Ammonia and Manganese Increase Arginine Uptake in Cultured Astrocytes

Recent work has suggested a possible role for nitric oxide (NO) in the development of hepatic encephalopathy (HE). In this study, we examined the effect of ammonia and manganese, factors implicated in the pathogenesis of HE, on the transport of arginine (a precursor of NO) into primary cultures of astrocytes. Treatment with 5 mM ammonia for 1–4 days produced a maximal (53%) increase in L-arginine uptake at 3 days when compared to untreated cells. Kinetic analysis following 4-day treatment with 5 mM ammonia revealed an 82% increase in the V_max and a 61% increase in the K_m, value. Similar analysis with 100 M manganese showed a 101% increase in V_max and a 131% increase in the K_m value. These results suggest that both manganese and ammonia alter L-arginine uptake by modifying the transporter for arginine. A decrease of 32% in the non-saturable component of L-arginine transport was also observed following treatment with ammonia. When cultures were treated separately with 5 mM ammonia and 100 M manganese for 2 days, the uptake of L-arginine increased by 41% and 57%, respectively. Combined exposure led to no further increase in uptake. Our results suggest that ammonia and manganese may contribute to the pathogenesis of HE by influencing arginine transport and thus possibly NO synthesis in astrocytes.     

Combined Effects of Ammonia and Manganese on Astrocytes in Culture

Ammonia has been strongly implicated in the pathogenesis of hepatic encephalopathy (HE), and astrocytes appear to be the primary target of ammonia neurotoxicity. Recent work has shown that manganese also plays a role in the pathogenesis of HE and causes astrocyte morphologic and functional changes similar to ammonia. We therefore investigated whether a combination of these compounds could produce additive/synergistic effects. Cultured astrocytes treated with 5 mM ammonia (NH₄Cl) along with 100 M manganese acetate (MnAc) for 3 h showed a 55–65% increase in free radical production over ammonia or manganese alone (P < 0.05). There was also a 50% decrease in the mitochondrial membrane potential ( _m) at 24 h following treatment with NH₄Cl (5 mM) plus MnAc (50 M) Pt< 0.05), as compared to ammonia or manganese alone. Astrocytes treated with ammonia or manganese alone for 24 h showed no cell death, as determined by LDH release and light microscopic examination. However, cultures treated with ammonia plus manganese showed 80–90 necrotic cell death as estimated by light microscopy and 59 cell death as determined by LDH release. LDH release by ammonia plus manganese was blocked by the antioxidant superoxide dismutase (25 units/ml) as well as by the nitric oxide synthase inhibitor N-nitro-L-argininemethyl ester (500 M). In conclusion, ammonia plus manganese exert additive/synergetic effects on the induction free radicals, mitochondrial inner membrane depolarization and cellular integrity, which may contribute to the tissue injury associated with chronic forms of HE.Special issue dedicated to Lawrence. F. Eng.     

Comparison of Effects of DL-Threo-β-Benzyloxyaspartate (DL-TBOA) and L-Trans-Pyrrolidine-2,4-Dicarboxylate (t-2,4-PDC) on Uptake and Release of [3H]D-Aspartate in Astrocytes and Glutamatergic Neurons

Uptake and release processes in cerebellar astrocytes and granule neurons (glutamatergic) for glutamate were investigated by the use of [³H]D-aspartate, a non-metabolizable glutamate analog. The effects of DL-threo--benzyloxyaspartate (DL-TBOA) and L-trans-pyrrolidine-2,4-dicarboxylate (t-2,4-PDC) on uptake and release of [³H]D-aspartate were studied. Both compounds inhibited potently uptake of [³H]D-aspartate in neurons and astrocytes (IC₅₀ values 10-100 M), DL-TBOA being slightly more potent than t-2,4-PDC. Release of preloaded [³H]D-aspartate from neurons or astrocytes could be stimulated by addition of excess t-2,4-PDC whereas addition of DL-TBOA had no effect on [³H]D-aspartate efflux. Moreover, DL-TBOA inhibited significantly the depolarization-induced (55 mM KCl) release of preloaded [³H]D-aspartate in the neurons. The results reflect the fact that DL-TBOA is not transported by the glutamate carriers while t-2,4-PDC is a substrate which may heteroexchange with [³H]D-aspartate. It is suggested that DL-TBOA may be used to selectively inhibit depolarization coupled glutamate release mediated by reversal of the carriers.     

Effect of Ammonia and Methionine Sulfoximine on myo-Inositol Transport in Cultured Astrocytes

Ammonia causes astrocyte swelling which is abrogated by methionine sulfoximine (MSO). Since myo-inositol is an important osmolyte, we investigated the effects of ammonia and MSO on myo-inositol flux in cultured astrocytes for periods up to 72 hours. Uptake of myo-inositol was significantly decreased by 26.7 (P < 0.05) and 39.3 (P lt; 0.006) percent after 48 hours of exposure to 5 or 10 mM ammonia, respectively. The maximum rate of uptake was 14.0 ± 0.5 nmol/hour/mg protein which was reduced to 7.45 ± 0.27 and 7.02 ± 0.57 nmoles/hour/mg protein by 5 or 10 mM ammonia, respectively. The Kms by Michaelis-Menten equation for the control, and in the presence of 5, or 10 mM ammonia were 32.5 ± 4.52, 44.4 ± 5.82, and 39.3 ± 7.0 M, respectively. Kms by Hanes-Woolf plot for the control, 5, or 10 mM ammonia were 25, 45, and 40 M, respectively. Treatment of astrocytes with either 5 or 10 mM NH₄Cl for 6 hours caused a decrease in myo-inositol content by 66% and 58%, respectively. MSO (3 mM) partially diminished the ammonia-induced inhibition of myo-inositol uptake and decreased myo-inositol content by 31% after 24 hours. Additionally, ammonia increased myo-inositol efflux briefly through the fast efflux component but had little effect on myo-inositol efflux through the slow efflux component of astrocytes exposed to ammonia for up to 72 hours. Predominantly decreased myo-inositol influx coupled with brief efflux through the fast component may represent an adaptive response to diminish the extent of ammonia-induced astrocyte swelling.     

Effects of ammonia on high affinity glutamate uptake and glutamate transporter EAAT3 expression in cultured rat cerebellar granule cells

Increased levels of extracellular glutamate are a consistent feature of hepatic encephalopathy (HE) associated with liver failure and other hyperammonemic pathologies. Reduction of glutamate uptake has been described in ammonia-exposed cultured astrocytes, synaptosomes, and in animal models of hyperammonemia. In the present study, we examine the effects of pathophysiological concentrations of ammonia on D-aspartate (a non-metabolizable analog of glutamate) uptake by cultured rat cerebellar granule neurons. Exposure of these cells to ammonia resulted in time-dependent (24% reduction at 24h and 60% reduction at 5 days, P<0.001) and dose-dependent (21, 37, and 57% reduction at 1, 2.5, and 5mM for 5 days, P<0.01) suppression of D-aspartate uptake. Kinetic analyses revealed significant decreases in the velocity of uptake (V(max)) (37% decrease at 2.5mM NH(4)Cl, P<0.05 and 52% decrease at 5mM NH(4)Cl, P<0.001) as well as significant reductions in K(m) values (25% reduction at 2.5mM NH(4)Cl, P<0.05 and 45% reduction at 5mM NH(4)Cl, P<0.001). Western blotting, on the other hand, showed no significant changes in the neuronal glutamate transporter EAAC1/EAAT3 protein, the only glutamate transporter currently known to be expressed by these cells. In addition, 1H combined with 13C-NMR spectroscopy studies using the stable isotope [1-13C]-glucose demonstrated a significant increase in intracellular glutamate levels derived from the oxidative metabolism of glucose, rather than from the deamidation of exogenous glutamine in cultured granule neurons exposed to ammonia. The present study provides evidence that the effects of ammonia on glutamate uptake are not solely an astrocytic phenomenon and that unlike the astrocytic glutamate transporter counterpart, EAAT3 protein expression in cultured cerebellar granule cells is not down-regulated when exposed to ammonia. Decrease of glutamate uptake in these cellular preparations may afford an additional regulatory mechanism aimed at controlling intracellular levels of glutamate and ultimately the releasable pool of glutamate in neurons.     

Three years of continuous measurements of atmospheric ammonia concentrations over a forest stand at the Höglwald site in southern Bavaria

Over a period of 3 years (1995 – 1997), atmospheric ammonia (NH₃) concentrations were measured 3 m above a spruce stand using a continuous-flow annular denuder at the Höglwald site near Munich, Bavaria. The annual average ammonia concentration was between 2.2 and 2.9 g NH₃ m^–3. The highest hourly average values occurred at the end of each year. In December 1995 the peak value was achieved with 183 g NH₃ m^–3. More than 50% of the hourly average means of the ammonia concentration were lower than 2 g NH₃ m^–3 and only fewer than 5% of the hourly average concentrations higher than 10 g NH₃ m^–3. The ammonia concentration course indicated a pronounced diurnal variation, with higher concentrations in the late morning and lower concentrations during the night. Often a sudden increase of the ammonia concentration was detected in the early morning with first sun exposure of the spruce crown and sinking humidity, indicating a reemission of ammonia from the canopy to the atmosphere.     

Effects of NH<Subscript>4</Subscript>CL application and removal on astrocytes and endothelial cells

Background

Hepatic encephalopathy (HE) is a complex disorder associated with increased ammonia levels in the brain. Although astrocytes are believed to be the principal cells affected in hyperammonemia (HA), endothelial cells (ECs) may also play an important role by contributing to the vasogenic effect of HA.

Methods

Following acute application and removal of NH₄Cl on astrocytes and endothelial cells, we analyzed pH changes, using fluorescence imaging with BCECF/AM, and changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i), employing fluorescence imaging with Fura-2/AM. Using confocal microscopy, changes in cell volume were observed accompanied by changes of [Ca²⁺]_i in astrocytes and ECs.

Results

Exposure of astrocytes and ECs to 1 – 20 mM NH₄Cl resulted in rapid concentration-dependent alkalinization of cytoplasm followed by slow recovery. Removal of the NH₄Cl led to rapid concentration-dependent acidification, again followed by slow recovery. Following the application of NH₄Cl, a transient, concentration-dependent rise in [Ca²⁺]_i in astrocytes was observed. This was due to the release of Ca²⁺ from intracellular stores, since the response was abolished by emptying intracellular stores with thapsigargin and ATP, and was still present in the Ca²⁺-free bathing solution. The removal of NH₄Cl also led to a transient concentration-dependent rise in [Ca²⁺]_i that resulted from Ca²⁺ release from cytoplasmic proteins, since removing Ca²⁺ from the bathing solution and emptying intracellular Ca²⁺ stores did not eliminate the rise. Similar results were obtained from experiments on ECs. Following acute application and removal of NH₄Cl no significant changes in astrocyte volume were detected; however, an increase of EC volume was observed after the administration of NH₄Cl, and EC shrinkage was demonstrated after the acute removal of NH₄Cl.

Conclusions

This study reveals new data which may give a more complete insight into the mechanism of development and treatment of HE.

     

Lactate Contributes to Ammonia-Mediated Astroglial Dysfunction During Hyperammonemia

Even though ammonia is considered to underlie nervous system symptoms of dysfunction during hyperammonemia, lactate, which increases as a metabolic consequence of high ammonia levels, might also be a contributing factor. The data presented here show that NH₄Cl (5 mM) mediates astroglial cell swelling, and that treatment with NH₄Cl or lactate (25 mM) causes rearrangements of actin filaments and reduces astroglial glutamate uptake capacity. Co-application with BaCl₂, which blocks astroglial uptake of NH₄ ⁺, prevents NH₄Cl-mediated cell swelling and rearrangement of actin filaments, but does not reduce NH₄Cl-induced glutamate uptake capacity inhibition. Neither NH₄Cl nor lactate affected glutamate uptake or protein expression in microglial cultures, indicating that astroglial cells are more susceptible to the neurotoxic affects of ammonia. Our results suggest that ammonium underlies brain edema, but that lactate can contribute to some of the cellular dysfunctions associated with elevated cerebral levels of ammonia.     

The Possible Role of Glutamate Uptake in Metaphit-Induced Seizures

In a study of the possible mechanism of action of metaphit and phencyclidine in the brain, the uptake of glutamate at the luminal side of the blood-brain barrier (BBB) was studied by means of an in situ brain perfusion technique in normal guinea pigs and in those pretreated with metaphit. Metaphit, an isothiocyanate analog of phencyclidine (PCP), induces time-dependent epileptogenic changes in the electroencephalogram in guinea pig, reaching a maximum 18–24 h after metaphit administration (50 mg/kg IP). In metaphit-pretreated animals a significant reduction of glutamate BBB uptake was found, in comparison with that of controls. Reduction of glutamate transport from blood to brain ranged from 77% to 79% in all brain structures studied. This inhibition was probably due to changes in the properties of saturable components responsible for transport of glutamate across the BBB. Kinetic measurements revealed a saturable amino acid influx into the parietal cortex, caudate nucleus, and hippocampus, with a Km between 3.1 and 5.1 M, and the V_max ranging from 14.3 to 27.8 pmol^–1 g^–1. The nonsaturable component, K_id, was statistically different from zero, ranging from 1.47 to 2.00 M min^–1 g^–1. Influx of glutamate into the brain was not altered in the presence of 1 mM D-aspartate, but it was significantly inhibited in the presence of 1 mM L-aspartate. We conclude that the cerebrovascular permeability of circulating glutamate is due to the presence of a higher-capacity saturable receptor and/or a carrier-mediated transport system (75%) and also a low-capacity diffusion transport system (25%) for the glutamate located at the luminal side of the BBB. The glutamate transport system is probably fully saturated at physiological plasma glutamate concentrations.     

Bradyrhizobium japonicum subspecies (USDA 110 and 26) characterized by fixed-nitrogen uptake and symbiotic indoleacetic acid production

Two dissimilar subspecies ofBradyrhizobium japonicum (USDA 110 and 26) differ in ammonia (NH₃) assimilation and symbiotic indoleacetic acid (IAA) production. Free-living cultures of type-strain USDA 26 grow on NH₃ as a sole N source and take up an NH₃ analog, methylamine, whereas USDA strain 110 does neither. Although both strains nodulate soybean effectively, root nodules infected with symbiont 26 contain 0.3–1.1 g IAA per gram fresh weight. Nodules infected by tryptophan catabolic variants 4b and 20d, derived from strain 26, also elicit an increased IAA content, two- to fourfold (2.0–3.9 g · g^–1). In contrast, nodules infected with the dissimilar subspecies (strains 110 and 123) contain significantly less IAA.     

Potassium modulation of methionine uptake in astrocytes in vitro

Methionine participates in a large variety of metabolic pathways in brain, and its transport may play an important regulatory role. The properties of methionine uptake were examined in a preparation of neonatal rat brain astrocytes. Uptake is linear for 15 minutes, up to 2.5 M. At steady state conditions, methionine is concentrated 30–50-fold. Measured methionine homoexchange accounts for a significant fraction of uptake at concentrations greater than 10 M. We recently reported that methionine uptake is decreased by elevations in extracellular K⁺. Potassium induced efflux cannot account for this apparent effect; and thus for concentrations less than 2.5M, and for short times of incubation, measured rates of methionine uptake represent unidirectional flux. At extracellular concentrations of K⁺ equal to 6.9 mM, the apparentV _max of methionine transport is 182 pmol/min/mg protein, and theK _m is 1.3 M. Where K⁺ is shifted to 11.9 mM, theK _m remains unchanged, and theV _max is reduced by half.     

Effects of chronic exposure to ammonia on glutamate and glutamine interconversion and compartmentation in homogeneous primary cultures of mouse astrocytes

Accumulation of radioactivity was studied in primary cultures of mouse astrocytes as a function of time of exposure (4–60 min) to 50 M glutamate and 200 M glutamine (initial concentrations), of whicheither glutamateor glutamine was¹⁴C-labeled. Both the glutamate pool and the glutamine pool were compartmentalized. Initially, by far the major intracellular glutamate pool (90%) was derived from extracellular glutamate and could be converted to glutamine. This allowed a rather accurate determination of metabolic flux from glutamate to glutamine, which under control conditions amounted to 2.0–2.2 nmol/min per mg protein. After chronic exposure to 3 mM ammonia for 3 days this flux was significantly increased to 3.1–3.6 nmol/min per mg protein. Acute exposure to ammonia caused a smaller, apparent increase, which was not statistically significant. The glutamine content was compartmentalized at all stages of the incubation. It consisted of at least two different pools. One of these was accessible to extracellular glutamine and could be converted to intracellular glutamate (constituting a sizeable fraction of the total glutamate pool after longer incubation), whereas the other constituted endogenously derived glutamine, formed from accumulated glutamate. The specific activity of the precursor pool for glutamate synthesis could not beaccurately determined and relatively exact fluxes therefore not be calculated. There was, however, no evidence that chronic exposure to ammonia decreases the rate of glutamine hydrolysis.     

Ammonia uptake and retention in some cyanobacteria

The internal pool of ammonia in strains of unicellular and filamentous cyanobacteria was found to be 6–12 nmol·mg^-1 protein. In nitrate grown Anacystis nidulans R-2 the pool size averaged 12 nmol·mg^-1 protein, which corresponds to 2.3 mM, and was little affected by N-source or medium pH during growth. Cells from NH ₄ ⁺ -limited continuous culture contained comparable pools, and cell yield was independent of medium pH (7.2–8.5). The internal pool was not bound to macromolecules. The pool fell transiently to about one-third within 2 h on shifting cells to N-free medium, but was slowly regenerated over 24 h.Added ammonia was removed from solution by illuminated cell suspensions at a linear rate, adequate to supply biosynthetic needs, to residual concentrations less than 5 M. An apparent K _m of less than 1 M can be inferred. Uptake rates were independent of N-source during growth, and of assay pH over the range 6.2–8.7. Bicarbonate was needed for uptake, but the rate of uptake was not influenced by the simultaneous presence of NaNO₃ (10 mM) or CH₃NH₃Cl (0.15 mM). Uptake was energydependent, and was eliminated in dark, anaerobic conditions or by the addition of protonophores. Uptake was also strongly inhibited by dicyclohexylearbodiimide, an ATPase inhibitor, by — SH reagents and methionine sulfoximine, suggesting that interference with energy supply or with ammonia metabolism prevented further entry into the cells.Non-standard abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCCD dicyclohexylcarbodiimide - DCMU dichlorophenyl dimethylurea - NEM N-ethylmaleimide - pCMB p-chloromercuribenzoate - MSX L-methionine Dl-sulfoximine     

Cooxidation of naphthalene and other polycyclic aromatic hydrocarbons by the nitrifying bacterium,Nitrosomonas europaea

The soil nitrifying bacterium Nitrosomonas europaea has shown the ability to transform cometabolically naphthalene as well as other 2- and 3-ringed polycyclic aromatic hydrocarbons (PAHs) to more oxidized products. All of the observed enzymatic reactions were inhibited by acetylene, a selective inhibitor of ammonia monooxygenase (AMO). A strong inhibitory effect of naphthalene on ammonia oxidation by N. europaea was observed. Naphthalene was readily oxidized by N. europaea and 2-naphthol was detected as a major product (85%) of naphthalene oxidation. The maximum naphthol production rate was 1.65 nmole/mg protein-min in the presence of 240 M naphthalene and 10 mM NH₄ ⁺. Our results demonstrate that the oxidation between ammonia and naphthalene showed a partial competitive inhibition. The relative ratio of naphthalene and ammonia oxidation, depending on naphthalene concentrations, demonstrated that the naphthalene was oxidized 2200-fold slower than ammonia at lower concentration of naphthalene (15 M) whereas naphthalene was oxidized only 100-fold slower than ammonia oxidation. NH₄ ⁺- and N₂H₄-dependent O₂ uptake measurement demonstrated irreversible inhibitory effects of the naphthalene and subsequent oxidation products on AMO and HAO activity.     

Permeability of ammonia,methylamine and ethylamine in the cyanobacterium,Synechococcus R-2 (Anacystis nidulans) PCC 7942

Summary Permeabilities of ammonia (NH₃), methylamine (CH₃NH₂) and ethylamine (CH₃CH₂NH₂) in the cyanobacterium (cyanophyte)Synechococcus R-2 (Anacystis nidulans) have been measured. Based on net uptake rates of DCMU (dichlorophenyldimethylurea) treated cells, the permeability of ammonia was 6.44±1.22 m sec^–1 (n=13). The permeabilities of methylamine and ethylamine, based on steady-state¹⁴C labeling were more than ten times that of ammonia (P _methylamine=84.6±9.47 m sec^–1 (76),P _ethylamine=109±11 m sec^–1 (55)). The apparent permeabilities based on net uptake rates of methylamine and ethylamine uptake were significantly lower, but this effect was partially reversible by ammonia, suggesting that net amine fluxes are rate limited by proton fluxes to an upper limit of about 700 nmol m^–2 sec^–1. Increasing concentrations of amines in alkaline conditions partially dissipated the pH gradient across the cell membrane, and this property could be used to calculate the relative permeabilities of different amines. The ratio of ethylamine to methylamine permeabilities was not significantly different from that calculated from the direct measurements of permeabilities; ammonia was much less effective in dissipating the pH gradient across the cell membrane than methylamine or ethylamine. An apparent permeability of ammonia of 5.7±0.9 m sec^–1 could be calculated from the permeability ratio of ammonia to methylamine and the experimentally measured permeability of methylamine. The permeability properties of ammonia and methylamine are very different; this poses problems in the interpretation of experiments where¹⁴C-methylamine is used as an ammonia analogue.     

Effect of light intensity on ammonia assimilation in maize leaves

The effect of light on the metabolism of ammonia was studied by subjecting detached maize leaves to 150 or 1350 mol m^–2 s^–1 PAR during incubation with the leaf base in 2 mM ¹⁵NH₄Cl. After up to 60 min, leaves were extracted. Ammonia, glutamine, glycine, serine, alanine, and aspartate were separated by isothermal distillation and ion exchange chromatography. ¹⁵N enrichments were analyzed by emission spectroscopy. The uptake of ammonium chloride did not influence CO₂ assimilation (8.3 and 17.4 mol m^–1 s^–1 at 150 and 1350 mol m^–2 s^–1 PAR, respectively). Leaves kept at high light intensity contained more serine and less alanine than leaves from low light treatments. Within 1 h of incubation the enrichment of ammonia extracted from leaves rose to approximately 20% ¹⁵N. In the high light regime the amino acids contained up to 15% ¹⁵N, whereas in low light ¹⁵N enrichments were small (up to 6%). The kinetics of ¹⁵N incorporation indicated that NH₃ was firstly assimilated into glutamine and then into glutamate. After 15 min ¹⁵N was also found in glycine, serine and alanine. At high light intensity nearly half of the ¹⁵N was incorporated in glycine. On the other hand, at low light intensity alanine was the predominant ¹⁵N sink. It is concluded that light influences ammonia assimilation at the glutamine synthetase reaction.     

Biosynthesis of glycoproteins in Candida albicans: Biochemical characterization of dolichol phosphate glucose synthase

A mixed membrane fraction isolated from C. albicans yeast cells catalyzed the transfer of glucose from UDP-Glc into three classes of endogenous acceptors: glucolipid, glycoprotein and lipid-linked oligosaccharides. About 80 of the total radioactivity transferred into these products corresponded to the glucolipid which was identified as dolichol phosphate glucose by several criteria. The remainder was detected in about equal proportions in the other two fractions. Conditions that stimulated or inhibited glucolipid synthesis did not affect the extent of glycoprotein labeling. The synthesis of dolichol phosphate glucose exhibited a K_mof 104 M UDP-Glc and was stimulated by Mg²⁺but not by Mn²⁺or Ca²⁺. The latter cations were, however, better stimulators of glycoprotein labeling than Mg²⁺. Most nucleotides strongly inhibited the synthesis of dolichol phosphate glucose, UMP being a competitive inhibitor with a K_iof 100 M. The dolichol phosphate glucose synthase reaction was reversed about 57 by 0.62 mM UDP but not by UMP.     

Ammonia assimilation and oxygen evolution by a reconstituted chloroplast system in the presence of 2-oxoglutarate and glutamate

(Ammonia plus 2-oxoglutarate)-dependent O₂ evolution by intact chloroplasts was enhanced three- to five fold by 2 mM L- and D-malate, attaining rates of 9–15 μmol mg^-1 Chl h^-1. Succinate and fumarate also promoted activity but D-aspartate and, in the presence of aminooxyacetate, L-aspartate inhibited the malate-promoted rate. A reconstituted chloroplast system supported (ammonia plus 2-oxoglutarate)-dependent O₂ evolution at rates of 6-11 μmol mg^-1 Chl h^-1 in the presence of MgCl₂, NADP(H), ADP plus Pi (or ATP), ferredoxin and L-glutamate. The concentrations of L-glutamate and ATP required to support 0.5 V _max were 5 mM and 0.25 mM, respectively. When the reaction was initiated with NH₄Cl, O₂ evolution was preceded by a lag phase before attaining a constant rate. The lag phase was shortened by addition of low concentrations of L-glutamine or by preincubating in the dark in the presence of glutamate, ATP and NH₄Cl. Oxygen evolution was inhibited by 2 mM azaserine and, provided it was added initially, 2 mM methionine sulphoximine. The (ammonia plus 2-oxoglutarate)-dependent O₂ evolution was attributed to the synthesis of glutamine from NH₄Cl and glutamate which reacted with 2-oxoglutarate in a reaction catalysed by ferredoxin-specific glutamate synthase using H₂O as the ultimate electron donor. The lag phase was attributed to the establishment of a steady-state pool of glutamine. L-Malate did not affect the activity of the reconstituted system.     

Differential effects of ammonia and β-methylene-dl-aspartate on metabolism of glutamate and related amino acids by astrocytes and neurons in primary culture

The effects of ammonium chloride (3 mM) and -methylene-dl-aspartate (BMA; 5 mM) (an inhibitor of aspartate aminotransferase, a key enzyme of the malate-aspartate shuttle (MAS)) on the metabolism of glutamate and related amino acids were studied in primary cultures of astrocytes and neurons. Both ammonia and BMA inhibited¹⁴CO₂ production from [U-¹⁴C]-and [1-¹⁴C]glutamate by astrocytes and neurons and their effects were partially additive. Acute treatment of astrocytes with ammonia (but not BMA) increased astrocytic glutamine. Acute treatment of astrocytes with ammonia or BMA decreased astrocytic glutamate and aspartate (both are key components of the MAS). Acute treatment of neurons with ammonia decreased neuronal aspartate and glutamine and did not apparently affect the efflux of aspartate from neurons. However, acute BMA treatment of neurons led to decreased neuronal glutamate and glutamine and apparently reduced the efflux of aspartate and glutamine from neurons. The data are consistent with the notion that both ammonia and BMA may inhibit the MAS although BMA may also directly inhibit cellular glutamate uptake. Additionally, these results also suggest that ammonia and BMA exert differential effects on astroglial and neuronal glutamate metabolism.This paper is dedicated to Professor E. Kvamme. Dr. Kvamme has conducted numerous pioneering studies on the regulation of the metabolism of glutamine, glutamate and ammonia in nervous and other tissues (see Refs. 1 and 3 for a complete discussion and citation of his many papers). Many important ideas in this exciting field of research have emerged from the work carried out in his laboratory.     

Ammonia Attenuates LPS-Induced Upregulation of Pro-Inflammatory Cytokine mRNA in Co-Cultured Astrocytes and Microglia

Hepatic encephalopathy (HE) is associated with cerebral microglia activation. Ammonia, a major toxin of HE, activates microglia in vitro but does not trigger pro-inflammatory cytokine synthesis. In the present study we analysed effects of ammonia on lipopolysaccharide (LPS)-induced upregulation of microglia activation and cytokine mRNA as well as on cytokine secretion in mono-cultured microglia and co-cultured astrocytes and microglia. In mono-cultured microglia LPS (100 ng/ml, 18 h) strongly elevated mRNA levels of the microglia activation marker CD14 and the pro-inflammatory cytokines IL-1α/β, IL-6 and TNF-α. NH₄Cl (5 mmol/l) had no effect on LPS-induced upregulation of CD14, IL-1α/β and IL-6 mRNA but enhanced LPS-induced upregulation of TNF-α mRNA in mono-cultured microglia. In co-cultured astrocytes and microglia, however, LPS-induced upregulation of IL-1α/β, TNF-α, IL-6, CD14 but not of IL-10, IL-12A/B or TGFβ_1?3 mRNA was attenuated by NH₄Cl. LPS-induced upregulation of IL-1α/β, IL-6 and TNF-α was also diminished by the TGR5-ligands allopregnanolone and taurolithocholic acid in mono-cultured microglia. NH₄Cl also attenuated LPS-induced release of MCP-1, IL-6 and IL-10 in mono-cultured microglia. mRNA level of surrogate marker for microglia activation (CD14) and for the anti-inflammatory M2-type microglia (CD163, CXCL1, CXCL2) were also elevated in post mortem brain tissue taken from the fusiforme gyrus of patients with liver cirrhosis and HE. The findings suggest that ammonia attenuates LPS-induced microglia reactivity in an astrocyte-dependent way. One may speculate that these anti-inflammatory effects of ammonia may be triggered by neurosteroids derived from astrocytes and may account for absence of microglia reactivity in cerebral cortex of cirrhotic patients with HE.