Jan Albrecht

Glutamine : A trojan horse in ammonia neurotoxicity

Mechanisms involved in hepatic encephalopathy still remain to be defined. Nonetheless, it is well recognized that ammonia is a major factor in its pathogenesis, and that the astrocyte represents a major target of its CNS toxicity. In vivo and in vitro studies have shown that ammonia evokes oxidative/nitrosative stress, mitochondrial abnormalities (the mitochondrial permeability transition, MPT) and astrocyte swelling, a major component of the brain edema associated with fulminant hepatic failure. How ammonia brings about these changes in astrocytes is not well understood. It has long been accepted that the conversion of glutamate to glutamine, catalyzed by glutamine synthetase, a cytoplasmic enzyme largely localized to astrocytes in brain, represented the principal means of cerebral ammonia detoxification. Yet, the “benign” aspect of glutamine synthesis has been questioned. This article highlights evidence that, at elevated levels, glutamine is indeed a noxious agent. We also propose a mechanism by which glutamine executes its toxic effects in astrocytes, the “Trojan horse” hypothesis. Much of the newly synthesized glutamine is subsequently metabolized in mitochondria by phosphate‐activated glutaminase, yielding glutamate and ammonia. In this manner, glutamine (the Trojan horse) is transported in excess from the cytoplasm to mitochondria serving as a carrier of ammonia. We propose that it is the glutamine‐derived ammonia within mitochondria that interferes with mitochondrial function giving rise to excessive production of free radicals and induction of the MPT, two phenomena known to bring about astrocyte dysfunction, including cell swelling. Future therapeutic approaches might include controlling excessive transport of newly synthesized glutamine to mitochondria and its subsequent hydrolysis. (HEPATOLOGY 2006;44:788–794.)

Hepatic encephalopathy: molecular mechanisms underlying the clinical syndrome.

Hepatic encephalopathy (HE) and portal-systemic encephalopathy (PSE) are the terms used interchangeably to describe a complex neuropsychiatric syndrome associated with acute or chronic hepatocellular failure, increased portal systemic shunting of blood, or both. Hepatic encephalopathy complicating acute liver failure is referred to as fulminant hepatic failure (FHF). The clinical manifestations of HE or PSE range from minimal changes in personality and motor activity, to overt deterioration of intellectual function, decreased consciousness and coma, and appear to reflect primarily a variable imbalance between excitatory and inhibitory neurotransmission. Pathogenic mechanisms that may be responsible for HE have been extensively investigated using animal models of HE, or cultures of CNS cells treated with neuroactive substances that have been implicated in HE. Of the many compounds that accumulate in the circulation as a consequence of impaired liver function, ammonia is considered to play an important role in the onset of HE. Acute ammonia neurotoxicity, which may be a cause of seizures in FHF, is excitotoxic in nature, being associated with increased synaptic release of glutamate (Glu), the major excitatory neurotransmitter of the brain, and subsequent overactivation of the ionotropic Glu receptors, mainly the N-methyl-D-aspartate (NMDA) receptors. Hepatic encephalopathy complicating chronic liver failure appears to be associated with a shift in the balance between inhibitory and excitatory neurotransmission towards a net increase of inhibitory neurotransmission, as a consequence of at least two factors. The first is down-regulation of Glu receptors resulting in decreased glutamatergic tone. The down-regulation follows excessive extrasynaptic accumulation of Glu resulting from its impaired re-uptake into nerve endings and astrocytes. Liver failure inactivates the Glu transporter GLT-1 in astrocytes. The second factor is an increase in inhibitory neurotransmission by gamma-aminobutyric acid (GABA) due to (a) increased brain levels of natural benzodiazepines; (b) increased availability of GABA at GABA-A receptors, due to enhanced synaptic release of the amino acid; (c) direct interaction of modestly increased levels of ammonia with the GABA-A-benzodiazepine receptor complex; and (d) ammonia-induced up-regulation of astrocytic peripheral benzodiazepine receptors (PBZR). Brain ammonia is metabolised in astrocytes to glutamine (Gln), an osmolyte, and increased Gln accumulation in these cells may contribute to cytotoxic brain edema, which often complicates FHF. Glutamine efflux from the brain is an event that facilitates plasma-to-brain transport of aromatic amino acids. Tryptophan and tyrosine are direct precursors of the aminergic inhibitory neurotransmitters, serotonin and dopamine, respectively. Changes in serotonin and dopamine and their receptors may contribute to some of the motor manifestations of HE. Finally, oxindole, a recently discovered tryptophan metabolite with strong sedative and hypotensive properties, has been shown to accumulate in cirrhotic patients and animal models of HE.

Experimental models of hepatic encephalopathy: ISHEN guidelines.

Objectives of the International Society for Hepatic Encephalopathy and Nitrogen Metabolism Commission were to identify well‐characterized animal models of hepatic encephalopathy (HE) and to highlight areas of animal modelling of the disorder that are in need of development. Features essential to HE modelling were identified. The best‐characterized animal models of HE in acute liver failure, the so‐called Type A HE, were found to be the hepatic devascularized rat and the rat with thioacetamide‐induced toxic liver injury. In case of chronic liver failure, surgical models in the rat involving end‐to‐side portacaval anastomosis or bile duct ligation were considered to best model minimal/mild (Type B) HE. Unfortunately, at this time, there are no satisfactory animal models of Type C HE resulting from end‐stage alcoholic liver disease or viral hepatitis, the most common aetiologies encountered in patients. The commission highlighted the urgent need for such models and of improved models of HE in chronic liver failure in general as well as a need for models of post‐transplant neuropsychiatric disorders. Studies of HE pathophysiology at the cellular and molecular level continue to benefit from in vitro and or ex vivo models involving brain slices or exposure of cultured cells (principally cultured astrocytes) to toxins such as ammonia, manganese and pro‐inflammatory cytokines. More attention could be paid in the future to in vitro models involving the neurovascular unit, microglia and neuronal co‐cultures in relation to HE pathogenesis.

Role of retinal glial cells in neurotransmitter uptake and metabolism

In addition to photoreceptors and neurons, glial cells (in particular Müller cells) contribute to the removal and metabolization of neurotransmitters in the neural retina. This review summarizes the present knowledge regarding the role of retinal glial cells in the uptake of glutamate, N-acetylaspartylglutamate, gamma-aminobutyric acid, glycine, and d-serine, as well as the degradation and removal of purinergic receptor agonists. Some major pathways of glutamate metabolism in Müller cells are described; these pathways are involved in the glutamate-glutamine cycle of the retina, in the defense against oxidative and nitrosative stress via the production of glutathione, and in the production of substrates for the neuronal energy metabolism. In addition, the developmental regulation of the major glial glutamate transporter, GLAST, and of the glia-specific enzyme glutamine synthetase is described, as well as the importance of a malfunction and even reversal of glial glutamate transporters, and a downregulation of the glutamine synthetase, as pathogenic factors in different retinopathies.

Taurine interaction with neurotransmitter receptors in the CNS: an update.

Taurine appears to have multiple functions in the brain participating both in volume regulation and neurotransmission. In the latter context it may exert its actions by serving as an agonist at receptors of the GABAergic and glycinergic neurotransmitter systems. Its interaction with GABAA and GABAB receptors as well as with glycine receptors is reviewed and the physiological relevance of such interactions is evaluated. The question as to whether local extracellular concentrations of taurine are likely to reach the threshold level for the pertinent receptor populations cannot presently be answered satisfactorily. Hence more sophisticated analytical methods are warranted in order to obtain a definite answer to this important question.

Methylmercury induces oxidative injury, alterations in permeability and glutamine transport in cultured astrocytes

The neurotoxicity of high levels of methylmercury (MeHg) is well established both in humans and experimental animals. Astrocytes accumulate MeHg and play a prominent role in mediating MeHg toxicity in the central nervous system (CNS). Although the precise mechanisms of MeHg neurotoxicity are ill-defined, oxidative stress and altered mitochondrial and cell membrane permeability appear to be critical factors in its pathogenesis. The present study examined the effects of MeHg treatment on oxidative injury, mitochondrial inner membrane potential, glutamine uptake and expression of glutamine transporters in primary astrocyte cultures. MeHg caused a significant increase in F(2)-isoprostanes (F(2)-IsoPs), lipid peroxidation biomarkers of oxidative damage, in astrocyte cultures treated with 5 or 10 microM MeHg for 1 or 6 h. Consistent with this observation, MeHg induced a concentration-dependant reduction in the inner mitochondrial membrane potential (DeltaPsi(m)), as assessed by the potentiometric dye, tetramethylrhodamine ethyl ester (TMRE). Our results demonstrate that DeltaPsi(m) is a very sensitive endpoint for MeHg toxicity, since significant reductions were observed after only 1 h exposure to concentrations of MeHg as low as 1 microM. MeHg pretreatment (1, 5 and 10 microM) for 30 min also inhibited the net uptake of glutamine ((3)H-glutamine) measured at 1 min and 5 min. Expression of the mRNA coding the glutamine transporters, SNAT3/SN1 and ASCT2, was inhibited only at the highest (10 microM) MeHg concentration, suggesting that the reduction in glutamine uptake observed after 30 min treatment with lower concentrations of MeHg (1 and 5 microM) was not due to inhibition of transcription. Taken together, these studies demonstrate that MeHg exposure is associated with increased mitochondrial membrane permeability, alterations in glutamine/glutamate cycling, increased ROS formation and consequent oxidative injury. Ultimately, MeHg initiates multiple additive or synergistic disruptive mechanisms that lead to cellular dysfunction and cell death.

Regulation of pH in the mammalian central nervous system under normal and pathological conditions: facts and hypotheses.

The maintenance of pH homeostasis in the CNS is of key importance for proper execution and regulation of neurotransmission, and deviations from this homeostasis are a crucial factor in the mechanism underlying a spectrum of pathological conditions. The first few sections of the review are devoted to the brain operating under normal conditions. The article commences with an overview of how extrinsic factors modelling the brain at work: neurotransmitters, depolarising stimuli (potassium and voltage changes) and cyclic nucleotides as major signal transducing vehicles affect pH in the CNS. Further, consequences of pH alterations on the major aspects of CNS function and metabolism are outlined. Next, the major cellular events involved in the transport, sequestration, metabolic production and buffering of protons that are common to all the mammalian cells, including the CNS cells. Since CNS function reflects tight interaction between astrocytes and neurons, the pH regulatory events pertinent to either cell type are discussed: overwhelming evidence implicates astrocytes as a key player in pH homeostasis in the brain. The different classes of membrane proteins involved in proton shuttling are listed and their mechanisms of action are given. These include: the Na+/H+ exchanger, different classes of bicarbonate transporters acting in a sodium-dependent- or -independent mode, monocarboxylic acid transporters and the vacuolar-type proton ATPase. A separate section is devoted to carbonic anhydrase, which is represented by multiple isoenzymes capable of pH buffering both in the cell interior and in the extracellular space. Next, impairment of pH regulation and compensatory responses occurring in brain affected by different pathologies: hypoxia/ischemia, epilepsy, hyperammonemic encephalopathies, cerebral tumours and HIV will be described. The review is limited to facts and plausible hypotheses pertaining to phenomena directly involved in pH regulation: changes in pH that accompany metabolic stress but have no distinct implications for the pH regulatory mechanisms are not dealt with. In most cases, the vast body of knowledge derived from in vitro studies remains to be verified in in vivo settings.

Roles of glutamine in neurotransmission

Glutamine (Gln) is found abundantly in the central nervous system (CNS) where it participates in a variety of metabolic pathways. Its major role in the brain is that of a precursor of the neurotransmitter amino acids: the excitatory amino acids, glutamate (Glu) and aspartate (Asp), and the inhibitory amino acid, γ-amino butyric acid (GABA). The precursor-product relationship between Gln and Glu/GABA in the brain relates to the intercellular compartmentalization of the Gln/Glu(GABA) cycle (GGC). Gln is synthesized from Glu and ammonia in astrocytes, in a reaction catalyzed by Gln synthetase (GS), which, in the CNS, is almost exclusively located in astrocytes (Martinez-Hernandez et al., 1977). Newly synthesized Gln is transferred to neurons and hydrolyzed by phosphate-activated glutaminase (PAG) to give rise to Glu, a portion of which may be decarboxylated to GABA or transaminated to Asp. There is a rich body of evidence which indicates that a significant proportion of the Glu, Asp and GABA derived from Gln feed the synaptic, neurotransmitter pools of the amino acids. Depolarization-induced-, calcium- and PAG activity-dependent releases of Gln-derived Glu, GABA and Asp have been observed in CNS preparations in vitro and in the brain in situ. Immunocytochemical studies in brain slices have documented Gln transfer from astrocytes to neurons as well as the location of Gln-derived Glu, GABA and Asp in the synaptic terminals. Patch-clamp studies in brain slices and astrocyte/neuron co-cultures have provided functional evidence that uninterrupted Gln synthesis in astrocytes and its transport to neurons, as mediated by specific carriers, promotes glutamatergic and GABA-ergic transmission. Gln entry into the neuronal compartment is facilitated by its abundance in the extracellular spaces relative to other amino acids. Gln also appears to affect neurotransmission directly by interacting with the NMDA class of Glu receptors. Transmission may also be modulated by alterations in cell membrane polarity related to the electrogenic nature of Gln transport or to uncoupled ion conductances in the neuronal or glial cell membranes elicited by Gln transporters. In addition, Gln appears to modulate the synthesis of the gaseous messenger, nitric oxide (NO), by controlling the supply to the cells of its precursor, arginine. Disturbances of Gln metabolism and/or transport contribute to changes in Glu-ergic or GABA-ergic transmission associated with different pathological conditions of the brain, which are best recognized in epilepsy, hepatic encephalopathy and manganese encephalopathy.

Roles of neuroactive amino acids in ammonia neurotoxicity

Many neurologic disorders are related to congenital or acquired hyperammonemia (HA). Advanced symptoms of HA range from seizures in acute stages to stupor and coma in more chronic conditions, manifesting variable imbalance between the inhibitory and excitatory neurotransmission. Evidence obtained with the use of experimental HA models suggests that acute neurotoxic effects of ammonia are mediated by overactivation of ionotropic glutamate (GLU) receptors, mainly the N‐methyl‐D‐aspartate (NMDA) receptors, and to a lesser degree the KA/AMPA receptors. NMDA receptor–mediated neurotoxicity may be potentiated by impaired control of their function by metabotropic GLU receptors, which are inactivated by ammonia. Prolonged overactivation of the NMDA receptors upon extended ammonia exposure causes their downregulation. The GLU receptor changes may be related to their excessive exposure to extrasynaptic GLU. Ammonia promotes GLU accumulation in the extrasynaptic space by enhancing its release from neurons, and/or by decreasing its reuptake to the nerve endings and astrocytes, where the effect results from inactivation (downregulation) of the astrocytic glutamate transporter GLT1. Excitotoxic effects of ammonia are augmented by increased synthesis of nitric oxide (NO), which is associated with NMDA receptor activation and/or increased synaptic transport of arginine (ARG). A shift toward neural inhibition is promoted by positive modulation of the γ‐aminobutyric acid (GABA)ergic tone resulting from excessive accumulation in the brain of endogenous central benzodiazepine receptor agonists, and from upregulation of astrocytic peripheral benzodiazepine receptors leading to elevated levels of prognenelone‐derived neurosteroids, which positively modulate the GABA(A) receptor complex. Inhibitory neurotransmission may also be favored by enhanced release from astrocytes of an inhibitory amino acid, taurine. J. Neurosci. Res. 51:133–138, 1998. © 1998 Wiley‐Liss, Inc.

Glutamine as a pathogenic factor in hepatic encephalopathy

Hepatic encephalopathy (HE) results from acute or chronic liver dysfunction and is associated with hyperammonemia. Ammonium ions penetrate from blood to brain, where they form glutamine (Gln) in the reaction with glutamate catalyzed by an astroglia‐specific enzyme, glutamine synthetase (GS). Experimental data suggest that many manifestations of HE can be ascribed to increased Gln synthesis and accumulation in the brain. In HE resulting from acute liver failure (“fulminant hepatic failure”), the osmotic action of Gln appears to be in a large degree responsible for cerebral edema and edema‐associated disturbances of cerebral blood flow and ionic homeostasis. In chronic HE not accompanied by cerebral edema, Gln contributes to impairment of cerebral energy metabolism, and its increased transport from brain to the periphery accelerates the blood‐to‐brain transport of aromatic amino acids, of which tryptophen (Trp) is converted to metabolites directly implicated in HE. Most of the evidence that Gln participates in pathological events has been derived from their disappearance or amelioration in HE rats in which the cerebral Gln content was reduced by treatment with a GS inhibitor, methionine sulfoximine. J. Neurosci. Res. 65:1–5, 2001.