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A protein induced by NGF in PC12 cells is stored in secretory vesicles and released through the regulated pathway. 总被引:6,自引:0,他引:6
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We have previously described the isolation of a cDNA clone corresponding to an mRNA rapidly induced to high levels in PC12 cells by treatment with NGF. We report here the complete amino acid sequence of the protein (named VGF8a) as deduced by nucleotide sequencing of overlapping cDNA clones. VGF8a is particularly rich in proline residues and has a conspicuous number of short stretches of basic amino acid residues which may represent potential targets for proteolytic cleavage. Antibodies directed against recombinant VGF8a-beta-galactosidase fusion proteins were used for immunofluorescent staining of the protein in PC12 cells as well as for its localization, by Western blot analysis, in subfractions of cell homogenates. We demonstrate that in PC12 cells, VGF8a protein is stored in secretory vesicles and is released in response to a variety of stimuli that are known to induce the regulated secretion of neurotransmitters. 相似文献
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蛇毒神经生长因子诱导PC12细胞分化超微结构的观察 总被引:1,自引:0,他引:1
目的观察蛇毒神经生长因子(Nerve growth factor,NGF)诱导大鼠肾上腺嗜铬细胞瘤细胞系(Pheo—chromocytoma cells。PC12)细胞分化后,细胞超微结构的改变。方法取对数生长期PC12细胞接种24孔板,设200ng/ml NGF实验组和对照组。培养72h,离心,分别收集细胞制成电镜标本,镜下观察各组细胞超微结构的改变。结果与对照组细胞相比,实验组细胞长出大量突起.并且胞质的细胞器逐渐消失.出现较多的脂滴。结论广西眼镜蛇毒神经生长因子可以促进PC12细胞增殖.并诱导其向神经样细胞分化,长出突触。 相似文献
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A rise of cytosolic Ca2+ is induced by NGF in rat pheochromocytoma PC12 and bovine chromaffin cells investigated (both in suspension and while attached to polyornithine-coated glass slides) by fluorescence techniques (with quin-2 and fura-2). The effect of NGF on [Ca2+]i is delayed (30-40 s of lag phase), slow (t1/2 = 40 s), relatively small (+50-75%) and persistent (over 10 min). It is due to Ca2+ influx (requires extracellular Ca2+ greater than 10 microM) through a pathway different from the voltage-gated Ca2+ channel, possibly accompanied by intracellular Ca2+ redistribution, and might play a messenger role in NGF action. 相似文献
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NGF诱导PC12细胞分化的研究 总被引:17,自引:0,他引:17
动物实验表明,生理浓度的乙醇在脑发育过程中,不但可以影响神经细胞的数量,还可协同增强NGF诱导PC12细胞形态和功能上的分化,分化的PC12细胞具有与交感神经元相似的性状特征。用100mmol/L乙醇和50ng/mlNGF联合诱导可建立PC12细胞分化模型,为以神经细胞为研究对象的实验提供一种获得神经细胞的方法。 相似文献
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Proadrenomedullin N-terminal 20 peptide (PAMP) is a novel hypotensive peptide found in the N-terminal portion of the precursor of adrenomedullin (AM). Although PAMP and AM originate from the same precursor and exert both a potent hypotensive action, they seem to control blood pressure through different mechanisms. To gain new insight into the anticholinergic actions of PAMP, we determined the effects of PAMP on the tyrosine hydroxylase (TH)- and dopamine beta-hydroxylase (DBH) mRNA expression in the rat pheochromocytoma cell line PC12 stimulated by nicotine. PAMP (> or =1 microM) significantly inhibited the nicotine-induced increases of TH- and DBH mRNA expression in a concentration-dependent manner. Also, PAMP at the concentrations (> or =1 microM) significantly inhibited nicotine-induced cyclic adenosine monophosphate (cAMP) production. These results indicate that the anticholinergic hypotensive actions of PAMP can be explained, at least in part, by its inhibition of the expression of mRNAs coding for catecholamine-synthesizing enzymes, and that the inhibitory effect is mediated by the cAMP/protein kinase A pathway. 相似文献
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Identification and characterization of mRNAs regulated by nerve growth factor in PC12 cells. 总被引:37,自引:9,他引:28
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Differential screening of cDNA libraries was used to detect and prepare probes for mRNAs that are regulated in PC12 rat pheochromocytoma cells by long-term (2-week) treatment with nerve growth factor (NGF). In response to NGF, PC12 cells change from a chromaffin cell-like to a sympathetic-neuron-like phenotype. Thus, one aim of this study was to identify NGF-regulated mRNAs that may be associated with the attainment of neuronal properties. Eight NGF-regulated mRNAs are described. Five of these increase 3- to 10-fold and three decrease 2- to 10-fold after long-term NGF exposure. Each mRNA was characterized with respect to the time course of the NGF response, regulation by agents other than NGF, and rat tissue distribution. Partial sequences of the cDNAs were used to search for homologies to known sequences. Homology analysis revealed that one mRNA (increased 10-fold) encodes the peptide thymosin beta 4 and a second mRNA (decreased 2-fold) encodes tyrosine hydroxylase. Another of the increased mRNAs was very abundant in sympathetic ganglia, barely detectable in brain and adrenals, and undetectable in all other tissues surveyed. One of the decreased mRNAs, by contrast, was very abundant in the adrenals and nearly absent in the sympathetic ganglia. With the exception of fibroblast growth factor, which is the only other agent known to mimic the differentiating effects of NGF on PC12 cells, none of the treatments tested (epidermal growth factor, insulin, dibutyryl cyclic AMP, dexamethasone, phorbol ester, and depolarization) reproduced the regulation observed with NGF. These and additional findings suggest that the NGF-regulated mRNAs may play roles in the establishment of the neuronal phenotype and that the probes described here will be useful to study the mechanism of action of NGF and the development and differentiation of neurons. 相似文献
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The effect of taxol, an experimental anti-tumor drug which exerts its effect through an action on the microtubular system, has been followed on the pheochromocytoma clonal cell line (PC12). Cells exposed to this drug, within 2–3 days, cease division and detach from the substratum. When nerve growth factor (NGF) is also present, PC 12 cells not only survive taxol treatment, but they also grow neuntes much larger than those emerging from cells incubated with NGF alone. At the ultrastructural examination, NGF-taxol-treated cells show the presence of an exceedingly large number of microtubules filling the cytoplasm even at the expense of other filamentous structures. These findings show that massive microtubule accumulation induced by taxol leads to rapid mitotic arrest, but is not sufficient to induce morphological differentiation of PC12 cells which requires also the presence of NGF. 相似文献
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Farida Sohrabji Lloyd A. Greene Rajesh C. Miranda C. Dominique Toran-Allerand 《Developmental neurobiology》1994,25(8):974-988
Recent work has shown that estrogen receptor mRNA and protein co-localize with neurotrophin receptor systems in the developing basal forebrain. In the present study we examined the potential for reciprocal regulation of estrogen and neurotrophin receptor systems by their ligands in a prototypical neurotrophin target, the PC12 cell. using in situ hybridization histochemistry, RT-PCR and a modified nuclear exchange assay, we found both estrogen receptor mRNA and estrogen binding in PC12 cells. Moreover, while estrogen binding was relatively low in naive PC12 cells, long-term exposure to NGF enhanced estrogen binding in these cells by sixfold. Furthermore, concurrent exposure to estrogen and NGF receptor mRNAs deifferentially regulated the expression of the two NGF receptor mRNAs. The expression of trkA mRNA was up-regulated, while p75NGFR mRNA was down-regulated transiently. The present data indicate that NGF may increase neuronal sensitivity to estrogen, and that estrogen, by differentially regulating p75NGFR and trkA mRNA, may alter the ratio fo the two NGF receptors, and, conseuqnetly, neurotrophin responsivity. In view of the widespread co-localization of estrogen and neurotrophin receptor systems in the developing CNS, the reciprocal regulation of these receptor systems by NGF and estrogen may have important implications for processes governing neural maturation and the maintenance of neural funciton. 1994 John Wiley & Sons, Inc. 相似文献
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D'Alessio A Cerchia L Amelio I Incoronato M Condorelli G de Franciscis V 《Cellular signalling》2007,19(6):1193-1200
The balance between specific signals from different growth factors dictates the biological response of mammalian cells including cell proliferation, differentiation and survival. PC12 cells represent a model of choice to compare the signalling of differentiative growth factors, as NGF, and of mitogenic growth factors, as EGF. In these cells the prolonged activity of the ERK kinase dictates the decision of cells to differentiate. Here we focused on the cytosolic tyrosine phosphatase Shp2 as an established regulator of the Ras-ERK cascade, to elucidate its involvement in determining the stimulation-dependent PC12 cell fate. To this end, we generated PC12 derived cell lines that express the interfering mutant of Shp2 under a tetracycline-inducible promoter. Our findings show that Shp2 participates to the opposite effects induced in PC12 cells by EGF and NGF and that the interactions with the multidocking Gab2 protein mediate such effects. 相似文献
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目的和方法:采用全细胞膜片钳技术观察神经生长因子(NGF)分化后的PC12细胞对乙酰胆碱(ACh)的敏感性,并对ACh诱发电流(IACh)的特性进行分析。结果:NGF处理后的PC12乐仅形态上向交感神经元分化,而且具有电学兴奋性,它对ACh敏感性比未分化前显著提高。药理学鉴定表明PC12上的IACh是由烟碱受体(nAChR)引起的,具有明显的失敏特性。宏观IACh呈内向整流和浓度依赖性。结论:PC12细胞培养方便,同源性好,加入NGF后向交感神经元分化,且其具有神经元烟碱受体,可以作为交感神经元烟碱受体研究的很好的模型系统。 相似文献
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S Belia S Fulle S Antonioli A M Salvatore G Fano 《Physiological chemistry and physics and medical NMR》1991,23(1):35-41
The aim of this work is to examine if the membrane-bound Phospholipase C system is correlated with the differentiative action of Nerve Growth Factor in PC12 cells. We found that the activity of membrane-bound Phospholipase C system increased with the presence of Nerve Growth Factor at two different phases. The early phase occurs during the first minutes after the formation of Nerve Growth Factor-receptor complex and it is completed within one hour. The later phase starts two hours after Nerve Growth Factor introduction and lasts for at least a total of 48 hours. The inositol-triphosphate levels measured in intact cells by radioimmunoassay show the same pattern of activation. We think that this dual response to Nerve Growth Factor could be due to either a double separated activation of the same enzyme or the presence of two different forms of membrane-bound Phospholipase C. 相似文献
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A series of cyclic glycerophosphates and their deoxy analogues were tested for induction of neuronal outgrowth in PC12 cells. Under chronic presence of a cyclic phosphate PC12 cells developed distinct isles of neuronal networks which covered up to 20% of the culture area, while alpha and beta glycerophosphates (the negative control compounds) did not induce any neuronal outgrowth. Distinct isles of neuronal networks were also observed upon short term application (i.e. 2 pulses of 3 hours each at day 1 and day 4) of the tested cyclic phosphates in contrast to an analogous short term exposure to NGF which was abortive. Analysis of tyrosine phosphorylation indicated a battery of phosphorylated proteins after several minutes of application of the cyclic phosphates, among which was an ERK protein of approximately 63 kD (possibly ERK7). Nerve rescue experiments were carried out with NGF differentiated PC12 cells where NGF was replaced with either 1,2 or 1,3 cyclic propanediolphosphate (1,2 cPP and 1,3 cPP) for 7 days. A distinct dose dependent preservation of neuronal network by these compounds was observed. In the control cultures NGF deprivation resulted in massive neuronal retraction and cell death. Preliminary experiments indicated that the nerve rescue by the cyclic phosphates involves the increase in the level of CASPase 6. The above findings suggest that cyclic glycerophosphates and their analogues may bear important physiological and pharmacological implications which are currently under investigation. 相似文献
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Rab22 is a small GTPase that is localized on early endosomes and regulates early endosomal sorting. This study reports that Rab22 promotes nerve growth factor (NGF) signaling-dependent neurite outgrowth and gene expression in PC12 cells by sorting NGF and the activated/phosphorylated receptor (pTrkA) into signaling endosomes to sustain signal transduction in the cell. NGF binding induces the endocytosis of pTrkA into Rab22-containing endosomes. Knockdown of Rab22 via small hairpin RNA (shRNA) blocks NGF-induced pTrkA endocytosis into the endosomes and gene expression (VGF) and neurite outgrowth. Overexpression of human Rab22 can rescue the inhibitory effects of the Rab22 shRNA, suggesting a specific Rab22 function in NGF signal transduction, rather than off-target effects. Furthermore, the Rab22 effector, Rabex-5, is necessary for NGF-induced neurite outgrowth and gene expression, as evidenced by the inhibitory effect of shRNA-mediated knockdown of Rabex-5. Disruption of the Rab22-Rabex-5 interaction via overexpression of the Rab22-binding domain of Rabex-5 in the cell also blocks NGF-induced neurite outgrowth, suggesting a critical role of Rab22-Rabex-5 interaction in the biogenesis of NGF-signaling endosomes to sustain the signal for neurite outgrowth. These data provide the first evidence for an early endosomal Rab GTPase as a positive regulator of NGF signal transduction and cell differentiation. 相似文献
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Elod Kortvely Szilvia Varszegi Arpad Palfi Karoly Gulya 《The journal of histochemistry and cytochemistry》2003,51(4):541-544
We investigated the intracellular distribution of the mRNAs corresponding to the three non-allelic CaM genes in cultured hippocampal cells by in situ hybridization with digoxigenin-labeled gene-specific riboprobes. In neurons the perikaryon was heavily stained and strong dendritic mRNA targeting was detected for all three CaM genes. The color labeling exhibited a punctate distribution, suggesting that CaM mRNAs are transported in RNA granules. Immunocytochemistry for S100 demonstrated that glial cells express CaM mRNAs at a very low level. A minority of the cultured cells were negative for either labeling. 相似文献
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Wang Q Maniati M Jabado O Pavlaki M Troy CM Greene LA Stefanis L 《Cell death and differentiation》2006,13(1):75-83
Caspase 2 has been implicated in trophic deprivation-induced neuronal death. We have shown that overexpression of the caspase 2-binding protein RAIDD induces neuronal apoptosis, acting synergistically with trophic deprivation. Currently, we examine the role of endogenous RAIDD in apoptosis of PC12 cells and sympathetic neurons. Expression of a truncated caspase recruitment domain-only form of caspase 2, which presumably disrupts the RAIDD interaction with endogenous caspase 2, attenuated trophic deprivation-induced apoptosis. Furthermore, downregulation of RAIDD by small interfering RNA led to inhibition of trophic deprivation-induced death, whereas death induced by DNA damage, which is not caspase 2-mediated, was not inhibited. Therefore, RAIDD, likely through interaction with caspase 2, is involved in trophic deprivation-induced neuronal apoptosis. This is the first demonstration of the involvement of RAIDD in apoptosis, and provides further support for the idea that apoptotic pathways in the same system may differ depending on the initiating stimulus. 相似文献
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D S Hartman M McCormack R Schubenel C Hertel 《The Journal of biological chemistry》1992,267(34):24516-24522
Rat pheochromocytoma (PC12) cells express two distinct nerve growth factor receptors (NGFRs), p75NGFR and trkA (p140trk). In addition to these receptors, by using 125I-mNGF affinity labeling and BS3 chemical cross-linking of PC12 cell protein, we have identified two additional trkA protein bands with apparent molecular weights of 220,000 and 300,000. These bands contain trkA, but were not immunoprecipitated by p75NGFR-specific antisera, suggesting that they do not represent trkA/p75NGFR protein complexes. The 220-kDa trkA band apparently represents trkA with alternate post-translational modification. The appearance of the 300-kDa trkA band was dependent on cross-linker concentration and could be diminished in the presence of reducing agents, suggesting that it represents a trkA dimer. All trkA bands were phosphorylated on tyrosine residues when bound to mNGF, suggesting that they participate in NGF-induced signal transduction. NGF binding kinetics to all three trkA bands were indistinguishable, with slow dissociation rates, and a slow association rate that required approximately 1 h to reach equilibrium levels at 4 degrees C. All three trkA bands bound the related neurotrophin brain-derived neurotrophic factor and neurotrophin-3 with a profile characteristic of trkA. 相似文献