Cultured C6 Glioma Cells Research Articles

Protective roles of glutathione in the toxicity of mercury and cadmium compounds to C6 glioma cells

The neurotoxic effects of mercury (II) chloride, methylmercury (MeHg) and cadmium chloride on astrocytes were modelled using C6 glioma cell cultures. All three compounds were cytotoxic to these cells with an order of potency of cadmium > MeHg > mercury chloride. Addition of reduced glutathione (GSH) to the media protected the cells in all three cases, whereas depletion of GSH with l-buthionine- S,R-sulfoximine enhanced the toxicity of cadmium and mercury chloride but not MeHg. The effects of subcytotoxic concentrations of these compounds on intracellular GSH levels were assessed using chlorobimane staining. All three showed a similar type of effect—an initial depletion of GSH followed by an increase to levels greater than in untreated cells. For mercury chloride-exposed cells (0.37–3.7 μM), the initial depletion occurred over 4 hr with the cells recovering by 7 hr and increases in the GSH content seen after 24 hr. With cadmium or MeHg (0.04–4 μM), the initial depletion was more protracted, with treated cells having less GSH than control cells for 4–7 hr. Glutathione S-transferase activity in the cells was increased after 24 hr of exposure to all three metals to about 200% of control values. These results show that some components of the glutathione system in C6 glioma cells are activated after exposure to heavy metal compounds

Experimental brain glioma: growth arrest and destruction by a blood-group-related tetrasaccharide.

A synthetic tetrasaccharide (TS4), structurally related to blood groups, inhibited the proliferation of the C6 glioma cells in culture and the growth of tumors formed after intracerebral transplantation of C6 cells. TS4-treated tumors were substantially smaller than controls, as expected from TS4 cytostatic action on C6 glioma cells in culture. However, in vivo treatment also caused extensive tumor destruction. This effect appeared to be caused by indirectly, either by activation of natural killer cells, cytotoxic lymphocytes, or by inhibition of tumor vascularization. Enhanced antigenicity of TS4-treated glioma may be related to the increased expression of connexin 43 observed in glioma cell cultures treated with the oligosaccharide. Because concentrations of up to 20 mg/ml of TS4 were not toxic for normal neuronal or glial cells, specific oligosaccharides such as TS4 offer the possibility of selective tumor treatment.

Selective inhibition of cholesterol biosynthesis in brain cells by squalestatin 1.

The effect of squalestatin 1 (SQ) on squalene synthase and other enzymes utilizing farnesyl pyrophosphate (F-P-P) as substrate was evaluated by in vitro enzymological and in vivo metabolic labeling experiments to determine if the drug selectively inhibited cholesterol biosynthesis in brain cells. Direct in vitro enzyme studies with membrane fractions from primary cultures of embryonic rat brain (IC50 = 37 nM), pig brain (IC50 = 21 nM), and C6 glioma cells (IC50 = 35 nM) demonstrated that SQ potently inhibited squalene synthase activity but had no effect on the long-chain cis-isoprenyltransferase catalyzing the conversion of F-P-P to polyprenyl pyrophosphate (Poly-P-P), the precursor of dolichyl phosphate (Dol-P). SQ also had no effect on F-P-P synthase; the conversion of [3H]F-P-P to geranylgeranyl pyrophosphate (GG-P-P) catalyzed by partially purified GG-P-P synthase from bovine brain; the enzymatic farnesylation of recombinant H-p21ras by rat brain farnesyltransferase; or the enzymatic geranylgeranylation of recombinant Rab 1A, catalyzed by rat brain geranylgeranyltransferase. Consistent with SQ selectively blocking the synthesis of squalene, when C6 glial cells were metabolically labeled with [3H]mevalonolactone, the drug inhibited the incorporation of the labeled precursor into squalene and cholesterol (IC50 = 3-5 microM) but either had no effect or slightly stimulated the labeling of Dol-P, ubiquinone (CoQ), and isoprenylated proteins. These results indicate that SQ blocks cholesterol biosynthesis in brain cells by selectively inhibiting squalene synthase.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the Chondroitin Sulfate Proteoglycans of Amyloid Precursor (Appican) and Amyloid Precursor‐Like Protein 2

The Alzheimer amyloid precursor (APP) protein is a member of a family of glycoproteins that includes the amyloid precursor-like proteins (APLPs). Previously, we showed that in C6 glioma cell cultures, secreted APP nexin II occurs as the core protein of a chondroitin sulfate proteoglycan (CSPG). Here, we report that among seven untransfected cell lines, expression of secreted APP CSPG was restricted to two cell lines of neural origin, namely, C6 glioma and Neuro-2a neuroblastoma (N2a) cells. Addition of dibutyryl cyclic AMP in N2a cultures, a treatment that induces the neuronal phenotype in these cells, resulted in a significant reduction in the amount of the secreted APP CSPG, although secretion of APP was only marginally affected. Growth in the presence of serum increased the size of the secreted APP CSPG, suggesting that the number and/or length of the chondroitin sulfate (CS) chains attached to the core APP varies with growth conditions. Extensive mapping with epitope-specific antibodies suggested that a CS chain is attached within or proximal to the A beta sequence of APP. In contrast to the restricted expression of the APP CSPG, expression of secreted APLP2 CSPGs was observed in all cell lines examined. After chondroitinase treatment, two core proteins of approximately 100 and 110 kDa were obtained that reacted with an APLP2-specific antiserum, suggesting that non-transfected cell lines contain at least two endogenous APLP2 CSPGs, probably derived by alternative splicing of the APLP2 KPI domain. The fraction of the APLP2 proteins in the CSPG form was dependent on the particular cell line examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of the MTT assay for estimating toxicity in primary astrocyte and C6 glioma cell cultures

Primary cultures of rat cortical astrocytes were exposed to 25 neurotoxic and non-neurotoxic compounds for 24 hr over a concentration range of 0.001 to 1000 μg/ml and the effects were quantified using the MTT assay. EC 50 values were obtained for nine of the compounds in the tested range, while increases in MTT conversion were seen at sub-cytotoxic concentrations for 12 compounds. The concentrations causing activation of this system were found, with some exceptions (notably organotin compounds) to be similar to those previously reported to cause increases in expression of the astrocyte marker glial fibrillary acidic protein. MTT conversion was also measured in the C6 cell line and, with some exceptions, the response to toxicants was found to be the same as that in the primary cultures of astrocytes. However, this was only the case when the C6 cells were pretreated for 48 hr with 0.5 m M dibutyryl cyclic AMP. This study represents the first direct comparison between primary astrocytes and C6 cells using a broad range of chemical neurotoxicants.

Cytotoxic effects of sigma ligands: sigma receptor-mediated alterations in cellular morphology and viability

The morphological effects of several neuroleptics as well as other novel and prototypic sigma ligands were examined by addition to cultures of C6 glioma cells. Sigma ligands caused loss of processes, assumption of spherical shape, and cessation of cell division. The time course and magnitude of this effect were dependent on the concentration of sigma ligand. Continued exposure to sigma compounds ultimately resulted in cell death. However, the morphological effect was reversible when sigma ligand was removed shortly after rounding. The potency of compounds to produce these effects generally correlated with binding affinity at sigma receptors of C6 glioma cell membranes labeled with [3H](+)-pentazocine. At a concentration of 100 microM, haloperidol, reduced haloperidol, fluphenazine, perphenazine, trifluoperazine, BD737, LR172, BD1008, and SH344 produced significant effects in 3-6 hr of exposure. Other compounds, such as trifluperidol, thioridazine, and (-)-butaclamol, produced significant effects by 24 hr of exposure. Despite the requirement of micromolar concentrations of ligand (some compounds were effective at 30 microM), the effect showed a remarkable specificity for compounds exhibiting sigma receptor binding affinity. Neuroleptics lacking potent sigma affinity [e.g., (-)-sulpiride, (+)-butaclamol, and clozapine] and other compounds that lack significant sigma affinity but that are agonists or antagonists at dopamine, serotonin, adrenergic, glutamate, phencyclidine, GABA, opiate, or muscarinic cholinergic receptors were without effect on cellular morphology at concentrations up to 300 microM over a period of 72 hr. Likewise, blockers and activators of Na+, K+, and Ca2+ channels and a monoamine oxidase inhibitor devoid of sigma affinity were without effect. Interestingly, 1,3-di-o-tolylguanidine (DTG), (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [(+)-3-PPP], (+)-pentazocine, (+)-cyclazocine, and other sigma-active benzomorphans and morphinans appeared inactive in up to 72 hr of culture. However, these compounds interacted synergistically with a subeffective dose of BD737 (30 microM) to produce effects usually in 6 hr or less. Also, the pH of the culture medium had a profound effect on the activity of sigma compounds. Increasing the pH from the normal range of 7.2-7.4 to pH 8.3-8.5 shifted the dose curves (30, 100, 300 microM) for all sigma compounds to the left. Under these conditions, DTG, (+)-3-PPP, and benzomorphans produced effects in 24 hr or less.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of Hyperosmolar Mannitol on the Delivery of High Molecular Weight Substances Entrapped in Liposomes to the Brain and Cultured C6 Glioma Cells.

The effect of intravenous hyperosmolar mannitol on delivery of liposomes to the brain was investigated. Pure CAMP phosphodiesterase was iodinated and entrapped in either dehydration-rehydration vesicles or small unilamellar vesicles composed of egg phosphatidyl-choline, cholesterol, and sulfatides. A time-course study of liposomal delivery to the brain after hyperosmolar mannitol administration showed increased delivery to the brain of dehydration-rehydration vesicles and of small unilamellar vesicles compared with their respective normal saline controls. Clearance of both free or liposomally-entrapped enzyme from the brain was slower under hyperosmolar conditions. Studies with FITC-BSA or morphine-BSA entrapped in small unilamellar vesicles further confirmed the delivery of liposomal contents to the brain, liver, and cultured glioma cells under hyperosmolar conditions. These data show that 2M mannitol is able to reversibly disrupt the blood-brain barrier and thereby increase the delivery of both free and liposomally-entrapped substances to the brain.

Glucose stimulates IGF-I gene expression in C6 glioma cells.

Glucose stimulates expression of the insulin-like growth factor I (IGF-I) gene in cultured C6 glioma cells. This stimulation is specific, as the expression of other genes, including those encoding hypoxanthine guanine phosphoribosyl transferase (HPRT) and ubiquitin, is not similarly affected by glucose. IGF-I gene expression is also stimulated by lactate, suggesting that the stimulatory effect is mediated by a product of glycolysis. Additional results indicate that the abundance of IGF-I mRNA is considerably higher in stationary confluent cells than in log-phase growing cells. This regulation is also specific for IGF-I, as HPRT mRNA is regulated in the opposite direction.

Inhibition of plasma membrane Ca2+-atpase pump activity in cultured c6 glioma cells by halothane and xenon

We have compared the effect of two inhalational anesthetics, halothane and xenon, on Ca(2+)-ATPase (PMCA) pumping activity in plasma membrane vesicles prepared from cultured rat C6 glioma cells. Halothane, at concentrations ranging from 0.5 to 1.75 vol% (equivalent to 0.5 to 1.6 MAC), significantly inhibited Ca2+ uptake (transport) by plasma membrane vesicles in a dose-related fashion. Xenon, at partial pressures ranging from 0.5 to 1.5 atm (equivalent to 0.5 to 1.6 MAC), similarly inhibited PMCA pumping activity. Additive effects on suppression of PMCA pump activity were observed when C6 cell plasma membrane vesicles were exposed to increasing partial pressures of xenon in the presence of halothane (1 vol%). Halothane also inhibited PMCA pumping in cells from two other lines of neural origin, B104 (rat neuroblastoma) and PC12 (rat pheochromocytoma). Studies described in this report support the thesis that PMCA in cells of neural origin is inhibited by quite different inhalational anesthetics at clinically relevant concentrations.

Interaction of high molecular weight kininogen with plasminogen inhibits binding of plasminogen to cell surfaces

Recent studies have shown that high molecular weight kininogen (HK) inhibits the binding of plasmin to platelets and thereby inhibits platelet activation by plasmin. We hypothesized that these results might be explained by HK binding to plasmin(ogen) in solution. Using ligand blotting, we showed that 125I-plasminogen (Pg) binds to immobilized HK and that 125I-HK binds to immobilized Pg. HK partially inhibited 125I-Pg binding to lysine-Sepharose. Non-covalent binding of HK to Pg in solution was demonstrated using the cross-linking agent, bis(sulfosuccinimidyl)suberate (BS 3) . The cross-linking agent stabilized a 180kDa complex as determined by SDS-PAGE. The 180kDa complex was not observed in the absence of BS 3 or when ϵ-amino caproic acid was added. In addition, excess unlabelled Pg or HK inhibited the binding of each radiolabelled protein to the other. The equilibrium dissociation constant (K D) for the binding of Pg to HK in solution was 0.8 μmol/L, as determined by BS 3 cross-linking. Binding of Pg to C6 glioma cells in culture was inhibited by HK in a concentration-dependent manner with an IC 50 of approximately 0.4 μmol/L. Similar inhibition was observed using human umbilical vein endothelial cells (IC 50≈0.5 μmol/L). Binding of 125I-HK to C6 glioma cells was apparently saturable, zinc-dependent and inhibited by Pg (IC 50≈0.9 μmol/L). The results presented here suggest that HK inhibits Pg binding to cell surfaces primarily by forming a complex with Pg in solution. Pg binding to HK may affect the localization of these proteins in the vascular compartment.

Expression of 25(OH) vitamin D3 24-hydroxylase gene in glial cells.

The expression of the 25(OH) vitamin D3 24-hydroxylase gene was studied in C6 glioma and rat primary glial cell culture. The expression of the 25(OH)D3 24-hydroxylase gene was not detected in C6 glioma or glial cells cultured in a serum-free medium. However, the 25(OH)D3 24-hydroxylase mRNA was induced in a dose-dependent manner in cells treated with 1,25(OH)2D3. These findings provide further evidence for an involvement of vitamin D3 metabolites in brain function.

Hypoxic effects on glutamate uptake in cultured glial cells

Hypoxic effects on glutamate uptake and ATP content in glial cells were investigated by using cultured C6 glioma cells. Mild regressive changes were found depending on the duration of the hypoxic insult, but necrosis or detachment of the cells from the substratum was rarely observed. Glutamate uptake was relatively well preserved after a short hypoxic insult, while a marked decrease in glutamate uptake was observed after hypoxia of long duration. The uptake of sucrose was reduced in a similar pattern to glutamate uptake. Hypoxic insult resulted in a significant reduction of the ATP content in glial cells. Therefore, the decrease in glutamate uptake by glial cells under hypoxia is likely to be due to ATP dependency, and not to the failure of a specific glutamate uptake system, but the failure of a general uptake of the glial cells owing to the energy-dependent membrane dysfunction by ATP depletion. These findings suggest that there are phased changes of astrocytic functions in a hypoxic condition, a preservative phase in the initial stages and then a dysfunctional phase in the later stages of hypoxia.

σ Receptor-active neuroleptics are cytotoxic to C6 glioma cells in culture

When exposed to neuroleptics (100 μM), C6 glioma cells exhibited marked changes in cell morphology, accompanied by cessation of cell division and ultimately cell death. The degree of activity generally correlated with binding affinity at σ sites: fluphenazine = perphenazine = haloperidol = reduced haloperidol > pimozide = spiperone ⪢ ⪢ (−)-sulpiride. Sigma-inactive compounds (100 μM) which block dopamine, serotonin, phencyclidine, muscarinic receptors and adrenoceptors showed no effect. These results may suggest a role of σ binding sites in maintenance of cell viability.

Comparative 31P and 1H NMR studies on rat astrocytes and C6 glioma cells in culture

Rat astroglial cells in primary culture (95% enrichment) and C6 glioma cells were adapted to grow on microcarrier beads. In vivo 31P NMR spectra were collected from cell-covered beads perfused in the NMR tube. The NMR-visible phosphorylated metabolite contents of both cell types were determined using saturation factors calculated from the values of longitudinal relaxation times determined for C6 cells using progressive saturation experiments. On the other hand, the amounts of phosphorylated metabolites in cells were determined from proton decoupled 31P NMR spectra of cell perchloric acid extracts. The results indicate that the NTP and Pi contents of the normal and tumoral cells were similar, whereas the PCr level was higher in C6 cells and the NDP and phosphomonoester levels higher in astrocytes. The comparison of 1H NMR spectra of cell perchloric acid extracts evidenced larger inositol and alanine contents in C6 cells, whereas larger taurine and choline (and choline derivatives) contents were found in astrocytes. The Glu/Gln ratio was very different, 3.5 and 1 in C6 cells and astrocytes, respectively. In both cases, the more intense resonance in the 1H NMR spectrum was assigned to glycine. Based on the comparison of the metabolite content of a tumoral and a normal cell of glial origin, this work emphasizes the usefulness of a multinuclear NMR study in characterizing intrinsic differences between normal and tumoral cells.

Gene expression of aromatic L-amino acid decarboxylase in cultured rat glial cells.

Northern blot hybridization was performed to detect aromatic L-amino acid decarboxylase (AADC) mRNA in primary cultures of astrocytes and C6 glioma cells. The cDNA probe for rat AADC was generated by reverse transcription from rat adrenal gland total RNA and was amplified by the polymerase chain reaction method. AADC mRNA from cultured astrocytes and C6 glioma cells was present as a single band, 2.2 kbp in size, that comigrated with the RNA from rat kidney. Western immunoblot showed a single protein band at 52 kDa for AADC enzyme protein. These findings demonstrate that AADC is expressed in rat glial cells.

Intercellular calcium signaling via gap junctions in glioma cells.

Calcium signaling in C6 glioma cells in culture was examined with digital fluorescence video microscopy. C6 cells express low levels of the gap junction protein connexin43 and have correspondingly weak gap junctional communication as evidenced by dye coupling (Naus, C. C. G., J. F. Bechberger, S. Caveney, and J. X. Wilson. 1991. Neurosci. Lett. 126:33-36). Transfection of C6 cells with the cDNA encoding connexin43 resulted in clones with increased expression of connexin43 mRNA and protein and increased dye coupling, as well as markedly reduced rates of proliferation (Zhu, D., S. Caveney, G. M. Kidder, and C. C. Naus. 1991. Proc. Natl. Acad. Sci. USA. 88:1883-1887; Naus, C. C. G., D. Zhu, S. Todd, and G. M. Kidder. 1992. Cell Mol. Neurobiol. 12:163-175). Mechanical stimulation of a single cell in a culture of non-transfected C6 cells induced a wave of increased intracellular calcium concentration ([Ca2+]i) that showed little or no communication to adjacent cells. By contrast, mechanical stimulation of a single cell in cultures of C6 clones expressing transfected connexin43 cDNA induced a Ca2+ wave that was communicated to multiple surrounding cells, and the extent of communication was proportional to the level of expression of the connexin43 cDNA. These results provide direct evidence that intercellular Ca2+ signaling occurs via gap junctions. Ca2+ signaling through gap junctions may provide a means for the coordinated regulation of cellular function, including cell growth and differentiation.

Polyunsaturated fatty acid incorporation into plasmalogens in plasma membrane of glioma cells is preceded temporally by acylation in microsomes

Plasmalogens (1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) are major phospholipids in many tissues and cells, particularly of neural origin. Using cultured C6 glioma cells and subcellular fractions isolated on Percoll gradients we investigated selectivity for esterification of several polyunsaturated fatty acids (PUFA) in the sn-2 position of plasmalogens compared to [1-14C]hexadecanol, representative of de novo synthesis of the ether-linked sn-1 position. In whole cells at a final concentration of 105 microM PUFA, 2-4 nmol plasmalogen/mg protein was labeled in 4 h and 10-14 nmol in 24 h, representing 8-15% and 35-50%, respectively, of initial plasmalogen mass. Incorporation of label from hexadecanol was lower than PUFA incorporation (20:5(n-3) greater than 20:4(n-6) greater than 18:3(n-3) much greater than 18:2(n-6)) suggesting deacylation-reacylation at the sn-2 position. Plasmalogens accounted for 50% of total cell ethanolamine phospholipids and 75% in plasma membrane. Using a novel, improved method for extraction of subcellular fractions containing Percoll, plasma membrane also was enriched in plasmalogen relative to microsomes (107.4 +/- 5.2 vs. 40.0 +/- 2.9 nmol/mg protein). Selectivity for esterification at the sn-2 position of plasmalogens with respect to chain length and unsaturation of the fatty acyl chain was similar in both subcellular fractions and reflected that of whole cells. Labeling of plasma membrane with PUFA and fatty alcohol lagged behind that of microsomes. Chase experiments in cells prelabeled with [1-14C]18:3(n-3) for 2 h showed no significant reduction of label in plasmalogen of any subcellular fraction although accumulation of label in the microsomal fraction was slowed initially. Reduction of plasmalogen label (40-50%) did occur in microsomes and plasma membrane when cells prelabeled for 24 h were switched to chase medium with or without chase fatty acid. Our data suggest that esterification of PUFA to plasmalogen may occur at the endoplasmic reticulum with subsequent translocation to plasma membrane resulting in accumulation of relatively stable pools of plasmalogen that are not readily accessible for deacylation-reacylation exchange with newly appearing PUFA. Alternatively, deacylation-reacylation may occur in a more stable phospholipid pool within the plasma membrane but would involve a slower process than at the endoplasmic reticulum.

A histochemical study of the activity of nonspecific cholinesterase in C6 glioma cells in culture.

The activity of nonspecific cholinesterase (nChE) was revealed histochemically in a small number of C6 glioma cells under conditions of spontaneous growth in culture. The nChE-positive cells (nChE+) first appeared in 4-day-old cultures. They occurred solitarily or in small groups with a cluster- or chain-like arrangement. The incidence of nChE+ cells increased with in vitro age and/or higher population densities of cultures but it never exceeded 3% of the whole population. These results indicate the necessity of histochemical as well as biochemical data on nChE activity in glioma cells both in situ and in culture for the discovery of the heterogeneous distribution of this enzyme. db-cAMP induced a decrease in the proportion of nChE+ cells to total number of cells. These changes suggest that the original C6 glioma cell seed stock contains a small subclone of less differentiated cells exhibiting nChE activity. The greater number of nChE+ cells in older cultures is probably due to recovery and multiplication of initially scarce subclone cells. C6 glioma cells in culture form a suitable model for investigation of changes in nChE activity of glial cells provided that a histochemical method of detection is applied.

Changes in cell cycle and chromatin distribution in C6 glioma cells treated by dibutyryl cyclic AMP.

C6 glioma cells in culture were treated with 1 mM dibutyryl cyclic AMP (Db-cAMP) for 5, 8, 24 and 72 h. The cells were labelled with [3H]-thymidine before either the end, or the beginning, of the Db-cAMP treatment. The cell cycle passage was monitored by the simultaneous determination of DNA content and DNA synthesis in propidium iodide stained autoradiograms. The data revealed an early (t less than or equal to 3-8 h) and moderate inhibitory effect of Db-cAMP on all phases of the cell cycle except mitosis; some cells (2%) were completely blocked in the S phase. Later (8 less than t less than 24-72 h), the cycling of a substantial part of the population became inhibited in G1 phase. Microdensitometric texture analysis of Feulgen-stained nuclei, performed 24 h after administration of Db-cAMP, showed a higher inhomogeneity of the DNA distribution in cell nuclei, caused by the condensation of a part of the chromatin. This may reflect either changes in genome expression taking part in the process of cAMP induced differentiation or transit of some cells into quiescent G0 or S0 phases.

Hexose uptake in primary cultures of bovine brain microvessel endothelial cells. II. Effects of conditioned media from astroglial and glioma cells

Regulation of glucose uptake by an astroglial cell secreted factor(s) was studied in primary cultures of brain microvessel endothelial cells (BMECs). Uptake of a non-metabolizable glucose analog, 3- O-[ 3H]methyl- d-glucose ([ 3H]3MG), was measured after the BMECs were treated with media conditioned by primary cultures of rat astrocytes (Astrocyte Conditioned Media: ACM) or rat C6 glioma cells (Glioma Cell Conditioned Media: GCM). Uptake of [ 3H]3MG was significantly increased by ACM (30–50%)and GCM (60–200%) treatments, whereas conditioned medium from 3T3 fibroblasts (3T3) caused no significant effect. The elevation in [ 3H]3MG uptake increased with increasing time of exposure of BMECs to these conditioned media (CM), and the effect was shown to be reversible. Glucose depletion of CM was shown not to be a factor. The presence of cycloheximide, a protein synthesis inhibitor, during treatment of the BMECs with ACM and GCM blocked the increase in [ 3H]3MG uptake by the cells. These results suggested that ACM or GCM treatment elevated de novo synthesis of brain-type glucose transporter (GLUT1). Indeed, enhanced GLUT1 expression by these treatments in BMECs was demonstrated directly by enzyme-linked immunosorbent assay (ELISA) using antibodies against human GLUT1. After trypsinization of ACM and GCM, both conditioned media still induced significant stimulation of [ 3H]3MG uptake by BMECs. A significant increase in [ 3H]3MG uptake was also observed when ACM or GCM was exposed to BMECs through a dialysis membrane with a molecular weight cutoff of 1000. To examine whether the effects were specific to brain endothelial cells, [ 3H]3MG uptake experiments were performed employing aortic endothelial cells (AECs), pulmonary microvessel endothelial cells (PMECs), and 3T3 cells. ACM treatment did not alter 3MG uptake by these cells, suggesting that the ACM effect was specific to BMECs. On the other hand, [ 3H]3MG uptake by AECs and PMECs treated with GCM was significantly enhanced. The present study demonstrated that some factor(s) of relatively small molecular weight, which was released from astrocytes or glioma cells, stimulated glucose uptake by enhancing GLUT1 synthesis in BMECs.