Muscarinic receptor regulation of osmosensitive taurine transport in human SH-SY5Y neuroblastoma cells

The ability of G protein‐coupled receptors to regulate osmosensitive uptake of the organic osmolyte, taurine, into human SH‐SY5Y neuroblastoma cells has been examined. When monitored under isotonic conditions and in the presence of physiologically relevant taurine concentrations (1–100 μM), taurine influx was mediated exclusively by a Na⁺‐dependent, high‐affinity (K_m = 2.5 μM) saturable transport mechanism (V_max = 0.087 nmol/mg protein/min). Reductions in osmolarity of > 20% (attained under conditions of a constant NaCl concentration) resulted in an inhibition of taurine influx (> 30%) that could be attributed to a reduction in V_max, whereas the K_m for uptake remained unchanged. Inclusion of the muscarinic cholinergic agonist, oxotremorine‐M (Oxo‐M), also resulted in an attenuation of taurine influx (EC₅₀～0.7 μM). Although Oxo‐M‐mediated inhibition of taurine uptake could be observed under isotonic conditions (～25–30%), the magnitude of inhibition was significantly enhanced by hypotonicity (～55–60%), a result that also reflected a reduction in the V_max, but not the K_m, for taurine transport. Oxo‐M‐mediated inhibition of taurine uptake was dependent upon the availability of extracellular Ca²⁺ but was independent of protein kinase C activity. In addition to Oxo‐M, inclusion of either thrombin or sphingosine 1‐phosphate also attenuated volume‐dependent taurine uptake. The ability of Oxo‐M to inhibit the influx of taurine was attenuated by 4‐[(2‐butyl‐6,7‐dichloro‐2‐cyclopentyl‐2,3‐dihydro‐1‐oxo‐1H‐inden‐5‐yl)oxy]butanoic acid, an inhibitor of the volume‐sensitive organic osmolyte and anion channel. 4‐[(2‐Butyl‐6,7‐dichloro‐2‐cyclopentyl‐2,3‐dihydro‐1‐oxo‐1H‐inden‐5‐yl)oxy]butanoic acid also prevented receptor‐mediated changes in the efflux and influx of K⁺ under hypoosmotic conditions. The results suggest that muscarinic receptor activation can regulate both the volume‐dependent efflux and uptake of taurine and that these events may be functionally coupled.     

Characterization of nuclear T3 receptors in human neuroblastoma cells SH-SY5Y: Effect of differentiation with sodium butyrate and nerve growth factor

Nuclear receptors for the thyroid hormone triiodothyronine (T₃) have been identified in vivo in brain tissues and in vitro in mouse and rat neuroblastoma and glioma cells. The present study characterizes nuclear T₃ receptors in human neuroblastoma SH-SY5Y cells and compares their levels before and after differentiation. Undifferentiated cells, grown in DME/HAM F-12 medium supplemented with 10% fetal calf serum, show an abundant single type of nuclear receptor, indicated by a straight Scatchard plot, with aK _d of 0.11 nmol/l. After treatment with sodium butyrate (0.5 mM for 4 days) or NGF (2 nM for 6 days), the cells showed neuronal-like patterns (extension of neurites, slowing of growth, increased tyrosine hydroxylase activity), with a decrease in the number of nuclear T₃ receptors. As sodium butyrate and NGF treatments differentiate neuroblastoma SH-SY5Y cells, these data suggest a down-regulation of T₃ receptors with cell maturation.     

Muscarinic cholinergic receptors activate both inhibitory and stimulatory growth mechanisms in NIH3T3 cells.

Activation of G(q) protein-coupled receptors can either stimulate or inhibit cell growth. Previously, these opposite effects were explained by differences in the cell models. Here we show that activation of m3 muscarinic acetylcholine receptors ectopically expressed in NIH3T3 cells can cause stimulation and inhibition of growth in the same cell. A clonal cell line was selected from cells that formed foci agonist dependently (3T3/m3 cells). In quiescent 3T3/m3 cells, carbachol stimulated DNA synthesis. In contrast, when 3T3/m3 cells were growing, either due to the presence of serum or after transformation with oncogenic v-src, carbachol inhibited growth. This inhibition was not due to reduction of extracellular signal-regulated kinase activity because carbachol induced extracellular signal-regulated kinase phosphorylation in both quiescent and growing 3T3/m3 cells. Investigating the cell cycle mechanisms involved in growth inhibition, we found that carbachol treatment decreased cyclin D1 levels, increased p21(cip1) expression, and led to hypophosphorylation of the retinoblastoma gene product (Rb). Proteasome inhibitors blocked the carbachol-induced degradation of cyclin D1. Effects on p21(cip1) were blocked by a protein kinase C inhibitor. Thus, m3 muscarinic acetylcholine receptors couple to both growth-stimulatory and -inhibitory signaling pathways in NIH3T3 cells, and the observed effects of receptor activation depend on the context of cellular growth.     

Insulin-like growth factor I rescues SH-SY5Y human neuroblastoma cells from hyperosmotic induced programmed cell death

Insulin-like growth factor I (IGF-I) and the type I IGF receptor are widely distributed in developing and adult mammalian nervous systems. In vitro, IGF-I is a mitogen for primary neurons and also for cells from the SH-SY5Y human neuroblastoma cell line, a well-characterized model system of neuronal growth. In the current study, we examined the effects of osmotic stress on SH-SY5Y cell viability and the mechanism by which IGF-I serves as a neuronal osmoprotectant. Within 24 hr, exposure of SH-SY5Y cells to hyperosmotic serum-free media decreased (1) the number of viable cells, (2) the rate of ³H-thymidine incorporation, and (3) cell cycle progression. The inclusion of 10 nM IGF-I with hyperosmotic media prevented the loss of cell viability. The osmoprotective effects of IGF-I were inhibited by α-IR_J, a blocking antibody of the type I IGF receptor. The observed loss of SH-SY5Y cell viability following hyperosmotic shock was due to an induction of programmed cell death as determined by flow cytometry and gel electrophoresis. Our results suggest that IGF-I can protect SH-SY5Y cells from hyperosmotic induced programmed cell death. © 1996 Wiley-Liss, Inc.     

聚乳酸微柱阵列型拓扑结构基底上SH-SY5Y细胞VEGF和IL-8的分泌及表达

以紫外光光刻、硅蚀刻及复制模塑技术制备了聚乳酸 (PLLA) 微柱阵列型拓扑结构基底,考察了SH-SY5Y人神经母细胞瘤细胞在拓扑结构基底上的生长及血管内皮生长因子 (VEGF) 和白细胞介素-8 (IL-8) 的分泌及表达。细胞的形态及铺展采用扫描电子显微图像进行分析,细胞在拓扑结构基底上生长24 h后的VEGF及IL-8分泌量采用ELISA进行检测,VEGF及IL-8在mRNA水平的表达量以实时定量PCR进行评价。实验中成功制备了微柱名义直径为2 μm和4 μm、微柱名义间距为2 μm和7 μm的4种拓扑结构基底。研究发现,SH-SY5Y细胞在2-2 μm (微柱名义直径-名义间距)、4-2 μm、4-7 μm拓扑结构基底上的VEGF和/或IL-8分泌量和表达较之PLLA平面基底上相应值出现上调,而在2-7 μm拓扑结构基底上VEGF及IL-8二者均表现出表达和分泌量大幅度和明显的上调。与在PLLA平面基底上相比,SH-SY5Y细胞在拓扑结构基底上表现出细胞形态 (铺展面积及圆度) 的明显变化,细胞VEGF及IL-8分泌量和表达的上调伴随细胞铺展面积的明显降低。结果表明微柱阵列型拓扑结构是影响SH-SY5Y细胞VEGF、IL-8分泌及表达的重要微环境因素,VEGF和IL-8可能构成SH-SY5Y细胞在拓扑结构基底上生长的重要分泌物标志。     

Glutathione intensifies gliotoxin-induced cytotoxicity in human neuroblastoma SH-SY5Y cells

Gliotoxin is a fungal second metabolite produced by diverse species that can be found in compost, stored crops, moist animal feed and sawdust. The role of glutathione in gliotoxin-induced toxicity was studied in order to elucidate the toxic mechanisms leading to neurite degeneration and cell death in differentiated human neuroblastoma (SH-SY5Y) cells. After 72 h of exposure to gliotoxin, moderate cytotoxicity was induced at 0.1 μmol/L, which was more severe at higher concentrations. A reduction in the number of neurites per cell was also observed. By decreasing the level of intracellular glutathione with l-buthionine-sulfoxamine (BSO) a specific inhibitor of glutathione synthesis, the cytotoxic effect of gliotoxin was significantly attenuated. The gliotoxin-induced cytotoxicity was also slightly reduced by the antioxidant vitamin C. However, the neurite degenerative effect was not altered by BSO, or by vitamin C. A concentration-dependent increase in the ratio between oxidized and reduced forms of glutathione, as well as the total intracellular glutathione levels, was noted after exposure to gliotoxin. The increase of glutathione was also reflected in western blot analyses showing a tendency for the regulatory subunit of γ-glutamylcysteine synthetase to be upregulated. In addition, the activity of glutathione reductase was slightly increased in gliotoxin-exposed cells. These results indicate that glutathione promotes gliotoxin-induced cytotoxicity, probably by reducing the ETP (epipolythiodioxopiperazine) disulfide bridge to the dithiol form.     

Inhibition by 2,4-toluene diisocyanate of the calcium signaling of neuronal nicotinic acetylcholine receptors in human neuroblastoma SH-SY5Y cells

Summary Toluene diisocyanate (TDI) is widely used as a chemical intermediate in the production of polyurethane products such as foams, coatings, and elastomers. In exposed workers, chronic inhalation of TDI has resulted in significant decreases in lung function. TDI-induced asthma is related to its disturbance of acetylcholine in most affected workers but the actions of TDI on nicotinic acetylcholine receptors (nAChR) are unclear. In order to understand the role of TDI acting on nAChR, we used human neuroblastoma SH-SY5Y cells to investigate the effects of TDI on cytosolic free calcium concentration ([Ca ) changes under the stimulation of nAChR. The results showed that TDI was capable of inhibiting the [Ca rise induced by nicotinic ligands, epibatidine, DMPP and nicotine. The inhibition was remained, even increased after chronic treatment of TDI. Our study of TDI acting on human nAChR suggests a possibility that the human nerve system plays some role in the toxicity of TDI in the pulmonary system.     

Rosiglitazone protects human neuroblastoma SH-SY5Y cells against acetaldehyde-induced cytotoxicity

Acetaldehyde, an inhibitor of mitochondrial function, has been widely used as a neurotoxin because it elicits a severe Parkinson's disease-like syndrome with elevation of the intracellular reactive oxygen species level and apoptosis. Rosiglitazone, a peroxisome proliferator-activated receptor-gamma agonist, has been known to show various non-hypoglycemic effects, including anti-inflammatory, anti-atherogenic, and anti-apoptotic. In this study, we investigated the protective effects of rosiglitazone on acetaldehyde-induced apoptosis in human neuroblastoma SH-SY5Y cells and attempted to examine its mechanism. Acetaldehyde-induced apoptosis was moderately reversed by rosiglitazone treatment. Our results suggest that the protective effects of rosiglitazone on acetaldehyde-induced apoptosis may be ascribed to ability to induce the expression of anti-oxidant enzymes and to regulate Bcl-2 and Bax expression. These data indicate that rosiglitazone may provide a useful therapeutic strategy for the prevention of progressive neurodegenerative disease such as Parkinson's disease.     

Mechanism of nitric oxide-induced apoptosis in human neuroblastoma SH-SY5Y cells

We have attempted to elucidate the precise mechanism of nitric oxide (NO)-induced apoptotic neuronal cell death. Enzymatic cleavages of DEVD-AFC, VDVAD-AFC, and LEHD-AFC (specific substrates for caspase-3-like protease (caspase-3 and -7), caspase-2, and caspase-9, respectively) were observed by treatment with NO. Western blot analysis showed that pro-forms of caspase-2, -3, -6, and -7 are decreased during apoptosis. Interestingly, Ac-DEVD-CHO, a caspase-3-like protease inhibitor, blocked not only the decreases in caspase-2 and -7, but also the formation of p17 from p20 in caspase-3 induced by NO, suggesting that caspase-3 exists upstream of caspase-2 and -7. Bongkrekic acid, a potent inhibitor of mitochondrial permeability transition, specifically blocked both the loss of mitochondrial membrane potential and subsequent DNA fragmentation in response to NO. Thus, NO results in neuronal apoptosis through the sequential loss of mitochondrial membrane potential, caspase activation, and degradation of inhibitor of caspase-activated DNase (CAD) (CAD activation).     

Multiple forms of human dopamine beta-hydroxylase in SH-SY5Y neuroblastoma cells

Dopamine beta-hydroxylase exists as three forms in human neuroblastoma (SH-SY5Y) cells. The membrane-bound form of the hydroxylase contains three different species with apparent relative molecular weights of 73,000, 77,000, and 82,000. The intracellular soluble form of dopamine beta-hydroxylase was present as a single species with an apparent molecular weight of 73,000. Pulse-chase experiments showed that membranous dopamine beta-hydroxylase contains two subunit forms of 73,000 and 77,000 after short chase times. The soluble hydroxylase was synthesized as a single species of 73,000 at approximately the same rate as the lower molecular weight species of the membranous enzyme. A constitutively secreted third form of the enzyme with an intermediate apparent molecular weight also incorporated [35S]sulfate, whereas no significant amount of [35S]sulfate was observed in the cellular forms of the enzyme. The [35S]sulfate was incorporated on N-linked oligosaccharides. Approximately 12% of the enzyme is released constitutively within 1 h. These results demonstrate that neuronal cells have the ability to constitutively secrete a specific form of dopamine beta-hydroxylase which may contribute to the levels of this enzyme found in plasma.     

BCL-2 is upregulated in human SH-SY5Y neuroblastoma cells differentiated by overexpression of fibroblast growth factor 1.

Fibroblast growth factor 1 (FGF1) is a multipotent factor in the development and differentiation of the central nervous system. Recent studies in PC12 cells attribute these effects to high endogenous FGF1 expression. To examine the differentiation mechanisms induced by FGF1, we performed studies in SH-SY5Y human neuroblastoma cells. We monitored the impact of FGF1 overexpression in SH-SY5Y either after addition of exogenous FGF1 and heparin or after stable transfection with the FGF1 eukaryotic expression vector. Under both conditions, the FGF1 endogenous rise caused SH-SY5Y cell differentiation with morphological changes (appearance of neuritic extensions), increased GAP-43 gene expression, decreased of N-myc gene expression, and prolonged long-term survival in serum-free media. These modifications were correlated with Bcl-2 upregulation. These results suggest that there is a link between the endogenous FGF1 signaling pathway and Bcl-2 in neuronal survival modulation.     

Antipsychotic drugs increase N-acetylaspartate and N-acetylaspartylglutamate in SH-SY5Y human neuroblastoma cells

N -Acetylaspartate (NAA) and N -acetylaspartylglutamate (NAAG) are related neuronal metabolites associated with the diagnosis and treatment of schizophrenia. NAA is a valuable marker of neuronal viability in magnetic resonance spectroscopy, a technique which has consistently shown NAA levels to be modestly decreased in the brains of schizophrenia patients. However, there are conflicting reports on the changes in brain NAA levels after treatment with antipsychotic drugs, which exert their therapeutic effects in part by blocking dopamine D₂ receptors. NAAG is reported to be an agonist of the metabotropic glutamate 2/3 receptor, which is linked to neurotransmitter release modulation, including glutamate release. Alterations in NAAG metabolism have been implicated in the development of schizophrenia possibly via dysregulation of glutamate neurotransmission. In the present study we have used high performance liquid chromatography to determine the effects of the antipsychotic drugs haloperidol and clozapine on NAA and NAAG levels in SH-SY5Y human neuroblastoma cells, a model system used to test the responses of dopaminergic neurons in vitro . The results indicate that the antipsychotic drugs haloperidol and clozapine increase both NAA and NAAG levels in SH-SY5Y cells in a dose and time dependant manner, providing evidence that NAA and NAAG metabolism in neurons is responsive to antipsychotic drug treatment.     

Inhibition of insulin-like growth factor-1 receptor and IRS-2 signaling by ethanol in SH-SY5Y neuroblastoma cells

The effect of ethanol on insulin-like growth factor-1 (IGF-I)-mediated signal transduction and functional activation in neuronal cells was examined. In human SH-SY5Y neuroblastoma cells, ethanol inhibited tyrosine autophosphorylation of the IGF-I receptor. This corresponded to the inhibition of IGF-I-induced phosphorylation of p42/p44 mitogen-activated/extracellular signal-regulated protein kinase (MAPK) by ethanol. Insulin-related substrate-2 (IRS-2) and focal adhesion kinase phosphorylation were reduced in the presence of ethanol, which corresponded to the prevention of lamellipodia formation (30 min). By contrast, ethanol had no effect on Shc phosphorylation when measured up to 1 h, and did not affect the association of Grb-2 with Shc. Neurite formation at 24 h was similarly unaffected by ethanol. The data indicate that the IGF-I receptor is a target for ethanol in SH-SY5Y cells However, there is diversity in the sensitivity of signaling elements within the IGF-I receptor tyrosine kinase signaling cascades to ethanol, which can be related to the inhibition of specific functional events in neuronal activation.     

Vanadate protects human neuroblastoma SH-SY5Y cells against peroxynitrite-induced cell death

We investigated the effect of vanadate, a tyrosine phosphatase inhibitor, on cell death induced by peroxynitrite in human neuroblastoma SH-SY5Y cells. Vanadate prevented cell death induced by 3-morpholinosydnonimine (SIN-1), a peroxynitrite donor; whereas SIN-1-induced cell death was not prevented by neither okadaic acid, an inhibitor of serine/threonine phosphatases 1 and 2A, nor cyclosporin A, an inhibitor of serine/threonine phosphatase 2B. Vanadate did not prevent cell death induced by N-ethyl-2-(1-ethyl-hydroxy-2-nitrosohydrazino)-ethanamine, a nitric oxide donor. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), did not block the protective effect of vanadate, suggesting that the protective effect of vanadate is independent on PI3-kinase. Vanadate increased tyrosine phosphorylation of several proteins including the focal adhesion protein p130 Crk-associated substrate (p130(cas)). By the treatment with SIN-1, the endogenous association of p130(cas) and Crk was disrupted, and the association was restored by vanadate treatment. These results suggest that disruption of tyrosine phosphorylation signaling may be critical for peroxynitrite-induced cell death, and that vanadate prevents cell death at least in part through the enhancement in tyrosine phosphorylation of the proteins including p130(cas).     

Increased epidermal growth factor receptor in multidrug-resistant human neuroblastoma cells

Multidrug-resistant human neuroblastoma cell lines obtained by selection with vincristine or actinomycin D from two independent clonal lines, SH-SY5Y and MC-IXC, have 3- to 30-fold more cell surface epidermal growth factor (EGF) receptors than the drug-sensitive parental cells as indicated by EGF binding assays and immunoprecipitation, affinity-labeling, and phosphorylation studies. Reversion to drug sensitivity in one line was accompanied by a return to the parental level of EGF receptor. SH-EP cells, a clone derived from the same neuroblastoma cell line as SH-SY5Y but which displays melanocyte rather than neuronal lineage markers, also express significantly more EGF receptor than SH-SY5Y cells. By nucleic acid hybridization analysis with a molecularly cloned probe, increased receptor level in multidrug-resistant cells was shown to be the result of higher levels of EGF receptor mRNA in drug-resistant than in drug-sensitive cells. The increased steady state amount of specific RNA did not result from amplification of receptor-encoding genes. A small difference was observed in the electrophoretic mobility under denaturing conditions of EGF receptor immunoprecipitated from drug-resistant and drug-sensitive cells. Quantitative and qualitative modulation of the EGF receptor might reflect alterations in the transformation and/or differentiation phenotype of the resistant cells or might result from unknown selective pressures associated with the development of multidrug resistance.     

Transport and toxic mechanism for aluminum citrate in human neuroblastoma SH-SY5Y cells

Aluminum (Al) is thought to be a risk factor for neurodegenerative disorders, but the molecular mechanism has been not clarified yet. In this study, we examined how a transport system handled transport of Al citrate, the major Al species in brain, and effect of Al citrate treatment on expression of the transporter and on susceptibility to oxidative stress in human neuroblastoma SH-SY5Y cells. Uptake of Al citrate by the cells was temperature- and concentration-dependent, and inwardly-directed Na(+)-gradient-independent. Simultaneous application and preloading of L-cystine or L-glutamate inhibited and stimulated, respectively, the Al citrate uptake by SH-SY5Y cells, demonstrating kinetically that Na(+)-independent L-cystine/L-glutamate exchanger, system Xc(-), is involved in its uptake. When the cells were treated with Al citrate, but not citrate, for 2 weeks, but not a day, the expression of the transporter was decreased. Although the cell viability and glutathione content of the cells were not altered by the treatment with Al citrate alone, the number of dead cells among the Al citrate-treated cells increased on exposure to oxidative stress caused by a glucose deprivation/reperfusion treatment. These findings demonstrate that Al citrate is a substrate for system Xc(-), and that chronic treatment with Al citrate causes downregulation of the transporter and increases the vulnerability of the cells to oxidative stress without a direct effect on the viability or GSH content.