External acidification leads to formation of reactive oxygen species in rat brain synaptosomes

Formation of reactive oxygen species in rat brain synaptosomes upon acidification of the incubation medium has been studied using a fluorescent probe DCFDA. It is shown that lowering the pH from 7.4 to 7.0 and 6.0 leads to a progressive increase in fluorescence that is indicative of oxidative stress. The effect is observed regardless of the presence of calcium ions in the incubation medium. In experiments without synaptosomes, acidification of the medium causes quenching of the fluorescence of DCF (pre-oxidized dye). This testifies that the fluorescence rise in synaptosomes is indeed associated with enhanced production of reactive oxygen species. Thus, even a small reduction of external pH to 7.0 is sufficient to cause oxidative stress in brain synaptosomes.     

Glutamate induces formation of free radicals in rat brain synaptosomes

The influence of glutamate and agonists of its ionotropic receptors on free radical formation in rat brain synaptosomes was investigated using the fluorescent dye DCFDA. Glutamate at concentrations of 100 μM and 1 mM increased the production of reactive oxygen species. This phenomenon was eliminated by removing calcium from the incubation medium. Addition of NMDA (100 μM) or kainate (100 μM) to a suspension of synaptosomes also led to free radical formation. The influence of glutamate receptor agonists was blocked by the specific antagonists MK-801 and NBQX. Thus, activation of NMDA and AMPA/kainate receptors can lead to oxidative stress in neuronal presynaptic endings.     

The regulation of cytosolic pH in isolated presynaptic nerve terminals from rat brain

Cytosolic pH (pHi) was measured in presynaptic nerve terminals isolated from rat brain (synaptosomes) using a fluorescent pH indicator, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The synaptosomes were loaded with BCECF by incubation with the membrane-permanent acetoxy-methyl ester derivative of BCECF, which is hydrolyzed by intracellular esterases to the parent compound. pHi was estimated by calibrating the fluorescence signal after permeabilizing the synaptosomal membrane by two different methods. Synaptosomes loaded with 15-90 microM BCECF were estimated to have a pHi of 6.94 +/- 0.02 (mean +/- standard error; n = 54) if the fluorescence signal was calibrated after permeabilizing with digitonin; a similar value was obtained using synaptosomes loaded with 10 times less BCECF (6.9 +/- 0.1; n = 5). When the fluorescence signal was calibrated by permeabilizing the synaptosomal membrane to H+ with gramicidin and nigericin, pHi was estimated to be 7.19 +/- 0.03 (n = 12). With the latter method, pHi = 6.95 +/- 0.09 (n = 14) when the synaptosomes were loaded with 10 times less BCECF. Thus, pHi in synaptosomes was approximately 7.0 and could be more precisely monitored using the digitonin calibration method at higher BCECF concentrations. When synaptosomes were incubated in medium containing 20 mM NH4Cl and then diluted into NH4Cl-free medium, pHi immediately acidified to a level of approximately 6.6. After the acidification, pHi recovered over a period of a few minutes. The buffering capacity of the synaptosomes was estimated to be approximately 50 mM/pH unit. Recovery was substantially slowed by incubation in an Na-free medium, by the addition of amiloride (KI = 3 microM), and by abolition of the Nao/Nai gradient. pHi and its recovery after acidification were not affected by incubation in an HCO3-containing medium; disulfonic stilbene anion transport inhibitors (SITS and DIDS, 1 mM) and replacement of Cl with methylsulfonate did not affect the rate of recovery of pHi. It appears that an Na+/H+ antiporter is the primary regulator of pHi in mammalian brain nerve terminals.     

Effects of peroxide on the fluorescence of the Ca2+ probe Fluo 3 and the pH probe BCECF

Fluorescence probes are invaluable tools in monitoring intracellular ion concentrations. They have also been used for studying how reactive oxygen species alter these concentrations and yet there are no studies indicating how reactive oxygen species directly affect the characteristics of the probes. Our concern was that if reactive oxygen were to affect characteristics of these probes, these measurements would be inconsequential. Therefore, we examined the effects of peroxide on the Ca²⁺-sensitive dye Fluo 3 and the pH sensitive dye BCECF. Peroxide concentrations below 10 mM did not alter fluorescence or binding characteristics of either dye. Since the concentrations of peroxide used in most pathophysiological experiments are in the micromolar range, we conclude that these probes are appropriate for monitoring the effects of peroxide on intracellular ion concentrations.     

Estimation of the mitochondrial calcium pool in rat brain synaptosomes using Rhod-2 AM fluorescent dye

The literature data on the role of synaptic mitochondria in the regulation of the cytosolic calcium level are contradictory. In the present paper calcium storage by mitochondria in rat brain synaptosomes using the fluorescent dye Rhod-2 has been investigated. The addition of 60 mM KCl increases Rhod-2 fluorescence. This effect is completely abolished by replacing K⁺ with Na⁺ or withdrawing Ca²⁺ from the incubation medium. A proton ionophore, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone, and a mixture of rotenone/oligomycin mitochondrial toxins cause a two-fold decrease in Rhod-2 fluorescence. Thapsigargin, an inhibitor of endoplasmic reticulum ATPase (1 μM), but not bafilomycin, an inhibitor of ATPase in synaptic vesicles (500 nM) also leads to a mitochondrial calcium influx. The addition of calcium to synaptosomes with the retained plasma membrane potential increased Rhod-2 fluorescence; however, this effect is insensitive to carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. We have shown that mitochondria can serve as a calcium store in synaptosomes only in the case of a high cytosolic concentration of calcium.     

Effect of the Ca ionophore A-23187 on the plasmatic and mitochondrial potentials of the brain synaptosomes in rats: fluorescence measurements

The applicability of the potential-sensitive dye diS-C3-(5) for the study of A23187 + Ca2+ induced plasma membrane hyperpolarization was tested in rat brain synaptosomes. An appropriate dye synaptosome ratio was chosen for the fluorescence titration dye in Ca-free Krebs-Ringer solution. The fluorescence intensity of the probe was increased upon the addition of Ca2+ (1 microM) to the synaptosomes in the presence of A23187 (1 microM). The effect of Ca2+ + A23187 persisted in a Na+-free medium or when Na+ channels were inhibited by tetrodotoxin as well as in high K+-depolarized synaptosomes (75 microM KCl). In the presence of oligomycin or a protonophore (1 microM) the effect of Ca2+ + A23187 was suppressed. This suggests that the A23187-induced fluorescence increase is due to a depolarization of intrasynaptosomal mitochondria. Therefore, the use of the dye diS-C3-(5) for the study of Ca-induced hyperpolarization does not seem to be feasible unless a quantitative model of changes in fluorescence related to the plasma and mitochondrial membrane potentials is elaborated.     

Calcium permeability of synaptosomes induced by alpha-latrotoxin

The paper deals with characteristics of ionic alpha-latrotoxin-induced permeability of rat brain synaptosomes. It has been shown that the addition of alpha-latrotoxin to synaptosomes in the Ca2+-containing media resulted in an extensive and rapid uptake of 45Ca2+ in synaptosomes. alpha -Latrotoxin was not able to enhance the 22Na+ and 86Rb+ uptake or efflux in the Ca2+-containing and Ca2+- and Mg2+-free media. The dye di-O-C3 was used to monitor the membrane potential changes as a consequence of alpha-latrotoxin treatment of synaptosomes. It has been found that alpha-latrotoxin increased synaptosomal fluorescence in the Ca2+-containing media, but failed to induce any increase of fluorescence in Ca2+- and Mg2+-free media. It has been also shown that the calcium uptake induced by alpha-latrotoxin depends on free calcium concentration in synaptosomes. Toxin-induced calcium flows are shown to be of the vector character.     

The effect of pH on the viability of hypothermically stored rat hepatocytes

Hepatocytes isolated from the rat liver were stored for up to 72 hr at 4 degrees C in a tissue culture medium (Liebovitz-15) at different pH values to determine how pH affects hepatocyte viability. This is a model to simulate cold storage of livers for transplantation and determine the optimal pH for maintenance of liver cell function. The cells were stored in the absence of oxygen. At the end of cold storage the percentage of the total cellular LDH released into the extracellular medium was used as a measure of hepatocyte viability. Also, lactate dehydrogenase (LDH) release was determined in hepatocytes incubated at normothermia (37 degrees C) for 90 min following 72 hr of cold storage. The results demonstrate that hepatocytes tolerate a wide range of pH values in the storage medium and that only about 10% of the total LDH was released from hepatocytes stored up to 72 hr at pH's from 5.0 to 8.0. Normothermic incubation, however, demonstrated that the pH of the storage medium affected viability. After 48 hr of storage only hepatocytes stored at pH values from 7.0 to 8.0 remained viable (LDH release similar to that of freshly incubated hepatocytes = 28 +/- 7.2%). After 72 hr of storage and 90 min of normothermic incubation, hepatocytes incubated at all pH values studied were nonviable (greater than 60% release of LDH). These results suggest that the optimal pH for storage of hepatocytes at 4 degrees C is near neutrality (7.0 to 7.4).     

Mitochondrial dysfunction mediated by quinone oxidation products of dopamine: Implications in dopamine cytotoxicity and pathogenesis of Parkinson's disease

The study has demonstrated that dopamine induces membrane depolarization and a loss of phosphorylation capacity in dose-dependent manner in isolated rat brain mitochondria during extended in vitro incubation and the phenomena are not prevented by oxyradical scavengers or metal chelators. Dopamine effects on brain mitochondria are, however, markedly prevented by reduced glutathione and N-acetyl cysteine and promoted by tyrosinase present in the incubation medium. The results imply that quinone oxidation products of dopamine are involved in mitochondrial damage under this condition. When PC12 cells are exposed to dopamine in varying concentrations (100-400μM) for up to 24h, a pronounced impairment of mitochondrial bio-energetic functions at several levels is observed along with a significant (nearly 40%) loss of cell viability with features of apoptotic nuclear changes and increased activities of caspase 3 and caspase 9 and all these effects of dopamine are remarkably prevented by N-acetyl cysteine. N-acetyl cysteine also blocks nearly completely the dopamine induced increase in reactive oxygen species production and the formation of quinoprotein adducts in mitochondrial fraction within PC12 cells and also the accumulation of quinone products in the culture medium. Clorgyline, an inhibitor of MAO-A, markedly decreases the formation of reactive oxygen species in PC12 cells upon dopamine exposure but has only mild protective actions against quinoprotein adduct formation, mitochondrial dysfunctions, cell death and caspase activation induced by dopamine. The results have indicated that quinone oxidation products and not reactive oxygen species are primarily involved in cytotoxic effects of dopamine and the mitochondrial impairment plays a central role in the latter process. The data have clear implications in the pathogenesis of Parkinson's disease.     

Physiologically relevant concentrations of NaCl and KCl increase DNA photocleavage by an N-substituted 9-aminomethylanthracene dye

This paper describes the synthesis of a new 9-aminomethylanthracene dye N-substituted with a pyridinylpolyamine side chain (4). The effects of NaCl and KCl on anthracene/DNA interactions were then studied, with the goal of simulating the conditions of high ionic strength that a DNA photosensitizer might encounter in the cell nucleus (~150 mM of NaCl and 260 mM of KCl). As exemplified by methylene blue (5), the expected effect of increasing ionic strength is to decrease DNA binding and photocleavage yields. In contrast, the addition of 150 mM of NaCl in combination with 260 mM of KCl to photocleavage reactions containing micromolar concentrations of 4 triggers the conversion of supercoiled, nicked, and linear forms of pUC19 plasmid into a highly degraded band of DNA fragments (350 nm hν, pH 7.0). Circular dichroism spectra point to a correlation between salt-induced unwinding of the DNA helix and the increase in DNA photocleavage yields. The results of circular dichroism, UV-vis absorption, fluorescence emission, thermal denaturation, and photocleavage inhibition experiments suggest that the combination of salts causes a change in the DNA binding mode of 4 from intercalation to an external interaction. This in turn leads to an increase in the anthracene-sensitized production of DNA-damaging reactive oxygen species.     

Y-27632 induces calcium-independent glutamate release in rat brain synaptosomes by a mechanism distinct from exocytosis

The Rho-kinase inhibitor Y-27632 is known to induce nonsecretory exocytosis in PC12 cells; its influence on central synapses has not been studied. We show that in brain synaptosomes, Y-27632 at 100 μM caused [¹⁴C]glutamate release without depolarization of the plasma membrane [the membrane potential was measured with a fluorescent dye DiSC3(5)]. Y-27632 induced an increase in acridine orange fluorescence but did not affect the fluorescence of FM2-10. Probably, the Rho-kinase inhibitor decreases the pH gradient in synaptic vesicles without inducing exocytosis. Dissipation of the gradient leads to leakage of neurotransmitters to the cytosol and their pumping out by plasma membrane transporters. Our results suggest the involvement of the Rho-dependent branch of intracellular signaling in regulation of the pH gradient in synaptic vesicles.     

The role of external and matrix pH in mitochondrial reactive oxygen species generation

Reactive oxygen species (ROS) generation in mitochondria as a side product of electron and proton transport through the inner membrane is important for normal cell operation as well as development of pathology. Matrix and cytosol alkalization stabilizes semiquinone radical, a potential superoxide producer, and we hypothesized that proton deficiency under the excess of electron donors enhances reactive oxygen species generation. We tested this hypothesis by measuring pH dependence of reactive oxygen species released by mitochondria. The experiments were performed in the media with pH varying from 6 to 8 in the presence of complex II substrate succinate or under more physiological conditions with complex I substrates glutamate and malate. Matrix pH was manipulated by inorganic phosphate, nigericine, and low concentrations of uncoupler or valinomycin. We found that high pH strongly increased the rate of free radical generation in all of the conditions studied, even when DeltapH=0 in the presence of nigericin. In the absence of inorganic phosphate, when the matrix was the most alkaline, pH shift in the medium above 7 induced permeability transition accompanied by the decrease of ROS production. ROS production increase induced by the alkalization of medium was observed with intact respiring mitochondria as well as in the presence of complex I inhibitor rotenone, which enhanced reactive oxygen species release. The phenomena revealed in this report are important for understanding mechanisms governing mitochondrial production of reactive oxygen species, in particular that related with uncoupling proteins.     

Influence of pH on sulfhydryl groups and fluidity of the mitochondrial membrane

Fluidity of the red blood cell membrane decreases as pH changes from 8 to 7.5. In rat liver mitochondrial (RLM) membrane fluidity precipitously declines as pH drops from 7.35 toward 7.0. With dithionitrobenzoate (Nbs2), reaction rates of mitochondrial -SH groups from rat liver and heart (RHM) and in beef heart submitochondrial particles are reduced at pH 7.0 as compared to 7.35. Similar results are obtained with the lipophilic fluorescence dye monobromobimane (MB). Bromobimane Q (MQ), which predominantly labels superficially located -SH groups, does not detect differences in -SH reaction rate between pH 7.35 and 7.0. Oligomycin diminishes the amount of reactive -SH groups in RLM titrated with Nbs2 only at pH 7.35, whereas with MB a decrease caused by oligomycin is found at pH 7.35 and pH 7.0. With MQ, an increase in reaction rate is observed for both pH values after pretreatment with oligomycin. Using 4-maleimido-TEMPO mobilization of -SH groups is found with oligomycin at pH 7.0, whereas at pH 7.35 they are immobilized. Phosphate significantly stimulates reaction rates of -SH groups at pH 7.0 in RHM and RLM. In RHM inhibition of succinate oxidation by oxaloacetate as well as the efflux of NAD(P)H is enhanced at pH 7.0, indicating increased permeability in both directions. Decreases in pH, fluidity, and thiol reactivity are important factors in hypoxic/ischemic membrane damage.     

Biodegradation of reactive textile dye Red BLI by an isolated bacterium Pseudomonas sp. SUK1

A novel bacterial strain capable of decolorizing reactive textile dye Red BLI is isolated from the soil sample collected from contaminated sites of textile industry from Solapur, India. The bacterial isolate was identified as Pseudomonas sp. SUK1 on the basis of 16S rDNA analysis. The Pseudomonas sp. SUK1 decolorized Red BLI (50 mg l(-1)) 99.28% within 1h under static anoxic condition at pH range from 6.5 to 7.0 and 30 degrees C. This strain has ability to decolorize various reactive textile dyes. UV-Vis spectroscopy, FTIR and TLC analysis of samples before and after dye decolorization in culture medium confirmed decolorization of Red BLI. A significant increase in the activities of aminopyrine N-demethylase and NADH-DCIP reductase in cells obtained after decolorization indicates involvement of these enzymes in the decolorization process. Phytotoxicity testing with the seeds of Sorghum vulgare and Phaseolus mungo, showed more sensitivity towards the dye, while the products obtained after dye decolorization does not have any inhibitory effects.     

Effect of osmolar concentration of culture media on exocytosis in isolated presynaptic nerve endings of rat brain

The effect of hypotonic and hypertonic shock on exocytosis in rat brain synaptosomes was studied using the fluorescent dye acridine orange. It was shown that an increase in medium osmolarity leads to calcium-independent exocytosis. The response of the probe was directly proportional to the amount of osmolithes added. A decrease in medium osmolarity to 230 mOsm led to an increase of acridine orange fluorescence, which is comparable with exocytosis occurring by the action of 15 mM KCl. This effect was independent of calcium concentration. It is assumed that, under hypotonic shock, part of neurotransmitters are released from the vesicular pool.     

Synaptosomal response to oxidative stress: effect of vinpocetine

It has been suggested that reactive oxygen species (ROS) play a role in the neuronal damage occurring in ischemic injury and neurodegenerative disorders and that their neutralization by antioxidant drugs may delay or minimize neurodegeneration. In the present study we examine whether vinpocetine can act as an antioxidant and prevent the formation of ROS and lipid peroxidation in rat brain synaptosomes. After ascorbate/Fe²⁺ treatment a significant increase in oxygen consumption (about 5-fold) and thiobarbituric acid reactive substances (TBARS) formation (about 7-fold) occurred as compared to control conditions. Vinpocetine inhibited the ascorbate/Fe²⁺ stimulated consumption of oxygen and TBARS accumulation, an indicator of lipid peroxidation, in a concentration-dependent manner. The ROS formation was also prevented by vinpocetine. Oxidative stress increased significantly the fluorescence of the probes 2',7'-dichlorodihydrofluorescein (DCFH₂-DA) (about 6-fold) and dihydrorhodamine (DHR) 123 (about 10-fold), which is indicative of intrasynaptosomal ROS generation. Vinpocetine at 100 μM concentration decreased the fluorescence of DCFH₂-DA and DHR 123 by about 50% and 83%, respectively. We conclude that the antioxidant effect of vinpocetine might contribute to the protective role exerted by the drug in reducing neuronal damage in pathological situations.     

Synaptosomal response to oxidative stress: Effect of vinpocetine

It has been suggested that reactive oxygen species (ROS) play a role in the neuronal damage occurring in ischemic injury and neurodegenerative disorders and that their neutralization by antioxidant drugs may delay or minimize neurodegeneration. In the present study we examine whether vinpocetine can act as an antioxidant and prevent the formation of ROS and lipid peroxidation in rat brain synaptosomes. After ascorbate/Fe²⁺ treatment a significant increase in oxygen consumption (about 5-fold) and thiobarbituric acid reactive substances (TBARS) formation (about 7-fold) occurred as compared to control conditions. Vinpocetine inhibited the ascorbate/Fe²⁺ stimulated consumption of oxygen and TBARS accumulation, an indicator of lipid peroxidation, in a concentration-dependent manner. The ROS formation was also prevented by vinpocetine. Oxidative stress increased significantly the fluorescence of the probes 2′,7′-dichlorodihydrofluorescein (DCFH₂-DA) (about 6-fold) and dihydrorhodamine (DHR) 123 (about 10-fold), which is indicative of intrasynaptosomal ROS generation. Vinpocetine at 100 μM concentration decreased the fluorescence of DCFH₂-DA and DHR 123 by about 50% and 83%, respectively. We conclude that the antioxidant effect of vinpocetine might contribute to the protective role exerted by the drug in reducing neuronal damage in pathological situations.     

Mitochondrial dysfunction mediated by quinone oxidation products of dopamine: Implications in dopamine cytotoxicity and pathogenesis of Parkinson's disease

The study has demonstrated that dopamine induces membrane depolarization and a loss of phosphorylation capacity in dose-dependent manner in isolated rat brain mitochondria during extended in vitro incubation and the phenomena are not prevented by oxyradical scavengers or metal chelators. Dopamine effects on brain mitochondria are, however, markedly prevented by reduced glutathione and N-acetyl cysteine and promoted by tyrosinase present in the incubation medium. The results imply that quinone oxidation products of dopamine are involved in mitochondrial damage under this condition. When PC12 cells are exposed to dopamine in varying concentrations (100-400 μM) for up to 24 h, a pronounced impairment of mitochondrial bio-energetic functions at several levels is observed along with a significant (nearly 40%) loss of cell viability with features of apoptotic nuclear changes and increased activities of caspase 3 and caspase 9 and all these effects of dopamine are remarkably prevented by N-acetyl cysteine. N-acetyl cysteine also blocks nearly completely the dopamine induced increase in reactive oxygen species production and the formation of quinoprotein adducts in mitochondrial fraction within PC12 cells and also the accumulation of quinone products in the culture medium. Clorgyline, an inhibitor of MAO-A, markedly decreases the formation of reactive oxygen species in PC12 cells upon dopamine exposure but has only mild protective actions against quinoprotein adduct formation, mitochondrial dysfunctions, cell death and caspase activation induced by dopamine. The results have indicated that quinone oxidation products and not reactive oxygen species are primarily involved in cytotoxic effects of dopamine and the mitochondrial impairment plays a central role in the latter process. The data have clear implications in the pathogenesis of Parkinson's disease.     

Hypotonic swelling activates the Na*/H* exchange in rat brain synaptosomes

The influence of hypotonic swelling and hypertonic shrinking on cytosolic pH in synaptosomes was investigated. It was shown that decreasing the osmolarity of incubation medium to 230 mOsm leads to alkalization and increasing the osmolarity of incubation medium to 810 mOsm leads to acidification. Alkalization was inhibited by amiloride, indicating the involvement of the Na+/H+ exchanger. The acidification of cytosol upon hypertonic shrinking was insensitive, to amiloride and the inhibitor of Na+, K+, Cl- cotransport bumetanide. Thus, the Na+/H+ exchange in synaptosomes is activated by hypotonic swelling but not hypertonic shrinking, in contrast with erythrocytes and lymphocytes, which have been investigated earlier.     

Enrichment of Escherichia coli proteins by column chromatography on reactive dye columns

The reliable identification and analysis of the low abundance proteins expressed by a cell remains a key challenge in the study of cellular proteomes. The analysis of low abundance proteins is a particular problem when using two-dimensional gel electrophoresis (2-DE) to resolve the cellular proteins since the technology is unable to display the wide dynamic range of protein levels typically synthesized by cells. We have investigated the use of reactive dye compounds for the enrichment of low abundance cellular proteins prior to analysis by 2-DE. The capacity of reactive dye compounds to bind specific protein species was used as the basis for a general chromatographic tool for protein enrichment. Six reactive dye compounds were investigated in detail for the analysis of Escherichia coli proteins. Whole bacterial cell lysates were passed down columns prepared with the reactive dye compounds. The bound proteins were eluted with 1.5 M NaCl and analyzed by 2-DE. Distinctive protein profiles were observed for the bound proteins recovered from the different reactive dye compounds. Selected proteins enriched by these methods were identified by peptide mass mapping. The enrichment procedure developed using reactive dye compounds were used to investigate acid-induced changes in the proteome of E. coli grown at either pH 7.0 or pH 5.8. Increased levels of expression were observed for a number of proteins (for example, GdhA, PanC, ProC, TkrA, EF-TS and YodA) were observed for E. coli grown at pH 5.8. Five identified proteins (AroG, FabI, GlyA, PurA and EF-Tu) showed reduced levels of synthesis for bacteria grown at pH 5.8 compared to pH 7.0. In the case of PanC and FabI the altered expression profiles were only reliably demonstrated using the enrichment protocols. One theme emerging from these data was that the expression of proteins concerned with one-carbon metabolism was perturbed at pH 5.8, which may point to a previously unrecognized affect of low pH stress on the physiology of E. coli cells. We conclude that the prefractionation of cell lysates on reactive dye columns will serve as a valuable generic tool for the analysis of low abundance proteins expressed by both prokaryotic and eukaryotic cells.