Further Characterization of Dopamine Release by Permeabilized PC 12 Cells

Rat pheochromocytoma cells (PC12) permeabilized with staphylococcal alpha-toxin release [3H]dopamine after addition of micromolar Ca2+. This does not require additional Mg2+-ATP (in contrast to bovine adrenal medullary chromaffin cells). We also observed Ca2+-dependent [3H]-dopamine release from digitonin-permeabilized PC12 cells. Permeabilization with alpha-toxin or digitonin and stimulation of the cells were done consecutively to wash out endogenous Mg2+-ATP. During permeabilization, ATP was removed effectively from the cytoplasm by both agents but the cells released [3H]dopamine in response to micromolar Ca2+ alone. Replacement by chloride of glutamate, which could sustain mitochondrial ATP production in permeabilized cells, does not significantly alter catecholamine release induced by Ca2+. However, Mg2+ without ATP augments the Ca2+-induced release. The release was unaltered by thiol-, hydroxyl-, or calmodulin-interfering substances. Thus Mg2+-ATP, calmodulin, or proteins containing -SH or -OH groups are not necessary for exocytosis in permeabilized PC12 cells.     

Protection against hydrogen peroxide-induced cytotoxicity in PC12 cells by scutellarin

The present study investigated the protective actions of the antioxidant scutellarin against the cytotoxicity produced by exposure to H2O2 in PC12 cells. This was done by assaying for MTT (3,(4,5-dimethylthiazole-2-yl)2,5-diphenyl-tetrazolium bromide) reduction and lactate dehydrogenase (LDH) release. Reactive oxygen species (ROS) and Ca2+ in cells were evaluated by fluorescent microplate reader using DCFH and Fura 2-AM, respectively, as probes. Lipid peroxidation was quantified using thiobarbituric acid-reactive substances (TBARS). Mitochondrial membrane potential (MMP) was assessed by the retention of rhodamine123 (Rh123), a specific fluorescent cationic dye that is readily sequestered by active mitochondria, depending on their transmembrane potential. The DNA content and percentage of apoptosis were monitored with flow cytometry. Vitamin E, a potent antioxidant, was employed as a comparative agent. Preincubation of PC12 cells with scutellarin prevented cytotoxicity induced by H2O2. Intracellular accumulation of ROS, Ca2+ and products of lipid peroxidation, resulting from H2O2 were significantly reduced by scutellarin. Incubation of cells with H2O2 caused a marked decrease in MMP, which was significantly inhibited by scutellarin. PC12 cells treated with H2O2 underwent apoptotic death as determined by flow cytometric assay. The percentage of this H2O2-induced apoptosis in the cells was decreased in the presence of different concentrations of scutellarin. Scutellarin exhibited significantly higher potency compared to the antioxidant vitamin E. The present findings showed that scutellarin attenuated H2O2-induced cytotoxicity, intracellular accumulation of ROS and Ca2+, lipid peroxidation, and loss of MMP and DNA, which may represent the cellular mechanisms for its neuroprotective action.     

Protective effects of Paeonia lactiflora pall on hydrogen peroxide-induced apoptosis in PC12 cells

This study was conducted to examine the antioxidative and neuroprotective effects of Paeonia lactiflora pall (PLE). Total phenolic content of PLE was 89.65 mg of gallic acid equivalent per gram of PLE. IC(50) values for reducing power, hydrogen peroxide scavenging activity, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity were 297.57, 3.33, and 32.74 microg, respectively. The protective effect of PLE against H(2)O(2)-induced oxidative damage to PC12 cells was investigated by an 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction assay and lactate dehydrogenase (LDH) release assay. After 2 h of cell exposure to 0.5 mM H(2)O(2), a marked reduction in cell survival was observed. However, this reduction was significantly prevented by 10-100 microg/ml of PLE. H(2)O(2) also induced severe apoptosis of the PC12 cells, which was indicated by a flow cytometric analysis. Interestingly, the H(2)O(2)-stressed PC12 cells that had been incubated with PLE had greatly suppressed apoptosis. The results suggest that PLE could be a candidate for a new antioxidant against neuronal diseases.     

Contribution of Intracellular Ca<Superscript>2+</Superscript> Concentration and Protein Dephosphorylation to the Induction of Dopamine Release from PC12 cells by the Green Odor Compound Hexanal

n-Hexanal (hexanal), a straight-chain six-carbon aldehyde, is mainly present in plants. Hexanal strongly affects the release of dopamine from rat striatal slices and rat pheochromocytoma (PC12) cells. In this study, we attempted to clarify the mechanism underlying the regulation of dopamine release by hexanal by using PC12 cells, which have the ability to synthesize, store, and release dopamine. The stimulation of PC12 cells with hexanal enhanced dopamine release in a time- and dose-dependent manner. Dopamine release was partially inhibited by pretreatment of the cells with BAPTA-AM, a cell-permeable Ca²⁺ chelator. In addition, the intracellular Ca²⁺ concentration was found to slowly increase after hexanal stimulation. Furthermore, the Src tyrosine kinase inhibitor PP2 partially inhibited hexanal-induced dopamine release. However, the levels of phosphorylated proteins decreased after hexanal stimulation. Hexanal stimulated the release of only a small amount of dopamine from reserpine-treated PC12 cells, in which the vesicular dopamine was depleted. These findings suggest that both an increase in the intracellular Ca²⁺ concentration and the dephosphorylation of phosphorylated proteins might be required for hexanal-stimulated release of dopamine, and that the dopamine released because of hexanal stimulation mainly comes from the dopamine vesicles. This study showed the cellular events that occurred in PC12 cells after stimulation of hexanal. Furthermore, it is important to examine the relationship between the cellular functions and the physiological effects of hexanal on dopamine release.     

The effects of dihydropyridines on neurotransmitter release from cultured neuronal cells

Depolarizing stimuli increase the release of transmitter substances from cultured PC12 pheochromocytoma cells and reaggregate cultures of mouse mesencephalic dopamine neurones. We measured the stimulated release of (3H) norepinephrine and (3H) dopamine from these systems respectively. In the cultured mouse dopaminergic neurones, several organic calcium channel blockers including nitrendipine, D-600, verapamil and diltiazem were unable to inhibit potassium-evoked transmitter release. However, release was blocked by 3 mM cobalt. The novel dihydropyridine calcium channel agonist BAY K8644 also had no effect on basal or evoked dopamine release. In contrast, BAY K8644 greatly stimulated the potassium-evoked release of (3H) norepinephrine from PC12 cells. The BAY K8644 enhanced release could be blocked by the dihydropyridine antagonist nitrendipine. These results indicate that while stimulus-secretion coupling in the PC12 cell line involves dihydropyridine sensitive calcium channels, this is not the case in primary cultured neurones.     

Non-genomic modulation of dopamine release by bisphenol-A in PC12 cells

An endocrine disruptor chemical, bisphenol-A (BPA), is reported to have several short-term actions in various tissues and/or cells; however, the mechanisms of these actions have not been fully elucidated. We investigated short-term actions evoked by BPA in pheochromocytoma PC12 cells. BPA elicited dopamine release in PC12 cells in a dose-dependent manner. A selective N-type calcium channel antagonist (omega-conotoxin GVIA) and a ryanodine receptor blocker (ryanodine) inhibited the BPA-induced dopamine release. The expression of ryanodine receptor mRNA was detected by RT-PCR in PC12 cells. Subsequently, in order to prove whether membrane receptors participate in BPA-evoked dopamine release, a guanine nucleotide-binding protein inhibitor [guanosine 5'-(beta-thio) diphosphate], cyclic AMP antagonist (Rp-cAMPS) or protein kinase A inhibitor (H7 or H89) was added to PC12 cells prior to BPA-treatment. All of these agents suppressed BPA-evoked dopamine release, indicating that multiple signaling pathways may be involved in BPA-evoked dopamine release in PC12 cells. In conclusion, we demonstrated that BPA induced dopamine release in a non-genomic manner through guanine nucleotide-binding protein and N-type calcium channels. These findings illustrate a novel function of BPA and suggest that exposure to BPA influences the function of dopaminergic neurons.     

Hydrogen peroxide induces loss of dopamine transporter activity: a calcium-dependent oxidative mechanism

H2O2 dose dependently inhibited dopamine uptake in PC12 cells and in striatal synaptosomes. Treatment with H2O2 resulted in a reversible reduction in Vmax, with no effect on its Km value. This suppressive effect of H2O2 could be relieved by reducing agents (dithiothreitol and cysteine). Furthermore, an oxidizer (dithiodipyridine) also markedly suppressed the dopamine transporter (DAT). Oxidative stress therefore might contribute to the action of H2O2. H2O2 appeared to modify DAT at both extracellular and intracellular sites because cumene-H2O2 (a radical generator mostly restricted to plasma membranes) at high concentrations also slightly suppressed DAT activity and the intracellular overexpression of catalase ameliorated the inhibitory effect of H2O2. Internalization was unlikely to be involved because concanavalin A, which blocked endocytosis, did not prevent the H2O2-evoked inhibition of DAT activity. Interestingly, H2O2 treatment evoked a Ca2+ influx in PC12 cells. Moreover, removal of external calcium by EGTA or reduction in the intracellular calcium level using BAPTA-AM reversed the inhibitory effect of H2O2. Conversely, depletion of intracellular calcium stores using thapsigargin did not affect the reduction in DAT activity by H2O2. Collectively, our results indicate that the DAT, one of the most important proteins controlling the dopaminergic system, is also a redox sensor. In addition, H2O2 might suppress the DAT by a Ca2+-dependent oxidative pathway.     

Aqueous extract of the Chinese medicine, Danggui-Shaoyao-San, inhibits apoptosis in hydrogen peroxide-induced PC12 cells by preventing cytochrome c release and inactivating of caspase cascade

Danggui-Shaoyao-San (DSS), a traditional Chinese medicine used for centuries for the enhancement of women's health, has been shown to display therapeutic efficacy on senile dementia. In the present study, using a rat pheochromocytoma (PC12) cell line, the effect of DSS on hydrogen peroxide (H2O2) induced apoptosis was studied. The apoptosis in H2O2-induced PC12 cells was accompanied by downregulation of Bcl-2, upregulation of Bax, the release of mitochondrial cytochrome c into cytosol, and sequential activation of caspase-9 and -3. DSS was able to suppress all these changes and eventually protected against H2O2-induced apoptosis. Taken together, these results suggest that treatment of PC12 cells with DSS can block H2O2-induced apoptosis by the regulation of Bcl-2 family members, as well as suppression of cytochrome c release and caspase cascade activation.     

Salidroside inhibits H2O2-induced apoptosis in PC12 cells by preventing cytochrome c release and inactivating of caspase cascade

We used a rat pheochromocytoma (PC12) cell line to study the effects of salidroside on hydrogen peroxide (H(2)O(2))-induced apoptosis. In PC12 cells, H(2)O(2)-induced apoptosis was accompanied by the down-regulation of Bcl-2, the up-regulation of Bax, the release of mitochondrial cytochrome c to cytosol, and the activation of caspase-3, -8 and -9. However, salidroside suppressed the down-regulation of Bcl-2, the up-regulation of Bax and the release of mitochondrial cytochrome c to cytosol. Moreover, salidroside attenuated caspase-3, -8 and -9 activation, and eventually protected cells against H(2)O(2)-induced apoptosis. Taken together, these results suggest that treatment of PC12 cells with salidroside can block H(2)O(2)-induced apoptosis by regulating Bcl-2 family members and by suppressing cytochrome c release and caspase cascade activation.     

Ginsenoside Rg1 protects against hydrogen peroxide-induced cell death in PC12 cells via inhibiting NF-κB activation

Oxidative stress is a major cause in neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), and cerebral ischemia. Ginsenoside Rg1, a natural product extracted from Panax ginseng C.A. Meyer, has been reported to exert notable neuroprotective activities, which partly ascribed to its antioxidative activity. However, its molecular mechanism against oxidative stress induced by exogenous hydrogen peroxide (H(2)O(2)) remained unclear. In this study, we investigated its effect on H(2)O(2)-induced cell death and explored possible signaling pathway in PC12 cells. We proved that pretreatment with Rg1 at concentrations of 0.1-10 μM remarkably reduced the cytotoxicity induced by 400 μM of H(2)O(2) in PC12 cells by MTT and Hoechst and PI double staining assay. Of note, we demonstrated the activation of NF-κB signaling pathway induced by H(2)O(2) thoroughly in PC12 cells, and Rg1 suppressed phosphorylation and nuclear translocation of NF-κB/p65, phosphorylation and degradation of inhibitor protein of κB (IκB) as well as the phosphorylation of IκB-kinase complex (IKK) by western blotting or indirect immunofluorescence assay. Besides, Rg1 also inhibited the activation of Akt and the extracellular signal-regulated kinase 1/2 (ERK1/2). Furthermore, the protection of Rg1 on H(2)O(2)-injured PC12 cells was attenuated by pretreatment with two NF-κB pathway inhibitors (JSH-23 or BOT-64). In conclusion, our results suggest that Rg1 could rescue the cell injury by H(2)O(2) via down-regulation NF-κB signaling pathway as well as Akt and ERK1/2 activation, which put new evidence on the neuroprotective mechanism of Rg1 against the oxidative stress and the regulatory role of H(2)O(2) in NF-κB pathway in PC12 cells.     

A water extract of Curcuma longa L. (Zingiberaceae) rescues PC12 cell death caused by pyrogallol or hypoxia/reoxygenation and attenuates hydrogen peroxide induced injury in PC12 cells

A number of studies indicate that free radicals are involved in the neurodegeneration in Alzheimer's disease (AD). The role of superoxide anion (O2*-) in neuronal cell injury induced by reactive oxygen species (ROS) was examined in PC12 cells using pyrogallol (1,2,3-benzenetrior), a donor to release O2*-. Pyrogallol induced PC12 cell death at concentrations, which evidently increased intracellular O2*-, as assessed by O2*- sensitive fluorescent precursor hydroethidine (HEt). A water extract of Curcuma longa L. (Zingiberaceae) (CLE), having O2*- scavenging activity rescued PC12 cells from pyrogallol-induced cell death. Hypoxia/reoxygenation injury of PC12 cells was also blocked by CLE. The present study was also conducted to examine the effect of CLE on H2O2 -induced toxicity in rat pheochromocytoma line PC12 by measuring cell lesion, level of lipid peroxidation and antioxidant enzyme activities. Following 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 increased production of malondialdehyde (MDA) were found. Pretreatment of the cells with CLE (0.5-10 microg/ml) prior to H2O2 exposure significantly elevated the cell survival, antioxidant enzyme activities and decreased the level of MDA. The above-mentioned neuroprotective effects are also observed with tacrine (THA, 1 microM), suggesting that the neuroprotective effects of cholinesterase inhibitor might partly contribute to the clinical efficacy in AD treatment. Further understanding of the underlying mechanism of the protective effects of these radical scavengers reducing intracellular O2*- on neuronal cell death may lead to development of new therapeutic treatments for hypoxic/ischemic brain injury.     

Neuroprotective effects of tetramethylpyrazine on hydrogen peroxide-induced apoptosis in PC12 cells

In the present study, we investigated the effects of tetramethylpyrazine (TMP) on hydrogen peroxide (H2O2)-induced apoptosis in PC12 cells. The apoptosis in H2O2-induced PC12 cells was accompanied by a decrease in Bcl-2/Bax protein ratio, release of cytochrome c to cytosol and the activation of caspase-3. TMP not only suppressed the down-regulation of Bcl-2, up-regulation of Bax and the release of mitochondrial cytochrome c to cytosol, but also attenuated caspase-3 activation and eventually protected against H2O2-induced apoptosis. These results indicated that TMP blocked H2O2-induced apoptosis by the regulation of Bcl-2 family members, suppression of cytochrome c release, and caspase cascade activation in PC12 cells.     

Leptinotoxin-h Action in Synaptosomes and Neurosecretory Cells: Stimulation of Neurotransmitter Release

Guinea pig brain cortex synaptosomes and neurosecretory PC12 cells were loaded with [3H]3,4-dihydroxyphenylethylamine ([3H]DA, [3H]dopamine) and then exposed to leptinotoxin-h (LPTx) (purified and partially purified preparations, obtained from the hemolymph of Leptinotarsa haldemani). In a Ca2+-containing Ringer medium the toxin induced prompt and massive release of the neurotransmitter. Half-maximal effects were obtained at concentrations estimated of approximately 3 X 10(-11) M for synaptosomes, and 1.5 X 10(-10) M for PC12 cells. Release responses in the two experimental systems investigated were dependent to different extents on the Ca2+ concentration in the medium. In synaptosomes clear, although slow, release of [3H]DA was elicited by the toxin even in Ca2+-free, EGTA-containing medium, provided that high (in the 10(-10) M range) concentrations were used; near-maximal responses were observed at 10(-5)M Ca2+. In contrast, the toxin-induced release from PC12 cells was appreciable only at 3 X 10(-5) M Ca2+, and was maximal at 2 X 10(-4) M and above. In both synaptosomes and PC12 cells Sr2+ and Ba2+ could substitute for Ca2+; Co2+ was inhibitory, whereas Mn2+ failed to modify the release induced by the toxin in Ca2+-containing medium. Organic blockers of the voltage-dependent Ca2+ channel (verapamil and nitrendipine) and calmodulin blocking drugs (trifluoperazine and calmidazolium) failed to inhibit the toxin-induced release of [3H]DA. LPTx induced profound morphological effects. Synaptosomes treated in the Ca2+-containing medium exhibited fusion of synaptic vesicles, formation of numerous infoldings and large cisternae, and alterations of mitochondria. In the Ca2+-free medium the effects were similar, except that their appearance was delayed, and mitochondria were well preserved. Swelling was observed in PC12 cells, accompanied by enlargement of the Golgi area, accumulation of multivesicular bodies, mitochondrial alterations, and decreased number of secretion granules (Ca2+-containing medium). Morphometric analyses revealed a good correlation between the decrease of both synaptic vesicles (synaptosomes) and neurosecretory granules (PC12 cells), and the release of [3H]DA measured biochemically. This is a good indication that the release effect of the toxin is due to stimulation of exocytosis. Taken as a whole, these results confirm the similarity of the effects of LPTx with alpha-latrotoxin of the black widow spider venom, mentioned in the companion article. However, differences in effect and target specificity suggest that the two toxins are specific to separate binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Introduction of Macromolecules into Bovine Adrenal Medullary Chromaffin Cells and Rat Pheochromocytoma Cells (PC12) by Permeabilization with Streptolysin O: Inhibitory Effect of Tetanus Toxin on Catecholamine Secretion

Conditions are described for controlled plasma membrane permeabilization of rat pheochromocytoma cells (PC12) and cultured bovine adrenal chromaffin cells by streptolysin O (SLO). The transmembrane pores created by SLO invoke rapid efflux of intracellular 86Rb+ and ATP, and also permit passive diffusion of proteins, including immunoglobulins, into the cells. SLO-permeabilized PC12 cells release [3H]dopamine in response to micromolar concentrations of free Ca2+. Permeabilized adrenal chromaffin cells present a similar exocytotic response to Ca2+ in the presence of Mg2+/ATP. Permeabilized PC12 cells accumulate antibodies against synaptophysin and calmodulin, but neither antibody reduces the Ca2+-dependent secretory response. Reduced tetanus toxin, although ineffective when applied to intact chromaffin cells, inhibits Ca2+-induced exocytosis by both types of permeabilized cells studied. Omission of dithiothreitol, toxin inactivation by boiling, or preincubation with neutralizing antibodies abolishes the inhibitory effect. The data indicate that plasma membrane permeabilization by streptolysin O is a useful tool to probe and define cellular components that are involved in the final steps of exocytosis.     

Antioxidation activity of tetrahydrobiopterin in pheochromocytoma PC 12 cells

Rat pheochromocytoma PC 12 cells are susceptible to the oxidative toxicity caused by H2O2, nitrofurantoin, dopamine, and xanthine/xanthine oxidase reaction. The cytotoxicities of these agents are greatly reduced by the simultaneous presence of 0.1 mM tetrahydrobiopterin (BH4), 3 units/ml horseradish peroxidase, 0.2 mM NADH, and 0.1 units/ml sheep liver dihydropteridine reductase (DHPR). Individually, BH4, NADH and DHPR have no protection against H2O2 toxicity in PC 12 cells. Peroxidase alone offers 58% of protection if cells are incubated in the medium but only 3% in Dulbecco's phosphate buffered saline. The efficiency of the BH4-mediated antioxidation system in PC 12 cells is equal to or better than ascorbic acid and catalase, depending on the source of the reactive O2 species (ROS). The reactions responsible for the BH4-antioxidation system may consist of the non-enzymatic and the peroxidase-catalyzed reduction of H2O2 to H2O by BH4 and the regeneration of BH4 by DHPR using NADH as the cofactor. The components of this defence mechanism against ROS are all normal cellular constituents and are ubiquitous in nature. This DHPR-catalyzed redox cycling of BH4 may constitute an as yet little-known antioxidation system in mammalian cells.     

Galpha(12) and galpha(13) inhibit Ca(2+)-dependent exocytosis through Rho/Rho-associated kinase-dependent pathway

The release of neurotransmitters is known to be regulated by activation of heterotrimeric G protein-coupled receptors, although precise mechanisms have not yet been elucidated. To assess the role of the G(12) family of heterotrimeric G proteins in the regulation of neurotransmitter release, we established PC12 cell lines that expressed constitutively active Galpha(12) or Galpha(13) using an isopropyl-beta-D-thiogalactoside-inducible expression system. In the cells, expression of constitutively active Galpha(12) or Galpha(13) inhibited the high K(+)-evoked [(3)H]dopamine release without any effect on the high K(+)-induced increase in intracellular Ca(2+) concentration. A Ca(2+) ionophore ionomycin-induced [(3)H]dopamine release was also inhibited by the expression of active Galpha(12) or Galpha(13). These inhibitory effects of Galpha(12) and Galpha(13) on [(3)H]dopamine release were mimicked by the expression of constitutively active RhoA. In addition, Y-27632, and inhibitor of Rho-associated kinase, a downstream Rho effector, completely abolished the inhibition of [(3)H]dopamine release by Galpha(12), Galpha(13), and RhoA. These results indicate that Ca(2+)-dependent exocytosis is regulated by Galpha(12) and Galpha(13) through a Rho/Rho-associated kinase-dependent pathway.     

Mechanism for manganese enhancement of dopamine-induced oxidative DNA damage and neuronal cell death

Although the cause of dopaminergic cell death in Parkinson's disease is still poorly understood, there is accumulating evidence suggesting that metal ions can be involved in the processes. We investigated the effect of manganese on cell death and DNA damage in PC12 cells treated with dopamine. Mn(II) enhanced cell death induced by dopamine. Mn(II) also increased the 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) contents of DNA in PC12 cells treated with dopamine. To clarify the mechanism of cellular DNA damage, we investigated DNA damage induced by dopamine and Mn(II) using (32)P-labeled DNA fragments. Mn(II) enhanced Cu(II)-dependent DNA damage by dopamine. The Mn(II)-enhanced DNA damage was greatly increased by NADH. Piperidine and formamidopyrimidine-DNA glycosylase treatment induced cleavage sites mainly at T and G of the 5'-TG-3' sequence, respectively. Bathocuproine, a Cu(I) chelator, and catalase inhibited the DNA damage. Oxygen consumption and UV-visible spectroscopic measurements showed that Mn(II) enhanced autoxidation of dopamine with H(2)O(2) formation. These results suggest that reactive species derived from the reaction of H(2)O(2) with Cu(I) participates in Mn(II)-enhanced DNA damage by dopamine plus Cu(II). Therefore, it is concluded that oxidative DNA damage induced by dopamine in the presence of Mn(II), NADH, and Cu(II) is possibly linked to the degeneration of dopaminergic neurons.     

An endogenous metabolite of dopamine, 3,4-dihydroxyphenylethanol, acts as a unique cytoprotective agent against oxidative stress-induced injury

A natural compound contained in olive oil, 3,4-dihydroxyphenylethanol (DOPE), is also known as an endogenous metabolite of dopamine. The role of DOPE in oxidative stress-induced cell damage was investigated using differentiated PC12 cells. Superoxide (O(2)(-)) and H(2)O(2) induced a dose-dependent leakage of lactate dehydrogenase (LDH) and decreased cell viability denoted by MTT assay. While O(2)(-) -induced cell damage was not affected by DOPE, pretreatment of the cells with DOPE dose-dependently prevented the leakage of LDH induced by H(2)O(2). In these cells, augmented activity of catalase was demonstrated, while the levels of glutathione and glutathione peroxidase activity remained unchanged. The effect of DOPE was abolished when an inhibitor of catalase 3-amino-l, 2,4-triazole, was included in the medium. DOPE also protected against cell damage induced by H(2)O(2), and Fe(2+). In the hydroxyl radical ((.-)OH) assay using p-nitroso-N, N-dimethylaniline (PNDA), oxidation of PNDA by (.-)OH generated by the Fenton reaction was significantly attenuated in the presence of DOPE. By an electron spin resonance spin trapping study that represents the direct activity of DOPE to scavenge (.-)OH, however, limited scavenging activity was demonstrated for DOPE. Taken together, DOPE may act as a unique cytoprotective compound in nerve tissue subjected to oxidative stress.     

Puerarin protects differentiated PC12 cells from H₂O₂-induced apoptosis through the PI3K/Akt signalling pathway

Oxidative stress has been implicated as a major mechanism underlying the pathogenesis of neurodegenerative disorders. ROS (reactive oxygen species) can cause cell death via apoptosis. NGF (nerve growth factor) differentiated rat PC12 cells have been extensively used to study the differentiation and apoptosis of neurons. This study has investigated the protective effects of puerarin in H2O2-induced apoptosis of differentiated PC12 cells, and the possible molecular mechanisms involved. Differentiated PC12 cells were incubated with 700 μM H2O2 in the absence or presence of different doses of puerarin (4, 8 and 16 μM). Apoptosis was assessed by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) analysis and Annexin V-PI (propidium iodide) double staining flow cytometry. Protein levels of phospho-Akt and phospho-BAD (Bcl-2/Bcl-XL-antagonist, causing cell death) were assayed by Western blotting. After stimulation with H2O2 for 18 h, the viability of differentiated PC12 cells decreased significantly and a large number of cells underwent apoptosis. Differentiated PC12 cells were rescued from H2O2-induced apoptosis at different concentrations of puerarin in a dose-dependent manner. This was through increased production of phospho-Akt and phospho-BAD, an effect that could be reversed by wortmannin, an inhibitor of PI3K (phosphoinositide 3-kinase). The results suggest that puerarin may have neuroprotective effect through activation of the PI3K/Akt signalling pathway.