Hyperammonemia causes protein oxidation and enhanced proteasomal activity in response to mitochondria-mediated oxidative stress in rat primary astrocytes

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Abstract

Hyperammonemia, as a consequence of severe liver failure, is strongly associated with the neurological syndrome hepatic encephalopathy (HE) whereby excessive ammonia is metabolized by astrocytes, followed by cell and brain swelling in vivo. In the present study we were able to show that ammonia treatment of primary astrocytes in vitro is followed by cell swelling and a loss of cell viability at higher ammonia concentrations. Lower ammonia concentrations are accompanied by mitochondria-derived oxidative stress, as demonstrated by using inhibitors of mitochondrial glutaminase I, 143B-rho (0) cells and isolated mitochondria. The oxidative stress generated by mitochondria is accompanied by protein oxidation. In further studies we could show, that an activation of the proteasomal system takes place during ammonia exposure and protects cells. The proteasome acitvation can be blocked by antioxidants or by inhibitors of enzymes of glutamine metabolism. We conclude that oxidative stress-mediated proteasomal activation is important for survival of astroglial cells.

Introduction

Acute as well as chronic liver diseases are among the most challenging tasks of gastroenterology. One of the reasons for that is the complication of the primary disease process in the liver by numerous metabolic disturbances throughout the organism. One of the most dramatic changes is the accumulation of various toxic products, among them ammonia. Hyperammonemia might accompany acute as well as chronic liver diseases and leads to a symptom called hepatic encephalopathy (HE1). This is accompanied by failure of energy metabolism [1] and disturbances in neurotransmission in the brain [2], [3]. One of the major features of hepatic encephalopathy is the swelling of the brain, perhaps leading to a fatal outcome [4]. This swelling is based on the reaction of astroglial cells, taking up the ammonia and metabolizing it [5], [6], [7]. It has been shown that pathophysiological levels of ammonia cause nitrosative- and oxidative stress as well and that astrocytes appear to be particularly vulnerable [8], [9]. Protein tyrosine nitration (PTN) was found in ammonia-exposed astrocyte cultures and in vivo. PTN was dependent on increased intracellular calcium levels, degradation of the NFκB inhibitor IκB, and activation of NO synthase (iNOS) [10]. Already early studies on ammonia-induced oxidative stress reported about increased lipid peroxidation in hyperammonemic mice [10], [11] and in ammonia-treated murine astrocyte cultures [12]. Recently, the production of free radicals was detected in response to ammonia exposure of astrocytes, which was reversible by using of l-methionine sulfoximine, a potent glutamine synthetase (GS) inhibitor [13]. It was suggested that production of reactive oxygen species (ROS) might be glutamine-mediated since GS is highly enriched in astrocytes [14]. Subsequent studies showed glutamine-induced formation of free radicals in cultured astrocytes [15]. This effect was attenuated after inhibiting the mitochondrial phosphate activated glutaminase I (PAG) which catalyzes the hydrolysis of glutamine [15], [16] and which is located in mitochondria [17], [18]. The released ammonia may accumulate in mitochondria, a likely source for oxidative stress. Furthermore, mitochondrial permeability transition (MPT) was found to be associated with elevated glutamine- and ammonia-levels and the production of free radicals [15], [16], [19], [20], [21], [22]. These findings are supported by the hypothesis that hyperammonemia linked ROS production is highly associated with the glutamine–glutamate-cycle and is generated in mitochondria. On the other hand, ammonia exposure is always accompanied by astrocyte swelling. Chan et al. [23] and Schliess et al. [24] have demonstrated, that swelling per sè might be accompanied by an increase in the generation of reactive oxidant and nitrogen species.

On the other hand, the quantification of reactive oxidant formation was frequently performed, but metabolic consequences in the swollen astrocytes were scarily detected. Therefore, only limited information about the consequences of oxidative stress towards intracellular cell components is available. This refers especially to the consequences of the oxidative stress on the intracellular protein pool and the proteolytic reaction to such protein oxidation during hyperammoniemia. In general oxidatively damaged proteins are targeted towards the major intracellular degradation machinery, the proteasomal system [25], [26], [27], [28], [29], [30], [31].

The present study addresses to the oxidative protein damage generated by high ammonia levels, which is a common feature of HE, and the response of the proteolytic system in astrocytes. Further, the origin of the reactive oxidant formation was investigated by using an mtDNA depleted strain and isolated mitochondria.

Section snippets

Cell cultures

Primary astrocytes were prepared from 3 days old Wistar rats as described by McCarthy and de Vellis [32] with slight modifications. In brief, brains were removed aseptically, washed and homogenized in icecold HBSS using pipettes. The cells were transferred into 75 culture flasks and grown for 2 weeks in glutamine-free DMEM low glucose medium containing 10% fetal calf serum, 1% Penicillin/Streptomycin and 1% Glutamine. The mixed glial cell culture was shaked overnight at 250 rpm to remove

Effects of ammonia on astrocyte volume and viability

To investigate the role of ammonia in the oxidation of the intracellular protein pool we isolated astrocytes from rat brain using the method of McCarthy and de Vellis [32]. As demonstrated in Fig. 1a, pure ⩾98% astrocyte cell cultures could be provided. For the hyperammonemic exposure, besides the control media we used media containing three different NH4Cl concentrations, namely 5, 15, 50 mM. The change of the pH value by less than ±0.025 was negligible. Astrocyte cell cultures were exposed to

Discussion

Oxidative stress caused by the pathophysiological process of HE like hyperammonemia and the developing symptoms have been investigated in the recent years to some degree [10], [13], [20], [21], [22]. Especially the intracellular oxidative status of astrocytes became more and more relevant since astrocytes are known to be the major site for ammonia detoxification [14], [41].

A variety of symptoms in the pathophysiological process of HE is accompanied by the occurrence of oxidative stress. A

Acknowledgments

We thank Dr. B. Görg and Dr. F. Schliess from the Department of Experimental Hepatology at the Heinrich-Heine-University in Düsseldorf for the supply with the hypoosmotic medium. Also, we thank Tobias Gremmel and Dr. Peter Schröder for the kind support in the oxygen electrode measurements. Dr. R. Schins has kindly provided the osteosarkoma cell line 143B and the deficient cell strain 143B-rho (0). Additionally, we thank Dr. Hu Li for her advice in 143B-specific cell culture treatment. T.G. was

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