Glial cell growth in culture: Influence of living cell substrata

The role of the microenvironment in the growth of glial cells in culture has been the topic of ongoing research in this laboratory. Recently, we reported a study on the contribution of fibroblast cell substratum and extracellular matrix in glial cell growth. In the present study we report data concerning a) the influence of a neuronal-enriched living substratum from chick embryo on the growth of glial cells derived from chick embryonic brain and plated onto the substratum; b) the influence of dissociated cells derived from chick embryonic brain on the growth of established glial cells in culture, and c) the influence of dissociated cells derived from adult rat spinal cord on the growth of established glial cells from newborn rat in culture. The activities of glutamine synthetase (GS) and 2, 3-cyclic nucleotide 3-phosphohydrolase (CNP) were the biochemical probes determined for astrocytes and oligodendrocytes, respectively. We found that glial growth as assessed by both enzyme activities, was enhanced when a nervous tissue derived cell population was plated onto a glial-enriched substratum, whereas glial growth was inhibited when the neuronal-enriched population was the cell substratum.Special Issue dedicated to Dr. Elizabeth Roboz-Einstein.     

Differential responsiveness of late passage C-6 glial cells and advanced passages of astrocytes derived from aged mouse cerebral hemispheres to cytokines and growth factor: Glutamine synthetase activity

In this study, we were interested to compare the responsiveness to growth factors, NGF, b-FGF and EGF and cytokines, IL1β, and TNF-α, in late passages (74–79) C6 glial cells committed astrocytes and astrocytes of advanced passages (26–28) in cultures derived from aged mouse cerebral hemispheres (MACH). Cultures were grown in either DMEM or chemically defined medium (CDM/TIPS) in order to test the effects of growth factors or cytokines. The activity of glutamine synthetase (GS), a marker for astrocytes, was used as a test parameter. We found that treatment with growth factors increased GS activity in both glial cell culture systems with the exception of EGF in C-6 glial cells. Treatment with cytokines markedly decreased GS activity in the late passage C6 glial cells whereas only TNF-α had a similar effect on MACH astrocytes. In view of the generally opposite effects of growth factors and cytokines on GS activity, we-speculate that these molecules which are also endogenously present in glial cells may play a role in the maintenance of cellular homeostasis.     

Induction of an oligodendroglial enzyme in C-6 glioma cells maintained at high density or in serum-free medium.

The relationship between cell density and the activity of 2':3'-cyclic nucleotide 3'-phosphohydrolase (CNP), an enzyme believed to be specific to oligodendroglial cells and myelin in the brain, has been studied in cultured C-6 glioma cells. Over a 12-day period, the specific activity of CNP underwent a 4-fold increase in conjunction with an increase in the cell density (total protein/flask) and a decline in the growth rate of the cultures. In contrast, the specific activity of Na+,K+-ATPase was not influenced by cell density. Experiments with cultures seeded at different initial densities indicated that the increase in CNP activity coincided with the attainment of a specific cell density rather than with the length of time that the cells were maintained in culture. Arrest of cell proliferation in non-confluent C-6 cells by means of thymidine blockade was not sufficient to cause an increase in the activity of CNP; however, removal of serum from the culture medium resulted in a 3-fold induction of the enzyme in the absence of a high degree of cell contact. The induction of CNP in cells maintained in serum-free medium paralleled the development of a series of distinct morphological changes reminiscent of glial differentiation, which occurred within 48 hours after removal of the serum. Inhibition of protein synthesis by cycloheximide prevented the induction of CNP in serum-free cultures. The demonstration that an enhancement of an oligodendroglial characteristic in C-6 glioma cells can be obtained by growing the cells to high density or by removing serum from the medium, provides further support for the suggestion that these cells may be analogous to the glial stem cells present in the developing brain.     

Type I collagen preparations inhibit DNA synthesis in glial cells of the peripheral nervous system

The mechanisms underlying cessation of glial proliferation in the developing peripheral nervous system are obscure. One possibility, as yet little explored, is that mitotic inhibitory signals play a part in regulating glial cell numbers. In this study we demonstrate that type I collagen preparations from several different sources can inhibit the rate of DNA synthesis in purified populations of enteric glia and both short-term and long-term secondary Schwann cells in dissociated cell cultures. When these cells are grown on gelled or dried type I collagen substrata, they proliferate at substantially lower rates than on polylysine substrata. In contrast, type III or V collagen preparations do not inhibit glial DNA synthesis and laminin, fibronectin, type IV collagen, and secreted matrix from bovine corneal endothelial cells all stimulate thymidine incorporation. The inhibitory effect is not observed with heat denatured type I collagen preparations, but is seen equally in serum-containing medium, in medium containing fibronectin-free serum, or in serum-free medium, suggesting that the interaction of collagen with the cells requires structurally intact collagen molecules and does not occur via intermediary linkage to fibronectin. The inhibition on collagen is accompanied by a shape change from a more flattened morphology to a narrow spindle form. The labeling index of a rat Schwannoma cell line, 33B, is not inhibited on type I collagen substrata. These results demonstrate that type I collagen preparations inhibit the DNA synthesis levels of early postnatal peripheral glial cells in vitro. It remains to be determined whether this effect occurs via direct collagen-cell membrane interactions or whether it depends on accessory molecules, perhaps present in the collagen preparations themselves, since these are not purified to absolute homogeneity.     

Cultured neonatal rat oligodendrocytes are enriched in acid hydrolase activities

We measured the activity of several acid hydrolases in oligodendrocyte and mixed glial (predominantly astrocytic) cell cultures prepared from neonatal rat cerebra. When compared with the mixed glial cultures, the cultured oligodendrocytes exhibited higher levels for all the hydrolases when activities were normalized to protein content. When enzymic activities were examined as a function of DNA content, oligodendrocytic -l-fucosidase, -d-glucuronidase, arylsulfatase, and N-acetyl--d-glucosaminidase were higher than in mixed glial cultures, whereas the activities of -d-glucosidase, -d-galactosidase and acid phosphatase were not elevated. These differences could not be accounted for by the fetal bovine serum present in the culture medium. The enrichment in acid hydrolase specific activities in the oligodendrocytes may be associated with a rapid turnover of at least some of the extensive myelin-like membranes formed by these cultured cells. Alternatively, the enrichment of acid hydrolase activity in the oligodendrocytes may be associated with intracellular vesicles of lysosomal origin which may play a role in myelin-like membrane assembly. Exactly which of the above two processes, or possible combinations thereof, is responsible for the present finding is not known.     

Relation of Cholesterol to Oligodendroglial Differentiation in C-6 Glial Cells

Abstract: The relation of cellular cholesterol content to a biochemical expression of oligodendroglial differentiation was studied in cultured C-6 glial cells. Induction of the oligodendroglial marker enzyme 2′: 3′-cyclic nucleotide 3′-phosphohydrolase (CNP) was determined after alteration of the sterol content of cellular membranes by exposure to compactin, a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol synthesis. The sterol content and as a consequence, the sterol/phospholipid molar ratio of C-6 glial cells were decreased by treating the cells, in 10% lipoprotein-poor serum, with various concentrations of compactin for 24 h. The degrees of sterol depletion thus produced were maintained for 48 h after removal of the compactin if the cells were maintained in serum-free medium, the culture conditions necessary for induction of CNP in untreated cells. Forty-eight hours after removal of serum, no induction of CNP occurred in cells previously treated with 0.5 μg/ml of compactin, whereas untreated cells exhibited a three- to fourfold increase in CNP activity. Intermediate degrees of sterol depletion resulted in intermediate degrees of inhibition of the CNP induction. Moreover, the morphological expressions of glial differentiation observed in the untreated cells did not occur in the sterol-depleted cells. That the effect of compactin on the induction of CNP relates to depletion of sterol was indicated by the finding that when low-density lipoprotein was added to the compactin-treated cells, the induction of CNP, the morphological expressions of differentiation and the sterol/phospholipid molar ratios were preserved. The degree of sterol depletion that totally prevented the induction of CNP had no effect on (Na⁺+ K⁺)-activated ATPase activity, total protein synthesis and cell viability. The data define a critical role for sterol in oligodendroglial differentiation in this model system.     

Expression and extinction of glial properties in glioma X neuroblastoma cell hybrids

Rat glioma cells (clone C₆TK⁻) were hybridized with mouse neuroblastoma cells (clone NA), and 18 primary and secondary hybrid clones containing one chromosome set from each parent were isolated. The hybrids were assayed for the glial marker enzymes 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) and glycerol-3-phosphate dehydrogenase (GPDH). In many of the hybrid clones, the levels of CNP and GPDH were reduced to 5–20% of the activity of C₆TK⁻, as has been observed in other classes of glial X non-glial cell hybrids. In some hybrid clones, however, GPDH and CNP were expressed at high activity. Rat (glial) GPDH activity was not reduced in these clones, but mouse GPDH activity remained low, and was not “de-repressed” or “activated”. This suggests that the controls governing differentiation in neuroblastoma cells and extinction in hybrids may differ in some important details. There was a strong positive correlation between the specific activities of CNP and GPDH in the hybrid clones, suggesting that a mechanism regulates the activity of these two glial enzymes coordinately.     

Responsiveness of acetylcholinesterase and butyrylcholinesterase activities in neural cells to age and cell density in culture

Acetylcholinesterase activity in neuroblastoma and C-6 glial cells, maintained in monolayer culture, decreased with increasing age and cell density (cells/mm²). Butyrylcholinesterase activity in C-6 glial cells did not change with age, but increased slowly with increasing cell density. AChE reached peak activity at a lower density in neuroblastoma than in C-6 glial cells. The data suggest either that AChE in both cell types is responsive to cell-cell contact or that different enzymes are involved.     

Responsiveness of actylcholinesterase and butyrylcholinesterase activities in neural cells to age and cell density in culture.

Acetylcholinesterase activity in neuroblastoma and C-6 glial cells, maintained in monolayer culture, decreased with increasing age and cell density (cells/mm²). Butyrylcholinesterase activity in C-6 glial cells did not change with age, but increased slowly with increasing cell density. AChE reached peak activity at a lower density in neuroblastoma than in C-6 glial cells. The data suggest either that AChE in both cell types is responsive to cell-cell contact or that different enzymes are involved.     

Modulation of protein synthesis and secretion by substratum in primary cultures of rat hepatocytes

Hepatocytes isolated by perfusion of adult rat liver and cultured on substrata consisting of one or more of the major components of the liver biomatrix (fibronectin, laminin, type IV collagen) have been examined for the synthesis of defined proteins. Under these conditions, tyrosine amino transferase, a marker of hepatocyte function, is maintained at similar levels in response to dexamethasone over 5 days in culture on each substratum, and total cellular protein synthesis remains constant. By contrast, there is a rapid decrease in synthesis and secretion of albumin and a 3-7-fold increase in synthesis and secretion of alpha-fetoprotein which are most marked on a laminin substratum, but least evident on type IV collagen, and an increased synthesis of fibronectin and type IV collagen. The newly synthesized matrix proteins are present in the cell layer as well as in cell secretions. The enhanced synthesis of fibronectin is less in cells seeded onto a fibronectin substratum than on laminin or type IV collagen substrata, and its synthesis by hepatocytes seeded onto a mixed substratum of laminin and fibronectin is down-regulated by fibronectin in a dose-related manner. Similarly, type IV collagen synthesis is less when the cells are seeded on the homologous matrix protein substratum than on heterologous substrata. These results indicate that hepatocytes cultured in serum-free medium on substrata composed of components of the liver biomatrix maintain certain functions of the differentiated state (tyrosine amino transferase), lose others (albumin secretion) and switch to increased synthesis of matrix components as well as fetal markers such as alpha-fetoprotein. The magnitude of these effects depends on the substratum on which the hepatocytes are cultured.     

Sialic acid masked membrane antigens of clonal functional glial cells

Antisera have been prepared to clonal cultured rat astrocytic cells (C₆ cells) and clonal human astrocytic cells (CHB₄ cells). When cultured glial cells are grown in the presence of neuraminidase the serological activity of the cell membrane with homologous antiserum is increased extensively. A similar activation of serological activity can be effected by incubating washed harvested cells with this enzyme. Membrane bound N-acetyl-neuraminic acid is extensively reduced by either incubation. The activity responsible for promoting the enhanced serological activity co-chromatographs and co-electrophoreses with neuraminidase. Proteolytic digestion, unlike neuraminidase treatment, was unable to enhance the serological activity of cultured glial cells.     

Isolation and characterization of Chinese hamster ovary cell variants defective in adhesion to fibronectin-coated collagen

Variant clones of Chinese hamster ovary (CHO) cells were selected for reduced adhesion to serum-coated tissue culture plates. These clones also displayed reduced adhesion to substrata composed of collagen layers coated with bovine serum or with fibronectin (cold-insoluble globulin). Wild-type (WT) and adhesion variant (ADv) cells grew at comparable rates in suspension culture, but the adhesion variants could not be grown in monolayer culture because of their inability to attach to the substratum. The adhesion deficit in these cells was not corrected by raising the concentration of divalent cations or of serum to levels 10-fold greater than those normally utilized in cell culture. However, both WT and ADv clones could adhere, spread, and attain a normal CHO morphology on substrata coated with concanavalin A or poly-L- lysine. In addition, the adhesion variants could attach to substrata coated with "footpad" material (substratum-attached material) derived from monolayers of human diploid fibroblasts or WT CHO cells. These observations suggest that the variant clones may have a cell surface defect that prevents them from utilizing exogeneous fibronectin as an adhesion-promoting ligand; however the variants seem to have normal cytoskeletal and metabolic capacities that allow them to attach and spread on substrata coated with alternative ligands. These variants should be extremely useful in studying the molecular basis of cell adhesion.     

Trypsinization of chick glial cells before seeding: Effects on energy metabolism enzymes and glutamine synthetase

In order to test the possible involvement of surface proteins on some metabolical aspects of chick glial cell differentiation in culture, perturbations were induced on the glial cell surface membrane by limited trypsinization before seeding. The developmental changes of enzymes involved in the energy metabolism of the cell: malate dehydrogenase (MDH), glutamate dehydrogenase (GDH), hexokinase (HK), lactate dehydrogenase (LDH), enolase as well as glutamine synthetase (GS) were determined in trypsin treated cells and controls. The total protein and DNA content per dish was higher in treated cells than in controls, however the protein ratio towards DNA remained unchanged. The levels of GS, GDH, LDH, and enolase activities were significantly enhanced after trypsin treatment of the cells compared to controls. The enhanced value of total LDH activity is essentially the result of the increase of M subunit containing isoenzymes. Considering that a higher level of GS activity characterizes some maturation of the glial cells (as observed during the maturation of the chick brain) it is apparent that modifications of cell surface located factors, by trypsin treatment, induce differentiation phenomena at the functional state of the glial cells in culture. This may indicate that interactions located at the cell surface are involved in the modulation of key enzymes of the energy metabolism pathway.     

Biochemical characteristics of C-6 glial cells

C-6 glial cells were studied in culture with respect to morphological and biochemical changes under several experimental conditions. Doubling time was 33 hr for cells plated at either 0.5 or 1.0×10⁶ cells per flask. Markedly reduced cell growth and astrocyte-like appearance were observed following dibutyryl cyclic AMP (DBcAMP) treatment. An inverse relationship between cell density and DNA, RNA, and protein content per cell was observed. AChE and BuChE activities were also inversely related to cell density, and treatment with DBcAMP increased enzyme activity, but did not alter the cell density relationship. Uptake of³H-norepinephrine also decreased with increasing cell density. In DBcAMP-treated cells,³H-NE uptake was markedly lower than in nontreated controls, and cortisol treatment decreased the uptake of³H-NE in DBcAMP-treated cells further still. We interpreted the foregoing changes to indicate that cellular activity is cell density-dependent.     

Catecholaminergic Expression in 2N27 Immortal Neural Cell Line Is Enhanced by Glial-Derived Factors

The goal of this study was to examine the responsiveness of an immortalized catecholaminergic neuronal line, 2N27, to various growth factors and identify those which promote catecholaminergic expression. 2N27 is a newly established neural cell line derived from fetal rat mesencephalic tissue and, thus, contains tyrosine hydroxylase (TH), a reliable marker for catecholaminergic neurons. Using TH activity as a biochemical index, we examined the responsiveness to both recognized trophic factors (NGF, TGF- and basic- and acidic-FGF) as well as novel, glia-derived factors present in conditioned media from several glial sources. The glial cells included MACH, a normal cell line derived from aged mouse cerebral hemispheres NBCC, normal glia derived from newborn mouse cerebral hemispheres; and C-6 glioma cells, 2B clone, passage 72, predominately astrocytes. Cells were cultured in the presence of added factors from 0 to 3 days in vitro (DIV) and were harvested on day 4. We found that 2N27 neural cells responded differentially to growth factors. No change was observed in TH activity in response to NGF, TH activity even decreased in response to b-FGF ad TGF- addition to the culture medium. However, a dose dependent increase in TH activity was observed following treatment with a-FGF and the increase to a-FGF was associated to an increase in cell proliferation as compared to TH increase by cAMP associated to differentiation. However, the 2N27 cells responded with a marked increase in TH when cultured in the glial cell conditioned media. We conclude that immortal cells require a variety of microenvironmental signals to maintain their phenotype.     

Glutamine synthetase expression in rat oligodendrocytes in culture: regulation by hormones and growth factors.

Glutamine synthetase (GS, EC 6.3.1.2.) has long been considered as a protein specific for astrocytes in the brain, but recently GS immunoreactivity has been reported in oligodendrocytes both in mixed primary glial cell cultures and in vivo. We have investigated its expression and regulation in "pure" oligodendrocyte cultures. "Pure" oligodendrocyte secondary cultures were derived from newborn rat brain primary cultures enriched in oligodendrocytes as described by Besnard et al. (1987) and were grown in chemically defined medium. These cultures contain more than 90% galactocerebroside-positive oligodendrocytes and produce "myelin" membranes (Fressinaud et al., 1990) after 6-10 days in subcultures (30-35 days, total time in culture). The presence of GS in oligodendrocytes from both primary glial cell cultures and "pure" oligodendrocyte cultures was confirmed by double immunostaining with a rabbit antisheep GS and guinea pig antirat brain myelin 2', 3'-cyclic nucleotide 3'-phosphodiesterase. In "pure" oligodendrocyte cultures, about half of cells were labeled with anti-GS antibody. Furthermore, on the immunoblot performed with a rabbit antisheep GS, the GS protein in "pure" oligodendrocyte secondary cultures was visualized as a single band with an apparent molecular mass of about 43 kDa. In contrast, two protein bands for GS were observed in cultured astrocytes. On the immunoblot performed with a rabbit antichick GS, two immunopositive protein bands were observed: a major one migrating as the purified adult chick brain GS and a minor one with a lower molecular mass. Two similar immunoreactive bands were also observed in pure rat astrocyte cultures. Compared to pure rat astrocyte cultures, "pure" oligodendrocyte cultures of the same age displayed an unexpectedly high GS specific activity that could not be explained by astrocytic contamination of the cultures (less than 5%). As for cultured astrocytes, treatment of oligodendrocyte cultures with dibutyryl-adenosine 3':5'-cyclic monophosphate, triiodothyronine, or hydrocortisone increased significantly GS specific activity. Interestingly, epidermal growth factor, basic fibroblast growth factor, and platelet-derived growth factor that increase the GS activity in astrocytes do not affect this activity in oligodendrocytes. Thus we confirm the finding of Warringa et al. (1988) that GS is also expressed in oligodendrocytes. We show that its activity is regulated similarly in astrocytes and oligodendrocytes by hormones, but that it is regulated differently by growth factors in these two cell types.     

Primary cultures of hepatocytes in serum and hormone-free medium: Identification of conditions which stimulate an in vivo-like induction of G6PD

Summary Recent results from several laboratories suggest that complex interactions between hormones and dietary carbohydrate may be responsible for regulating the induction of several hepatic lipogenic enzymes. Elucidation of these interactions requires the ability to culture hepatocytes for several days in serum-free medium where the hormones or carbohydrate or both present is strictly controlled. The functional response of primary adult rat hepatocytes was examined in a medium without exposure to serum, hormones, or carbohydrates and on three substrata commonly used to culture cells in a defined medium. Hepatocytes cultured on a floating collagen gel in which is embedded a nylon mesh possess cell attachment and morphologic characteristics superior to either cells cultured on a collagen-coated or fibronectin(Fn)-coated substratum. Cells cultured on the gel-mesh system retain insulin responsivity, as measured by protein synthesis rates and glucose-6-phosphate dehydrogenase (G6PD) induction, for at least 6 d in culture. Under these conditions, insulin, dexamethasone, and fructose increase G6PD specific activity to levels comparble to that seen in an induced animal. Hepatocytes cultured on the gel-mesh system tolerate restricted medium conditions better than cells cultured on collagen or Fn-coated substratum, and remain viable for sufficient times to allow, for the first time, full expression and maximal induction (i.e. like in vivo), of G6PD in cultured cells. This system represents a satisfactory model for in vivo liver metabolism and a superior system for studying the effects of hormones and metabolites on G6PD levels, as well as other nutritional-hormonally regulated enzymes.     

Nerve growth factor inhibits osmotic swelling of rat retinal glial (Müller) and bipolar cells by inducing glial cytokine release

Osmotic swelling of neurons and glial cells contributes to the development of retinal edema and neurodegeneration. We show that nerve growth factor (NGF) inhibits the swelling of glial (Müller) and bipolar cells in rat retinal slices induced by barium‐containing hypoosmotic solution. NGF also reduced Müller and bipolar cell swelling in the post‐ischemic retina. On the other hand, NGF prevented the swelling of freshly isolated Müller cells, but not of isolated bipolar cells, suggesting that NGF induces a release of factors from Müller cells that inhibit bipolar cell swelling in retinal slices. The inhibitory effect of NGF on Müller cell swelling was mediated by activation of TrkA (the receptor tyrosine kinase A), but not p75^NTR, and was prevented by blockers of metabotropic glutamate, P2Y₁, adenosine A₁, and fibroblast growth factor receptors. Basic fibroblast growth factor fully inhibited the swelling of freshly isolated Müller cells, but only partially the swelling of isolated bipolar cells. In addition, glial cell line‐derived neurotrophic factor and transforming growth factor‐β1, but not epidermal growth factor and platelet‐derived growth factor, reduced the swelling of bipolar cells. Both Müller and bipolar cells displayed TrkA immunoreactivity, while Müller cells were also immunostained for p75^NTR and NGF. The data suggest that the neuroprotective effect of NGF in the retina is in part mediated by prevention of the cytotoxic glial and bipolar cell swelling.

     

Estrogen (17β-Estradiol) Enhances Glutamine Synthetase Activity in C6-Glioma Cells

Glutamine synthetase (GS) is the major glutamine-forming enzyme of vertebrates and is accepted to be a marker of astroglial cells. Maturation of astroglial cells is characterized by an increase of GS activity, and the regulation of this enzyme is the topic of many publications. Because of the fundamental role of the GS in controlling brain glutamate and glutamine level, it is essential to understand the mechanism of expression of this enzyme. To our knowledge, the effect of estrogen (17β-estradiol) on GS activity in glial cells has not been reported. We examined the effect of treatment with estrogen on glutamine synthetase enzyme activity in glial cells. C6-glioma cells in later passage have many astrocytic characteristics and provided a convenient and well-established model system. We adapted a colorimetric method to measure GS-catalyzed γ-glutamyltransferase (GT) activity in C6-glioma cells. The assay monitors GT activity of glutamine synthetase by following the absorbance of the product γ-glutamyl hydroxamate at 540 nm. We observed that, the absorbance of γ-glutamyl hydroxamate significantly increased in estrogen treated cells (0.13±0.03), as compared to untreated cells (0.058±0.015). Estrogen also significantly increased concentration of glutamine in C6-glioma cells as measured by fluorometric assay. In addition, western blot analysis showed that estrogen significantly increased the amount of glutamine synthetase compared to control. This estrogen effect could have important physiological implications on cerebral glutamate and glutamine metabolism.