Inhibition of glutamine synthetase during ammonia exposure in rainbow trout indicates a high reserve capacity to prevent brain ammonia toxicity

Abstract

SUMMARYGlutamine synthetase (GSase), the enzyme that catalyses the conversion of glutamate and ammonia to glutamine, is present at high levels in vertebrate brain tissue and is thought to protect the brain from elevated ammonia concentrations. We tested the hypothesis that high brain GSase activity is critical in preventing accumulation of brain ammonia and glutamate during ammonia loading in the ammonia-intolerant rainbow trout. Trout pre-injected with saline or the GSase inhibitor methionine sulfoximine (MSOX, 6mgkg -1 ), were exposed to 0, 670 or 1000moll -1 NH 4 Cl in the water for 24 and 96h. Brain ammonia levels were 3-to 6-fold higher in ammonia-exposed fish relative to control fish and MSOX treatment did not alter this. Brain GSase activity was unaffected by ammonia exposure, while MSOX inhibited GSase activity by ~75%. Brain glutamate levels were lower and glutamine levels were higher in fish exposed to ammonia relative to controls. While MSOX treatment had little impact on brain glutamate, glutamine levels were significantly reduced by 96h. With ammonia treatment, significant changes in the concentration of multiple other brain amino acids occurred and these changes were mostly reversed or eliminated with MSOX. Overall the changes in amino acid levels suggest that multiple enzymatic pathways can supply glutamate for the production of glutamine via GSase during ammonia exposure and that alternative transaminase pathways can be recruited for ammonia detoxification. Plasma cortisol levels increased 7-to 15-fold at 24h in response to ammonia and MSOX did not exacerbate this stress response. These findings indicate that rainbow trout possess a relatively large reserve capacity for ammonia detoxification and for preventing glutamate accumulation during hyperammonaemic conditions.

Sensitivity of ventilation and brain metabolism to ammonia exposure in rainbow trout,Oncorhynchus mykiss

“…Ammonia readily crosses the blood-brain barrier in teleost fish, because when blood ammonia levels increase during HEA, intracellular ammonia concentrations in the brain tissue also increase. While this has been widely documented in tropical fish (reviewed by Ip et al, 2001;Chew et al, 2005), it has also been seen in rainbow trout Sanderson et al, 2010). Furthermore, mRNA expressions of the Rhbg and Rhcg1 glycoproteins have been detected in trout brain (Nawata et al, 2007); these appear to function as NH 3 channels (Nawata et al, 2010) and their expression decreases after 48h of HEA exposure (Nawata et al, 2007).…”

“…The whole tissue sampling was finished within 2min. (Sanderson et al, 2010)]. They were then kept individually in darkened plexiglass boxes with aeration and flowing water for 48h to allow the effects of MSOX to develop.…”

“…In a separate experiment, the time course of changes in brain T Amm and ventilation were followed closely during acute HEA challenge. Methionine sulfoxamine (MSOX), a well-established inhibitor of GSase in previous fish studies Veauvy et al, 2005;Wee et al, 2007;Sanderson et al, 2010) was also employed in both acute and chronic exposures, together with measurements of GSase activity, and concentrations of T Amm , glutamine, and glutamate in brain tissue. These tests examined how ammonia detoxification in the brain interacts with the ventilatory responses to ammonia.…”

Feeding through your gills and turning a toxicant into a resource: how the dogfish shark scavenges ammonia from its environment

“…Different animals were used in the two treatments, and standard 6 h flux tests were run with 200 µmol l −1 ammonia (added as NH 4 HCO 3 ) in the water. The MSOX dose (60 mg kg −1 ) was approximately 10-fold greater than routinely used in teleosts (Sanderson et al, 2010;Zhang et al, 2013), but glutamine synthetase is more abundant in elasmobranchs (Kajimura et al, 2006). In a preliminary experiment with one animal, this dose proved not to cause mortality but did cause a slow decline in urea-N excretion rate after 48 h. Therefore, the 6 h tests were run at 35-41 h after injection, a time selected to avoid any secondary consequences due to internal urea declines.…”

Features of nitrogen metabolism in fishes

“…In particular, the study of the effectiveness of glutamatedependent mechanisms intraorganic neutralize the ammonia in some species of fish, a long time can survive in arid environments where the gill system of clearance of nitrogenous products of catabolism are not fully functioning (Anderson et al 2002), promotes the formation of science-based policy in selection of high-value fish species that are resistant to adverse hydrochemical conditions (Wright et al 2007;Sanderson et al 2010). In addition, attract the attention that the fish eggs, along with gill and kidney epithelium, characterized by high expression of specific transport proteins involved in mediated transport of ammonia (Nakada et al 2007).…”