Lowering ambient or core body temperature elevates striatal MPP+ levels and enhances toxicity to dopamine neurons in MPTP-treated mice

The neuroprotective effects of lowering body temperature have been well documented in various models of neuronal injury. The present study investigated the effects a lower ambient or core body temperature would have on damage to striatal dopamine (DA) neurons produced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Mice received systemic MPTP treatment at two different temperatures, 4 degrees C and 22 degrees C. MPTP-treated mice maintained at 4 degrees C demonstrated (1) a greater hypothermic response, (2) a significant reduction in striatal DA content and tyrosine hydroxylase (TH) activity, and (3) significantly greater striatal 1-methyl-4-phenylpyridinium (MPP+) levels, as compared to mice dosed with MPTP at room temperature. Parallel studies with methamphetamine (METH) were conducted since temperature appears to play a pivotal role in the mediation of damage to DA neurons by this CNS stimulant in rodents. As previously reported, METH-induced hyperthermia and the subsequent loss of striatal DA content were attenuated in animals dosed at 4 degrees C. We also evaluated the effects a hypothermic state induced by pharmacological agents would have on striatal neurochemistry and MPP+ levels following MPTP treatment. Concurrent administration of MK-801 or 8-OHDPAT increased the striatal MPP+ levels following MPTP treatment. However, only 8-OHDPAT potentiated the MPTP-induced decrements of striatal DA content and TH activity; MK-801 did not affect MPTP decreases in these striatal markers of dopaminergic damage. Altogether, these findings indicate that temperature has a profound effect on striatal MPP+ levels and MPTP-induced damage to DA neurons in mice.     

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyride neurotoxicity is attenuated in mice overexpressing Bcl-2

The proto-oncogene Bcl-2 rescues cells from a wide variety of insults. Recent evidence suggests that Bcl-2 protects against free radicals and that it increases mitochondrial calcium-buffering capacity. The neurotoxicity of 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyride (MPTP) is thought to involve both mitochondrial dysfunction and free radical generation. We therefore investigated MPTP neurotoxicity in both Bcl-2 overexpressing mice and littermate controls. MPTP-induced depletion of dopamine and loss of [3H]mazindol binding were significantly attenuated in Bcl-2 overexpressing mice. Protection was more profound with an acute dosing regimen than with daily MPTP administration over 5 d. 1-Methyl-4-phenylpyridinium (MPP+) levels after MPTP administration were similar in Bcl-2 overexpressing mice and littermates. Bcl-2 blocked MPP+-induced activation of caspases. MPTP-induced increases in free 3-nitrotyrosine levels were blocked in Bcl-2 overexpressing mice. These results indicate that Bcl-2 overexpression protects against MPTP neurotoxicity by mechanisms that may involve both antioxidant activity and inhibition of apoptotic pathways.     

Stimulation of glutamate uptake and Na,K-ATPase activity in rat astrocytes exposed to ischemia-like insults

The postsynaptic actions of glutamate are rapidly terminated by high affinity glutamate uptake into glial cells. In this study we demonstrate the stimulation of both glutamate uptake and Na,K-ATPase activity in rat astrocyte cultures in response to sublethal ischemia-like insults. Primary cultures of neonatal rat cortical astrocytes were subjected to hypoxia, or to serum- and glucose-free medium, or to both conditions (ischemia). Cell death was assessed by propidium iodide staining of cell nuclei. To measure sodium pump activity and glutamate uptake, 3H-glutamate and 86Rb were both simultaneously added to the cell culture in the presence or absence of 2 mM ouabain. Na,K-ATPase activity was defined as ouabain-sensitive 86Rb uptake. Concomitant transient increases (2-3 times above control levels) of both Na,K-ATPase and glutamate transporter activities were observed in astrocytes after 4-24 h of hypoxia, 4 h of glucose deprivation, and 2-4 h of ischemia. A 24 h ischemia caused a profound loss of both activities in parallel with significant cell death. The addition of 5 mM glucose to the cells after 4 h ischemia prevented the loss of both sodium pump activity and glutamate uptake and rescued astrocytes from death observed at the end of 24 h ischemia. Reoxygenation after the 4 h ischemic event caused the selective inhibition of Na,K-ATPase activity. The observed increases in Na,K-ATPase activity and glutamate uptake in cultured astrocytes subjected to sublethal ischemia-like insults may model an important functional response of astrocytes in vivo by which they attempt to maintain ion and glutamate homeostasis under restricted energy and oxygen supply.     

Effects of ageing on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxic effects on striatum and brainstem in the rat

In 3- and 18-month-old male Wistar rats, levels of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), ascorbic acid (AA), dehydroascorbic acid (DHAA), noradrenaline (NA), uric acid, glutathione (GSH) and 1-methyl-4-phenylpyridinium ion (MPP+) were determined by HPLC in the striatum and/or in the brainstem 24 h after single injections of MPTP (12-35 mg/kg i.p.). Aged rats had lower baseline levels of AA and GSH, compared to young rats. In aged rats, MPTP 35 mg/kg induced a 70% death rate and a decrease in striatal DOPAC/DA ratio which was significantly correlated to MPP+ concentrations (r = -0.840, P < 0.005); in addition, MPTP did not increase AA oxidation. In the brainstem, the MPTP-induced decrease in NA levels and increase in uric acid levels were significantly correlated to the MPP+ concentrations (r = -0.709, P < 0.05, and r = +0.888, P < 0.001, respectively). In conclusion, evidence is given of a mechanism of toxicity of MPTP involving oxidative stress produced by xanthine oxidase; in addition, in aged rats the neuronal antioxidant system (levels of AA and GSH) is considerably lower than in young rats and may play an enabling role in the MPTP age-related neurotoxic effects on striatum and brainstem.     

Tyrosine-533 of rat dopamine transporter: involvement in interactions with 1-methyl-4-phenylpyridinium and cocaine

To improve our understanding of structure-function relationships for neurotransmitter transporters, we performed site-directed mutagenesis of the rat dopamine transporter (DAT) and assessed the functions of the mutants in transiently-expressing COS cells. Tyrosine-533 of rat DAT lies in the 11th transmembrane region, where the corresponding amino acid of human DAT is phenylalanine. Alanine substitution of tyrosine-533 (Y533A) conferred an increased affinity for 1-methyl-4-phenylpyridinium (MPP+). Phenylalanine substitution of tyrosine-533 (Y533F) increased the velocity of MPP+ uptake but decreased DAT's affinity for MPP+. Cocaine's potency in inhibiting dopamine uptake was unchanged with Y533A, but increased with Y533F. Differences in the uptake kinetics and inhibitory potency of cocaine between rat and human DATs were similar to the differences observed between the wild-type and Y533F mutants DATs. Tyrosine-533 may be important for the DAT function and for species differences in transporter functions, including differential sensitivities to cocaine and 1-methyl-1,2,3,6-tetrahydropyridine (MPTP) in humans and rats.     

Cytotoxic effects of MPTP on SH-SY5Y human neuroblastoma cells

Morphological and metabolic endpoints were used to evaluate MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) toxicity to SH-SY5Y human neuroblastoma cells. After 8 hours of exposure, MPTP was found to affect cell viability only at a very high concentration (3 x 10(-3) M), but its metabolite MPP+ could decrease viability at 10(-4) M. MPTP, via its metabolite MPP+, inhibited NADH dehydrogenase activity when concentrations exceeded 10(-4) M (for MPP+ 10(-5)M). The Ki were 2.4 x 10(-3) M and 3 x 10(-4)M for MPTP and MPP+, respectively. MPTP at concentrations greater than 10(-4) M altered cell morphology as early as one hour after exposure. These changes included formation of cell surface blebs and attenuated neurites. After 8 hours at 10(-3) M and 24 hrs at 10(-4) M, MPTP caused ultrastructural changes of mitochondria with increased electron-density of the matrix and disorganization of cristae, as well as abnormal aggregation of filamentous material of the cytoskeleton. Because these changes of structure and function took place at concentrations lower than those needed to affect cell viability, they may play a role in MPTP neurotoxicity in SH-SY5Y cell culture.     

A role for glutamate and its receptors in the regulation of oligodendrocyte development in cerebellar tissue slices

We tested the hypothesis that the neurotransmitter glutamate would influence glial proliferation and differentiation in a cytoarchitecturally intact system. Postnatal day 6 cerebellar slices were maintained in organotypic culture and treated with glutamate receptor agonists or antagonists. After dissociation, cells were stained with antibodies for different oligodendrocyte developmentally regulated antigens. Treatment of the slices with the glutamate receptor agonists kainate or alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid significantly decreased the percentage of LB1(+), NG2(+) and O4(+) cells, and their bromodeoxyuridine labeling index. The non-N-methyl-D-aspartate glutamate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione increased the percentage and bromodeoxyuridine labeling of LB1(+), NG2(+) and O4(+) cells. In intact slices, RNA levels of the oligodendrocyte gene for 2',3'-cyclic nucleotide 3'-phosphodiesterase were decreased by kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and increased by 6,7-dinitroquinoxaline-2,3-dione. The percentage of astrocytes was not modified by kainate, alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or 6, 7-dinitroquinoxaline-2,3-dione. Treatment with the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonopentanoic acid did not alter the percentage of O4(+) cells, nor their proliferation. Incubation with the gamma-aminobutyric acid receptor antagonist bicuculline did not modify the percentage of LB1(+), A2B5(+) and O4(+) cells. In purified cerebellar oligodendrocyte progenitor cells, glutamate receptor agonists blocked K+ currents, and inhibited cell proliferation and lineage progression. The K+ channel blocker tetraethylammonium also inhibited oligodendrocyte progenitor cell proliferation. These findings indicate that in rat cerebellar tissue slices: (i) glutamate specifically modulates oligodendrocyte but not astrocyte development through selective activation of alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, and (ii) cell depolarization and blockage of voltage-dependent K+ channels is likely to be the triggering mechanism.     

A study of the precision of confocal, ratiometric, Fura-2-based [Ca2+] measurements

Increased ammonia has been considered a key factor in the pathogenesis of hepatic encephalopathy. The high concentration of ammonia interferes with oxidative metabolism in the brain through an inhibitory effect on the tricarboxylic acid cycle (TCA). Inhibition of the TCA cycle may result in depletion of ATP. Due to the involvement of astrocytes in brain detoxification of ammonia, these cells are good candidates for studying ammonia's effect on energy stores in the brain. C6-glioma cells, which have altered glycolytic rates, may show greater sensitivity to the toxicity of ammonium chloride than astrocytes. To study the effect of ammonium chloride on energy storage of both astrocytes and C6-glioma, we observed the acute and chronic effects of NH4Cl (7.5 or 15 mM) on the metabolism of isolated astrocytes and C6-glioma cells. Primary astrocytes were isolated from the cerebral hemispheres of 1-2 day old Sprague-Dawley rats, and C6-glioma cells were purchased from the American Type Culture Collection (ATCC). Following treatment of the cells with ammonia, glucose, lactate, glutamate, ATP, and PCr were assayed. Our data showed that at 15 min following treatment with NH4Cl, there were no significant differences in the concentration of metabolites measured in astrocytes. However, following 15 min of treatment with NH4Cl, the concentration of some metabolites, for example, ATP and lactate, changed significantly in C6-glioma cells. We have shown that 24 h of treatment was sufficient time to see significant biochemical changes but not morphological changes in either cell type. Simultaneous biochemical and morphological changes were observed 48 h following treatment in C6-glioma cells and at 9-10 days following treatment in primary astrocytes. In primary astrocytes at 24 h following treatment, glucose utilization increased. This high utilization of glucose was in accordance with the increase in lactate and glutamate production and the decrease in ATP and PCr formation. In C6-glioma cells the utilization of glucose increased but this high utilization of glucose was consistent with a significant decrease in the concentration of lactate, glutamate and ATP.     

Dopamine transporter is required for in vivo MPTP neurotoxicity: evidence from mice lacking the transporter

The neurotoxic effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was tested on mice lacking the dopamine (DA) transporter (DAT-/- mice). Striatal tissue DA content and glial fibrillary acidic protein (GFAP) mRNA expression were assessed as markers of MPTP neurotoxicity. MPTP (30 mg/kg, s.c., b.i.d.) produced an 87% decrease in tissue DA levels and a 29-fold increase in the level of GFAP mRNA in the striatum of wild-type animals 48 h after administration. Conversely, there were no significant changes in either parameter in DAT-/- mice. Heterozygotes demonstrated partial sensitivity to MPTP administration as shown by an intermediate value (48%) of tissue DA loss. Direct intrastriatal infusion of the active metabolite of MPTP, 1-methyl-4-phenylpyridinium (MPP+; 10 mM), via a microdialysis probe produced a massive efflux of DA in wild-type mice (>320-fold). In the DAT-/- mice the same treatment produced a much smaller increase in extracellular DA (sixfold), which is likely secondary to tissue damage due to the implantation of the dialysis probe. These observations show that the DAT is a mandatory component for expression of MPTP toxicity in vivo.     

A new concept of cotreatment with human growth hormone and menotropins in ovulation induction protocols

Inhibition of Na+/K+ ATPase by cardiac glycosides has been shown to potentiate toxic effects of excitatory amino acids and mitochondrial poisons in neurons in vitro. The present study tested the hypothesis that the systemic administration of the cardiac glycoside, digoxin, potentiates effects of the dopamine neurotoxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) in vivo. Mice were injected with digoxin (1 mg/kg) or vehicle followed by MPTP (20 mg/kg) or saline 1 h later. After 1 or 8 days, mice were euthanized and dopamine levels in the striatum were measured by high-performance liquid chromatography with electrochemical detection. MPTP caused a significant 35-45% reduction in striatal dopamine levels compared to those in control mice. However, pretreatment with digoxin completely prevented the MPTP-induced dopamine depletion. This result was unexpected and suggests that cardiac glycosides may protect against MPTP neurotoxicity.     

Protective effects of the antiparkinsonian drugs talipexole and pramipexole against 1-methyl-4-phenylpyridinium-induced apoptotic death in human neuroblastoma SH-SY5Y cells

Treatment of human neuroblastoma SH-SY5Y cells with 1 mM 1-methyl-4-phenylpyridinium (MPP+) for 3 days induced production of reactive oxygen species (ROS), followed by caspase-3 activation, cleavage of poly(ADP-ribose) polymerase (PARP), and apoptotic cell death with DNA fragmentation and characteristic morphological changes (condensed chromatin and fragmented nuclei). Simultaneous treatment with 1 mM talipexole slightly inhibited the MPP+-induced ROS production and apoptotic cell death. In contrast, pretreatment with 1 mM talipexole for 4 days markedly protected the cells against MPP+-induced apoptosis. However, this protective effect might not be mediated by dopamine receptors. The talipexole pretreatment induced an increase in antiapoptotic Bcl-2 protein level but had no effect on levels of proapoptotic Bax, Bak, and Bad. It also inhibited MPP+-induced ROS production, p53 expression, and cleavages of caspase-3 and PARP. Similarly, pramipexole pretreatment increased Bcl-2 and inhibited MPP+-induced apoptosis. Although pretreatment with bromocriptine also had a protective effect against MPP+-induced apoptosis, it had no effect on the protein levels of Bcl-2 family members. On the other hand, N6,2'-O-dibutyryl cAMP or calphostin C induced a decreased Bcl-2 level and enhanced MPP+-induced cell death. These results suggest that talipexole has dual actions: (1) it directly scavenges ROS, affording slight protection against MPP+-induced apoptosis, and (2) it induces Bcl-2 expression, thereby affording more potent protection, if it is administrated before MPP+. Pramipexole has similar effects, whereas bromocriptine seems to exhibit the former but not the latter effect.     

Deletion polymorphism in the angiotensin-converting enzyme gene as a thrombophilic risk factor after hip arthroplasty

Recent work has suggested a possible role for nitric oxide (NO) in the development of hepatic encephalopathy (HE). In this study, we examined the effect of ammonia and manganese, factors implicated in the pathogenesis of HE, on the transport of arginine (a precursor of NO) into primary cultures of astrocytes. Treatment with 5 mM ammonia for 1-4 days produced a maximal (53%) increase in L-arginine uptake at 3 days when compared to untreated cells. Kinetic analysis following 4-day treatment with 5 mM ammonia revealed an 82% increase in the Vmax and a 61% increase in the Km value. Similar analysis with 100 microM manganese showed a 101% increase in Vmax and a 131% increase in the Km value. These results suggest that both manganese and ammonia alter L-arginine uptake by modifying the transporter for arginine. A decrease of 32% in the non-saturable component of L-arginine transport was also observed following treatment with ammonia. When cultures were treated separately with 5 mM ammonia and 100 microM manganese for 2 days, the uptake of L-arginine increased by 41% and 57%, respectively. Combined exposure led to no further increase in uptake. Our results suggest that ammonia and manganese may contribute to the pathogenesis of HE by influencing arginine transport and thus possibly NO synthesis in astrocytes.     

Identifying crash involvement among older drivers: agreement between self-report and state records

Methamphetamine (METH)- and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity is thought to be associated with the formation of free radicals. Since evidence suggests that melatonin may act as a free radical scavenger and antioxidant, the present study was undertaken to investigate the effect of melatonin on METH- and MPTP-induced neurotoxicity. In addition, the effect of melatonin on METH-induced locomotor sensitization was investigated. The administration of METH (5 mg kg(-1) x 3) or MPTP (20 mg kg(-1) x 3) to Swiss Webster mice resulted in 45-57% depletion in the content of striatal dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, and 57-59% depletion in dopamine transporter binding sites. The administration of melatonin (10 mg kg(-1)) before each of the three injections of the neurotoxic agents (on day 1), and thereafter for two additional days, afforded a full protection against METH-induced depletion of dopamine and its metabolites and dopamine transporter binding sites. In addition, melatonin significantly diminished METH-induced hyperthermia. However, the treatment with melatonin had no significant effect on MPTP-induced depletion of the dopaminergic markers tested. In the set of behavioral experiments, we found that the administration of 1 mg kg(-1) METH to Swiss Webster mice for 5 days resulted in marked locomotor sensitization to a subsequent challenge injection of METH, as well as context-dependent sensitization (conditioning). The pretreatment with melatonin (10 mg kg(-1)) prevented neither the sensitized response to METH nor the development of conditioned locomotion. Results of the present study indicate that melatonin has a differential effect on the dopaminergic neurotoxicity produced by METH and MPTP. Since it is postulated that METH-induced hyperthermia is related to its neurotoxic effect, while regulation of body temperature is unrelated to MPTP-induced neurotoxicity or METH-induced locomotor sensitization, the protective effect of melatonin observed in the present study may be due primarily to diminishing METH-induced hyperthermia.     

Astrocytic glutamate uptake and prion protein expression

Factors influencing glutamate uptake by astrocytes may indirectly influence neuronal survival. Elevated extracellular glutamate may be excitotoxic or may exacerbate neurodegeneration in various neurological diseases. By using a cell culture model, we have investigated the influence of astrocytic prion protein (PrPc) expression on glutamate uptake. Type 1 astrocytes expressing PrPc have a higher rate of Na+-dependent glutamate uptake than PrPc-deficient type 1 astrocytes. This difference is exacerbated when serum free media is used to culture the astrocytes. Further analysis suggested that a decrease in substrate affinity is responsible for the sensitivity of PrP-deficient astrocytic glutamate uptake to culture conditions. PrPc has been shown to bind copper. Greater sensitivity of cells to copper concentrations may be responsible for the decreased substrate affinity observed. PrPc-deficient cerebellar cells are more sensitive to glutamate toxicity in the presence of copper. These results show that glutamate uptake from astrocytes is dependent on PrPc expression which in turn may be related to copper metabolism.     

Interstitial cells of Cajal: mediators of communication between circular and longitudinal muscle layers of canine colon

Autopsy studies of patients with AIDS dementia have shown neuronal loss consistent with a neurotoxic component of this disease. In vitro studies suggest that viral products or cytokines from HIV-infected macrophages (Mphi) may modulate or directly mediate excitotoxic cell death of neurons. Mphi differentiated from peripheral mononuclear blood cultures were infected with HIV, and conditioned media (CM) were harvested from these cultures. Exposure of SK-N-MC (neuroblastoma) cells to CM from HIV-infected Mphi for 4, 24 or > or = 48 h resulted in a mean suppression of 12-34% of the glutamate transport Vmax with no appreciable change in transport Km. An astrocytoma tumor cell, U373MG, showed similar CM-mediated glutamate uptake suppression. Changes were evident in total and Na+-dependent glutamate uptake, with significantly more suppression of Na+-dependent uptake. Similar effects were seen with the nonmetabolizable transporter agonist D-aspartate, indicating that the effect was on transport and not metabolism. No suppression was seen with CM from uninfected Mphi or Mphi infected with heat-inactivated HIV. The magnitude of uptake suppression was not correlated with CM p24 values, and removal of CM virions by ultracentrifugation and immunoprecipitation did not alter the uptake-suppressive properties of infected Mphi CM. Uptake suppression was seen when Mphi were infected with Mphi-tropic strains HIV(SF162), HIV(JR-CSF), HIV(NFN-SX) and a Mphi-tropic patient isolate, but not the lymphotropic strain HIV(LAI). HIV-infected Mphi may produce substances which suppress neuronal and glial glutamate neurotransmitter uptake, resulting in higher extracellular glutamate levels and leading possibly to deficits in cell signaling and neurotoxicity.     

MPP(+)-like neurotoxicity of a pyridinium metabolite derived from haloperidol: in vivo microdialysis and in vitro mitochondrial studies

Intracerebral (intrastriatal, intranigral and intracortical) microdialysis studies were conducted in conscious rats to investigate the comparative dopaminergic and serotonergic neurotoxic potential of the pyridinium metabolite 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]pyridinium (HPP+), derived from the extensively used neuroleptic agent haloperidol and 1-methyl-4-phenylpyridinium (MPP+), the pyridinium metabolite derived from the parkinsonian inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Although HPP+ was less potent than MPP+ on the dopaminergic system, the two compounds displayed comparable toxic effects on the serotonergic system. HPP+ also proved to be a weaker inhibitor of mitochondrial respiration than MPP+ in vivo as measured by increases in extracellular lactate levels. On the other hand, HPP+ was a more potent inhibitor of mitochondrial respiration in vitro than MPP+, with IC50 values of 12 microM (HPP+) and 160 microM (MPP+). Quantitative estimations established that the concentrations of the more hydrophobic HPP+ in the brain tissues surrounding the microdialysis probe were less than those of MPP+ after comparable perfusions. Consequently, the inherent toxicity of HPP+ relative to MPP+ may be greater than suggested by the results observed in the microdialysis experiments. These data support previous speculations that HPP+ may contribute to some of the persistent extrapyramidal side effects associated with chronic haloperidol treatment.     

Regulation of the glial Na+-dependent glutamate transporters by cyclic AMP analogs and neurons

Sodium-dependent transport into astrocytes is critical for maintaining the extracellular concentrations of glutamate below toxic levels in the central nervous system. In this study, the expression of the glial glutamate transporters GLT-1 and GLAST was studied in primary cultures derived from cortical tissue. In primary astrocytes, GLAST protein levels were approximately one half of those observed in cortical tissue, but GLT-1 protein was present at very low levels compared with cortical tissue. Maintenance of these astrocytes in medium supplemented with dibutyryl-cAMP (dbcAMP) caused a dramatic change in cell morphology, increased GLT-1 and GLAST mRNA levels approximately 5-fold, increased GLAST protein approximately 2-fold, and increased GLT-1 protein >/=8-20-fold. These increases in protein expression were accompanied by 2-fold increases in the Vmax and Km values for Na+-dependent L-[3H]glutamate transport activity. Although GLT-1 is sensitive to inhibition by dihydrokainate in heterologous expression systems, no dihydrokainate sensitivity was observed in astrocyte cultures that expressed GLT-1. Biotinylation with a membrane-impermeant reagent, separation of the biotinylated/cell surface proteins, and subsequent Western blotting demonstrated that both GLT-1 and GLAST were present at the cell surface. Coculturing of astrocytes with neurons also induced expression of GLT-1, which colocalized with the glial specific marker, glial fibrillary acidic protein. Neurons induced a small increase in GLAST protein. Several studies were performed to examine the mechanism by which neurons regulate expression of the glial transporters. Three different protein kinase A (PKA) antagonists did not block the effect of neurons on glial expression of GLT-1 protein, but the addition of dbcAMP to mixed cultures of neurons and astrocytes did not cause GLT-1 protein to increase further. This suggests that neurons do not regulate GLT-1 by activation of PKA but that neurons and dbcAMP regulate GLT-1 protein through convergent pathways. As was observed with GLT-1, the increases in GLAST protein observed in cocultures were not blocked by PKA antagonists, but unlike GLT-1, the addition of dbcAMP to mixed cultures of neurons and astrocytes caused GLAST protein to increase approximately 2-fold. Neurons separated from astrocytes with a semipermeable membrane increased GLT-1 protein, indicating that the effect of neurons was mediated by a diffusible molecule. Treatment of cocultures with high concentrations of either N-methyl-D-aspartate or glutamate killed the neurons, caused GLT-1 protein to decrease, and caused GLAST protein to increase. These studies suggest that GLT-1 and GLAST protein are regulated independently in astrocyte cultures and that a diffusible molecule secreted by neurons induces expression of GLT-1 in astrocytes.     

Net glutamate release from astrocytes is not induced by extracellular potassium concentrations attainable in brain

Elevated extracellular potassium concentration ([K+]e) has been shown to induce reversal of glial Na+-dependent glutamate uptake in whole-cell patch clamp preparations. It is uncertain, however, whether elevated [K+]e similarly induces a net glutamate efflux from intact cells with a physiological intracellular milieu. To answer this question, astrocyte cultures prepared from rat and mouse cortices were incubated in medium with elevated [K+]e (by equimolar substitution of K+ for Na+), and glutamate accumulation was measured by HPLC. With [K+]e elevations to 60 mM, medium glutamate concentrations did not increase during incubation periods of 5-120 min. By contrast, 45 min of combined inhibition of glycolytic and oxidative ATP production increased medium glutamate concentrations 50-100-fold. Similar results were obtained in both rat and mouse cultures. Studies were also performed using astrocytes loaded with the nonmetabolized glutamate tracer D-aspartate, and parallel results were obtained; no increase in medium D-aspartate content resulted from [K+]e elevation up to 90 mM, whereas a large increase occurred during inhibition of energy metabolism. These results suggest that a net efflux of glutamate from intact astrocytes is not induced by any [K+]e attainable in brain.     

Inhibitory effect of ginseng total saponins on glutamate-induced swelling of cultured astrocytes

The effects of ginseng total saponins (GTS) on L-glutamate-induced swelling of cultured astrocytes from rat brain were studied. Following exposure to 0.5 mM glutamate for 1 h, the intracellular water space (as measured by [3H]O-methyl-D-glucose uptake) of astrocytes increased three-fold with a morphological change: the disappearance of cellular processes. Simultaneous addition of GTS with glutamate reduced the astrocytic swelling in a dose-dependent manner. GTS at 0.5 mg/ml did not affect the viability of astrocytes for up to 18 h, which was determined by a colorimetric assay for cellular growth and survival. These data suggest that GTS prevents the cell swelling of astrocytes induced by glutamate.