Rat C6 Glial Cells Research Articles

Mechanisms of cholesterol synthesis inhibition by D-glucosamine.

The amino sugar D-glucosamine possesses antitumor activity which is thought to depend in part upon its ability to impair cholesterol biosynthesis and damage cellular membranes. The present study examined the effect of glucosamine on acetate utilization for lipid and sterol synthesis in rat C6 glial tumor cells. At cytotoxic concentrations, the amino sugar inhibited [14C]acetate incorporation into nonesterified sterols and lipids but increased the flow of label into cholesteryl esters. A comparison of the rates of acetate utilization for glucosamine metabolism (N-acetylation) and sterol and lipid synthesis suggested that glucosamine might act by competing for a common cytosolic pool of acetyl CoA. The inhibition of lipid and sterol synthesis, however, remained constant over a wide range of extracellular acetate concentrations. These results suggest that, if glucosamine acts by restricting the supply of acetate for these biosynthetic processes, it probably inhibits a step prior to the formation of acetyl CoA. Alternative mechanisms are discussed.

Detection of two chromatin proteins which bind specifically to the 5'-flanking region of the rat prolactin gene.

We employed a protein gel blotting procedure to search for nuclear proteins from rat pituitary cells that bind preferentially to the 5'-flanking region of the rat prolactin gene. By gel blots of chromatin proteins from GH3 rat pituitary tumor cells with a 32P-labeled prolactin genomic clone, we detected two major binding proteins with molecular weights of approximately 44,000 and 48,000, designated NP44 and NP48, respectively. Both NP44 and NP48 are minor chromatin proteins which are extracted at low salt concentrations (0.4 M NaCl) and exhibit a range of slightly acidic isoelectric variants. NP44 and NP48 were detected at similar levels in chromatin extracts of GH3 cells, the prolactin-negative GC cell variant of the GH3 cells, and normal rat pituitary tissue. Considerably lower levels of these two proteins were found in chromatin extracts from rat liver and rat C6 glial cells. NP44 and NP48 exhibit DNA sequence specificity, as evidenced by their strong binding to the upstream flanking region of the prolactin gene, but only very weak binding to plasmid DNA, rat prolactin or growth hormone cDNAs, or upstream flanking regions of two other rat genes. By analyzing subclones of a rat prolactin genomic clone, we established that NP44 and NP48 bind to at least two sites, which are located between 0.4 and 2.0 kilobases (region I) and between 2.0 and 4.8 kilobases (region II) upstream of the transcription initiation site. These findings are discussed in the context of a possible functional association between the strong binding of NP44 and NP48 to the prolactin 5'-flanking region and pituitary-specific expression of the prolactin gene.

Detection of two chromatin proteins which bind specifically to the 5'-flanking region of the rat prolactin gene.

We employed a protein gel blotting procedure to search for nuclear proteins from rat pituitary cells that bind preferentially to the 5'-flanking region of the rat prolactin gene. By gel blots of chromatin proteins from GH3 rat pituitary tumor cells with a 32P-labeled prolactin genomic clone, we detected two major binding proteins with molecular weights of approximately 44,000 and 48,000, designated NP44 and NP48, respectively. Both NP44 and NP48 are minor chromatin proteins which are extracted at low salt concentrations (0.4 M NaCl) and exhibit a range of slightly acidic isoelectric variants. NP44 and NP48 were detected at similar levels in chromatin extracts of GH3 cells, the prolactin-negative GC cell variant of the GH3 cells, and normal rat pituitary tissue. Considerably lower levels of these two proteins were found in chromatin extracts from rat liver and rat C6 glial cells. NP44 and NP48 exhibit DNA sequence specificity, as evidenced by their strong binding to the upstream flanking region of the prolactin gene, but only very weak binding to plasmid DNA, rat prolactin or growth hormone cDNAs, or upstream flanking regions of two other rat genes. By analyzing subclones of a rat prolactin genomic clone, we established that NP44 and NP48 bind to at least two sites, which are located between 0.4 and 2.0 kilobases (region I) and between 2.0 and 4.8 kilobases (region II) upstream of the transcription initiation site. These findings are discussed in the context of a possible functional association between the strong binding of NP44 and NP48 to the prolactin 5'-flanking region and pituitary-specific expression of the prolactin gene.

Expression of differentiated functions in heterokaryons between skeletal myocytes, adrenal cells, fibroblasts and glial cells

The regulation of both muscle and adrenal functions was examined in heterokaryons formed by fusing differentiated chick skeletal myocytes to Y1 mouse adrenal cells. Mouse fast skeletal myosin light chain one (LC1) synthesis was induced and acetylcholine receptor expression was maintained at muscle control levels. Steroid secretion, although reduced compared with Y1 × Y1 adrenal homokaryon control fusions, was nonetheless maintained at relatively high levels. Steroid secretion in the myocyte × adrenal heterokaryons was constitutively expressed and was not increased by exposure to either adrenocorticotrophic hormone or db-cAMP. The population of heterokaryons was thus simultaneously expressing both muscle and adrenal functions. The steroid secretion in these heterokaryons was compared to that in heterokaryons formed by fusing Y1 adrenal cells to either chick skin fibroblasts or rat C6 glial cells. Both of these sets of heterokaryons exhibited low baseline levels of steroid secretion that were inducible to control values by ACTH. These results extend previous observations showing that heterokaryons are functionally very different than cell hybrids, and exhibit a variety of phenotypic interactions. Although fibroblasts suppress muscle functions in heterokaryons, they are permissive for adrenal functions. C6 glial cells are permissive for both adrenal and muscle functions, and along with several other neurectodermal derivatives contain an inducible skeletal myosin light chain gene. Finally, myocytes and Y1 adrenal cells are mutually permissive for their differentiated functions, and Y1 adrenal cells contain an inducible myosin light chain gene.

Virus-host cell interaction in rat C6 glial cell cultures persistently infected with herpes simplex virus.

The virus-host cell interaction in a rat C6 glial cell line persistently infected with herpes simplex virus type 1 (HSV1) was examined. The C6 cultures infected at an MOI of 10 or 0.01 exhibited virally induced cytopathology and produced infectious virus and viral antigens for 108 and 50 days, respectively. Infectious virus was continually present in the supernatant fluids of persistently infected cultures, with only a minority of the cells having viral CPE or viral antigens at any given time. There was no obvious pattern of periodic cyclical viral replication in these cultures; no predictable regularity in the fluctuations of virus production was evident. 'Cured' cultures were as susceptible to re-infection with HSV1 as previously uninfected cultures; thus, there was no selection of a subpopulation of C6 cells resistant to subsequent challenge with HSV1. The virus recovered from persistently infected cultures was more infectious for C6 cells relative to HeLa cells than was the original virus stock. Significant levels of interferon were not detected in HSV1-infected C6 cultures during either the acute or persistent phases of infection or in 'cured' cultures.

In vitro stimulation of cell growth by hepatic stimulator substance.

An extract of normal weanling or adult regenerating rat liver called hepatic stimulator substance (HSS) stimulates incorporation of tritiated thymidine ([3H]dThd) into liver DNA. In vivo, it is organ specific, species nonspecific, displays diurnal rhythm, and has a molecular weight of approximately 10,000-20,000. It has now been further characterized in vitro. HSS retained organ specificity, stimulating normal adult hepatocytes in primary culture, the HTC, PLC, and MH1C1 hepatoma cell lines, and the PRC rat liver cell line. It did not stimulate human or mouse lymphocytes, 3T3 mouse fibroblasts, Y1BS1 mouse adrenal cells, Chinese hamster ovary cells, rat hypernephroma cells, C6 rat glial cells, or L1210 mouse leukemic cells. More detailed studies of the response of HTC cells to HSS revealed a dose-dependent increase in [3H]dThd incorporation (up to 30-fold) with a response to as little as 140-ng purified extract/ml culture medium. Autoradiography confirmed that the thymidine was taken up by nuclear DNA. This represented a true stimulation of growth as shown by an almost twofold increase in cell number after 6 days of exposure to low doses of HSS.

Substrates of Nuclear Protein Kinases in Rat C6 Glial Cell Cultures

Publisher Summary Nuclear proteins of confluent rat C6 glial cell cultures are very highly phosphorylated. Their separation after labeling with Na 2 H 32 PO 4 by SDS-polyacrylamide gel electrophoresis and identification by autoradiography has allowed researchers to identify between 250 and 300 individual phosphorylated peptides. The chemical nature and function of most of these peptides are unknown and it can be assumed that most of the phosphorylations are of constitutive nature. This chapter identifies a number of the better characterized nuclear proteins as phosphoproteins, for example, histones, HMG 14 and 17, and RNA polymerase 11, and illustrates that that the phosphorylation/dephosphorylation state of these proteins is either subject to effector agent regulation or occurs preferentially at certain loci of the chromatin which differ in their transcriptional activities.

Glial-released proteins in clonal cultures and their modulation by hydrocortisone

Rat glial C6 cells release into the culture medium a reproducible spectrum of soluble proteins of 12 major peaks over a broad molecular weight range as determined by fractionation on SDS-gel electrophoresis. Exposing C6 monolayers to hydrocortisone (HC) results in a selective alteration in the pattern of glial-released protein (GRP). The selective HC-induced increase or decrease in GRP peaks is specific to HC in that 17 beta-estradiol, dibutyryl cyclic AMP, isoproterenol and melatonin exert either no detectable or a qualitatively different influence on the GRP pattern. The HC influence is dose dependent over a physiological range of concentrations from 10(-9) to 10(-6) M. Differences in culture age and in subclones of C6 can influence both the normal and the HC-induced pattern of GRP. The origin of th GRP is unknown, but pattern reproducibility, viability tests, surface labeling studies and metabolic labeling studies of soluble and particulate compartment proteins and glycoproteins support the position that cell lysis is not an important source of GRP. More importantly, these studies indicate that GRP and HC-induced changes in GRP pattern are physiologically significant aspects of glial cell behavior.

Mechanism of action of cholera toxin: effect of receptor density and multivalent binding on activation of adenylate cyclase.

Choleragen (cholera toxin) activates adenylate cyclase in HeLa cells, which contain less than 15,000 toxin receptors per cell, in a time- and concentration-dependent manner. Activation is blocked by the addition of the oligosaccharide chain of the ganglioside GM1, the receptor for the toxin. When the cells are preincubated with choleragen at 4 degrees C and then incubated with oligosaccharide at 37 degrees C, adenylate cyclase is activated less than 10%. When the preincubation phase is above 18 degrees C, adenylate cyclase becomes activated and the amount of activation depends on the time of preincubation. This inhibitory effect of the oligosaccharide is also observed with human lymphocytes and rat glial C6 cells but not with Friend erythroleukemic and mouse neuroblastoma N18 cells. The latter two cell lines have large numbers ot toxin receptors, whereas the former two cell lines have few receptors. When the number of toxin receptors in HeLa and C6 cells is increased by treating the cells with GM1, activation of adenylate cyclase by choleragen is no longer blocked by the oligosaccharide. The oligosaccharide has a corresponding effect on the displacement of bound 125I-choleragen. When bound to cells at 4 degrees C, most of the radiotoxin is displaced from HeLa, C6, and lymphocytes but not from Friend, N18, or HeLa cells pretreated with GM1. In untreated HeLa cells, dissociation of toxin-receptor complexes by the oligosaccharide depends on the time and temperature of complex formation; above 18 degrees C, the toxin rapidly becomes stably bound to the cells. The inhibitory effect of GM1 oligosaccharide us reversible, as, once it is removed, the small amount of toxin that remains bound can activate adenylate cyclase. These results are consistent with a model in which choleragen, which is multivalent, must bind to several GM1 molecules on the cell surface in order to subsequently activate adenylate cyclase. Lateral mobility of toxin-receptor complexes may be required only to achieve multivalent binding in cells with few receptors.

Mechanism of action of cholera toxin: studies on the lag period.

The lag period for activation of adenylate cyclase by choleragen was shorter in mouse neuroblastoma N18 cells than in rat glial C6 cells. N18 cells have 500-fold more toxin receptors than C6 cells. Treatment of C6 cells with ganglioside GM1 increased the number of toxin receptors and decreased the lag phase. Choleragen concentration also effected the lag phase, which increased as the toxin concentration and the amount of toxin bound decreased. The concentration, however, required for half-maximal activation of adenylate cyclase depended on the exposure time; at 1.5, 24, and 48 hr, the values were 200, 1.1, and 0.35 PM, respectively. Under the latter conditions, each cell was exposed to 84 molecules to toxin. The length of the lag period was temperature-dependent. When exposed to choleragen at 37, 24, and 20 degrees C, C6 cells began to accumulate cyclic AMP after 50, 90, and 180 min, respectively. In GM1-treated cells, the corresponding times were 35, 60, and 120 min. Cells treated with toxin at 15 degrees C for up to 22 hr did not accumulate cAMP, whereas above this temperature they did. Antiserum to choleragen, when added prior to choleragen, completely blocked the activation of adenylate cyclase. When added after the toxin, the antitoxin lost its inhibitory capability in a time and temperature-dependent manner. Cells, however, could be preincubated with toxin at 15 degrees C, and the antitoxin was completely effective when added before the cells were warmed up. Finally, cells exposed to choleragen for less than 10 min at 37 degrees C accumulated cyclic AMP when shifted to 15 degrees C. Under optimum conditions at 37 degrees C, the minimum lag period for adenylate cyclase activation in these cells was 10 min. These findings suggest that the lag period for choleragen action represents a temperature-dependent transmembrane event, during which the toxin (or its active component) gains access to adenylate cyclase.

Uptake of diflubenzuron ( N-[[(4-chlorophenyl)amino] carbonyl]-2,6-difluorobenzamide) by rat C6 glial cells in vitro

The uptake, subcellular localization, and metabolic fate of [ 14C]diflubenzuron, N-[[(4-chlorophenyl)amino]carbonyl]-2,6-difluorobenzamide, in cultured rat C6 glial cells have been characterized. Confluent monolayer cultures display a concentration-dependent uptake of the labeled insecticide. Experiments conducted in the presence and absence of dimethyl sulfoxide (DMSO) showed that 1.2% DMSO in the growth medium had no significant effect on the rate of incorporation of radioactivity. Diflubenzuron was not metabolized to any measurable extent and more than 98% of the administered compound could be recovered from whole cells and the culture medium by extraction with diethyl ether. As much as 95% of the cell-associated radioactivity was localized in the particulate fraction. Pulse-chase experiments revealed that less than 10% of the [ 14C]diflubenzuron incorporated during 24 hr of incubation was released into the growth medium over a period of 48 hr. This study in conjunction with our previous investigation (A. C. Stoolmiller, Gen. Pharmacol. 9, 11, 1978) indicates that diflubenzuron is neither cytotoxic nor does it inhibit the synthesis of complex carbohydrates in animal cells.

Induction of cholera toxin receptors in cultured cells by butyric acid.

Exposure of HeLa cells to sodium butyrate caused an increase in choleragen (cholera toxin) receptors as measured by increased binding of 125I-choleragen to the intact cells. The process was dependent on time and butyrate concentration; maximal increases (over 40-fold) were observed at 48 h and 5 mM sodium butyrate. Other short chain fatty acids were less effective in elevating choleragen receptors in the order: butyrate greater than pentanoate greater than hexanoate greater than propionate. Acetate and isobutyrate had no effect. The increase in toxin receptors caused by butyrate was reversible and occurred in serum-free medium. The affinity of choleragen for control and butyrate-treated HeLa cells appeared to be similar. Butyrate also induced an elevation in choleragen receptors in rat C6 glial and Friend erythroleukemic cells but not in a butyrate-resistant HeLa mutant. The increase observed in Friend cells paralleled the increase in ganglioside GM1 (galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide), the reported choleragen receptor. Although no GM1 could be detected in untreated Hela cells, small amounts were found in cells exposed to butyrate.

Effect of hydrocortisone on cell morphology in C6 cells.

Hydrocortisone has been found to induce cell spreading in rat glial C6 cells by 24 hours after its addition. This spreading phenomenon is correlated with an increase in the fraction of the peripheral cytoplasm occupied by microfilaments. Cytochalasin B causes disorganization of microfilaments in the peripheral cytoplasm of the cells. Additionally, it also prevents cell spreading in response to hormonal stimulation. High levels of calcium prevent recovery of normal microfilament organization and cell spreading following removal of cytochalasin B, but have no effect on normal microfilament organization alone. Additionally both the hydrocortisone induced spreading of C6 cells and increases in peripheral microfilaments are shown to be dependent on RNA ans protein synthesis. The levels of protein co-electrophoresing with actin are not effected by hydrocortisone.

Reversible inhibition of the norepinephrine induction of lactate dehydrogenase by cytochalasin B in rat glial C6 cells

The cyclic AMP mediated induction of lactate dehydrogenase (LDH: E.C. 1.1.1.27) activity by norepinephrine in the rat glial cell line C6 is inhibited by cytochalasin B. Doses of 5, 15, and 25 microgram/ml of cytochalasin B inhibited the induction equally. Twenty-five microgram/ml of cytochalasin B inhibited the induction reversibly, and had no effect on basal enzyme level. No effect of cytochalasin B on general protein synthesis was found, nor did it increase the rate of decline of enzyme activity in deinduced cells. It therefore appears to block LDH induction by selectivity inhibiting its synthesis. Cytochalasin B had no effect on the transient (intracellular and extracelllular) rise in cyclic AMP generated in response to norepinephrine treatment. Cytochalasin B was effective when added during the transcription dependent phase (first 3 hours) but not during the translation dependent phase (after 3 hours) of LDH induction. The suggestion is discussed that cytochalasin B inhibits one of the early events of the inductive process.

Polymerising ability of C6 glial cell microtubule protein decays much faster than its colchicine-binding activity

BECAUSE of its high degree of specificity1, the binding of colchicine to tubulin has become a powerful tool for the quantification and subcellular localisation of the protein. The mechanism of the binding reaction is not yet completely understood, but several of its characteristics are well documented. One of these is the decay of binding capacity with time, which follows the kinetics of a firstorder reaction2,3 and is accentuated at elevated temperatures4. Although correlation between this rate of decay and the loss of other characteristic functions of tubulin, such as its ability to polymerise into microtubules, has not been directly established by experiment, carefully con trolled measurements of the decay of colchicine binding seem to be useful for estimating the stability of the tubulin molecules per se. In contrast, the decay rate of the polymerisability of tubulin preparations should depend not only on the stability of the tubulin molecules themselves but also on that of other factors that may be essential for polymerisation5–7. As part of our recently initiated8,9 attempt to study the mechanisms regulating microtubule assembly in cultured cells, we report here that tubulin preparations from rat glial C6 cells exhibit a decay rate of colchicine-binding activity that is quite different from the decay rate of polymerisability.

Molecular forms of acetylcholinesterase: their de novo synthesis in mouse neuroblastoma cells.

Rat mouse AChE molecular forms are indistinguishable with respect to their sedimentation coefficients and their evolutive proportions during brain maturation. Among rat or mouse erythrocytes, rat C6 glial cells, and mouse 2A and NS 20 neuroblastoma cells, only neuroblastoma cells showed both the ES and HS molecular forms with a 1:1 proportion for NS 20 cells. All these cells lack a third molecular form (16S), which is present in rat and mouse superior cervical ganglia. After irreversible inhibition of pre-existing NS 20 neuroblastoma AchE, the ES form is first synthesized (de novo synthesis). The HS form begins to appear after a lag time of several hours and represents, 24 h after inhibition, only 15% of the total recovered activity, which is near the initial level. The initial relative proportions return by 2 to 3 days after inhibition. The recovery of the HS form is, for the most part, blocked by actinomycin D, which does not block the recovery of activity itself, which remains as an ES form. It seems that integration of the ES form into the HS form more probably depends on the synthesis of a new messenger RNA, which is required for the synthesis of either new AChE polypeptide chain, polymerization initiating protein or activating enzyme.

SUBSTRATE AND INHIBITOR‐RELATED CHARACTERISTICS OF MONOAMINE OXIDASE IN C6 RAT GLIAL CELLS

AbstractCultured C6 rat glial cells preferentially deaminated 5‐hydroxytryptamine, tryptamine, dopamine and tyramine in comparison to phenylethylamine and benzylamine. Deamination of all substrates was uniformly sensitive to inhibition by clorgyline and relatively insensitive to deprenyl. These data together with the observations of simple sigmoid curves for the inhibition of tyramine deamination by both inhibitors suggest that C6 glial cells contain mainly monoamine oxidase type A, which previously had been suggested to be primarily an intraneuronal MAO type. As these findings are in agreement with other studies of brain MA0 activity in mitochondria separated from neuronal vs glial cell preparations, they help explain why MA0 activity measured with some substrates may be little affected by lesions or by drugs producing nerve ending degeneration.

Rat interferon inhibits steroid-inducible glycerol 3-phosphate dehydrogenase synthesis in a rat glial cell line

The treatment of animal cells by an interferon (IF) from homologous species modifies the cellular translation apparatus and renders the cells incapable of translating viral mRNA. Several authors interested in studying this problem of discriminatory translation of mRNA in cells pretreated with interferon have utilized acellular protein synthesis. In certain systems the discrimination is total [l] , in others it is partial [2,3]. One possible approach to this question would be to utilize hormonal induction of an enzyme in cell lines which have retained differentiated characteristics. In a previous paper [4], we have shown that pretreatment of Hepatoma Tissue Culture (HTC) cells by rat and mouse IF inhibits the induction of tyrosine aminotransferase (TAT) by dexamethasone and that this inhibition depends on the concentration of IF. In these cells, the induced synthesis of TAT requires the neosynthesis of the corresponding mRNA [51. We report there the effect of pretreating rat C6 glial cells [6] with rat IF on the induction of cytoplasmic glycerol 3-phosphate dehydrogenase (GPDH) using hydrocortisone hemisuccinate (HCH) [7] as inducer.