AMMONIUM ION INHIBITION OF EVOKED RELEASE OF ENDOGENOUS GLUTAMATE FROM HIPPOCAMPAL SLICES

Abstract— The effect of pathophysiological levels (2-5 m m ) of ammonium chloride on the efflux of endogenous and exogenous [¹⁴C]glutamate from hippocampal slices was studied. The evoked release of glutamate which occurs dring tissue depolarization with 56 m m -KCl was greatly reduced when the tissue had been exposed to NH₄Cl for 40–80 min. This effect was seen whether or not glutamine (0.5 m m ) was present in the incubation medium. The effect was completely reversible. The spontaneous efflux and the evoked release of [¹⁴C]glutamate was, on the contrary, completely unaltered after exposure of the slice to ammonium ions. Nigher (20–36 m m ) amounts of NH₄Cl evoked a release of [¹⁴C]glutamate from the crude mitochondrial fraction, as did high concentrations of KCl. The results are discussed in relation to the compartmentation of glutamate metabolism and the pathogenesis of hepatic coma.     

Increase in the Content of Quinolinic Acid in Cerebrospinal Fluid and Frontal Cortex of Patients with Hepatic Failure

Abstract: Quinolinic acid (QUIN), an excitotoxic tryptophan metabolite, has been identified and measured in human cerebrospinal fluid (CSF) using a mass-fragmentographic method. Furthermore, its content has been evaluated in frontal cortex obtained at autopsy from the cadavers of patients who died after hepatic coma. During the coma, the concentration of QUIN in the CSF was 152 ± 38 pmol ml^-1. In contrast, the concentration in control patients affected by different pathologies was 22 ± 7 pmol ml^-1. In the frontal cortex of patients who died after episodes of hepatic encephalopathy, the content of QUIN was three times higher than in controls (2.6 ± 0.6 versus 0.80 ± 0.08 nmol/g wet weight). As a result of these investigations we are now able to extend our previous observations on the increase of QUIN in the brains of rats used as experimental models of hepatic encephalopathy to man. QUIN should therefore be added to the list of compounds possibly involved in the pathogenesis and symptomatology of brain disorders associated with liver failure.     

Portacaval Shunting and Hyperammonemia Stimulate the Uptake of l-[3H]Arginine but Not of l-[3H]Nitroarginine into Rat Brain Synaptosomes

Abstract: Elevated activities of nitric oxide synthase (NOS) have been reported previously in the brains of portacaval-shunted (PCS) rats, a model of chronic hepatic encephalopathy (HE). As l -arginine availability for nitric oxide synthesis depends on a specific uptake mechanism in neurons, we studied the kinetics of l -[³H]-arginine uptake into synaptosomes prepared from the brains of PCS rats. Results demonstrate that l -arginine uptake is significantly increased in cerebellum (60%; p < 0.01), cerebral cortex (42%; p < 0.01), hippocampus (56%; p < 0.01), and striatum (51%; p < 0.01) of PCS rats compared with sham-operated controls. Hyperammonemia in the absence of portacaval shunting also stimulated the transport of l -[³H]arginine; kinetic analysis revealed that the elevated uptake was due to increased uptake capacity ( V _max) without any change in affinity ( K _m). Incubation of cerebellar synaptosomes with ammonium acetate for 10 min caused a dose-dependent stimulation of l -[³H]arginine uptake. Neither portacaval shunting nor hyperammonemia had any significant effect on the synaptosomal uptake of N ^G-nitro- l -[³H]arginine. These studies demonstrate that increased NOS activity observed in experimental HE may result from increased availability of l -arginine resulting from a direct stimulatory effect of ammonia on l -arginine transport.     

Quinolinic Acid In Vivo Synthesis Rates, Extracellular Concentrations, and Intercompartmental Distributions in Normal and Immune-Activated Brain as Determined by Multiple-Isotope Microdialysis

Abstract: Quinolinic acid (QUIN) kills neurons by activation of NMDA receptors that are accessed via the extracellular fluid (ECF). In vivo microdialysis was employed to quantify the dynamics of ECF QUIN levels. [¹³C₇]QUIN was perfused through the probe for in vivo calibration to accurately quantify ECF QUIN concentrations. Osmotic pumps infused [²H₃]QUIN subcutaneously to quantify blood contributions to ECF and tissue levels. Local QUIN production rates and influx and efflux rates across the blood-brain barrier were calculated from the extraction fraction of [¹³C₇]QUIN, probe geometry, tissue diffusion coefficients, the extracellular volume fraction, and [²H₃]QUIN/QUIN ratios in blood and dialysates. In normal brain, 85% of ECF QUIN levels (110 n M ) originated from blood, whereas 59% of tissue homogenate QUIN (130 pmol/g) originated from local de novo synthesis. During systemic immune activation (intraperitoneal injection of endotoxin), blood QUIN levels increased (10.2-fold) and caused a rise in homogenate (10.8-fold) and ECF (18.5-fold) QUIN levels with an increase in the proportions of QUIN derived from blood. During CNS inflammation (local infusion of endotoxin), increases in brain homogenate (246-fold) and ECF (66-fold) QUIN levels occurred because of an increase in local synthesis rate (146-fold) and a reduction in efflux/influx ratio (by 53%). These results demonstrate that brain homogenate measures are a reflection of ECF concentrations, although there are quantitative differences in the values obtained. The mechanisms that maintain ECF QUIN levels at low values cannot do so when there are large increases in local brain synthesis or when there are large elevations in blood QUIN concentrations.     

Releases of Norepinephrine and Dopamine in Ventriculocisternal Perfusions in Hepatectomized and Laparotomized Rats

Abstract: The completely hepatectomized rat has frequently been used as a model to study changes in the economy of norepinephrine (NE) and dopamine (DA) in hepatic coma. Hypothermia characteristically develops in hepatectomized rats and also occurs in patients in hepatic coma and is associated with improved survival in both. The aims of the present study were to measure both release and uptake of NE and release of DA in brain in warm (37°C) and cool (30–32°C) rats at 3–5 h after laparotomy or hepatectomy. Ventriculocisternal perfusions of the brain were performed on rats under basal conditions and during releases evoked by 40 m M K⁺. Basal releases of NE and DA and evoked release of DA were greater in the warm hepatectomized rats than in all other groups. In some studies, 10⁻⁵ M amitriptyline was added to the perfusates to assess whether neuronal uptake was changed after hepatectomy. Uptake of released NE was equally robust in cool hepatectomized as in cool laparotomized rats but could not be measured in warm hepatectomized rats because of amitriptyline toxicity in these rats. Decreases in NE and increases in DA content were found in most areas of the brain after perfusion. Increased releases of NE and DA may contribute to the pathogenesis of hepatic encephalopathy.     

Metabolism of [5-3H]Kynurenine in the Rat Brain In Vivo: Evidence for the Existence of a Functional Kynurenine Pathway

Abstract: The incorporation of tritium label into quinolinic acid (QUIN), kynurenic acid (KYNA), and other kynurenine (KYN) pathway metabolites was studied in normal and QUIN-lesioned rat striata after a focal injection of [5-³H]KYN in vivo. The time course of metabolite accumulation was examined 15 min to 4 h after injection of [5-³H]KYN, and the concentration dependence of KYN metabolism was studied in rats killed 2 h after injection of 1.5–1,500 µ M [5-³H]KYN. Labeled QUIN, KYNA, 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid, and xanthurenic acid (XA) were recovered from the striatum in every experiment. Following injection of 15 µ M [5-³H]KYN, a lesion-induced increase in KYN metabolism was noted. Thus, the proportional recoveries of [³H]KYNA (5.0 vs. 1.8%), [³H]3-HK (20.9 vs. 4.5%), [³H]XA (1.5 vs. 0.4%), and [³H]QUIN (3.6 vs. 0.6%) were markedly elevated in the lesioned striatum. Increases in KYN metabolism in lesioned tissue were evident at all time points and KYN concentrations used. Lesion-induced increases of the activities of kynurenine-3-hydroxylase (3.6-fold), kynureninase (7.6-fold), kynurenine aminotransferase (1.8-fold), and 3-hydroxyanthranilic acid oxygenase (4.2-fold) likely contributed to the enhanced flux through the pathway in the lesioned striatum. These data provide evidence for the existence of a functional KYN pathway in the normal rat brain and for a substantial increase in flux after neuronal ablation. This method should be of value for in vivo studies of cerebral KYN pathway function and dysfunction.     

Redistribution of Imipramine from Regions of the Brain Under the Influence of Circulating Specific Antibodies

Abstract: The kinetics of brain-to-blood redistribution of imipramine (IMI) was assessed in nine brain regions of control rats and rats given anti-tricyclic antidepressant (anti-TCA) antibody. Two antibodies were given intravenously 6 min after intravenous [³H]IMI (1 nmol/kg). One was a murine monoclonal IgG₁ ( K _a = 3.8 × 10⁷ M ⁻¹) at an IgG/IMI molar ratio of 1,000 (IgG1,000), and the other was a sheep polyclonal IgG (TAb; K _a = 1.3 × 10¹⁰ M ⁻¹) at IgG/IMI molar ratios of 1, 10, and 100 (TAb1, TAb10, and TAb100). In the control rats, IMI was rapidly taken up by the brain ( C _max at 5 min) with no significant differences among the brain regions (4.1 ± 0.4 to 5.4 ± 0.6 pmol/g), and brain IMI then declined monoexponentially with a half-life of 44.2 min (cerebellum) to 77.3 min (hippocampus). The greatest IMI content was in the frontal cortex and the lowest in the cerebellum. The antibodies (except TAb1) stimulated the extent and rate of IMI redistribution from all the brain regions depending on the immunoreactive capacity ( NK _a) of the antibody. The antibody with the highest NK _a (TAb100) had the greatest effect. The fraction of IMI removed from the brain was 58–74%, and the redistribution half-life was 7.9–15.6 min; the mean residence time was reduced by 66–75% (11.8–23.9 min). These results demonstrate that circulating anti-TCA IgG rapidly and reliably removes IMI from the brain, indicating that immunotoxicotherapy could be an efficient procedure for accelerating the removal of TCA from the brain.     

Increased levels of pregnenolone and its neuroactive metabolite allopregnanolone in autopsied brain tissue from cirrhotic patients who died in hepatic coma

It has been suggested that neurosteroids with agonist properties at the central GABA-A receptor are implicated in the pathogenesis of hepatic encephalopathy (HE) in chronic liver disease. In order to address this issue, gas chromatography/mass spectrometry was used to measure the neurosteroids pregnenolone, allopregnanolone, and tetrahydrodeoxycorticosterone (THDOC) in postmortem brain tissue from controls, cirrhotic patients who died without HE, a patient who died in uremic coma, and cirrhotic patients who died in hepatic coma. Exposure of rat cerebral cortical membranes to brain extracts from hepatic coma patients resulted in a 53% (p < 0.001) increase in binding of [3H]muscimol, a GABA-A receptor ligand. Subsequent GC/MS analysis showed that concentrations of the GABA-A receptor agonist neurosteroid allopregnanolone were significantly increased in brain tissue from hepatic coma patients compared to patients without HE or controls (p < 0.001). Brain allopregnanolone concentrations were significantly correlated with the magnitude of induction of [3H]muscimol binding (r2 = 0.82, p < 0.0001). Concentrations of allopregnanolone comparable to those observed in hepatic coma brains are pathophysiologically relevant. Concentrations of the neurosteroid precursor pregnenolone were also increased in brain tissue from hepatic coma patients, while those of a second neurosteroid THDOC were below the levels of detection in all groups. Brain concentrations of benzodiazepine receptor ligands estimated by radioreceptor assay were not significantly increased in cirrhotic patients with or without hepatic coma. These findings suggest that increased levels of allopregnanolone rather than "endogenous benzodiazepines" offer a cogent explanation for the phenomenon of "increased GABAergic tone" previously proposed in HE.     

Glycolate metabolism in cyanobacteria. III. Nitrogen controls excretion and metabolism of glycolate in Anabaena cylindrica

The effect of nitrogen on excretion and metabolism of glycolate in Anabaena cylindrica (CCAP 1403/2a) was studied. Glycidate, an inhibitor of glutamate:glyoxylate aminotransferase (EC 2.6.1.4), reduced the L-methionine-DL-sulfoximine-induced NH₄⁺ release by ca 40%, while net CO₂ fixation and C₂H₂ reduction were not lowered. This indicates that at least a part of the glyoxylate synthesized in A. cylindrica is metabolized via glycine to serine. Addition of NH₄Cl or glutamate to the medium reduced the excretion of glycolate. At pH 9, under air, NH₄Cl reduced the excretion by 10–30% and under high pO2 (0.03 kPa CO₂ in O₂) by about 80–90%. At pH 7.5, under high pO₂, NH₄Cl and glulamate reduced the excretion by about 40 and 80%, respectively. Also, the presence of NH₄Cl stimulated the animation of glyoxylate under such conditions as shown by an increased glycine pool and a decreased glutamate pool. We suggest that nitrogen regulates the capacity of A. cylindrica to retain and recycle glycolate intracellularly and that glutamate serves as an amino donor in the conversion of glyoxylate to glycine.     

THE EFFECT OF DEFICIENCY OF THIAMINE ON THE METABOLISM OF [U-14C]GLUCOSE AND [U-14C]RIBOSE AND THE LEVELS OF AMINO ACIDS IN RAT BRAIN

Abstract— The incorporation of ¹⁴C into amino acids of the brain was determined at different times after injection of [U-¹⁴C]glucose and [U-¹⁴C]ribose to rats maintained on thiamine-supplemented and thiamine-deficient diets for 22 days.
The ¹⁴C-content of amino acids in the brain of thiamine-deficient rats decreased at times 2–10 min after injection of [U-¹⁴C]glucose. but it increased at 2 min and decreased at times 5–10 min after injection of [U-¹⁴C]ribose.
The results of labelling of amino acids indicated that the activities in vivo of the thiamine pyrophosphate requiring enzymes, pyruvate oxidase, a-oxoglutarate dehydrogenase and transketolase were similar in the two groups. It was suggested that the observed decrease in the labelling of amino acids was due to one or more of the following factors: (i) a decrease in the activities of glycolytic enzymes catalysing the conversion of glucose into triose phosphate; (ii) a decrease in the transport of substrate to the active site of the enzymes; or (iii) altered neurohistopathology of the brain.
Thiamine deficiency in rats showed a 5% decrease in glutamate ( P < 0–05), 46% decrease in threonine (P < 0001) and 16% increase in glycine ( P < 0–01) content of the brain.     

Region-Selective Decreases in Densities of [3H]Tryptamine Binding Sites in Autopsied Brain Tissue from Cirrhotic Patients with Hepatic Encephalopathy

Abstract: The distribution of [³H]tryptamine binding sites, in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy (HE) and an equal number of age-matched control subjects free from hepatic, neurological, or psychiatric disorder, was investigated. Scatchard analysis demonstrated a heterogeneous distribution for this binding site, with the highest density being observed in hippocampus ≫ frontal cortex = caudate nucleus > temporal cortex = cerebellum. When comparing [³H]-tryptamine binding site densities in control brain tissue with that in brain tissue from patients with HE, significant decreases in densities were observed in the frontal cortex (by 56%, p < 0.001), hippocampus (by 43%, p < 0.001), and caudate nucleus (by 41%, p < 0.01) of the HE group. Binding site affinities were within normal limits. The findings of decreased densities of [³H]tryptamine binding sites taken in conjunction with previous reports of increased CSF and brain tryptamine concentrations in HE suggest a pathogenic role for this neuroactive amine in HE resulting from chronic liver failure.     

Translocation of NH4+ in oilseed rape plants in relation to glutamine synthetase isogene expression and activity

Translocation of NH₄⁺ was studied in relation to the expression of three glutamine synthetase (GS, EC 6.3.1.2) isogenes and total GS activity in roots and leaves of hydroponically grown oilseed rape ( Brassica napus ). The concentration of NH₄⁺ in the stem xylem sap of NO₃⁻-fed plants was 0.55–0.70 m M , which was ≈60% higher than that in plants deprived of external nitrogen for 2 days. In NH₄⁺-fed plants, xylem NH₄⁺ concentrations increased linearly both with time of exposure to NH₄⁺ and with increasing external NH₄⁺ concentration. The maximum xylem NH₄⁺ concentration was 8 m M , corresponding to 11% of the nitrogen translocated in the xylem. In the leaf apoplastic solution, the NH₄⁺ concentration increased from 0.03 m M in N-deprived plants to 0.20 m M in N-replete plants. The corresponding values for leaf tissue water were 0.33 and 1.24 m M , respectively. The addition of either NO₃⁻ or NH₄⁺ to N-starved plants induced both cytosolic gs isogene expression and GS activity in the roots. In N-replete plants, gs isogene expression and GS activity were repressed, probably due to carbon limitations, thereby protecting the roots against the excessive drainage of photosynthates. Repressed gs isogene expression and GS activity under N-replete conditions caused enhanced NH₄⁺ translocation to the shoots.     

Severity of Hyperammonemic Encephalopathy Correlates with Brain Ammonia Level and Saturation of Glutamine Synthetase In Vivo

Abstract: Correlation among in vivo glutamine synthetase (GS) activity, brain ammonia and glutamine concentrations, and severity of encephalopathy was examined in hyperammonemic rats to obtain quantitative information on the capacity of GS to control these metabolites implicated in the etiology of hepatic encephalopathy. Awake rats were observed for neurobehavioral impairments after ammonium acetate infusion to attain a steady-state blood ammonia concentration of 0.9 (group A) or 1.3 µmol/g (group B). As encephalopathy progressed from grade III to IV, brain ammonia concentration increased from 1.9 to 3.3 µmol/g and then decreased to 1.3 µmol/g on recovery to grade III. In contrast, brain glutamine concentration was 26, 23, and 21 µmol/g, respectively. NH₄⁺-infused rats pretreated with l -methionine dl -sulfoximine reached grade IV when brain ammonia and glutamine concentrations were 3.0 and 5.5 µmol/g, respectively; severity of encephalopathy correlates with brain ammonia, but not glutamine. In vivo GS activity, measured by NMR, was 6.8 ± 0.7 µmol/h/g for group A and 6.2 ± 0.6 µmol/h/g for group B. Hence, the in vivo activity, shown previously to increase with blood ammonia over a range of 0.4–0.64 µmol/g, approaches saturation at blood ammonia >0.9 µmol/g. This is likely to be the major cause of the observed accumulation of brain ammonia and the onset of grade IV encephalopathy.     

Disposition of venlafaxine enantiomers in rats with hepatic encephalopathy after chronic drug treatment

Portacaval shunted (PCS) rats, a model of hepatic encephalopathy, and control animals were administered racemic venlafaxine for 14 days (10 mg/kg). The levels of the S- and R-enantiomers and the S/R-enantiomer ratios of venlafaxine and its metabolites were assessed by an enantiomer-selective chromatographic assay in serum, brain parenchyma, and brain dialysate of both groups. Higher levels of the S- and R-enantiomers of venlafaxine were found in serum and brain of PCS vs. normal rats (median values of S- and R-venlafaxine in serum: 290 and 201 nM in PCS; 97 and 66 nM in normal rats; median values of S- and R-venlafaxine in cortex: 956 and 939 nM in PCS; 357 and 318 nM in normal rats). Interestingly, similar S/R-venlafaxine ratios were observed in PCS and normal rats both in serum (S/R = 1.4) and brain compartments (S/R = l.0-1.1). These findings may have clinical relevance for the safety of venlafaxine in chronic hepatic encephalopathy.     

Long-term ammonia exposure of turbot: effects on plasma parameters

Turbot juveniles were exposed to four ammonia concentrations [0·17 (L), 0·34 (M), 0·73 (MH) and 0·88 (H) mg l⁻¹ NH₃-N] for different exposure durations (28 days minimum to 84 days). Their physiological status and growth performances were compared to a control group [0·004 (C) mg l⁻¹ NH₃-N]. No growth was observed in the H group, and by day 57, mass increase in the MH group was only 15% of that in group C. During the first month growth in the L group was similar to that in control group while it was lower (33%) in the M group; afterwards the L and M groups had a similar growth (half that of controls). Accumulation of total ammonia nitrogen (TA-N) in plasma was dependent on ambient ammonia concentrations. Plasma urea levels in ammonia-exposed fish were lower, similar or greater than in controls (depending on ammonia concentration or exposure duration). Osmolarity, Cl^– and Na⁺ plasma concentrations were stable in the L and M groups. The increases in Na⁺, Cl^–, K⁺ and total Ca concentrations observed by the end of the experiment in the H and MH groups suggest that fish failed to adapt. There was an initial rise in plasma cortisol in all ammonia-exposed groups followed by a return to basal level (1·7–4 ng ml⁻¹) in the L and M groups. In group MH, plasma cortisol peaked at 42 ng ml⁻¹ by day 14, and after a decline at c . 1 month (14 ng ml⁻¹), it rose again.     

Specific Activity of Brain Palmitoyl-CoA Pool Provides Rates of Incorporation of Palmitate in Brain Phospholipids in Awake Rats

Abstract: In vivo rates of palmitate incorporation into brain phospholipids were measured in awake rats following programmed intravenous infusion of unesterified [9,10-³H]palmitate to maintain constant plasma specific activity. Animals were killed after 2–10 min of infusion by microwave irradiation and analyzed for tracer distribution in brain phospholipid and phospholipid precursor, i.e., brain unesterified palmitate and palmitoyl-CoA, pools. [9,10-³H]Palmitate incorporation into brain phospholipids was linear with time and rapid, with >50% of brain tracer in choline-containing glycerophospholipids at 2 min of infusion. However, tracer specific activity in brain phospholipid precursor pools was low and averaged only 1.6–1.8% of plasma unesterified palmitate specific activity. Correction for brain palmitoyl-CoA specific activity increased the calculated rate of palmitate incorporation into brain phospholipids (0.52 nmol/s/g) by ∼60-fold. The results suggest that palmitate incorporation and turnover in brain phospholipids are far more rapid than generally assumed and that this rapid turnover dilutes tracer specific activity in brain palmitoyl-CoA pool owing to release and recycling of unlabeled fatty acid from phospholipid breakdown.     

Effect of Ammonia on Brain Serotonin Metabolism in Relation to Function in the Portacaval Shunted Rat

Four weeks following portacaval anastomosis (PCA) in the rat, severe liver atrophy, sustained hyperammonemia, and increased plasma and brain tryptophan are observed. Administration of ammonium acetate (NH4Ac) to rats with PCA precipitates severe signs of hepatic encephalopathy (HE) (loss of righting reflex progressing to loss of consciousness and ultimately deep coma). To evaluate the relationship between the deterioration of neurological status in HE and serotonin (5-HT) metabolism, the levels of 5-HT, its precursor 5-hydroxytryptophan, and its major metabolite 5-hydroxy-indole-3-acetic acid (5-HIAA) were measured by HPLC with ion-pairing and electrochemical detection in three well-defined areas of the cerebral cortex: anterior cingulate, piriform and entorhinal, and frontoparietal; as well as in the caudate-putamen, the raphe nuclei, and the locus ceruleus in rats with PCA at different stages of HE, before and after injection of NH4Ac, as well as in sham-operated controls. The results demonstrate increased 5-HIAA/5-HT ratios after PCA and NH4Ac loading, suggesting increased 5-HT turnover in the brains of these animals. However, these changes do not appear to be related to the precipitation of coma as no significant difference in 5-HT turnover was observed between precoma and coma stages of HE. Increased 5-HT turnover in brain of shunted rats may be related to early symptoms of HE such as altered sleep patterns and disorders of motor coordination.     

Numerical abundance of myxomycetes (myxogastrids) in soils in the West of England

Abstract A most-probable-number technique was used to quantify the abundance of myxomycetes (myxogastrids) in soil samples taken from 23 'Sites of Special Scientific Interest' and one other site in the West of England. Associated organisms and soil conditions were also recorded. Sixteen of the 24 sites yielded myxomycete plasmodium-forming units (PFUs) in numbers averaging 230 cm⁻³ fresh soil. Where detectable in samples of grassland soil, the numbers ranged from 20–2750 cm⁻³, in woodland soil from 20–800 cm⁻³, and in sand dunes from 80–400 cm⁻³. Repeated sampling revealed changing numbers at single sites. The abundance of PFUs was correlated positively with numbers of soil amoebae, ciliates and nematodes, and with levels of magnesium and soil density, and negatively with organic matter content and levels of NH₄-nitrogen and phosphate. Each PFU probably corresponded to one or several uninucleate cells in the soil rather than to a plasmodium.