1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine- and 1-Methyl-4-(2''-Ethylphenyl)-1,2,3,6-Tetrahydropyridine-Induced Toxicity in PC 12 Cells: Role of Monoamine Oxidase A

The toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-(2'-ethylphenyl)-1,2,3,6-tetrahydropyridine (2'Et-MPTP), and their corresponding pyridinium species was studied in the rat pheochromocytoma PC12 cell line. MPTP and its analogues are known to be metabolized by monoamine oxidase (MAO) to dihydropyridinium intermediates which are further transformed, either enzymatically or spontaneously, into pyridinium species. MAO activity in PC12 cells is almost exclusively of the A form, and 2'Et-MPTP is a good substrate for both MAO-A and MAO-B. In contrast, MPTP is a poor substrate for MAO-A, but a good substrate for MAO-B. 2'Et-MPTP caused considerably more cell death than MPTP in the PC12 cells. However, 1-methyl-4-(2'-ethylphenyl)pyridinium and 1-methyl-4-phenylpyridinium, the corresponding pyridinium species formed from 2'Et-MPTP and MPTP, respectively, were equipotent as toxins. The toxic effects of the tetrahydropyridines and their corresponding pyridiniums were both concentration- and time-dependent. Measurements of the levels of the pyridinium species formed and the remaining tetrahydropyridine in the media indicated that 2'Et-MPTP was converted about five to seven times more readily into its toxic pyridinium species than was MPTP. There was, moreover, an excellent correlation between amount of pyridinium formed and cell death. There was also a parallel between the capacity of clorgyline and pargyline, irreversible MAO inhibitors, to decrease the formation of the pyridinium species and their capacity to protect against the toxic actions of the tetrahydropyridines. These data are consistent with the concept that the MAO-A-dependent formation of the pyridinium species from the tetrahydropyridine is a prerequisite for toxicity in PC12 cells.     

Potentiation by the Tetraphenylboron Anion of the Effects of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine and Its Pyridinium Metabolite

The 1-methyl-4-phenylpyridinium species (MPP+) is the four-electron oxidation product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and is widely assumed to be the actual neurotoxic species responsible for the MPTP-induced destruction of dopaminergic neurons. MPTP is oxidized by the enzyme monoamine oxidase-B to a dihydropyridinium intermediate which is oxidized further to MPP+, an effective inhibitor of the oxidation of the Complex I substrates glutamate/malate in isolated mitochondrial preparations. In the present study, the tetraphenylboron anion (TPB) greatly potentiated the inhibitory effects of MPP+ and other selected pyridinium species on glutamate/malate respiration in isolated mouse liver mitochondria. At 10 microM TPB, the potentiation ranged from approximately 50-fold to greater than 1,000-fold for the several pyridinium species tested. In other experiments, TPB greatly enhanced the accumulation of [3H]MPP+ by isolated mitochondrial preparations. This facilitation by TPB of MPP+ accumulation into mitochondria explains, at least in part, the potentiation by TPB of the above-mentioned inhibition of mitochondrial respiration. Moreover, TPB addition increased the amount of lactate formed during the incubation of mouse neostriatal tissue slices with MPTP and other tetrahydropyridines. The administration of TPB also potentiated the dopaminergic neurotoxicity of MPTP in male Swiss-Webster mice. All of these observations, taken together, are consistent with the premise that the inhibitory effect of MPP+ on mitochondrial respiration within dopaminergic neurons is the ultimate mechanism to explain MPTP-induced neurotoxicity.     

l-DOPA Cytotoxicity to PC12 Cells in Culture Is via Its Autoxidation

Abstract: The mechanism of cytotoxicity of l -DOPA was studied in the rat pheochromocytoma PC12 cell line. The cytotoxicity of l -DOPA to PC12 cells was time and concentration dependent. Carbidopa, which inhibited the conversion of l -DOPA to dopamine, did not protect against l -DOPA cytotoxicity in PC12 cells. Furthermore, clorgyline, a selective inhibitor of monoamine oxidase type A, and pargyline, an inhibitor of both monoamine oxidase types A and B, both did not have an effect on l -DOPA toxicity. These findings suggest that cytotoxicity was not due to dopamine formed from l -DOPA. Catalase or superoxide dismutase each partially protected against l -DOPA toxicity in PC12 cells. In combination, the effects were synergistic and provided almost total protection against cytotoxicity. 6-Cyano-7-nitroquinoxaline-2,3-dione, an antagonist of non-NMDA receptors, did not protect against l -DOPA toxicity. These data suggest that toxicity of l -DOPA is most likely due to the action of free radicals formed as a result of its autoxidation. Furthermore, these findings suggest that patients on long-term l -DOPA therapy are potentially at risk from the toxic intermediates formed as a result of its autoxidation.     

Evidence that the Loss of the Voltage-Dependent Mg2+ Block at the N-Methyl-D-Aspartate Receptor Underlies Receptor Activation During Inhibition of Neuronal Metabolism

Both phosphointermediate- and vacuolar-type (P- and V-type, respectively) ATPase activities found in cholinergic synaptic vesicles isolated from electric organ are immunoprecipitated by a monoclonal antibody to the SV2 epitope characteristic of synaptic vesicles. The two activities can be distinguished by assay in the absence and presence of vanadate, an inhibitor of the P-type ATPase. Each ATPase has two overlapping activity maxima between pH 5.5 and 9.5 and is inhibited by fluoride and fluorescein isothiocyanate. The P-type ATPase hydrolyzes ATP and dATP best among common nucleotides, and activity is supported well by Mg2+, Mn2+, or Co2+ but not by Ca2+, Cd2+, or Zn2+. It is stimulated by hyposmotic lysis, detergent solubilization, and some mitochondrial uncouplers. Kinetic analysis revealed two Michaelis constants for MgATP of 28 microM and 3.1 mM, and the native enzyme is proposed to be a dimer of 110-kDa subunits. The V-type ATPase hydrolyzes all common nucleoside triphosphates, and Mg2+, Ca2+, Cd2+, Mn2+, and Zn2+ all support activity effectively. Active transport of acetylcholine (ACh) also is supported by various nucleoside triphosphates in the presence of Ca2+ or Mg2+, and the Km for MgATP is 170 microM. The V-type ATPase is stimulated by mitochondrial uncouplers, but only at concentrations significantly above those required to inhibit ACh active uptake. Kinetic analysis of the V-type ATPase revealed two Michaelis constants for MgATP of approximately 26 microM and 2.0 mM. The V-type ATPase and ACh active transport were inhibited by 84 and 160 pmol of bafilomycin A1/mg of vesicle protein, respectively, from which it is estimated that only one or two V-type ATPase proton pumps are present per synaptic vesicle. The presence of presumably contaminating Na+,K(+)-ATPase in the synaptic vesicle preparation is demonstrated.     

Alteration in Neuronal-Glial Metabolism of Glutamate by the Neurotoxin Kainic Acid

The effect of the excitotoxin kainic acid on glutamate and glutamine metabolism was studied in cerebellar slices incubated with D-[2-14C]glucose, [U-14C]gamma-aminobutyric acid, [3H]acetate, [U-14C]glutamate, and [U-14C]glutamine as precursors. Kainic acid (1 mM) strongly inhibited the labeling of glutamine relative to that of glutamate from all precursors except [2-14C]glucose and [U-14C]glutamine. Kainic acid did not inhibit glutamine synthetase directly. The data indicate that in the cerebellum kainic acid inhibits the synthesis of glutamine from the small pool of glutamate that is thought to be associated with glial cells. Kainic acid also markedly stimulated the efflux of glutamate from cerebellar slices and this release was not sensitive to tetrodotoxin. Kainic acid stimulated efflux of both glucose- and acetate-labeled glutamate. In contrast, veratridine released glucose-labeled glutamate preferentially via a tetrodotoxin-sensitive mechanism. Kainic acid did not release [U-14C]glutamate from synaptosomal fractions. These results suggest that the bulk of the glutamate released from cerebellar slices by kainic acid comes from nonsynaptic pools.     

Induction of Glutamine Synthetase by Dibutyryl Cyclic AMP in C-6 Glioma Cells

Glutamine synthetase was found to be increased in C-6 glioma cells as a result of increasing culture passage and N-6,2'-O-dibutyryl cyclic AMP (dbcAMP) treatment. At low passage dbcAMP produced a 2.5-fold increase in glutamine synthetase activity per unit of cellular protein. At high passage control glutamine synthetase was approximately double that seen at low passage, but dbcAMP produced an additional 65% increase. Lactate dehydrogenase activity was also increased by dbcAMP treatment at both low and high passage, but culture passage produced no change in the lactate dehydrogenase. With increasing culture passage, the ratio of cellular protein to DNA doubled. Therefore, expression of data per unit of protein tended to minimize the apparent changes in activity. The maximum increase in glutamine synthetase activity produced by both dbcAMP and increasing culture passage and expressed on a DNA basis was 5.6-fold. The increase in glutamine synthetase activity was generally linear during the first 20 h of drug treatment, after which enzyme activity remained nearly constant up to 72 h. Ninety percent or more of the dbcAMP remained in the medium at the end of 48-h exposure of cells to dbcAMP. 8-br-Cyclic AMP also increased glutamine synthetase activity of C-6-cels, but n-butyrate did not. Isoproterenol, which increases cyclic AMP in C-6-cells, increased glutamine synthetase activity. The effect of isoproterenol on glutamine synthetase was inhibited by the beta-adrenergic blocking agent sotalol. Cycloheximide (10 micrograms/ml) inhibited the dbcAMP effect on glutamine synthetase activity and also decreased the control enzyme activity by 60%.     

Particle-Induced Pulmonary Acute Phase Response Correlates with Neutrophil Influx Linking Inhaled Particles and Cardiovascular Risk

Background

Particulate air pollution is associated with cardiovascular disease. Acute phase response is causally linked to cardiovascular disease. Here, we propose that particle-induced pulmonary acute phase response provides an underlying mechanism for particle-induced cardiovascular risk.

Methods

We analysed the mRNA expression of Serum Amyloid A (Saa3) in lung tissue from female C57BL/6J mice exposed to different particles including nanomaterials (carbon black and titanium dioxide nanoparticles, multi- and single walled carbon nanotubes), diesel exhaust particles and airborne dust collected at a biofuel plant. Mice were exposed to single or multiple doses of particles by inhalation or intratracheal instillation and pulmonary mRNA expression of Saa3 was determined at different time points of up to 4 weeks after exposure. Also hepatic mRNA expression of Saa3, SAA3 protein levels in broncheoalveolar lavage fluid and in plasma and high density lipoprotein levels in plasma were determined in mice exposed to multiwalled carbon nanotubes.

Results

Pulmonary exposure to particles strongly increased Saa3 mRNA levels in lung tissue and elevated SAA3 protein levels in broncheoalveolar lavage fluid and plasma, whereas hepatic Saa3 levels were much less affected. Pulmonary Saa3 expression correlated with the number of neutrophils in BAL across different dosing regimens, doses and time points.

Conclusions

Pulmonary acute phase response may constitute a direct link between particle inhalation and risk of cardiovascular disease. We propose that the particle-induced pulmonary acute phase response may predict risk for cardiovascular disease.     

Large-Scale Production of Nanographite by Tube-Shear Exfoliation in Water

The number of applications based on graphene, few-layer graphene, and nanographite is rapidly increasing. A large-scale process for production of these materials is critically needed to achieve cost-effective commercial products. Here, we present a novel process to mechanically exfoliate industrial quantities of nanographite from graphite in an aqueous environment with low energy consumption and at controlled shear conditions. This process, based on hydrodynamic tube shearing, produced nanometer-thick and micrometer-wide flakes of nanographite with a production rate exceeding 500 gh^-1 with an energy consumption about 10 Whg^-1. In addition, to facilitate large-area coating, we show that the nanographite can be mixed with nanofibrillated cellulose in the process to form highly conductive, robust and environmentally friendly composites. This composite has a sheet resistance below 1.75 Ω/sq and an electrical resistivity of 1.39×10^-4 Ωm and may find use in several applications, from supercapacitors and batteries to printed electronics and solar cells. A batch of 100 liter was processed in less than 4 hours. The design of the process allow scaling to even larger volumes and the low energy consumption indicates a low-cost process.