Neurobiology of Lipids (ISSN 1683-5506) Vol 9 article 1


HOME	ABOUT	FEEDBACK	RECENT CONTENT	PUBLISH IN NoL	ARCHIVE WITH US	HELP MENU

Neurobiol Lipids 9, 1 (18 March 2010)
find out how to cite this article

original research:
GLUTAMINE SYNTHETASE ACTIVITY AND AMBIENT L-[¹⁴C]GLUTAMATE IN CHOLESTEROL-DEFICIENT RAT BRAIN NERVE TERMINALS

Tatiana Borisova

The Department of Neurochemistry, Palladin Institute of Biochemistry, NAS of Ukraine, 9 Leontovicha Street, Kiev, 01601, Ukraine

Corresponding author: Tatiana Borisova, The Department of Neurochemistry, Palladin Institute of Biochemistry, NAS of Ukraine, 9 Leontovicha Street, Kiev, 01601,Ukraine; Tel:+38044 2343254; Fax:+38044 2796365; E-mail: tborisov[at]biochem.kiev.ua

Submitted: 2 february 2010 Accepted for Publication: 11 March 2010 Published: 18 March, 2010
Copyright © 2010 T Borisova, Licensee Neurobiology of Lipids

Article view and respond options:

GOOGLE SCHOLAR CITATION DOAJ CITATION FULL TEXT ACROBAT .PDF SUBMIT E-LETTER AUTHORPUBMED

GOOGLE SCHOLAR CITATION	DOAJ CITATION	FULL TEXT	ACROBAT .PDF	SUBMIT E-LETTER	AUTHORPUBMED

ARTICLE NAVIGATION MENU

ABSTRACT

INTRODUCTION

MATERIALS & METHODS

RESULTS & DISCUSSION

FIGURE 1A

FIGURE 1B

FIGURE 2

REFERENCES

ABSTRACT

Glutamate is not only the predominant excitatory neurotransmitter in the mammalian CNS but also a potent neurotoxin. The excessive activation of glutamate receptors can cause exitotoxicity and cell death both in vitro and in vivo. The low level of ambient glutamate is important for the brain’s spontaneous activity and proper synaptic transmission. The study was focused on the assessment of the effects of glutamine synthetase inhibitor, L-methionine sulfoximine (MSO), on the level of ambient L-[¹⁴C]glutamate in rat brain nerve terminals treated with the cholesterol acceptor methyl-b-cyclodextrin (MbCD) (15 mM), which reduced the synaptosomal cholesterol content by 25.0 ± 3.0 %. It was revealed that the inhibitor per se decreased transporter - mediated uptake of L-[¹⁴C]glutamate by synaptosomes in dose-dependent manner. Therefore, we applied the non-transportable competitive inhibitor of glutamate transporters DL-threo-b-benzyloxyaspartate (DL-TBOA) to eliminate the contribution of transporter-mediated glutamate uptake to the value of ambient L-[¹⁴C]glutamate during the inhibition of the activity of glutamine synthetase. It was shown that the combined application of 200 mM DL-TBOA and 1.5 mM MSO increased the extracellular L-[¹⁴C]glutamate level from 0.193 ± 0.013 nmol/mg protein to 0.52 ± 0.02 nmol/mg protein in control and from 0.282 ± 0.013 nmol/mg protein to 0.62 ± 0.02 nmol/mg protein in MbCD – treated synaptosomes. Thus, the inhibition of glutamine synthetase activity increased the level of ambient L-[¹⁴C]glutamate in control and cholesterol-deficient synaptosomes, and this level was higher under condition of cholesterol deficiency than in control irrespective of the presence or absence of glutamine synthetase inhibitor.

Key words: cholesterol, methyl-beta-cyclodextrin, MbCD, L-glutamate, the extracellular level, glutamine synthetase, L-methionine sulfoximine, rat brain synaptosomes.

INTRODUCTION

Glutamate is the prevailing neurotransmitter of excitatory signals in the mammalian CNS. It is suggested that under physiological conditions neuronal and glial glutamate uptake effectively limits glutamate spillover, so the most synapses function independently, and only a small amount of glutamate is able to diffuse away to activate neighboring synapses [1]. The low level of ambient glutamate is important for the brain’s spontaneous activity and proper synaptic transmission. Ambient glutamate can interact with post-, pre- and extra- synaptic receptors to modulate transmitter release. The excessive activation of glutamate receptors can cause exitotoxicity and cell death both in vitro and in vivo.

The amino acid, glutamine, serves an important role as intermediary in both the biosynthesis and metabolism of glutamate [2]. The synthesis of glutamine from glutamate is catalyzed by the cytoplasmic enzyme glutamine synthetase. Initially, glutamine synthetase activity was found in glial cells [3]. Later, this enzyme was detected in nerve terminals but the specific activity of glutamine synthetase was several times lower in neurons in comparison with that observed in astrocytes [4, 5]. Released glutamate taken up from the synaptic cleft should be "inactivated" in this way [2]. Also, glutamine synthesis is important for the detoxification of ammonia. Ammonia is a well-known toxic substance for the CNS, especially when its levels exceed the antitoxic capacity of the brain cells. It should be noted that the brain lacks a complete urea cycle [6, 7]. Therefore, the importance of correct function of glutamine synthetase and the glutamine/glutamate cycle during this detoxifying step in the brain is clear. Glutamine has very low affinity for glutamate receptors and does not interfere with synaptic signalling even at the high concentration (0.3 mM), at which it normally occurs in the brain extracellular fluid, as shown by Erecińska et al. [6]. Most studies on glutamate-glutamine interrelationships have used synaptosomes, acutely prepared slices, or dispersed cell cultures in conjunction with biochemical assays [3-9].

Cholesterol is the major sterol component in most mammalian membranes. A characteristic of the cholesterol molecule is its planar structure, which is relatively rigid. When presents in a membrane at a high concentration, cholesterol increases its mechanical strength, while keeping the membrane fluid [10]. Cholesterol is very important for the maintenance of synapse organization, processes of synaptogenesis and synaptic vesicles recycling [11-15]. There is a lot of data that cholesterol depletion influences the basic characteristics of synaptic transmission, such as the distribution of channels and proteins involved in exocytosis [16-23], permeability of voltage-gated calcium and potassium channels [24-30], localization and trafficking/internalization of neurotransmitter receptors [31-33], activity of specific plasma membrane transporters [34].

EXPERIMENTAL PROCEDURES

ISOLATION OF RAT BRAIN SYNAPTOSOMES

Wistar rats (males, age of ~ 2 months, 100–120 g body weight) were maintained in accordance with the European Guidelines and International Laws and Policies. The cerebral hemispheres of decapitated animals were rapidly removed and homogenized in ice-cold 0.32 M sucrose, 5 mM HEPES-NaOH, pH 7.4 and 0.2 mM EDTA. Synaptosomes were prepared by differential and Ficoll-400 density gradient centrifugation of rat brain homogenate according to the method of Cotman [35] with slight modifications. All manipulations were performed at 4°C. The synaptosomal suspensions were used in experiments during 2–4 h after isolation. The standard salt solution was oxygenated with O₂ for 1 hour and contained (in mM): NaCl 126; KCl 5; MgCl₂ 1.4; NaH₂PO₄ 1.0; HEPES 20; pH 7.4 and D-Glucose 10. The Ca²⁺-supplemented medium contained 2 mM CaCl₂. Protein concentration was measured as described by Larson [36].

EXTRACTION OF CHOLESTEROL FROM SYNAPTOSOMES

Treatment with methyl-b-cyclodextrin (MbCD) (30 min, 37^oC) were carried out in standard oxygenated salt solution, then suspension was washed with 10 volumes of ice-cold standard salt solution, sedimented and pellet was resuspended in this solution to a final concentration of 1 mg protein/ml and immediately used for release experiments. Control synaptosomes were simultaneously incubated without MbCD for 30 min at 37^oC, and then also subjected to washing procedure similarly to MbCD experiments. MbCD complexed with cholesterol (15 mM MbCD and 2.3 mM cholesterol) was prepared as described in [37].

UPTAKE EXPERIMENTS

Uptake of L-[¹⁴C]glutamate by synaptosomes was measured as follows: control samples (125 ml of the suspension, 0.2 mg of protein/ml) were pre-incubated in standard salt solution for 10 min at 37°C. Uptake was initiated by the addition of 10 mM L-glutamate supplemented with 420 nM L-[¹⁴C]glutamate (0.1 mCi/ml), incubated for 0–20 min at 37°C and then rapidly sedimented in a microcentrifuge (20 s at 10,000 × g). L-[¹⁴C]glutamate uptake was measured as a decrease in radioactivity of supernatant and an increase in radioactivity of pellet in aliquots of supernatant (100 μl) and pellets by liquid scintillation counting with scintillation cocktail ACS (1.5 ml). Nonspecific binding of the neurotransmitter was evaluated in cool samples sedimented immediately after addition of radiolabeled glutamate.

RELEASE EXPERIMENTS

Control, MbCD- or MbCD/cholesterol- treated synaptosomes were diluted in standard salt solution to 2 mg of protein/ml and after pre-incubation for 10 min at 37°C were loaded with L-[¹⁴C]glutamic acid (1 nmol/mg of protein, 238 mCi/mmol ) in Ca²⁺-supplemented oxygenated standard salt solution for 10 min. After loading, the suspension was washed with 10 volumes of ice-cold oxygenated standard salt solution; pellet was resuspended in this solution to a final concentration of 1 mg protein/ml and immediately used for release experiments. Release of L-[¹⁴C]glutamate from synaptosomes was performed according to following method: samples (125 ml of the suspension, 0.5 mg of protein/ml) were incubated for different time intervals within the range 0–30 min at 37°C and rapidly sedimented in a microcentrifuge (20 s at 10,000 × g). Release was measured in the aliquots of supernatants (100 ml) and pellets by liquid scintillation counting with scintillation cocktail ACS (1.5 ml) and was expressed as percentage of total amount of radiolabeled neurotransmitter incorporated. The extracellular basal level of glutamate in synaptosomes remained unchanged within the range of synaptosomal protein concentration from 0.25 to 2 mg of protein/ml.

Experiments with glutamine synthetase blocker L-methionine sulfoximine (MSO) were carried out using two protocols: synaptosomes were pre-incubated with 1.5 mM MSO for 10 min before L-[¹⁴C]glutamate loading procedure or L-[¹⁴C]glutamate loaded synaptosomal suspension was treated with 1.5 mM MSO for 10 min before release measurements.

STATISTICAL ANALYSIS

Results were expressed as mean ± S.E.M. of n independent experiments. Difference between two groups was compared by two-tailed Student's t-test. Differences were considered significant at Р≤0.05.

MATERIALS

EGTA, HEPES, MbCD, were purchased from Sigma (U.S.A.). Ficoll 400, L-[¹⁴C]glutamate, aqueous counting scintillant (ACS) were from Amersham (UK). DL-TBOA (DL-threo-beta-benzyloxyaspartate) was purchased from Tocris, L-methionine sulfoximine from Fluka. Analytical grade salts were from Reachim (Ukraine).

RESULTS AND DISCUSSION

EFFECTS OF MSO ON GLUTAMATE UPTAKE

The specific activity of the cytoplasmic enzyme glutamine synthetase, which ensures the reaction that transforms glutamate in glutamine, is several times lower in neurons in comparison with that observes in astrocytes [4, 5]. To analyze the contribution of glutamine synthetase to the establishment and maintaining of the level of ambient glutamate, the metabolic glutamine synthetase blocker L-methionine sulfoximine (MSO) was applied to rat brain synaptosomes. Since glutamine synthetase utilizes cytosolic glutamate, it is suggested that the inhibition of this enzyme can influence transporter-mediated L-[¹⁴C]glutamate uptake by nerve terminals. Thus, the first set of the experiments was focused on the effects of MSO on L-[¹⁴C]glutamate uptake. It was demonstrated that MSO decreased L-[¹⁴C]glutamate uptake in a dose-dependent manner. As shown Figure 1, Panel A, 1.5 mM MSO lowered the initial velocity of L-[¹⁴C]glutamate uptake (10 mM) by 4.0 % and 15 mM MSO did so by 7.0 % that was equal to 3 ± 0.04 nmol x min-1 x mg-1 of protein in control, 2.88 ± 0.04 nmol x min^-1 x mg^-1 of protein in the presence of 1.5 mM MSO and 2.79 ± 0.04 nmol x min^-1 x mg^-1 of protein in the presence of 15 mM MSO (Р≤0.05, Student's t-test, n=4). The accumulation of L-[¹⁴C]glutamate (10 mM) for 10 min was also changed in the presence of the inhibitor. 1.5 mM MSO attenuated the accumulation by 7.0 %, whereas 15 mM MSO did so by 20.0 % that was equal to 10.5 ± 0.2 nmol x mg^-1 of protein in control, 9.7 ± 0.2 nmol x mg^-1 of protein in the presence of 1.5 mM MSO and 8.4 ± 0.2 nmol x mg^-1 of protein in the presence of 15 mM MSO (Р≤0.05, Student's t-test, n=4) (Figure 1, Panel B).

FIGURE 1A
The dose-dependent effect of MSO on the initial velocity of L-[¹⁴C]glutamate (10 mM) uptake

Figure 1a, Panel A: The dose-dependent effect of MSO on the initial velocity of L-[14C]glutamate (10 microM) uptake. T Borisova. Neurobiol. Lipids Vol. 9, 1 (2010) http://neurobiologyoflipids.org/content/9/1

Note: you may need to resize your browser window for better view of Figure 1A

FIGURE 1 LEGEND

Figure 1, Panel A, The dose-dependent effect of MSO on the initial velocity of L-[¹⁴C]glutamate (10 mM) uptake. Panel B, The dose-dependent effect of MSO on the accumulation of L-[¹⁴C]glutamate (10 mM) in synaptosomes. Synaptosomes (125 ml of the suspension, 0.2 mg of protein/ml) were pre-incubated with the inhibitor in standard salt solution for 10 min at 37°C. Uptake was initiated by the addition of 10 mM L-glutamate supplemented with 420 nM L-[¹⁴C]glutamate (0.1 mCi/ml), incubated at 37°C for 0–2 min for the measurements of the initial velocity of L-[¹⁴C]glutamate uptake (A) and for 0-10 min for the measurements of the accumulation of L-[¹⁴C]glutamate (B), then rapidly sedimented in a microcentrifuge (20 s at 10,000 × g). L-[¹⁴C]glutamate uptake was measured as a decrease in radioactivity of supernatant and an increase in radioactivity of pellet in aliquots of supernatant (100 ml) and pellets by liquid scintillation counting with scintillation cocktail ACS (1.5 ml). Data are means ± S.E.M. of four independent experiments, each performed in triplicate.

FIGURE 1B
The dose-dependent effect of MSO on the accumulation of L-[¹⁴C]glutamate (10 mM) in synaptosomes

Figure 1b, Panel B: The dose-dependent effect of MSO on the accumulation of L-[14C]glutamate (10 microM) in synaptosomes. T Borisova, Neurobiol. Lipids Vol. 9, 1 (2010) http://neurobiologyoflipids.org/content/9/1

Note: you may need to resize your browser window for better view of Figure 1B

To assay for the changes in ambient L-[¹⁴C]glutamate in cholesterol - deficient nerve terminals, a cholesterol acceptor methyl-b-cyclodextrin (MbCD) was applied. Recently, we have shown that the treatment of synaptosomes with 15 mM MbCD at 37°C for 30 min followed by the washing procedure reduced the synaptosomal cholesterol level by 25.0±3.0 % as compared with intact synaptosomes [38]. It was also shown that cholesterol deficiency significantly attenuated transporter-mediated glutamate uptake [30, 34, 39]. Moreover, we have demonstrated that the extracellular level of L-[¹⁴C]glutamate increased in cholesterol depleted synaptosomes and consisted of 0.193 ± 0.013 nmol/mg protein in control and 0.282 ± 0.013 nmol/mg protein in 15mM MbCD - treated synaptosomes (Р≤0.05, Student's t-test, n=8) [39].

Application of 1.5 mM MSO to synaptosomes preliminary loaded with L-[¹⁴C]glutamate (see Methods) revealed that in the presence of the inhibitor, the extracellular L-[¹⁴C]glutamate level was higher under conditions of cholesterol deficiency as compared to control. Moreover, the treatment with MSO extended the difference in ambient glutamate between control and cholesterol-depleted synaptosomes. Thus, the enhanced level of ambient L-[¹⁴C]glutamate in cholesterol deficiency was not originated from the alteration in the activity of glutamine synthetase. The increased level of extracellular radioactivity in the presence of MSO also showed that namely ambient L-[¹⁴C]glutamate (but not L-[¹⁴C]glutamine) was augmented in MbCD–treated synaptosomes. Above experiments were carried out with MSO, which was applied to synaptosomes preliminary loaded with L-[¹⁴C]glutamate. Studying MSO effects with the other experimental protocol, when 1.5 mM MSO was applied before L-[¹⁴C]glutamate loading procedure (see Methods), we also revealed that the extracelular level of the neurotransmitter was higher after cholesterol depletion than in control. The experiments performed according to both protocols showed the enhanced extracellular glutamate level in the presence of MSO. Thus, the treatment with MSO before and after L-[¹⁴C]glutamate loading did not mask the difference in the level of ambient glutamate between control and cholesterol-depleted synaptosomes. It was suggested that the inhibition of the enzyme activity was not the main cause of enhanced ambient glutamate in cholesterol deficiency.

Taking into account the following facts: (i) the above data on MSO - mediated inhibition of L-[¹⁴C]glutamate uptake; (ii) a decrease in L-[¹⁴C]glutamate uptake under conditions of cholesterol deficiency; it is rational to use the competitive non-transportable inhibitor of glutamate transporters DL-TBOA to eliminate the contribution of transporter-mediated glutamate uptake, when evaluate the ambient L-[¹⁴C]glutamate level in the presence of MSO.

EFFECTS OF DL-TBOA AND MSO ON THE EXTRACELLULAR LEVEL OF GLUTAMATE IN CONTROL AND CHOLESTEROL-DEPLETED SYNAPTOSOMES

In the next series of the experiments, the level of ambient L-[¹⁴C]glutamate was evaluated during the combined application of DL-TBOA and MSO in control and cholesterol-depleted synaptosomes. As shown Figure 2, the extracellular L-[¹⁴C]glutamate level measured in the presence of 200 mM DL-TBOA and 1.5 mM MSO was higher in cholesterol-deficient synaptosomes and was equal to 0.52 ± 0.02 nmol/mg protein in control and 0.62 ± 0.02 nmol/mg protein in MbCD–treated synaptosomes (Р≤0.05, Student's t-test, n=8). Thus, MSO in the presence of DL-TBOA caused a 2.69 - times increase in the level of ambient L-[¹⁴C]glutamate, whereas under the conditions of cholesterol deficiency this value was equal to 2.19-times.

FIGURE 2
The extracellular L-[¹⁴C]glutamate level of control (empty bar) and cholesterol-depleted synaptosomes (shaded bar)

Figure 2: The extracellular L-[14C]glutamate level of control and cholesterol-depleted synaptosomes. T Borisova. Neurobiol. Lipids Vol. 9, 1 (2010) http://neurobiologyoflipids.org/content/9/1

Note: you may need to resize your browser window for better view of Figure 2

FIGURE 2 LEGEND

Figure 2 The extracellular L-[¹⁴C]glutamate level of control (empty bar) and cholesterol-depleted synaptosomes (shaded bar). Synaptosomes were incubated without MbCD (control experiments) or with 15 mM MbCD (extraction of cholesterol) at 37°C during 30 min followed by washing. Control and MbCD-treated synaptosomes were loaded with L-[¹⁴C]glutamic acid (1 nmol/mg of protein, 238 mCi/mmol) in Ca²⁺-supplemented oxygenated standard salt solution. After loading, the extracellular level of L-[¹⁴C]glutamate was measured according to following method: samples (125 ml of the suspension, 0.5 mg of protein/ml) were preincubated for 10 min at 37°C with MSO, then DL-TBOA was added and the preparations were incubated further for 6 min at 37°C and rapidly sedimented in a microcentrifuge. L-[¹⁴C]glutamate radioactivity in the supernatants and pellets was determined as described in Materials and Methods. Total synaptosomal L-[¹⁴C]glutamate content was equal to 200000±15000 cpm/mg protein. Data are means ± S.E.M. of eight independent experiments, each performed in triplicate. Data are compared by Student`s t-test. *, Р≤0.05 as compared to control synaptosomes.

It should be noted that we revealed only insignificant changes in extracellular L-[¹⁴C]glutamate in synaptosomes treated with 15mM MbCD complexed with cholesterol (2.3 mM) (followed by the washing of the complex) as compared to untreated control. Thus, it was expected that an increase in the ambient glutamate level in MbCD-treated synaptosomes was a result of depletion of membrane cholesterol, but not the effect of MbCD per se irrespective to cholesterol accepting capacity.

The uncertainty in glutamine synthetase experiments was the possible contamination of synaptosomal fraction obtained by density Ficoll gradient centrifugation by astrocytes or gliosomes up to ~5-10 % [40, 41, 42, 43, 44]. However, we suggested that this contamination was of no consequence and could not be taken into consideration in the measurements of the extracellular level and uptake of glutamate in synaptosomes. Indeed, in our experiments, cholesterol depletion caused a considerable decrease in the activity of glutamate transporters, whereas Tsai et al. [45] demonstrated an increase in transporter-mediated glutamate uptake in astrocytes. In this context, if glial fragments significantly contributed to glutamate uptake in our experiments, the latter should be higher after cholesterol depletion, but this did not correspond to our experimental data, where we showed a significant decrease in uptake under tested conditions. In contrast to uptake and release assay, glial fragment contamination may merit consideration in glutamine synthetase experiments. It should be kept in mind that glutamine synthetase activity was several times higher in astrocytes than in neurons [4, 5], thereby contributing inaccuracy to the measurements in synaptosomes. One question remained unanswered in this set of the experiments: whether MSO-evoked alterations in the extracellular glutamate level of cholesterol-deficient synaptosomes were caused by inhibition of glutamine synthetase activity of nerve terminals and/or glial fragments. Our results showed that the application of MSO caused a dose-dependent decrease in synaptosomal glutamate uptake (Figure 1). Whereas in glial cells, the effect of the inhibitor on the intracellular glutamate concentration was opposite to that found in synaptosomes [46, 47]. Thus, we suggested that above glutamine synthetase experiments could be considered as adequate, thereby reflecting processes occurred in nerve terminals, but not in glial fragment.

A decrease in the ability of MSO to augment the level of ambient L-[¹⁴C]glutamate after cholesterol depletion showed that the activity of glutamine synthetase might be suppressed under the conditions of cholesterol deficiency. This fact is in accordance with the data of Tsai et al. [45] on the reduction of the activity of glutamine synthetase in cultured astrocytes after cholesterol reduction. The inhibition of glutamine synthetase activity in cholesterol deficiency may have harmful physiological consequences, because if ammonia is not sufficiently detoxified, its concentration increase pathologically, neuron and astrocyte functions deteriorate, resulting in damage and cell death. It has been shown clearly that acute ammonia toxicity and liver failure lead to excitotoxicity as a result of activation of N-methyl-D-aspartate (NMDA) receptors in the brain [48].

Using MSO and DL-TBOA, we have demonstrated that the inhibition of glutamine synthetase activity increased the level of ambient L-[¹⁴C]glutamate in control and cholesterol-deficient synaptosomes. Also, the treatment with MSO and DL-TBOA did not eliminate the difference in ambient glutamate between control and cholesterol-deficient synaptosomes, which was maintained at a higher level under conditions of cholesterol deficiency than in control, and thus, changing enzyme activity was not the main cause of an increase in extracellular glutamate in cholesterol deficiency.

REFERENCES
Please note: web enhanced references below provide no registration free access to documents

1. Vizi E.S. Role of high-affinity receptors and membrane transporters in nonsynaptic communication and drug action in the central nervous system. Pharmacol. Rev. 2000; 52: 63-89 [ PubMed ][ Back2Text ].

2. Fonnum F. Glutamate: a neurotransmitter in mammalian brain. J. Neurochem. 1984; 42: 1–11 [ PubMed ][ Back2Text ].

3. Martinez-Hernandez A, Bell KP, Norenberg MD. Glutamine synthetase: glial localization in brain. Science. 1977; 195 (4284):1356–1358 [ PubMed ][ Back2Text ].

4. Patel A. J., Hunt A., Tahourdin C. S. M. Regulation of in vivo glutamine synthetase activity by glucocorticoids in the developing rat brain. Dev. Brain Res. 1983; 10: 83-91 [ PubMed ][ Back2Text ].

5. Tansey F.A., Farooq M., Cammer W. Glutamine Synthetase in Oligodendrocytes and Astrocytes: New Biochemical and Immunocytochemical Evidence. J. Neurochem. 1991; 56: 266-272 [ PubMed ][ Back2Text ].

6. Erecińska M., Silver IA. Metabolism and role of glutamate in mammalian brain. Prog Neurobiol. 1990; 35(4): 245-296 [ PubMed ][ Back2Text ].

7. Acosta G., Fernández M., Roselló D., Tomaro M., Balestrasse K., Lemberg A. Glutamine synthetase activity and glutamate uptake in hippocampus and frontal cortex in portal hypertensive rats. World J Gastroenterol 2009; 15(23): 2893-2899 [ PubMed ][ Back2Text ].

8. Laake J.H., Slyngstad T.A., Haug F. M. S., Ottersen O.P. Glutamine from Glial Cells Is Essential for the Maintenance of the Nerve Terminal Pool of Glutamate: Immunogold Evidence from Hippocampal Slice Cultures. J. Neurochem. 1995; 65: 871-881 [ PubMed ][ Back2Text ].

9. Zou J., Wanga Y-X.,Dou F-F., Lu H-Z., Maa Z-W., Lu P-H., Xu X-M. Glutamine synthetase down-regulation reduces astrocyte protection against glutamate excitotoxicity to neurons. Neurochemistry Int. doi:10.1016/j.neuint.2009.12.021 2010 Mar; 56(4): 577-84 Epub 2010 Jan 12 [ PubMed ][ Back2Text ].

10. Jadot M., Andrianaivo F., Dubois F., Wattiaux R. Effects of methylcyclodextrin on lysosomes. Eur J Biochem 2001; 268: 1392-1399 [ PubMed ][ Back2Text ].

11. Koudinov AR, Koudinova NV. Cholesterol homeostasis failure as a unifying cause of synaptic degeneration. J Neurol Sci 2005; 15:233-40 [ PubMed ][ Back2Text ].

12. Pfrieger FW. Cholesterol homeostasis and function in neurons of the central nervous system. Cell Mol Life Sci 2003; 60: 1158-1171 [ PubMed ][ Back2Text ].

13. Salaün C., James D.J., Chamberlain L.H.. Lipid rafts and the regulation of exocytosis. Traffic 2004; 5: 1-10 [ PubMed ][ Back2Text ].

14. Wasser C.R., Ertunc M., Liu X., Kavalali E.T. Cholesterol-dependent balance between evoked and spontaneous vesicle recycling. J.Physiol. 2007; 579(2): 413-429 [ PubMed ][ Back2Text ].

15. Lang T., Bruns D., Wenzel D., Riedel D., Holroyd P., Thiele C., Jahn R. SNAREs are concentrated in cholesterol-dependent clusters that define docking and fusion sites for exocytosis. EMBO J. 2001; 20: 2202-2213 [ PubMed ][ Back2Text ].

16. Lange Y, Ye J, Rigney M, Steck TL. Regulation of endoplasmic reticulum cholesterol by plasma membrane cholesterol. J Lipid Res 1999; 40: 2264-2269 [ PubMed ][ Back2Text ].

17. Chattopadhyay A, Paila YD. Lipid-protein interactions, regulation and dysfunction of brain cholesterol. Biochem Biophys Res Commun 2007; 354: 627-633 [ PubMed ][ Back2Text ].

18. Churchward MA, Rogasevskaia T, Hofgen J, Bau J, Coorseen JR. Cholesterol facilitates the native mechanism of Ca²⁺-triggerted membrane fusion. J Cell Sci 2005;118: 4833-4848 [ PubMed ][ Back2Text ].

19. Zamir O, Charlton MP. Cholesterol and synaptic transmitter release at crayfish neuromuscular junctions. J Physiol 2006; 571: 83-99 [ PubMed ][ Back2Text ].

20. Cho WJ, Jeremic A, Jin H, Ren G, Jena BP. Neuronal fusion pore assembly requires membrane cholesterol. Cell Biol Int 2007; 31: 1301-1308 [ PubMed ][ Back2Text ].

21. Dietschy JM, Turley SD. Cholesterol metabolism in the brain. Curr Opin Lipidol 2001;12:105-112 [ PubMed ][ Back2Text ].

22. Gil C, Soler-Jover A, Blasi J. Aguilera synaptic proteins and SNARE complexes are localized in lipid rafts from brain synaptosomes. Biochem Biophys Res Commun 2005; 329: 117-124 [ PubMed ][ Back2Text ].

23. Launikonis BS, Stephenson DG. Effects of membrane cholesterol manipulation on excitation-contraction coupling in skeletal muscle of the toad. J Physiol. 2001; 534: 71-85 [ PubMed ][ Back2Text ].

24. Hajdú P, Varga Z, Pieri C, Panyi G, Gáspár R Jr. Cholesterol modifies the gating of Kv1.3 in human T lymphocytes. Pflugers Arch 2003; 445: 674-682 [ PubMed ][ Back2Text ].

25. Hill W, An B, Johnson J. Endogenously expressed epithelial sodium channel is present in lipid rafts in A6 cells. J Biol Chem 2002; 277: 33541-33544 [ PubMed ][ Back2Text ].

26. Taverna E, Saba E, Rowe J, Francolini M, Clementi F, Rosa P. Role of lipid microdomains in P/Q-type calcium channel (Cav2.1) clustering and function in presynaptic membranes. J Biol Chem 2004; 279: 5127-5134 [ PubMed ][ Back2Text ].

27. Murtazia R, Kovbasnjuk O, Donowitz M. X. Li: Na⁺/H⁺ exchanger NHE3 3 activity and trafficking are lipid raft-dependent. J Biol Chem 2006;281: 17845-17855 [ PubMed ][ Back2Text ].

28. Kato N, Nakanishi M, Hirashima N. Cholesterol depletion inhibits store-operated calcium currents and exocytotic membrane fusion in RBL-2H3 cells. Biochemistry 2003; 42: 11808-11814 [ PubMed ][ Back2Text ].

29. Martens JR, O`Connell K, Tamkun M. Targeting of ion channels to membrane microdomains: localization of Kv channels to lipid rafts. Trends in Pharmacological Sciences 2004; 25: 16-21 [ PubMed ][ Back2Text ].

30. Jennings LJ, Xu QW, Firth TA, Nelson MT, Mawe GM. Cholesterol inhibits spontaneous action potentials and calcium currents in guinea pig gallbladder smooth muscle. Am J Physiol 1999; 277: 1017-1026 [ PubMed ][ Back2Text ].

31. Barrantes FJ. Lipid matters: nicotinic acetylcholine receptor-lipid interactions (Review). Mol Membr Biol 2002; 19: 277-284 [ PubMed ][ Back2Text ].

32. Burger K, Gimpl G, Fahrenholz F. Regulation of receptor function by cholesterol. Cell Mol Life Sci 2000; 57: 1577-1592 [ PubMed ][ Back2Text ].

33. Eroglu C, Brugger B, Wieland F, Sinning I. Glutamate-binding affinity of Drosophila metabotropic glutamate receptor is modulated by association with lipid rafts. Proc Natl Acad Sci USA 2003; 100: 10219-10224 [ PubMed ][ Back2Text ].

34. Butchbach M, Tian G, Guo H, Lin C G. Association of excitatory amino acid transporters,especially EAAT2,with cholesterol-rich lipid raft microdomains. J Biol Chem 2004; 279: 34388-34396 [ PubMed ][ Back2Text ].

35. Cotman CW. Isolation of synaptosomal and synaptic plasma membrane fractions. Methods Enzymol 1974; 31: 445-452 [ PubMed ][ Back2Text ].

36. Larson E, Howlett B, Jagendorf A. Artificial reductant enhancement of the Lowry method for protein determination. Anal Biochemistry 1986; 155: 243-248 [ PubMed ][ Back2Text ].

37. Klein U., Gimpl G., Fahrenholz F. Alteration of the myometrial plasma cholesterol content with beta-cyclodextrin modulates the binding affinity of the oxytocin receptor. Biochemistry 1995; 34: 13784-13793 [ PubMed ][ Back2Text ].

38. Sivko R., Krisanova N., Borisova T. Reduced cholesterol content and the effects of inhibitors on Na⁺-dependent glutamate transport in rat brain nerve terminals. Neurobiol. Lipids 2009; 8: http://neurobiologyoflipids.org/content/8/2/ [ FullText ][ Back2Text ].

39. Borisova T., Krisanova N., Sivko R., Borysov A. Cholesterol depletion attenuates tonic release but increases the ambient level of glutamate in rat brain synaptosomes. Neurochemistry International, 2010; 56(3): 466-478. Epub 2009 Dec 16, doi:10.1016/j.neuint.2009.12.006 [ PubMed ][ Back2Text ].

40. Cotman C. W., Matthews D.A. Synaptic plasma membranes from rat brain synaprtosomes: isolation and partial characterization. Biochim. Biophys. Acta 1971; 249: 380-394 [ PubMed ][ Back2Text ].

41. Gorracci G., Blomstrand C., Arienti G., Hamberger A., Porcellati G. Base-exchange enzymic system for the synthesis of phospholipids in neuronal and glial cells and their subfractions: a possible marker for neuronal membranes. J. Neurochem. 1973; 20: 1167-1180 [ PubMed ][ Back2Text ].

42. Henn F.A., Anderson D.J., Rustad D.G. Glial contamination of synaptosomal fractions. Brain Res. 1976; 101: 341–344 [ PubMed ][ Back2Text ].

43. Subbalakshmi G. Y. C. V., Murthy Ch. R. K. Isolation of astrocytes, neurons, and synaptosomes of rat brain cortex. Neurochem. Res. 1985; 10: 239-250 [ PubMed ][ Back2Text ].

44. Crispino M., Capano C.P., Aiello A., Iannetti E., Cupello A., Giuditta A. Messenger RNAs in synaptosomal fraction from rat brain. Brain Res. Mol. Brain Res. 2001; 97(2): 171-176 [ PubMed ][ Back2Text ].

45. Tsai H.I., Tsai L.H., Chen M.Y., Chou Y.C. Cholesterol deficiency perturbs actin signaling and glutamate homeostasis in hippocampal astrocytes. Brain Res. 2006; 1104: 27-38 [ PubMed ][ Back2Text ].

46. Ohnishi M., Watanabe Y., Shibuya T. Potentiation of excitotoxicity by glutamate uptake inhibitor rather than glutamine synthetase inhibitor. Jpn. J. Pharmacol. 1995; 68: 315-321 [ PubMed ][ Back2Text ].

47. Makani S., Zagha E. Out of the cleft: the source a target of extra-synaptic glutamate in the CA1 region the hippocampus. J.Physiol. 2007; 582(2): 479–480 [ PubMed ][ Back2Text ].

48. Rodrigo R., Cauli O., Boix J., ElMlili N., Agusti A., Felipo V. Role of NMDA receptors in acute liver failure and ammonia toxicity: Therapeutical implications. Neurochemistry Int. 2009; 55 (1-3): 113-118 [ PubMed ][ Back2Text ].


CLICK HERE OR PRESS <CTRL><D> TO BOOKMARK THIS ARTICLE

This article should be cited in the following way:

Borisova T. Glutamine synthetase activity and ambient glutamate in cholesterol-deficient rat brain nerve terminals. Neurobiol. Lipids Vol. 9, 1 (2010), Published online March 18, 2010, Available at: http://neurobiologyoflipids.org/content/9/1/

Please note: Because Neurobiology of Lipids is published online only, the articles are identified with an article number rather than with traditional (printed) page numbers. Adobe Acrobat (.PDF) reprint, however, allows citation with page numbers, and serves the algorithmic needs of tenure committees to count published print pages.


HOME	ABOUT	FEEDBACK	RECENT CONTENT	SUBJECT COLLECTIONS	PUBLISH WITH US	HELP MENU