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GENERAL ARTICLES

The Effects of Hypothermia on a Cloned Human Brain Glutamate Transporter (hGLT-1) Expressed in Chinese Hamster Ovary Cells: -[3H]L-Glutamate Uptake Study

Department of Anesthesiology and Critical Care Medicine, Tokyo Medical and Dental University, Tokyo, Japan

Address correspondence and reprint requests to Fumio Sakai, MD, PhD, Department of Anesthesiology and Critical Care Medicine, Tokyo Medical and Dental University, 5-45, Yushima 1-chome, Bunkyo-ku, Tokyo, 113-8519 Japan.

	Abstract

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Hypothermia provides neuroprotection that inhibits increases in extracellular glutamate concentration during ischemia; however, the effect of hypothermia on the glutamate transporter is uncertain. A human glial glutamate transporter (hGLT-1) cDNA, isolated by screening a cDNA, library was cloned and stably transfected into Chinese hamster ovary cells. We assessed the effects of temperature on transporter activity in [³H]L-glutamate flux experiments at 23, 32, and 37°C. Hypothermia of 23°C and 32°C decreased [³H]L-glutamate uptake at 60 min, to 76.7% ± 7.3% (P < 0.05, n = 5) and 70.7% ± 7.5% (P < 0.05, n = 5) of uptake at 37°C, respectively. Reversed uptake of preloaded [³H]L-glutamate via hGLT-1 was not observed at any temperature. The specific uptakes (Q₁₀ values) for 37°C to 32°C and 32°C to 23°C at 30 min were 3.48 and 2.37, whereas they were 2.17 and 0.91, respectively, for 60 min. These changes suggest that hypothermia attenuates uptake of extracellular glutamate via hGLT-1 in a temperature- and time-dependent manner.

Implications: Under certain pathologic conditions, including cerebral ischemia and traumatic brain injury, glutamate neurotoxicity may initially be propagated by hypothermia due to relative failure of glutamate uptake via Human Glial Glutamate Transporter before a subsequent recovery of uptake.

	Introduction

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Some patients undergoing cardiac, cerebral, or thoracic vascular surgery requiring cardiopulmonary bypass are exposed to mild to moderate hypothermia to reduce the risk of cerebral ischemia. A key event in the cascade that leads to neuronal damage during hypoxia or anoxia is the extracellular accumulation of glutamate in the brain (1). Glutamate is the major excitatory neurotransmitter in the brain. An increased extracellular glutamate concentration leads to activation of glutamate receptors, causing uncontrolled calcium influx, adenosine triphosphate loss, activation of proteases and lipases, and, ultimately, cell damage and death (2). The carriers that play a major role in the uptake of extracellular glutamate are Na⁺-dependent glutamate transporters. These carriers are widely distributed in the brain and spinal cord, and they act to maintain extracellular glutamate concentrations below neurotoxic levels.

Five homologous types of the glutamate transporter—GLAST, GLT-1, EAAC1, EAAT4, and EAAT5—have been cloned from various species. EAAC1 and EAAT4 apparently are exclusive to neurons, whereas GLAST is found in both neurons and astroglia (3,4). GLT-1, specific to astroglia (4), is an important determinant of clearance of free glutamate from the synaptic cleft (5,6) that acts to prevent abnormally increased extracellular glutamate concentrations (7). Recent observations indicate that GLT-1 can be expressed in neurons in experimental situations, which suggests that the cellular localization of GLT-1 may change under certain pathologic conditions (8). Previous studies on the change in extracellular glutamate concentration during hypothermia have been based on tissue slices and synaptosome preparations, which likely reflect the combined activation of multiple subtypes of transporters, receptors, ion channels, and pumps.

The cloning of glutamate transporter cDNAs offers the first opportunity to independently investigate individual members of this family after expression in heterologous systems. In preliminary experiments using [3H]L-glutamate, we found that the endogenous transport activity of L-glutamate in Chinese hamster ovary (CHO) cells was less than that of human embryonic kidney 293 cells or baby hamster kidney cells. Thus, in a heterologous system using CHO cells, we investigated the temperature-related properties of hGLT-1. Although increases of extracellular glutamate during anoxia has been reported to decrease in microdialysis experiments under hypothermia (9), little is known about changes in glutamate uptake or the time course of glutamate transport activities.

	Methods

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A GLT-1 cDNA (1.7 kbp) clone was isolated by the polymerase chain reaction (PCR) from an adult human cerebral cortex cDNA library (Clontech, Palo Alto, CA). PCR, including denaturation (1 min, 94°C), annealing (2 min, 55°C), and extension (3 min, 72°C) for 35 cycles, followed by further incubation for 7 min at 72°C, was performed in 50 µL of reaction mixtures that contained oligonucleotide primers at 0.3 µM each, the adult human cerebral cortex cDNA library, 200 µM of each deoxynucleotide, and Vent DNA polymerase 5 units (NEB, Beverly, MA). Two primers, 5'-AGACCATGGCATCTACGGAA-3' and 5'-TTACCATAGGATACGCTGGG-3' submitted to the GenBank/EMBL/DDBJ Data Bank (10), were designed. PCR products were subcloned into a pUC18 vector (Stratagene, La Jolla, CA) after digestion with Sma I and were amplified by transformation of Epicurian Coli XL1-Blue MRF'Kan supercompetent cells (Stratagene). The human GLT-1 (hGLT-1) cDNA was ligated into a eukaryotic expression vector (pcDNA3.1); this plasmid was transfected into CHO cells using the calcium phosphate method (11). Clonal cell lines expressing the highest level of hGLT-1 and exhibiting significant [3H]L-glutamate transporting above the basal level were selected for subsequent experiments.

Unaltered (intact) CHO cells and stable hGLT-1–transfected CHO cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco BRL, Grand Island, NY) supplemented with 10% fetal calf serum, 100 U/mL penicillin G, 100 µg/mL streptomycin, and 250 ng/mL amphotericin B in a 5% CO₂ atmosphere in a humidified incubator at 37°C. The transfected CHO cells were selected using 600 µg/mL Geneticin (Gibco-BRL). Cells were grown in 24-well plates, and wells containing 104 to 105 cells were used.

The culture medium in 24-well plates was exchanged with 90 µL of HEPES-Tris buffer solution containing or lacking Na⁺ (in mM): NaCl or choline chloride 140, KCl 2.5, CaCl₂ 1.2, MgCl₂ 1.2, K₂HPO₄ 1.2, HEPES 10, Tris 5, and dextrose 10. Samples were divided to 9 µL on 96-well plates and were preincubated at 37°C. The reaction was started by adding 1 µL of a 1-µM stock solution of [3H]L-glutamate to 9 µL of samples to achieve a final glutamate concentration of 0.1 µM. After samples were incubated for 3, 5, 10, 30, or 60 min at 37°C, 32°C, or 23°C, the solution was removed from the well and quickly washed twice with ice-cold fresh buffer to reduce the nonspecific association of radiolabel. To each well, 10 µL of 0.5% Triton and 90 µL of Microscint 20 (Packard Instrument, Meriden, CT) were added, and radioactivity was measured using TopCount (Packard). The amount of glutamate taken up by the cells by incubation was normalized by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) analysis (12), which reflects mitochondrial succinate dehydrogenase activity and thus is a general indicator of the respiratory status of the cells. Because CHO cells take up or adsorb [3H]L-glutamate independent of Na⁺ on the cell surface, Na⁺-dependent uptake of [3H]L-glutamate was calculated as the difference between the amount of radioactivity obtained using normal buffers containing Na⁺ and that of Na⁺-free buffer in which NaCl was replaced with choline chloride. After measurement of Na⁺-dependent radioactivity in each sample, the difference between the amount of Na⁺-dependent radioactivity accumulated by CHO cells expressing hGLT-1 and that shown by unaltered CHO cells was regarded as hGLT-1–specific transport activity.

We investigated the effect of temperature on release of [3H]L-glutamate in CHO cells expressing hGLT-1 or intact CHO cells to assess the contribution of changes in uptake compared with the possibility of changes in release. The same cell lines used for uptake studies were used in this study. Cells were loaded with 100 µL of HEPES-Tris buffer solution containing 0.1 µM [3H]L-glutamate for 60 min at 37°C, then washed twice in ice-cold buffer solution to remove extracellular [3H]L-glutamate. Immediately after washing, CHO cells were added and resuspended in 100 µL of Na⁺-free buffer solution at 37°C, 32°C, or 23°C. Release of [3H]L-glutamate was determined after 0.5, 1, 3, 5, or 10 min of exposure to buffer medium, supernatants were removed and counted for tritium. The cells were scraped on the coverslips, scintillant was added, and the residual accumulated [3H]L-glutamate determined. Values were expressed as a percentage of total cell tritium content. Release evoked by normal buffer solution was determined in parallel at the same time intervals.

All data are expressed as means ± SEM. For statistical analysis of data, one-way analysis of variance with a post hoc Scheffé F or paired t-test was used. A P value <0.05 was considered statistically significant.

Temperature-dependent effects were expressed as the Q₁₀ of [3H]L-glutamate release; that is, the factor by which the activity of the transporter varies per 10°C change in temperature. The Q₁₀ was calculated as described below, where X₂ equals the experimental value at the higher absolute temperature (T₂) and X₁ equals the experimental value at the lower absolute temperature (T_I): Q₁₀ = (X₂/X₁)^{[10/(T_2–T1)]} (13).

	Results

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Total accumulation of [3H]L-glutamate in CHO cells expressing hGLT-1 was greater than nonspecific accumulation of [3H]L-glutamate in intact CHO cells, depending on temperature and duration of incubation (Fig. 1a and b). Exposure to hypothermia (23°C and 32°C) for 60 min decreased specific accumulation of [3H]L-glutamate to 76.7% ± 7.3% (P < 0.05, n = 5) and 70.7% ± 7.5% (P < 0.05, n = 5) of the control (37°C) (Fig. 1c). Between 30 and 60 min, specific accumulation of [3H]L-glutamate via hGLT-1 followed a steep time-dependent curve. At 30 min, the Q₁₀ values calculated for 37°C to 32°C and for 32°C to 23°C were 3.48 and 2.37, respectively; at 60 min, they were 2.17 and 0.91, respectively. Release of preloaded [3H]L-glutamate through CHO cells expressing hGLT-1 was the same as or less than that of intact CHO cells for each temperature range (Fig. 2 a and b). Release of [3H]L-glutamate via hGLT-1 in Na⁺-containing buffer solution was less than that seen in Na⁺-free buffer solution at 37°C and 32°C (P < 0.01, n = 5) but not 23°C (n = 5) (Fig. 2).

View larger version (22K): [in this window] [in a new window]   Figure 1. The effect of hypothermia on the time course of [3H]L-glutamate uptake. The accumulation of [3H]L-glutamate ([3H]L-Glu) was measured by assaying aliquots of solution from multiwell plates containing Chinese hamster ovary (CHO) cells expressing a human glial glutamate transporter (hGLT-1) at indicated times (3–60 min) after the addition of 0.1 µM [3H]L-Glu. Na+-independent accumulation of [3H]L-Glu was determined in a medium in which Na+ was replaced by choline. To exclude [3H]L-Glu adherent to the cell surface or simply diffused into cells, this volume was subtracted from the accumulation of [3H]L-Glu using Na+-containing buffer solution. Similar experiments were performed using unaltered CHO cells, including an endogenous glutamate transporter, to yield specific [3H]L-Glu uptake via hGLT-1. A, Total accumulation in CHO cells expressing hGLT-1. b, Nonspecific accumulation in unaltered CHO cells. C, hGLT-1–specific accumulation. Values represent the means ± SEM of five wells, calculated for each time period. The result shown is typical of the results recorded in three experiments.

View larger version (23K): [in this window] [in a new window]   Figure 2. The effect of hypothermia on the time course of preloaded [3H]L-glutamate ([3H]L-Glu) release from Chinese hamster ovary (CHO) cells expressing a human glial glutamate transporter (hGLT-1) and intact CHO cells. Release of [3H]L-Glu from samples was measured for 0.5–10 min in Na+-free buffer solution (a) and Na+-containing buffer solution (b). Release of [3H]L-Glu is expressed as a percentage of total cell content. * P < 0.05, ** P < 0.01, P < 0.05, P < 0.01 compared with the control value from intact CHO cells. Values represent the mean ± SEM calculated for each time period.

	Discussion

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In measuring the amount of radiolabeled glutamate in cells with increasing duration of exposure, contributions of changes in uptake compared with possible changes in release must be assessed. In hypoxia/anoxia, the glutamate transporter loses its concentrating power because of disruption of cellular ionic gradients; running in reverse, the transporter deposits glutamate into the extracellular space, resulting in irreversible neuronal damage. We studied the release of [3H]L-glutamate with no L-glutamate presented externally to the preloaded cells; in this situation, the uptake carrier can only run backward (16). Nevertheless, such release of [3H]L-glutamate was not detected; indeed, the release of preloaded [3H]L-glutamate from CHO cells expressing hGLT-1 was significantly less than that from intact CHO cells. Similarly, the release of [3H]L-glutamate in Na⁺-containing buffer solution was less than that in Na⁺-free buffer solution. These results indicate that hGLT-1 expressed in CHO cells preserves uptake activity of L-glutamate, but not reverse uptake, beyond 60 min. We believe that the data presented do not include any potential contribution of L-glutamate release, although we could not exclude potential effects of the metabolism of L-glutamate with increasing time.

Over the ranges from 37°C to 32°C and 32°C to 23°C, the Q₁₀ values calculated from the uptake of [3H]L-glutamate for 30 min were 3.48 and 2.37; those for 60 minutes were 2.17 and 0.91, respectively. In general, flux through a channel is relatively insensitive to temperature, with the Q₁₀ values typically being <1.5 because of low-energy barriers associated with ionic diffusion (17–19). The Q₁₀ value for the coupled uptake current of glutamate (approximately 3) is more consistent with the energy requirement for the large conformational transitions that occur during each transport cycle (20). The Q₁₀ values we obtained indicate that [3H]L-glutamate uptake by hGLT-1 is temperature-dependent, as reported in a previous study in rat dorsal spinal cord (21). We also demonstrated that accumulation of [3H]L-glutamate via hGLT-1 was initially reduced by the induction of hypothermia (23°C and 32°C), subsequently accelerating after 60 min, indicating time-dependent recovery. Specific uptake from 32°C to 23°C at 60 minutes, characterized by the Q₁₀ value of 0.91, suggests that the [3H]L-glutamate transport activity of hGLT-1 may not necessarily depend on temperatures <32°C, which argues against a threshold near that temperature.

The mechanisms underlying the observed initial reduction of [3H]L-glutamate accumulation are unknown, although there are two possible explanations. One involves physical properties of cell membranes; decreases in temperature shift the lipid bilayer from a fluid to a gel phase (21). GLT-1 requires either posttranslational processing or coassembly with an interacting protein for activity. Furthermore, GLT-1 is retained in a subcellular compartment with trafficking to the cell surface (22). Such loss of membrane fluidity would affect both integral and peripheral proteins associated with the transport activity of GLT-1. The other possible explanation is up- or down-regulation of GLT-1. Transient global ischemia causes nearly synchronous down-regulation of GLT-1 mRNA at 3 hours and GLT-1 protein at 6 hours in the rat hippocampal CA1 pyramidal layer, with a return to control levels by 24 hours (23). In contrast, increases in GLT-1 expression in astrocytes are a relatively gradual processes requiring several days (22). Anderson et al. (24) reported that cerebral ischemia induced an increase in the density of excitatory amino acid transporter binding sites in hippocampus and in neocortical regions that persisted for 48 hours after ischemia. Interpretation of the data obtained is difficult because the normal modulatory machinery is presumably absent in heterologous systems; however, our results are not likely to be reflections of either up- or down-regulation of GLT-1.

Net extracellular glutamate concentration represents a balance between the release of glutamate and its removal by uptake and/or diffusion. Hypothermia would reduce Ca²⁺-dependent synaptic glutamate release by suppression of neuronal electrophysiologic activity and Ca²⁺-independent glutamate release via glutamate transporters by decreased energy use in hypothermia due to decreases in both electrophysiologic activity and homeostatic functions required to maintain cellular integrity. Therefore, hypothermia may conversely lead to the initial increase of extracellular glutamate concentration under such pathologic circumstances as cerebral ischemia or traumatic brain injury, due to relative failure of glutamate uptake. Especially during hypothermia of a short period such as CPB, the excessive elevation of extracellular glutamate concentration might lead to glutamate neurotoxicity because glutamate receptors are minimally affected by mild hypothermia (25). Such scenarios are inconsistent with the long-lasting protection observed in hypothermia and the prolonged inhibition of elevation of extracellular glutamate levels observed in vivo. Further study is required to clarify the effects of hypothermia on the glutamate transporter for periods longer than 60 minutes.

In conclusion, mild and moderate hypothermia decreased glutamate uptake by hGLT-1 in a temperature- and time-dependent manner, indicating that intraoperative hypothermia may transiently decrease the uptake of extracellular glutamate. However, time- but not temperature-dependent recovery of glutamate uptake via hGLT-1 may represent another mechanism tending to restrict elevations of extracellular glutamate concentration during hypothermia.

	Acknowledgments

 
We express our gratitude to Kohei Sawada, PhD, Toshihide Hashimoto, PhD, Yoshiaki Furuya, MS, Tsukuba Research Laboratories, and Eisai Co., Ltd., Tsukuba, Ibaraki, Japan, for technical assistance and valuable comments in preparing the manuscript.

	Footnotes

 
This work was presented at the 44th annual meeting of the Japan Society of Anesthesiology, April 1997, Niigata, Japan.

	References

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