Na-K-Cl Cotransporter-1 in the Mechanism of Ammonia-induced Astrocyte Swelling

Department of Pathology and Biochemistry, University of Miami School of Medicine and Veterans Affairs Medical Center, Miami, Florida 33101, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 11/2008; 283(49):33874-82. DOI: 10.1074/jbc.M804016200
Source: PubMed


Brain edema and the consequent increase in intracranial pressure and brain herniation are major complications of acute liver failure (fulminant hepatic failure) and a major cause of death in this condition. Ammonia has been strongly implicated as an important factor, and astrocyte swelling appears to be primarily responsible for the edema. Ammonia is known to cause cell swelling in cultured astrocytes, although the means by which this occurs has not been fully elucidated. A disturbance in one or more of these systems may result in loss of ion homeostasis and cell swelling. In particular, activation of the Na-K-Cl cotransporter (NKCC1) has been shown to be involved in cell swelling in several neurological disorders. We therefore examined the effect of ammonia on NKCC activity and its potential role in the swelling of astrocytes. Cultured astrocytes were exposed to ammonia (NH(4)Cl; 5 mm), and NKCC activity was measured. Ammonia increased NKCC activity at 24 h. Inhibition of this activity by bumetanide diminished ammonia-induced astrocyte swelling. Ammonia also increased total as well as phosphorylated NKCC1. Treatment with cyclohexamide, a potent inhibitor of protein synthesis, diminished NKCC1 protein expression and NKCC activity. Since ammonia is known to induce oxidative/nitrosative stress, and antioxidants and nitric-oxide synthase inhibition diminish astrocyte swelling, we also examined whether ammonia caused oxidation and/or nitration of NKCC1. Cultures exposed to ammonia increased the state of oxidation and nitration of NKCC1, whereas the antioxidants N-nitro-l-arginine methyl ester and uric acid all significantly diminished NKCC activity. These agents also reduced phosphorylated NKCC1 expression. These results suggest that activation of NKCC1 is an important factor in the mediation of astrocyte swelling by ammonia and that such activation appears to be mediated by NKCC1 abundance as well as by its oxidation/nitration and phosphorylation.

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Available from: Arumugam R Jayakumar
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    • "There are studies as early as 2005 reporting the NKCC1 and cell volume regulation in particular reference to astrocytes (Chen and Sun, 2005). Similar study conducted elsewhere show ammonia induced astrocyte swelling was associated with increase in the activity of NKCC1 (Jayakumar et al., 2008a). Treatment of astrocytes with manganese resulted in the oxidation of NKCC1 after 6 h contrast to the immediate effect observed with oxidants. "
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    ABSTRACT: Manganese has shown to be involved in astrocyte swelling. Several factors such as transporters, exchangers and ion channels are attributed to astrocyte swelling as a result in the deregulation of cell volume. Products of oxidation and nitration has been implied to be involved in the pathophysiology of swelling, however the direct link and mechanism of manganese induced astrocyte swelling has not been fully elucidated. In the current study, we used rat primary astrocyte cultures to investigate the activation of Na-K-Cl cotransporter-1 (NKCC1) a downstream mechanism for free radical induced astrocyte swelling as a result of manganese toxicity. Our results showed manganese, oxidants and NO donors as potent inducer of oxidation and nitration of NKCC1. Our results further confirmed that manganese (50μM) increased the total protein, phosphorylation and activity of NKCC1 as well as cell volume (p<0.05 vs. control). NKCC1 inhibitor (Bumetanide), NKCC1-siRNA, antioxidants; DMTU, MnTBAP, tempol, catalase and Vit-E, NOS inhibitor; L-NAME, peroxinitrite scavenger; uric acid all significantly reversed the effects of NKCC1 activation (p<0.05). From the current investigation we infer that manganese or oxidants and NO induced activation, oxidation/nitration of NKCC1 play an important role in the astrocyte swelling. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Mar 2015 · Brain research
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    • "We have shown earlier that NH4+ activates NKCC1 in astrocytes, resulting in a substantial increase in the intracellular sodium concentration [28], [29]. NKCC1 plays a central role in the swelling of astrocytes [60], and has also been linked to NH4+/NH3-induced cell swelling [61]. Application of bumetanide, which efficiently blocks NKCC1 and related sodium elevations in astrocytes in hippocampal slice preparations [28], did not reduce NH4+/NH3-induced calcium signals in the present study. "
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    ABSTRACT: Increased brain ammonium (NH4+/NH3) plays a central role in the manifestation of hepatic encephalopathy (HE), a complex syndrome associated with neurological and psychiatric alterations, which is primarily a disorder of astrocytes. Here, we analysed the influence of NH4+/NH3 on the calcium concentration of astrocytes in situ and studied the underlying mechanisms of NH4+/NH3-evoked calcium changes, employing fluorescence imaging with Fura-2 in acute tissue slices derived from different regions of the mouse brain. In the hippocampal stratum radiatum, perfusion with 5 mM NH4+/NH3 for 30 minutes caused a transient calcium increase in about 40% of astrocytes lasting about 10 minutes. Furthermore, the vast majority of astrocytes (∼90%) experienced a persistent calcium increase by ∼50 nM. This persistent increase was already evoked at concentrations of 1-2 mM NH4+/NH3, developed within 10-20 minutes and was maintained as long as the NH4+/NH3 was present. Qualitatively similar changes were observed in astrocytes of different neocortical regions as well as in cerebellar Bergmann glia. Inhibition of glutamine synthetase resulted in significantly larger calcium increases in response to NH4+/NH3, indicating that glutamine accumulation was not a primary cause. Calcium increases were not mimicked by changes in intracellular pH. Pharmacological inhibition of voltage-gated sodium channels, sodium-potassium-chloride-cotransporters (NKCC), the reverse mode of sodium/calcium exchange (NCX), AMPA- or mGluR5-receptors did not dampen NH4+/NH3-induced calcium increases. They were, however, significantly reduced by inhibition of NMDA receptors and depletion of intracellular calcium stores. Taken together, our measurements show that sustained exposure to NH4+/NH3 causes a sustained increase in intracellular calcium in astrocytes in situ, which is partly dependent on NMDA receptor activation and on release of calcium from intracellular stores. Our study furthermore suggests that dysbalance of astrocyte calcium homeostasis under hyperammonemic conditions is a widespread phenomenon, which might contribute to the disturbance of neurotransmission during HE.
    Full-text · Article · Aug 2014 · PLoS ONE
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    • "Impaired neurotransmission in HE is in a great part because of the disturbances in astrocytic functions (Albrecht 2005). HE, hyperammonemia or exposure of astrocytes to ammonia, impairs the expression and the function of crucial ion and neurotransmitter transporting moieties in the astrocytic membrane, among these the glutamate transporters GLT-1 (Norenberg et al. 1997) and GLAST (Chan et al. 2000), the inward rectifying potassium channel Kir4.1 (Obara-Michlewska et al. 2011), and the Na–K–Cl cotransporter (Jayakumar et al. 2008). These changes elicit neurotransmitter and ion imbalance in the synaptic cleft resulting in synaptic dysfunction, best exemplified by disinhibition by increased extracellular K + of neuronal circuits, which is mediated by overactivation of neuronal Na–K–Cl cotransporter (Rangroo Thrane et al. 2013). "
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    ABSTRACT: Read the full article ‘Decreased astrocytic thrombospondin‐1 secretion after chronic ammonia treatment reduces the level of synaptic proteins: in vitro and in vivo studies’ on page 333
    Full-text · Article · Aug 2014 · Journal of Neurochemistry
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