Ammonia increases nitric oxide, free Zn2+, and metallothionein mRNA expression in cultured rat astrocytes

Clinic for Gastroenterology, Hepatology and Infectiology.
Biological Chemistry (Impact Factor: 3.27). 09/2011; 392(12). DOI: 10.1515/BC-2011-199
Source: PubMed


Abstract Ammonia is a major player in the pathogenesis of hepatic encephalopathy (HE) and affects astrocyte function by triggering a self-amplifying cycle between osmotic and oxidative stress. We recently demonstrated that hypoosmotic astrocyte swelling rapidly stimulates nitric oxide (NO) production and increases intracellular free Zn(2+) concentration ([Zn(2+)](i)). Here we report effects of ammonia on [Zn(2+)](i) homeostasis and NO synthesis. In cultured rat astrocytes NH(4)Cl (5 mmol/l) increased within 6h both, cytosolic and mitochondrial [Zn(2+)]. The [Zn(2+)](i) increase was transient and was mimicked by the non-metabolizable CH(3)NH(3)Cl, and was dependent on NO-formation, as evidenced by the sensitivity towards the NOS inhibitor L-NMMA. The NH(4)Cl-induced NO-formation was sensitive to the Ca(2+)-chelator BAPTA-AM and both, the increase of NO and [Zn(2+)](i) were blocked by the NMDA receptor antagonist MK-801. The NH(4)Cl-triggered increase of [Zn(2+)](i) was followed by a Zn(2+)-dependent nuclear appearance of the transcription factor MTF-1 and metallothionein mRNA induction. Metallothionein mRNA was also increased in vivo in rat cerebral cortex 6h after an NH(4)Ac challenge. NH(4)Cl increased peripheral-type benzodiazepine receptor (PBR) protein expression, whereas PBR mRNA levels were decreased in a Zn(2+)-independent manner. The Zn(2+)-dependent upregulation of metallothionein following ammonia intoxication may reflect a cytoprotective response, whereas the increase in PBR expression may augment HE development.

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    ABSTRACT: Increased l-Arg (Arg) uptake to astrocytes and neurons is thought to contribute to enhanced nitric oxide (NO) synthesis and oxidative/nitrosative stress associated with hyperammonemia (HA). Recently we had shown that HA increases the expression in the brain of y(+)LAT2, an isoform of the y(+)L heteromeric transporter which promotes [(3)H]Arg efflux form brain cells in the presence of l-glutamine (Gln) (Zielińska et al., 2011). In this study, we demonstrate that a significant proportion of [(3)H]Arg uptake to cultured cortical astrocytes is likewise mediated by system y(+)L, in addition to the uptake showing characteristics of systems y(+), B(0+) and b(0+). However, stimulation of [(3)H]Arg uptake by treatment with 5mM ammonium chloride ("ammonia") for 48h could be solely ascribed to the y(+)L-mediated component of the uptake. Ammonia treatment increased the expression of the brain specific y(+)L isoform, y(+)LAT2, both at the mRNA and protein level, and silencing of the Slc7a6 gene coding for y(+)LAT2 protein specifically reduced the ammonia-induced [(3)H]Arg uptake. This study suggests an important role of y(+)LAT2 in the modulation of NO synthesis in the ammonia-exposed astrocytes.
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    ABSTRACT: Glutathione and thioredoxin are complementary antioxidants in the protection of mammalian tissues against oxidative-nitrosative stress (ONS), and ONS is a principal cause of symptoms of hepatic encephalopathy (HE) associated with acute liver failure (ALF). We compared the activities of the thioredoxin system components: thioredoxin (Trx), thioredoxin reductase (TrxR) and the expression of the thioredoxin-interacting protein, and of the key glutathione metabolizing enzyme, glutathione peroxidase (GPx) in the cerebral cortex of rats with ALF induced by thioacetamide (TAA). ALF increased the Trx and TrxR activity without affecting Trip protein expression, but decreased GPx activity in the brains of TAA-treated rats. The total antioxidant capacity (TAC) of the brain was increased by ALF suggesting that upregulation of the thioredoxin may act towards compensating impaired protection by the glutathione system. Intraperitoneal administration of L-histidine (His), an amino acid that was earlier reported to prevent acute liver failure-induced mitochondrial impairment and brain edema, abrogated most of the acute liver failure-induced changes of both antioxidant systems, and significantly increased TAC of both the control and ALF-affected brain. These observations provide further support for the concept of that His has a potential to serve as a therapeutic antioxidant in HE. Most of the enzyme activity changes evoked by His or ALF were not well correlated with alterations in their expression at the mRNA level, suggesting complex translational or posttranslational mechanisms of their modulation, which deserve further investigations.
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