Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 130
Filter
1.
J Biol Chem ; 291(44): 22970-22976, 2016 10 28.
Article in English | MEDLINE | ID: mdl-27624933

ABSTRACT

When treated with nerve growth factor, PC12 cells will differentiate over the course of several days. Here, we have followed changes during differentiation in the cellular levels of phosphoinositide-specific phospholipase Cß (PLCß) and its activator, Gαq, which together mediate Ca2+ release. We also followed changes in the level of the novel PLCß binding partner TRAX (translin-associated factor X), which promotes RNA-induced gene silencing. We find that the level of PLCß increases 4-fold within 24 h, whereas Gαq increases only 1.4-fold, and this increase occurs ∼24 h later than PLCß. Alternately, the level of TRAX remains constant over the 72 h tested. When PLCß1 or TRAX is down-regulated, differentiation does not occur. The impact of PLCß on differentiation appears independent of Gαq as down-regulating Gαq at constant PLCß does not affect differentiation. Förster resonance energy transfer studies after PLCß association with its partners indicate that PLCß induced soon after nerve growth factor treatment associates with TRAX rather than Gαq Functional measurements of Ca2+ signals to assess the activity of PLCß-Gαq complexes and measurements of the reversal of siRNA(GAPDH) to assess the activity of PLCß-TRAX complexes additionally suggest that the newly synthesized PLCß associates with TRAX to impact RNA-induced silencing. Taken together, our studies show that PLCß, through its ability to bind TRAX and reverse RNA silencing of specific genes, plays a key role in switching PC12 cells to their differentiated state.


Subject(s)
Cell Differentiation , PC12 Cells/enzymology , Phospholipase C beta/metabolism , Animals , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , GTP-Binding Protein alpha Subunits, Gq-G11/genetics , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , PC12 Cells/cytology , Phospholipase C beta/genetics , Protein Binding , Rats
2.
Am J Chin Med ; 41(6): 1377-92, 2013.
Article in English | MEDLINE | ID: mdl-24228607

ABSTRACT

The natural product Laetispicine ( N -isobutyl-(3,4-methylendioxyphenyl)-2E, 4E, 9E-undecatrienoamide), was isolated from the Piper laetispicum C. DC and screened, for its antidepressant activity and antinociceptive effects. Structure-functional activities of five natural products indicated that biological activity is dependent on double bonds present within the benzene ring and a conjugated double bond located at positions 2-3 and 4-5 in the molecular structure. To further understand the structural-activity relationship of Laetispicine as a new potent and safe antidepressant, the structural-activity relationship of 39 analogs of Laetispicine were synthetized and tested in forced swimming tests in mice whilst also in protective effects against glutamate or H 2 O 2 induced apoptosis in PC12 cells. The results show that the compound 30 - N -isobutyl-11-(4-chlorophenyl) undeca-2E,4E,9E-trienamide exhibited the same activity as the parental compound Laetispicine, and furthermore, the effective dose of this compound is lower than Laetispicine. Therefore, the compound 30 might be a potentially useful therapy in the treatment of depression. For structure, the conjugated double bonds located at 2-3, 4-5 and isolated double bonds from benzene ring are necessary for the antidepressant activities no matter the different length of carbon chain; the isobutyl connected with acylamino also are necessary; and the benzodioxole moiety is replaceable, the halogen atom in phenyl ring at the para-position could enhance this kind of activity.


Subject(s)
Antidepressive Agents , Apoptosis/drug effects , Benzodioxoles/chemistry , Benzodioxoles/pharmacology , Depression/drug therapy , PC12 Cells/pathology , Phytotherapy , Piper , Amides/pharmacology , Amides/therapeutic use , Animals , Benzodioxoles/chemical synthesis , Benzodioxoles/therapeutic use , Cell Survival/drug effects , Cells, Cultured , Depression/psychology , Fatty Acids, Unsaturated/pharmacology , Fatty Acids, Unsaturated/therapeutic use , L-Lactate Dehydrogenase/metabolism , Mice , Molecular Structure , PC12 Cells/enzymology , Rats , Stress, Psychological/drug therapy , Structure-Activity Relationship , Swimming/psychology
3.
Cell Signal ; 25(12): 2634-45, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24012499

ABSTRACT

Western blot analysis demonstrated that PC-12 cells express monomeric and dimeric forms of serine racemase (m-SR, d-SR) and that 1321N1 cells express m-SR. Quantitative RT-PCR and functional studies demonstrated that PC-12 cells express homomeric and heteromeric forms of nicotinic acetylcholine receptors (nAChR) while 1321N1 cells primarily express the α7-nAChR subtype. The effect of nAChR agonists and antagonists on SR activity and expression was examined by following concentration-dependent changes in intracellular d-Ser levels and SR protein expression. Incubation with (S)-nicotine increased d-Ser levels, which were attenuated by the α7-nAChR antagonist methyllycaconitine (MLA). Treatment of PC-12 cells with mecamylamine (MEC) produced a bimodal reduction of d-Ser reflecting MEC inhibition of homomeric and heteromeric nAChRs, while a unimodal curve was observed with 1321N1 cells, reflecting predominant expression of α7-nAChR. The nAChR subtype selectivity was probed using α7-nAChR selective inhibitors MLA and (R,S)-dehydronorketamine and α3ß4-nAChR specific inhibitor AT-1001. The compounds reduced d-Ser in PC-12 cells, but only MLA and (R,S)-dehydronorketamine were effective in 1321N1 cells. Incubation of PC-12 and 1321N1 cells with (S)-nicotine, MEC and AT-1001 did not affect m-SR or d-SR expression, while MLA and (R,S)-dehydronorketamine increased m-SR expression but not SR mRNA levels. Treatment with cycloheximide indicated that increased m-SR was due to de novo protein synthesis associated with phospho-active forms of ERK1/2, MARCKS, Akt and rapamycin-sensitive mTOR. This effect was attenuated by treatment with the pharmacological inhibitors U0126, LY294002 and rapamycin, which selectively block the activation of ERK1/2, Akt and mTOR, respectively, and siRNAs directed against ERK1/2, Akt and mTOR. We propose that nAChR-associated changes in Ca(2+) flux affect SR activity, but not expression, and that MLA and (R,S)-dehydronorketamine bind to allosteric sites on the α7-nAChR and promote multiple signaling cascades that converge at mTOR to increase m-SR levels.


Subject(s)
Aconitine/analogs & derivatives , Mecamylamine/pharmacology , Nicotinic Antagonists/pharmacology , PC12 Cells/drug effects , Racemases and Epimerases/genetics , Racemases and Epimerases/metabolism , Receptors, Nicotinic/metabolism , Aconitine/pharmacology , Animals , Cell Line , Cell Line, Tumor , Gene Expression Regulation/drug effects , Humans , Nicotine/metabolism , PC12 Cells/enzymology , PC12 Cells/metabolism , Rats , Receptors, Nicotinic/genetics , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/metabolism
4.
J Neurosci Res ; 91(2): 273-84, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23161662

ABSTRACT

During the neurodegenerative process in several brain diseases, oxidative stress is known to play important roles in disease severity and evolution. Although early events of stress, such as increased lipid peroxidation and decreased superoxide dismutase, are known to characterize early onsets of these diseases, little is known about the events that participate in maintaining the chronic evolving phase influencing the disease progression in neurons. Here, we used differentiated PC12 cells to identify premitochondrial and postmitochondrial events occurring during the oxidative stress cascade leading to apoptosis. Our data indicate that an acute and strong oxidative impulse (500 µM H(2)O(2), 30 min) can induce, in this model, a 24-hr self-evolving stress, which advances from a premitochondrial phase characterized by lysosomes and cathepsin B and D translocations to cytosol and early mitochondrial membrane hyperpolarization. This phase lasts for about 5 hr and is followed by a postmitochondrial phase distinguished by mitochondrial membrane depolarization, reactive oxygen species increase, caspase-9 and caspase-3 activations, and apoptosis. Inhibition of cathepsins B and D suggests that cells can be protected at the premitochondrial phase of stress evolution and that new cathepsins regulators, such as glycosaminoglycans mimetics, can be considered as new therapeutic prototypes for neurodegeneration. Insofar as early oxidative stress markers have been related to the early onset of neurodegeneration, strategies protecting cells at the premitochondrial phase of oxidative stress may have important therapeutic applications.


Subject(s)
Mitochondria/metabolism , Oxidative Stress/physiology , Reactive Oxygen Species/metabolism , Aconitate Hydratase/metabolism , Animals , Apoptosis/drug effects , Caspase 3/metabolism , Caspase 9/metabolism , Cathepsin D/metabolism , Cathepsin E/metabolism , Cell Survival/drug effects , Enzyme Inhibitors/pharmacology , Hydrogen Peroxide/pharmacology , Lipid Peroxidation/drug effects , Lysosomes/drug effects , Lysosomes/metabolism , Malondialdehyde/metabolism , Mitochondria/drug effects , Oxidation-Reduction/drug effects , Oxidative Stress/drug effects , PC12 Cells/drug effects , PC12 Cells/enzymology , Rats , Superoxide Dismutase/metabolism , Time Factors
5.
J Neural Transm (Vienna) ; 119(11): 1327-42, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22392058

ABSTRACT

Aripiprazole is the only atypical antipsychotic drug known to cause the phosphorylation of AMP-activated protein kinase (AMPK) in PC12 cells. However, the molecular mechanisms underlying this phosphorylation in aripiprazole-treated PC12 cells have not yet been clarified. Here, using PC12 cells, we show that these cells incubated for 24 h with aripiprazole at 50 µM and 25 mM glucose underwent a decrease in their NAD⁺/NADH ratio. Aripiprazole suppressed cytochrome c oxidase (COX) activity but enhanced the activities of pyruvate dehydrogenase (PDH), citrate synthase and Complex I. The changes in enzyme activities coincided well with those in NADH, NAD⁺, and NAD⁺/NADH ratio. However, the bioenergetic peril judged by the lowered COX activity might not be accompanied by excessive occurrence of apoptotic cell death in aripiprazole-treated cells, because the mitochondrial membrane potential was not decreased, but rather increased. On the other hand, when PC12 cells were incubated for 24 h with clozapine at 50 µM and 25 mM glucose, the NAD⁺/NADH ratio did not change. Also, the COX activity was decreased; and the PDH activity was enhanced. These results suggest that aripiprazole-treated PC12 cells responded to the bioenergetic peril more effectively than the clozapine-treated ones to return the ATP biosynthesis back toward its ordinary level. This finding might be related to the fact that aripiprazole alone causes phosphorylation of AMPK in PC12 cells.


Subject(s)
Antipsychotic Agents/pharmacology , Carbon/metabolism , Clozapine/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Glycolysis/drug effects , Piperazines/pharmacology , Quinolones/pharmacology , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Animals , Apoptosis/drug effects , Aripiprazole , Cell Survival/drug effects , Dihydrolipoamide Dehydrogenase/genetics , Dihydrolipoamide Dehydrogenase/metabolism , Dose-Response Relationship, Drug , Electron Transport Complex IV/metabolism , Extracellular Fluid/drug effects , Glucose/pharmacology , Hypoxanthine Phosphoribosyltransferase/genetics , Hypoxanthine Phosphoribosyltransferase/metabolism , Isocitrate Dehydrogenase/genetics , Isocitrate Dehydrogenase/metabolism , Ketone Oxidoreductases/genetics , Ketone Oxidoreductases/metabolism , Lactic Acid/metabolism , Membrane Potential, Mitochondrial/drug effects , NAD/metabolism , Oxidoreductases/genetics , Oxidoreductases/metabolism , PC12 Cells/drug effects , PC12 Cells/enzymology , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Pyruvate Dehydrogenase Acetyl-Transferring Kinase , Pyruvic Acid/metabolism , RNA, Messenger/metabolism , Rats , Time Factors
6.
BMB Rep ; 44(5): 312-6, 2011 May.
Article in English | MEDLINE | ID: mdl-21615985

ABSTRACT

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its toxic metabolite 1-methyl-4-phenylpyridium ion (MPP(+)) have been shown to induce Parkinson's disease-like symptoms as well as neurotoxicity in humans and animal species. Recently, we reported that maintenance of redox balance and cellular defense against oxidative damage are primary functions of the novel antioxidant enzyme cytosolic NADP(+) -dependent isocitrate dehydrogenase (IDPc). In this study, we examined the role of IDPc in cellular defense against MPP(+) -induced oxidative injury using PC12 cells transfected with IDPc small interfering RNA (siRNA). Our results demonstrate that MPP(+) -mediated disruption of cellular redox status, oxidative damage to cells, and apoptotic cell death were significantly enhanced by knockdown of IDPc.


Subject(s)
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/pharmacology , Cytosol/enzymology , Isocitrate Dehydrogenase/metabolism , NADP/metabolism , Oxidative Stress , PC12 Cells/drug effects , PC12 Cells/enzymology , Animals , Apoptosis/drug effects , Gene Knockdown Techniques , Isocitrate Dehydrogenase/genetics , Oxidation-Reduction , PC12 Cells/cytology , RNA, Small Interfering/metabolism , Rats
7.
Acta Biochim Pol ; 57(4): 589-96, 2010.
Article in English | MEDLINE | ID: mdl-21165344

ABSTRACT

Microsomal glutathione transferase 1 (MGST1) possesses glutathione transferase and peroxidase activities and is active in biotransformation of xenobiotics and in defense against oxidative stress. To assess MGST1 role in the development and functioning of PC12 cells, we constructed a cell line with reduced MGST1 (PC12_M). Real-time PCR and immunoblot assays showed MGST1 expression lowered to 60 % and immunocytochemical analyses demonstrated an altered concentration and distribution of the enzyme. PC12_M cells revealed a larger tendency to grow in clusters, weaker adhesion, irregular shape of bodies, short neurite outgrowth and higher percentage of necrotic cells (34 %). The total GSTs activity determined with non-specific substrate CDNB (1-chloro-2,4-dinitrobenzene) decreased by 15-20 %, whereas that with DCNB (2,4-dichloro-1-nitrobenzene), a substrate more specific for cytosolic GSTs, was similar to the one in control cells. This suggests that reduction of MGST1 cannot be compensated by other glutathione transferases. In PC12_M cells the total glutathione content was higher by 15-20 %, whereas the GSSG/GSH ratio was lower than in control cells. Moreover, the laminin-dependent migration rate was much faster in control cells than in PC12_M, suggesting some alterations in the metastatic potential of the line with suppressed MGST1. The amount of MAP kinases (p38, JNK, ERK1/2) was elevated in PC12_M cells but their phosphorylation level declined. Microarray analysis showed changed expression of several genes, which may be linked with differentiation and necrosis of PC12_M cells. Our data suggest that MGST1 could be an important regulator of PC12 cells development and might have significant effects on cell growth and proliferation, probably through altered expression of genes with different biological function.


Subject(s)
Glutathione Transferase/deficiency , Glutathione Transferase/metabolism , PC12 Cells/enzymology , Animals , Down-Regulation , Glutathione Transferase/genetics , RNA, Messenger/metabolism , Rats
8.
Neurotox Res ; 16(3): 186-93, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19526285

ABSTRACT

We previously demonstrated that spare respiratory capacity of the TCA cycle enzyme alpha-ketoglutarate dehydrogenase (KGDH) was completely abolished upon increasing levels of MAO-B activity in a dopaminergic cell model system (Kumar et al., J Biol Chem 278:46432-46439, 2003). MAO-B mediated increases in H(2)O(2) also appeared to result in direct oxidative inhibition of both mitochondrial complex I and aconitase. In order to elucidate the contribution that each of these components exerts over metabolic respiratory control as well as the impact of MAO-B elevation on their spare respiratory capacities, we performed metabolic respiratory control analysis. In addition to KGDH, we assessed the activities and substrate-mediated respiration of complex I, pyruvate dehydrogenase (PDH), succinate dehydrogenase (SDH), and mitochondrial aconitase in the absence and presence of complex-specific inhibitors in specific and mixed substrate conditions in mitochondria from our MAO-B elevated cells versus controls. Data from this study indicates that Complex I and KGDH are the most sensitive to inhibition by MAO-B mediated H(2)O(2) generation, and could be instrumental in determining the fate of mitochondrial metabolism in this cellular PD model system.


Subject(s)
Gene Expression Regulation, Enzymologic/physiology , Mitochondria/enzymology , Monoamine Oxidase/metabolism , Aconitate Hydratase/metabolism , Animals , Cell Respiration/drug effects , Cell Respiration/physiology , Doxycycline/pharmacology , Electron Transport Complex I/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Hydrogen Peroxide/pharmacology , Insecticides/pharmacology , Ketoglutarate Dehydrogenase Complex/metabolism , Ketone Oxidoreductases/metabolism , Mitochondria/drug effects , NADH, NADPH Oxidoreductases/metabolism , Oxidants/pharmacology , Oxygen Consumption/drug effects , Oxygen Consumption/physiology , PC12 Cells/drug effects , PC12 Cells/enzymology , Quinone Reductases , Rats , Rotenone/pharmacology , Succinate Dehydrogenase/metabolism
9.
Neuroscience ; 158(4): 1436-45, 2009 Feb 18.
Article in English | MEDLINE | ID: mdl-19095046

ABSTRACT

Intermittent hypoxia (IH) is a major pathological factor in the development of neural deficits associated with sleep-disordered breathing. Here we demonstrate that IH lasting 2 or 30 days, but not sustained hypoxia (SH) of the same duration, was accompanied by several posttranslational modifications of the large subunit of RNA polymerase II, Rpb1, including hydroxylation of proline 1465, phosphorylation of serine 5 residues within the C-terminal domain, and nondegradative ubiquitylation. These modifications were found to occur in two regions of the brain, hippocampal region CA1 and the prefrontal cortex, but not in neocortex, brainstem and CA3 region of hippocampus. We also found that mice exposed to 14 or 30 days of IH, but not SH, demonstrated cognitive deficits in behavioral assays. Furthermore, by using the pheochromocytoma-derived PC12 cell line, we showed that, under in vitro IH conditions, induction of Rpb1 hydroxylation, phosphorylation, and ubiquitylation required that the von Hippel-Lindau protein be present. We hypothesize that the observed modifications of Rpb1 participate in regulating the expression of genes involved in mediating cognitive deficits evoked by chronic IH.


Subject(s)
Gene Expression Regulation/physiology , Hippocampus/enzymology , Hypoxia/pathology , Prefrontal Cortex/enzymology , RNA Polymerase II/metabolism , Animals , Carrier Proteins/metabolism , Cullin Proteins/metabolism , Hypoxia/physiopathology , Learning Disabilities/etiology , Male , Maze Learning/physiology , Mice , Mice, Inbred C57BL , PC12 Cells/enzymology , PC12 Cells/pathology , RNA Polymerase II/genetics , Rats , Retinol-Binding Proteins, Cellular/metabolism , Serine/metabolism , Time Factors , Von Hippel-Lindau Tumor Suppressor Protein/metabolism
10.
J Neurochem ; 104(2): 409-19, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17949411

ABSTRACT

Besides its role in terminating acetylcholine-mediated neurotransmission, acetylcholinesterase (AChE) is found to be expressed and participate in the process of apoptosis in various cell types. However, the mechanisms underlying AChE up-regulation in neuronal cells remain elusive. Herein we demonstrated that glycogen synthase kinase-3beta (GSK3beta) mediates induced AChE-S expression during apoptosis. In this study, A23187 and thapsigargin (TG) were employed to induce apoptosis in neuroendocrine PC12 cells. The results showed that exposure of PC12 cells to A23187 and TG up-regulated AChE activity significantly. The same treatment also led to activation of GSK3beta. Two different inhibitors of GSK3beta (lithium and GSK3beta-specific inhibitor VIII) could block A23187- or TG-induced up-regulation of AChE activity, AChE-S mRNA level and protein expression. However, lithium could not inhibit the induction of AChE-R mRNA and protein under similar conditions. Taken together, our results show that GSK3beta is specifically involved in the induction of AChE-S expression in PC12 cells during apoptosis.


Subject(s)
Acetylcholinesterase/metabolism , Apoptosis/physiology , Glycogen Synthase Kinase 3/metabolism , Synapses/enzymology , Acetylcholinesterase/genetics , Animals , Apoptosis/drug effects , Calcimycin/pharmacology , Drug Interactions , Enzyme Inhibitors/pharmacology , Gene Expression/drug effects , Glycogen Synthase Kinase 3 beta , Iodides , Ionophores/pharmacology , Lithium Chloride/pharmacology , PC12 Cells/cytology , PC12 Cells/drug effects , PC12 Cells/enzymology , RNA, Messenger/metabolism , Rats , Synapses/drug effects , Thapsigargin/pharmacology , Up-Regulation/drug effects
11.
J Vet Sci ; 8(4): 377-82, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17993752

ABSTRACT

Mesenchymal stem cells (MSCs) secrete a variety of neuroregulatory molecules, such as nerve growth factor, brain-derived neurotrophic factor, and glial cell-derived neurotrophic factor, which upregulate tyrosine hydroxylase (TH) gene expression in PC12 cells. Enhancing TH gene expression is a critical step for treatment of Parkinson's disease (PD). The objective of this study was to assess the effects of co-culturing PC12 cells with MSCs from feline bone marrow on TH protein expression. We divided the study into three groups: an MSC group, a PC12 cell group, and the combined MSC + PC12 cell group (the co-culture group). All cells were cultured in DMEM-HG medium supplemented with 10% fetal bovine serum for three days. Thereafter, the cells were examined using western blot analysis and immunocytochemistry. In western blots, the co-culture group demonstrated a stronger signal at 60 kDa than the PC12 cell group (p < 0.001). TH was not expressed in the MSC group, either in western blot or immunocytochemistry. Thus, the MSCs of feline bone marrow can up-regulate TH expression in PC12 cells. This implies a new role for MSCs in the neurodegenerative disease process.


Subject(s)
Cats/physiology , Gene Expression Regulation, Enzymologic , Mesenchymal Stem Cells/cytology , PC12 Cells/enzymology , Tyrosine 3-Monooxygenase/metabolism , Animals , Antigens, Surface/metabolism , Blotting, Western , Cell Culture Techniques , Cells, Cultured , Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/metabolism , Immunohistochemistry , Mesenchymal Stem Cells/metabolism , Microscopy, Phase-Contrast , PC12 Cells/cytology , Rats
12.
Toxicol Appl Pharmacol ; 216(2): 309-18, 2006 Oct 15.
Article in English | MEDLINE | ID: mdl-16843508

ABSTRACT

An excessive and sustained increase in reactive oxygen species (ROS) production and oxidative stress have been implicated in the pathogenesis of many diseases. In the present study, we have demonstrated that 4-hydroxynonenal (4-HNE), a product of lipid peroxidation, alters glutathione (GSH) pools and induces oxidative stress in PC12 cells in culture. This increase was accompanied by alterations in subcellular ROS and glutathione (GSH) metabolisms. The GSH homeostasis was affected as both mitochondrial and extramitochondrial GSH levels, GSH peroxidase and glutathione reductase activities were inhibited and glutathione S-transferase (GST) activity was increased after 4-HNE treatment. A concentration- and time-dependent increase in cytochrome P450 2E1 (CYP 2E1) activity in the mitochondria and postmitochondrial supernatant was also observed. 4-HNE-induced oxidative stress also caused an increase in the expression of GSTA4-4, CYP2E1 and Hsp70 proteins in the mitochondria. Increased oxidative stress in PC12 cells initiated apoptosis as indicated by the release of mitochondrial cytochrome c, activation of poly-(ADP-ribose) polymerase (PARP), DNA fragmentation and decreased expression of antiapoptotic Bcl-2 proteins. Mitochondrial respiratory and redox functions also appeared to be affected markedly by 4-HNE treatment. These results suggest that HNE-induced oxidative stress and apoptosis might be associated with altered mitochondrial functions and a compromised GSH metabolism and ROS clearance.


Subject(s)
Aldehydes/toxicity , Apoptosis/drug effects , Cysteine Proteinase Inhibitors/toxicity , Cytochrome P-450 CYP2E1/metabolism , Glutathione Transferase/drug effects , Mitochondria/drug effects , PC12 Cells/drug effects , Animals , Cytochromes c/metabolism , DNA Fragmentation , Dose-Response Relationship, Drug , Glutathione/metabolism , Glutathione Peroxidase/antagonists & inhibitors , Glutathione Peroxidase/drug effects , Glutathione Peroxidase/metabolism , Glutathione Reductase/antagonists & inhibitors , Glutathione Reductase/drug effects , Glutathione Reductase/metabolism , Glutathione Transferase/metabolism , HSP70 Heat-Shock Proteins/metabolism , Mitochondria/metabolism , Oxidative Stress/drug effects , PC12 Cells/enzymology , PC12 Cells/pathology , Poly Adenosine Diphosphate Ribose/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Rats , Reactive Oxygen Species/metabolism
13.
Toxicol Appl Pharmacol ; 215(2): 218-27, 2006 Sep 01.
Article in English | MEDLINE | ID: mdl-16603213

ABSTRACT

Disulfiram (an alcohol-aversive drug) and related compounds are known to provoke several side effects involving behavioral and neurological complications. N,N-diethyldithiocarbamate (DDC) is considered as one of the main toxic species of disulfiram and acts as an inhibitor of superoxide dismutase. Since arachidonic acid (AA) formation is regulated by reactive oxygen species (ROS) and related to toxicity in neuronal cells, we investigated the effects of DDC on AA release and expression of the alpha type of cytosolic phospholipase A(2) (cPLA(2)alpha) in PC12 cells. Treatment with 80-120 microM DDC that causes a moderate increase in ROS levels without cell toxicity stimulated cPLA(2)alpha mRNA and its protein expression. The expression was mediated by extracellular-signal-regulated kinase (ERK1/2), one of the mitogen-activated protein kinases. Treatment with N(G) nitro-L-arginine methyl ester (an inhibitor of nitric oxide synthase, 1 mM) and oxy-hemoglobin (a scavenger of nitric oxide, 2 mg/mL) abolished the DDC-induced responses (ERK1/2 phosphorylation and cPLA(2)alpha expression). We also showed DDC-induced up-regulation of the mRNA expression of lipocortin 1, an inhibitor of PLA(2). Furthermore, DDC treatment of the cells enhanced Ca(2+)-ionophore-induced AA release in 30 min, although the effect was limited. Changes in AA metabolism in DDC-treated cells may have a potential role in mediating neurotoxic actions of disulfiram. In this study, we show the first to demonstrate the up-regulation of cPLA(2)alpha expression by DDC treatment in neuronal cells.


Subject(s)
Antiviral Agents/pharmacology , Ditiocarb/pharmacology , Nitric Oxide/metabolism , PC12 Cells/drug effects , Phospholipases A/metabolism , Reactive Oxygen Species/metabolism , Animals , Annexin A1/genetics , Annexin A1/metabolism , Arachidonic Acid/metabolism , Cell Survival/drug effects , Cytosol/drug effects , Cytosol/enzymology , Drug Antagonism , Gene Expression/drug effects , Group IV Phospholipases A2 , Mitogen-Activated Protein Kinase 3/metabolism , NG-Nitroarginine Methyl Ester/pharmacology , Oxyhemoglobins/pharmacology , PC12 Cells/enzymology , PC12 Cells/pathology , Phospholipases A/genetics , RNA, Messenger/metabolism , Rats , Superoxide Dismutase/metabolism , Up-Regulation
14.
J Neurochem ; 93(5): 1157-67, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15934936

ABSTRACT

Our previous studies have shown that the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) prevents neuronal cell death caused by several neurotoxins. The present study sought to determine the neuroprotective effect of EGCG when it is administered after the induction of cell damage ('neurorescue'). In an attempt to imitate a progressive mode of death, PC12 cells were initially subjected to serum-starvation conditions for a period of 1 or 3 days before administration of EGCG (0.1-10 microM) for up to 3 days. In spite of the high percentage of cell death, single or repetitive administration of EGCG (1 microM) significantly attenuated cell death. The neurorescue effect of EGCG was abolished by pre-treatment with the protein kinase C inhibitor GF109203X (2.5 microM), suggesting the involvement of the protein kinase C pathway in neurorescue by the drug. This is consistent with the rapid (15 min) translocation of the protein kinase C alpha isoform to the cell membrane in response to EGCG. The correlative neurite outgrowth activity of EGCG on PC12 cells may also contribute to its neurorescue effect. The present findings suggest that EGCG may have a positive impact on aging and neurodegenerative diseases to retard or perhaps even reverse the accelerated rate of neuronal degeneration.


Subject(s)
Catechin/analogs & derivatives , Catechin/pharmacology , Culture Media, Serum-Free/pharmacology , Flavonoids/pharmacology , Neurites/physiology , Neuroprotective Agents/pharmacology , PC12 Cells/drug effects , Phenols/pharmacology , Animals , Biological Transport/drug effects , Cell Death/drug effects , Cell Differentiation/drug effects , Cell Membrane/enzymology , Enzyme Activation/drug effects , Neurites/drug effects , PC12 Cells/enzymology , PC12 Cells/pathology , PC12 Cells/physiology , Polyphenols , Protein Kinase C/metabolism , Protein Kinase C/physiology , Protein Kinase C-alpha , Rats
15.
Phytother Res ; 18(6): 488-93, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15287076

ABSTRACT

The Paeng-Jo-Yeon-Nyeon-Baek-Ja-In-Hwan (PJBH) prescription is a dried decoctum consisting of a mixture of 18 medicinal herbs that include Semen Biotae, Fructus Torilis seu cnidii, Fructus Rubi, Herba Dendrobii, Radix Morindae officinalis, Cortex Eucommiae, Radix Aspragi, Radix Polygalae, Radix Dipsaci, Ramulus Cinnamomi, Rhizoma Acori graminei, Rhizoma Alismatis, Rhizoma Dioscoreae, Radix Ginseng, Radix Rehmanniae preparata, Fructus corni, Fructus Schisandrae and Herba Cistanches. The effect of PJBH extracts on H2O2-induced toxicity in the rat pheochromocytoma line PC12 was examined by measurements of cell lesion, level of lipid peroxidation and antioxidant enzyme activities, since free radicals are involved in neurodegeneration in Alzheimer's disease (AD). After a 30 min exposure of the cells to H2O2 (150 microM), a marked decrease in cell survival, activities of glutathione peroxidase and catalase as well as an increased production of malondialdehyde (MDA) were found. Pretreatment of the cells with PJBH (0.5-10 microg/ml) prior to H2O2 exposure significantly elevated cell survival, antioxidant enzyme activities and resulted in a decrease in the level of MDA. The effects of the PJBH on hydrogen peroxide-induced injury in PC12 cells were also examined. PJBH had a remarkable elevating effect on catalase and GSH-Px activities as well as cell survival, suggesting that cytoprotective effects of the PJBH are involved in stimulation against intermediate concentrations of H2O2-induced PC12 cell injury. The above-mentioned neuroprotective effects were also compared with the effect of tacrine. The results suggest that PJBH has potential for use as a novel neuronal therapeutic agent.


Subject(s)
Alzheimer Disease/prevention & control , Cholinesterase Inhibitors/pharmacology , Drugs, Chinese Herbal/pharmacology , Phytotherapy , Plants, Medicinal , Animals , Cell Survival/drug effects , Cholinesterase Inhibitors/administration & dosage , Cholinesterase Inhibitors/therapeutic use , Drugs, Chinese Herbal/administration & dosage , Drugs, Chinese Herbal/therapeutic use , Fruit , Glutathione Peroxidase/metabolism , Humans , Hydrogen Peroxide , Korea , Medicine, Traditional , PC12 Cells/drug effects , PC12 Cells/enzymology , PC12 Cells/metabolism , PC12 Cells/pathology , Plant Leaves , Plant Roots , Plant Stems , Rats , Rhizome , Seeds
16.
J Neurosci ; 24(30): 6650-8, 2004 Jul 28.
Article in English | MEDLINE | ID: mdl-15282267

ABSTRACT

Neurotrophins, such as NGF and BDNF, activate Trk receptor tyrosine kinases through receptor dimerization at the cell surface followed by autophosphorylation and intracellular signaling. It has been shown that activation of Trk receptor tyrosine kinases can also occur via a G-protein-coupled receptor (GPCR) mechanism, without involvement of neurotrophins. Two GPCR ligands, adenosine and pituitary adenylate cyclase-activating polypeptide (PACAP), can activate Trk receptor activity to increase the survival of neural cells through stimulation of Akt activity. To investigate the mechanism of Trk receptor transactivation, we have examined the localization of Trk receptors in PC12 cells and primary neurons after treatment with adenosine agonists and PACAP. In contrast to neurotrophin treatment, Trk receptors were sensitive to transcriptional and translational inhibitors, and they were found predominantly in intracellular locations particularly associated with Golgi membranes. Biotinylation and immunostaining experiments confirm that most of the transactivated Trk receptors are found in intracellular membranes. These results indicate that there are alternative modes of activating Trk receptor tyrosine kinases in the absence of neurotrophin binding at the cell surface and that receptor signaling may occur and persist inside of neuronal cells.


Subject(s)
Adenosine/analogs & derivatives , Adenosine/pharmacology , Golgi Apparatus/enzymology , Intracellular Membranes/enzymology , Nerve Growth Factors/pharmacology , Neuropeptides/pharmacology , Neurotransmitter Agents/pharmacology , Phenethylamines/pharmacology , Receptor, trkA/drug effects , Receptor, trkB/drug effects , Receptors, G-Protein-Coupled/drug effects , Animals , Brefeldin A/pharmacology , Chromones/pharmacology , Cycloheximide/pharmacology , Dactinomycin/pharmacology , Enzyme Activation/drug effects , Morpholines/pharmacology , Nucleic Acid Synthesis Inhibitors/pharmacology , PC12 Cells/drug effects , PC12 Cells/enzymology , Phosphatidylinositol 3-Kinases/physiology , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation/drug effects , Pituitary Adenylate Cyclase-Activating Polypeptide , Prosencephalon/enzymology , Protein Biosynthesis/drug effects , Protein Processing, Post-Translational/drug effects , Protein Serine-Threonine Kinases/physiology , Protein Synthesis Inhibitors/pharmacology , Proto-Oncogene Proteins/physiology , Proto-Oncogene Proteins c-akt , Rats , Rats, Sprague-Dawley , Receptor, trkA/metabolism , Receptor, trkB/metabolism , Receptors, G-Protein-Coupled/physiology , Signal Transduction/drug effects , Transcription, Genetic/drug effects
17.
Planta Med ; 70(5): 427-31, 2004 May.
Article in English | MEDLINE | ID: mdl-15124087

ABSTRACT

The present study investigated the protective effects of the antioxidant scutellarin against oxidative toxicity induced by glutamate in PC12 cells. Vitamin E, a classical antioxidant was employed as a comparative agent. Incubation of PC12 cells with 10 mM glutamate resulted in significant cytotoxity as evaluated by the MTT and lactate dehydrogenase (LDH) assays, decreases of GSSG reductase activity, disturbance of the cell redox state as indicated by the GSH/GSSG ratio, and accumulation of intracellular reactive oxygen species (ROS) and lipid peroxidation products. Scutellarin at 0.1, 1 and 10 microM significantly protected against the cytoxicity and production of ROS and lipid peroxidation induced by glutamate. Scutellarin did not prevent the reduction of cellular GSH levels, but it up-regulated GSSG reductase activity, thus preventing an increase in cellular GSSG levels, and concomitantly improved the cell redox status. Our data also show that the protective effects of scutellarin against glutamate-induced oxidative toxicity are more potent than that of vitamin E. These results demonstrate that scutellarin can protect PC12 cells from oxidative glutamate toxicity by scavenging ROS, inhibiting lipid peroxidation and improving the cell redox status, and may reduce the cellular damage in pathological conditions associated with excessive glutamate release.


Subject(s)
Antioxidants/pharmacology , Apigenin , Asteraceae , Flavonoids/pharmacology , Glucuronates/pharmacology , Glutathione Disulfide/pharmacology , Phytotherapy , Plant Extracts/pharmacology , Protective Agents/pharmacology , Animals , Antioxidants/administration & dosage , Antioxidants/therapeutic use , Apoptosis/drug effects , Dose-Response Relationship, Drug , Drug Synergism , Flavonoids/administration & dosage , Flavonoids/therapeutic use , Glucuronates/administration & dosage , Glucuronates/therapeutic use , Lipid Peroxidation/drug effects , PC12 Cells/drug effects , PC12 Cells/enzymology , Plant Extracts/administration & dosage , Plant Extracts/therapeutic use , Protective Agents/administration & dosage , Protective Agents/therapeutic use , Rats , Vitamin E/administration & dosage , Vitamin E/pharmacology , Vitamin E/therapeutic use
18.
Toxicol Lett ; 147(1): 63-72, 2004 Feb 28.
Article in English | MEDLINE | ID: mdl-14700529

ABSTRACT

Acute exposure to trimethyltin (TMT) causes neuronal degeneration in the hippocampus, amygdala, pyriform cortex, and neocortex [Am. J. Pathol. 97 (1979) 59]. Despite extensive efforts elucidating neuropathological changes and behavioral deficits following TMT exposure, only a limited amount of work has examined the molecular signaling mechanisms that lead to these changes. The present paper demonstrates that TMT impairs neurite outgrowth and cell viability in an in vitro model of neuronal development. The decrease in cell viability is paralleled by a decrease in cell body size, an increase in DNA fragmentation, activation of caspase-9, and cleavage of the caspase substrate poly-ADP ribose polymerase (PARP). These results suggest that TMT induces apoptosis. Pharmacological inhibition of caspase activity, p38 stress-responsive protein kinase activity, or oxidative stress prevented TMT-induced cell death. This work provides the first evidence for a TMT-initiated apoptotic pathway requiring oxidative stress, caspase activation, and p38 protein kinase activity.


Subject(s)
Apoptosis/drug effects , Caspases/biosynthesis , Mitogen-Activated Protein Kinases/biosynthesis , Oxidative Stress , PC12 Cells/drug effects , Trimethyltin Compounds/toxicity , Amino Acid Chloromethyl Ketones/pharmacology , Animals , Blotting, Western , Caspase 9 , Caspase Inhibitors , Cell Size/drug effects , Cell Survival/drug effects , Cysteine Proteinase Inhibitors/pharmacology , DNA Fragmentation , Dose-Response Relationship, Drug , Neurites/drug effects , Neurites/pathology , PC12 Cells/enzymology , Poly(ADP-ribose) Polymerases/metabolism , Rats , p38 Mitogen-Activated Protein Kinases
19.
Gene Expr ; 11(3-4): 117-24, 2003.
Article in English | MEDLINE | ID: mdl-14686785

ABSTRACT

Clonal pheochromocytoma cell lines overexpressing cytochrome P450 2D6 (CYP2D6) were established. CYP2D6 was localized in the endoplasmic reticulum, and its enzymatic activity in the microsomal fraction was confirmed by using high performance liquid chromatography analysis with [guanidine-14C]debrisoquine as a substrate. Overexpression of CYP2D6 protected both actively dividing and differentiated cells against the toxic effects of 1-methyl-4-phenylpyridinium ion at the concentration range of 20-40 microM, as assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The production of reactive oxygen species in the mitochondria was suppressed. The cytotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine was unchanged in both actively dividing and differentiated cells overexpressing CYP2D6 versus mock-transfected controls at concentrations up to 500 microM. These results suggest that the lowered enzyme activity of CYP2D6 in individuals termed "poor metabolizers" may represent a risk factor from exposure to select neurotoxicants.


Subject(s)
1-Methyl-4-phenylpyridinium/toxicity , Cytochrome P-450 CYP2D6/genetics , Gene Expression Regulation, Enzymologic/physiology , Herbicides/toxicity , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/pharmacology , Animals , Cell Differentiation/drug effects , Cell Division/drug effects , Cytochrome P-450 CYP2D6/metabolism , Humans , Mitochondria/metabolism , PC12 Cells/cytology , PC12 Cells/enzymology , Rats , Reactive Oxygen Species/metabolism
20.
Brain Res ; 980(1): 71-9, 2003 Aug 01.
Article in English | MEDLINE | ID: mdl-12865160

ABSTRACT

We sought to further elucidate signal transduction pathways for the I(1)-imidazoline receptor in PC12 cells and their interaction with the well-characterized signaling events triggered by nerve growth factor (NGF) in these cells. Stimulation of the I(1)-imidazoline receptor with moxonidine, a centrally acting antihypertensive, increased by greater than two-fold the proportion of ERK-1 and ERK-2 in the phosphorylated active form. Similarly, NGF elicited a five-fold increase in activated ERKs. Surprisingly, treatment of NGF-treated cells with moxonidine completely reversed activation of ERK. Moxonidine-induced inhibition of ERK activation in NGF-treated cells was dose-dependent, followed a limited time course and could be blocked by the I(1)-antagonist efaroxan. These data suggested possible deactivation of ERK by specific phosphatases. Therefore, we assayed levels of MKP-2, a dual specificity phosphatase whose substrates include ERK. Moxonidine and NGF both increased levels of MKP-2 by three-fold. These effects were additive, as both agents together increased MKP-2 by a total of six-fold. Moxonidine-induced induction of MKP-2 was time- and dose-dependent and could be blocked by the I(1)-antagonist efaroxan or by D609, an inhibitor of phosphatidylcholine-selective phospholipase C known to block downstream signaling events coupled to I(1)-receptors. Thus, I(1)-receptors can abrogate the primary signaling cascade activated by NGF, most likely by increasing levels of a specific phosphatase to return dually phosphorylated ERK to its unphosphorylated state.


Subject(s)
Mitogen-Activated Protein Kinases/metabolism , Nerve Growth Factor/metabolism , PC12 Cells/drug effects , PC12 Cells/enzymology , Phosphoric Monoester Hydrolases/metabolism , Receptors, Drug/metabolism , Animals , Antihypertensive Agents/pharmacology , Blotting, Western , Dose-Response Relationship, Drug , Imidazoles/pharmacology , Imidazoline Receptors , Rats , Receptors, Drug/agonists , Signal Transduction
SELECTION OF CITATIONS
SEARCH DETAIL
...