Brain alanine formation as an ammonia-scavenging pathway during hyperammonemia:effects of glutamine synthetase inhibition in rats and astrocyte-neuron co-cultures

Hyperammonemia is a major etiological toxic factor in the development of hepatic encephalopathy. Brain ammonia detoxification occurs primarily in astrocytes by glutamine synthetase (GS), and it has been proposed that elevated glutamine levels during hyperammonemia lead to astrocyte swelling and cerebral edema. However, ammonia may also be detoxified by the concerted action of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALAT) leading to trapping of ammonia in alanine, which in vivo likely leaves the brain. Our aim was to investigate whether the GS inhibitor methionine sulfoximine (MSO) enhances incorporation of (15)NH(4)(+) in alanine during acute hyperammonemia. We observed a fourfold increased amount of (15)NH(4) incorporation in brain alanine in rats treated with MSO. Furthermore, co-cultures of neurons and astrocytes exposed to (15)NH(4)Cl in the absence or presence of MSO demonstrated a dose-dependent incorporation of (15)NH(4) into alanine together with increased (15)N incorporation in glutamate. These findings provide evidence that ammonia is detoxified by the concerted action of GDH and ALAT both in vivo and in vitro, a mechanism that is accelerated in the presence of MSO thereby reducing the glutamine level in brain. Thus, GS could be a potential drug target in the treatment of hyperammonemia in patients with hepatic encephalopathy

Effect of glutamine synthetase inhibition on brain and interorgan ammonia metabolism in bile duct ligated rats

Ammonia has a key role in the development of hepatic encephalopathy (HE). In the brain, glutamine synthetase (GS) rapidly converts blood-borne ammonia into glutamine which in high concentrations may cause mitochondrial dysfunction and osmolytic brain edema. In astrocyte-neuron cocultures and brains of healthy rats, inhibition of GS by methionine sulfoximine (MSO) reduced glutamine synthesis and increased alanine synthesis. Here, we investigate effects of MSO on brain and interorgan ammonia metabolism in sham and bile duct ligated (BDL) rats. Concentrations of glutamine, glutamate, alanine, and aspartate and incorporation of (15)NH(4)(+) into these amino acids in brain, liver, muscle, kidney, and plasma were similar in sham and BDL rats treated with saline. Methionine sulfoximine reduced glutamine concentrations in liver, kidney, and plasma but not in brain and muscle; MSO reduced incorporation of (15)NH(4)(+) into glutamine in all tissues. It did not affect alanine concentrations in any of the tissues but plasma alanine concentration increased; incorporation of (15)NH(4)(+) into alanine was increased in brain in sham and BDL rats and in kidney in sham rats. It inhibited GS in all tissues examined but only in brain was an increased incorporation of (15)N-ammonia into alanine observed. Liver and kidney were important for metabolizing blood-borne ammonia

Reducing peripheral inflammation with infliximab reduces neuroinflammation and improves cognition in rats with hepatic encephalopathy.

Inflammation contributes to cognitive impairment in patients with hepatic encephalopathy (HE). However, the process by which peripheral inflammation results in cognitive impairment remains unclear. In animal models, neuroinflammation and altered neurotransmission mediate cognitive impairment. Taking into account these data, we hypothesized that in rats with HE: (1) peripheral inflammation is a main contributor to neuroinflammation; (2) neuroinflammation in hippocampus impairs spatial learning by altering AMPA and/or NMDA receptors membrane expression; (3) reducing peripheral inflammation with infliximab (anti-TNF-a) would improve spatial learning; (4) this would be associated with reduced neuroinflammation and normalization of the membrane expression of glutamate receptors. The aims of this work were to assess these hypotheses. We analyzed in rats with portacaval shunt (PCS) and control rats, treated or not with infliximab: (a) peripheral inflammation by measuring prostaglandin E2, IL10, IL-17, and IL-6; (b) neuroinflammation in hippocampus by analyzing microglial activation and the content of TNF-a and IL-1b; (c) AMPA and NMDA receptors membrane expression in hippocampus; and (d) spatial learning in the Radial and Morris water mazes. We assessed the effects of treatment with infliximab on peripheral inflammation, on neuroinflammation and AMPA and NMDA receptors membrane expression in hippocampus and on spatial learning and memory. PCS rats show increased serum prostaglandin E2, IL-17, and IL-6 and reduced IL-10 levels, indicating increased peripheral inflammation. PCS rats also show microglial activation and increased nuclear NF-kB and expression of TNF-a and IL-1b in hippocampus. This was associated with altered AMPA and NMDA receptors membrane expression in hippocampus and impaired spatial learning and memory in the radial and Morris water maze. Treatment with infliximab reduces peripheral inflammation in PCS rats, normalizing prostaglandin E2, IL-17, IL-6, and IL-10 levels in serum. Infliximab also prevents neuroinflammation, reduces microglial activation, translocates NF-kB into nucleoli and normalizes TNF-a and IL-1b content in hippocampus. This was associated with normalization of AMPA receptors membrane expression in hippocampus and of spatial learning and memory. The results suggest that peripheral inflammation contributes to spatial learning impairment in PCS rats. Treatment with anti-TNF-a could be a new therapeutic approach to improve cognitive function in patients with HE