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.     

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.     

Early and late passage C-6 glial cell growth: Similarities with primary glial cells in culture

Earlier studies in our laboratory have shown that C-6 glial cells in culture exhibit astrocytic properties with increasing cell passage. In this study, we tested the responsiveness of early and late passage C-6 glial cells to various cultures conditions: culture substrata (collagen, poly-L-lysine, plastic), or supplements for the culture medium, DMEM, [fetal calf, or heat inactivated (HI) serum, or media conditioned from mouse neuroblastoma cells (NBCM) or primary chick embryo cultured neurons (NCM)]. Glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP), astrocytic and oligodendrocytic glial markers, were used. Cell numer and protein content increased exponentially with days in culture regardless of the type of the substratum or cell passage. Differences in cell morphology among the three types of substratum were also reflected on GS activity, which rose by three-fold on culture day 3 for cells grown on collagen; thereafter, GS profiles were similar for all substrata. This early rise in GS is interpreted to reflect differential cell adhesion processes on the substrata; specifically, cell adhesion on the collagen stimulated differentiation into astrocytic phenotype.Analogous to immature glia cells in primary cultures, early passage C-6 glial cells responded to neuronal factors supplied either from NCM or NBCM by expressing reduced GS activity, the astrocytic marker and enhanced CNP activity, the oligodendrocytic marker. Thus, early passage cells can be induced to express either astrocytic or oligodendrocytic phenotype. In accordance with our previous reports on primary glial cells, late passage C-6 cells exhibit their usual astrocytic behavior, responding to serum factors with GS activity. Moreover, whereas NCM or NBCM alone markedly lowered GS activity, a combination with serum restored activity. The present findings confirm our previous observations and further establish the C-6 glial cells as a reliable model to study immature glia.Special issue dedicated to Dr. Paola S. Timiras.     

Metabolic consequences of drug-induced inhibition of the pentose phosphate pathway in neuroblastoma and glioma cells.

6-Aminonicotinamide leads to a considerable accumulation of 6-phosphogluconate, which is 3 times higher in C-6 glial cells than it is in C-1300 neuroblastoma cells. Dephosphorylation of the accumulated 6-phosphogluconate causes a rise of intracellular gluconate, which can be released from the cells. The higher dephosphorylating capacity of neuroblastoma cells leads to an intracellular gluconate content which is 4 times that found in C-6 glial cells. Although 6-phosphogluconate is a potent competitive inhibitor of glucose phosphate isomerase, no reduction of glycolytic flux and ATP content in stationary phase neuroblastoma cells was found in contrast to observations in C-6 glial cells. Morphological changes are only found in C-6 glial cells during the experimental period.     

Effect of an unnatural phospholipid base analog,N-isopropylethanolamine,on 3-hydroxy-3-methylglutaryl coenzyme a reductase in cultured glial and neuronal cells

Cultured C-6 glial and neuroblastoma cells were utilized to study the effect of the unnatural amino alcohol, N-isopropylethanolamine, on the microsomal enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase. Growth of both cell types in the presence of the compound was accompanied in 24 hr by a decrease in reductase activity to 25–35% of activity in control cells. The effect was accompanied by a comparable decrease in the rate of cholesterol synthesis. However, no comparable change occurred in cell growth, fatty acid synthetase activity, or in total protein synthesis from [³H]leucine. The data suggest that the polar head groups of microsomal membrane phospholipids play an important role in the regulation of reductase activity.     

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.     

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.     

Two-Site Immunoradiometric Assay of Chicken Acetylcholinesterase: Active and Inactive Molecular Forms in Brain and Muscle

Abstract: Several monoclonal antibodies were raised against chicken acetylcholinesterase (AChE; EC 3.1.1.7). Some of these antibodies react with quail AChE but not with AChEs from nonavian vertebrates or invertebrates and not with butyrylcholinesterase. They may be classified in several mutually compatible groups, i.e., that can bind simultaneously to the monomeric form of AChE. Most antibodies recognize a peptidic domain that does not exist in mammalian AChE and that may be digested by trypsin without loss of activity or dissociation of quaternary structure. The only exception is the antibody C-131, which is conformation dependent and preferentially recognizes active AChE. We have set up two-site immunoradiometric assays, using an immobilized capture antibody, C-6 or C-131, and a radiolabeled antibody, ¹²⁵I-C-54. The C-6/C-54 assay quantifies the totality of inactive and active AChE subunits: It detects 10^?3 Ellman unit (～40 pg of protein) and yields a linear response up to at least 25 10^?3 Ellman units. An analysis of gradient fractions, using C-6/C-54 and C-131/C-54 assays as well as activity determination, shows that the A₁₂ and G₄ forms are exclusively composed of active subunits, whereas inactive molecules cosediment with the active G₂ and G₁ forms. Both active and inactive G₂ and G₁ forms are amphiphilic, as indicated by the influence of detergents on their sedimentation coefficients and Stokes radii. In brain, the proportion of inactive forms decreases from 40% at embryonic day 11 (E11) to 20% at birth [day 1 (D1)]. In muscle, we observed no inactive AChE at E11 and a small proportion of inactive G₁ at D1. The proportion of inactive forms was much higher in cultured myotubes, obtained from E11 myoblasts. These results show that the proportion of inactive AChE depends on the tissue and varies during development. Thus, the cells seem to control actively the acquisition of AChE activity, as well as the formation of the various oligomeric forms.     

Regulation of acetylcholinesterase activity by retinoic acid in a human neuroblastoma cell line

The ability of retinoic acid (RA) to modulate acetylcholinesterase (AChE) activity in a human neuroblastoma cell line (LN-N-5) was examined. The specific activity of AChE was significantly increased 3 days after exposure of LA-N-5 to RA and reached its maximum values after 9 or more days of culturing. Dose-response experiments demonstrated that large increases of AChE occurred at RA concentrations between 10(-7) and 10(-6) M with maximum AChE values detected at 10(-6)-10(-5) M. Increased AChE activity paralleled neurite outgrowth in LA-N-5 cultures. These findings demonstrate that RA can regulate specific AChE activity in human neuroblastoma cells in a manner consistent with neuronal maturation.     

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.     

乙酰胆碱酯酶的非经典功能及其与A1zheimer's疾病的关系

乙酰胆碱酯酶（acetylcholinesterase,AChE)是主要存在于神经系统的一种水解酶,其经典功能是水解神经递质乙酰胆碱,从而终止神经冲动的传递。但是近年来,研究者发现许多证据表明它具有“非经典”的新功能,引起了人们的关注。除了水解神经递质乙酰胆碱的经典功能外,AChE对神经细胞的分化、迁移,突触的形成,造血系细胞和肿瘤细胞的增殖与分化调控也有作用。最近的研究结果显示：AChE可能在细胞凋亡过程中起重要作用,这对于认识Alzheimer‘s疾病（AD）的发病机理又有新的进步。     

The role of nerve growth factor in vitro in cell resistance to 6-hydroxydopamine toxicity.

Cell resistance to the catecholaminergic neurotoxin 6-hydroxydopamine was studied in various cell lines: human neuroblastoma lines SK-N-MC. SK-N-SH, and SK-N-SH-SY5Y; and non-neuroblastoma lines CHO-K1, S-180, C-6, and L-M, the latter three of which synthesize nerve growth factor. Cells were treated one day after seeding for 1 h with 6OHDA. Cytotoxicity of the drug was quantified as the percent live cells, determined by the trypan blue exclusion test, 24 h after treatment. At 100 μg/ml, 6OHDA lethal toxicity was confined mainly to neuroblastoma cells. However, drug specificity was dependent not only on cell type, but also on cell density and presence of NGF. Thus, the non-neuroblastoma cell strain S-180-A, which produces less NGF than its parent line S-180, lost resistance to toxicity at low cell density, and even at high density was less resistant than SK-N-MC neuroblastoma cells. Moreover, when mouse β-NGF (500 BU/ml) was administered to human neuroblastoma clones SY5Y and IN one day after seeding for 24 h before drug treatment, the cell survival rate increased significantly, although only SY5Y cells were protected by a lower concentration (1 BU/ml) of exogenous NGF. Finally, cell line S-180 became susceptible to 6OHDA killing when incubated one hour with high titer anti-mouse β-NGF immediately prior to drug treatment, whereas cell line C-6 did not. NGF was therefore proposed to have an important, though not determinative, role in cell resistance to 6OHDA toxicity.     

HETEROGENEITY OF ACETYLCHOLINESTERASE IN NEUROBLASTOMA

Abstract– Multiple forms of acetylcholine hydrolyzing activity have been observed in Triton X-100 treated homogenates of mouse neuroblastoma cells. All these forms appear to be the true acetylcholinesterase, AChE (EC 3.1.17): they are substrate-inhibited; hydrolyze acetylcholine > acetyl-β-methylcholine ≫ butrylcholine and are preferentially inhibited by specific AChE inhibitors. Almost all of the cell AChE activity is membrane associated, but readily 'solubilized' by Triton X-100 and as such appears free of membrane contamination. With the aid of affinity chromatography the 'solubilized' neuroblastoma AChE has been partially purified (490-fold) to a specific activity of 34,300 nmol/min/mg protein.
Four active neuroblastoma AChE species appear upon acrylamide gel electrophoresis (with MWs of 64,000; 116,000; 186,000 and 284,000) while three species (4S, 6S and 9.6S) have been found upon sucrose gradient sedimentation analysis. We have determined that the 4S form migrates on acrylamide as the 116,000 MW species and the 9.6S form contains, in equal amounts, the 186,000 and 284,000 MW acrylamide species. Numerous active AChE forms are seen on Sepharose 6B chromatography. From comparing the crude, 4S, 9.6S and partially purified AChEs on acrylamide gels, sucrose gradients and Sepharose, mouse neuroblastoma appears to contain active AChE units which are capable of multiple types of dissociation and reassociation. An attempt is made to correlate all the observed AChE forms as well as relate this data to that known about AChE obtained from other sources.     

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.     

Regeneration of acetylcholinesterase in clonal neuroblastoma-glioma hybrid NG108-15 cells after soman inhibition: effect of glycyl-l-glutamine

Acetylcholinesterase (AChE) in the clonal NG108-15 cell line has been previously characterized. This cell line represents an in vitro system to study AChE regulation and effects of chemical compounds that may alter AChE activity. Recently, glycyl-L-glutamine (GLG) was demonstrated to function as a neurotrophic factor for maintenance of AChE content in cat denervated superior cervical ganglion cells. In the present study, regeneration of AChE activity in cultures of undifferentiated NG108-15 cells after soman inhibition was investigated in the presence and absence of GLG. Cells were treated with soman (5.5 × 10^–6 M) for 15 min and then washed to remove excess soman. Culture medium containing either GLG (10^–6, 10^–5, or 10^–4M) or glycyl-L-glutamic acid (10^–6 M) was added to cultures after soman treatment and remained in the medium until cell harvest. Cells were physically detached at various times after soman treatment and specific AChE activity was determined. After soman, AChE activity dramatically decreased to less than 1% of untreated cellular activity at 1 hr. AChE activity gradully increased after 5 hr, while untreated cell AChE activity was regained 20 hr after soman. The t_1/2 for AChE regeneration was approximately 10 hr. GLG did not increase the rate of AChE regeneration after soman inhibition. These results indicate that GLG is not a directly acting neurotrophic factor for AChE synthesis in NG108-15 cells after chemical AChE inactivation.Abbreviations AChE acetylcholinesterase - NG108-15 cell neuroblastoma-glioma 108-15 cell - DMEM Dulbecco's modified Eagles minimal essential medium - FBS fetal bovine serum - GLGA glycyl-L-glutamic acid - L-GA L-glutamic acid - GLG glycyl-L-glutamine - GD soman The opinions or assertions contained herein are the private views of the authors and are not to be construed as reflecting the view of the Department of the Army or the Department of the Army or the Department of Defense.     

A ROLE FOR MICROTUBULES IN THE REGULATION OF 3-HYDROXY-3-METHYLGLUTARYL COENZYME A REDUCTASE AND CHOLESTEROL BIOSYNTHESIS IN CULTURED GLIAL CELLS 1

—Cultured C-6 glial cells were utilized to evaluate the effect of the antimicrotubular drug, Colcemid, on 3-hydroxy-3-melhylglutaryl coenzyme A (HMG-CoA) reductase and cholesterol synthesis in cultured C-6 glial cells. The data indicate that Colcemid causes a marked inhibition of cholesterol synthesis (from [¹⁴C]acetate or ³H₂O) in these cells. A concentration of 0.5 μM led to a 50% lower rate of synthesis after 2 h and an 80–85% lower rate after 12 h or longer. That the effect of Colcemid is mediated at the level of HMG-CoA reductase was shown by defining closely coordinate temporal and quantitative changes in the activity of this enzyme under identical conditions. No comparable change in cell growth or in total protein synthesis accompanied the effect of Colcemid. The drug did lead to a decrease in the rate of DNA synthesis (from [³H]thymidine) but this effect was preceded by the decrease in the rate of cholesterol synthesis. Marked changes in glial cell shape were induced by exposure to Colcemid, and the temporal and quantitative aspects of these changes appeared to closely parallel the effects on reductase activily and cholesterol synthesis. The dala suggest that microtubules are involved in the regulation of HMG-CoA reductase and cholesterol synthesis in mammalian cells and that there are important interrelations between microtubules, glial differentiation and cholesterol synthesis.     

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.     

Ultrastructural localization of acetylcholinesterase

Summary A method is described allowing localization of acetylcholinesterase (AChE) by both light and electron microscopy. During the reaction lead thio-diacetyl is decomposed, and therefore precipitated as PbS in the presence of native-SH group produced by the hydrolysis of acetylthiocholine perchlorate. The reaction takes place at neutral pH, since improves the sensitivity of AChE localizations. Application of the method to parasympathetic neurons showed that AChE was mainly localized in the rough endoplasmic reticulum of the perikaryons. No reaction was visible in glial cells. AChE was also localized on the plasma membrane of parasympathetic neurons. In mouse embryo muscles AChE activity was seen to be high and was not yet restricted to the synaptic area. The well developed Schwann cells accompanying the neurites displayed constant AChE activity on their plasma membrane.Supported by a grant of INSERM C.R.L. N⁰ 79-5-318-6     

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.