Combined Application of Glutamate Transporter Inhibitors and Hypothermia Discriminates Principal Constituent Processes Involved in Glutamate Homo- and Heteroexchange in Brain Nerve Terminals

Abstract

Deep and profound hypothermia is successfully practiced in the prevention of ischemic stroke consequences and aortic arch cardiac surgery accompanied by reduction of cerebral circulation. Hypothermia is a current neuroprotection standard in hypoxic/ischemic encephalopathy. Drug–hypothermia administration is proposed as a new approach in pharmacotherapy for neonatal seizures. Also, hypothermia is useful as neuroprotective approach in long-term interplanetary space missions. We recently revealed gradual dynamics of hypothermia-induced decrease in transporter-mediated release and uptake of L-[¹⁴C]glutamate in presynaptic rat brain nerve terminals (synaptosomes), thereby confirming potent unspecific neuroprotective effect of hypothermia. Glutamate homo- and heteroexchange are significant mechanisms involved in the maintenance of the extracellular glutamate level in nerve terminals. We have analyzed whether glutamate homo- and heteroexchange in nerve terminals is temperature sensitive. In this study we showed that synaptosomal glutamate-induced L-[¹⁴C]glutamate release (homoexchange) and D-aspartate- and DL-threo-β-hydroxyaspartate-induced L-[¹⁴C]glutamate release (heteroexchange) gradually decreased from deep (27°C) to profound (17°C) hypothermia with dynamics similar to that of glutamate transporter reversal. Interestingly, ambient L-[¹⁴C]glutamate concentration in the nerve terminal preparations remained unaltered during hypothermia administration. Therefore, we demonstrated that glutamate homo- and heteroexchange decreased from deep to profound hypothermia thereby preventing further elevation of extracellular glutamate. Hypothermia uncovered the principal processes contributing to glutamate homo- and heteroexchange in nerve terminals and the maintenance of definite ambient glutamate concentration. Additionally, we showed that glutamate transporter reversal can be nonpathological and occurs under physiological conditions at least as a part of homo- and heteroexchange mechanisms.

Introduction

Glutamate is a principal amino acid excitatory neurotransmitter in the mammalian central nervous system implicated in ordinary brain functioning, for example, cognition, memorizing, and learning. Abnormalities of glutamate transport and extracellular homeostasis are associated with neuropathologies and are common features of the pathogenesis of major diseases, for example, Alzheimer's and Parkinson's disease, schizophrenia, epilepsy, and also brain trauma.

Fusion of the synaptic vesicles with the plasma membrane during exocytosis and glutamate release from presynaptic nerve terminals results in a temporal increase in the extracellular glutamate concentrations in the synaptic cleft and receptor-mediated activation of signaling pathways. Termination of glutamatergic neurotransmission, prevention receptor overstimulation and development of excitotoxicity occurs through glutamate removal from the synaptic cleft by high-affinity Na⁺-dependent glutamate transporters, the integral membrane proteins. The latest are able to perform not only uptake of extracellular glutamate but also its heteroexchange, that is, exchange of external and internal substrates with the ratio 1:1 (Pines and Kanner, 1990; Volterra et al., 1996; Zerangue and Kavanaugh, 1996; Zhou et al., 2014).

Proper balance of oppositely directed processes (transporter-mediated uptake vs. transporter reversal and tonic (unstimulated) release of glutamate) maintains definite extracellular glutamate concentration between exocytotic events and is a critical factor for synaptic transmission. Studying glutamate/L-[¹⁴C]glutamate exchange in rat brain nerve terminals, we revealed permanent extracellular/intracellular glutamate turnover (Borisova, 2016; Borisova et al., 2016). However, contribution of glutamate homo- and heteroexchange to the establishment of the definite extracellular glutamate level in nerve terminals was not exactly clear.

Estimates of the extracellular glutamate concentrations ranged over three orders of magnitude from 0.02 μM up to 20 μM and depended on the methodology of the measurements (Moussawi et al., 2011). In stroke, cerebral hypoxia/ischemia, traumatic brain injury, etc., excessive ambient glutamate provokes development of neurotoxicity (Grewer et al., 2008). Ambient glutamate concentration during the early phase of energy deprivation is mainly increased due to weak uptake and intensive reverse transport (Jabaudon et al., 2000). Heteroexchange can be used as a tool to monitor reverse glutamate transport (Jabaudon et al., 2000). Theoretical calculations demonstrated that heteroexchange and transporter-mediated release of glutamate have a common rate-limiting step in the transport process. Zhou et al. (2014) revealed unexplained lack of correspondence between the distribution of EAAT2 type glutamate transporters and EAAT2-mediated transport activity. The authors have suggested that glutamate heteroexchange is significantly faster than uptake, and heteroexchange is preferred because of high intracellular glutamate level in nerve terminals (Zhou et al., 2014). A theoretic two-substrate kinetic model described glutamate transport reversal was proposed (Makarov et al., 2013). The calculations have showed that transporter-mediated release is the main component of glutamate heteroexchange mechanism; however, this suggestion is not proven in the experiments because of absence of the available experimental approach to perform such diversification. Therefore, constituent processes involved in homo- and heteroexchange of glutamate is not yet clear in details.

Transporter-mediated glutamate release from nerve terminals is one of the main mechanisms underlying an increase in the ambient glutamate concentration and development of neurotoxicity under hypoxic/ischemic conditions, and so its attenuation is one of the main strategies in neuroprotection. In this context, therapeutic hypothermia is a potent unspecific neuroprotectant that can modulate glutamate transporter activity and decrease significantly pathological transporter-mediated glutamate release from nerve terminals. Hypothermia was defined as mild (>32°C), moderate (28–32°C), deep (20–28°C), or profound (5–20°C) (Liu and Yenari, 2009). Deep hypothermia is used in cardiac surgery with cardiac arrest for the operation duration and also for brain protection during selectively reduced cerebral perfusion; therapeutic mild and moderate hypothermia are applicable for focal or global ischemia (Liu and Yenari, 2009).

Recently, we revealed different dynamics of hypothermia-induced decrease in transporter-mediated release of L-[¹⁴C]glutamate as compared with the tonic one in rat brain nerve terminals (synaptosomes) (Pastukhov et al., 2016). In this study, deep (27°C) and profound (17°C) hypothermia has been administrated for the analysis of temperature sensitivity of glutamate Homo- and heteroexchange in nerve terminals. Hypothermia was used not only as unspecific neuroprotectant but also as a methodological approach for discrimination of constituent processes involved in maintaining ambient glutamate level. The dynamics of hypothermia-induced changes in homoexchange (glutamate-induced L-[¹⁴C]glutamate release) and heteroexchange (D-aspartate- and DL-threo-β-hydroxyaspartate (DL-THA))-induced L-[¹⁴C]glutamate release in nerve terminals (Fig. 1A), and also net tonic release of L-[¹⁴C]glutamate measured in the presence of DL-threo-β-benzyloxyaspartate (DL-TBOA) (Fig. 1B) were analyzed. The experiments were performed using synaptosomes which retain all characteristics of intact nerve terminals and are one of the best systems to explore the relationship between the structure of a protein, its biochemical properties, and physiological role (Sudhof, 2004).

FIG. 1.

Experimental design. Homo- and heteroexchange (A) and net tonic release of glutamate (B).

Methods and Materials

Ethics statement

Wistar male rats with body weight between 300 and 250 g, 2 months of age, were kept using animal facilities of the Palladin Institute of Biochemistry NAS of Ukraine. They were housed at 22–23°C in temperature-controlled rooms where water and dry food pellets were provided ad libitum. All experimental procedures were performed in accordance with the Helsinki Declaration (“Scientific Requirements and Research Protocols” and “Research Ethics Committees”). The experimental protocols were approved by the Palladin Institute's Animal Care and Use Committee (Protocol from 19/09/2011). Before removing the brain, the rats were quickly decapitated (Pozdnyakova et al., 2014). All animal studies were carried out in agreement with the ARRIVE guidelines for reporting experiments involving animal (Kilkenny et al., 2010; McGrath et al., 2010). The total number of animals used in this research was 34. Namely, 15 animals were used for glutamate-induced L-[¹⁴C]glutamate release, homoexchange; 15 animals – for aspartate-induced L-[¹⁴C]glutamate release, heteroexchange; 4 animals – for DL-THA-induced L-[¹⁴C]glutamate release, heteroexchange; several parameters, that is, L-[¹⁴C]glutamate uptake and the ambient L-[¹⁴C]glutamate level were examined simultaneously using the same synaptosomal preparations.

Procedure of isolation of rat brain nerve terminal (synaptosomes)

The rat brain cerebral hemispheres were quickly removed from the skull of decapitated animals and homogenized in ice-cold solution containing sucrose (0.32 M), HEPES-NaOH (5 mM) pH 7.4, and EDTA (0.2 mM). One synaptosomal preparation was isolated from one animal. The synaptosomes were obtained by differential centrifugation and centrifugation in Ficoll-400 density gradient of brain homogenate in accordance to the method of Cotman (1974) with minor corrections (Borisova, 2014; Borisova and Himmelreich, 2005). All isolation procedures were done out at +4°C. The synaptosomal preparations were suitable to be used in the experiments during 2–4 hours after isolation. The standard salt solution was oxygenated before the experiments and contained NaCl 126 mM; KCl 5 mM; MgCl₂ 2.0 mM; NaH₂PO₄ 1.0 mM; HEPES 20 mM, pH 7.4; and D-glucose 10 mM. Ca²⁺-supplemented medium contained CaCl₂ at a concentration of 2 mM. Ca²⁺-free medium contained EGTA at a concentration of 1 mM and Ca²⁺ was not added. The concentration of proteins was calculated in accordance to Larson et al. (1986).

In vitro hypothermia treatment

Hypothermia conditions were classified according to Liu and Yenar (2007). The experiments were carried out using following approaches. In one series of the experiments, the synaptosomes in the standard salt solution at +4°C obtained according to abovementioned Cotman's method were warmed up at 17°C, 27°C, and 37°C and incubated for 8 min before starting L-[¹⁴C]glutamate uptake and release experiments. In another experiment, all synaptosomes were first warmed up to 37°C for 8 min and then cooled to reach 17°C, 27°C, and incubated at definite temperature regimes for 8 min.

Assessment of L-[¹⁴C]glutamate uptake by nerve terminals

The uptake of L-[¹⁴C]glutamate was analyzed using the synaptosomal suspension (125 μL; of the suspension, 0.4 mg of protein per mL) that was preliminarily incubated in the standard salt solution at 17°C, 27°C, or 37°C for 8 min. The uptake was initiated by the application of L-glutamate at a concentration of 10 μM supplemented with L-[¹⁴C]glutamate (420 nM, 0.1 μCi/mL). The synaptosomes were incubated at 17°C, 27°C, or 37°C for 1 min, then immediately sedimented in a microcentrifuge at 10,000 g for 20 s. The L-[¹⁴C]glutamate uptake was calculated as a decrease in radioactivity in the supernatant and an increase in radioactivity of the SDS-treated pellets of synaptosomal aliquots (100 μL). L-[¹⁴C]glutamate uptake was determined by the method of liquid scintillation counting with scintillation cocktail, aqueous counting scintillant (ACS) (1.5 mL) (Borisova, 2013; Soldatkin et al., 2015). Data were collected from independent experiments using different synaptosomal preparations. “n” was the value of L-[¹⁴C]glutamate uptake measured using one synaptosomal preparation isolated from one animal during one experimental day. Data are shown as mean ± SEM.

Assessment of L-[¹⁴C]glutamate release from nerve terminals

The synaptosomes for release purposes were used at a concentration of 2 mg of protein/ml. After preliminary incubation at 37°C for 10 min they were loaded with L-[¹⁴C]glutamate (1 nmol/mg of protein, 238 mCi/mmol) in Ca²⁺-supplemented standard salt solution at 37°C for 10 min. Then the synaptosomal suspensions were washed with ice-cold standard salt solution (10 volumes) and centrifuged. The synaptosomal pellets were suspended in the ice-cold standard salt solution to a concentration of 1 mg protein/ml and without delay used in the release experiments. The release of L-[¹⁴C]glutamate from the synaptosomes was performed in Ca²⁺-free incubation medium The synaptosomal samples (125 μL of the suspension, 0.5 mg of protein/mL) were preincubated at different temperature regimes, that is, 17°C, 27°C, or 37°C for 8 min, and then the inhibitors were added, and the synaptosomes were further incubated with them for 6 min and sedimented in a microcentrifuge at 10,000 g for 20 s. The release was measured in the supernatant (100 μL) and pellets of the synaptosomal aliquots using the method of liquid scintillation counting with scintillation cocktail ACS (1.5 mL). The total synaptosomal L-[¹⁴C]glutamate content was 200,000 ± 15,000 cpm/mg protein. The glutamate transporter inhibitor DL-TBOA was added to the synaptosomes at zero time point. Data were collected from several independent experiments using different synaptosomal preparations, where “n” was the value of L-[¹⁴C]glutamate release measured using one synaptosomal preparation isolated from one animal during one experimental day. Data are shown as mean ± SEM.

Statistical analyses

The results of the experiments were expressed as a mean ± SEM of n independent experiments. All data were analyzed by one-way analysis of variance (ANOVA). Post hoc analyses were made using Tukey's test. The experimental unit used in statistical analysis was the value of L-[¹⁴C]glutamate release or uptake measured using one synaptosomal preparation isolated from one animal. The accepted level of significance was set as p < 0.05. All statistical analyses were performed using Microsoft Excel.

Materials

HEPES, EDTA, EGTA, DL-TBOA, DL-THA, and the analytical grade salts were purchased from Sigma; Ficoll-400 and ACS were obtained from Amersham; L-[¹⁴C]glutamate was purchased from Amersham and PerkinElmer.

Results

Homo- and heteroexchange of L-[¹⁴C]glutamate in nerve terminals under conditions of deep and profound hypothermia

Hypothermia-induced changes in neurotransmitter transport processes can be adequately analyzed using radiolabeled neurotransmitters because fluorescent and enzymatic methods are temperature sensitive themselves. To assess hypothermia influence on glutamate homoexchange, the release of radiolabeled L-[¹⁴C]glutamate from L-[¹⁴C]glutamate-preloaded synaptosomes was measured in Ca²⁺-free media after the application of nonradiolabeled glutamate. In other words, the substitution of preloaded synaptosomal L-[¹⁴C]glutamate by extracellular nonradiolabeled glutamate (glutamate-induced L-[¹⁴C]glutamate release) was recorded. Heteroexchange of L-[¹⁴C]glutamate in the synaptosomes was analyzed in Ca²⁺-free media using transportable inhibitors of glutamate transporters, D-aspartate, and DL-THA.

The ambient level of L-[¹⁴C]glutamate in the preparations of nerve terminals was not changed significantly under hypothermia conditions and consisted of approximately 25% of total synaptosomal label at 37°C, 27°C, and 17°C. To simplify further explanations, measured radioactivity was marked in bold type and the main constituents that determined the extracellular L-[¹⁴C]glutamate level were written as follows: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} { \bf{L}} & - \left[ {^{{ \bf{14}}}{ \bf{C}}} \right] { \bf{glutamat}}{{ \bf{e}}_{{ \bf{extracellular}} \ ( { \bf{measured}} ) }} \\\quad & = { \rm{L - }} \left[ {^{{ \rm{14}}}{ \rm{C}}} \right] { \rm{glutamat}}{{ \rm{e}}_{{ \rm{ ( tonic \ release ) }}}}\\\quad & { \rm{ + L - }} \left[ {^{{ \rm{14}}}{ \rm{C}}} \right] { \rm{glutamat}}{{ \rm{e}}_{{ \rm{ ( transporter \ reversal \ release ) }}}}\\ \quad & { \rm{ - L - }} \left[ {^{{ \rm{14}}}{ \rm{C}}} \right] { \rm{glutamat}}{{ \rm{e}}_{{ \rm{ ( uptake ) }}}} \end{align*} \end{document}

Glutamate-induced L-[¹⁴C]glutamate release from nerve terminals under conditions of deep and profound hypothermia

Competitive nontransportable inhibitor of glutamate transporters DL-TBOA at a concentration of 200 μM inhibited the initial rate of synaptosomal L-[¹⁴C]glutamate uptake by approximately 80% ± 2.7% (F(1,28) = 819.1, p < 0.001, n = 15). Glutamate (100 μM) added to the incubation media of the synaptosomes stimulated the release of preloaded L-[¹⁴C]glutamate by means of homoexchange. The latest parameter was calculated as difference between the values of L-[¹⁴C]glutamate_{extracellular} before glutamate (100 μM) addition to the synaptosomes and after their incubation with glutamate for 6 min. DL-TBOA application for 1 min decreased glutamate-induced L-[¹⁴C]glutamate release from nerve terminals thereby reflecting contribution of glutamate transporter to homoexchange process (Fig. 2). Hence, DL-TBOA administration partially prevented glutamate homoexchange process due to inhibition of glutamate transporter functioning.

FIG. 2.

Glutamate (100 μM)-induced release of L-[¹⁴C]glutamate from nerve terminals for 6 min in the control (the first column) and after preliminary administration of DL-TBOA (200 μM) (the second column). Data are mean ± SEM of 15 independent experiments. The experimental unit was the value of L-[¹⁴C]glutamate release measured using one synaptosomal preparation isolated from one animal. Data are compared by one-way ANOVA and post hoc Tukey's test. **p ≤ 0.01 as compared with the value of L-[¹⁴C]glutamate release without DL-TBOA.

Glutamate-induced L-[¹⁴C]glutamate release decreased gradually in deep and profound hypothermia that was equal to 31.7% ± 2.3% of total synaptosomal label in the control at 37°C, 22.9% ± 1.8% of total synaptosomal label at 27°C, and 12.9% ± 2.1% of total synaptosomal label at 17°C (F(2,42) = 235.2, p < 0.01, n = 15) (Fig. 3A).

FIG. 3.

L-[¹⁴C]glutamate release from nerve terminals in response to the application of 100 μM glutamate (A), 100 μM D-aspartate (B), and 100 μM DL-THA (C) at 37°C (the first columns), 27°C (the second columns), and 17°C (the third columns). Data are mean ± SEM of 15 (A, B), 4 (C) independent experiments. The experimental unit was the value of L-[¹⁴C]glutamate release measured using one synaptosomal preparation isolated from one animal. Data are compared by one-way ANOVA and post hoc Tukey's test. **p ≤ 0.01 as compared with the control at 37°C.

D-aspartate-induced L-[¹⁴C]glutamate release from nerve terminals under conditions of deep and profound hypothermia

D-aspartate is a substrate of the glutamate transporters. Its application at a concentration of 100 μM to L-[¹⁴C]glutamate-preloaded synaptosomes resulted in significant L-[¹⁴C]glutamate release that consisted of 25.9% ± 2.2% of total synaptosomal label at 6 min time point (Fig. 3B). Heteroexchange of L-[¹⁴C]glutamate decreased gradually in deep and profound hypothermia and was equal to 20.4% ± 2.0% of total synaptosomal label at 27°C and 11.2% ± 1.2% of total synaptosomal label at 17°C (F(2,42) = 170.5, p < 0.01, n = 15) (Fig. 3B). Therefore, D-aspartate-induced L-[¹⁴C]glutamate release decreased gradually in deep and profound hypothermia.

DL-THA-induced L-[¹⁴C]glutamate release from nerve terminals under conditions of deep and profound hypothermia

L-[¹⁴C]glutamate release by means of heteroexchange with a competitive transportable inhibitor of glutamate transporters DL-THA (100 μM) was decreased at 6 min time point from 21.5% ± 0.9% of total accumulated label at 37°C up to 16.1% ± 0.5% of total accumulated label at 27°C, and 6.1% ± 0.5% of total accumulated label at 17°C (F(2,9) = 226.9, p < 0.01, n = 4) (Fig. 3C). Dynamics of hypothermia-induced changes in DL-THA-induced heteroexchange is in accord with above data on glutamate- and D-aspartate-induced release of L-[¹⁴C]glutamate from the synaptosomes.

Discussion

Complete justification of the mechanisms determining glutamate homo- and heteroexchange still remains unclear despite its principal significance for maintenance of the extracellular glutamate level and a lot of theoretical and experimental data regarding glutamate transporter functioning. Theoretical calculations predict that homo- and heteroexchange have a common rate-limiting step with transporter-mediated glutamate release (Jabaudon et al., 2000; Makarov et al., 2013). Glutamate transporter reversal is an acknowledged release mechanism that caused excitotoxicity under conditions of energy deprivation. The question arises as to whether or not transporter-mediated glutamate release occurs under normal physiological conditions. There is lack of experimental evidences of physiological significance of glutamate transporter reversal (Grewer et al., 2008). Glutamate can be released through transporters during physiological depolarization of the plasma membrane thereby contributing to neurotransmission regulation (Grewer et al., 2008). Membrane depolarization leads to a tremendous temporal increase in the extracellular concentrations of glutamate released by means of exocytosis, and so transporters are competent to start working with homoexchange mechanism also. Calculations of Makarov et al. (2013) have shown that the Michaelis–Menten kinetics, which is accepted in transporter studies, does not include transporter reversal, and it completely neglects the possibility of equilibrium between the substrate concentrations on both sides of the membrane. A complex two-substrate kinetic model that included transporter reversal was developed. One transporter substrate can release another one already accumulated inside the cell (Makarov et al., 2013), and when both substrates are in equilibrium, the addition of one of them leads to reequilibrium and release of the second substrate. We recently demonstrated the importance and effectiveness of permanent extracellular/intracellular glutamate turnover in nerve terminals (Borisova and Borysov, 2016; Borisova et al., 2016). Rapid transporter-mediated extracellular/intercellular glutamate turnover in nerve terminals was shown in this study. We substituted intracellular synaptosomal L-[¹⁴C]glutamate with glutamate, D-aspartate, and DL-THA under normal and hypothermia conditions.

The main finding of this study is the fact that homo- and heteroexchange are sensitive to hypothermia. This fact is not obvious a priori because the constituents involved in homo- and heteroexchange are oppositely directed transport processes, all of which are decreased in hypothermia. In particular, hypothermia gradually attenuated both transporter-mediated glutamate uptake (inward glutamate transport) and transporter-mediated and tonic release of glutamate (outward glutamate transport) (Pastukhov et al., 2016). In nerve terminals, the released neurotransmitter is immediately deleted from the extracellular space by glutamate transporters. In this context, complete inhibition of L-[¹⁴C]glutamate uptake by D-aspartate, DL-THA, and glutamate uncovers net values of transporter-mediated and tonic L-[¹⁴C]glutamate release. We suggested that net tonic L-[¹⁴C]glutamate release should be negligibly low when we started homoexchange process by the addition of 100 μM glutamate because of the small concentration gradient between the [glutamate]_{intracellular} and [glutamate]_{extracellular}. Presumably, net tonic L-[¹⁴C]glutamate release contributed insignificantly to both processes. Extracellular glutamate initiated homoexchange process and simultaneously activated presynaptic glutamate receptors (Borisova et al., 2016), thereby inducing additional L-[¹⁴C]glutamate release, and therefore L-[¹⁴C]glutamate homoexchange values (Fig. 3A) were higher as compared with heteroexchange ones (Fig. 3B, C).

Importantly, dynamics of hypothermia-induced changes of homo- and heteroexchange resembles dynamics of depolarization-induced transporter-mediated release of glutamate from nerve terminals. By analogy with homo- and heteroexchange, the latest gradually decrease with decrease in temperature. This fact can be considered as an experimental confirmation of the suggestion that the main principal mechanism involved in glutamate homo- and heteroexchange is transporter reversal. So, hypothermia can be considered in this study as a methodological approach discriminating different components of glutamate homo- and heteroexchange process. In this context, glutamate transporter reversal occurs under physiological conditions as a part of homo- and heteroexchange mechanism, and so is nonpathological under these conditions. Also, the central role of glutamate transporters in permanent efficient glutamate turnover across the plasma membrane of nerve terminals was confirmed. Administration of DL-TBOA inhibited glutamate transporter functioning and so glutamate homoexchange (Fig. 2). Importantly, the ambient level of L-[¹⁴C]glutamate in nerve terminals depended on the concentration of exogenously added glutamate, D-aspartate, and DL-THA in both norm and hypothermia.

Hypothermia can be accidental due to inadequate environmental exposure, and intentional due to usage of special medical conditions for therapeutic temperature modulation. Therapeutic hypothermia can prevent the development of neurological consequences and significantly improve rates of long-term neurological survival after out-of-hospital sudden cardiac arrest (Chiota et al., 2011), in cardiac surgery of the aortic arch, where it facilitates operations by the reduction of cerebral circulation, and in therapy of ischemic stroke, traumatic brain injury (Urbano and Oddo, 2012), and subarachnoid hemorrhage (Seule et al., 2009). Hypothermia is the current neuroprotection standard in hypoxic/ischemic encephalopathy. Recent finding underlined the urgent requirement of investigation of interactions between drugs, neuroprotectants, and hypothermia before administration in neonates for pharmacotherapy of neonatal seizures (Donovan et al., 2016). Also, hypothermia is considered as new neuroprotective approach in long-term interplanetary space missions. Ultraprofound (<5°C) hypothermia is under investigation for rapid cooling of trauma victims (Liu and Yenari, 2009). So, it is an urgent necessity to acquire knowledge on hypothermia-induced changes in nonpathological processes in nerve terminals. Identification of the exact mechanisms of the neuroprotective effects of hypothermia and hypothermia-sensitive pathological process in nerve terminals, and also justification of optimal temperature regime are the main principal tasks to further promote this perspective approach.

Our data on hypothermia-induced changes in glutamate homo- and heteroexchange are of value for therapeutic hypothermia prognosis because they reflect how “healthy” nerve terminals do react on excess of extracellular glutamate during lowering of temperature. Translating in vivo, this glutamate excess can originate from synaptic vesicle exocytosis and pathological hypoxia-induced transporter-mediated release from neighboring nerve terminals.

In conclusion, we showed clearly that nerve terminal reaction on excess of extracellular glutamate (homoexchange) and aspartate (heteroexchange) decreased gradually from deep to profound hypothermia, thereby preventing further elevation of extracellular glutamate, and thus demonstrating neuroprotective feature. Similarity in gradual character of dynamics of hypothermia-induced changes in glutamate homo- and heteroexchange with those in transporter-mediated glutamate release from nerve terminals confirmed the prevalence of transporter reversal constituent in glutamate homo- and heteroexchange mechanisms. Transporter-mediated glutamate release can be nonpathological and can occur under physiological conditions at least as a part of homo- and heteroexchange mechanism.

Footnotes

Acknowledgments

The authors would like to thank K. Paliienko, Dr. N. Pozdnyakova, Dr. N. Krisanova, and M. Dudarenko for assistance in the experimental work.

This work was supported by Projects of National Academy of Sciences of Ukraine within Programs: “Molecular and Cellular Biotechnology for Medicine, Industry, and Agriculture,” “Space Research Program,” “Sensors for Medicine, Ecology, Industry, and Technology,” “DFFD,” and “Project F76/72.”

Authors' Contributions

Isolation of synaptosomes and L-[¹⁴C]glutamate uptake and release experiments were performed by A.P.; data analysis by A.P. and T.B., and article written by T.B.

Ethical Approval

All procedures were conducted according to the Declaration of Helsinki (“Scientific Requirements and Research Protocols” and “Research Ethics Committees”). Experimental protocols were approved by the Animal Care and Use Committee of the Palladin Institute of Biochemistry (Protocol from 17/12-2015).

Author Disclosure Statement

The authors declare that they have no competing interests.

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Combined Application of Glutamate Transporter Inhibitors and Hypothermia Discriminates Principal Constituent Processes Involved in Glutamate Homo- and Heteroexchange in Brain Nerve Terminals

Abstract

Introduction

Methods and Materials

Ethics statement

Procedure of isolation of rat brain nerve terminal (synaptosomes)

In vitro hypothermia treatment

Assessment of L-[14C]glutamate uptake by nerve terminals

Assessment of L-[14C]glutamate release from nerve terminals

Statistical analyses

Materials

Results

Homo- and heteroexchange of L-[14C]glutamate in nerve terminals under conditions of deep and profound hypothermia

Glutamate-induced L-[14C]glutamate release from nerve terminals under conditions of deep and profound hypothermia

D-aspartate-induced L-[14C]glutamate release from nerve terminals under conditions of deep and profound hypothermia

DL-THA-induced L-[14C]glutamate release from nerve terminals under conditions of deep and profound hypothermia

Discussion

Footnotes

Acknowledgments

Authors' Contributions

Ethical Approval

Author Disclosure Statement

References

Assessment of L-[¹⁴C]glutamate uptake by nerve terminals

Assessment of L-[¹⁴C]glutamate release from nerve terminals

Homo- and heteroexchange of L-[¹⁴C]glutamate in nerve terminals under conditions of deep and profound hypothermia

Glutamate-induced L-[¹⁴C]glutamate release from nerve terminals under conditions of deep and profound hypothermia

D-aspartate-induced L-[¹⁴C]glutamate release from nerve terminals under conditions of deep and profound hypothermia

DL-THA-induced L-[¹⁴C]glutamate release from nerve terminals under conditions of deep and profound hypothermia