scholarly journals 2235 15N-Leucine transport across the blood brain barrier is significantly impaired in the glutamine synthetase-inhibited brain

2018 ◽  
Vol 2 (S1) ◽  
pp. 1-1
Author(s):  
Shaun E. Gruenbaum ◽  
Roni Dhaher ◽  
Kevin Behar ◽  
Hitten Zaveri ◽  
Mark Erfe ◽  
...  
Keyword(s):  
Amino Acid ◽  
Glutamine Synthetase ◽  
Dentate Gyrus ◽  
Extracellular Fluid ◽  
Ultra Performance Liquid Chromatography ◽  
Methionine Sulfoximine ◽  
Causative Role ◽  
Immune System Dysfunction ◽  
Tightly Coupled ◽  
The Brain

OBJECTIVES/SPECIFIC AIMS: Astroglial glutamine synthetase (GS), which metabolizes glutamate and ammonia to glutamine, is critical for the detoxification of brain ammonia, clearance of synaptic glutamate, and production of brain glutamine. Perturbations in the expression and activity of GS are thought to play a causative role in the pathogenesis of several conditions of abnormal neurotransmission. Although the long-term consequences of GS inhibition on amino acid homeostasis in the brain are unknown, it is thought that amino acid influx in the brain is tightly coupled with glutamine efflux via the L-type amino acid transporter. Both glutamine and leucine serve many critical functions in the brain including protein synthesis, gene expression, insulin regulation, and immune signaling. The objective of this study was to determine the effects of chronic GS inhibition with methionine sulfoximine (MSO) on glutamine and leucine homeostasis in the brain. METHODS/STUDY POPULATION: In total, 12 rats were surgically implanted with microdialysis guide cannulas in the bilateral dentate gyrus. Rats were randomly divided for surgical implantation of either a MSO (n=6) or phosphate buffer saline (PBS; n=6) pump in the right dentate gyrus. After 7 days, bilateral microdialysis probes were placed under brief isoflurane anesthesia, and microdialysis flow was established by infusing 0.5 µL/min of artificial extracellular fluid. Dialysate samples were collected every 30 minutes for the duration of the experiment. A 113 mM 15N-Leucine (3.6 mL/h) and 2 M 2–13C-sodium acetate (0.0633 μL/g/min for t=0–5 min, 0.0316 μL/g/min for t=5–10 min, and 0.0253 μL/g/min for t>10 min) solution was infused intravenously for 300 minutes. The EZ:Faast Free Amino Acid analysis kit and ultra-performance liquid chromatography/tandem mass spectrometry was used for quantification of amino acids in the dialysate fluid. RESULTS/ANTICIPATED RESULTS: At baseline (t=0 h), the concentrations of glutamine were significantly lower in MSO-treated rats (p<0.001) in the ipsilateral (GS-inhibited) hippocampus. There were no differences in glutamine concentrations between MSO and PBS-treated rats in the contralateral hippocampus. In PBS-treated rats, there was a significant increase in 15N-leucine between t=0 hour and t=5 hour in the contralateral (p<0.05) and ipsilateral (p<0.05) hippocampus. In MSO-treated rats, there was a significant increase in 15N-leucine between t=0 and t=5 hours in the contralateral (p<0.05) hippocampus, but not in the ipsilateral hippocampus (p=ns.). DISCUSSION/SIGNIFICANCE OF IMPACT: This study demonstrated for the first time that basal glutamine concentrations are low in areas of the brain where GS is acutely inhibited, and that leucine uptake in these brain areas are markedly decreased. Perturbations in glutamine and leucine homeostasis have been implicated in several disease processes including diabetes, obesity, liver disease, immune system dysfunction, epilepsy, and cancer, and the glutamine-dependent leucine influx in the brain may be a novel and important therapeutic target to treat these conditions.

scholarly journals Asymmetrical Transport of Amino Acids across the Blood—Brain Barrier in Humans

1990 ◽  
Vol 10 (5) ◽  
pp. 698-706 ◽  
Author(s):  
G. Moos Knudsen ◽  
K. D. Pettigrew ◽  
C. S. Patlak ◽  
M. M. Hertz ◽  
O. B. Paulson
Keyword(s):  
Amino Acids ◽  
Endothelial Cell ◽  
Amino Acid ◽  
Blood Brain Barrier ◽  
Extracellular Fluid ◽  
Brain Barrier ◽  
Reverse Direction ◽  
Single Membrane ◽  
Blood Brain ◽  
The Brain

Blood–brain barrier permeability to four large neutral and one basic amino acid was studied in 30 patients with the double indicator technique. The resultant 64 venous outflow curves were analyzed by means of two models that take tracer backflux and capillary heterogeneity into account. The first model considers the blood–brain barrier as a double membrane where amino acids from plasma enter the endothelial cell. When an endothelial cell volume of 0.001 ml/g was assumed, permeability from the blood into the endothelial cell was, for most amino acids, about 10–20 times larger than the permeability for the reverse direction. The second model assumes that the amino acids, after intracarotid injection, cross a single membrane barrier and enter a well-mixed compartment, the brain extracellular fluid, i.e., the endothelial cell is assumed to behave as a single membrane. With this model, for large neutral amino acids, the permeability out of the extracellular fluid space back to the blood was between 8 to 12 times higher than the permeability from the blood into the brain. Such a difference in permeabilities across the blood–brain barrier can almost entirely be ascribed to the effect of a nonlinear transport system combined with a relatively small brain amino acid metabolism. The significance of the possible presence of an energy-dependent A system at the abluminal side of the blood–brain barrier is discussed and related to the present findings. For both models, calculation of brain extraction by simple peak extraction values underestimates true unidirectional brain uptake by 17–40%. This raises methodological problems when estimating blood to brain transfer of amino acids with this traditional in vivo method.

scholarly journals Effect of Glutamine Synthetase Inhibition on Brain and Interorgan Ammonia Metabolism in Bile Duct Ligated Rats

2013 ◽  
Vol 34 (3) ◽  
pp. 460-466 ◽  
Author(s):  
Andreas W Fries ◽  
Sherry Dadsetan ◽  
Susanne Keiding ◽  
Lasse K Bak ◽  
Arne Schousboe ◽  
...  
Keyword(s):  
Amino Acids ◽  
Bile Duct ◽  
Glutamine Synthetase ◽  
Methionine Sulfoximine ◽  
Ammonia Metabolism ◽  
Liver And Kidney ◽  
Glutamine Synthesis ◽  
Alanine Synthesis ◽  
High Concentrations ◽  
The Brain

Ammonia has a key role in the development of hepatic encephalopathy (HE). In the brain, glutamine synthetase (GS) rapidly converts blood-borne ammonia into glutamine which in high concentrations may cause mitochondrial dysfunction and osmolytic brain edema. In astrocyte-neuron cocultures and brains of healthy rats, inhibition of GS by methionine sulfoximine (MSO) reduced glutamine synthesis and increased alanine synthesis. Here, we investigate effects of MSO on brain and interorgan ammonia metabolism in sham and bile duct ligated (BDL) rats. Concentrations of glutamine, glutamate, alanine, and aspartate and incorporation of 15NH4+ into these amino acids in brain, liver, muscle, kidney, and plasma were similar in sham and BDL rats treated with saline. Methionine sulfoximine reduced glutamine concentrations in liver, kidney, and plasma but not in brain and muscle; MSO reduced incorporation of 15NH4+ into glutamine in all tissues. It did not affect alanine concentrations in any of the tissues but plasma alanine concentration increased; incorporation of 15NH4+ into alanine was increased in brain in sham and BDL rats and in kidney in sham rats. It inhibited GS in all tissues examined but only in brain was an increased incorporation of 15N-ammonia into alanine observed. Liver and kidney were important for metabolizing blood-borne ammonia.

scholarly journals Brain Alanine Formation as an Ammonia-Scavenging Pathway during Hyperammonemia: Effects of Glutamine Synthetase Inhibition in Rats and Astrocyte—Neuron Co-Cultures

2013 ◽  
Vol 33 (8) ◽  
pp. 1235-1241 ◽  
Author(s):  
Sherry Dadsetan ◽  
Eva Kukolj ◽  
Lasse K Bak ◽  
Michael SØrensen ◽  
Peter Ott ◽  
...  
Keyword(s):  
Hepatic Encephalopathy ◽  
Glutamine Synthetase ◽  
Drug Target ◽  
Methionine Sulfoximine ◽  
Concerted Action ◽  
N Incorporation ◽  
Dose Dependent ◽  
The Brain

Hyperammonemia is a major etiological toxic factor in the development of hepatic encephalopathy. Brain ammonia detoxification occurs primarily in astrocytes by glutamine synthetase (GS), and it has been proposed that elevated glutamine levels during hyperammonemia lead to astrocyte swelling and cerebral edema. However, ammonia may also be detoxified by the concerted action of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALAT) leading to trapping of ammonia in alanine, which in vivo likely leaves the brain. Our aim was to investigate whether the GS inhibitor methionine sulfoximine (MSO) enhances incorporation of 15NH4+ in alanine during acute hyperammonemia. We observed a fourfold increased amount of 15NH4 incorporation in brain alanine in rats treated with MSO. Furthermore, co-cultures of neurons and astrocytes exposed to 15NH4Cl in the absence or presence of MSO demonstrated a dose-dependent incorporation of 15NH4 into alanine together with increased 15N incorporation in glutamate. These findings provide evidence that ammonia is detoxified by the concerted action of GDH and ALAT both in vivo and in vitro, a mechanism that is accelerated in the presence of MSO thereby reducing the glutamine level in brain. Thus, GS could be a potential drug target in the treatment of hyperammonemia in patients with hepatic encephalopathy.

Effects of Methionine Sulfoximine and Glycine on Nitrogen Metabolism of Maize Leaves in the Light

1983 ◽  
Vol 10 (2) ◽  
pp. 187 ◽  
Author(s):  
MG Berger ◽  
HP Fock
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Glutamine Synthetase ◽  
Nitrogen Cycling ◽  
Co2 Assimilation ◽  
Methionine Sulfoximine ◽  
Co2 Evolution ◽  
Ammonia Content ◽  
Ammonia Accumulation ◽  
Maize Leaves

Detached maize leaves with their cut bases in water or in solutions containing 15 mM [14C, 15N]glycine, 15 mM [14C]glutamate, 5 mM methionine sulfoximine (an inhibitor of glutamine synthetase) or the appropriate amino acid plus inhibitor, were incubated for up to 135 min in the light. The concentrations and the 15N content of ammonia and of amino acids involved in photorespiratory nitrogen cycling were determined. Incubation with methionine sulfoximine or glycine increased the ammonia content significantly, whereas glutamate showed no effect. The nitrogen of glycine was metabolized into serine and ammonia. Ammonia was first recycled into glutamine, and then into glutamate. The glycine carbon skeleton served as a precursor for serine. Based on the data for ammonia accumulation the minimum rate of photorespiratory CO2 evolution in maize leaves was estimated to be about 1% of the rate of CO2 assimilation.

Immunoelectron microscope detection of C-type natriuretic peptide in normal human atrial tissue

1993 ◽  
Vol 51 ◽  
pp. 388-389
Author(s):  
Chi-Ming Wei ◽  
Margaret Hukee ◽  
Christopher G.A. McGregor ◽  
John C. Burnett
Keyword(s):  
Amino Acid ◽  
Natriuretic Peptide ◽  
Vascular Tone ◽  
Cardiac Tissue ◽  
Ring Structure ◽  
Terminal Amino Acid ◽  
Amino Acid Peptide ◽  
Normal Human ◽  
Terminal Amino ◽  
The Brain

C-type natriuretic peptide (CNP) is a newly identified peptide that is structurally related to atrial (ANP) and brain natriuretic peptide (BNP). CNP exists as a 22-amino acid peptide and like ANP and BNP has a 17-amino acid ring formed by a disulfide bond. Unlike these two previously identified cardiac peptides, CNP lacks the COOH-terminal amino acid extension from the ring structure. ANP, BNP and CNP decrease cardiac preload, but unlike ANP and BNP, CNP is not natriuretic. While ANP and BNP have been localized to the heart, recent investigations have failed to detect CNP mRNA in the myocardium although small concentrations of CNP are detectable in the porcine myocardium. While originally localized to the brain, recent investigations have localized CNP to endothelial cells consistent with a paracrine role for CNP in the control of vascular tone. While CNP has been detected in cardiac tissue by radioimmunoassay, no studies have demonstrated CNP localization in normal human heart by immunoelectron microscopy.

scholarly journals Post-Stroke Social Isolation Reduces Cell Proliferation in the Dentate Gyrus and Alters miRNA Profiles in the Aged Female Mice Brain

2020 ◽  
Vol 22 (1) ◽  
pp. 99
Author(s):  
Aleah Holmes ◽  
Yan Xu ◽  
Juneyoung Lee ◽  
Michael E. Maniskas ◽  
Liang Zhu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Dentate Gyrus ◽  
Social Isolation ◽  
Census Data ◽  
Elderly Women ◽  
Stroke Recovery ◽  
Tissue Loss ◽  
Female Mice ◽  
Post Stroke ◽  
The Brain

Social isolation and loneliness are risk factors for stroke. Elderly women are more likely to be isolated. Census data shows that in homeowners over the age of 65, women are much more likely to live alone. However, the underlying mechanisms of the detrimental effects of isolation have not been well studied in older females. In this study, we hypothesized that isolation impairs post-stroke recovery in aged female mice, leading to dysregulated microRNAs (miRNAs) in the brain, including those previously shown to be involved in response to social isolation (SI). Aged C57BL/6 female mice were subjected to a 60-min middle cerebral artery occlusion and were randomly assigned to either single housing (SI) or continued pair housing (PH) immediately after stroke for 15 days. SI immediately after stroke led to significantly more brain tissue loss after stroke and higher mortality. Furthermore, SI significantly delayed motor and sensory recovery and worsened cognitive function, compared to PH. A decrease in cell proliferation was seen in the dentate gyrus of SI mice assessed by bromodeoxyuridine (BrdU) labeling. miRNAome data analysis revealed changes in several miRNAs in the brain, such as miR-297a-3p and miR-200c-3p, which are known to regulate pathways involved in cell proliferation. In conclusion, our data suggest that SI can lead to a poor post-stroke recovery in aged females and dysregulation of miRNAs and reduced hippocampal cell proliferation.

Association of the brain anion exchanger, AE3, with the repeat domain of ankyrin

1993 ◽  
Vol 105 (4) ◽  
pp. 1137-1142 ◽  
Author(s):  
C.W. Morgans ◽  
R.R. Kopito
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Anion Exchanger ◽  
Deletion Mutant ◽  
Tandem Repeats ◽  
Structural Homology ◽  
Terminal Domain ◽  
Repeat Domain ◽  
The Brain

The 89 kDa NH2-terminal domain of erythrocyte ankyrin is composed almost entirely of 22 tandem repeats of a 33 amino acid sequence and constitutes the binding site for the cytoplasmic NH2-terminal domain of the erythrocyte anion exchanger, AE1. We have developed an assay to evaluate the in vivo interaction between a fragment of ankyrin corresponding to this domain (ANK90) and two non-erythroid anion exchangers, AE2 and AE3, that share considerable structural homology with AE1. Association was assessed by co-immunoprecipitation of ANK90-anion exchanger complexes from detergent extracts of cells cotransfected with plasmids encoding the ankyrin fragment and the anion exchanger or mutants thereof. ANK90 was co-immunoprecipitated with AE1 but not with an AE1 deletion mutant lacking the cytoplasmic NH2-terminal domain. Using this assay, we show that the brain anion exchanger AE3, but not the closely related homologue, AE2, is capable of binding to ankyrin.

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