UPTAKE AND RELEASE OF TAURINE FROM CEREBRAL CORTEX SLICES AND THEIR SUBCELLULAR COMPARTMENTS

—Cortex slices, synaptosomes and C-6 glioma cells were used to study [³⁵S]taurine uptake and its electrically-stimulated release. After exposure to taurine at two concentrations, the synaptosome preparation subsequently derived from the slices contained 41% of the particle-bound taurine and 16% of the total in the tissue. The uptake of [¹⁴C]GABA by C-6 glioma cells was inhibited 3-fold more by β-alanine than by l -DABA, whilst synaptosome preparations showed the opposite pattern, l -DABA being 2 or 3 times more effective than β-alanine. [³⁵S]Taurine uptake inhibition by l -DABA was low for synaptosomes and C-6 glioma, whereas β-alanine showed considerable effect on C-6 glioma (41%) and slices of white matter (ependyma; 50%). Synaptosome preparations showed little effect with β-alanine. When 30 min rather than 5 min incubations were employed, β-alanine depressed [³⁵S]taurine uptake by cortex slices by 30%. Taurine was taken up by a calcium-dependent mechanism and subcellular fractionation indicated that the synaptosome fraction showed losses commensurate with the net taurine release when low stimulation currents were used.     

Aluminum increases levels of beta-amyloid and ubiquitin in neuroblastoma but not in glioma cells

Several epidemiological studies suggest the involvement of aluminum (Al) in the pathogenesis of Alzheimer's disease (AD). There is an increase in the levels of Abeta and ubiquitin in the pathological lesions of AD. Therefore, we have investigated whether aluminum (Al) treatment alters the levels of Abeta and ubiquitin in murine neuroblastoma (NBP2) and rat glioma (C-6) cell cultures. At a low concentration (10 microM), aluminum sulfate stimulated the level of immunoreactive Abeta and ubiquitin in NBP2 cells without changing the levels of the amyloid precursor protein (APP). However, at higher concentrations (100 and 500 microM), aluminum failed to elicit any significant effect on beta-amyloid, whereas ubiquitin levels continued to increase. No changes in the Abeta and ubiquitin content were found in the C-6 glioma cells following treatment with Al at any of the concentrations tested. Exposure of cells to aluminum salts did not alter the rate of proliferation in either of the two cell lines. These data suggest that one of the mechanisms by which Al may play a role in AD is by promoting the formation of Abeta and ubiquitin in neurons.     

Metabolic changes in fruits of the tomato dx mutant

The tomato DWARF cytochrome P450 protein catalyzes the C-6 oxidation of 6-deoxo-castasterone to castasterone. The d(x) mutant does not produce a functional DWARF enzyme, and d(x) shoots display severe symptoms of brassinosteroid-deficiency. However, fruits express the CYP85A3 protein which compensates for the deficiency of the DWARF protein and produce bioactive brassinosteroids. Here, we report on the metabolic characterization of d(x) fruits. Fruit size, fresh weight, and pigment content were not altered. However, d(x) fruits showed reduced dry mass content. Levels of starch and various sugars were reduced, amino acid levels were elevated. BR application to d(x) leaves partially normalized dry mass content, sugar and amino acid levels in d(x) fruits. The data demonstrate that brassinosteroid in shoots is required for fruit development in tomato.     

Glycolytic metabolism in cultured cells of the nervous system

Pyruvate and lactate efflux from C-6 glioma cells has been found to be regulated by both the medium glucose concentration and the medium concentration of the two acids. Each moves down a concentration gradient until the extracellular level is in equilibrium with the intracellular. Long-term growth studies demonstrated that the cells preferentially utilize glucose but that once it is depleted, they will take up first pyruvate, followed by lactate, for further metabolism. Changes in the intracellular levels of the two metabolites correspond to those seen in the medium. The rate of glycogen breakdown parallels that of medium glucose ultilization. Preliminary results with the C-1300 neuroblastoma cells showed pyruvate and lactate efflux rates comparable to those of the glioma cells.     

Polyploid rat glioma cells. Production, oscillations of membrane potential and response to neurohormones

Due to their small size, electrophysiological investigation of C6 rat glioma cells by intracellular recording with microelectrodes is very difficult. In order to facilitate such electrophysiological studies, the degree of ploidy of C6 cells was increased in three successive steps. At each step complementary drug-resistant mutants of C6 were fused and hybrid cell clones isolated in selective culture medium. Cell volume was measured by electronic cell sizing and DNA content per cell assessed by impulse cytofluorimetry. In the final polyploid line, C6-4-2, both parameters were found to be increased 6-fold in comparison with the original cell line C6. In response to β-adrenergic agonists and prostaglandin E₁ the intracellular concentration of cAMP was raised in C6-4-2 cells. Stable membrane potentials could be recorded from the polyploid glioma cells much more easily than from the original C6 cells. The plasma membrane of the C6-4-2 cells could hyperpolarize spontaneously with a frequency of about 1 cycle/min. Elevation of the concentration of Ca²⁺ ions (but not other earth alkali ions) in the medium induced the onset of the spontaneous hyperpolarizations. The membranes of the C6-4-2 cells also hyperpolarized in response to acetylcholine, γ-aminobutyric acid (GABA) and glutamate.     

Glycolytic metabolism in cultured cells of the nervous system

The effects of thiamine deficiency and of the antithiamine drug pyrithiamine on the C-6 glioma and the C-1300 neuroblastoma cell lines have been studied. Thiamine deficiency increased the doubling time of the neuroblastoma cells without affecting that of the glioma cells. Pyrithiamine prevented both cell lines from doubling even once. (hiamine deficiency had only slight effects on intracellular pyruvate and lactate levels or on efflux rates for the acids, but pyrithiamine treatment resulted in large increases in both the intracellular levels and the efflux in both cell lines. For comparison, the pyruvate and lactate levels in mouse brain were measured. The levels from thiamine-deficient mouse brain were essentially unchanged from controls while pyrithiamine treatment caused a significant elevation only of the pyruvate concentration.     

Uptake of taurine into subcellular fractions of C-6 glioma cells.

Subcellular fractions from cultured C-6 glioma cells prepared by methods similar to those for crude synaptosomal fractions of rat cerebral cortex accumulated [³⁵S]taurine as did intact glioma cells. Thus, the accumulation of taurine was dependent on temperature and sodium concentration and sensitive to osmotic shock. The kinetic properties of this uptake are characterized by an apparent K_m, of about 25 μm, The properties of taurine uptake into subcellular fractions from C-6 glioma cells were compared with those of crude synaptosomal fractions and differences could be observed in temperature sensitivity and with metabolic inhibitors, which were less potent in the glioma preparation. Equilibrium density gradient centrifugation of subcellular fractions from glioma cells revealed that particles containing [³⁵S]taurine sediment to a lower buoyant density than mitochondria. But on co-sedimentation of subcellular fractions from glioma cells with synaptosomal fractions derived from cerebral cortex, differences in the buoyant density between these two preparations could be found. The findings support the possibility of a contamination of synaptosomal fractions with subcellular fractions derived from glial origin.     

Inhibitory effects of carotenoids and retinoids on the in vitro growth of rat C-6 glioma cells

The inhibitory effects of 4 retinoids, namely, retinal (Ral), retinoic acid (RA), retinyl acetate (RAc), and retinyl palmitate (RP), and 3 carotenoid including beta-carotene (BCT), lycopene (LCP), and crocetin (CCT) on the growth and DNA synthesis of rat C-6 glioma cells were studied. All the retinoids and carotenoids caused reduction of plating efficiency and inhibition of the cellular growth. RA was the most potent inhibitor of plating efficiency, followed in decreasing order by RAc, Ral, LCP, RP, BCT, and CCT. The effects of various doses of retinoids and carotenoids on the inhibition of DNA synthesis were clearly demonstrated in the growing C-6 glioma cells, whereas negligible effects of these compounds on the RNA and protein synthesis were observed. These results suggested that retinoids or carotenoids are biologically active as anti-tumor agents against brain tumor cells in culture, while carotenoids appeared to be less active.     

Local protein accumulation during gene activation

Relative values for the dry mass in puff-forming regions of Drosophila hydei salivary gland chromosomes were established with a Leitz double beam interference microscope. All measurements were made after RNA digestion.Optical path differences per unit area of the induced puffs 2-48C and 4-81B (temperature induced) and a cytoplasm-free background were recorded. In each of the nuclei used for these measurements, the same procedure was applied to two reference regions in the vicinity of the puff, region 2, 47A-48B and region 4, 81C-82C respectively. For comparison of the dry mass values of a puff region at various time intervals after the onset of puff induction, ratios of the optical path differences of the puff region over that of the reference region were calculated.These ratios were established at 5, 10, 30, 60 and 120 min after the onset of a temperature treatment and compared with the ratio in non-treated animals. From the data it can be concluded that the dry mass in the induced puffs increases gradually up to 30 min. At this time the dry mass ratio for puff 2-48C has reached a value of 150% and that of puff 4-81B, 210%. It is concluded that this increase in dry mass is due almost entirely to a local accumulation of non-histone protein.     

Rapid Cellular Regulation of D-Glucose Transport in Cultured Neural Cells

Previous studies have revealed two different kinds of regulation of glucose utilization in cell lines derived from the nervous system (Keller et al., 1981). We found glucose metabolism of C-6 glioma cells to be limited and regulated by membrane transport. In contrast, glucose utilization of C-1300 neuroblastoma (N2A) cells was limited by the known regulatory enzymes of the Embden-Meyerhof pathway. Under the given experimental conditions the "membrane-limited" C-6 glioma cells were characterized by periodically changing glucose transport rates and very low intracellular glucose concentrations, which remained constant in spite of widely differing transport rates. These findings suggest the close functional coupling between transport and phosphorylation required for the regulation of glucose transport by cellular metabolic needs.     

Glycolytic metabolism in cultured cells of the nervous system IV. The effects of thiamine deficiency on thiamine levels,metabolites and thiamine-dependent enzymes of the C-6 glioma and C-1300 neuroblastoma cell lines.

Summary C-6 glioma and C-1300 neuroblastoma cells were cultured in thiamine deficient and control media. Thiamine levels, transketolase and pyruvate decarboxylase activities, and high energy phosphate metabolites were all measured in deficient and control cells. Thiamine levels in the deficient cells were found to be below the level of detectability. Pyruvate decarboxylase activity was more susceptible to thiamine deficiency in both cell lines than transketolase. In spite of the large decrease in pyruvate decarboxylase activity, high energy phosphate metabolites were not decreased in either cell line. These data indicate that C-6 glioma and C-1300 neuroblastoma cells have the capacity to maintain normal energy metabolites in the presence of large changes in thiamine levels and thiamine dependent enzyme activity.Supported in part by USPHS grant AA 01391.     

Aluminum-induced oxidative events in cell lines: glioma are more responsive than neuroblastoma.

Aluminum, a trivalent cation unable to undergo redox reactions, has been linked to many diseases such as dialysis dementia and microcytic anemia without iron deficiency. It has also been implicated in Alzheimer's disease although this is controversial. Because cell death due to oxidative injury is suspected to be a contributory factor in many neurological diseases and aluminum neurotoxicity, glioma (C-6) and neuroblastoma (NBP2) cells were utilized to assess early changes in oxidative parameters consequent to a 48-h exposure to aluminum sulfate. A 500-microM concentration of this salt produced a significant increase in reactive oxygen species (ROS) production and a significant decrease in glutathione (GSH) content in glioma cells. However, the same concentration of the aluminum salt did not lead to any significant changes in the neuroblastoma cells. Mitochondrial respiratory activity in glioma cells was also found to be significantly higher in the aluminum treated cells. As judged by morin-metal complex formation, aluminum can enter glioma cells much more readily than neuroblastoma cells. Thus, it is possible that the cerebral target following an acute exposure to aluminum may be glial rather than neuronal.     

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.     

Vimentin: a phosphoprotein under hormonal regulation

Past studies of norepinephrine-stimulated protein phosphorylation in intact C-6 glioma cells had identified a 58,000 molecular weight, 5.7 isoelectric point protein (58K-5.7) as a cyclic AMP-dependent phosphoprotein and had shown that 58K-5.7 was one of the most abundant proteins of the nuclear fraction. Initial experiments of present studies showed that the 58K-5.7 protein remained with the nuclear ghost, or matrix structure, after removal of chromatin. Based on the size, acidity, abundance, nonsolubilization by nonionic detergent and salt, and solubilization by urea, the hypothesis was advanced that the 58K-5.7 protein was the vimentin-type intermediate filament protein. The hypothesis was tested by two types of immunochemical experiments. Antisera against hamster vimentin reacted selectively with only the 58K- 5.7 protein in polyacrylamide gels of urea-solubilized cellular residues (i.e., nonionic detergent and 0.6 M salt-insoluble material) as determined by immunoautoradiography. Antisera against the pure 58K- 5.7 protein of C-6 cells bound selectively to a fibrous array of cellular material typical of vimentin filaments as determined by indirect immunofluorescence. It is concluded that the 58K-5.7 protein is vimentin.     

Glial Fibrillary Acidic Protein: Norepinephrine Stimulated Phosphorylation in Intact C-6 Glioma Cells

Abstract: Coelectrophoresis in two-dimensional gels of rat glial fibrillary acidic protein (GFA) and ³²P-labeled whole cell extracts of rat C-6 glioma cells showed that the GFA migrated in close proximity to a previously noted phosphoprotein, 50K-6.1, of these cells. GFA electrophoresed as a 50K polypeptide with at least four charge variants, the most acidic of which coelectrophoresed with 50K-6.1. Exposure of the C-6 cultures to dibutyryl cyclic AMP (dbcAMP) for 48 h increased the relative abundance of the endogenous polypeptide associated with 50K-6.1 by threefold, consistent with the hypothesis that 50K-6.1 was GFA. Norepinephrine stimulated 50K-6.1 phosphorylation 3.2-fold in dbcAMP-induced cultures. Peptide mapping with V8 protease and subtilisin was used to test the hypothesis that GFA and 50K-6.1 were identical polypeptides. With V8 protease, the peptides generated from the [³⁵S]methionine labeled putative GFA spot of the C-6 cells were indistinguishable from the stained bands derived from authentic GFA in mixed samples of the two proteins. Likewise, the ³⁵S-labeled acidic satellite to the putative GFA spot also yielded a peptide map that matched that of the authentic GFA. ³²P-labeled peptides derived from the 50K-6.1 protein were a subset of those from authentic GFA. With three subtilisin concentrations, ³²P-labeled 50K-6.1 was degraded to peptides which were again a subset of the stained GFA peptides. A cytoskeletal fraction from ³²P-labeled C-6 cells contained a 50K phosphoprotein. Pep-, tide mapping with V8 protease produced a ³²P-peptide pattern which was a subset of that from authentic GFA. The pattern closely resembled the ³²P-peptide pattern for the 50K-6.1 protein from 2-dimensional gels of whole cell extract. It was concluded that the protein 50K-6.1 is a phosphorylated form of GFA and that GFA is a phosphoprotein whose phosphòrylation is stimulated by norepinephrine in C-6 glioma cells.     

The metabolism of estradiol 17-sulfate by pheochromocytoma tissue

The metabolism of estradiol 17-sulfate by subcellular localization enzymes of pheochromocytoma tissue obtained from a 41-year old female was investigated. In any incubations under the presence of NADH and NADPH, metabolites hydroxylated at the C-2, C-4, C-6 beta, C-7 alpha and C-7 beta positions were produced. These hydroxylations are considered to occur without cleavage of the sulfate group. The 2-hydroxylation at the substrate concentration of 100 microM by mitochondria, microsomes and cytosol fractions occurred at rates of 141, 222 and 167 pmol/mg protein/30 min, respectively; the corresponding rates for the 4-hydroxylation were 24, 40 and 38 pmol/mg protein/30 min. Mitochondrial 2- and 4-hydroxylations were enhanced by addition of calcium ion (Ca2+) into the incubation medium.     

Forskolin induction of S-100 protein in glioma and hybrid cells

The S-100 protein level in mouse neuroblastoma (N18TG-2 and NIE-115), rat glioma (C6, C6BU-1, and C6V-1), and hybrid (NG108-15, 140-3, 141-B, NBr10A, NBr20A, NCB20, and NX3IT) cells was determined with a sensitive enzyme immunoassay system that uses a rabbit antibody to bovine brain S-100 protein. S-100 protein was detected in glioma but not in neuroblastoma cells. All seven hybrid cells derived from neuroblastoma and glioma or other types of cells were found to possess a very little or undetectable S-100 protein. The induction of S-100 protein level in prestationary phase cultures of glioma C6BU-1 cells was examined by forskolin, which was a highly specific activator of adenylate cyclase of the cells and produced morphological differentiation. After incubation with 10 microM forskolin for 48 hr, the S-100 protein level increased 2-2.5-fold in C6BU-1 glioma cells whose mean control level was 60 +/- 26 ng/mg protein (+/- SD). The forskolin induction of S-100 protein in the cells was dose dependent, and the concentration of forskolin required for 50% activation of S-100 protein was about 0.6 microM. The increase by forskolin was initiated from 10-15 hr after incubation with it and was inhibited with cycloheximide and actinomycin D. In NG108-15 hybrid cells the induction of S-100 protein was also observed by forskolin as well as prostaglandin (PG) E1 plus theophylline which are known to activate adenylate cyclase of the cells. The results indicate that S-100 protein biosynthesis is genetically controlled in these clonal cells, and that S-100 protein can be regulated in a cAMP-dependent fashion in prestationary cultures.     

3 beta-Hydroxysteroid dehydrogenase in human placental microsomes and mitochondria: co-solubilization of androstene and pregnene activities

3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) was solubilized from human term-placental microsomes and mitochondria using the non-ionic detergent, polyoxyethylene 20 cetyl ether (BrijR-58). Electron photomicrographs showed microsomes and mitochondria well disrupted by the detergent. The pregnene (C-21) and androstene (C-19) activities co-solubilized over a range (0.04-0.44) of BrijR-58/protein (B/P) concentration ratios (w/w). Optimal solubilization of the C-19 and C-21 activities were 63.3 +/- 2.6% (mean +/- SEM) from mitochondria (B/P ratio 0.37) and 71.8 +/- 2.1% from microsomes (B/P ratio 0.34). Detergent treatment of microsomes and mitochondria--varying time (5-90 min, pH 7.4) or varying pH (6.0-7.8, 90 min)--yielded C-19 activities identical with C-21 activities. The C-21/C-19 specific activity ratios of 3 beta-HSD in particulate, solubilized and chromatographed preparations were 2.28 +/- 0.16 (mean +/- SEM) for mitochondria and 1.97 +/- 0.07 for microsomes. 3 beta-HSD molecular weight estimates were 208,000 (microsomes) and 220,000 (mitochondria). These studies argue that a single protein is responsible for both the C-19 and C-21 activities of 3 beta-HSD and that this protein is the same in microsomes and mitochondria.     

High performance liquid chromatography analysis of 1,25-dihydroxyvitamin D3 receptor in malignant cells. Correlation of effects on cell proliferation and receptor concentration

Malignant cells were assayed for 1,25(OH)2D3 receptors and for the effects of 1,25(OH)2D3 on cell proliferation. The established lines studied were human promyelocytic leukemia (HL-60), T-cell lymphocytic leukemias (Molt-4, RPMI-8402, CEM), mouse leukemia (L1210), breast cancers (HT-39 and MCF-7) and a glioma (C-6) cultures. A TSK 3000 SW (0.75 X 60 cm) HPLC size exclusion column was used to characterize specific 1,25(OH)2D3 binding. We show for the first time that this column is capable of resolving the 3.2-3.5S 1,25(OH)2D3 mammalian receptor (Rs = 32 A) from the 5.5-6.0S form of the mammalian serum 25(OH)D3 transport receptor (Rs = 40 A). The molecular size of the 1,25(OH)2D3 receptors from these cancer cell lines was identical to that from rabbit intestine. HT-39, HL-60, MCF-7, Molt-4, C-6, RPMI-8402 and L1210 cells demonstrated specific 1,25(OH)2[3H]D3 binding (120, 90, 80, 45, 30 and 18 fmoles of sites/mg protein, respectively). Receptors were not detected in the CEM line. 1,25(OH)2D3 inhibited cell proliferation of HT-39, HL-60, MCF-7 and Molt-4 cells by 20% to 70%. In contrast, mouse leukemia (L1210) cells were stimulated to proliferate by this hormone. Proliferation of RPMI and CEM cells was not affected by 1,25(OH)2D3. We demonstrate that size-exclusion HPLC of 1,25(OH)2D3 binding proteins from mammalian intestine and cancer cells provided a rapid method for identification of specific 1,25(OH)2D3 receptors. Furthermore, in the cells studied, the presence and concentration of 1,25(OH)2D3 receptors qualitatively predicted the potency of this hormone to alter cell proliferation. We believe this assay will be useful for rapid analysis of human tumor receptor concentrations.     

Characterization of 3-[(123)I]iodo-L-alpha-methyl tyrosine transport in astrocytes of neonatal rats

3-[(123)I]Iodo-L-alpha-methyl tyrosine ((123)I-IMT) is used for diagnosis and monitoring of brain tumours by means of single-photon emission tomography. As recently shown, (123)I-IMT is predominantly mediated into rat C6 glioma cells by sodium-independent system L for large neutral amino acids. Until now, (123)I-IMT transport in non-neoplastic glial cells has not been examined. Therefore, the aim of this study was to examine the cellular pathways and precise transport kinetics of (123)I-IMT uptake into astrocytes of neonatal rats. In particular sodium-independent (123)I-IMT transport into neonatal astrocytes was compared with sodium-independent (123)I-IMT uptake into neoplastic rat C6 glioma cells. Competitive inhibition experiments showed that (123)I-IMT is exclusively transported via sodium-independent system L into the neonatal astrocytes (92%). Kinetic analysis of sodium-independent (123)I-IMT uptake into neonatal astrocytes and into C6 glioma cells revealed apparent Michaelis constants K(M) = 13.9 +/- 0.5 microM and K(M) = 33.9 +/- 4.1 microM, respectively, which are in the same range of K(M) values as those recently determined for amino acid transport into neoplastic and non-neoplastic glial cells. Indeed, the K(M) values in the micromolar range correspond to the expression of the LAT-1 subunit of system L both in the neonatal astrocytes and in C6 glioma cells. However, sodium-independent maximum transport velocities (V(max)) differed significantly between neonatal astrocytes and C6 glioma cells (11.1 +/- 0.3 and 39.9 +/- 3.3 nmol/mg protein/10 min, respectively).