Rat Pheochromocytoma Cell Line Research Articles

Synthesis and in vitro evaluation of novel 1,2,4-triazine derivatives as neuroprotective agents

The role of novel triazine derivatives against oxidative stress exerted by hydrogen peroxide on differentiated rat pheochromocytoma (PC12) cell line was examined and a consistent protection from H 2O 2-induced cell death, associated with a marked reduction in caspase-3 activation, was observed. Moreover, activation of NF-κB, a known regulator of a host of genes that involves in specific stress and inflammatory responses by H 2O 2, was greatly impaired by triazine pretreatment in differentiated PC12 cells. Neuroprotective effect of such compounds may represent a promising approach for treatment of neurodegenerative diseases.

Inhibition of Hydrogen Sulfide Generation Contributes to 1-Methy-4-Phenylpyridinium Ion-Induced Neurotoxicity

Reactive oxygen species (ROS) overproduction contributes to the neurotoxicity of 1-methy-4-phenylpyridinium ion (MPP(+)). Increasing studies have shown that hydrogen sulfide (H(2)S) is an endogenous antioxidant gas. We have hypothesized that MPP(+)-caused neurotoxicity may involve the imbalance of proportion to this endogenous protective antioxidant gas. The aim of this study is to evaluate whether MPP(+) disturbs H(2)S synthesis in PC12 cells, a clonal rat pheochromocytoma cell line, and whether disturbance of H(2)S generation induced by MPP(+) is an underlying mechanism of MPP(+)-induced neurotoxicity. We show that exposure of PC12 cells to MPP(+) causes a significant decrease in H(2)S generation and results in remarkable cell damage. We find that cystathionine-β-synthetase (CBS) is catalyzed in PC12 cells to generate H(2)S, and that both expression and activity of CBS are inhibited by MPP(+) treatment. Exposure of sodium hydrosulfide (NaHS), a donor of H(2)S, extenuates MPP(+)-induced cytotoxicity and ROS accumulation in PC12 cells, while inhibition of CBS by amino-oxyacetate (AOAA) exacerbates the effects of MPP(+). These results indicate that MPP(+) neurotoxicity involves reduction of H(2)S production, which is caused by inhibition of CBS. This study provides novel insights into cell death observed in neurodegenerative disease such as Parkinson's disease.

Suppression of Nitric Oxide Implicated in the Protective Effect of Echinacoside on H2O2-Induced PC12 Cell Injury

We investigated whether suppression of nitric oxide (NO) implicated in the protective effect of echinacoside (ECH), a phenylethanoid glycoside, on H2O2-induced injury to the rat pheochromocytoma cell line (PC12 cells). Data show that application of ECH to H2O2-injured PC12 cells (HIPCs) increased cell viability and decreased the necrotic ratio. Laser scanning confocal microscopic (LSCM) analysis suggested that ECH exerted an inhibitory effect on the formation of NO. In addition, RT-PCR analysis revealed that ECH down-regulated p65 and iNOS mRNA expressions in HIPCs. In summary, suppression of NO is related to the protective effect of ECH on HIPCs.

Stimulation of neuronal neurite outgrowth using functionalized carbon nanotubes

Low concentrations (0.11–1.7 µg ml − 1) of functionalized carbon nanotubes (CNTs), which are multi-walled CNTs modifiedby amino groups, when added with nerve growth factor (NGF), promotedoutgrowth of neuronal neurites in dorsal root ganglion (DRG) neurons and ratpheochromocytoma cell line PC12h cells in culture media. The quantity of activeextracellular signal-regulated kinase (ERK) was higher after the addition of both 0.85 µg ml − 1 CNTs and NGF than that with NGF alone. CNTs increased the number of cells withneurite outgrowth in DRG neurons and PC12h cells after the inhibition of the ERKsignaling pathway using a mitogen-activated protein kinase (MAPK)/ERK kinase (MEK)inhibitor. Active ERK proteins were detected in MEK inhibitor-treated neurons after theaddition of CNTs to the culture medium. These results demonstrate that CNTs maystimulate neurite outgrowth by activation of the ERK signaling pathway. Thus, CNTs arebiocompatible and are promising candidates for biological applications and devices.

Rab13 Regulates Neurite Outgrowth in PC12 Cells through Its Effector Protein, JRAB/MICAL-L2

Neurite outgrowth is the first step in the processes of neuronal differentiation and regeneration and leads to synaptic polarization and plasticity. Rab13 small G protein shows an increased mRNA expression level during neuronal regeneration; it is therefore thought to be involved in this process. We previously identified JRAB (junctional Rab13-binding protein)/MICAL-L2 (molecules interacting with CasL-like 2) as a novel Rab13 effector protein. Here, we show that Rab13 regulates neurite outgrowth in the rat pheochromocytoma cell line PC12 through an interaction with JRAB/MICAL-L2. The expression of JRAB/MICAL-L2 alone inhibits neurite outgrowth, whereas coexpression of the dominant active form of Rab13 rescues this effect. We also demonstrate an intramolecular interaction between the N-terminal calponin-homology (CH) and LIM domains of JRAB/MICAL-L2 and the C-terminal coiled-coil domain. Finally, we show that the binding of Rab13 to JRAB/MICAL-L2 stimulates the interaction of JRAB/MICAL-L2 with actinin-4, an actin-binding protein, which localizes to the cell body and the tips of the neurites in PC12 cells. These results suggest that Rab13 and JRAB/MICAL-L2 may act to transfer actinin-4 from the cell body to the tips of neurites, where actinin-4 is involved in the reorganization of the actin cytoskeleton which results in neurite outgrowth.

Effect of Urotensin II on PC12 Rat Pheochromocytoma Cells

Urotensin II (U-II), initially identified as a cyclic peptide from fish urophysis, acts both as a strong vasoconstrictor and vasodilator in the vasculature via its receptor, G-protein coupled receptor 14. In addition, U-II and its receptor are co-expressed in the adrenal medulla, as well as in human pheochromocytomas, suggesting that this peptide may have some function in chromaffin cells. However, the precise role of U-II in these cells is unknown. In the present study, we initially demonstrate that U-II and its receptors mRNA are co-expressed in the rat pheochromocytoma cell line PC12. Moreover, U-II has not effect on tyrosine hydroxylase (TH), the rate-limiting enzyme involved in the biosynthesis of catecholamine, in terms of enzyme activity or at the mRNA level. However, U-II does induce an increase in the phosphorylation of TH specifically at Ser31 without affecting phosphorylation at the two other sites (Ser19 and Ser40). U-II also markedly activates extracellular signal-regulated kinases (ERKs) and p38, but not Jun N-terminal kinase. Blockade of the epidermal growth factor (EGF) receptor by AG1478 significantly reduces activation of ERK, suggesting that EGF receptor transactivation could act upstream of the ERK pathway in PC12 cells. Furthermore, U-II significantly increases dopamine secretion from PC12 cells. Finally, we show that U-II induced significant DNA synthesis in a ERKs and P38 mitogen-activated protein kinase-dependent manner. The results obtained indicate that U-II may exert its effects as a neuromodulator in chromaffin cells.

Ligand-Dependent Activation of the Chimeric Tumor Necrosis Factor Receptor-Amyloid Precursor Protein (APP) Reveals Increased APP Processing and Suppressed Neuronal Differentiation

The β-amyloid precursor protein (APP) is a type I transmembrane protein whose functions and metabolic processing have been implicated in the pathogenesis of Alzheimer’s disease (AD). APP’s physical resemblance as a glycosylated receptor and the presence of several conserved motifs which are characteristics of a membrane-associated receptor has prompted interest to study and understand the role of downstream signaling events mediated by the activation of APP during both physiological and pathological conditions. Efforts to elucidate the mechanisms of APP signaling have not been conclusive. In order to further characterize the intracellular signaling activities of APP, we have constructed a chimeric APP receptor which is made up of the extracellular domain of human tumor necrosis factor receptor superfamily, member 1B (TNFRSF1B), the membrane-spanning segment of human APP₇₇₀ isoform’s β-cleaved carboxyl-terminal fragment (C99), and the green fluorescent protein (GFP) tagged to the carboxyl-terminus of C99. The mammalian rat pheochromocytoma (PC12) cell line is used as the cellular model for transfection of this <under>T</under>NFRSF1B-<under>A</under>PP-G<under>F</under>P (TAF) fusion receptor. Ligand-specific stimulation of this TAF receptor using human recombinant TNF reveals the induction of cytoplasmic TAF phosphorylation on Thr743 (numbering for APP₇₇₀ isoform) and an elevated release of APP intracellular domains (AICDs). Time-lapse microscopy shows the trafficking of GFP-tagged AICDs into the nucleus upon TAF receptor activation. The increased presence of AICDs is believed to suppress neuronal differentiation of PC12 cells in response to nerve growth factor treatment, by negatively regulating the levels of p53, cyclin D1 and phosphorylation of the signal transducer and activator of transcription STAT3.

Melanocortin 1 receptor mutations impact differentially on signalling to the cAMP and the ERK mitogen-activated protein kinase pathways

Melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor expressed in melanocytes, is a major determinant of skin pigmentation, phototype and cancer risk. MC1R activates cAMP and mitogen-activated protein kinase ERK1/ERK2 signalling. When expressed in rat pheochromocytoma cell line cells, the R151C, R160W and D294H MC1R variants associated with melanoma and impaired cAMP signalling mediated ERK activation and ERK-dependent, agonist-induced neurite outgrowth comparable with wild-type. Dose–response curves for ERK activation and cAMP production indicated higher sensitivity of the ERK response. Thus, the melanoma-associated MC1R mutations impact differently on cAMP and ERK signalling, suggesting that cAMP is not responsible for functional coupling of MC1R to the ERK cascade.

Atomic force microscopy analysis of central nervous system cell morphology on silicon carbide and diamond substrates

Brain machine interface (BMI) devices offer a platform that can be used to assist people with extreme disabilities, such as amyotrophic lateral sclerosis (ALS) and Parkinson's disease. Silicon (Si) has been the material of choice used for the manufacture of BMI devices due to its mechanical strength, its electrical properties and multiple fabrication techniques; however, chronically implanted BMI devices have usually failed within months of implantation due to biocompatibility issues and the fact that Si does not withstand the harsh environment of the body. Single crystal cubic silicon carbide (3C-SiC) and nanocrystalline diamond (NCD) are semiconductor materials that have previously shown good biocompatibility with skin and bone cells. Like Si, these materials have excellent physical characteristics, good electrical properties, but unlike Si, they are chemically inert. We have performed a study to evaluate the general biocompatibility levels of all of these materials through the use of in vitro techniques. H4 human neuroglioma and PC12 rat pheochromocytoma cell lines were used for the study, and polystyrene (PSt) and amorphous glass were used as controls or for morphological comparison. MTT [3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide] assays were performed to determine general cell viability with each substrate and atomic force microscopy (AFM) was used to quantify the general cell morphology on the substrate surface along with the substrate permissiveness to lamellipodia extension. 3C-SiC was the only substrate tested to have good viability and superior lamellipodia permissiveness with both cell lines, while NCD showed a good level of viability with the neural H4 line but a poor viability with the PC12 line and lower permissiveness than 3C-SiC. Explanations pertaining to the performance of each substrate with both cell lines are presented and discussed along with future work that must be performed to further evaluate specific cell reactions on these substrates.

Genomic response to Wnt signalling is highly context-dependent — Evidence from DNA microarray and chromatin immunoprecipitation screens of Wnt/TCF targets

Wnt proteins are important regulators of embryonic development, and dysregulated Wnt signalling is involved in the oncogenesis of several human cancers. Our knowledge of the downstream target genes is limited, however. We used a chromatin immunoprecipitation-based assay to isolate and characterize the actual gene segments through which Wnt-activatable transcription factors, TCFs, regulate transcription and an Affymetrix microarray analysis to study the global transcriptional response to the Wnt3a ligand. The anti-β-catenin immunoprecipitation of DNA–protein complexes from mouse NIH3T3 fibroblasts expressing a fusion protein of β-catenin and TCF7 resulted in the identification of 92 genes as putative TCF targets. GeneChip assays of gene expression performed on NIH3T3 cells and the rat pheochromocytoma cell line PC12 revealed 355 genes in NIH3T3 and 129 genes in the PC12 cells with marked changes in expression after Wnt3a stimulus. Only 2 Wnt-regulated genes were shared by both cell lines. Surprisingly, Disabled-2 was the only gene identified by the chromatin immunoprecipitation approach that displayed a marked change in expression in the GeneChip assay. Taken together, our approaches give an insight into the complex context-dependent nature of Wnt pathway transcriptional responses and identify Disabled-2 as a potential new direct target for Wnt signalling.

Protective Effects of Echinacoside, One of the Phenylethanoid Glycosides, on H2O2-Induced Cytotoxicity in PC12 Cells

We have investigated the protective effects of echinacoside (ECH), one of the phenylethanoid glycosides, on H(2)O(2)-induced cytotoxicity in the rat pheochromocytoma cell line (PC12 cells). Our data show that application of ECH to H(2)O(2)-injured PC12 cells (HIPCs) increased cell viability and decreased the apoptotic ratio. Flow cytometry (FCM) and laser scanning confocal microscopy (LSCM) analysis suggested that ECH exerted its inhibitory effects on the formation of reactive oxygen species (ROS) and the accumulation of intracellular free Ca(2+) ([Ca(2+)]i). In addition, ECH elevated the mitochondrial membrane potential (MMP) in HIPCs. Furthermore, Western blot analysis revealed that ECH prevented an H(2)O(2)-induced increase of the Bax/Bcl-2 ratio by down-regulating Bax protein expression and upregulating Bcl-2 protein expression. In summary, ECH showed significant neuroprotective effects on HIPCs through the mitochondrial apoptotic pathway, and could be a potential candidate for intervention in neurodegenerative diseases such as Alzheimer's and Parkinson's disease.

Clozapine protects PC-12 cells from death due to oxidative stress induced by hydrogen peroxide via a cell-type specific mechanism involving inhibition of extracellular signal-regulated kinase phosphorylation

Recent evidence suggests that some atypical antipsychotic drugs may protect against oxidative stress and consequent neurodegeneration by mechanisms that remain unclear. Using the neuron-like rat pheochromocytoma (PC-12) cell line, Clozapine and N-desmethylclozapine were tested for their ability to protect against cell death due to oxidative stress induced by hydrogen peroxide (H 2O 2). These drugs demonstrated significant protection of PC-12 cells, as measured by both the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) and Alamar Blue cell viability assays. However, neither viability assay detected a protective effect of Clozapine on human embryonic kidney (HEK293), rat primary cortical neurons, or human neuroblastoma (SH-SY5Y) exposed to H 2O 2 treatment. The mechanism of protection involves a PC-12 cell-specific differential response to H 2O 2 treatment vs. the other cell lines. Pre-treatment with 250 µM or 125 µM diethyldithiocarbamate (DETC), a superoxide dismutase (SOD) inhibitor, unexpectedly showed protection of the PC-12 cells from H 2O 2 treatment. Western blots revealed that Clozapine, N-desmethylclozapine, and DETC reduce the phosphorylation of extracellular signal-regulated kinase (ERK) that is caused by H 2O 2 exposure in PC-12 cells. In both HEK293 and SH-SY5Y cells, H 2O 2 exposure did not increase ERK phosphorylation over control, demonstrating a different response to H 2O 2 vs. PC-12 cells, and explaining why Clozapine could not protect these cells. Also, U0126, a specific MEK inhibitor, was able to protect PC-12 cells from H 2O 2 exposure, showing that inhibiting ERK phosphorylation is sufficient to provide protection. Cumulatively, these results indicate that Clozapine, N-desmethylclozapine, DETC, and U0126 protect PC-12 cells by blocking the cell-type specific H 2O 2 induced increase in ERK phosphorylation.

Characterization and cellular uptake of platinum anticancer drugs encapsulated in apoferritin

Clinical application of platinum-based anticancer drugs is largely limited by severe general toxicity and drug resistance. Drug delivery systems with tumor-targeting potential are highly desired for improving the efficacy and applicability of these drugs. This study describes an alternative strategy for the delivery of platinum drugs (cisplatin, carboplatin and oxaliplatin) by encapsulating each of them in the cavity of apoferritin (AFt). The encapsulation was achieved through manipulating the pH-dependent unfolding-refolding process of AFt at pH 2.0 and 7.4, respectively, in saturated drug solution. UV-vis spectrometry, circular dichroism spectrometry, dynamic light scattering, and inductively coupled plasma mass spectrometry were used to characterize the AFt-drug complexes. The loading capacity of AFt varies with respective drugs and the structural integrity of the protein shell remains intact after encapsulation. In vitro assays on the rat pheochromocytoma cell line (PC12) show that AFt-cisplatin inhibits the cells in a slow but sustaining mode and the cellular uptake of platinum is enhanced by AFt. AFt-carboplatin and AFt-oxaliplatin complexes only exhibit a marginal cytotoxicity towards this cell line under similar concentrations.

Regulation of the outgrowth position of neurites in cell body and the direction of their elongation for PC12 cells in three dimensional hydrogel culture system with feeble flow of the solvent

A rat pheochromocytoma cell line (PC12) was encapsulated in a water-insoluble hydroxyethyl methacrylate-methyl methacrylate copolymer by interfacial precipitation from a polyethylene glycol 200 solution into phosphate-buffered saline. The resulting capsules (660 ± 44 μm in diameter; 84 ± 27 μm wall thickness) contained viable PC12 cells in a spheroidal arrangement, much like tumour spheroids, the latter grown on surfaces unsuitable for cell attachment. In these spheroids, the viable cells formed a band approximately 100 μm thick, surrounding an inner core of necrotic cells. A similar arrangement was seen 14, 28 and 42 days after encapsulation, with capsules maintained in an in vitro tissue culture environment; the annular ring was roughly constant in size, although the packing density appeared to increase over the 6 week observation period. During the first 4 weeks, when measurements were made the encapsulated cells converted a tetrazolium dye (MTT) into an insoluble formazan product, in a time-after-encapsulation-dependent manner. This indicated that PC12 cells retained viability despite encapsulation and an ability to increase (at least in part) their metabolic capacity, presumably by a combination of proliferation and altered cellular activity. The encapsulated PC12 cells also secreted dopamine when incubated in a high potassium release medium but not in a low potassium, conventional tissue culture medium (RPMI 1640). Consistent with the MTT results, the amount of dopamine released was also dependent on the time after encapsulation, as well as the cell density at the time of encapsulation.

Proteasome Inhibitor Associated Neuropathy Is Mechanism Based

Background: Peripheral neuropathy is a common side effect of chemotherapeutics, particularly taxanes, platinums, vinca alkaloids, and proteasome inhibitors (PIs). There are currently three chemical scaffolds of PIs in clinical development: leucine boronates, lactacystin analogs, and epoximicin derivatives. Among these, only a leucine boronate, VELCADE, is currently approved for use in humans. Approximately 39% of patients treated with VELCADE develop peripheral neuropathy. Preclinical data suggest that peripheral neuropathy may develop as a consequence of functional effects in the dorsal root ganglia (Silverman et al. 2006; Toxicologic Pathology 34:989). Moreover, in vitro data suggests that these effects may be the result of a reorganization of cytoskeletal elements and an accumulation of ubiquitinated proteins in the neuronal cytoplasm (Csizmadia et al. 2008; Neurotoxicology 29:232). In vivo and in vitro data suggest that PI-associated neuropathy develops following treatment with PIs of differing chemical scaffolds, consistent with a mechanism based effect. A better understanding of the mechanism underlying PI-associated peripheral neuropathy could potentially lead to improved management strategies for this adverse event while maintaining therapeutic benefit.Aims:To investigate if PI-associated neuropathy is mechanism based (a consequence of proteasome inhibition), andIf mechanism based, to further understand the mechanism by which proteasome inhibition results in peripheral neuropathy.Methods: The effects of treating in vitro neuronal cell cultures (PC-12 rat pheochromocytoma cell line treated with nerve growth factor [NGF] in order to induce neuronal differentiation) with leucine boronate, lactacystin analog, or epoximicin PIs, as well as with taxol and cisplatinum, were examined by Western immunoblot and immunohistochemistry specific for ubiquitinated proteins. Additionally, the morphologic effects on the peripheral nervous system of mice treated with leucine boronate or lactacystin analog PIs were examined.Results: Treatment of PC-12 cells with leucine boronate, lactacystin analog or epoximicin PIs but not with taxol or cisplatinum resulted in perinuclear accumulations of ubiquitinated proteins. In in vivo models, morphologic lesions were observed in the peripheral nervous system of mice treated with both leucine boronate and lactacystin analog PIs. In a time course study with a prototypical PI, dorsal root ganglia was identified as the primary target leading to secondary peripheral nerve degeneration. Routine hematoxylin and eosin histologic analysis showed an accumulation of eosinophilic material within the dorsal root ganglia neurons. Immunohistochemistry showed these accumulations to contain ubiquitinated proteins, consistent with the in vitro observations. The in vivo effects were similar following treatment with either a leucine boronate or a lactacystin analog PI.Summary: Our in vitro and in vivo studies identified a morphologic effect which among the chemotherapeutics which cause peripheral neuropathy is uniquely associated with PIs. Moreover, these findings were seen regardless of the chemical scaffold.

Nerve growth factor regulates the expression of the cholinergic locus and the high-affinity choline transporter via the Akt/PKB signaling pathway.

Nerve growth factor (NGF) is a trophic and survival factor for cholinergic neurons, and it induces the expression of several genes that are essential for synthesis and storage of acetylcholine (ACh), specifically choline acetyltransferase, vesicular ACh transporter (VAChT), and choline transporter. We have found previously that the phosphatidylinositol 3'-kinase pathway, but not the MEK/MAPK pathway, is the mediator of NGF-induced cholinergic differentiation. Here we demonstrate, in the rat pheochromocytoma cell line PC12 and in primary mouse neuronal cultures, that NGF-evoked up-regulation of these three cholinergic-specific genes is mediated by the anti-apoptotic signaling molecule Akt/protein kinase B. Inhibition of Akt activation by the pharmacological inhibitor 1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO), or by a peptide fragment derived from the proto-oncogene TLC1, eliminated NGF-stimulated increases in cholinergic gene expression, as demonstrated by RT-PCR and reporter gene assays. Moreover, treatment with HIMO reversed NGF-evoked increases in choline acetyltransferase activity and ACh production. In co-transfection assays with the reporter construct, a dominant-negative Akt plasmid and Akt1-specific small interfering RNA also attenuated NGF-induced cholinergic promoter activity. Our data indicate that, in addition to its well-described role in promoting neuronal survival, Akt can also mediate signals necessary for neurochemical differentiation.

A food‐derived synergist of NGF signaling: identification of protein tyrosine phosphatase 1B as a key regulator of NGF receptor‐initiated signal transduction

Neurotrophins, such as the nerve growth factor (NGF), play an essential role in the growth, development, survival and functional maintenance of neurons in the central and peripheral systems. They also prevent neuronal cell death under various stressful conditions, such as ischemia and neurodegenerative disorders. NGF induces cell differentiation and neurite outgrowth by binding with and activating the NGF receptor tyrosine kinase followed by activation of a variety of signaling cascades. We have investigated the NGF-dependent neuritogenesis enhancer potential of a food-derived small molecule contained in Brassica vegetables and identified the protein tyrosine phosphatase (PTP) 1B as a key regulator of the NGF receptor-initiated signal transduction. Based on an extensive screening of Brassica vegetable extracts for the neuritogenic-promoting activity in the rat pheochromocytoma cell line PC12, we found the Japanese horseradish, wasabi (Wasabia japonica, syn. Eutrema wasabi), as the richest source and identified 6-methylsulfinylhexyl isothiocyanate (6-HITC), an analogue of sulforaphane isolated from broccoli, as one of the major neuritogenic enhancers in the wasabi. 6-HITC strongly enhanced the neurite outgrowth and neurofilament expression elicited by a low-concentration of NGF that alone was insufficient to induce neuronal differentiation. 6-HITC also facilitated the sustained-phosphorylation of the extracellular signal-regulated kinase and the autophosphorylation of the NGF receptor TrkA. It was found that PTP1B act as a phosphatase capable of dephosphorylating Tyr-490 of TrkA and was inactivated by 6-HITC in a redox-dependent manner. The identification of PTP1B as a regulator of NGF signaling may provide new clues about the chemoprotective potential of food components, such as isothiocyanates.

Myelin-forming ability of Schwann cell-like cells induced from rat adipose-derived stem cells in vitro

Although Schwann cell (SC) transplantation can enhance peripheral and central nerve repair experimentally, it is difficult to generate sufficient SC quickly for clinical application. So alternative cell systems for SC are desired. SC-like cells induced from adipose-derived stem cells (ADSC) may be one of the ideal alternative cell systems for SC. However, myelin-forming ability, which is the most important characteristics and function of SC, has not been investigated in SC-like cells from ADSC up to now. In this experiment, ADSC were harvested from rat inguinal fat pad. Rat ADSC were fibroblast-like in shape, almost all the cells expressed mesodermal marker fibronectin, and only few cells expressed neural stem cell marker nestin. A mixture of glial growth factors (Heregulin, bFGF, PDGF and forskolin) could induce rat ADSC into SC-like cells. SC-like cells were spindle-like in shape and expressed glial markers GFAP and S100, similar to genuine SC. When intracellular cAMP was increased, SC-like cells could express myelin protein p0. More importantly, when co-cultured with rat pheochromocytoma cell line (PC12 cells), SC-like cells could induce the differentiation of PC12 cells rapidly and form myelin structures with PC12 cells in vitro. Our data further demonstrated that SC-like cells from ADSC were able to form myelins and these cells may benefit the treatment of peripheral and central nerve injuries.

Role of TRP channels and NCX in mediating hypoxia-induced [Ca2+]i elevation in PC12 cells

Mammalian cells require a constant O2 supply to produce adequate energy, and sustained hypoxia can kill cells. Mammals therefore have evolved sophisticated mechanisms to allow their cells to adapt to hypoxia. In this study, we investigated the role of TRP channels and the Na+-Ca2+ exchanger (NCX) in mediating hypoxia-induced [Ca2+]i elevation in a model of the O2-sensing rat pheochromocytoma (PC12) cell line by using Ca2+ imaging and molecular biological approaches. Non-selective cation channels, such as TRPC1, 3 and 6, were found to be functionally expressed in PC12 cells. They mediated Ca2+ entry when cells were exposed to acute hypoxia (PO2 of 15 mmHg), in addition to Ca2+ entry via VGCCs. Blockage of TRPCs by 2APB and SKF96365 could significantly reduce hypoxia-mediated [Ca2+]i elevation. Suramin and U73122 attenuated the hypoxia-induced [Ca2+]i elevation, implying the involvement of the G-protein and PLC pathways in the hypoxic response. In addition to TRPCs and VGCCs, NCX also contributed to the hypoxia-induced [Ca2+]i elevation, and blockade of NCX by KBR7943 could significantly decrease the hypoxia-induced [Ca2+]i elevation. Our results suggest that the activation of TRP by hypoxia could lead to NCX reversal; furthermore, membrane depolarization and TRPCs may play a primary role in mediating the hypoxic response in PC12 cells.

Salidroside inhibits H&amp;lt;sub&amp;gt;2&amp;lt;/sub&amp;gt;O&amp;lt;sub&amp;gt;2&amp;lt;/sub&amp;gt;-induced apoptosis in PC 12 cells by preventing cytochrome &amp;lt;italic&amp;gt;c&amp;lt;/italic&amp;gt; release and inactivating of caspase cascade

We used a rat pheochromocytoma (PC 12) cell line to study the effects of salidroside on hydrogen peroxide (H2O2)-induced apoptosis. In PC12 cells, H2O2-induced apoptosis was accompanied by the down-regulation of Bcl-2, the up-regulation of Bax, the release of mitochondrial cytochrome c to cytosol, and the activation of caspase-3, -8 and -9. However, salidroside suppressed the down-regulation of Bcl-2, the up-regulation of Bax and the release of mitochondrial cytochrome c to cytosol. Moreover, salidroside attenuated caspase-3, -8 and -9 activation, and eventually protected cells against H2O2-induced apoptosis. Taken together, these results suggest that treatment of PC12 cells with salidroside can block H2O2-induced apoptosis by regulating Bcl-2 family members and by suppressing cytochrome c release and caspase cascade activation.