Sample records for rat pc12 pheochromocytoma

  1. Effect of Inhalational Anesthetics on Cytotoxicity and Intracellular Calcium Differently in Rat Pheochromocytoma Cells (PC12)

    Qiujun WANG; Kezhong LI; Shanglong YAO


    Isoflurane, a commonly used inhaled anesthetic, induces apoptosis in rat pheochromo-cytoma cells (PC12) in a concentration- and time-dependent manner with unknown mechanism. We hypothesized that isoflurane induced apoptosis by causing abnormal calcium release from the endo-plasmic reticulum (ER) via activation of inositol 1,4,5-trisphosphate (IP3) receptors. Alzheimer's pre-senilin-1 (PS1) mutation increased activity of IP3 receptors and therefore rendered cells vulnerable to isoflurane-induced cytotoxicity. Sevoflurane and desflurane had less ability to disrupt intraceUular calcium homeostasis and thus being less potent to cause cytotoxicity. This study examined and com- pared the cytotoxic effects of various inhaled anesthetics on PC12 cells transfected with the Alz- heimer's mutated Psi (L286V) and the disruption of intracellular calcium homeostasis. PC12 cells transfected with wild type (WT) and mutated PS1 (L286V) were treated with equivalent of 1 MAC of isoflurane, sevoflurane and desflurane for 12 h. MTT reduction and LDH release assays were per- formed to evaluate cell viability. Changes of calcium concentration in cytosolic space ([Ca2+]c) were determined after exposing different types of cells to various inhalational anesthetics. The effects of IP3 receptor antagonist xestospongin C on isoflurane-induced cytotoxicity and calcium release from the ER in L286V PC12 cells were also determined. The results showed that isoflurane at 1 MAC for 12 h induced cytoxicity in L286V but not WT PC12 cells, which was also associated with greater and faster elevation of peak [Ca2+]c in L286V than in the WT cells. Xestospongin C significantly amelio- rated isoflurane cytotoxicity in L286V cells, as well as inhibited the calcium release from the ER in L286V cells. Sevoflurane and desflurane at equivalent exposure to isoflurane did not induce similar cytotoxicity or elevation of peak [Ca2+]c in L286V PC12 cells. These results suggested that isoflurane induced cytoxicity by

  2. Activation of glycogen phosphorylase in rat pheochromocytoma PC12 cells and isolated hepatocytes by organophosphates.

    Kauffman, F C; Davis, L H; Whittaker, M


    Several organophosphates including diisopropylfluorophosphonate (DPF) and a variety of compounds used as chemical warfare agents produced dose- and time-dependent increases in phosphorylase-a, the phosphorylated form of glycogen phosphorylase in rat pheochromocytoma cells, PC12, and isolated hepatocytes. Increases in phosphorylase-a did not occur in cells exposed to the carbamates, physostigmine or pyridostigmine, or to O-ethyl S-2-diisopropylaminoethylmethyl-phosphonathiolate (VX), an organophosphate which is protonated at physiological pH. When extracellular pH was increased to pH 8, VX acted like the other organophosphates and increased phosphorylase-a activity. The possibility that organophosphates increase phosphorylase-a in intact cells by releasing Ca2+ from intracellular binding sites is supported by the following findings: organophosphate-induced increases in phosphorylase-a did not correlate with changes in cyclic AMP in the two cell types studied; in PC12 cells, increases in this activity occurred in the absence of extracellular calcium and were not inhibited by the calcium channel blocker, verapamil; fluorescence of the calcium sensitive dye, Quin-2, in PC12 cells preloaded with the acetoxymethyl ester of the dye was increased by soman; finally, addition of the calcium ionophore, A23187, to PC12 cells maintained in calcium-free medium caused sarin-stimulated phosphorylase-a activity to return rapidly to basal levels. Collectively, these data argue strongly that organophosphates increase phosphorylase-a activity in intact cells via a novel mechanism involving release of calcium from intracellular binding sites.

  3. Nerve growth factor activates calcium-insensitive protein kinase C-epsilon in PC-12 rat pheochromocytoma cells.

    Ohmichi, M; Zhu, G.; Saltiel, A R


    Protein kinase C (PKC) family members were examined in PC-12 rat pheochromocytoma cells to evaluate their role in the action of nerve growth factor (NGF). Immunoblot analysis of whole cell lysates using antibodies against various PKC isoforms revealed that PC-12 cells contained PKC-alpha, -delta, -epsilon and zeta. Assay of the protein kinase activity in these different anti-PKC immunoprecipitates demonstrated that NGF stimulated the kinase activity of PKC-epsilon, but not PKC-alpha, -delta a...

  4. The endothelin-2/vasoactive intestinal contractor gene: expression and promoter activity in PC12 rat pheochromocytoma cells.

    Saida, K; Uchide, T; Usui, A; Gao, X D; Tomizuka, N; Oka, S; Masuda, H


    In order to understand the physiological roles of vasoactive intestinal contractor (VIC)/endothelin-2 (ET-2), we examined the expression of this peptide by specific reverse transcriptase polymerase chain reaction (RT-PCR) analysis and found that PC12 rat pheochromocytoma cells express the VIC gene. The 5'-flanking 1.0 kilo base pair (kb) region of the mouse VIC gene is sufficient to express a secreted alkaline phosphatase (SEAP) reporter gene in transiently transfected PC12 cells. The 1.0 kb promoter region may contain cis-acting elements that determine the rate of the VIC gene transcription in PC12 cells.

  5. A rapidly inactivating Ca2(+)-dependent K+ current in pheochromocytoma cells (PC12) of the rat.

    Pun, R Y; Behbehani, M M


    The membrane electrical properties of undifferentiated pheochromocytoma cells of the rat (PC12) were studied using both current- and voltage-clamp techniques with the use of low-resistance blunt-tipped micropipettes (patch electrodes). In the presence of tetrodotoxin (TTX, 2-3 microM), a spike-like wave form with a prominent after-hyperpolarization (AHP) was recorded following brief (less than 10 ms) depolarizing current pulses. The inorganic divalent cations, Cd2+ (0.5 mM), Mn2+ (4 mM), and 0 mM Ca2+/4 mM Mg2+ solution prolonged the duration, attenuated the AHP, slowed the rate of repolarization, and slightly enhanced the amplitude of this wave form. A rapidly inactivating outward current was recorded in over 70% of the cells under voltage-clamp conditions. This transient current was elicited at about -30 mV, and was blocked by tetraethylammonium (5 mM), inorganic divalent cations (Cd2+, 0.5 mM; Mn2+, 4 mM; Ba2+, 3 mM), and removal of Ca2+ (0 mM Ca2+/4 mM Mg2+) from the local perfusion medium. In addition, 4-aminopyridine (5 mM), which blocks the transient outward K+ current IA in a variety of excitable cells, did not have any appreciable effect on this rapidly inactivating current. Moreover, it was possible to elicit the current at a holding potential of -40 mV. The reversal potential of this current was -90 mV, and shifted positively when extracellular K+ concentrations were elevated. It is concluded that PC12 cells have a rapidly inactivating Ca2(+)-dependent K+ current. A possible explanation for the transient nature of this current may be the presence of an effective intracellular Ca2+ buffering (uptake or extrusion) system.

  6. Subcellular localization of WD40 repeat 1 protein in PC12 rat pheochromocytoma cells.

    Shin, Dong Hoon; Lee, Eunju; Chung, Yoon Hee; Mun, Ga Hee; Park, Ji yeong; Lomax, Margaret I; Oh, Seung Ha


    The dynamics of actin filament protein is crucial for various physiological processes of the cells. Among the proteins correlating with actin dynamics, a novel 67-kDa WD40 repeat protein 1 (WDR1) was the vertebrate homologue of actin-interacting protein 1 (Aip1). Even though previous studies have provided the clues on the function of WDR1 in specific organs under pathological conditions, the exact subcellular localization of WDR1 is not known. Therefore, in the present study, we undertook to determine the distribution of WDR1 within PC12 pheochromocytoma cells (PC12 cells) using light and electron microscopic techniques. Double immunocytochemistry clearly showed that WDR1 immunoreactivities (IRs) were co-localized with anti-actin antibody, suggesting the involvement of WDR1 in actin dynamics. WDR1 immunoreactivities (IRs) in PC12 cells showed different distribution patterns as nerve growth factor (NGF) concentrations varied. During active proliferation, the distribution of WDR1 IRs seemed to be similar to those found in cortical actin patches, whereas WDR1 IR was observed in cytoplasmic actin cables after PC12 cells were induced to differentiate by treating with NGF. Though further studies are necessary to determine the function of WDR1, the current data represents a first step towards the in vitro study of WDR1 protein.

  7. Phosphorylation-dependent regulation of phospholipase D2 by protein kinase C delta in rat Pheochromocytoma PC12 cells.

    Han, Jung Min; Kim, Jae Ho; Lee, Byoung Dae; Lee, Sang Do; Kim, Yong; Jung, Yon Woo; Lee, Sukmook; Cho, Wonhwa; Ohba, Motoi; Kuroki, Toshio; Suh, Pann-Ghill; Ryu, Sung Ho


    Many studies have shown that protein kinase C (PKC) is an important physiological regulator of phospholipase D (PLD). However, the role of PKC in agonist-induced PLD activation has been mainly investigated with a focus on the PLD1, which is one of the two PLD isoenzymes (PLD1 and PLD2) cloned to date. Since the expression of PLD2 significantly enhanced phorbol 12-myristate 13-acetate (PMA)- or bradykinin-induced PLD activity in rat pheochromocytoma PC12 cells, we investigated the regulatory mechanism of PLD2 in PC12 cells. Two different PKC inhibitors, GF109203X and Ro-31-8220, completely blocked PMA-induced PLD2 activation. In addition, specific inhibition of PKC delta by rottlerin prevented PLD2 activation in PMA-stimulated PC12 cells. Concomitant with PLD2 activation, PLD2 became phosphorylated upon PMA or bradykinin treatment of PC12 cells. Moreover, rottlerin blocked PMA- or bradykinin-induced PLD2 phosphorylation in PC12 cells. Expression of a kinase-deficient mutant of PKC delta using adenovirus-mediated gene transfer inhibited the phosphorylation and activation of PLD2 induced by PMA in PC12 cells, suggesting the phosphorylation-dependent regulation of PLD2 mediated by PKC delta kinase activity in PC12 cells. PKC delta co-immunoprecipitated with PLD2 from PC12 cell extracts, and associated with PLD2 in vitro in a PMA-dependent manner. Phospho-PLD2 immunoprecipitated from PMA-treated PC12 cells and PLD2 phosphorylated in vitro by PKC delta were resolved by two-dimensional phosphopeptide mapping and compared. At least seven phosphopeptides co-migrated, indicating the direct phosphorylation of PLD2 by PKC delta inside the cells. Immunocytochemical studies of PC12 cells revealed that after treatment with PMA, PKC delta was translocated from the cytosol to the plasma membrane where PLD2 is mainly localized. These results suggest that PKC delta-dependent direct phosphorylation plays an important role in the regulation of PLD2 activity in PC12 cells.

  8. Urocortin stimulates tyrosine hydroxylase activity via the cAMP/protein kinase a pathway in rat Pheochromocytoma PC12 cells.

    Nanmoku, Toru; Takekoshi, Kazuhiro; Fukuda, Toshiyuki; Isobe, Kazumasa; Shibuya, Shunsuke; Kawakami, Yasushi

    Urocortin is a novel mammalian member of the corticotrophin releasing factor (CRF)-related peptides. We have investigated the expression, mechanism of action and second messenger for urocortin in rat pheochromocytoma PC12 cells. We initially confirmed the expression of urocortin and CRF-R2beta, which is thought to be an endogenous receptor for urocortin, in PC12 cells. We also demonstrate that urocortin (> or = 1 nM) significantly elevates the level of cAMP in these cells. Moreover, alpha-helical CRF-(9-41), a more specific antagonist of CRF-R2 than CRF-R1 and the adenylate cyclase inhibitor SQ22536, inhibited the urocortin-induced increase in the level of cAMP. Thus, urocortin may exert its physiological role in chromaffin cells via CRF-R2beta coupling to adenylate cyclase. Urocortin (> or = 1 nM) significantly increased the mRNA level and activity of tyrosine hydroxylase (TH), a rate-limiting enzyme in the biosynthesis of catecholamine. Furthermore, urocortin-induced changes in TH-mRNA and activity were inhibited by H89 (a PKA inhibitor) and SQ22536 as well as alpha-helical CRF-(9-41). However, urocortin did not affect DNA synthesis or catecholamine secretion in these cells. In conclusion, we have demonstrated that urocortin stimulates catecholamine biosynthesis via the cAMP/protein kinase A pathway in PC12 cells, where both urocortin and its receptor, CRF-R2, are expressed.

  9. Toxic effect of zinc nanoscale metal-organic frameworks on rat pheochromocytoma (PC12) cells in vitro

    Ren, Fei, E-mail: [Department of Pharmacy, Nanfang Hospital, Southern Medical University, Guangzhou 510515 (China); Yang, Baochun; Cai, Jing [Department of Pharmacy, Nanfang Hospital, Southern Medical University, Guangzhou 510515 (China); Jiang, Yaodong [Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou 510515 (China); Xu, Jun [Department of Health Economy Administration, Nanfang Hospital, Southern Medical University, Guangzhou 510515 (China); Wang, Shan [Department of Pharmacy, Winthrop University Hospital, Mineola, NY 11501 (United States)


    Highlights: • Metal-organic frameworks (MOFs) represent a newborn family of hybrid materials. • MOFs have already shown promise in a number of biological applications. • The biological applications of MOFs raise concerns for potential cytotoxicity. • Substantial information about MOF's neurotoxicity is still quite scarce. • This study reveals for the first time the interaction of MOFs with neural cells. - Abstract: Metal-organic frameworks (MOFs) possess unique properties desirable for delivery of drugs and gaseous therapeutics, but their uncharacterized interactions with cells raise increasing concerns of their safety in such biomedical applications. We evaluated the adverse effects of zinc nanoscale MOFs on the cell morphology, cytoskeleton, cell viability and expression of neurotrophin signaling pathway-associated GAP-43 protein in rat pheochromocytoma PC12 cells. At the concentration of 25 μg/ml, zinc MOFs did not significantly affect morphology, viability and membrane integrity of the cells. But at higher concentrations (over 100 μg/ml), MOFs exhibited a time- and concentration-dependent cytotoxicity, indicating their entry into the cells via endocytosis where they release Zn{sup 2+} into the cytosol to cause increased intracellular concentration of Zn{sup 2+}. We demonstrated that the toxicity of MOFs was associated with a disrupted cellular zinc homeostasis and down-regulation of GAP-43 protein, which might be the underlying mechanism for the improved differentiation in PC12 cells. These findings highlight the importance of cytotoxic evaluation of the MOFs before their biomedical application.

  10. Toxic effect of zinc nanoscale metal-organic frameworks on rat pheochromocytoma (PC12) cells in vitro.

    Ren, Fei; Yang, Baochun; Cai, Jing; Jiang, Yaodong; Xu, Jun; Wang, Shan


    Metal-organic frameworks (MOFs) possess unique properties desirable for delivery of drugs and gaseous therapeutics, but their uncharacterized interactions with cells raise increasing concerns of their safety in such biomedical applications. We evaluated the adverse effects of zinc nanoscale MOFs on the cell morphology, cytoskeleton, cell viability and expression of neurotrophin signaling pathway-associated GAP-43 protein in rat pheochromocytoma PC12 cells. At the concentration of 25 μg/ml, zinc MOFs did not significantly affect morphology, viability and membrane integrity of the cells. But at higher concentrations (over 100 μg/ml), MOFs exhibited a time- and concentration-dependent cytotoxicity, indicating their entry into the cells via endocytosis where they release Zn(2+) into the cytosol to cause increased intracellular concentration of Zn(2+). We demonstrated that the toxicity of MOFs was associated with a disrupted cellular zinc homeostasis and down-regulation of GAP-43 protein, which might be the underlying mechanism for the improved differentiation in PC12 cells. These findings highlight the importance of cytotoxic evaluation of the MOFs before their biomedical application.

  11. Endosomes and lysosomes are involved in early steps of Tl(III)-mediated apoptosis in rat pheochromocytoma (PC12) cells.

    Hanzel, Cecilia E; Almeira Gubiani, María F; Verstraeten, Sandra V


    The mechanisms that mediate thallium (Tl) toxicity are still not completely understood. The exposure of rat pheochromocytoma (PC12) cells to Tl(I) or Tl(III) activates both mitochondrial (Tl(I) and Tl(III)) and extrinsic (Tl(III)) pathways of apoptosis. In this work we evaluated the hypothesis that the effects of Tl(III) may be mediated by the damage to lysosomes, where it might be incorporated following the route of iron uptake. PC12 cells exposed for 3 h to 100 μM Tl(III) presented marked endosomal acidification, effect that was absent when cells were incubated in a serum-free medium and that was fully recovered when the latter was supplemented with transferrin. After 6 h of incubation the colocalization of cathepsins D and B with the lysosomal marker Lamp-1 was decreased together with an increase in the total activity of the enzymes. A permanent damage to lysosomes after 18 h of exposure was evidenced from the impairment of acridine orange uptake. Cathepsin D caused the cleavage of pro-apoptotic protein BID that is involved in the activation of the intrinsic pathway of apoptosis. Supporting that, BID cleavage and the activation of caspase 3 by Tl(III) were fully prevented when cells were preincubated with cathepsin D inhibitor (pepstatin A) and only partially prevented when cathepsin B inhibitor (E64d) was used. None of these inhibitors affected BID cleavage or caspase 3 activation in Tl(I)-treated cells. Together, experimental results support the role of Tl(III) uptake by the acidic cell compartments and their involvement in the early steps of Tl(III)-mediated PC12 cells apoptosis.

  12. Physiological and morphological studies of rat pheochromocytoma cells (PC12) chemically fused and grown in culture.

    O'Lague, P. H.; Huttner, S L


    Cell fusion induced by polyethylene glycol has been used to produce in culture giant multinucleate PC12 cells (up to 300 micron in diameter compared to 10-20 micron for unfused cells). Fused cells, like their unfused counterparts, were found to express various neuronal properties. They contained catecholamines. In the presence of nerve growth factor they extended long processes and expressed Na+, Ca2+, and K+ conductances generally associated with excitable cells. In the absence of nerve grow...

  13. Physiological and morphological studies of rat pheochromocytoma cells (PC12) chemically fused and grown in culture.

    O'Lague, P H; Huttner, S L


    Cell fusion induced by polyethylene glycol has been used to produce in culture giant multinucleate PC12 cells (up to 300 micron in diameter compared to 10-20 micron for unfused cells). Fused cells, like their unfused counterparts, were found to express various neuronal properties. They contained catecholamines. In the presence of nerve growth factor they extended long processes and expressed Na+, Ca2+, and K+ conductances generally associated with excitable cells. In the absence of nerve growth factor these cells neither grew long processes nor generated Na+-spikes. Other neuronal properties were also observed.

  14. The influence of magnetic fields exposure on neurite outgrowth in PC12 rat pheochromocytoma cells

    Fan, W.; Ding, J.; Duan, W.; Zhu, Y. M.


    The aim of present work was to investigate the influence of magnetic fields exposure on neurite outgrowth in PC12 cells. The neurite number per cell, length of neurites and directions of neurite growth with respect to the direction of the magnetic field were analyzed after exposure to 50 Hz electromagnetic field for 96 h. A promotion was observed under a weak field (0.23 mT), as the average number of neurites per cell increased to 2.38±0.06 compared to 1.91±0.07 neurites/cell of the control dishes, while inhibition and directional outgrowth was evident under a relatively stronger field (1.32 mT). Our work shows that biological systems can be very sensitive to the strength of electromagnetic field.

  15. The influence of magnetic fields exposure on neurite outgrowth in PC12 rat pheochromocytoma cells

    Fan, W.; Ding, J. E-mail:; Duan, W.; Zhu, Y.M


    The aim of present work was to investigate the influence of magnetic fields exposure on neurite outgrowth in PC12 cells. The neurite number per cell, length of neurites and directions of neurite growth with respect to the direction of the magnetic field were analyzed after exposure to 50 Hz electromagnetic field for 96 h. A promotion was observed under a weak field (0.23 mT), as the average number of neurites per cell increased to 2.38{+-}0.06 compared to 1.91{+-}0.07 neurites/cell of the control dishes, while inhibition and directional outgrowth was evident under a relatively stronger field (1.32 mT). Our work shows that biological systems can be very sensitive to the strength of electromagnetic field.


    Mn is a neurotoxin that leads to a syndrome resembling Parkinson's disease after prolonged exposure to high concentrations. Our laboratory has been investigating the mechanism by which Mn induces neuronal cell death. To accomplish this, we have utilized rat pheochromocytom...

  17. Photosensitizer-induced fluorescence of the rat adrenal gland and rat pheochromocytoma cells (PC 12) by meso-tetra(hydroxyphenyl)chlorin (mTHPC)

    Colombo-Benkmann, Mario; Muhm, Markus; Gahlen, Johannes; Heym, Christine; Senninger, Norbert


    Rat adrenal glands exhibit an intense mTHPC-induced fluorescence. The objective of our study was the identification of adrenal cells exhibiting mTHPC-induced fluorescence under normal conditions and under stimulation of adrenal proliferation by reserpine. Furthermore mTHPC-uptake of rat pheochromocytoma (PC 12) cells was investigated. Four male Wistar rats received 0.5 mg mTHPC/kg iv 48 hours before perfusion. Furthermore four rats received reserpine (2 mg/kg im od), bromo-deoxy-uridine (BrdU; 50 mg/kg ip od) each for one week and mTHPC (0.5 mg/kg) 48 hours before perfusion. BrdU was detected immunohistochemically. PC 12-cells were incubated with 0.5 mg mTHPC/l culture medium for 24 or 48 hours. Cells and tissues were examined by fluorescence microscopy. The adrenal cortex exhibited an intense mTHPC-induced fluorescence. The adrenal medulla fluoresced faintly. Reserpine increased fluorescence of intramedullary cells, not coinciding with adrenal proliferation. Cortical fluorescence remained unchanged. PC 12-cells lying singly or in small groups and differentiating cells showed a more intense mTHPC- induced fluorescence than confluent cells. Differences of cortical and medullary uptake of mTHPC are independent of proliferation and may be explained by lipophilia of mTHPC, since adrenocytes have an uptake mechanism for cholesterol. The difference of mTHPC-uptake between PC 12-cells and chromaffin cells implicate the possibility of photodynamic applications for medullary neoplasia.

  18. Epidermal growth factor-receptor interaction in rat pheochromocytoma (PC12) and human epidermoid A431 cells: Biochemical and ultrastructural studies

    Laat, S.W. de; Boonstra, J.; Mummery, C.L.; Defize, L.; Leunissen, J.; Verkleij, A.J.


    Pheochromocytoma cells (clone PC12) have specific plasmamembrane receptors for both epidermal growth factor (EGF) and nerve growth factor (NGF). These growth factors have however, opposite biological effects in PC12 cells; EGF acts mitogenically, while NGF induces differentiation and causes arrest o

  19. Interleukin 1 expression is inducible by nerve growth factor in PC12 pheochromocytoma cells.

    Alheim, K; Andersson, C; Tingsborg, S; Ziolkowska, M; Schultzberg, M. (Marianne); Bartfai, T


    Expression of the cytokine interleukin 1 alpha (IL-1 alpha) was demonstrated in the rat PC12 pheochromocytoma cell line by (i) immunohistochemistry using rabbit polyclonal antisera raised against the recombinant murine IL-1 alpha, (ii) an ELISA, and (iii) a specific cell conversion bioassay based on the use of LBRM33-1A5 cells. IL-1 alpha mRNA was demonstrated in the PC12 cells, by PCR amplification. Constitutive expression of IL-1 alpha in PC12 cells was demonstrated in all experiments, alth...

  20. Nuclear translocation of phosphatidylinositol 3-kinase in rat pheochromocytoma PC 12 cells after treatment with nerve growth factor.

    Neri, L M; Milani, D; Bertolaso, L; Stroscio, M; Bertagnolo, V; Capitani, S


    Immunocytochemical analysis of PI 3-kinase localization in PC 12 cells demonstrates that the enzyme translocates to the nucleus after cell treatment with differentiating doses of NGF. The association of PI 3-kinase to the nucleus occurs rapidly (within minutes) and increases with the time of exposure of NGF. We suggest that PI-3 kinase specific localization may determine the production of novel phosphoinositides in cell compartments targeted to effect diverse cell responses. The nuclear translocation is consistent with accumulating data on the existence of a nuclear inositol lipid cycle which could also include 3-phosphorylated inositides, participating to the modulation of the cell response to extracellular stimuli.

  1. Phosphodiesterase 2 negatively regulates adenosine-induced transcription of the tyrosine hydroxylase gene in PC12 rat pheochromocytoma cells.

    Makuch, Edyta; Kuropatwa, Marianna; Kurowska, Ewa; Ciekot, Jaroslaw; Klopotowska, Dagmara; Matuszyk, Janusz


    Adenosine induces expression of the tyrosine hydroxylase (TH) gene in PC12 cells. However, it is suggested that atrial natriuretic peptide (ANP) inhibits expression of this gene. Using real-time PCR and luciferase reporter assays we found that ANP significantly decreases the adenosine-induced transcription of the TH gene. Results of measurements of cyclic nucleotide concentrations indicated that ANP-induced accumulation of cGMP inhibits the adenosine-induced increase in cAMP level. Using selective phosphodiesterase 2 (PDE2) inhibitors and a synthetic cGMP analog activating PDE2, we found that PDE2 is involved in coupling the ANP-triggered signal to the cAMP metabolism. We have established that ANP-induced elevated levels of cGMP as well as cGMP analog stimulate hydrolytic activity of PDE2, leading to inhibition of adenosine-induced transcription of the TH gene. We conclude that ANP mediates negative regulation of TH gene expression via stimulation of PDE2-dependent cAMP breakdown in PC12 cells.

  2. Chorein Sensitive Dopamine Release from Pheochromocytoma (PC12 Cells

    Sabina Honisch


    Full Text Available Background: Chorein, a protein supporting activation of phosphoinositide 3 kinase (PI3K, participates in the regulation of actin polymerization and cell survival. A loss of function mutation of the chorein encoding gene VPS13A (vacuolar protein sorting-associated protein 13A leads to chorea-acanthocytosis (ChAc, a neurodegenerative disorder with simultaneous erythrocyte akanthocytosis. In blood platelets chorein deficiency has been shown to compromise expression of vesicle-associated membrane protein 8 (VAMP8 and thus degranulation. The present study explored whether chorein is similarly involved in VAMP8 expression and dopamine release of pheochromocytoma (PC12 cells. Methods: Chorein was down-regulated by silencing in PC12 cells. Transmission electron microscopy was employed to quantify the number of vesicles, RT-PCR to determine transcript levels, Western blotting to quantify protein expression and ELISA to determine dopamine release. Results: Chorein silencing significantly reduced the number of vesicles, VAMP8 transcript levels and VAMP8 protein abundance. Increase of extracellular K+ from 5 mM to 40 mM resulted in marked stimulation of dopamine release, an effect significantly blunted by chorein silencing. Conclusions: Chorein deficiency down-regulates VAMP8 expression, vesicle numbers and dopamine release in pheochromocytoma cells.

  3. Phytoestrogens genistein and daidzin enhance the acetylcholinesterase activity of the rat pheochromocytoma cell line PC12 by binding to the estrogen receptor

    Isoda, Hiroko; Talorete, Terence P. N.; Kimura, Momoko; Maekawa, Takaaki; Inamori, Yuhei; Nakajima, Nobuyoshi; Seki, Humitake


    Some compounds derived from plants have been known to possess estrogenic properties and can thus alter the physiology of higher organisms. Genistein and daidzin are examples of these phytoestrogens, which have recently been the subject of extensive research. In this study, genistein and daidzin were found to enhance the acetylcholinesterase (AChE) activity of the rat neuronal cell line PC12 at concentrations as low as 0.08 μM by binding to the estrogen receptor (ER). Results have shown that t...

  4. Phytoestrogens genistein and daidzin enhance the acetylcholinesterase activity of the rat pheochromocytoma cell line PC12 by binding to the estrogen receptor.

    Isoda, Hiroko; Talorete, Terence P N; Kimura, Momoko; Maekawa, Takaaki; Inamori, Yuhei; Nakajima, Nobuyoshi; Seki, Humitake


    Some compounds derived from plants have been known to possess estrogenic properties and can thus alter the physiology of higher organisms. Genistein and daidzin are examples of these phytoestrogens, which have recently been the subject of extensive research. In this study, genistein and daidzin were found to enhance the acetylcholinesterase (AChE) activity of the rat neuronal cell line PC12 at concentrations as low as 0.08 muM by binding to the estrogen receptor (ER). Results have shown that this enhancement was effectively blocked by the known estrogen receptor antagonist tamoxifen, indicating the involvement of the ER in AChE induction. That genistein and daidzin are estrogenic were confirmed in a cell proliferation assay using the human breast cancer cell line MCF7. This proliferation was also blocked by tamoxifen, again indicating the involvement of the ER. On the other hand, incubating the PC12 cells in increasing concentrations of 17 beta-estradiol (E2) did not lead to enhanced AChE activity, even in the presence of genistein or daidzin. This suggests that mere binding of an estrogenic compound to the ER does not necessarily lead to enhanced AChE activity. Moreover, the effect of the phytoestrogens on AChE activity cannot be expressed in the presence of E2 since they either could not compete with the natural ligand in binding to the ER or that E2 down-regulates its own receptor. This study clearly suggests that genistein and daidzin enhance AChE activityin PC12 cells by binding to the ER; however, the actual mechanism of enhancement is not known.


    Differential modulation of catecholamines by chlorotriazine herbicides in pheochromocytoma (PC12) cells in vitro.Das PC, McElroy WK, Cooper RL.Curriculum in Toxicology, University of North Carolina, Chapel Hill 27599, USA.Epidemiological, wildlife, and lab...

  6. Arachidonic acid release from PC12 pheochromocytoma cells is regulated by I1-imidazoline receptors.

    Ernsberger, P


    Rat PC 12 pheochromocytoma cells lack alpha2-adrenergic receptors but express plasma membrane I1-imidazoline receptors. In response to the I1-agonist moxonidine, diglycerides are generated via phosphatidylcholine-selective phospholipase C, and prostaglandin E2 is released. This report characterizes I-receptor-mediated release of arachidonic acid, the precursor to the prostaglandins. PC12 cells were incubated with [3H]arachidonic acid for 24 h and superfused with 0.01% bovine serum albumin in Krebs' physiological buffer at 1 ml/min. Calcium ionophore increased arachidonic acid release only marginally, implying that in PC12 cells arachidonic acid release is not driven by calcium. The I1-agonist moxonidine at concentrations between 10 nM and 1.0 microM rapidly elicited up to two-fold increases in [3H]arachidonic acid release. Guanabenz, a potent alpha2-agonist and I2-ligand, had no effect. The selective I1-antagonist efaroxan blocked the action of moxonidine. The phospholipase A2 inhibitor aristolochic acid had no effect, suggesting that arachidonic acid release may be through an indirect pathway, possibly involving diglycerides. Thus, I1-imidazoline receptors in PC12 cells are coupled to arachidonic acid release through an as yet unknown pathway.

  7. Nucleofection of Rat Pheochromocytoma PC-12 Cells with Human Mutated Beta-Amyloid Precursor Protein Gene (APP-sw) Leads to Reduced Viability, Autophagy-Like Process, and Increased Expression and Secretion of Beta Amyloid

    Beata Pająk; Elżbieta Kania; Arkadiusz Orzechowski


    Pheochromocytoma PC-12 cells are immune to physiological stimuli directed to evoke programmed cell death. Besides, metabolic inhibitors are incapable of sensitizing PC-12 cells to extrinsic or intrinsic apoptosis unless they are used in toxic concentrations. Surprisingly, these cells become receptive to cell deletion after human APP-sw gene expression. We observed reduced cell viability in GFP vector + APP-sw-nucleofected cells (drop by 36%) but not in GFP vector − or GFP vector + APP-wt-nucl...

  8. Rectification of Acetylcholine-Elicited Currents in PC12 Pheochromocytoma Cells

    Ifune, C. K.; Steinbach, J. H.


    The current-voltage (I-V) relationship for acetylcholine-elicited currents in the rat pheochromocytoma cell line PC12 is nonlinear. Two voltage-dependent processes that could account for the whole-cell current rectification were examined, receptor channel gating and single receptor channel permeation. We found that both factors are involved in the rectification of the whole-cell currents. The voltage dependence of channel gating determines the shape of the I-V curve at negative potentials. The single-channel I-V relationship is inwardly rectifying and largely responsible for the characteristic shape of the whole-cell I-V curve at positive potentials. The rectification of the single-channel currents is produced by the voltage-dependent block of outward currents by intracellular Mg2+ ions.


    ABSTRACTPotential Mechanisms Responsible for Chlorotriazine-induced Changes in Catecholamine Metabolism in Pheochromocytoma (PC12) Cells*PARIKSHIT C. DAS1, WILLIAM K. McELROY2 , AND RALPH L. COOPER2+ 1Curriculum in Toxicology, University of North Carolina, Chape...

  10. Internalization of nerve growth factor by pheochromocytoma PC12 cells: absence of transfer to the nucleus.

    Rohrer, H; Schäfer, T; Korsching, S; Thoenen, H


    The intracellular distribution of 125I-labeled nerve growth factor (NGF) in rat pheochromocytoma PC12 cells was studied by quantitative electron microscopic (EM) autoradiography and by subcellular fractionation. PC12 cells were grown as monolayer cultures in medium supplemented with serum in the presence of 125I-NGF. EM autoradiography showed that 125I-NGF was localized at the plasma membrane and cytoplasmic compartments but did not accumulate in the nuclear chromatin or in the nuclear membrane compartment of cells analyzed after 1 hr and 1, 2, and 8 d of incubation with 125I-NGF. 125I-NGF also was not detected in nuclear subcellular fractions prepared from cells grown in serum-supplemented medium either in suspension for 1 d or in monolayer cultures for 1 to 8 d. In contrast, and in confirmation of the results of Yankner and Shooter (Yankner, B. A., and E. M. Shooter (1979) Pro. Natl. Acad. Sci. U. S. A. 76: 1269-1273), about 60% of the cell-bound 125I-NGF was found in the nuclear pellet after cell fractionation if the cells had been kept previously in suspension for 1 d in phosphate-buffered saline supplemented with 0.2% glucose, 0.1% bovine serum albumin, and 125I-NGF. The ultrastructure of PC12 cells grown under such conditions, however, revealed signs of varying degrees of damage. Autoradiography of the nuclear pellet from these cells showed the grains to be located mainly over damaged nuclei or over cell debris between nuclei. It is concluded that NGF, after binding to specific receptors at the plasma membrane, is transferred to membrane-confined cytoplasmic compartments but does not have to be transferred further to the nuclear membrane or to the nuclear chromatin as a prerequisite for its physiological action.

  11. Induction of calbindin-D 28K gene and protein expression by physiological stimuli but not in calcium-mediated degeneration in rat PC12 pheochromocytoma cells.

    Vyas, S; Michel, P P; Copin, M C; Biguet, N F; Thomasset, M; Agid, Y


    To understand the role of calbindin-D 28K in neuronal degeneration, we examined its expression in differentiated PC12 cells in response to calcium intoxication, using the ionophore A23187 treatment, that results in cell degeneration and death. We first established that calbindin-D 28K is expressed in PC12 cells. The amounts of calbindin-D 28K mRNA and protein were increased by the differentiation factors, NGF and retinoic acid, but not by vitamin D3. Calbindin-D 28K expression was also significantly up-regulated by stimuli (depolarization, low concentrations of Ca2+ ionophore A23187) which increase intracellular calcium levels within the physiological range. In contrast, the calbindin-D 28K mRNA and protein concentrations were not modulated by high concentrations of A23187, which resulted in cell degeneration and death. Experiments with the antisense oligonucleotides showed that, although the calbindin-D 28K protein levels were decreased significantly, the progression of degenerative changes induced by calcium via A23187, was not altered.

  12. Neuronal growth inhibitory factor inhibits pheochromo-cytoma PC12 in vitro


    Neuronal growth inhibitory factor (GIF),named Metaliothioneins-Ⅲ (MT-Ⅲ), is the first protein validated to be capable of inhibiting the growth of neurons in nervous system. We have detected the effects of recombinant GIF on the growth of neuroblastoma SY5Y and pheochromocytoma PC12 by the MTT (Thiazolyl blue) reduction assay. Recombinant GIF inhibited PC12 in vitro; the inhibitory rate was about 25% when GIF was at 100 mg/L; and the inhibitory rate was about 50% when GIF was at 300 mg/L. It is shown that PC12 could serve as a proper model for detecting neuronal growth inhibitory activity of GIF. Recombinant GIF did not inhibit neuroblastoma SY5Y in vitro, a common model of neuroma; it is also shown that GIF could not inhibit neuromata extensively. The reason for GIF inhibiting PC12 may be that PC12 have some properties of cholinergic neuron. It must play an important role in discovering the mechanism of GIF's neuronal growth inhibitory activity.``

  13. Nucleofection of rat pheochromocytoma PC-12 cells with human mutated beta-amyloid precursor protein gene (APP-sw) leads to reduced viability, autophagy-like process, and increased expression and secretion of beta amyloid.

    Pająk, Beata; Kania, Elżbieta; Orzechowski, Arkadiusz


    Pheochromocytoma PC-12 cells are immune to physiological stimuli directed to evoke programmed cell death. Besides, metabolic inhibitors are incapable of sensitizing PC-12 cells to extrinsic or intrinsic apoptosis unless they are used in toxic concentrations. Surprisingly, these cells become receptive to cell deletion after human APP-sw gene expression. We observed reduced cell viability in GFP vector + APP-sw-nucleofected cells (drop by 36%) but not in GFP vector - or GFP vector + APP-wt-nucleofected cells. Lower viability was accompanied by higher expression of Aβ 1-16 and elevated secretion of Aβ 1-40 (in average 53.58 pg/mL). At the ultrastructural level autophagy-like process was demonstrated to occur in APP-sw-nucleofected cells with numerous autophagosomes and multivesicular bodies but without autolysosomes. Human APP-sw gene is harmful to PC-12 cells and cells are additionally driven to incomplete autophagy-like process. When stimulated by TRAIL or nystatin, CLU protein expression accompanies early phase of autophagy.

  14. Nucleofection of Rat Pheochromocytoma PC-12 Cells with Human Mutated Beta-Amyloid Precursor Protein Gene (APP-sw Leads to Reduced Viability, Autophagy-Like Process, and Increased Expression and Secretion of Beta Amyloid

    Beata Pająk


    Full Text Available Pheochromocytoma PC-12 cells are immune to physiological stimuli directed to evoke programmed cell death. Besides, metabolic inhibitors are incapable of sensitizing PC-12 cells to extrinsic or intrinsic apoptosis unless they are used in toxic concentrations. Surprisingly, these cells become receptive to cell deletion after human APP-sw gene expression. We observed reduced cell viability in GFP vector + APP-sw-nucleofected cells (drop by 36% but not in GFP vector − or GFP vector + APP-wt-nucleofected cells. Lower viability was accompanied by higher expression of Aβ 1-16 and elevated secretion of Aβ 1-40 (in average 53.58 pg/mL. At the ultrastructural level autophagy-like process was demonstrated to occur in APP-sw-nucleofected cells with numerous autophagosomes and multivesicular bodies but without autolysosomes. Human APP-sw gene is harmful to PC-12 cells and cells are additionally driven to incomplete autophagy-like process. When stimulated by TRAIL or nystatin, CLU protein expression accompanies early phase of autophagy.

  15. Cytotoxic, Genotoxic, and Neurotoxic Effects of Mg, Pb, and Fe on Pheochromocytoma (PC-12) Cells

    Sanders, Talia; Liu, Yi-Ming; Tchounwou, Paul B.


    Metals such as lead (Pb), magnesium (Mg), and iron (Fe) are ubiquitous in the environment as a result of natural occurrence and anthropogenic activities. Although Mg, Fe and others are considered essential elements, high level of exposure has been associated with severe adverse health effects including cardiovascular, hematological, nephrotoxic, hepatotoxic, and neurologic abnormalities in humans. In the present study we hypothesized that Mg, Pb, and Fe are cytotoxic, genotoxic and neurotoxic, and their toxicity is mediated through oxidative stress and alteration in protein expression. To test the hypothesis, we used the pheochromocytoma (PC-12) cell line as a neuro cell model and performed the LDH assay for cell viability, Comet assay for DNA damage, Western blot for oxidative stress, and HPLC-MS to assess the concentration levels of neurological biomarkers such as glutamate, dopamine (DA), and 3-methoxytyramine (3-MT). The results of this study clearly show that Mg, Pb, and Fe, respectively in the form of MgSO4, Pb(NO3)2, FeCl2, and FeCl3 induce cytotoxicity, oxidative stress, and genotoxicity in PC-12 cells. In addition, exposure to these metallic compounds caused significant changes in the concentration levels of glutamate, dopamine, and 3-MT in PC-12 cells. Taken together the findings suggest that MgSO4, Pb(NO3)2, FeCl2, and FeCl3 have the potential to induce substantial toxicity to PC-12 cells. PMID:24942330

  16. Analysis of Pheochromocytoma (PC12) Membrane Potential under the Exposure to Millimeter-wave Radiation

    Mizuno, M.; Hirata, A.; Kawase, K.; Otani, C.; Nagatsuma, T.


    Non-thermal effects of millimeter wave (MMW) on Pheochromocytoma (PC12) were studied by potential measurement with a voltage sensitive dye (DiBAC4(3)). Cells were irradiated at fixed frequencies of 30, 40, 60, 76GHz as well as sweeping frequency between 10 and 100 GHz by an MMW generator based on a uni-traveling-carrier photodiode (UTC-PD), the most widely tunable MMW source. However there were no significant changes in membrane potential between MMW-irradiated and control cells. The results suggest that MMW irradiation in the range from 10 to 100GHz appears to be safe for ordinary PC12 cells under non-thermal conditions.

  17. Identification of coffee components that stimulate dopamine release from pheochromocytoma cells (PC-12).

    Walker, J; Rohm, B; Lang, R; Pariza, M W; Hofmann, T; Somoza, V


    Coffee and caffeine are known to affect the limbic system, but data on the influence of coffee and coffee constituents on neurotransmitter release is limited. We investigated dopamine release and Ca(2+)-mobilization in pheochromocytoma cells (PC-12 cells) after stimulation with two lyophilized coffee beverages prepared from either Coffea arabica (AR) or Coffea canephora var. robusta (RB) beans and constituents thereof. Both coffee lyophilizates showed effects in dilutions between 1:100 and 1:10,000. To identify the active coffee compound, coffee constituents were tested in beverage and plasma representative concentrations. Caffeine, trigonelline, N-methylpyridinium, chlorogenic acid, catechol, pyrogallol and 5-hydroxytryptamides increased calcium signaling and dopamine release, although with different efficacies. While N-methylpyridinium stimulated the Ca(2+)-mobilization most potently (EC(200): 0.14±0.29μM), treatment of the cells with pyrogallol (EC(200): 48±14nM) or 5-hydroxytryptamides (EC(200): 10±3nM) lead to the most pronounced effect on dopamine release. In contrast, no effect was seen for the reconstituted biomimetic mixture. We therefore conclude that each of the coffee constituents tested stimulated the dopamine release in PC-12 cells. Since no effect was found for their biomimetic mixture, we hypothesize other coffee constituents being responsible for the dopamine release demonstrated for AR and RB coffee brews. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. Release of chromaffin granule glycoproteins and proteoglycans from potassium-stimulated PC12 pheochromocytoma cells.

    Salton, S R; Margolis, R U; Margolis, R K


    Cultured PC12 pheochromocytoma cells were labeled with [3H]glucosamine, and the glycoproteins and proteoglycans released following potassium-induced depolarization were fractionated and characterized. Exposure of PC12 cells for 20 min to a high concentration of potassium (51.5 mM in Krebs-Ringers-HEPES buffer) results in an approximately sixfold increase in the release of labeled glycoproteins and proteoglycans, compared to incubation in physiological levels of potassium (6 mM). The released complex carbohydrates include chromogranins, dopamine beta-hydroxylase, and two chondroitin sulfate/heparan sulfate proteoglycan fractions, which together account for 7.4% of the soluble cell radioactivity. The chromogranins contained galactosyl(beta 1 leads to 3)N-acetylgalactosamine, as well as several mono- and disialyl O-glycosidically-linked oligosaccharides, and the tetrasaccharide AcNeu(alpha 2 leads to 3)Gal(beta 1 leads to 3)[AcNeu(alpha 2 leads to 6)] GalNAcol, obtained by alkaline borohydride treatment of the chromogranin glycopeptides, accounted for almost half of the total chromogranin labeling. The proteoglycan fractions varied in their relative proportions of chondroitin sulfate (23-68%), heparan sulfate (16-23%), and glycoprotein oligosaccharides (16-54%), which are of the tri- and tetraantennary and O-glycosidic types. As previously found in the case of proteoglycans from bovine chromaffin granules, the more acidic species has a considerably higher proportion of carbohydrate in the form of sulfated glycosaminoglycans.

  19. A subcellular distribution of estrogen receptor-alpha is changed during artificially induced senescence of PC12 pheochromocytoma cells.

    Lee, Eunju; Mun, Ga Hee; Oh, Chang Seok; Chung, Yoon Hee; Cha, Choong Lk; Lee, Young Soo; Shin, Dong Hoon


    Although estrogen has been considered as a sex hormone for decades, recent reports suggest that estrogen might modulate the development and physiological function of the brain. In addition, the subcellular localization of estrogen receptors (ERs) has shown their presence within both the perinuclear cytoplasm and nuclei, suggesting that these ERs may differ functionally. We, therefore, assayed changes in the subcellular localization of ER-alpha immunoreactivity (IR) in rat pheochromocytoma PC12 cells during the artificial senescence induced by the telomerase inhibitor, 3'-azido-3'-deoxythymidine (AZT). After 2 months of culture with AZT, PC12 cells showed morphological and biochemical characteristics of cellular senescence. In the cells showing artificial senescence, the ER-alpha IR was mainly localized within the cytoplasm, whereas in control cells, ER-alpha IR was found only in the nuclei. Since senescence was induced by AZT, which inhibits the action of telomerase whenever the cells divide, the change in subcellular distribution of ER-alpha IR may be correlated with the length of the telomere.

  20. Overexpression of the human ubiquitin E3 ligase CUL4A alleviates hypoxia-reoxygenation injury in pheochromocytoma (PC12) cells

    Tan, Can [Department of Histology and Embryology, School of Basic Medical Sciences, Central South University, 172 Tong Zipo Road, Changsha 410013 (China); Zhang, Li-Yang [Key Laboratory of Carcinogenesis and Cancer Invasion of Ministry of Education, Cancer Research Institute, Central South University, 110 Xiang Ya Road, Changsha 410078 (China); Chen, Hong [Department of Developmental Biology, School of Biological Science and Technology, Central South University, 172 Tong Zipo Road, Changsha 410013 (China); Xiao, Ling [Department of Histology and Embryology, School of Basic Medical Sciences, Central South University, 172 Tong Zipo Road, Changsha 410013 (China); Liu, Xian-Peng, E-mail: [Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130-3932 (United States); Zhang, Jian-Xiang, E-mail: [Department of Histology and Embryology, School of Basic Medical Sciences, Central South University, 172 Tong Zipo Road, Changsha 410013 (China); Department of Developmental Biology, School of Biological Science and Technology, Central South University, 172 Tong Zipo Road, Changsha 410013 (China)


    Highlights: Black-Right-Pointing-Pointer Overexpression of human CUL4A (hCUL4A) in PC12 cells. Black-Right-Pointing-Pointer The effects of hCUL4A on hypoxia-reoxygenation injury were investigated. Black-Right-Pointing-Pointer hCUL4A suppresses apoptosis and DNA damage and thus promotes cell survival. Black-Right-Pointing-Pointer hCUL4A regulates apoptosis-related proteins and cell cycle regulators. -- Abstract: The ubiquitin E3 ligase CUL4A plays important roles in diverse cellular processes including carcinogenesis and proliferation. It has been reported that the expression of CUL4A can be induced by hypoxic-ischemic injury. However, the effect of elevated expression of CUL4A on hypoxia-reoxygenation injury is currently unclear. In this study, human CUL4A (hCUL4A) was expressed in rat pheochromocytoma (PC12) cells using adenoviral vector-mediated gene transfer, and the effects of hCUL4A expression on hypoxia-reoxygenation injury were investigated. In PC12 cells subjected to hypoxia and reoxygenation, we found that hCUL4A suppresses apoptosis and DNA damage by regulating apoptosis-related proteins and cell cycle regulators (Bcl-2, caspase-3, p53 and p27); consequently, hCUL4A promotes cell survival. Taken together, our results reveal the beneficial effects of hCUL4A in PC12 cells upon hypoxia-reoxygenation injury.

  1. Characterization of RNA interference in rat PC12 cells

    Thonberg, Håkan; Schéele, Camilla C; Dahlgren, Cecilia


    Double-stranded RNA can initiate post transcriptional gene silencing in mammalian cell cultures via a mechanism known as RNA interference (RNAi). The sequence-specific degradation of homologous mRNA is triggered by 21-nucleotide RNA-duplexes termed short interfering RNA (siRNA). The homologous...... of the rat Golgi-ER protein 95 kDa (GERp95), an Argonaute family protein, by siRNA methodology. After GERp95-ablation, sequential knockdown of NPY by siRNA was shown to be impaired. Thus, we report that the GERp95 protein is functionally required for RNAi targeting NPY in rat PC12 cells....

  2. In vitro effects of fetal rat cerebrospinal fluid on viability and neuronal differentiation of PC12 cells

    Nabiuni Mohammad


    Full Text Available Abstract Background Fetal cerebrospinal fluid (CSF contains many neurotrophic and growth factors and has been shown to be capable of supporting viability, proliferation and differentiation of primary cortical progenitor cells. Rat pheochromocytoma PC12 cells have been widely used as an in vitro model of neuronal differentiation since they differentiate into sympathetic neuron-like cells in response to growth factors. This study aimed to establish whether PC12 cells were responsive to fetal CSF and therefore whether they might be used to investigate CSF physiology in a stable cell line lacking the time-specific response patterns of primary cells previously described. Methods In vitro assays of viability, proliferation and differentiation were carried out after incubation of PC12 cells in media with and without addition of fetal rat CSF. An MTT tetrazolium assay was used to assess cell viability and/or cell proliferation. Expression of neural differentiation markers (MAP-2 and β-III tubulin was determined by immunocytochemistry. Formation and growth of neurites was measured by image analysis. Results PC12 cells differentiate into neuronal cell types when exposed to bFGF. Viability and cell proliferation of PC12 cells cultured in CSF-supplemented medium from E18 rat fetuses were significantly elevated relative to the control group. Neuronal-like outgrowths from cells appeared following the application of bFGF or CSF from E17 and E19 fetuses but not E18 or E20 CSF. Beta-III tubulin was expressed in PC12 cells cultured in any media except that supplemented with E18 CSF. MAP-2 expression was found in control cultures and in those with E17 and E19 CSF. MAP2 was located in neurites except in E17 CSF when the whole cell was positive. Conclusions Fetal rat CSF supports viability and stimulates proliferation and neurogenic differentiation of PC12 cells in an age-dependent way, suggesting that CSF composition changes with age. This feature may be important

  3. Plasminogen activator inhibitor-1 aids survival of neurites on neurons derived from pheochromocytoma (PC-12) cells.

    Soeda, Shinji; Imatoh, Takuya; Ochiai, Takashi; Koyanagi, Satoru; Shimeno, Hiroshi


    Plasminogen activator inhibitor-1 is a serpin that regulates the activities of plasminogen activators. However, its physiological roles in the CNS are incompletely understood. We have found that plasminogen activator inhibitor-1 has a novel biological function in the CNS: the contribution to survival of neurites on neurons. PC-12 cells treated with nerve growth factor differentiated into neurons and formed a network of neurites. In a serum-free culture medium, these neurites disappeared within 24 h. The addition of plasminogen activator inhibitor-1 prevented the disintegration of the neuronal networks, while the addition of the serpin inhibitors aprotinin and antipain did not. The plasminogen activator inhibitor-1 maintained or promoted the phosphorylated state of extracellular signal-regulated kinase (ERK), but not of protein kinase B (Akt). These results are the first evidence that plasminogen activator inhibitor-1 in the CNS acts to maintain the morphology of neurites via activation of the ERK-related pathway in the neurons.

  4. Satureja khuzestanica attenuates apoptosis in hyperglycemic PC12 cells and spinal cord of diabetic rats.

    Kaeidi, Ayat; Esmaeili-Mahani, Saeed; Abbasnejad, Mehdi; Sheibani, Vahid; Rasoulian, Bahram; Hajializadeh, Zahra; Pasban-Aliabadi, Hamzeh


    Several studies have indicated the involvement of oxidative stress and high glucose-induced cell death in the development of diabetic neuropathy. Satureja khuzestanica has been recommended in the literature as a remedy for the treatment of diabetes, and also contains antioxidant agents. Here, we investigated the possible neuroprotective effects of Satureja khuzestanica extract (SKE) on in vitro and in vivo models of diabetic neuropathy pain. High-glucose-induced damage to pheochromocytoma (PC12) cells and in streptozotocin-induced diabetic rats was studied. Tail-flick and rotarod treadmill tests were used to access nociceptive threshold and motor coordination, respectively. Cell viability was determined by MTT assay. Activated caspase 3 and Bax/Bcl-2 ratio-biochemical markers of apoptosis-were evaluated using immunoblotting. We found that elevating the glucose in the medium (to 4× normal) increased cell toxicity and caspase-3 activation in PC12 cells. Incubation with SKE (200 and 250 μg/ml) decreased cell damage. Furthermore, the diabetic rats developed neuropathy, which was evident from thermal hyperalgesia and motor deficit. Administering SKE at a daily dose of between 50 and 200 mg/kg to the diabetic animals for 3 weeks ameliorated hyperglycemia, weight loss, hyperalgesia, and motor deficit, inhibited caspase 3 activation, and decreased the Bax/Bcl-2 ratio. The results suggest that SKE exerts neuroprotective effects against hyperglycemia-induced cellular damage. The mechanisms of these effects may be related to (at least in part) the prevention of neural apoptosis, and the results suggest that Satureja has the therapeutic potential to attenuate side effects of diabetes, such as neuropathy.

  5. The urokinase plasminogen activator receptor (UPAR) is preferentially induced by nerve growth factor in PC12 pheochromocytoma cells and is required for NGF-driven differentiation.

    Farias-Eisner, R; Vician, L; Silver, A; Reddy, S; Rabbani, S A; Herschman, H R


    Nerve growth factor (NGF)-driven differentiation of PC12 pheochromocytoma cells is a well studied model used both to identify molecular, biochemical, and physiological correlates of neurotrophin-driven neuronal differentiation and to determine the causal nature of specific events in this differentiation process. Although epidermal growth factor (EGF) elicits many of the same early biochemical and molecular changes in PC12 cells observed in response to NGF, EGF does not induce molecular or morphological differentiation of PC12 cells. The identification of genes whose expression is differentially regulated by NGF versus EGF in PC12 cells has, therefore, been considered a source of potential insight into the molecular specificity of neurotrophin-driven neuronal differentiation. A "second generation" representational difference analysis procedure now identifies the urokinase plasminogen activator receptor (UPAR) as a gene that is much more extensively induced by NGF than by EGF in PC12 cells. Both an antisense oligonucleotide for the UPAR mRNA and an antibody directed against UPAR protein block NGF-induced morphological and biochemical differentiation of PC12 cells; NGF-induced UPAR expression is required for subsequent NGF-driven differentiation.


    莫永炎; 陈瑗; 周玫; 张宝


    在神经系统的生长发育过程中,星形胶质细胞对神经元生长发育的作用是一项重要的研究课题。本文以体外培养的SD大鼠大脑皮质星形胶质细胞与PC12神经元按不同细胞数目比例(50:1~1:1)共同培养,并用其制备的条件培养液培养PC12细胞,用快速灵敏的MTT比色法测定PC12神经元的细胞活力,用光学相差显微镜观察PC12细胞形态学变化。结果显示,星形胶质细胞条件培养液可增强PC12细胞活力(MTT测定的 OD值由0.255±0.012提高到0.510±0.036,P<0.001,且细胞折光性较对照组强),却不能促使PC12神经元突起的生出。将星形胶质细胞与PC12细胞按30:1~1:1的比例共同培养时,既可提高PC12细胞折光性和光晕又可促使其突起的生长;如按50:1~40:1的比例共同培养时,只观察到提高PC12细胞折光性和光晕,而无促使其突起生长发育的作用。本文结果提示,PC12神经元细胞活力的提高与星形胶质细胞分泌到条件培养液中的可溶性因子有关,而PC12神经元突起生长发育可能是和与星形胶质细胞的直接接触以及二者的细胞数且比有关。%To investigate effects of cultured astrocytes from Sprague Dawley rat cerebral cortex on the development of PC12 cellsderived from rat pheochromocytoma, PC12 cells were cocultured with astrocyte according to different astrocytes/neurons ratio(50:1~1:1) , or with serum-free conditioned medium of astrocytes(ACM). The vitality of PC12 cells was measured by sensi-tive MTT method and their morphologic features were observed by Olympus light microscope. The results showed: (1) WhenPC12 cells were cultured with ACM, compared with the control group, the vitality of PC12 cells was increased significantly (0.255+0. 012 vs 0. 510±0. 036, P<0. 001) and the morphological changes were not obvious in the experimental group. (2) WhenPC12 cells were cocultured with astrocyte in the ratio of 30: 1

  7. A "classical" homodimeric erythropoietin receptor is essential for the antiapoptotic effects of erythropoietin on differentiated neuroblastoma SH-SY5Y and pheochromocytoma PC-12 cells.

    Um, Moonkyoung; Gross, Alec W; Lodish, Harvey F


    The hematopoietic cytokine erythropoietin (Epo) exerts cytoprotective effects on several types of neuronal cells both in vivo and in culture. Detailed molecular mechanisms underlying this phenomenon have not been elucidated and even the identity of the cytoprotective Epo receptors in neuronal cells is controversial. Here we show that Epo prevents staurosporine-induced apoptosis of differentiated human neuroblastoma SH-SY5Y cells, and activates the STAT5, AKT and MAPK signaling pathways. Differentiated SH-SY5Y cells have fewer than 50 high affinity Epo surface binding sites per cell, which could not be detected by standard assays measuring binding of 125I-labeled Epo. However, by measuring endocytosis of 125I-Epo, we could reliably quantify very small numbers of high-affinity Epo surface binding sites. Using SH-SY5Y cells stably expressing an Epo receptor (EpoR) shRNA and thus lacking detectable EpoR expression, we show that high affinity binding of Epo to these neuronal cells is mediated by the hematopoietic EpoR, and that this EpoR is also essential for the antiapoptotic activity of Epo. In contrast, a mutant Epo that has an intact binding site 1 but a non-functional binding site 2 and hence binds only to one cell surface EpoR molecule ("site 2" Epo mutant) displays significantly lower antiapoptotic activity than wild-type Epo. Furthermore, expression of the GM-CSF/IL-3/IL-5 receptor common beta chain, which was proposed to be responsible for the cytoprotective activity of Epo on certain types of neuronal cells, was undetectable in differentiated SH-SY5Y cells. Epo also alleviated staurosporine-induced apoptosis of rat PC-12 pheochromocytoma cells while the R103A "site 2" Epo mutant did not, and we could not detect expression of the common beta chain in PC-12 cells. Together our results indicate that Epo exerts its antiapoptotic effects on differentiated SH-SY5Y and PC-12 cells through the standard stoichiometry of one molecule of Epo binding to two EpoR subunits

  8. Effects of cultured astrocytes from rat cerebral cortex onthe neurite development of PC12 cells%星形神经胶质细胞对PC12神经元突起生长发育的影响

    莫永炎; 邵紫韫; 陈瑗; 周玫; 张宝


    背景:星形细胞对神经元有提供营养、支持及调节突触活性作用,但它对神经元发育的影响还尚不清楚.目的:探讨体外培养的Sprague Dawley大鼠大脑皮质星形细胞对PC12神经元突起生长发育的作用.设计:完全随机设计,对照实验研究.方法:以培养的星形细胞与PC12神经元按不同细胞数目比例(50:1~1:1)共同培养,并用其制备的条件培养液培养PG12细胞.主要观察指标:用快速灵敏的MTT'比色法测定PC12神经元的细胞活力,用光学相差显微镜观察PC12细胞形态学变化.结果:①星形细胞条件培养液可增强PG12细胞活力(MTT测定的A值由0.255±0.012提高到0.510±0.036,P<0.001),却不能促使PC12神经元突起的生出.②当将星形细胞与PC12细胞按30:1~1:1的比例共同培养时,既可提高PC12细胞折光性和光晕又可促使其突起的生长;但按50:1~40:1的比例共同培养时,只观察到提高PC12细胞折光性和光晕,而无促使其突起生长发育的作用.结论:PC12神经元细胞活力的提高与星形细胞分泌到条件培养液中的可溶性因子有关,而PC12神经元突起生长发育可能是和与星形细胞的直接接触以及二者的细胞数目比有关.%BACKGROUND: Although astrocytes are kown to provide structural andtrophic support to neurons and modulate synaptic activity, their role is farfrom being completely understood.OBJECTIVE: To investigate effects of cultured astrocytes fromSprague-Dawley rat cerebral cortex on the neurite development of PC12 cellsderived from rat pheochromocytoma.DESIGN: Completely randomized controlled trial.METHODS: PC12 cells were co-cultured with astrocyte according to dif-ferent astrocytes/neurons ratio(50: 1 -1: 1), or cultured with serum-freeconditioned medium of astrocytes (ACM).MAIN OUTCOME MEASURES: The vitality of PC12 cells was measuredby sensitive MTT method and their morphologic features were observed byOlympus light microscope.RFSULTS: When PC

  9. IgG anti-GalNAc-GD1a antibody inhibits the voltage-dependent calcium channel currents in PC12 pheochromocytoma cells.

    Nakatani, Yoshihiko; Nagaoka, Takumi; Hotta, Sayako; Utsunomiya, Iku; Yoshino, Hiide; Miyatake, Tadashi; Hoshi, Keiko; Taguchi, Kyoji


    We investigated the effects of IgG anti-GalNAc-GD1a antibodies, produced by immunizing rabbits with GalNAc-GD1a, on the voltage-dependent calcium channel (VDCCs) currents in nerve growth factor (NGF)-differentiated PC12 pheochromocytoma cells. VDCCs currents in NGF-differentiated PC12 cells were recorded using the whole-cell patch-clamp technique. Immunized rabbit serum that had a high titer of anti-GalNAc-GD1a antibodies inhibited the VDCCs currents in the NGF-differentiated PC12 cells (36.0+/-9.6% reduction). The inhibitory effect of this serum was reversed to some degree within 3-4 min by washing with bath solution. Similarly, application of purified IgG from rabbit serum immunized with GalNAc-GD1a significantly inhibited the VDCCs currents in PC12 cells (30.6+/-2.5% reduction), and this inhibition was recovered by washing with bath solution. Furthermore, the inhibitory effect was also observed in the GalNAc-GD1a affinity column binding fraction (reduction of 31.1+/-9.85%), while the GalNAc-GD1a affinity column pass-through fraction attenuated the inhibitory effect on VDCCs currents. Normal rabbit serum and normal rabbit IgG did not affect the VDCCs currents in the PC12 cells. In an immunocytochemical study using fluorescence staining, the PC12 cells were stained using GalNAc-GD1a binding fraction. These results indicate that anti-GalNAc-GD1a antibodies inhibit the VDCCs currents in NGF-differentiated PC12 cells.

  10. Multipotent Neural Crest Stem Cell-Like Cells from Rat Vibrissa Dermal Papilla Induce Neuronal Differentiation of PC12 Cells

    Meiying Li


    Full Text Available Bone marrow mesenchymal stem cells (BMSCs transplants have been approved for treating central nervous system (CNS injuries and diseases; however, their clinical applications are limited. Here, we model the therapeutic potential of dermal papilla cells (DPCs in vitro. DPCs were isolated from rat vibrissae and characterized by immunocytofluorescence, RT-PCR, and multidifferentiation assays. We examined whether these cells could secrete neurotrophic factors (NTFs by using cocultures of rat pheochromocytoma cells (PC12 with conditioned medium and ELISA assay. DPCs expressed Sox10, P75, Nestin, Sox9, and differentiated into adipocytes, osteoblasts, smooth muscle cells, and neurons under specific inducing conditions. The DPC-conditioned medium (DPC-CM induced neuronal differentiation of PC12 cells and promoted neurite outgrowth. Results of ELISA assay showed that compared to BMSCs, DPCs secreted more brain-derived neurotrophic factor (BDNF and glial cell line-derived neurotrophic factor (GDNF. Moreover, we observed that, compared with the total DPC population, sphere-forming DPCs expressed higher levels of Nestin and P75 and secreted greater amounts of GDNF. The DPCs from craniofacial hair follicle papilla may be a new and promising source for treating CNS injuries and diseases.

  11. Comparative Study on the Protective Effects of Salidroside and Hypoxic Preconditioning for Attenuating Anoxia-Induced Apoptosis in Pheochromocytoma (PC12) Cells.

    Hu, Yao; Lv, Xiumei; Zhang, Jing; Meng, Xianli


    BACKGROUND Hypoxia is an important sign that can result from body injuries or a special condition such as being at a high altitude or deep water diving. In the current studies, hypoxic preconditioning (HPC) plays a key role in reducing hypoxia-induced apoptosis. We aimed to study the pharmacologic preconditioning effects of salidroside versus those of HPC in hypoxia-/anoxia-induced apoptosis in PC12 cells (pheochromocytoma). MATERIAL AND METHODS PC12 cells were treated by different experimental conditions: control condition, hypoxia condition, HPC condition, low-/middle-/high-dose condition of salidroside, cyclosporine A (CsA), and oratractyloside (ATR). The cell viability, lactate dehydrogenase (LDH) activity, apoptosis, mitochondrial membrane potential (MMP), intracellular Ca2+, caspase-3 activity, and expression of Bcl-2 were detected in PC12 cells after the hypoxia treatment. Salidroside, extracted from the traditional Chinese herb Rhodiola rosea L, plays an essential role in reducing hypoxia-induced apoptosis in PC12 cells by the mitochondrial pathway. RESULTS Salidroside decreased the apoptosis and increased the viability of hypoxia-induced PC12 cells more effectively than HPC Moreover, salidroside markedly stabilized MMP and intracellular Ca2+, reduced or inhibited LDH and caspase-3 activity, and up-regulated Bcl-2; CsA and ATR showed corresponding function. CONCLUSIONS Salidroside administration restrains apoptosis induced by hypoxia in PC12 cells. The protective effects are mediated by preservation of mitochondrial integrity and MMP to inhibit the excessive Ca2+ influx and caspase-3 activity and to promote the Bcl-2 expression, providing a potential clinical and effective therapeutic mechanism to reduce deaths from ischemic or hypoxic injury.

  12. Neurite outgrowth stimulatory effects of myco synthesized AuNPs from Hericium erinaceus (Bull.: Fr.) Pers. on pheochromocytoma (PC-12) cells.

    Raman, Jegadeesh; Lakshmanan, Hariprasath; John, Priscilla A; Zhijian, Chan; Periasamy, Vengadesh; David, Pamela; Naidu, Murali; Sabaratnam, Vikineswary


    Hericium erinaceus has been reported to have a wide range of medicinal properties such as stimulation of neurite outgrowth, promotion of functional recovery of axonotmetic peroneal nerve injury, antioxidant, antihypertensive, and antidiabetic properties. In recent years, the green synthesis of gold nanoparticles (AuNPs) has attracted intense interest due to the potential use in biomedical applications. The aim of this study was to investigate the effects of AuNPs from aqueous extract of H. erinaceus on neurite outgrowth of rat pheochromocytoma (PC-12) cells. The formation of AuNPs was characterized by UV-visible spectrum, energy dispersive X-ray (EDX), field-emission scanning electron microscope (FESEM), transmission electron microscopy (TEM), particle size distribution, and Fourier transform-infrared spectroscopy (FTIR). Furthermore, the neurite extension study of synthesized AuNPs was evaluated by in vitro assay. The AuNPs exhibited maximum absorbance between 510 and 600 nm in UV-visible spectrum. FESEM and TEM images showed the existence of nanoparticles with sizes of 20-40 nm. FTIR measurements were carried out to identify the possible biomolecules responsible for capping and efficient stabilization of the nanoparticles. The purity and the crystalline properties were confirmed by EDX diffraction analysis, which showed strong signals with energy peaks in the range of 2-2.4 keV, indicating the existence of gold atoms. The synthesized AuNPs showed significant neurite extension on PC-12 cells. Nerve growth factor 50 ng/mL was used as a positive control. Treatment with different concentrations (nanograms) of AuNPs resulted in neuronal differentiation and neuronal elongation. AuNPs induced maximum neurite outgrowth of 13% at 600 ng/mL concentration. In this study, the AuNPs synthesis was achieved by a simple, low-cost, and rapid bioreduction approach. AuNPs were shown to have potential neuronal differentiation and stimulated neurite outgrowth. The water

  13. Components of Goutengsan in Rat Plasma by Microdialysis Sampling and Its Protection on Aβ1–42-Induced PC12 Cells Injury

    Hou-Cai Huang


    Full Text Available Goutengsan, a Chinese herbal formula, potential protection on Alzheimer’s disease (AD has been less reported. In current study, we investigated the protection of Goutengsan on Aβ1–42-induced pheochromocytoma-derived cells (PC12. Furthermore, the components from Goutengsan in rat plasma were identified by microdialysis (MD for in vivo sampling. Meanwhile, the protection of components identified was also verified. At last, we found that Goutengsan has a potential protective effect on Aβ1–42-induced PC12 cells via reducing cells damage and increasing cells vitality as well as six components (pachymic acid, liquiritin, rhynchophylline, isorhynchophylline, corynoxeine, and isocorynoxeine which may be effective components. This study helps to understand the treatment of Goutengsan for AD and would facilitate the clinical and further studies for this formula.

  14. Distinctive effects of rat fibroblast growth factor-2 isoforms on PC12 and Schwann cells.

    Müller-Ostermeyer, F; Claus, P; Grothe, C


    Fibroblast growth factor-2 (FGF-2) is an important modulator of cell growth and differentiation and stimulates cell survival of various cells including neurons. Rat FGF-2 occurs in three isoforms, a low molecular weight 18 kD and two high molecular weight forms (21, 23 kD), representing alternative translation products from a single mRNA. The 18 kD isoform shows mainly cytoplasmatic localization, whereas the 21/23 kD FGF-2 are localized in the nucleus. In addition, the FGF-2 isoforms are differentially regulated in the sensory ganglia and peripheral nerve following nerve injury and in the adrenal medulla during post-natal development and after hormonal stimuli. The distinct intracellular distribution and differential regulation of the different FGF-2 isoforms indicate that they have unique biological roles, however, little is known about the biological effects of the high molecular weight FGF-2 isoforms. Immortalized Schwann cells and PC12 cells, which stably overexpress the different FGF-2 isoforms, showed that the different endogenous-overexpressed FGF-2 isoforms lead to dramatic modifications in cell proliferation and survival, when tested in serum-free and serum-containing medium. In contrast, application of recombinant FGF-2 isoforms on normal PC12 and immortalized Schwann cells results in similar biological effects on the proliferation and survival of the cells. Furthermore, we investigated the potential regulatory effects of endogenous-overexpressed and exogenous-applied FGF-2 isoforms on the mRNA level of the FGF-2 receptors and, additionally, on the tyrosin hydroxylase mRNA expression in PC12 cells.

  15. Mechanism of morphine on proliferation of pheochromocytoma cells%吗啡对 PC12细胞增殖的抑制作用及机制初探

    陈伟强; 刘付宁; 刘玲; 何惠燕; 曹铭辉


    Objective To investigate the effects of morphine on roliferation of pheochromocytoma cell and its mechanism. Methods PC12 cell cultures were exposed to 10,20,30 μmol/L morphine. The inhibition of the cell proliferation was determined by MTT assay. The level of p38MAPK was detected by immunoblotting. Results The proliferation of PC12 cells were inhibited after morphine exposure for 48 h.Immunoblotting showed that morphine increased the phosphorylation of p38MAPK was increased in stimulation of monphine. SB203580 , the inhibitor of p38MAPK , reduced inhibition the proliferation of PC12 cells by morphine. Conclusion These results suggest that morphine can significantly inhibits the proliferation of PC12 cells by an increase in p38MAPK phosphorylation.%目的:探讨吗啡对大鼠嗜铬细胞瘤 PC12细胞增殖的抑制作用及其分子机制。方法 PC12细胞体外培养,将吗啡加入细胞培养液中使吗啡浓度分别稀释至10、20、30μmol/L ,然后分别与 PC12细胞一同孵育,四甲基偶氮噻唑蓝法(MTT)观察吗啡对细胞增殖的抑制作用;免疫印迹法检测PC12细胞p38MAPK 表达及磷酸化水平。结果不同浓度吗啡对PC12细胞增殖有明显的抑制作用,抑制率为17.66%~31.05%;并且吗啡可以诱导PC12细胞p38MAPK 磷酸化水平升高,而p38MAPK 通路阻断剂SB203580可以抑制吗啡诱导 PC12细胞 p38MAPK 磷酸化水平升高,减轻吗啡对 PC12细胞增殖的抑制。结论吗啡可以明显抑制 PC12细胞的增殖,升高p38MAPK 的磷酸化水平可能是其重要分子机制之一。

  16. 麝香酮对谷氨酸所致PC12细胞损伤的保护及作用机制研究%Protective effects and machanism of muskone on pheochromocytoma cell injure induced by glutamate

    孙蓉; 张作平; 黄伟; 吕丽莉; 尹建伟


    Objective: To explore the mechanism of glutamate (Glu)-induced PC 12 cell apoptosis and the protective effect of muscone. Method: PC 12 cells were randomly divided into six groups; normal group (Normal) , model group injured by glutamate (Glu) , nimodipine group (Nim) and muskone groups (Mus) of high,middle and low doses. The PC12 cells were pretreated with or without different concentrations of muskone for 30 min and then exposed to glutamate. MTT assay for cell survival, flow cytometric detection of apoptotic cells, DCF assay for reactive oxygen species (ROS) and flow cytomet ric assay was performed to determine the mitochondrial membrane potential in PC12 cell. Result; PC12 cell damage, the concentrations of [ Ca~(2+) ] , and apoptosis induced by Glu were decreased after being administrated with paeoniflorin. Conclusion: Muskone inhibited Glu-induced apoptosis in PC12 cells. The mechanism is related to inhibiting intracellular Ca~(2+) overload and maintaining mitochondral membrance potential.%目的:考察麝香酮对谷氨酸引起PC12细胞损伤的保护作用及作用机制.方法:将大鼠嗜铬细胞瘤细胞株(pheochromocytoma,cells,PC12)分为正常组、模型组、麝香酮10.0,1.0,0.1 μmol·L~(-1)高、中、低剂量组和1μmol·L~(-1)尼莫地平组,用500μmol·L~(-1)谷氨酸造成PC12细胞损伤,采用药物预处理给药方法,MTT法测定细胞存活率,流式细胞仪测定细胞凋亡率,采用Fura3/AM为荧光指示剂,用Vector~2 1420多标记免疫分析仪研究麝香酮对谷氨酸所致损伤PC12细胞内Ca~(2+)的作用,流式细胞仪检测线粒体跨膜电位,考察不同浓度麝香酮对PC12细胞的保护作用.结果:麝香酮可明显提高谷氨酸诱导的PC12细胞还原能力,抑制该细胞LDH的释放,提高细胞存活率;抑制兴奋性氨基酸所引起的细胞内Ca~(2+)含量的升高;降低PC12细胞的凋亡百分率,并呈剂量相关性.结论:麝香酮可抑制谷氨酸诱导的PC12细胞凋亡,其作用

  17. Toluene-induced, Ca2+-dependent vesicular catecholamine release in rat PC12 cells

    Westerink, R.H.S.; Vijverberg, H.P.M.


    Acute effects of toluene on vesicular catecholamine release from intact PC12 phaeochromocytoma cells have been investigated using carbon fiber microelectrode amperometry. The frequency of vesicles released is low under basal conditions and is enhanced by depolarization. Toluene causes an increase in

  18. Toluene-induced, Ca2+-dependent vesicular catecholamine release in rat PC12 cells

    Westerink, R.H.S.|info:eu-repo/dai/nl/239425952; Vijverberg, H.P.M.


    Acute effects of toluene on vesicular catecholamine release from intact PC12 phaeochromocytoma cells have been investigated using carbon fiber microelectrode amperometry. The frequency of vesicles released is low under basal conditions and is enhanced by depolarization. Toluene causes an increase in

  19. Cyclic AMP activates the mitogen-activated protein kinase cascade in PC12 cells

    Frödin, M; Peraldi, P; Van Obberghen, E


    reported. In rat pheochromocytoma PC12 cells, we demonstrate here a stimulation of the MAP kinase isozyme extracellular signal-regulated kinase 1 (ERK1) following elevation of intracellular cAMP after exposure of the cells to isobutylmethylxanthine, cholera toxin, forskolin, or cAMP-analogues. cAMP acted...

  20. Neuroprotective effects of dimerumic acid and deferricoprogen from Monascus purpureus NTU 568-fermented rice against 6-hydroxydopamine-induced oxidative stress and apoptosis in differentiated pheochromocytoma PC-12 cells.

    Tseng, Wei-Ting; Hsu, Ya-Wen; Pan, Tzu-Ming


    Context Oxidative stress plays a key role in neurodegenerative disorders, including Parkinson's disease (PD). Rice fermented with Monascus purpureus Went (Monascaceae) NTU 568 (red mould rice) was found to contain antioxidants, including dimerumic acid (DMA) and deferricoprogen (DFC). Objective The effects of DMA and DFC on 6-hydroxydopamine (6-OHDA)-induced cytotoxicity and potential protective mechanisms in differentiated PC-12 pheochromocytoma cells were investigated. Materials and methods DMA (0-60 μM) or DFC (0-10 μM) was co-treated with 6-OHDA (200 μM, 24 h exposure) in differentiated PC-12 cells. Cell viability and intercellular reactive oxygen species (ROS) were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assays, respectively. Cell apoptosis was determined by DNA fragmentation analysis and propidium iodide staining by flow cytometry. Western blot analysis was used to measure the levels of cell protein expression. Results DMA and DFC significantly increased cell viability to 72% and 81% in 6-OHDA-induced differentiated PC-12 cell cultures, respectively. Furthermore, DMA and DFC reduced 6-OHDA-induced formation of extracellular and intercellular ROS by 25% and 20%, respectively, and decreased NADPH oxidase-2 expression in differentiated PC-12 cells. DMA and DFC inhibited 6-OHDA-induced apoptosis and decreased activation of caspase-3 via regulation of Bcl-2-associated X protein (Bax) and Bcl-2 protein expression in differentiated PC-12 cells. Conclusion DMA and DFC may protect against 6-OHDA toxicity by inhibiting ROS formation and apoptosis. These results showed that the metabolites from M. purpureus NTU 568 fermentation were potential therapeutic agents for PD induced by oxidative damage and should be encouraged for further research.

  1. Postconditioning mitigates cell death following oxygen and glucose deprivation in PC12 cells and forebrain reperfusion injury in rats.

    Lin, Han-Chen; Narasimhan, Purnima; Liu, Shin-Yun; Chan, Pak H; Lai, I-Rue


    Postconditioning mitigates ischemia-induced cellular damage via a modified reperfusion procedure. Mitochondrial permeability transition (MPT) is an important pathophysiological change in reperfusion injury. This study explores the role of MPT modulation underlying hypoxic postconditioning (HPoC) in PC12 cells and studies the neuroprotective effects of ischemic postconditioning (IPoC) on rats. Oxygen-glucose deprivation (OGD) was performed for 10 hr on PC12 cells. HPoC was induced by three cycles of 10-min reoxygenation/10-min rehypoxia after OGD. The MPT inhibitor N-methyl-4-isoleucine cyclosporine (NIM811) and the MPT inducer carboxyatractyloside (CATR) were administered to selective groups before OGD. Cellular death was evaluated by flow cytometry and Western blot analysis. JC-1 fluorescence signal was used to estimate the mitochondrial membrane potential (△Ψm ). Transient global cerebral ischemia (tGCI) was induced via the two-vessel occlusion and hypotension method in male Sprague Dawley rats. IPoC was induced by three cycles of 10-sec reperfusion/10-sec reocclusion after index ischemia. HPoC and NIM811 administration attenuated cell death, cytochrome c release, and caspase-3 activity and maintained △Ψm of PC12 cells after OGD. The addition of CATR negated the protection conferred by HPoC. IPoC reduced neuronal degeneration and cytochrome c release and cleaved caspase-9 expression of hippocampal CA1 neurons in rats after tGCI. HPoC protected PC12 cells against OGD by modulating the MPT. IPoC attenuated degeneration of hippocampal neurons after cerebral ischemia.


    We demonstrated recently that 6 days of exposure to nanomolar concentrations (3-10 nM) of methylmercury (MeHg) during nerve growth factor (NGF) induced PC12 cell differentiation reduced the amplitude and density of voltage-gated sodium and calcium currents. In the present study,...

  3. Protective effect of serotonin derivatives on glucose-induced damage in PC12 rat pheochromocytoma cells.

    Piga, Rosaria; Naito, Yuji; Kokura, Satoshi; Handa, Osamu; Yoshikawa, Toshikazu


    Oxidative damage is believed to be associated with ageing, cancer and several degenerative diseases. Previous reports have shown that safflower-seed extract and its major antioxidant constituents, serotonin hydroxycinnamic amides, possess a powerful free radical-scavenging and antioxidative activity, paying particular attention to atherosclerotic reactive oxygen species (ROS)-related dysfunctions. In the present report, we examined a still unknown cell-based mechanism of serotonin derivatives against ROS-related neuronal damage, phenomena that represent a crucial event in neurodegenerative diseases. Serotonin derivatives N-(p-coumaroyl)serotonin and N-feruloylserotonin exerted a protective effect on high glucose-induced cell death, inhibited the activation of caspase-3 which represents the last and crucial step within the cascade of events leading to apoptosis, and inhibited the overproduction of the mitochondrial superoxide, which represents the most dangerous radical produced by hyperglycaemia, by acting as scavengers of the superoxide radical. In addition, serotonin derivative concentration inside the cells and inside the mitochondria was increased in a time-dependent manner. Since recent studies support the assertion that mitochondrial dysfunctions related to oxidative damage are the major contributors to neurodegenerative diseases, these preliminary cell-based results identify a mitochondria-targeted antioxidant property of serotonin derivatives that could represent a novel therapeutic approach against the neuronal disorders and complications related to ROS.


    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  5. Tl(I) and Tl(III) alter the expression of EGF-dependent signals and cyclins required for pheochromocytoma (PC12) cell-cycle resumption and progression.

    Pino, María T L; Verstraeten, Sandra V


    The effects of thallium [Tl(I) and Tl(III)] on the PC12 cell cycle were evaluated without (EGF(-)) or with (EGF(+)) media supplementation with epidermal growth factor (EGF). The following markers of cell-cycle phases were analyzed: cyclin D1 (G1 ); E2F-1, cyclin E and cytosolic p21 (G1 →S transition); nuclear PCNA and cyclin A (S); and cyclin B1 (G2). The amount of cells in each phase and the activation of the signaling cascade triggered by EGF were also analyzed. Tl(I) and Tl(III) (5-100 μM) caused dissimilar effects on PC12 cell proliferation. In EGF(-) cells, Tl(I) increased the expression of G1 →S transition markers and nuclear PCNA, without affecting cyclin A or cyclin B1. In addition to those, cyclin B1 was also increased in EGF(+) cells. In EGF(-) cells, Tl(III) increased the expression of cyclin D1, all the G1→S and S phase markers and cyclin B1. In EGF(+) cells, Tl(III) increased cyclin D1 expression and decreased all the markers of G1 →S transition and the S phase. Even when these cations did not induce the activation of EGF receptor (EGFR) in EGF(-) cells, they promoted the phosphorylation of ERK1/2 and Akt. In the presence of EGF, the cations anticipated EGFR phosphorylation without affecting the kinetics of EGF-dependent ERK1/2 and Akt phosphorylation. Altogether, results indicate that Tl(I) promoted cell proliferation in both EGF(-) and EGF(+) cells. In contrast, Tl(III) promoted the proliferation of EGF(-) cells but delayed it in EGF(+) cells, which may be related to the toxic effects of this cation in PC12 cells. Copyright © 2014 John Wiley & Sons, Ltd.

  6. Geniposide inhibits CoCl_2-induced PC12 cells death via the mitochondrial pathway

    GUO Li-xia; LIU Jian-hui; XIA Zhi-ning


    Background A number of studies have shown that oxidative stress and mitochondrial involvement are major triggering factors in the development of neurodegenerative diseases. Cobalt chloride (CoCl_2)-induced cell death in PC12 cells may serve a simple and convenient in vitro model of hypoxia-induced neuronal cytotoxicity. To explore the effect of geniposide on CoCl_2 which induced cytotoxicity and mitochondrial function in rat pheochromocytoma PC12 cells, we analyzed the influence of geniposide on the expression of apoptosis-related proteins. Methods PC12 cells and RNAi PC12 cells were treated with 0, 12.5, 25, 50, 100 μmol/L geniposide for 12 hours and then exposure to 400 μmol/L CoCl_2 for 12 hours. Cell viability, cell morphology, and expression of Bcl-2, Bax, P53 and caspase-9 were determined using Western blotting. Results Pretreatment with geniposide markedly improved the cells viability and morphology, decreased the expression of Bax, P53 and caspase-9, and increased the expression of Bcl-2 in PC12 cells challenged by CoCl_2. However, in the RNAi PC12 cells, geniposide had no significant effect on the expression of these proteins. Conclusion Geniposide protects PC12 cells from CoCl_2 involved in mitochondrial mediated apoptosis, and GLP-1 R might play a critical role in the neuroprotection of geniposide in PC12 cells.

  7. Pheochromocytoma in rats with multiple endocrine neoplasia (MENX) shares gene expression patterns with human pheochromocytoma

    Molatore, Sara; Liyanarachchi, Sandya; Irmler, Martin; Perren, Aurel; Mannelli, Massimo; Ercolino, Tonino; Beuschlein, Felix; Jarzab, Barbara; Wloch, Jan; Ziaja, Jacek; Zoubaa, Saida; Neff, Frauke; Beckers, Johannes; Höfler, Heinz; Atkinson, Michael J.; Pellegata, Natalia S.


    Pheochromocytomas are rare neoplasias of neural crest origin arising from chromaffin cells of the adrenal medulla and sympathetic ganglia (extra-adrenal pheochromocytoma). Pheochromocytoma that develop in rats homozygous for a loss-of-function mutation in p27Kip1 (MENX syndrome) show a clear progression from hyperplasia to tumor, offering the possibility to gain insight into tumor pathobiology. We compared the gene-expression signatures of both adrenomedullary hyperplasia and pheochromocytoma with normal rat adrenal medulla. Hyperplasia and tumor show very similar transcriptome profiles, indicating early determination of the tumorigenic signature. Overrepresentation of developmentally regulated neural genes was a feature of the rat lesions. Quantitative RT-PCR validated the up-regulation of 11 genes, including some involved in neural development: Cdkn2a, Cdkn2c, Neurod1, Gal, Bmp7, and Phox2a. Overexpression of these genes precedes histological changes in affected adrenal glands. Their presence at early stages of tumorigenesis indicates they are not acquired during progression and may be a result of the lack of functional p27Kip1. Adrenal and extra-adrenal pheochromocytoma development clearly follows diverged molecular pathways in MENX rats. To correlate these findings to human pheochromocytoma, we studied nine genes overexpressed in the rat lesions in 46 sporadic and familial human pheochromocytomas. The expression of GAL, DGKH, BMP7, PHOX2A, L1CAM, TCTE1, EBF3, SOX4, and HASH1 was up-regulated, although with different frequencies. Immunohistochemical staining detected high L1CAM expression selectively in 27 human pheochromocytomas but not in 140 nonchromaffin neuroendocrine tumors. These studies reveal clues to the molecular pathways involved in rat and human pheochromocytoma and identify previously unexplored biomarkers for clinical use. PMID:20937862

  8. Differential repression by the transcription factor REST/NRSF of the various Ca2+ signalling mechanisms in pheochromocytoma PC12 cells.

    Ariano, P; Zamburlin, P; D'Alessandro, R; Meldolesi, J; Lovisolo, D


    Expression of the nerve cell phenotype is orchestrated by the REST/NRSF transcription repressor, working on hundreds of genes recognized at a specific regulatory binding sequence. Most PC12 clones, the most frequently employed neuronal model, maintain low levels of REST; however a few, defective of neurosecretion, express high levels. To investigate the role of REST in Ca2+ signalling we studied the [Ca2+](i) changes in single cells of four clones, two wild-type and two defective, pre-treated for 5 days with NGF. We focused on Ca2+ influxes induced by depolarization and ATP. Only a subpopulation ( approximately 15%) of the defective, high REST cells responded to depolarization (Ca(V) expression approximately 10%). The ATP-induced intracellular Ca2+ release was little changed, whereas influx via ionotropic P2X receptors was decreased, in agreement with the decreased expression of P2X2 receptors. The percentage of defective cells expressing store-operated calcium entry (SOCE) following ATP stimulation was also lower. The responses of the defective clones were little affected by their differentiated state. In conclusion, our results revealed important new aspects of REST control of Ca2+ homeostasis, of potential physiological importance. The mechanisms of this control remain to be investigated. 2010 Elsevier Ltd. All rights reserved.

  9. Pheochromocytoma

    Raju A Gopal


    Pheochromocytoma arises from adrenal medulla, which actually comprises about 10 % of adrenal gland. Sympathochromaffin system is the major neuroendocrine system in the body, it contains 2 components...

  10. Regulatory roles of bone morphogenetic proteins and glucocorticoids in catecholamine production by rat pheochromocytoma cells.

    Kano, Yoshihiro; Otsuka, Fumio; Takeda, Masaya; Suzuki, Jiro; Inagaki, Kenichi; Miyoshi, Tomoko; Miyamoto, Manabu; Otani, Hiroyuki; Ogura, Toshio; Makino, Hirofumi


    We here report a new physiological system that governs catecholamine synthesis involving bone morphogenetic proteins (BMPs) and activin in the rat pheochromocytoma cell line, PC12. BMP type I receptors, including activin receptor-like kinase-2 (ALK-2) (also referred to as ActRIA) and ALK-3 (BMPRIA), both type II receptors, ActRII and BMPRII, as well as the ligands BMP-2, -4, and -7 and inhibin/activin subunits were expressed in PC12 cells. PC12 cells predominantly secrete dopamine, whereas noradrenaline and adrenaline production is negligible. BMP-2, -4, -6, and -7 and activin A each suppressed dopamine and cAMP synthesis in a dose-dependent fashion. The BMP ligands also decreased 3,4-dihydroxyphenylalanine decarboxylase mRNA expression, whereas activin suppressed tyrosine hydroxylase expression. BMPs induced both Smad1/5/8 phosphorylation and Tlx2-Luc activation, whereas activin stimulated 3TP-Luc activity and p38 MAPK phosphorylation. ERK signaling was not affected by BMPs or activin. Dexamethasone enhanced catecholamine synthesis, accompanying increases in tyrosine hydroxylase and 3,4-dihydroxyphenylalanine decarboxylase transcription without cAMP accumulation. In the presence of dexamethasone, BMPs and activin failed to reduce dopamine as well as cAMP production. In addition, dexamethasone modulated mitotic suppression of PC12 induced by BMPs in a ligand-dependent manner. Furthermore, intracellular BMP signaling was markedly suppressed by dexamethasone treatment and the expression of ALK-2, ALK-3, and BMPRII was significantly inhibited by dexamethasone. Collectively, the endogenous BMP/activin system plays a key role in the regulation of catecholamine production. Controlling activity of the BMP system may be critical for glucocorticoid-induced catecholamine synthesis by adrenomedullar cells.

  11. Pheochromocytoma

    Raju A Gopal


    Full Text Available Pheochromocytoma arises from adrenal medulla, which actually comprises about 10 % of adrenal gland. Sympathochromaffin system is the major neuroendocrine system in the body, it contains 2 components. Symapthetic neurons and chromaffin cells in adrenal medulla.

  12. Microwave Exposure Impairs Synaptic Plasticity in the Rat Hippocampus and PC12 Cells through Over-activation of the NMDA Receptor Signaling Pathway

    XIONG Lu; DONG Ji; YAO Bin Wei; ZHAO Li; PENG Rui Yun; SUN Cheng Feng; ZHANG Jing; GAO Ya Bing; WANG Li Feng; ZUO Hong Yan; WANG Shui Ming; ZHOU Hong Mei; XU Xin Ping


    Objective The aim of this study is to investigate whether microwave exposure would affect the N-methyl-D-aspartate receptor (NMDAR) signaling pathway to establish whether this plays a role in synaptic plasticity impairment. Methods 48 male Wistar rats were exposed to 30 mW/cm² microwave for 10 min every other day for three times. Hippocampal structure was observed through H&E staining and transmission electron microscope. PC12 cells were exposed to 30 mW/cm² microwave for 5 min and the synapse morphology was visualized with scanning electron microscope and atomic force microscope. The release of amino acid neurotransmitters and calcium influx were detected. The expressions of several key NMDAR signaling molecules were evaluated. Results Microwave exposure caused injury in rat hippocampal structure and PC12 cells, especially the structure and quantity of synapses. The ratio of glutamic acid and gamma-aminobutyric acid neurotransmitters was increased and the intracellular calcium level was elevated in PC12 cells. A significant change in NMDAR subunits (NR1, NR2A, and NR2B) and related signaling molecules (Ca2+/calmodulin-dependent kinase II gamma and phosphorylated cAMP-response element binding protein) were examined. Conclusion 30 mW/cm² microwave exposure resulted in alterations of synaptic structure, amino acid neurotransmitter release and calcium influx. NMDAR signaling molecules were closely associated with impaired synaptic plasticity.

  13. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces expression of p27(kip¹) and FoxO3a in female rat cerebral cortex and PC12 cells.

    Xu, Guangfei; Liu, Jiao; Yoshimoto, Katsuhiko; Chen, Gang; Iwata, Takeo; Mizusawa, Noriko; Duan, Zhiqing; Wan, Chunhua; Jiang, Junkang


    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent toxin that alters normal brain development, producing cognitive disability and motor dysfunction. Previous studies in rats have proved that female rats are more sensitive to TCDD lethality than male ones. Recent studies have shown that TCDD induces cell cycle arrest and apoptosis, but the regulatory proteins involved in these processes have yet to be elucidated. In this study, we constructed an acute TCDD injury female rat model, and investigated the effects of TCDD on apoptosis and expression of cell cycle regulators, forkhead box class O 3a (FoxO3a) and p27(kip1), in the central nervous system (CNS). Increased levels of active caspase-3 were observed in the cerebral cortex of female rats treated with TCDD, suggesting that TCDD-induced apoptosis occurs in the CNS. The terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling assay showed that apoptosis primarily occurred in neurons. Furthermore, Western blot analysis, reverse transcription-polymerase chain reaction, and immunohistochemistry showed a significant up-regulation of FoxO3a and p27(kip1) in the cerebral cortex. Immunofluorescent labeling indicated that FoxO3a and p27(kip1) were predominantly localized in apoptotic neurons, but not in astrocytes. In vitro experiments using PC12, a rat neuron-like pheochromocytoma cell line, also revealed that TCDD induced apoptosis and an increase in FoxO3a and p27(kip1) expression. Furthermore, knockdown of FoxO3a expression inhibited p27(kip1) transcription and TCDD-induced apoptosis. Based on our data, induction of FoxO3a may play an important role in TCDD-induced neurotoxicity.

  14. Pheochromocytoma.

    Pederson, Lee C; Lee, Jeffrey E


    Pheochromocytoma is a rare tumor, but it represents a potentially curable form of hypertension. In patients with inherited pheochromocytoma, benign and bilateral tumors are more common. The diagnosis of pheochromocytoma rests in biochemical confirmation of catecholamine excess. Plasma-free metanephrine levels are arguably the most sensitive and specific test for the biochemical diagnosis of pheochromocytoma in high-risk patient populations. A timed 24-hour urine collection for total catecholamines and metabolic products (eg, vanillylmandelic acid and metanephrines) is the favored confirmatory test. Localization is most commonly accomplished with high-resolution computed tomography imaging, but magnetic resonance imaging can also be used. If both of these imaging modalities are nonlocalizing or equivocal, then radiolabeled meta-iodobenzylguanidine or somatostatin can be used to identify an adrenal or extra-adrenal tumor (paraganglioma). These imaging modalities can be used in the evaluation of patients with suspected or confirmed recurrent or metastatic disease. Systemic therapies for the treatment of patients with recurrent or metastatic disease have been disappointing. Radiation therapy is best applied for palliative relief of pain associated with bony metastases. In the absence of radiographic evidence for local tumor invasion, laparoscopic resection of small- to medium-sized (Hipple-Lindau syndrome or multiple endocrine neoplasia type 2 setting in which the cumulative incidence of clinical bilateral tumors is high, a cortical-sparing approach may minimize the risk of Addisonian complications.

  15. 人参皂苷对紫外线诱导PC12细胞损伤及凋亡的保护作用%Study of ginsenoside on injury and apoptosis of pheochromocytoma(PC12) cells induced by ultraviolet



    目的研究人参皂苷(GRS)对神经元分化细胞株(PC12)损伤及凋亡的影响.方法采用神经细胞培养和流式细胞仪技术,以不同强度紫外线诱导PC12细胞损伤作用作为细胞模型,观察紫外线对PC12细胞的影响,以及不同剂量GRS对PC12损伤的保护作用.结果 PC12照射后死亡方式之一是凋亡,吖啶橙和溴化锭(AO)染色荧光显微镜计数凋亡细胞百分率和DNA梯状带(DNA ladder)检测结果发现细胞凋亡有时间和剂量上的依赖性,GRS对紫外线诱导的细胞凋亡,也有一定的拮抗作用.结论 GRS能拮抗紫外线诱导的PC12细胞凋亡,对紫外线引起的细胞凋亡有一定的保护作用.

  16. Rat Nasal Respiratory Mucosa-Derived Ectomesenchymal Stem Cells Differentiate into Schwann-Like Cells Promoting the Differentiation of PC12 Cells and Forming Myelin In Vitro

    Jian Zhang


    Full Text Available Schwann cell (SC transplantation as a cell-based therapy can enhance peripheral and central nerve repair experimentally, but it is limited by the donor site morbidity for clinical application. We investigated weather respiratory mucosa stem cells (REMSCs, a kind of ectomesenchymal stem cells (EMSCs, isolated from rat nasal septum can differentiate into functional Schwann-like cells (SC-like cells. REMSCs proliferated quickly in vitro and expressed the neural crest markers (nestin, vimentin, SOX10, and CD44. Treated with a mixture of glial growth factors for 7 days, REMSCs differentiated into SC-like cells. The differentiated REMSCs (dREMSCs exhibited a spindle-like morphology similar to SC cells. Immunocytochemical staining and Western blotting indicated that SC-like cells expressed the glial markers (GFAP, S100β, Galc, and P75 and CNPase. When cocultured with dREMSCs for 5 days, PC12 cells differentiated into mature neuron-like cells with long neurites. More importantly, dREMSCs could form myelin structures with the neurites of PC12 cells at 21 days in vitro. Our data indicated that REMSCs, a kind of EMSCs, could differentiate into SC-like cells and have the ability to promote the differentiation of PC12 cells and form myelin in vitro.

  17. Effects of (-)-sesamin on 6-hydroxydopamine-induced neurotoxicity in PC12 cells and dopaminergic neuronal cells of Parkinson's disease rat models.

    Park, Hyun Jin; Zhao, Ting Ting; Lee, Kyung Sook; Lee, Seung Ho; Shin, Keon Sung; Park, Keun Hong; Choi, Hyun Sook; Lee, Myung Koo


    The present study investigated the effects of (-)-sesamin on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity using PC12 cells and dopaminergic neuronal cells of 6-OHDA-lesioned rat model of Parkinson's disease (PD). In PC12 cells, treatment with (-)-sesamin (25 µM) reduced 6-OHDA (100 µM)-induced cell death and induced transient extracellular signal-regulated kinase (ERK1/2) phosphorylation and Bad phosphorylation at Ser112 (BadSer112). In contrast, sustained ERK1/2 phosphorylation, p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK1/2) phosphorylation, and cleaved-caspase-3 activity, all of which were induced by 6-OHDA (100 µM), were inhibited by treatment with (-)-sesamin (25 µM). Furthermore, co-treatment with (-)-sesamin (30 mg/kg, p.o.) once a day for 28 days significantly increased the number of tyrosine hydroxylase-immunopositive neuronal cells and the levels of dopamine, norepinephrine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid in the substantia nigra-striatum of 6-OHDA-lesioned rat model of PD with or without L-DOPA treatment. These results suggest that (-)-sesamin protects 6-OHDA-induced cytotoxicity via the activation of transient ERK1/2-BadSer112 system and the inhibition of sustained ERK-p38MAPK-JNK1/2-caspase-3 system in PC12 cells. (-)-Sesamin also shows protective effects on long-term L-DOPA therapy in dopaminergic neuronal cells of PD rat models. (-)-Sesamin may serve as adjuvant therapeutics in PD.

  18. Phycocyanobilin promotes PC12 cell survival and modulates immune and inflammatory genes and oxidative stress markers in acute cerebral hypoperfusion in rats

    Marín-Prida, Javier [Centre for Research and Biological Evaluations (CEIEB), Institute of Pharmacy and Food, University of Havana, Ave. 23 e/ 214 y 222, La Lisa, PO Box: 430, Havana (Cuba); Pavón-Fuentes, Nancy [International Centre for Neurological Restoration (CIREN), Ave. 25 e/ 158 y 160, Playa, PO Box: 11300, Havana (Cuba); Llópiz-Arzuaga, Alexey; Fernández-Massó, Julio R. [Centre for Genetic Engineering and Biotechnology (CIGB), Ave. 31 e/158 y 190, Playa, PO Box: 6162, Havana (Cuba); Delgado-Roche, Liván [Centre for Research and Biological Evaluations (CEIEB), Institute of Pharmacy and Food, University of Havana, Ave. 23 e/ 214 y 222, La Lisa, PO Box: 430, Havana (Cuba); Mendoza-Marí, Yssel; Santana, Seydi Pedroso; Cruz-Ramírez, Alieski; Valenzuela-Silva, Carmen; Nazábal-Gálvez, Marcelo; Cintado-Benítez, Alberto [Centre for Genetic Engineering and Biotechnology (CIGB), Ave. 31 e/158 y 190, Playa, PO Box: 6162, Havana (Cuba); Pardo-Andreu, Gilberto L. [Centre for Research and Biological Evaluations (CEIEB), Institute of Pharmacy and Food, University of Havana, Ave. 23 e/ 214 y 222, La Lisa, PO Box: 430, Havana (Cuba); Polentarutti, Nadia [Istituto Clinico Humanitas (IRCCS), Rozzano (Italy); Riva, Federica [Department of Veterinary Science and Public Health (DIVET), University of Milano (Italy); Pentón-Arias, Eduardo [Centre for Genetic Engineering and Biotechnology (CIGB), Ave. 31 e/158 y 190, Playa, PO Box: 6162, Havana (Cuba); Pentón-Rol, Giselle [Centre for Genetic Engineering and Biotechnology (CIGB), Ave. 31 e/158 y 190, Playa, PO Box: 6162, Havana (Cuba)


    Since the inflammatory response and oxidative stress are involved in the stroke cascade, we evaluated here the effects of Phycocyanobilin (PCB, the C-Phycocyanin linked tetrapyrrole) on PC12 cell survival, the gene expression and the oxidative status of hypoperfused rat brain. After the permanent bilateral common carotid arteries occlusion (BCCAo), the animals were treated with saline or PCB, taking samples 24 h post-surgery. Global gene expression was analyzed with GeneChip Rat Gene ST 1.1 from Affymetrix; the expression of particular genes was assessed by the Fast SYBR Green RT-PCR Master Mix and Bioplex methods; and redox markers (MDA, PP, CAT, SOD) were evaluated spectrophotometrically. The PCB treatment prevented the H{sub 2}O{sub 2} and glutamate induced PC12 cell injury assessed by the MTT assay, and modulated 190 genes (93 up- and 97 down-regulated) associated to several immunological and inflammatory processes in BCCAo rats. Furthermore, PCB positively modulated 19 genes mostly related to a detrimental pro-inflammatory environment and counteracted the oxidative imbalance in the treated BCCAo animals. Our results support the view of an effective influence of PCB on major inflammatory mediators in acute cerebral hypoperfusion. These results suggest that PCB has a potential to be a treatment for ischemic stroke for which further studies are needed. - Highlights: • Phycocyanobilin (PCB) prevents H{sub 2}O{sub 2} and glutamate induced PC12 cell viability loss. • Anterior cortex and striatum are highly vulnerable to cerebral hypoperfusion (CH). • PCB modulates 190 genes associated to inflammation in acute CH. • PCB regulates 19 genes mostly related to a detrimental pro-inflammatory environment. • PCB restores redox and immune balances showing promise as potential stroke therapy.

  19. ProNGF derived from rat sciatic nerves downregulates neurite elongation and axon specification in PC12 cells

    Anna Sofia Trigos


    Full Text Available Several reports have shown that a sciatic nerve conditioned media (CM causes neuronal-like differentiation in PC12 cells. This differentiation is featured by neurite outgrowth, which are exclusively dendrites, without axon or sodium current induction. In previous studies, our group reported that the CM supplemented with a generic inhibitor for tyrosine kinase receptors (k252a enhanced the CM-induced morphological differentiation upregulating neurite outgrowth, axonal formation and sodium current elicitation. Sodium currents were also induced by depletion of endogenous proNGF from the CM (pNGFd-CM. Given that sodium currents, neurite outgrowth and axon specification are important features of neuronal differentiation, in the current manuscript, first we investigated if proNGF was hindering the full PC12 cell neuronal-like differentiation. Second, we studied the effects of exogenous wild type (pNGFwt and mutated (pNGFmut proNGF isoforms over sodium currents and, whether or not their addition to the pNGFd-CM would prevent sodium current elicitation. Third, we investigated if proNGF was exerting its negative regulation through the sortilin receptor, and for this, the proNGF action was blocked with neurotensin (NT, a factor known to compete with proNGF for sortilin. Thereby, here we show that pNGFd-CM enhanced cell differentiation, cell proportion with long neurites, total neurite length, induced axonal formation and sodium current elicitation. Interestingly, treatment of PC12 cells with wild type or mutated proNGF isoforms elicited sodium currents. Supplementing pNGFd-CM with pNGFmut reduced 35% the sodium currents. On the other hand, pNGFd-CM+pNGFwt induced larger sodium currents than pNGFd-CM. Finally, treatments with CM supplemented with NT showed that sortilin was mediating proNGF negative regulation, since its blocking induced similar effects than the pNGFd-CM treatment. Altogether, our results suggest that proNGF within the CM, is one of the

  20. Potassium current inhibition by nonselective cation channel-mediated sodium entry in rat pheochromocytoma (PC-12) cells.

    Strübing, C; J Hescheler


    Under physiological conditions, nonselective cation (NSC) channels mediate the entry of cations into cells, the most important being Na+ and Ca2+. In contrast to the Ca(2+)-dependent signaling mechanisms, little is known about the consequences and the spatial distribution of intracellular [Na+] elevation. In this study we demonstrate that Na+ entry, during the opening of ATP-activated NSC channels, leads to an inhibition of voltage-dependent K+ currents (IK) in cromaffin-like undifferentiated...


    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  2. Effects of scutellarin on PKCγ in PC12 cell injury induced by oxygen and glucose deprivation

    Wei XU; Ruo-peng ZHA; Wen-yi WANG; Yi-ping WANG


    Aim: To evaluate the neuroprotective effect and mechanisms of scutellarin (Scu)against PC12 cell injury after oxygen and glucose deprivation followed by reperfusion (OGD-Rep). Methods: Undifferentiated rat pheochromocytoma PC12 cells, exposed to oxygen and glucose deprivation followed by reperfusion (OGD-Rep), used as an in vitro model of ischemia/reperfusion. Cell survival was evalu-ated by diphenyltetrazolium bromide (MTT) assay and the amount of LDH release was determined using assay kits. [Ca2+]1 was monitored using a fluorescent Ca2+-sensitive dye Fura-2 acetoxymethyl ester. Cell apoptosis was detected by a DNA ladder and by flow cytometric detection. The expression of protein kinase C (PKC)γ was determined using both RT-PCR and Western blotting. The translocation of PKCγ was assayed by subcellular fractionation and Western blotting.Results: OGD-Rep injury significantly elevated the level of LDH release, [Ca2+]1,mRNA expression and the translocation of PKCγ compared in the PC12 cells with those of the normal group. Scu (10-100 μmol/L) exerted a protective effect against OGD-Rep injury by reducing LDH release, [Ca2+]1, the percent of apoptosis, and the translocation of PKCγ. Conclusion: Scu inhibits the increase of [Ca2+]1 and the activation of PKCγ, exerting protective effects against PC12 cell injury induced by OGD-Rep.

  3. Involvement of stathmin 1 in the neurotrophic effects of PACAP in PC12 cells.

    Dejda, Agnieszka; Chan, Philippe; Seaborn, Tommy; Coquet, Laurent; Jouenne, Thierry; Fournier, Alain; Vaudry, Hubert; Vaudry, David


    Rat pheochromocytoma PC12 cells have been widely used to investigate the neurotrophic activities of pituitary adenylate cyclase-activating polypeptide (PACAP). In particular, PACAP has been shown to promote differentiation and to inhibit apoptosis of PC12 cells. In order to identify the mechanisms mediating these effects, we sought for proteins that are phosphorylated upon PACAP treatment. High-performance liquid chromatography and 2D gel electrophoresis analysis, coupled with mass spectrometry, revealed that stathmin 1 is strongly phosphorylated within only 5 min of exposure to PACAP. Western blot experiments confirmed that PACAP induced a robust phosphorylation of stathmin 1 in a time-dependent manner. On the other hand, PACAP decreased stathmin 1 gene expression. Investigations of the signaling mechanisms known to be activated by PACAP revealed that phosphorylation of stathmin 1 was mainly mediated through the protein kinase A and mitogen-activated protein kinase pathways. Blockage of stathmin 1 expression with small interfering RNA did not affect PC12 cell differentiation induced by PACAP but reduced the ability of the peptide to inhibit caspase 3 activity and significantly decreased its neuroprotective action. Taken together, these data demonstrate that stathmin 1 is involved in the neurotrophic effect of PACAP in PC12 cells.

  4. Protective effects of red wine flavonols on 4-hydroxynonenal-induced apoptosis in PC12 cells.

    Jang, Young Jin; Kang, Nam Joo; Lee, Ki Won; Lee, Hyong Joo


    There is accumulating evidence that a moderate consumption of red wine has health benefits, such as the inhibition of neurodegenerative diseases. Although this is generally attributed to resveratrol, the protective mechanisms and the active substance(s) remain unclear. We examined whether and how red wine extract (RWE) and red wine flavonols quercetin and myricetin inhibited 4-hydroxynonenal (HNE)-induced apoptosis of rat pheochromocytoma PC12 cells. RWE attenuated HNE-induced PC12 cell death in a dose-dependent manner. HNE induced cleavage of poly(ADP-ribose) polymerase, which is involved in DNA repair in the nucleus, and this was inhibited by RWE treatment. Treatment with RWE also inhibited HNE-induced nuclear condensation in PC12 cells. Data of 2',7'-dichlorofluorescin diacetate showed that RWE protected against apoptosis of PC12 cells by attenuating intracellular reactive oxygen species. The cytoprotective effects on HNE-induced cell death were stronger for quercetin and myricetin than for resveratrol. HNE-induced nuclear condensation was attenuated by quercetin and myricetin. These results suggest that the neuroprotective potential of red wine is attributable to flavonols rather than to resveratrol.

  5. Induction of neuritogenesis in PC12 cells by a pulsed electromagnetic field via MEK-ERK1/2 signaling.

    Kudo, Tada-aki; Kanetaka, Hiroyasu; Shimizu, Yoshinaka; Abe, Toshihiko; Mori, Hitoshi; Mori, Kazumi; Suzuki, Eizaburo; Takagi, Toshiyuki; Izumi, Shin-ichi


    We examined the regulation of neuritogenesis by a pulsed electromagnetic field (PEMF) in rat PC12 pheochromocytoma cells, which can be induced to differentiate into neuron-like cells with elongated neurites by inducers such as nerve growth factor (NGF). Plated PC12 cells were exposed to a single PEMF (central magnetic flux density, 700 mT; frequency, 0.172 Hz) for up to 12 h per day and were then evaluated for extent of neuritogenesis or acetylcholine esterase (AChE) activity. To analyze the mechanism underlying the effect of the PEMF on the cells, its effects on intracellular signaling were examined using the ERK kinase (MEK) inhibitors PD098059 and U0126 (U0124 was used as a negative control for U0126). The number of neurite-bearing PC12 cells and AChE activity increased after PEMF exposure without the addition of other inducers of neuritogenesis. Additionally, PEMF exposure induced sustained activation of ERK1/2 in PC12 cells, but not in NR8383 rat alveolar macrophages. Furthermore, U0126 strongly inhibited PEMF-dependent ERK1/2 activation and neuritogenesis. The PEMF-dependent neuritogenesis was also suppressed by PD098059, but not U0124. These results suggest that PEMF stimulation independently induced neuritogenesis and that activation of MEK-ERK1/2 signaling was induced by a cell-type-dependent mechanism required for PEMF-dependent neuritogenesis in PC12 cells.

  6. Protective Effect of Tetrahydroxystilbene Glucoside on 6-OHDA-Induced Apoptosis in PC12 Cells through the ROS-NO Pathway

    Tao, Lizhen; Li, Xiaofeng; Zhang, Lingling; Tian, Jiyu; Li, Xiaobing; Sun, Xin; Li, Xuefen; Jiang, Lin; Zhang, Xiaojun; Chen, Jianzong


    Oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, such as Parkinson's disease. The molecule, 2,3,5,4′-tetrahydr- oxystilbene-2-O-β-D-glucoside (TSG), is a potent antioxidant derived from the Chinese herb, Polygonum multiflorum Thunb. In this study, we investigated the protective effect of TSG against 6-hydroxydopamine-induced apoptosis in rat adrenal pheochromocytoma PC12 cells and the possible mechanisms. Our data demonstrated that TSG significantly ...

  7. Effect of a Nerve Graft Substitute Single-walled Carbon Nanotubes on Rat Pheochromocytoma Cells

    WU Qian; ZHANG Juan; HU Zheqin


    Carbon nanotubes (CNTs) were extensively explored for their beneficial use in nervous system tissue engineering. However, an important concern regarding the use of CNTs is their toxicity during the interaction between cells and the nano particles. The rat pheochromocytoma cell line (PC12) was co-cultured with three types of single-walled carbon nanotubes (SWNTs), purified raw SWNTs (C), hydroxyl purified SWNTs (C-OH) and carboxyl purified SWNTs (C-COOH) at 25 µg/mL and 100 µg/ml. The experimental results revealed that SWNTs at the concentration below 100 µg/mL did not affect the cell viability. Notably, powerful antioxidant system in nerous system tissue is able to counteract with the toxicity of CNTs, which is characterized by the prominently enhanced expression of main antioxidant enzymes (catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-S-transferase (GST)). Therefore, we believe that CNTs can be good candidates for the fabrication of biomedical scaffolds for the nerve tissue repair.

  8. Early response of glutathione- and thioredoxin-dependent antioxidant defense systems to Tl(I)- and Tl(III)-mediated oxidative stress in adherent pheochromocytoma (PC12adh) cells.

    Puga Molina, Lis C; Salvatierra Fréchou, Damiana M; Verstraeten, Sandra V


    Thallium (Tl) is a toxic heavy metal that causes oxidative stress both in vitro and in vivo. In this work, we evaluated the production of oxygen (ROS)- and nitrogen (RNS)-reactive species in adherent PC12 (PC12adh) cells exposed for 0.5-6 h to Tl(I) or Tl(III) (10-100 µM). In this system, Tl(I) induced mostly H2O2 generation while Tl(III) induced H2O2 and ONOO(·-) generation. Both cations enhanced iNOS expression and activity, and decreased CuZnSOD expression but without affecting its activity. Tl(I) increased MnSOD expression and activity but Tl(III) decreased them. NADPH oxidase (NOX) activity remained unaffected throughout the period assessed. Oxidant levels returned to baseline values after 6 h of incubation, suggesting a response of the antioxidant defense system to the oxidative insult imposed by the cations. Tl also affected the glutathione-dependent system: while Tl(III) increased glutathione peroxidase (GPx) expression and activity, Tl(I) and Tl(III) decreased glutathione reductase (GR) expression. However, GR activity was mildly enhanced by Tl(III). Finally, thioredoxin-dependent system was evaluated. Only Tl(I) increased 2-Cys peroxiredoxins (2-Cys Prx) expression, although both cations increased their activity. Tl(I) increased cytosolic thioredoxin reductase (TrxR1) and decreased mitochondrial (TrxR2) expression. Tl(III) had a biphasic effect on TrxR1 expression and slightly increased TrxR2 expression. Despite of this, both cations increased total TrxR activity. Obtained results suggest that in Tl(I)-exposed PC12adh cells, there is an early response to oxidative stress mainly by GSH-dependent system while in Tl(III)-treated cells both GSH- and Trx-dependent systems are involved.

  9. Group IIA secretory phospholipase A2 stimulates exocytosis and neurotransmitter release in pheochromocytoma-12 cells and cultured rat hippocampal neurons.

    Wei, S; Ong, W Y; Thwin, M M; Fong, C W; Farooqui, A A; Gopalakrishnakone, P; Hong, W


    Recent evidence shows that secretory phospholipase A2 (sPLA2) may play a role in membrane fusion and fission, and may thus affect neurotransmission. The present study therefore aimed to elucidate the effects of sPLA2 on vesicle exocytosis. External application of group IIA sPLA2 (purified crotoxin subunit B or purified human synovial sPLA2) caused an immediate increase in exocytosis and neurotransmitter release in pheochromocytoma-12 (PC12) cells, detected by carbon fiber electrodes placed near the cells, or by changes in membrane capacitance of the cells. EGTA and a specific inhibitor of sPLA2 activity, 12-epi-scalaradial, abolished the increase in neurotransmitter release, indicating that the effect of sPLA2 was dependent on calcium and sPLA2 enzymatic activity. A similar increase in neurotransmitter release was also observed in hippocampal neurons after external application of sPLA2, as detected by changes in membrane capacitance of the neurons. In contrast to external application, internal application of sPLA2 to PC12 cells and neurons produced blockade of neurotransmitter release. Our recent studies showed high levels of sPLA2 activity in the normal rat hippocampus, medulla oblongata and cerebral neocortex. The sPLA2 activity in the hippocampus was significantly increased, after kainate-induced neuronal injury. The observed effects of sPLA2 on neurotransmitter release in this study may therefore have a physiological, as well as a pathological role.

  10. Propofol prevents autophagic cell death following oxygen and glucose deprivation in PC12 cells and cerebral ischemia-reperfusion injury in rats.

    Derong Cui

    Full Text Available BACKGROUND: Propofol exerts protective effects on neuronal cells, in part through the inhibition of programmed cell death. Autophagic cell death is a type of programmed cell death that plays elusive roles in controlling neuronal damage and metabolic homeostasis. We therefore studied whether propofol could attenuate the formation of autophagosomes, and if so, whether the inhibition of autophagic cell death mediates the neuroprotective effects observed with propofol. METHODOLOGY/PRINCIPAL FINDINGS: The cell model was established by depriving the cells of oxygen and glucose (OGD for 6 hours, and the rat model of ischemia was introduced by a transient two-vessel occlusion for 10 minutes. Transmission electron microscopy (TEM revealed that the formation of autophagosomes and autolysosomes in both neuronal PC12 cells and pyramidal rat hippocampal neurons after respective OGD and ischemia/reperfusion (I/R insults. A western blot analysis revealed that the autophagy-related proteins, such as microtubule-associated protein 1 light chain 3 (LC3-II, Beclin-1 and class III PI3K, were also increased accordingly, but cytoprotective Bcl-2 protein was decreased. The negative effects of OGD and I/R, including the formation of autophagosomes and autolysosomes, the increase in LC3-II, Beclin-1 and class III PI3K expression and the decline in Bcl-2 production were all inhibited by propofol and specific inhibitors of autophagy, such as 3-methyladenine (3-MA, LY294002 and Bafilomycin A1 (Baf,. Furthermore, in vitro OGD cultures and in vivo I/R rats showed an increase in cell survival following the administration of propofol, as assessed by an MTT assay or histochemical analyses. CONCLUSIONS/SIGNIFICANCE: Our data suggest that propofol can markedly attenuate autophagic processes via the decreased expression of autophagy-related proteins in vitro and in vivo. This inhibition improves cell survival, which provides a novel explanation for the pleiotropic effects of

  11. Protective effect of arctigenin on ethanol-induced neurotoxicity in PC12 cells.

    Huang, Jia; Xiao, Lan; Wei, Jing-Xiang; Shu, Ya-Hai; Fang, Shi-Qi; Wang, Yong-Tang; Lu, Xiu-Min


    As a neurotropic substance, ethanol can damage nerve cells through an increase in the production of free radicals, interference of neurotrophic factor signaling pathways, activation of endogenous apoptotic signals and other molecular mechanisms. Previous studies have revealed that a number of natural drugs extracted from plants offer protection of nerve cells from damage. Among these, arctigenin (ATG) is a lignine extracted from Arctium lappa (L.), which has been found to exert a neuroprotective effect on scopolamine‑induced memory deficits in mice with Alzheimer's disease and glutamate-induced neurotoxicity in primary neurons. As a result, it may offer beneficial effects on ethanol-induced neurotoxicity. However, the effects of ATG on ethanol‑induced nerve damage remain to be elucidated. To address this issue, the present study used rat pheochromocytoma PC12 cells to investigate the neuroprotective effects of ATG on ethanol-induced cell damage by performing an MTT reduction assay, cell cycle analysis, Hoechst33342/propidium iodide fluorescence staining and flow cytometry to examine apoptosis. The results showed that 10 µM ATG effectively promoted the proliferation of damaged cells, and increased the distribution ratio of the cells at the G2/M and S phases (P<0.05). In addition, the apoptosis and necrosis of the PC12 cells were significantly decreased following treatment with ATG. Therefore, it was concluded that 10 µM ATG had a protective effect on ethanol‑induced injury in PC12 cells.

  12. The role of cAMP in nerve growth factor-promoted neurite outgrowth in PC12 cells


    Nerve growth factor (NGF)-mediated neurite outgrowth in rat pheochromocytoma PC12 cells has been described to be synergistically potentiated by the simultaneous addition of dibutyryl cAMP. To elucidate further the role of cAMP in NGF-induced neurite outgrowth we have used the adenylate cyclase activator forskolin, cAMP, and a set of chemically modified cAMP analogues, including the adenosine cyclic 3',5'-phosphorothioates (cAMPS) (Rp)-cAMPS and (Sp)-cAMPS. These diastereomers have differentia...

  13. Protective effect of whey protein hydrolysates against hydrogen peroxide-induced oxidative stress on PC12 cells.

    Zhang, Qiu-Xiang; Ling, Yu-Fang; Sun, Zhen; Zhang, Li; Yu, Hui-Xin; Kamau, Samuel Mburu; Lu, Rong-Rong


    The protective effect of whey protein hydrolysates (WPHs) against H(2)O(2)-induced oxidative damage on rat pheochromocytoma line 12 (PC12) cells was studied. Whey protein was hydrolyzed by pepsin and trypsin and purified by macrospore absorption resins. PC12 cells were pretreated with WPHs (from 369 to 1,980 Da) at different concentrations for 2 h, then washed and incubated with 100 μM H(2)O(2) in the presence of WPHs for another 24 h. With 100-400 μg WPH/ml the viable cells increased by 20-30 % when incubated with H(2)O(2) suggesting that they may play a role as antioxidant in foods.

  14. Synthesis of cystine C60 derivative and its protective effects on hydrogen peroxide-induced apoptosis in PC12 cells

    Zhen Hu; Hai Ping Xing; Zhou Zhu; Wei Wang; Wen Chao Guan


    Oxidative stress has been considered as a major cause of cellular injuries in a variety of clinical abnormalities, especially prominent in neural diseases. One of the usable ways to prevent the reactive oxygen species (ROS)-mediated cellular injury is dietary or pharmaceutical augmentation of some free radical scavenger. Water-soluble amino-fullerene is a novel compound that behaves as a free radical scavenger with excellent biology consistent. In the present study, we have synthesized and characterized a novel cystine C60 derivative for the first time, and investigated the effects on hydrogen peroxide-induced oxidative stress and apoptotic death in cultured rat pheochromocytoma (PC12) cells. PC12 cells treated with hydrogen peroxide underwent apoptotic death as determined by MTT, PI/Hoechst 33342 staining and flow cytometry analysis. These results suggested that cystine C60 derivative has the potential to prevent oxidative stress-induced cell death and has no evident toxicity.

  15. The effects of functional magnetic nanotubes with incorporated nerve growth factor in neuronal differentiation of PC12 cells

    Xie Jining; Chen Linfeng; Varadan, Vijay K [Nanomaterials and Nanotubes Research Laboratory, College of Engineering, University of Arkansas, Fayetteville, AR 72701 (United States); Yancey, Justin; Srivatsan, Malathi [Department of Biological Sciences, Arkansas State University, State University, AR 72467 (United States)], E-mail:, E-mail:


    In this in vitro study the efficiency of magnetic nanotubes to bind with nerve growth factor (NGF) and the ability of NGF-incorporated magnetic nanotubes to release the bound NGF are investigated using rat pheochromocytoma cells (PC12 cells). It is found that functional magnetic nanotubes with NGF incorporation enabled the differentiation of PC12 cells into neurons exhibiting growth cones and neurite outgrowth. Microscope observations show that filopodia extending from neuron growth cones were in close proximity to the NGF-incorporated magnetic nanotubes, at times appearing to extend towards or into them. These results show that magnetic nanotubes can be used as a delivery vehicle for NGF and thus may be exploited in attempts to treat neurodegenerative disorders such as Parkinson's disease with neurotrophins. Further neurite outgrowth can be controlled by manipulating magnetic nanotubes with external magnetic fields, thus helping in directed regeneration.

  16. Beneficial effect of astragalosides on stroke condition using PC12 cells under oxygen glucose deprivation and reperfusion.

    Chiu, Bi-Ying; Chang, Ching-Ping; Lin, Jia-Wei; Yu, Jung-Sheng; Liu, Wen-Pin; Hsu, Yao-Chin; Lin, Mao-Tsun


    Astragalosides (AST) are reported to be neuroprotective in focal cerebral ischemic models in vivo. In this study, the direct effect of AST against oxygen and glucose deprivation (OGD) including neuronal injury and the underlying mechanisms in vitro were investigated. 5 h OGD followed by 24 h of reperfusion [adding back oxygen and glucose (OGD-R)] was used to induce in vitro ischemia reperfusion injury in differentiated rat pheochromocytoma PC12 cells. AST (1, 100, and 200 µg/mL) were added to the culture after 5 h of the OGD ischemic insult and was present during the reoxygenation phases. A key finding was that OGD-R decreased cell viability, increased lactate dehydrogenase, increased reactive oxygen species, apoptosis, autophagy, functional impairment of mitochondria, and endoplasmic reticulum stress in PC12 cells, all of which AST treatment significantly reduced. In addition, AST attenuated OGD-R-induced cell loss through P38 MAPK activation a neuroprotective effect blunted by SB203580, a specific inhibitor of P38 MAPK. Our data suggest that both apoptosis and autophagy are important characteristics of OGD-R-induced PC12 death and that treating PC12 cells with AST blocked OGD-R-induced apoptosis and autophagy by suppressing intracellular oxidative stress, functional impairment of mitochondria, and endoplasmic reticulum stress. Our data provide identification of AST that can concomitantly inhibit multiple cells death pathways following OGD injuries in neural cells.

  17. Bcl-2 protects against FCCP-induced apoptosis and mitochondrial membrane potential depolarization in PC12 cells.

    Dispersyn, G; Nuydens, R; Connors, R; Borgers, M; Geerts, H


    This report addresses the relation between Bcl-2 and mitochondrial membrane potential (DeltaPsi(m)) in apoptotic cell death. Rat pheochromocytoma (PC12) cells are differentiated into neuron-like cells with nerve growth factor (NGF). It is known that Bcl-2 can attenuate apoptosis induced by deprivation of neurotrophic factor. The protective effect of Bcl-2 has been correlated with preservation of DeltaPsi(m). Protonophores, such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), collapse the proton gradient across the mitochondrial inner membrane, resulting in a complete abolition of the mitochondrial membrane potential. Based on the analysis of morphology, of phosphatidylserine exposure and of nuclear fragmentation we conclude that FCCP induces apoptosis in PC12 cells, which can be prevented by overexpression of Bcl-2. To determine whether the cytoprotective effect of Bcl-2 is due to stabilization of DeltaPsi(m), we investigated the effect of Bcl-2 on changes in DeltaPsi(m), induced by FCCP in PC12 cells. We showed that treatment with FCCP induced a reduction in DeltaPsi(m), as assessed with the lipophilic cationic membrane potential-sensitive dye JC-1, and that Bcl-2 protects against FCCP-induced changes in NGF differentiated PC12 cells. Our data indicate that Bcl-2 protects against FCCP-induced cell death by stabilizing DeltaPsi(m).

  18. Effect of simulated microgravity on oxidation-sensitive gene expression in PC12 cells

    Kwon, Ohwon; Sartor, Maureen; Tomlinson, Craig R.; Millard, Ronald W.; Olah, Mark E.; Sankovic, John M.; Banerjee, Rupak K.


    Oxygen utilization by and oxygen dependence of cellular processes may be different in biological systems that are exposed to microgravity (micro-g). A baseline in which cellular changes in oxygen sensitive molecular processes occur during micro-g conditions would be important to pursue this question. The objective of this research is to analyze oxidation-sensitive gene expression in a model cell line [rat pheochromocytoma (PC12)] under simulated micro-g conditions. The PC12 cell line is well characterized in its response to oxygen, and is widely recognized as a sensitive model for studying the responses of oxygen-sensitive molecular and cellular processes. This study uses the rotating wall vessel bioreactor (RWV) designed at NASA to simulate micro-g. Gene expression in PC12 cells in response to micro-g was analyzed by DNA microarray technology. The microarray analysis of PC12 cells cultured for 4 days under simulated micro-g under standardized oxygen environment conditions revealed more than 100 genes whose expression levels were changed at least twofold (up-regulation of 65 genes and down-regulation of 39 genes) compared with those from cells in the unit gravity (unit-g) control. This study observed that genes involved in the oxidoreductase activity category were most significantly differentially expressed under micro-g conditions. Also, known oxidation-sensitive transcription factors such as hypoxia-inducible factor-2α, c-myc, and the peroxisome proliferator-activated receptor-γ were changed significantly. Our initial results from the gene expression microarray studies may provide a context in which to evaluate the effect of varying oxygen environments on the background of differential gene regulation of biological processes under variable gravity conditions. PMID:19081771

  19. Effects of huperzine A on secretion of nerve growth factor in cultured rat cortical astrocytes and neurite outgrowth in rat PC12 cells

    Li-li TANG; Rui WANG; Xi-can TANG


    Aim: To study the effects of huperzine A (HupA) on neuritogenic activity and the expression of nerve growth factor (NGF). Methods: After being treated with 10 μmol/L HupA, neurite outgrowth of PC12 cells was observed and counted under phase-contrast microscopy. Mitogenic activity was assayed by [3H]thymidine incorporation. Cell cytotoxicity was evaluated by lactate dehydrogenase (LDH)release. AChE activity, mRNA and protein expression were measured by the Ellman's method, RT-PCR, and Western blot, respectively. NGF mRNA and protein levels were determined by RT-PCR and ELISA assays. Results: Treatment of PC 12 cells with 10 μmol/L HupA for 48 h markedly increased the number of neuritebearing cells, but caused no significant alteration in cell viability or other signs of cytotoxicity. In addition to inhibiting AChE activity, 10 μmol/L HupA also increased the mRNA and protein levels of this enzyme. In addition, following 2 h exposure of the astrocytes to 10 μmol/L HupA, there was a significant up-regulation of mRNA for NGF and P75 low-affinity NGF receptor. The protein level of NGF was also increased after 24 h treatment with HupA. Conclusion: Our findings demonstrate for the first time that HupA has a direct or indirect neurotrophic activity, which might be beneficial in treatment of neurodegenerative disorders such as Alzheimer disease.

  20. Essential Oils from the Medicinal Herbs Upregulate Dopamine Transporter in Rat Pheochromocytoma Cells.

    Choi, Min Sun; Choi, Bang-sub; Kim, Sang Heon; Pak, Sok Cheon; Jang, Chul Ho; Chin, Young-Won; Kim, Young-Mi; Kim, Dong-il; Jeon, Songhee; Koo, Byung-Soo


    The dopamine transporter (DAT) protein, a component of the dopamine system, undergoes adaptive neurobiological changes from drug abuse. Prevention of relapse and reduction of withdrawal symptoms are still the major limitations in the current pharmacological treatments of drug addiction. The present study aimed to investigate the effects of essential oils extracted from Elsholtzia ciliata, Shinchim, Angelicae gigantis Radix, and Eugenia caryophyllata, well-known traditional Korean medicines for addiction, on the modulation of dopamine system in amphetamine-treated cells and to explore the possible mechanism underlying its therapeutic effect. The potential cytotoxic effect of essential oils was evaluated in PC12 rat pheochromocytoma cells using cell viability assays. Quantification of DAT, p-CREB, p-MAPK, and p-Akt was done by immunoblotting. DAT was significantly reduced in cells treated with 50 μM of amphetamine in a time-dependent manner. No significant toxicity of essential oils from Elsholtzia ciliata and Shinchim was observed at doses of 10, 25, and 50 μg/mL. However, essential oils from A. gigantis Radix at a dose of 100 μg/mL and E. caryophyllata at doses of 50 and 100 μg/mL showed cytotoxicity. Treatment with GBR 12909, a highly selective DAT inhibitor, significantly increased DAT expression compared with that of amphetamine only by enhancing phosphorylation of mitogen-activated protein kinases (MAPK) and Akt. In addition, essential oils effectively induced hyperphosphorylation of cyclic-AMP response element-binding protein (CREB), MAPK, and Akt, which resulted in DAT upregulation. Our study implies that the essential oils may rehabilitate brain dopamine function through increased DAT availability in abstinent former drug users.

  1. Protective effects of fractions from Artemisia biennis hydro-ethanolic extract against doxorubicin-induced oxidative stress and apoptosis in PC12 cells

    Mahdi Mojarrab


    Full Text Available Objective(s: This study was designed to indicate whether different fractions from Artemisia biennis hydroethanolic extract could provide cytoprotection against oxidative stress and apoptosis induced by doxorubicin (DOX in rat pheochromocytoma cell line (PC12. Material and Methods:Cell viability was determined by MTT assay. Also, activation of caspase-3 and superoxide dismutase were evaluated by spectrophotometry. Detection of reactive oxygen species (ROS and measurement of mitochondrial membrane potential (MMP were performed by flowcytometry. Results:  Treatment of PC12 cells with DOX reduced viability dose dependently. For evaluation of the effect of fractions (A-G on DOX-induced cytotoxicity, PC12 cells were pretreated for 24 hr with the A. biennis fractions and then cells were treated with DOX.  The fractions C and D increased PC12 cells viability significantly compared to DOX treated cells.  Moreover, pretreatment with fractions C and D for 24 hr attenuated DOX-mediated apoptosis and the anti-apoptotic action of A. biennis fractions was partially dependent on inhibition of caspase 3 activity and also increasing the  mitochondrial membrane potential (MMP. Selected A. biennis fractions also suppressed the generation of ROS and increased superoxide dismutase (SOD activity. Conclusion: Taken together our observation indicated that subtoxic concentration of aforementioned fractions of A. biennis hydroetanolic extract has protective effect against apoptosis induced by DOX in PC12 cell. The results highlighted that fractions C and D may exert cytoprotective effects through their antioxidant actions.

  2. Reactive oxygen species scavenging activities in a chemiluminescence model and neuroprotection in rat pheochromocytoma cells by astaxanthin, beta-carotene, and canthaxanthin.

    Chang, Chi-Sen; Chang, Chia-Lin; Lai, Guia-Hung


    The objective of this study was to determine chemiluminescence (CL) antioxidant activities and neuroprotective effects of astaxanthin, beta-carotene (β-carotene), and canthaxanthin on undifferentiated rat pheochromocytoma (PC12) cells. We performed three CL antioxidant assays, and the three carotenoids showed varying degrees of antioxidant activity, with astaxanthin exhibiting the highest antioxidant activity than the other two samples. Results of a pyrogallol-luminol assay revealed β-carotene to have higher antioxidant activity than canthaxanthin, whereas cupric sulfate-Phen-Vc-hydrogen peroxide (H₂O₂) assay showed canthaxanthin to have higher antioxidant activity than β-carotene. Luminol-H₂O₂ assay showed the antioxidant activity series as canthaxanthin > β-carotene at 62.5-1000 μg/mL and β-carotene > canthaxanthin at 1000-4000 μg/mL. Astaxanthin exhibited partial neuroprotective activity against H₂O₂ and the strongest neuroprotective activity against amyloid beta-peptide(25-35) [(Aβ)(25-35)]-induced undifferentiated PC12 cell deaths at 0.5-5.0 μM. Canthaxanthin showed partial neuroprotective activity in Aβ(25-35)-induced undifferentiated PC12 cell deaths at 1.0-5.0 μM. Astaxanthin protected undifferentiated PC12 cells from the damaging effects of H₂O₂ and Aβ(25-35) by the following ways: (1) scavenging superoxide anion radicals, hydroxyl radicals, and H₂O₂; (2) securing cell viability; (3) suppressing the production of reactive oxygen species; and (4) eliminating calcium ion influx. Our results conclusively show that astaxanthin has the merit as a potential neuron protectant.

  3. Neurotrophic Effect of Citrus Auraptene: Neuritogenic Activity in PC12 Cells

    Mitsunari Nakajima


    Full Text Available The activation of extracellular signal-regulated kinases (ERK leads to a number of cellular changes associated with the development of long-term memory. Using cultured cortical neurons, we previously showed that the n-hexane extract prepared from the peels of Citrus grandis (Kawachi bankan induces the activation of ERK1/2 and that one of the compounds with this ability in the extract is 3,5,6,7,8,3′,4′-heptamethoxyflavone (HMF, a Citrus polymethoxyflavone. In fact, we found that HMF has the ability to rescue mice from drug-induced learning impairment. This hexane extract contains auraptene (AUR, a coumarin derivative with a monoterpene unit, together with HMF. The present study was designed to investigate the effect of AUR in vitro. Our results show that 1 AUR had the ability to induce the activation of ERK1/2 in not only cortical neurons but also the rat pheochromocytoma cell line (PC12 cells, which is a model system for studies on neuronal proliferation and differentiation; and 2 AUR had the ability to promote neurite outgrowth from PC12 cells.

  4. Activated RET/PTC oncogene elicits immediate early and delayed response genes in PC12 cells.

    Califano, D; Monaco, C; de Vita, G; D'Alessio, A; Dathan, N A; Possenti, R; Vecchio, G; Fusco, A; Santoro, M; de Franciscis, V


    The expression of the receptor-like tyrosine kinase RET is associated with tumors, tissues or cell lines of neural crest origin. In addition RET products (Ret) are involved in determining cell fate during the differentiation of the enteric nervous system and during renal organogenesis. However, as yet, no direct evidence exists to indicate that the Ret kinase activity might interfere in a specific way with cellular differentiation, or proliferation, of a neural crest derived cell line. By using two constitutively activated forms of RET (RET/PTC1 and RET/PTC3) in transient transfection experiments, we have obtained evidence that active RET could reprogramme the gene expression pattern in the rat pheochromocytoma PC12 cell line. Transcription driven by gene promoters, such as NGFI-A and vgf, which belong, respectively, to primary and delayed response genes to nerve growth factor (NGF), and by the neuron-specific enolase (NSE) promoter, is rapidly induced by the expression of activated RET oncogenes. This induction is not elicited in other non neural derived cell types tested. We also demonstrate that endogenous ras activity is required for RET induction of these neural markers. Finally, in the RET/PTC transfected PC12 cells, NGF is unable to induce further their transcription. This suggests that RET/PTC could share an intracellular signalling pathway with the NGF-receptor.

  5. New physiological function of secoiridoids: neuritogenic activity in PC12h cells.

    Chiba, Kenzo; Yamazaki, Matsumi; Kikuchi, Masafumi; Kakuda, Rie; Kikuchi, Masao


    Previously, we have reported that geniposide isolated from an extract of Gardenia fructus has neuritogenic activity in PC12h cells, a subclone of rat pheochromocytoma cells. Furthermore, we have indicated that several geniposide-related iridoid compounds also had similar potent neuritogenic activity. In this study, we have examined the effects of various secoiridoid compounds [K-1, sweroside; K-2, swertiamarin; K-3, gentiopicroside; K-4, 6'-O-β-D: -glucopyranosylsweroside; K-5, 6'-O-β-D: -glucopyranosylgentiopicroside; K-6, 6'-O-β-D: -glucopyranosylswertiamarin; K-7, 5'-O-β-D: -glucopyranosylamarogentin; K-8, 5'-O-β-D: -glucopyranosylamaroswertin; H-1, n-butyl vogeloside; H-2, n-butyl epivogeloside; H-3, (7S)-secologanin butyl methyl acetal; H-4, (7R)-secologanin butyl methyl acetal; H-5, secologanin dimethyl acetal] isolated from various medicinal herbs. The secoiridoids H-1, H-2, H-3, H-4, and H-5 induced significant neurite outgrowth. Among these H-series compounds, H-2 was the most potent neuritogenic compound. Among the K-series compounds, K-1, K-2, K-3, and K-8 showed the most potent activity. These results suggest that secoiridoids have neuritogenic activity in PC12h cells and that these secoiridoid compounds are promising starting compounds for the development of neurotrophic factor-like and iridoid compounds.

  6. Antinociceptive Effect of Intrathecal Microencapsulated Human Pheochromocytoma Cell in a Rat Model of Bone Cancer Pain

    Xiao Li


    Full Text Available Human pheochromocytoma cells, which are demonstrated to contain and release met-enkephalin and norepinephrine, may be a promising resource for cell therapy in cancer-induced intractable pain. Intrathecal injection of alginate-poly (l lysine-alginate (APA microencapsulated human pheochromocytoma cells leads to antinociceptive effect in a rat model of bone cancer pain, and this effect was blocked by opioid antagonist naloxone and alpha 2-adrenergic antagonist rauwolscine. Neurochemical changes of cerebrospinal fluid are in accordance with the analgesic responses. Taken together, these data support that human pheochromocytoma cell implant-induced antinociception was mediated by met-enkephalin and norepinephrine secreted from the cell implants and acting at spinal receptors. Spinal implantation of microencapsulated human pheochromocytoma cells may provide an alternative approach for the therapy of chronic intractable pain.

  7. Radiofrequency radiation at 1950 MHz (UMTS) does not affect key cellular endpoints in neuron-like PC12 cells.

    Zeni, Olga; Sannino, Anna; Sarti, Maurizio; Romeo, Stefania; Massa, Rita; Scarfì, Maria R


    In this study, rat pheochromocytoma (PC12) cells were exposed, as a model of neuron-like cells, to 1950 MHz radiofrequency (RF) radiation with a signal used by the 3G wireless technology of the Universal Mobile Telecommunications System (UMTS) to assess possible adverse effects. RF exposure for 24 h at a specific absorption rate (SAR) of 10 W/kg was carried out in a waveguide system under accurately controlled environmental and dosimetric parameters. DNA integrity, cell viability, and apoptosis were investigated as cellular endpoints relevant for carcinogenesis and other diseases of the central nervous system. Very sensitive biological assays were employed to assess the effects immediately after RF exposure and 24 h later, as demonstrated by the cellular response elicited in PC12 cells using positive control treatments provided for each assay. In our experimental conditions, 24 h of RF exposure at a carrier frequency and modulation scheme typical of a UMTS signal was not able to elicit any effect in the selected cellular endpoints in undifferentiated PC12 cells, despite the application of a higher SAR value than those applied in the majority of the studies reported in the literature. Copyright © 2012 Wiley Periodicals, Inc.

  8. Inhibitory effects of multiwall carbon nanotubes with high iron impurity on viability and neuronal differentiation in cultured PC12 cells.

    Meng, Li; Jiang, Aihua; Chen, Rui; Li, Chen-zhong; Wang, Liming; Qu, Ying; Wang, Peng; Zhao, Yuliang; Chen, Chunying


    The increasing use of carbon nanotubes (CNTs) in biomedical applications has garnered a great concern on their potential negative effects to human health. CNTs have been reported to potentially disrupt normal neuronal function and they were speculated to accumulate and cause brain damage, although a lot of distinct and exceptional properties and potential wide applications have been associated with this material in neurobiology. Fe impurities strapped inside the CNTs may be partially responsible for neurotoxicity generation. In the present study, we selected rat pheochromocytoma (PC12) cells to investigate and compare the effects of two kinds of multiwall carbon nanotubes (MWCNTs) with different concentrations of Fe impurities which usually come from the massive production of CNTs by chemical vapor deposition. Exposure to Fe-high MWCNTs can reduce cell viability and increase cytoskeletal disruption of undifferentiated PC12 cells, diminish the ability to form mature neurites, and then adversely influence the neuronal dopaminergic phenotype in NGF-treated PC-12 cells. The present results highlight the critical role of iron residue in the adverse response to MWCNTs exposure in neural cells. These findings provide useful information for understanding the toxicity and safe application of carbon nanotubes.

  9. Subcellular Distribution of S-Nitrosylated H-Ras in Differentiated and Undifferentiated PC12 Cells during Hypoxia.

    Barbakadze, Tamar; Goloshvili, Galina; Narmania, Nana; Zhuravliova, Elene; Mikeladze, David


    Hypoxia or exposure to excessive reactive oxygen or nitrogen species could induce S-nitrosylation of various target proteins, including GTPases of the Ras-superfamily. Under hypoxic conditions, the Ras-protein is translocated to the cytosol and interacts with the Golgi complex, endoplasmic reticulum, mitochondria. The mobility/translocation of Ras depend on the cells oxidative status. However, the importance of relocated Snitrosylated- H-Ras (NO-H-Ras) in proliferation/differentiation processes is not completely understood. We have determined the content of soluble- and membrane-bound-NO-HRas in differentiated (D) and undifferentiated (ND) rat pheochromocytoma (PC12) cells under hypoxic and normoxic conditions. In our experimental study, we analyzed NO-H-Ras levels under hypoxic/normoxic conditions in membrane and soluble fractions of ND and D PC12 cells with/without nitric oxide donor, sodium nitroprusside (SNP) treatment. Cells were analyzed by the S-nitrosylated kit, immunoprecipitation, and Western blot. We assessed the action of NO-H-Ras on oxidative metabolism of isolated mitochondria by determining mitochondrial hydrogen peroxide generation via the scopoletin oxidation method and ATPproduction as estimated by the luminometric method. Hypoxia did not influence nitrosylation of soluble H-Ras in ND PC12 cells. Under hypoxic conditions, the nitrosylation of soluble-H-Ras greatly decreased in D PC12 cells. SNP didn't change the levels of nitrosylation of soluble-H-Ras, in either hypoxic or normoxic conditions. On the other hand, hypoxia, per se, did not affect the nitrosylation of membrane-bound-H-Ras in D and ND PC12 cells. SNP-dependent nitrosylation of membrane-bound-H-Ras greatly increased in D PC12 cells. Both unmodified normal and mutated H-Ras enhanced the mitochondrial synthesis of ATP, whereas the stimulatory effects on ATP synthesis were eliminated after S-nitrosylation of H-Ras. According to the results, it may be proposed that hypoxia can decrease S


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  11. Hydroxytyrosol increases norepinephrine transporter function in pheochromocytoma cells

    Luzon-Toro, Berta [Institute of Parasitology and Biomedicine ' Lopez-Neyra' , Spanish National Research Council (CSIC), 18100 Granada (Spain); Geerlings, Arjan [Puleva Biotech, 18004 Granada (Spain); Hilfiker, Sabine [Institute of Parasitology and Biomedicine ' Lopez-Neyra' , Spanish National Research Council (CSIC), 18100 Granada (Spain)], E-mail:


    Introduction: The norepinephrine transporter is responsible for the intracellular uptake of {sup 131}I- iodometaiodobenzylguanidine ({sup 131}I-MIBG), which is used for the diagnostic localization and treatment of pheochromocytomas as well as other tumors such as neuroblastomas and carcinoids. This agent is variably delivered into tumor cells by the norepinephrine transporter, but few studies have shown treatments that work to increase norepinephrine transporter activity. The objective of the present study was to test the possible beneficial effects of hydroxytyrosol in enhancing norepinephrine transporter function, which may have implications for its combined use with {sup 131}I-MIBG in the diagnosis and treatment of pheochromocytomas. Methods: Rat pheochromocytoma PC12 cells were labeled with [{sup 3}H]-norepinephrine in the presence or absence of different concentrations of hydroxytyrosol, a naturally occurring compound with strong antioxidant properties, followed by measurements of uptake and release of radiolabeled norepinephrine. Results: Hydroxytyrosol pronouncedly increased norepinephrine transporter activity, with the rapid onset excluding effects on norepinephrine transporter expression levels. Concomitant with increased norepinephrine transporter activity, hydroxytyrosol caused a decrease of both spontaneous and evoked norepinephrine release, indicating that it affects pre-existing plasma membrane-associated norepinephrine transporter, rather than the incorporation of novel norepinephrine transporter molecules into the plasma membrane. Conclusion: Hydroxytyrosol potently enhances norepinephrine transporter activity in pheochromocytoma PC12 cells, suggesting that combinatorial therapy employing hydroxytyrosol may improve the effectiveness of {sup 131}I-MIBG as a diagnosis and treatment modality.

  12. Characterization of the Rat GAL2R Promoter: Positive Role of ETS-1 in Regulation of the Rat GAL2R Gene in PC12 Cells.

    Yang, Yutao; Liu, Li; Luo, Hanjiang; Li, Yueting; Li, Hui; Xu, Zhi-Qing David


    Galanin receptor 2 (GAL2R) is a G protein-coupled receptor for the neuropeptide galanin that regulates many important physiological functions and pathological processes. To investigate the molecular mechanism governing GAL2R gene transcription, the rat GAL2R promoter was isolated and analyzed. We found that the region from -320 to -300 of the GAL2R promoter contains two putative ETS-1 elements and plays an important role in regulating GAL2R promoter activity. We also showed that transcription factor ETS-1 bound to this region in vitro and in vivo. Overexpression of ETS-1 significantly increased GAL2R promoter activity and transcription of the GAL2R gene, whereas knockdown of ETS-1 produced the opposite effects. In addition, we showed that ETS-1 recruited co-activator p300 to the GAL2R promoter. These data indicate a role for ETS-1 in the control of the GAL2R gene expression and provide a basis for understanding the transcriptional regulation of the GAL2R gene.

  13. Influence of Panax quinquefolium saponins on increased intracellular Ca2+ in PC12 cells

    Lixin Guan; Xiudong Jin; Yanhui Chu; Yufei Zhang; Yan Wu; Xin Yi; Fengguo Zhai; Mengquan Li


    BACKGROUND: Previous studies have demonstrated that intracellular Ca2+ ([Ca2+]i) overload,excitotoxicity, free radical injury, and nitric oxide toxicity are involved in mechanisms of neuronal death in the ischemic brain.OBJECTIVE: To investigate the influence of Panax quinquefolium saponins (PQS) on multiple factors-induced Ca2+ overload in the rat pheochromocytoma (PC12) cell line.DESIGN, TIME AND SETTING: Intergroup comparison, in vitro study. The experiment was performed at the Heilongjiang Key Laboratory of Anti-fibrosis Biotherapy, Mudanjiang Medical University between November 2007 and April 2008.MATERIALS: In vitro cultured PC12 cells in the logarithmic phase were assigned into blank control, model, and drug treatment groups (10 μmol/L nimodipine; 40 μg/L, 100 μg/L, and 250 μg/L PQS). Nimodipine was purchased from Jiangsu Yangtze River Pharmacy Group Co.,China; PQS (purity > 95%, HLPC grade) was provided by School of Basic Medical Sciences, Jilin University. Caffeine, Na2S204, L-glutamic acid (Glu), Fura-2/AM, and calcium ionophore A23187 were purchased from Sigma, USA.METHODS: PC12 cells in the model and drug treatment groups were separately incubated in glucose-free Hank's buffered saline solution + Na2S2O4, (2 mmol/L) for 6 hours, Glu (200 μ mol/L)plus A23187 (0.05 μmol/L) for 6 hours, KCI (50 mmol/L) for 1 hour, and caffeine (5 mmol/L) for 3 hours to establish models of intracellular Ca2+ overload induced by oxygen and glucose deprivation, Glu, A23187, high K+, or caffeine. In addition, control cells were incubated in high-glucose DMEM culture medium.MAIN OUTCOME MEASURES: [Ca2+]i changes in PC12 cells exposed to oxygen-glucose deprivation, Glu, A23187, high K+, or caffeine were detected using spectrofluorometer.RESULTS: PQS blocked the [Ca2+]i increase induced by oxygen-glucose deprivation, Glu,A23187, high K+, or caffeine. In particular, high-dose PQS was most effective (P < 0.01). PQS significantly inhibited Glu- or caffeine-induced [Ca2+]i

  14. Protective effect of tetrahydroxystilbene glucoside on 6-OHDA-induced apoptosis in PC12 cells through the ROS-NO pathway.

    Lizhen Tao

    Full Text Available Oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, such as Parkinson's disease. The molecule, 2,3,5,4'-tetrahydr- oxystilbene-2-O-β-D-glucoside (TSG, is a potent antioxidant derived from the Chinese herb, Polygonum multiflorum Thunb. In this study, we investigated the protective effect of TSG against 6-hydroxydopamine-induced apoptosis in rat adrenal pheochromocytoma PC12 cells and the possible mechanisms. Our data demonstrated that TSG significantly reversed the 6-hydroxydopamine-induced decrease in cell viability, prevented 6-hydroxydopamine-induced changes in condensed nuclei and decreased the percentage of apoptotic cells in a dose-dependent manner. In addition, TSG slowed the accumulation of intracellular reactive oxygen species and nitric oxide, counteracted the overexpression of inducible nitric oxide syntheses as well as neuronal nitric oxide syntheses, and also reduced the level of protein-bound 3-nitrotyrosine. These results demonstrate that the protective effects of TSG on rat adrenal pheochromocytoma PC12 cells are mediated, at least in part, by the ROS-NO pathway. Our results indicate that TSG may be effective in providing protection against neurodegenerative diseases associated with oxidative stress.

  15. Protective effect of tetrahydroxystilbene glucoside on 6-OHDA-induced apoptosis in PC12 cells through the ROS-NO pathway.

    Tao, Lizhen; Li, Xiaofeng; Zhang, Lingling; Tian, Jiyu; Li, Xiaobing; Sun, Xin; Li, Xuefen; Jiang, Lin; Zhang, Xiaojun; Chen, Jianzong


    Oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, such as Parkinson's disease. The molecule, 2,3,5,4'-tetrahydr- oxystilbene-2-O-β-D-glucoside (TSG), is a potent antioxidant derived from the Chinese herb, Polygonum multiflorum Thunb. In this study, we investigated the protective effect of TSG against 6-hydroxydopamine-induced apoptosis in rat adrenal pheochromocytoma PC12 cells and the possible mechanisms. Our data demonstrated that TSG significantly reversed the 6-hydroxydopamine-induced decrease in cell viability, prevented 6-hydroxydopamine-induced changes in condensed nuclei and decreased the percentage of apoptotic cells in a dose-dependent manner. In addition, TSG slowed the accumulation of intracellular reactive oxygen species and nitric oxide, counteracted the overexpression of inducible nitric oxide syntheses as well as neuronal nitric oxide syntheses, and also reduced the level of protein-bound 3-nitrotyrosine. These results demonstrate that the protective effects of TSG on rat adrenal pheochromocytoma PC12 cells are mediated, at least in part, by the ROS-NO pathway. Our results indicate that TSG may be effective in providing protection against neurodegenerative diseases associated with oxidative stress.

  16. Neurotropin promotes NGF signaling through interaction of GM1 ganglioside with Trk neurotrophin receptor in PC12 cells.

    Fukuda, Yu; Fukui, Takao; Hikichi, Chika; Ishikawa, Tomomasa; Murate, Kenichiro; Adachi, Takeshi; Imai, Hideki; Fukuhara, Koki; Ueda, Akihiro; Kaplan, Allen P; Mutoh, Tatsuro


    Activation of the high-affinity nerve growth factor (NGF) receptor Trk occurs through multiple processes consisted of translocation and clustering within the plasma membrane lipid rafts, dimerization and autophosphorylation. Here we found that a nonprotein extract of inflamed rabbit skin inoculated with vaccinia virus (Neurotropin(®)) enhanced efficiency of NGF signaling. In rat pheochromocytoma PC12 cells overexpressing Trk (PCtrk cells), Neurotropin augmented insufficient neurite outgrowth observed at suboptimal concentration of NGF (2ng/mL) in a manner depending on Trk kinase activity. Cellular exposure to Neurotropin resulted in an accumulation of Trk-GM1 complexes without affecting dimerization or phosphorylation states of Trk. Following NGF stimulation, Neurotropin significantly facilitated the time course of NGF-induced Trk autophosphorylation. These observations provide a unique mechanism controlling efficiency of NGF signaling, and raise the therapeutic potential of Neurotropin for various neurological conditions associated with neurotrophin dysfunction.

  17. AMP N1-Oxide, a Unique Compound of Royal Jelly, Induces Neurite Outgrowth from PC12 Vells via Signaling by Protein Kinase A Independent of that by Mitogen-Activated Protein Kinase

    Noriko Hattori


    Full Text Available Earlier we identified adenosine monophosphate (AMP N1-oxide as a unique compound of royal jelly (RJ that induces neurite outgrowth (neuritegenesis from cultured rat pheochromocytoma PC12 cells via the adenosine A2A receptor. Now, we found that AMP N1-oxide stimulated the phosphorylation of not only mitogen-activated protein kinase (MAPK but also that of cAMP/calcium-response element-binding protein (CREB in a dose-dependent manner. Inhibition of MAPK activation by a MEK inhibitor, PD98059, did not influence the AMP N1-oxide-induced neuritegenesis, whereas that of protein kinase A (PKA by a selective inhibitor, KT5720, significantly reduced neurite outgrowth. AMP N1-oxide also had the activity of suppressing the growth of PC12 cells, which correlated well with the neurite outgrowth-promoting activity. KT5720 restored the growth of AMP N1-oxide-treated PC12 cells. It is well known that nerve growth factor suppresses proliferation of PC12 cells before causing stimulation of neuronal differentiation. Thus, AMP N1-oxide elicited neuronal differentiation of PC12 cells, as evidenced by generation of neurites, and inhibited cell growth through adenosine A2A receptor-mediated PKA signaling, which may be responsible for characteristic actions of RJ.

  18. 红藻氨酸诱导PC12细胞凋亡及阿魏酸对神经元的保护作用%Ferulic acid protects against apoptosis of PC12 cells induced by kainic acid

    陈勤; 叶海燕; 陈逸青; 余嗣明


    AIM:To investigate the effect of ferulic acid (FA) on the apoptosis of PC12 cells induced by kainic acid (KA) in vitro.METHODS:In order to establish an Alzheimer disease neuronal cell model,the rat pheochromocytoma cell line PC12 was treated with KA at a concentration of 50 μmol/L.These model neurons were divided into KA model group and3 groups treated with FA at doses of 25,50 and 100 μmol/L,respectively.At the same time,normal group was established without KA pretreatment.The viability of the PC12 cells was detected by MTT assay.The expression of Bcl-2,Bax and cytochrome C (Cyt C) was determined by immunocytochemical method.Apoptotic rate of the PC12 cells was measured by flow cytometry with annexin V/PI double staining.The protein levels of Bcl-2,Bax and Cyt C were analyzed by Western blotting.RESULTS:The cell survival rate,the expression of Bcl-2 and the ratio of Bcl-2 to Bax in KA model group were significantly decreased (P <0.01),while the expression of Bax and Cyt C was obviously increased com pared with normal control group (P <0.01).The apoptotie rate in KA model group was obviously increased compared with normal control group (P <0.01) After the intervention of FA,the cell survival rates were increased and the apoptotic rates were decreased.Furthermore,the positive rate and expression of Bcl-2,and the ratio of Bcl-2 to Bax in each dose of FA treatment group were significantly increased,while the expression of Bax and Cyt C in each dose group was significantly reduced as compared with KA model group (P < 0.05 or P < 0.01).CONCLUSION:KA obviously induces apoptosis of PC12 cells.FA had obvious protective effect on PC12 cells against the toxicity of KA.FA blocks endogenous apoptic pathway through inhibiting the expression of Bax and Cyt C and increasing the expression of Bcl-2 and the ratio of Bcl-2/Bax,thus improving the survival rate of PC12 cells.%目的:探讨阿魏酸(ferulic acid,FA)对红藻氨酸(kainic acid,KA)诱导的PC12细胞凋亡

  19. Icariin Prevents Amyloid Beta-Induced Apoptosis via the PI3K/Akt Pathway in PC-12 Cells

    Dongdong Zhang


    Full Text Available Icariin is a prenylated flavonol glycoside derived from the Chinese herb Epimedium sagittatum that exerts a variety of pharmacological activities and shows promise in the treatment and prevention of Alzheimer’s disease. In this study, we investigated the neuroprotective effects of icariin against amyloid beta protein fragment 25–35 (Aβ25–35 induced neurotoxicity in cultured rat pheochromocytoma PC12 cells and explored potential underlying mechanisms. Our results showed that icariin dose-dependently increased cell viability and decreased Aβ25–35-induced apoptosis, as assessed by MTT assay and Annexin V/propidium iodide staining, respectively. Results of western blot analysis revealed that the selective phosphatidylinositol 3-kinase (PI3K inhibitor LY294002 suppressed icariin-induced Akt phosphorylation, suggesting that the protective effects of icariin are associated with activation of the PI3K/Akt signaling pathway. LY294002 also blocked the icariin-induced downregulation of proapoptotic factors Bax and caspase-3 and upregulation of antiapoptotic factor Bcl-2 in Aβ25–35-treated PC12 cells. These findings provide further evidence for the clinical efficacy of icariin in the treatment of Alzheimer’s disease.

  20. Tyrosine hydroxylase is short-term regulated by the ubiquitin-proteasome system in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats: possible implications in hypertension.

    Nadia A Congo Carbajosa

    Full Text Available Aberrations in the ubiquitin-proteasome system (UPS are implicated in the pathogenesis of various diseases. Tyrosine hydroxylase (TH, the rate-limiting enzyme in catecholamines biosynthesis, is involved in hypertension development. In this study we investigated whether UPS regulated TH turnover in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats (SHR and whether this system was impaired in hypertension. PC12 cells were exposed to proteasome or lysosome inhibitors and TH protein level evaluated by Western blot. Lactacystin, a proteasome inhibitor, induced an increase of 86 ± 15% in TH levels after 30 min of incubation, then it started to decrease up to 6 h to reach control levels and finally it rose up to 35.2 ± 8.5% after 24 h. Bafilomycin, a lysosome inhibitor, did not alter TH protein levels during short times, but it increased TH by 92 ± 22% above basal after 6 h treatment. Before degradation proteasome substrates are labeled by conjugation with ubiquitin. Efficacy of proteasome inhibition on TH turnover was evidenced by accumulation of ubiquitinylated TH after 30 min. Further, the inhibition of proteasome increased the quantity of TH phosphorylated at Ser40, which is essential for TH activity, by 2.7 ± 0.3 fold above basal. TH protein level was upregulated in neurons from hypothalami and brainstem of SHR when the proteasome was inhibited during 30 min, supporting that neuronal TH is also short-term regulated by the proteasome. Since the increased TH levels reported in hypertension may result from proteasome dysfunction, we evaluate proteasome activity. Proteasome activity was significantly reduced by 67 ± 4% in hypothalamic and brainstem neurons from SHR while its protein levels did not change. Present findings show that TH is regulated by the UPS. The impairment in proteasome activity observed in SHR neurons may be one of the causes of the increased TH protein levels reported in hypertension.

  1. Lycium europaeum fruit extract: antiproliferative activity on A549 human lung carcinoma cells and PC12 rat adrenal medulla cancer cells and assessment of its cytotoxicity on cerebellum granule cells.

    Ghali, Wafa; Vaudry, David; Jouenne, Thierry; Marzouki, Mohamed Nejib


    Cancer is a major worldwide health problem and one of the leading causes of death either in developed or developing countries. Plant extracts and derivatives have always been used for various disease treatments and many anticancer agents issued from plants and vegetables are clinically recognized and used all over the world. Lycium europaeum (Solanaceae) also called "wolfberry" was known since ancient times in the Mediterranean area as a medicinal plant and used in several traditional remedies. The Lycium species capacity of reducing the incidence of cancer and also of halting or reserving the growth of cancer was reported by traditional healers. In this study, the antiproliferative capacity, protective properties, and antioxidant activity of the hydro-alcoholic fruit extract of Lycium europaeum were investigated. Results showed that Lycium extract exhibits the ability to reduce cancer cell viability, inhibits proliferation, and induces apoptosis in A549 human lung cancer cells and PC12 rat adrenal medulla cancer cells, in a concentration- and time-dependent manner. Cytotoxic effect on normal rat cerebellum granule cells was assessed to be nonsignificant. Results also showed that Lycium fruit extract protected lipids, proteins, and DNA against oxidative stress damages induced by H2O2 via scavenging reactive oxygen species.

  2. 外源性H2S通过调节β-位淀粉样前体蛋白裂解酶1表达对PC12细胞APP/Ap代谢的影响%Effect of Exogenous Hydrogen Sulfide on Amyloid Precursor Protein/β-amyloid Processing Through Regulating Expression of β-site Amyloid Precursor Protein Cleaving Enzyme 1 in Pheochromocytoma Cells

    代政伟; 孟涛; 晏勇


    目的 观察外源性硫化氮对啥格细胞瘤细胞p一位淀粉样前体蛋白裂解酶1(BACE1)的调节作用,进而探讨其对淀粉样前体蛋白/β-位淀粉样蛋白代谢途径的影响.方法 用硫氮化钠作外源性HZS供体,实脸设空白对照组、NaHS 50 μmol/L组、NaHS 100μmol/L组和NaHS 200 μmol/L组,按分组浓度处理PC12细胞24 h后,RT-PCR和Western blot法检测细胞内BACEI mRNA及蛋白表达,并用Western blot法继而检测APP代谢过程中关健蛋白APP,C99,C83表达变化,ELISA法检浏细胞培养液中Aβ40和Aβ42水平.结果 NaHS在实脸浓度范围内从基因与蛋白两个水平上呈剂量依赖性下调BACEI表达,并下调C99,Aβ40和Aβ42蛋白表达,上调C83蛋白,各NaHS组分别与对照组比校,差别均有统计学意义(P0.05).结论 外源性HZS具有通过调节PC12细胞BACEI表达下调APP/Ap代谢的作用.%Objective To observe the effect of exogenous hydrogen sulfide on β-site amyioid precursor protein cleaving enzyme 1 ( BACE1 ) and the amyloid precursor protein/β-amyloid (APP/Aβ) processing in pheochromocytoma (PC12) cells. Methods PC12 cells were divided into 4 groups:blank control group,NaHS 50 μmol/L group, NaHS100 μmol/L group and NaHS 200 .μmol/L group. Four groups were treated with 0,50,100 or 200 μmol/L sodium hydrosulfide( NaHS, the homer of exogenous hydrogen sulfide ), respectively. RT-PCR and Western blot were used to detect the levels of BACE1 mRNA and protein expression. Western blot was also used to detect the levels of key proteins in the metabolic process of APP,including APP,C99 and C83. ELISA method was used to analyze the levels of Aβ40 and Aβ42 in cellular culture medium. Results Compared with blank control group, NaHS significantly and dose-dependently decreased BACE1 mRNA and protein expression within experimental concentration rages in NaHS groups. So did the C99, Aβ40 and Aβ42 proteins( all P < 0. 05 ). On the contrary, C83 protein significantly increased

  3. A study of the effects of flux density and frequency of pulsed electromagnetic field on neurite outgrowth in PC12 cells.

    Zhang, Yang; Ding, Jun; Duan, Wei


    The aim of this study was to investigate the influence of pulsed electromagnetic fields with various flux densities and frequencies on neurite outgrowth in PC12 rat pheochromocytoma cells. We have studied the percentage of neurite-bearing cells, average length of neurites and directivity of neurite outgrowth in PC12 cells cultured for 96 hours in the presence of nerve growth factor (NGF). PC12 cells were exposed to 50 Hz pulsed electromagnetic fields with a flux density of 1.37 mT, 0.19 mT and 0.016 mT respectively. The field was generated through a Helmholtz coil pair housed in one incubator and the control samples were placed in another identical incubator. It was found that exposure to both a relatively high flux density (1.37 mT) and a medium flux density (0.19 mT) inhibited the percentage of neurite-bearing cells and promoted neurite length significantly. Exposure to high flux density (1.37 mT) also resulted in nearly 20% enhancement of neurite directivity along the field direction. However, exposure to low flux density field (0.016 mT) had no detectable effect on neurite outgrowth. We also studied the effect of frequency at the constant flux density of 1.37 mT. In the range from 1 approximately 100 Hz, only 50 and 70 Hz pulse frequencies had significant effects on neurite outgrowth. Our study has shown that neurite outgrowth in PC12 cells is sensitive to flux density and frequency of pulsed electromagnetic field.

  4. 利福平对鱼藤酮诱导的分化PC12细胞线粒体损伤的保护作用%Rifampicin protects rotenone-induced mitochondrial damage in differentiated PC12 cells

    陈世文; 孙元林; 曾志芬; 陶恩祥


    Objective To explore the effects ofrifampicin(RFP)on the cell viability.reactive oxygen species(ROS)formation,the change of mitochondrial transmembrance potential(△ψm)and cell apoptosis induced by rotenone(Rot)in differentiated PC12 cells.Methods Rot was added to make a model ofParkinson's disease in rat pheochromocytoma(PCI2)cells in the presence of RFP.Cell viability was determined by MTT assay.Change of △ψm and cell apoptosis were measured by fluorescence microscope and flow cytometry respectively.Results Compared with control group and 300 μmol/L RFP group,cell viability was significantly decreased but depolarization of △ψm,ROS formation and cell apoptosis rate were significantly increased in 2.5 μmol/L Rot group.Compared with 2.5 μmol/L Rot group,RFP(100,200 and 300 μmol/L)pretreated groups,cell viability was significantly increased,but depolarization of △ψm、ROS formation and cell apoptosis rate were significantly decreased in a dose-dependent manner.Conclusion RFP may protect the damage induced by Rot in differentiated PC12 by reducing depolarization of △ψm and ROS formation in a dose-dependent manner.%目的 探讨利福平(RFP)对鱼藤酮(Rot)诱导的分化PC12细胞活性、胞内活性氧(ROS)水平、线粒体膜电位(AWm)及凋亡的影响. 方法 利用Rot诱导的分化PC12细胞建立帕金森病(PD)细胞模型,应用不同浓度RFP(100、200、300 μmol/L)预先干预,分别采用MTT法检测PC12细胞活性、流式细胞仪检测胞内ROS生成量、荧光显微镜和流式细胞仪检测△ψm及凋亡的变化.结果 2.5 μmol/L Rot可使PC12细胞活性降低.ROS生成、△ψm去极化程度和细胞凋亡率增加;100、200、300 μmol/L RFP预处理对上述变化有抑制作用,且浓度越大,作用越明显. 结论 RFP可能通过稳定△ψm、降低细胞内ROS生成来对抗Rot对分化PC12细胞的损伤,且这种作用呈浓度依赖性.

  5. 醒脑启智胶囊药物血清对PC12细胞缺氧损伤的保护作用%Protective role of xingnao qizhi capsule in hypoxia injury of PC12 cell

    杨牧祥; 于文涛; 田元祥; 王少贤


    .42,12.71,6.35,3.18 g/kg组分别为82.9%,75.6%,65.9%,53.7%.结论:醒脑启智药物血清能明显减轻PC12细胞的缺氧损伤,可增强细胞活力,降低乳酸脱氢酶活性,并呈现一定的剂量依赖关系.%BACKGROUND: The onset of vascular dementia (VD) is closely related to brain ischemic-hypoxia. Previous research has proved that behavioral disturbance can be obviously attenuated by xingnao qizhi capsule in VD rats; however, it remains unclear whether the improvement of VD is due to the protection on ischemic cells.OBJECTIVE: Pheochromocytoma PC12 cells were cultured in vitro and exposed to Na2S2O4 to induce hypoxia injury. Then serum pharmacological technique was used to investigate the protection of xingnao qizhi drug serum on hypoxia-injured PC12 cells.DESIGN: Randomized controlled study.SETTING: School of Traditional Chinese Medicine of Hebei Medical College.PARTICIPANTS: This study was conduced at the Experimental Animal Laboratory and Cell Culture Laboratory, School of Traditional Chinese Medicine of Hebei Medical College, between June and December 2003.Forty SD rats were randomized into 5 groups, namely serum control group (n=12) and xingnao qizhi serum 25.42, 12.71, 6.35 and 3.18 g/kg groups (n=7). PC12 cell line was purchased form the Cell Center of Chinese Academy of Medical Sciences.serum 25.42, 12.71, 6.35 and 3.18 g/kg groups were exposed through gastric lavage to xingnao qizhi of corresponding dosages (composed of wolfberwhile normal saline was given by gastric lavage to rats in control group in dosage of 10 mL/kg, twice a day at an interval of 12 hours for 3 consecutive days. One hour after the last administration, femoral blood samples were collected when rats were deeply anaesthetized and serum was sepainto 6 groups. Cells in control group were cultured with control serum while in experimental model groups cells were exposed to Na2S2O4 to induce hypoxia injury 1 hour later. Cells in Xingnao Qizhi 25.42, 12.71,6.35 and 3.18 g/kg groups were cultured in 5

  6. PC12 differentiation on biopolymer nanostructures

    Cecchini, Marco [Scuola Normale Superiore and NEST-CNR-INFM, Pisa (Italy); Bumma, Giorgia [Scuola Normale Superiore and Italian Institute of Technology, Pisa (Italy); Serresi, Michela [Scuola Normale Superiore and Italian Institute of Technology, Pisa (Italy); Beltram, Fabio [Scuola Normale Superiore and NEST-CNR-INFM, Pisa (Italy)


    The study of nervous system regeneration and axonal outgrowth control are relevant in several research areas, like neurophysiology or biomedical engineering. Among the elements that control neuron dynamics, the host substrate topography is a key parameter in determining cell differentiation. We present time-lapse experiments to analyze the differentiation dynamics of PC12 cells on nanopatterned biocompatible substrates. 200 nm depth gratings were fabricated on tissue-culture polystyrene substrates by nanoimprint lithography; different linewidths and pitches were compared down to 500 nm and 1000 nm, respectively. PC12 cells were cultured on these substrates and, following NGF administration to the medium, body morphology, cell movement and neuritogenesis were monitored at different time periods. In addition to demonstrating guided differentiation, our studies show complex time variations in body morphology and axon length, and guided cell movement. We show unstable synaptic connections and cell-body polarization, and the competition between topographical guidance and cell-cell interactions.

  7. PC12 differentiation on biopolymer nanostructures

    Cecchini, Marco; Bumma, Giorgia; Serresi, Michela; Beltram, Fabio


    The study of nervous system regeneration and axonal outgrowth control are relevant in several research areas, like neurophysiology or biomedical engineering. Among the elements that control neuron dynamics, the host substrate topography is a key parameter in determining cell differentiation. We present time-lapse experiments to analyze the differentiation dynamics of PC12 cells on nanopatterned biocompatible substrates. 200 nm depth gratings were fabricated on tissue-culture polystyrene substrates by nanoimprint lithography; different linewidths and pitches were compared down to 500 nm and 1000 nm, respectively. PC12 cells were cultured on these substrates and, following NGF administration to the medium, body morphology, cell movement and neuritogenesis were monitored at different time periods. In addition to demonstrating guided differentiation, our studies show complex time variations in body morphology and axon length, and guided cell movement. We show unstable synaptic connections and cell-body polarization, and the competition between topographical guidance and cell-cell interactions.

  8. PC12 polarity on biopolymer nanogratings

    Cecchini, M; Ferrari, A; Beltram, F [Scuola Normale Superiore and NEST-CNR-INFM, Pisa (Italy); Scuola Normale Superiore and Italian Institute of Technology, Pisa (Italy)], E-mail:


    Cell differentiation properties are strongly entangled with the morphology and physical properties of the extracellular environment. A complete understanding of this interaction needs artificial scaffolds with controlled nano-/micro-topography. We induced specific topographies by nanoimprint lithography (NIL) on tissue culture polystyrene (TCPS) dishes substrates and, using light microscopy and high-magnification scanning-electron-microscopy, quantitatively compared the changes in PC12 differentiation phenotype induced by the periodicity of the nanopatterns. This analysis revealed that nanogratings reduce the number of neurites produced by PC12 cells upon treatment with NGF and that neuronal bipolarity correlated with an increased stretching of the cell body and a reduced length of the cell neuronal protrusions.

  9. Preconditioning with Gua Lou Gui Zhi decoction enhances H2O2-induced Nrf2/HO-1 activation in PC12 cells.

    Mao, Jingjie; Li, Zuanfang; Lin, Ruhui; Zhu, Xiaoqin; Lin, Jiumao; Peng, Jun; Chen, Lidian


    Spasticity is common in various central neurological conditions, including after a stroke. Such spasticity may cause additional problems, and often becomes a primary concern for afflicted individuals. A number of studies have identified nuclear factor (erythroid-derived 2)-like 2 (Nrf2) as a key regulator in the adaptive survival response to oxidative stress. Elevated expression of Nrf2, combined with heme oxygenase 1 (HO-1) resistance, in the central nervous system is known to elicit key internal and external oxidation protection. Gua Lou Gui Zhi decoction (GLGZD) is a popular traditional Chinese formula with a long history of clinical use in China for the treatment of muscular spasticity following a stroke, epilepsy or a spinal cord injury. However, the mechanism underlying the efficacy of the medicine remains unclear. In the present study, the antioxidative effects of GLGZD were evaluated and the underlying molecular mechanisms were investigated, using hydrogen peroxide (H2O2)-induced rat pheochromocytoma cells (PC12 cells) as an in vitro oxidative stress model of neural cells. Upon application of different concentrations of GLGZD, a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay and ATP measurement were conducted to assess the impact on PC12 cell proliferation. In addition, inverted microscopy observations, and the MTT and ATP assessments, revealed that GLGZD attenuated H2O2-induced oxidative damage and signaling repression in PC12 cells. Furthermore, the mRNA and protein expression levels of Nrf2 and HO-1, which are associated with oxidative stress, were analyzed using reverse transcription quantitative polymerase chain reaction (PCR) and confocal microscopy. Confocal microscopy observations, as well as the quantitative PCR assay, revealed that GLGZD exerted a neuroprotective function against H2O2-induced oxidative damage in PC12 cells. Therefore, the results demonstrated that GLGZD protected PC12 cells injured by H2O2, which may be

  10. Triptolide Inhibited Cytotoxicity of Differentiated PC12 Cells Induced by Amyloid-Beta₂₅₋₃₅ via the Autophagy Pathway.

    Pengjuan Xu

    Full Text Available Evidence shows that an abnormal deposition of amyloid beta-peptide25-35 (Aβ25-35 was the primary cause of the pathogenesis of Alzheimer's disease (AD. And the elimination of Aβ25-35 is considered an important target for the treatment of AD. Triptolide (TP, isolated from Tripterygium wilfordii Hook.f. (TWHF, has been shown to possess a broad spectrum of biological profiles, including neurotrophic and neuroprotective effects. In our study investigating the effect and potential mechanism of triptolide on cytotoxicity of differentiated rat pheochromocytoma cell line (the PC12 cell line is often used as a neuronal developmental model induced by Amyloid-Beta25-35 (Aβ25-35, we used 3-(4, 5-dimethylthiazol-2-yl-2, 5- diphenyltetrazolium bromide (MTT assay, flow cytometry, Western blot, and acridine orange staining to detect whether triptolide could inhibit Aβ25-35-induced cell apoptosis. We focused on the potential role of the autophagy pathway in Aβ25-35-treated differentiated PC12 cells. Our experiments show that cell viability is significantly decreased, and the apoptosis increased in Aβ25-35-treated differentiated PC12 cells. Meanwhile, Aβ25-35 treatment increased the expression of microtubule-associated protein light chain 3 II (LC3 II, which indicates an activation of autophagy. However, triptolide could protect differentiated PC12 cells against Aβ25-35-induced cytotoxicity and attenuate Aβ25-35-induced differentiated PC12 cell apoptosis. Triptolide could also suppress the level of autophagy. In order to assess the effect of autophagy on the protective effects of triptolide in differentiated PC12 cells treated with Aβ25-35, we used 3-Methyladenine (3-MA, an autophagy inhibitor and rapamycin (an autophagy activator. MTT assay showed that 3-MA elevated cell viability compared with the Aβ25-35-treated group and rapamycin inhibits the protection of triptolide. These results suggest that triptolide will repair the neurological damage in AD

  11. Extracellular α-synuclein leads to microtubule destabilization via GSK-3β-dependent Tau phosphorylation in PC12 cells.

    Magdalena Gąssowska

    Full Text Available α-Synuclein (ASN plays an important role in pathogenesis of Parkinson's disease (PD and other neurodegenerative disorders. Novel and most interesting data showed elevated tauopathy in PD and suggested relationship between ASN and Tau protein. However, the mechanism of ASN-evoked Tau protein modification is not fully elucidated. In this study we investigated the role of extracellular ASN in Tau hyperphosphorylation in rat pheochromocytoma (PC12 cells and the involvement of glycogen synthase kinase-3β (GSK-3β and cyclin-dependent kinase 5 (CDK5 in ASN-dependent Tau modification. Our results indicated that exogenously added ASN increases Tau phosphorylation at Ser396. Accordingly, the GSK-3β inhibitor (SB-216763 prevented ASN-evoked Tau hyperphosphorylation, but the CDK5 inhibitor had no effect. Moreover, western blot analysis showed that ASN affected GSK-3β via increasing of protein level and activation of this enzyme. GSK-3β activity evaluated by its phosphorylation status assay showed that ASN significantly increased the phosphorylation of this enzyme at Tyr216 with parallel decrease in phosphorylation at Ser9, indicative of stimulation of GSK-3β activity. Moreover, the effect of ASN on microtubule (MT destabilization and cell death with simultaneous the involvement of GSK-3β in these processes were analyzed. ASN treatment increased the amount of free tubulin and concomitantly reduced the amount of polymerized tubulin and SB-216763 suppressed these ASN-induced changes in tubulin, indicating that GSK-3β is involved in ASN-evoked MT destabilization. ASN-induced apoptotic processes lead to decrease in PC12 cells viability and SB-216763 protected those cells against ASN-evoked cytotoxicity. Concluding, extracellular ASN is involved in GSK-3β-dependent Tau hyperphosphorylation, which leads to microtubule destabilization. GSK-3β inhibition may be an effective strategy for protecting against ASN-induced cytotoxicity.

  12. Lead Intoxication Synergies of the Ethanol-Induced Toxic Responses in Neuronal Cells--PC12.

    Kumar, V; Tripathi, V K; Jahan, S; Agrawal, M; Pandey, A; Khanna, V K; Pant, A B


    Lead (Pb)-induced neurodegeneration and its link with widespread neurobehavioral changes are well documented. Experimental evidences suggest that ethanol could enhance the absorption of metals in the body, and alcohol consumption may increase the susceptibility to metal intoxication in the brain. However, the underlying mechanism of ethanol action in affecting metal toxicity in brain cells is poorly understood. Thus, an attempt was made to investigate the modulatory effect of ethanol on Pb intoxication in PC12 cells, a rat pheochromocytoma. Cells were co-exposed to biological safe doses of Pb (10 μM) and ethanol (200 mM), and data were compared to the response of cells which received independent exposure to these chemicals at similar doses. Ethanol (200 mM) exposure significantly aggravated the Pb-induced alterations in the end points associated with oxidative stress and apoptosis. The finding confirms the involvement of reactive oxygen species (ROS)-mediated oxidative stress, and impairment of mitochondrial membrane potential, which subsequently facilitate the translocation of triggering proteins between cytoplasm and mitochondria. We further confirmed the apoptotic changes due to induction of mitochondria-mediated caspase cascade. These cellular changes were found to recover significantly, if the cells are exposed to N-acetyl cysteine (NAC), a known antioxidant. Our data suggest that ethanol may potentiate Pb-induced cellular damage in brain cells, but such damaging effects could be recovered by inhibition of ROS generation. These results open up further possibilities for the design of new therapeutics based on antioxidants to prevent neurodegeneration and associated health problems.

  13. Antioxidative effects of berberine pre-treatment on hydrogen peroxide-induced PC12 cell toxicity

    Daohua Xu; Chenhui Zhou


    Oxidative stress has been implicated in the pathogenesis of Alzheimer's disease.Oxidative damage could be prevented by augmenting the endogenous defense capacity against oxidative stress by antioxidant intake.As an effective alkaloid component of Chinese herbal medicine Rhizoma coptidis extract,berberine exhibits antioxidative properties and ameliorates memory impairment in a rat model of Alzheimer's disease.The present study investigated the protective effects of berberine on H2O2-induced PC12 cell toxicity.Results demonstrated that berbedne protects PC12 cells from H2O2-induced apoptosis and increases PC12 cell viability.Lactate dehydrogenase release,reactive oxygen content,and malonyl dialdehyde levels were significantly decreased(P < 0.01).The protective effects of berberine on H2O2-induced PC12 cell toxicity were achieved via the antioxidative effects of berberine.

  14. Cooperative cytotoxic activity of Zn and Cu in bovine serum albumin-conjugated ZnS/CuS nano-composites in PC12 cancer cells

    Wang, Hua-Jie, E-mail:; Yu, Xue-Hong; Wang, Cai-Feng; Cao, Ying, E-mail: [Henan Normal University, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, College of Chemistry and Chemical Engineering (China)


    Series of self-assembled and mono-dispersed bovine serum albumin (BSA)-conjugated ZnS/CuS nano-composites with different Zn/Cu ratios had been successfully synthesized by a combination method of the biomimetic synthesis and ion-exchange strategy under the gentle conditions. High-resolution transmission electron microscopy observation, Fourier transform infrared spectra and zeta potential analysis demonstrated that BSA-conjugated ZnS/CuS nano-composites with well dispersity had the hierarchical structure and BSA was a key factor to control the morphology and surface electro-negativity of final products. The real-time monitoring by atomic absorption spectroscopy and powder X-ray diffraction revealed that the Zn/Cu ratio of nano-composites could be controlled by adjusting the ion-exchange time. In addition, the metabolic and morphological assays indicated that the metabolic proliferation and spread of rat pheochromocytoma (PC12) cells could be inhibited by nano-composites, with the high anti-cancer activity at a low concentration (4 ppm). What were more important, Zn and Cu in nano-composites exhibited a positive cooperativity at inhibiting cancer cell functions. The microscope observation and biochemical marker analysis clearly revealed that the nano-composites-included lipid peroxidation and disintegration of membrane led to the death of PC12 cells. Summarily, the present study substantiated the potential of BSA-conjugated ZnS/CuS nano-composites as anti-cancer drug.

  15. Suppression of p75 neurotrophin receptor surface expression with intrabodies influences Bcl-xL mRNA expression and neurite outgrowth in PC12 cells.

    Congcong Zhang

    Full Text Available BACKGROUND: Although p75 neurotrophin receptor (p75NTR is the first neurotrophin receptor isolated, its diverse physiological functions and signaling have remained elusive for many years. Loss-of-function phenotypic analyses for p75NTR were mainly focused at the genetic level; however these approaches were impacted by off-target effect, insufficient stability, unspecific stress response or alternative active splicing products. In this study, p75NTR surface expression was suppressed for the first time at the protein level by endoplasmic reticulum (ER retained intrabodies. RESULTS: Three monoclonal recombinant antibody fragments (scFv with affinities in the low nanomolar range to murine p75NTR were isolated by antibody phage display. To suppress p75NTR cell surface expression, the encoding genes of these scFvs extended by the ER retention peptide KDEL were transiently transfected into the neuron-like rat pheochromocytoma cell line PC12 and the mouse neuroblastoma x mouse spinal cord hybrid cell line NSC19. The ER retained intrabody construct, SH325-G7-KDEL, mediated a downregulation of p75NTR cell surface expression as shown by flow cytometry. This effect was maintained over a period of at least eight days without activating an unfolded protein response (UPR. Moreover, the ER retention of p75NTR resulted in downregulation of mRNA levels of the anti-apoptotic protein Bcl-xL as well as in strong inhibition of NGF-induced neurite outgrowth in PC12 cells. CONCLUSION: The ER retained intrabody SH325-G7-KDEL not only induces phenotypic knockdown of this p75NTR but also p75NTR-associated cellular responses in PC12 cells.

  16. Nrdp1 Increases Ischemia Induced Primary Rat Cerebral Cortical Neurons and Pheochromocytoma Cells Apoptosis Via Downregulation of HIF-1α Protein

    Yuan Zhang


    Full Text Available Neuregulin receptor degradation protein-1 (Nrdp1 is an E3 ubiquitin ligase that targets proteins for degradation and regulates cell growth, apoptosis and oxidative stress in various cell types. We have previously shown that Nrdp1 is implicated in ischemic cardiomyocyte death. In this study, we investigated the change of Nrdp1 expression in ischemic neurons and its role in ischemic neuronal injury. Primary rat cerebral cortical neurons and pheochromocytoma (PC12 cells were infected with adenoviral constructs expressing Nrdp1 gene or its siRNA before exposing to oxygen-glucose deprivation (OGD treatment. Our data showed that Nrdp1 was upregulated in ischemic brain tissue 3 h after middle cerebral artery occlusion (MCAO and in OGD-treated neurons. Of note, Nrdp1 overexpression by Ad-Nrdp1 enhanced OGD-induced neuron apoptosis, while knockdown of Nrdp1 with siRNA attenuated this effect, implicating a role of Nrdp1 in ischemic neuron injury. Moreover, Nrdp1 upregulation is accompanied by increased protein ubiquitylation and decreased protein levels of ubiquitin-specific protease 8 (USP8 in OGD-treated neurons, which led to a suppressed interaction between USP8 and HIF-1α and subsequently a reduction in HIF-1α protein accumulation in neurons under OGD conditions. In conclusion, our data support an important role of Nrdp1 upregulation in ischemic neuronal death, and suppressing the interaction between USP8 and HIF-1α and consequently the hypoxic adaptive response of neurons may account for this detrimental effect.

  17. Acrylamide decreased dopamine levels and increased 3-nitrotyrosine (3-NT) levels in PC 12 cells.

    Tareke, Eden; Lyn-Cook, Beverly D; Duhart, Helen; Newport, Glenn; Ali, Syed


    Acrylamide is a chemical known to produce neurotoxicity in animals, as well as in humans. The mechanism of acrylamide-induced neurotoxicity is not fully known. However, recent studies have revealed that acrylamide affects the dopaminergic system. Therefore, the aim of this study was to investigate the effect of acrylamide on dopamine (DA) and the metabolites, 3,4-dihydroxy phenylacetic acid (DOPAC) and homovanillicacid (HVA), levels in Pheochromocytoma (PC 12) cells. In addition, the generation of peroxynitrite (ONOO(-)), measured by 3-nitrotyrosine (3-NT), was investigated as a possible mechanism in acrylamide-induced neurotoxicity. HPLC-coupled to electrochemical detection (ECD) was used to determine DA, DOPAC, HVA and 3-NT levels. Acrylamide (0.01-5mM) exposure produced a dose- and time (1-42h)-dependent decrease in DA levels. The decrease (P<0.05) in DA levels was noted at 24h after exposure to acrylamide. The study also revealed that 3-NT levels in PC 12 increased as a result of treatment with acrylamide. Thus, these data suggest that acrylamide-induced decrease in DA levels in PC 12 cells may be associated with peroxynitrite formation, measured as 3-NT levels.

  18. Mechanism for desensitization of beta-adrenergic receptor-stimulated lipolysis in adipocytes from rats harboring pheochromocytoma.

    Prokocimer, P G; Maze, M; Vickery, R G; Hoffman, B B


    Prolonged stimulation of beta-adrenergic receptors with catecholamines leads to desensitization of their ability to activate cAMP accumulation. However, little is known about the relationship between these changes and possible alterations in physiological responses. We have used isolated adipocytes prepared from NEDH rats harboring pheochromocytomas, a norepinephrine-secreting tumor, to address this question. As expected, there was a decrease in the ability of isoproterenol to maximally activate cAMP accumulation in adipocytes from rat harboring pheochromocytoma [323 +/- 107 vs. 707 +/- 145 pmol/10(5) cells.min (mean +/- SD) in controls]. This change was associated with an increase in the EC50 of isoproterenol for activation of cAMP-dependent protein kinase (5.8 X 10(-8) vs. 2.4 X 10(-8) M in controls) and a decrease in maximal activation of the kinase (38 +/- 16% vs. 77 +/- 14% in controls). For lipolysis there was a loss in sensitivity to isoproterenol but no change in maximal lipolytic rate in the adipocytes from rats harboring pheochromocytoma. For both groups there was a similar relationship between kinase activation and lipolysis; maximal lipolysis had already occurred for protein kinase-A activity ratios less than 30%. Therefore, the blunted cAMP response in adipocytes from rats harboring pheochromocytoma did not impair the maximal lipolytic rate. These results demonstrate that adipocytes can efficiently maintain maximal lipolysis in a desensitized state because of considerable reserve in the biochemical cascade leading to the lipolytic response. In addition, our findings demonstrate that there are no regulatory changes induced by prolonged exposure to catecholamines that are distal to cAMP accumulation.

  19. Activation of p90 Rsk1 is sufficient for differentiation of PC12 cells

    Silverman, Eran; Frödin, Morten; Gammeltoft, Steen;


    We investigated the role of Rsk proteins in the nerve growth factor (NGF) signaling pathway in PC12 cells. When rat Rsk1 or murine Rsk2 proteins were transiently expressed, NGF treatment (100 ng/ml for 3 days) caused three- and fivefold increases in Rsk1 and Rsk2 activities, respectively. Increas...

  20. The high-affinity D2/D3 agonist D512 protects PC12 cells from 6-OHDA-induced apoptotic cell death and rescues dopaminergic neurons in the MPTP mouse model of Parkinson's disease.

    Shah, Mrudang; Rajagopalan, Subramanian; Xu, Liping; Voshavar, Chandrashekhar; Shurubor, Yevgeniya; Beal, Flint; Andersen, Julie K; Dutta, Aloke K


    In this study, in vitro and in vivo experiments were carried out with the high-affinity multifunctional D2/D3 agonist D-512 to explore its potential neuroprotective effects in models of Parkinson's disease and the potential mechanism(s) underlying such properties. Pre-treatment with D-512 in vitro was found to rescue rat adrenal Pheochromocytoma PC12 cells from toxicity induced by 6-hydroxydopamine administration in a dose-dependent manner. Neuroprotection was found to coincide with reductions in intracellular reactive oxygen species, lipid peroxidation, and DNA damage. In vivo, pre-treatment with 0.5 mg/kg D-512 was protective against neurodegenerative phenotypes associated with systemic administration of MPTP, including losses in striatal dopamine, reductions in numbers of DAergic neurons in the substantia nigra (SN), and locomotor dysfunction. These observations strongly suggest that the multifunctional drug D-512 may constitute a novel viable therapy for Parkinson's disease.

  1. The Study of Protection and Mechanism Effect of CJY Flavanoid on the Beta-amy-loid Peptide 25-35-induced Injury in PC12%黄酮类化合物对 Aβ25-35介导的 PC12细胞损伤的保护及机制研究



    目的:探讨黄酮类化合物(CJY)对β-淀粉样蛋白25-35(Aβ25-35)介导的 PC12细胞(大鼠嗜铬细胞瘤细胞)氧化损伤的保护作用及其可能机制。方法:对 PC12细胞采用不同浓度的 Aβ25-35进行损伤,MTT 法测定PC12细胞的损伤率和存活率;采用 SOD 试剂盒检测 Aβ25-35对 PC12细胞的氧化损伤;使用 DCFH-DA 结合活细胞高内涵成像系统检测细胞内 ROS 的水平。结果:经终浓度为10μM的 Aβ25-35对 PC12细胞损伤后,可降低细胞的存活率,而浓度为5μM,10μM,50μM的 CJY 呈浓度依赖性地提高 PC12细胞的存活率,并明显改善细胞形态。Aβ25-35可触发细胞内的氧化应激反应,造成细胞氧化损伤,提高细胞内 ROS 水平,降低细胞内 SOD 的活性;CJY 则降低细胞内 ROS 水平,升高 SOD 的活性,具有明显的抗氧化损伤作用。结论:通过离体实验证明黄酮类化合物CJY 对 Aβ25-35诱导的 PC12细胞损伤有保护作用,为进一步研究黄酮类化合物在阿尔茨海默病中的应用提供实验依据。%Objective:To explore the the protective effects and the potential mechanism of CJY flavanoids on inju-ries induced by the beta-amyloid peptide25-35( Aβ25-35 )in rat pheochromocytoma cells( PC12 cells). Methods:The Aβ25-35 with different concentrations were used to cause injuries in PC12 cells by the same way. The damage and survival rate of PC12 cells were detected by MTT as well as Oxidative damage by SOD and the content level of ROS in cells by DCFH-DA combined with living cells high content imaging system. Results:The injuries induced by the Aβ25-35 with the concentration of 10 μM resulted in the significant decrease of the survival rate of PC12 cells. CJY flavonoids(concentra-tions:5 μM,10 μM,50 μM)could markedly improve damage cells form and the survival rate of PC12 cells,which was dose-dependent. The oxidative stress in cells could be triggered by the Aβ25

  2. Human adipose tissue-derived multilineage progenitor cells exposed to oxidative stress induce neurite outgrowth in PC12 cells through p38 MAPK signaling

    Moriyama Mariko


    Full Text Available Abstract Background Adipose tissues contain populations of pluripotent mesenchymal stem cells that also secrete various cytokines and growth factors to support repair of damaged tissues. In this study, we examined the role of oxidative stress on human adipose-derived multilineage progenitor cells (hADMPCs in neurite outgrowth in cells of the rat pheochromocytoma cell line (PC12. Results We found that glutathione depletion in hADMPCs, caused by treatment with buthionine sulfoximine (BSO, resulted in the promotion of neurite outgrowth in PC12 cells through upregulation of bone morphogenetic protein 2 (BMP2 and fibroblast growth factor 2 (FGF2 transcription in, and secretion from, hADMPCs. Addition of N-acetylcysteine, a precursor of the intracellular antioxidant glutathione, suppressed the BSO-mediated upregulation of BMP2 and FGF2. Moreover, BSO treatment caused phosphorylation of p38 MAPK in hADMPCs. Inhibition of p38 MAPK was sufficient to suppress BMP2 and FGF2 expression, while this expression was significantly upregulated by overexpression of a constitutively active form of MKK6, which is an upstream molecule from p38 MAPK. Conclusions Our results clearly suggest that glutathione depletion, followed by accumulation of reactive oxygen species, stimulates the activation of p38 MAPK and subsequent expression of BMP2 and FGF2 in hADMPCs. Thus, transplantation of hADMPCs into neurodegenerative lesions such as stroke and Parkinson’s disease, in which the transplanted hADMPCs are exposed to oxidative stress, can be the basis for simple and safe therapies.

  3. Upregulation of glutathione peroxidase-1 expression and activity by glial cell line-derived neurotrophic factor promotes high-level protection of PC12 cells against 6-hydroxydopamine and hydrogen peroxide toxicities.

    Gharib, Ehsan; Gardaneh, Mossa; Shojaei, Sahar


    We examined the impact of strong co-presence and function of glutathione peroxidase-1 (GPX-1) and glial cell line-derived neurotrophic factor (GDNF) on protecting the rat dopaminergic pheochromocytoma cell line PC12 against 6-hydroxydopamine (6-OHDA) and hydrogen peroxide (H₂O₂) toxicities. Primarily, GPX-1 over-expression by PC12 cells infected with pLV-GPX1 lentivirus vectors significantly increased cell survival against 6-OHDA toxicity (pcells with astro-CM of GDNF-over-secreting astrocytes (Test astro-CM) significantly induced GPX-1 expression, peroxidase enzymatic activity, and intra-cellular glutathione (GSH) levels. These changes paralleled with protection of 90% of GDNF⁺/GPX1⁺ PC12 cells against toxicity, a rate that was 37% up from their un-infected un-treated (GDNF⁻/GPX1⁻) controls (pcells that received only Control astro-CM (GPX⁺/GDNF⁻) (pcell groups, increased cell survival against either compound was further confirmed by increased live cell counts measured by double staining. Following depletion of intra-cellular GSH, only 46% of pLV-GPX1 cells survived 6-OHDA toxicity, whereas over 70% of them were saved upon GDNF treatment (pcells and maximized by addition of GDNF. Comparison analyses established correlations between GPX-1-GDNF co-presence and both enhanced cell protection and diminished levels of activated caspase-3. Our data collectively indicate that GDNF is capable of inducing anti-oxidant activities of intra-cellular GPX-1 and that growth-promoting potential of GDNF and anti-oxidant properties of GPX-1 can, in concert, maximize survival of dopaminergic neurons.

  4. Cocaine induces a differential dose-dependent alteration in the expression profile of immediate early genes, transcription factors, and caspases in PC12 cells: a possible mechanism of neurotoxic damage in cocaine addiction.

    Imam, Syed Z; Duhart, Helen M; Skinner, John T; Ali, Syed F


    Cocaine is a widely used drug of abuse and psychostimulant that acts on the central nervous system by blocking the dopamine reuptake sites. PC12 cells, a rat pheochromocytoma clonal line, in the presence of nerve growth factor (NGF), multiply and differentiate into competent neurons that can synthesize, store, and secrete the neurotransmitter dopamine (DA). In the present study, we evaluated the effect of increasing doses of cocaine on the expression of immediate early genes (IEGs), c-fos and c-jun, and closely related transcription factors, SP-1 and NF-kbeta, at 24 h after the exposure to cocaine (50, 100, 200, 500, 1000, 2500 microM) in NGF-differentiated PC12 cells. Cocaine (50-500 microM) resulted in significant induction of the expression of c-fos, c-jun, SP-1, and NF-kbeta. However, higher concentrations of cocaine (1000 and 2500 microM) resulted in the downregulation of these expressions after 24 h. To further understand the role of dose-dependent changes in the mechanisms of cell death, we evaluated the protein expression of apoptotic markers. A concentration-dependent increase in the expression of caspase-9 and -3 was observed up to 500 microM cocaine. However, the higher dose did not show any expression. We also evaluated the effect of increasing doses of cocaine on DA concentration and the expression of dopamine transporter (DAT). A significant dose-dependent decrease in the concentration of DA as well as the expression of DAT was observed 24 h after the exposure of PC12 cells to cocaine. Therefore, in the present study, we reported that cocaine has both upstream and downstream regulatory actions on some IEGs and transcription factors that can regulate the mechanism of cell death, and these effects on gene expression are independent of its action on the dopaminergic system.

  5. Inhibition of nerve growth factor-induced neurite outgrowth from PC12 cells by dexamethasone: signaling pathways through the glucocorticoid receptor and phosphorylated Akt and ERK1/2.

    Kazuki Terada

    Full Text Available Glucocorticoids are important mediators of the stress response and are commonly employed as drugs for the suppression of immune rejection after organ transplantation. Previous investigations uncovered the possibility of mood depression in patients undergoing long-term treatment with synthetic glucocorticoids, including dexamethasone (DEX. Exogenous glucocorticoids and their synthetic derivatives can also adversely affect the development of the central nervous system. Although neurite extension from rat pheochromocytoma-derived PC12 cells and a variety of primary neurons is stimulated by nerve growth factor (NGF, and signaling pathways triggered by the binding of NGF to tyrosine kinase receptor type 1 (TrkA function in both neurite outgrowth and neuronal survival, the effect of DEX on the activation of regulatory proteins and pathways downstream of TrkA has not been well characterized. To analyze the influence of DEX on NGF-induced neurite outgrowth and signaling, PC12 cells, a widely utilized model of neuronal differentiation, were pretreated with the glucocorticoid prior to NGF induction. NGF-induced neurite outgrowth was attenuated by pretreatment with DEX, even in the absence of DEX after the addition of NGF. Moreover, DEX suppressed the phosphorylation of Akt and extracellular-regulated kinase 1/2 (ERK1/2 in the neurite outgrowth signaling cascade initiated by NGF. Finally, the glucocorticoid receptor (GR antagonist, RU38486, counteracted the inhibitory effect of DEX pretreatment, not only on the phosphorylation of Akt and ERK1/2, but also on neurite extension from PC12 cells. These results suggest that DEX binding to the GR impairs NGF-promoted neurite outgrowth by interfering with the activation/phosphorylation of Akt and ERK1/2. These novel findings are likely to be useful for elucidating the central nervous system depressive mechanism(s of action of DEX and other glucocorticoids.

  6. Light-Mediated Kinetic Control Reveals the Temporal Effect of the Raf/MEK/ERK Pathway in PC12 Cell Neurite Outgrowth

    Zhang, Kai; Duan, Liting; Ong, Qunxiang; Lin, Ziliang; Varman, Pooja Mahendra; Sung, Kijung; Cui, Bianxiao


    It has been proposed that differential activation kinetics allows cells to use a common set of signaling pathways to specify distinct cellular outcomes. For example, nerve growth factor (NGF) and epidermal growth factor (EGF) induce different activation kinetics of the Raf/MEK/ERK signaling pathway and result in differentiation and proliferation, respectively. However, a direct and quantitative linkage between the temporal profile of Raf/MEK/ERK activation and the cellular outputs has not been established due to a lack of means to precisely perturb its signaling kinetics. Here, we construct a light-gated protein-protein interaction system to regulate the activation pattern of the Raf/MEK/ERK signaling pathway. Light-induced activation of the Raf/MEK/ERK cascade leads to significant neurite outgrowth in rat PC12 pheochromocytoma cell lines in the absence of growth factors. Compared with NGF stimulation, light stimulation induces longer but fewer neurites. Intermittent on/off illumination reveals that cells achieve maximum neurite outgrowth if the off-time duration per cycle is shorter than 45 min. Overall, light-mediated kinetic control enables precise dissection of the temporal dimension within the intracellular signal transduction network. PMID:24667437

  7. Bcl-xS and Bax induce different apoptotic pathways in PC12 cells.

    Lindenboim, L; Yuan, J; Stein, R


    Apoptosis is regulated by the action of the Bcl-2 family of proteins, which includes anti- and pro-apoptotic members such as Bcl-xS and Bax. These proteins may differ from each other in structure, mechanism of action and interactions with anti-apoptotic signaling. The mechanism whereby Bax induces cell death has been studied in some cellular systems, but the mechanism of Bcl-xS-induced apoptosis is largely unknown. In this study we investigated and compared the apoptotic effects of Bcl-xS and Bax in the pheochromocytoma cell line, PC12 (a useful model system for studying neuronal apoptosis), and the extent to which they are protected by the survival factor, nerve growth factor (NGF). PC12 cells express endogenous Bcl-xS, Bax and Bcl-xL proteins. Subcellular fractionation revealed that Bax is presented mainly in the cytosolic and the heavy membrane fractions, Bcl-xS is present only in the cytosol, and the anti-apoptotic protein Bcl-xL is located mainly in the heavy membrane fraction. In contrast to the cytosolic localization of endogenous Bcl-xS, the exogenously overexpressed Bcl-xS is localized to the mitochondria. Overexpression of Bcl-xS or Bax induces cell death in the transfected cells. The cell death induced by overexpression of Bcl-xS was inhibited by coexpression of Bcl-xS with Bcl-2 or Bcl-xL, or by treatment with the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoro-methylketone (Z-VAD-FMK) or with NGF. The Bcl-2 mutants deltaC22, which lacks the transmembrane domain, and G145A (mI-3) were able to inhibit the death-inducing effect of Bcl-xS. These results therefore suggest that the apoptotic pathway induced by overexpression of Bcl-xS in PC12 cells can be controlled by Bcl-2 and Bcl-xL, is mediated by caspases, and can be inhibited by the NGF signaling pathway. The Bax-induced cell death was inhibited by co-expression of Bax with Bcl-2 or Bcl-xL, but was not inhibited by Z-VAD-FMK, NGF, or the Bcl-2 ml-3 or deltaC22 mutants. These

  8. Aspartame-induced apoptosis in PC12 cells.

    Horio, Yukari; Sun, Yongkun; Liu, Chuang; Saito, Takeshi; Kurasaki, Masaaki


    Aspartame is an artificial sweetner added to many low-calorie foods. The safety of aspartame remains controversial even though there are many studies on its risks. In this study, to understand the physiological effects of trace amounts of artificial sweetners on cells, the effects of aspartame on apoptosis were investigated using a PC12 cell system. In addition, the mechanism of apoptosis induced by aspartame in PC12 cells and effects on apoptotic factors such as cytochrome c, apoptosis-inducing factor, and caspase family proteins were studied by Western blotting and RT-PCR. Aspartame-induced apoptosis in PC12 cells in a dose-dependent manner. In addition, aspartame exposure increased the expressions of caspases 8 and 9, and cytochrome c. These results indicate that aspartame induces apoptosis mainly via mitochondrial pathway involved in apoptosis due to oxigen toxicity.

  9. Volume regulation in rat pheochromocytoma cultured cells submitted to hypoosmotic conditions.

    Delpire, E; Cornet, M; Gilles, R


    The mechanisms at work in cell volume regulation have been studied in PC12 cultured cells. Results show, for the first time to our knowledge, that the volume readjustment process occurring after application of a hypoosmotic saline is sensitive to amiloride, IBMX and forskoline. The process is also inhibited by quinine hydrochloride and trifluoperazine. Volume readjustment is concomtant with a decrease in K+ and Cl- intracellular levels. The decrease in K+ level can be related to an assymetrical change in the fluxes in and out of the ion as shown by flux kinetics studies using Rb86. These results are interpreted considering that the control of the activity of the ion channel pathways associated with volume readjustment in PC12 cells may implicate the Ca(2+)-calmodulin - cAMP system.

  10. Gengnianchun recipe inhibits apoptosis of pheochromocytoma cells from beta-amyloid 25-35 insult, better than monotherapies and their compounds

    Jun Li; Wenjun Wang; Dajin Li; Wenjiang Zhou


    This study aims to determine and compare the protective effects of Gengnianchun recipe drug serum and compounds of its representative drug monotherapies against sympathetic nerve pheochromocytoma cell line PC12 cells damaged by beta-amyloid 25-35 at the cellular apoptosis and related signal pathway levels. PC12 cells cultured with medicated rat serum showed enhanced cell viability and reduced cellular apoptosis rates compared with those of monotherapies and their compounds. Furthermore, Gengnianchun recipe up-regulated expressions of anti-apoptotic protein Bcl-2, estrogen receptor-beta and phosphorylated extracellular-signal-regulated kinase 1/2; and down-regulated expressions of pro-apoptotic proteins Bax and caspase-3. Gengnianchun recipe was superior to representative drug monotherapies, such as paeoniflorin, berberine, timosaponin A-III, icariine and their compounds in protecting PC12 cells. Mitogen-activated protein kinase blocker and estrogen receptor antagonist were found to reverse the above effects of Gengnianchun recipe. The experimental findings indicate that, Gengnianchun recipe protects PC12 cells from beta-amyloid 25-35 insult; its inhibitory effect on apoptosis may be achieved through the mitogen-activated protein kinase and estrogen receptor pathways.

  11. Induction of cytoprotective autophagy in PC-12 cells by cadmium

    Wang, Qiwen [College of Veterinary Medicine, Yangzhou University, Yangzhou 225009 (China); Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009 (China); Bijie Pilot Area Research Institute of Bijie University, Bijie 551700 (China); Zhu, Jiaqiao; Zhang, Kangbao; Jiang, Chenyang; Wang, Yi; Yuan, Yan; Bian, Jianchun; Liu, Xuezhong; Gu, Jianhong [College of Veterinary Medicine, Yangzhou University, Yangzhou 225009 (China); Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009 (China); Liu, Zongping, E-mail: [College of Veterinary Medicine, Yangzhou University, Yangzhou 225009 (China); Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009 (China)


    Highlights: •Cadmium can promote early upregulation of autophagy in PC-12 cells. •Autophagy precedes apoptosis in cadmium-treated PC-12 cells. •Cadmium-induced autophagy is cytoprotective in PC-12 cells. •Class III PI3K/beclin-1/Bcl-2 signaling pathway plays a positive role in cadmium-triggered autophagy. -- Abstract: Laboratory data have demonstrated that cadmium (Cd) may induce neuronal apoptosis. However, little is known about the role of autophagy in neurons. In this study, cell viability decreased in a dose- and time-dependent manner after treatment with Cd in PC-12 cells. As cells were exposed to Cd, the levels of LC3-II proteins became elevated, specific punctate distribution of endogenous LC3-II increased, and numerous autophagosomes appeared, which suggest that Cd induced a high level of autophagy. In the late stages of autophagy, an increase in the apoptosis ratio was observed. Likewise, pre-treatment with chloroquine (an autophagic inhibitor) and rapamycin (an autophagic inducer) resulted in an increased and decreased percentage of apoptosis in contrast to other Cd-treated groups, respectively. The results indicate that autophagy delayed apoptosis in Cd-treated PC-12 cells. Furthermore, co-treatment of cells with chloroquine reduced autophagy and cell activity. However, rapamycin had an opposite effect on autophagy and cell activity. Moreover, class III PI3 K/beclin-1/Bcl-2 signaling pathways served a function in Cd-induced autophagy. The findings suggest that Cd can induce cytoprotective autophagy by activating class III PI3 K/beclin-1/Bcl-2 signaling pathways. In sum, this study strongly suggests that autophagy may serve a positive function in the reduction of Cd-induced cytotoxicity.

  12. NFAT1 and NFAT3 cooperate with HDAC4 during regulation of alternative splicing of PMCA isoforms in PC12 cells.

    Michalina Kosiorek

    Full Text Available The bulk of human genes undergo alternative splicing (AS upon response to physiological stimuli. AS is a great source of protein diversity and biological processes and is associated with the development of many diseases. Pheochromocytoma is a neuroendocrine tumor, characterized by an excessive Ca2+-dependent secretion of catecholamines. This underlines the importance of balanced control of calcium transport via regulation of gene expression pattern, including different calcium transport systems, such as plasma membrane Ca2+-ATPases (PMCAs, abundantly expressed in pheochromocytoma chromaffin cells (PC12 cells. PMCAs are encoded by four genes (Atp2b1, Atp2b2, Atp2b3, Atp2b4, whose transcript products undergo alternative splicing giving almost 30 variants.In this scientific report, we propose a novel mechanism of regulation of PMCA alternative splicing in PC12 cells through cooperation of the nuclear factor of activated T-cells (NFAT and histone deacetylases (HDACs. Luciferase assays showed increased activity of NFAT in PC12 cells, which was associated with altered expression of PMCA. RT-PCR experiments suggested that inhibition of the transcriptional activity of NFAT might result in the rearrangement of PMCA splicing variants in PC12 cells. NFAT inhibition led to dominant expression of 2x/c, 3x/a and 4x/a PMCA variants, while in untreated cells the 2w,z/b, 3z,x/b,c,e,f, and 4x/b variants were found as well. Furthermore, chromatin immunoprecipitation experiments showed that NFAT1-HDAC4 or NFAT3-HDAC4 complexes might be involved in regulation of PMCA2x splicing variant generation.We suggest that the influence of NFAT/HDAC on PMCA isoform composition might be important for altered dopamine secretion by PC12 cells.

  13. Astroglia overexpressing heme oxygenase-1 predispose co-cultured PC12 cells to oxidative injury.

    Song, Linyang; Song, Wei; Schipper, Hyman M


    The mechanisms responsible for the progressive degeneration of dopaminergic neurons and pathologic iron deposition in the substantia nigra pars compacta of patients with Parkinson's disease (PD) remain unclear. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in the oxidative degradation of heme to ferrous iron, carbon monoxide, and biliverdin, is upregulated in affected PD astroglia and may contribute to abnormal mitochondrial iron sequestration in these cells. To determine whether glial HO-1 hyper-expression is toxic to neuronal compartments, we co-cultured dopaminergic PC12 cells atop monolayers of human (h) HO-1 transfected, sham-transfected, or non-transfected primary rat astroglia. We observed that PC12 cells grown atop hHO-1 transfected astrocytes, but not the astroglia themselves, were significantly more susceptible to dopamine (1 microM) + H(2)O(2) (1 microM)-induced death (assessed by nuclear ethidium monoazide bromide staining and anti-tyrosine hydroxylase immunofluorescence microscopy) relative to control preparations. In the experimental group, PC12 cell death was attenuated significantly by the administration of the HO inhibitor, SnMP (1.5 microM), the antioxidant, ascorbate (200 microM), or the iron chelators, deferoxamine (400 microM), and phenanthroline (100 microM). Exposure to conditioned media derived from HO-1 transfected astrocytes also augmented PC12 cell killing in response to dopamine (1 microM) + H(2)O(2) (1 microM) relative to control media. In PD brain, overexpression of HO-1 in nigral astroglia and accompanying iron liberation may facilitate the bioactivation of dopamine to neurotoxic free radical intermediates and predispose nearby neuronal constituents to oxidative damage. (c) 2007 Wiley-Liss, Inc.

  14. Melatonin inhibits maneb-induced aggregation of alpha-synuclein in rat pheochromocytoma cells.

    Ishido, Masami


    Melatonin, a secretory product of the pineal gland, is involved in the regulation of circadian and seasonal rhythms, in oncostasis, and in inducing osteoblast differentiation. Furthermore, melatonin is a scavenger of a number of reactive oxygen and reactive nitrogen species both in vitro and in vivo. In this study, the antioxidant nature of melatonin was shown to prevent cultured neural cells from apoptosis induced by endocrine-disrupting chemical, maneb. The neurotoxicity of the fungicide, maneb (1 microg/mL), on the PC12 cells was elicited through apoptotic cell death, concomitant with aggregation of alpha-synuclein, a feature of Parkinson's disease. Activation of caspase-3/7 was associated with this process. A fluorescence rationing technique using a mitochondrial dye revealed that maneb altered the mitochondrial membrane potential of the neural cells. However, melatonin (1 nm) largely prevented the neural cells from the neural toxicant by inhibition of both caspase-3/7 activation and disruption of the mitochondrial transmembrane potential. Furthermore, aggregation of alpha-synuclein by maneb was also inhibited by melatonin. Thus, melatonin prevents maneb-induced neurodegeneration at a nighttime physiological blood concentration, most likely by inhibiting the aggregation of alpha-synuclein as well as preventing mitochondrial dysfunction in PC 12 cells.

  15. Hypergravity Stimulation Enhances PC12 Neuron-Like Cell Differentiation

    Giada Graziana Genchi


    Full Text Available Altered gravity is a strong physical cue able to elicit different cellular responses, representing a largely uninvestigated opportunity for tissue engineering/regenerative medicine applications. Our recent studies have shown that both proliferation and differentiation of C2C12 skeletal muscle cells can be enhanced by hypergravity treatment; given these results, PC12 neuron-like cells were chosen to test the hypothesis that hypergravity stimulation might also affect the behavior of neuronal cells, in particular promoting an enhanced differentiated phenotype. PC12 cells were thus cultured under differentiating conditions for either 12 h or 72 h before being stimulated with different values of hypergravity (50 g and 150 g. Effects of hypergravity were evaluated at transcriptional level 1 h and 48 h after the stimulation, and at protein level 48 h from hypergravity exposure, to assess its influence on neurite development over increasing differentiation times. PC12 differentiation resulted strongly affected by the hypergravity treatments; in particular, neurite length was significantly enhanced after exposure to high acceleration values. The achieved results suggest that hypergravity might induce a faster and higher neuronal differentiation and encourage further investigations on the potential of hypergravity in the preparation of cellular constructs for regenerative medicine and tissue engineering purposes.

  16. Mutant alpha-synuclein and autophagy in PC12 cells

    Kangyong Liu; Chunfeng Liu; Chuancheng Ren; Yaping Yang; Liwei Shen; Xuezhong Li; Fen Wang; Zhenghong Qin


    Several studies have demonstrated that overexpression of mutant α-synuclein in PC12 cells is related to occurrence of autophagy.The present study established mutant a-synuclein (A30P)-transfected PC12 cells and treated them with the autophagy inducer rapamycin and autophagy inhibitor wortmannin, respectively.Results demonstrated that mutant o-synuclein resulted in cell death via autophagy and involved α-synuclein accumulation, membrane lipid oxidation, and loss of plasma membrane integrity.Mutant α-synuclein (A30P) also mediated toxicity of1-methyl-4-phenylpyridinium ion.Moreover, rapamycin inhibited a-synuclein aggregation, while wortmannin promoted o-synuclein aggregation and cell death.To further determine the role of autophagy due to mutant a-synuclein, the present study measured expression of microtubule-associated protein light chain 3.Results revealed that wortmannin and 1-methyl-4-phenylpyridinium ion inhibited expression of microtubule-associated protein light chain 3,while rapamycin promoted its expression.These findings suggested that abnormal aggregation of a-synuclein induced autophagic programmed cell death in PC12 cells.

  17. Mechanisms of rotenone-induced neurotoxicity in PC 12 cells

    Wei Han; Lizhong Sun; Jiafeng Chen; Ming Chang; Hongyan Huo; Linsen Hu


    BACKGROUND: Rotenone-induced neurotoxicity in PC 12 cells has been widely used to study the pathogenesis of Parkinson's disease. However, the precise mechanisms underlying rotenone-induced dopaminergic neuronal degeneration in Parkinson's disease remains unclear.OBJECTIVE: To establish rotenone-induced neurotoxicity in PC 12 cells, and to investigate the possible action pathways to rotenone-induced neural cell injury at the protein level.DESIGN, TIME AND SETTING: A controlled proteomics study was performed at the Department of Nearology, First Hospital, Jilin University between March 2006 and March 2007.MATERIALS: PC 12 cells were obtained from Shanghai Cell Bank of Chinese Academy of Sciences, China.Rotenone was provided by Sigma, USA.METHODS: PC 12 cells in logarithmic growth phase were treated under experimental and control conditions, respectively. A total of 0.5 μ mol/L rotenone, or the same amount of Dulbecco's modified eagle's medium (DMEM), was added in the experimental and control conditions, respectively.MAIN OUTCOME MEASURES: Following 72 hours of rotenone treatment, cellular survival rate was determined by methyl thiazolyl tetrazolium assay, and apoptotic changes were detected by Hoechst 33342 staining. Total cellular protein was extracted to acquire differential protein expression data utilizing two-dimensional differential in-gel electrophoresis. To identify differential protein spots, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) was used.RESULTS: In the MTT assay, the experimental condition induced significantly less cell survival compared to the control condition (P < 0.01 ). Hoechst 33342 staining revealed a larger number of apoptotic cells under the experimental condition compared to the control condition (P < 0.01 ), as determined by the presence of nuclear condensation, pyknosis, and nuclear fragmentation. Two-dimensional electrophoresis results showed that the differential expression of protein

  18. Lack of effects of typical and atypical antipsychotics in DARPP-32 and NCS-1 levels in PC12 cells overexpressing NCS-1

    Reis Helton J


    Full Text Available Abstract Background Schizophrenia is the major psychiatry disorder, which the exact cause remains unknown. However, it is well known that dopamine-mediated neurotransmission imbalance is associated with this pathology and the main target of antipsychotics is the dopamine receptor D2. Recently, it was described alteration in levels of two dopamine signaling related proteins in schizophrenic prefrontal cortex (PFC: Neuronal Calcium Sensor-1 (NCS-1 and DARPP-32. NCS-1, which is upregulated in PFC of schizophrenics, inhibits D2 internalization. DARPP-32, which is decreased in PFC of schizophrenics, is a key downstream effector in transducing dopamine signaling. We previously demonstrated that antipsychotics do not change levels of both proteins in rat's brain. However, since NCS-1 and DARPP-32 levels are not altered in wild type rats, we treated wild type PC12 cells (PC12 WT and PC12 cells stably overexpressing NCS-1 (PC12 Clone with antipsychotics to investigate if NCS-1 upregulation modulates DARPP-32 expression in response to antipsychotics treatment. Results We chronically treated both PC12 WT and PC12 Clone cells with typical (Haloperidol or atypical (Clozapine and Risperidone antipsychotics for 14 days. Using western blot technique we observed that there is no change in NCS-1 and DARPP-32 protein levels in both PC12 WT and PC12 Clone cells after typical and atypical antipsychotic treatments. Conclusions Because we observed no alteration in NCS-1 and DARPP-32 levels in both PC12 WT and Clone cells treated with typical or atypical antipsychotics, we suggest that the alteration in levels of both proteins in schizophrenic's PFC is related to psychopathology but not with antipsychotic treatment.

  19. 益母草碱对缺糖/缺氧损伤PC12细胞的保护作用%Protective effect of leonurine on PC12 cell injury induced by the deprivation of oxygen/glucose

    袁楠; 王训翠; 李晓祥; 李庆林


    Objective To study the protective effect of the hydrochloride leonurine on deprivation of oxygen/glucose injury in rat adrenal chromium tumor cell line ( PC12 ). Methods We used the sodium dithionite ( Na2S2O4 ) + sugar-free in PC12 cells to bulid glucose deprivation/hypoxia injury model ( the oxygen and glucose,deprivation,OGD ). Cell viability was measured by MTT assay,flow cytometry detection was used to determine the oxygen content of active cells in each group. Results Compared with model group, the 25 μmol · L ~' hydrochloride leonurine significantly improved cells viability, reducing intracellular reactive oxygen species. Conclusion The hydrochloric leonurine has a significant protective effect on PC12 cell hypoglycemia/hypoxia injury model .%目的 研究盐酸益母草碱对缺糖/缺氧损伤大鼠肾上腺嗜铬瘤细胞株(PC12)的保护作用.方法 采用连二亚硫酸钠(Na2S2O4)+缺糖建立PC12细胞缺糖/缺氧损伤模型(oxygen and glucose deprivation,OGD),MTT法检测各组细胞存活率,流式细胞仪检测各组细胞内活性氧含量.结果 与模型组相比,25 μmol·L-1盐酸益母草碱显著提高细胞存活率,降低细胞内的活性氧含量.结论 盐酸益母草碱对PC12 细胞缺糖/缺氧损伤模型有明显的保护作用.

  20. Cyclophilin A affects Bcl-2 and Bax expression following beta-amyloid fragment 25-35-induced injury to PC12 cells

    Li Cheng; Chaodong Zhang


    BACKGROUND: Cyclophilin A can protect neurons against oxidative stress.OBJECTIVE: To investigate the effect of cyclophilin A on Bcl-2 and Bax protein expression in pheochromocytoma (PCI2) cells treated with beta-amyloid fragment 25-35 (A β25-35), and to verify the protection pathway ofcyclophilin A.DESIGN, TIME AND SETTING: The initial experiment was performed at the Laboratory of Department of Neurology, First Clinical College, China Medical University from November 2006 to July 2007.MATERIALS: PCI2 cells were cultured at the Cell Center of Peking Union Medical College. A β25-35 (Sigma, USA), antibodies of Bcl-2 and Bax (Wuhan Boster, China), and recombinant human cyclophilin A (Biomol, USA) were used in this study.METHODS: PC12 cells were divided into three groups. Cells in the control group were incubated in culture medium. Cells in the Aβ25-35 injury group were incubated in medium containing a final concentration of 10 μ mol/L of Aβ25-35. Cells in the cyclophilin A group were incubated in medium containing a final concentration of 10 nmol/L of cyclophilin A for 30 minutes, and then treated with 10 μmol/L Aβ25-35. MAIN OUTCOME MEASURES: After 24 hours of culture, immunohistochemistry was used to detect Bcl-2 and Bax expression in PC12 cells. Annexin-V flow cytometry was employed to measure the apoptosis rate of PC12 cells. The MTT method was applied to examine the survival rate of PC12 cells.RESULTS: Bcl-2 expression decreased, whereas Bax expression increased in PCI2 cells treated with Aβ25-35 (t = 2.277, 5.957, P<0.05). However, in PC12 cells treated with Aβ25-35 and cyclophilin A, Bcl-2 expression increased and Bax expression decreased (t = 4.497, 2.531, P < 0.05). The survival rate of PC12 cells significantly decreased and the apoptosis rate increased (t=8.509, 22.886, P < 0.05) following Aβ25-35 treatment. Cyclophilin A enhanced the survival rate of PC12 cells to Aβ25-35-induced apoptosis (t = 4.895, 10.042, P< 0.05).CONCLUSION: Cyclophilin A can

  1. Modulation of vesicular catecholamine release from rat PC12 cells

    Westerink, R.H.S.


    Intercellular communication is of vital importance for the nervous system, since the nervous system is the main coordinating system in animals. Nerve cell communication is initiated by the release of chemical messengers, neurotransmitters, from the presynaptic nerve cell. The neurotransmitters, such

  2. Yokukansan, a kampo medicine, protects PC12 cells from glutamate-induced death by augmenting gene expression of cystine/glutamate antiporter system Xc-.

    Hitomi Kanno

    Full Text Available Effects of the kampo medicine yokukansan on gene expression of the cystine/glutamate antiporter system Xc-, which protects against glutamate-induced cytotoxicity, were examined in Pheochromocytoma cells (PC12 cells. Yokukansan inhibited glutamate-induced PC12 cell death. Similar cytoprotective effects were found in Uncaria hook. Experiments to clarify the active compounds revealed that geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook, had cytoprotective effects. These components enhanced gene expressions of system Xc- subunits xCT and 4F2hc, and also ameliorated the glutamate-induced decrease in glutathione levels. These results suggest that the cytoprotective effect of yokukansan may be attributed to geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook.

  3. Yokukansan, a kampo medicine, protects PC12 cells from glutamate-induced death by augmenting gene expression of cystine/glutamate antiporter system Xc-.

    Kanno, Hitomi; Kawakami, Zenji; Mizoguchi, Kazushige; Ikarashi, Yasushi; Kase, Yoshio


    Effects of the kampo medicine yokukansan on gene expression of the cystine/glutamate antiporter system Xc-, which protects against glutamate-induced cytotoxicity, were examined in Pheochromocytoma cells (PC12 cells). Yokukansan inhibited glutamate-induced PC12 cell death. Similar cytoprotective effects were found in Uncaria hook. Experiments to clarify the active compounds revealed that geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook, had cytoprotective effects. These components enhanced gene expressions of system Xc- subunits xCT and 4F2hc, and also ameliorated the glutamate-induced decrease in glutathione levels. These results suggest that the cytoprotective effect of yokukansan may be attributed to geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook.

  4. Relationship between apoptosis and pErk in manganese-treated PC12 cell line%锰致PC12细胞凋亡作用及与p- Erk关系

    徐文; 刘红; 闫文; 杨银书; 卢娟; 徐强; 董大海; 缪珊; 李金翠; 高琨; 高丽莉; 侯顺利; 左晶


    目的 以鼠嗜铬神经瘤细胞(PC12)为模型,筛选锰对神经细胞增殖抑制作用的时间及剂量,观察锰作用下PC12细胞的细胞形态学、生化指标改变和磷酸化的胞外信号调节激酶(p - Erk)的表达.方法 用200、400、600、800 μmol/L MnCl2的培养液,分别作用对数生长期PC12细胞1、2、3、4d后,四甲基偶氮塞唑蓝(MTT)筛选锰的细胞毒性剂量;透射电镜观察细胞形态学变化;琼脂糖凝胶电泳检测MnCl2对PCl2细胞基因组DNA的影响.免疫印迹法( western blot)检测p- Erk.结果 MTT显示200~800 μmol/L MnCl2作用1、2、3、4d对PC12有显著的抑制作用,呈剂量和时间依赖趋势,600 μmol/L MnCl2作用4d对PC12的抑制率50%;600 μrnol/L MnCl2作用4d电镜可见细胞凋亡,同样条件下细胞DNA碎片化;Western blot显示600μml/oL MnCl2作用1、2、3、4d可见p- Erk2逐渐降低,其中作用2d时较对照降低75%(n=3,P<0.05),200、400、600 μmol/L MnCl2分别作用4d时,p- Erk亦逐渐降低,当400 μmol/L MnCl2作用4d时较对照明降低78%(n=3,P<0.01);使用Erk通路MEK1/2特异性阻制剂PD98059实验结果表明:锰可能通过MEK1/2磷酸化下游的Erk,下调p- Erk.结论 锰对PC12细胞的毒性作用可能是通过关闭胞外信号调节激酶ErK通路诱导细胞凋亡.%Objective To observe apoptosis related cell morphology, biochemical changes and phosphrylations of phos-phoralated extracellularc signal-regulated kinase(p-Erk)in pheochromocytoma cells(PC12) exposed to manganese at different concentration and exposure time. Methods PC12 cells in logarithm growth period were incubated in culture media with 200,400,600,and 800μmol/L manganese(MnCl2)for 1,2,3 and 4 days,respectively. The cell viability was examined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasolium bromide (MTT) and morphological changes of PC12 cells were investigated with transmission electron microscope. Agarose gel electrophoresis was adopted to

  5. Apoptotic effect of cobalt chloride on PC12 cells%氯化钴对PC12细胞的凋亡作用

    曾季平; 王立祥; 胡晓燕; 于清水; 秦文; 崔行


    目的确定氯化钴(CoCl2)对PC12细胞的影响.方法构建CoCl2诱导的PC12细胞模型,检测CoCl2对PC12细胞的毒性作用;将Caspases的抑制基因p35转染PC12细胞得到可稳定表达p35基因的细胞株PC12/p35,检测p35对CoCl2诱导的PC12细胞的作用;检测Caspases特异性多肽抑制剂Z-VAD-FMK对CoCl2诱导的PC12细胞的作用.结果分别以100、300、500、700和1 000 μmol/L CoCl2诱导PC12细胞24 h或以500μmol/L CoCl2分别诱导PC12细胞12、24、36、48和60 h后,PC12细胞存活率均明显下降(P<0.01),并与CoCl2诱导的时间和浓度呈正相关;细胞亚显微结构检测,流式细胞分析和DNA片段化结果也表明500 μmol/L CoCl2诱导PC12细胞24 h可使PC12细胞出现明显凋亡特征.构建可稳定表达Caspase蛋白酶抑制基因p35的PC12细胞株,或分别加入50和100 μmol/L Caspases多肽抑制剂Z-VAD-FMK预处理PC12细胞1 h后,以500μmol/L CoCl2诱导PC12细胞24 h,形态学观察结果,细胞存活率检测和流式细胞分析均表明p35基因和Z-VAD-FMK均可有效地抑制CoCl2诱导的PC12细胞凋亡(P<0.01).结论CoCl2可诱导PC12细胞凋亡.

  6. 15-Deoxy-Δ12,14-Prostaglandin J2 Protects PC12 cells from LPS-Induced Cell Death Through Nrf2 pathway in PPAR-γ Dependent Manner

    Fariba Khodagholi


    Full Text Available Introduction:The inflammatory response requires a coordinated integration of various signaling pathway including cyclooxygenase (COX.COX catalyzes the formation of prostaglandins from arachidonic acid. Among prostaglandins, 15-Deoxy-D12,14-prostaglandin J2 (15d-PGJ2,an endogenous ligand of Peroxisome proliferator-activated receptor-gamma (PPAR-γ,has been demonstrated to have anti-inflammatory actions.In this study,we investigated whether 15d-PGJ2 as a PPAR-γ ligand could exert neuroprotective effects in rat pheochromocytoma (PC12 cells in PPAR-γ dependent manner. Methods: In our experiment, using PC12 cells, the levels of NF-κB, Nrf2, γ-glutamylcysteine synthetase (γ-GCS, hemeoxygenase (HO-1 and apoptosis factors were determined using Western blot in different groups. Also cell viability was determined by the conventional MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide reduction assay and two staining involved Hoechst staining and Acridine Ordange/Ethidiume Bromide staining respectively. Results: Our results show that NS-398, a selective COX-2 inhibitor and 15d-PGJ2, a natural potent ligand of PPAR-γ, were neuroprotective through modulation of at least three different, but related pathways and molecules, including NF-κB and Nrf2 signaling pathway. Our data showed that 15d-PGJ2 and NS-398 induced Nrf2 signaling pathway and its downstream factors such as HO-1 and γ-GCS, while 15d-PGJ2 and NS-398 decreased NF-κB level. Interestingly, the observed protective effects were mediated through PPAR-γ-dependent mechanisms, as they reversed by GW9662, an irreversible antagonist of PPAR-γ receptor. Discussion: Thus we conclude that 15d-PGJ2 as well as NS-398 exert anti cell death effect in a PPAR-γ dependent mechanisms.

  7. Comparison of Monoamine Oxidase Inhibitors in Decreasing Production of the Autotoxic Dopamine Metabolite 3,4-Dihydroxyphenylacetaldehyde in PC12 Cells.

    Goldstein, David S; Jinsmaa, Yunden; Sullivan, Patti; Holmes, Courtney; Kopin, Irwin J; Sharabi, Yehonatan


    According to the catecholaldehyde hypothesis, the toxic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) contributes to the loss of nigrostriatal dopaminergic neurons in Parkinson's disease. Monoamine oxidase-A (MAO-A) catalyzes the conversion of intraneuronal dopamine to DOPAL and may serve as a therapeutic target. The "cheese effect"-paroxysmal hypertension evoked by tyramine-containing foodstuffs-limits clinical use of irreversible MAO-A inhibitors. Combined MAO-A/B inhibition decreases DOPAL production in rat pheochromocytoma PC12 cells, but whether reversible MAO-A inhibitors or MAO-B inhibitors decrease endogenous DOPAL production is unknown. We compared the potencies of MAO inhibitors in attenuating DOPAL production and examined possible secondary effects on dopamine storage, constitutive release, synthesis, and auto-oxidation. Catechol concentrations were measured in cells and medium after incubation with the irreversible MAO-A inhibitor clorgyline, three reversible MAO-A inhibitors, or the MAO-B inhibitors selegiline or rasagiline for 180 minutes. Reversible MAO-A inhibitors were generally ineffective, whereas clorgyline (1 nM), rasagiline (500 nM), and selegiline (500 nM) decreased DOPAL levels in the cells and medium. All three drugs also increased dopamine and norepinephrine, decreased 3,4-dihydroxyphenylalanine, and increased cysteinyl-dopamine concentrations in the medium, suggesting increased vesicular uptake and constitutive release, decreased dopamine synthesis, and increased dopamine spontaneous oxidation. In conclusion, clorgyline, rasagiline, and selegiline decrease production of endogenous DOPAL. At relatively high concentrations, the latter drugs probably lose their selectivity for MAO-B. Possibly offsetting increased formation of potentially toxic oxidation products and decreased formation of DOPAL might account for the failure of large clinical trials of MAO-B inhibitors to demonstrate slowing of neurodegeneration in Parkinson

  8. Lead exposure in pheochromocytoma cells induces persistent changes in amyloid precursor protein gene methylation patterns.

    Li, Yuan-Yuan; Chen, Tian; Wan, Yanjian; Xu, Shun-qing


    It has been suggested that lead (Pb) exposure in early life may increase amyloid precursor protein (APP) expression and promote the pathogenesis of Alzheimer's disease in old age. The current study examined whether the DNA methylation patterns of APP gene in rat pheochromocytoma (PC12) cells changed after Pb acetate exposure. Undifferentiated PC12 cells were exposed to three doses of Pb acetate (50, 250, and 500 nM) and one control for 2 days or 1 week. The methylation patterns of APP promoter and global DNA methylation were analyzed. The DNA methyltransferase 1 (DNMT1) expression and the level of amyloid β peptide (Aβ) were also investigated. The results showed that the exposure of the three concentrations of Pb acetate could make the APP promoter hypomethylated. The global DNA methylation level and the expression of DNMT1 were changed in the 500 nM group after 2 days exposure and in the 250 and 500 nM group after 7 days exposure. Thus, Pb may exert neurotoxic effects through mechanisms that alter the global and promoter methylation patterns of APP gene. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012. Copyright © 2010 Wiley Periodicals, Inc.

  9. Inhibition of Corydalis decumbens Alkaloids on Hydrogen Peroxideinduced Apoptosis of PC12 Cells through Down-regulating Caspase-3 Expression

    YAN Ren-jie; YANG Yi-fang; LUO Yong-ming; WU Chun-zhen


    Objective To extract alkaloids from Corydalis decumbens (AsCD) by supercritical CO2 fluid extraction (SFE) and to evaluate protective effects of AsCD against hydrogen peroxide (H2O2)-induced apoptosis in rat PC12 cells.Methods AsCD were extracted by SFE and oxidative damage PC12 cells model was induced by H2O2.The survival rate of the cells was determined by MTT assay; Lactate dehydrogenase release was determined by ultraviolet spectrophotometry; Flow cytometry was used to detect apoptosis; Caspase-3 mRNA and protein were determined by real-time PCR and Western blotting assay,respectively.Results AsCD remarkably reduced the cytotoxicity,prevented membrane damage,and inhibited cell apoptosis.AsCD inhibited Caspase-3 mRNA and protein expression induced by H2O2 in PC12 cells.Conclusion AsCD possess protective effects against H2O2-induced apoptosis in PC12 cells,and the mechanism of AsCD responsible to the inhibition of apoptosis is possibly attributed to thedown-regulating Caspase-3 expression.AsCD might be useful in the treatment of oxidative stress-related neurodegenerative diseases.

  10. Identification of the alternative spliced form of the alpha 2/delta subunit of voltage sensitive Ca2+ channels expressed in PC12 cells.

    Gilad, B; Shenkar, N; Halevi, S; Trus, M; Atlas, D


    The alpha 2/delta subunit of voltage sensitive Ca2+ channels expressed in PC12 has been cloned and partially sequenced. The message observed in Northern blot analysis displays a 7.5 kb transcript, identical in size to mRNA of rabbit skeletal muscle and rat brain. The nucleotide sequence of the cloned alpha 2 subunit of the PC12 specific cDNA is > 99% identical to rat brain sequence and 85% to skeletal muscle. Reverse-transcriptase-polymerase chain reaction (RT-PCR) of the alternative splicing region identifies two deleted regions of 57 bp and 21 bp in PC12 expressed alpha 2/delta transcript. The alternative variant alpha 2e of alpha 2/delta subunit which is expressed in PC12 cells was previously identified in human embryonic kidney (HEK293) cells. RT-PCR analysis show two different sized alternative PCR fragments in rat lung and none in rat spleen, kidney and intestine. Antibodies prepared against a 19 amino acid peptide within the alternative spliced region effectively inhibits [3H]dopamine release in PC12 cells. This implies that the alternatively spliced region is positioned extracellularly and is involved in regulation of the L-type Ca2+ channel-mediated transmitter release.

  11. Hydrogen peroxide induces the activation of the phospholipase C-γ1 survival pathway in PC12 cells: protective role in apoptosis

    Wenli Yuan; Jiazhi Guo; Xingguo Li; Zhirong Zou; Guangxue Chen; Jun Sun; Tinghua Wang; Di Lu


    It has been reported that phospholipase C-γ1 (PLC-γ1) plays an important protective role in hydrogen peroxide (H2O2)-induced pheochromocytoma (PC) 12 cells death. However, most studies have used high doses of H2O2 and the downstream targets of PLC-γ1 activation remain to be identified. The present study was designed to examine the roles of PLC-γ1 signaling pathway in the apoptosis of PC12 cells induced by low dose of H2O2, as well as the downstream factors involved in this pathway. Low-dose treatment of H2O2 resulted in PLC-γ1 tyrosine phosphorylation in a time-dependent manner and H2O2 killed the PC12 cells by inducing necrosis. In contrast, pretreatment of PC12 cells with U73122, a specific inhibitor of PLC, markedly increased the percentage of dead cells. The mode of cell death was converted to apoptosis as determined by Hoechst/PI nuclear staining and fluorescence microscopy. Western blot analysis demonstrated that the expression of Bcl-2 protein and the activation of pro-caspase-3 were not significantly affected by low dose of H2O2 alone. However, after pretreatment with U73122, Bcl-2 protein expression was dramatically decreased and the activation of pro-caspase-3 was sig-nificantly increased. We concluded that PLC-γ1 plays an important protective role in H2O2-induced PC12 cells death. Bcl-2 and caspase-3 probably participate in the signaling pathway as downstream factors.

  12. (-)Schisandrin B ameliorates paraquat-induced oxidative stress by suppressing glutathione depletion and enhancing glutathione recovery in differentiated PC12 cells.

    Lam, Philip Y; Ko, Kam Ming


    Exposure to paraquat (PQ; N,N'-dimethyl-4-4'-bipyridium), a potent herbicide, can lead to neuronal cell death and increased risk of Parkinson's disease because of oxidative stress. In this study, we investigated the effect of (-)schisandrin B [(-)Sch B, a potent enantiomer of schisandrin B] on PQ-induced cell injury in differentiated pheochromocytoma cells (PC12). PQ treatment caused cell injury in PC12 cells, as indicated by the significant increase in lactate dehydrogenase (LDH) leakage. Pretreatment with (-)Sch B (5 μM) protected against PQ-induced toxicity in PC12 cells, as evidenced by the significant decrease in LDH leakage. (-)Sch B induced the cytochrome P-450-mediated reactive oxygen species generation in differentiated PC12 cells. The cytoprotection afforded by (-)Sch B pretreatment was associated with an increase in cellular reduced glutathione (GSH) level as well as the enhancement of γ-glutamylcysteine ligase (GCL) and glutathione reductase (GR) activity in PQ-challenged cells. Both GCL and GR inhibitors abrogated the cytoprotective effect of (-)Sch B in PQ-challenged cells. The biochemical mechanism underlying the GSH-enhancing effect of (-)Sch B was further investigated in PC12 cells subjected to an acute peroxide challenge. Although the initial GSH depletion induced by peroxide was reduced through GR-catalyzed regeneration of GSH in (-)Sch B-pretreated cells, the later enhanced GSH recovery was mainly mediated by GCL-catalyzed GSH synthesis. The results suggest that (-)Sch B treatment may increase the resistance of dopaminergic cells against PQ-induced oxidative stress through reducing the extent of oxidant-induced GSH depletion and enhancing the subsequent GSH recovery.

  13. Design and fabrication of a microplatform for the proximity effect study of localized ELF-EMF on the growth of in vitro HeLa and PC-12 cells

    Chen, Y. C.; Chen, C. C.; Tu, W.; Cheng, Y. T.; Tseng, F. G.


    This paper presents a platform technology with experimental results that show the scientists and biologists a way to rapidly investigate and analyze the biological effects of localized extremely low frequency (ELF) electromagnetic field (EMF) on living cells. The proximity effect of the localized ELF-EMF on living cells is revealed using the bio-compatible microplatform on which an on-glass inductive coil array, the source of the localized ELF-EMF in micro scale, is designed, fabricated and operated with a field strength of 1.2 ± 0.1 mT at 60 Hz for cell culturing study. After a 72 h ELF-EMF exposure, HeLa (human cervical cancer) and PC-12 (rat pheochromocytoma) cells exhibit about 18.4% and 12.9% cell proliferation rate reduction, respectively. Furthermore, according to the presented dynamic model, the reduction of the proliferation can be attributed to the interference of signal transduction processes due to the tangential currents induced around the cells.

  14. The PERK-eIF2α signaling pathway is involved in TCDD-induced ER stress in PC12 cells.

    Duan, Zhiqing; Zhao, Jianya; Fan, Xikang; Tang, Cuiying; Liang, Lingwei; Nie, Xiaoke; Liu, Jiao; Wu, Qiyun; Xu, Guangfei


    Studies have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces apoptotic cell death in neuronal cells. However, whether this is the result of endoplasmic reticulum (ER) stress-mediated apoptosis remains unknown. In this study, we determined whether ER stress plays a role in the TCDD-induced apoptosis of pheochromocytoma (PC12) cells and primary neurons. PC12 cells were exposed to different TCDD concentrations (1, 10, 100, 200, or 500nM) for varying lengths of time (1, 3, 6, 12, or 24h). TCDD concentrations much higher than 10nM (100, 200, or 500nM) markedly increased glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP) levels, which are hallmarks of ER stress. We also evaluated the effects of TCDD on ER morphology in PC12 cells and primary neurons that were treated with different TCDD concentrations (1, 10, 50, or 200nM) for 24h. Ultrastructural ER alterations were observed with transmission electron microscopy in PC12 cells and primary neurons treated with high concentrations of TCDD. Furthermore, TCDD-induced ER stress significantly promoted the activation of the PKR-like ER kinase (PERK), a sensor for the unfolded protein response (UPR), and its downstream target eukaryotic translation initiation factor 2 α (eIF2α); in contrast, TCDD did not appear to affect inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6), two other UPR sensors. Importantly, TCDD significantly inhibited eIF2α phosphorylation and triggered apoptosis in PC12 cells after 6-24h of treatment. Salubrinal, which activates the PERK-eIF2α pathway, significantly enhanced eIF2α phosphorylation in PC12 cells and attenuated the TCDD-induced cell death. In contrast, knocking down eIF2α using small interfering RNA markedly enhanced TCDD-induced cell death. Together, these results indicate that the PERK-eIF2α pathway plays an important role in TCDD-induced ER stress and apoptosis in PC12 cells.

  15. Rho激酶抑制剂诱导PC12PC12Adh细胞突起生长的差异比较%Differences of neurite outgrowth induced by rho kinase inhibitors between in PC12 cell line and PC12 Adh cell line

    却翎; 段为钢; 张陆勇; 江振洲


    PC12 cell line is one of the basic tool cell lines used in neural differentiation research.The cell line is able to grow neurite by exposure to rho kinase ( ROCK) inhibitors.Recently, American Type Culture Collection ( ATCC) provided PC12 cell line and PC12 Adh cell line.The main aim of the study was to clarify the differences of the two cell lines in neurite outgrowth induced by ROCK inhibitors.PC12 cell line and PC12 Adh cell line (from ATCC) were treated with nerve growth factor (NGF,1000 ng/mL) or ROCK inhibitors (Y27632 of 33 μmol/L and fasudil of 33 μmol/L) for 1 -4 days.NGF was able to promote neurite outgrowth both in PC12 cell line and PC12 Adh cell line,while ROCK inhibitors was able to do so only in PC12 Adh cell line.These results suggested that PC12 Adh cell line is more suitable for ROCK inhibitors study in neural differentiation.%PC12细胞是研究神经分化最常用的细胞之一.在rho激酶(ROCK)抑制剂的作用下,PC12细胞能够长出神经样突起.最近,美国菌种保存中心(ATCC)同时提供PC12细胞和PC12 Adh细胞.研究的主要目的是观察ROCK抑制剂诱导这2种细胞长突起是否存在差异.PC12细胞和PC12Adh细胞按照ATCc方法进行培养,用神经生长因子(NGF,1 000 ng/mL)或ROCK抑制剂(33 μmol/L Y27632,33 μmol/L法舒地尔)处理细胞1~4 d.结果发现NGF能够诱导这2种细胞生长突起,而ROCK抑制剂只诱导PC12Adh细胞长突起,对PC12细胞不明显.因此,ROCK抑制剂诱导这2种细胞突起生长存在明显差异,PC12Adh细胞更适合用于ROCK抑制剂的神经诱导分化实验.

  16. KCl stimulation increases norepinephrine transporter function in PC12 cells.

    Mandela, Prashant; Ordway, Gregory A


    The norepinephrine transporter (NET) plays a pivotal role in terminating noradrenergic signaling and conserving norepinephrine (NE) through the process of re-uptake. Recent evidence suggests a close association between NE release and regulation of NET function. The present study evaluated the relationship between release and uptake, and the cellular mechanisms that govern these processes. KCl stimulation of PC12 cells robustly increased [3H]NE uptake via the NET and simultaneously increased [3H]NE release. KCl-stimulated increases in uptake and release were dependent on Ca2+. Treatment of cells with phorbol-12-myristate-13-acetate (PMA) or okadaic acid decreased [3H]NE uptake but did not block KCl-stimulated increases in [3H]NE uptake. In contrast, PMA increased [3H]NE release and augmented KCl-stimulated release, while okadaic acid had no effects on release. Inhibition of Ca2+-activated signaling cascades with KN93 (a Ca2+ calmodulin-dependent kinase inhibitor), or ML7 and ML9 (myosin light chain kinase inhibitors), reduced [3H]NE uptake and blocked KCl-stimulated increases in uptake. In contrast, KN93, ML7 and ML9 had no effect on KCl-stimulated [3H]NE release. KCl-stimulated increases in [3H]NE uptake were independent of transporter trafficking to the plasma membrane. While increases in both NE release and uptake mediated by KCl stimulation require Ca2+, different intracellular mechanisms mediate these two events.

  17. Platycodin D induced apoptosis and autophagy in PC-12 cells through mitochondrial dysfunction pathway

    Zeng, Chuan-Chuan; Zhang, Cheng; Yao, Jun-Hua; Lai, Shang-Hai; Han, Bing-Jie; Li, Wei; Tang, Bing; Wan, Dan; Liu, Yun-Jun


    In this article, the in vitro cytotoxicity of platycodin D was evaluated in human PC-12, SGC-7901, BEL-7402, HeLa and A549 cancer cell lines. PC-12 cells were sensitive to platycodin D treatment, with an IC50 value of 13.5 ± 1.2 μM. Morphological and comet assays showed that platycodin D effectively induced apoptosis in PC-12 cells. Platycodin D increased the levels of reactive oxygen species (ROS) and induced a decrease in mitochondrial membrane potential. Platycodin D induced cell cycle arrest at the G0/G1 phase in the PC-12 cell line. Platycodin D can induce autophagy. In addition, platycodin D can down-regulate the expression of Bcl-2 and Bcl-x, and up-regulate the levels of Bid protein in the PC-12 cells. The results demonstrated that platycodin D induced PC-12 cell apoptosis through a ROS-mediated mitochondrial dysfunction pathway.

  18. Translationally Controlled Tumor Protein Stimulates Dopamine Release from PC12 Cells via Ca(2+)-Independent Phospholipase A₂ Pathways.

    Seo, Jihui; Maeng, Jeehye; Kim, Hwa-Jung


    The translationally controlled tumor protein (TCTP), initially identified as a tumor- and growth-related protein, is also known as a histamine-releasing factor (HRF). TCTP is widely distributed in the neuronal systems, but its function is largely uncharacterized. Here, we report a novel function of TCTP in the neurotransmitter release from a neurosecretory, pheochromocytoma (PC12) cells. Treatment with recombinant TCTP (rTCTP) enhanced both basal and depolarization (50 mM KCl)-evoked [³H]dopamine release in concentration- and time-dependent manners. Interestingly, even though rTCTP induced the increase in intracellular calcium levels ([Ca(2+)]i), the rTCTP-driven effect on dopamine release was mediated by a Ca(2+)-independent pathway, as evidenced by the fact that Ca(2+)-modulating agents such as Ca(2+) chelators and a voltage-gated L-type Ca(2+)-channel blocker did not produce any changes in rTCTP-evoked dopamine release. In a study to investigate the involvement of phospholipase A₂ (PLA₂) in rTCTP-induced dopamine release, the inhibitor for Ca(2+)-independent PLA₂ (iPLA₂) produced a significant inhibitory effect on rTCTP-induced dopamine release, whereas this release was not significantly inhibited by Ca(2+)-dependent cytosolic PLA₂ (cPLA₂) and secretory PLA₂ (sPLA₂) inhibitors. We found that rTCTP-induced dopamine release from neuronal PC12 cells was modulated by a Ca(2+)-independent mechanism that involved PLA₂ in the process, suggesting the regulatory role of TCTP in the neuronal functions.

  19. Translationally Controlled Tumor Protein Stimulates Dopamine Release from PC12 Cells via Ca2+-Independent Phospholipase A2 Pathways

    Jihui Seo


    Full Text Available The translationally controlled tumor protein (TCTP, initially identified as a tumor- and growth-related protein, is also known as a histamine-releasing factor (HRF. TCTP is widely distributed in the neuronal systems, but its function is largely uncharacterized. Here, we report a novel function of TCTP in the neurotransmitter release from a neurosecretory, pheochromocytoma (PC12 cells. Treatment with recombinant TCTP (rTCTP enhanced both basal and depolarization (50 mM KCl-evoked [3H]dopamine release in concentration- and time-dependent manners. Interestingly, even though rTCTP induced the increase in intracellular calcium levels ([Ca2+]i, the rTCTP-driven effect on dopamine release was mediated by a Ca2+-independent pathway, as evidenced by the fact that Ca2+-modulating agents such as Ca2+ chelators and a voltage-gated L-type Ca2+-channel blocker did not produce any changes in rTCTP-evoked dopamine release. In a study to investigate the involvement of phospholipase A2 (PLA2 in rTCTP-induced dopamine release, the inhibitor for Ca2+-independent PLA2 (iPLA2 produced a significant inhibitory effect on rTCTP-induced dopamine release, whereas this release was not significantly inhibited by Ca2+-dependent cytosolic PLA2 (cPLA2 and secretory PLA2 (sPLA2 inhibitors. We found that rTCTP-induced dopamine release from neuronal PC12 cells was modulated by a Ca2+-independent mechanism that involved PLA2 in the process, suggesting the regulatory role of TCTP in the neuronal functions.

  20. Developmental neurotoxicity of different pesticides in PC-12 cells in vitro.

    Christen, Verena; Rusconi, Manuel; Crettaz, Pierre; Fent, Karl


    The detection of developmental neurotoxicity (DNT) of chemicals has high relevance for protection of human health. However, DNT of many pesticides is only little known. Furthermore, validated in vitro systems for assessment of DNT are not well established. Here we employed the rat phaeochromocytoma cell line PC-12 to evaluate DNT of 18 frequently used pesticides of different classes, including neonicotinoids, pyrethroids, organophosphates, organochlorines, as well as quaternary ammonium compounds, the organic compound used in pesticides, piperonyl butoxide, as well as the insect repellent diethyltoluamide (DEET). We determined the outgrowth of neurites in PC-12 cells co-treated with nerve growth factor and different concentrations of biocides for 5days. Furthermore, we determined transcriptional alterations of selected genes that may be associated with DNT, such as camk2α and camk2β, gap-43, neurofilament-h, tubulin-α and tubulin-β. Strong and dose- dependent inhibition of neurite outgrowth was induced by azamethiphos and chlorpyrifos, and dieldrin and heptachlor, which was correlated with up-regulation of gap-43. No or only weak effects on neurite outgrowth and transcriptional alterations occurred for neonicotinoids acetamiprid, clothianidin, imidacloprid and thiamethoxam, the pyrethroids λ-cyhalothrin, cyfluthrin, deltamethrin, and permethrin, the biocidal disinfectants C12-C14-alkyl(ethylbenzyl)dimethylammonium (BAC), benzalkonium chloride and barquat (dimethyl benzyl ammonium chloride), and piperonyl butoxide and DEET. Our study confirms potential developmental neurotoxicity of some pesticides and provides first evidence that azamethiphos has the potential to act as a developmental neurotoxic compound. We also demonstrate that inhibition of neurite outgrowth and transcriptional alterations of gap-43 expression correlate, which suggests the employment of gap-43 expression as a biomarker for detection and initial evaluation of potential DNT of chemicals

  1. Beneficial effect of (-)schisandrin B against 3-nitropropionic acid-induced cell death in PC12 cells.

    Lam, Philip Y; Ko, Kam Ming


    Huntington's disease (HD) is characterized by the dysfunction of mitochondrial energy metabolism, which is associated with the functional impairment of succinate dehydrogenase (mitochondrial complex II), and pyruvate dehydrogenase (PDH). Treatment with 3-nitropropionic acid (3-NP), a potent irreversible inhibitor of succinate dehydrogenase, replicates most of the pathophysiological features of HD. In the present study, we investigated the effect of (-)schisandrin B [(-)Sch B, a potent enantiomer of schisandrin B] on 3-NP-induced cell injury in rat differentiated neuronal PC12 cells. The 3-NP caused cell necrosis, as assessed by lactate dehydrogenase (LDH) leakage, and mitochondrion-dependent cell apoptosis, as assessed by caspase-3 and caspase-9 activation, in differentiated PC12 cells. The cytotoxicity induced by 3-NP was associated with a depletion of cellular reduced glutathione (GSH) as well as the activation of redox-sensitive c-Jun N-terminal kinase (JNK) pathway and the inhibition of PDH. (-)Sch B pretreatment (5 and 15 μM) significantly reduced the extent of necrotic and apoptotic cell death in 3-NP-challenged cells. The cytoprotection afforded by (-)Sch B pretreatment was associated with the attenuation of 3-NP-induced GSH depletion as well as JNK activation and PDH inhibition. (-)Sch B pretreatment enhanced cellular glutathione redox status and ameliorated the 3-NP-induced cellular energy crisis, presumably by suppressing the activated JNK-mediated PDH inhibition, thereby protecting against necrotic and apoptotic cell death in differentiated PC12 cells.

  2. Erythropoietin prevents PC12 cells from beta-amyloid-induced apoptosis via PI3K⁄Akt pathway

    Zhi-Kun Sun


    Full Text Available Abstract Background Several studies indicated that Erythropoietin (Epo may provide remarkable neuroprotection in some neurological diseases. It also showed the significant decrease of Epo immunoreactivity in the cerebral cortex and hippocampus in aged rats, suggesting the role of Epo in the pathogenesis of age-related neurodegenerative diseases such as AD. Methods The protective effect of Epo was studied in differentiated PC12 cells treated with Abeta. The viability of the cells, the apoptosis of the cells and the level of Bax, Bcl-2, cleaved caspase-3 and cleaved PARP expression were detected by MTT, Hoechst 33258 staining and Western blotting respectively. Results 20 μM Abeta (25-35 could induce a decreased viability and a increased apoptosis in PC12 cell in a time-dependent manner. However, 20 μM Abeta (35-25 had no effect on cell viability and apoptosis. Western blot analysis also showed that Abeta(25-35 treatment could decrease the expression of Bcl-2 (P P P P (25-35 (P P Conclusions Epo prevented cell injuries in PC12 cells exposed to the Abeta(25-35 and this effect may depend on the PI3K⁄Akt pathway. Our study provided an important evidence for the potential application of Epo in the therapy of Alzheimer's disease.

  3. Reversible effects of vitamins C and E combination on oxidative stress-induced apoptosis in melamine-treated PC12 cells.

    An, L; Li, Z; Zhang, T


    Due to its high nitrogen content, melamine was deliberately added to raw milk for increasing the apparent protein content. Previous studies showed that melamine-induced apoptosis and oxidative damage on PC12 cells and rats' hippocampus. Several evidences suggested that vitamin antioxidant reduced oxidative stress and improved organic function. Whether treatments with antioxidant vitamins C or E, otherwise combination of them can attenuate oxidative stress after melamine administration remains to be elucidated. In this study, the reversible effects of vitamin antioxidants was investigated on melamine-induced neurotoxicity in cultured PC12 cells, an in vitro model of neuronal cells. When comparing vitamin C and E, the combination of both statistically increased PC12 cells viability. The results further showed that vitamin complex has effectively reduced the formation of reaction oxygen species, decreased the level of malondialdehyde, and elevated the activities of antioxidative enzymes. Hoechst 33342 staining and flow cytometric analysis of apoptosis showed that vitamin combination treatment effectively prevented PC12 cells from this melamine-induced apoptosis. It revealed the apoptotic nuclear features of the melamine-induced cell death. Additionally, a combination treatment of vitamins effectively inhibited apoptosis via blocking the increased activation of caspase-3. In summary, the vitamin E and C combination treatment could rescue PC12 cells from the injury induced by melamine through the downregulation of oxidative stress and prevention of melamine-induced apoptosis.

  4. von Hippel-Lindau protein induces hypoxia-regulated arrest of tyrosine hydroxylase transcript elongation in pheochromocytoma cells.

    Kroll, S L; Paulding, W R; Schnell, P O; Barton, M C; Conaway, J W; Conaway, R C; Czyzyk-Krzeska, M F


    Rat pheochromocytoma (PC12) cells were stably transfected with either wild type or mutated human von Hippel-Lindau tumor suppressor protein (hpVHL). These proteins have opposing effects on regulating expression of the gene encoding tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. Whereas wild type hpVHL represses levels of TH mRNA and protein 5-fold, a truncated pVHL mutant, pVHL(1-115), induces accumulation of TH mRNA and protein 3-fold. hpVHL-induced inhibition of TH gene expression does not involve either a decrease in TH mRNA stability or repression of TH promoter activity. However, repression results from inhibition of RNA elongation at a downstream region of the TH gene. This elongation pause is accompanied by hpVHL sequestration in the nuclear extracts of elongins B and C, regulatory components of the transcription elongation heterotrimer SIII (elongin A/B/C). Hypoxia, a physiological stimulus for TH gene expression, alleviates the elongation block. A truncated pVHL mutant, pVHL(1-115), stimulates TH gene expression by increasing the efficiency of TH transcript elongation. This is the first report showing pVHL-dependent regulation of specific transcript elongation in vivo, as well as dominant negative activity of pVHL mutants in pheochromocytoma cells.

  5. Differentiation of PC12 cells induced by total saponin of panax ginseng%人参总皂苷对PC12细胞分化的影响

    李晗宇; 王顺和; 郑娟; 姜蓉


    目的:研究人参总皂苷(Total saponin ofpanax ginseng,TSPG)诱导PC12细胞神经元性分化的作用.方法:体外培养PC12细胞,观察PC12细胞在TSPG的影响下细胞发生的形态学改变.诱导48 h透射电镜观察突触连接形成情况,72 h利用免疫细胞化学技术,观察TSPG作用后PC12细胞向MAP-2(Microtubule-associatedprotein 2)阳性细胞分化的情况.结果:TSPG作用后的PC12细胞其突起长度和细胞直径较对照组明显增长和增大[长度由(13.95±2.59)μm增长至(30.33±3.82)μm;直径由(8.25±1.82)μm提高到(14.33±2.84)μm,P<0.05].TSPG能促进PC12细胞形成突触连接.TSPG组表达MAP2的阳性细胞率明显高于对照组(由4.55%提高到12.22%,P<0.05).结论:TSPG具有诱导PC12细胞神经元性分化的作用.%Objective: To study the potential ahility of TSPG ( Total saponin of panax ginseng ) to induce the neuronal differentiation of PC12 cells. Methods : PC12 cells were cultured in vitro , and their morphological changes under the TSPG were observed. The synaptic linkage were ohserved by transmitting electronic microscope after the PC12 cells were exposed to TSPG for 48 h and the expression of MAP-2 was detected by immunocytochemistry after 72 h. Results : The length of neurite and max diameter of PC12 cells in TSPG group were obviously longer and bigger than those in control group ((13.95 ± 2.59) μm to ( 30.33 ± 3.82) μ m; ( 8.25 ± 1.82) μm to ( 14.33 ± 2.84) μm , P < 0.05 ) TSPG could induce PC12 cells to form the synaptic linkage.compared with the control group , the numher of MAP-2 positive cells was significantly increased in TSPC group (4.55%to 12.22% ,P < 0.05 ) .Conclusion : TSPG can induce the neuronal differentiation of PC12 cells.

  6. Exposure of PC12 cells to NGF/ethanol results in accelerated differentiation and altered gene expression

    White, K.R.; Wooten, M.W. (Auburn Univ., AL (United States))


    The role of alcohols in affecting neuromorphogenesis was investigated in the pheochromocytoma cell line, PC12. The effect of ethanol at physiological concentrations in this system leads to accelerated neurite extension in the presence of suboptimal concentrations of NGF. Accelerated morphological differentiation was dependent upon the side chain length of the alcohol and not inhibited by pyrazole. Ethanol/NGF induced neurite extension can be blocked with 50nM of K252a, but not sphingozine, H7, H89, genistein or okadaic acid. Changes in the expression of 17 NGF-induced and/or neuronal transcripts were examined in relationship to time of NGF/ethanol exposure; dose of NGF/ethanol; and side-chain length of NFG/alcohol. The authors studies indicate that ethanol potentiates the effects of NFG and subsequent neurogenesis through both protein kinase C and cAMP-independent pathways. In addition, these data show that ethanol is capable of altering gene expression in a specific manner.


    M. A. Abiodun


    Full Text Available A clinical case of arterial hypertension (AH in patient with family history of pheochromocytoma is described. Patient has no classical clinical signs and imaging phenotype of pheochromocytoma, but there are a number of warnings – family history of pheochromocytoma, prevalence of humoral-metabolic regulation and reduced reaction to the respiratory test, CT-signs of nodular hyperplasia of left adrenal gland – which may indicate its possible manifestations in the future, and therefore the monitoring is required.

  8. The co-culture system of MSCs and injured PC12 in vitro could inhibit the apoptosis of PC12%骨髓基质干细胞与受损PC12细胞的共育体系减少PC12的凋亡

    周进; 罗晓光; 张朝东


    目的 建立体外骨髓基质干细胞(MSCs)与Aβ1-40损伤PC12的共育体系,探讨共育体系抑制Aβ1-40致PC12凋亡的效应与可能机制.方法 分别培养MSCs与PC12,以Aβ1-40刺激PC12后,用转移筛网转移PC12.实验分A组:正常培养的PC12+MSCs共育;B组:Aβ1-40刺激的PC12+MSCs共育;C组:正常PC12的培养上清+MSCs;D组:受损PC12上清+MSCs;E组:普通1640培养的MSCs.用PI和Annexin-V进行细胞的双染凋亡检测及电镜检测PC12凋亡,以ELISA方法检测各组中上清液的TGF-β、NGF、BDNF、bFGF含量.结果 骨髓基质干细胞与Aβ1-40损伤PC12共育组即B组凋亡细胞数最少(B组46.17%±8.28%,对照组86.39%±9.34%,F=61.637,P<0.01),ELISA结果显示各组均能检测到bFGF,B组上清中bFGF分泌最高[B组(598.76±41.32)pg/ml,对照组(296.43±47.86)pg/ml,F=24.15,P<0.01)],各组均检测到TGF-β、NGF和BDNF分泌,但差异无统计学意义.结论 MSCs与Aβ1-40刺激的PC12的共育体系能减少受损PC12的凋亡.

  9. BDNF-TrkB pathway mediates neuroprotection of hydrogen sulfide against formaldehyde-induced toxicity to PC12 cells.

    Jia-Mei Jiang

    Full Text Available Formaldehyde (FA is a common environmental contaminant that has toxic effects on the central nervous system (CNS. Our previous data demonstrated that hydrogen sulfide (H2S, the third endogenous gaseous mediator, has protective effects against FA-induced neurotoxicity. As is known to all, Brain-derived neurotropic factor (BDNF, a member of the neurotrophin gene family, mediates its neuroprotective properties via various intracellular signaling pathways triggered by activating the tyrosine kinase receptor B (TrkB. Intriguingly, our previous data have illustrated the upregulatory role of H2S on BDNF protein expression in the hippocampus of rats. Therefore, in this study, we hypothesized that H2S provides neuroprotection against FA toxicity by regulating BDNF-TrkB pathway. In the present study, we found that NaHS, a donor of H2S, upregulated the level of BDNF protein in PC12 cells, and significantly rescued FA-induced downregulation of BDNF levels. Furthermore, we found that pretreatment of PC12 cells with K252a, an inhibitor of the BDNF receptor TrkB, markedly reversed the inhibition of NaHS on FA-induced cytotoxicity and ablated the protective effects of NaHS on FA-induced oxidative stress, including the accumulation of intracellular reactive oxygen species (ROS, 4-hydroxy-2-trans-nonenal (4-HNE, and malondialdehyde (MDA. We also showed that K252a abolished the inhibition of NaHS on FA-induced apoptosis, as well as the activation of caspase-3 in PC12 cells. In addition, K252a reversed the protection of H2S against FA-induced downregulation of Bcl-2 protein expression and upregulation of Bax protein expression in PC12 cells. These data indicate that the BDNF-TrkB pathway mediates the neuroprotection of H2S against FA-induced cytotoxicity, oxidative stress and apoptosis in PC12 cells. These findings provide a novel mechanism underlying the protection of H2S against FA-induced neurotoxicity.

  10. Serum-free culture conditions for serial subculture of undifferentiated PC12 cells.

    Ohnuma, Kiyoshi; Hayashi, Yohei; Furue, Miho; Kaneko, Kunihiko; Asashima, Makoto


    PC12 cells, a widely used model neuronal cell line, are usually cultured in serum-supplemented medium. This report describes a serum-free medium for the culture of PC12 cells. PC12 cells grown in the two media types had similar growth rates and released dopamine in response to high potassium-induced calcium elevation. However, the levels of dopamine and of dopamine release in cells cultured in the serum-free medium were less than 10% of that in cells cultured in serum-supplemented medium. Dopamine levels recovered within 10 days if cells were returned to serum-supplemented medium, but dopamine release could not be recovered. Nerve growth factor (NGF) induced similar responses in PC12 cells cultured in both media, including phosphorylation of extracellular signal-regulated protein kinases and neurite extension. Transferrin was necessary for survival of neurite-bearing PC12 cells subcultured in serum-free medium and insulin promoted the cells proliferation. Ten days culture with NGF produced a similar increase in neurofilament expression and acetylcholinesterase activity in both media. These results suggest that PC12 in the hormonally defined serum-free media are qualitatively the same as those cultured in serum-supplemented media, and therefore this new culture protocol should enable more precise studies of PC12 cells culture in the absence of confounding unknown factors.

  11. Regulation of NGF-driven neurite outgrowth by Ins(1,4,5)P3 kinase is specifically associated with the two isoenzymes Itpka and Itpkb in a model of PC12 cells.

    Koenig, Sandra; Moreau, Colette; Dupont, Geneviève; Scoumanne, Ariane; Erneux, Christophe


    Four inositol phosphate kinases catalyze phosphorylation of the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3 ] to inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4 ]: these enzymes comprise three isoenzymes of inositol 1,4,5-trisphosphate 3-kinase (Itpk), referred to as Itpka, Itpkb and Itpkc, and the inositol polyphosphate multikinase (IPMK). The four enzymes that act on Ins(1,4,5)P3 are all expressed in rat pheochromocytoma PC12 cells, a model that is used to study neurite outgrowth induced by nerve growth factor (NGF). We compared the effect of over-expression of the four GFP-tagged kinases on NGF-induced neurite outgrowth. Our data show that over-expression of the Itpka and Itpkb isoforms inhibits NGF-induced neurite outgrowth, but over-expression of Itpkc and IPMK does not. Surprisingly, over-expression of the N-terminal F-actin binding domain of Itpka, which lacks catalytic activity, was as effective at inhibiting neurite outgrowth as the full-length enzyme. Neurite length was also significantly decreased in cells over-expressing Itpka and Itpkb but not Itpkc or IPMK. This result did not depend on the over-expression level of any of the kinases. PC12 cells over-expressing GFP-tagged kinase-dead mutants Itpka/b have shorter neurites than GFP control cells. The decrease in neurite length was never as pronounced as observed with wild-type GFP-tagged Itpka/b. Finally, the percentage of neurite-bearing cells was increased in cells over-expressing the membranous type I Ins(1,4,5)P3 5-phosphatase. We conclude that Itpka and Itpkb inhibit neurite outgrowth through both F-actin binding and localized Ins(1,4,5)P3 3-kinase activity. Itpkc and IPMK do not influence neurite outgrowth or neurite length in this model.

  12. PC12细胞转染人GPx-1基因后的抗氧化损伤作用%Anti-oxidative damage effect on PC12 cells transfected with human glutathione peroxidase 1 gene

    马琳; 王辉; 王淑荣; 陈蓉; 郑俩燕


    Objective To study the protective effect and molecular mechanism of glutathione peroxidase 1 (GPx-1) high expression on PC12 cell damage mediated by hydrogen peroxide (H2O2). Methods PC12 cells were transfected with plncx plasmid containing human GPx-1 gene and plncx plasmid, and a PC12 cell line was established that could stably express human GPx-1 gene and plncx plasmid through continuous screening. Two means of intervention, H2O2 and sodium selenite + H2O2, were given to PC12, GPx-1-PC12 and plncx-PC12 cells, respectively. GPx activity of cells in each group was detected with GPx activity detection kit; the survival and apoptosis of cells were detected by MTT method and flow cytometer, respectively. The expression of NF-κBp65 was detected by immunohistochemistry technique. Results GPx activity of GPx-PC12 group cells was obviously higher than that of controls. Intervention of H2O2 and sodium selenite + H2O2 did not significantly affect the cell survival rate in PC12 and plncx-PC12 groups (P>0. 05). The cell survival rate was higher in GPx-1-PC12 group than in PC12 and plncx-PC12 groups (P0.05),GPx-1-PC12PC12、plncx-PC12组细胞存活率明显增高(P<0.01);GPx-1-PC12PC12、plncx-PC12细胞早期和晚期凋亡率明显下降,存在统计学差异(P<0.01);GPx-1-PC12PC12细胞NF-κBp65表达明显减少(P<0.01).结论 GPx-1基因高表达对H2O2所致的PC12细胞氧化损伤有很好的保护作用;GPx-1可抑制PC12细胞氧化损伤时凋亡的发生及NF-κB的表达.

  13. Effects of Long-term exposure of Gelatinated and Non-gelatinated Cadmium Telluride Quantum Dots on Differentiated PC12 cells

    Prasad, Babu R


    Abstract Background The inherent toxicity of unmodified Quantum Dots (QDs) is a major hindrance to their use in biological applications. To make them more potent as neuroprosthetic and neurotherapeutic agents, thioglycolic acid (TGA) capped CdTe QDs, were coated with a gelatine layer and investigated in this study with differentiated pheochromocytoma 12 (PC12) cells. The QD - cell interactions were investigated after incubation periods of up to 17 days by MTT and APOTOX-Glo Triplex assays along with using confocal microscopy. Results Long term exposure (up to 17 days) to gelatinated TGA-capped CdTe QDs of PC12 cells in the course of differentiation and after neurites were grown resulted in dramatically reduced cytotoxicity compared to non-gelatinated TGA-capped CdTe QDs. Conclusion The toxicity mechanism of QDs was identified as caspase-mediated apoptosis as a result of cadmium leaking from the core of QDs. It was therefore concluded that the gelatine capping on the surface of QDs acts as a barrier towards the leaking of toxic ions from the core QDs in the long term (up to 17 days).

  14. Effects of long-term exposure of gelatinated and non-gelatinated cadmium telluride quantum dots on differentiated PC12 cells

    Prasad Babu R


    Full Text Available Abstract Background The inherent toxicity of unmodified Quantum Dots (QDs is a major hindrance to their use in biological applications. To make them more potent as neuroprosthetic and neurotherapeutic agents, thioglycolic acid (TGA capped CdTe QDs, were coated with a gelatine layer and investigated in this study with differentiated pheochromocytoma 12 (PC12 cells. The QD - cell interactions were investigated after incubation periods of up to 17 days by MTT and APOTOX-Glo Triplex assays along with using confocal microscopy. Results Long term exposure (up to 17 days to gelatinated TGA-capped CdTe QDs of PC12 cells in the course of differentiation and after neurites were grown resulted in dramatically reduced cytotoxicity compared to non-gelatinated TGA-capped CdTe QDs. Conclusion The toxicity mechanism of QDs was identified as caspase-mediated apoptosis as a result of cadmium leaking from the core of QDs. It was therefore concluded that the gelatine capping on the surface of QDs acts as a barrier towards the leaking of toxic ions from the core QDs in the long term (up to 17 days.

  15. Inhibition by anandamide of 6-hydroxydopamine-induced cell death in PC12 cells.

    Mnich, Katarzyna


    6-hydroxydopamine (6-OHDA) is a selective neurotoxin that is widely used to investigate cell death and protective strategies in models of Parkinson\\'s disease. Here, we investigated the effects of the endogenous cannabinoid, anandamide, on 6-OHDA-induced toxicity in rat adrenal phaeochromocytoma PC12 cells. Morphological analysis and caspase-3 activity assay revealed that anandamide inhibited 6-OHDA-induced apoptosis. The protection was not affected by antagonists of either cannabinoid receptors (CB(1) or CB(2)) or the vanilloid receptor TRPV1. Anandamide-dependent protection was reduced by pretreatment with LY294002 (inhibitor of phosphatidylinositol 3-kinase, PI3K) and unaffected by U0126 (inhibitor of extracellularly-regulated kinase). Interestingly, phosphorylation of c-Jun-NH2-terminal kinase (JNK) in cells exposed to 6-OHDA was strongly reduced by anandamide pre-treatment. Furthermore, 6-OHDA induced c-Jun activation and increased Bim expression, both of which were inhibited by anandamide. Together, these data demonstrate antiapoptotic effects of anandamide and also suggest a role for activation of PI3K and inhibition of JNK signalling in anandamide-mediated protection against 6-OHDA.

  16. Interventional effect of phycocyanin on mitochondrial membrane potential and activity of PC12 cells after hypoxia/reoxygenation

    Nan Jiang; Yunliang Guo; Hongbing Chen


    BACKGROUND: Phycocyanin can relieve decrease of mitochondrial membrane potential through reducing production of active oxygen so as to protect neurons after hypoxia/reoxygenation.OBJECTIVE: To observe the effect of phycocyanin on activity of PC12 cells and mitochondrial membrane potential after hypoxia/reoxygenation.DESIGN: Randomized controlled study.SETTING: Cerebrovascular Disease Institute of Affiliated Hospital, Medical College of Qingdao University.MATERIALS: The experiment was carried out at the Key Laboratory of Prevention and Cure for cerebropathia in Shandong Province from October to December 2005. PC12 cells, rat chromaffin tumor cells,were provided by Storage Center of Wuhan University; phycocyanin was provided by Ocean Institute of Academia Sinica; Thiazoyl blue tetrazolium bromide (MTT) and rhodamine 123 were purchased from Sigma Company, USA; RPMI-1640 medium, fetal bovine serum and equine serum were purchased from Gibco Company, USA.METHODS: ① Culture of PC12 cells: PC12 cells were put into RPMI-1640 medium which contained 100 g/L heat inactivation equine serum and 0.05 volume fraction of fetal bovine serum and incubated in CO2 incubator at 37 ℃. Number of cells was regulated to 4 × 105 L-1, and cells were inoculated at 96-well culture plate.The final volume was 100 μL. ② Model establishing and grouping: Cultured PC12 cells were randomly divided into three groups: phycocyanin group, model control group and non-hypoxia group. At 24 hours before hypoxia, culture solution in phycocyanin group was added with phycocyanin so as to make sure the final concentration of 3 g/L, but cells in model control group did not add with phycocyanin. Cells in non-hypoxia group were also randomly divided into adding phycocyanin group (the final concentration of 3 g/L) and non-adding phycocyanin group. Cells in model control group and phycocyanin group were cultured with hypoxia for 1 hour and reoxygenation for 1, 2 and 3 hours; meanwhile, cells in non

  17. Polysaccharides purified from Cordyceps cicadae protects PC12 cells against glutamate-induced oxidative damage.

    Olatunji, Opeyemi J; Feng, Yan; Olatunji, Oyenike O; Tang, Jian; Wei, Yuan; Ouyang, Zhen; Su, Zhaoliang


    Two polysaccharides CPA-1 and CPB-2 were isolated purified from Cordyceps cicadae by hot water extraction, ethanol precipitation and purification using anion exchange and gel filtration chromatography. Preliminary structural characterization of CPA-1 and CPB-2 were performed. The protective effect of CPA-1 and CPB-2 against glutamate-induced oxidative toxicity in PC12 cells was analyzed. The results indicated that pretreatment of PC12 cells with CPA-1 and CPB-2 significantly increased cell survival, Ca(2+) overload and ROS generation. CPA-1 and CPB-2 also markedly up-regulated the antioxidant status of pretreated PC12 cells. Our results suggested that Cordyceps cicadae polysaccharides can protect PC12 cells against glutamate excitotoxicity and might serve as therapeutic agents for neuronal disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Signal transduction pathway of nitric oxide inducing PC12 cell death


    Objective: To study signal transduction pathway of nitric oxideinducing death of PC12 cells.Methods: Cell survival rate was measured with MTT assay, and caspase-3 activity with caspase-3 assay kits after PC12 cells were incubated with sodium nitroprusside (SNP), caspase-3 inhibitor Ⅱ plus SNP or p38 inhibitor-SB203580 plus SNP.Results: SNP induced death of PC12 cells in dose- and time-dependent manner and enhanced caspase-3 activity gradually. Both caspase-3 inhibitor Ⅱ and SB203580 reduced cell death, but SB203580 reduced caspase-3 activity significantly.Conclusions: NO may induce death of PC12 cells through activation of p38 and caspase-3.

  19. Adenovirus-mediated transfection with glucose transporter 3 suppresses PC12 cell apoptosis following ischemic injury

    Junliang Li; Xinke Xu; Shanyi Zhang; Meiguang Zheng; Zhonghua Wu; Yinlun Weng; Leping Ouyang; Jian Yu; Fangcheng Li


    In this study, we investigated the effects of adenovirus-mediated transfection of PC12 cells with glucose transporter 3 after ischemic injury. The results of flow cytometry and TUNEL showed that exogenous glucose transporter 3 significantly suppressed PC12 cell apoptosis induced by ischemic injury. The results of isotopic scintiscan and western blot assays showed that, the glucose uptake rate was significantly increased and nuclear factor kappaB expression was significantly decreased after adenovirus-mediated transfection of ischemic PC12 cells with glucose transporter 3. These results suggest that adenovirus-mediated transfection of cells with glucose transporter 3 elevates the energy metabolism of PC12 cells with ischemic injury, and inhibits cell apoptosis.

  20. Baicalin inhibits colistin sulfate-induced apoptosis of PC12 cells******

    Hong Jiang; Pengfei Lv; Jichang Li; Hongjun Wang; Tiezhong Zhou; Yingzi Liu; Wei Lin


    Baicalin, a type of flavonoid extracted from the dried root of Scutel aria baicalensis georgi, has been shown to effectively inhibit cellapoptosis. Therefore, we assumed that baicalin would suppress colistin sulfate-induced neuronal apoptosis. PC12 cells exposed to colistin sulfate (62.5-500 μg/mL) for 24 hours resulted in PC12 cellapoptosis. In addition, caspase-3 activity, lactate dehydrogenase level and free radical content increased in a dose-dependent manner. Subsequently, PC12 cells were pretreated with baicalin (25, 50 and 100 μg/mL), and exposed to 125 μg/mL colistin sulfate. cellmorphology markedly changed, and cellviability increased. Moreover, caspase-3 activity, lac-tate dehydrogenase level and free radical content decreased. Results indicated that baicalin inhib-ited colistin sulfate-induced PC12 cellapoptosis by suppressing free radical injury, and reducing caspase-3 activity and lactate dehydrogenase activity.

  1. The protective effect of tetramethylpyrazine (TMP) against PC12 cells damages

    Xin-ruiCHENG; LanSUN; LiZHANG; Juan-juanHU; Guan-HuaDU


    AIM: To discover the protective effects of tetramethylpyrazine (TMP) against PC12 cells damages and explore its protective mechanisms. METHODS: We established three in vitro models to investigate the protective effects of TMP against the injuries. In both of glutamate and natrium azide-induced PC12 injuries, the action of TMP on the cell viability was measured by MTT assay. The LDH efflux was measured by the assay kit, production

  2. Serum containing Tongqiaohuoxue decoction suppresses glutamate-induced PC12 cell injury☆

    Wang, Ning; Deng, Yi; Wei, Wei; Song, Lihua; Wang, Yan


    Glutamate application is an established method of inducing PC12 cell injury. PC12 cells were cultured with serum containing Tongqiaohuoxue decoction consisting of moschus, Carthamus tinctorius, Rhizoma chuanxiong, Semen pruni persicae, and Radix Paeoniae Rubra. After 24 hours of co-cultivation, glutamate (12.5 mM) was added to the culture medium. We found that serum containing Tongqiaohuoxue decoction prevented the increase in reactive oxygen species, and the decreases in superoxide dismutase...

  3. Melatonin attenuates 1-methyl-4-phenylpyridinium-induced PC12 cell death

    Jin-feng BAO; Ren-gang WU; Xiao-ping ZHANG; Yan SONG; Chang-ling LI


    Aim: To explore the effect of melatonin on PC12 cell death induced by 1-methyl-4-phenylpyridinium (MPP+). Methods: MTT assay, lactate dehydrogenase (LDH)efflux assay, and immunohistochemistry methods were used to measure neurotoxicity of PC 12 cells treated acutely with MPP+ in low glucose and high glucose conditions, and to assess the neuroprotective effect of melatonin on PC 12 cell death induced by MPP+. Results: In a low glucose condition, MPP+ significantly induced PC 12 cell death, which showed time and concentration dependence. In a serum-free low glucose condition, the percentages of viability of cells treated with MPP+ for 12, 24, 48, 72, and 96 h were 85.1%, 75.4%, 64.9%, 28.15%, and 9%, respectively. The level of LDH in the culture medium increased and tyrosine hydroxylase positive (TH+) cell count decreased. However, in a serum-free high glucose condition, MPP+ did not significantly induce PC12 cell death compared with control at various concentrations and time regimens. When the cells were preincubated with melatonin 250 μmol/L for 48, 72, and 96 h in a serum-free low glucose condition, cell survival rate significantly increased to 78.1%, 58.8%, and 31.6%, respectively. Melatonin abolished the LDH leakage of cells treated with MPP+ and increased TH+ cells count. Conclusion: MPP+ caused concentrationdependent PC12 cell death. The level of glucose was an important factor to MPP+induced dopaminergic PC12 cell death. Low glucose level could potentiate MPP+toxicity, while high glucose level could reduce the toxicity. In addition, melatonin attenuated PC12 cell death induced by MPP+.

  4. Investigation of Apoptosis Induction in Differentiated PC-12 Cells after Exposure to Hydrostatic Pressure

    S. Sadri


    Full Text Available Objective: Hydrostatic pressure is crucial component of cell environment andfundamental physical quantity, also it is the main factor of both cell integrity andfunction. Pressure variation disorder, beyond physiological limits, may lead topathological states. In this study, we examined the effect of hydrostatic pressureon apoptosis induction, viability, morphology, adhesion potency to substrate andmigration of differentiated PC-12 cells.Materials and Methods: PC-12 as a neuronal cell line maintained in RPMI1640 culture medium supplemented with 10% fetal bovine serum. Staurosporinewas used for differentiating of mitotic PC-12 cells to post mitotic anddifferentiated neuronal cells. Exclusion Dye was used for viability assay, totalneurite length of each cell as well as morphometry. TUNEL staining was alsoperformed for apoptosis detection, adhesion potency of cells to substrate andevaluation of cell migration.Results: Hydrostatic pressure, over physiological limits, induced apoptosis indifferentiated PC-12 cells. It changed cell viability gradually and reduction happenedsignificantly after 24 hours (p<0.05. In compare to the control group, hydrostaticpressure reduced total neurite length, adhesion potency to substrate and migrationof cells in the examined group (p<0.05.Conclusion: Hydrostatic pressure induced apoptosis in differentiated PC-12 cellsas a result of inappropriate interaction between cells and substrate. We proposethat apoptosis in differentiated PC-12 cells may be an anoikis causing to lose theattachment to the substrate.

  5. Neurotoxic effect of rotenone on dopaminergic neuron PC12 in vitro

    SAI Yan; WU Qiang; LE Wei-dong; DONG Zhao-jun


    Objective: To investigate the mechanism of oxidative stress in rotenone neurotoxicity to dopaminergic neuron PC12. Methods: High differentiated PC12 cells as dopaminergic neurons were treated by different concentrations of rotenone. The morphology was observed with inverted phase contrast microscope and transmission electron microscope. Cell viability and proliferation inhibition were assessed by MTT. SOD and MDA were detected with biochemical assay. And the specific fluorescent probe (DCFDA) was used to examine ROS in PC12 cells. Results: After treated with rotenone for 24 h, most of the PC12 cells became smaller and rounder. The process of axon was reduced, shortened or broken in a time and concentration dependent manner. The mitochondrial structure and metabolism were changed. Endoplasmic reticulum expanded and the free ribosome increased. Compared with the control group, cell proliferation inhibition increased and cell viability decreased. SOD increased and MDA decreased. The intensity of fluorescence was more obvious in PC12 cells treated by rotenone compared with control group. Conclusion: Rotenone is neurotoxic to cultured dopaminergic neuron PC12. Rotenone might exert this effect through the metabolism of oxidative stress on the pathogenesis of the neuron.

  6. Analysis of the role of nerve growth factor in promoting cell survival during endoplasmic reticulum stress in PC12 cells.

    Shimoke, Koji; Sasaya, Harue; Ikeuchi, Toshihiko


    Nerve growth factor (NGF) was first described by Rita Levi-Montalcini in the early 1960s from her studies of peripheral neurons. It has since been reported that NGF has the potential to elongate neurites or to prevent apoptosis via specific intracellular mechanisms. It has further been reported that as a component of these mechanisms, NGF binds to a specific receptor, TrkA, and thereby contributes to peripheral nerve cell functions or neuronal functions. It is noteworthy in this regard that pheochromocytoma 12 (PC12) cells express TrkA and respond to neurite outgrowth or anti-apoptotic signals by binding to NGF. Hence, PC12 cells have been used as an in vitro model system for the study of neuronal functions. It has been reported that endoplasmic reticulum (ER) stress is involved in neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's disease. The common link with regard to ER stress is that the neuronal cells die in these pathologies via specific intracellular mechanisms. This type of cell death, if it is apoptotic in nature, is termed ER stress-mediated apoptosis. In the process of ER stress-mediated apoptosis, the cleavage of pro-caspase-12 residing on the ER and the expression of glucose-regulated protein 78 (GRP78) can be observed. The expression of GRP78 protein is a characteristic of an unfolded protein response (UPR) via specific signal transduction pathways mediated by the unfolded protein response element (UPRE) in the upstream region of the grp78 gene so on. In ER stress-mediated apoptosis, a caspase cascade is also observed. To further clarify the mechanisms underlying ER stress-mediated apoptosis, a better understanding of the UPR is therefore important. In our current study, we describe a method for detecting gene induction via the UPR, focusing on GRP78 and caspase activities as the measurement end-points. The information generated by our method will accelerate our understanding of the pathophysiological processes leading

  7. Preparation, characterization of nanoceria and its protection on oxidative damage of PC12 cells%CeO2的制备、表征及对 PC12细胞氧化损伤的保护作用

    刘纯青; 申林林; 栾艺; 刘娜


    Objective To prepare nanoceria ( CeO2 ) and to investigate the protective effect on oxidative damage of Pheo-chromocytoma cell (PC12).Methods Homogenous precipitation method was used to prepare the nanoceria.XRD and TEM were used to characterize and determine the size of CeO2 .250 mmol/L rough concentration of CeO2 was added into PC12 cell culture fluid.Cells were induced and damaged by 20 μmol/L H2 O2 .And oxidative stress damage model of PC12 cell was set up.The cell survival rate of control group, CeO2 +H2 O group and H2 O2 group was measured by MTT method, re-spectively.The antioxidative protection of CeO2 to the PC12 damaged by H2 O2 was evaluated.Results The size of nanocer-ia was 5-10 nm.According to the results of MTT method, it was shown that the cell survival rate of H2 O2 group was (74.61 ±3.97)%.And the cell survival rate of CeO2 +H2 O group was (99.21 ±4.01)%, which was significantly high-er than H2O2 group (P<0.05).Conclusion Nanoceria would have the protective effect on the PC12 which was oxidative damaged by H2 O2 .%目的:制备纳米氧化铈( nanoceria,CeO2),初步探讨其对肾上腺嗜铬细胞瘤细胞PC12氧化损伤的保护作用。方法采用均相沉淀法制备纳米CeO2,对其晶粒大小和晶粒形貌进行表征。将粗浓度为250 mmol/L的CeO2加入PC12细胞培养液,通过20μmol/L H2 O2诱导细胞损伤,建立PC12细胞的氧化应激损伤模型,采用MTT法检测对照组、CeO2组、CeO2+H2 O2组,H2 O2组的细胞活性,测定CeO2对PC12细胞的保护作用。结果采用均相沉淀的方法合成粒子直径为5~10 nm的CeO2。 MTT实验显示H2 O2组细胞存活率为(74.61±3.97)%,CeO2+H2O2组的细胞存活率为(99.21±4.01)%,明显高于H2O2组(P<0.05)。结论 CeO2对H2O2造成的氧化损伤细胞具有保护作用。

  8. Effects of Nogo-A Silencing on TNF-α and IL-6 Secretion and TH Downregulation in Lipopolysaccharide-Stimulated PC12 Cells

    Jianbin Zhong


    Full Text Available Parkinson’s disease (PD is a common degenerative disease that lacks efficient treatment. Myelin-associated neurite outgrowth inhibitor A (Nogo-A is relevant with inhibition of nerve regeneration and may play vital role in pathogenesis of PD. The study aimed to establish the shRNA expression plasmids of Nogo-A gene and explore the regulatory effects of Nogo-A silencing on the expression of inflammation factor tumor necrosis factor-alpha (TNF-alpha and interleukin-6 (IL-6 as well as tyrosine hydroxylase (TH in lipopolysaccharide- (LPS- stimulated rat PC12 cells. The results showed that both mRNA and protein levels of Nogo-A in pGenesil-nogoA-shRNA group were downregulated. The viabilities of PC12 cells decreased with increase of LPS concentrations. LPS significantly increased the supernatant TNF-alpha and IL-6 concentrations and reduced TH protein expression in PC12 cells, while silencing Nogo-A could block these effects. These results suggested that LPS can activate PC12 cells to secrete inflammatory cytokines and lower the TH expression, which can be regulated by Nogo-A gene silencing. Nogo-A silencing might provide new ideas for PD treatment in the future.

  9. Pheochromocytoma in Urologic Practice

    Waingankar, Nikhil; Bratslavsky, Gennady; Jimenez, Camilo; Russo, Paul; Kutikov, Alexander


    Context Pheochromocytoma is regularly encountered in urological practice and requires a thoughtful and careful clinical approach. Objective To review clinical aspects of management of pheochromocytoma in urologic practice. Evidence Acquisition A systematic review of English-language literature was performed through year 2015 using the Medline database. Manuscripts were selected with consensus of the coauthors and evaluated using the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) criteria. Evidence Synthesis Findings and recommendations of the evaluated manuscripts are discussed with an emphasis on the description of presentation, diagnosis, evaluation, and perioperative care. Conclusion In addition to surgical expertise, appropriate management of pheochromocytoma in urologic practice requires nuanced understanding of pathophysiology, genetics, and endocrinological principles. When skillfully managed, the vast majority of patients with pheochromocytoma should expect an excellent prognosis. Patient Summary In this article we review the clinical approach to patients with pheochromocytoma, a tumor that stems from the innermost part of the adrenal gland and that often secretes excessive amounts of powerful hormones such as noradrenaline and adrenaline. Significant expertise is required to appropriately manage patients with these tumors. Take Home Message In addition to surgical expertise, appropriate management of pheochromocytoma in urologic practice requires nuanced understanding of pathophysiology, genetics, and endocrinological principles. When skillfully managed, vast majority of patients with pheochromocytoma should expect an excellent prognosis. PMID:28078330

  10. Edaravone protects PC12 cells from ischemic-like injury via attenuating the damage to mitochondria

    SONG Ying; LI Meng; LI Ji-cheng; WEI Er-qing


    Background: Edaravone had been validated to effectively protect against ischemic injuries. In this study, we investigated the protective effect of edaravone by observing the effects on anti-apoptosis, regulation of Bcl-2/Bax protein expression and recovering from damage to mitochondria after OGD (oxygen-glucose deprivation)-reperfusion. Methods: Viability of PC 12cells which were injured at different time of OGD injury, was quantified by measuring MTT (2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) staining. In addition, PC 12 cells' viability was also quantified after their preincubation in different concentration of edaravone for 30 min followed by (OGD). Furthermore, apoptotic population of PC12 cells that reinsulted from OGD-reperfusion with or without preincubation with edaravone was determined by flow cytometer analysis,electron microscope and Hoechst/PI staining. Finally, change of Bcl-2/Bax protein expression was detected by Westem blot.Results: (1) The viability of PC 12 cells decreased with time (1~12 h) after OGD. We regarded the model of OGD 2 h, then replacing DMEM (Dulbecco's Modified Eagle's Medium) for another 24 h as an OGD-reperfusion in this research. Furthermore,most PC12 cells were in the state of apoptosis after OGD-reperfusion. (2) The viability of PC12 cells preincubated with edaravone at high concentrations (1,0.1, 0.01 μmol/L) increased significantly with edaravone protecting PC 12 cells from apoptosis after OGD-reperfusion injury. (3) Furthermore, edaravone attenuates the damage of OGD-reperfusion on mitochondria and regulated Bcl-2/Bax protein imbalance expression after OGD-reperfusion. Conclusion: Neuroprotective effects of edaravone on ischemic or other brain injuries may be partly mediated through inhibition of Bcl-2/Bax apoptotic pathways by recovering from the damage of mitochondria.

  11. Assessment of the neurotoxic potential of exposure to 50Hz extremely low frequency electromagnetic fields (ELF-EMF) in naïve and chemically stressed PC12 cells.

    de Groot, Martje W G D M; Kock, Marjolijn D M; Westerink, Remco H S


    Increasing exposure to extremely low frequency electromagnetic fields (ELF-EMF), generated by power lines and electric appliances, raises concern about potential adverse health effects of ELF-EMF. The central nervous system is expected to be particularly vulnerable to ELF-EMF as its function strongly depends on electrical excitability. We therefore investigated effects of acute (30min) and sub-chronic (48h) exposure to 50Hz ELF-EMF on naïve and chemically stressed pheochromocytoma (PC12) cells. The latter have higher levels of iron and/or reactive oxygen species (ROS) and display increased vulnerability to environmental insults. Effects of ELF-EMF on Ca(2+)-homeostasis, ROS production and membrane integrity were assessed using Fura-2 single cell fluorescence microscopy, H2-DCFDA and CFDA assays, respectively. Our data demonstrate that acute exposure of naïve PC12 cells to 50Hz ELF-EMF up to 1000μT fails to affect basal or depolarization-evoked [Ca(2+)]i. Moreover, sub-chronic ELF-EMF exposure up to 1000μT has no consistent effects on Ca(2+)-homeostasis in naïve PC12 cells and does not affect ROS production and membrane integrity. Notably, in chemically stressed PC12 cells both acute and sub-chronic ELF-EMF exposure also failed to exert consistent effects on Ca(2+)-homeostasis, ROS production and membrane integrity. Our combined findings thus indicate that exposure to 50Hz ELF-EMF up to 1000μT, i.e. 10,000 times above background exposure, does not induce neurotoxic effects in vitro, neither in naïve nor in chemically stressed PC12 cells. Though our data require confirmation, e.g. in developing neuronal cells in vitro or (developing) animals, it appears that the neurotoxic risk of ELF-EMF exposure is limited.

  12. Proteases inhibition assessment on PC12 and NGF treated cells after oxygen and glucose deprivation reveals a distinct role for aspartyl proteases.

    Aristidis Kritis

    Full Text Available Hypoxia is a severe stressful condition and induces cell death leading to neuronal loss both to the developing and adult nervous system. Central theme to cellular death is the activation of different classes of proteases such as caspases calpains and cathepsins. In the present study we investigated the involvement of these proteases, in the hypoxia-induced PC12 cell death. Rat PC12 is a model cell line for experimentation relevant to the nervous system and several protocols have been developed for either lethal hypoxia (oxygen and glucose deprivation OGD or ischemic preconditioning (IPS. Nerve Growth Factor (NGF treated PC12 differentiate to a sympathetic phenotype, expressing neurites and excitability. Lethal hypoxia was established by exposing undifferentiated and NGF-treated PC12 cells to a mixture of N(2/CO(2 (93:5% in DMEM depleted of glucose and sodium pyruvate for 16 h. The involvement of caspases, calpains and lysosomal cathepsins D and E to the cell death induced by lethal OGD was investigated employing protease specific inhibitors such as z-VAD-fmk for the caspases, MDL28170 for the calpains and pepstatin A for the cathepsins D and E. Our findings show that pepstatin A provides statistically significant protection from cell death of both naive and NGF treated PC12 cells exposed to lethal OGD. We propose that apart from the established processes of apoptosis and necrosis that are integral components of lethal OGD, the activation of cathepsins D and E launches additional cell death pathways in which these proteases are key partners.



    Objective To investigate integration and expression of adeno-associated virus (AAV) vectors in neuronal PC12 cells,the result of which can be applied in further gene therapy of diseases of the central nervous system. Methods Human neurotrophin-3(hNT3)genes were inserted into AAV vectors. Then the recombinat AAV plasmids were encapsidated as recombinant virions. PC12 cells were transfected with the virions and the positive cells were selected by G418. The transfection positive (hNT3 modified)PC12 cells were cultured for several generations and the cellular genomic DNA and total RNA were extracted. We investigated the integration locus of AAV vectors by Southern blot and transcript situation of foreign genes by dot blot. Results The hybridization tests showed that AAV introduced foreign genes were stably integrated, but at random locus, and robustly transcribed in hNT3 modified PC12cells. Conclusion AAV vectors can serve as high efficiency vectors of target genes in neuronal PC12 cells.

  14. Intracellular dopamine oxidation mediates rotenone-induced apoptosis in PC12 cells

    Hua-qing LIU; Xing-zu ZHU; En-qi WENG


    Aim: To study the role of dopamine (DA) in rotenone-induced neurotoxicity in PC12 cells. Methods: Cell viability was assessed by detecting the leakage of lactate dehydrogenase (LDH) into the medium. Apoptosis rate was measured by flow cytometry. Caspase-3-1ike activity was measured by fluorescence assay using the probe Ac-DEVD-AMC. The level of intracellular hydrogen peroxide and other peroxides in PC12 cells were quantified by loading cells with 2'-7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) in fluorescence assay. Lactic acid was measured spectrophotometrically. The DA levels in PC12 cells were determined by HPLC-ECD. Results: A 48-h incubation of PC12 cells with rotenone caused an apoptotic cell death and elevated intracellular reactive oxygen species (ROS) and lactic acid accumulation. Intracellular DA depletion with reserpine significantly attenuated rotenone-induced ROS accumulation and apoptotic cell death. No change was found in rotenone-induced ROS accumulation when cells were co-treated with deprenyl. Brief treatment with reserpine at the end of rotenone treatment had no effect on rotenone-induced neurotoxicity. However,when cells were first incubated with deprenyl, a monoamine oxidase-B inhibitor for 30 min then co-incubated with rotenone plus deprenyl, a brief treatment with reserpine enhanced cell injury. Conclusion: Rotenone-induced apoptosis in PC 12 cells was mediated by intracellular dopamine oxidation.

  15. Rho family GTPase Chp/RhoV induces PC12 apoptotic cell death via JNK activation.

    Shepelev, Mikhail V; Chernoff, Jonathan; Korobko, Igor V


    Rho GTPases regulate numerous cellular processes including apoptosis. Chp/RhoV is an atypical Rho GTPase which functions are poorly understood. Here we investigated the role of Chp in regulation of cell viability using PC12 cells with inducible expression of Chp as a model. We found that expression of Chp results in apoptosis in PC12 cells. Chp-induced apoptosis was accompanied by activation of JNK signaling and both death receptor-mediated and mitochondrial apoptotic pathways as justified by caspase-8 and caspase-9 activation, respectively. Moreover, inhibition of JNK by SP600125 rescued PC12 cells from Chp-triggered cell death and attenuated activation of caspases-9 and -3/7 suggesting that activation of JNK mediates pro-apoptotic effect of Chp. Expression of Chp resulted in increased phosphorylation of c-Jun in PC12 cells, and Chp expression in HE K293 cells upregulated AP-1-dependent transcription in a JNK-dependent manner. Together results of our study reveal the role of Chp GTPase as a putative regulator of JNK-dependent apoptotic death in PC12 cells, similarly to previously described pro-apoptotic activity of the related Cdc42 and Rac1 GTPases.

  16. Daidzin protects PC12 cells from serum deprivation-induced apoptosis.

    Ji, Zhao-Ning; Liu, Guo-Qing


    This article examines the effect of daidzin on PC12 cell apoptosis induced by serum-free medium. PC12 cell survival was measured by MTT assay. The DNA content and percentage of apoptosis were monitored by flow cytometry and DNA fragmentation was analyzed by agarose gel electrophoresis. The results showed that serum-free (12 h) medium induced apoptosis in PC12 cells. When the cells had been treated with daidzin (0.1, 1 microM) for 12 h, the percentage of PC12 cell apoptosis was significantly decreased to 12.21 and 4.24% from 91.94% in the group with serum deprivation, and DNA fragmentation was prevented. Daidzin (0.01-10 microM) attenuated the cytotoxic effect of sodium cyanide (20 mM), glutamate (0.5 mM) and sodium nitroprusside (0.5 mM) in a manner dependent on concentration. The results suggested that daidzin prevented PC12 cell from serum free-induced apoptosis.

  17. Protective effects of Gynostemma pentaphyllum polysaccharides on PC12 cells impaired by MPP(+).

    Deng, Qi; Yang, Xinling


    The aim of the present study was to explore the neuroprotective effects of Gynostemma pentaphyllum polysaccharides (GP) in a 1-methyl-4-phenylpyridiniumion (MPP(+))-induced cellular model of Parkinson's disease (PD) and the underlying mechanisms. Our results indicated that exposure of PC12 cells to 1mM MPP(+) significantly decreased the cell viability when examined by MTT assay, LDH assay, and annexin-V-FITC and propidium iodide (PI) apoptosis detection assays. MPP(+)-induced apoptosis in PC12 cells was accompanied by an increased Bax/Bcl-2 ratio, release of mitochondrial cytochrome c into the cytosol, activation of caspase-3/9 and cleavage of poly (ADP-ribose) polymerase (PARP). However, pretreatment of PC12 cells with 50μg/ml GP prior to MPP(+) exposure effectively attenuated the cytotoxicity and improved cell viability via inhibiting elevated Bax/Bcl-2 ratio, as well as the release of cytosolic cytochrome c. Furthermore, GP was effective in attenuating caspase-3/9 activation and cleavage of PARP in MPP(+)-exposed PC12 cells. These results suggest that the GP has protective effects against MPP(+)-induced neuronal apoptosis in PC12 cells by suppressing apoptosis-related protein, and therefore, might likely be a promising candidate for the treatment of Parkinson's disease (PD). Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Phase II enzyme induction by a carotenoid, lutein, in a PC12D neuronal cell line

    Miyake, Seiji [Laboratory of Retinal Cell Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Wakasa Seikatsu Co., Ltd., 134 Chudoujiminami-cho, Shimogyo-ku, Kyoto 600-8813 (Japan); Kobayashi, Saori [Wakasa Seikatsu Co., Ltd., 134 Chudoujiminami-cho, Shimogyo-ku, Kyoto 600-8813 (Japan); Tsubota, Kazuo [Laboratory of Retinal Cell Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Ozawa, Yoko, E-mail: [Laboratory of Retinal Cell Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan)


    Highlights: • Lutein reduced ROS levels in a PC12D neuronal cell line. • Lutein induced mRNAs of phase II antioxidative enzymes in PC12D neuronal cells. • Lutein increased protein levels of HO-1, SOD2, and NQO-1 in PC12D neuronal cells. • Lutein had no effect on intranuclear Nrf2 levels in PC12D neuronal cells. • Lutein did not activate potential upstream Nrf2 nuclear translocation pathways. - Abstract: The mechanism by which lutein, a carotenoid, acts as an antioxidant in retinal cells is still not fully understood. Here, lutein treatment of a neuronal cell line (PC12D) immediately resulted in reduced intracellular ROS levels, implying that it has a direct role in ROS scavenging. Significantly, lutein treatment also induced phase II antioxidative enzyme expression, probably via a nuclear factor-like 2 (Nrf2) independent pathway. This latter mechanism could explain why lutein acts diversely to protect against oxidative/cytotoxic stress, and why it is physiologically involved in the human neural tissue, such as the retina.

  19. Neurite outgrowth resistance to rho kinase inhibitors in PC12 Adh cell.

    Yin, Hua; Hou, Xiaolin; Tao, Tingrui; Lv, Xiaoman; Zhang, Luyong; Duan, Weigang


    Rho kinase (ROCK) inhibitor is a promising agent for neural injury disorders, which mechanism is associated with neurite outgrowth. However, neurite outgrowth resistance occurred when PC12 Adh cell was treated with ROCK inhibitors for a longer time. PC12 Adh cells were treated with ROCK inhibitor Y27632 or NGF for different durations. Neurite outgrowth resistance occurred when PC12 Adh cell exposed to Y27632 (33 µM) for 3 or more days, but not happen when exposed to nerve growth factor (NGF, 100 ng/mL). The gene expression in the PC12 Adh cells treated with Y27632 (33 µM) or NGF (100 ng/mL) for 2 or 4 days was assayed by gene microarray, and the reliability of the results were confirmed by real-time RT-PCR. Cluster analysis proved that the gene expression profile of PC12 Adh cell treated with Y27632 for 4 days was different from that treated with Y27632 for 2 days and those treated with NGF for 2 and 4 days, respectively. Pathway analysis hinted that the neurite outgrowth resistance could be associated with up-regulation of inflammatory pathways, especially rno04610 (complement and coagulation cascades), and down-regulation of cell cycle pathways, especially rno04110.

  20. NGF-mediated transcriptional targets of p53 in PC12 neuronal differentiation

    Labhart Paul


    Full Text Available Abstract Background p53 is recognized as a critical regulator of the cell cycle and apoptosis. Mounting evidence also suggests a role for p53 in differentiation of cells including neuronal precursors. We studied the transcriptional role of p53 during nerve growth factor-induced differentiation of the PC12 line into neuron-like cells. We hypothesized that p53 contributed to PC12 differentiation through the regulation of gene targets distinct from its known transcriptional targets for apoptosis or DNA repair. Results Using a genome-wide chromatin immunoprecipitation cloning technique, we identified and validated 14 novel p53-regulated genes following NGF treatment. The data show p53 protein was transcriptionally activated and contributed to NGF-mediated neurite outgrowth during differentiation of PC12 cells. Furthermore, we describe stimulus-specific regulation of a subset of these target genes by p53. The most salient differentiation-relevant target genes included wnt7b involved in dendritic extension and the tfcp2l4/grhl3 grainyhead homolog implicated in ectodermal development. Additional targets included brk, sdk2, sesn3, txnl2, dusp5, pon3, lect1, pkcbpb15 and other genes. Conclusion Within the PC12 neuronal context, putative p53-occupied genomic loci spanned the entire Rattus norvegicus genome upon NGF treatment. We conclude that receptor-mediated p53 transcriptional activity is involved in PC12 differentiation and may suggest a contributory role for p53 in neuronal development.

  1. Long-term exposure of CdTe quantum dots on PC12 cellular activity and the determination of optimum non-toxic concentrations for biological use

    Gérard Valérie A


    Full Text Available Abstract Background The unique and tuneable photonic properties of Quantum Dots (QDs have made them potentially useful tools for imaging biological entities. However, QDs though attractive diagnostic and therapeutic tools, have a major disadvantage due to their inherent cytotoxic nature. The cellular interaction, uptake and resultant toxic influence of CdTe QDs (gelatinised and non-gelatinised Thioglycolic acid (TGA capped have been investigated with pheochromocytoma 12 (PC12 cells. In conjunction to their analysis by confocal microscopy, the QD - cell interplay was explored as the QD concentrations were varied over extended (up to 72 hours co-incubation times. Coupled to this investigation, cell viability, DNA quantification and cell proliferation assays were also performed to compare and contrast the various factors leading to cell stress and ultimately death. Results Thioglycolic acid (TGA stabilised CdTe QDs (gel and non - gel were co-incubated with PC12 cells and investigated as to how their presence influenced cell behaviour and function. Cell morphology was analysed as the QD concentrations were varied over co-incubations up to 72 hours. The QDs were found to be excellent fluorophores, illuminating the cytoplasm of the cells and no deleterious effects were witnessed at concentrations of ~10-9 M. Three assays were utilised to probe how individual cell functions (viability, DNA quantification and proliferation were affected by the presence of the QDs at various concentrations and incubation times. Cell response was found to not only be concentration dependant but also influenced by the surface environment of the QDs. Gelatine capping on the surface acts as a barrier towards the leaking of toxic atoms, thus reducing the negative impact of the QDs. Conclusion This study has shown that under the correct conditions, QDs can be routinely used for the imaging of PC12 cells with minimal adverse effects. We have found that PC12 cells are highly

  2. Long-term exposure of CdTe quantum dots on PC12 cellular activity and the determination of optimum non-toxic concentrations for biological use

    Prasad, Babu R


    Abstract Background The unique and tuneable photonic properties of Quantum Dots (QDs) have made them potentially useful tools for imaging biological entities. However, QDs though attractive diagnostic and therapeutic tools, have a major disadvantage due to their inherent cytotoxic nature. The cellular interaction, uptake and resultant toxic influence of CdTe QDs (gelatinised and non-gelatinised Thioglycolic acid (TGA) capped) have been investigated with pheochromocytoma 12 (PC12) cells. In conjunction to their analysis by confocal microscopy, the QD - cell interplay was explored as the QD concentrations were varied over extended (up to 72 hours) co-incubation times. Coupled to this investigation, cell viability, DNA quantification and cell proliferation assays were also performed to compare and contrast the various factors leading to cell stress and ultimately death. Results Thioglycolic acid (TGA) stabilised CdTe QDs (gel and non - gel) were co-incubated with PC12 cells and investigated as to how their presence influenced cell behaviour and function. Cell morphology was analysed as the QD concentrations were varied over co-incubations up to 72 hours. The QDs were found to be excellent fluorophores, illuminating the cytoplasm of the cells and no deleterious effects were witnessed at concentrations of ~10-9 M. Three assays were utilised to probe how individual cell functions (viability, DNA quantification and proliferation) were affected by the presence of the QDs at various concentrations and incubation times. Cell response was found to not only be concentration dependant but also influenced by the surface environment of the QDs. Gelatine capping on the surface acts as a barrier towards the leaking of toxic atoms, thus reducing the negative impact of the QDs. Conclusion This study has shown that under the correct conditions, QDs can be routinely used for the imaging of PC12 cells with minimal adverse effects. We have found that PC12 cells are highly susceptible to

  3. Familial Pheochromocytomas and Paragangliomas

    King, Kathryn S.; Pacak, Karel


    Pheochromocytomas and paragangliomas are neural crest cell tumors of the adrenal medulla and parasympathetic/sympathetic ganglia, respectively, that are often associated with catecholamine production. Genetic research over the years has led to our current understanding of the association 13 susceptibility genes with the development of these tumors. Most of the susceptibility genes are now associated with specific clinical presentations, biochemical makeup, tumor location, and associated neoplasms. Recent scientific advances have highlighted the role of somatic mutations in the development of pheochromocytoma/paraganglioma as well as the usefulness of immunohistochemistry in triaging genetic testing. We can now approach genetic testing in pheochromocytoma/paraganglioma patients in a very organized scientific way allowing for the reduction of both the financial and emotional burden on the patient. The discovery of genetic predispositions to the development of pheochromocytoma/paraganglioma not only facilitates better understanding of these tumors but will also lead to improved diagnosis and treatment of this disease. PMID:23933153

  4. Reactive oxygen species regulated mitochondria-mediated apoptosis in PC12 cells exposed to chlorpyrifos

    Lee, Jeong Eun [Department of Pharmacology, College of Medicine, Hanyang University, Seoul (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Park, Jae Hyeon [Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of); Shin, In Chul [Department of Pharmacology, College of Medicine, Hanyang University, Seoul (Korea, Republic of); Koh, Hyun Chul, E-mail: [Department of Pharmacology, College of Medicine, Hanyang University, Seoul (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of)


    Reactive oxidative species (ROS) generated by environmental toxicants including pesticides could be one of the factors underlying the neuronal cell damage in neurodegenerative diseases. In this study we found that chlorpyrifos (CPF) induced apoptosis in dopaminergic neuronal components of PC12 cells as demonstrated by the activation of caspases and nuclear condensation. Furthermore, CPF also reduced the tyrosine hydroxylase-positive immunoreactivity in substantia nigra of the rat. In addition, CPF induced inhibition of mitochondrial complex I activity. Importantly, N-acetyl cysteine (NAC) treatment effectively blocked apoptosis via the caspase-9 and caspase-3 pathways while NAC attenuated the inhibition of mitochondrial complex I activity as well as the oxidative metabolism of dopamine (DA). These results demonstrated that CPF-induced apoptosis was involved in mitochondrial dysfunction through the production of ROS. In the response of cellular antioxidant systems to CPF, we found that CPF treatment increased HO-1 expression while the expression of CuZnSOD and MnSOD was reduced. In addition, we found that CPF treatment activated MAPK pathways, including ERK 1/2, the JNK, and the p38 MAP kinase in a time-dependent manner. NAC treatment abolished MAPK phosphorylation caused by CPF, indicating that ROS are upstream signals of MAPK. Interestingly, MAPK inhibitors abolished cytotoxicity and reduced ROS generation by CPF treatment. Our results demonstrate that CPF induced neuronal cell death in part through MAPK activation via ROS generation, suggesting its potential to generate oxidative stress via mitochondrial damage and its involvement in oxidative stress-related neurodegenerative disease. -- Highlights: ► Chlorpyrifos induces apoptosis. ► Chlorpyrifos inhibits mitochondrial complex I activity. ► ROS is involved in chlorpyrifos-induced apoptosis. ► Chlorpyrifos affects cellular antioxidant systems. ► Chlorpyrifos-induced apoptosis mediates activation of MAPK.

  5. Effect of formaldehyde on endogenous hydrogen sulfide production in PC12 cells%甲醛对PC12细胞内源性H2S生成的影响

    尹蔚兰; 何剑琴; 方恒荣; 杨丝丝; 胡弼; 唐小卿


    目的 观察甲醛对PC12细胞内源性硫化氢(hydrogen sulfide,H2S)生成的影响,以探讨甲醛损伤PC12细胞的新机制.方法 RT-PCR方法检测PC12细胞的胱硫醚-β-合酶 (cystathionine-beta-synthase,CBS) mRNA 的表达;亚甲基蓝分光光度计法检测PC12细胞CBS活性及PC12细胞内源性H2S的含量;MTT法观察PC12细胞的存活率.结果 甲醛可以抑制PC12细胞CBS的表达及其活性,减少内源性H2S的生成;甲醛可以明显地降低PC12细胞的存活率.结论 甲醛能抑制CBS的表达和活性,减少内源性H2S生成,这可能与其损伤PC12细胞有关.%Aim To observe the effect of formaldehyde on the production of endogenous hydrogen sulfide ( H2S ) in PC12 cells and to explore the novel molecular mechanisms of formaldehyde-induced damage in PC12 cells. Methods The expression of cystathionine-beta-synthase ( CBS ) mRNA in PC12 cells was determined by RT-PCR. The production of H2S and the activity of CBS in PC12 cells were detected by methylene blue spectrophotometric method. The survival of PC12 cells was observed by MTT assay. Results Formaldehyde inhibited the expression of CBS mRNA and the activity of CBS, and decreased the production of endogenous H2S in PC12 cells. Formaldehyde significantly reduced the survival rate of PC12 cells. Conclusion Formaldehyde inhibits the expression and activity of CBS, as well as endogenous H2S production in PC12 cells, which may be associated with formaldehyde-induced PC12 cells damage.

  6. Effect of 1-methyl, 4-phenyl pyridium on endogenous hydrogen sulfide production in PC12 cells%MPP+对PC12细胞内源性H2S生成的影响

    唐小卿; 范黎黎; 杨春涛; 申新田; 赵静; 李玉娟; 郭瑞鲜; 冯鉴强


    目的:观察1-甲基4-苯基吡啶离子 (MPP+)对PC12细胞内源性硫化氢(H2S)生成的影响,以探讨MPP+损伤PC12细胞的新机制.方法:RT-PCR方法检测PC12细胞胱硫醚-β-合酶(CBS)mRNA 的表达;亚甲基蓝分光光度计法检测PC12细胞内源性H2S的含量及PC12细胞CBS活性;台盼蓝拒染色法观察PC12细胞的存活率.结果:MPP+ 可以抑制PC12细胞CBS的表达及其活性,减少内源性H2S的生成;MPP+可以明显地降低PC12细胞的存活率;H2S的供体硫氢化钠(NaHS)对MPP+诱导的PC12细胞损伤具有显著的拮抗作用.结论:MPP+能抑制CBS的表达和活性,减少内源性H2S生成,这可能与其损伤PC12细胞的机制有关.

  7. Minocycline protects the apoptosis of PC12 cells induced by 1-methyl-4-phenylpyridinium


    Objective: To explore the protective effect of minocycline on the apoptosis of cellular parkinsonism models induced by MPP+ . Methods: Using PC12 cells as the apoptotic model of dopaminergic neurons, MC and MPP+ were added into the culture medium of PC12 cells, and using MTT to assay the cell viability and metabolic state; The cells apoptosis was assayed by electrophoresis method and using flow cytometry FACS to assay the apoptosis ratio. Results: Added the MPP+ to get the concentration of 10μmol/L, the cellular parkinsonism model of apoptosis had been prepared. The pre-treatment of MC (100 μmol/L) could significantly increase the PC12 cell viability. The apoptosis ratio of MC + MPP+ group was significantly lower than that of MPP+ group, but was still significantly higher than that of control group. Conclusion: MC may protect the cell apoptosis induced by MPP+ to some extent.

  8. Serum containing Tongqiaohuoxue decoction suppresses glutamate-induced PC12 cell injury.

    Wang, Ning; Deng, Yi; Wei, Wei; Song, Lihua; Wang, Yan


    Glutamate application is an established method of inducing PC12 cell injury. PC12 cells were cultured with serum containing Tongqiaohuoxue decoction consisting of moschus, Carthamus tinctorius, Rhizoma chuanxiong, Semen pruni persicae, and Radix Paeoniae Rubra. After 24 hours of co-cultivation, glutamate (12.5 mM) was added to the culture medium. We found that serum containing Tongqiaohuoxue decoction prevented the increase in reactive oxygen species, and the decreases in superoxide dismutase and Na(+)-K(+)-ATPase activity, induced by glutamate. It also reduced the concentration of malondialdehyde, enhanced the mitochondrial transmembrane potential, inhibited the elevation of cellular calcium, and decreased phosphorylation of calmodulin-dependent protein kinase II. Thus, serum containing Tongqiaohuoxue decoction had protective effects on cell proliferation and membrane permeability in glutamate-injured PC12 cells.

  9. Serum containing Tongqiaohuoxue decoction suppresses glutamate-induced PC12 cell injury

    Ning Wang; Yi Deng; Wei Wei; Lihua Song; Yan Wang


    Glutamate application is an established method of inducing PC12 cell injury. PC12 cells were cultured with serum containing Tongqiaohuoxue decoction consisting of moschus, Carthamus tinctorius, Rhizoma chuanxiong, Semen pruni persicae, and Radix Paeoniae Rubra. After 24 hours of co-cultivation, glutamate (12.5 mM) was added to the culture medium. We found that serum containing Tongqiaohuoxue decoction prevented the increase in reactive oxygen species, and the decreases in superoxide dismutase and Na+-K+-ATPase activity, induced by glutamate. It also reduced the concentration of malondialdehyde, enhanced the mitochondrial transmembrane potential, inhibited the elevation of cellular calcium, and decreased phosphorylation of calmodulin-dependent protein kinase II. Thus, serum containing Tongqiaohuoxue decoction had protective effects on cell proliferation and membrane permeability in glutamate-injured PC12 cells.

  10. Protective effects of Ginkgo biloba extract on 6-hydroxydopamine-induced apoptosis in PC12 cells

    Jie Wang; Yanbo Cheng; Jiale Yin; Qian Lu; Xingshun Xu; Xiaoxing Yin


    The present study analyzed the protective effects of Ginkgo biloba extract against 6-hydroxydopamine-induced PC12 cell apoptosis in a model of Parkinson's disease. The results showed that Ginkgo biloba extract had a potent cytoprotective action and inhibited apoptosis of PC12 cells induced by 6-hydroxydopamine. Ginkgo biloba extract decreased the ratio of Bax to Bcl-2 and markedly inhibited the activation of p53 and caspase-3. These experimental findings indicate that Ginkgo biloba extract may significantly reduce the effects of oxidative stress induced by 6-hydroxydopamine in PC12 cells and suppress cell apoptosis. The potential effects of Ginkgo biloba extract might be greater than those of levodopa in the treatment of Parkinson's disease.

  11. Cytoprotective effects of fisetin against hypoxia-induced cell death in PC12 cells.

    Chen, Pei-Yi; Ho, Yi-Ru; Wu, Ming-Jiuan; Huang, Shun-Ping; Chen, Po-Kong; Tai, Mi-Hsueh; Ho, Chi-Tang; Yen, Jui-Hung


    Fisetin (3,7,3',4'-tetrahydroxyflavone), a flavonol compound of flavonoids, exhibits a broad spectrum of biological activities including anti-oxidant, anti-inflammatory, anti-cancer and neuroprotective effects. The aim of this study is to investigate the cytoprotective effect of fisetin and the underlying molecular mechanism against hypoxia-induced cell death in PC12 cells. The results of this study showed that fisetin significantly restored the cell viability of PC12 cells under both cobalt chloride (CoCl₂)- and low oxygen-induced hypoxic conditions. Treatment with fisetin successfully reduced the CoCl₂-mediated reactive oxygen species (ROS) production, which was accompanied by an increase in the cell viability of PC12 cells. Furthermore, we found that treatment of PC12 cells with fisetin markedly upregulated hypoxia-inducible factor 1α (HIF-1α), its nuclear accumulation and the hypoxia-response element (HRE)-driven transcriptional activation. The fisetin-mediated cytoprotection during CoCl₂ exposure was significantly attenuated through the administration of HIF-1α siRNA. Moreover, we demonstrated that MAPK/ERK kinase 1/2 (MEK1/2), p38 MAPK and phosphatidylinositol 3-kinase (PI3 K) inhibitors significantly blocked the increase in cell survival that was induced by fisetin treatment under hypoxic conditions. Consistently, increased phosphorylation of ERK, p38 and Akt proteins was observed in PC12 cells treated with fisetin. However, the fisetin-induced HRE-driven transcription was not affected by inhibition of these kinase signaling pathways. Current results reveal for the first time that fisetin promotes cell survival and protects against hypoxia-induced cell death through ROS scavenging and the activation of HIF1α-, MAPK/ERK-, p38 MAPK- and PI3 K/Akt-dependent signaling pathways in PC12 cells.

  12. DA-9801 promotes neurite outgrowth via ERK1/2-CREB pathway in PC12 cells.

    Won, Jong Hoon; Ahn, Kyong Hoon; Back, Moon Jung; Ha, Hae Chan; Jang, Ji Min; Kim, Ha Hyung; Choi, Sang-Zin; Son, Miwon; Kim, Dae Kyong


    In the present study, we examined the mechanisms underlying the effect of DA-9801 on neurite outgrowth. We found that DA-9801 elicits its effects via the mitogen-activated protein kinase (MEK) extracellular signal-regulated kinase (ERK)1/2-cAMP response element-binding protein (CREB) pathway. DA-9801, an extract from a mixture of Dioscorea japonica and Dioscorea nipponica, was reported to promote neurite outgrowth in PC12 cells. The effects of DA-9801 on cell viability and expression of neuronal markers were evaluated in PC12 cells. To investigate DA-9801 action, specific inhibitors targeting the ERK signaling cascade were used. No cytotoxicity was observed in PC12 cells at DA-9801 concentrations of less than 30 µg/mL. In the presence of nerve growth factor (NGF, 2 ng/mL), DA-9801 promoted neurite outgrowth and increased the relative mRNA levels of neurofilament-L (NF-L), a marker of neuronal differentiation. The Raf-1 inhibitor GW5074 and MEK inhibitor PD98059 significantly attenuated DA-9801-induced neurite outgrowth. Additionally, the MEK1 and MEK2 inhibitor SL327 significantly attenuated the increase in the percentage of neurite-bearing PC12 cells induced by DA-9801 treatment. Conversely, the selective p38 mitogen-activated protein kinase inhibitor SB203580 did not attenuate the DA-9801 treatment-induced increase in the percentage of neurite-bearing PC12 cells. DA-9801 enhanced the phosphorylation of ERK1/2 and CREB in PC12 cells incubated with and without NGF. Pretreatment with PD98059 blocked the DA-9801-induced phosphorylation of ERK1/2 and CREB. In conclusion, DA-9801 induces neurite outgrowth by affecting the ERK1/2-CREB signaling pathway. Insights into the mechanism underlying this effect of DA-9801 may suggest novel potential strategies for the treatment of peripheral neuropathy.

  13. 靶向微管解聚蛋白stathmin siRNA对Aβ1-42诱发PC12细胞损伤的保护作用%Protective effect of stathmin siRNA on Aβ1-42 induced injury in PC12 cells

    林芳; 高萍; 刘昕阳; 和婷; 张惠中


    目的:探讨微管解聚蛋白stathmin siRNA对β淀粉样蛋白片段(Aβ1-42)诱导PC12细胞损伤的保护作用.方法:构建靶向stathmin基因的shRNA真核表达载体;然后用脂质体转染的方法将重组质粒转染至大鼠嗜铬细胞瘤PC12细胞,用G418筛选得到稳定转染的细胞;通过RT-PCR和Western印迹方法分析stathmin基因mRNA及蛋白表达水平,筛选表达最低的稳定细胞株.用不同质量浓度的Aβ1-42诱导稳定细胞株,用MTT试验观察筛选稳定细胞的增殖活性,流式细胞仪检测细胞凋亡情况.结果:与对照组比较,转染了重组载体pSilencer4.1-s的细胞中stathmin核酸和蛋白水平的表达均显著下调.与Aβ1-42处理的PC12细胞(63.88±2.36)和PC12-parental细胞(65.78±5.74)比较,Aβ1-42处理的PC12-s细胞(78.57±5.22)的存活率明显增加.用Aβ1-42处理过的PC12细胞(28.31±1.65)和PC12-parental细胞(26.65±3.64)的凋亡比用Aβ1-42处理过的PC12-s细胞(17.77±2.37)的凋亡明显增加.结论:Stathmin基因的下调对Aβ1-42诱导PC12细胞损伤具有保护作用,为阿尔茨海默病的治疗探索了一条新策略.%Objective: To determine the effect of stathmin on the injury induced by amyloid beta-protein 1-42 (A^l-42) in rat's phaeochromocytoma cells (PC 12 cells). Methods: The shRNA expressing vectors to target stathmin gene were designed and constructed. The recombinant plasmids were transfected into PC12 cells, and the stable transfectants were selected by G418 and treated by A($l-42. RT-PCR and Western blot were used to analyze the expression levels of stathmin mRNA and protein, respectively. MTT assay was performed to detect the proliferation of the stable transfectants. The changes of apoptosis were detected by flow cytometry. Results: Compared with control cells, stathmin expression was obviously decreased in PC12 cells with transfected pSilencer4.1-s plasmid.Compared with PC12 cells (63.88?.36) and PC12-parental cells (65.78?.74) treated

  14. Identification of a Novel Rat NR2B Subunit Gene Promoter Region Variant and Its Association with Microwave-Induced Neuron Impairment.

    Wang, Li-Feng; Tian, Da-Wei; Li, Hai-Juan; Gao, Ya-Bing; Wang, Chang-Zhen; Zhao, Li; Zuo, Hong-Yan; Dong, Ji; Qiao, Si-Mo; Zou, Yong; Xiong, Lu; Zhou, Hong-Mei; Yang, Yue-Feng; Peng, Rui-Yun; Hu, Xiang-Jun


    Microwave radiation has been implicated in cognitive dysfunction and neuronal injury in animal models and in human investigations; however, the mechanism of these effects is unclear. In this study, single nucleotide polymorphism (SNP) sites in the rat GRIN2B promoter region were screened. The associations of these SNPs with microwave-induced rat brain dysfunction and with rat pheochromocytoma-12 (PC12) cell function were investigated. Wistar rats (n = 160) were exposed to microwave radiation (30 mW/cm(2) for 5 min/day, 5 days/week, over a period of 2 months). Screening of the GRIN2B promoter region revealed a stable C-to-T variant at nucleotide position -217 that was not induced by microwave exposure. The learning and memory ability, amino acid contents in the hippocampus and cerebrospinal fluid, and NR2B expression were then investigated in the different genotypes. Following microwave exposure, NR2B protein expression decreased, while the Glu contents in the hippocampus and CSF increased, and memory impairment was observed in the TT genotype but not the CC and CT genotypes. In PC12 cells, the effects of the T allele were more pronounced than those of the C allele on transcription factor binding ability, transcriptional activity, NR2B mRNA, and protein expression. These effects may be related to the detrimental role of the T allele and the protective role of the C allele in rat brain function and PC12 cells exposed to microwave radiation.

  15. Effects of methylmercury on the differentiation and survival of PC12 cells%甲基汞对PC12细胞分化及活性的影响

    李永进; 毕晓颖; 宋春梅; 马洪波; 王长文; 李志超


    目的研究氯化甲基汞对PC12细胞活性和分化的影响. 方法培养PC12细胞,用不同浓度的甲基汞作用于PC12细胞,用倒置显微镜观察细胞形态学变化,通过MTT检测PC12细胞活性. 结果氯化甲基汞能抑制PC12细胞突起生长,降低细胞活性. 结论氯化甲基汞可抑制PC12细胞分化和活性.


    Zhurishkina, E V; Lapina, I M; Ivanen, D R; Stepanov, S I; Shvetsova, S V; Shavarda, A L; Giliano, N Ya; Kulminskaya, A A


    The aim of the research was to investigate cytotoxicity of fucoidans on mammals cells. Three different samples of fucoidans were isolated from mechanically grounded brown algae Laminaria digitata and Fucus ve- siculosus. The sample F2 that differed from the others by higher sulfatation level and suppression of HeLa G-63 line culture growth was taken for further study in cell lines HeLa G-63, ECV 304 and PC 12. We have shown that fucoidan preparation F2 inhibits proliferation and induces cell death in a dose- and time-dependent manner for all investigated cell lines. Neuroendocrine tumor rat cell line PC 12 appeared to be the most sensitive to fucoidan treatment whereas endothelial human cells ECV 304 were the least sensitive.

  17. 人Slitrk1基因对PC12细胞增殖和分化的影响%The effect of human Slitrk1 gene on proliferation and differentiation of PC12 cells

    靳雁斌; 吴燕; 王晓文; 吴海涛; 景孝堂; 刘毅; 范文红; 范明


    目的 研究人Slitrk1基因过表达对PC12细胞增殖和分化的影响.方法 PCR扩增人Slitrk1 CDS,克隆到pcDNA4/myc-His A载体,构建重组质粒pcDNA4/St1,分别以空载体和重组质粒转染PC12细胞,RT-PCR法鉴定出阳性转染细胞,观察空载体转染的PC12细胞(pcDNA4/PC12)和过表达Slitrk1的PC12细胞(ST1/PC12)的表型并用MTT法检测细胞增殖情况.结果 与空载体转染的PC12细胞相比,过表达Slitrk1基因的细胞增殖速度明显减慢.pcDNA4/PC12与野生型PC12细胞表型一致,均为悬浮成团生长,细胞少有突起,而ST1/PC12细胞大部分贴壁生长,突起多见,呈分化状态.结论过表达人Slitrk1基因能够抑制PC12细胞增殖,促进细胞的贴壁及突起长出.人Slitrk1基因可能有促进PC12细胞神经分化的作用.


    徐阳曦; 杨铁峥; 张学荣; 陈缨; 覃柳燕; 张钦乐; 舒雨雁


    目的:观察胎盘免疫调节因子(placenta factor,PF)对PC12细胞生长的影响.方法:培养PC12细胞,用MTT法观察PF对体外无血清培养PC12细胞存活率的影响及PF对低血清培养PC12细胞增殖、分化的影响.结果:对无血清培养的PC12细胞加入不同浓度的PF培养44 h后, PF在浓度为50、25、12.5、6.25 mg/L时可以显著提高无血清培养PC12细胞的存活率(P<0.05).对低血清培养的PC12细胞加入不同浓度的PF培养68 h后,PF在浓度为12.5 mg/L和6.25 mg/L时可以显著提高PC12细胞的数量(P<0.01).对低血清培养的PC12细胞加入不同浓度的PF培养10 d,PF在浓度为3.13 mg/L和1.56 mg/L作用第6天时,细胞长出突起.结论:PF能提高无血清培养PC12细胞的存活率,促进PC12细胞增殖,诱导PC12细胞分化.

  19. 生物发光法测定PC12细胞微量ATP含量%Detection of ATP content in PC12 cells by bioluminescence method

    邓晓红; 许予明; 张苏明; 许康


    目的研究生物发光法测定PC12细胞内腺苷三磷酸(ATP)含量的可行性及短暂缺氧血清剥夺再灌注后ATP含量的动态变化.方法将PC12细胞随机分为正常对照组、缺氧组, 测定短暂缺氧血清剥夺再灌注后不同时间点的ATP含量和细胞活性, 对缺氧组ATP含量与细胞活性进行相关性分析.结果与正常对照组相比, 缺氧血清剥夺15 min再灌注1 h后的ATP含量、细胞活性显著降低, 差异具有显著性意义(P<0.05);缺氧血清剥夺15 min再灌注6 h后基本恢复至正常水平.缺氧组ATP含量与细胞活性呈显著正相关(r=0.904, P<0.05).结论生物发光法测定PC12细胞内ATP含量有较高的准确性.短暂缺氧血清剥夺再灌注后, 存在低能量状态, 且可完全恢复.

  20. Enhanced PC12 cells proliferation with self-assembled S-layer proteins scaffolds.

    Babolmorad, Ghazal; Emtiazi, Giti; Ghaedi, Kamran; Jodeiri, Mohamad


    Finding 3D biocompatible and biodegradable scaffold is important in tissue engineering which plays a critical role in transplanting methods. Several biomaterials, such as poly-L-lactide, poly(lactic-co-glycolic acid), poly(L-lactic acid)/poly(lactide-co-glycolide), alginate, collagen gel, and so on, have been applied as scaffold to culture cells in 3D environment. The most significant problem of the synthetic materials is lack of biocompatibility and bioactivity. Herein, self assemble S-layer proteins are used as a scaffold for PC12 cells culturing. For this purpose, S-layer protein was extracted from Bacillus coagulans HN68. Then, extracted S-layer was studied by SDS page and AFM. Using MTS test and Immunochemistry staining methods, the effect of self assembled S-layer scaffold on proliferation of PC12 cells was assayed. This study provides that S-layer could be an appropriate scaffold for PC12 cells culturing. Even though poly-L-ornithine is a common scaffold in PC12 cells culturing, the results show that (PLO)/S-layer is more protective.

  1. Crocin suppresses tumor necrosis factor-alpha-induced cell death of neuronally differentiated PC-12 cells.

    Soeda, S; Ochiai, T; Paopong, L; Tanaka, H; Shoyama, Y; Shimeno, H


    Crocus sativus L. is used in Chinese traditional medicine to treat some disorders of the central nervous system. Crocin is an ethanol-extractable component of Crocus sativus L.; it is reported to prevent ethanol-induced impairment of learning and memory in mice. In this study, we demonstrate that crocin suppresses the effect of tumor necrosis factor (TNF)-alpha on neuronally differentiated PC-12 cells. PC-12 cells dead from exposure to TNF-alpha show apoptotic morphological changes and DNA fragmentation. These hallmark features of cell death did not appear in cells treated in the co-presence of 10 microM crocin. Moreover, crocin suppressed the TNF-alpha-induced expression of Bcl-Xs and LICE mRNAs and simultaneously restored the cytokine-induced reduction of Bcl-X(L) mRNA expression. The modulating effects of crocin on the expression of Bcl-2 family proteins led to a marked reduction of a TNF-alpha-induced release of cytochrome c from the mitochondria. Crocin also blocked the cytochrome c-induced activation of caspase-3. To learn how crocin exhibits these anti-apoptotic actions in PC-12 cells, we tested the effect of crocin on PC-12 cell death induced by daunorubicin. We found that crocin inhibited the effect of daunorubicin as well. Our findings suggest that crocin inhibits neuronal cell death induced by both internal and external apoptotic stimuli.

  2. Effects of epigallocatechin gallate on ultra-violet-induced cell death in PC12 cells

    Takahashi, Hideo; Seki, Sakiko; Sakamoto, Naotaka; Nakagawa, Shigeki [Nihon Univ., Tokyo (Japan). School of Medicine


    We examined the effects of catechin on ultra-violet-induced cell death in PC12 cells. PC12 cells were irradiated by ultra-violet C (254 nm) (UVC). We found that the lactate dehydrogenase (LDH) activities in culture media and lipid peroxide in PC12 cells, which indicate cell death and cell membrane damage, respectively, were increased by UVC irradiation in a time-dependent manner. Cell death was gradually stimulated for 9 hours of cultivation after a UVC irradiation period of 10 or 30 min. Epigallocatechin gallate (EGCG), which is one of the main catechins found in green tea, suppressed the increase in LDH activity in culture medium and also inhibited the formation of lipid peroxide. I{kappa}B, a member of the cell death signaling system, was phosphorylated at 1 hour after 10 min of UVC irradiation. Stimulation of phosphorylation of I{kappa}B by UVC was suppressed by the addition of EGCG. We concluded that EGCG protects the PC12 cell from cell damage caused by UVC irradiation. (author)

  3. Curcumin Protects β-Lactoglobulin Fibril Formation and Fibril-Induced Neurotoxicity in PC12 Cells.

    Mansooreh Mazaheri

    Full Text Available In this study the β-lactoglobulin fibrillation, in the presence or absence of lead ions, aflatoxin M1 and curcumin, was evaluated using ThT fluorescence, Circular dichroism spectroscopy and atomic force microscopy. To investigate the toxicity of the different form of β-Lg fibrils, in the presence or absence of above toxins and curcumin, we monitored changes in the level of reactive oxygen species and morphology of the differentiated neuron-like PC12 cells. The cell viability, cell body area, average neurite length, neurite width, number of primary neurites, percent of bipolar cells and node/primary neurite ratios were used to assess the growth and complexity of PC12 cells exposed to different form of β-Lg fibrils. Incubation of β-Lg with curcumin resulted in a significant decrease in ROS levels even in the presence of lead ions and aflatoxin M1. The β-Lg fibrils formed in the presence of lead ions and aflatoxin M1 attenuated the growth and complexity of PC12 cells compared with other form of β-Lg fibrils. However, the adverse effects of these toxins and protein fibrils were negated in the presence of curcumin. Furthermore, the antioxidant and inhibitory effects of curcumin protected PC12 cells against fibril neurotoxicity and enhanced their survival. Thus, curcumin may provide a protective effect toward β-Lg, and perhaps other protein, fibrils mediated neurotoxicity.

  4. Activation of muscarinic receptors inhibits glutamate-induced GSK-3β overactivation in PC12 cells

    Ke MA; Li-min YANG; Hong-zhuan CHEN; Yang LU


    Aim:To investigate the actions of the muscarinic agonist carbachol on glutamate-induced neurotoxicity in PC12 cells,and the underlying mechanisms.Methods:PC12 cells were treated with different concentrations of glutamate for 24 or 48 h.The cell viability was measured using MTT assay,and the expression and activation of GSK-3β were detected with Western blot.β-Catenin translocation was detected using immunofluorescence.Luciferase reporter assay and real-time PCR were used to analyze the transcriptional activity of β-catenin.Results:Glutamate (1,3,and 10 mmol/L) induced PC12 cell death in a dose-dependent manner.Moreover,treatment of the cells with glutamate (1 mmol/L) caused significant overactivation of GSK-3β and prevented β-catenin translocation to the nucleus.Pretreatment with carbachol (0.01 μmol/L) blocked glutamate-induced cell death and GSK-3β overactivation,and markedly enhanced β-catenin transcriptional activity.Conclusion:Activation of muscarinic receptors exerts neuroprotection in PC12 cells by attenuating glutamate-induced GSK-3β overactivation,suggesting potential benefits of muscarinic agonists for Alzheimer's disease.

  5. Betulinic Acid Induces Apoptosis in Differentiated PC12 Cells Via ROS-Mediated Mitochondrial Pathway.

    Wang, Xi; Lu, Xiaocheng; Zhu, Ronglan; Zhang, Kaixin; Li, Shuai; Chen, Zhongjun; Li, Lixin


    Betulinic acid (BA), a pentacyclic triterpene of natural origin, has been demonstrated to have varied biologic activities including anti-viral, anti-inflammatory, and anti-malarial effects; it has also been found to induce apoptosis in many types of cancer. However, little is known about the effect of BA on normal cells. In this study, the effects of BA on normal neuronal cell apoptosis and the mechanisms involved were studied using differentiated PC12 cells as a model. Treatment with 50 μM BA for 24 h apparently induced PC12 cell apoptosis. In the early stage of apoptosis, the level of intracellular reactive oxygen species (ROS) increased. Afterwards, the loss of the mitochondrial membrane potential, the release of cytochrome c and the activation of caspase-3 occurred. Treatment with antioxidants could significantly reduce BA-induced PC12 cell apoptosis. In conclusion, we report for the first time that BA induced the mitochondrial apoptotic pathway in differentiated PC12 cells through ROS.

  6. 甲基汞对PC12细胞分化及活性的影响

    李永进; 毕晓颖; 宋春梅; 马洪波; 王长文; 李志超



  7. Genetic aspects of pheochromocytoma.

    Kolačkov, Katarzyna; Tupikowski, Krzysztof; Bednarek-Tupikowska, Grażyna


    Pheochromocytomas are derived from chromaffin cells of the adrenal medulla which synthesize and secrete catecholamines, thus affecting the cardiovascular system and metabolic processes. Pheochromocytoma is a tumor of the following multicarcinoma hereditary syndromes: type 2 multiple endocrine neoplasia, von Hippel-Lindau disease, type 1 neurofibromatosis and the pheochromocytomas/paragangliomas syndrome. Pheochromocytomas are relatively rare, and because of non-specific manifestation of these tumors and the possible lack of signs and symptoms for extended periods of time, the diagnosis may be delayed, which may, in turn, lead to death. Pheochromocytomas may occur sporadically. However, due to the frequent incidence of hereditary forms of these cancers, the presymptomatic genetic testing of family members with a positive family history is indicated, thus allowing for selecting people with higher risk of cancer. Early detection of the syndrome and the coexisting tumors (which may be malignant) may lead to a correct diagnosis, regular surveillance, preventive examinations and implementation of appropriate early treatment. Recent examinations have shown significant involvement of RET, VHL, NF1, SDHB and SDHD as well as the newly discovered KIF1Bβ, TMEM127 and MAX genes in pathogenesis of these tumors. The microarray-gene expression studies, based on the analysis of cellular pathways, have revealed two distinct clusters indicating two different routes of tumorgenesis. The genotype-phenotype correlations are still being studied and future research can give us clearer information about the function of these genes, which may prove crucial from the clinical point of view.

  8. Retroperitoneoscopic adrenalectomy in pheochromocytoma

    Marcelo Hisano


    Full Text Available Since the first laparoscopic adrenalectomy, the technique has evolved and it has become the standard of care for many adrenal diseases, including pheochromocytoma. Two laparoscopic accesses to the adrenal have been developed: transperitoneal and retroperitoneal. Retroperitoneoscopic adrenalectomy may be recommended for the treatment of pheochromocytoma with the same peri-operative outcomes of the transperitoneal approach because it allows direct access to the adrenal glands without increasing the operative risks. Although technically more demanding than the transperitoneal approach, retroperitoneoscopy can shorten the mean operative time, which is critical for cases with pheochromocytoma where minimizing the potential for intra-operative hemodynamic changes is essential. Blood loss and the convalescence time can be also shortened by this approach. There is no absolute indication for either the transperitoneal or retroperitoneal approach; however, the latter procedure may be the best option for patients who have undergone previous abdominal surgery and obese patients. Also, retroperitoneoscopic adrenalectomy is a good alternative for treating cases with inherited pheochromocytomas, such as multiple endocrine neoplasia type 2A, in which the pheochromocytoma is highly prevalent and frequently occurs bilaterally.

  9. Susceptibility of naïve and differentiated PC12 cells to Japanese encephalitis virus infection.

    Li, Jian-Ri; Wu, Chih-Cheng; Chang, Cheng-Yi; Ou, Yen-Chuan; Lin, Shih-Yi; Wang, Ya-Yu; Chen, Wen-Ying; Raung, Shue-Ling; Liao, Su-Lan; Chen, Chun-Jung


    Japanese encephalitis is a mosquito-borne disease caused by Japanese encephalitis virus (JEV) infection. Although JEV infects and replicates in cells with multiple tissue origins, neurons are the preferential cells for JEV infection. Currently, the identities of JEV cell tropism are largely unclear. To gain better insight into the underlying identities of JEV cell tropism, this study was designed to compare the JEV cell tropism with naïve or differentiated PC12 cells. Through nerve growth factor-differentiated PC12 cells, we discovered that JEV efficiently replicated in differentiated PC12 cells rather than naïve cells. Mechanistic studies revealed that viral adsorption/attachment seemed not to be a crucial factor. Supporting data showed that antagonizing postreceptor intracellular signaling of interferons, along with the activation of suppressor of cytokine signaling-3 (SOCS3) expression and protein tyrosine phosphatase activity, were apparent in differentiated PC12 cells after JEV infection. Independent of differentiating inducing agents, the upregulation of SOCS3 expression and protein tyrosine phosphatase activity, as well as preferential JEV tropism, were common in JEV-infected differentiated PC12 cells. Using cultured primary neurons, JEV efficiently replicated in embryonic neurons rather than adult neurons, and the preference was accompanied by higher SOCS3 expression and protein tyrosine phosphatase activity. Given that both SOCS3 and protein tyrosine phosphatases have been implicated in the process of neuronal differentiation, JEV infection seems to not only create an antagonizing strategy to escape host's interferon antiviral response but also takes advantage of cellular machinery to favor its replication. Taken together, current findings imply that dynamic changes within cellular regulators of antiviral machinery could be accompanied by events of neuronal differentiation, thus concurrently playing roles in the control of JEV cell tropism and

  10. In vitro toxicity of nanosized copper particles in PC12 cells induced by oxidative stress

    Xu Pengjuan; Xu Jing; Liu Shichang [Nankai University, College of Medicine (China); Ren, Guogang [University of Hertfordshire, School of Aerospace Engineering (United Kingdom); Yang Zhuo, E-mail: [Nankai University, College of Medicine (China)


    Recent evidence suggests that some nanomaterials, which are widely used in many fields, have health effects. In order to investigate the cytotoxicity induced by nanosized copper particles (nano-Cu), PC12 cells, which were widely used as an in vitro model for the neuron research, were treated with different concentrations (0, 1, 10, 30, and 100 {mu}g/mL) of nano-Cu. The cell viability was determined by measurement of the reduction product of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). The oxidative stress induced by nano-Cu and its possible mechanism were studied in relation to the generation of reactive oxygen species (ROS) and the cellular activity of superoxide dismutase (SOD). Results showed that incubation of PC12 cells with increasing concentrations of nano-Cu induced a decrease of cell viability in a concentration- and time-dependent manner. In addition, flow cytometry assay using Annexin V-FITC/PI staining was used to investigate the mode of nano-Cu-induced cell death and quantified the percentage of apoptotic cells. Results showed that nano-Cu induced the significant apoptosis in PC12 cells. Meanwhile, intracellular accumulation of ROS was increased with the increased concentrations of nano-Cu and it was associated with decreased SOD activity, which was probably due to protect effects against the oxidative stress in PC12 cells. Results suggested that both excessive intracellular ROS and decreased SOD contributed to nano-Cu-induced cytotoxicity. In other words, the increasing of oxidative stress was a key mechanism in PC12 apoptosis induced by nano-Cu.

  11. Enhanced proliferation of PC12 neural cells on untreated, nanotextured glass coverslips

    Islam, Muhymin; Atmaramani, Rahul; Mukherjee, Siddhartha; Ghosh, Santaneel; Iqbal, Samir M.


    Traumatic injury to the central nervous system is a significant health problem. There is no effective treatment available partly because of the complexity of the system. Implementation of multifunctional micro- and nano-device based combinatorial therapeutics can provide biocompatible and tunable approaches to perform on-demand release of specific drugs. This can help the damaged cells to improve neuronal survival, regeneration of axons, and their reconnection to appropriate targets. Nano-topological features induced rapid cell growth is especially important towards the design of effective platforms to facilitate damaged neural circuit reconstruction. In this study, for the first time, feasibility of neuron-like PC12 cell growth on untreated and easy to prepare nanotextured surfaces has been carried out. The PC12 neuron-like cells were cultured on micro reactive ion etched nanotextured glass coverslips. The effect of nanotextured topology as physical cue for the growth of PC12 cells was observed exclusively, eliminating the possible influence(s) of the enhanced concentration of coated materials on the surface. The cell density was observed to increase by almost 200% on nanotextured coverslips compared to plain coverslips. The morphology study indicated that PC12 cell attachment and growth on the nanotextured substrates did not launch any apoptotic machinery of the cell. Less than 5% cells deformed and depicted condensed nuclei with apoptotic bodies on nanotextured surfaces which is typical for the normal cell handling and culture. Enhanced PC12 cell proliferation by such novel and easy to prepare substrates is not only attractive for neurite outgrowth and guidance, but may be used to increase the affinity of similar cancerous cells (ex: B35 neuroblastoma) and rapid proliferation thereafter—towards the development of combinatorial theranostics to diagnose and treat aggressive cancers like neuroblastoma.

  12. The Epidemiology of Pheochromocytoma

    Ebbehoj, A; Søndergaard, Esben; Trolle, Christian

    Pheochromocytoma is a rare disease but frequently poses a diagnostic dilemma due to the unspecific symptoms and its potentially life-threatening nature. There is a perception of an increase in the incidence of pheocromocytomas in recent years, but no data on time trends exist. We obtained data from...... The Danish National Registry of Patients, The Danish Registry of Causes of Death, and The National Pathology Registry for all persons registered with pheochromocytoma in 1977–2015. Health records were reviewed to validate the diagnosis for all patients in the Northern and Central Regions of Denmark...... diagnosed in 1977–2006 in a secondary analysis using the Wilcoxon–Mann–Whitney test.We identified 183 confirmed cases of pheochromocytoma. A significant increasing trend (P

  13. Pheochromocytoma after Cesarean Section

    Naghshineh, Elham; Shahraki, Azar Danesh; Sheikhalian, Somaye; Hashemi, Leila


    Pheochromocytoma is a catecholamine-producing tumor. There are a very few reported cases of clinical pheochromocytoma. Here, we report a 27-year-old woman para 1 live 1 with chief complaint of headache, confusion, nausea, and vomiting 2 days after cesarean section. She was anxious and had palpitation. On physical examination, fever, tachycardia, tachypnea, high blood pressure, and right thyroid nodule were found. She was managed as pregnancy-induced hypertension at first. In laboratory data, epinephrine, norepinephrine, metanephrine, normetanephrine, and vanillylmandelic acid were increased in 24 h urine collection. An adrenal mass was detected in abdominal computed tomography. Regarding clinical and paraclinical findings, pheochromocytoma was diagnosed. The patient received medical treatment, but it was not effective; hence, she underwent adrenalectomy. PMID:27076898

  14. Pheochromocytoma after cesarean section

    Elham Naghshineh


    Full Text Available Pheochromocytoma is a catecholamine-producing tumor. There are a very few reported cases of clinical pheochromocytoma. Here, we report a 27-year-old woman para 1 live 1 with chief complaint of headache, confusion, nausea, and vomiting 2 days after cesarean section. She was anxious and had palpitation. On physical examination, fever, tachycardia, tachypnea, high blood pressure, and right thyroid nodule were found. She was managed as pregnancy-induced hypertension at first. In laboratory data, epinephrine, norepinephrine, metanephrine, normetanephrine, and vanillylmandelic acid were increased in 24 h urine collection. An adrenal mass was detected in abdominal computed tomography. Regarding clinical and paraclinical findings, pheochromocytoma was diagnosed. The patient received medical treatment, but it was not effective; hence, she underwent adrenalectomy.

  15. Hexabromocyclododecane inhibits depolarization-induced increase in intracellular calcium levels and neurotransmitter release in PC12 cells.

    Dingemans, Milou M L; Heusinkveld, Harm J; de Groot, Aart; Bergman, Ake; van den Berg, Martin; Westerink, Remco H S


    Environmental levels of the brominated flame retardant (BFR) hexabromocyclododecane (HBCD) have been increasing. HBCD has been shown to cause adverse effects on learning and behavior in mice, as well as on dopamine uptake in rat synaptosomes and synaptic vesicles. For other BFRs, alterations in the intracellular Ca(2+) homeostasis have been observed. Therefore, the aim of this study was to investigate whether the technical HBCD mixture and individual stereoisomers affect the intracellular Ca(2+) concentration ([Ca(2+)](i)) in a neuroendocrine in vitro model (PC12 cells). [Ca(2+)](i) and vesicular catecholamine release were measured using respectively single-cell Fura-2 imaging and amperometry. Exposure of PC12 cells to the technical HBCD mixture or individual stereoisomers did neither affect basal [Ca(2+)](i), nor the frequency of basal neurotransmitter release. However, exposure to HBCD (0-20 microM) did cause a dose-dependent reduction of a subsequent depolarization-evoked increase in [Ca(2+)](i). This effect was apparent only when HBCD was applied at least 5 min before depolarization (maximum effect after 20 min exposure). The effects of alpha- and beta-HBCD were comparable to that of the technical mixture, whereas the inhibitory effect of gamma-HBCD was larger. Using specific blockers of L-, N- or P/Q-type voltage-gated Ca(2+) channels (VGCCs) it was shown that the inhibitory effect of HBCD is not VGCC-specific. Additionally, the number of cells showing depolarization-evoked neurotransmitter release was markedly reduced following HBCD exposure. Summarizing, HBCD inhibits depolarization-evoked [Ca(2+)](i) and neurotransmitter release. As increasing HBCD levels should be anticipated, these findings justify additional efforts to establish an adequate exposure, hazard and risk assessment.

  16. The ethanol response gene Cab45 can modulate the impairment elicited by ethanol and ultraviolet in PC12 cells

    Yunfeng Zhu; Quanli Wang; Wangru Xu; Sha Li


    High consumption of ethanolic beverages facilitates neurodegeneration,but the mechanism of this process still remained elusive.Suppression subtractive hybridization (SSH) is a technique for detection of rare transcripts.With SSH approach,we identified one ethanol response gene Cab45,which was down-regulated by ethanol with time-dependent manner in B104 cells.The full-length sequence of Cab45 gene was obtained by 5'-RACE (5'Rapid Amplification of cDNA Ends) for the first time in rat.Based on the sequence of deduced amino acid of rat Cab45,the alignment was conducted with its counterparts in different species and displayed a high conservation.Using different tissues in rat and cell lines,Cab45 was characterized by a ubiquitous expression and differentiation dependent down-regulation.Given that ethanol facilitates some cell differentiation,we hypothesize that Cab45 is involved in ethanol-mediated differentiation.With transient transfection,the function of Cab45 was investigated by up-regulation and down-regulation in PC12 cells.Ethanol treatment and UV exposure were conducted subsequently and cell proliferations were detected by MTT (Methyl Thiazolyl Tetrazolium) approach.It revealed that the up-regulation of Cab45 modulated the impairment elicited by ethanol and UV in transfected cells.As a member of new calcium binding protein family,the exact role of Cab45 still remains unclear.

  17. Structure and function of the ssDNA aptamer specifically recognizing differentiated PC12 cells%特异识别已分化PC12细胞的ssDNA适体的结构和功能研究

    王成龙; 邵宁生; 张明; 杨光; 张达矜; 丁红梅; 王会信


    目的:研究特异识别已分化PC12细胞的ssDNA适体的二级结构及截短前后的适体与已分化PC12细胞的结合能力.方法:利用RNA structure 3.5软件对截短前后的适体的空间结构进行模建,体外合成这些截短适体,利用流式细胞术和荧光显微镜研究适体与已分化PC12细胞的结合能力.结果:单茎环结构是适体与已分化PC12细胞结合的二级结构;截短后的适体与已分化PC12细胞的结合能力明显大于与未分化PC12细胞结合能力;AP17-38与已分化PC12细胞结合的荧光强度明显大于与未分化PC12细胞结合的荧光强度;截短前后的适体均能够特异识别混合物中已分化PC12细胞.结论:截短后的适体能够保留适体的空间结构,而且与已分化PC12细胞的亲合力不会降低.

  18. Effect of Calmodulin on the Differrentiation and Migration of PC12 Cells%钙调蛋白在PC12细胞分化和迁移中的作用

    袁俊; 李朝军


    To investigate the roles of calmodulin during neuronal differentiation and migration,we checked PC12 cells by immunofluorescence staining and single cell tracking assay after NGF treatment. We found that calmodulin showed a dense distribution pattern in top of PC12 cells. Only a small percentage of the cells grown in W7 treatment cells. A single cell tracking experiment showed that calmodulin in PC12 cells could increase cell motility. The data suggested that calmodulin may play an important role in differentiation and migration of PC12 cells.%PC12 细胞经神经生长因子 (NGF)作用后,利用免疫荧光染色、单个细胞迁移率检测等方法,研究了钙调蛋白在PC12 细胞中的分布以及钙调蛋白对PC12 细胞分化和迁移的影响.免疫荧光染色结果表明,钙调蛋白在PC12细胞突起的顶端处呈密集分布.加入钙调蛋白抑制剂W7的细胞仅有少量长出突起.单个细胞迁移率检测表明钙调蛋白可能促进PC12 细胞迁移.提示钙调蛋白可能在PC12细胞的分化和迁移过程中发挥作用.

  19. The Epidemiology of Pheochromocytoma

    Ladefoged Ebbehøj, Andreas


    Pheochromocytoma and catecholamine secreting paraganglioma (PPGL) are exceedingly rare endocrine tumours, but remain a frequent diagnostic dilemma due to their potential life-threatening nature. Reliable data on the epidemiology of PPGL is lacking and no time trends in incidence rates (IR) have...

  20. Characterization of docking and fusion of synaptic-like microvesicles in PC12 cells using TIRFM


    Neurotransmitters are released by the fusion of synaptic vesicles with presynaptic membrane, which has been extensively studied. The analysis of single vesicle fusion kinetics reveals that there exist fusion modes of "kiss and run" and "kiss and stay" which may be favored by neurons especially during strong firing beside full fusion. Pre-fusion steps of translocation, docking and priming along the exocytotic pathway play important roles in neurotransmitter release and its regulation. In the present report, we used dual-color imaging of VAMP2-pHluorin and VAChT-TDimer2 under total internal reflection fluorescence microscope (TIRFM) to monitor the docking and fusion of synaptic-like microvesicles (SLMVs) in PC12 cells stimulated by high K+. Our results show that "kiss and run" is a dominative fusion mode in PC12 cells under high K+-challenge, and the dwell time of SLMVs is prolonged by the high K+ stimulation that suggests an enhancement of vesicle priming.

  1. Cell cycle markers have different expression and localization patterns in neuron-like PC12 cells and primary hippocampal neurons.

    Negis, Yesim; Unal, Aysegul Yildiz; Korulu, Sirin; Karabay, Arzu


    Neuron-like PC12 cells are extensively used in place of neurons in published studies. Aim of this paper has been to compare mRNA and protein expressions of cell cycle markers; cyclinA, B, D, E; Cdk1, 2 and 4; and p27 in post-mitotic primary hippocampal neurons, mitotically active PC12 cells and NGF-differentiated post-mitotic PC12 cells. Contrary to PC12 cells, in neurons, the presence of all these markers was detected only at mRNA level; except for cyclinA, cyclinE and Cdk4, which were detectable also at protein levels. In both NGF-treated PC12 cells and neurons, cyclinE was localized only in the nucleus. In NGF-treated PC12 cells cyclinD and Cdk4 were localized in the nucleus while, in neurons cyclinD expression was not detectable; Cdk4 was localized in the cytoplasm. In neurons, cyclinA was nuclear, whereas in NGF-treated PC12 cells, it was localized in the cell body and along the processes. These results suggest that PC12 cells and primary neurons are different in terms of cell cycle protein expressions and localizations. Thus, it may not be very appropriate to use these cells as neuronal model system in order to understand neuronal physiological activities, upstream of where may lie cell cycle activation triggered events.

  2. Protective Effect of Eecdysterone on the PC12 Cell CytotoxicityInduced by beta-amyloid 25-35

    YANGSu-Fen; WUZhong-Jun; YANGZheng-Qin; LIYu; WuQin; ZHOUQi-Xin; SHIJing-Shan


    Objective. To study the effect of ecdysterone (ECR) on beta - amyloid peptide fragment 25-35 ( Aβ25-35 )-induced PC12 cell cytotoxicity, and further to expore its mechanism. Methods: PC12 survial was monitored by LDH release and 3-(4, 5-dimethylthiazol-yl-2, 5-diphenyhetrazolium bromide (MTT) assays. The content of malondi-

  3. The role of ribosylated-BSA in regulating PC12 cell viability.

    Kuo, Tsun-Yung; Huang, Chuen-Lin; Yang, Jung-Mou; Huang, Wei-Jung; Huang, Nai-Kuei; Chen, Yue-Wen; Lin, Ren-Jye; Yang, Ying-Chen


    Glycation, one of the post-translational modifications, is known to influence protein structure and biological function. Advanced glycation end products (AGEs) have been shown to cause pathologies of diabetes. Glycation levels in patients with Alzheimer's disease (AD) are higher than in normal people. However, whether the glycation of susceptible proteins is a triggering event for cell damage or simply a result remains to be elucidated. In this study, we demonstrated that ribose-conjugated BSA (Rib-BSA) directly induces PC12 cell death in a dose- and time-dependent manner. The IC(50) is 4.6 μM. Unlike glucose-incubated BSA, Rib-BSA rapidly forms cytotoxic AGEs. PC12 is vulnerable to Rib-BSA. However, fructose can induce AGE formation, although no effect on cell survival was observed. This effect of Rib-BSA is reversed by pretreatment of pioglitazone and rosiglitazone, which belongs to thiazolidinediones (TZDs) and are peroxisome proliferator-activated receptor (PPAR-γ) ligands. Moreover, Rib-BSA upregulates inducible nitric oxide synthase (iNOS), cycloxygenase 2 (COX-2) expression, and p-38 phosphorylation and leaves extracellular regulated protein1/2 (ERK1/2) phosphorylation unchanged. The Rib-BSA-induced signaling changes are blocked by rosiglitazone and confirmed by PPAR-γ small-interfering RNA transfection. The reduction of cell survival by Rib-BSA is blocked by the iNOS inhibitor and p38 inhibitor. No effect on cell survival was observed using the COX-2 inhibitor. Consequently, these results show that Rib-BSA directly inducing PC12 cell death is a triggering event and TZDs protect PC12 cell from Rib-BSA damage. Signaling molecules, such as PPAR-γ, P38, and iNOS, are involved in Rib-BSA-mediated cytotoxicity.

  4. Protective effect of telmisartan against oxidative damage induced by high glucose in neuronal PC12 cell.

    Eslami, Habib; Sharifi, Ali M; Rahimi, Hamzeh; Rahati, Maryam


    Telmisartan is an angiotensin II type 1 receptor blocker and partial agonist of peroxisome proliferator-activated receptor gamma (PPAR-γ). Here, we investigated the protective capacity of telmisartan against high glucose (HG)-elicited oxidative damage in PC12 cells. The activity of lactate dehydrogenase (LDH), NADPH oxidase (NOX), superoxide dismutase (SOD), catalase (CAT) as well as the levels of malondialdehyde (MDA), glutathione (GSH), intracellular reactive oxygen species (ROS), cell viability and DNA fragmentation were measured in HG-treated PC12 cells with and without telmisartan co-treatment. Moreover, the direct antioxidant effect of telmisartan was determined by 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay and protein expression of Bax, Bcl-2, cleaved caspase-3 and NOX subunit p47phox by western blotting. Telmisartan exhibited antioxidant activity in the ABTS assay with the IC50 value of 37.5 μM. Pretreatment of PC12 cells with telmisartan, prior to HG exposure, was associated with a marked diminution in cleaved caspase-3 expression, DNA fragmentation, Bax/Bcl-2 ratio, intracellular ROS and MDA levels. Additionally, the cell viability, GSH level, SOD and CAT activity were notably elevated by telmisartan, whereas the activity and the protein expression of NADPH oxidase subunit p47phox were attenuated. Interestingly, co-treatment with GW9662, a PPAR-γ antagonist, partially inhibited the beneficial effects of telmisartan. These findings suggest that telmisartan has protective effects on HG-induced neurotoxicity in PC12 cells, which may be related to its antioxidant action and inhibition of NADPH oxidase. Furthermore, the results show that PPAR-γ activation is involved in the neuroprotective effects of telmisartan. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  5. Nitric oxide promotes nicotine-triggered ERK signaling via redox reactions in PC12 cells.

    Miyamoto, Yoshiaki; Sakai, Ryosuke; Maeda, Chiharu; Takata, Tsuyoshi; Ihara, Hideshi; Tsuchiya, Yukihiro; Watanabe, Yasuo


    Nitric oxide (NO), produced by neuronal NO synthase (nNOS), serves as a signaling molecule with diverse biological responses in the central nervous system (CNS). In the present study, we demonstrated that nNOS expression enhances the nicotine-triggered activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in nNOS-transfected PC12 (NPC12) cells. Treatment with nicotine increased the phosphorylation level of ERK1/2 in the NPC12 cells as compared with that in control PC12 cells. However, nicotine treatment failed to enhance ERK1/2 phosphorylation when NPC12 cells were pretreated with several selective inhibitors of NOS, the nicotinic acetylcholine receptors, L-type voltage-dependent Ca(2+) channels, protein kinase C, Src, epidermal growth factor receptor, and MEK. The nicotine-induced ERK1/2 phosphorylation in PC12 cells was observed by their pretreatment with a NO donor. Moreover, the enhancement of nicotine-induced ERK1/2 phosphorylation in the NPC12 cells was regulated by intracellular glutathione levels, but not by the soluble guanylate cyclase-cGMP-protein kinase G signaling. Meanwhile, depolarization stimulated ERK1/2 phosphorylation in both PC12 and NPC12 cells. Taken together, these findings suggest that nicotine modulates NO-dependent redox condition; the resulting calcium influx, would increase ERK1/2 phosphorylation in nNOS expressing cells. Blockade of NO pathway may be selective target to reduce ERK1/2 phosphorylation via attenuation of the nicotine responses in the CNS.

  6. Damaged PC12 cells in Transwell culture system for promoting the transdifferentiation of bone marrow stromal stem cells into neuron-like cells%Transwell培养体系中受损PC12细胞促进骨髓间质干细胞向神经元样细胞的转分化

    周进; 罗晓光; 张朝东


    BACKGROUND: Drug treatment has unsatisfactory effect on Alzheimer disease, while many studies have indicated that the transplantation of bone marrow stromal cells (MSCs) is effective on Parkinson disease, cerebral ischemia, etc., but the mechanism is still unclear.OBJECTIVE: To imitate transplantation environment by co-culture of amyloid β1-40 (Aβ1-40) damaged PC12 cells and MSCs, observe the effect of bi-directional information feedback in the microenvironment on the transdifferentiation of MSCs to nerve cells, and observe its protective effect on the apoptosis of damaged PC12 cells.DESTGN: A comparative observation.SETTING: Department of Neurology, China Medical University.MATERIALS: SD rats of 2-3 weeks after birth either male or female were used. PC12 cell lines were purchased from the Institute of Cell Biology, Chinese Academy of Sciences; neuro-specific enolase (1:50, Boster, Wuhan);Methyl-thiazol-tetrazolium (MTT) 15 μL (terminal concentration of 0.5 g/L).METHODS: The experiment was carried out in the Experimental Center (provincial experimental animal center) of China Medical University from June to July in 2004. Bilateral femurs were aseptically removed from 1 SD rat, and MSCs were identified using CD44 antibody immunofiuorescently. PC12 was used to replace nerve cells. The PC12 cells were stimulated by Aβ1-40 then transferred by Transwell. There were 5 groups: Group A: normally cultured PC12 co-cultured with MSCs; Group B: Aβ1-40 stimulated PC12 co-cultured with MSCs; Group C: normal PC12 supernatant+MSCs; Group D: damaged PC12 supernatant+MSCs; Group E: MSCs cultured with common medium 1640.MAIN OUTCOME MEASURES:Routine immunohistochemical staining was performed. NSE positive cells were observed under inverted fluorescence microscope, 10 visual fields (200×) were randomly selected to count the positive cells. MTT metabolic rate was used to detect the proliferation of MSCs in each group. The differences of measurement data were compared using the

  7. Roles of notch signaling in rotenone-induced apoptosis of PC12 cells%Notch信号通路在鱼藤酮诱导的PC12细胞凋亡中的作用

    张媚; 何亮; 李俊; 张临洪


    Objective To elucidate the roles of Notch Signaling in rotenone-induced apoptosis of PC12 cells.Methods PC12 ceils were treated with rotenone (5 μmol/L).Apoptosis of PC12 cells and activation of Notch signaling were observed.PC12 cells were pretreated with N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butvlester ( DAPT,10 μmol/L) before incubation with rotenone.The effect of Notch signaling on apoptosis of PC12 cells in response to rotenone was evaluated and the relationship between Notch signaling and rotenone-induced apoptosis of PC12 cells was analyzed.Results Rotenone significantly induced apoptosis of PC12 cells [( 35.6 ± 5.4 ) %,P < 0.05] and increased Caspase-3 activity (0.52 ±0.15,P <0.05 ).At the same time,rotenone activated Notch/Jagged1 signaling in PC12 cells.However,inhibition of Notch signaling by DAPT not only decreased apoptosis of PC12 cells [(9.8 ±3.1 ) %,P < 0.05],but also diminished Caspase-3 activity (0.16 ± 0.06,P < 0.05 ).Conclusion Activation of Notch signaling could be one of the main mechanisms for rotenone-induced apoptosis of PC12 cells.%目的 探讨Notch信号通路在鱼藤酮诱导PC12细胞凋亡中的作用机制.方法 用鱼藤酮(5 μmol/L)处理PC12细胞,检测PC12细胞的凋亡和Notch信号通路的活化状态.同时以Notch信号抑制剂(2S)-N-N-(3,5-二氟苯乙酰基)-L-丙氨酰-2-苯基甘氨酸叔丁酯(DAPT,10 μmol/L)处理上述PC12细胞,观察Notch信号通路抑制后PC12细胞对鱼藤酮处理的凋亡反应,分析Notch信号通路与鱼藤酮诱导PC12细胞凋亡之间的关系.结果 鱼藤酮显著诱导PC12细胞凋亡[(35.6±5.4)%,P<0.05]和半胱氨酰天冬氨酸特异性蛋白酶-3(Caspase-3)活性升高(0.52±0.15,P<0.05),并活化其Notch/Jagged1信号通路.DAPT显著抑制鱼藤酮诱导的Notch/Jagged1信号通路活化并降低PC12细胞凋亡率[(9.8±3.1)%,P<0.05]和Caspase-3活性(0.16 ±0.06,P<0.05).结论 Notch信号通路活化是鱼藤酮诱导PC

  8. Codonopsis pilosula (Franch) Nannftotal alkaloids potentiate neurite outgrowth induced by nerve growth factor in PC12 cells

    LIUJian-Hui; BAOYong-Ming; SONGJi-Jun; ANLi-Jia


    AIM:To explore the effect of Codonopsis pilosula (Franch) Nannf total alkaloids (DSA) on differentiation inducedby nerve growth factor (NGF) in PC12 cells. METHODS: After culturing PC12 cells with DSA in the presence orabsence of NGF, neurite outgrowth in PC12 cells and correlated protein kinases were assayed. RESULTS: DSAalone did not exhibit neuritogenic activity, but caused a significant enhancement of NGF (2 μg/L)-induced neuriteoutgrowth in PC12 cells, and increased the phosphorylation of mitogen-activated protein kinase (MAPK).Furthermore, this enhancing effect was completely blocked by a specific MAPK kinase inhibitor, PD98059.CONCLUSION: DSA enhanced the NGF-induced neurite outgrowth in PC12 cells by amplifying an up-streamstep of the MAPK-dependent signaling pathway.

  9. VAMP-2 promotes neurite elongation and SNAP-25A increases neurite sprouting in PC12 cells.

    Shirasu, M; Kimura, K; Kataoka, M; Takahashi, M; Okajima, S; Kawaguchi, S; Hirasawa, Y; Ide, C; Mizoguchi, A


    Recent studies suggest that the soluble N-ethylmaleimide-sensitive factor attached protein (SNAP) receptor (SNARE)-mediated membrane fusion system is involved in vesicle fusion in the plasma membrane that allows expansion for neurite elongation. There have been several reports analyzing the effects of neurite outgrowth by inhibition of SNAREs. In this study, we took the opposite approach by overexpressing green fluorescent protein (GFP)-fusion SNAREs, including VAMP-2, SNAP-25A, and syntaxin1A, in PC12 cells to investigate the role of SNAREs in the neurite outgrowth of PC12 cells. Neurite outgrowth analysis demonstrated that: (1) GFP-VAMP-2 increased the length of individual neurites, without changing the number of neurites per cell; (2) GFP-SNAP-25A increased the number of neurites per cell, with no change in the length of the individual neurites. In both cases, the total length of neurites per cell was increased; (3) GFP-syntaxin1A resulted in no significant change, either in neurite length, or in the number of neurites per cell. These findings suggest that when overexpressed in PC12 cells, VAMP-2 can promote neurite elongation, while SNAP-25A can stimulate neurite sprouting. On the other hand, overexpression of syntaxin1A neither promotes nor inhibits neurite outgrowth. Thus VAMP-2 and SNAP-25A play different roles in neurite elongation and sprouting.

  10. Activin A prevents neuron-like PC12 cell apoptosis after oxygen-glucose deprivation

    Guihua Xu; Zhongxin Xu; Jinting He; Hongliang Guo; Chunli Mei; Jiaoqi Wang; Zhongshu Li; Han Chen; Jing Mang; Hong Yang


    In this study, PC12 cells were induced to differentiate into neuron-like cells using nerve growth factor, and were subjected to oxygen-glucose deprivation. Cells were treated with 0, 10, 20, 30, 50, 100 ng/mL exogenous Activin A. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay and Hoechst 33324 staining showed that the survival percentage of PC12 cells significantly decreased and the rate of apoptosis significantly increased after oxygen-glucose deprivation. Exogenous Activin A significantly increased the survival percentage of PC12 cells in a dose-dependent manner. Reverse transcription-PCR results revealed a significant increase in Activin receptor IIA, Smad3 and Smad4 mRNA levels, which are key sites in the Activin A/Smads signaling pathway, in neuron-like cells subjected to oxygen-glucose deprivation, while mRNA expression of the apoptosis-regulation gene caspase-3 decreased. Our experimental findings indicate that exogenous Activin A plays an anti-apoptotic role and protects neurons by means of activating the Activin A/Smads signaling pathway.

  11. Oxidative stress-mediated cytotoxicity of zirconia nanoparticles on PC12 and N2a cells

    Asadpour, Elham [Shiraz University of Medical Sciences, Anesthesiology and Critical Care Research Center (Iran, Islamic Republic of); Sadeghnia, Hamid R. [Mashhad University of Medical Sciences, Department of Pharmacology, Faculty of Medicine (Iran, Islamic Republic of); Ghorbani, Ahmad [Mashhad University of Medical Sciences, Pharmacological Research Center of Medicinal Plants (Iran, Islamic Republic of); Sedaghat, Mehran, E-mail: [Mashhad University of Medical Sciences, Department of Neurosurgery (Iran, Islamic Republic of); Boroushaki, Mohammad T., E-mail: [Mashhad University of Medical Sciences, Department of Pharmacology, Faculty of Medicine (Iran, Islamic Republic of)


    In recent years, there is a growing interest in the application of nanoparticles like zirconium dioxide (zirconia <100 nm), for many purposes. Since a comprehensive study on the toxic effects of zirconia has not been done, we decided to investigate the effects of zirconia nanoparticles on cultured PC12 and N2a cells. In this study, cytotoxic effect of different concentrations of zirconia nanoparticles at three different time intervals were evaluated using MTT and ROS (reactive oxygen species) assays. Also, Lipid peroxidation, glutathione (GSH) content changes, and DNA damage were measured. Zirconia nanoparticles caused a significant reduction in cell viability and GSH content of the cells, and induce a significant increase in intracellular ROS and MDA content of PC12 and N2a cells. Moreover, it increases the percentage of DNA tail of treated cells as compared with control group. Zirconia nanoparticles have cytotoxic and genotoxic effects in PC12 and N2a cells in a time and concentration-dependent manner in concentration more than 31 µg/mL.

  12. Cell metabolomics reveals the neurotoxicity mechanism of cadmium in PC12 cells.

    Zong, Li; Xing, Junpeng; Liu, Shu; Liu, Zhiqiang; Song, Fengrui


    The heavy metals such as cadmium (Cd) can induce neurotoxicity. Extensive studies about the effects of Cd on human health have been reported, however, a systematic investigation on the molecular mechanisms of the effects of Cd on central nervous system is still needed. In this paper, the neuronal PC-12 cells were treated with a series of concentrations of CdCl2 for 48h. Then the cytotoxicity was evaluated by MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. The IC15 value (15% inhibiting concentration) was selected for further mechanism studies. After PC-12 cells incubated with CdCl2 at a dose of IC15 for 48h, the intracellular and extracellular metabolites were profiled using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS)-based cell metabolomics approach. As found, the effects of the heavy metal Cd produced on the PC-12 cell viability were dose-dependent. The metabolic changes were involved in the glycolysis and gluconeogenesis, biopterin metabolism, tryptophan metabolism, tyrosine metabolism, glycerophospholipid metabolism, and fatty acids beta-oxidation. These could cause the perturbation of cell membrane, redox balance, energy supply, cellular detoxification, further affecting the cellular proliferation and apoptosis and other cellular activities. Copyright © 2017. Published by Elsevier Inc.

  13. Autophagy Alleviates Melamine-Induced Cell Death in PC12 Cells Via Decreasing ROS Level.

    Wang, Hui; Gao, Na; Li, Zhigui; Yang, Zhuo; Zhang, Tao


    Since melamine was illegally added to raw milk for increasing the apparent protein content, such a scandal has not been quite blown out. Previous studies showed that melamine induced apoptosis and oxidative damage in both in vivo and in vitro experiments. It is well known that autophagy is closely related to oxidative stress. In the present study, we examined whether autophagy played an important role in protecting PC12 cells, which were damaged by melamine. Immunofluorescence assay showed that melamine enhanced the number of punctuate dot, indicating the increase of autophagosomes. Western blot assay presented that melamine significantly elevated the expression level of autophagy markers including LC3-II/LC3-I ratio, beclin-1, and Atg 7. Rapamycin further enhanced the effect, whereas 3-methyadenine (3-MA) inhibited it. MTT assay exhibited that rapamycin significantly enhanced the cell viability (P melamine-treated PC12 cells (P melamine-treated PC12 cells (P melamine-induced cell death via inhibiting the excessive generation of ROS. Regulating autophagy may become a new targeted therapy to relieve the damage induced by melamine.

  14. Luteolin induces microRNA-132 expression and modulates neurite outgrowth in PC12 cells.

    Lian-Fang Lin

    Full Text Available Luteolin (3',4',5,7-tetrahydroxyflavone, a food-derived flavonoid, has been reported to exert neurotrophic properties that are associated with its capacity to promote neuronal survival and neurite outgrowth. In this study, we report for the first time that luteolin induces the persistent expression of microRNA-132 (miR-132 in PC12 cells. The correlation between miR-132 knockdown and a decrease in luteolin-mediated neurite outgrowth may indicate a mechanistic link by which miR-132 functions as a mediator for neuritogenesis. Furthermore, we find that luteolin led to the phosphorylation and activation of cAMP response element binding protein (CREB, which is associated with the up-regulation of miR-132 and neurite outgrowth. Moreover, luteolin-induced CREB activation, miR-132 expression and neurite outgrowth were inhibited by adenylate cyclase, protein kinase A (PKA and MAPK/ERK kinase 1/2 (MEK1/2 inhibitors but not by protein kinase C (PKC or calcium/calmodulin-dependent protein kinase II (CaMK II inhibitors. Consistently, we find that luteolin treatment increases ERK phosphorylation and PKA activity in PC12 cells. These results show that luteolin induces the up-regulation of miR-132, which serves as an important regulator for neurotrophic actions, mainly acting through the activation of cAMP/PKA- and ERK-dependent CREB signaling pathways in PC12 cells.

  15. Effect of nerve regeneration factor on differentiation of PC12 cells and its signaling pathway

    DING Fei; QIANG Liang; LIU Mei; GU Xingxing; GU Xiaosong


    The effects of nerve regeneration factor (NRF) on neuronal differentiation of PC12 cells and its signaling pathway are investigated by morphological observation and immunofluorescent cytochemical method, and the activity of ERK1/2 in NRF-treated PC12 cells in absence of serum is also studied by immuno-coprecipitation and Western blot analysis. The MEK1/2-specific inhibitor U0126, the broad-spectrum protein kinase C (PKC) inhibitor G6983 and tyrosine protein kinase (TPK) inhibitor genistein were used to determine the roles of the activation of ERK1/2 by NRF and the involvement of certain kinds of PKC or TPK receptor in this activation process. The results show that U0126 and G6983 inhibit the activation of ERK1/2 by NRF to different extents, while genistein has no effect on it, demonstrating that NRF remarkably induces neuronal differentiation of PC12 cells through activating ERK1/2 in a dose-dependent and time-dependent manner.

  16. Neuronal differentiation of PC12 cells induced by sciatic nerve and optic nerve conditioned medium

    DU Chan; YANG De-mei; ZHANG Pei-xun; DENG Lei; JIANG Bao-guo


    Background Previous work has shown that optic nerve and sciatic nerve conditional medium had neurotrophic activity on neurons. In order to find if the optic nerve conditioned media (CM) had a similar activity to make PC12 cells differentiate as sciatic nerve CM did, we explored the neurotrophic activity in optic nerve CM in the same in vitro system and compared the neurotrophin expression levels in optic and sciatic nerves under both conditions.Methods PC12 cells were used to examine the effects of neurotrophins secreted by the sciatic nerve and optic nerve. RT-PCR and real-time QPCR showed that the sciatic nerve and optic nerve produced a range of neurotrophins including nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3).Results The effects of sciatic nerve and optic nerve CM on neurite outgrowth were tested against a range of neurotrophins, and they had different neuritogenic activities. Only NGF and sciatic nerve CM had obvious neuritogenic activities, although the concentration of NGF in the sciatic nerve CM was very low.Conclusions Our experiment showed that sciatic nerve CM had a higher neurotrophic activity on PC12 cells than optic nerve CM. These results suggested that peripheral nervous system (PNS) and central nervous system (CNS) had different expression levels of neurotrophin, which may in part explain the lack of ability to regenerate the CNS.

  17. Selective alterations of transcription factors in MPP+-induced neurotoxicity in PC12 cells.

    Xu, Z; Cawthon, D; McCastlain, K A; Duhart, H M; Newport, G D; Fang, H; Patterson, T A; Slikker, W; Ali, S F


    MPP(+) (1-methyl-4-phenylpyridinium; the active metabolite of the neurotoxin MPTP (1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine)) depletes dopamine (DA) content and elicits cell death in PC12 cells. However, the mechanism of MPP(+)-induced neurotoxicity is still unclear. In this study, the dose response and time-course of MPP(+)-induced DA depletion and decreased cell viability were determined in nerve growth factor (NGF)-differentiated PC12 cells. The alteration of transcription factors (TFs) induced by MPP(+) from a selected dose level and time point was then evaluated using protein/DNA-binding arrays. K-means clustering analysis identified four patterns of protein/DNA-binding changes. Three of the 28 TFs identified in PC12 cells increased by 100% (p53, PRE, Smad SBE) and 2 decreased by 50% (HSE, RXR(DR1)) of control with MPP(+) treatment. In addition, three TFs decreased within the range of 33-50% (TFIID, E2F1, CREB) and two TFs increased within the range of 50-100% (PAX-5, Stat4). An electrophoretic mobility shift assay (EMSA) was used to confirm the changes of p53 and HSE. The observed changes in TFs correlated with the alterations of DA and cell viability. The data indicates that selective transcription factors are involved in MPP(+)-induced neurotoxicity and it provides mechanistic information that may be applicable to animal studies with MPTP and clinical studies of Parkinson's disease.

  18. Resveratrol Protects PC12 Cell against 6-OHDA Damage via CXCR4 Signaling Pathway

    Jing Zhang


    Full Text Available Resveratrol, herbal nonflavonoid polyphenolic compound naturally derived from grapes, has long been acknowledged to possess extensive biological and pharmacological properties including antioxidant and anti-inflammatory ones and may exert a neuroprotective effect on neuronal damage in neurodegenerative diseases. However, the underlying molecular mechanisms remain undefined. In the present study, we intended to investigate the neuroprotective effects of resveratrol against 6-OHDA-induced neurotoxicity of PC12 cells and further explore the possible mechanisms involved. For this purpose, PC12 cells were exposed to 6-OHDA in the presence of resveratrol (0, 12.5, 25, and 50 μM. The results showed that resveratrol increased cell viability, alleviated the MMP reduction, and reduced the number of apoptotic cells as measured by MTT assay, JC-1 staining, and Hoechst/PI double staining (all p<0.01. Immunofluorescent staining and Western blotting revealed that resveratrol averts 6-OHDA induced CXCR4 upregulation (p<0.01. Our results demonstrated that resveratrol could effectively protect PC12 cells from 6-OHDA-induced oxidative stress and apoptosis via CXCR4 signaling pathway.

  19. 神经干细胞对去血清诱导PC12细胞凋亡的作用%Effect of Neural Stem Cells on Apoptosis of PC12 Cells Induced by Serum Deprivation

    李香琴; 刘天庆; 朱蕾蕾; 葛丹; 崔占峰


    肾上腺嗜铬细胞瘤细胞(PC12 cells)去血清后,其凋亡与多巴胺能神经元的凋亡有许多共同之处,今通过研究神经干细胞(NSCs)对去血清诱导的PC12细胞凋亡的作用,进一步为NSCs移植治疗神经系统疾病提供相应的实验和理论依据.将正常培养的PC12细胞与NSCs以不同的方式进行去血清共培养,观察PC12细胞的形态,检测PC12细胞的活性,计算PC12细胞的存活率,同时检测培养基中胶质细胞源神经营养因子(GDNF)的浓度,分析不同培养方式下NSCs对去血清诱导凋亡的PC12细胞的作用以及NSCs与去血清诱导凋亡的PC12细胞共培养后,其分泌GDNF的能力.结果表明:①去血清诱导PC12细胞凋亡呈时间依赖性,去血清72 h后,PC12细胞存活的比率为44.25%;②NSCs培养基对去血清诱导凋亡的PC12细胞没有明显的保护作用;③NSCs培养上清及NSCs对去血清诱导凋亡的PC12细胞具有保护作用;④NSCs与去血清诱导凋亡的PC12细胞共培养后,分泌GDNF的能力增强.

  20. PGN激活BV2内吞Aβ寡聚体后对PC12的影响%Influences of peptidoglycan on PC12 cells after endocytosis of amyioid protein β oligomers in activated BV2 cells

    姜新; 谢明; 白丽娟; 陈晓虹; 马恩龙


    探讨前炎性介质肽聚糖(peptidoglycan,PGN)激活BV2细胞内吞β淀粉样蛋白(amyloid protein β,Aβ)1~42寡聚体后对PC12的影响.方法采用细胞株传代法培养PC12细胞及BV2细胞,分别替代神经元细胞和小胶质细胞;用转移筛网进行PC12、BV2细胞共育培养;制备Aβ1-42寡聚体;分别采用MTT方法检测PC12细胞抑制率、Western blotting方法检测各组PC12细胞tau( pS396)蛋白表达情况、流式细胞仪检测PC12细胞凋亡.结果Aβ寡聚体、Aβ寡聚体作用BV2细胞后及PGN作用BV2细胞后均能抑制PC12细胞增殖、增加PC12细胞tau( pS396)表达量、增加PC12细胞凋亡率;而PGN激活BV2细胞内吞Aβ寡聚体后对PC12细胞增殖抑制、tau (pS396)表达量、PC12细胞凋亡率与前面三种情况比较明显增加.结论Aβ寡聚体可引起PC12细胞的细胞抑制率,tau蛋白异常磷酸化水平,细胞凋亡的增多;PGN激活BV2细胞内吞Aβ后,加重Aβ寡聚体引起PC12细胞的细胞抑制率,tau蛋白异常磷酸化水平,细胞凋亡的增多.

  1. 人工合成多肽FGL对神经细胞模型PC12细胞增殖与凋亡的影响%Effects of synthetic peptides FG loop on PC12 cells proliferation and apoptosis

    付洪龙; 马学晓; 于腾波; 陈伯华; 李宁


    背景:FGL是NCAM的核心活性多肽片段,可直接作用于成纤维细胞生长因子受体1,激活NCAM的信号传导途径.目的:观察FGL人工合成多肽联合培养对PC12细胞增殖和凋亡的作用.方法:将培养的PC12细胞分为对照组和实验组,实验组预先加入1%的FGL多肽溶液.分别于培养1,3,5,7,9 d采用细胞计数试剂8法检测细胞增殖情况.将PC12细胞分为正常组、实验组和损伤组,损伤组加入H2O2刺激16 h.实验组加入H2O2与FGL人工合成多肽刺激16 h,流式细胞仪检测细胞凋亡,荧光定量PCR法检测PC12中的核转录因子κB mRNA表达.结果与结论:FGL人工合成多肽与PC12复合培养细胞生长良好,可明显促进PC12细胞的活性并且减低PC12 细胞凋亡并可明显降低凋亡模型中PC12细胞核转录因子κB基因的表达.说明FGL多肽可以明显促进PC12细胞增殖,并可以抑制PC12细胞凋亡.%BACKGROUND: FG loop (FGL) is a core active peptide fragment of neural cell adhesion molecule (NCAM), which can directly act on fibroblast growth factor receptor 1 (FGFR1) to activate NCAM signal pathway.OBJECTIVE: To observe the effects of synthetic peptides FGL on PC12 cells proliferation and apoptosis.METHODS: ①PC12 cells proliferation and apoptosis: The cultured PC12 cells were divided into control group and experiment group. The experimental group was added with 1% FGL peptide solution. The control group was pre-coated with poly-lysine plates. The cells were cultured 1, 3, 5, 7, 9 d respectively to detect cell proliferation by using Cell Counting Kit-8. ②PC12 apoptosis and nuclear factor kappa B mRNA detection: The PC12 cells were divided into normal group, experimental group and injury group. H2O2 was added into the injury group for 16 hours stimulation. In the experimental group, H2O2 and FGL were used for 16 hours stimulation. The cell apoptosis were detected by flow cytometry; mRNA expression of nuclear factor kappa B was detected by quantitative

  2. Antagonism of astragalus on PC12 cells apoptosis induced by manganese%黄芪对锰致PC12细胞凋亡的拮抗作用

    陈斯琦; 黄国香; 李莉; 宋世震



  3. P38信号通路在Aβ介导的PC12细胞损伤中的作用%P38 signaling pathway participates in PC12 cell injury induced by Aβ

    徐芳; 魏桂荣


    目的 探讨P38信号通路在Aβ介导的PC12细胞损伤中的作用.方法 体外培养PC12细胞,不同浓度的Aβ处理PC12细胞.用MTT法检测Aβ对PC12细胞活力的影响,免疫印迹法检测P38通路活化水平,并观察P38通路抑制剂SB203580对细胞活力的影响.结果 Aβ处理后,PC12细胞活力随Aβ浓度的增高而逐渐下降(P<0.05);Aβ处理后P38表达水平从4 h开始升高,12 h 达到高峰,并一直持续到24 h;SB203580预处理能够明显抑制P38信号通路活化,并对PC12细胞起到保护作用.结论 P38信号通路活化参与Aβ介导的PC12细胞损伤.

  4. Predictive Characteristics of Malignant Pheochromocytoma

    Park, Junsoo; Song, Cheryn; Park, Myungchan; Yoo, Sangjun; Park, Se Jun; Hong, SeokJun; Hong, Bumsik; Kim, Choung-Soo; Ahn, Hanjong


    Purpose The prognosis of patients with malignant pheochromocytoma is poor, but the predictive factors are not well understood. We aimed to identify the clinical characteristics predictive of malignancy after initial surgical removal in patients with pheochromocytoma. Materials and Methods We retrospectively reviewed the records of 152 patients diagnosed with pheochromocytoma, including 5 (3.3%) with metastasis at the time of the initial surgical excision and 12 (7.9%) who developed metastasis...

  5. Protective Effects of Periploca forrestii Schltr. Root Extract Against Cytotoxicity Induced by Hydrogen Peroxide in PC12 Cells%黑骨藤对PC12细胞氧化损伤的保护研究

    张贵源; 龚莉莉; 余跃生; 王恒; 陆玉炯; 杨再昌


    [Objective] The aim was to study the protective effects of Periploca forrestii Schltr.root extract against cytotoxicity induced by hydrogen peroxide in PC12 cells.[Method] The model of PC12 cells toxicity induced by hydrogen peroxide was established and the protective effects of Periploca forrestii Schltr.root extract on PC12 cells were detected.[Result] Compared to negative control,Periploca forrestii Schltr.root extract could protect PC12 cells at concentration ranges of 16-128 ug/mL at a dose-response manner.[Conclusion] Periploca forrestii Schltr.root extract showed anti-oxidative effect on PC12 cells.%[目的]研究黑骨藤提取物对H2O2诱导的PC12氧化损伤的保护作用。[方法]使用H2O2氧化压力诱导PC12出现氧化损伤,检测黑骨藤乙醇提取物对PC12细胞的保护作用。[结果]与模型对照组相比,黑骨藤乙醇提取物浓度在16~128μg/mL时,能显著提高PC12细胞的存活率,呈典型的量效关系。[结论]黑骨藤乙醇提取物具有抗氧化损伤作用。

  6. Rat NPFF(1) receptor-mediated signaling: functional comparison of neuropeptide FF (NPFF), FMRFamide and PFR(Tic)amide.

    Chen, Jin-Chung; Lee, Wei-Hsin; Chen, Pei-Chun; Tseng, Ching-Ping; Huang, Eagle Yi-Kung


    Neuropeptide FF (NPFF) participates in many physiological functions associated with opioids in the mammalian CNS. We established a pheochromocytoma PC-12 cell line clone stably expressing rat NPFF1 receptors. Both NPFF and FMRFamide activated NPFF1 receptors to couple with Gi/o protein and inhibited adenylyl cyclase activity in PC-12/rNPFF1 cells, but there were no effects on MAPKs (ERK1/2 and p38 MAPK) or PI3K/Akt pathway. FMRFamide also inhibited DARPP-32/Thr34 phosphorylation in the presence of forskolin. Similarly, PFR(Tic)amide, a 'super-agonist' of NPFF receptors, inhibited the production of cAMP and slightly decreased DARPP-32/Thr34 phosphorylation in PC-12/rNPFF1 cells. Intracerebroventricular injections of PFR(Tic)amide blocked behavioral sensitization of locomotor activity to amphetamine, and intrathecal injection of PFR(Tic)amide caused a dose-dependent antinociception in vivo in rats. Thus, "over-activation" of NPFF receptors by PFR(Tic)amide induced different bio-effects from those induced by NPFF in vivo.

  7. Undiagnosed pheochromocytoma: the anesthesiologist nightmare

    Myklejord, Duane J


    .... High urinary and plasma catecholamines suggested the presence of pheochromocytoma. Three weeks of oral phenoxybenzamine therapy subsequently allowed uneventful induction of anesthesia and open adrenalectomy...

  8. A superoxide anion-scavenger, 1,3-selenazolidin-4-one suppresses serum deprivation-induced apoptosis in PC12 cells by activating MAP kinase

    Nishina, Atsuyoshi, E-mail: [Yonezawa Women' s Junior College, 6-15-1 Tohrimachi, Yonezawa, Yamagata 992-0025 (Japan); Kimura, Hirokazu; Kozawa, Kunihisa [Gunma Prefectural Institute of Public Health and Environmental Sciences, 378 Kamioki, Maebashi, Gunma 371-0052 (Japan); Sommen, Geoffroy [Lonza Braine SA, Chaussee de Tubize 297, B-1420 Braine l' Alleud (Belgium); Nakamura, Takao [Department of Biomedical Information Engineering, Graduate School of Medical Science, Yamagata University, Yamagata 990-9585 (Japan); Heimgartner, Heinz [University of Zuerich, Institut of Organic Chemistry, Winterthurerstrasse 190, CH-8057 Zuerich (Switzerland); Koketsu, Mamoru [Department of Materials Science and Technology, Faculty of Engineering, Gifu University, Gifu 501-1193 (Japan); Furukawa, Shoei [Laboratory of Molecular Biology, Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, Gifu 502-8585 (Japan)


    Synthetic organic selenium compounds, such as ebselen, may show glutathione peroxidase-like antioxidant activity and have a neurotrophic effect. We synthesized 1,3-selenazolidin-4-ones, new types of synthetic organic selenium compounds (five-member ring compounds), to study their possible applications as antioxidants or neurotrophic-like molecules. Their superoxide radical scavenging effects were assessed using the quantitative, highly sensitive method of real-time kinetic chemiluminescence. At 166 {mu}M, the O{sub 2}{sup -} scavenging activity of 1,3-selenazolidin-4-ones ranged from 0 to 66.2%. 2-[3-(4-Methoxyphenyl)-4-oxo-1,3-selenazolidin-2-ylidene]malononitrile (compound b) showed the strongest superoxide anion-scavenging activity among the 6 kinds of 2-methylene-1,3-selenazolidin-4-ones examined. Compound b had a 50% inhibitory concentration (IC{sub 50}) at 92.4 {mu}M and acted as an effective and potentially useful O{sub 2}{sup -} scavenger in vitro. The effect of compound b on rat pheochromocytome cell line PC12 cells was compared with that of ebselen or nerve growth factor (NGF) by use of the MTT [3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay. When ebselen was added at 100 {mu}M or more, toxicity toward PC12 cells was evident. On the contrary, compound b suppressed serum deprivation-induced apoptosis in PC12 cells more effectively at a concentration of 100 {mu}M. The activity of compound b to phosphorylate mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) 1/2 (MAP kinase) in PC12 cells was higher than that of ebselen, and the former at 100 {mu}M induced the phosphorylation of MAP kinase to a degree similar to that induced by NGF. From these results, we conclude that this superoxide anion-scavenger, compound b, suppressed serum deprivation-induced apoptosis by promoting the phosphorylation of MAP kinase. -- Highlights: Black-Right-Pointing-Pointer We newly synthesized 1,3-selenazolidin-4-ones to

  9. Protective effect of curcumin on dopamine-induced apoptosis in PC12 cells%姜黄素对多巴胺诱导PC12细胞凋亡的拮抗作用

    王翔宇; 梅元武; 杨红卫


    目的 探讨姜黄素(Cur)对多巴胺氧化应激损伤PC12细胞的保护作用.方法 以多巴胺(DA)损伤PC12细胞为氧化应激损伤的模型,采用MTT法检测细胞增殖状况,Hoechst染色检测细胞核形态,碘化丙啶(PI)染色流式细胞术(FCM)检测细胞凋亡,罗丹明123(Rh123)染色FCM检测细胞线粒体膜电位(MMP).结果 多巴胺(50~800 μmol/L,24 h)呈浓度依赖性地损伤PCI2细胞,姜黄素(10~40μmol/L)对PC12细胞无毒性作用,但呈浓度依赖性地减轻DA对PC12细胞的毒性作用,抑制DA诱导PC12细胞凋亡,减轻DA对PC12 MMP的降低作用.结论 姜黄素可抑制DA诱导PC12细胞凋亡,对DA氧化应激损伤神经元具有保护作用,其机制可能与姜黄素抑制MMP的降低有关.%Objective To explore the protective effect of curcumin (Cur) on apoptosis of PC12 cells induced by dopamine (DA) oxidative stress. Methods Using DA to induce PC12 cell damage, the model of oxidative stress-induced neuron damage was established. Proliferation of PC12 cells was observed by 3-[4 ,5-dimethylthiazolyl ]-2 ,5-diphenyltetrazolium bromide (MTT) assay. Morphology of the nucleus was observed by Hoechst staining. Apoptosis of PC12 cells was detected by propidium iodide(PI) staining flow cytometry (FCM). The mitochondrial membrane potential (△Ψm) was determined by Rh123 staining flow cytometry. Results ① DA damaged PC12 cells in a concentrationdependent manner after application of DA (50-800 μmol/L) for 24 h, and MTT assay showed that Cur ( 10-40 μmol/L)had no toxic effect on PC12 cells. ② Cur reduced the toxic effect induced by DA on PC12 cells in a concentration-dependent manner. ③ Cur reducing the DA-induced toxic effect on PC12 cells was confirmed by cellular morphologic experiments. ④ PI staining FCM showed that Cur inhibited cellular apoptosis induced by DA and this effect was also confirmed by Hoechst staining. ⑤ The level of △Ψm was significantly decreased in PC12 cells after 100 and 200

  10. Effect of insulin on 1-methyl-4-phenylpyridinium ion-induced apoptosis of PC12 cells

    Liping Guo; Jian Wang; Yuping Jiang


    BACKGROUND: Insulin receptor (IR) expression in the substantia nigra of patients with Parkinson disease (PD) is not only significantly lower than that in the substantia nigra of normal persons of the same age, but also significantly lower than that in other regions in brain of himself/herself. It suggests that the abnormal effect of insulin receptor-mediated insulin, as a neurotrophic factor, is very possibly related to the loss of dopaminergic neurons in the substantia nigra and striatum in patients with Parkinson disease.OBJECTIVE: To observe the interventional effect of insulin on 1-methyl-4-phenylpyridinium ion (MPP+)-induced apoptosis of PC12.DESIGN: Controlled observation.SETTINGS: Department of Neurology, Beijing China-Japan Friendship Hospital; Department of Neurology,Huashan Hospital Affiliated to Fudan University.MATERIALS: PC12 cells were provided by the Cell Bank, Shanghai Institute of Cell Biology, Chinese Academyof Science. MPP+, MTT, HOECHST 33258 (Invitrogen Life Technologies), reverse transcription-polymerase chain reaction (RT-PCR) reagent (Takara Shuzo Co., Ltd.), flow cytometer (Bacton Dickionson, San Jose, CA),enzyme labelling instrument (Bio-Tek, Winooski, VT) and PCR circulation instrument (Takara Shuzo Co., Ltd)were used in this study.METHODS: This study was carried out in the Department of Neurology, Huashan Hospital Affiliated to Fudan University during June 2003 to August 2004. ① Cell culture and experimental grouping: PC12 cells were cultured according to the method from Peng et al, then were randomized into 3 groups; blank control group, MPP+ group and insulin group. ② Detection of relative survival rate of cells: The relative survival rate of cells at different MPP+ final concentrations (0, 50, 100, 200, 300, 1 000 μmol/L) and at different culture time (0, 4, 8, 12, 18, 24hours) in the 300 μmol/L MPP+ group and different concentrations of insulin (0, 15, 50, 100 nmol/L) in the insulin group was detected with MTT method according

  11. Activated microglia cause reversible apoptosis of pheochromocytoma cells, inducing their cell death by phagocytosis.

    Hornik, Tamara C; Vilalta, Anna; Brown, Guy C


    Some apoptotic processes, such as phosphatidylserine exposure, are potentially reversible and do not necessarily lead to cell death. However, phosphatidylserine exposure can induce phagocytosis of a cell, resulting in cell death by phagocytosis: phagoptosis. Phagoptosis of neurons by microglia might contribute to neuropathology, whereas phagoptosis of tumour cells by macrophages might limit cancer. Here, we examined the mechanisms by which BV-2 microglia killed co-cultured pheochromocytoma (PC12) cells that were either undifferentiated or differentiated into neuronal cells. We found that microglia activated by lipopolysaccharide rapidly phagocytosed PC12 cells. Activated microglia caused reversible phosphatidylserine exposure on and reversible caspase activation in PC12 cells, and caspase inhibition prevented phosphatidylserine exposur and decreased subsequent phagocytosis. Nitric oxide was necessary and sufficient to induce the reversible phosphatidylserine exposure and phagocytosis. The PC12 cells were not dead at the time they were phagocytised, and inhibition of their phagocytosis left viable cells. Cell loss was inhibited by blocking phagocytosis mediated by phosphatidylserine, MFG-E8, vitronectin receptors or P2Y6 receptors. Thus, activated microglia can induce reversible apoptosis of target cells, which is insufficient to cause apoptotic cell death, but sufficient to induce their phagocytosis and therefore cell death by phagoptosis.

  12. The interaction of manganese nanoparticles with PC-12 cells induces dopamine depletion.

    Hussain, Saber M; Javorina, Amanda K; Schrand, Amanda M; Duhart, Helen M; Ali, Syed F; Schlager, John J


    This investigation was designed to determine whether nano-sized manganese oxide (Mn-40 nm) particles would induce dopamine (DA) depletion in a cultured neuronal phenotype, PC-12 cells, similar to free ionic manganese (Mn(2+)). Cells were exposed to Mn-40 nm, Mn(2+) (acetate), or known cytotoxic silver nanoparticles (Ag-15 nm) for 24 h. Phase-contrast microscopy studies show that Mn-40 nm or Mn(2+) exposure did not greatly change morphology of PC-12 cells. However, Ag-15 nm and AgNO(3) produce cell shrinkage and irregular membrane borders compared to control cells. Further microscopic studies at higher resolution demonstrated that Mn-40 nm nanoparticles and agglomerates were effectively internalized by PC-12 cells. Mitochondrial reduction activity, a sensitive measure of particle and metal cytotoxicity, showed only moderate toxicity for Mn-40 nm compared to similar Ag-15 nm and Mn(2+) doses. Mn-40 nm and Mn(2+) dose dependently depleted DA and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), while Ag-15 nm only significantly reduced DA and DOPAC at concentrations of 50 mug/ml. Therefore, the DA depletion of Mn-40 nm was most similar to Mn(2+), which is known to induce concentration-dependent DA depletion. There was a significant increase (> 10-fold) in reactive oxygen species (ROS) with Mn-40 nm exposure, suggesting that increased ROS levels may participate in DA depletion. These results clearly demonstrate that nanoscale manganese can deplete DA, DOPAC, and HVA in a dose-dependent manner. Further study is required to evaluate the specific intracellular distribution of Mn-40 nm nanoparticles, metal dissolution rates in cells and cellular matrices, if DA depletion is induced in vivo, and the propensity of Mn nanoparticles to cross the blood-brain barrier or be selectively uptaken by nasal epithelium.

  13. The Study of the Transfection Efficiency of PC12 Cells Transfeced by Cationic Liposomes Lipofectamine2000%阳离子脂质体Lipofectamine2000对PC12细胞转染效率的研究

    杨云廷; 付崇罗


    通过改变转染试剂及DNA用量和转染时间等影响转染效率的重要因素来实现阳离子脂质体lipofectamineTM2000(lipo2000)对PC12细胞的高效转染.结果表明,lipo2000转染PC12细胞的最佳转染条件:lipo2000用量为5μL,DNA 2μg,转染时间为6 h,这种条件下的PC12细胞转染率高达40%,且未影响细胞的正常分泌,这为应用PC12细胞进行神经生物学研究提供了基础资料.

  14. Cholecystokinin-2 receptor mediated gene expression in neuronal PC12 cells

    Hansen, Thomas v O; Borup, Rehannah; Marstrand, Troels;


    Cholecystokinin (CCK) is abundantly expressed in the CNS, in which it regulates feeding behavior and long-term memory. Moreover, CCK has been implicated in mental disorders, such as anxiety and schizophrenia. Despite its manifest physiological and pathophysiological role, the molecular targets...... could be identified. Comparison with forskolin- and nerve growth factor (NGF)-treated PC12 cells showed that CCK induced a separate set of target genes. Taken together, we propose that neuronal CCK may have a role in the regulation of the circadian rhythm, the metabolism of cerebral cholesterol...

  15. Recycling of intact dense core vesicles in neurites of NGF-treated PC12 cells.

    Bauer, Roslyn A; Khera, Rebecca S; Lieber, Janet L; Angleson, Joseph K


    Exocytic fusion in neuroendocrine cells does not always result in complete release of the peptide contents from dense core vesicles (DCVs). In this study, we use fluorescence imaging and immunoelectron microscopy to examine the retention, endocytosis and recycling of chromogranin B in DCVs of NGF-treated PC12 cells. Our results indicate that DCVs retained and retrieved an intact core that was available for subsequent exocytic release. The endocytic process was inhibited by cyclosporine A or by substitution of extracellular Ca(2+) with Ba(2+) and the total recycling time was less than 5 min.

  16. Protective Effect of Cordycepin on PC12 Cells Injury Induced by β-amyloid Protein%虫草素对β-淀粉样蛋白诱导PC12细胞损伤的保护作用

    赵国华; 韩笑


    目的 应用Aβ25-35孵育PC12细胞制作细胞损伤模型,探讨虫草素对PC12细胞损伤的保护作用.方法 将不同组别PC12细胞用Aβ25-35诱导48 h后,采用MTT法测定细胞活力,LDH法测定细胞膜通透性,化学比色法测定细胞中SOD活性和MDA含量,Western blot法检测PC12细胞中Caspase-3的表达.结果 与模型组相比,两种剂量的虫草素均使细胞存活率显著增加,细胞培养上清中LDH含量显著减少,细胞中SOD活性显著升高,mDA含量显著下降,并明显降低PC12细胞Caspase-3的表达.结论 虫草素对Aβ325-35诱导的PC12细胞死亡、氧化损伤和细胞凋亡有明显保护作用.

  17. 硫化氢对H2O2损伤PC12细胞的保护作用%Protective Effect of Hydrogen Sulfide on H2O2-Induced Damage in PC12 Cells

    杨丝丝; 姜志胜; 唐小卿


    目的 探讨硫化氢对氧化应激损伤PC12细胞的保护作用.方法 以H2O2损伤PC12细胞为氧化应激损伤的模型,甲氮甲唑蓝法检测细胞增殖状况;Hoechst荧光染色观察细胞形态和核形态;碘化丙啶染色、流式细胞术检测细胞凋亡.结果 硫化氢可明显减少H2O2诱导的PC12细胞核呈浓染致密的固缩形态或颗粒状荧光的细胞数;200和400 μmol/L硫化氢均可降低200和400 μmol/L H2 O2作用24 h后对PC12细胞生长的抑制率,明显抑制200和400 μmol/L H2O2作用24 h后对PC12细胞凋亡的诱导作用.结论 硫化氢对氧化应激损伤PC12细胞具有保护作用.

  18. Differentiation of PC12 Cells Results in Enhanced VIP Expression and Prolonged Rhythmic Expression of Clock Genes

    Pretzmann, C.P.; Fahrenkrug, J.; Georg, B.


    PC12 cultures lasted only one 24-h period, while in differentiated cultures, the rhythms continued for at least 3 days. Thus, neuronal differentiation provided PC12 cells the ability to maintain rhythmicity for an extended period. Both vasoactive intestinal polypeptide (VIP) and its receptor VPAC(2......) are expressed in the suprachiasmatic nucleus (SCN), and in agreement with VIP signaling being crucial for maintenance of rhythmicity, we found both VIP and VPAC(2) mRNA increased after differentiation of PC12 cells. Pituitary adenylate cyclase activating polypeptide (PACAP) exerts time- and concentration-dependent......To examine for circadian rhythmicity, the messenger RNA (mRNA) amount of the clock genes Per1 and Per2 was measured in undifferentiated and nerve-growth-factor-differentiated PC12 cells harvested every fourth hour. Serum shock was needed to induce circadian oscillations, which in undifferentiated...

  19. EGb-761 Attenuates the Anti-proliferative Activity of Fluoride via DDK1 in PC-12 Cells.

    Zhang, Cai-Yi; Chen, Rui; Wang, Fen; Ren, Chao; Zhang, Peng; Li, Qian; Li, Hui-Hua; Guo, Ke-Tai; Geng, De-Qin; Liu, Chun-Feng


    EGb-761 is commonly used as a treatment for ischemic brain injury, neurodegenerative diseases and some types of tumors (Christen and Maixent, in Cell Mol Biol 48(6):601-611, 2002). However, it is unclear whether EGb-761 affects the proliferation of cells exposed to fluoride. In this study, the proliferation and apoptosis of PC-12 cells exposed to fluoride were investigated and EGb-761 was used to protect PC-12 cells against the effects of fluoride. We found that the canonical Wnt signaling pathway was involved in the anti-proliferation of PC-12 cells exposed to fluoride. Furthermore, the results also showed that EGb-761 could attenuate the anti-proliferative activity of fluoride via DDK1 in PC-12 cells. This study may provide a new method for protecting against the inhibition of cell proliferation induced by fluoride.


    张红; 王芳; 刘湧; 王建东


    [目的]探讨灵芝孢子提取物对Lactacystin诱导PC12细胞损伤的保护作用.[方法]体外培养PC12细胞,建立Lactacystin诱导PC12细胞损伤的模型,观察细胞形态,用CCK-8法检测细胞活力,annexinV-FITC/PI流式细胞法检测细胞调亡.[结果]用不同浓度的灵芝孢子提取物处理PC12细胞时,细胞存活率与对照几乎一致,并未表现出细胞毒性.经20 μmol/L的Lactacystin处理24h后,PC12细胞活力比对照组降低,仅为对照组的63.2%.模型组经不同浓度的是芝孢子提取物预处理后,细胞活力明显提高,其保护作用随浓度升高而升高.Lactacystin诱导PC12细胞凋亡,处理24h后细胞凋亡率为48.86%,经灵芝孢子提取物处理后,细胞凋亡率明显降低.[结论]Lactacystin 能诱PC12细胞凋亡,灵芝孢子提取物能对Lactacystin致PC12细胞损伤有一定的保护作用.%[Objective] To investigate the effect of Ganodenna lucidum spore extract (GLS extract) on PC12 cells damage iduced by Lactacystin. [Methods] The toxicity model was established by treating PC12 cells with Lactacystin in vitro. Observed the morphological changes of cells by microscope. Cell viability was assayed with CCk-8 method. The cell apoptotic rate was measured by flow cytometry (FCM) method. [Results] After being treated with different concentrations of GLS extract, the viability of PC12 cells as same as the cell control group tested by CCK-8 assay. After being treated with Lactacystin (20? Mol/L) for 24 h, the viability of PC12 cells depressed and the viable cells was only 63.2% when compared with cell control group. Pretreated with GLS extract at different concentrations, the cell viabilities were all increased significantly. The protective effect on cells increased with its concentration increased. Lactacystin induced apoptosis in PC12 cells, and the apoptotic rate was 48.86% after 24 h of treatment Pretreated with GLS extract, the cell apoptotic rates reduced. [Conclusion] Lactacystin

  1. Inhibition of Hydrogen Peroxide-induced PC12 Cell Apoptosis by Modafinil%莫达非尼抑制过氧化氢诱导的PC12细胞凋亡

    易宏伟; 刘国卿


    目的:研究莫达非尼(modafinil)对过氧化氢诱导的PC12细胞凋亡的抑制作用,并对其机制进行探讨.方法:以噻唑兰(MTT)法测定PC12细胞存活率;用流式细胞术检测PC12细胞凋亡的百分率;测定细胞内罗丹明123(Rhodamine123)的荧光强度,反映细胞线粒体膜电位的改变.结果:莫达非尼能抑制过氧化氢(200 μmol/L)、硝普钠(500μmol/L)和连二亚硫酸钠(2 mmol/L)对PC12细胞的损伤.过氧化氢(200 μmol/L)24h可以诱导PC12细胞凋亡(凋亡率为32.65%).莫达非尼(15,7.5 μmol/L)显著降低凋亡细胞的百分率(凋亡百分率分别为7.95%、15.46%).并且莫达非尼可以抑制过氧化氢引起的线粒体膜电位降低.结论:莫达非尼对PC12细胞有保护作用.能抑制过氧化氢诱导的PC12细胞凋亡,这一作用可能与其抑制过氧化氢引起的线粒体膜电位降低有关.%AIM: To study the inhibition of hydrogen peroxide-induced PC12 cell apoptosis by modafinil and its mechanism. METHOD: PC12 cell survival was measured by MTT assay. The percentage of apoptosis was monitored with flow cytometry. By measuring the intracellular rhodamine123(Rh123)fluorescence density,we evaluated the change of PC12 cell mitochondrial membrane potential (mmp) induced by hydrogen peroxide. RESULT:Modafinil attenuated the cytotoxic effect of hydrogen peroxide (200 μmol/L),sodium nitoprusside (500 μmol/L) and sodium dithionite (2mmol/L). Hydrogen peroxide (200 μmol/L,24 h) induced apoptosis in PC12 cells.PC12 cells were preconditioned with modafinil, then the percentage of apoptosis in the cells was decreased significantly to 7.95%,15.46%,and 17.52% from 32.65%. Modafinil inhibited the mmp decrease in PC12 cells induced by hydrogen peroxide. CONCLUSION:Modafinil prevented PC12 cell from apoptosis induced by hydrogen peroxide,and the mechanism might be associated with its effect of increasing the mitochondrial membrane potential of PC12 cell.

  2. Neuroprotective Effects of Exogenous Activin A on Oxygen-Glucose Deprivation in PC12 Cells

    Zhong-Xin Xu


    Full Text Available Ischemic cerebrovascular disease is one of the most common causes of death in the World. Exogenous activin A (ActA protects neurons against toxicity and plays a central role in regulating the brain’s response to injury. In the present study, we investigated the mechanisms involved in the neuroprotective effects of ActA in a model of hypoxic-ischemic brain disease. We found that ActA could effectively increase the survival rate of PC12 cells and relieve oxygen-glucose deprivation (OGD damage. To clarify the neuroprotective mechanisms of ActA, the effects of ActA on the ActA/Smad pathway and on the up-regulation of inducible nitric oxide synthase (NOS and superoxide dismutase (SOD were investigated using OGD in PC12 cells. The results showed that ActA could increase the expression of activin receptor IIA (ActRIIA, Smad3 and Smad4 and that 50 ng/mL and 100 ng/mL of ActA could reduce NO levels and increase SOD activity by 78.9% and 79.9%, respectively. These results suggested that the neuroprotective effects of ActA in ischemia could be related to the activation of the ActA/Smad signaling pathway and to its anti-oxidant activities.

  3. Protective Effects of Costunolide against Hydrogen Peroxide-Induced Injury in PC12 Cells

    Chong-Un Cheong


    Full Text Available Oxidative stress-mediated cellular injury has been considered as a major cause of neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases. The scavenging of reactive oxygen species (ROS mediated by antioxidants may be a potential strategy for retarding the diseases’ progression. Costunolide (CS is a well-known sesquiterpene lactone, used as a popular herbal remedy, which possesses anti-inflammatory and antioxidant activity. This study aimed to investigate the protective role of CS against the cytotoxicity induced by hydrogen peroxide (H2O2 and to elucidate potential protective mechanisms in PC12 cells. The results showed that the treatment of PC12 cells with CS prior to H2O2 exposure effectively increased the cell viability. Furthermore, it decreased the intracellular ROS, stabilized the mitochondria membrane potential (MMP, and reduced apoptosis-related protein such as caspase 3. In addition, CS treatment attenuated the cell injury by H2O2 through the inhibition of phosphorylation of p38 and the extracellular signal-regulated kinase (ERK. These results demonstrated that CS is promising as a potential therapeutic candidate for neurodegenerative diseases resulting from oxidative damage and further research on this topic should be encouraged.

  4. Histone deacetylase inhibitor attenuates neurotoxicity of clioquinol in PC12 cells.

    Fukui, Takao; Asakura, Kunihiko; Hikichi, Chika; Ishikawa, Tomomasa; Murai, Rie; Hirota, Seiko; Murate, Ken-Ichiro; Kizawa, Madoko; Ueda, Akihiro; Ito, Shinji; Mutoh, Tatsuro


    Clioquinol is considered to be a causative agent of subacute myelo-optico neuropathy (SMON), although the pathogenesis of SMON is yet to be elucidated. We have previously shown that clioquinol inhibits nerve growth factor (NGF)-induced Trk autophosphorylation in PC12 cells transformed with human Trk cDNA. To explore the further mechanism of neuronal damage by clioquinol, we evaluated the acetylation status of histones in PC12 cells. Clioquinol reduced the level of histone acetylation, and the histone deacetylase (HDAC) inhibitor Trichostatin A upregulated acetylated histones and prevented the neuronal cell damage caused by clioquinol. In addition, treatment with HDAC inhibitor decreased neurite retraction and restored the inhibition of NGF-induced Trk autophosphorylation by clioquinol. Thus, clioquinol induced neuronal cell death via deacetylation of histones, and HDAC inhibitor alleviates the neurotoxicity of clioquinol. Clioquinol is now used as a potential medicine for malignancies and neurodegenerative diseases. Therefore, HDAC inhibitors can be used as a candidate medicine for the prevention of its side effects on neuronal cells.

  5. JWA protein binds to α-tubulin in PC12 cells

    CHEN Hairong; LI Aiqun; LI Aiping; ZHOU Jianwei


    Our previous study elucidated that JWA protein was a newly identified microtubule-associated protein (MAP), which combined to and co-localized with β-tubulin.In the present study, we designed a series of experiments to explore if any interactions between JWA protein and α-tubulin existed and how JWA protein would functionally link to α-tubulin, especially in cell mitosis. Results of coimmunoprecipitation, gene transfection and immunofluorescence microscopy from PC12 and HEK293 cells provided strong evidence for a linkage between JWA protein and α-tubulin. Our data showed that JWA protein bound to α-tubulin stably no matter whether α-tubulin was polymerized or not. In addition, by using antisense oligonucleotides, cell cycle blocking agents and hypothermia disposal techniques,we also found the interaction between JWA protein and α-tubulin. The further analysis using flow cytometry and confocal microscopy showed that both proteins co-existed in PC12 cells and were independent on the cell cycle. In conclusion, JWA protein is a newly identified microtubuleassociated protein, binds to α-tubulin, and probably plays an important role in regulation of microtubular stability.

  6. Toxicity induced by cumene hydroperoxide in PC12 cells: protective role of thiol donors.

    Vimard, F; Saucet, M; Nicole, O; Feuilloley, M; Duval, D


    Oxidative shock and production of reactive oxygen species are known to play a major role in situations leading to neuron degeneration, but the precise mechanisms responsible for cell degeneration remain uncertain. In the present article, we have studied in PC 12 cells the effect of cumene hydroxyperoxide on both cell metabolism and morphology. We observed that relatively low concentrations of the drug (100 μM) led to a significant decrease in the cellular content of ATP and reduced glutathione as well as to a decreased mitochondrial potential. These metabolic alterations were followed by an important increase in intracellular free calcium and membrane disruption and death. In parallel, we observed profound changes in cell morphology with a shortening of cell extensions, the formation of ruffles and blebs at the cell surface, and a progressive detachment of the cells from the surface of the culture flasks. We also showed that addition of thiol donors such as N-acetylcysteine or β-mercaptoethanol, which were able to enhance cell glutathione content, almost completely protected PC 12 cells from the toxic action of cumene hydroperoxide whereas pretreatment by buthionine sulfoximine, a selective inhibitor of GSH synthesis, enhanced its action.

  7. Inhibition of ROCK2 expression protects against methamphetamine-induced neurotoxicity in PC12 cells.

    Yang, Xingyi; Liu, Yunyun; Liu, Chao; Xie, Weibing; Huang, Enping; Huang, Weiye; Wang, Jiawen; Chen, Ling; Wang, Huipin; Qiu, Pingming; Xu, Jingtao; Zhang, Fu; Wang, Huijun


    Methamphetamine is a type of psychoactive drug. It is well known that neurotoxicity caused by Methamphetamine(METH) can damage the nervous system and lead to apoptosis and cell loss of dopaminergic neurons. ROCK2 is a prominent target for gene therapy because its inhibition has proved to have a protective effect in various cell lines and pathophysiological conditions. Although several of the negative effects of METH on the dopaminergic system have been studied, the protective molecular mechanisms and the effective treatment of METH-induced apoptosis remain to be clarified. We hypothesized that ROCK2 is involved in METH-induced apoptosis. We tested our hypothesis using RT-PCR and western blotting to analyze whether silencing of ROCK2 with small interfering RNA (siROCK2) could reduce damage and apoptosis in PC12 cells after METH exposure. Increases in viability and cytomorphological changes were detected by MTT assay and bright field microscopy after pretreatment of METH-treated PC12 cells with 100 nM siROCK2. Apoptosis decreased significantly after ROCK2 silencing, as shown by Annexin V and TUNEL staining. The results show that ROCK2 is a possible gene target for therapeutics in METH-induced neurotoxicity in vitro, providing a foundation for future in vivo research.

  8. Functional-dependent and size-dependent uptake of nanoparticles in PC12

    Sakai, N; Matsui, Y; Nakayama, A; Yoneda, M [Department of Environment Engineering, Graduate School of Engineering, Kyoto University, 4 Kyotodaigaku Katsura, Nishikyo-ku, Kyoto 6158540 (Japan); Tsuda, A, E-mail: [Department of Environmental Health, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115 (United States)


    It is suggested that the uptake of nanoparticles is changed by the particle size or the surface modification. In this study, we quantified the uptake of nanoparticles in PC12 cells exposed Quantum Dots with different surface modification or fluorescent polystyrene particles with different particle size. The PC12 cells were exposed three types of the Quantum Dots (carboxyl base-functionalized, amino base-functionalized or non-base-functionalized) or three types of the fluorescent particles (22 nm, 100 nm or 1000 nm) for 3 hours. The uptake of the nanoparticles was quantified with a spectrofluorophotometer. The carboxyl base-functionalized Quantum Dots were considerably taken up by the cells than the non-base-functionalized Quantum Dots. Conversely, the amino base-functionalized Quantum Dots were taken up by the cells less frequently than the non-base-functionalized Quantum Dots. The particle number of the 22 nm-nanoparticles taken up by the cells was about 53 times higher than the 100 nm-particles. However, the particle weight of the 100 nm-particles taken up by the cells was higher than that of the 22 nm-nanoparticles. The 1000 nm-particles were adhered to the cell membrane, but they were little taken up by the cells. We concluded that nanoparticles can be taken up nerve cells in functional-dependent and size-dependent manners.

  9. Antibody-mediated inhibition of Nogo-A signaling promotes neurite growth in PC-12 cells

    Iman K Yazdi


    Full Text Available The use of a monoclonal antibody to block the neurite outgrowth inhibitor Nogo-A has been of great interest for promoting axonal recovery as a treatment for spinal cord injury. While several cellular and non-cellular assays have been developed to quantify the bioactive effects of Nogo-A signaling, demand still exists for the development of a reliable approach to characterize the effectiveness of the anti-Nogo-A antibody. In this study, we developed and validated a novel cell-based approach to facilitate the biological quantification of a Nogo-A antibody using PC-12 cells as an in vitro neuronal cell model. Changes in the mRNA levels of the neuronal differentiation markers, growth-associated protein 43 and neurofilament light-polypeptide, suggest that activation of the Nogo-A pathway suppresses axonal growth and dendrite formation in the tested cell line. We found that application of anti-Nogo-A monoclonal antibody can significantly enhance the neuronal maturity of PC-12 cells by blocking the Nogo-A inhibitory effects, providing enhanced effects on neural maturity at the molecular level. No adverse effects were observed on cell viability.

  10. The protective effect of the insulin against paraquat-induced PC12 cells%胰岛素对百草枯诱导的PC12细胞的保护作用

    邬剑军; 蒋雨平; 张晨


    Objective To observe dopamine (DA) D2R protein expression in the cultured PC 12 cells and the effect of insulin on the survival rate,morphology,and DA of paraquat (PQ)-induced PC12 cells. Methods Immunoprecipitate Western blotting method was performed to observe DA D2R protein expression in PC 12 cells,MTT assay was used to analyze the changes in viability and morphology of PC 12 ceils which were exposed to different concentrations of PQ and insulin. Results (1) DA D2R protein was expressed in PC 12 ceils. (2) Normal PCI2 cells bodies showed fusiform shape and the synapses were in-tegrity. The cells which were exposed to the 600 μmol/L PQ became ball-like, vacuolar degeneration oc-urred,and the synapse became shorter or disappeared. But the morphology of PC12 cells had a little difference between the normal PC12 and the insulin groups,except that the cells were fusiform shape or a-nomalism but not round shape,and the synapses grew. (3) With the increase of the concentration of PQ, the viability of the cells was decreased. Insulin increased the viability of the ceils which were exposed to 600 μmol/L PQ. Insulin elevated DA concentration both in the normal PC12 cells and those exposed to 600 μmol/L PQ,but there was no significant difference ( P > 0.05 ). Conclusion Insulin could protect the PC12 dopaminergic neurons from injury induced by PQ.%目的 观察培养的PC12细胞中多巴胺D2受体(DA D2R)蛋白的表达,胰岛素对百草枯(paraquat,PQ)诱导的PC12细胞形态学、生存率和DA含量的影响.方法 应用免疫沉淀Western印迹分析技术对培养的PC12细胞中DA D2R表达的检测;应用二甲基噻唑二苯基四唑溴盐(MTT)法,观察PC12细胞暴露于不同浓度PQ组和胰岛素预先干预后再暴露于PQ组后细胞形态学和生存率的改变;应用酶联免疫吸附试验(ELISA)方法检测空白对照PC12细胞组、PQ干预组、胰岛素组和胰岛素加PQ组PC12细胞上清液DA的浓度.结果 (1)Western法检测到培养的PC

  11. Panaxynol induces neurite outgrowth in PC12D cells via cAMP- and MAP kinase-dependent mechanisms.

    Wang, Ze-Jian; Nie, Bao-Ming; Chen, Hong-Zhuan; Lu, Yang


    Panaxynol, a polyacetylene ((3R)-heptadeca-1,9-diene-4,6-diyn-3-ol; syn. falcarinol), was isolated from the lipophilic fractions of Panax notoginseng, a Chinese traditional medicinal plant. In the present study, we reported the neurotrophic effects of panaxynol on PC12D cells and mechanism involved in neurite outgrowth of the cells. Panaxynol could morphologically promote neurite outgrowth in PC12D cells, concentration-dependently reduce cell division and up-regulate molecular marker (MAP1B) expression in PC12D cells. Panaxynol induces the elevation of intracellular cAMP in PC12D cells. The neurite outgrowth in PC12D cells induced by panaxynol could be inhibited by the protein kinase A inhibitor RpcAMPS and by MAP kinase kinase 1/2 inhibitor U0126. These observations reveal that panaxynol could induce the differentiation of PC12D cells in a process similar to but distinct from that of NGF and the panaxynol's effects were via cAMP- and MAP kinase-dependent mechanisms.

  12. Evaluation of antidepressant effect and protective effect against corticosterone-induced PC12 cell injury of icariin%淫羊藿苷抗抑郁及对皮质酮致PC12细胞损伤的保护作用研究

    石翠格; 李慧; 王丽丽; 王宁; 王介东; 张树成; 徐志卿


    Objective:To investigate the antidepressant effect and the protective effect against corticosterone-induced PC12 cell neurotoxity of icariin. Methods:The antidepressant effects of icariin were evaluated by the tail suspension test in mice and the forced swimming tests in rats and mice. The animals were divided into four groups randomly:control group, low dose icariin group, high dose icariin group and amitriptyline group. The effects of icariin on immobility time were measured in the forced swimming test in rats and mice, together with the tail suspension test in mice. Likewise, PC12 cell injury model was induced by corticosterone and the protective effect of icariin on the cell viability was measured by MTT assay. Results:Icariin could signiifcantly decrease the immobility time in tail suspension test in mice, as well as the forced swimming tests in rats and mice, with the signiifcant differences when compared with the control group. In addition, icariin could improve the survival rate of PC12 cells injuried by corticosterone. Conclusion:Icariin possessed antidepressant effect and its antidepressant effect maybe involve in the protective effects of neural cells.%目的::研究淫羊藿苷的抗抑郁及对皮质酮致PC12细胞损伤的保护作用。方法:采用大/小鼠强迫游泳、小鼠悬尾三种实验模型,将动物随机分为对照组、淫羊藿苷低剂量组、淫羊藿苷高剂量组、阿米替林组,观察药物对大/小鼠强迫游泳不动时间、小鼠悬尾不动时间的影响;并在细胞水平建立皮质酮损伤PC12细胞模型,观察淫羊藿苷的细胞保护作用。结果:在大/小鼠强迫游泳和小鼠悬尾实验中,淫羊藿苷可显著缩短大/小鼠的强迫游泳不动时间和小鼠悬尾不动时间,与对照组相比差异有统计学意义(P <0.05);在皮质酮损伤PC12细胞的模型上,淫羊藿苷可显著提高PC12细胞的存活率,拮抗皮质酮诱导的细胞损伤作用。结

  13. 他莫昔芬介导小胶质细胞 LRRK-2表达对炎症刺激 PC12细胞的影响%Effect of Tamoxifen on Inflammation-Stimulated PC12 Cells Mediated by LRRK-2 Gene Expression of Microglia

    卢奎; 吴文军; 周敏; 黎捷; 钟健强; 张成


    Objective To study the effect of tamoxifen on the expressions of leucine-rich repeat kinase 2(LRRK-2) gene of microglia and related inflammatory factors .Methods Primary microglia was divided into 4 groups including control group,lipopolysaccharide(LPS) group,LPS plus tamoxifen group and LPS plus LRRK-2 group.LPS-activated microglia was regulated by tamoxifen .PC12 cells of rats were co-cultured with microglia for 24 hours in each group .The expressions of LRRK-2 gene and iNOS were detected by Western Blot .The levels of tumor necrosis factor α( TNF-α) and interleukin 1β(IL-1β) were tested by enzyme-linked immunosorbent assay ( ELISA).The apoptosis/survival ratio of PC12 cells was determined by Hochest staining .Results Tamoxifen down-regulated the LRRK-2 gene expression of microglia by LPS stimulation,inhibited the release of iNOS ,TNF-αand IL-1βby suppressing LRRK-2 gene,and decreased the co-cultured inflammatory expression of microglia and PC12 cells as well as apoptosis/survival ratio of PC12 cells.Conclusion LRRK-2 might be an upstream-regulated target for the release of iNOS in microglia .Tamoxifen can inhibit the activation of microglia by regulation of LRRK-2 gene,which might alleviate the inflammation-associated dopamine neuron injury .%目的:探讨他莫昔芬对小胶质细胞的富含亮氨酸重复序列激酶2( LRRK2)基因及相关炎症因子表达的影响。方法取原代小胶质细胞分为CON组、脂多糖( LPS)组、LPS+他莫昔芬组、LPS+LRRK2组;以LPS刺激激活小胶质细胞,用他莫昔芬调控小胶质细胞激活;将各组小胶质细胞与大鼠肾上腺嗜铬瘤细胞( PC12)细胞共培养24 h;Western Blot法检测小胶质细胞的LRRK2、一氧化氮合成酶( iNOS )表达,酶联免疫吸附法( ELISA)测定TNF-α、IL-1β水平;Hochest染核法检测PC12细胞凋亡/存活比。结果他莫昔芬抑制LPS刺激后小胶质细胞的LRRK2表达,通过抑制LRRK2

  14. Inhibition of Voltage-Gated Calcium Channels After Subchronic and Repeated Exposure of PC12 Cells to Different Classes of Insecticides.

    Meijer, Marieke; Brandsema, Joske A R; Nieuwenhuis, Desirée; Wijnolts, Fiona M J; Dingemans, Milou M L; Westerink, Remco H S


    We previously demonstrated that acute inhibition of voltage-gated calcium channels (VGCCs) is a common mode of action for (sub)micromolar concentrations of chemicals, including insecticides. However, because human exposure to chemicals is usually chronic and repeated, we investigated if selected insecticides from different chemical classes (organochlorines, organophosphates, pyrethroids, carbamates, and neonicotinoids) also disturb calcium homeostasis after subchronic (24 h) exposure and after a subsequent (repeated) acute exposure. Effects on calcium homeostasis were investigated with single-cell fluorescence (Fura-2) imaging of PC12 cells. Cells were depolarized with high-K(+) saline to study effects of subchronic or repeated exposure on VGCC-mediated Ca(2+) influx. The results demonstrate that except for carbaryl and imidacloprid, all selected insecticides inhibited depolarization (K(+))-evoked Ca(2+) influx after subchronic exposure (IC50's: approximately 1-10 µM) in PC12 cells. These inhibitory effects were not or only slowly reversible. Moreover, repeated exposure augmented the inhibition of the K(+)-evoked increase in intracellular calcium concentration induced by subchronic exposure to cypermethrin, chlorpyrifos, chlorpyrifos-oxon, and endosulfan (IC50's: approximately 0.1-4 µM). In rat primary cortical cultures, acute and repeated chlorpyrifos exposure also augmented inhibition of VGCCs compared with subchronic exposure. In conclusion, compared with subchronic exposure, repeated exposure increases the potency of insecticides to inhibit VGCCs. However, the potency of insecticides to inhibit VGCCs upon repeated exposure was comparable with the inhibition previously observed following acute exposure, with the exception of chlorpyrifos. The data suggest that an acute exposure paradigm is sufficient for screening chemicals for effects on VGCCs and that PC12 cells are a sensitive model for detection of effects on VGCCs. © The Author 2015. Published by Oxford

  15. Ac-cel, a novel antioxidant, protects against hydrogen peroxide-induced injury in PC12 cells via attenuation of mitochondrial dysfunction.

    Guo, Xianjun; Chen, Yuting; Liu, Qunfang; Wu, Jian; Wang, Luoyi; Tang, Xican; Zhao, Weimin; Zhang, Haiyan


    Oxidative stress has been implicated in pathophysiology of many neurodegenerative diseases (ND) and increased oxidative stress is closely associated with mitochondrial dysfunction. As a result, looking for potent antioxidants, especially those targeting mitochondria, has become an attractive strategy in ND therapy. In this study, we explored protective effects and potential mechanism of Ac-cel, a novel compound, against hydrogen peroxide (H(2)O(2))-induced injury in PC12 cells. Pretreatment of PC12 cells with Ac-cel prior to 24 h of H(2)O(2) exposure markedly attenuated cytotoxicity induced by H(2)O(2) as evidenced by morphological changes and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Ac-cel also exhibited potent antiapoptotic effect demonstrated by results of annexin V and PI staining. The above beneficial effects of Ac-cel were accompanied by improved mitochondrial function, reduced caspase-3 cleavage as well as upregulated ratio of Bcl-2/Bax protein expression. Moreover, Ac-cel pretreatment markedly reversed intracellular reactive oxygen species (ROS) accumulation following 30 min of H(2)O(2) exposure in PC12 cells. Further, subcellular investigation indicated that Ac-cel significantly reduced production of mitochondrial ROS in isolated rat cortical mitochondria. Taken together, the present study, for the first time, reports that Ac-cel pretreatment inhibits H(2)O(2)-stimulated early accumulation of intracellular ROS possibly via reducing mitochondrial ROS production directly and leads to subsequent preservation of mitochondrial function. These results indicate that Ac-cel is a potential drug candidate for treatment of oxidative stress-associated ND.

  16. 三七总皂苷保护PC12细胞对抗过氧化氢损伤的作用%Effect of Panax notoginseng Saponins Protect PC12 Cell Against Hydrogen Peroxide Induced Damage

    夏星; 钟振国; 冯丹霞


    目的:考察三七总皂苷在PC12细胞中对过氧化氢引起损伤的细胞保护作用.方法:采用MTT法考察0.01~100mg·L-1三七总皂苷对正常PC12细胞活力的影响,并在H2O2刺激的PC12细胞中考察0.1~10 mg·L-1的三七总皂苷对细胞活力的影响,通过对细胞活力的考察选择三七总皂苷合适的实验剂量,测定三七总皂苷0.1,1 mg·L-1对H2O2刺激的PC12细胞中活性氧水平、过氧化氢酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活力的影响以及对丙二醛(MDA)含量的影响.结果:三七总皂苷在0.1~10 mg·L-1浓度能提高正常PC12细胞活力,并能显著提高H202刺激的PC12细胞活力(P<0.01).0.1,1mg·L-12个剂量的三七总皂苷均能显著减少H2O2刺激的PC12细胞中活性氧水平,提高SOD活力(P <0.001)和GSH-Px活力(P <0.001),并减少MDA生成(P <0.001).结论:三七总皂苷能通过增强细胞内抗氧化酶活力,保护PC12细胞对抗氧化损伤.%Objective: To investigate the protective effect of Panax notoginseng saponins ( PNS) on hydrogen peroxide ( H2O2 ) induced oxidative damage in PC 12 cell. Method: MTT method was employed to determine the viability in PNS (0.01-100 mg·L1) treated PC12 cell and PNS (0. 1-10 mg ·L-1) plus H2O2 treated PC12 cell. Thereafter, intracellular reactive oxygen species amounts, activity of SOD, GSH-Px, as well as MDA concentration were determined at optimal dosage of PNS selected based on MTT experiment. Result; PC12 cell viability was increased by PNS at 0. 1-10 mg ·L-1, and the viability of H2O2 treated PC12 cell was increased by PNS at 0. 1-10 mg ·L-1. Furthermore, PNS at 0. 1 -1. 0 mg ·L-1 significantly decreased intracellular reactive oxygen species levels, as well as enhanced SOD activity (P < 0. 001) and GSH-Px activity ( P < 0. 001). But MDA concentration was decreased significantly (P < 0. 05 ). Conclusion: PNS can protect PC12 cell against H2O2 induced damage via enhancement of intracellular antioxidant

  17. Apoptosis of PC12 cell Induced by Amyloid Beta-peptide Fragment 25-35%β-淀粉样蛋白25-35片段诱导PC12细胞凋亡

    罗蔓; 谢瑞满


    目的探讨β-淀粉样蛋白25-35片段(Aβ25-35)对体外培养的PC12细胞的毒性作用机制.方法用四甲基偶氮唑蓝(MTT)代谢率检测,光镜吖啶橙荧光染色术,透射电镜以及流式细胞仪技术研究Aβ25-35损伤PC12细胞的途径.结果用Aβ25-35处理PC12细胞24 h,Aβ25-35剂量依赖性地引起PC12细胞的MTT代谢率减少,荧光染色及电镜观察发现经Aβ25-35处理的PC12细胞表现出凋亡细胞的特征,流式细胞仪检测发现,对照组20μmol/L及50 μmol/L的Aβ25-35组PC12细胞的凋亡率分别为0.08%±0.01%,14.8%±1.13%,25.9%±2.34%.结论Aβ对PC12细胞的损伤主要通过细胞凋亡的途径.


    王维维; 贾凤菊; 宋宁; 谢俊霞; 姜宏


    Objective To investigate the influence of phthalandione fucoidan (PHF) on PC12 cell viability and the action of PHF on the cells.Methods MTT assay was used to detect the effect of different-concentration PHF on survival rate of PC12 cells.Results Lower doses of PHF (0.001 g/L and 0.010 g/L) had no effect on the cell viability.However,higher doses of PHF (0.100,1.000,10.000 g/L) could significantly reduce the cell viability,the most reduction was noted in the group of 10.000 g/L PHF.Conclusion Low doses of PHF do not affect the survival of PC 12 cells,and high doses result in dose-dependent toxic effect.%目的 通过观察苯甲酰化褐藻多糖(PHF)对PC12细胞存活率的影响,探讨PHF对神经细胞的作用.方法 采用四甲基偶氮唑盐(MTT)方法,检测不同浓度PHF作用下PC12细胞的存活率.结果 低剂量的PHF(0.001、0.010 g/L)对PC12细胞的存活率没有影响;较高剂量的PHF(0.100、1.000、10.000 g/L)可以显著降低PC12细胞的存活率,10.000 g/L PHF处理组细胞的存活率下降最明显.结论 低剂量的PHF不影响PC12细胞的存活率,而较高浓度的PHF能显著降低PC12细胞的存活率,产生剂量依赖性的毒性作用.

  19. 曲古抑菌素A对缺糖/缺氧损伤PC12细胞的保护作用%Protective effect of TSA on PC12 cell injury induced by the deprivation of oxygen/glucose

    越茂松; 黄琼; 王进京; 季秋虹; 季煜华


    Aim To study the protective effects of his-tone deacetylase inhibitor ( HDACi ) , trichostatin A ( TSA ), on oxygen/glucose deprivation ( OGD ) inju-ried PC 12 cells and its underlying mechanism. Methods PC 12 OGD injury model was established by using cobalt chloride ( CoCl2 ) and glucose-free medium , and treated by 1-640 nmol · L-1 TSA. Cell viability was measured by MTT assay, cell apoptosis and necrosis was observed by PI and Hoechst staining, and flow cytometry detection and fluorescence microscope were applied to determine the reactive oxygen species in each group. Results Compared with control group, 80 nmol · L-1 TSA significantly improved cell viability and reduced intracellular reactive oxygen species ( ROS ). Conclusions TSA protects the OGD injured PC 12 cells. The possible underlying mechanisms may be related to maintaining or increasing the acetylation level of the energy metabolism enzymes, whereby keeping the PC12 cells from the injury induced by OGD.%目的 研究组蛋白去乙酰化酶抑制剂曲古抑菌素A(trichostatin A,TSA)对PC12大鼠肾上腺嗜铬细胞瘤细胞缺糖/缺氧损伤的保护作用及其可能的作用机制.方法 建立PC12细胞缺糖/缺氧 (oxygen and glucose deprivation,OGD) 损伤模型,1-640 nmol·L-1 TSA处理细胞,噻唑蓝(MTT)检测对PC12细胞活性的影响,Propidium iodide (PI)和Hoechst 33258染色检测细胞凋亡与坏死,荧光显微镜和流式细胞仪检测各组细胞内活性氧(ROS)的含量.结果 与模型组相比,80 nmol·L-1 TSA可明显提高ODG PC12的细胞存活率,降低细胞内的ROS含量(P<0.05).结论 TSA对OGD损伤的PC12细胞具有保护作用,其机制可能是通过增加细胞内能量代谢中各种酶的乙酰化水平来应对缺血损伤.

  20. [Pheochromocytoma and von Recklinghausen's disease].

    Rabii, R; Fekak, H; Moufid, K; Joual, A; Bennani, S; el Mrini, M; Benjelloun, S


    The association between von Recklinghausen's disease and pheochromocytoma is present about 10% of cases. We report a case of 49 years old women who presented with elevated blood pressure and von Recklinghausen's neurofibromatosis. Laboratory examination showed a marked level in the urinary excretion of cathecholamine. The computed tomography showed a right adrenal tumor suggesting a pheochromocytoma. The adrenalectomy was realised by transabdominal approach and the histological examination confirmed a benign pheochromocytoma. The authors discuss the pathogenetic hypothesis of this rare pathological association, the diagnostic methods and the therapeutic procedure.

  1. Enhanced neurite outgrowth of PC-12 cells on graphene-monolayer-coated substrates as biomimetic cues

    Lee, Jong Ho; Shin, Yong Cheol; Jin, Oh Seong; Han, Dong-Wook; Kang, Seok Hee; Hong, Suck Won; Kim, Jong Man


    Neurons are electrically excitable cells that transmit and process information in the nervous system. Recently, the differentiation of human neural stem cells to neurons has been shown to be enhanced on graphene substrates, and differentiated neurons have been shown to be able to still carry electrical signals when stimulated by graphene electrodes. Graphene films grown by using chemical vapor deposition were transferred onto glass coverslips by using the scooping method and were then coated with fetal bovine serum for a neuronal cell culture. The graphene substrates as biomimetic cues have been shown to enhance the neurite outgrowth of PC-12 cells. Our findings suggest that graphene has a unique surface property that can promote neuronal cells, which should open tremendous opportunities in neuroscience, neural engineering and regenerative medicine.

  2. Preparation of a hydrophobic polythiophene film to improve protein adsorption and proliferation of PC 12 cells.

    Li, Da-Feng; Wang, Hua-Jie; Fu, Jian-Xi; Wang, Wei; Jia, Xue-Shun; Wang, Jin-Ye


    High quality films of polythiophene with different alkyl side chains were successfully synthesized by a novel method in the presence of sodium dodecylbenzenesulonate (SDBS) under N2 atmosphere on the PTFE (polytetrafluorethylene) substrate. The as-prepared films were characterized by scanning electron microscopy (SEM), conductivity measurement, and water contact angle measurement. The morphologies of the films were homogeneous with micro-/nanostructures, and their conductivities were high enough for biomedical applications. Hydrophobicity of the films could be adjusted easily by inducing alkyl side chains with different length, which could control protein adsorption in succession. Hydrophobic polythiophene film with a long alkyl side chain had a higher ability of protein adsorption and PC 12 cell proliferation. The biocompatibility study of the synthesized films in vitro proved that the synthesized films were not cytotoxic to two cell lines used and could support cell attachment and proliferation well. Polythiophenes films prepared by in-situ deposition will be good candidates for biomedical applications.

  3. Silence of Synaptotagmin VII inhibits release of dense core vesicles in PC12 cells


    Synaptotagmin VII (Syt VII), which has a higher Ca2+ affinity and slower disassembly kinetics with lipid than Syt I and Syt IX, was regarded as being uninvolved in synaptic vesicle (SV) exocytosis but instead possibly as a calcium sensor for the slower kinetic phase of dense core vesicles (DCVs) release. By using high temporal resolution capacitance and amperometry measurements, it was demonstrated that the knockdown of endogenous Syt VII attenuated the fusion of DCV with the plasma membrane, reduced the amplitude of the exocytotic burst of the Ca2+-triggered DCV release without affecting the slope of the sustained component, and blocked the fusion pore expansion. This suggests that Syt VII is the Ca2+ sensor of DCV fusion machinery and is an essential factor for the establishment and maintenance of the pool size of releasable DCVs in PC12 cells.

  4. Rab3D is critical for secretory granule maturation in PC12 cells.

    Tanja Kögel

    Full Text Available Neuropeptide- and hormone-containing secretory granules (SGs are synthesized at the trans-Golgi network (TGN as immature secretory granules (ISGs and complete their maturation in the F-actin-rich cell cortex. This maturation process is characterized by acidification-dependent processing of cargo proteins, condensation of the SG matrix and removal of membrane and proteins not destined to mature secretory granules (MSGs. Here we addressed a potential role of Rab3 isoforms in these maturation steps by expressing their nucleotide-binding deficient mutants in PC12 cells. Our data show that the presence of Rab3D(N135I decreases the restriction of maturing SGs to the F-actin-rich cell cortex, blocks the removal of the endoprotease furin from SGs and impedes the processing of the luminal SG protein secretogranin II. This strongly suggests that Rab3D is implicated in the subcellular localization and maturation of ISGs.

  5. The role of the cytoskeleton in volume regulation and beading transitions in PC12 neurites

    Fernandez, Pablo


    We present investigations on volume regulation and beading shape transitions in PC12 neurites conducted using a flow-chamber technique. By disrupting the cell cytoskeleton with specific drugs we investigate the role of its individual components in the volume regulation response. We find that microtubule disruption increases both swelling rate and maximum volume attained, but does not affect the ability of the neurite to recover its initial volume. In addition, investigation of axonal beading --also known as pearling instability-- provides additional clues on the mechanical state of the neurite. We conclude that the initial swelling phase is mechanically slowed down by microtubules, while the volume recovery is driven by passive diffusion of osmolites. Our experiments provide a framework to investigate the role of cytoskeletal mechanics in volume homeostasis.

  6. Pheochromocytomas and secreting paragangliomas

    Gimenez-Roqueplo Anne-Paule


    Full Text Available Abstract Catecholamine-producing tumors may arise in the adrenal medulla (pheochromocytomas or in extraadrenal chromaffin cells (secreting paragangliomas. Their prevalence is about 0.1% in patients with hypertension and 4% in patients with a fortuitously discovered adrenal mass. An increase in the production of catecholamines causes symptoms (mainly headaches, palpitations and excess sweating and signs (mainly hypertension, weight loss and diabetes reflecting the effects of epinephrine and norepinephrine on α- and β-adrenergic receptors. Catecholamine-producing tumors mimic paroxysmal conditions with hypertension and/or cardiac rhythm disorders, including panic attacks, in which sympathetic activation linked to anxiety reproduces the same signs and symptoms. These tumors may be sporadic or part of any of several genetic diseases: familial pheochromocytoma-paraganglioma syndromes, multiple endocrine neoplasia type 2, neurofibromatosis 1 and von Hippel-Lindau disease. Familial cases are diagnosed earlier and are more frequently bilateral and recurring than sporadic cases. The most specific and sensitive diagnostic test for the tumor is the determination of plasma or urinary metanephrines. The tumor can be located by computed tomography, magnetic resonance imaging and metaiodobenzylguanidine scintigraphy. Treatment requires resection of the tumor, generally by laparoscopic surgery. About 10% of tumors are malignant either at first operation or during follow-up, malignancy being diagnosed by the presence of lymph node, visceral or bone metastases. Recurrences and malignancy are more frequent in cases with large or extraadrenal tumors. Patients, especially those with familial or extraadrenal tumors, should be followed-up indefinitely.

  7. Boolean Modeling Reveals the Necessity of Transcriptional Regulation for Bistability in PC12 Cell Differentiation.

    Offermann, Barbara; Knauer, Steffen; Singh, Amit; Fernández-Cachón, María L; Klose, Martin; Kowar, Silke; Busch, Hauke; Boerries, Melanie


    The nerve growth factor NGF has been shown to cause cell fate decisions toward either differentiation or proliferation depending on the relative activity of downstream pERK, pAKT, or pJNK signaling. However, how these protein signals are translated into and fed back from transcriptional activity to complete cellular differentiation over a time span of hours to days is still an open question. Comparing the time-resolved transcriptome response of NGF- or EGF-stimulated PC12 cells over 24 h in combination with protein and phenotype data we inferred a dynamic Boolean model capturing the temporal sequence of protein signaling, transcriptional response and subsequent autocrine feedback. Network topology was optimized by fitting the model to time-resolved transcriptome data under MEK, PI3K, or JNK inhibition. The integrated model confirmed the parallel use of MAPK/ERK, PI3K/AKT, and JNK/JUN for PC12 cell differentiation. Redundancy of cell signaling is demonstrated from the inhibition of the different MAPK pathways. As suggested in silico and confirmed in vitro, differentiation was substantially suppressed under JNK inhibition, yet delayed only under MEK/ERK inhibition. Most importantly, we found that positive transcriptional feedback induces bistability in the cell fate switch. De novo gene expression was necessary to activate autocrine feedback that caused Urokinase-Type Plasminogen Activator (uPA) Receptor signaling to perpetuate the MAPK activity, finally resulting in the expression of late, differentiation related genes. Thus, the cellular decision toward differentiation depends on the establishment of a transcriptome-induced positive feedback between protein signaling and gene expression thereby constituting a robust control between proliferation and differentiation.

  8. Organic Photovoltaics and Bioelectrodes Providing Electrical Stimulation for PC12 Cell Differentiation and Neurite Outgrowth.

    Hsiao, Yu-Sheng; Liao, Yan-Hao; Chen, Huan-Lin; Chen, Peilin; Chen, Fang-Chung


    Current bioelectronic medicines for neurological therapies generally involve treatment with a bioelectronic system comprising a power supply unit and a bioelectrode device. Further integration of wireless and self-powered units is of practical importance for implantable bioelectronics. In this study, we developed biocompatible organic photovoltaics (OPVs) for serving as wireless electrical power supply units that can be operated under illumination with near-infrared (NIR) light, and organic bioelectronic interface (OBEI) electrode devices as neural stimulation electrodes. The OPV/OBEI integrated system is capable to provide electrical stimulation (ES) as a means of enhancing neuron-like PC12 cell differentiation and neurite outgrowth. For the OPV design, we prepared devices incorporating two photoactive material systems--β-carotene/N,N'-dioctyl-3,4,9,10-perylenedicarboximide (β-carotene/PTCDI-C8) and poly(3-hexylthiophene)/phenyl-C61-butyric acid methyl ester (P3HT/PCBM)--that exhibited open circuit voltages of 0.11 and 0.49 V, respectively, under NIR light LED (NLED) illumination. Then, we connected OBEI devices with different electrode gaps, incorporating biocompatible poly(hydroxymethylated-3,4-ethylenedioxythiophene), to OPVs to precisely tailor the direct current electric field conditions during the culturing of PC12 cells. This NIR light-driven OPV/OBEI system could be engineered to provide tunable control over the electric field (from 220 to 980 mV mm(-1)) to promote 64% enhancement in the neurite length, direct the neurite orientation on chips, or both. The OPV/OBEI integrated systems under NIR illumination appear to function as effective power delivery platforms that should meet the requirements for wirelessly offering medical ES to a portion of the nervous system; they might also be a key technology for the development of next-generation implantable bioelectronics.

  9. CEPO对6-OHDA诱导PC12细胞损伤的保护作用%Experimental exploration on the protective effect of CEPO on 6-hydroxydopamine-induced injury of PC12 cells

    贾钰; 雷万龙; 欧阳丽斯; 马宇昕; 陈嘉昌; 阳桂香; 李幽兰; 刘冰冰; 穆淑花; 陈思


    Objective To investigate the effect and possible mechanisms of carbamylated erythropoietin (CEPO) in inhibiting the injury of PC12 cells induced by 6-hydroxydopamine (6-OHDA). Methods PC12 cells were divided into the three groups: control group, 6-OHDA group and 6-OHDA + CEPO group. The PC12 cells viability was measured by CCK8 assay. Flow cytometry (FCM) was used to determine apoptosis rate of PC12 cells. The expression of cleaved caspase-3 was detected by western-blotting. The expression of Bcl-2 and Bax mRNA in PC12 cells were measured by Reverse transcriptase polymerase chain reaction (RT-PCR). Results CCK8 assay showed that the cell viability of PC12 cells treated with 200μmol 6-OHDA decreased to 56.70±7.86%, while 40U CEPO treatment increased the cell viability to (87.9±5.3)% (P<0.05). Flow cytometry demonstrated that CEPO treatment significantly inhibited the apoptosis of PC12 cells induced by 6-OHDA (P<0.05). RT-PCR results showed that the expression of Bcl-2 and Bax mRNA in PC12 cells were significantly upregulated and downregulated by CEPO as compared with 6-OHDA treated group (P< 0.05 ). Western-blotting showed CEPO treatment markedly induced the downregulation of cleaved caspase-3 expression compared to 6-OHDA treatment alone (P<0.05). Conclusion CEPO protects PC12 cells from injury and apoptosis induced by 6-OHDA. The protective effect of CEPO might be executed by upregulation of Bcl-2 and downregulation of Bax and Caspase-3 expression in PC12 cells.%目的 探讨氨甲酰化促红细胞生成素(CEPO)对6-羟基多巴胺(6-hydroxydopamine,6-OHDA)诱导PC12细胞损伤及凋亡的保护作用及其可能机制.方法 借助CCK8、流式细胞(Flow cytometry,FCM)、Western-blotting和逆转录PCR(RT-PCR)技术检测PC12模型细胞相关指标的变化,实验数据以SPSS15软件统计分析.结果 CCK8结果显示6-OHDA处理能够显著降低PC12模型细胞的存活率,而CEPO处理对其变化显示抑制作用;FCM技术探察结果显示,6

  10. Primary investigation of methamphetamine-induced toxicity in PC12 cells%甲基苯丙胺对PC12细胞的毒性损伤作用

    李丽增; 王慧君; 兰江维; 岳霞; 刘超


    Objective To investigate the mechanism of methamphetamine (METH)-induced toxicity in PC12 cells. Methods PC12 cells were treated with METH for 24 h at the doses of 0,05,1.0,15,2.0, or 2.5 mmol/L. The morphological changes of the cells were observed under inverted microscope after the treatment. MTT assay and flow cytometry were used to assess the cell viability and apoptotic rates, respectively, and the level of nitric oxide (NO) was measured by enzyme reduction method. Results The PC12 cells exposed to METH were morphologically featured by cell shrinkage, dendrite disruption and disappearance of cell reticular formation. METH exposure caused a dose-dependent reduction in the cell viability (P<0.01), resulting in also increased cell apoptotic rate and significant elevation of NO in the cell culture supernatant (P<0.05). Conclusion METH exposure induces cytotoxicity and injury of differentiated PC12 cells, leading to decreased cell viability and increased cell apoptosis and NO level. Cell apoptosis and excessive NO production are involved in METH-induced cytotoxicity.%目的 探讨甲基苯丙胺(METH)对PC12细胞的毒性损伤作用,为进一步研究METH神经毒性作用机制提供基础.方法 采用已分化PC12细胞为体外神经元模型,分别用浓度0、0.5、1.0、1.5、2.0、2.5 mmol/L的METH处理PC12细胞24 h,倒置显微镜下观察细胞形态改变;MTT法检测细胞存活率;流式细胞术分析细胞凋亡率;硝酸还原酶法检测细胞培养液NO含量.结果 以0mmol/L为对照组,经0.5~2.5mmol/LMETH处理的PC12细胞,24h倒置显微镜下可见PC12细胞胞体变圆,神经突起变短、断裂至消失,神经网络逐渐消失,细胞边缘不清,可呈毛刺样;MTT法检测PC12细胞活力随METH浓度的升高而逐渐下降,与对照组相比均有显著性差异(P<0.01);细胞凋亡率在0.5~1.5 mmol/L浓度范围内逐渐升高,至2.0mmol/L时凋亡率较前下降,但与对照组相比均升高,且与对照

  11. Set up Alzheimer's Disease Cell Apoptosis Model with PC-12 Cell Induced by Aβ25-35

    王亚利; 王中卫; 宋潍; 杨林


    Objective: To prepare an apoptosis cell model of Alzheimer Disease ( AD ) by PC-12 cells treated with β-amyloid protein (Aβ). Methods: PC-12 cells were incubated with differ-ent concentrations of Aβ25-35 for different duration in vitro. The cell viability was detected by MTT as-say. Morphological features of apoptosis were analyzed with Hoechst 33258/Prorlidium iodide dual staining, The level of intracellular free calcium ([Ca2+]i) was calculated by Fura-2 / AM fluorescence ratio imaging. Results: ① The viability of PC-12 cells was significantly decreased in prolrrrtion to concentration of Aβ25-35 and duration of exposure to Aβ25-35. ②The apoplotic cells appeared in a time and concentration-dependent manner, and the maximal apoptosis happened at 48 h after execute to 20μmol/ L of Aβ25-35 and 36 h to 30μmol/ L. Cell death reached the peak at 12-24 h later than the apoptotic peak. ③([Ca2+]i) of PC-12 cells was increased in prolrrrtion to duration of exposure to the same concentration of Aβ25-35. The time of the highest increase rate of [Ca2+]i was about 12 h earlier than that of apoptosis. Conclusion : An AD cell model using the PC-12 cells induced with Aβ25-35 displays aseries of chanqes related to apoptosis, which may be related to elevation of [Ca2+]i.

  12. The cannabinoid beta-caryophyllene (BCP) induces neuritogenesis in PC12 cells by a cannabinoid-receptor-independent mechanism.

    Santos, Neife Aparecida Guinaim; Martins, Nádia Maria; Sisti, Flávia Malvestio; Fernandes, Laís Silva; Ferreira, Rafaela Scalco; de Freitas, Osvaldo; Santos, Antônio Cardozo


    Beta-caryophyllene (BCP) is a phytocannabinoid whose neuroprotective activity has been mainly associated with selective activation of cannabinoid-type-2 (CB2) receptors, inhibition of microglial activation and decrease of inflammation. Here, we addressed the potential of BCP to induce neuritogenesis in PC12 cells, a model system for primary neuronal cells that express trkA receptors, respond to NGF and do not express CB2 receptors. We demonstrated that BCP increases the survival and activates the NGF-specific receptor trkA in NGF-deprived PC12 cells, without increasing the expression of NGF itself. The neuritogenic effect of BCP in PC12 cells was abolished by k252a, an inhibitor of the NGF-specific receptor trkA. Accordingly, BCP did not induce neuritogenesis in SH-SY5Y neuroblastoma cells, a neuronal model that does not express trkA receptors and do not respond to NGF. Additionally, we demonstrated that BCP increases the expression of axonal-plasticity-associated proteins (GAP-43, synapsin and synaptophysin) in PC12 cells. It is known that these proteins are up-regulated by NGF in neurons and neuron-like cells, such as PC12 cells. Altogether, these findings suggest that BCP activates trka receptors and induces neuritogenesis by a mechanism independent of NGF or cannabinoid receptors. This is the first study to show such effects of BCP and their beneficial role in neurodegenerative processes should be further investigated.

  13. Efficient GSH delivery using PAMAM-GSH into MPP-induced PC12 cellular model for Parkinson’s disease

    Sun, Hong-Ji; Wang, Yan; Hao, Tong; Wang, Chang-Yong; Wang, Qi-Yu; Jiang, Xiao-Xia


    Glutathione (GSH) depletion has been an important contributor to the dysfunction of dopamine neurons. Polyamidoamine-GSH (PAMAM-GSH) was synthesized and the delivery effect of GSH into PC12 cells was tested. MTT assessment for cytotoxicity and reactive oxygen species (ROS) as well as nitrite oxide (NO) and intracelluar superoxide dismutase (SOD) detection for antioxidative ability were performed. Furthermore, the antiapoptotic ability was analysed by assessing caspase-3, JNK1/2 and Erk1/2 expression. Our data indicated that PAMAM-GSH is an effective agent to replenish GSH into PC12 cells. PAMAM-GSH developed its antioxidative and protective ability for 1-methyl-4-phenylpyridinium (MPP)-induced PC12 cells by reducing the intracellular levels of ROS and SOD activity as well as decreasing the release of NO. Meanwhile, PAMAM-GSH could inhibit caspase-3 activation and might show its antiapoptotic ability to MPP-induced PC12 cells through JNK2/Erk1/2 pathway. In summary, these studies suggest that PAMAM-GSH conjugate has an intrinsic ability to penetrate PC12 cells and deliver GSH into these cells which may provide a new strategy for clinical applications in the treatment of Parkinson’s disease. PMID:27699060

  14. Role of Notch-1 signaling pathway in PC12 cell apoptosis induced by amyloid beta-peptide (25-35)

    Huimin Liang; Yaozhou Zhang; Xiaoyan Shi; Tianxiang Wei; Jiyu Lou


    Recent studies have demonstrated that Notch-1 expression is increased in the hippocampus of Alzheimer’s disease patients. We speculate that Notch-1 signaling may be involved in PC12 cell apoptosis induced by amyloid beta-peptide (25-35) (Aβ25-35). In the present study, PC12 cells were cultured with different doses (0, 0.1, 1.0, 10 and 100 nmol/L) of N-[N-(3,5-Dilfuorophen-acetyl)-L-alanyl]-S-phenylglycine t-butyl ester, a Notch-1 signaling pathway inhibitor, for 30 minutes. Then cultured cells were induced with Aβ25-35 for 48 hours. Pretreatment of PC12 cells with high doses of N-[N-(3,5-Dilfuorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (> 10 nmol/L) prolonged the survival of PC12 cells after Aβ25-35 induction, decreased the expression of apoptosis-related proteins caspase-3, -8, -9, increased the activity of oxidative stress-related su-peroxide dismutase and catalase, inhibited the production of active oxygen, and reduced nuclear factor kappa B expression. This study indicates that the Notch-1 signaling pathway plays a pivotal role in Aβ25-35-induced PC12 apoptosis.

  15. Stages of Pheochromocytoma and Paraganglioma

    ... foods high in tyramine (such as red wine, chocolate, and cheese). Tests that examine the blood and ... up tests will be needed. There are different types of treatment for patients with pheochromocytoma or paraganglioma. ...

  16. High Sensitive and Selective Sensing of Hydrogen Peroxide Released from Pheochromocytoma Cells Based on Pt-Au Bimetallic Nanoparticles Electrodeposited on Reduced Graphene Sheets

    Guangxia Yu


    Full Text Available In this study, a high sensitive and selective hydrogen peroxide (H2O2 sensor was successfully constructed with Pt-Au bimetallic nanoparticles (Pt-Au NPs/reduced graphene sheets (rGSs hybrid films. Various molar ratios of Au to Pt and different electrodeposition conditions were evaluated to control the morphology and electrocatalytic activity of the Pt-Au bimetallic nanoparticles. Upon optimal conditions, wide linear ranges from 1 µM to 1.78 mM and 1.78 mM to 16.8 mM were obtained, with a detection limit as low as 0.31 µM. Besides, due to the synergetic effects of the bimetallic NPs and rGSs, the amperometric H2O2 sensor could operate at a low potential of 0 V. Under this potential, not only common anodic interferences induced from ascorbic acid, uric acid and dopamine, but also the cathodic interference induced from endogenous O2 could be effectively avoided. Furthermore, with rat pheochromocytoma cells (PC 12 as model, the proposed sensor had been successfully used in the detection of H2O2 released from the cancer cells. This method with wide linear ranges and excellent selectivity can provide a promising alternative for H2O2 monitoring in vivo in the fields of physiology, pathology and diagnosis.

  17. High sensitive and selective sensing of hydrogen peroxide released from pheochromocytoma cells based on Pt-Au bimetallic nanoparticles electrodeposited on reduced graphene sheets.

    Yu, Guangxia; Wu, Weixiang; Pan, Xiaoqi; Zhao, Qiang; Wei, Xiaoyun; Lu, Qing


    In this study, a high sensitive and selective hydrogen peroxide (H2O2) sensor was successfully constructed with Pt-Au bimetallic nanoparticles (Pt-Au NPs)/reduced graphene sheets (rGSs) hybrid films. Various molar ratios of Au to Pt and different electrodeposition conditions were evaluated to control the morphology and electrocatalytic activity of the Pt-Au bimetallic nanoparticles. Upon optimal conditions, wide linear ranges from 1 µM to 1.78 mM and 1.78 mM to 16.8 mM were obtained, with a detection limit as low as 0.31 µM. Besides, due to the synergetic effects of the bimetallic NPs and rGSs, the amperometric H2O2 sensor could operate at a low potential of 0 V. Under this potential, not only common anodic interferences induced from ascorbic acid, uric acid and dopamine, but also the cathodic interference induced from endogenous O2 could be effectively avoided. Furthermore, with rat pheochromocytoma cells (PC 12) as model, the proposed sensor had been successfully used in the detection of H2O2 released from the cancer cells. This method with wide linear ranges and excellent selectivity can provide a promising alternative for H2O2 monitoring in vivo in the fields of physiology, pathology and diagnosis.

  18. [Pheochromocytomas as adrenal gland incidentalomas].

    Cerović, Snezana; Cizmić, Milica; Milović, Novak; Ajdinović, Boris; Brajusković, Goran


    Adrenal incidentalomas are a heterogeneous group of pathological entities, including benign or malignant adrenocortical or medullary tumors, hormonally active or inactive lesions, which are identified incidentally during the examination of nonadrenal-related abdominal complaints. About 1.5% to 23% of adrenal incidentalomas are pheochromocytomas. Composite pheochromocytoma is a rare tumour of adrenal medulla with divergente clinical course. This type of pheochromocytoma is designated "composite" or "mixed," depending on whether pheochromocytoma and nonpheochromocytoma components show the same embryologic origin. Nonpheochromocytoma components found in the composite pheochromocytoma include ganglioneuroma, ganglioneuroblastoma, neuroblastoma, and malignant schwannoma. The biologic behavior of composite pheochromocytomas may be as difficult to predict as more traditional pheochromocytomas; based on the number of cases reported to date the presence of areas resembling ganglioneuroblastoma or neuroblastoma does not necessary indicate a poor prognosis. Some may behave in a malignant fashion with metastasis by a component of the tumour which has neural features. Pheochromocytomas and paragangliomas are well-defined entities. Some of their nonsporadic associations and unusual morphological appearances are not universally appreciated. We report on a rare association of left adrenal CP, with typical right adrenal phochromocytoma and retroperitoneal paraganglioma, and a review of literature. We analyzed the clinical and immunohistochemical features in a 24-year-old woman with composite pheochromocytoma localized in the left adrenal gland and associated with blood pressure of 200/140 mmHg. Abdominal computed tomography and 131-J MIBG revealed a 65 x 60 mm mass in the right adrenal gland, but no revealed 45 x 40 mm retroperitoneal mass and 20 x 20 mm mass in the left adrenal region. Serum and urinary adrenaline levels were high, and catecholamine levels in the blood sample of

  19. Diverse clinical manifestations of pheochromocytomas.

    Badui, E; Mancilla, R; Szymanski, J J; Garcia-Rubi, D; Estañol, B


    Many difficulties are encountered by clinicians in attempting to diagnose pheochromocytomas. We describe several patients with unusual clinical features. These include sudden death, cerebral hemorrhage, refractory congestive heart failure, acute abdominal pain, and hypercalcemia. In 2 patients, the rare association of this tumor and pregnancy was observed. Two subjects had sudden death, 1 during a pneumoencephalogram and another during an epidural block. The clinicians should be aware of these manifestations of pheochromocytomas.

  20. Hydrogen sulfide protects PC 12 cells against H2O2-induced damage%硫化氢对抗过氧化氢对PC12细胞的损伤作用

    杨春涛; 郭瑞鲜; 杨丝丝; 冯鉴强; 唐小卿


    目的 探讨硫化氢(hydrogen sulfide,H2S)对抗过氧化氢(hydrogen peroxide,H2O2)对PC12细胞的损伤作用及有关机制.方法 应用H2O2在PC12细胞建立氧化应激损伤的实验模型;应用甲氮甲唑蓝(MTT)法检测细胞存活率,碘化丙啶(PI)染色流式细胞技术(FCM)检测细胞凋亡率,罗丹明123(Rhodamine 123,Rh123)染色FCM检测细胞线粒体膜电位(mitochondrial membrane potential,MMP),双氢罗丹明123染色FCM检测细胞内活性氧(reactive oxygen species,ROS)的含量.应用硫化氢钠(sodium hydrosulfide,NaHS)作为H2S的供体.结果 200 μmol和400μmol H2O2作用PC12细胞24 h均使细胞的存活率明显降低及凋亡率显著增加,200 μmol H2O2引起PC12细胞的MMP明显降低及ROS生成显著增多.当NaHS与H2O2(200或400μmol/L)共同作用于PC12细胞时,NaHS(100~400 μmol/L)浓度依赖性的阻断H2O2引起PC12细胞的存活率降低及细胞凋亡率增加.400 μmol NaHS明显地阻断200 μmol H2O2引起PC12细胞的MMP降低及ROS增多.结论 H2S能明显地保护PC12细胞对抗H2O2引起的损伤,阻断MMP降低及ROS生成可能是H2S的细胞保护机制之一.

  1. Hydrogen peroxide preconditioning protects PC12 cells against apoptosis induced by oxidative stress%过氧化氢预处理对抗氧化应激诱导的PC12细胞凋亡

    唐小卿; 陈静; 唐二虎; 冯鉴强; 陈培熹


    Oxidative stress can induce significant cell death by apoptosis. We explore whether prior exposure to H2O2(H2O2 preconditioning) protects PC12 cells against the apoptotic consequences of subsequent oxidative damages and what role the ATP sensitive potassium (KATP) channels play in the preconditioning protection. PC12 cells were preconditioned with 90 min exposure to H2O2 at 10 μmol/L, followed by 24-h recovery and subsequent exposures to different concentrations (20, 30, 50 and 100 μmol/L) of H2O2 for 24 h respectively. We used PI staining flow cytometry (FCM) to observe the apoptosis of PC12 cells. It was shown that 24-h exposures to H2O2 at 20, 30, 50 and 100 μmol/L respectively induced substantial cell apoptosis, which was greatly prevented in the preconditioning cells, indicating that H2O2 preconditioning protected PC 12 cells against apoptosis induced by H2O2. Administration of pinacidil (10 μmol/L), an KATP channel activator, significantly attenuated the apoptosis of PC12 cells induced by H2O2 at 30 and 50μmol/L for 24 h respectively. Glybenclamide (10 μmol/L), a KATP channel inhibitor, significantly suppressed or abolished the protective effects caused by the pinacidil but not by H2O2 preconditioning. However, when both H2O2 preconditioning and pinacidil were coapplied, their protection against the apoptosis of PC12 cells was much stronger than that of the individual one of them. These results suggest that H2O2 preconditioning protects PC12 cells against apoptosis and that the activation of KATP channels is not involved in, but synergetically enhances adaptive protection of H2O2 preconditioning.%氧化应激可明显地诱导细胞凋亡.本研究旨在探讨H2O2预处理能否对H2O2诱导的PC12细胞凋亡产生保护作用及ATP敏感性K+(ATP-sensitive potassium,KATP)通道在其中的作用.采用PI染色流式细胞仪(flow cytometry,FCM)检测PC12细胞凋亡.结果表明,经10μmol/L H2O2预处理90 min的PC12细胞,分别在20、30、50和100

  2. The effect of an electrically conductive carbon nanotube/collagen composite on neurite outgrowth of PC12 cells.

    Cho, Youngnam; Borgens, Richard Ben


    We report the preparation of an electrically conductive composite composed of collagen and carbon nanotubes (CNTs) and its use as a substrate for the in vitro growth of PC12 cells. Morphological observation by scanning electron microscopy (SEM) indicated the homogenous dispersion of CNTs in the collagen matrix. Four-point probe and cyclic voltammogram studies demonstrated the enhanced electroactivity and a lowered electrical resistivity of the resulting composites even at low loadings (collagen matrix. SEM and immunofluorescent images have indicated that the morphological features of PC12 cells were dominantly influenced by electrical potential. Greater neurite extension was preferentially induced on the exposure of electrical stimulation by facilitating the differentiation of PC12 cells into neurons indicated by more significant filopodium extension. These electrically conductive, biocompatible CNT/collagen composites could be of benefit for the development of novel neural electrodes, enhancing the growth, differentiation, and branching of neurons in an electrically driven way.

  3. microRNA regulatory mechanism by which PLLA aligned nanofibers influence PC12 cell differentiation

    Yu, Yadong; Lü, Xiaoying; Ding, Fei


    Objective. Aligned nanofibers (AFs) are regarded as promising biomaterials in nerve tissue engineering. However, a full understanding of the biocompatibility of AFs at the molecular level is still challenging. Therefore, the present study focused on identifying the microRNA (miRNA)-mediated regulatory mechanism by which poly-L-lactic acid (PLLA) AFs influence PC12 cell differentiation. Approach. Firstly, the effects of PLLA random nanofibers (RFs)/AFs and PLLA films (control) on the biological responses of PC12 cells that are associated with neuronal differentiation were examined. Then, SOLiD sequencing and cDNA microarray were employed to profile the expressions of miRNAs and mRNAs. The target genes of the misregulated miRNAs were predicted and compared with the mRNA profile data. Functions of the matched target genes (the intersection between the predicted target genes and the experimentally-determined, misregulated genes) were analyzed. Main results. The results revealed that neurites spread in various directions in control and RF groups. In the AF group, most neurites extended in parallel with each other. The glucose consumption and lactic acid production in the RF and AF groups were higher than those in the control group. Compared with the control group, 42 and 94 miRNAs were significantly dysregulated in the RF and AF groups, respectively. By comparing the predicted target genes with the mRNA profile data, five and 87 matched target genes were found in the RF and AF groups, respectively. Three of the matched target genes in the AF group were found to be associated with neuronal differentiation, whereas none had this association in the RF group. The PLLA AFs induced the dysregulation of miRNAs that regulate many biological functions, including axonal guidance, lipid metabolism and long-term potentiation. In particular, two miRNA-matched target gene-biological function modules associated with neuronal differentiation were identified as follows: (1) miR-23b, mi

  4. Pheochromocytoma and paraganglioma

    Kantorovich, Vitaly; Pacak, Karel


    Pheochromocytoma is a very special kind of tumor full of duplicity. On the one hand it represents its own microworld with unique clinical, biochemical and pathological features, while on the other it constitutes a tremendously significant part of whole body system, playing a vital role for practically every organ system. It has a very special character – sometimes like a child it can be sweet and predictable, while at times it can behave like a deadly wild beast, crashing and tearing everything on its path in a fierce rage. It also consists of the amazingly intelligent neuroendocrine cells that possess a magical ability to make miraculous substances of many kinds. But most of all, it is a system that is able to drive our curiosity and the itch of “Cogito, ergo sum” to limitless depths and year by year it still amazes us with new and unexpected discoveries that move our understanding of multiple pathways and metabolic events closer to the ultimate truth. Recent discoveries of succinate dehydrogenase (SHD) and prolyl hydroxylase (PHD) mutations, for example, propelled our understanding of neuroendocrine tumorigenesis as a whole, as well as physiology of mitochondrial respiratory chain and phenomenon of pseudohypoxia in particular. Good old discoveries make their way from dusty repositories to shine with new meaning, appropriate for the current level of knowledge. This acquired wisdom makes us better physicians – knowing the specific expression makeup of catecholamine transporters, GLUTs and SRIFs allows for better tailored imaging and therapeutic manipulations. There are still long ways to go, keeping in mind that pheochromocytoma is but so very special, and we are optimistic and expect many great things to come. PMID:20541673

  5. Cerebrolysin protects PC12 cells from CoCl2-induced hypoxia employing GSK3β signaling.

    Hartwig, Kerstin; Fackler, Viktoria; Jaksch-Bogensperger, Heidi; Winter, Stefan; Furtner, Tanja; Couillard-Despres, Sebastien; Meier, Dieter; Moessler, Herbert; Aigner, Ludwig


    Cerebrolysin (EVER Neuro Pharma GmbH, Austria) is a peptidergic drug indicated for clinical use in stroke, traumatic brain injury and dementia. The therapeutic effect of Cerebrolysin is thought to ensure from its neurotrophic activity, which shares some properties with naturally occurring neurotrophic factors. However, the exact mechanism of action of Cerebrolysin is yet to be fully deciphered. This study aimed to investigate the neuroprotective effect of Cerebrolysin in a widely used in vitro model of hypoxia-induced neuronal cytotoxicity, namely cobalt chloride (CoCl2)-treatment of PC12 cells. CoCl2-cytotoxicity was indicated by a reduced cell-diameter, cell shrinkage, increased pro-apoptotic Caspase-activities and a decreased metabolic activity. Cerebrolysin maintained the cell-diameter of CoCl2-treated naïve PC12 cells, decreased the activation of Caspase 3/7 in CoCl2-stressed naïve PC12 cells and restored the cells' metabolic activity in CoCl2-impaired naïve and differentiated PC12 cells. Cerebrolysin treatment also decreased the levels of superoxide observed after exposure to CoCl2. Investigating the mechanism of action, we could demonstrate that Cerebrolysin application to CoCl2-stressed PC12 cells increased the phosphorylation of GSK3β, resulting in the inhibition of GSK3β. This might become clinically relevant for Alzheimer's disease, since GSK3β activity has been linked to the production of amyloid beta. Taken together, Cerebrolysin was found to have neuroprotective effects in CoCl2-induced cytotoxicity in PC12 cells.

  6. Arecoline Induces Neurotoxicity to PC12 Cells: Involvement in ER Stress and Disturbance of Endogenous H2S Generation.

    Jiang, Jia-Mei; Wang, Li; Gu, Hong-Feng; Wu, Keng; Xiao, Fan; Chen, Ying; Guo, Run-Min; Tang, Xiao-Qing


    Arecoline is a major alkaloid of areca nut and has been effect on central nervous system. Although arecoline-induced neurotoxicity has been reported, the possible underlying neurotoxic mechanisms have not yet been elucidated. Increasing evidences have shown that both excessive endoplasmic reticulum (ER) stress and disturbance of hydrogen sulfide (H2S) production are involved in the pathophysiology of numerous neurodegenerative diseases. Here, the purpose of present study was to verify whether ER stress and the disturbance of endogenous H2S generation are also involved in arecoline-caused neurotoxicity. We found that treatment of PC12 cells with arecoline induced the down-regulation of cells viability and up-regulation of apoptosis and the activity of caspase-3, indicating the neurotoxic role of arecoline to PC12 cells. In addition, arecoline also increased the expression of Bax (pro-apoptotic protein) and attenuated the expression of Bcl-2 (anti-apoptotic protein) in PC12 cells. Simultaneously, arecoline caused excessive ER stress in PC12 cells, as evidenced by the up-regulations of Glucose-regulated protein 78 (GRP78), CCAAT/enhancer binding protein homologous protein (CHOP), and Cleaved caspase-12 expressions. Notably, the level of H2S in the culture supernatant and the expressions of cystathionine β-synthase and 3-mercaptopyruvate sulfurtransferase (two major enzymes for endogenous H2S generation in PC12 cells) were also reduced by arecoline treatment. These results indicate that arecoline-caused neurotoxicity to PC12 cells is involved in ER stress and disturbance of endogenous H2S generation and suggest that the modulation of ER stress and endogenous H2S generation may be potential therapeutic approach in treatment of arecoline-caused neurotoxicity.

  7. Dystrophin Dp71f associates with the beta1-integrin adhesion complex to modulate PC12 cell adhesion.

    Cerna, Joel; Cerecedo, Doris; Ortega, Arturo; García-Sierra, Francisco; Centeno, Federico; Garrido, Efrain; Mornet, Dominique; Cisneros, Bulmaro


    Dystrophin Dp71 is the main product of the Duchenne muscular dystrophy gene in the brain; however, its function is unknown. To study the role of Dp71 in neuronal cells, we previously generated by antisense treatment PC12 neuronal cell clones with decreased Dp71 expression (antisense-Dp71 cells). PC12 cells express two different splicing isoforms of Dp71, a cytoplasmic variant called Dp71f and a nuclear isoform called Dp71d. We previously reported that antisense-Dp71 cells display deficient adhesion to substrate and reduced immunostaining of beta1-integrin in the cell area contacting the substrate. In this study, we isolated additional antisense-Dp71 clones to analyze in detail the potential involvement of Dp71f isoform with the beta1-integrin adhesion system of PC12 cells. Immunofluorescence analyses as well as immunoprecipitation assays demonstrated that the PC12 cell beta1-integrin adhesion complex is composed of beta1-integrin, talin, paxillin, alpha-actinin, FAK and actin. In addition, our results showed that Dp71f associates with most of the beta1-integrin complex components (beta1-integrin, FAK, alpha-actinin, talin and actin). In the antisense-Dp71 cells, the deficiency of Dp71 provokes a significant reduction of the beta1-integrin adhesion complex and, consequently, the deficient adhesion of these cells to laminin. In vitro binding experiments confirmed the interaction of Dp71f with FAK and beta1-integrin. Our data indicate that Dp71f is a structural component of the beta1-integrin adhesion complex of PC12 cells that modulates PC12 cell adhesion by conferring proper complex assembly and/or maintenance.

  8. Lysophosphatidylinositol causes neurite retraction via GPR55, G13 and RhoA in PC12 cells.

    Yutaro Obara

    Full Text Available GPR55 was recently identified as a putative receptor for certain cannabinoids, and lysophosphatidylinositol (LPI. Recently, the role of cannabinoids as GPR55 agonists has been disputed by a number of reports, in part, because studies investigating GPR55 often utilized overexpression systems, such as the GPR55-overexpressing HEK293 cells, which make it difficult to deduce the physiological role of endogenous GPR55. In the present study, we found that PC12 cells, a neural model cell line, express endogenous GPR55, and by using these cells, we were able to examine the role of endogenous GPR55. Although GPR55 mRNA and protein were expressed in PC12 cells, neither CB(1 nor CB(2 mRNA was expressed in these cells. GPR55 was predominantly localized on the plasma membrane in undifferentiated PC12 cells. However, GPR55 was also localized in the growth cones or the ruffled border in differentiated PC12 cells, suggesting a potential role for GPR55 in the regulation of neurite elongation. LPI increased intracellular Ca(2+ concentration and RhoA activity, and induced ERK1/2 phosphorylation, whereas endogenous and synthetic cannabinoids did not, thereby suggesting that cannabinoids are not GPR55 agonists. LPI also caused neurite retraction in a time-dependent manner accompanied by the loss of neurofilament light chain and redistribution of actin in PC12 cells differentiated by NGF. This LPI-induced neurite retraction was found to be G(q-independent and G(13-dependent. Furthermore, inactivation of RhoA function via C3 toxin and GPR55 siRNA knockdown prevented LPI-induced neurite retraction. These results suggest that LPI, and not cannabinoids, causes neurite retraction in differentiated PC12 cells via a GPR55, G(13 and RhoA signaling pathway.

  9. 提高Lipofectamine2000对PC12细胞转染效率的研究%Study of enhancement of the transfection efficiency in PC12 cells transfected with Lipofectamine 2000



    目的 探讨提高PC12细胞转染效率的方法.方法 细胞培养板用10 μg/mL Ⅰ型牛胶原蛋白包被,通过在转染过程中加入9 μmol/L趋溶酶体试剂氯喹及8μmol/L聚胺类试剂亚精胺,同时调整Lipofectamine2000与DNA用量的比例和转染时间.考察DNA与Lipofectamine2000的比例对PC12细胞转染效率的影响,转染时间对PC12细胞转染效率的影响,氯喹、亚精胺用量对PC12细胞转染效率的影响,氯喹、亚精胺对PC12细胞活性的影响,氯喹、亚精胺对PC12细胞神经轴突生长的影响及与4种常用脂质体转染试剂对PC12细胞转染效率的比较.结果 ①对PC12细胞转染,DNA:Lipofectamine2000用量比例应控制在1∶4.②当转染时间分别为1、2、4、8、24 h时,PC12细胞转染率分别为35.5%、37.9% 、40.5%、40.3%及38.6%,4h达到最大转染效率.③氯喹、亚精胺加入浓度的增加,PC12细胞转染效率随之增加,当氯喹、亚精胺加入终浓度分别增至9μmol/L和8μmol/L时,PC12细胞的转染效率最高,转染效率为40.5%.④加入终浓度为9μmol/L氯喹与8μmol/L亚精胺前、后MTT试验吸光度(590 nm)分别为(0.466±0.042)与(0.451±0.038),差异无统计学意义(P>0.05),对PC12细胞的活性无影响.⑤加入氯喹、亚精胺前、后,PC12细胞的神经轴突生长数量与长度差异无统计学意义(P>0.05).⑥FuGENE、PolyJet、Lipofectamine LTX and Plus、Lipofectamine2000 4种脂质体转染试剂对PC12细胞转染效率分别为10.5%、8.6%、11.8%及15.3%,本法PC12细胞转染率为40.5%,差异有高度统计学意义(P<0.01).结论 加入氯喹及亚精胺,实现阳离子脂质体Lipofectamine2000对PC12细胞的高效转染,为PC12细胞进行神经细胞基因功能及开发遗传病治疗方案等生物学研究提供了一种安全、廉价的新方法.

  10. Real-time monitoring of intracellular signal transduction in PC12 cells by non-adiabatic tapered optical fiber biosensor

    Zibaii, M. I.; Latifi, H.; Asadollahi, A.; Noraeipoor, Z.; Dargahi, L.


    Real-time observation of intracellular process of signal transduction is very useful for biomedical and pharmaceutical applications as well as for basic research work of cell biology. For feasible and reagentless observation of intracellular alterations in real time, we examined the use of a nonadiabatic tapered optical fiber (NATOF) biosensor for monitoring of intracellular signal transduction that was mainly translocation of protein kinase C via refractive index change in PC12 cells adhered on tapered fiber sensor without any indicator reagent. PC12 cells were stimulated with KCl . Our results suggest that complex intracellular reactions could be real-time monitored and characterized by NATOF biosensor.

  11. Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35★

    Yan, Mingmin; Mao, Shanping; Dong, Huimin; Liu, Baohui; Zhang, Qian; PAN, GAOFENG; Fu, Zhiping


    PC12 cell injury was induced using 20 μM amyloid β-protein 25–35 to establish a model of Alzheimer's disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25–35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical...

  12. Mercury induced the Accumulation of Amyloid Beta (Aβ) in PC12 Cells: The Role of Production and Degradation of Aβ

    Song, Ji-Won; Choi, Byung-Sun


    Extracellular accumulation of amyloid beta protein (Aβ) plays a central role in Alzheimer’s disease (AD). Some metals, such as copper, lead, and aluminum can affect the Aβ accumulation in the brain. However, the effect of mercury on Aβ accumulation in the brain is not clear. Thus, this study was proposed to estimate whether mercury concentration affects Aβ accumulation in PC12 cells. We treated 10, 100, and 1000 nM HgCl2 (Hg) or CH3HgCl2 (MeHg) for 48 hr in PC12 cells. After treatment, Aβ40 i...

  13. Effect of Tinospora cordifolia on the reduction of ultraviolet radiation-induced cytotoxicity and DNA damage in PC12 cells

    Masuma, Runa; Okuno, Tsutomu; Choudhuri, Mohammad Shahabuddin Kabir; Saito, Takeshi; Kurasaki, Masaaki


    The safety of Tinospora cordifolia and its potential to protect against ultraviolet radiation-induced cytotoxicity and DNA damage in PC12 cells were investigated. To evaluate the safety of T. cordifolia, cell viability and agarose gel electrophoresis were carried out using PC12 cells treated with 0 to 100g mL(-1) of methanol extract of T. cordifolia. T. cordifolia extracts did not show cytotoxicity ranging 0 to 100g mL(-1). In addition, T. cordifolia extracts significantly increased cell viab...

  14. Antidepressant-like Effect of Cell-free Filtrate of Sodium Ferulate-induced and Differentioned PC12 Cell Lysates%阿魏酸钠诱导分化的PC12细胞裂解液的无细胞滤液的抗抑郁样效果

    廖铭能; 于立坚; 张永平; 马润娣; 张霄瑜; 于廷曦


    阿魏酸(ferulic acid,FA)是一种广泛存在的低毒酚酸,阿魏酸钠(sodium ferulate,SF)则是其钠盐.先前的研究已经证实,阿魏酸钠具有显著的神经保护和神经发生增强作用及抗抑郁效果.该研究的目的在于探讨阿魏酸钠诱导分化的PC12细胞裂解液的无细胞滤液可能的抗抑郁效果.PC12细胞在含80 μmol/L阿魏酸钠的DMEM培养基中孵育6 d,无菌条件下制备阿魏酸钠诱导分化的PC12细胞液的无细胞滤液,测定PC12细胞裂解液无细胞滤液中残留的阿魏酸钠量.以慢性不可预期的多种刺激制造大鼠抑郁模型,用行为学、形态学、免疫组织化学和BrdU掺入等方法观察并检测阿魏酸钠诱导分化的PC12细胞裂解液的无细胞滤液对慢性应激大鼠抑郁模型行为学、海马的组织病理学、海马和大脑皮质的神经生长因子(nerve growth factor,NGF)及脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)的表达及神经发生的影响.实验证实,阿魏酸钠诱导分化的PC12细胞裂解液的无细胞滤液能改善抑郁症样模型大鼠的行为学障碍,上调其海马和大脑皮质NGF和BNDF的表达,增加海马神经干细胞/神经前体细胞的增殖.由此可见,阿魏酸钠诱导分化的PC12细胞裂解液的无细胞滤液有明显的抗抑郁效果,而其抗抑郁效果可能源自它的上调NGF和BNDF以及其增强神经发生作用.%Ferulic acid (FA), 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid, is one of the most common phenolic acids with low toxicity, and sodium ferulate (SF) is its sodium salt.Our previous work demonstrates that SF has significant neuroprotective and neurogenesis-enhancing actions and antidepressant-like effects.The aim of this study was to investigate a potential antidepressant-like effect of cell-free filtrate of sodium ferulate-induced and differentioned PC 12 cell lysates (SFIDPC 12CL) in the chronic mild stress (CMS)-induced depression-like model rats.PC

  15. Renal infarction associated with adrenal pheochromocytoma.

    Thewjitcharoen, Yotsapon; Atikankul, Taywin; Sunthornyothin, Sarat


    The coexistence of pheochromocytoma and renal artery stenosis had been reported occasionally from the possible mechanism of catecholoamine-induced vasospasm and extrinsic compression of renal artery in some reported cases. However, renal infarction caused by pheochromocytoma is an uncommon phenomenon. Herein, we report an interesting case of adrenal pheochromocytoma associated with renal artery thrombosis, which should be included in the differential diagnosis of pheochromocytoma patients who present with abdominal pain.

  16. From Childhood Migraine Headache to Pheochromocytoma

    Y. M. Hazimeh


    Full Text Available Pheochromocytoma may have multiple clinical manifestations including paroxysmal hypertension, tachycardia, sweating, nausea, and headache (Phillips et al., 2002. Migraine has some of the manifestations seen with pheochromocytoma. We describe a patient who had a history of migraine headaches since childhood and was found to have pheochromocytoma. Resection of her tumor significantly improved her headache. The diagnoses of pheochromocytoma subsequently lead to diagnosing her with medullary thyroid cancer (MTC and multiple endocrine neoplasia type 2A (MEN-2A.

  17. Carbon Fiber Ultramicrodic Electrode Electrodeposited with Over-Oxidized Polypyrrole for Amperometric Detection of Vesicular Exocytosis from Pheochromocytoma Cell

    Wang, Li; Xu, Huiren; Song, Yilin; Luo, Jinping; Xu, Shengwei; Zhang, Song; Liu, Juntao; Cai, Xinxia


    Vesicular exocytosis is ubiquitous, but it is difficult to detect within the cells' communication mechanism. For this purpose, a 2 μm ultramicrodic carbon fiber electrode was fabricated in this work based on electrodeposition with over-oxidized polypyrrole nanoparticle (PPyox-CFE), which was applied successfully for real-time monitoring of quantal exocytosis from individual pheochromocytoma (PC12) cells. PPyox-CFE was evaluated by dopamine (DA) solutions through cyclic voltammetry and amperometry electrochemical methods, and results revealed that PPyox-CFE improved the detection limit of DA. In particular, the sensitivity of DA was improved to 24.55 μA·μM−1·μm−2 using the PPyox-CFE. The ultramicrodic electrode combined with the patch-clamp system was used to detect vesicular exocytosis of DA from individual PC12 cells with 60 mM K+ stimulation. A total of 287 spikes released from 7 PC12 cells were statistically analyzed. The current amplitude (Imax) and the released charge (Q) of the amperometric spikes from the DA release by a stimulated PC12 cell is 45.1 ± 12.5 pA and 0.18 ± 0.04 pC, respectively. Furthermore, on average ∼562,000 molecules were released in each vesicular exocytosis. PPyox-CFE, with its capability of detecting vesicular exocytosis, has potential application in neuron communication research. PMID:25569759

  18. Methamphetamine-induced dopaminergic neurotoxicity and production of peroxynitrite are potentiated in nerve growth factor differentiated pheochromocytoma 12 cells.

    Imam, Syed Z; Newport, Glenn D; Duhart, Helen M; Islam, Fakhrul; Slikker, William; Ali, Syed F


    Methamphetamine (METH) is a widely abused psychomotor stimulant known to cause dopaminergic neurotoxicity in rodents, nonhuman primates, and humans. METH administration selectively damages the dopaminergic nerve terminals, which is hypothesized to be due to release of dopamine from synaptic vesicles within the terminals. This process is believed to be mediated by the production of free radicals. The current study evaluates METH-induced dopaminergic toxicity in pheochromocytoma 12 (PC12) cells cultured in the presence or absence of nerve growth factor (NGF). Dopaminergic changes and the formation of 3-nitrotyrosine (3-NT), a marker for peroxynitrite production, were studied in PC12 cell cultures grown in the presence or absence of NGF after different doses of METH (100-1,000 microM). METH exposure did not cause significant alterations in cell viability and did not produce significant dopaminergic changes or 3-NT production in PC12 cells grown in NGF-negative media after 24 hours. However, cell viability of PC12 cells grown in NGF-positive media was decreased by 45%, and significant dose-dependent dopaminergic alteration and 3-NT production were observed 24 hours after exposure to METH. The current study supports the hypothesis that METH acts at the dopaminergic nerve terminals and produces dopaminergic damage by the production of free radical peroxynitrite.

  19. Carbon Fiber Ultramicrodic Electrode Electrodeposited with Over-Oxidized Polypyrrole for Amperometric Detection of Vesicular Exocytosis from Pheochromocytoma Cell

    Li Wang


    Full Text Available Vesicular exocytosis is ubiquitous, but it is difficult to detect within the cells’ communication mechanism. For this purpose, a 2 µm ultramicrodic carbon fiber electrode was fabricated in this work based on electrodeposition with over-oxidized polypyrrole nanoparticle (PPyox-CFE, which was applied successfully for real-time monitoring of quantal exocytosis from individual pheochromocytoma (PC12 cells. PPyox-CFE was evaluated by dopamine (DA solutions through cyclic voltammetry and amperometry electrochemical methods, and results revealed that PPyox-CFE improved the detection limit of DA. In particular, the sensitivity of DA was improved to 24.55 µA·µM−1·µm−2 using the PPyox-CFE. The ultramicrodic electrode combined with the patch-clamp system was used to detect vesicular exocytosis of DA from individual PC12 cells with 60 mM K+ stimulation. A total of 287 spikes released from 7 PC12 cells were statistically analyzed. The current amplitude (Imax and the released charge (Q of the amperometric spikes from the DA release by a stimulated PC12 cell is 45.1 ± 12.5 pA and 0.18 ± 0.04 pC, respectively. Furthermore, on average ~562,000 molecules were released in each vesicular exocytosis. PPyox-CFE, with its capability of detecting vesicular exocytosis, has potential application in neuron communication research.

  20. Cytoprotection of 13 Pakistan Medicinal Plants on Neurotoxicity of PC12 Cells%13种巴基斯坦草药对PC12细胞保护作用的研究

    梁岚; 刘新民; 孙丽华; 徐淑萍; 杨艳艳; 赵明耀; Ahsana Dar; Iqbal Choudhary; Dr. Bina; Dr. Shaheen


    目的:选择13种巴基斯坦传统草药,观察其对PC12细胞增殖的影响,并探讨其对H202、谷氨酸诱导PC12细胞损伤的保护作用,为寻找神经细胞保护作用新药提供实验依据.方法:体外培养PC12细胞,应用不同浓度的H202、GLU,分别建立神经细胞损伤模型,采用MTT法检测细胞存活率.结果:虎爪豆、诃子等能明显促进PC12细胞增殖作用(P<0.05);在H202 150μmol· L-1损伤模型中,药物组和模型组相比,罗勒、仙人掌、芦笋、灯油藤、诃子等能显著减轻H202的细胞损伤作用(P<0.05);在GLU 30mmol·L-1诱导PC12细胞损伤,与模型组比较,凤仙花、仙人掌、虎爪豆、诃子等均显示出损伤保护作用(P<0.05).其中,诃子不仅能促进PC12细胞增殖,且在两种PC12细胞损伤模型中均具有较强的损伤保护作用.结论:具有促进PC12细胞增殖的药物为诃子、罗勒、虎爪豆;具有对抗H202损伤作用的药物有:诃子、凤仙花、仙人掌、芦笋、灯油藤、罗勒;具有减弱GLU诱导PC12细胞损伤的药物为:虎爪豆、诃子、凤仙花、仙人掌、芦笋、甘草;诃子在3种模型中均表现出较强的细胞损伤保护作用.本研究结果为神经退行性疾病药物的开发和选择提供了初步的实验依据.%This study aimed to investigate the cytoprotection of 20 extracts from 13 Pakistan medicinal plants on PC12 cells injured by H2O2 and glutamate (GLU). Different concentrations of H2O2 and GLU were used to establish the toxicity models on PC12 cells. Cell viability was determined by MTT assay. The results showed that the extracts of Mucuna pruriens L and Terminalia chebula had significantly proliferative effects on PC12 cells (P<0.05). Additionally, extracts of Lawsonia alba, Opuntia dillenii Haw., Celastrus panic ulata, Terminalia chebula showed markedly cytoprotection against H2O2-induced cell death (P<0.05). Similar results were observed in GLU injured model, which showed that

  1. Luteolin modulates 6-hydroxydopamine-induced transcriptional changes of stress response pathways in PC12 cells.

    Ling-Wei Hu

    Full Text Available The neurotoxin 6-hydroxydopamine (6-OHDA, which causes transcriptional changes associated with oxidative and proteotoxic stress, has been widely used to generate an experimental model of Parkinson's disease. The food-derived compound luteolin has multi-target actions including antioxidant, anti-inflammatory and neurotrophic activities. The aim of this study is to investigate how luteolin affects 6-OHDA-mediated stress response pathways. The results showed that when PC12 cells were pre-treated with luteolin (20 µM 30 min prior to 6-OHDA (100 µM exposure, 6-OHDA-induced ROS overproduction, cytotoxicity, caspase-3 activation, and mRNA expression of BIM, TRB3 and GADD34 were significantly attenuated. Moreover, 6-OHDA-mediated cell cycle arrest and transcription of p53 target genes, p21, GADD45α and PUMA, were reduced by luteolin. Luteolin also significantly down-regulated 6-OHDA-mediated unfolded protein response (UPR, leading to decreases in phospho-eIF2α, ATF4, GRP78 and CHOP. In addition, luteolin attenuated 6-OHDA-induced Nrf2-mediated HO-1 and GCLC. Taken together, these results suggest that diminishing intracellular ROS formation and down-regulation of p53, UPR and Nrf2-ARE pathways may be involved in the neuroprotective effect of luteolin.

  2. Roscovitine increases intracellular calcium release and capacitative calcium entry in PC12 cells.

    Choi, Ho Sook; Chung, Sul-Hee


    Cyclin-dependent kinase 5 (Cdk5), which is activated by the non-cyclin regulator p35 or p39, is a proline-directed serine/threonine kinase that is implicated in many physiological and pathological processes. Here, we studied calcium signaling using the fluorescent cytosolic calcium indicator, Fura-4, in NGF-differentiated PC12 cells treated with roscovitine, a Cdk5 inhibitor. As compared to the control cells, the roscovitine-treated cells significantly potentiated intracellular calcium release by membrane depolarization (high K(+)) or through thapsigargin. In addition, roscovitine increased the magnitude of capacitative calcium entry (CCE), i.e., a calcium influx mechanism triggered by the depletion of intracellular calcium stores. Notably, roscovitine markedly slowed the rate of Ca(2+) removal from the plasma membrane. These results suggest that Cdk5 regulates intracellular calcium homeostasis and that the dysregulation of Cdk5 may contribute to disease pathogenesis by perturbing cellular Ca(2+) signaling. (c) 2009 Elsevier Ireland Ltd. All rights reserved.

  3. Evidence of increased reactive species formation by retinol, but not retinoic acid, in PC12 cells.

    Gelain, Daniel Pens; Moreira, José Claudio Fonseca


    The biological effects of vitamin A (retinol) are generally ascribed to the activation of nuclear retinoid receptors by retinoic acid (RA), considered the most biologically active retinoid. However, it is not established whether the cytotoxic effects of vitamin A are due to retinoid receptors activation by RA. Vitamin A-related toxicity is associated with cellular redox modifications, often leading to severe oxidative damage, but the role of RA in this effect is also uncertain. We therefore studied the formation of intracellular reactive species induced by retinol and retinoic acid in PC12 cells, using an in vitro dichlorofluorescein (DCFH) fluorescence real-time assay. We observed that retinol, but not retinoic acid, induced a steady increase in DCF-based fluorescence over 60 min of incubation, and this increase was reversed by antioxidant (N-acetyl-cysteine and alpha-tocopherol) pre-treatment. This effect was also inhibited by the iron chelator 1,10-phenantroline and the impermeable calcium chelator EGTA. These results suggest that vitamin A-associated cytotoxicity is probably related to an oxidant mechanism dependent on iron and calcium, and the formation of intracellular reactive species is related to retinol, but not to RA.

  4. Schisandrin B protects PC12 cells against oxidative stress of neurodegenerative diseases.

    Jiang, En-Ping; Li, He; Yu, Chun-Rong; Yu, Chun-Yan; Jing, Shu; Sun, Hong-Xia; Wang, Chun-Mei; Fan, Xin-Tian; Chen, Jian-Guang; Wang, Sen


    Increasing evidence places Schisandrin B (Sch B) at an important position in nerve protection, indicating that Sch B might play a positive role in the therapy of neurodegenerative diseases. However, there is little information on it. Our studies showed that pretreatment with Sch B could reduce lactate dehydrogenase, malondialdehyde, and reactive oxygen species release and significantly increase the cell viability and the superoxide dismutase level. Sch B (10 μM) markedly inhibited cell apoptosis, whereas LY294002 (20 μM), a phosphatidylinositol-3 kinase inhibitor, blocked the antiapoptotic effect. More importantly, Sch B (10 μM) increased the phosphoprotein kinase B/protein kinase B (Akt) and B-cell lymphoma-2/Bcl-2 associated X protein ratios on preincubation with cells for 2 h, which was then inhibited by LY294002 (20 μM). Results indicate that Sch B can protect PC12 cells from apoptosis by activating the phosphatidylinositol-3 kinase/Akt signaling pathway and may emerge as a potential drug for neurodegenerative diseases.

  5. Dynamics of dynamin during clathrin mediated endocytosis in PC12 cells.

    Joshua Z Rappoport

    Full Text Available BACKGROUND: Members of the dynamin super-family of GTPases are involved in disparate cellular pathways. Dynamin1 and dynamin2 have been implicated in clathrin-mediated endocytosis. While some models suggest that dynamin functions specifically at the point of vesicle fission, evidence also exists for a role prior to fission during vesicle formation and it is unknown if there is a role for dynamin after vesicle fission. Although dynamin2 is ubiquitously expressed, dynamin1 is restricted to the nervous system. These two structurally similar endocytic accessory proteins have not been studied in cells that endogenously express both. METHODOLOGY/PRINCIPAL FINDINGS: The present study quantitatively assesses the dynamics of dynamin1 and dynamin2 during clathrin-mediated endocytosis in PC12 cells, which endogenously express both proteins. Both dynamin isoforms co-localized with clathrin and showed sharp increases in fluorescence intensity immediately prior to internalization of the nascent clathrin-coated vesicle. The fluorescence intensity of both proteins then decreased with two time constants. The slower time constant closely matched the time constant for the decrease of clathrin intensity and likely represents vesicle movement away from the membrane. The faster rate may reflect release of dynamin at the neck of nascent vesicle following GTP hydrolysis. CONCLUSIONS/SIGNIFICANCE: This study analyses the role of dynamin in clathrin-mediated endocytosis in a model for cellular neuroscience and these results may provide direct evidence for the existence of two populations of dynamin associated with nascent clathrin-coated vesicles.

  6. Two distinct signaling pathways regulate peroxynitrite-induced apoptosis in PC12 cells.

    Shacka, J J; Sahawneh, M A; Gonzalez, J D; Ye, Y-Z; D'Alessandro, T L; Estévez, A G


    The mechanisms of peroxynitrite-induced apoptosis are not fully understood. We report here that peroxynitrite-induced apoptosis of PC12 cells requires the simultaneous activation of p38 and JNK MAP kinase, which in turn activates the intrinsic apoptotic pathway, as evidenced by Bax translocation to the mitochondria, cytochrome c release to the cytoplasm and activation of caspases, leading to cell death. Peroxynitrite induces inactivation of the Akt pathway. Furthermore, overexpression of constitutively active Akt inhibits both peroxynitrite-induced Bax translocation and cell death. Peroxynitrite-induced death was prevented by overexpression of Bcl-2 and by cyclosporin A, implicating the involvement of the intrinsic apoptotic pathway. Selective inhibition of mixed lineage kinase (MLK), p38 or JNK does not attenuate the decrease in Akt phosphorylation showing that inactivation of the Akt pathway occurs independently of the MLK/MAPK pathway. Together, these results reveal that peroxynitrite-induced activation of the intrinsic apoptotic pathway involves interactions with the MLK/MAPK and Akt signaling pathways.

  7. Partitioning and Exocytosis of Secretory Granules during Division of PC12 Cells

    Nickolay Vassilev Bukoreshtliev


    Full Text Available The biogenesis, maturation, and exocytosis of secretory granules in interphase cells have been well documented, whereas the distribution and exocytosis of these hormone-storing organelles during cell division have received little attention. By combining ultrastructural analyses and time-lapse microscopy, we here show that, in dividing PC12 cells, the prominent peripheral localization of secretory granules is retained during prophase but clearly reduced during prometaphase, ending up with only few peripherally localized secretory granules in metaphase cells. During anaphase and telophase, secretory granules exhibited a pronounced movement towards the cell midzone and, evidently, their tracks colocalized with spindle microtubules. During cytokinesis, secretory granules were excluded from the midbody and accumulated at the bases of the intercellular bridge. Furthermore, by measuring exocytosis at the single granule level, we showed, that during all stages of cell division, secretory granules were competent for regulated exocytosis. In conclusion, our data shed new light on the complex molecular machinery of secretory granule redistribution during cell division, which facilitates their release from the F-actin-rich cortex and active transport along spindle microtubules.

  8. Toosendanin interferes with pore formation of botulinum toxin type A in PC12 cell membrane

    Mu-feng LI; Yu-liang SHI


    Aim: Botulinum neurotoxins (BoNT) abort the process of neurotransmitter release at presynaptic motor nerve terminals, causing muscle paralysis. The ability of botulinum toxin to produce its effect is dependent on the ability of the light chain to cleave the SNARE proteins associated with transmitter release. Translocation of the light chain protease through the heavy chain-formed channel is a pivotal step in the intoxication process. Toosendanin (TSN), a triterpenoid derivative extracted from a Chinese traditional medicine, has been demonstrated to be an effective cure for experimental botulism. This study was designed to explore the antibotulismic mechanisms of toosendanin. Methods: The inside-out singlechannel recording patch-clamp technique was used to record the BoNT/A-induced currents in the presence and absence of TSN. Results: Channel formation was delayed and the sizes of the channels were reduced in the TSN-treated PC12cell membrane. Conclusion: The antibotulismic effect of TSN might occur via interference with toxin translocation.

  9. Effects of hydrogen sulfide on apoptosis of PC12 cells induced by β-amyloid%硫化氢对β-淀粉样蛋白诱导PC12细胞凋亡的影响

    陈秀琴; 唐小卿; 李景田; 赵春梅; 冯鉴强; 陈培熹


    目的 探讨硫化氢(hydrogen sulfide,H2S)对抗β-淀粉样蛋白(β-amylmoid)诱导PC12细胞凋亡的作用及机制.方法 应用硫化氢钠(sodium hydrosulfide,NaHS)作为H2S的供体,甲氮甲唑蓝(MTT)法检测细胞存活率,碘化丙啶(PI)染色流式细胞技术(FCM)检测细胞凋亡率,Hoechst染色检测细胞凋亡的形态学变化,罗丹明123(Rhodamine123,Rh123)染色FCM检测细胞线粒体膜电位(mitochondrial membrane potential,MMP),双氢罗丹明123染色FCM检测细胞内活性氧(reactive oxygen species,ROS)的含量.结果 淀粉样多肽β25-35(Aβ25-35)引起PC12细胞的存活率显著降低及凋亡率明显增大,同时引起PC12细胞的MMP明显降低及ROS生成率显著增加.当NaHS与AB25-35共同作用于PC12细胞时,NaHS浓度依赖性地阻断20 μmol/L Aβ25-35引起PC12细胞的存活率降低,100 μmol/L NaHS显著地降低20 μmol/LAβ25-35引起PC12细胞的凋亡率并阻断Aβ25-35引起的MMP降低及ROS升高.结论 H2S的供体NaHS具有细胞保护作用,能对抗Aβ25-35引起的PC12细胞凋亡,此作用可能与其阻断MMP降低及ROS生成增多有关.

  10. Inhibitive Effect of Magnolol Against MPTP-Induced Apoptosis in PC12 Cells%厚朴酚对MPTP诱导PC12细胞凋亡的抑制作用

    鄢印根; 林泉峰; 徐焱; 黄小丽


    Objective:To investigate the inhibition of magnolol on the apoptosis of PC 12 cell induced by MPTP and its mechanism. Method: Magnolol (final concentration 0.1, 10 μmol·L-1) and /or MPTP ( final concentration 50, 100, 150, 200 μmol·L-1) were added into the culture of PC12 cells. The cell viability was detected with MTT method, apoptosis was measured by flow cytometry and Bcl-2/Bax ratio was detected by western blot. Result: Cell viability was declined sharply by MPTP with different concentrations, the pre-treatment of magnolol 0. 1-10 μmol·L-1 could significantly increase the PC12 cell viability, apoptosis and the decrease in the Bcl-2/Bax ratio. Conclusion: Magnolol may inhibit the cell damage induced by MPTP and the mechanism is likely related to inhibition to expression of Bcl-2/Bax.%探讨厚朴酚( mangolol,Mag)对1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)诱导PC12细胞凋亡的抑制作用及可能作用机制.方法 将厚朴酚(终浓度0.1,10 μmol· L-1)或/和MPTP(终浓度50,100,150,200 μmol·L-1)加入到培养的PC12细胞中.四甲基偶氮唑盐法(MTT法)检测细胞增殖活性,用流式细胞仪检测细胞凋亡率,以及Western -blot法检测Bax和Bcl-2的蛋白表达变化.结果 在加入不同浓度的MPTP处理细胞24 h,细胞增殖活性渐次降低,而厚朴酚0.1~10μmol·L-1预处理1h可明显减轻MPTP导致的PC12细胞的损伤,抑制细胞凋亡,以及Bcl-2/Bax比值的改变.结论 厚朴酚对MPTP诱导的PC12细胞凋亡损伤有一定的抑制作用,其作用机制涉及到影响细胞内Bcl-2/Bax蛋白的表达.

  11. Primary meningeal pheochromocytoma: a case report

    Yoon, Il Ju; Suh, Hyoung Sim [Myung-Ji St. Mary' s Hospital, Seoul (Korea, Republic of); Kim, Sung Nam [Green Cross Reference Lab, Seoul (Korea, Republic of)


    Pheochromocytoma is a rare endocrine tumor arising from the chromaffin tissue, and it is able to produce and secrete catecholamines. Lymph nodes, liver, lung and bone are the most frequent sites of metastasis. We report here on a case of pheochromocytoma arising from the dura in a patient who was surgically treated for bilateral pheochromocytoma five years previously.

  12. 硫化氢对MPP+诱导PC12细胞氧化应激损伤的保护作用%Protective Effect of Hydrogen Sulfide on MPP+ -Induced Oxidative Stress Damage in PC12 Cells

    尹蔚兰; 何剑琴; 唐国华; 张恺芳; 唐小卿


    目的 探讨硫化氢对MPP+诱导PC12细胞氧化应激损伤的保护作用.方法 以MPP+损伤PC12细胞作为帕金森病的细胞模型,甲氮甲唑蓝(MTT)法检测细胞存活率;丙酮酸二硝基幕腙比色法检测细胞上清液乳酸脱氢酶(LDH)漏出量;硫代巴比妥法检测细胞上清液中丙二醛(MDA)浓度;双氢罗丹明123染色FCM检测细胞内活性氧水平变化;应用硫化氢钠(sodium hydrosulfide,NaHS)作为H2S的供体.结果 200 μmol/L和400 μmol/L硫氢化钠呈浓度依赖性阻断MPP+ 引起PC12细胞存活率的降低;400 μmol/L硫氢化钠能明显抑制400μmol/L MPP+诱导的PC12细胞LDH的漏出,以及抑制MDA和活性氧的产生.结论 硫化氢对MPP+诱导PC12细胞的氧化应激损伤具有保护作用.

  13. Neuroprotection of geniposide against hydrogen peroxide induced PC12 cells injury: involvement of PI3 kinase signal pathway

    Jianhui LIU; Fei YIN; Lixia GUO; Xiaohong DENG; Yinhe HU


    Aim:Oxidative stress plays a critical role in the pathogenic cascade leading to neuronal degeneration in AD.Consequently,the induction of endogenous antioxidative proteins by antioxidants seems to be a very reasonable strategy for delaying the disease's progression.In previous work,we identified the neurotrophic and neuroprotective effects of geniposide,which result from the activation of glucagon-like peptide 1 receptor (GLP-1R).In this study,we explore the role of PI3 kinase sig-naling pathway in the neuroprotection of geniposide in PC12 cells.Methods: Cell viability was determined by MTr assay.Apoptosis was detected by Hoechst and PI double staining.The protein expression of Bcl-2 and phosphorylation of Akt308,Akt473,GSK-3β,and PDK1 was measured by Western blot.Results: Geniposide induced the expression of the antiapoptotic protein Bcl-2,which inhibited apoptosis in PC12 cells induced by H2O2,and this effect could be inhibited by preincubation with LY294002,a selective inhibitor of PI3K.Further-more,geniposide enhanced the phosphorylation of Akt308,Akt473,GSK-3β and PDK1 under conditions of oxidative stress.Conclusion: These results demonstrate that the PI3K signaling pathway is involved in the neuroprotection of geniposide in PC12 cells against the oxidative damage induced by H202 in PC12 cells.

  14. Chemically-induced oxidative stress increases the vulnerability of PC12 cells to rotenone-induced toxicity

    de Groot, Martje W G D M; Westerink, Remco H S


    In vitro models, including the widely used PC12 cell line, can increase insight into cellular and molecular mechanisms underlying neurodegenerative processes. An important determinant for the vulnerability of cells for chemical insults may be the endogenous level of oxidative stress. To test this hy

  15. Luteolin enhances cholinergic activities in PC12 cells through ERK1/2 and PI3K/Akt pathways.

    El Omri, Abdelfatteh; Han, Junkyu; Kawada, Kiyokazu; Ben Abdrabbah, Manef; Isoda, Hiroko


    Luteolin, a 3', 4', 5, 7-tetrahydroxyflavone, is an active compound in Rosmarinus officinalis (Lamiacea), and has been reported to exert several benefits in neuronal cells. However cholinergic-induced activities of luteolin still remain unknown. Neuronal differentiation encompasses an elaborate developmental program which plays a key role in the development of the nervous system. The advent of several cell lines, like PC12 cells, able to differentiate in culture proved to be the turning point for gaining and understanding of molecular neuroscience. In this work, we investigated the ability of luteolin to induce PC12 cell differentiation and its effect on cholinergic activities. Our findings showed that luteolin treatment significantly induced neurite outgrowth extension, enhanced acetylcholinesterase (AChE) activity, known as neuronal differentiation marker, and increased the level of total choline and acetylcholine in PC12 cells. In addition, luteolin persistently, activated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt; while the addition of pharmacological MEK/ERK1/2 inhibitor (U0126) and PI3k/Akt inhibitor (LY294002) attenuated luteolin-induced AChE activity and neurite outgrowth in PC12 cells. The above findings suggest that luteolin induces neurite outgrowth and enhanced cholinergic activities, at least in part, through the activation of ERK1/2 and Akt signaling.

  16. Protective effects of veskamide, enferamide, becatamide, and oretamide on H2O2-induced apoptosis of PC-12 cells

    Veskamide, enferamide, becatamide, and oretamide are phenolic amides whose analogues are found in plants. In this study, the four amides were prepared by chemical synthesis and their protective effects on H(2)O(2)-induced apoptosis in PC-12 cells were investigated. The syntheses were relatively si...


    The effects of pertussis toxin, an uncoupler of Gi protein from adenylate cyclase, and luzindole, a competitive inhibitor of melatonin receptor binding, were examined for their ability to inhibit melatonin-induced suppression of PC12 cell growth. Both agents inhibited the mela...

  18. Chemically-induced oxidative stress increases the vulnerability of PC12 cells to rotenone-induced toxicity

    de Groot, Martje W G D M|info:eu-repo/dai/nl/411965328; Westerink, Remco H S|info:eu-repo/dai/nl/239425952

    In vitro models, including the widely used PC12 cell line, can increase insight into cellular and molecular mechanisms underlying neurodegenerative processes. An important determinant for the vulnerability of cells for chemical insults may be the endogenous level of oxidative stress. To test this


    MAGNETIC FIELD INFLUENCE ON NGF-STIMULATED NEURITE OUTGROWTH IN PC-12 CELLS: EFFECT OF PAINT FUMES. C. F. Blackman1, D. E. House2*, S. G. Benane3*, A. Ubeda4, M.A. TrilIo4. 1 National Health and Environmental Effects Research Laboratory, EPA,Research Triangle Park, North Caro...

  20. Neuroprotective effects of the citrus flavanones against H2O2-induced cytotoxicity in PC12 cells.

    Hwang, Sam-Long; Yen, Gow-Chin


    The citrus flavanones hesperidin, hesperetin, and neohesperidin are known to exhibit antioxidant activities and could traverse the blood-brain barrier. H2O2 formation induces cellular oxidative stress associated with neurodegenerative diseases. In this study, protective effects of pretreatments (6 h) with hesperidin, hesperetin, and neohesperidin (0.8, 4, 20, and 50 microM) on H2O2-induced (400 microM, 16 h) neurotoxicity in PC12 cells were evaluated. The results showed that hesperetin, hesperidin, and neohesperidin, at all test concentrations, significantly ( p neohesperidin-treated cells) and the increase of caspase-3 activity in H2O2-induced PC12 cells. Meanwhile, hesperidin and hesperetin attenuated decreases of glutathione peroxidase and glutathione reductase activities and decreased DNA damage in H2O2-induced PC12 cells. These results first demonstrate that the citrus flavanones hesperidin, hesperetin, and neohesperidin, even at physiological concentrations, have neuroprotective effects against H2O2-induced cytotoxicity in PC12 cells. These dietary antioxidants are potential candidates for use in the intervention for neurodegenerative diseases.

  1. Protective Effect of Ecdysterone on PC12 Cells Cytotoxicity Induced by Beta-amyloid25-35

    YANG Su-fen; WU Zhong-jun; YANG Zheng-qin; WU Qin; GONG Qi-hai; ZHOU Qi-xin; SHI Jing-shan


    Objective: To examine the protective effect of ecdysterone (ECR) against beta-amyloid peptide fragment25-35 (Aβ25-35)-induced PC12 cells cytotoxicity, and to further explore its mechanism. Methods:Experimental PC12 cells were divided into the Aβ group (treated by Aβ25-35 100 μmol/L), the blank group (untreated), the positive control group (treated by Vit E 100 μmol/L after induction) and the ECR treated groups (treated by ECR with different concentrations of 1, 50 and 100 μmol/L). The damaged and survival condition of PC12 cells in various groups was monitored by lactate dehydrogenase (LDH) release and MTT assay. The content of malondialdehyde (MDA) was measured by fluorometric assay to indicate the lipid peroxidation. And the antioxidant enzymes activities in PC12 cells, including superoxide dismutases(SOD), catalase (CAT) and glutathione peroxidase(GSH-Px), were detected respectively. Results: After PC12 cells were treated with Aβ25-35 ( 100 μmol/L) for 24 hrs, they revealed a great decrease in MTT absorbance and activity of antioxidant enzymes, including SOD, CAT and GSH-Px as well as a significant increase of LDH activity and MDA content in PC12 cells (P<0.01). When the cells was pretreated with 1-100 μmol/L ECR for 24 hrs before Aβ25-35 treatment, the above-mentioned cytotoxic effect of Aβ25-35 could be significantly attenuated dose-dependently, for ECR 50 μmol/L, P<0.05 and for ECR 100 μmol/L, P<0.01. Moreover, ECR also showed significant inhibition on the Aβ25-35 induced decrease of SOD and GSH-Px activity, but not on that of CAT. Conclusion: ECR could protect PC12 cells from cytotoxicity of Aβ25-35, and the protective mechanism might be related to the increase of SOD and GSH-Px activities and the decrease of MDA resulting from the ECR-pretreatment.

  2. Hydrogen sulfide inhibits beta-amyloid peptide-induced apoptosis in PC12 cells and the underlying mechanisms

    Xiuqin Chen; Jingtian Li; Jinhui Zou; Bailing Li; Meng Wang


    BACKGROUND: Studies have demonstrated that hydrogen sulfide (H2S) levels are 55% lower in brains of Alzheimer's disease (AD) patients than in age-matched normal individuals, which suggests that H2S might be involved in some aspects of AD pathogenesis.OBJECTIVE: To observe the protective mechanisms of varied concentrations of H2S against β -amyloid-peptide (A β) induced apoptosis in pheochromoytoma (PC12) cells, and to analyze the pathway of action.DESIGN, TIME AND SETTING: A controlled, observational, in vitro experiment was performed at Nenrophysiology Laboratory in Zhougshan Medical School, Sun Yat-sen University between July 2006 and May 2007.MATERIALS: PC12 cells were provided by the Animal Experimental Center of Medical School of Sun Yat-sen University. Glybenclamide, rhodamine123, and dihydrorhodamine123 were purchased from Sigma (USA).METHODS: PCI2 cells were incubated at 37℃ in a 5% CO2-enriched incubator with RPMI-1640 medium, supplemented with 5% horse-serum and 10% fetal bovine serum. Cells in logarithmic growth curves received different treatment: The PC12 cells were maintains at 37℃ with the original medium, then incubated in A β 25-35, sodium hydrosulfide (NariS), glybenclamide, NailS+ A β 25-35, or pretreated with glybenelamide 30 minutes prior to administration of and A β 25-35, respectively. MAIN OUTCOME MEASURES: (1) The survival rate of PC12 cells was detected by MTT assay and Hoechst staining. (2) The apoptosis rate of PC12 cells was detected utilizing flow cytometry with propidium iodide staining, and morphological changes of apoptotic cells were observed. (3) The mitochondrial membrane potential was detected by Rhodamine 123-combined flow cytometry. (4) The intracellular reactive oxygen species content was detected by dihydrorhodamine123-combined flow cytometry. RESULTS: A β 25-35 induced significantly decreased viability and increased percentage of apoptosis in PC 12 cells, as well as dissipated mitochondrial membrane potential

  3. Effects of exendin-4 on methylglyoxal-induced oxidative stress in PC12 cells%Exendin-4对甲基乙二醛诱导PC12细胞氧化应激的影响

    周清; 王燕萍; 刘小莺; 刘晓红; 潘晓东; 陈洲; 刘礼斌


    目的:探讨肠促胰岛素类似物(Ex-4)对甲基乙二醛(MG)诱导PC12细胞氧化应激的影响及其机制.方法:传代培养PC12细胞,不同浓度MG(0、0.25、0.50、0.75、1.0、2.0 mmol/1)处理PC12细胞12~48 h,或用不同浓度Ex-4(25、50、100、200 nmol/L)预处理24h后加用MG(0.75 mmol/L)干预24h后,MTT比色法检测细胞存活率;荧光探针法检测活性氧(ROS)含量,黄嘌呤氧化酶法检测超氧化物歧化酶(SOD)活力.Ex-4(100 nmol/L)预处理PC12细胞24h加用MG(0.75 mmol/L)干预1h后,Western blot检测蛋白P-IκB-α、IκB-α表达情况.结果:随着MG浓度的增加和作用时间的延长,PC12细胞存活率逐渐降低;加用不同浓度Ex-4预处理后,PC12细胞存活率较单独MG处理组逐渐升高.100 nmol/L的Ex-4预处理PC12细胞后,ROS表达量较MG单独处理组下降65.30%(P< 0.01);NAC预处理组(阳性对照)ROS表达量下降107.40% (P< 0.01);Ex-4预处理组SOD活力增加5.30 U/mg prot(P<0.01);NAC预处理组SOD活力增加8.53 U/mg prot(P<0.01).Ex-4预处理组P-IκB-α/IκB-α表达比例下降25.50%(P<0.01);NAC预处理组P-IκB-α/IκB-α表达比例下降35.14%(P< 0.01).结论:Ex-4浓度依赖性地增加MG诱导的PC12细胞的存活率.Ex-4能够减轻MG诱导的PC12细胞的氧化应激,其机制可能涉及抑制蛋白IκB-α的活化.

  4. 甲基苯丙胺致PC12细胞的差异蛋白表达%Differentially expressed proteins in PC12 cells induced by methylamphetamine

    李丽增; 王慧君; 兰江维; 刘超


    目的 探讨甲基苯丙胺( METH)作用于PC12细胞后的蛋白质表达变化.方法 METH2.5 mmol· L-1作用PC12细胞24 h后,提取细胞总蛋白质,丙酮沉淀法纯化蛋白,Braford法对蛋白质进行定量,并对蛋白质进行双向凝胶电泳,Image scannerⅢ透射扫描仪获取凝胶电泳图谱.应用Image Master 7.0软件对获得的双向凝胶电泳图谱进行差异性蛋白质点分析,并对相应差异蛋白点用高端基质辅助激光解析-飞行时间( MALDI-TOF)串联质谱仪进行差异蛋白质鉴定.结果 应用双向凝胶电泳结合质谱分析技术,METH作用PC12细胞24h后,共鉴定出18个差异表达蛋白质点,其中8个差异蛋白点在METH作用后表达增强,10个蛋白点表达减弱.这些蛋白质主要包括细胞骨架相关蛋白、分子伴侣、氧化应激和凋亡相关的蛋白以及与能量代谢相关的酶类.结论 METH诱导PC12细胞18个蛋白差异表达.%OBJECTIVE To investigate differentially expressed proteins of PC 12 cells exposed to methylamphetamine ( METH ). METHODS PC 12 cells were treated with METH 2.5 mmol·L-1 for 24 h. Total proteins were extracted for two dimensional gel electrophoresis (2-DE). Impurities were removed by acetone precipitation and the quantity of proteins was fixed by Braford method. Proteins were separated by two-dimensional gel electrophoresis (2-DE) , and the 2-DE patterns were obtained by transmission scan with Image scanner HI. The scanned patterns were then analyzed by ImageMaster 7. 0 software to search the differentially expressed proteins. Those protein spots were cutted and then identified by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) tandem mass spectrum. RESULTS After PC 12 cells were cultured with METH for 24 h, eighteen differentially expressed proteins were identified in PC 12 cells, eight of which were up-regulated and 10 down-regulated. According to functions of proteins, these proteins were divided into 4 categories

  5. Pituitary adenylate cyclase activating polypeptide modulates catecholamine storage and exocytosis in PC12 cells.

    Yan Dong

    Full Text Available A number of efforts have been made to understand how pituitary adenylate cyclase activating polypeptide (PACAP functions as a neurotrophic and neuroprotective factor in Parkinson's disease (PD. Recently its effects on neurotransmission and underlying mechanisms have generated interest. In the present study, we investigate the effects of PACAP on catecholamine storage and secretion in PC12 cells with amperometry and transmission electron microscopy (TEM. PACAP increases quantal release induced by high K+ without significantly regulating the frequency of vesicle fusion events. TEM data indicate that the increased volume of the vesicle is mainly the result of enlargement of the fluidic space around the dense core. Moreover, the number of docked vesicles isn't modulated by PACAP. When cells are acutely treated with L-DOPA, the vesicular volume and quantal release both increase dramatically. It is likely that the characteristics of amperometric spikes from L-DOPA treated cells are associated with increased volume of individual vesicles rather than a direct effect on the mechanics of exocytosis. Treatment with PACAP versus L-DOPA results in different profiles of the dynamics of exocytosis. Release via the fusion pore prior to full exocytosis was observed with the same frequency following treatment with PACAP and L-DOPA. However, release events have a shorter duration and higher average current after PACAP treatment compared to L-DOPA. Furthermore, PACAP reduced the proportion of spikes having rapid decay time and shortened the decay time of both fast and slow spikes. In contrast, the distributions of the amperometric spike decay for both fast and slow spikes were shifted to longer time following L-DOPA treatment. Compared to L-DOPA, PACAP may produce multiple favorable effects on dopaminergic neurons, including protecting dopaminergic neurons against neurodegeneration and potentially regulating dopamine storage and release, making it a promising

  6. Azole fungicides disturb intracellular Ca2+ in an additive manner in dopaminergic PC12 cells.

    Heusinkveld, Harm J; Molendijk, Jeffrey; van den Berg, Martin; Westerink, Remco H S


    Humans are exposed to complex mixtures of pesticides and other compounds, mainly via food. Azole fungicides are broad spectrum antifungal compounds used in agriculture and in human and veterinary medicine. The mechanism of antifungal action relies on inhibition of CYP51, resulting in inhibition of fungal cell growth. Known adverse health effects of azole fungicides are mainly linked to CYP inhibition. Additionally, azole fungicide-induced neurotoxicity has been reported, though the underlying mechanism(s) are largely unknown. We therefore investigated the effects of a group of six azole fungicides (imazalil, flusilazole, fluconazole, tebuconazole, triadimefon, and cyproconazole) on cell viability using a combined alamar Blue/CFDA-AM assay and on oxidative stress using a H2-DCFDA fluorescent assay. As calcium plays a pivotal role in neuronal survival and functioning, effects of these six azole fungicides and binary and quaternary mixtures of azole fungicides on the intracellular calcium concentration ([Ca(2+)]i) were investigated using single-cell fluorescence microscopy in dopaminergic PC12 cells loaded with the calcium-sensitive fluorescent dye Fura-2. Only modest changes in cell viability and ROS production were observed. However, five out of six azole fungicides induced a nonspecific inhibition of voltage-gated calcium channels (VGCCs), though with varying potency. Experiments using binary IC20 and quaternary IC10 mixtures indicated that the inhibitory effects on VGCCs are additive. The combined findings demonstrate modulation of intracellular Ca(2+) via inhibition of VGCCs as a novel mode of action of azole fungicides. Furthermore, mixtures of azole fungicides display additivity, illustrating the need to take mixture effects into account in human risk assessment.

  7. Silencing of nicotinamide nucleotide transhydrogenase impairs cellular redox homeostasis and energy metabolism in PC12 cells.

    Yin, Fei; Sancheti, Harsh; Cadenas, Enrique


    Mitochondrial NADPH generation is largely dependent on the inner-membrane nicotinamide nucleotide transhydrogenase (NNT), which catalyzes the reduction of NADP(+) to NADPH utilizing the proton gradient as the driving force and NADH as the electron donor. Small interfering RNA (siRNA) silencing of NNT in PC12 cells results in decreased cellular NADPH levels, altered redox status of the cell in terms of decreased GSH/GSSG ratios and increased H(2)O(2) levels, thus leading to an increased redox potential (a more oxidized redox state). NNT knockdown results in a decrease of oxidative phosphorylation while anaerobic glycolysis levels remain unchanged. Decreased oxidative phosphorylation was associated with a) inhibition of mitochondrial pyruvate dehydrogenase (PDH) and succinyl-CoA:3-oxoacid CoA transferase (SCOT) activity; b) reduction of NADH availability, c) decline of mitochondrial membrane potential, and d) decrease of ATP levels. Moreover, the alteration of redox status actually precedes the impairment of mitochondrial bioenergetics. A possible mechanism could be that the activation of the redox-sensitive c-Jun N-terminal kinase (JNK) and its translocation to the mitochondrion leads to the inhibition of PDH (upon phosphorylation) and induction of intrinsic apoptosis, resulting in decreased cell viability. This study supports the notion that oxidized cellular redox state and decline in cellular bioenergetics - as a consequence of NNT knockdown - cannot be viewed as independent events, but rather as an interdependent relationship coordinated by the mitochondrial energy-redox axis. Disruption of electron flux from fuel substrates to redox components due to NNT suppression induces not only mitochondrial dysfunction but also cellular disorders through redox-sensitive signaling.

  8. Neurotrophic effect of citrus 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone: promotion of neurite outgrowth via cAMP/PKA/CREB pathway in PC12 cells.

    Hui-Chi Lai

    Full Text Available 5-Hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5-OH-HxMF, a hydroxylated polymethoxyflavone, is found exclusively in the Citrus genus, particularly in the peels of sweet orange. In this research, we report the first investigation of the neurotrophic effects and mechanism of 5-OH-HxMF in PC12 pheochromocytoma cells. We found that 5-OH-HxMF can effectively induce PC12 neurite outgrowth accompanied with the expression of neuronal differentiation marker protein growth-associated protein-43(GAP-43. 5-OH-HxMF caused the enhancement of cyclic AMP response element binding protein (CREB phosphorylation, c-fos gene expression and CRE-mediated transcription, which was inhibited by 2-naphthol AS-E phosphate (KG-501, a specific antagonist for the CREB-CBP complex formation. Moreover, 5-OH-HxMF-induced both CRE transcription activity and neurite outgrowth were inhibited by adenylate cyclase and protein kinase A (PKA inhibitor, but not MEK1/2, protein kinase C (PKC, phosphatidylinositol 3-kinase (PI3K or calcium/calmodulin-dependent protein kinase (CaMK inhibitor. Consistently, 5-OH-HxMF treatment increased the intracellular cAMP level and downstream component, PKA activity. We also found that addition of K252a, a TrKA antagonist, significantly inhibited NGF- but not 5-OH-HxMF-induced neurite outgrowth. These results reveal for the first time that 5-OH-HxMF is an effective neurotrophic agent and its effect is mainly through a cAMP/PKA-dependent, but TrKA-independent, signaling pathway coupling with CRE-mediated gene transcription. A PKC-dependent and CREB-independent pathway was also involved in its neurotrophic action.

  9. 新合成腺苷结构类似物B2对无血清培养PC12细胞损伤的保护作用%Protective effect of new adenosine analog B2 against serum deprivation-induced PC12 cell injury

    孙静; 李敏; 康瑞霞; 石建功; 张建军


    研究新合成腺苷结构类似物B2对无血清培养所致PC12细胞损伤的保护作用,并对其机制进行初步探讨.采用放射性配基3H-MSX-2与腺苷A2A受体竞争结合法检测B2与大鼠纹状体腺苷A2A受体的亲和力;MTT法检测B2对无血清培养PC12细胞存活率的影响;用荧光探针DCFDA检测细胞内活性氧(ROS)含量变化.放射性配基受体竞争结合实验求得B2与大鼠脑纹状体A2A受体结合的K值为0.37 μmol·L-1.B2 (0.1、1、10和100 μmol·L-1)可使去血清培养24h的PC12细胞存活率由模型组的49.6%分别上升至63.3%、74.9%、86.3%、88.1%.合并使用0.1 μmol·L-1 SCH 58261使B2 (0.1~10 μmol·L-1)的作用分别下降16.1%,24.0%和19.8%.去血清培养24 h使PC12细胞内ROS含量升高为对照组的3.5倍,B2(1~100 μmol·L-1)可使胞内荧光强度分别降低为对照组的3.1倍、2.4倍和1.5倍.B2对无血清培养所致PC12细胞损伤有明显的保护作用,该作用与腺苷A2A受体相关,同时可显著降低去血清培养时细胞内活性氧自由基的过度生成,可能是其产生保护作用的机制之一.%This study is to investigate the effect of compound B2 on the damage of PC 12 cells induced by serum deprivation and to explore its related mechanisms. The binding characteristics of B2 to rat striatum adenosine A2A receptor was studied by radioligand 3H-MSX-2 binding assay. Cell viability was detected by MTT assay. ROS formation was measured after DCFDA fluorescent staining. B2 has affinity to rat adenosine A2A receptor (Ki = 0.37 umol-L-1). B2 remarkably increased PC 12 cell survival rate in serum deprivation-induced PC 12 cells. The percentage of serum deprivation-induced death of PC 12 was 49.6%, and the treatment of B2 (0.1-100 umolL-1) increased the cell viability to 63.3%, 74.9%, 86.3% and 88.1%, respectively. Adenosine A2A receptor antagonist SCH 58261 could significantly block the protective effect of B2. The cell viability with 0

  10. Malignant Catatonia Mimicking Pheochromocytoma

    Sophia Wong


    Full Text Available Malignant catatonia is an unusual and highly fatal neuropsychiatric condition which can present with clinical and biochemical manifestations similar to those of pheochromocytoma. Differentiating between the two diseases is essential as management options greatly diverge. We describe a case of malignant catatonia in a 20-year-old male who presented with concurrent psychotic symptoms and autonomic instability, with markedly increased 24-hour urinary levels of norepinephrine at 1752 nmol/day (normal, 89–470 nmol/day, epinephrine at 1045 nmol/day (normal, <160 nmol/day, and dopamine at 7.9 μmol/day (normal, 0.4–3.3 μmol/day. The patient was treated with multiple sessions of electroconvulsive therapy, which led to complete clinical resolution. Repeat urine collections within weeks of this presenting event revealed normalization or near normalization of his catecholamine and metanephrine levels. Malignant catatonia should be considered in the differential diagnosis of the hypercatecholamine state, particularly in a patient who also exhibits concurrent catatonic features.

  11. [Pheochromocytoma. Report of 10 cases].

    Rabii, R; Joual, A; Rais, H; Bennani, S; el Mrini, M; Benjelloun, S


    We report 10 cases of adrenal pheochromocytoma seen over a period 15-years. A female predominance was noted (8 women/2 men). Patients were aged between 16-46 years with a mean of 34 years. Clinical manifestations consisted of hypertension observed in all cases, with vasomotor symptoms (90%). Time to consultation was prolonged (mean: 23 months). CT scan performed in 7 cases showed pheochromocytoma in all cases, located on the right side in 6 cases, while one pheochromocytoma was located in Zukerkandal organ. All patients were operated via anterior approach and adrenalectomy was performed. A favourable course was observed in 90% of cases with normalisation blood pressure. One death was noted. Histological examination showed no malignancy in all cases.

  12. NGF诱导PC12细胞分化的神经细胞行为观察%Observation on differentiated neuron behaviour induced by NGF in cultured PC12 cells

    丁明杰; 史学义; 张娓; 丁一


    Aim:To study the cell division, neurite extension, migration of neurons differentiated by NGF in subcultured PC12 cells.Methods: The Giemsa staining, methyl green-pyronin staining and proliferating cell nuclear antigen immunohistochemical assay were used to study the subcultured PC12 cells.Results: The differentiated neuron appeared different patterns of amitotic figures such as transverse sever, longitudinal split, etc. The proliferating neurons divided in an asymmetric way morphologically and chemically. The differentiated neuron appeared an obvious movement of cell migration and neurite extension.Conclusion: The neurons differentiated by NGF in cultured PC12 cells can divide in amitotic way.The differentiated neuron can form junction by cell migration, neurite extension or cytoplasmic bridge.%目的:观察PC12细胞分化的神经细胞的分裂、突起延伸、迁徙和分化等细胞行为。方法:采用Giemsa、甲绿-派郎宁染色和增殖细胞核抗原(PCNA)免疫组织化学染色方法观察培养的PC12细胞。结果:培养分化的神经细胞呈现横裂、纵裂等不同方式的无丝分裂像;分裂中的神经细胞可呈现形态及化学不对称性;分化的神经细胞有明显的细胞迁徙运动和突起延伸。结论:NGF诱导PC12细胞分化的神经细胞能以无丝分裂方式增殖;分化神经元可以通过细胞迁徙、突起延伸及共同胞质桥等方式相连接。

  13. Experimental Study on the Inhibitory Effects of Vitamin E Against Manganese-induced Cytotoxicity in PC12 Cells%维生素E抑制锰诱导的PC12细胞毒性的实验研究

    王迪雅; 蔡同建; 赵芳; 姚婷; 陈景元; 骆文静


    目的 观察维生素E对锰诱导的PC12细胞毒性的抑制效应,从而为锰神经毒性的防护提供可能的途径.方法 MTT、TUNEL、姬姆萨染色、流式细胞仪检测细胞毒性;试剂盒检测MDA含量以及T-SOD、MnSOD活性的变化. 结果 锰可以诱导PC12细胞氧化应激水平显著增高,显示为MDA含量的增高和T-SOD、MnSOD活性代偿性升高.维生素E可以抑制锰诱导的PC12细胞毒性,表现为其可抑制锰诱导的细胞活力的下降以及凋亡率的升高. 结论 维生素E作为抗氧化剂可以抑制由锰诱导的PC12细胞的一系列细胞毒性,补充维生素可作为一种防护锰毒性的有效手段.%Objective To study the inhibitory effects of vitamin E on manganese- induced cytotoxicity in PC12 cells and to provide a potential new way against manganese — induced neurotoxicity. Methods The cytotoxicity was determined by MTT assay, TUNEL, Giemsa staining and flow cytometry, while the MDA content, T- SOD and MnSOD activities were detected by kits. Results Manganese induced enhanced oxidative stress, as shown by the elevation of MDA content and compensatory increase of T— SOD and MnSOD activities. The supplement of vitamin E could inhibit manganese- induced cytotoxicity in that it could antagonize the decrease of cell viability and increase of cell apoptosis rate caused by manganese. Conclusions As a typical antioxidant, vitamin E can inhibit manganese- induced cytotoxicity in PC12 cells. Vitamin E supplement can be an effective way against manganese toxicity.

  14. 外源性 ATP 诱导 PC12细胞的膜孔形成%Exogenous ATP induces formation of membrane pore in PC12 cells

    沈慧; 尹雅玲; 李超堃; 赵红岗; 马洁; 李东亮


    AIM:To investigate the formation of membrane pore in PC 12 cells induced by exogenous adenosine triphosphate ( ATP) and to identify the key molecular targets .METHODS:PC12 cells were treated with different concen-trations of ATP to establish the injury model .The morphological change was observed under an inverted phase -contrast mi-croscope.The viability of the PC12 cells was measured by CCK-8 assay.Fluorescent dye YO-PRO-1 was used to detect the membrane permeability.The expression of P2X7 receptor and pannexin 1 (Panx1) at mRNA and protein levels was as-sessed by real-time PCR and Western blotting .RESULTS:After exposed to ATP (1 mmol/L, 3 mmol/L and 5 mmol/L) for 3 h, the PC12 cells became edematous , and the number of adherent cells decreased gradually in a dose-dependent man-ner .The cell viabilities in 3 mmol/L ATP group and 5 mmol/L ATP group were significantly decreased compared with con-trol group (P0. 05).The expression of P2X7 receptor at mRNA and protein levels was significantly increased (P0.05) when PC12 cells were exposed to ATP for 3 h.CONCLUSION:Extracellular ATP at high concentration may induce membrane pore formation with the expression and activation of P 2X7 receptor in PC12 cells.%目的:探讨外源性三磷酸腺苷( ATP)诱导PC12细胞的膜孔形成及关键分子靶标。方法:用不同浓度的ATP处理培养的PC12细胞,采用倒置相差显微镜观察形态,CCK-8法检测细胞存活率,YO-PRO-1染色检测细胞膜通透性,Western blotting和real-time PCR检测P2X7受体和pannexin 1(Panx1)表达的变化。结果:(1)ATP(1 mmol/L、3 mmol/L、5 mmol/L)作用3 h,可见随着ATP浓度升高,PC12细胞变圆,脱壁细胞增多;当ATP浓度为3 mmol/L或5 mmol/L时,PC12细胞活力较对照组显著下降(P<0.05)。(2)不同浓度的ATP(0、1、3、5 mmol/L)作用1 h,PC12细胞摄入YO-PRO-1的荧光强度随着浓度增加而增加;同一浓度的ATP作用不同时间(15

  15. Fluorescence labelling of lipid raft′s special proteins and its functions in living PC12 cell%脂筏特异蛋白荧光标记及在PC12细胞中的功能

    曹惠雅; 李臣鸿



  16. Effects of an autophagy/lysosomal pathway induced by 6-hydroxydopamine in PC12 cells%6-羟基多巴胺致PC12细胞损伤机制中自噬的影响

    周生奎; 程言博; 耿润潼; 陈浩; 刘涵; 徐兴顺; 耿德勤


    Objective To investigated the role of the autophagy lysosomal pathway in PD cells and the possible molecular mechanisms. Methods A dopaminergic neuronal injury model was induced by 6-OHDA in PC12 cells . Autophagosomes in PC12 cells were examined by transmission electronmicro-scopy( TEM ). The expression of LC3- Ⅱ , Cathepsin B were assayed by western blot analysis. Results TEM revealed that the autophagosomes were increased in PC12 cells after 6-OHDA treatment and appeared apoptosis. The LC3-Ⅱ (2h:52.57 ±2.27,4h:56.83 ±3.51,6h:73.43 ±5.41,12h:103.90 ±2.57,24h: 100.40 ±3.91 )and Cathepsin B expression ( model group: 113.80 ± 4.46; normal group 35.89 ± 3.40) were increased after 6-OH DA treatments (P < 0.05 or P < 0.01 ). Conclusion The results indicate that autophagy lysosome pathway is involved in 6-OHDA-induced cell death in PC12 cells.%目的 研究自噬在帕金森病(PD)细胞模型中的作用及可能的机制.方法 体外培养的PC12细胞加入6-羟基多巴(6-OHDA)诱导多巴胺能神经元损伤模型.利用透射电镜观察PC12细胞中自噬的激活,免疫印迹法检测LC3-Ⅱ、Cathepsin B蛋白的表达.结果 电镜下观察到6-OHDA可使PC12细胞内自噬体增多,并出现了凋亡特征.6-OHDA作用2h(灰度比:52.57±2.27),4h(灰度比:56.83±3.51),6h(灰度比:73.43±5.41),12h(灰度比:103.90±2.57),24h(灰度比:100.40±3.91)时LC3-Ⅱ表达逐渐升高,与正常对照组(42.10±2.05)比较差异有统计学意义(P<0.05),模型组Cathepsin B(113.80±4.46)表达与正常对照组(35.89±3.40)比较明显增加(P<0.01),与模型组相比,广谱蛋白酶抑制剂UTI组(57.69±4.24)降低Cathepsin B表达(P<0.01).结论 自噬/溶酶体途径参与PC12细胞的死亡过程:6-OHDA诱导自噬过度激活,LC3-Ⅱ与Cathepsin B表达增加,促进细胞死亡.

  17. 刺五加苷B对MPP+诱导PC12细胞损伤ERK通路的影响%The effect of Eleutheroside B on ERK1/2 of MPP+-induced PC12 cells

    董杨; 刘树民; 安丽凤; 卢芳; 唐波; 周世慧


    目的 观察刺五加苷B对MPP+诱导损伤的PC12细胞ERK1/2蛋白及相关转录因子表达的影响,探究其神经保护作用机制.方法 以MPP+损伤的PC12细胞为模型组,以正常PC12细胞为对照组,Western blot法检测细胞ERK1/2表达及磷酸化水平,荧光定量PCR法检测c-fos和c-jun mRNA 的表达水平.结果 模型组ERK1/2磷酸化水平较对照组显著降低(P<0.01).c-fos和c-jun表达明显上调,差异有统计学意义;给予刺五加苷B(10 mg·L-1)后,受损细胞ERK1/2的磷酸化水平明显升高,c-fos 和c-jun基因的过表达水平降低.结论 刺五加苷B对MPP+诱导的PC12细胞损伤的保护作用机制可能与提高ERK1/2蛋白磷酸化水平.避免诱发c-fos和c-jun基闪过表达有关.

  18. A genome-wide signature of glucocorticoid receptor binding in neuronal PC12 cells

    Polman J Annelies E


    Full Text Available Abstract Background Glucocorticoids, secreted by the adrenals in response to stress, profoundly affect structure and plasticity of neurons. Glucocorticoid action in neurons is mediated by glucocorticoid receptors (GR that operate as transcription factors in the regulation of gene expression and either bind directly to genomic glucocorticoid response elements (GREs or indirectly to the genome via interactions with bound transcription factors. These two modes of action, respectively called transactivation and transrepression, result in the regulation of a wide variety of genes important for neuronal function. The objective of the present study was to identify genome-wide glucocorticoid receptor binding sites in neuronal PC12 cells using Chromatin ImmunoPrecipitation combined with next generation sequencing (ChIP-Seq. Results In total we identified 1183 genomic binding sites of GR, the majority of which were novel and not identified in other ChIP-Seq studies on GR binding. More than half (58% of the binding sites contained a GRE. The remaining 42% of the GBS did not harbour a GRE and therefore likely bind GR via an intermediate transcription factor tethering GR to the DNA. While the GRE-containing binding sites were more often located nearby genes involved in general cell functions and processes such as apoptosis, cell motion, protein dimerization activity and vasculature development, the binding sites without a GRE were located nearby genes with a clear role in neuronal processes such as neuron projection morphogenesis, neuron projection regeneration, synaptic transmission and catecholamine biosynthetic process. A closer look at the sequence of the GR binding sites revealed the presence of several motifs for transcription factors that are highly divergent from those previously linked to GR-signaling, including Gabpa, Prrx2, Zfp281, Gata1 and Zbtb3. These transcription factors may represent novel crosstalk partners of GR in a neuronal context

  19. [Acute cardiac failure in pheochromocytoma.

    Jønler, Morten; Munk, Kim


    Pheochromocytoma (P) is an endocrine catecholamine-secreting tumor. Classical symptoms like hypertension, attacks of sweating, palpitations, headache and palor are related to catecholamine discharge. We provide a case of P in a 71 year-old man presenting with acute cardiac failure, severe reduction...

  20. Undiagnosed pheochromocytoma: the anesthesiologist nightmare.

    Myklejord, Duane J


    A male, 62 years of age, presented to the operating room for the removal of a right adrenal mass. Induction of anesthesia triggered a severe hypertensive crisis resistant to high doses of nitroprusside, nitroglycerin and labetalol. The crisis was ultimately resolved with the administration of 5 mg bolus of phentolamine. Surgery was canceled, the patient was transported to the intensive care unit with a continuous drip of phentolamine. High urinary and plasma catecholamines suggested the presence of pheochromocytoma. Three weeks of oral phenoxybenzamine therapy subsequently allowed uneventful induction of anesthesia and open adrenalectomy. Pathologic examination of the resected adrenal tissue confirmed the presence of pheochromocytoma. Anesthetic drugs can exacerbate the life-threatening cardiovascular effects of catecholamines secreted by pheochromocytomas. Treating patients preoperatively with alpha-adrenergic blockade is helpful for reducing intraoperative hypertensive episodes. Postoperative administration of inotropic agents to correct hypotension due to catecholamine withdrawal may be required. Management of patients with pheochromocytoma remains a challenge for the anesthesiologist, despite the advent of new drugs and techniques.

  1. Expression and purification of recombinant vesicular glutamate transporter VGLUT1 using PC12 cells and High Five insect cells

    Andersen Søren S.L.


    Full Text Available In synaptic vesicles, the estimated concentration of the excitatory amino acid glutamate is 100-150 mM. It was recently discovered that VGLUT1, previously characterized as an inorganic phosphate transporter (BNPI with 9-11 predicted transmembrane spanning domains, is capable of transporting glutamate. The expression and His-tag based purification of recombinant VGLUT1 from PC12 cells and High Five insect cells is described. Significantly better virus and protein expression was obtained using High Five rather than Sf9 insect cells. PC12 cell expressed VGLUT1 is functional but not the Baculovirus expressed protein. The lack of functionality of the Baculovirus expressed VGLUT1 is discussed. The data indicate that VGLUT1 readily oligomerizes/dimerizes. The data are discussed in the context of developing this system further in order to reconstitute vesicular glutamate uptake in vitro using lipid-detergent vesicles.

  2. 蛋白酶体功能障碍对多巴胺能PC12细胞的特异性损伤%Specific Damage of Ubiquitin-Proteasome System Dysfunction to Dopaminergic PC12 Cells

    周海燕; 戚辰; 范国华; 王刚; 张宇红; 陈生弟


    目的探讨蛋白酶体抑制剂lactacystin对多巴胺能PC12细胞的特异性损伤.方法不同浓度的lactacystin(1、5、10、15和20μmoL/L)分别处理多巴胺能PC12细胞和胶质瘤U251细胞24 h,MTT法检测细胞活力;50 μmol/L的lactacystin处理U251细胞24 h,MTT法检测细胞活力;10、20 μmol/L lactacystin处理PC12细胞,Western Blot检测细胞内多泛素化蛋白含量;单胺氧化酶B抑制剂selegiline(500μmol/L)和特异性酪氨酸羟化酶抑制剂α-MT(1 mmol/L)提前4 h预处理PC12细胞,再与10 μmol/Llactacystin共同作用24 h,MTT法检测细胞活力,Western Blot检测多泛素化蛋白含量.结果Lactacystin呈剂量依赖性损伤多巴胺能PC12细胞,其对胶质瘤U251细胞无毒性作用,而且其毒性与细胞内多泛素化蛋白生成增加相关.用以增加细胞内多巴胺含量的selegiline和用以减少细胞内多巴胺含量的α-MT都导致lactacystin毒性增强.结论蛋白酶体功能障碍特异性损伤多巴胺能细胞,而其特征性的神经递质多巴胺在其易感性中的作用复杂.

  3. Combined Effects of 50 Hz Magnetic Field and Magnetic Nanoparticles on the Proliferation and Apoptosis of PC12 Cells

    JIA Hong Li; WANG Chao; LI Yue; LU Yan; WANG Ping Ping; PAN Wei Dong; SONG Tao


    ObjectiveTo investigate the bioeffects of extremely low frequency (ELF) magnetic field (MF) (50 Hz, 400μT) and magnetic nanoparticles (MNPs) via cytotoxicity and apoptosis assays on PC12 cells. MethodsMNPs modified by SiO2 (MNP-SiO2) were characterized by transmission electron microscopy (TEM), dynamic light scattering and hysteresis loop measurement.PC12 cells were administrated with MNP-SiO2 with or without MF exposure for 48 h. Cytotoxicity and apoptosis were evaluated with MTT assay and annexin V-FITC/PI staining, respectively. The morphology and uptake of MNP-SiO2 were determined by TEM. MF simulation was performed by Ansoft Maxwell based on the finite element method. ResultsMNP-SiO2 were identified as~20nm (diameter) ferromagnetic particles. MNP-SiO2reduced cell viability in a dose-dependent manner. MF also reduced cell viability with increasing concentrations of MNP-SiO2. MNP-SiO2 alone did not cause apoptosis in PC12 cells; instead, the proportion of apoptotic cells increased significantly under MF exposure and increasing doses of MNP-SiO2. MNP-SiO2 could be ingested andthen cause a slight change in cellmorphology. ConclusionCombined exposure of MF and MNP-SiO2 resulted in remarkable cytotoxicity and increased apoptosis in PC12 cells. The results suggested that MF exposure couldstrengthen the MF of MNPs, which may enhance the bioeffects of ELF MF.

  4. Early Cellular Responses of Purine Nucleoside-mediated Protection of Hypoxia-induced Injuries of Neuronal PC12 Cells

    Bettina Tomaselli


    Full Text Available Hypoxia in brain may lead to cell death by apoptosis and necrosis. In parallel adenosine, a powerful endogenous neuroprotectant is formed. We wanted to investigate the effect of adenosine and its purine nucleoside relatives, inosine and guanosine on early cellular responses to hypoxia. O2-sensitive neuronal PC12-cells were subjected to chemical hypoxia induced with rotenone, an inhibitor of mitochondrial complex I. Loss of viability after hypoxic insult was impressively rescued by adenosine, guanosine and inosine. PC12-cells mainly express the A2A adenosine receptor. Its inhibition with a specific antagonist (CSC induced cell death of PC12-cells, which could be salvaged by adenosine but not with guanosine or inosine. We have previously demonstrated the important role of mitogen activated protein kinases 1/2 (p42/44 MAPK in purine-mediated rescue. In this study we were interested in the involvement of protein kinases whose activities mediate these processes, including protein kinase A (PKA, phosphoinositide 3-kinase (PI3-K and protein kinase C-related kinases (PRK 1/2. Pharmacological inhibition of PKA and PI3-K increased hypoxia-induced toxicity and likewise also affected the rescue by purine nucleosides. Nerve growth factor (NGF and purine nucleosides induced an activation of PRK 1/2, which to our knowledge indicates for the first time that these kinases are potentially involved in purine nucleoside-mediated rescue of hypoxic neuronal cells. Results suggest that A2A receptor expressing cells are mainly dependent on the purine nucleoside adenosine for their rescue after hypoxic insult. In addition to PKA, PI3-K is an important effector molecule in A2A-mediated signaling and for the rescue of PC12-cells after hypoxic insult.

  5. Extracellular ATP activates NFAT-dependent gene expression in neuronal PC12 cells via P2X receptors

    Becker Walter


    Full Text Available Abstract Background Treatment of neuronal PC12 cells with ATP induces depolarisation and increases intracellular calcium levels via purinergic receptors. In many cell types, sustained elevation of intracellular calcium levels cause changes in gene expression via activation of the transcription factor NFAT (nuclear factor of activated T cells. We have therefore characterised the signalling pathway by which ATP regulates NFAT-dependent gene expression in PC12 cells. Results The activation of NFAT transcriptional activity by extracellular ATP was characterised with the help of reporter gene assays. Treatment of PC12 cells with ATP elicited a dose-dependent increase in luciferase activity (EC50 = 78 μM. UTP, 4-benzoylbenzoyl ATP and α,β-methylene ATP did not mimic the effect of ATP, which was abolished by treatment with the P2X receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS. This pharmacological characterisation provides evidence for a critical role of ionotropic P2X receptors. Blockade of L-type voltage-dependent calcium channels by nifedipine reduced the response of NFAT to ATP, indicating that a depolarisation-mediated calcium influx was required for maximal NFAT activation. Inhibition of store-operated calcium entry by the pyrazole derivative BTP2 also diminished ATP-dependent NFAT activation. Furthermore, ATP-induced NFAT activation was associated with the activation of the mitogen-activated protein kinases ERK1/2. Finally, treatment with ATP increased the levels of the NFAT target transcripts, RCAN1-4 (regulator of calcineurin and BDNF (brain derived neurotrophic factor. Conclusion The present data show that ATP induces NFAT-dependent changes in gene expression in PC12 cells by acting on P2X receptors. Maximal NFAT activation depends on both depolarisation-induced calcium influx and store-operated calcium entry and requires the activity of the protein phosphatase calcineurin and the mitogen-activated protein

  6. Protective effect of glutathione proxidase 1 overexpression on amyloid β protein-mediated injury of PC12 cells%谷胱甘肽过氧化物酶1高表达对β淀粉样蛋白介导PC12细胞损伤的保护作用

    马琳; 王辉; 王淑荣; 陈志斌; 张海英


    Objective To study the molecular mechanism underlying amyloid β(Aβ) protein-mediated Aizheimer's disease and the protective effect of glutathione proxidase 1(GPX1) overexpres-sion on Aβ protein-mediated injury of PC12 cells. Methods PC12 cells, transfected into plticx plasmids and blank vector plasmids containing human GPX1 gene,were divided into PC12 group, GPX1-PC12 group and plncx-PC12 group,and intervened with Aβ25-35 and sodium selenite+Aβ25-35, respectively. Their survival rate was measured by MTT assay. Expression of pCREB was detected by immunohistochemistry. Results No significant difference was found in the survival rate of PC12 ceils between PC1 2 and pincx-PC12 groups(P>0. 05). The survival rate of PC12 cells was significantly higher in GPX1-PC12 group than in plncx-PC12 group(P<0. 01). The positive rate of pCREB protein was significantly lower in plncx-PC12 and GPX1-PC12 groups, especially in plncx-PC12 group than in GPX1-PC12 group after intervention with Aβ25-35 (P<0. 05) and significantly higher in plncx-PC12 and GPX1-PC12 grous,especially in GPX1-PC12 group than in plncx-PC12 group after intervention with sodium selenite+ Aβ25-35 (P<0, 01). Conclusion GPX1 gene overexpression can effectively protect PC12 cells against Aβ25-35-mediated injury. Aβ25-35 can down-regulate the expression of pCREB in PC12 cells. Both GPX1 and pCREB participate in protection of PC12 cells,thus reducing Aβ25-35-induced injury of PC12 cells.%目的 探讨β淀粉样蛋白(Aβ)所致阿尔茨海默病发病的分子机制,以及谷胱甘肽过氧化物酶1(GPX1)高表达时对其所致细胞损伤的保护作用.方法 将PC12细胞转染含人GPX1基因的plncx质粒及空载体plncx质粒,对PC12、GPX1-PC12、plncx-PC123组细胞,分别给予Aβ25-35和亚硒酸钠+Aβ25-35 2种干预方式,MTT法检测细胞的存活情况,免疫细胞化学法观察磷酸化环磷酸腺苷反应元件结合蛋白(pCREB)的表达情况.结果 PC12组与plncX-PC12组细胞存

  7. Deep hypothermia-enhanced autophagy protects PC12 cells against oxygen glucose deprivation via a mitochondrial pathway.

    Tang, Dang; Wang, Cheng; Gao, Yongjun; Pu, Jun; Long, Jiang; Xu, Wei


    Deep hypothermia is known for its organ-preservation properties, which is introduced into surgical operations on the brain and heart, providing both safety in stopping circulation as well as an attractive bloodless operative field. However, the molecular mechanisms have not been clearly identified. This study was undertaken to determine the influence of deep hypothermia on neural apoptosis and the potential mechanism of these effects in PC12 cells following oxygen-glucose deprivation. Deep hypothermia (18°C) was given to PC12 cells while the model of oxygen-glucose deprivation (OGD) induction for 1h. After 24h of reperfusion, the results showed that deep hypothermia decreased the neural apoptosis, and significantly suppressed overexpression of Bax, CytC, Caspase 3, Caspase 9 and cleaved PARP-1, and inhibited the reduction of Bcl-2 expression. While deep hypothermia increased the LC3II/LC3I and Beclin 1, an autophagy marker, which can be inhibited by 3-methyladenine (3-MA), indicating that deep hypothermia-enhanced autophagy ameliorated apoptotic cell death in PC12 cells subjected to OGD. Based on these findings we propose that deep hypothermia protects against neural apoptosis after the induction of OGD by attenuating the mitochondrial apoptosis pathway, moreover, the mechanism of these antiapoptosis effects is related to the enhancement of autophagy, which autophagy might provide a means of neuroprotection against OGD.

  8. Quercetin and sesamin protect neuronal PC12 cells from high-glucose-induced oxidation, nitrosative stress, and apoptosis.

    Bournival, Julie; Francoeur, Marc-André; Renaud, Justine; Martinoli, Maria-Grazia


    Complications of diabetes are now well-known to affect sensory, motor, and autonomic nerves. Diabetes is also thought to be involved in neurodegenerative processes characteristic of several neurodegenerative diseases. Indeed, it has been acknowledged recently that hyperglycemia-induced oxidative stress contributes to numerous cellular reactions typical of central nervous system deterioration. The goal of the present study was to evaluate the effects of the polyphenol quercetin and the lignan sesamin on high-glucose (HG)-induced oxidative damage in an in vitro model of dopaminergic neurons, neuronal PC12 cells. When incubated with HG (13.5 mg/mL), neuronal PC12 cells showed a significant increase of cellular death. Our results revealed that quercetin and sesamin defend neuronal PC12 cells from HG-induced cellular demise. An elevated level of reactive oxygen and nitrogen species is a consequence of improved oxidative stress after HG administration, and we demonstrated that this production diminishes with quercetin and sesamin treatment. We also found that quercetin and sesamin elicited an increment of superoxide dismutase activity. DNA fragmentation, Bax/Bcl-2 ratio, nuclear translocation of apoptosis-inducing factor, as well as poly(adenosine diphosphate [ADP]-ribose) polymerase cleavage were significantly reduced by quercetin and sesamin administration, affirming their antiapoptotic features. Also, HG treatment impacted caspase-3 cleavage, supporting caspase-3-dependent pathways as mechanisms of apoptotic death. Our results indicate a powerful role for these natural dietary compounds and emphasize preventive or complementary nutritional strategies for diabetes control.

  9. Effect of Tinospora cordifolia on the reduction of ultraviolet radiation-induced cytotoxicity and DNA damage in PC12 cells.

    Masuma, Runa; Okuno, Tsutomu; Kabir Choudhuri, Mohammad Shahabuddin; Saito, Takeshi; Kurasaki, Masaaki


    The safety of Tinospora cordifolia and its potential to protect against ultraviolet radiation-induced cytotoxicity and DNA damage in PC12 cells were investigated. To evaluate the safety of T. cordifolia, cell viability and agarose gel electrophoresis were carried out using PC12 cells treated with 0 to 100 μg mL(-1) of methanol extract of T. cordifolia. T. cordifolia extracts did not show cytotoxicity ranging 0 to 100 μg mL(-1). In addition, T. cordifolia extracts significantly increased cell viability at 1 ng, 10 ng and 1 μg mL(-1) concentrations in serum-deprived medium compared to control. To confirm the protective role against UV-induced damage, PC12 cells alone or in the presence of 10 ng, 100 ng, or 1 μg mL(-1) of T. cordifolia extract were exposed to 250, 270 and 290 nm of UV radiation, which corresponded to doses of 120, 150 and 300 mJ cm(-2), respectively. Treatment with T. cordifolia extracts significantly increased the cell survival rate irradiated at 290 nm. In addition, T. cordifolia extracts significantly reduced cyclobutane pyrimidine dimer formation induced by UV irradiation at all wavelengths. In conclusion, T. cordifolia is not toxic and safe for cells. Our findings can support its application as phototherapy in the medical sector.

  10. High Glucose-Induced PC12 Cell Death by Increasing Glutamate Production and Decreasing Methyl Group Metabolism

    Minjiang Chen


    Full Text Available Objective. High glucose- (HG- induced neuronal cell death is responsible for the development of diabetic neuropathy. However, the effect of HG on metabolism in neuronal cells is still unclear. Materials and Methods. The neural-crest derived PC12 cells were cultured for 72 h in the HG (75 mM or control (25 mM groups. We used NMR-based metabolomics to examine both intracellular and extracellular metabolic changes in HG-treated PC12 cells. Results. We found that the reduction in intracellular lactate may be due to excreting more lactate into the extracellular medium under HG condition. HG also induced the changes of other energy-related metabolites, such as an increased succinate and creatine phosphate. Our results also reveal that the synthesis of glutamate from the branched-chain amino acids (isoleucine and valine may be enhanced under HG. Increased levels of intracellular alanine, phenylalanine, myoinositol, and choline were observed in HG-treated PC12 cells. In addition, HG-induced decreases in intracellular dimethylamine, dimethylglycine, and 3-methylhistidine may indicate a downregulation of methyl group metabolism. Conclusions. Our metabolomic results suggest that HG-induced neuronal cell death may be attributed to a series of metabolic changes, involving energy metabolism, amino acids metabolism, osmoregulation and membrane metabolism, and methyl group metabolism.

  11. 6-OHDA Induces Cycle Reentry and Apoetosis of PC12 Cells through Activation of ERK1/2 Signaling Pathway

    Zhentao ZHANG; Tao WANG; Xuebing CAO; Shenggang SUN; Lan WANG


    This study investigated the effect and mechanism of cell cycle reentry induced by 6-hydrodopamine (6-OHDA) in PCI2 cells.By using neural differentiated PCI2 cells treated with 6-OHDA,the apoptosis model of dopaminergic neurons was established.Cell viability was measured by MTT.Cell apoptosis and the distribution of cell cycle were assessed by flow cytometry.Western blot was used to detect the activation of extracellular regulator kinasel/2 (ERK1/2) pathway and the phosphorylation of retinoblastoma protein (RB).Our results showed that after PC12 cells were treated wtih 6-OHDA,the viability of PC12 cells was declined in a concentration-dependent manner.Flow cytometry revealed that 6-OHDA could increase the apoptosis ratio of PC12 cells in a time-dependent manner.The percentage of cells in G0/G1 phase of cell cycle was decreased and that in S phase and G2/M phase increased.Simultaneously,ERK1/2 pathway was activated and phos- phorylated RB increased.It was concluded that 6-OHDA could induce cell cycle reentry of dopa-minergic neurons through the activation of ERK1/2 pathway and RB phosphorylation.The aberrant cell cycle reentry contributes to the apoptosis of dopaminergic neurons.

  12. Neuroprotective effect of Eleutheroside B on 1-methyl-4-phenylpyridinium ion-induced apoptosis in PC12 cells

    Fang Lu; Yang Dong; Laijun Deng; Shumin Liu; Shihui Zhou; Lifeng An; Bo Tang


    Apoptosis and viability of PC12 cells following 1-methyl-4-phenylpyridinium ion (MPP+)-induced injury were monitored by flow cytometry, following Annexin V-propidium iodide double labeling, and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, respectively. The release of lactate dehydrogenase, superoxide dismutase activity and levels of malondialdehyde were determined by UV spectrophotometry. The changes in mitochondrial membrane potential and the intracellular concentration of calcium were determined by flow cytometry, and the activity of caspase-3 was monitored by western blot. According to cell viability and apoptosis studies, MPP+-induced apoptosis in PC12 cells was inhibited in the presence of 10 μg/mL of Eleutheroside B. Our results indicate that the neuroprotective effect of Eleutheroside B, following MPP+-induced apoptosis in PC12 cells, involves increasing the anti-oxidative stress capacity of cells, maintaining the high-energy state of mitochondrial membrane potential, reducing intracellular calcium concentration and inhibiting caspase-3 activity.

  13. Nitric oxide enhances increase in cytosolic Ca(2+) and promotes nicotine-triggered MAPK pathway in PC12 cells.

    Kajiwara, Aya; Tsuchiya, Yukihiro; Takata, Tsuyoshi; Nyunoya, Mayumi; Nozaki, Naohito; Ihara, Hideshi; Watanabe, Yasuo


    The purpose of this study was to investigate the roles of neuronal nitric oxide synthase (nNOS), Ca(2+)/calmodulin (CaM)-dependent protein kinases (CaMKs), and protein kinase C (PKC) in nicotine-induced extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) activation. Treatment with nicotine stimulated ERK1/2 and p38 MAPK phosphorylation in the PC12 cells expressing nNOS (NPC12 cells) as compared with that in control PC12 cells. An inhibitor of L-type voltage-sensitive Ca(2+) channel suppressed the nicotine-induced phosphorylation of p38 MAPK. The inhibition of CaMK-kinase, the upstream activator of CaMKI and CaMKIV, did not inhibit the enhanced their phosphorylation. ERK1/2 phosphorylation was attenuated by inhibitors of p38 MAPK, PKC, and MAPK-kinase 1/2, indicating the involvement of these protein kinases upstream of ERK1/2. Furthermore, we found that nNOS expression enhances the nicotine-induced increase in the intracellular concentration of Ca(2+), using the Ca(2+)-sensitive fluorescent probe Fura2. These data suggest that NO promotes nicotine-triggered Ca(2+) transient in PC12 cells to activate possibly CaMKII, leading to sequential phosphorylation of p38 MAPK and ERK1/2.

  14. Stabilization of Nrf2 protein by D3T provides protection against ethanol-induced apoptosis in PC12 cells.

    Jian Dong

    Full Text Available Previous studies have demonstrated that maternal ethanol exposure induces a moderate increase in Nrf2 protein expression in mouse embryos. Pretreatment with the Nrf2 inducer, 3H-1, 2-dithiole-3-thione (D3T, significantly increases the Nrf2 protein levels and prevents apoptosis in ethanol-exposed embryos. The present study, using PC12 cells, was designed to determine whether increased Nrf2 stability is a mechanism by which D3T enhances Nrf2 activation and subsequent antioxidant protection. Ethanol and D3T treatment resulted in a significant accumulation of Nrf2 protein in PC 12 cells. CHX chase analysis has shown that ethanol treatment delayed the degradation of Nrf2 protein in PC12 cells. A significantly greater decrease in Nrf2 protein degradation was observed in the cells treated with D3T alone or with both ethanol and D3T. In addition, D3T treatment significantly reduced ethanol-induced apoptosis. These results demonstrate that the stabilization of Nrf2 protein by D3T confers protection against ethanol-induced apoptosis.

  15. The induction of tuftelin expression in PC12 cell line during hypoxia and NGF-induced differentiation.

    Leiser, Yoav; Silverstein, Nechama; Blumenfeld, Anat; Shilo, Dekel; Haze, Amir; Rosenfeld, Eli; Shay, Boaz; Tabakman, Rinat; Lecht, Shimon; Lazarovici, Philip; Deutsch, Dan


    The tuftelin protein isoforms undergo post-translation modifications, and are ubiquitously expressed in various tissues in embryos, adults, and tumors. Developmental and pathological studies suggested an apparent correlation between oxygen deprivation and tuftelin expression. The aim of the study was therefore to investigate the effect of a pathological insult (hypoxia) and a physiological growth factor (NGF), which antagonistically regulate HIF1 expression, on tuftelin expression using the neuronal PC12 cell model. In the present study, we first demonstrated the expression of tuftelin in PC12 cells, providing an experimental system to investigate the pathophysiological role of tuftelin. Furthermore, we demonstrated the induction of tuftelin during hypoxia by oxygen deprivation and during chemical hypoxia by cobalt chloride. Down-regulation of HIF1α mRNA blocked hypoxia-induced HIF1α expression, and reduced by 89% hypoxia-induced tuftelin expression. In mice, intraperitoneal injection of cobalt chloride significantly induced tuftelin mRNA and protein expression in the brain. During NGF-mediated PC12 differentiation, tuftelin expression was significantly induced in correlation with neurite outgrowth. This induction was partially blocked by K252a, a selective antagonist of the NGF receptor TrkA, indicating the involvement of the TrkA-signaling pathways in tuftelin induction by NGF. Revealing the physiological role of tuftelin will clarify mechanisms related to the "hypoxic genome," and NGF-induced neurotrophic and angiogenic effects.

  16. Protective Effect of Tetramethylpyrazine on Caffeine-induced PC12 Cell Injury%川芎嗪对咖啡因引起的PC12细胞损伤的保护作用

    王佳; 高峰; 张春兵


    目的:分析川芎嗪对咖啡因引起大鼠肾上腺嗜铬细胞瘤克隆化细胞株PC12细胞损伤的保护作用,探讨川芎嗪治疗脑缺血-再灌注损伤的机制。方法制备咖啡因细胞损伤模型,通过CCK-8法活细胞检测、流式细胞术线粒体膜电位测定、Western-blot检测高迁移率族蛋白B1(HMGB1)、酶联免疫吸附测定(ELISA)检测氧化应激指标观察咖啡因的毒性及川芎嗪的保护作用。结果川芎嗪预处理后,PC12细胞的存活数显著提高,细胞线粒体膜电位提高,HMGB1表达显著降低,超氧化物歧化酶( SOD)上调,乳酸脱氢酶( LDH)和丙二醛( MDA)下调,谷胱甘肽( GSH)升高。结论川芎嗪对咖啡因引起的PC12细胞损伤有显著的保护作用,其保护作用可能与川芎嗪抑制细胞凋亡、调节炎症性递质表达水平及氧化应激反应相关。%Objective To analyze whether tetramethylpyrazine could protect PC12 cells from injuries induced by caffeine,and to explore the mechanism of tetramethyipyrazine in the treatment of cerebral ischemia-reperfusion injury. Methods Caffeine was added to induce apoptosis of PC12 cells. Cytotoxicity was detected by CCK-8 assay. The electric potential of mitochondrial membrane was determined by flow cytometry. HMGB1 was detected by Western blotting. Oxidative stress was detected by ELISA. We observed toxicity of caffeine and the protective effects of tetramethylpyrazine. Results After the pre-treatment,tetramethylpyrazine significantly improved PC12 cell survival. Mitochondrial membrane potential was increased,the expression of HMGB1 decreased,SOD increased,LDH and MDA decreased,and GSH elevated. Conclusion Tetramethylpyrazine exerts a significant protective effect on PC12 cell injury caused by caffeine. The protective effect may be related to inhibition of apoptosis and regulation of the expression level of mediators involved in inflammation and oxidative stress.

  17. ACTH-Secreting Pheochromocytoma. Case report

    N S Kuznetsov


    Full Text Available Ectopic hormone-secreting pheochromocytomas are rare. Only case reports exist in the literature. Despite the large number of guides on diagnosis and treatment of pheochromocytoma, and Cushing syndrome, the extreme rarity of ectopic ACTH-syndrome caused by pheochromocytoma, and complexity of clinical cause numerous diagnostic errors leading to treatment failure. Therefore, we belive it appropriate to share our experience of this group of patients.

  18. Effects of serum containing natural cerebrolysin on glucose-regulated protein 78 and CCAAT enhancer-binding protein homologous protein expression in neuronal PC12 cells following tunicamycin-induced endoplasmic reticulum stress

    Zhengzhi Wu; Ming Li; Andrew C.J. HuangO; Xiuqing Jia; Yinghong Li; Manyin Chen


    BACKGROUND: Glucose-regulated protein 78 (GRP78), a marker of endoplasmic reticulum stress, can prolong cell survival. Alternatively, CCAAT enhancer-binding protein homologous protein (CHOP), a transcription factor specific for endopiasmic reticulum stress, can cause cell cycle arrest and cell apoptosis.OBJECTIVE: To study the protective effects of serum containing natural cerebrolysin on endoplasmic reticulum stress in tunicamycin-induced neuronal PC12 cells, and analyze the influence on GRP78 and CHOP expressions.DESIGN, TIME AND SETTING: A parallel controlled study was performed at the Institute of Integrated Western and Traditional Chinese Medicine, Shenzhen Hospital, Southern Medical University, between March 2006 and August 2008.MATERIALS: Adult Sprague-Dawley rats were perfused with natural Cerebrelysin aqueous extract (0.185 g/kg/d) to produce serum containing natural Cerebrolysin. Physiological saline was used to produce blank serum. PC12 cell line was provided by Shanghai Institute of Cell Biology, Chinese Academy of Science. Tunicamycin was provided by Sigma (St. Louis, USA), and natural Cerebrolysin, containing ginseng, rhizoma gastrodiae, and gingko leaf (1:2:2), by Shengzhen Institute of Integrated Western and Traditional Chinese Medicine.METHODS: PC12 cells were treated with DMEM culture media containing 10% blank serum (normal control group), tunicamycin (1μg/mL; model group), and 5%, 10%, and 15% serum containing natural cerebrolysin and tunicamycin (1μg/mL; low-, moderate-, and high-dose serum containing natural cerebrelysin groups), for 2 hours.MAIN OUTCOME MEASURES: PC12 cells were treated with tunicamycin for 48 hours after which apoptosis was measured using the TUNEL method to calculate apoptotic index. GRP78 expression was detected using immunocytochemistry. After 24 hours of treatment with tunicamycin, GRP78 and CHOP mRNA expressions were measured using RT-PCR.RESULTS: The apoptotic index and CHOP mRNA expression were in the model group

  19. Estrogen receptor ERα36 gene silencing impacts the expression of growth-associated protein in PC12 cells%雌激素受体ERα36基因沉默对PC12细胞生长相关蛋白表达的影响

    嵇志红; 邹萍; 邹伟


    Objective To investigate the effects of ERα36 (a novel subtype of estrogen receptor alpha) on growth and proliferation in PC12 cells via examining the expression of growth-associated protein in differentiated PC12 cells after ERα36 gene silencing.Methods Transfection of ERα36-shRNA plasmid into PC12 cells was performed to establish the ERoα36 gene silencing cells model (PC12-36L1,PC12-36L2).Immunocytofluorescence was used to examine the expression of ERα36,and Western blot was used to analyze the expression of PCNA,cyclinD1 and MAPK in the PC12 cells.Results ① ERα36 was expressed in both cell types(PC12-36C1,PC12-36L1 and PC12-36L2).Compared with PC12-36C1,PC12-36L2 cells(OD value were respectively 0.95±0.05,0.78±0.10),PC12-36L1 cells significantly decreased expression of ERα36(OD value 0.47±0.12,P<0.01).② Compared with PC12 and PC12-36C1 cells,PC12-36L1 cells were significantly higher expression of PCNA,CyclinD1 and p-MAPK(P<0.01)(OD value of PCNA,CyclinD1 and p-MAPK:PC12 cells were respectively 1.00±0.05,1.00± ±0.11,1.00±0.05,PC12-36C1 cells were respectively 1.09±0.15,0.92±0.23,1.12± 0.08,PC12-36L1 cells were respectively 1.74±0.12,2.20±0.25,1.77±0.06).Conclusion ERα36 gene silencing can promote the growth and proliferation in PC12 cells.It suggests that the lower expression of ERα36 may be related to the diseases in nervous system such as brain tumor.%目的 通过检测新型雌激素受体(ERα36)基因沉默后PC12高分化细胞中生长相关蛋白的表达,探讨ERα36对PC12细胞生长增殖的影响.方法 利用ERα36-shRNA质粒转染细胞建立ERα36基因沉默细胞模型(PC12-36L1,PC12-36L2),免疫细胞荧光法检测ERα36的表达,Western blot检测PC12细胞中增殖细胞核抗原(PCNA)、细胞周期蛋白D1(CyclinD1)和丝裂原激活的蛋白激酶(MAPK)等生长相关蛋白的表达变化.结果 ①三个细胞株PC12-36C1(转染空质粒)、PC 12-36L1和PC12-36L2中均有ERα36表达;与PC12-36C1、PC12

  20. 阿米洛利通过自噬-溶酶体途径保护PC12细胞%Amiloride protects PC12 cells from MPP + induced injury via autophagy-lysosome pathway

    张润娉; 李建平


    Objective:To evaluate the neuroprotective effects of amiloride; a non-selective blockers of acid-sensing ion channels,on PC12 cells and to view the influence of autophagy-lysosome pathway (ALP).Methods:PC12 cells were tested with methy-phenylpyridi(MPP+),while giving amiloride as interventiong:The cells viability was analyzed by MTT assay; The cells injury was assessed by lactate dehydrogenase (LDH) assay;Flow cytometry was used to study the apoptotic;Western blot analysis was used to study the autophagic mechanisms.Results:MPP + medium treatment resulted in significantly lower survival rate,the supernatant of LDH leakage was significantly higher,the apoptosis rate increased,the expression of LC3-Ⅱ was inhibited while induced more expression of LAMP2a ; but amiloride effectively improved the cell survival rate,reduced LDH leakage rate,inhibited apoptosis,and upregulated LC3-Ⅱ protein,which is associated decrease expression of LAMP2a.Conclusion:Amiloride can protect PC12 cells against MPP +-induced cell death by autophagic,through inhibition of acid-sensing ion channels activity.%目的:研究酸敏感离子通道阻断剂阿米洛利对大鼠肾上腺嗜铬细胞瘤PC12细胞株的保护作用及其对自噬-溶酶体通路的影响.方法:在PC12细胞模型上,观察甲基苯基吡啶(methy-phenylpyridi,MPP+)处理对细胞存活率(MTT检测)、乳酸脱氢酶(LDH)漏出率、细胞凋亡率的影响,并观察阿米洛利对MPP+所致细胞死亡的影响;蛋白质印迹法检测大自噬通路标志蛋白LC3和分子伴侣介导的自噬通路标志蛋白LAMP2a表达水平的变化.结果:MPP+导致细胞存活率明显降低,上清液中LDH漏出率明显升高,细胞凋亡率升高,抑制大自噬通路标志蛋白LC3-Ⅱ的表达,同时上调了分子伴侣介导的自噬通路标志性蛋白LAMP2a的表达水平;而阿米洛利可提高细胞存活率,减少上清液中LDH漏出率,降低细胞凋亡率,并能在激活大自噬通路的同时下调分

  1. 用FCM法测定YLSP对Aβ25-35诱导PC12细胞凋亡影响的观察




  2. 甘草苷对一氧化氮诱导的PC12细胞损伤的保护作用

    孙国庆; 罗正里


    目的:应用NO诱导PC12细胞制作细胞损伤模型,探讨甘草苷对PC12细胞损伤的保护作用。方法甘草苷各剂量的PC12细胞用SNAP诱导48 h后,采用MTT法测定细胞活力,LDH法测定细胞膜通透性,化学比色法测定细胞中超氧化物歧化酶( SOD)活性和丙二醛( MDA)含量,Annexin V-FITC/PI检测PC12细胞凋亡率。结果与模型组相比,甘草苷可以使 PC12细胞存活率显著增加,细胞培养上清中 LDH 含量显著减少,SOD活性显著升高,MDA含量显著下降,并明显降低PC12细胞凋亡率。结论甘草苷对NO诱导的PC12细胞凋亡有明显保护作用。

  3. A dopamine-secreting pheochromocytoma.

    Yasunari, K; Kohno, M; Minami, M; Kano, H; Ohhira, M; Nakamura, K; Yoshikawa, J


    We describe a patient with pheochromocytoma, which secretes dopamine. He was admitted to hospital because of chronic diarrhea. After surgical resection of the tumor, dramatic cessation of the diarrhea and blood pressure elevation were observed. Decreased expression of dopamine beta-hydroxylase in the tumor was considered a possible mechanism of producing a pathophysiological concentration of dopamine. This case shows that excessive excretion of dopamine, a vasodilative hormone, may affect blood pressure.

  4. Pelvic Pheochromocytoma Mimicking as Urinary Bladder Pheochromocytoma: Looking Beyond the Obvious

    Santosh Kumar


    Full Text Available Pheochromocytomas located outside the adrenal glands are called paragangliomas. A pelvic location is rare, the most common location for a paraganglioma being the retroperitoneal space. Paragangliomas arise from neural crest cells. Pelvic pheochromocytomas may mimic urinary bladder pheochromocytomas on imaging studies. Patients may present with hypertensive crisis during micturition. We present a 26-year-old female who presented to us with accelerated hypertension with episodes of severe headache and palpitation during micturition. Based on imaging studies, she was diagnosed to have a urinary bladder pheochromocytoma. However, on exploration, the patient was found to have an extravesical pheochromocytoma arising from the left posterolateral pelvic wall, which was excised while preserving the bladder. We present this case report as pelvic pheochromocytomas can mimic bladder pheochromocytomas and are difficult to differentiate on radiological imaging and can lead to inadvertent cystectomy.

  5. A retrospective study of pheochromocytoma

    Larigani B


    Full Text Available Pheochromocytoma is a rare disease. A retrospective study of the signs and clinical course of this disorder was performed by evaluating medical records. Our fidings indicate that the prevalence of pheochromocytoma was equal in men and women, and most patients (56% were in their second and third decades of life. In 10% of the cases, the disease was bilateral, and in 13% it was outside the adrenal (totally para-aortic. The tumor was more common on the right side (8%, and 3.5% were familial. Almost all cases had a history of hypertension and hypertensive crises. Attack-like episodes of clinical symptoms and signs and hypertension were observed in 98%, headache in 71% and profuse perspiration in 68% of the cases. An abdominal mass was palapated in 13% of the cases, 26% had overt diabetes, 23% had ECG changes. Malignancy was observed in 4%, with metastases to the liver (n=2 lung (n=1 and spine (n=1. In the latter four cases, the metastic lesion was histologically proven to be pheochromocytoma. In three of the 28 female cases, the first hypertensive crisis occurred during pregnancy causing abortion in one case.

  6. Protective effects of ginkgo biloba leaves extract on peroxide-induced oxidative stress damage in PC12 cells

    Weiqiang Chen; Taiping Hu; Ying Liu


    BACKGROUND: Extracts of ginkgo biloba leaves (EGB) and its metabolites have been reported to enhance brain function and nerve behavior. It has also been hypothesized that they can protect neurons from oxidative stress.OBJECTIVE: To investigate protective effects of EGB on peroxide (H2O2)-induced oxidative stress damage in PC12 cells.DESIGN: Observational contrast study.SETTING: Department of Pathophysiology, Guangdong Pharmacological College.MATERIALS: EGB was provided by Xi'an Fujie Biotechnological Development Company; 1640 culture medium, methylthiazolyl tetrazolium (MTT), trypsin and dimathyl sulfoxide (DMSO) by Sigma Company;PC 12 cell strain by Cell Center of Medical College of Zhongshan University; calf serum by Hangzhou Sijiqing Bioengineering Company; lactate dehydrogenase (LDH) kit by Nanjing Jiancheng Bioengineering Research Institute.METHODS: The experiment was carried out in Department of Cell Biology of Guangdong Pharmacological College from June to December 2005.①Cell culture: PC 12 cells were cultured in 1640 medium containing 200 g/L fetal calf serum. The cells were diluted to 1×107 L-1 and washed every two days. Those cells were used to experiment until they grew in logarithm on solid wall.②Grouping and intervention: PC 12 cells(1×108L-1) were plated in 96-well plates with the density of 200 μL/hole and divided into three groups:normal control group (routinely adding media), H2O2 group (treating with media and H2O2 for 20 hours) and EGB group (adding media, 100 μ mol/L EGB and 100 μmol/L H2O2).③MTT assay: PC12 cells (1×108L-1) were plated in 96-well plates and divided into three groups with 8 holes for each group. Under sterile condition, cells were added with 5 g/L MTT (100 μ L) and cultured for 4 hours. And then, 200 μL DMSO fluid was added and shaken for 30 minutes until blue crystal products formed were dissolved soundly.④Experimental evaluation: Absorbance (A) at 630 nm was measured and LDH activity was measured at the same

  7. Eriocaulon buergerianum extract protects PC12 cells and neurons in zebrafish against 6-hydroxydopamine-induced damage

    Lin Zhixiu


    Full Text Available Abstract Background Ericaulon buergerianum (Gujingcao is an ophthalmic, anti-inflammatory and antimicrobial Chinese medicinal herb. This study aims to investigate the neuroprotective effects of Ericaulon buergerianum ethanol extract (EBE and to elucidate its underlying action mechanism. Methods The viability of dopaminergic (DA neuron in zebrafish was examined by anti-tyrosine hydroxylase (TH immunostaining. The locomotor activity of zebrafish was assessed with a digital video tracking system. The viability and cellular damage of the PC12 cells were determined by MTT and LDH assays respectively. The nuclear morphological changes in apoptotic cells were evaluated with DNA staining by Hoechst 33342 dye. Intracellular nitric oxide (NO was quantified by DAF-FM diacetate staining. The expression of inducible nitric oxide synthase (iNOS was determined by Western blot. Results EBE inhibited the 6-OHDA-induced decrease in total distance of movement in zebrafish. Pretreatments of EBE (25, 50, 100 and 200 μg/ml increased the viability of 6-OHDA-damaged PC12 cells in a dose dependent manner. Protection against 6-OHDA-induced nuclear fragmentation and accumulation of apoptotic bodies was also observed in EBE pretreated cells. Anti-oxidative (inhibition of NO production and iNOS expression in PC12 cells in vitro activities of EBE are related to its neuroprotective effects in 6-OHDA-induced DA neuron damage. Conclusion EBE exhibited significant neuroprotective activities in zebrafish, including recovery of dopaminergic neuron loss caused by 6-OHDA in a dose-dependent manner in vivo, inhibition of 6-OHDA-induced decrease of total distance in movement in zebrafish. The iNOS-NO pathway may be involved.

  8. Cajaninstilbene Acid Prevents Corticosterone-Induced Apoptosis in PC12 Cells by Inhibiting the Mitochondrial Apoptotic Pathway

    Bao-Ping Jiang


    Full Text Available Background/Aims: Cajaninstilbene acid (3-hydroxy-4-prenyl-5-methoxystilben-2 -carboxylic acid, CSA, a natural stilbene isolated from the leaves of Cajanus cajan, has attracted considerable attention for its wide range of pharmacological activities. This study investigated whether CSA protects against corticosterone (CORT-induced injury in PC12 cells and examined the potential mechanisms underlying this protective effect. Methods: Cell viability and cytotoxicity were detected using a 3-(4,5-desethyithiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay and a lactate dehydrogenase (LDH assay kit, respectively. PC12 cell apoptosis was measured using Hoechst 33342 staining and a DNA fragmentation assay kit, and intracellular Ca2+ concentrations were assessed by fluorescent labelling. Next, the mitochondrial permeability transition pores (mPTPs and mitochondrial membrane potentials (∆Ψm were detected using a colorimetric mPTP detection kit and a 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolyl-carbocyanine iodide (JC-1 kit, respectively. Finally, cytochrome c, caspase-3 and inhibitor of caspase-activated deoxyribonuclease (ICAD expression levels were monitored by western blot analysis. Results: Treatment with 100 µmol/l CORT induced cytotoxicity in PC12 cells. However, CSA dose-dependently increased cell viability and decreased LDH release as well as CORT-induced apoptosis. Mechanistically, compared with the CORT-treated group, CSA strongly attenuated intracellular Ca2+ overload and restored mitochondrial functions, including mPTPs and ∆Ψm. Furthermore, the down-regulation of cytochrome c and ICAD protein expression and the blockage of caspase-3 activity were observed upon CSA treatment. Conclusions: In summary, our data are the first to show that the in vitro antidepressant-like effect of CSA may be attributed to the cytoprotection of neurons and that such neuroprotective mechanisms are correlated with intracellular Ca2+ homeostasis

  9. ErbB receptors and PKC regulate PC12 neuronal-like differentiation and sodium current elicitation.

    García, L; Castillo, C; Carballo, J; Rodríguez, Y; Forsyth, P; Medina, R; Martínez, J C; Longart, M


    Excitability, neurite outgrowth and their specification are very important features in the establishment of neuronal differentiation. We have studied a conditioned medium (CM) from sciatic nerve which is able to induce a neuronal-like differentiation of PC12 cells. Previously, we have demonstrated that supplementing this CM with a generic inhibitor (k252a), which mainly inhibits tropomyosin-related kinase receptors (Trk receptors) and protein kinase C (PKC), caused neurite elongation, sodium current induction and axon development. In the present work, we are showing that the enhancement of neurite length and induction of sodium currents induced by CM+k252a were prevented by ErbB receptor inhibition. Additionally, we demonstrated that specific inhibition of PKC produced a similar effect to that exerted by k252a in CM-treated cells, specifically by increasing the percentage of differentiated cells with long neurites and inducing sodium currents. Moreover, CM changed the mRNA levels for ErbB2 and ErbB3 increasing them 6- and 36-folds respectively compared to their control. The inclusion of k252a with CM changed the ErbB1, ErbB2 and ErbB3 mRNA proportions increasing those eight-, seven- and fivefolds respectively. From this point, it is clear that appropriate ErbB receptor levels and PKC inhibition are necessary to enhance the effect of the CM in inducing the neuronal-like differentiation of PC12 cells. In summary, we demonstrated the involvement of ErbB receptors in the regulation of neurite elongation and sodium current induction in PC12 cells and propose that these processes could be initiated by ErbB receptors followed by a fine regulation of PKC signaling. These findings might implicate a novel interplay between ErbB receptors and PKC in the regulation of these molecular mechanisms.

  10. Repeated Glucose Deprivation/Reperfusion Induced PC-12 Cell Death through the Involvement of FOXO Transcription Factor

    Han, Na; Kim, You Jeong; Park, Su Min; Kim, Seung Man; Lee, Ji Suk; Jung, Hye Sook; Lee, Eun Ju; Kim, Tae Kyoon; Kim, Tae Nyun; Kwon, Min Jeong; Lee, Soon Hee; Rhee, Byoung Doo


    Background Cognitive impairment and brain damage in diabetes is suggested to be associated with hypoglycemia. The mechanisms of hypoglycemia-induced neural death and apoptosis are not clear and reperfusion injury may be involved. Recent studies show that glucose deprivation/reperfusion induced more neuronal cell death than glucose deprivation itself. The forkhead box O (FOXO) transcription factors are implicated in the regulation of cell apoptosis and survival, but their role in neuronal cells remains unclear. We examined the role of FOXO transcription factors and the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt and apoptosis-related signaling pathways in PC-12 cells exposed to repeated glucose deprivation/reperfusion. Methods PC-12 cells were exposed to control (Dulbecco's Modified Eagle Medium [DMEM] containing 25 mM glucose) or glucose deprivation/reperfusion (DMEM with 0 mM glucose for 6 hours and then DMEM with 25 mM glucose for 18 hours) for 5 days. MTT assay and Western blot analysis were performed for cell viability, apoptosis, and the expression of survival signaling pathways. FOXO3/4',6-diamidino-2-phenylindole staining was done to ascertain the involvement of FOXO transcription factors in glucose deprivation/reperfusion conditions. Results Compared to PC-12 cells not exposed to hypoglycemia, cells exposed to glucose deprivation/reperfusion showed a reduction of cell viability, decreased expression of phosphorylated Akt and Bcl-2, and an increase of cleaved caspase-3 expression. Of note, FOXO3 protein was localized in the nuclei of glucose deprivation/reperfusion cells but not in the control cells. Conclusion Repeated glucose deprivation/reperfusion caused the neuronal cell death. Activated FOXO3 via the PI3K/Akt pathway in repeated glucose deprivation/reperfusion was involved in genes related to apoptosis.

  11. Expression changes of dopaminergic system-related genes in PC12 cells induced by manganese, silver, or copper nanoparticles.

    Wang, Jianyong; Rahman, Mohammed F; Duhart, Helen M; Newport, Glenn D; Patterson, Tucker A; Murdock, Richard C; Hussain, Saber M; Schlager, John J; Ali, Syed F


    Nanoparticles have received a great deal of attention for producing new engineering applications due to their novel physicochemical characteristics. However, the broad application of nanomaterials has also produced concern for nanoparticle toxicity due to increased exposure from large-scale industry production. This study was conducted to investigate the potential neurotoxicity of manganese (Mn), silver (Ag), and copper (Cu) nanoparticles using the dopaminergic neuronal cell line, PC12. Selective genes associated with the dopaminergic system were investigated for expression changes and their correlation with dopamine depletion. PC12 cells were treated with 10 microg/ml Mn-40 nm, Ag-15 nm, or Cu-90 nm nanoparticles for 24 h. Cu-90 nanoparticles induced dopamine depletion in PC12 cells, which is similar to the effect induced by Mn-40 shown in a previous study. The expression of 11 genes associated with the dopaminergic system was examined using real-time RT-PCR. The expression of Txnrd1 was up-regulated after the Cu-90 treatment and the expression of Gpx1 was down-regulated after Ag-15 or Cu-90 treatment. These alterations are consistent with the oxidative stress induced by metal nanoparticles. Mn-40 induced a down-regulation of the expression of Th; Cu-90 induced an up-regulation of the expression of Maoa. This indicates that besides the oxidation mechanism, enzymatic alterations may also play important roles in the induced dopamine depletion. Mn-40 also induced a down-regulation of the expression of Park2; while the expression of Snca was up-regulated after Mn-40 or Cu-90 treatment. These data suggest that Mn and Cu nanoparticles-induced dopaminergic neurotoxicity may share some common mechanisms associated with neurodegeneration.

  12. Aβ寡聚体预处理的BV2细胞对PC12细胞凋亡的影响%Effect of BV2 pre-treated with Aβ1 ~42 oligimers upon PC12 cell apoptosis

    蔺慕会; 徐忠信; 陈晓虹


    Objective To study the effect of BV2 upon PC 12 before or after it was pre-treated with Aβ1-42 oligim-ers( ADDLs) ,in order to investigate microglia's injury to neuron. Methods In control group ( Aβ+ PC12) ,PC12 cells were treated with Aβ1-42 oligimers of different concentrations. In experimental group( Aβ + BV2 + PC12) ,BV2 cells were pre-cultured with Aβ1-42 oligimers of the same concentration and then co-cultured with PC12 cells through transwells. PC12 cells' inhibition ratio was tested by MTT,cell apoptosis was tested by flow cytometry assay,and tau( pS396) protein expression was tested by Western blot. Results ADDLs of all concentrations could induce PC12 cells apoptosis and PC12 cells' apoptosis increased with ADDLs' concentrations increased. PC 12 cells' inhibition ratio, cell apoptosis and tau (pS396) protein expression were increased with Aβ concentration increased in all group. Compared with control group, the change was more apparent in experimental group. The difference between two group with same Aβ concentration group was significant(P <0.05). Conclusion BV2 cells can significantly attenuates PC 12 cells' inhibition,tau(pS396)protein expression and apoptosis caused by Aβ1-42 oligimers.%目的 研究Aβ寡聚体(Aβ-derived diffusible ligands,ADDLs)干预的BV2细胞对PC12细胞凋亡的影响,进而探讨小胶质细胞对神经元损伤的作用.方法 分别用不同浓度Aβ1~42寡聚体作用于PC12细胞(大鼠肾上腺嗜铬细胞瘤细胞)作为对照组( Aβ+ PC12);用相应浓度的Aβ1 ~42寡聚体预处理BV2细胞,然后通过转移筛网与PC12细胞共育作为实验组(Aβ+ BV2+ PC12).应用MTT方法检测PC12细胞抑制率,流式细胞仪检测细胞凋亡,Western blot方法观察PC12细胞tau、tau( pS396)蛋白表达的变化.结果 所有浓度的ADDLs均可导致PC12细胞凋亡,且PC12细胞凋亡表现为ADDL浓度依赖性关系,即随着Aβ浓度增加PC12抑制率、细胞凋亡、tau( pS396)表达也明显增加,其中实验组PC

  13. Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35

    Mingmin Yan; Shanping Mao; Huimin Dong; Baohui Liu; Qian Zhang; Gaofeng Pan; Zhiping Fu


    PC12 cell injury was induced using 20 μM amyloid β-protein 25-35 to establish a model of Alzheimer's disease.The cells were then treated with 5, 10, and 25 μM Schisandrin B.Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25-35 gradually increased and the rate of apoptosis gradually decreased.Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased.Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change.These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25-35 in a dose-dependent manner.This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein.PC12 cell injury was induced using 20 μM amyloid β-protein 25-35 to establish a model of Alzheimer's disease.The cells were then treated with 5, 10, and 25 μM Schisandrin B.Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25-35 gradually increased and the rate of apoptosis gradually decreased.Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased.Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change.These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25

  14. 木豆叶醇提物对皮质酮诱导的PC12细胞损伤的保护作用%Protective Effect of Alcohol Extract of Cajanus cajan on Corticosterone-induced Lesion in Cultured PC12 Cells

    姜保平; 刘亚旻; 李宗阳; 宋波; 潘瑞乐


    建立皮质酮诱导的PC12细胞损伤模型并观察木豆叶醇提物及不同组分对皮质酮损伤PC12细胞的保护作用.以100μ mol/L的皮质酮诱导PC12细胞损伤;损伤后的PC12细胞与木豆叶醇提物及不同组分孵育24h,通过形态学观察、MTT检测、LDH测定,研究各组分对皮质酮损伤PC12细胞的保护作用.结果表明,PC12细胞与皮质酮孵育48 h后细胞存活率明显降低,而LDH水平显著升高.而加入木豆叶醇提物及各组分时上述效果明显减轻,且存在明显的剂量依赖关系.从以上结果可知,木豆叶醇提物及不同组分对皮质酮损伤的PC12细胞均有保护作用,且醇提物的效果最好.%The aim of this paper was to establish the corticosterone-induced damage model in PC12 cells and study the protective activity of alcohol extracts and its fractions from Cajanus cajan on corticosterone-induced PC12 cells. PC12 cells were exposed to different concentrations of corticosterone to determine the optimum concentration of establishing the damage model,and then the injured PC12 cells were incubated with the alcohol extract and its fractions for 24 h. After that the neuroprotective effect on injured PC12 cells was investigated by morphological observation, LDH detection and MTT determination. The results showed that the survival rate of PC12 cells and the cells exposed to corticosterone (100 (μmol/L) for 48 h decreased obviously,with an increase in lactate dehydrogenase (LDH) level,but the effect became weak when the alcohol extracts and its fractions from the C. cajan was added to the PC 12 cells,and it was in a dose-dependent manner. The author suggests that the alcohol extract and its fractions from C. cajan can generate a neuroprotective effect on injured PC 12 cells induced by corticosterone,and that of alcohol extract is the most significant.

  15. Nuclear medicine imaging of pheochromocytoma and neuroblastoma

    Sisson, J.C.; Shulkin, B.L. [Michigan Health Systems Univ., Ann Arbor, MI (United States). Div. of Nuclear Medicine, Dept. of Internal Medicine


    Both pheochromocytomas and neuroblastomas can now be identified and located with a high level of accuracy. Scintigraphy with MIBG has become an indispensable diagnostic method for defining the extent and location of many if not most pheochromocytomas. To define the stage, to document the course and to evaluate the response to therapies in patients with neuroblastoma, imaging with MIBG is now essential.

  16. Minocycline attenuates both OGD-induced HMGB1 release and HMGB1-induced cell death in ischemic neuronal injury in PC12 cells

    Kikuchi, Kiyoshi [Division of Laboratory and Vascular Medicine, Field of Cardiovascular and Respiratory Disorders, Department of Advanced Therapeutics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8520 (Japan); Department of Neurosurgery, Omuta City General Hospital, 2-19-1 Takarazaka, Omuta-City, Fukuoka 836-8567 (Japan); Kawahara, Ko-ichi; Biswas, Kamal Krishna; Ito, Takashi [Division of Laboratory and Vascular Medicine, Field of Cardiovascular and Respiratory Disorders, Department of Advanced Therapeutics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8520 (Japan); Tancharoen, Salunya [Department of Pharmacology, Faculty of Dentistry, Mahidol University, 6 Yothe Rd., Rajthevee Bangkok 10400 (Thailand); Morimoto, Yoko [Department of Periodontology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Matsuda, Fumiyo [Division of Physical Therapy, School of Health Sciences, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8560 (Japan); Oyama, Yoko; Takenouchi, Kazunori [Division of Laboratory and Vascular Medicine, Field of Cardiovascular and Respiratory Disorders, Department of Advanced Therapeutics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8520 (Japan); Miura, Naoki [Laboratory of Veterinary Diagnostic Imaging, Department of Veterinary Medicine, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065 (Japan); Arimura, Noboru; Nawa, Yuko; Meng, Xiaojie; Shrestha, Binita; Arimura, Shinichiro [Division of Laboratory and Vascular Medicine, Field of Cardiovascular and Respiratory Disorders, Department of Advanced Therapeutics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8520 (Japan); and others


    High mobility group box-1 (HMGB1), a non-histone DNA-binding protein, is massively released into the extracellular space from neuronal cells after ischemic insult and exacerbates brain tissue damage in rats. Minocycline is a semisynthetic second-generation tetracycline antibiotic which has recently been shown to be a promising neuroprotective agent. In this study, we found that minocycline inhibited HMGB1 release in oxygen-glucose deprivation (OGD)-treated PC12 cells and triggered the activation of p38mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases (ERK1/2). The ERK kinase (MEK)1/2 inhibitor U-0126 and p38MAPK inhibitor SB203580 blocked HMGB1 release in response to OGD. Furthermore, HMGB1 triggered cell death in a dose-dependent fashion. Minocycline significantly rescued HMGB1-induced cell death in a dose-dependent manner. In light of recent observations as well as the good safety profile of minocycline in humans, we propose that minocycline might play a potent neuroprotective role through the inhibition of HMGB1-induced neuronal cell death in cerebral infarction.

  17. Enantioselective induction of cytotoxicity by o,p'-DDD in PC12 cells: implications of chirality in risk assessment of POPs metabolites.

    Wang, Cui; Li, Zhuoyu; Zhang, Quan; Zhao, Meirong; Liu, Weiping


    The increased release of chiral persistent organic pollutants (POPs) into the environment has resulted in more attention to the role of enantioselectivity in the fate and ecotoxicological effects of these compounds. Although the enantioselectivity of chiral POPs has been considered in previous studies, little effort has been expended to discern the enantiospecific effects of chiral POPs metabolites, which may impede comprehensive risk assessments of these chemicals. In the present study, o,p'-DDD, the chiral metabolite of o,p'-DDT, was used as a model chiral metabolite. First, a preferential chiral separation at 100% ethanol was employed to obtain a pure enantiomer. The enantioselective cytotoxicity of o,p'-DDD in rat cells (PC12) was evaluated by detecting activation of the cellular apoptosis and oxidative stress systems and microarray analysis. We have documented for the first time that R-(+)-o,p'-DDD increases apoptosis by selectively disturbing the oxidative system (enzymes and molecules) and regulating the transcription of Aven, Bid, Cideb and Tp53. By comparing the data from the present study to data derived from the parent compound, we concluded that the R-enantiomer is the more detrimental stereostructure for both o,p'-DDT and o,p'-DDD. This observed stereostructural effect is in line with the structure-activity relationship formulated at other structural levels. Biological activities of the chiral metabolites are likely to occur in the same absolute configuration between chiral POPs and their metabolites provided that they have the similar stereostructures.

  18. Protein kinase C is a target for diverse developmental neurotoxicants: transcriptional responses to chlorpyrifos, diazinon, dieldrin and divalent nickel in PC12 cells.

    Slotkin, Theodore A; Seidler, Frederic J


    Unrelated developmental neurotoxicants can elicit similar functional outcomes, whereas agents in the same class may differ. We compared two organophosphate insecticides (chlorpyrifos, diazinon) with an organochlorine (dieldrin) and a metal (Ni(2+)) for similarities and differences in their effects on gene expression encoding subtypes of protein kinase C and their modulators, a cell signaling cascade that integrates the actions of neurotrophic factors involved in brain development. We conducted evaluations in PC12 cells, a model for neuronal development, with each agent introduced at 30 microM for 24 or 72 h, treatments devoid of cytotoxicity. Chlorpyrifos evoked by far the largest effect, with widespread upregulation of multiple genes; the effects were greater during neurodifferentiation than when cells were exposed prior to differentiation. Diazinon had smaller and less widespread effects, consistent with its lesser long-term impact on synaptic function and behavior noted for in vivo exposures in developing rats. Surprisingly, the effects of diazinon, dieldrin and Ni(2+) showed basic similarities despite the fact that all three come from different classes of toxicants. Our findings provide some of the first evidence for a specific mechanistic cascade contributing to the cholinesterase-independent developmental neurotoxicant actions of chlorpyrifos and its differences from diazinon, while at the same time identifying mechanistic convergence between otherwise unrelated toxicants that provides predictions about common neurodevelopmental outcomes. These results further show how combined use of cell cultures and microarray technology can guide future in vivo work on diverse developmental neurotoxicants.

  19. Induction of aromatic L-amino acid decarboxylase mRNA by interleukin-1 beta and prostaglandin E2 in PC12 cells.

    Li, X M; Juorio, A V; Boulton, A A


    Aromatic 1-amino acid decarboxylase (AADC) is involved in the synthesis of the putative neurotransmitters dopamine (DA), norepinephrine (NA) and 5-hydroxytryptamine (5-HT). We report here that the gene expression of AADC can be regulated by interleukin (IL) 1-beta and prostaglandin (PG) E2 in PC12 cells. The cells were treated with different doses of IL 1-beta and PGE2 for 3 days. Slot blot hybridization was performed to detect AADC mRNA and Western immunoblot to detect AADC protein. The cDNA probe for rat AADC was generated by the PCR method. IL 1-beta and PGE2 produced a dose- and time-dependent up-regulation in AADC mRNA levels (up to 200% of the control values) which was followed by a stable increase in AADC protein. The data further support the suggestion that AADC is a regulated enzyme and that the regulation occurs at the level of gene expression. Because IL-1 is synthesized, and acts locally, within the brain to influence neuronal and glial functions, it has been proposed to be a mediator with both beneficial and detrimental responses to inflammation and injury. The regulation of AADC by IL-1 may indicate a possible involvement for AADC in neuronal injury and recovery. Since IL-1 promotes PGE2 formation, its effects may be occurring by increasing level of PGE2.

  20. Pheochromocytoma in neurofibromatosis type 1 during pregnancy.

    Remón-Ruiz, Pablo; Aliaga-Verdugo, Alberto; Guerrero-Vázquez, Raquel


    Pregnant women with neurofibromatosis type 1 (NF-1) have increased complications during gestation, including hypertensive disorders that are sometimes caused by pheochromocytoma. Pheochromocytoma is an extremely rare condition during pregnancy, and the main clinical manifestation is hypertension. If not properly treated, pheochromocytoma has high maternal and fetal mortality rates. Early recognition and adequate clinical management before delivery have led to better outcomes in the last few decades. Despite the association of NF-1 and pheochromocytoma, there are few clinical reports of these two conditions in pregnant patients. We present a rare case of pheochromocytoma diagnosed during pregnancy in a patient with NF-1, and we describe the treatment and the obstetric and fetal outcomes. We also review other medical conditions related to NF-1 that complicated this patient's pregnancy.

  1. SRB法检测没食子酸乙酯对PC12细胞的毒性作用

    黄金兰; 田新; 景鑫; 吴登攀


    目的:研究没食子酸乙酯对PC12细胞的毒性作用. 方法:SRB法体外检测没食子酸乙酯对PC12细胞的毒性. 结果:不同浓度的没食子酸乙酯对PC12细胞的增值均有不同程度的抑制作用,且呈剂量依赖性. 结论:1.25μg/mL~5μg/mL浓度范围没食子酸乙酯对PC12细胞的毒害最小.

  2. Clinical Experiences of Pheochromocytoma in Korea

    Kim, Kwang Hyun; Chung, Jae Seung; Kim, Won Tae; Oh, Cheol Kyu; Chae, Yun Byung; Yu, Ho Song; Ham, Won Sik


    Purpose We report herein 119 patients with pheochromocytoma at our institute over the last 23 years. Materials and Methods Between 1986 and 2009, 119 patients were diagnosed with pheochromocytoma at our institute. We reviewed the medical records of these patients. Results Of 119 patients, 45 were male and 74 were female, and mean age was 43.83 ± 13.49 years. Forty-three patients (36.1%) were diagnosed incidentally, and 8 patients (6.7%) were found to have familial pheochromocytoma. The mean dimension of the tumors was 5.89 ± 3.18 cm. 4 patients had bilateral tumors; three of these patients were found to have familial pheochromocytoma and 1 patient was diagnosed with malignant pheochromocytoma. A total of eight patients (6.7%) were found to have malignant pheochromocytoma. In 1 patient, metastasis to a lymph node was found at the time of diagnosis. Metastases were found at a mean of 49 ± 25.83 (6-75) months after surgery in the other seven patients. 6 patients died of malignant pheochromocytoma at a mean of 31 ± 28.71 months (1-81) after diagnosis, and the other 2 patients survived for 15 and 24 months, respectively. Conclusion Approximately 35% of patients with pheochromocytoma are diagnosed incidentally, and the number of detected cases is increasing. Although familial pheochromocytoma was found only in 6.7% of the patients, genetic testing should be considered in all patients, especially in patients with a family history, young age, or multifocal, bilateral, extra-adrenal, or malignant tumors. Given that malignant pheochromocytomas are frequently diagnosed during the follow-up period, long-term follow-up is necessary to confirm the absence of recurrence or metastasis. PMID:21155034

  3. Neuroprotective effects of Activin A on endoplasmic reticulum stress-mediated apoptotic and autophagic PC12 cell death

    Long-xing Xue


    Full Text Available Activin A, a member of the transforming growth factor-beta superfamily, plays a neuroprotective role in multiple neurological diseases. Endoplasmic reticulum (ER stress-mediated apoptotic and autophagic cell death is implicated in a wide range of diseases, including cerebral ischemia and neurodegenerative diseases. Thapsigargin was used to induce PC12 cell death, and Activin A was used for intervention. Our results showed that Activin A significantly inhibited morphological changes in thapsigargin-induced apoptotic cells, and the expression of apoptosis-associated proteins [cleaved-caspase-12, C/EBP homologous protein (CHOP and cleaved-caspase-3] and biomarkers of autophagy (Beclin-1 and light chain 3, and downregulated the expression of thapsigargin-induced ER stress-associated proteins [inositol requiring enzyme-1 (IRE1, tumor necrosis factor receptor-associated factor 2 (TRAF2, apoptosis signal-regulating kinase 1 (ASK1, c-Jun N-terminal kinase (JNK and p38]. The inhibition of thapsigargin-induced cell death was concentration-dependent. These findings suggest that administration of Activin A protects PC12 cells against ER stress-mediated apoptotic and autophagic cell death by inhibiting the activation of the IRE1-TRAF2-ASK1-JNK/p38 cascade.

  4. Heat shock protein 70 protects PC12 cells against ischemia-hypoxia/reoxygenation by maintaining intracellular Ca2+homeostasis

    Yuan Liu; Xue-chun Wang; Dan Hu; Shu-ran Huang; Qing-shu Li; Zhi Li; Yan Qu


    Heat shock protein 70 (HSP70) maintains Ca2+homeostasis in PC12 cells, which may protect against apoptosis;however, the mechanisms of neuroprotection are unclear. Therefore, in this study, we examined Ca2+levels in PC12 cells transfected with an exogenous lentiviral HSP70 gene expression construct, and we subsequently subjected the cells to ischemia-hypoxia/reoxygenation injury. HSP70 overex-pression increased neuronal viability and ATPase activity, and it decreased cellular reactive oxygen species levels and intracellular Ca2+concentration after hypoxia/reoxygenation. HSP70 overexpression enhanced the protein and mRNA expression levels of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA), but it decreased the protein and mRNA levels of inositol 1,4,5-trisphosphate receptor (IP3R), thereby leading to decreased intracellular Ca2+concentration after ischemia-hypoxia/reoxygenation. These results suggest that exogenous HSP70 protects against ischemia-hypoxia/reoxygenation injury, at least in part, by maintaining cellular Ca2+homeostasis, by upregulating SERCA expression and by downregulating IP3R expression.

  5. Astragaloside IV Attenuates Glutamate-Induced Neurotoxicity in PC12 Cells through Raf-MEK-ERK Pathway.

    Rongcai Yue

    Full Text Available Astragaloside IV (AGS-IV is a main active ingredient of Astragalus membranaceus Bunge, a medicinal herb prescribed as an immunostimulant, hepatoprotective, antiperspirant, a diuretic or a tonic as documented in Chinese Materia Medica. In the present study, we employed a high-throughput comparative proteomic approach based on 2D-nano-LC-MS/MS to investigate the possible mechanism of action involved in the neuroprotective effect of AGS-IV agains