Neurobiology of Lipids (ISSN 1683-5506) Vol.8 article 2 -- R Sivko, N Krisanova, T Borisova


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Neurobiol Lipids 8, 2 (28 February 2009)
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REDUCED CHOLESTEROL CONTENT AND THE EFFECTS OF INHIBITORS ON Na⁺-DEPENDENT GLUTAMATE TRANSPORT IN RAT BRAIN NERVE TERMINALS

Roman Sivko, Natalia Krisanova, 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: Accepted for Publication: Published online: 28 February, 2009 | Article readership
Copyright © 2009 R Sivko and colleagues, Licensee Neurobiology of Lipids

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ABSTRACT

INTRODUCTION

MATERIALS & METHODS

RESULTS & DISCUSSION

FIGURE 1 & FIGURE 2

FIGURE 3 & FIGURE 4

FIGURE 5 & FIGURE 6

REFERENCES

ABSTRACT

The influence of the inhibitors DL-threo-b-benzyloxyaspartate (DL-TBOA) and DL-threo-b-hydroxyaspartate (DL-THA) on Na⁺-dependent glutamate transport was investigated in synaptosomes losing one quarter of membrane cholesterol after half an hour treatment by 15 mM methyl-b-cyclodextrin (MbCD). Despite significant decrease in the initial velocity of glutamate uptake (49 ± 4% at 100 mM L-[¹⁴C]glutamate), cholesterol-depleted synaptosomes retained the ability to accumulate and keep the neurotransmitter inside during loading with L-[¹⁴C]glutamate reaching concentration of 1.2 ± 0,1 nmol/mg of protein in control and 1.1±0,1 nmol/mg of protein in treated synaptosomes. After cholesterol extraction, stimulated by depolarization transporter-mediated release of preloaded L-[¹⁴C]glutamate from synaptosomes became more sensitive to DL-TBOA (100 mM), which inhibited release by 40 ± 4 % of total in control and 55 ± 4 % after application of MbCD. L-[¹⁴C]glutamate uptake by treated synaptosomes demonstrated similar sensitivity with controls to DL-TBOA and DL-THA. We suggested that an increase in the inhibitory effects of DL-TBOA on transporter-mediated glutamate release uncovered a decrease in the activity of reverse transporters in cholesterol-depleted synaptosomes, which might remain unnoticeable in the absence of the inhibitor.

Key words: cholesterol, methyl-beta-cyclodextrin, L-glutamate, Na⁺-dependent transporters, uptake, transporter-mediated release, rat brain synaptosomes.

INTRODUCTION

Cholesterol is an essential constituent and the main sterol component in most mammalian membranes, playing a profound role in membrane-associated processes and cellular functions [1-11]. It is very important that most of membrane proteins, including receptors, pumps, ion channels and transporters need proper level of membrane cholesterol for normal functioning and are affected by altering cholesterol content [12-23]. It also relates to high affinity Na⁺-dependent glutamate transporters that are plasma membrane proteins with 8 putative transmembrane domains. These proteins accomplish glutamate uptake, which is the mechanism responsible for the maintenance of low extracellular concentration of glutamate [24-29]. Glutamate transporters utilize the Na⁺/K⁺ electrochemical gradient as the driving force for uptake. Maintaining glutamate at low extracellular concentration is necessary to protect neurons from excitotoxic injury, abnormal glutamatergic synaptic physiology, impaired behavioral conditioning and learning. Glutamate transporters provide not only uptake but can also contribute to glutamate release, which is so-called transporter-mediated release. Conditions of ATP depletion, elevated extracellular K⁺ and reduction of extracellular Na⁺ thermodynamically favor reversal of uptake. Transporter reversal is the dominant mechanism of glutamate release in cerebral ischemia and hypoxia [24-29].

Despite the great importance of cholesterol for nerve cell functioning and extensive research on this subject, the exact roles and mechanisms of cholesterol action in neuronal context still remain unclear. The main aim of the present study was to assess whether depletion of membrane cholesterol changed the effects of glutamate transporter inhibitors on Na⁺-dependent release and uptake of glutamate in isolated nerve terminals.

MATERIALS AND METHODS

ISOLATION OF RAT BRAIN SYNAPTOSOMES

Young male adult Wistar rats (appx. 3 month old, 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 [30] with slight modifications, namely the standard salt solution contained (in mM): NaCl 126; KCl 5; MgCl₂ 1.4; NaH₂PO₄ 1.0; HEPES 20; pH 7.4 and d-glucose10. The Ca²⁺-supplemented medium contained 2 mM CaCl₂. The Ca²⁺-free medium contained 1 mM EGTA and no added Ca²⁺. All manipulations were performed at 4°C. The synaptosomal suspensions were used in experiments during 2–4 h after isolation. Protein concentration was measured as described by Larson [31].

RELEASE EXPERIMENTS

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 (500 nM, 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 μl of the suspension, 0.5 mg of protein/mL) were incubated for 0–3 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 μl) by liquid scintillation counting with scintillation cocktail ACS (1.5 mL) and was expressed as percentage of total amount of radiolabeled neurotransmitter incorporated. Release of the neurotransmitter from synaptosomes incubated without stimulating agents was used for assay of basal release. Stimulated release of neurotransmitter was calculated by subtracting the basal value from the value of total release.

Results were expressed as mean ± S.E.M. values. Statistical analysis used two-tailed Student's t-test. Differences were considered significant when Р≤0.05.

UPTAKE EXPERIMENTS

Uptake of L-[¹⁴C]glutamate by synaptosomes was measured as follows: samples (125 μl 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 μM L-glutamate supplemented with 420 nM L-[¹⁴C]glutamate (0.1 μCi/mL), incubated for 0–20 min at 37°C and then rapidly sedimented in a microcentrifuge (20 s at 10,000×g). Uptake was measured 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 cooling samples sedimented immediately after addition of radiolabeled glutamate. Results were expressed as mean ± S.E.M. values. Statistical analysis used two-tailed Student's t-test. Differences were considered significant when Р≤0.05.

CHOLESTEROL EXTRACTION, RELOADING AND DETERMINATION

Synaptosomes were suspended in oxygenated ice-cold standard salt solution, incubated without or with methyl-b-cyclodextrin (MbCD) in different concentrations at 37°С for 35 min. After MbCD treatment the suspension was washed with 10 volumes of ice-cold standard salt solution and then centrifuged. The supernatant was completely removed and synaptosomal pellets were resuspended in the appropriate buffer to obtain protein concentration of 2 mg of protein/ml. To determine whether the MbCD induced effects were a result of cholesterol depletion, synaptosomes were also treated with 15mM MbCD complexed with 2.3 mM cholesterol. Cholesterol content was determined using method described by Findlay and Evans [32] in aliquots of the untreated (control), MbCD-treated and MbCD/cholesterol complex –treated samples.

MATERIALS

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

RESULTS AND DISCUSSION

MODULATION OF MEMBRANE CHOELSTEROL CONTENT BY METHYL-b-CYCLODEXTRIN

Cholesterol depletion of brain synaptosomes, plasma membrane vesicles, rat primary cortical cultures, cell lines is routinely used to evaluate the physiological role of cholesterol in neuronal function. In this way, common methodological approach is treatment with cyclodextrins, which are a family of cyclic oligosaccharides composed of a lipophilic cavity and hydrophilic outer surface. Methyl-b-cyclodextrin (MbCD) is known as an effective cholesterol-depleting agent that contains seven a-(1,4) linked glycosyl - units [33-38]. We efficiently modulated cholesterol content of rat brain synaptosomes using MbCD and MbCD-cholesterol complex (2.3 mM cholesterol in 15 mM MbCD) as control. It was demonstrated that treatment of synaptosomes with 5 mM MbCD at 37⁰C for 35 min followed by washing of synaptosomes from MbCD reduced cholesterol level by 7.0±3.0 %, whereas 15; 30; 60 mM MbCD did so by 25.0±3.0 %; 45±4.0 %; 73.0±4.0%, respectively, as compared with untreated control synaptosomes (Р≤0.05, n=4) (Fig.1, first-fifth columns). Synaptosomal cholesterol content was not significantly altered after application of 15 mM MbCD complexed with 2.3 mM cholesterol at 37°C for 35 min (Fig.1, last columns). It was concluded that treatment of synaptosomes with MbCD considerably reduced cholesterol level in a dose-dependent manner, whereas application of cholesterol-MbCD complex did not significantly affect cholesterol content.

FIGURE 1
Treatment of synaptosomes by MbCD resulted in a reduction of cholesterol level. Influence of MbCD is dose-dependent. *, Р≤0.05 as compared to control

Figure 1: Treatment of synaptosomes by MCD resulted in a reduction of cholesterol level. Influence of MbCD is dose-dependent. *, Р≤0.05 as compared to control - 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 Vol. 8, 2 (2009)

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THE EFFECTS OF DL-THREO-b-BENZYLOXYASPARTATE AND DL-THREO-b-HYDROXYASPARTATE ON Na⁺-DEPENDENT GLUTAMATE TRANSPORT IN CHOLESTEROL-DEPLETED SYNAPTOSOMES

Depletion of membrane cholesterol resulted in a significant dose-dependent reduction in L-[¹⁴C] glutamate uptake by rat brain synaptosomes. Treatment of synaptosomes with 15mM MbCD at 37^oC for 35 min followed by washing of synaptosomes from MbCD caused a decrease in the initial velocity of uptake of 10 mM L-[¹⁴C]glutamate and 100 mM L-[¹⁴C]glutamate by 42 ± 4 % and 49 ± 4 %, respectively, as compared to controls (Р≤0.05, n=8). Lowering concentration of MbCD up to 5 mM resulted in an attenuation of the initial velocity of synaptosomal uptake of 10 mM L-[¹⁴C]glutamate and 100 mM L-[¹⁴C]glutamate by 19 ± 4 % and 20 ± 4 % (Р≤0.05, n=8) (Fig.2). Synaptosomes were also treated with 15 mM MbCD complexed with cholesterol (2.3 mM) to determine whether the observed MbCD-induced effects were a result of cholesterol extraction. Application of MbCD-cholesterol complex caused insignificant changes in L-[¹⁴C]glutamate uptake by synaptosomes as compared to controls showing that decreased uptake we have registered after MbCD treatment was associated with depletion of membrane cholesterol, but not with effect of MbCD per se irrespective to cholesterol accepting capacity (Fig. 2).

FIGURE 2
100mM L-[¹⁴C]glutamate uptake by isolated nerve terminals after extraction of membrane cholesterol by MbCD and cholesterol-MbCD complex (shaded bars). *, Р≤0.05 as compared to control

Figure 2: 100uM L-[14C]glutamate uptake by isolated nerve terminals after extraction of membrane cholesterol by MbCD and cholesterol-MbCD complex (shaded bars). *, Р≤0.05 as compared to control - 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 Vol. 8, 2 (2009)

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As it was mentioned in the introduction section, glutamate transporters provided not only uptake but could also contribute to non-vesicular glutamate release, which occurred from cytosolic pool of the neurotransmitter. Figure 3 shows glutamate release from nerve terminals mediated by glutamate transporter reversal during depolarization of the plasma membrane by 35 mM KCl.

FIGURE 3
Depolarization-evoked release of cytosolic glutamate from synaptosomes

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In the next set of experiments we analyzed transporter-mediated release of the neuromediator in control and 15 mM MbCD-treated synaptosomes using methodological protocol of preliminary loading of radiolabeled L-[¹⁴C]glutamate into synaptosomes. Taking into account profound effects of cholesterol depletion on glutamate uptake it seems reasonable to compare L-[¹⁴C]glutamate loading procedures in control and cholesterol-depleted synaptosomes. We have shown that intracellular L-[¹⁴C]glutamate accumulated inside of synaptosomes for 10 min did not change as a result of cholesterol depletion and consisted of 1.2 ± 0.1 nmol/mg of protein in control and 1.1± 0.1 nmol/mg of protein in cholesterol-depleted synaptosomes. Thus, treated synaptosomes were shown to retain similar ability in L-[¹⁴C]glutamate keeping and storage despite a drastic decrease in the activity of glutamate uptake. Figure 4 shows a similar amount of L-[¹⁴C]glutamate inside of control and MbCD-treated synaptosomes. The estimated parameter made synaptosomal preparation appropriate for further investigation of L-[¹⁴C]glutamate release process.

FIGURE 4
Loading of control and 15 mM MbCD–treated synaptosomes with L-[¹⁴C]glutamate, which included 10 min incubation with the neuromediator followed by the washing procedure

Figure 4: Loading of control and 15 mM MCD–treated synaptosomes with L-[14C]glutamate, which included 10 min incubation with the neuromediator followed by the washing procedure - 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 Vol. 8, 2 (2009)

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The effects of cholesterol depletion on high KCl-stimulated release of preloaded L-[¹⁴C]glutamate from synaptosomes was investigated in Ca²⁺-free medium. The value of this Ca²⁺-independent L-[¹⁴C]glutamate release for 6 min was insignificantly decreased after MbCD treatment and consisted of 14.7±2.0 % of total label in control and 14.1±2.0 % of total label in MbCD–treated synaptosomes. We examined the effects of the nonransportable inhibitor DL-threo-b-benzyloxyaspartate (DL-TBOA) on depolarization-stimulated Ca²⁺-independent L-[¹⁴C]glutamate release and have found that DL-TBOA inhibited L-[¹⁴C]glutamate release both in control and after MbCD treatment. The inhibitory effects of 100 mM DL-TBOA on L-[¹⁴C]glutamate release was significantly higher after cholesterol depletion and consisted of 40 ± 4 % of total label in control and 56 ± 4 % of total label in treated synaptosomes (Figure 5). One of the possible causes that lead to an increase in the inhibitory effect of DL-TBOA after depletion of membrane cholesterol may be alterations in the affinity of transporters to the inhibitor. To clarify this suggestion, the study assessed DL-TBOA and also the competitive transported inhibitor of glutamate transporters DL-threo-b-hydroxyaspartate (DL-THA) for abilities to affect L-[¹⁴C]glutamate uptake process. Recently, we have determined IC₅₀ values for DL-TBOA and DL-THA calculated on the basis of curves of non-linear regression kinetic analysis [39, 40]. In this study the data on L-[¹⁴C]glutamate uptake (10 mM) showed that inhibition caused by 10 mM DL-TBOA or 10 mM DL-THA in control synaptosomes and after application of 15 mM MbCD related to untreated and treated controls, respectively, were not significantly different and consisted of 54 ± 4% and 56 ± 4%, respectively (Figure 6). Thus, before and after cholesterol extraction glutamate transporters demonstrated similar sensitivity to the competitive non-transported and transported inhibitors DL-TBOA and DL-THA.

FIGURE 5
Inhibition of Ca²⁺-independent glutamate release by DL-TBOA after extraction of membrane cholesterol with 15 mM MbCD. *, Р≤0.05 as compared to control

Figure 5: Inhibition of Ca2+-independent glutamate release by DL-TBOA after extraction of membrane cholesterol with 15 mM MbCD. *, Р≤0.05 as compared to control - 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 Vol. 8, 2 (2009)

FIGURE 6
Inhibition of L-[¹⁴C]glutamate uptake by DL-ТВОА and DL-ТНА in untreated synaptosomes and after application of MbCD. *, Р≤0.05 as compared to control

Figure 6: Inhibition of L-[14C]glutamate uptake by DL-ТВОА and DL-ТНА in untreated synaptosomes and after application of MbCD. *, Р≤0.05 as compared to control - 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 Vol. 8, 2 (2009)

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It should be noted that according to the methodological approach we used an increase in the extracellular L-[¹⁴C]glutamate level in response to depolarization of the plasma membrane as a measure of Ca²⁺-independent release. This extracellular glutamate may be simultaneously utilized by glutamate transporters by means of uptake, thereby lowering release value. However, we preliminary demonstrated that depolarization of the plasma membrane by 35 mM KCl decreased a half of glutamate uptake. Also, the presence of 100 mM DL-TBOA and 100 mM DL-THA in the incubation media attenuated uptake by 78 ± 4% in control and 80 ± 5% after cholesterol depletion, showing that contribution of uptake into an increase in extracellular glutamate may be neglected when study release process. Taking into account the above-mentioned data we suggested that an increase in the inhibitory effects of DL-TBOA on transporter-mediated glutamate release evoked by depolarization of the plasma membrane in cholesterol-depleted synaptosomes unmasked a decrease in the activity of reverse transporters under these conditions, which remained covered when studied directly release of glutamate in the absence of the inhibitor.

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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 Vol. 8, 2 (2009), Published online February 28, 2009, Available at: http://neurobiologyoflipids.org/content/8/2/

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