Hypo-osmotic swelling modifies glutamate-glutamine cycle in the cerebral cortex and in astrocyte cultures

Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, New York, USA.
Journal of Neurochemistry (Impact Factor: 4.28). 07/2011; 118(1):140-52. DOI: 10.1111/j.1471-4159.2011.07289.x
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J. Neurochem. (2011) 10.1111/j.1471-4159.2011.07289.x
In our previous work, we found that perfusion of the rat cerebral cortex with hypo-osmotic medium triggers massive release of the excitatory amino acid L-glutamate but decreases extracellular levels of L-glutamine (R. E. Haskew-Layton et al., PLoS ONE, 3: e3543). The release of glutamate was linked to activation of volume-regulated anion channels, whereas mechanism(s) responsible for alterations in extracellular glutamine remained unclear. When mannitol was added to the hypo-osmotic medium to reverse reductions in osmolarity, changes in microdialysate levels of glutamine were prevented, indicating an involvement of cellular swelling. As the main source of brain glutamine is astrocytic synthesis and export, we explored the impact of hypo-osmotic medium on glutamine synthesis and transport in rat primary astrocyte cultures. In astrocytes, a 40% reduction in medium osmolarity moderately stimulated the release of L-[3H]glutamine by ∼twofold and produced no changes in L-[3H]glutamine uptake. In comparison, hypo-osmotic medium stimulated the release of glutamate (traced with D-[3H]aspartate) by more than 20-fold. In whole-cell enzymatic assays, we discovered that hypo-osmotic medium caused a 20% inhibition of astrocytic conversion of L-[3H]glutamate into L-[3H]glutamine by glutamine synthetase. Using an HPLC assay, we further found a 35% reduction in intracellular levels of endogenous glutamine. Overall, our findings suggest that cellular swelling (i) inhibits astrocytic glutamine synthetase activity, and (ii) reduces substrate availability for this enzyme because of the activation of volume-regulated anion channels. These combined effects likely lead to reductions in astrocytic glutamine export in vivo and may partially explain occurrence of hyperexcitability and seizures in human hyponatremia.

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Available from: Alexander A Mongin, Dec 01, 2014
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    • "Effect of hypo-osmotic media on intracellular amino acid levels and transmembrane amino acid fluxes in primary astrocytes Cultured astrocytes, when exposed to hypo-osmotic media, actively regulate their volume via loss of intracellular osmotically active molecules, including a number of cytosolic amino acids (Pasantes Morales and Schousboe 1988; Kimelberg et al. 1990; Olson 1999; Hyzinski-Garcia et al. 2011). We started this study by comparing the effect of exposure to media with different osmolarities on the intracellular content of five major endogenous amino acids. "
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    ABSTRACT: Hyponatremia and several other CNS pathologies are associated with substantial astrocytic swelling. To counteract cell swelling, astrocytes lose intracellular osmolytes, including l-glutamate and taurine, through volume regulated anion channel (VRAC). In vitro, when swollen by exposure to hypo-osmotic medium, astrocytes lose endogenous taurine faster, paradoxically, than l-glutamate or l-aspartate. Here, we explored the mechanisms responsible for differences between the rates of osmolyte release in primary rat astrocyte cultures. In radiotracer assays, hypo-osmotic efflux of preloaded [14C]taurine was indistinguishable from d-[3H]aspartate and only 30-40% faster than l-[3H]glutamate. However, when we used HPLC to measure the endogenous intracellular amino acid content, hypo-osmotic loss of taurine was ~5-fold greater than l-glutamate, and no loss of l-aspartate was detected. The dramatic difference between loss of endogenous taurine and glutamate was eliminated after inhibition of both glutamate reuptake (with 300 μM TBOA) and glutamate synthesis by aminotransferases (with 1 mM aminooxyacetic acid, AOA). Treatment with TBOA+AOA made reductions in the intracellular taurine and l-glutamate levels approximately equal. Taken together, these data suggest that swollen astrocytes actively conserve intracellular glutamate via reuptake and de novo synthesis. Our findings likely also explain why in animal models of acute hyponatremia, extracellular levels of taurine are dramatically elevated with minimal impact on extracellular l-glutamate. This article is protected by copyright. All rights reserved.
    Journal of Neurochemistry 01/2015; 135(1):176-185. DOI:10.1111/jnc.13229 · 4.28 Impact Factor
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    • "Primary astrocyte cultures and microglial cells were prepared from brain cortical tissue of newborn Sprague–Dawley rats as previously described [34] [45]. One-day-old pups were euthanized by rapid decapitation and cortical tissue was dissected from meninges, hippocampi, and basal ganglia and minced with small scissors. "
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    ABSTRACT: The contribution of oxidative stress to ischemic brain damage is well established. Nevertheless, for unknown reasons, several clinically tested antioxidant therapies failed to show benefits in human stroke. Based on our previous in vitro work, we hypothesized that the neuroprotective potency of antioxidants is related to their ability to limit release of the excitotoxic amino acids, glutamate and aspartate. We explored the effects of two antioxidants, tempol and edaravone, on amino acid release in the brain cortex, in a rat model of transient occlusion of the middle cerebral artery (MCAo). Amino acid levels were quantified using a microdialysis approach, with the probe positioned in the ischemic penumbra as verified by a laser Doppler technique. Two-hour MCAo triggered a dramatic increase in the levels of glutamate, aspartate, taurine and alanine. Microdialysate delivery of 10mM tempol reduced the amino acid release by 60-80%, while matching levels of edaravone had no effect. In line with these latter data, an intracerebroventricular injection of tempol but not edaravone (500 nmols each, 15 minutes prior to MCAo) reduced infarction volumes by ~50% and improved neurobehavioral outcomes. In vitro assays showed that tempol was superior in removing superoxide anion, whereas edaravone was more potent in scavenging hydrogen peroxide, hydroxyl radical, and peroxynitrite. Overall, our data suggests that the neuroprotective properties of tempol are likely related to its ability to reduce tissue levels of the superoxide anion and pathological glutamate release, and, in such a way, limit progression of brain infarction within ischemic penumbra. These new findings may be instrumental in developing new antioxidant therapies for treatment of stroke.
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    • "Astrocytes are also responsible for clearing extracellular glutamate, the main neurotransmitter of excitatory signaling in the mammalian CNS, due to their high-affinity glutamate transporters: GLAST (EAAT1) and GLT1 (EAAT2) [8], [9], [10], [11], [12], [13], [14]. Once taken up by astrocytes, glutamate is converted into glutamine by glutamine synthetase (GS – EC [15], [16], [17], [18]. Glutamate uptake is also important for maintaining the levels of glutathione (GSH), a major antioxidant molecule in the brain [15], [16], [19], [20]. "
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    ABSTRACT: Astrocytes, a major class of glial cells, regulate neurotransmitter systems, synaptic processing, ion homeostasis, antioxidant defenses and energy metabolism. Astrocyte cultures derived from rodent brains have been extensively used to characterize astrocytes' biochemical, pharmacological and morphological properties. The aims of this study were to develop a protocol for routine preparation and to characterize a primary astrocyte culture from the brains of adult (90 days old) Wistar rats. For this we used enzymatic digestion (trypsin and papain) and mechanical dissociation. Medium exchange occurred from 24 h after obtaining a culture and after, twice a week up to reach the confluence (around the 4(th) to 5(th) week). Under basal conditions, adult astrocytes presented a polygonal to fusiform and flat morphology. Furthermore, approximately 95% the cells were positive for the main glial markers, including GFAP, glutamate transporters, glutamine synthetase and S100B. Moreover, the astrocytes were able to take up glucose and glutamate. Adult astrocytes were also able to respond to acute H2O2 exposure, which led to an increase in reactive oxygen species (ROS) levels and a decrease in glutamate uptake. The antioxidant compound resveratrol was able to protect adult astrocytes from oxidative damage. A response of adult astrocytes to an inflammatory stimulus with LPS was also observed. Changes in the actin cytoskeleton were induced in stimulated astrocytes, most likely by a mechanism dependent on MAPK and Rho A signaling pathways. Taken together, these findings indicate that the culture model described in this study exhibits the biochemical and physiological properties of astrocytes and may be useful for elucidating the mechanisms related to the adult brain, exploring changes between neonatal and adult astrocytes, as well as investigating compounds involved in cytotoxicity and cytoprotection.
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