Astrocytic Transporter Research Articles

Regulation of serotonin transporter gene expression in human glial cells by growth factors

The aims of this study were to identify monoamine transporters expressed in human glial cells, and to examine the regulation of their expression by stress-related growth factors. The expression of serotonin transporter mRNA was detected by reverse transcriptase-polymerase chain reaction in normal human astrocytes, whereas the dopamine transporter (DAT) and the norepinephrine transporter (NET) were not detected. The cDNA sequence of the “glial” serotonin transporter in astrocytes was consistent with that reported for the “neuronal” serotonin transporter (SERT). Moreover, we also demonstrated SERT expression in glial fibrillary acidic protein-positive cells by immunocytochemical staining in normal human astrocytes. Serotonin transporter gene expression was also detected in glioma-derived cell lines (A172, KG-1-C and KGK). Addition of basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF) for 2 days increased serotonin transporter gene expression in astrocytes and JAR (human choriocarcinoma cell line). Basic fibroblast growth factor, but not epidermal growth factor, increased specific [ 3H]serotonin uptake in astrocytes in a time (1–4 days)- and concentration (20–100 ng/ml)-dependent manner. The expression of genes for basic fibroblast growth factor and epidermal growth factor receptors was detected in astrocytes. These findings suggest that the expression of the serotonin transporter in human glial cells is positively regulated by basic fibroblast growth factor.

Methylmercury inhibits the in vitro uptake of the glutathione precursor, cystine, in astrocytes, but not in neurons

Maintenance of adequate intracellular glutathione (GSH) levels is vital for intracellular defense against oxidative damage. The toxic effects of methylmercury (MeHg) are attributable, at least in part, to elevated levels of reactive oxygen species, and thus decreases in GSH synthesis may increase methylmercury toxicity. Astrocytes have recently been proposed to play an essential role in providing GSH precursors to neurons. Therefore, cystine transport, a prerequisite to GSH production, was characterized in cultured astrocytes and neurons, and the effects of methylmercury on this transport were assessed. Astrocytes and neurons both possessed temperature dependent transport systems for cystine. Astrocytes accumulated cystine by Na +-independent (X C−) and -dependent (X AG−) systems while neurons used exclusively Na +-independent systems. Inhibition of the X AG− transport system decreased cystine transport in astrocytes to levels equivalent to those in sodium-depleted conditions, suggesting that cystine is carried by a glutamate/aspartate transporter in astrocytes. Inhibition of the multifunction ectoenzyme/amino acid transporter γ-glutamyltranspeptidase (GGT) decreased cystine transport in both neurons and astrocytes. Inhibition of System X C− with quisqualate also decreased cystine uptake in both astrocytes and neurons. These data demonstrate that cultured astrocytes accumulate cystine via three independent mechanisms, System X AG−, System X C−, and GGT, while cultured hippocampal neurons use System X C− and GGT exclusively. Inhibition of cystine uptake in astrocytes by methylmercury appears to be due to actions on the System X AG− transporter.

Characterization of 3‐[123I]iodo‐L‐α‐methyl tyrosine transport in astrocytes of neonatal rats

3-[(123)I]Iodo-L-alpha-methyl tyrosine ((123)I-IMT) is used for diagnosis and monitoring of brain tumours by means of single-photon emission tomography. As recently shown, (123)I-IMT is predominantly mediated into rat C6 glioma cells by sodium-independent system L for large neutral amino acids. Until now, (123)I-IMT transport in non-neoplastic glial cells has not been examined. Therefore, the aim of this study was to examine the cellular pathways and precise transport kinetics of (123)I-IMT uptake into astrocytes of neonatal rats. In particular sodium-independent (123)I-IMT transport into neonatal astrocytes was compared with sodium-independent (123)I-IMT uptake into neoplastic rat C6 glioma cells. Competitive inhibition experiments showed that (123)I-IMT is exclusively transported via sodium-independent system L into the neonatal astrocytes (92%). Kinetic analysis of sodium-independent (123)I-IMT uptake into neonatal astrocytes and into C6 glioma cells revealed apparent Michaelis constants K(M) = 13.9 +/- 0.5 microM and K(M) = 33.9 +/- 4.1 microM, respectively, which are in the same range of K(M) values as those recently determined for amino acid transport into neoplastic and non-neoplastic glial cells. Indeed, the K(M) values in the micromolar range correspond to the expression of the LAT-1 subunit of system L both in the neonatal astrocytes and in C6 glioma cells. However, sodium-independent maximum transport velocities (V(max)) differed significantly between neonatal astrocytes and C6 glioma cells (11.1 +/- 0.3 and 39.9 +/- 3.3 nmol/mg protein/10 min, respectively).

Polyamine uptake in cultured astrocytes: characterization and modulation by protein kinases.

The properties and regulation of the polyamine transport system in brain are still poorly understood. The present study shows, for the first time, the existence of a polyamine transport system in cerebellar astrocytes and suggests that polyamine uptake is mediated by a single and saturable high-affinity transport system for putrescine, spermine, and spermidine (K:(m) = 3.2, 1.2, and 1.8 microM:, respectively). Although substitution of NaCl by choline chloride produced a decrease in the putrescine, spermine, and spermidine uptake, it seems that polyamine transport in cerebellar astrocytes is not mediated by an Na(+) cotransport as in the presence of Na(+) and cholinium, polyamine uptake was much lower than when measured in a sucrose-based medium. On the other hand, ouabain, gramicidin (a Na(+) ionophore), and ionomycin (a Ca(2+) ionophore) produced a strong inhibition of polyamine uptake, suggesting that membrane potential could have an important role in the functioning of the astroglial polyamine uptake system. Moreover, protein kinase C inhibition produced an enhancement of polyamine uptake, whereas stimulation of protein kinase C with phorbol esters inhibited polyamine uptake. Alternatively, the tyrosine kinase inhibitor genistein caused a marked reduction in the uptake. No effects on polyamine uptake were observed with inhibitors and activators of cyclic AMP-dependent protein kinase or when Ca(2+)/calmodulin-dependent protein kinase II was inhibited with KN-62. These results suggest that the polyamine uptake system in cerebellar astrocytes could be modulated by protein kinase C and tyrosine kinase activities.

Amygdala‐Kindling and Pentylenetetrazole‐Induced Seizures in Glutamate Transporter GLAST‐Deficient Mice

Purpose: A number of previous ncurophysiological studies have indicated that the glutamatergic system is important in the induction of epileptiform activity and the dcvelopment of epileptogenesis. Clutamate transport is the primary mechanism of inactivation of syiiaptically released glutamate. GLAST is classified BS an astrocytic transporter and occurs in high concentrations in the ccrebcllum. The pathophysiologic rolc of GLAST in epilepsy is not known in detail. To investigate the role of thc astroglial glutamatc transporter GLAST in epileptogenesis, we compared amygdalu‐kindling and pentylenctetrazolc (PTZ) induced seizures in GLAST‐deficient mice (GLAST(‐/‐)) wild‐type mice (GLAST(+/+)), and maternal C57Black6/J mice (C57). Methods: Kindling: All mice were between 10–1 I weeks of age. Under diethyl ether anesthesia, a chronic tripolar electrode w taxically implanted into the left amygdala (CS7: n = 6, GLAST(+/+): n = 3, CLAST(‐I‐): n = 5). All rats underwent kindling stimulation once daily that coiisistcd of 2 second duration of 100 Hz monophasic square pulses at the intensity of thc afterdischarge (AD) threshold. The seizure responses of thc kindled mice were classified as follows: stage 0, no response; stage 1, mouth and facial movement; stage 2, forclimb clonus; stage 3, bilateral forclimb clonus and tail up. The numbers of stimulations and AD durations required eliciting stage I or stage 2 werc compared, and the mean AD duration, latency and acizurc duration or five consecutive stagc 3 scizures (generalized scizures) were compared in each type of mouse. PTZ: PTZ was injected i.p. at a dose of SO mg/kg (GLAST(+/+): n = 13, GLAST(‐/‐): n = I3), and each mousc was observed for 30 min after the injection. Seizure rcsponses to PTZ werc rated as follows: stage 0, no response; stage I, isolated twitches; stagc 2, tonic‐chic convulsion; stage 3, tonic extension or death. Results: Kindling: No significant differcnces wcre scen in the first responses of amygdala‐kindled GLAST knockout mice. Nor were any significant differences seen in kindling seizure development. The mean AD duration, latency, and scizure duration of 5 consccutive stage 3 seizures in GLAST(‐/‐), GLAST(+/+), and C57 were compared. No significant differences were seen in AD duration and latency, but the generalized seizure duration (stage 3) of the AM‐kindled scizures in the GLAST(‐/‐) was significantly (approximately 35%) longer than in CS7. PTZ: GLAST(‐/‐) showed more severe scizurc stages of PTZ induced generalized seizures than GLAST(+/+) (p < 0.02, MannWhitney rank sum test), and seizure latency was significantly shortcr in the mutant mice (p < 0.005, unpaired t‐test). Conclusions: The results of present study clearly demonstrate that GLAST has different roles according to the type of epilepsy. In amygdala‐kindling, a limbic partial epilepsy model with secondary generalization, GLAST seems to have little role in seizure induction or the development of epileptogenesis in the limbic focus. Secondarily generalized seizures, on the other hand, may be modulated by GLAST, since the duration of bilateral forelimb clonus was found to be significantly longer in GLAST knockout mice. Several previous experiments have suggested significant increases in GLAST mRNA expression after amygdala‐kindled seizures or systemic administration of kainic acid. In contrast to kindled seizures, GLAST is closely related to the neuronal mcchanism underling seizure induction and severity in PTZ‐induced seizures, a primary generalized epilepsy model. GLAST may participate in the modulation of generalized tonicchic seizure activity in the cerebral cortex, because GLAST is widely localized i n the cerebral cortex, and the basic mechanism underling PTZ‐induced seizures is rclated to thalamocortical circuitry. Although no spontaneous seizures or EEG paroxysmal discharges were observed in GLAST knockout mice, it is concluded that GLAST is involved in the molecular mechanism of genetic idiopathic generalized epilepsy.

VIQ‐PIQ Discrepancies in Partial Epilepsy: On the Relation to Lat‐ eralities of Focal MRI Lesions, P3 Peaks, and Focal Spikes.

Purpose: A number of previous ncurophysiological studies have indicated that the glutamatergic system is important in the induction of epileptiform activity and the dcvelopment of epileptogenesis. Clutamate transport is the primary mechanism of inactivation of syiiaptically released glutamate. GLAST is classified BS an astrocytic transporter and occurs in high concentrations in the ccrebcllum. The pathophysiologic rolc of GLAST in epilepsy is not known in detail. To investigate the role of thc astroglial glutamatc transporter GLAST in epileptogenesis, we compared amygdalu‐kindling and pentylenctetrazolc (PTZ) induced seizures in GLAST‐deficient mice (GLAST(‐/‐)) wild‐type mice (GLAST(+/+)), and maternal C57Black6/J mice (C57). Purpose: Subtest IQ such as verbal IQ (VIQ) and performance IQ (PIQ) in WAIS or WISC are thought to represent neuropsychological functions of the left and right hemispheres, respectively. The P300 (P3) event‐related potential reflects cognitive processes. We do not ye1 know the brain site of P3 origin or how epileptogenic foci (EF) influ‐ ence P3 potentials. To examine neuropsychological influence by partial epilepsy (PE), we studied VIQ‐PIQ discrepancies in PE in relation to lateralities of focal MRI lesions, P3 peaks, and EF. Methods: Thirteen patients showed VIQ‐PIQ discrepancies significant at the p7lt;O.O5 level, represented by a>l2‐point spread for the WAIS in adults, and a 15‐point spread in the WISC in children. We evoked P3 potentials in the individuals with discrepant IQ differences by asking them to keep a mental count of rare tones, including introduction of oddbail tones. EEGs were recorded by the international 10–20 system and P3 peaks were shown in a topographical view by offline analysis. Patients were divided into normal and abnormal groups according to MRI findings, and were examined for the laterali‐ ties of the dominant side in subtest IQ (conventionally, we regarded higher VIQ as left hemisphere dominant and higher PIQ as right hemisphere dominant), P3 peaks, and EF. We did not correlate results with lert or right handedness. Results: Five patients (38.5%) were in the normal group and 8 patients (61.5%) were in the abnormal group. Concordance of the lateralities in P3 peaks and dominant side in subtest IQ was shown in 1 patient (20%) in the normal group and 5 patients (62.5%) in the abnormal group. In the normal group, all patients showed contralateral P3 peak shift to EF, and all except I patient showed contralateral P3 peak shift to the dominant side in subtest IQ. The other 3 patients in the abnormal group showed unilateral focal cortical dysplasias (FCD), ipsilateral P3 shift, and contralateral dominant side in subtest IQ to the focal MRI lesions. Conclusion: In our partial epilepsy series with VIQ‐PIQ discrepancies, concordance of the lateralities in P3 peaks and dominant side in subtest IQ was shown in < half of the patients. Epileptogenic foci seem to have 3 different grades of influence on P3 peak shift and dominant side in subtest IQ according to the severities of accompanying focal MRI lesions: 1. Without MRI lesions, EF can make P3 peak shift contralaterally, but the dominant side in the subtest IQ shift ipsilaterally; 2. With less severe focal MRI lesions such as hippocampal atrophy etc., EF can make not only P3 peaks but also the dominant side in the subtest IQ shift contralaterally; 3. With severe focal MRI lesions such as FCD, EF can make the dominant side in the subtest IQ shift contralaterally, but the P3 peak may shift ipsilaterally. Epileptogenic foci without MRI lesions seem to control ipsilateral P3 potentials. MRI lesions render a hemisphere unlikely to become dominant, but epileptogenic foci can coexist with apparently normal neuropsychological function.

Excitatory amino acid transporter 1 and 2 immunoreactivity in the spinal cord in amyotrophic lateral sclerosis.

The spinal cord of 20 patients with amyotrophic lateral sclerosis (ALS) and 5 patients with lower motor neuron disease (LMND) were investigated immunohistochemically using anti-human excitatory amino acid transporter 1 (EAAT1) and EAAT2 antibodies which are the astrocytic transporters. The purpose of the study was to examine relationships between EAAT1 and EAAT2 immunoreactivity and degeneration of anterior horn neurons. Specimens from 20 patients without any neurological disease served as controls. In controls, spinal cord gray matter was densely immunostained by antibodies, whereas the white matter was generally not immunostained. In motor neuron disease (MND) patients, EAAT1 immunoreactivity was relatively well preserved in the gray matter despite neuronal loss of anterior horn cells. On the other hand, EAAT2 immunoreactivity in anterior horns correlated with the degree of neuronal loss of anterior horn cells: in the patients with mild neuronal depletion, anterior horns were densely immunostained by the antibody, whereas in the patients with severe neuronal loss, EAAT2 expression was markedly reduced. Degenerated anterior horn cells frequently showed a much denser EAAT1 and EAAT2 immunoreactivity around the surface of the neurons and their neuronal processes than that observed in normal-appearing neurons. There was no difference in the expression of EAAT1 and EAAT2 immunoreactivity between LMND and ALS patients. These findings suggest that in the early stage of degeneration of anterior horn cells, EAAT1 and EAAT2 immunoreactivity is preserved in the astrocytic foot directly attached to normal-appearing neurons, whereas levels of EAAT1 and EAAT2 protein rather increase in the astrocytic foot directly attached to degenerated anterior horn neurons; the latter effect most probably reduces the elevated glutamate level, compensates for the reduced function of astroglial glutamate transporters, or represents a condensation of EAAT1 and EAAT2 immunoreactivity secondary to loss of neurites and greater condensation of astrocytic processes. Thus, we demonstrate a difference in EAAT1 and EAAT2 immunoreactivity in different stages of progression in ALS, as a feature of the pathomechanism of this disease.

Methylmercury alters glutamate transport in astrocytes

Methylmercury (MeHg) is a significant environmental contaminant that will continue to pose great risk to human health. Considerable attention in the scientific and health policy fora is focused on the question of whether MeHg intake from a diet high in fish is associated with aberrant CNS function. A number of recent studies ( Kjellstrom et al., 1986: Kjellstrom, T., Kennedy, P., Wallis, S., Mantell, C., 1986. Physical and mental development of children with prenatal exposure to mercury from fish. Stage I: preliminary tests at age 4. Solna, Sweden. National Swedish Environmental Protection Board Report 3080, 1989: Kjellstrom, T., Kennedy, P., Wallis, S., Stewart, A., Friberg, L. et al., 1989. Physical and mental development of children with prenatal exposure to mercury from fish. Stage II: interviews and psychological tests at age 6. Solna, Sweden. National Swedish Environmental Protection Board Report 3642; McKeown-Eyssen et al., 1983: McKeown-Eyssen, G., Ruedy, J., Neims, A., 1983. Methylmercury exposure in Northern Quebec II: neurologic findings in children. American Journal of Epidemiology 118, 470–479; Grandjean et al., 1997: Grandjean, P., Weihe, P., White, R. F., Debes, F., Araki, S., Yokoyama, K., Murata, K., Sorensen, N., Dahl, R., Jorgensen, P. J., 1997. Cognitive deficit in 7-year-old children with prenatal exposure to methylmercury. Neurotoxicology and Teratology 19, 417–428) suggest that fetal exposure at levels attained by mothers eating fish regularly during pregnancy are associated with neurological deficits in their offspring. Astrocytes play a key role in MeHg-induced excitotoxicity. (1) MeHg preferentially accumulates in astrocytes. (2) MeHg potently and specifically inhibits glutamate uptake in astrocytes. (3) Neuronal dysfunction is secondary to disturbances in astrocytes. (4) Co-application of nontoxic concentrations of MeHg and glutamate leads to the typical appearance of neuronal lesions associated with excitotoxic stimulation. (5) MeHg induces swelling of astrocytes. These observations are fully consistent with MeHg-induced dysregulation of excitatory amino acid homeostasis, and indicate that a glutamate-mediated excitotoxic mechanism is involved. This manuscript details the role of astrocytes in mediating MeHg-induced excitotoxicity, and elaborates on the protective role afforded by metallothioneins (MTs) in attenuating MeHg cytotoxicity.

Isolation of Current Components and Partial Reaction Cycles in the Glial Glutamate Transporter EAAT2

The kinetic properties of the excitatory amino acid transporter EAAT2 were studied using rapid applications of L-glutamate to outside-out patches excised from transfected human embryonic kidney 293 cells. In the presence of the highly permeant anion SCN(-), pulses of glutamate rapidly activated transient anion channel currents mediated by the transporter. In the presence of the impermeant anion gluconate, glutamate pulses activated smaller currents predicted to result from stoichiometric flux of cotransported ions. Both anion and stoichiometric currents displayed similar kinetics, suggesting that anion channel gating and stoichiometric charge movements are linked to early transitions in the transport cycle. Transporter-mediated anion currents were recorded with ion and glutamate gradients favoring either unidirectional influx or exchange. Analysis of deactivation and recovery kinetics in these two conditions suggests that, after binding, translocation of substrate is more likely than unbinding under physiological conditions. The kinetic properties of EAAT2, the dominant glutamate transporter in brain astrocytes, distinguish it as an efficient sink for synaptically released glutamate.

GSH transport in human cerebrovascular endothelial cells and human astrocytes: evidence for luminal localization of Na +-dependent GSH transport in HCEC

The purpose of the present study was to identify and localize glutathione (GSH) transport in an in vitro tissue culture model of blood–brain barrier (BBB). The localization of Na +-dependent GSH transport in an immortalized cell line of human cerebrovascular endothelial cells (HCEC) and asymmetry of transport in Transwell™ studies were investigated. Initial studies with cultured HCEC established a significant (45%) Na +-dependency for GSH uptake in cultured HCEC pretreated with acivicin, an inhibitor of γ-glutamyltranspeptidase (GGT). Transendothelial electrical resistance (TEER) and uptake of [ 35 S ]GSH from luminal and abluminal fluids of HCEC were measured in Na +-containing and Na +-free (choline chloride) buffers using cells grown on gelatin-coated membrane filters. TEER of HCEC monolayers in regular medium was 40.1±8.0 Ω cm 2. Human astrocyte-conditioned medium (ACM) caused no change in TEER, but increased GGT activity approximately threefold when measured in cell lysates. Luminal and abluminal GSH uptake increased in a time-dependent fashion and were not affected by inhibition of GGT activity with acivicin. Sodium dependency was only observed for luminal uptake (Na +-containing 2.41±0.15 vs. Na +-free 0.96±0.03 pmol/30 min/million cells, p<0.001) but not for abluminal uptake (1.02±0.13 vs. 1.11±09, p>0.05). Apparent efflux via the luminal membrane was lower in the presence of sodium as compared to that without sodium, further suggesting that a Na +-dependent uptake process for GSH is operative at this membrane. GSH uptake and efflux were also demonstrated in neonatal rat and fetal human astrocytes, both exhibiting partial Na +-dependency of uptake. In conclusion, our results show for the first time, that HCEC and astrocytes take up GSH by both Na +-dependent and -independent mechanisms. The Na +-dependent GSH transport process in HCEC appears to be localized to luminal plasma membranes of HCEC.

Effect of dibutyryl cyclic AMP on the kinetics of myo-inositol transport in cultured astrocytes.

Dibutyryl cyclic AMP (dBcAMP) is known to induce maturation and differentiation in astrocytes. As myo-inositol is an important osmoregulator in astrocytes, we examined the effects of maturation and biochemical differentiation on the kinetic properties of myo-inositol transport. Treatment of astrocytes with dBcAMP significantly decreased the Vmax of myo-inositol uptake, but the effect on Km was not significant. The myo-inositol content of astrocytes was significantly decreased in cells treated for 5 days with dBcAMP as compared with untreated controls. Maximum suppression of myo-inositol uptake occurred 7 days after exposure of astrocytes to dBcAMP; this was gradually reversible when dBcAMP was removed from the medium. After exposure to hypertonic medium for 6 h, mRNA expression of the myo-inositol co-transporter was diminished by approximately 36% in astrocytes treated with dBcAMP as compared with untreated cells. It appears that myo-inositol transporters in astrocytes treated with dBcAMP are either decreased in number or inactivated during maturation and differentiation, suggesting that the stage of differentiation and biochemical maturation of astrocytes is an important factor in osmoregulation.

Lactate Transport by Cortical Synaptosomes from Adult Rat Brain: Characterization of Kinetics and Inhibitor Specificity

Since lactate released by glial cells may be a key substrate for energy in neurons, the kinetics for the uptake of L-[U-¹⁴C]lactate by cortical synaptic terminals from 7- to 8-week-old rat brain were determined. Lactate uptake was temperature-dependent, and increased by 64.9% at pH 6.2, and decreased by 43.4% at pH 8.2 relative to uptake at pH 7.3. Uptake of monocarboxylic acids was saturable with increasing substrate concentration. Eadie-Hofstee plots of the data gave evidence of two carrier-mediated uptake mechanisms with a high-affinity K_m of 0.66 mM and V_max of 3.66 mM for pyruvate, and a low-affinity system with a K_m of 9.9 mM for both lactate and pyruvate and V_max values of 16.6 and 23.1 nmol/30 s/mg protein for lactate and pyruvate, respectively. Saturable uptake was seen in the presence of 10 mM α-cyano-4-hydroxycinnamate. Lactate transport by synaptic terminals was much more sensitive to inhibition by sulfhydryl reagents than transport in astrocytes. Addition of 0.5 and 2 mM mersalyl decreased the uptake of 1 mM lactate by synaptic terminals by 59.3 and 66.37%, respectively. Pyruvate moderately decreased lactate transport, whereas 3-hydroxybutyrate had little effect. Quercetin, an inhibitor of lactate release, had little effect on the content of ¹⁴C lactate in synaptic terminals, supporting the concept that the majority of lactate produced within brain is from glial cells. Oxidation of L-[U-¹⁴C]lactate by synaptosomes was saturable, and yielded a K_m of 1.23 mM and a V_max of 116 nmol/h/mg protein. Overall the studies show that synaptic terminals from adult brain have a high capacity for transport and oxidation of lactate, consistent with the proposed role for this compound in metabolic trafficking in brain. Furthermore, the data provide kinetic evidence of two carrier-mediated mechanisms for monocarboxylic acid transport by synaptosomes and demonstrate that uptake of lactate by synaptic terminals is regulated differently than transport by astrocytes. Uptake of lactate by synaptic terminals also has differences from the systems described for neurons.

Glial Contribution to Glutamate Uptake at Schaffer Collateral–Commissural Synapses in the Hippocampus

Astrocytes in the hippocampus express high-affinity glutamate transporters that are important for lowering the concentration of extracellular glutamate after release at excitatory synapses. These transporters exhibit a permeability to chaotropic anions that is associated with transport, allowing their activity to be monitored in cell-fee patches when highly permeant anions are present. Astrocyte glutamate transporters are highly temperature sensitive, because L-glutamate-activated, anion-potentiated transporter currents in outside-out patches from these cells exhibited larger amplitudes and faster kinetics at 36 degreesC than at 24 degreesC. The cycling rate of these transporters was estimated by using paired applications of either L-glutamate or D-aspartate to measure the time necessary for the peak of the transporter current to recover from the steady-state level. Transporter currents in patches recovered with a time constant of 11.6 msec at 36 degreesC, suggesting that either the turnover rate of native transporters is much faster than previously reported for expressed EAAT2 transporters or the efficiency of these transporters is very low. Synaptically activated transporter currents persisted in astrocytes at physiological temperatures, although no evidence of these currents was found in CA1 pyramidal neurons in response to afferent stimulation. L-glutamate-gated transporter currents were also not detected in outside-out patches from pyramidal neurons. These results are consistent with the hypothesis that astrocyte transporters are responsible for taking up the majority of glutamate released at Schaffer collateral-commissural synapses in the hippocampus.

Preferential Utilization of Acetate by Astrocytes Is Attributable to Transport

Exogenous acetate is preferentially metabolized by astrocytes in the CNS, but the biochemical basis for this selectivity is unknown. We observed that rat cortical astrocytes produce 14CO2 from 0.2 mM [14C]acetate at a rate of 0.43 nmol/min per milligram of protein, 18 times faster than cortical synaptosomes. Subsequent studies examined whether this was attributable to cellular differences in the transport or metabolism of acetate. The activity of acetyl-CoA synthetase, the first enzymatic step in acetate utilization, was greater in synaptosomes than in astrocytes (5.0 and 2.9 nmol/min per milligram of protein), indicating that slower metabolism in synaptosomes cannot be attributed to lack of enzymatic activity. [14C]Acetate uptake in astrocytes is rapid and time-dependent and follows saturation kinetics (Vmax, 498 nmol/min per milligram of protein; Km, 9.3 mM). Uptake is inhibited stereospecifically by L-lactate as well as by pyruvate, fluoroacetate, propionate, and alpha-cyano-4-hydroxycinnamate (CHC). Preloading astrocytes with L-lactate or acetate, but not D-lactate, pyruvate, or glyoxylate, transaccelerates [14C]acetate uptake. Acetate uptake by astrocytes appears to be mediated by a carrier with properties similar to that of monocarboxylate transport. In contrast, studies with synaptosomes provided no evidence for time-dependent, saturable, transaccelerated, or CHC-inhibitable uptake of [14C]acetate. The high rate of transport in astrocytes compared with synaptosomes explains the rapid incorporation of [14C]acetate into brain glutamine over glutamate. These findings provide support for the use of acetate as a marker for glial metabolism and suggest that extracellular acetate in the brain generated from acetylcholine and ethanol metabolism is accumulated first by astrocytes.

Serotonin transporters in adult rat brain astrocytes revealed by [3H]5-HT uptake into glial plasmalemmal vesicles

Cultured astrocytes derived from neonatal rat brain exhibited high affinity, Na+-dependent, paroxetine and fluoxetine sensitive [3H] 5-HT uptake. Reverse transcriptase-PCR demonstrated that astrocytes in culture expressed messenger RNA for the cloned serotonin transporter protein which has been characterised as the neuronal serotonin transporter. Although the serotonin transporter in cultured astrocytes displayed a Km value approximately 10 times greater than found in adult brain synaptosomes, these observations indicated that astrocytes in vitro may express the same serotonin transporter as neurons. Reverse transcriptase-PCR demonstrated the presence of serotonin transporter mRNA in the adult rat cerebral cortex, suggesting that astrocytes in vivo may express low levels of this mRNA. To investigate whether astrocytes in the adult CNS express functional serotonin transporters, glial plasmalemmal vesicles were prepared from cerebral cortex, representing a subcellular fraction composed primarily of vesicles derived from astrocytes. These vesicles were characterised by [3H]-glutamate and [3H]-dopamine uptake and by immunoblot analysis, using glial and synaptic markers: glutamate synthase, SNAP-25 and synaptobrevin. [3H]5-HT was taken up into glial plasmalemmal vesicles in a high affinity (Km approximately 40 nM), Na + dependent, paroxetine-sensitive manner. The [3H]5-HT uptake capacity (Vmax ) in these vesicles was approximately one quarter of that observed in synaptosomes. These data indicate that astrocytes in culture and in vivo are capable of 5-HT uptake via the previously characterised ‘neuronal’ serotonin transporter.

Serotonin transporters in adult rat brain astrocytes revealed by [3H]5-HT uptake into glial plasmalemmal vesicles

Serotonin transporters in adult rat brain astrocytes revealed by [3H]5-HT uptake into glial plasmalemmal vesicles

4-hydroxynonenal, a lipid peroxidation product, impairs glutamate transport in cortical astrocytes.

Astrocytes possess plasma membrane glutamate transporters that rapidly remove glutamate from the extracellular milieu and thereby prevent excitotoxic injury to neurons. Cellular oxidative stress is increased in neural tissues in a variety of acute and chronic neurodegenerative conditions. Recent findings suggest that oxidative stress increases neuronal vulnerability to excitotoxicity and that membrane lipid peroxidation plays a key role in this process. We now report that 4-hydroxynonenal (HNE), an aldehydic product of membrane lipid peroxidation, impairs glutamate transport in cultured cortical astrocytes. Impairment of glutamate transport occurred within 1-3 h of exposure to HNE; FeSO4, an inducer of membrane lipid peroxidation, also impaired glutamate transport. Vitamin E prevented impairment of glutamate transport induced by FeSO4, but not that induced by HNE, consistent with HNE acting as an effector of lipid peroxidation-induced impairment of glutamate transport. Glutathione, which binds and thereby detoxifies HNE, prevented HNE from impairing glutamate transport. Western blot, immunoprecipitation, and immunocytochemical analyses using an antibody against HNE-protein conjugates provided evidence that HNE covalently binds to many different astrocytic proteins including the glutamate transporter GLT-1. Data further suggest that HNE promotes intermolecular cross-linking of GLT-1 monomers to form dimers. HNE also induced mitochondrial dysfunction and accumulation of peroxides in astrocytes. Impairment of glutamate transport and mitochondrial function occurred with sublethal concentrations of HNE, concentrations known to be generated in cells exposed to various oxidative insults. Collectively, our data suggest that HNE may be an important mediator of oxidative stress-induced impairment of astrocytic glutamate transport and may thereby play a role in promoting neuronal excitotoxicity.

Synaptic Activation of Glutamate Transporters in Hippocampal Astrocytes

Glutamate transporters in the CNS are expressed in neurons and glia and mediate high affinity, electrogenic uptake of extracellular glutamate. Although glia have the highest capacity for glutamate uptake, the amount of glutamate that reaches glial membranes following release and the rate that glial transporters bind and sequester transmitter is not known. We find that stimulation of Schaffer collateral/commissural fibers in hippocampal slices evokes glutamate transporter currents in CA1 astrocytes that activate rapidly, indicating that a significant amount of transmitter escapes the synaptic cleft shortly after release. Transporter currents in outside-out patches from astrocytes have faster kinetics than synaptically elicited currents, suggesting that the glutamate concentration attained at astrocytic membranes is lower but remains elevated for longer than in the synaptic cleft.

Glutamate transport and metabolism in astrocytes.

Glutamate uptake and metabolism was studied in cerebral cortical astrocytes. The expression of the astrocytic glutamate transporter GLAST was found to be stimulated by extracellular glutamate through activation of kainate receptors on the astrocytes. Energy metabolism and ammonia homeostasis are two important aspects of glutamate handling in astrocytes. It is well known that glutamate transport into astrocytes and glutamine formation are energy consuming processes. Furthermore, ammonia is required for glutamine production. On the other hand, glutamate metabolism through the tricarboxylic acid cycle is an energy and ammonia producing pathway. In the present study it was shown that at an extracellular glutamate concentration of 0.5 mM, high energy phosphates were reduced, and more than 50% of the glutamate carbon skeleton entered the tricarboxylic acid cycle to yield products like lactate, aspartate, and additionally glutamate and glutamine derived from tricarboxylic acid cycle intermediates. Entry into the cycle was not affected by the transaminase inhibitor aminooxyacetic acid, indicating that deamination is the major route for 2-oxoglutarate formation from glutamate. Synthesis of glutamate from 2-oxoglutarate, however, proceeded via transamination. In an earlier study it was shown that at glutamate concentrations at and below 0.2 mM, glutamine appears to be the major product and entry of glutamate into the tricarboxylic acid cycle is decreased 70% by aminooxyacetic acid. In an attempt to unify the above mentioned results, it is suggested that availability of ammonia and energy demands are major factors determining the metabolic fate of glutamate in astrocytes.

Cerebral astrocytes transport ascorbic acid and dehydroascorbic acid through distinct mechanisms regulated by cyclic AMP.

Cerebral ischemia and trauma lead to rapid increases in cerebral concentrations of cyclic AMP and dehydroascorbic acid (DHAA; oxidized vitamin C), depletion of intracellular ascorbic acid (AA; reduced vitamin C), and formation of reactive astrocytes. We investigated astrocytic transport of AA and DHAA and the effects of cyclic AMP on these transport systems. Primary cultures of astrocytes accumulated millimolar concentrations of intracellular AA when incubated in medium containing either AA or DHAA. AA uptake was Na+-dependent and inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), whereas DHAA uptake was Na+-independent and DIDS-insensitive. DHAA uptake was inhibited by cytochalasin B, D-glucose, and glucose analogues specific for facilitative hexose transporters. Once inside the cells, DHAA was reduced to AA. DHAA reduction greatly decreased astrocytic glutathione concentration. However, experiments with astrocytes that had been previously depleted of glutathione showed that DHAA reduction does not require physiological concentrations of glutathione. Astrocyte cultures were treated with a permeant analogue of cyclic AMP or forskolin, an activator of adenylyl cyclase, to induce cellular differentiation and thus provide in vitro models of reactive astrocytes. Cyclic AMP stimulated uptake of AA, DHAA, and 2-deoxyglucose. The effects of cyclic AMP required at least 12 h and were inhibited by cycloheximide, consistent with a requirement for de novo protein synthesis. Uptake and reduction of DHAA by astrocytes may be a recycling pathway that contributes to brain AA homeostasis. These results also indicate a role for cyclic AMP in accelerating the clearance and detoxification of DHAA in the brain.