Manganese induces sustained Ser40 phosphorylation and activation of tyrosine hydroxylase in PC12 cells

Manganese (Mn²⁺) is an essential metal involved in normal functioning of a range of physiological processes. However, occupational overexposure to Mn²⁺ causes neurotoxicity. The dopaminergic system is a particular target for Mn²⁺ neurotoxicity. Tyrosine hydroxylase (TH) is the rate limiting enzyme for dopamine synthesis and is regulated acutely by phosphorylation at Ser40 and chronically by protein synthesis. In this study we used pheochromocytoma 12 cells to investigate the effects of Mn²⁺ exposure on the phosphorylation and activity of TH. Mn²⁺ treatment for 24 h caused a sustained increase in Ser40 phosphorylation and TH activity at a concentration of 100 μM, without altering the level of TH protein or PC12 cell viability. Inhibition of protein kinase A and protein kinase C and protein kinases known to be involved in sustained phosphorylation of TH in response to other stimuli did not block the effects of Mn²⁺ on Ser40 phosphorylation. A substantial increase in H₂O₂ production occurred in response to 100 μM Mn²⁺. The antioxidant Trolox^TM completely inhibited H₂O₂ production but did not block TH phosphorylation at Ser40, indicating that oxidative stress was not involved. Sustained TH phosphorylation at Ser40 and the consequent activation of TH both occurred at low concentrations of Mn²⁺ and this provides a potential new mechanism for Mn²⁺-induced neuronal action that does not involve H₂O₂-mediated cell death.     

The roles of calcium and manganese ions in the in vitro conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene by lentil root membranes

Ca²⁺ and Mn²⁺ activate the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) by root microsomes of Vicia lens as they do in other similar systems. The preparation of microsomes in the presence of Mn²⁺ greatly increases their ability to convert ACC into ethylene, without addition of Mn²⁺ in the reaction mixture. Ca²⁺ does not have this property. The effect could not be attributed to Mn²⁺ entrapping into membrane vesicles (sonication followed by repelleting had no effect) but, possibly, in part to Mn²⁺-mediated binding to microsomes of a soluble factor favouring the conversion of ACC to C₂H₄. Although no direct correlation could be established in vitro between ethylene-forming-enzyme (EFE) and peroxidase activities, some soluble peroxidases might be this soluble factor. Mn²⁺ favoured attachment to membranes of some peroxidase activity from the soluble fraction and from commercial HRP and lipoxygenase. This binding effect of Mn₂₊ cannot be readily distinguished from its role in the generation of a chain of free radicals and in redox mechanisms.     

3-Hydroxypropyl amide formation from 1-aminocyclopropane-1-carboxylic acid by a cell-free ethylene-forming system from olive leaves

The low ethylene yield in a cell-free ethylene-forming system from olive tree leaves ( Olea europaea L. cv. Picual) was investigated. During the incubation, 1-aminocyclopropane-1-carboxylic acid (ACC) was extensively transformed into 3-hydroxypropyl amide (HPA). Enzyme extract, Mn²⁺ and oxygen are responsible for this reaction. Horseradish peroxidase (EC 1.11.1.7) can substitute for the enzyme extract in this reaction. HPA formation could be one reason for the poor in vitro conversion efficiency of ACC to ethylene.     

K+ATP channel opening prevents succinate-dependent H2O2 generation by plant mitochondria

The role of a recently identified K⁺_ATP channel in preventing H₂O₂ formation was examined in isolated pea stem mitochondria. The succinate-dependent H₂O₂ formation was progressively inhibited, when mitochondria were resuspended in media containing increasing concentration of KCl (from 0.05 to 0.15  M ). This inhibition was linked to a partial dissipation of the transmembrane electrical potential (ΔΨ) induced by KCl. Conversely, the malate plus glutamate-dependent H₂O₂ formation was not influenced. The succinate-sustained H₂O₂ generation was also unaffected by nigericin (a H⁺/K⁺ exchanger), but completely prevented by valinomycin (a K⁺ ionophore). In addition, cyclosporin A (a K⁺_ATP channel opener) inhibited this H₂O₂ formation, while ATP (an inhibitor of the channel opening) slightly increased it. The inhibitory effect of ATP was strongly stimulated in the presence of atractylate (an inhibitor of the adenine nucleotide translocase), thus suggesting that the receptor for ATP on the K⁺ channel faces the intermembrane space. Finally, the succinate-dependent H₂O₂ formation was partially prevented by phenylarsine oxide (a thiol oxidant).     

Calcium ion dependency of ethylene production in segments of primary roots of Zea mays

We investigated the effect of Ca²⁺ on ethylene production in 2-cm long apical segments from primary roots of corn ( Zea mays L., B73 × Missouri 17) seedlings. The seedlings were raised under different conditions of Ca²⁺ availability. Low-Ca and high-Ca seedlings were raised by soaking the grains and watering the seedlings with distilled water or 10 m M CaCl₂, respectively. Segments from high-Ca roots produced more than twice as much ethylene as segments from low-Ca roots. Indoleacetic acid (IAA; 1 μ M ) enhanced ethylene production in segments from both low-Ca and high-Ca roots but auxin-induced promotion of ethylene production was consistently higher in segments from high-Ca roots. Addition of I-aminocyclopropane-I-carboxylic acid (ACC) to root segments from low-Ca seedlings doubled total ethylene production and the rate of production remained fairly constant during a 24 h period of monitoring. In segments from high-Ca seedlings ACC also increased total ethylene production but most of the ethylene was produced within the first 6 h. The data suggest that Ca²⁺ enhances the conversion of ACC to ethylene. The terminal 2 mm of the root tip were found to be especially important to ethylene biosynthesis by apical segments and, experiments using ⁴⁵Ca²⁺ as tracer indicated that the apical 2 mm of the root is the region of strongest Ca²⁺ accumulation. Other cations such as Mn²⁺, Mg²⁺, and K⁺ could largely substitute for Ca²⁺. The significance of these findings is discussed with respect to recent evidence for gravity-induced Ca²⁺ redistribution and its relationship to the establishment of asymmetric growth during gravitropic curvature.     

Hydrogen Peroxide Induces a Long-Lasting Inhibition of the Ca2+-Dependent Glutamate Release in Cerebrocortical Synaptosomes Without Interfering with Cytosolic Ca2+

Abstract: We studied the action of H₂O₂ on the exocytosis of glutamate by cerebrocortical synaptosomes. The treatment of synaptosomes with H₂O₂ (50–150 µ M ) for a few minutes results in a long-lasting depression of the Ca²⁺-dependent exocytosis of glutamate, induced by KCl or by the K⁺-channel inhibitor 4-aminopyridine. The energy state of synaptosomes, as judged by the level of phosphocreatine and the ATP/ADP ratio, was not affected by H₂O₂, although a transient decrease was observed after the treatment. H₂O₂ did not promote peroxidation, as judged by the formation of malondialdehyde. In indo-1-loaded synaptosomes, the treatment with H₂O₂ did not modify significantly the KCl-induced increase of [Ca²⁺]_i. H₂O₂ inhibited exocytosis also when the latter was induced by increasing [Ca²⁺]_i with the Ca²⁺ ionophore ionomycin. The effects of H₂O₂ were unchanged in the presence of superoxide dismutase and the presence of the Fe³⁺ chelator deferoxamine. These results appear to indicate that H₂O₂, apparently without damaging the synaptosomes, induces a long-lasting inhibition of the exocytosis of glutamate by acting directly on the exocytotic process.     

Effects of 1-Methyl-4-Phenylpyridinium on Isolated Rat Brain Mitochondria: Evidence for a Primary Involvement of Energy Depletion

Abstract: The effects of 1-methyl-4-phenylpyridinium (MPP⁺) on the oxygen consumption, ATP production, H₂O₂ production, and mitochondrial NADH-CoQ₁ reductase (complex I) activity of isolated rat brain mitochondria were investigated. Using glutamate and malate as substrates, concentrations of 10–100 µ M MPP⁺ had no effect on state 4 (−ADP) respiration but decreased state 3 (+ADP) respiration and ATP production. Incubating mitochondria with ADP for 30 min after loading with varying concentrations of MPP⁺ produced a concentration-dependent decrease in H₂O₂ production. Incubation of mitochondria with ADP for 60 min after loading with 100 µ M MPP⁺ caused no loss of complex I activity after washing of MPP⁺ from the mitochondrial membranes. These data are consistent with MPP⁺ initially binding specifically to complex I and inhibiting both the flow of reducing equivalents and the production of H₂O₂ by the mitochondrial respiratory chain, without irreversibly damaging complex I. However, mitochondria incubated with H₂O₂ in the presence of Cu²⁺ ions showed decreased complex I activity. This study provides additional evidence that cellular damage initiated by MPP⁺ is due primarily to energy depletion caused by specific binding to complex I, any increased damage due to free radical production by mitochondria being a secondary effect.     

Oxidant Injury in PC12 Cells—A Possible Model of Calcium "Dysregulation" in Aging: II. Interactions with Membrane Lipids

Abstract: In a model recently developed to study the parameters altering vulnerability to oxidative stress, it was shown via image analysis that H₂O₂-exposed PC12 cells exhibited increased levels of intracellular Ca²⁺ (baseline), decreases in K⁺-stimulated Ca²⁺ levels (peak), and decreased poststimulation Ca²⁺ clearance (recovery). The present experiments were performed to determine if the response patterns in these parameters to oxidative stress would be altered after modification of membrane lipid composition induced by incubating the PC12 cells with 660 µ M cholesterol (CHL) in the presence or absence of 500 µ M sphingomyelin (SPH) before low (5 µ M ) or high (300 µ M ) H₂O₂ exposure. Neither CHL nor SPH had synergistic effects with high concentrations of H₂O₂ on baseline. However, CHL in the presence or absence of SPH reversed the effect of low concentrations of H₂O₂ on baseline. SPH decreased significantly the cell's ability to clear excess Ca²⁺ in the presence or absence of H₂O₂ and increased significantly the level of conjugated dienes (CDs). It is surprising that in the cells pretreated with CHL, the CD levels were not significantly different from controls. However, in the presence of SPH, the effects of CHL on CDs were altered. These results suggest that the ratios of membrane lipids could be of critical importance in determining the vulnerability to oxidative stress and Ca²⁺ translocation in membranes. This may be of critical importance in aging where there is increased membrane SPH and significant loss of calcium homeostasis.     

Hydrogen peroxide formation in cultured rose cells in response to UV-C radiation

Suspension-cultured rose ( Rosa damascena Mill. cv. Gloire de Guilan) cells irradiated with UV-C (254 nm. 558 J m⁻²) showed a transient production of H₂O₂ as measured by chemiluminescence of luminol in the presence of peroxidase (EC 1.1 1.1.7). The peak concentration of H₂O₂, which occurred at about 60–90 min after irradiation, was 8–9 μ M . The time course for the appearance of H₂O₂ matched that for UV–induced K⁺ efflux. Treatments that inhibited the UV-induced efflux of K⁺, including heat and overnight incubation with cycloheximide and diethylmaleate, also inhibited the appearance of H₂O₂. The converse was not always true, since catalase (EC 1.11.1.6. and salicylhydroxamic acid, which inhibited luminescence, did not stop K⁺ efflux. We conclude that H₂O₂ synthesis depends on K⁺ efflux. Because H₂.O₂ in the extracellular space is required for lignin synthesis in many plant tissues, we suggest that the UV–stimulated production of H₂O₂ is an integral part of a defensive lignin synthesis.     

Studies on the lactoperoxidase system: reaction kinetics and antibacterial activity using two methods for hydrogen peroxide generation

D.A. DIONYSIUS, P.A. GRIEVE AND A.C. VOS. 1992. Components of the lactoperoxidase system were measured during incubation in Isosensitest broth, with enzymatic (glucose oxidase, GO) or chemical (sodium carbonate peroxyhydrate, SCP) means to generate H₂O₂. When low levels of thiocyanate (SCN^-) were used in the GO system, H₂O₂ was detected and lactoperoxidase (LP) was inactivated when SCN^- was depleted. With 10-fold higher SCN^-, LP remained active and H₂O₂ was not detectable. The oxidation product of the LP reaction, most likely hypothiocyanite, was present in low concentrations. When SCP was used for the immediate generation of H₂O₂ in a system employing low SCN^-, half the LP activity was lost within minutes but thereafter it remained stable. Low concentrations of oxidation product were measured and H₂O₂ was not detected during the course of the experiment. At high SCN^- levels, relatively high concentrations of oxidation product were produced immediately, with H₂O₂ undetectable. The results suggest that the final product of the LP reaction depends on the method of H₂O₂ generation and the relative proportions of the substrates. Antibacterial activity of the two LPS was tested against an enterotoxigenic strain of Escherichia coli. Both systems showed bactericidal activity within 4 h incubation at 37°C.     

Temperature profile of oxygen activation and defense against oxygen toxicity in a psychrophilic member of the Bacteroidaceae

Abstract The temperature profiles have been determined for O₂ reduction by activating substrates for whole cells and cell extracts of the psychrophilic, obligately anaerobic bacterium, strain B6, belonging to the Bacteroidaceae. The profiles were similar whether the cells were grown at 15 or 1°C, and also for cells harvested in the exponential or stationary phase. The H₂O producing pyruvate oxidase displayed in cell-free extracts a considerably higher activity than the H₂O₂ producing NADH and NADPH oxidases at all temperatures in the range 30–1°C, and characteristically makes up a larger proportion of the total O₂ reduction capacity the lower the temperature. It thus seems that the O₂ scavenging property of the pyruvate oxidase, postulated to be utilized in a defense mechanism against the detrimental effects of the H₂O₂ producing pyridine nucleotide oxidases, is particularly well adapted to function at the low temperatures of the Barents Sea, from which this obligately anaerobic organism originates.     

Hydroxyl Radicals Generated In Vivo Kill Neurons in the Rat Spinal Cord: Electrophysiological, Histological, and Neurochemical Results

Abstract: We have used microdialysis to establish an experimental model to characterize mechanisms whereby released substances cause secondary damage in spinal cord injury. We use this model here to characterize damaging effects of the hydroxyl radical (OH') in vivo in the spinal cord. OH'was generatad in vivo by pumping H₂O₂ and FeCI₂/EDTA through parallel microdialysis fibers inserted into the spinal cord. These agents mixed in the tissue to produce OH'by Fenton's reaction. Two types of control experiments were also conducted, one administering only 5 m M H₂O₂ and the other only 0.5 m M FeCI₂/0.82 m M EDTA. During administration of these chemicals, electrical conduction was recorded as one test for deterioration. OH'blocked conduction completely in 2.5-5 h and Fe²⁺/EDTA partly blocked conduction, but H₂O₂ alone did not cause detectable blockage. Histological examination supported the hypothesis that neurons were killed by OH', as Fe²⁺/EDTA and H₂O₂ alone did not destroy significant numbers of neurons. OH', H₂O₂, and Fe²⁺ all caused gradual increases in extracellular amino acid levels. These results are consistent with Fe²⁺-catalyzed free radical generation playing a role in tissue damage upon spinal cord injury.     

Research note : Unsuitability of the MRS medium for the screening of hydrogen peroxide-producing lactic acid bacteria

The effect of MRS broth on the stability of hydrogen peroxide (H₂O₂) has been studied. Known concentrations (1–100 μg ml⁻¹) of H₂O₂ were prepared in distilled water, phosphate buffer (pH 7·0) and MRS broth (pH 6·2 and 3·9). H₂O₂ was very stable in aqueous and buffer solutions but it was rapidly degraded in MRS broth (pH 3·9). The presence of H₂O₂ in MRS broth (pH 6·2) could not be detected.     

Effect of broad-band ultraviolet and visible radiation on hydrogen peroxide formation by cultured rose cells

Broad-band radiation from a high-pressure Hg-vapor lamp, including ultraviolet wavelengths from 290 to 400 nm, blue, green and red wavelengths, did not induce the synthesis of H₂O₂ in cultured rose cells. This was in contrast to the effects of shortwave (254 nm) ultraviolet radiation, even though, like shortwave ultraviolet radiation, the UV-B component of the broadband radiation induced a striking K⁺ efflux from the cells, and this efflux has been associated with H₂O₂ synthesis in a previous report. The UV-A and visible wavelengths were shown to inhibit the synthesis of H₂O₂. This effect was associated with inhibition of peroxidase, an enzyme reported to be involved in the synthesis of H₂O₂ in cell walls. UV-B radiation inhibited the alternate pathway for mitochondrial electron transport, but there was no evidence that this effect contributed to the inhibition of H₂O₂ synthesis in cells treated with broad-band radiation.     

Galacto-oligosaccharide production using a recycling cell culture of Sterigmatomyces elviae CBS8119

Addition of small amounts of Fe²⁺, Zn²⁺, Cu²⁺ and thiamine-HCl to the culture medium was required for promoting the galacto-oligosaccharide (Gal-OS)-producing activity of Sterigmatomyces elviae CBS8119, when the concentration of yeast extract in the medium was lowered to 0·1 g l⁻¹. Galacto-oligosaccharide production using a recycling cell culture was performed in a medium containing 360 mg ml⁻¹ of lactose supplemented with optimal concentrations of Fe²⁺ (1·5 mg l⁻¹ of FeSO₄.7H₂O), Zn²⁺ (15 mg l⁻¹ of ZnSO₄.7H₂O), Cu²⁺ (0·5 mg l⁻¹ of CuSO₄.5H₂O) and thiamine-HCl (1 mg l⁻¹ ) . Galacto-oligosaccharide production was maintained at high levels during six cycles of production, with the amount of Gal-OS produced in each cycle being more than 216 mg ml⁻¹ (weight yield of more than 60%).     

The effect of hydrogen peroxide on spores of Clostridium perfringens

Dithiothreitol (DTT)-treated spores of Clostridium perfringens were much more sensitive to lysis by H₂O₂ in the presence of Cu²⁺ than untreated spores. Lysis was greatly inhibited by hydroxyl radical (.OH) scavengers such as thiourea, dimethylthiourea and dimethylsulfoxide, suggesting that lysis of spores by H₂O₂ involves formation of OH by Cu²⁺-catalysed decomposition of the peroxide. DTT-treated spores took up Cu²⁺ at almost the same rate and extent as did isolated cortical fragments. Hydrogen peroxide caused both the decrease in optical density and the hexosamine solubilization of cortical fragments which bound Cu²⁺.     

The oxidative stress response in Enterococcus faecalis: relationship between H2O2 tolerance and H2O2 stress proteins

The hydrogen peroxide (H₂O₂) stress response in Enterococcus faecalis ATCC19433 was investigated. A 2·4 mmol l⁻¹ H₂O₂ pretreatment conferred protection against a lethal concentration (45 mmol l⁻¹) of this agent. The relatively high concentrations of H₂O₂ used for adaptation and challenge treatments in Ent. faecalis emphasised the strong resistance towards oxidative stress in this species. Various stresses (NaCl, heat, ethanol, acidity and alkalinity) induced weak or strong H₂O₂ cross-protection. This paper describes the involvement of protein synthesis in the active response to lethal dose of H₂O₂, in addition to the impressive enhancement of synthesis of five H₂O₂ stress proteins. Combined results suggest that these proteins might play an important role in the H₂O₂ tolerance response.     

The regulation of Mn2+ and Cu2+ uptake in cells of the yeast Candida utilis grown in continuous culture

Abstract Transport of Mn²⁺ was repressed in Candida utilis cells grown in continuous culture in high-Mn²⁺ (100 μM Mn²⁺) medium as compared to cells grown in basic (0.45 μM Mn²⁺) and low-Mn²⁺ (< 0.05 μM Mn²⁺) media. In contrast, no repression of Cu²⁺ uptake occurred in high-Cu²⁺-grown (25 μM Cu²⁺) cells as compared to cells grown in basic medium (0.54 μM Cu²⁺). Cu²⁺-limited cells did not hyperaccumulate Cu²⁺ and there was not significant difference in initial uptake rates for all 3 Cu²⁺ conditions. Mn²⁺ uptake appears to be regulated by a mechanism sensitive to the external Mn²⁺ concentration, whereas Cu²⁺ transport is not governed in this way by the external Cu²⁺.     

Calcium regulation of exogenous and endogenous 1-aminocylopropane-1-carboxylic acid bioconversion to ethylene

Ethylene production and overall levels of free and conjugated 1-aminocyclopropane-1-carboxylic acid (ACC) were studied in parenchymatous tissues, excised from clmacteric apples ( Malus domestica Borkh. cv. Granny Smith) and infiltrated with an incubation medium containing 0, 1, 10 or 100 m M Ca²⁺, with or without exogenous ACC (2 m M ). Irrespective of whether exogenous ACC was applied or not, ethylene production was inhibited to the same extent (40%) by an apoplastic effect of 100 m M Ca²⁺. In the absence of external ACC, the inhibition was associated with an increase in total endogenous ACC and may be related to a reduction in the rate of the last step of ethylene pathway. This suggests that the ethylene-forming enzyme (EFE) is localized in the plasma membrane. Low Ca²⁺ concentrations (1 m M ) enhanced basal ethylene synthesis due to influx of Ca²⁺ into the cytosol, while overall concentrations of ACC in the tissue decreased. However, 1 m M Ca²⁺ did not stimulate ACC-dependent ethylene formation. Thus, Ca²⁺ influx may stimulate the translocation of endogenous ACC from synthesis or storage compartment (s) to reactive site(s) of the plasma membrane. The concentration of 10 m M Ca²⁺ had no effect on basal ethylene production and appears to represent a balance point between the stimulating and inhibiting effects of 1 and 100 m M Ca²⁺, respectively, Furthermore, the charge-times of exogenous ACC observed with 0, 1 and 10 m M Ca²⁺ suggest that EFE is located on the inner side of the plasma membrane.