Effect of ethanol on adenosine triphosphatase and enolase activities in rat brain and in cultured nerve cells

The effect of alcohol on enzymes involved in energy metabolism of nervous tissue were analyzed, in vivo after acute and chronic ethanol administration to rats and in vitro by addition of 50 mM and 100 mM ethanol to the medium of cultured nerve cells: chick neurons, chick glial cells, a neuronal cell line (MT17) and a glial tumoral cell line (C6). The parameters we measured were (Na⁺,K⁺), Mg²⁺ and ecto Ca²⁺,Mg²⁺ ATPase activities involved in transport phenomena and enolase activities (non neuronal NNE and neuron specific enolase NSE) as markers of nerve cell maturation. In vivo, after chronic ethanol administration (Na⁺,K⁺) ATPase activity was increased while Mg²⁺ dependent activity was not affected. Enolase activity was decreased. Acute ethanol administration decreased (Na⁺,K⁺) ATPase activity, while Mg²⁺ dependent activity was not affected. In cultured nerve cells ethanol effect was dose, time and cell type dependent; alterations of the cell membrane by trypsinization of the tissue before seeding modifies the effect of ethanol on the enzymes we analyzed. Our results suggest that alcohol effect on nerve cells depends mainly on the lipoprotein structure of the cell membranes which may have different properties from one cell type to another.     

Modulation of Mn2+ accumulation in cultured rat neuronal and astroglial cells

The effects of physiological concentrations of K⁺ on Mn²⁺ accumulation were compared in rat glial cells and neurons in culture. Increasing the K⁺ concentration in growth medium increased significantly the Mn²⁺ level of the cultivated cells, with glial cells more affected than neurons. Ethanol markedly increased the Mn²⁺ accumulation within glia but not within neurons while ouabaïn caused inhibition of Mn²⁺ uptake with neurons and glial cells. A modulation of the total protein synthesis by Mn²⁺ and ethanol level in the growth medium was observed with glial cells. These data suggest that the mechanisms involved in Mn²⁺ accumulation in glial cells are different from those present in neurons. Moreover, the results are consistent with the hypothesis that Mn²⁺ plays a regulatory role in glial cell metabolism.     

Effect of manganese on the development of glial cells cultured from prenatally alcohol exposed rats

Maternal alcohol abuse is known to produce retardation in brain maturation and brain functions. Using cultured glial cells as a model system to study these effects of alcohol we found an alcohol antagonizing property for manganese (Mn). Mn was added to the alcohol diet (MnCl₂ 25 mg/l of 20% v/v ethanol) of pregnant rats. Glial cells were cultured during 4 weeks from cortical brain cells of pups born to these mothers. Several biochemical parameters were examined: protein levels, enzymatic markers of glial cell maturation (enolase and glutamine synthetase), superoxide dismutase a scavenger of free radicals produced during alcohol degradation. The results were compared to appropriate controls. A beneficent effect of Mn was observed for the pups weight which was no more significantly different from the control values. Protein levels, enolase and glutamine synthetase activities were increased mainly during the proliferative period when Mn was added to the alcohol diet compared to the only alcohol treated animals. This Mn effect was not found for superoxide dismutase in cultured glial cells but exists in the total brain of the 2 week-old offspring. In the total 2 and 4 week-old brain the alcohol induced decrease of enolase and glutamine synthetase was also antagonized by the Mn suplementation. Our data suggest that Mn may act as a factor overcoming at least partially some aspects of alcohol induced retardation of nerve cell development.     

Development of glial cells cultured from prenatally alcohol treated rat brain: Effect of supplementation of the maternal alcohol diet with a grape extract

The aim of this work was to investigate the effect of supplementation of a maternal alcohol diet with a grape extract on glial cell development. Glial cells were cultured during 4 weeks from cortical brain cells of the new born offspring in DMEM medium supplemented with fetal calf serum. Enzymatic markers of nerve cell development were measured (enolase isoenzymes and glutamine synthetase). Since alcohol consumption produces free radicals the antioxidant system superoxide dismutase was also investigated. Compared to the decrease found in only alcohol treated animals, all parameters except neuron-specific enolase were antagonized and even stimulated after grape extract supplementation. The effect was more important after only 1 month than 3 months of treatment. Also in the total brain an alcohol antagonizing effect and a glutamine synthetase activation were found. Our data demonstrate that addition of a grape extract to the maternal alcohol diet may partially or completely overcome the alcohol induced retardation of glial cell development.     

Effect of hydrocortisone and thyroxine on ATPase activities of neuronal and glial cell lines in culture

The effect of hydrocortisone and thyroxine, on the activities of Ca²⁺-and Mg²⁺-ATPase was studied in cultured neuronal (clone M1) and glial (clones NN and C6) cell lines. For M1 and NN cells an increase in Ca²⁺-and Mg²⁺-ecto-ATPase activity was found when the cells were cultured during 4–6 days in presence of hydrocortisone or together with thyroxine. In the same conditions, a decrease in Ca²⁺-and Mg²⁺-ecto-ATPase activity was found for the C6 cells. In C6 cells the effect of hormones was more pronounced for the Mg²⁺-than for the Ca²⁺-ecto-ATPase activity. The observed decrease may be related to the tumoral origin of the C6 cells. The activity of (Na⁺, K⁺)-ATPase in all three cell lines increased in presence of hydrocortisone or together with thyroxine when the cells were cultured during 4–6 days, in presence of the hormones, whereas the total Mg²⁺-ATPase activity increased only after 6 days of treatment. Thyroxine alone has very few effect either on Ca²⁺-and Mg²⁺-ecto-ATPase, or on (Na⁺, K⁺)-and total Mg²⁺-ATPase activity. These observations are interpreted to indicate that hormones may modulate or induce enzymatic activities involved in active transport phenomena in nervous tissue.     

High levels of Mn2+ inhibit secretory pathway Ca2+/Mn2+‐ATPase (SPCA) activity and cause Golgi fragmentation in neurons and glia

Excess Mn²⁺ in humans causes a neurological disorder known as manganism, which shares symptoms with Parkinson's disease. However, the cellular mechanisms underlying Mn²⁺‐neurotoxicity and the involvement of Mn²⁺‐transporters in cellular homeostasis and repair are poorly understood and require further investigation. In this work, we have analyzed the effect of Mn²⁺ on neurons and glia from mice in primary cultures. Mn²⁺ overload compromised survival of both cell types, specifically affecting cellular integrity and Golgi organization, where the secretory pathway Ca²⁺/Mn²⁺‐ATPase is localized. This ATP‐driven Mn²⁺ transporter might take part in Mn²⁺ accumulation/detoxification at low loads of Mn²⁺, but its ATPase activity is inhibited at high concentration of Mn²⁺. Glial cells appear to be significantly more resistant to this toxicity than neurons and their presence in cocultures provided some protection to neurons against degeneration induced by Mn²⁺. Interestingly, the Mn²⁺ toxicity was partially reversed upon Mn²⁺ removal by wash out or by the addition of EDTA as a chelating agent, in particular in glial cells. These studies provide data on Mn²⁺ neurotoxicity and may contribute to explore new therapeutic approaches for reducing Mn²⁺ poisoning.     

Levels and sub-cellular distribution of physiologically important metal ions in neuronal cells cultured from chick embryo cerebral cortex

Mg²⁺, Ca²⁺, Mn²⁺, Zn²⁺, and Cu content of neurons from chick embryo cortex cultivated in chemically defined serum free growth medium was determined by energy dispersive X-ray fluorescence and atomic absorption spectroscopy. The intracellular volume of cultured neurons was determined to be 2.73 l/mg. Intracellular Mn²⁺, Fe²⁺, Zn²⁺, and Cu²⁺ in the cultivated neurons were 100–200 times the concentrations in the growth medium: Mg²⁺ and Ca²⁺ were 0.9 and 1.7 mM respectively, around 20 fold higher than in growth medium. Mg²⁺, Fe²⁺, Cu²⁺ and Zn²⁺ concentrations in neurons were in the range of ca. 300–600 M, approximately 2–3 times the values previously reported in glial cells; Ca²⁺ and Mn²⁺ content of the neurons were higher by 5 and 10 fold respectively compared to glial cells. In neurons, the subcellular distribution of Fe²⁺, Cu²⁺, and Mn²⁺ follows the rank order: cytosol>microsomes>mitochondria; for Zn²⁺ the distribution differs as following: cytosol >mitochondria>microsomes. Determination of the superoxide dismutase activities in the cultivated neurons indicated that the Mn²⁺ linked activity predominates whereas, the Cu-Zn dependent enzyme is dominant in glial cells. Enrichment of the culture medium with Mn²⁺ to 2.5 M enhanced the Mn-SOD by approximately 33% but Cu²⁺–Zn²⁺ enzyme activity was not modified. The high Mn²⁺ content, the capacity to accumulate Mn²⁺, and the predominancy of the Mn–SOD form observed in neurons is in accord with a fundamental functional role for this metal ion in this type of brain cells.     

Effects of Mn2+ and other divalent cations on adenylate cyclase activity in rat brain.

Abstract— Mn²⁺ caused an 8-to 16-fold stimulation of adenylate cyclase activity in homogenates as well as synaptosomcs. isolated synaptic membranes, and slices prepared from rat brain. The stimulation occurred at low concentrations of Mn²⁺. with a doubling of activity at 50-60μM. and was unaffected by a 60-fold excess of Mg²⁺. Whether or not Mg²⁺ was added, inclusion of a low concentration of Mn²⁺ reduced, but did not prevent the stimulation of adenylate cyclase caused by dopaminc in homogenates of corpus striatum. In contrast, Ca²⁺. at a concentration that had little effect on basal cyclase activity, completely prevented the stimulation by dopamine. The increase of cyclase activity produced by Mn²⁺ in brain homogenates was potentiated by F^?. Other ions, notably Hg²⁺. Pb²⁺. Cu²⁺ and Zn²⁺. in order of decreasing potency, inhibited both basal and Mn^2--stimulated cyclase activity. It is proposed that the effect of Mn²⁺ on adenylate cyclase activity may involve only the catalytic subunit of the enzyme, and that the mechanism is different from that by which either dopamine or F^? stimulates the enzyme. These results suggest that the effects of low concentrations of Mn²⁺ and certain other divalent metal ions on adenylate cyclase activity may be involved in their neuropsychiatrie or other toxic effects, and that such ions may also participate in normal physiological mechanisms involving cyclic nucleotides.     

Superoxide dismutase activity in rat brain during acute and chronic alcohol intoxication

The effect of acute and chronic ethanol administration on rat brain superoxide dismutase (SOD) activity was studied. Intraperitoneal injections of ethanol led to an inhibition of SOD activity. When ethanol was fed as the sole fluid, the SOD activity decreased progressively, reaching a plateau after 6 weeks of treatment. Withdrawal of ethanol produced a recovery of control values within 48 hr. SOD activity was also decreased in rats born from ethanol-drinking mothers. Inhibition of SOD activity by ethanol may allow an accumulation of cytotoxic O₂ ^– radicals; this may account for some nervous system disorders during alcohol intoxication.     

Evidence of metal binding activities of pentadecapeptide from Panax ginseng

A tetradecapeptide from ginseng (Panax ginseng) root showing anti-lipolytic activity in an isolated rat fat cell assay was chemically synthesized for analysis of metal binding activities in vitro. Binding activities against several metal ions were analysed by measuring mobility shifts during capillary zone electrophoresis experiments. The ginseng polypeptide (GPP) showed the greatest increase in effective molecular electrophoretic mobility in the presence of Mg²⁺. Mobility was also affected in the presence of La³⁺, Mn²⁺, Ca²⁺ and Zn²⁺ ions. Analysis with the dye Stains-all revealed GPP to possess a cation binding site similar to those in Ca²⁺-binding proteins. GPP thus appears to be a metal binding peptide. The results of this analysis suggested that GPP may perform its anti-lipolytic activities through an ability to modulate the level of free cellular Mg²⁺ and Mn²⁺ ions.     

Biochemical studies of citric acid production and accumulation by Aspergillus niger mutants

The biochemical rationale for the inhibition of citric acid fermentation by Aspergillus niger in the presence of Mn²⁺ ions has been investigated using high citric acid-yielding, Mn²⁺ ion-sensitive as well as Mn²⁺ ion-tolerant mutant strains of A. niger. In the presence of Mn²⁺ (1.5 mg/l), citric acid production by the Mn²⁺ ion-sensitive strain (KCU 520) was reduced by about 75% with no apparent effect on citric acid yield by the Mn²⁺ ion-tolerant mutant strain (GS-III) of A. niger. The significantly increased level of the Mn²⁺ ion-requiring NADP⁺-isocitrate dehydrogenase activity in KCU 520 cells and the lack of effect on the activity level of the enzyme in GS-III mutant cells by Mn²⁺ ions during fermentation seem to be responsible for the Mn²⁺ ion inhibition of citric acid production by the KCU 520 strain and the high citric acid yield by the mutant strain GS-III of A. niger even in the presence of Mn²⁺.     

The effect of ethanol and acetaldehyde on [14C]pantothenate incorporation into CoA in cultured rat liver parenchymal cells

The effect of ethanol on [¹⁴C]pantothenate incorporation into CoA and on total CoA levels was measured in 3-day-old primary cultures of adult rat liver parenchymal cells. Ethanol decreased the incorporation of radioactivity into CoA a maximum of 67%, 5 mm ethanol was saturating for the inhibitory effect and 0.2 mm ethanol was sufficient for half-saturation. This inhibitory effect did not result from a loss of CoA precursors or from cell death. Ethanol concentrations up to 10 mm did not decrease the ATP content of cells or the total protein content of cells which adhered to the incubation flask. Ethanol (5 mm) had no effect on the cyteine + cystine content of the cells. Intracellular pantothenate concentrations were not affected by 5 mm ethanol, and increasing the pantothenate concentration did not affect ethanol inhibition. Ethanol inhibition of [¹⁴C]pantothenate conversion to CoA could be fully reversed by rinsing the cells free of ethanol. The ethanol inhibition could also be fully reversed by addition of 4-methylpyrazole, indicating that ethanol must be oxidized via alcohol dehydrogenase to exert its inhibitory effect. Acetaldehyde, the immediate product of alcohol dehydrogenase, was also an inhibitor of the incorporation of [¹⁴C]pantothenate into CoA; the maximum inhibition was 63%. Acetaldehyde concentrations maintained between 18 and 103 μm inhibited incorporation by 57%. The inhibition by acetaldehyde did not correlate well with changes in the NADH and NAD⁺ ratio of the cells (as determined by measuring changes in the lactate-to-pyruvate ratio). The ability of glucagon, dibutyryl cAMP + theophylline, or dexamethasone to stimulate [¹⁴C]pantothenate conversion to CoA was not decreased by the addition of ethanol or acetaldehyde, indicating that ethanol inhibition does not occur by reversal of the cAMP-mediated regulatory mechanism for CoA biosynthesis.     

Ethanol production at 45°C by alginate-immobilized Kluyveromyces marxianus IMB3 during growth on lactose-containing media

The thermotolerant yeast strain Kluyveromyces marxianus IMB3 was immobilized in calcium alginate and this was used in batch-fed reactor systems to convert lactose (4?g/l) to ethanol. Production of ethanol by the free and immobilized biocatalyst in the presence and absence of Mn²⁺ was compared. In systems containing the free microorganism in the presence and absence of Mn²⁺, ethanol increased to a maximum of 8?g/l within 40 hours with no significant difference in production by both systems. Ethanol production by the immobilized system in the absence of Mn²⁺ increased to a maximum of 13?g/l within 40 hours and then decreased to 9?g/l within 80 hours. Ethanol production by the immobilized system in the presence of Mn²⁺ increased to 14?g/l within 60 hours and this decreased to 13?g/l at 80 hours. When all systems were re-fed at 80 hours, ethanol production by systems containing the free biocatalyst increased to a maximum of 3?g/l while the immobilized system in the presence of Mn²⁺ increased to a maximum of 12?g/l. Subsequent experiments involving re-feeding the system at shorter time intervals demonstrated that ethanol production by the immobilized system on lactose-containing media at 45?°C was far superior to ethanol production by the free biocatalyst.     

Uptake and metabolism of l-[3H]pyroglutamic acid in neuronal and glial cells in culture

The presence of an efficient uptake system for l-pyroglutamate was demonstrated in cultured glial cells originating from newborn rats. This compound is also transported by a high affinity uptake mechanism in neurons cultured from rat embryos cerebral hemispheres, but the V_max is 6 times lower than for glial cells. It is shown that l-pyroglutamate like l-glutamate is preferentially transported by glial cells, but with a V_max 40 to 60 times lower than for glutamate. The metabolism of l-pyroglutamate was also studied in cultured rat neuronal and glial cells, using l-[³H]pyroglutamate. Pyroglutamate, its metabolites and the various amino acids were separated by thin-layer electrophoresis. [³H]Pyroglutamate is more actively metabolised in glial cells than in neurons and glutamate is the main metabolite. Glutamate maximal specific activity is 4 times higher in glial than in neuronal cultures. It should also be noted that some [³H]pyroglutamate is transformed in [³H]GABA after longer incubation periods, but only in neurons. These results show the importance of glial cells for pyroglutamate uptake and metabolism in nervous tissue. They also suggest that pyroglutamate may interfere with glutamate neurotransmission in vivo.     

Use of a Vibrating Electrode to Measure Changes in Calcium Fluxes Across the Cell Membranes of Oxidatively Challenged Aplysia Nerve Cells

A self-referencing and non-invasive Ca²⁺-sensitive vibrating electrode was used to assess the effects of hydrogen peroxide-induced oxidative challenges on the efflux and influx of calcium across the plasma membrane of single nerve cells cultured from abdominal ganglion of Aplysia californica. A reduced net efflux of Ca²⁺ from the cell soma occurred immediately after the addition of hydrogen peroxide (0.0025 mM, 0.005 mM or 0.01 mM) to the culture medium, indicating damage to the cell membrane or Ca²⁺ transport mechanism. There then followed a marked efflux, the extent and duration of which was related to the concentration of hydrogen peroxide used and which may reflect compensatory activity by the Ca²⁺ regulatory mechanisms in the plasmalemma. No morphological changes were observed in cells challenged with 0.0025 mM hydrogen peroxide and the enhanced rate of Ca²⁺ efflux rapidly decreased to pre-exposure values. Sustained and enhanced Ca²⁺ effluxes from those cells exposed to 0.005 mM or 0.01 mM hydrogen peroxide were also consistent with regulatory pumping of Ca²⁺ out of the cell although contraction and blebbing of neurites and swelling of the soma may indicate that a proportion of the efflux arose from release of Ca²⁺ from disrupted intracellular stores. The vibrating electrode is a useful additional technique for the study of the pathogenesis of neurological conditions, as ionic fluxes across single nerve cells exposed to physiologically-relevant concentrations of free radicals can be monitored non-invasively for prolonged periods.     

Co2+ and Mn2+ uptake by crab nerve fibers in resting state and potassium depolarization

Transition metal ions, Mn²⁺ and Co²⁺, are incorporated into nerve fibers when they are applied externally. For nerve fibers in the resting state, however, extracellular and intracellular water may be distinguished by applying transition metal ions externally. NMR spectra of water protons from nerve fibers in high potassium media, which contain transition metal ions, consist of three or more components, reflecting a complex distribution of these ions around the nerve membranes. In the case of Co²⁺, three components may be identified.     

Ca<Superscript>2+</Superscript> channel currents of cortical neurons from pure and mixed cultures

Voltage-gated Ca²⁺ channels (VGCCs) are key regulators of many neuronal functions, and involved in multiple central nervous system diseases. In the last 30 years, a large number of injury and disease models have been established based on cultured neurons. Culture with serum develops a mixture of neurons and glial cells, while culture without serum develops pure neurons. Both of these neuronal-culture methods are widely used. However, the properties of Ca²⁺ currents in neurons from these two cultures have not been compared. In this study, we cultured rat cortical neurons in serum-containing or -free medium and then recorded the Ca²⁺ channel currents using patch-clamp technique. Our results showed that there were significant differences in the amplitude and activation properties of whole-cell Ca²⁺ channel currents, and of non-L-type Ca²⁺ channel currents between the neurons from these two culture systems. Our data suggested that the difference of whole-cell Ca²⁺ currents may result from the differences in non-L-type currents. Understanding of these properties will considerably advance studies of VGCCs in neurons from pure or mixed culture.     

Vesicular distribution of Secretory Pathway Ca<Superscript>2+</Superscript>-ATPase isoform 1 and a role in manganese detoxification in liver-derived polarized cells

Manganese is a trace element that is an essential co-factor in many enzymes critical to diverse biological pathways. However, excess Mn²⁺ leads to neurotoxicity, with psychiatric and motor dysfunction resembling parkinsonism. The liver is the main organ for Mn²⁺ detoxification by excretion into bile. Although many pathways of cellular Mn²⁺ uptake have been established, efflux mechanisms remain essentially undefined. In this study, we evaluated a potential role in Mn²⁺ detoxification by the Secretory Pathway Ca²⁺, Mn²⁺-ATPase in rat liver and a liver-derived cell model WIF-B that polarizes to distinct bile canalicular and sinusoidal domains in culture. Of two known isoforms, only secretory pathway Ca²⁺-ATPase isoform 1 (SPCA1) was expressed in liver and WIF-B cells. As previously observed in non-polarized cells, SPCA1 showed overlapping distribution with TGN38, consistent with Golgi/TGN localization. However, a prominent novel localization of SPCA1 to an endosomal population close to, but not on the basolateral membrane was also observed. This was confirmed by fractionation of rat liver homogenates which revealed dual distribution of SPCA1 to the Golgi/TGN and a fraction that included the early endosomal marker, EEA1. We suggest that this novel pool of endosomes may serve to sequester Mn²⁺ as it enters from the sinusoidal/basolateral domains. Isoform-specific partial knockdown of SPCA1 delayed cell growth and formation of canalicular domain by about 30% and diminished viability upon exposure to Mn²⁺. Conversely, overexpression of SPCA1 in HEK 293T cells conferred tolerance to Mn²⁺ toxicity. Taken together, our findings suggest a role for SPCA1 in Mn²⁺ detoxification in liver.     

Effects of divalent metal ions on the calcium pump and membrane phosphorylation in human red cells

In inside-out red cell membrane vesicles ATP-dependent calcium transport is activated by the divalent metal ions Mg²⁺, Mn²⁺, Co²⁺, Ni²⁺ and Fe²⁺. This activation is based on the formation of Me²⁺-ATP complexes which can serve as energy-donor substrates for the calcium pump, and probably, satisfy the requirement for free Me²⁺ in this transport process. Higher Me²⁺ concentrations inhibit calcium transport with various efficiencies. Mn²⁺ directly competes with Ca²⁺ at the transport site, while other divalent metal ions investigated have no such effect. The formation of the hydroxylamine-sensitive phosphorylated intermediate (EP) of the red cell membrane calcium pump from [γ-³²P]ATP is induced by Ca²⁺ while rapid dephosphorylation requires the presence of Mg²⁺. At higher concentrations Mn²⁺ and Ni²⁺ inhibit predominantly the formation of EP, while Co²⁺ and Fe²⁺ block dephosphorylation. The possible sites and nature of the divalent metal interactions with the red cell calcium pump are discussed. Hydroxylamine-insensitive membrane phosphorylation in inside-out vesicles from [γ-³²P]ATP is significantly stimulated by Mn²⁺ and Co²⁺, as compared to that produced by Mg²⁺, Fe²⁺ and Ni²⁺. Part of this labelling is found in phospholipids, especially in phosphatidylinositol. The results presented for the metal dependency of protein and lipid phosphorylation in red cell membranes may help in the characterization of ATP consumptions directly related to the calcium pump and those involved in various regulatory processes.