An In Vivo Model for Studying Function of Brain Tissue Temporarily Devoid of Glial Cell Metabolism: The Use of Fluorocitrate

The effect of intrastriatal injection of fluorocitrate on amino acid pattern, cell enzyme markers, and ultrastructural appearance was investigated. A dose of 1 nmol of fluorocitrate resulted in temporarily decreased levels of glutamine, glutamate, and aspartate, whereas the level of alanine was increased. The glutamine level was severely reduced after 4 h but was reversed after 24 h. The activity of different cellular enzyme markers did not change markedly after this dose. Ultrastructural changes in glial cells were observed, concomitant with the biochemical changes. A dose of greater than or equal to 2 nmol of fluorocitrate resulted in more marked and irreversible changes in amino acid levels. By 24-72 h after the injection of this dose, several marker enzyme activities decreased markedly. The ultrastructural changes affected the neurons as well as the glial cells and were not reversible. The use of microinjection of 1 nmol of fluorocitrate into the neostriatum of the rat to provide a model for studying transmitter amino acid metabolism in brain devoid of glial cell activity is discussed.     

Altered aconitase activity in hamster cells selected for resistance to fluorocitrate.

Two independent Chinese hamster ovary cell lines have been isolated in cell culture which exhibit resistance to the cytotoxic effects of fluorocitrate. Although the oxidation of citrate by wild type cell suspensions was markedly inhibited by 1 mM fluorocitrate drug-resistant cells oxidized citrate at approximately normal rates in the presence of the drug. The aconitase activity from the resistant cells was less sensitive to the inhibitory action of fluorocitrate $\text{in vitro}$ and showed altered heat stability properties when tested in heat inactivation experiments at 3 different temperatures. These results are consistent with the view that the resistant cell lines contain a structural gene mutation.     

The inhibition by fluorocitrate of rat liver mitochondrial and extramitochondrial aconitate hydratase

1. The effects of synthetic fluorocitrate were studied on: (a) the oxidation of citrate and cis-aconitate by rat liver mitochondria; (b) the activity of the aconitate hydratase found in the liver cell sap; (c) the activity of the aconitate hydratase solubilized from liver mitochondria. 2. Fluorocitrate was found to be a potent inhibitor of oxidation of citrate but only a weak inhibitor of oxidation of cis-aconitate: 6.7mum-fluorocitrate (containing 4% of the inhibitory isomer) caused 94% inhibition of the oxidation of citrate (2mm) whereas 1.0mm-fluorocitrate was necessary to provoke the same inhibition when cis-aconitate (2mm) was the substrate. The degree of inhibition varied in relation to the respiratory state of mitochondria when fluorocitrate was added. The inhibition could be partially reversed by cis-aconitate. 3. The aconitate hydratase extracted from the mitochondria was much less inhibited by fluorocitrate than was the mitochondria-bound enzyme, and the aconitate hydratase found in the cell sap was even less sensitive. 0.3mm-Fluorocitrate was required to cause 50% inhibition of the reaction citrate-->cis-aconitate, catalysed by the aconitate hydratase extracted from the mitochondria, and 1.2m-fluorocitrate for the extramitochondrial enzyme. For both enzymes the reaction citrate-->cis-aconitate was 2-3 times more sensitive to fluorocitrate than was the reaction isocitrate-->cis-aconitate. The inhibition was of the competitive type for both reactions.     

氟代柠檬酸对体外培养的神经胶质瘤细胞G422增殖的抑制效应

为研究氟代柠檬酸(Fluorocitrate)对体外培养的神经胶质瘤细胞生长的影响,采用MTT法研究不同的氟代柠檬酸浓度(0.0025mmol/L,0.005mmol/L,0.01 mmol/L,0.025mmol/L和0.1 mmol/L)和作用时间(36h,48h和60h)对神经胶质瘤细胞G422增殖的影响.结果发现:(1)氟代柠檬酸可抑制G422细胞的增殖,并且其抑制作用随氟代柠檬睃浓度的增加而增强;(2)高浓度(0.01 mmol/L,0.025 mmol/L和0.1 mmol/L)氟代柠檬酸对G422细胞的增殖抑制作用随作用时问的延长而增强:(3)低浓度(0.0025mmol/L和0.005mmol/L)氟代柠檬酸对G422细胞的增殖抑制作用不随作用时间的延长而改变.实验表明,氟代柠檬酸能够抑制神经胶质瘤细胞的增殖,其抑制能力随氟代柠檬酸浓度的增加和作用时间的延长而加强.     

Stable ornithine decarboxylase in a rat hepatoma cell line selected for resistance to alpha-difluoromethylornithine

Ornithine decarboxylase (ODC) is extremely unstable in mammalian cells. This unusual characteristic facilitates rapid fluctuations in the activity of this enzyme in response to variations in its biosynthesis. Unfortunately, very little is known about the mechanism or regulation of this ODC-specific proteolytic pathway. This study describes the production and characterization of a variant of the rat hepatoma HTC cell line that is strikingly deficient in this pathway. This cell variant was induced by selection for growth in stepwise increasing concentrations (up to 10 mM) of the irreversible ODC inhibitor, alpha-difluoromethylornithine (DFMO). Resistance to this inhibitor appears to result from a combination of elevated (10X) ODC biosynthesis and inhibited degradation, producing greater than a 2000-fold increase in the level of ODC protein. In these variant cells (DH23b) inhibition of protein synthesis by cycloheximide did not result in rapid loss of enzyme activity or ODC protein determined by radioimmunoassay. Pulse-chase studies with [35S]methionine confirmed that this enzyme was not preferentially degraded, even when spermidine was added to the media. ODC purified from the variant cells was found to be identical to the control cell enzyme in size, isoelectric point, substrate binding kinetics, and sensitivity to the inhibitor DFMO. Also, as in the control cells, a major fraction of the ODC molecules extracted from DH23b cells was shown to be phosphorylated on a serine residue. The inability to detect physical or kinetic differences between the parent and the variant cell ODC suggests that the unusual stability of ODC in this cell is associated with a defect in a cellular mechanism for ODC-specific degradation.     

The effect of fluorocitrate on transmitter amino acid release from rat striatal slices

In order to study the role of glutamine from glial cells for the synthesis of transmitter amino acids, the effect of the gliotoxic substance fluorocitrate on amino acid release from slices was investigated. In vivo treatment with 1 nmol fluorocitrate reduced the Ca²⁺ dependent K⁺ evoked release of endogenous glutamate and GABA from the slices, whereas the glutamine efflux decreased and alanine efflux increased. The K⁺ evoked release of [³H]d-aspartate increased during fluorocitrate treatment. The latter is consistent with an inhibited uptake ofd-aspartate into glial cells. Incubation of striatal slices with fluorocitrate (0.1 mM) decreased the glutamine efflux and increased the alanine efflux. Similar to the in vivo condition, fluorocitrate increased the K⁺ evoked [³H]d-asparate release, but the K⁺ evoked release of endogenous glutamate and GABA increased rather than decreased. The ratio between the K⁺ evoked release of exogenousd-aspartate to endogenous glutamate increased in both cases. The results suggest an important role of glial cells in the synthesis and inactivation of transmitter amino acids.Special Issue dedicated to Prof. Holger Hydén.     

Citrate enhances in vitro metastatic behaviours of PC-3M human prostate cancer cells: status of endogenous citrate and dependence on aconitase and fatty acid synthase

Prostate is a unique organ that produces and releases large amounts of citrate. This is reduced significantly in cancer and it is possible that citrate is (re)taken up and used as a metabolite to enhance cellular activity. The main purpose of this study was to determine how cytosolic citrate might affect in vitro metastatic cell behaviours (lateral motility, endocytosis and adhesion). Normal (PNT2-C2) and metastatic (PC-3M) human prostate cancer cells were used in a comparative approach. As regards intermediary metabolic enzymes, aconitase and fatty acid synthase, already implicated in prostate cancer, were evaluated. The level of intracellular citrate was significantly higher in PNT2-C2 cells under both control conditions and following preincubation in extracellular citrate. Supply of exogenous citrate enhanced endocytosis, lateral motility, decreased cell adhesion of PC-3M cells but failed to produce any effect on normal cells. Real-time PCR measurements showed that the mRNA levels of mitochondrial and cytosolic aconitases and fatty acid synthase were significantly higher in PC-3M cells. Correspondingly, aconitase activity was also higher in PC-3M cells. Using cerulenin (an inhibitor of fatty acid synthase), oxalomalate and fluorocitrate (inhibiting aconitases), we investigated the dependence of citrate-induced down-regulation of cellular adhesion on aconitase and fatty acid synthase activities. It was concluded: (1) that strongly metastatic PC-3M cells stored less/utilised more cytosolic citrate than the normal PNT2-C2 cells and (2) that cancer cells could metabolise cytoplasmic citrate via aconitase and fatty acid synthase to enhance their metastatic behaviour.     

Fluorocitrate inhibition of aconitate hydratase and the tricarboxylate carrier of rat liver mitochondria

1. The effect of biologically synthesized and purified fluorocitrate on the metabolism of tricarboxylate anions by isolated rat liver mitochondria was investigated, in relation to the claim by Eanes et al. (1972) that this fluoro compound inhibits the tricarboxylate carrier at concentrations at which it has little effect on the aconitate hydratase activity. 2. That the inhibitory action of fluorocitrate is at the level of the aconitate hydratase and not at the level of the tricarboxylate carrier is indicated by the following findings. Although the oxidation of citrate and cis-aconitate, but not that of isocitrate, was inhibited by fluorocitrate, the exchange of internal citrate for external citrate or l-malate was not. Had the tricarboxylate carrier been affected, these latter exchange reactions would have been inhibited. 3. By using aconitate hydratase solubilized from mitochondria it was found that with citrate as substrate the inhibition by fluorocitrate was partially competitive (K(i)=3.4x10(-8)m), whereas with cis-aconitate as substrate the inhibition was partially non-competitive (K(i)=3.0x10(-8)m).     

Effect of fluorocitrate on cerebral oxidation of lactate and glucose in freely moving rats

Glucose is the primary carbon source to enter the adult brain for catabolic and anabolic reactions. Some studies suggest that astrocytes may metabolize glucose to lactate; the latter serving as a preferential substrate for neurons, especially during neuronal activation. The current study utilizes the aconitase inhibitor fluorocitrate to differentially inhibit oxidative metabolism in glial cells in vivo. Oxidative metabolism of 14C-lactate and 14C-glucose was monitored in vivo using microdialysis and quantitating 14CO2 in the microdialysis eluate following pulse labeling of the interstitial glucose or lactate pool. After establishing a baseline oxidation rate, fluorocitrate was added to the perfusate. Neither lactate nor glucose oxidation was affected by 5 micromol/L fluorocitrate. However, 20 and 100 micromol/L fluorocitrate reduced lactate oxidation by 55 +/- 20% and 68 +/- 12%, respectively (p < 0.05 for both). Twenty and 100 micromol/L fluorocitrate reduced 14C-glucose oxidation by 50 +/- 14% (p < 0.05) and 24 +/- 19% (ns), respectively. Addition of non-radioactive lactate to (14)C-glucose plus fluorocitrate decreased 14C-glucose oxidation by an additional 29% and 38%, respectively. These results indicate that astrocytes oxidize about 50% of the interstitial lactate and about 35% of the glucose. By subtraction, neurons metabolize a maximum of 50% of the interstitial lactate and 65% of the interstitial glucose.     

Dehydrogenase activity of infected cells and biological properties of HIV-1 variants

Changes in dehydrogenase activity of some continuous cell lines were studied during the response to acute infection in vitro with HIV-1 variants having different biological features. Soon after infection, the dehydrogenase activity of infected cells increased, and this increase was greater and more prolonged with the HIV-1 r/h variant than with the HIV-1 s/l variant. Later stages showed decreased dehydrogenase activity of HIV-1-infected cells compared to the noninfected control; this is a manifestation of the cytodestructive effect of the virus. The changes increased monotonously (but not in direct proportion) with an increase in the infecting dose.     

Induction of stalk cell differentiation by cyclic-AMP in a susceptible variant of Dictyostelium discoideum.

The P-4 variant of Dictyostelium discoideum (DdH) was found to produce a great excess of stalk cells compared to the wild type DdH. If the vegetative cells of P-4 were repeatedly washed, the variant changed back to the wild type phenotype, and if cyclic-AMP was added to the washed P-4 cells, the variant character was restored. Furthermore, if the concentration of added cyclic-AMP was increased, it was possible to induce 100% stalk cells in P-4. Phosphodiesterase would cause the variant to change to the wild type, while 5-AMP and cyclic-nucleotides other than cyclic-AMP have no effect at all. Therefore it was concluded that cyclic-AMP plays a key role in stalk cell differentiation.A comparison between wild type DdH and the variant P-4 showed that DdH is ten times less sensitive to cyclic-AMP induction. They both produce the same amount of cyclic-AMP and extracellular phosphodiesterase, but the specific activity of P-4 cell-bound phosphodiesterase during development is significantly less than that in the DdH. One hypothesis that accounts for the P-4-DdH difference is that because of the lack of cell-bound phosphodiesterase, more cyclic-AMP enters the variant cells and hence more stalk cell differentiation.     

Characterization of a CV-1 cell cycle. III. Biophysical parameters.

Membrane preparations from three independently selected concanavalin A-resistant cell lines incorporated si$\text{g}$nificantly less GDP-[¹⁴C]mannose into lipid, oligosaccharide-lipid and protein fractions than preparations obtained from parental wild populations. The results from experiments with membranes from a revertant concanavalin A-resistant line more closely resembled the wild-type populations. The amount of mannose label incorporated into glycoprotein in the variant cells was higher than expected if it is assumed that the pathway GDP-mannose → mannolipid → oligosaccharide-lipid → mannoprotein is functioning in these cells. Evidence is presented to suggest that conversion of mannose label to fucose occurs in wild-type and variant cell lines and that this pathway may be of greater importance in the variant cells; this result could explain at least in part, the higher than expected levels of ¹⁴C-label in glycoprotein in the variant cell lines. The changes in the glycosyl transferase activities in these lectin-resistant cell lines are probably involved in determining the concanavalin A-resistant property and the accompanying complex phenotype exhibited by these variant cell lines.     

Isolation and characterization of a variant of B16-mouse melanoma resistant to MSH growth inhibition

A variant of B-16 F₁ mouse melanoma was selected for its ability to survive and replicate in the presence of melanocyte-stimulating hormone (MSH). Although the variant (MR-4) was completely resistant to growth inhibition by MSH, cyclic AMP was still able to block cell replication. Tyrosinase activity in MR-4 cells was considerably lower than in B-16 F₁ cells. MSH induced a twofold to three-fold increase in tyrosinase activity in both cell types, but the absolute activity in MR-4 remained significantly less than in the parental cells. MR-4 cells were also found to have a markedly depressed cyclic AMP-dependent protein kinase activity relative to B-16 F₁ cells. The protein kinase from both cell types was stimulated by cyclic AMP, but the level of MR-4 kinase activity at maximal cyclic AMP concentrations remained considerably lower than B-16 F₁ kinase activity under the same conditions. In both cell types adenylate cyclase activity was markedly stimulated by MSH. When equal numbers of viable F₁ and MR-4 cells were injected subcutaneously into C57/B1 mice, the MR-4 cells formed tumors earlier and killed the host sooner than the parental F₁ cells. We conclude that the biochemical alteration which allows MR-4 cells to replicate in the presence of MSH is a low level of tyrosinase activity, which in turn may be the result of low cyclic AMP-dependent protein kinase activity.     

Reconstitution of catecholamine-sensitive adenylate cyclase activity: interaction of components following cell-cell and membrane-cell fusion

Heterokaryons formed by the fusion of adenylate cyclase-deficient S49 cells and beta-adrenergic receptor-deficient B82 cells in the presence of cycloheximide display a catecholamine-sensitive adenylate cyclase activity. Similar complementation can be observed when receptor-replete membranes from the cyclase-deficient cell are fused with intact B82 cells. Using the cell fusion technique it can be demonstrated that the uncoupled S49 cell variant has a functional beta-adrenergic receptor but that this variant and the adenylate cyclase-deficient variant are not complementary. Hypothetically, both clones may share a common defect in regulatory components of adenylate cyclase or both may lack a specific coupling factor.     

Direct measurement of oxidative metabolism in the living brain by microdialysis: a review

This review summarizes microdialysis studies that address the question of which compounds serve as energy sources in the brain. Microdialysis was used to introduce ¹⁴C-labeled glucose, lactate, pyruvate, glutamate, glutamine, and acetate into the interstitial fluid of the brain to observe their metabolism to ¹⁴CO₂. Although glucose uptake from the systemic system supplies the carbon source for these compounds, compounds synthesized from glucose by the brain are subject to recycling including complete metabolism to CO₂. Therefore, the brain utilizes multiple compounds in its domain to provide the energy needed to fulfill its function. The physiological conditions controlling metabolism and the contribution of compartmentation into different brain regions, cell types, and subcellular spaces are still unresolved. The aconitase inhibitor fluorocitrate, with a lower inhibition threshold in glial cells, was used to identify the proportion of lactate and glucose that was oxidized in glial cells versus neurons. The fluorocitrate data suggest that glial and neuronal cells are capable of utilizing both lactate and glucose for energy metabolism.     

Increased adenosine deaminase synthesis and messenger RNA activity in deoxycoformycin-resistant cells

The basis for the increased adenosine deaminase activity in deoxycoformycin-resistant rat hepatoma cells was investigated. Three variant cell lines with different levels of adenosine deaminase activity showed increases in the relative rate of synthesis of the enzyme in vivo. No difference in the rate of degradation of the enzyme was seen between the parental cell line and one variant cell line which exhibits a 180-fold increase in adenosine deaminase activity. Polysomal RNA isolated from this variant exhibited a 175-fold increase in the ability to direct the synthesis of adenosine deaminase in vitro.     

Evidence that hydrogen peroxide generated by 365-nm UVA radiation is not important in mammalian cell killing

We compared measurements of cell survival and DNA single-strand breaks (SSBs) caused by hydrogen peroxide (H2O2) and UVA radiation (365-nm) in both a parental and a H2O2-resistant variant of the Chinese hamster ovary HA1 line derived by culturing cells in progressively higher concentrations of H2O2. Both RNA slot blot analysis and enzyme analysis confirmed that the variant possesses high levels of both catalase activity and mRNA. The variant was completely resistant to the lethal effects of H2O2 over the concentration range tested (up to 480 microM), whereas the parental strain showed less than 1% survival at this concentration. Similarly, the H2O2-resistant strain exhibited far fewer SSBs after exposure to H2O2 than the parental strain. Addition of o-phenanthroline to the parental cells during H2O2 exposure almost completely inhibited SSB induction, evidence that these SSBs are produced via the Fenton pathway of Haber-Weiss reactions. Very little difference was found between the variant and the parent after exposure to 365-nm radiation: only a minor difference in survival kinetics and no difference is SSB induction were observed between the two cell lines. These results are consistent with a hypothesis that most lethal events caused in cells by UVA occur by pathways that do not involve the H2O2 that is produced by sensitized reactions within the cells.     

The kinetics of ox kidney biliverdin reductase in the pre-steady state. Evidence that the dissociation of bilirubin is the rate-determining step.

Four mouse and two human tumour cell lines resistant to alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), were analysed for the activities of polyamine-biosynthetic and -biodegradative enzymes as well as for cellular polyamine contents. In all but one of these cell lines the resistance to DFMO was based on an overproduction of ODC. In a human myeloma cell line the resistance was based on a greatly enhanced arginase activity. Except for one L1210 variant cell line, all the resistant cell lines contained elevated S-adenosylmethionine decarboxylase activity. Similarly, all the resistant mouse, but not human, cell lines displayed enhanced spermidine and spermine synthase activities. Arginase activity was detected only in human cell lines. In both DFMO-resistant cell lines the activity of arginase was strikingly elevated. Of the biodegradative enzymes, polyamine oxidase activity was readily detectable in all mouse cells, but no measurable activity was found in the human cells. Spermidine/spermine N1-acetyltransferase activity was elevated in three out of four resistant mouse cell lines. Even though the concentration of spermidine was usually lower in the overproducer cells, this was compensated by an increased content of spermine. The two resistant human myeloma cells contained intracellular ornithine concentrations that were from more than 5 to more than 20 times higher than those in the parental cells.     

A Nonneuronal Isoform of Cell Adhesion Molecule L1: Tissue-Specific Expression and Functional Analysis

Abstract: The cell adhesion molecule L1 is a multifunctional protein in the nervous system characterizing cell adhesion, migration, and neurite outgrowth. In addition to full-length L1, we found an alternatively spliced variant lacking both the KGHHV sequence in the extracellular part and the RSLE sequence in the cytoplasmic part of L1. This L1 variant was expressed exclusively in nonneuronal cells such as Schwann cells, astrocytes, and oligodendrocytes, in contrast to the expression of the full-length L1 in neurons and cells of neuronal origin. To investigate the functions of the L1 variant, we established cell lines transfected with a cytoplasmic short L1 (L1cs) cDNA that lacks only the 12-bp segment encoding for the RSLE sequence. The promoting activities of homophilic cell adhesion, neurite outgrowth, and neuronal cell migration of L1cs-transfected cells (L4-2) were similar to those of full-length L1-transfected cells (L3-1), but the cell migratory activity of L4-2 itself was clearly lower than that of L3-1. In conclusion, the short form of L1 is a nonneuronal type, in contrast to the neuronal type of the full-length L1. Deletion of the four amino acids RSLE in the cytoplasmic region of L1 markedly reduced cell migratory activity, suggesting an importance of the RSLE sequence for the signaling events of neuronal migration mediated by L1.