Dependence of protective effect of α-tocopherol on its concentration and time of action on pc12 cells under conditions of oxidative stress

At the short-term incubation (0.5 and 1.5 h) of cells of the PC12 neuronal line with α-tocopherol, its protective effect against the cytotoxic hydrogen peroxide action was increased with rise of its concentration in samples; the protection was practically absent at action of nanomolar antioxidant concentrations, but was well expressed at its micromolar concentrations. These data agree with the concept that α-tocopherol increases the cell viability by reacting directly with free radicals, which leads to formation of the less reactive compounds deprived of non-paired electron. The evidence is obtained that at the long-term action on PC12 cells, α-tocopherol not only in micro-, but also in nanomolar concentrations increases significantly the cell viability under conditions of oxidative stress. As follows from the obtained data, an important role in realization of the α-tocopherol protective effect at the long-term incubation seems to be played by modulation by this antioxidant of activity of protein kinase activated by extracellular signaling, phosphatidylinosite 3-kinase, and protein kinase C.     

Antiapoptotic effect of alpha-tocopherol at micro- and nanomolar concentrations on cells of neuronal line PC12 according to data of flow cytometry

By the method of flow cytometry it has been shown that alpha-tocopherol at micromolar concentrations produces antiapoptotic effect on the PC12 neuronal line cells exposed to the toxic agent hydrogen peroxide at various terms of incubation with it. At the same time, alpha-tocopherol at nanomolar concentrations had protective (antiapoptotic) effect only after the long (18 h) preincubation of the PC12 cells with it prior to exposure to hydrogen peroxide. This seems to indicate that the alpha-tocopherol effect at these concentrations is mediated by a signal transduction system.     

Antiapoptotic effect of alpha-tocopherol at micro- and nanomolar concentrations on cells of neuronal line PC12 according to data of flow cytometry

By the method of flow cytometry it has been shown that alpha-tocopherol at micromolar concentrations produces antiapoptotic effect on PC12 neuronal line cells exposed to the toxic agent hydrogen peroxide at various terms of incubation with it. At the same time, α-tocopherol at nanomolar concentrations had protective (antiapoptotic) effect only after the long (18 h) preincubation of the PC12 cells with it prior to exposure to hydrogen peroxide. This seems to indicate that the α-tocopherol effect at these concentrations is mediated by a signal transduction system.     

Metabolic effects of ganglioside GM1 on PC12 cells in oxidative stress depend on modulation of activity of tyrosine kinase Trk of receptors

There have been obtained evidences that not only GM1, but also other main brain gangliosides (GD1a, GD1b, and GT1b) increase viability of cells of the neuronal line PC12 under action of H₂O₂. By the example of GM1 and GD1a, gangliosides have been shown to produce a protective effect on PC12 cells under conditions of oxidative stress both at micro- and nanomolar concentrations that are physiological concentrations of gangliosides in cerebrospinal fluid. For the first time, GM1 at nanomolar concentrations was shown to decrease the H₂O₂-induced formation of reactive oxygen species (ROS). It was found that in the presence of inhibitor of tyrosine kinase Trk of receptors K-252a, GM1 at concentrations of 10 μM and 10 nM lost its ability to produce such metabolic effects as a decrease of ROS accumulation and of the degree of oxidative inactivation of Na⁺,K⁺-ATPase in PC12 cells, as well as ceased to increase viability of these cells under conditions of oxidative stress. The dependence of protective and metabolic effects of gangliosides GM1 in PC12 cells treated with H₂O₂ on modulation of activity of activity of tyrosine kinase Trk receptors (i.e., from the same signal system) agrees with concept about the essential role of oxidant effect of GM1 in its increase of cell viability.     

GM1 Ganglioside Activates ERK1/2 and Akt Downstream of Trk Tyrosine Kinase and Protects PC12 Cells Against Hydrogen Peroxide Toxicity

Ganglioside GM1 at micro- and nanomolar concentrations was shown to increase the viability of pheochromocytoma PC12 cells exposed to hydrogen peroxide and diminish the accumulation of reactive oxygen species and oxidative inactivation of Na⁺,K⁺-ATPase, the effects of micromolar GM1 being more pronounced than those of nanomolar GM1. These effects of GM1 were abolished by Trk receptor tyrosine kinase inhibitor and diminished by MEK1/2, phosphoinositide 3-kinase and protein kinase C inhibitors. Hydrogen peroxide activates Trk tyrosine kinase; Akt and ERK1/2 are activated downstream of this protein kinase. GM1 was found to activate Trk receptor tyrosine kinase in PC12 cells. GM1 (100 nM and 10 µM) increased the basal activity of Akt, but did not change Akt activity in cells exposed to hydrogen peroxide. Basal ERK1/2 activity in PC12 cells was increased by GM1 at a concentration of 10 µM, but not at nanomolar concentrations. Activation of ERK1/2 by hydrogen peroxide was enhanced by GM1 at a concentration of 10 µM and to a lesser extent at a concentration of 100 nM. Thus, the protective and metabolic effects of GM1 ganglioside on PC12 cells exposed to hydrogen peroxide appear to depend on the activation of Trk receptor tyrosine kinase and downstream activation of Akt and ERK1/2.     

Stimulation by Gangliosides of Viability of Rat Brain Neurons and of Neuronal PC12 Cell Line under Conditions of Oxidative Stress

We studied effect of gangliosides on viability of brain neurons and neuronal PC12 cell line exposed to toxic concentrations of compounds activating free radical reactions. It is found that preincubation of cerebellar granule cells and PC12 cells with micromolar concentrations of ganglioside GM1 increases statistically significantly viability of these cells submitted to inductors of oxidative stress, such as hydrogen peroxide and the Fe²⁺-ascorbate system However, the effect of ganglioside GM1 in the PC12 cells failed to be revealed 1–2 days after treatment of the cells with trypsin, which indicates an importance of interaction of gangliosides with surface proteins for realization of their protective action. GM1, GD1a, and other gangliosides were shown to produce the neuroprotective effect on cerebellar granule cells in the presence of toxic glutamate concentrations. Not only micro-, but also nanomolar concentrations of these gangliosides increased statistically significantly the neuronal viability, although at micromolar concentrations this effect as a rule was more pronounced. The obtained data allow suggesting that the neuroprotective action of gangliosides is determined to a considerable degree by their ability to inhibit free-radical reactions in nerve cells.     

Molecular basis of vitamin E action. Tocotrienol potently inhibits glutamate-induced pp60(c-Src) kinase activation and death of HT4 neuronal cells

HT4 hippocampal neuronal cells were studied to compare the efficacy of tocopherols and tocotrienol to protect against glutamate-induced death. Tocotrienols were more effective than alpha-tocopherol in preventing glutamate-induced death. Uptake of tocotrienols from the culture medium was more efficient compared with that of alpha-tocopherol. Vitamin E molecules have potent antioxidant properties. Results show that at low concentrations, tocotrienols may have protected cells by an antioxidant-independent mechanism. Examination of signal transduction pathways revealed that protein tyrosine phosphorylation processes played a central role in the execution of death. Activation of pp60(c-Src) kinase and phosphorylation of ERK were observed in response to glutamate treatment. Nanomolar amounts of alpha-tocotrienol, but not alpha-tocopherol, blocked glutamate-induced death by suppressing glutamate-induced early activation of c-Src kinase. Overexpression of kinase-active c-Src sensitized cells to glutamate-induced death. Tocotrienol treatment prevented death of Src-overexpressing cells treated with glutamate. alpha-Tocotrienol did not influence activity of recombinant c-Src kinase suggesting that its mechanism of action may include regulation of SH domains. This study provides first evidence describing the molecular basis of tocotrienol action. At a concentration 4-10-fold lower than levels detected in plasma of supplemented humans, tocotrienol regulated unique signal transduction processes that were not sensitive to comparable concentrations of tocopherol.     

PC12 cells transfected with human mutant gene causing one of Alzheimer's disease forms have a high sensitivity to oxidative stress

Used in this work are PC12 cells transfected with human gene expressing amyloid precursor protein of beta-peptide and carrying the so-called "Swedish mutation" leading to the appearance of one Alzheimer's disease family forms. It has been shown that the PC12 cells transfected with this mutant gene, at action of various hydrogen peroxide concentrations, die to the significant greater degree than the used for comparison PC12 cells transfected with analogous human gene of the wild type or than vector-transfected cells. It has been found that ganglioside GM1 at micro- or nanomolar concentrations is able to increase viability of the PC12 cells transfected with the mutant gene causing a significant accumulation of endogenous amyloid beta-peptide. The obtained data confirm an important role of oxidative stress in injury and death of brain nerve cells in Alzheimer's disease.     

Cocoa procyanidins attenuate 4-hydroxynonenal-induced apoptosis of PC12 cells by directly inhibiting mitogen-activated protein kinase kinase 4 activity

Neurodegenerative disorders such as Alzheimer's disease (AD) are associated with oxidative stress, and it has been suggested that apoptosis is a crucial pathway in neuronal cell death in AD patients. 4-Hydroxynonenal (HNE), one of the aldehydic products of membrane lipid peroxidation, is reported to be elevated in the brains of AD patients and mediates the induction of neuronal apoptosis in the presence of oxidative stress. In this study, we investigated the HNE-induced apoptosis mechanism and the protective effects of the cocoa procyanidin fraction (CPF) and its major antioxidant procyanidin B2 against the apoptosis induced by HNE in rat pheochromocytoma (PC12) cells. HNE-induced nuclear condensation and increased sub-G1 fraction, both of which are markers of apoptotic cell death, were inhibited by CPF and procyanidin B2. Intracellular reactive oxygen species (ROS) accumulation was attenuated by pretreatment with CPF and procyanidin B2. CPF and procyanidin B2 also prevented HNE-induced poly(ADP-ribose) polymerase cleavage, antiapoptotic protein (Bcl-2 and Bcl-X_L) down-regulation, and caspase-3 activation. Activation of c-Jun N-terminal protein kinase (JNK) and mitogen-activated protein kinase kinase 4 (MKK4) was attenuated by CPF and procyanidin B2. Moreover, CPF and procyanidin B2 bound directly to MKK4 and inhibited its activity. Data obtained with SP600125, a selective inhibitor of JNK, revealed that JNK is involved in HNE-induced apoptosis through the inhibition of PARP cleavage and caspase-3 activation in PC12 cells. Collectively, these results indicate that CPF and procyanidin B2 protect PC12 cells against HNE-induced apoptosis by blocking MKK4 activity as well as ROS accumulation.     

Ecto-Protein Kinase and Surface Protein Phosphorylation in PC12 Cells: Interactions with Nerve Growth Factor

Abstract: The phosphorylation of surface proteins by ectoprotein kinase has been proposed to play a role in mechanisms underlying neuronal differentiation and their responsiveness to nerve growth factor (NGF). PC 12 clones represent an optimal model for investigating the mode of action of NGF in a homogeneous cell population. In the present study we obtained evidence that PC12 cells possess ectoprotein kinase and characterized the endogenous phosphorylation of its surface protein substrates. PC12 cells maintained in a chemically defined medium exhibited phosphorylation of proteins by [γ-³²P]ATP added to the medium at time points preceding the intracellular phosphorylation of proteins in cells labeled with ³²P_i. This activity was abolished by adding apyrase or trypsin to the medium but was not sensitive to addition of an excess of unlabeled P_i. As also expected from ecto-protein kinase activity, PC12 cells catalyzed the phosphorylation of an exogenous protein substrate added to the medium, dephospho-α-casein, and this activity competed with the endogenous phosphorylation for extracellular ATP. Based on these criteria, three protein components migrating in sodium dodecyl sulfate gels with apparent molecular weights of 105K, 39K, and 20K were identified as exclusive substrates of ecto-protein kinase in PC12 cells. Of the phosphate incorporated into these proteins from extracellular ATP, 75–87% was found in phosphothreonine. The phosphorylation of the 39K protein by ecto-protein kinase did not require Mg²⁺, implicating this activity in the previously demonstrated regulation of Ca²⁺-dependent, high-affinity norepinephrine uptake in PC12 cells by extracellular ATP. The protein kinase inhibitor K-252a inhibited both intra- and extracellular protein phosphorylation in intact PC12 cells. Its hydrophilic analogue K-252b, had only minimal effects on intracellular protein phosphorylation but readily inhibited the phosphorylation of specific substrates of ecto-protein kinase in PC12 cells incubated with extracellular ATP, suggesting the involvement of ecto-protein kinase in the reported inhibition of NGF-induced neurite extension by K-252b. Preincubation of PC12 cells with 50 ng/ml of NGF for 5 min stimulated the activity of ecto-protein kinase toward all its endogenous substrates. Exposure of PC12 cells to the same NGF concentration for 3 days revealed another substrate of ecto-protein kinase, a 53K protein, whose surface phosphorylation is expressed only after NGF-induced neuronal differentiation. In the concentration range (10–100 μM) at which 6-thioguanine blocked NGF-promoted neurite outgrowth in PC12 cells, 6-thioguanine effectively inhibited the phosphorylation of specific proteins by ecto-protein kinase. This study provides the basis for continued investigation of the involvement of ecto-protein kinase and its surface protein substrates in neuronal differentiation, neuritogenesis, and synaptogenesis.     

PC12 cells transfected with human mutant gene causing one of Alzheimer’s disease forms have a high sensitivity to oxidative stress

Used in this work are PC12 cells transfected with human gene expressing amyloid-precursor protein of β-peptide and carrying the so-called “Swedish mutation” leading to the appearance of one of Alzheimer’s disease family forms. It has been shown that the PC12 cells transfected with this mutant gene, at action of various hydrogen peroxide concentrations, die to the significant greater degree than the used for comparison PC12 cells transfected with analogous human gene of the wild type or than vector-transfected cells. It has been found that ganglioside GM1 at micro-or nanomolar concentrations is able to increase viability of the PC12 cells transfected with the mutant gene causing a significant accumulation of endogenous amyloid β-peptide. The obtained data confirm an important role of oxidative stress in injury and death of brain nerve cells in Alzheimer’s disease.     

Second-messenger control of catecholamine release from PC12 cells. Role of muscarinic receptors and nerve-growth-factor-induced cell differentiation.

The role of various intracellular signals and of their possible interactions in the control of neurotransmitter release was investigated in PC12 cells. To this purpose, agents that affect primarily the cytosolic concentration of Ca2+, [Ca2+]i (ionomycin, high K+), agents that affect cyclic AMP concentrations (forskolin; the adenosine analogue phenylisopropyladenosine; clonidine) and activators of protein kinase C (phorbol esters) were applied alone or in combination to either growing chromaffin-like PC12-cells, or to neuron-like PC12+ cells differentiated by treatment with NGF (nerve growth factor). In addition, the release effects of muscarinic-receptor stimulation (which causes increase in [Ca2+]i, activation of protein kinase C and decrease in cyclic AMP) were investigated. Two techniques were employed to measure catecholamine release: static incubation of [3H]dopamine-loaded cells, and perfusion incubation of unlabelled cells coupled to highly sensitive electrochemical detection of released catecholamines. The results obtained demonstrate that: (1) release from PC12 cells can be elicited by both raising [Ca2+]i and activating protein kinases (protein kinase C and, although to a much smaller extent, cyclic AMP-dependent protein kinase); and (2) these various control pathways interact extensively. Activation of muscarinic receptors by carbachol induced appreciable release responses, which appeared to be due to a synergistic interplay between [Ca2+]i and protein kinase C activation. The muscarinic-induced release responses tended to become inactivated rapidly, possibly by feedback desensitization of the receptor mediated by protein kinase C. Muscarinic inactivation was prevented (or reversed) by agents that increase, and accelerated by agents that decrease, cyclic AMP. Agents that stimulate release primarily through the Ca2+ pathway (ionomycin and high K+) were found to be equipotent in both PC12- and PC12+ cells, whereas the protein kinase C activator 12-O-tetradecanoyl-phorbol 13-acetate was approx. 10-fold less potent in PC12+ cells, when administered either alone or in combination with ionomycin. In contrast, the cell binding of phorbol esters was not greatly modified by NGF treatment. Thus control of neurotransmitter release from PC12 cells is changed by differentiation, with a diminished role of the mechanism mediated by protein kinase C.     

Inhibition of cell proliferation by alpha-tocopherol. Role of protein kinase C

The effect of alpha-tocopherol (vitamin E) on the proliferation of vascular smooth muscle cells (A7r5), human osteosarcoma cells (Saos-2), fibroblasts (Balb/3T3), and neuroblastoma cells (NB2A) has been studied. The proliferation of vascular smooth muscle cells was inhibited by physiologically relevant concentrations of alpha-tocopherol, neuroblastoma cells were only sensitive to higher alpha-tocopherol concentrations, and proliferation of the other cell lines was not inhibited. The inhibition of smooth muscle cell proliferation was specific for alpha-tocopherol. Trolox, phytol, and alpha-tocopherol esters had no effect. Proliferation of smooth muscle cells stimulated by platelet-derived growth factor or endothelin was completely sensitive to alpha-tocopherol. If smooth muscle cells were stimulated by fetal calf serum, proliferation was 50% inhibited by alpha-tocopherol. No effect of alpha-tocopherol was observed when proliferation of smooth muscle cells was stimulated by bombesin and lysophosphatidic acid. The possibility of an involvement of protein kinase C in the cell response to alpha-tocopherol was suggested by experiments with the isolated enzyme and supported by the 2- to 3-fold stimulation of phorbol ester binding induced by alpha-tocopherol in sensitive cells. Moreover, alpha-tocopherol also caused inhibition of protein kinase C translocation induced by phorbol esters and inhibition of the phosphorylation of its 80-kDa protein substrate in smooth muscle cells. A model is discussed by which alpha-tocopherol inhibits cell proliferation by interacting with the cytosolic protein kinase C, thus preventing its membrane translocation and activation.     

Induction of neurite outgrowth in PC12 cells by alpha -phenyl-N-tert-butylnitron through activation of protein kinase C and the Ras-extracellular signal-regulated kinase pathway.

The spin trap alpha-phenyl-N-tert-butylnitron (PBN) is widely used for studies of the biological effects of free radicals. We previously reported the protective effects of PBN against ischemia-reperfusion injury in gerbil hippocampus by its activation of extracellular signal-regulated kinase (ERK) and suppression of both stress-activated protein kinase and p38 mitogen-activated protein kinase. In the present study, we found that PBN induced neurite outgrowth accompanied by ERK activation in PC12 cells in a dose-dependent manner. The induction of neurite outgrowth was inhibited significantly not only by transient transfection of PC12 cells with dominant negative Ras, but also by treatment with mitogen-activated protein kinase/ERK kinase inhibitor PD98059. The activation of receptor tyrosine kinase TrkA was not involved in PBN-induced neurite outgrowth. A protein kinase C (PKC) inhibitor, GF109203X, was found to inhibit neurite outgrowth. The activation of PKCepsilon was observed after PBN stimulation. PBN-induced neurite outgrowth and ERK activation were counteracted by the thiol-based antioxidant N-acetylcysteine. From these results, it was concluded that PBN induced neurite outgrowth in PC12 cells through activation of the Ras-ERK pathway and PKC.     

Nerve Growth Factor-Stimulated Nuclear S6 Kinase in PC12 Cells

Abstract: Previous work has shown that nerve growth factor (NGF) stimulates the phosphorylation of the ribosomal protein S6 in PC12 cells. In this study, we show that S6 kinase activity is also present in purified PC12 cell nuclei. This activity was increased by treatment of the cells with NGF and, to a lesser extent, by treatment with epidermal growth factor. The NGF-stimulated activity was obtained from nuclear extracts and some of its characteristics described. The increase in activity was prevented by treatment of the cells with rapamycin or with wortmannin, and the overall activity could be precipitated by antibodies directed against the p85^S6K. These data indicate that p85^S6K is the NGF-stimulated S6 kinase in PC12 cell nuclei. The presence of S6 protein in the nucleus of PC12 cells has been confirmed and evidence is presented that suggests that it is identical to a protein called SMP reported some years ago.     

Effect of the Alzheimer amyloid fragment Abeta(25-35) on Akt/PKB kinase and survival of PC12 cells

The phosphatidylinositol 3 kinase (PI3K)-Akt/PKB pathway protects neurons from apoptosis caused by diverse stress stimuli. However, its protective role against the amyloid beta peptide (Abeta), a major constituent of Alzheimer's disease plaques, has not been studied. We investigated the effect of the Abeta-derived Abeta(25-35) peptide on apoptosis and on the Akt survival pathway in PC12 cells. Cells submitted to micromolar concentrations of Abeta(25-35) exhibited increased production of reactive oxygen species (ROS) and morphological alterations consistent with apoptosis. Akt1 was activated shortly after incubation with Abeta(25-35) and Abeta(1-40) with a kinetics different to that of nerve-derived growth factor. Akt1 activation was blocked by the PI3K inhibitor wortmannin. We tested the hypothesis that Akt1 might modify the vulnerability of neural cells to apoptosis induced by Abeta(25-35). Overexpression of an active version of Akt1 attenuated the apoptotic effect of Abeta(25-35) as determined by flow cytometry. Moreover, PC12 cells overexpressing a membrane-targeted N-myristylated fusion protein of enhanced green fluorescence protein (EGFP) and mouse Akt1 exhibited lower levels of ROS than control EGFP-transfected cells. The present findings demonstrate that Akt1 is activated in response to Abeta(25-35) in a PI3K-dependent manner and that active Akt1 protects PC12 cells against the pro-apoptotic action of this peptide.     

Inhibition of Glutamate-Induced Intensification of Free Radical Reactions by Gangliosides: Possible Role in Their Protective Effect in Rat Cerebellar Granule Cells and Brain Synaptosomes

The neurotoxic effect of exposure of rat cerebellar granule cells to glutamate (I00 M) is to a large extent prevented by incubation of neurons not only with micromolar, but even with nanomolar concentrations of gangliosides GM1, GD1b, and GT1b. GM1 was also shown to decrease significantly the per cent of dead neurons in culture after induction of lipid peroxidation. Exposure to glutamate was found to cause a significant decrease of the activity of Na⁺, K⁺-ATP-ase in rat brain cortex synaptosomes, but superoxide dismutase, alpha-tocopherol, or 10–100 nM GM1 practically prevented its action. Other data showing the ability of gangliosides to inhibit the intensification of free radical reactions by glutamate (based on the estimation of methemoglobin formation, SH group content, etc.) have been obtained. The results suggest that gangliosides are able to decrease the glutamate-induced activation of free radical reactions in nerve cells. This effect appears to contribute to their protective action against glutamate neurotoxicity.     

Nitric oxide inhibits prooxidant actions of uric acid during copper-mediated LDL oxidation

Interactions between uric acid and physiologically relevant fluxes of nitric oxide ((?)NO) during copper-mediated low-density lipoprotein (LDL) oxidation were evaluated. In the absence of (?)NO, a dual pro- and antioxidant action of uric acid was evident: low concentrations of uric acid enhanced lipid oxidation and alpha-tocopherol consumption, while its protective role was observed at higher concentrations. The prooxidant effects of uric acid were mostly related to its copper-reducing ability to form Cu(+), an initiator of lipid oxidation processes. While the prooxidant action of uric acid was completely inhibited by (?)NO, the antioxidant action of (?)NO was slightly counterbalanced by uric acid. Enhancement of alpha-tocopherol consumption by uric acid was inhibited in the presence of (?)NO while additive antioxidant effects between (?)NO and uric acid were observed in conditions where uric acid spared alpha-tocopherol. Altogether, these results suggest that in the artery wall, the (?)NO/uric acid pair may exert antioxidant actions on LDL, even if increased amounts of redox active copper were available at conditions favoring prooxidant activities of uric acid.     

Non-antioxidant molecular functions of alpha-tocopherol (vitamin E)

alpha-Tocopherol (the major vitamin E component) regulates key cellular events by mechanisms unrelated with its antioxidant function. Inhibition of protein kinase C (PKC) activity and vascular smooth muscle cell growth by alpha-tocopherol was first described by our group. Later, alpha-tocopherol was shown to inhibit PKC in various cell types with consequent inhibition of aggregation in platelets, of nitric oxide production in endothelial cells and of superoxide production in neutrophils and macrophages. alpha-Tocopherol diminishes adhesion molecule, collagenase and scavenger receptor (SR-A and CD36) expression and increases connective tissue growth factor expression.