Thrombin receptor peptides induce shape change in neonatal murine astrocytes in culture

Astrocytes appear star-shaped in the brain, increasingly so after injury. When astroglia are cultures in serum-containing medium, they exhibit a flat, fibroblast-like morphology. In serum-free medium, astrocytes become stellate, with many long processes. The serine protease α-thrombin mimics the effects of serum at subnanomolar concentractions, whereas the thrombin-inhibiting serpin, protease nexin I (PNI), reverses the thrombin effect. In our current experiements, murine neonatal spinal cord astrocytes became stellate after 4 hr in serum-free medium, while cortical astrocytes requires 12 hr in serum-free medium for stellation. Astrocytes from either region flattened after 60 min in medium containing 3.0 to 300 pM proteoloytically active human α-thrombin. After 12 hr in thrombin-containing medium, 98% of the astrocytes had a flattened morphology. No flattening occured if α-thrombin was replaced by γ-thrombin, which has its fibrinogen-recognition exosite disrupted. PNI added at 1 nM to serum-containing medium caused stellation after 3 hr, and astroglia were 50% stellate by 12 hr. The effect of thrombin was mimicked by a 7-amino acid peptide (TRP-7) action on astrocytes. These results indicate that astrocytes possess a cell surface receptor for thrombin, similar to that described for platelets, endothelial cells, and neurons. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Astrocytes in cell culture incorporate GM1 ganglioside

Ganglioside GM1 3H-labelled at the terminal galactose was added to astrocyte cell cultures. GM1 incorporation was studied in the two typical forms of astrocytes in cell culture of flat and stellate morphology. There was a strong time- and concentration-dependent increase in GM1 incorporation for both cell types of astrocytes. The incorporation of GM1 into the stellate form increased continuously up to 48 h (maximum time studied), while the incorporation into the flat form reached a plateau at the same time. After 2 h of GM1 incubation additional gangliosides appeared; the latter resulted from the metabolism of the GM1 incorporated, indicating that astrocytes in cell culture can biosynthesize more complex gangliosides. To confirm that GM1 was indeed incorporated into astrocytes, two other different approaches were used. Astrocyte cells treated with 3H-GM1 were visualized using autoradiography. The specific marker for GM1, rhodamine-labelled choleratoxin, was used to detect the incorporated GM1 using fluorescence microsocpy. In both cases GM1 treated cells were intensely labelled. These observations indicate that exogenous GM1 ganglioside can also be integrated into the astrocyte membranes as occurs in other types of cells and membranes.     

Neutral glycolipid and ganglioside composition of type-1 and type-2 astrocytes from rat cerebral hemisphere.

We reported previously that the major gangliosides in primary mixed-type astrocyte cultures are GM3 and GD3. To obtain more information regarding the exact distribution of glycosphingolipids in different types of astrocytes, we established a line of type-1 astrocytes that are characterized by a Ran-2 positive, broad flat morphology, and by the absence of binding to A2B5 antibodies. We also purified O-2A progenitor cells by immunopanning and cultured them in the presence of 10% newborn calf serum. They differentiated into type-2 astrocytes that were identified by immunostaining for each of GD3, A2B5, and GFAP. Using these cell cultures, we demonstrate that the major gangliosides were GM3 in type-1 astrocytes and GM3 and GD3 in type-2 astrocytes. In addition, a set of neutral glycolipids was identified based on the HP-TLC migration properties of CMH, CDH, CTH, and Glob, but the component distribution of these glycolipids is related to that of glycolipids of astrocytes. A marked increase in the expression of CTH and Glob was shown in type-2 astrocytes. The amount of neutral glycolipid-sugar was higher in the type-2 astrocytes than in the type-1 astrocytes. These results suggest that the increase in the total glycosphingolipid content and the change in the neutral glycolipid composition produced by type-2 astrocytes may be related to their biological functions and the cellular compositions.     

Morphological modulation of cultured rat brain astroglial cells: antagonism by ganglioside GM1

Secondary cultures of neonatal rat astroglial cells, maintained in a serum-free, chemically defined medium were treated with several agents thought to activate cyclic AMP-synthesizing systems. Dibutyryl cyclic AMP (dBcAMP), forskolin and cholera toxin promoted, within 2 h, the near-complete conversion of 1-day-old (D1) astroglial cells from a flat, epithelioid morphology to a stellate (star-shaped) morphology. With all 3 agents, cell susceptibility to morphological change declined with culture age, 5-day-old cultures failing to respond altogether. D1 cultures, after 48 h of treatment, had reverted to the flat morphology. Gangliosides reported to stimulate adenylate cyclase were also tested, using purified GM1 X GM1 failed to stimulate the conversion to stellate morphologies. GM1, however, did affect these astroglial cells by causing a block or reversal of their morphological response to dBcAMP, forskolin or cholera toxin. The GM1 response was specific for the intact ganglioside molecule, asialo GM1 and sialic acid having no effect. Gangliosides GD1a, GD1b and GT1b were also active, being effective at ca. 4-fold lower concentrations. The response to GM1 appeared to involve a direct interaction with the astroglial cell, rather than influencing either substratum or medium components.     

Gangliosides alter morphology and growth of astrocytes and increase the activity of choline acetyltransferase in cultures of dissociated septal cells

Administration of gangliosides has been reported to stimulate regeneration of motoneurons and of central dopaminergic and cholinergic neurons. To shed light on the mechanism by which gangliosides mediate the effects on cholinergic neurons, we studied their actions on cultures of cells dissociated from the septal area of fetal rat brains. These cultures contain cholinergic neurons, which, in vivo, give rise to the cholinergic septo-hippocampal pathway. Gangliosides produced prominent changes in the morphological appearance of the cultures. In contrast to control cultures, which contained many process-bearing cells and a confluent layer of flat cells, there were no flat cells in cultures grown in the presence of gangliosides (0.2 to 0.8 mg/ml of medium). Using immunocytochemical visualization of the astrocytic marker glial fibrillary acid protein, it was shown that all astrocytes in cultures grown in the presence of gangliosides exhibited the morphology of process-bearing cells, whereas in control cultures astrocytes represented the majority of the flat cells. Furthermore, gangliosides attenuated astrocytic proliferation. The effects of gangliosides apparently were not mediated by cAMP, since they could be differentiated from actions of forskolin, an activator of adenylate cyclase. Astrocytic growth and morphology were affected by ganglioside mixtures of various sources and composition and also by the pure gangliosides GM1 and GD1a, whereas lipid and carbohydrate components of gangliosides were ineffective. In contrast to the prominent effects on astrocytes, gangliosides failed to significantly alter survival or fiber growth of cholinergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of the N-acetylgalactosaminyltransferase inhibitor on cultured cerebral cells

The inhibitor preparation of the UDP-N-acetylgalactosamine: GM3, N-acetylgalactosaminyltransferase (EC 2.4.1.92) (GalNAc-T) produces effects on the neurons and the glial (astrocytes) cells of the cerebrum in culture. The effect in culture is evidenced by aneuritogenesis, deficiency in the GalNAc-T activity, and decrease in the content of gangliosides, proteins, and lipids. In isolated glial cells the effect is evidenced by cytoplasm vesiculation and premature cessation of proliferation compared with control culture. The pattern of gangliosides in the inhibited culture shows a decrease in the amount of GD1a with respect to GD3; this is compatible with the notion that the effect is due to an inhibitor of the GM2 synthase. The inhibitor effects are reverted when it is eliminated after 24 or 48 hr in the culture medium. © 1994 Wiley-Liss, Inc.     

Neuroimmunology of gangliosides in human neurons and glial cells in culture

Gangliosides (sialic-acid-bearing glycolipids) have received attention in recent years because of their role in cell recognition phenomena, synaptic transmission, memory generation, and nerve regeneration in the fields of neurosciences. It is suggested that each brain region or each neural cell type may contain a specific and characteristic set of gangliosides. We have investigated the immunocytochemical localization of several classes of gangliosides that include GM1, GM4, GD3, and GQ gangliosides on the cell surface of various cell types found in human neural cell cultures with antibodies specific for these gangliosides. Cell cultures were obtained from adult human brains and fetal human dorsal root ganglia and spinal cord and cultured in vitro for the period up to 6 months and utilized for the ganglioside immunocytochemistry. It was demonstrated that GM1 ganglioside was present in all galactocerebroside-positive oligodendrocytes and most of glial fibrillary acid protein (GFAP)-positive astrocytes (80%), most of neurofilament-positive neurons (80%), 50-70% of Schwann cells, and 5-10% of fibronectin-positive fibroblasts; GM4 ganglioside could be detected in all oligodendrocytes, 80% of astrocytes, and 50% of Schwann cells, while no staining was found in neurons or fibroblasts; GD3 ganglioside was present in all oligodendrocytes and 5-10% of astrocytes but not in neurons, Schwann cells, or fibroblasts; and all of fetal CNS neurons and approximately 80-90% of fetal dorsal root ganglia (DRG) neurons and a small percentage of astrocytes (10-20% in fetal and less than 1% in adult astrocytes) was labeled by A2B5 antibody which is specific for GQ ganglioside, while this antibody did not stain cell surface of oligodendrocytes, Schwann cells, or fibroblasts. Three classes of gangliosides, GM1, GM4, and GD3 were found to be definite components of fetal and adult human oligodendroglial plasma membrane, while GM1 and GM4 gangliosides were detected on the surface of most astrocytes. Only a minor population of astrocytes from both fetal and adult human CNS contained GD3 and GQ gangliosides. Two classes of gangliosides, GM1 and GQ, were detected on the surface of fetal human neurons. More than half of fetal Schwann cells reacted to GM1 and GM4 antibodies but did not to GD3 or GQ antibodies. We recognized the presence of a specific and characteristic set of gangliosides on the cell surface of different human neural cell types and these findings should facilitate further investigation of the precise biological activity of these gangliosides.     

Effect of ATP on astrocyte stellation is switched from suppressive to stimulatory during development

Adenosine 5′-triphosphate (ATP) functions as a neurotransmitter or neuromodulator in the brain. To understand the role of ATP during brain development, we investigated the effects of ATP on morphology of cultured astrocytes obtained from the cerebral cortices of embryonic day 18 (E18) and postnatal day 2 (PN2) rats. In E18 astrocytes, ATP (10–1000 μM) alone did not affect astrocyte morphology, but significantly suppressed astrocyte stellation induced by the β-adrenoceptor agonist isoproterenol or the membrane-permeable cyclic AMP analog dibutyryl cyclic AMP. The suppressive effect of ATP in embryonic astrocytes was selectively mimicked by P_2U purinoceptor agonists. ATP had no effect on stellation induced by the protein kinase C (PKC) activator phorbol ester. It is probable that ATP, via P_2U purinoceptors, suppresses cyclic AMP-dependent regulatory mechanism for stellation in embryonic astrocytes. On the other hand, PN2 astrocytes differentiated into stellate cells in response to ATP. The ATP-stimulated stellation in PN2 astrocytes was mimicked by adenosine, and blocked by P₁ purinoceptor antagonists. It is probable that ATP is broken down into adenosine, which stimulates P₁ purinoceptors, inducing stellation in postnatal astrocytes. These findings suggest that the effect of ATP on astrocyte stellation is switched from suppressive (P_2U purinoceptor-mediated) to stimulatory (P₁ purinoceptor-mediated) during late embryonic to neonatal stages. ATP may be a critical factor that determines timing of astrocyte differentiation during development.     

Astrocyte spreading in response to thrombin and lysophosphatidic acid is dependent on the Rho GTPase

Astrocytes are typically star shaped cells playing diverse roles in the function of the nervous system. In astrocyte cultures established from the cerebral hemispheres of newborn rats, the cells have generally a polygonal fibroblast-like morphology, but acquire a stellate shape upon serum removal. When the serine protease thrombin or the bioactive lipid lysophosphatidic acid is added, the stellate cells revert to the flat morphology. Here we show that the effect of these agents is mediated via activation of the small GTP-binding protein Rho. Neither thrombin nor lysophosphatidic acid induced spreading of astrocytes microinjected with C3 transferase, an exoenzyme which ADP-ribosylates and thereby inactivates Rho. In contrast, the response of cells injected with a dominant negative form of Rac was unaffected. In addition, the injection of active Rho into stellate astrocytes mimicked the effect of thrombin and lysophosphatidic acid and an injection of C3 into flat cells grown in serum induced stellation. The conversion from a stellate to a spread morphology upon activation of Rho resulted in the formation of stress fibers and focal adhesions which most probably are key events in establishing and stabilizing the altered cytoarchitecture. These results suggest that Rho plays a crucial role in determining the shape of astrocytes and thereby may modulate their interaction with neurons in vivo. GLIA 21:244–252, 1997. © 1997 Wiley-Liss, Inc.     

cAMP-induced stellation in primary astrocyte cultures with regional heterogeneity

It is well known that increased cAMP levels in cultured astrocytes can convert flat polygonal shaped astrocytes into process-bearing, stellate astrocytes. In this study, we have examined the possible existence of astrocyte regional heterogeneity in morphological changes in response to cAMP stimulation. Primary astrocyte cultures were prepared from six different regions of neonatal rat brains, including cerebral cortex, hippocampus, brain stem, mid brain, cerebellum, and hypothalamus. After about 2 weeks in culture, the astrocyte culture medium was changed to DMEM containing various concentrations of 8-CPT-cAMP, a membrane permeable cAMP analog, for 2 h. We found that 250 microM 8-CPT-cAMP produced a maximum effect causing >95% stellation in all regional astrocytes except hypothalamic astrocytes (56% stellation). At lower cAMP concentrations, cell stellation most effectively occurred in cerebellar astrocytes. To examine further the regional heterogeneity of astrocyte morphological changes, glutamate was added together with 8-CPT-cAMP to block cAMP-induced astrocyte stellation. Interestingly, glutamate blockage on cAMP-induced astrocyte stellation was brain region-specific in that cerebral and hippocampal astrocytes were effectively blocked by glutamate when compared to other regional astrocytes. Furthermore, glutamate inhibited isoproterenol-induced astrocyte stellation in a region-specific manner similarly as in cAMP-induced stellation. The present study demonstrates that astrocytes derived from different regions of the neonatal rat brain maintain different levels of morphological plasticity in culture.     

Astrocyte stellation in saline media lacking bicarbonate: possible relation to intracellular pH and tyrosine phosphorylation

Primary cultures of astrocytes exhibit a polygonal morphology, but on treatment with agents that increase cAMP they change to stellate cells. We found that astrocyte stellation also occurred on replacing the culture medium with saline buffered with HEPES. However, stellation did not occur when the medium was replaced with saline buffered with bicarbonate/CO(2) provided Ca(2+) was present. Since exposure of astrocytes to media lacking bicarbonate results in a decrease in intracellular pH (pH(i)) we sought evidence for an association between pH(i) and morphology. Astrocytic pH(i) was monitored for 60 min after transferring the cells to HEPES or bicarbonate-buffered saline. HEPES-induced stellation was associated with transient acidification which coincided with the morphological changes. Acidification was not observed in cells transferred to bicarbonate-saline. However when cytoplasmic acidification of cells in bicarbonate-saline was induced pharmacologically, rapid stellation occurred. Stellation induced by cAMP is reversed by activation of the RhoA pathway with lysophosphatidic acid (LPA). Here we found that LPA inhibited HEPES-induced stellation, but only with Ca(2+) present. Inhibition of stellation by LPA+Ca(2+) was associated with transient acidification followed by modest alkanization. A close association of tyrosine phosphorylation with stellation and pH(i) was observed. Thus incubation of astrocytes in HEPES-saline with orthovanadate to inhibit dephosphorylation abolished stellation and acidification; conversely incubation of cells in bicarbonate-saline with genistein to inhibit tyrosine kinases caused stellation and major acidification. Acidification may be one of several factors resulting in stellation, but it is not a necessary factor since stellation without acidification was observed in bicarbonate-saline lacking Ca(2+).     

Adenosine stimulates stellation of cultured rat cortical astrocytes

We investigated the effect of adenosine on astrocyte morphology by using cell cultures prepared from the cerebral cortices of neonatal rats. Cultured rat cortical astrocytes exhibited flattened, polygonal morphology in the absence of stimulation, but differentiated into process-bearing stellate cells in response to adenosine (1–1000 μM). Adenosine-induced astrocyte stellation was abolished by treatment with microtubule inhibitors, colchicine and paclitaxel, indicating the involvement of cytoskeletal elements. The effect of adenosine was mimicked by other adenosine receptor agonists, and blocked by adenosine receptor antagonists and guanosine 5′-O-(2-thiodiphosphate), indicating that the effect of adenosine is mediated by G protein-coupled adenosine receptors. Although adenosine receptors are known to be linked to adenylate cyclase or phospholipase C, adenosine did not change intracellular cyclic AMP level nor intracellular Ca²⁺ concentration in astrocytes. Alternatively, adenosine-induced stellation was abolished by tyrosine phosphatase inhibitors, orthovanadate and phenylarsine oxide, suggesting that adenosine causes astrocyte stellation through tyrosine dephosphorylation. Adenosine may function as a factor regulating astrocyte differentiation.     

Expression of gangliosides on glial and neuronal cells in normal and pathological adult human brain

Few studies have assessed the glycolipid phenotype of glial cells in the human central nervous system (CNS) in situ. We investigated by immunohistochemistry the expression and cellular distribution of a panel of gangliosides (GM1, GM2, acetyl-GM3, GD1a, GD1b, GD2, GD3, GT1b, GQ1b and the A2B5 antibody) in adult, human normal and pathological brain, namely multiple sclerosis (MS) and other neurological diseases (OND). In normal conditions, we found diffuse expression in the white matter of most gangliosides tested, with the exception of acetyl-GM3, GT1b and GQ1b. By double immunofluorescence with phenotypic markers, GM1 and GD1b were preferentially expressed on GFAP+ astrocytes, GD1a on NG2+ oligodendrocyte precursors, A2B5 immunostained both populations, while GD2 was selectively present on mature oligodendrocytes. In the gray matter, only GM1, GD2 and A2B5 were present on neuronal cells. Interestingly, those gangliosides present on astrocytes in normal conditions were preferentially expressed on NG2+ cells in chronic MS lesions and in OND. Selective expression of GT1b upon astrocytes and NG2+ cells was instead observed in MS lesions, but not in OND. The definition of the glycolipid phenotype of CNS glial cells may be useful to identify distinct biological glial subsets and provide insights on the potential autoantigenic role of gangliosides in CNS autoimmune diseases.     

Effects of exogenous GM1 and GD1a on S20Y neuroblastoma cells

The effects of exogenous GM1 and GD1a on S20Y murine neuroblastoma cells were assessed by monitoring morphology, tumorigenicity, mitotic index, and plating efficiency. S20Y cells were seeded at a density equivalent to 5 X 10(4) cells per 35-mm tissue culture dish; 38-42 hr after seeding (preconfluent stage) the cells were treated for 12 hr with 100 micrograms of ganglioside per ml of medium in which the serum content was reduced from 10% to 0.5%. Analysis of the cell lipids indicated that added ganglioside became tightly associated with the membrane during the 12-hr exposure. GM1 treatment resulted in increased projections on the cell surface and fine structures projecting from the cell processes. GD1a treatment resulted in a reduction in the cellular mitotic index. Plating efficiency was reduced by both GM1 and GD1a. Neither ganglioside affected tumorigenicity of the S20Y cells. Twelve hours after removal of the added ganglioside and exposure of the cells to normal medium, the ganglioside composition of the membranes from treated cells approached that of the controls, and the ganglioside-induced effects had been reversed. These results suggest that addition of specific gangliosides induces different cellular responses and that these changes are dependent upon the continued presence of the ganglioside.     

Ganglioside metabolism in the hippocampus after septal lesion in rats

The changes in ganglioside composition and metabolism of deafferentiated rat hippocampus were estimated after septal lesion. A significant decrease in total ganglioside concentration was found 7 days after the lesion. The reduced level of total gangliosides persisted at 17 and 25 days. Relative increase in the proportion of GD1b and GX (O-acetylated GT1b) and decrease in GM1 were found in hippocampus only at 25 days post-lesion. The incorporation of 3H-N-acetylmannoseamine into gangliosides was examined in rats whose hippocampi were lesioned 25 days prior to radioprecursor injection. Differences in the labeling pattern of total and individual gangliosides were found. Increases in the label in GM1, GD3, and GD1a and decreases in GT1b and GQ1b were found 10 hr after isotope injection. However, decreases in the specific activity of all gangliosides except GT1b and GQ1b were observed 24 hr after 3H-N-acetylomannosamine injection, suggesting the activated turnover of gangliosides in postlesioned hippocampus. The significance of these changes has been discussed in terms of cellular damage and repair in the hippocampal tissue.     

Gangliosides of cultured astroglia

Cultured astrocytes prepared from newborn rat brain and 13-day-old chick embryonic brain were analyzed qualitatively and quantitatively for ganglioside content. All preparations contained approximately the same total level: 2.4-3.4 micrograms N-acetylneuraminic acid (NeuAc)/mg protein. In contrast, the value for primary cultures of neurons from chick embryonic brain was 5.9. The non-hexosamine-containing species, GM3 and GD3, comprised 75-85% of the total in astroglial cultures, the remainder consisting mainly of structural types other than the gangliotetraose series; choleragenoid assay revealed the latter to be virtually absent or to comprise at most a few percent. Deficiency of gangliotetraose synthesizing ability was indicated by the very low level of UDP-GalNac:GM3 N-acetylgalactosaminyltransferase detected in the cells. Treatment of cultured astrocytes with astroglial growth factor 2 or dibutyryl cyclic AMP caused little if any change in quantity or pattern of gangliosides. The large majority of cells stained in a manner characteristic of astrocytes: positive for glial fibrillary acidic protein, negative for galactosyl ceramides. Staining with cholera toxin and anti-GM1 antibody was essentially negative, as was that with tetanus toxin, A2B5 monoclonal antibody, and antibody to GD3. All evidence thus points to cultured astrocytes of rat and chick brain containing appreciable gangliosides, most of which are GM3 and GD3 with the majority of the remainder comprising structures other than the gangliotetraose type.     

Protection of neuro-2a cells against calcium ionophore cytotoxicity by gangliosides.

Gangliosides are known to assert both neuritogenic and neuroprotective effects when applied to a variety of neuroblastoma and primary neuronal cultures. We have developed a model employing Neuro-2a neuroblastoma cells with Ca2+ ionophore A23187 as neurotoxic agent causing neurite retraction and eventual cell death. Gangliosides attenuated the toxicity of this substance, increasing both cell survival and neurite stability. In one series of experiments, cells were exposed to A23187 for 24 hr and then incubated in fresh medium (washout) for 18 hr; gangliosides were present at varying times. The paradigm in which cells were only preincubated (2 hr) with ganglioside provided no benefit, nor did incubation of the cells in both ionophore and ganglioside during the 24-hr exposure period. Significant protection was achieved by exposing the cells to ganglioside after washout of A23187, or continuously throughout the whole period. Bovine brain ganglioside mixture and the four major components (GM1, GD1a, GD1b, GT1b) applied individually were all effective. By contrast, GM3 and GM1-alcohol, a neutral derivative of GM1, provided little or no protection. Dichlorobenzamil, an inhibitor of the Na(+)-Ca2+ exchanger, tended to block the neurite stabilizing effect of gangliosides, suggesting that the mechanism might involve potentiation of this antiporter.     

Manganese stimulates stellation of cultured rat cortical astrocytes.

We investigated the effect of manganese on the morphology of cultured rat cortical astrocytes. Astrocytes exhibited flattened, polygonal morphology in the absence of stimulation, and differentiated into process-bearing stellate cells following exposure to MnCl(2). MnCl(2)-induced stellation was a reversible process, which depended on the presence of extracellular free manganese. MnCl(2)-induced stellation did not stop with the introduction of pertussis toxin, PD98059, SB203580, phorbol 12-myristat 13-acetate, SQ22536, or LY83583. Alternatively, MnCl(2)-induced stellation did stop when exposed to colchicine and sodium orthovanadate, suggesting the involvement of the cytoskeletal elements and orthovanadate-sensitive protein tyrosine phosphatase. MnCl(2) might function as a factor regulating astrocyte morphology.     

The composition of glycosphingolipids and the effect of neuraminidase on cultured glioma cell lines

The composition of glycosphingolipid on human cultured glioma cell line U 251 and rat glioma cell line C6 was analysed by high performance thin layer chromatography. As a result, the major gangliosides were simple gangliosides such as GM3 (U 251: 7.7%, C6: 84.3%), GM2 (U 251: 32.6%) and SPG (U 251: 30.0%) on glioma cells whereas the major neutral glycosphingolipids were CDH, CTH and globoside. After treatment with neuraminidase 2.92 nmol/mg dry weight and 3.73 nmol/mg dry weight of sialic acid were freed from U 251 cells and C6 cell, but only 8.11% (U 251 cell) and 11.24% (C 6 cell) of these sialic acids originated from glycolipid, and thus the major part of sialic acid might be released from glycoprotein of the cells. The gangliosides that react to neuraminidase are SPG, GD1a and GD1b in U 251 cells and are GM1a and little GM3 in C 6 cells. The biolabelling study using N-acetyl-14C-mannosamine as a precursor of sialic acid demonstrated that the precursor was mainly incorporated into both or either of GM3 and SPG in the acidic glycolipid fraction. In addition, no significant change on proliferation and morphology of glioma cells after neuraminidase treatment was observed in this study.     

Tissue oxygen levels control astrocyte movement and differentiation in developing retina

Astrocytes play a key role in the development of retinal vessels by detecting hypoxia in developing retina and secreting the hypoxia-induced angiogenic factor VEGF to induce vessel formation. The astrocytes which play this role are themselves spreading over the retina, just ahead of the growing vessels. To understand the mechanisms which keep astrocytes in this strategic 'just ahead' position we have studied the effects of hyperoxia and hypoxia on astrocyte differentiation and movement in situ in neonatal rat retina and in primary culture. Hyperoxia in situ inhibited the stellation of astrocytes, so that they persisted in a relatively unbranched form, which accumulated at the edge of their spreading population; hyperoxia permitted but did not accelerate migration. Conversely, hypoxia induced unstellated astrocytes to stellate within 6 h. If the hypoxia was abnormally severe, it caused the astrocytes to hyperstellate and slowed their spread. Astrocytes in primary culture did not change morphology or motility when challenged by hypoxia. When treated with medium conditioned by retina however, astrocytes became mobile and, if the medium was conditioned by hypoxic retina, became stellate. These results suggest that the oxygen released by retinal vessels maintains the mobility of astrocytes, via a diffusible factor released by other retinal cells. Conversely, naturally generated hypoxia of developing retina plays a triple role, inducing astrocytes to stellate, to end their migration and to produce VEGF, thereby inducing vessel formation. The induction of stellation is mediated by a diffusible factor released by other retinal cells. Thus hypoxia of the retina generated by neural maturation induces key events in both the differentiation of astrocytes and the formation of blood vessels.