Endothelial cell conditioned media mediated regulation of glutamine synthetase activity in glial cells (2025)

Glutamine synthetase enhances the clearance of extracellular glutamate by the neural retina

Journal of Neurochemistry, 2002

Clearance of synaptic glutamate by glial cells is required for the normal function of excitatory synapses and for prevention of neurotoxicity. Although the regulatory role of glial glutamate transporters in glutamate clearance is well established, little is known about the influence of glial glutamate metabolism on this process. This study examines whether glutamine synthetase (GS), a glial-specific enzyme that amidates glutamate to glutamine, affects the uptake of glutamate. Retinal explants were incubated in the presence of [ 14 C]glutamate and glutamate uptake was assessed by measurement of the amount of radioactively labeled molecules within the cells and the amount of [ 14 C]glutamine released to the medium. An increase in GS expression in Mü ller glial cells, caused by induction of the endogenous gene, did not affect the amount of glutamate accumulated within the cells, but led to a dramatic increase in the amount of glutamine released. This increase, which was directly correlated with the level of GS expression, was dependent on the presence of external sodium ions, and could be completely abolished by methionine sulfoximine, a specific inhibitor of GS activity. Our results demonstrate that GS activity significantly influences the uptake of glutamate by the neural retina and suggest that this enzyme may represent an important target for neuroprotective strategies.

Effect of glaucoma on the retinal glutamate/glutamine cycle activity

Faseb Journal, 2005

Glutamate-induced excitotoxicity has been proposed to mediate the death of retinal ganglion cells in glaucoma. The metabolic dependence of glutamatergic neurons upon glia via the glutamate/glutamine cycle to provide the precursor for neurotransmitter glutamate is well established. Thus, the aim of the present work was to study the retinal glutamate/glutamine activity in eyes with hypertension induced by intracameral injections of hyaluronic acid (HA). For this purpose, weekly injections of HA were performed unilaterally in the rat anterior chamber, whereas the contralateral eye was injected with saline solution. At 3 or 10 weeks of treatment, glutamate and glutamine uptake and release were assessed using [ 3 H]-glutamate and [ 3 H]-glutamine as radioligands, respectively. In addition, glutamine synthetase activity was assessed by a spectrophotometric assay, whereas glutaminase activity was measured through the conversion of [ 3 H]-glutamine to [ 3 H]-glutamate. At 3 weeks of treatment with HA, a significant decrease (P<0.01) in glutamate uptake and glutamine synthetase activity was observed. Glutamine uptake and release, as well as glutaminase activity, were significantly increased (P<0.01) in eyes injected with HA for 3 weeks compared with vehicle-injected eyes, whereas [ 3 H]-glutamate release did not change in hypertensive eyes. Only the changes in glutamine synthetase activity persisted at 10 weeks of treatment with HA. These results indicate a significant alteration in the retinal glutamate/glutamine cycle activity in hypertensive eyes. Since these changes preceded both functional and histological alterations induced by ocular hypertension, these results support the involvement of glutamate in glaucomatous neuropathy.

Expression of glutamate receptors on cultured cerebral endothelial cells

Journal of Neuroscience Research, 1998

Activation of glutamate receptors has been shown to mediate a large number of neuronal processes such as long-term potentiation and ischemic damage. In addition to neurons and glia, glutamate receptors may occur on cerebral endothelial cells (CECs). The aim of the present study was to determine which glutamate receptors are expressed in CECs and to demonstrate the functional presence of such channels. By using reverse transcriptase-polymerase chain reaction, we showed that primary cultures of rat CECs express N-methyl-D-aspartate (NMDA) receptors (NR1 subunit, which is necessary for the formation of functional NMDA receptors, and NR2A-C subunits),

In vitro evidence for the brain glutamate efflux hypothesis: Brain endothelial cells cocultured with astrocytes display a polarized brain-to-blood transport of glutamate

Glia, 2012

The concentration of the excitotoxic amino acid, L-glutamate, in brain interstitial fluid is tightly regulated by uptake transporters and metabolism in astrocytes and neurons. The aim of this study was to investigate the possible role of the blood-brain barrier endothelium in brain L-glutamate homeostasis. Transendothelial transport-and accumulation studies of 3 H-L-glutamate, 3 H-L-aspartate, and 3 H-D-aspartate in an electrically tight bovine endothelial/rat astrocyte blood-brain barrier coculture model were performed. After 6 days in culture, the endothelium displayed transendothelial resistance values of 1014 6 70 X cm 2 , and 14 C-D-mannitol permeability values of 0.88 6 0.13 3 10 26 cm s 21 . Unidirectional flux studies showed that L-aspartate and L-glutamate, but not D-aspartate, displayed polarized transport in the brain-to-blood direction, however, all three amino acids accumulated in the cocultures when applied from the abluminal side. The transcellular transport kinetics were characterized with a K m of 69 6 15 lM and a J max of 44 6 3.1 pmol min 21 cm 22 for L-aspartate and a K m of 138 6 49 lM and J max of 28 6 3.1 pmol min 21 cm 22 for L-glutamate. The EAAT inhibitor, DL-threo-ß-Benzyloxyaspartate, inhibited transendothelial brain-to-blood fluxes of L-glutamate and L-aspartate. Expression of EAAT-1 (Slc1a3), 22 (Slc1a2), and 23 (Slc1a1) mRNA in the endothelial cells was confirmed by conventional PCR and localization of EAAT-1 and 23 in endothelial cells was shown with immunofluorescence. Overall, the findings suggest that the blood-brain barrier itself may participate in regulating brain L-glutamate concentrations.

Glutamine Synthetase in Oligodendrocytes and Astrocytes: New Biochemical and Immunocytochemical Evidence

Journal of Neurochemistry, 1991

Abstract: The results of recent immunocytochemical experiments suggest that glutamine synthetase (GS) in the rat CNS may not be confined to astrocytes. In the present study, GS activity was assayed in oligodendrocytes isolated from bovine brain and in oligodendrocytes, astrocytes, and neurons isolated from rat forebrain, and the results were compared with new immunochemical data. Among the cells isolated from rat brain, astrocytes had the highest specific activities of GS, followed by oligodendrocytes. Oligodendrocytes isolated from white matter of bovine brain had GS specific activities almost fivefold higher than those in white matter homogenates. Immunocytochemical staining also showed the presence of GS in both oligodendrocytes and astrocytes in bovine forebrain, in three white-matter regions of rat brain, and in Vibratome sections as well as paraffin sections.

Glutamate accumulation by a clone of glial cells

Brain Research, 1974

High affinity transports of putative neurotransmitters have often been referred to as a partial evidence for their physiological role in nervous transmission: glutamate (Glu), aspartate and GABA are accumulated by high affinity systems in rat brain 1,9, and in spinal cord slicesL However, these transports do not appear to be specific to neuronal cells: radioactive Glu is accumulated in a synaptosomal population of rat brain 10,17 as well as in a cortical fraction enriched in glial cellsV, s. As the kinetic parameters of this amino acid uptake have not been determined in glia, it seemed to us that clonal nervous cells would provide a homogeneous preparation for such uptake studies. Moreover, they allow comparisons with non-nervous cell lines, such as fibroblasts. Thus, we have recently reported on a high affinity transport system for tryptophan, both in cultured glia/cells (clone C6) and fibroblasts; interestingly, the kinetic values and ion sensitivity were identical 3. These results are in agreement with those reported by Richelson and Thompson 15 where tyrosine, phenylalanine and choline appeared to be accumulated with about the same affinity in these two cell types.

Diabetes Reduces Glutamate Oxidation and Glutamine Synthesis in the Retina

Experimental Eye Research, 2000

Retinas of diabetic individuals develop early functional changes measurable by electrophysiological and psychometric testing. Using a rat model of diabetes, we previously identi®ed diabetes-induced alterations in metabolism of the neurotransmitter glutamate which may ultimately lead to accumulation of glutamate in the retina (Diabetes, 47: 815, 1998). We therefore investigated the function of enzymes that mediate the synthesis and breakdown of glutamate in retinas from rats made diabetic by injection of streptozotocin. De novo synthesis of nitrogen-containing amino acids including glutamate, glutamine and aspartate was assessed by measuring the rate of carbon ®xation in freshly dissected retinas, and was unchanged by diabetes. In contrast, the oxidation of glutamate was signi®cantly reduced in retinas from diabetic rats (62 %, P 5 0 . 05). Furthermore, diabetic retinas were less susceptible to inhibition of glutamate oxidation by the transaminase inhibitor aminoxyacetate (80 %, N.S.), compared to the signi®cant decrease seen in control rats (61 %, P 5 0 . 001). The activity and content of glutamine synthetase were also signi®cantly reduced in retinas from rats diabetic for 2±6 months [range of 48 % (P 5 0 . 005) to 83 % (P 5 0 . 05) compared to control]. The activity of glutamine synthetase was normalized by acute injections of insulin, but not by reducing blood sugar levels with injections of phlorizin. These results indicate two enzymatic abnormalities in the glutamate metabolism pathway in the retina during diabetes: transamination to a-ketoglutarate and amination to glutamine. The reduced ux through these pathways may be associated with the accumulation of glutamate. These results are also consistent with the possibility that some of the glial changes in the retina during diabetes may be caused by hypoinsulinemia rather than hyperglycemia.

News on glutamate receptors in glial cells

Trends in neurosciences, 1996

Glutamate (Glu) receptors convey most of the excitatory synaptic transmission in the mammalian CNS. Distinct Glu-receptor genes and different subtypes of glutamate-activated channels are expressed ubiquitously throughout the developing and mature brain in the two major macroglial cell types, astrocytes and oligodendrocytes. These glial receptors are found in acutely isolated cells and in brain slices, and are therefore functional in vivo. Glutamate receptors in glial cells are activated during neuronal activity, and their activation modulates gene expression in astrocytes and oligodendrocytes. The proliferation and differentiation of glial precursor cells are also regulated by activation of Glu receptors, suggesting that the excitatory transmitter might be one of the environmental signals that regulate glial-cell development.

Effect of Glucose Deprivation and Acute Glutamate Exposure in Cultured Retinal Cells

Experimental Neurology, 1998

The relationship between bioenergetics and the glutamate system was analyzed in a neuronal model of retinal cells in culture, submitted to glucose deprivation and exposed to glutamate for 2 h, and compared with exposure to glutamate in the presence of glucose. Under glucose deprivation, a reduction (about 1.1-fold) in the energy charge of the cells occurred, probably as a result of a decrement (by about 75%) in the cellular redox efficacy, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. In the absence of glucose, exposure of retinal cells to 10 M glutamate potentiated the reduction in the energy charge (by about 1.2-fold) and induced a significant increase in the uptake of 45 Ca 2؉ by the cells (1.3-fold), although no significant changes were observed in the presence of glucose. Under glucose deprivation, 100 M glutamate caused an irreversible cell membrane damage, as shown by the significant increase in lactate dehydrogenase (LDH) leakage (about 1.8-fold). A significant increase in membrane depolarization, measured by the reduction of [ 3 H]tetraphenylphosphonium ؉ ([ 3 H]TPP ؉) uptake, was also observed after glutamate exposure in the absence of glucose. In the presence of glucose, high glutamate concentrations (10 mM) induced a major increase in Ca 2؉ entry into the cells and membrane depolarization, without affecting the energy charge or cell survival. In contrast, in the absence of glucose, 10 mM glutamate did not alter Ca 2؉ accumulation by the cells and a smaller decrease in membrane potential occurred, as compared to 100 M glutamate exposure. Data shown in this study suggest that during a prolonged (2 h) and acute exposure to high glutamate (10 mM), under glucose deprivation conditions, the neuronal systems have ''adaptive'' mechanisms that allow the survival of cells. These findings may have implications in neuronal degeneration occurring during brain ischemia. 1998 Academic Press

Culture medium components modulate retina cell damage induced by glutamate, kainate or “chemical ischemia”

Neurochemistry International, 1998

The aim of this study was to determine whether culture-conditioned medium (CCM) can prevent neuronal damage caused by excitotoxicity or by "chemical ischemia" in cultured chick retina cells. Excitotoxic conditions were obtained by incubating retina cells with glutamate or kainate and "chemical ischemia" was induced by metabolic inhibition. In this case, cultures were briefly exposed to sodium cyanide, to block oxidative phosphorylation and iodoacetic acid, to block glycolysis. The assessment of neuronal injury was made spectrophotometrically by quantification of cellularly reduced MTT. Stimulation of retina cells with glutamate or kainate in serum deprived culture medium (BME-FCS), lead to a decrease in the MTT metabolism that was dependent on the time of exposure to the toxic agents. CCM prevented cell damage, either when present during the stimulation period or during the recovery period. This protection was more prominent in the case of kainate-induced neuronal death. "Chemical ischemia" also lead to a decrease of the MTT metabolism in a time-dependent manner and CCM protected retina cells from "ischemia"-induced lesions when present during the stimulation period and during the recovery period. The protective effect of CCM was partially decreased by the tyrosine kinase inhibitor, genistein, when the cells were stimulated with kainate, but not with glutamate, or when the cells were subjected to "chemical ischemia". CCM protected retina cells against both the acute and the delayed toxicity induced by either glutamate or kainate, or by "chemical ischemia", when present during both the insult and the recovery period. The presence of survival factors in the media may effectively inhibit the cell death signals generated by glutamate receptor activation or by "chemical ischemia".