causes a disturbance in the water homeostasis leading to brain edema. Importantly, water transport at BBB is regulated by water channels, aquaporins (AQPs), with AQP4 being expressed in astrocytic end-feet surrounding brain endothelium. Thus, the main goal of this work was to unravel the role of AQP4 under conditions of METH consumption. Our results show that METH (4× 10 mg/kg, 2 h apart, i.p.) interferes with AQP4 protein levels causing brain edema and BBB breakdown in both mice striatum and hippocampus, which culminated in locomotor and motivational impairment. Furthermore, these effects were prevented by pharmacological blockade of AQP4 with a specific inhibitor (TGN-020). Moreover, siRNA knockdown of this water channel protected astrocytes from METH-induced swelling and morphologic alterations. Herein, we unraveled AQP4 as a new therapeutic target to prevent the negative impact of METH.     

Hyperosmotic induction of aquaporin expression in rat astrocytes through a different MAPK pathway

Water homeostasis of the nervous system is important during neural signal transduction. Astrocytes are crucial in water transport in the central nervous system under both physiological and pathological conditions. To date, five aquaporins (AQP) have been found in rat brain astrocytes. Most studies have focused on AQP4 and AQP9, however, little is known about the expression of AQP3, ‐5, and ‐8 as well as their regulating mechanism in astrocytes. The expression patterns of AQP3, ‐5, and ‐8 in astrocytes exposed to hyperosmotic solutions were examined to clarify the roles of AQP3, ‐5, and ‐8 in astrocyte water movement. The expression of AQP4 and AQP9 under the same hyperosmotic conditions was also investigated. The AQP4 and AQP9 expressions continuously increased until 12 h after hyperosmotic solution exposure, whereas the AQP3, ‐5, and ‐8 expressions continued to increase until 6 h after hyperosmotic solution exposure. The different AQPs decreased at corresponding time points (24 h for AQP4 andAQP9; 12 h for AQP3, ‐5, and ‐8 after hyperosmotic solution exposure). The ERK inhibitor can attenuate the expression of AQP3, ‐5, and ‐8 after hyperosmotic solution exposure. The p38 inhibitor can inhibit the AQP4 and AQP9 expressions in cultured astrocytes. AQP expression is directly related to the extracellular hyperosmotic stimuli. Moreover, different AQPs can be regulated by a distinct MAPK signal transduction pathway. J. Cell. Biochem. 114: 111–119, 2012. © 2012 Wiley Periodicals, Inc.     

Protein kinase G is involved in ammonia-induced swelling of astrocytes

Ammonia-induced swelling of astrocytes is a primary cause of brain edema associated with acute hepatic encephalopathy. Previous studies have shown that ammonia transiently increases cGMP in brain in vivo and in cultured astrocytes in vitro . We hypothesized that protein kinase G (PKG), an enzyme activated by cGMP and implicated in regulation of cell shape, size, and/or volume in peripheral and CNS cells, may play a role in the ammonia-induced astrocytic volume increase. Treatment of cultured rat cortical astrocytes with 1 or 5 mM NH₄Cl (ammonia) for 24 h increased their cell volume by 50% and 80% above control, respectively, as measured by confocal imaging followed by 3D computational analysis. A cGMP analog, 8-(4-chlorophenylthio)-cGMP, increased the cell volume in control cells and potentiated the increase in 1 mM ammonia-treated cells. A soluble guanylate cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) abrogated, and a PKG inhibitor [8-(4-chlorophenylthio)-cGMP-thioate, Rp-isomer] dose-dependently reduced the cell volume-increasing effect of 5 mM ammonia. The results suggest that (i) PKG may play a permissive role in ammonia-induced astrocytic swelling and (ii) elevation of brain cGMP associated with acute exposure to ammonia in vivo may aggravate the ensuing brain edema.     

Calcineurin-NFATc signaling pathway regulates AQP2 expression in response to calcium signals and osmotic stress

The aquaporin (AQP)2 channel mediates the reabsorption of water in renal collecting ducts in response to arginine vasopressin (AVP) and hypertonicity. Here we show that AQP2 expression is induced not only by the tonicity-responsive enhancer binding protein (TonEBP)/nuclear factor of activated T cells (NFAT)5-mediated hypertonic stress response but also by the calcium-dependent calcineurin-NFATc pathway. The induction of AQP2 expression by the calcineurin-NFATc pathway can occur in the absence of TonEBP/NFAT5. Mutational and chromatin immunoprecipitation analyses revealed the existence of functional NFAT binding sites within the proximal AQP2 promoter responsible for regulation of AQP2 by NFATc proteins and TonEBP/NFAT5. Contrary to the notion that TonEBP/NFAT5 is the only Rel/NFAT family member regulated by tonicity, we found that hypertonicity promotes the nuclear translocation of NFATc proteins for the subsequent induction of AQP2 expression. Calcineurin activity was also found to be involved in the induction of TonEBP/NFAT5 expression by hypertonicity, thus further defining the signaling mechanisms that underlie the TonEBP/NFAT5 osmotic stress response pathway. The coordinate regulation of AQP2 expression by both osmotic stress and calcium signaling appears to provide a means to integrate diverse extracellular signals into optimal cellular responses. aquaporin; nuclear factor of activated T cells; tonicity-responsive enhancer binding protein; osmotic response     

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

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.     

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

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.     

Functional down-regulation of volume-regulated anion channels in AQP4 knockdown cultured rat cortical astrocytes

In the brain, the astroglial syncytium is crucially involved in the regulation of water homeostasis. Accumulating evidence indicates that a dysregulation of the astrocytic processes controlling water homeostasis has a pathogenetic role in several brain injuries. Here, we have analysed by RNA interference technology the functional interactions occurring between the most abundant water channel in the brain, aquaporin-4 (AQP4), and the swelling-activated Cl(-) current expressed by cultured rat cortical astrocytes. We show that in primary cultured rat cortical astrocytes transfected with control small interfering RNA (siRNA), hypotonic shock promotes an increase in cellular volume accompanied by augmented membrane conductance mediated by volume-regulated anion channels (VRAC). Conversely, astroglia in which AQP4 was knocked down (AQP4 KD) by transfection with AQP4 siRNA changed their morphology from polygonal to process-bearing, and displayed normal cell swelling but reduced VRAC activity. Pharmacological manipulations of actin cytoskeleton in rat astrocytes, and functional analysis in mouse astroglial cells, which retain their morphology upon knockdown of AQP4, suggest that stellation of AQP4 KD rat cortical astrocytes was not causally linked to reduction of VRAC current. Molecular analysis of possible candidates of swelling-activated Cl(-) current provided evidence that in AQP4 KD astrocytes, there was a down-regulation of chloride channel-2 (CIC-2), which, however, was not involved in VRAC conductance. Inclusion of ATP in the intracellular saline restored VRAC activity upon hypotonicity. Collectively, these results support the view that in cultured astroglial cells, plasma membrane proteins involved in cell volume homeostasis are assembled in a functional platform.     

Mitogen limitation and bone morphogenetic protein-4 promote neurogenesis in SFME cells, an EGF-dependent neural stem cell line

Serum-free mouse embryo (SFME) cells are an epidermal growth factor (EGF)-dependent established line derived from brains of 16-d-old Balb/c mouse embryos. SFME cells grow indefinitely in serum-free medium without replicative senescence, chromosomal abnormalities, or malignant transformation. SFME cells express nestin, a neural stem cell marker, under serum-free conditions. Exposure to serum or transforming growth factor β (TGF-β) leads to a marked increase in differentiation toward the astrocytic lineage with expression of glial fibrillary acidic protein and other astrocyte markers. In this study, we show that treatment of SFME cells with bone morphogenetic protein-4 (BMP-4), another member of the TGF-β family, led to differentiation toward a neuronal lineage under conditions of low mitogenic stimulation (0.5 ng/mL) by EGF and fibroblast growth factor. Maximum mitogenic stimulation with 50 ng/mL EGF blocked the BMP-4 effect on neuronal differentiation, but did not block TGF-β-induced expression of markers of the astrocytic lineage. BMP-4 treatment also enhanced the activity of the neuron-specific enolase (NSE) promoter in SFME-NSE-lacZ cells that carry the gene for bacterial β-galactosidase under the control of the NSE promoter. Extended BMP-4 treatment caused SFME cells to express a neuronal phenotype synthesizing gamma-aminobutyric acid. These results indicate that SFME cells have the capacity to generate both neurons and astrocytes in vitro, which resemble the behavior of EGF-dependent multipotential stem cells in the central nervous system, and establish a relationship between effects of BMP-4 and degree of mitogenic stimulation by other peptide growth factors.     

Progesterone Attenuates Aquaporin-4 Expression in an Astrocyte Model of Ischemia/Reperfusion

Previous studies have suggested that progesterone may be involved in neuroprotection by preventing brain edema. In this study, we assessed the effects of progesterone on aquaporin-4 (AQP4) expression in an ischemia/reperfusion model of cultured rat astrocytes, and further explored the possible role of the protein kinase C (PKC) pathway in this course. We evaluate primary culture astrocytes exposed to 4 h oxygen–glucose deprivation (OGD) followed by 24 h reperfusion (OGD4h/R24h) as a means of simulating cortex ischemia and reperfusion, and test the effect of progesterone on AQP4 expression in response to OGD4h/R24h. Besides, the cell viability was assessed by MTT reduction and lactate dehydrogenase release assay, accompanied by cell morphology survey. At a concentration of 1 and 2 μM, progesterone significantly attenuated AQP4 at the level of both protein and mRNA and ameliorated the cell viability of astrocytes from OGD/reperfusion injury. Moreover, this effect was blocked by the PKC inhibitor Ro31-8220, which was employed before the OGD. These results indicate that progesterone exerts the protective effects and attenuates AQP4 expression in an astrocyte model of ischemia/reperfusion depending on the PKC signal pathway.     

Attenuation of Acute Phase Injury in Rat Intracranial Hemorrhage by Cerebrolysin that Inhibits Brain Edema and Inflammatory Response

The outcome of intracerebral hemorrhage (ICH) is mainly determined by the volume of the hemorrhage core and the secondary brain damage to penumbral tissues due to brain swelling, microcirculation disturbance and inflammation. The present study aims to investigate the protective effects of cerebrolysin on brain edema and inhibition of the inflammation response surrounding the hematoma core in the acute stage after ICH. The ICH model was induced by administration of type VII bacterial collagenase into the stratum of adult rats, which were then randomly divided into three groups: ICH + saline; ICH + Cerebrolysin (5 ml/kg) and sham. Cerebrolysin or saline was administered intraperitoneally 1 h post surgery. Neurological scores, extent of brain edema content and Evans blue dye extravasation were recorded. The levels of pro-inflammatory factors (IL-1β, TNF-α and IL-6) were assayed by Real-time PCR and Elisa kits. Aquaporin-4 (AQP4) and tight junction proteins (TJPs; claudin-5, occludin and zonula occluden-1) expression were measured at multiple time points. The morphological and intercellular changes were characterized by Electron microscopy. It is found that cerebrolysin (5 ml/kg) improved the neurological behavior and reduced the ipsilateral brain water content and Evans blue dye extravasation. After cerebrolysin treated, the levels of pro-inflammatory factors and AQP4 in the peri-hematomal areas were markedly reduced and were accompanied with higher expression of TJPs. Electron microscopy showed the astrocytic swelling and concentrated chromatin in the ICH group and confirmed the cell junction changes. Thus, early cerebrolysin treatment ameliorates secondary injury after ICH and promotes behavioral performance during the acute phase by reducing brain edema, inflammatory response, and blood–brain barrier permeability.