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The effect of aminooxyacetic acid (AOAA), NH , phenylsuccinate (Phs), ketone bodies (KB) and glutamine (Gln), that might interfere with the biosynthesis of neurotransmitter glutamate on the K-evoked Ca-dependent release of d-[H]aspartate from rat cerebellar slices was studied. Therefore slices were preincubated in a Krebs-Ringer-bicarbonate-glucose (KR) buffer, loaded with d-[H]aspartate and superfused in the presence of Ca or when Ca was replaced by Mg or in some cases by EGTA. AOAA, NH and Phs increase the K-evoked Ca-dependent release of radioactivity by 30%, 68% and 188% compared to the control respectively indicating that these agents are inhibitors of the K-evoked Ca-dependent release of glutamate. KB and Gln had no effect on the Ca-dependent release of radioactivity. AOAA., NH, Phs and KB but not Gln increase the total release of radioactivity by 43%, 69%, 139%, and 37% respectively. AOAA, NH and KB but not Phs or Gln increase the Ca-independent release (Mg replacing Ca) of radioactivity by 71%, 71% and 108% respectively. The present results indicate that in the cerebellum: 1) Neurotransmitter glutamate is mostly synthesized through the phosphate activated glutaminase (PAG) reaction 2) It is further supported that glutamate released in a Ca-dependent manner before entering its pool in the cytosol has to move into the mitochondrial matrix. [ABSTRACT FROM AUTHOR]

To study the effect of agents interfering with the biosynthesis and/or the K-evoked Ca-dependent release of neurotransmitter glutamate, rat cerebral slices were preincubated with Krebs-Ringer-HEPES-glucose-glutamine buffer (KRH buffer), loaded with d-[H]aspartate and superfused with the preincubation medium in the presence or in the absence of Ca. The difference in radioactivity release divided by the basal release per min under the two conditions represented the K-evoked Ca-dependent release. The agents used were: 1) Aminooxyacetic acid (AOAA), the inhibitor of transaminases, 2) Leucine (Leu), the inhibitor of phosphate activated glutaminase (PAG), 3) NH, the inhibitor of PAG, 4) Phenylsuccinic acid (Phs), the inhibitor of the mitochondrial ketodicarboxylate carrier, 5) ketone bodies, the inhibitors of glycolysis, 6) the absence of glutamine, the substrate of PAG. The results show that Leu, NH, Phs and the absence of Gln significantly increase the K-evoked Ca-dependent release of radioactivity by 64%, 200%, 95% and 147% respectively, indicating that these agents are inhibitors of the K-evoked Ca-dependent release of glutamate. Ketone bodies and AOAA had no effect. These results indicate that the major if not the exclusive biosynthetic pathway of neurotransmitter glutamate in rat cerebral cortex is through the PAG reaction and support a model for the pathway followed by neurotransmitter glutamate i.e. glutamate formed outside the inner mitochondrial membrane has to enter the mitochondrial matrix or is formed within it from where it can be extruded to supply the transmitter pool in exchange of GABA. [ABSTRACT FROM AUTHOR]

Rat hippocampal slices preloaded with d-[H]aspartate, a non metabolizable analogue of l-glutamate, were superfused with artifical CSF. Depolarization was induced by 53.5 mM K, in the presence of Ca (1.3 mM) or Mg (5 mM) to determine the Ca dependent release. Haloperidol added in the superfusion medium at 100 μM reduced by about 60% the Ca dependent release of d-[H]aspartate. This drug at 20 μM or 100 μM inhibited the non-activated glutamate dehydrogenase (GDH) but had no effect on GDH activated by ADP (2 mM) or leucine (5 mM). In addition no effect was observed on phosphate activated glutaminase (PAG) in the presence either of 20 mM or 5 mM phosphate. These results indicate that the effect of haloperidol is exerted on presynaptic mechanisms regulating neurotransmitter release. [ABSTRACT FROM AUTHOR]

Background: Using telemedicine in the school setting in Greece, we screened a representative adolescent sample for MetS (International Diabetes Federation criteria) and explored its associations with anthropometric, sociodemographic and behavioural parameters., Materials and Methods: Cross-sectional data were obtained from 12- to 17-year-old high school students., Results: The prevalence of MetS in 1578 adolescents (mean age ± SD 14.4 ± 1.7 years) was 2.6% (3.4% among males; 2.0% among females), highest (4.3%) at age 13 years and lowest (1.3%) at 16 years. Adolescents with MetS had significantly higher mean body mass index (BMI) ± SD than those without MetS (30.2 ± 4.2 vs 21.3 ± 3.2 kg/m 2 , respectively; P < 0.001); among participants with obesity, 31.6% had MetS. Abdominal obesity, elevated triglycerides, low HDL-cholesterol, impaired fasting blood glucose (FBG) and elevated blood pressure (BP) were detected in 9.5%, 2.3%, 10.7%, 25.9% and 21.8% of participants, respectively. Additional analysis (modified NCEP:ATPIII youth criteria) demonstrated similar overall prevalence of MetS (2.9%). Statistically significant correlations were found between most anthropometric and MetS characteristics, with the exception of FBG, which was correlated only with systolic BP. BMI was strongly correlated with waist and hip circumferences (r = 0.818, P < 0.001; r = 0.825, P < 0.001, respectively). Single parenthood and older maternal age (>60 years) were risk factors for MetS. Although counterintuitive, body image distortion, body dissatisfaction and bullying about weight were more prevalent in normal weight girls., Conclusions: The overall prevalence of MetS was low but 12-fold higher when obesity was taken into account. Impaired FBG and elevated BP were the most prevailing features. Telemedicine services were used effectively in Greek schools for screening youth MetS., (© 2019 Stichting European Society for Clinical Investigation Journal Foundation.)

An in-silico model, of the glucocorticoid regulated glutamate release, in rat hippocampal tissue, is constructed. The model permits the pseudo-steady state estimation of various fluxes, experimentally impossible to measure, from a set of measured rates. Estimates of the astrocytic pyruvate carboxylase reaction and the neuronal TCA cycle rates are correlated with different dexamethasone concentrations, in order to extrapolate explicit kinetic equations. The model suggests that the observed effects of glucocorticoids can be attributed to the inhibitory actions of dexamethasone on two competing pathways, that of the neuronal TCA cycle and the biosynthetic pathway of neurotransmitter glutamate.

A flux analysis model for the metabolism of neurotransmitter glutamate is constructed, in order to study functional aspects of its metabolism. This work is based on the potassium [K(+)] evoked neurotransmitter glutamate released, as measured in a series of experiments of superfused rat or mouse brain preparations. These measurements are combined with data reported, concerning the metabolism of glutamate and its precursors, glutamine and glucose in rat cerebral cells in vivo. The proposed stoichiometry of the specific reaction network renders the model solvable. The classification procedure establishes that the measured fluxes are all balanceable and all non-measured fluxes can be calculated. The system is well posed with a condition number of 7.8536. The results emphasize the importance of phosphate activated glutaminase and aspartate aminotransferase in the metabolism of neurotransmitter glutamate. Reported data on the rate of the malate-aspartate shuttle, as well as the anaplerotic flux of the glial pyruvate carboxylase reaction are in agreement with the estimations calculated from the proposed model.

Dexamethasone (DEX) at physiologically elevated (stress) concentration (1 microM) decreased K(+)-evoked glutamate release from rat hippocampal slices under superfusion in the presence of Ca2+. On the contrary 10 microM DEX increased this K(+)-evoked glutamate release while 0.1 microM DEX had no effect. The glucocorticoid antagonist for the "classic" receptor, RU 486, completely reversed the effect of 1 microM DEX. Actinomycin D had no effect. Dexamethasone at 1 microM had no effect on the Ca2(+)-independent (10 mM Mg2+ replacing 1 mM Ca2+) K(+)-evoked glutamate release. Dexamethasone at 1 microM or 10 microM had no effect on the phosphate-activated glutaminase--the key enzyme for the biosynthesis of neurotransmitter glutamate. These results suggest that the effect of DEX on K(+)-evoked glutamate release: (i) depends on its concentration; (ii) is exerted on the Ca2(+)-dependent (neurotransmitter release), at least at physiological stress concentrations; and (iii) is exerted via the classical receptor but is nongenomic.

The fate of leucine nitrogen in the central nervous system was investigated by incubating rat cerebral cortex slices in the presence of 0.5 mM each of L-[15N]-leucine, L-isoleucine, and L-valine. Analysis of the slices and incubation media for free amino acids by gas chromatography-mass spectrometry revealed that the 15N from leucine is incorporated into isoleucine only. No 15N was detected in valine or any other amino acid. These results suggest that leucine, valine, and their corresponding aminotransferases may be compartmentalized in brain cerebral cortex.

Cetirizine, a peripheral H1 antagonist, was administered to patients with delayed pressure urticaria in a double-blind, placebo-controlled, crossover study. Efficacy in reducing pressure-induced wheals and flares was evaluated. Histologic changes were also assessed with the skin window technique in weight-induced wheals. Results showed a statistically significant reduction in weight-induced wheal area (p less than 0.01) after cetirizine therapy; this improvement was accompanied by a concomitant reduction in eosinophil recruitment as demonstrated by the skin window technique (p = 0.0029). Subsequently, 14 patients with delayed pressure urticaria underwent biopsy before and after 3 weeks of cetirizine therapy to evaluate the drug's histologic effects. A blinded observer performed the histologic studies. Weight-induced lesions showed a mixed inflammatory infiltrate, primarily polymorphonuclear (neutrophils and eosinophils), whereas the unchallenged skin sites were normal. Cell counts from pressure-induced lesions showed a significant reduction in eosinophils after cetirizine treatment.

Glucose is the major fuel of the adult brain under normal conditions. The most abundant free amino acids of the brain are: Glutamate, taurine N-acetylaspartate, glutamine, aspartate and 4-aminobutyrate. Deprivation of glucose from the incubation medium of brain cortex slices decreases mainly glutamate and glutamine content. Substitution of pyruvate for glucose maintains the content of aspartate and glutamate but not of glutamine. Therefore the entire glucose molecule is indispensable for the maintenance of the glutamine content. When branched chain amino acids, (BCAA), leucine, isoleucine and valine (0.5 mM) are added to the incubation medium in the presence of glucose the content of glutamine only is increased, all other amino acids are decreased. This suggests that BCAA spare protein as in muscle and produce glutamine. Branched chain amino acids in the presence of pyruvate do not spare protein but they still produce glutamine. Furthermore it seems that they potentiate the protein sparing effect of glucose in brain. In all these experiments glutamate and aspartate content in the incubation medium ranges from 3-10% of their total content (tissue plus media). On the other hand 40-60% of each of the other amino acids is found outside. The presence of 3-hydroxybutyrate and glucose in the incubation medium of brain cortex slices from normal rats spares protein but can maintain constant the content of glutamate, aspartate and glutamine. In addition 3-hydroxybutyrate blocks the release into the medium of glutamate and aspartate.

Based on the selective inhibition of glutamate release in cerebellar granule cells in primary cultures by the aspartate aminotransferase inhibitor, aminooxyacetic acid, and by the ketodicarboxylate carrier inhibitor, phenylsuccinate, a novel model for synthesis of transmitter glutamate is suggested: Glutamate is formed from glutamine in the mitochondrial intramembrane space by phosphate-activated glutaminase, transported across the inner membrane in exchange with aspartate, transaminated in the matrix to alpha-ketoglutarate, which via the ketodicarboxylate carrier is transferred to the cytoplasm, and transaminated to form transmitter glutamate. Such a mechanism would explain the functional role of aspartate aminotransferase in glutamatergic neurons.

Glutamate dehydrogenase (NAD) activity was measured in liver and diaphragm mitochondria from 48 h fasted rats. Kinetic studies were performed with diaphragm enzyme and the effects of L-leu, ADP and L-ala on the K(1)m and V(1)max for NH(4)+, and α-ketoglutarate were evaluated. L-leucine increases by 2-8 fold the V(1)max for all three substrates with no significant changes in the K(1)m. ADP increased by 3-7 fold the V(1)max for all three substrates and the K(1)m for NADH and α-ketoglutarate by 1.5-7.0 fold. L-alanine had no effect on either the V(1)max or the K(1)m of any substrate. The results suggest that muscle has the capacity to form glutamate through the glutamate dehydrogenase reaction and that L-leucine may stimulate this reaction in muscle.

In normal man, the fasting state is characterized by release of alanine and glutamine from muscle and in situ muscle catabolism of branched chain amino acids (lecucine, isoleucine, and valine). The alanine released by muscle is utilized by the liver for gluconeogenesis. Muscle nitrogen repletion occurs during protein feeding primarily by means of selective hepatic escape and muscle uptake of branched chain amino acids in ingested protein. In the diabetic, amino acid catabolism is exaggerated in the fasting state as reflected by increased uptake of alanine by the liver for gluconeogenesis and accelerated branched chain amino acid catabolism in muscle. After protein feeding, uptake of branched chain amino acids by muscle is reduced and these amino acids accumulate in increased amounts in arterial blood. Protein feeding also exaggerates the hyperglycemia of diabetes by causing an increase in hepatic glucose production. Diabetes is thus characterized by accelerated protein catabolism during fasting as well as diminished nitrogen repletion and hyperglycemia after protein feeding. The hyperketonemia of diabetes may however, have a restraining influence on protein catabolism thereby reducing alanine availability for gluconeogenesis.

Evoked release of glutamate and aspartate from cultured cerebellar granule cells was studied after preincubation of the cells in tissue culture medium with glucose (6.5 mM), glutamine (1.0 mM), D[3H] aspartate and in some cases aminooxyacetate (5.0 mM) or phenylsuccinate (5.0 mM). The release of endogenous amino acids and of D-[3H] aspartate was measured under physiological and depolarizing (56 mM KCl) conditions both in the presence and absence of calcium (1.0 mM), glutamine (1.0 mM), aminooxyacetate (5.0 mM) and phenylsuccinate (5.0 mM). The cellular content of glutamate and aspartate was also determined. Of the endogenous amino acids only glutamate was released in a transmitter fashion and newly synthesized glutamate was released preferentially to exogenously supplied D-[3H] aspartate, a marker for exogenous glutamate. Evoked release of endogenous glutamate was reduced or completely abolished by respectively, aminooxyacetate and phenylsuccinate. In contrast, the release of D-[3H] aspartate was increased reflecting an unaffected release of exogenous glutamate and an increased "psuedospecific radioactivity" of the glutamate transmitter pool. Since aminooxyacetate and phenylsuccinate inhibit respectively aspartate aminotransferase and mitochondrial keto-dicarboxylic acid transport it is concluded that replenishment of the glutamate transmitter pool from glutamine, formed in the mitochondrial compartment by the action of glutaminase requires the simultaneous operation of mitochondrial keto-dicarboxylic acid transport and aspartate aminotransferase which is localized both intra- and extra-mitochondrially. The purpose of the latter enzyme apparently is to catalyze both intra- and extra-mitochondrial transamination of alpha-ketoglutarate which is formed intramitochondrially from the glutamate carbon skeleton and transferred across the mitochondrial membrane to the cytosol where transmitter glutamate is formed.(ABSTRACT TRUNCATED AT 250 WORDS)