Chronic Ethanol Treatment Decreases [3H]Epibatidine and [3H]Nicotine Binding and Differentially Regulates mRNA Levels of Nicotinic Acetylcholine Receptor Subunits Expressed in M10 and SH-SY5Y Neuroblastoma Cells

Abstract: For a study of the underlying mechanisms of a possible interaction between ethanol and nicotinic receptors during ethanol dependence, the aim of this work was to investigate the effect of chronic ethanol exposure on nicotinic receptor subtypes in a transfected fibroblast cell line (M10 cells) stably expressing α4β2 nicotinic receptor subtype and an SH-SY5Y neuroblastoma cell line expressing α3, α5, α7, β2, and β4 nicotinic acetylcholine receptor (nAChR) subunits. A significant dose-related decrease (−30–80%) in number of [³H]nicotine binding sites was observed in ethanol-treated (25–240 m M ) compared with untreated M10 cells. Similarly, 4-day treatment with ethanol in concentrations relevant to chronic alcoholism (100 m M ) decreased the number of nicotinic receptor binding sites in the SH-SY5Y cells when measured using [³H]epibatidine. When M10 cells were chronically treated with nicotine, ethanol partly inhibited the up-regulation of nicotinic receptors when present in the cells together with nicotine. Chronic treatment for 4 days with 100 m M ethanol significantly decreased the mRNA level for the α3 nAChR subunit (−39%), while the mRNA levels for the α7 (+30%) and α4 (+22%) subunits were significantly increased. Chronic ethanol treatment did not affect the mRNA levels for the β2 nAChR subunit. Changes in the levels of nAChR protein and mRNA may have adaptive significance and be involved in the development of dependence, tolerance, and addiction to chronic ethanol and nicotine exposure. They also may be targets for therapeutic strategies in the treatment of ethanol and nicotine dependence.     

Involvement of Receptor Cycling and Receptor Reserve in Resensitization of Muscarinic Responses in SH-SY5Y Human Neuroblastoma Cells

Abstract: Preexposure of SH-SY5Y cells to the muscarinic agonist carbachol caused a rapid desensitization of subsequent carbachol-stimulated intracellular Ca²⁺ responses and a slower decrease in the number of receptors at the plasma membrane. Desensitization (to 30% of the control response) was maximal after 1 min of exposure to agonist, whereas the number of cell surface receptors reached a minimum (33% of control) only after 5 min. Following agonist washout, the recovery of response was complete within 12 min, whereas the recovery of surface receptor number reached a plateau at 65% of control after 30 min. Treatment with inhibitors of endocytosis (concanavalin A) or recycling (nigericin) did not affect rapid desensitization but did decrease resensitization, suggesting that receptor cycling is involved in resensitization. Experiments with the irreversible antagonist propylbenzilylcholine mustard demonstrated that the receptor reserve for the Ca²⁺ response to 1 m M carbachol is ∼50%. Removal of this receptor reserve led to a decrease in the rate of resensitization. We propose that the existence of a receptor reserve might explain the poor correlation between functional response and surface receptor number, and that one of its roles might be to permit rapid resensitization after a significant agonist-induced decrease in surface receptor number. The purpose of receptor cycling might be to allow dephosphorylation (and reactivation) of receptors that have become phosphorylated (and inactivated) in response to agonist stimulation, because the protein phosphatase inhibitor calyculin A significantly reduced resensitization.     

Retinoic Acid-Induced Differentiation of Human Neuroblastoma SH-SY5Y Cells Is Associated with Changes in the Abundance of G Proteins

Abstract: Western blot analysis, using subtype-specific anti-G protein antibodies, revealed the presence of the following G protein subunits in human neuroblastoma SH- SY5Y cells: Gaα, G_iα1, G_jα2, G_cα, G_zα, and Gβ. Differentiation of the cells by all-trans-retinoic acid (RA) treatment (10 μmol/L; 6 days) caused substantial alterations in the abundance of distinct G protein subunits. Concomitant with an enhanced expression of μ-opioid binding sites, the levels of the inhibitory G proteins G_iα1 and G_jα1 were found to be significantly increased. This coordinate up-reg- ulation is accompanied by functional changes in μ-opioid receptor-stimulated Iow-K_m GTPase, μ-receptor-mediated adenylate cyclase inhibition, and receptor-independent guanosine 5′-(βγ-imido)triphosphate [Gpp(NH)p; 10 nmol/ L]-mediated attenuation of adenylate cyclase activity. In contrast, increased levels of inhibitory G proteins had no effect on muscarinic cholinergic receptor-mediated adenylate cyclase inhibition. With respect to stimulatory receptor systems, a reciprocal regulation was observed for prosta- glandin E₁ (PGE₁) receptors and G_sα, the G protein subunit activating adenylate cyclase. RA treatment of SH-SY5Y cells increases both the number of PGE₁ binding sites and PGE₁ stimulated adenylate cyclase activity, but significantly reduced amounts of G_zα were found. This down- regulation is paralleled by a decrease in the stimulatory activity of G_zα as assessed in S49 cyc- reconstitution assays. However, the reduction in G_aα levels had no effect on both intrinsic and receptor-independent-activated [Gpp(NH)p or forskolin; 100 μtmol/L each] adenylate cyclase, suggesting that the amount of G_zα is in excess over the functional capacity of adenylate cyclase in SH-SY5Y cell membranes. Additional quantitative changes were found for G_zα, G_cα, and Gβ subunits. In contrast, neuronal differentiation in the presence of 12-O-tetradecanoylphor- bol 13-acetate (16 nmol/L; 6 days) failed to affect G protein abundance. Our results provide evidence for a specific RA effect on the abundance of distinct G protein sub- units in human SH-SY5Y neuroblastoma cells. These alterations might contribute to functional changes in transmembrane signaling pathways associated with RA-in- duced neuronal differentiation of the cells.     

Nicotinic Receptor-Mediated Release of Noradrenaline in the Human Neuroblastoma SH-SY5Y

Abstract: Dimethylphenylpiperazinium iodide (a nicotinic agonist) evokes noradrenaline release from human neuroblastoma SH-SY5Y cells that have been pretreated with 12- O -tetradecanoylphorbol 13-acetate for 8 min. This effect of dimethylphenylpiperazinium iodide was inhibited by 1 μ M mecamylamine but not by 1 μ M atropine, which suggests that SH-SY5Y cells express nicotinic receptors coupled to the release of noradrenaline. Dimethylphenylpiperazinium iodide-evoked release was enhanced by 5 μ M Bay K 8644 (an L-type calcium agonist) and inhibited by 1 μ M nifedipine. Dimethylphenylpiperazinium iodide depolarised SH-SY5Y cells and enhanced the level of intracellular calcium in cells loaded with fura 2. The effects of dimethylphenylpiperazinium iodide on noradrenaline release, depolarisation, and intracellular calcium levels were all inhibited by 1 μ M desmethylimipramine. The results of this study show that nicotinic receptors in SH-SY5Y cells stimulate noradrenaline release by activation of L-type calcium channels.     

Aluminium Inhibits Muscarinic Agonist-Induced Inositol 1,4,5-Trisphosphate Production and Calcium Mobilization in Permeabilized SH-SY5Y Human Neuroblastoma Cells

Abstract: The effects of aluminium (as Al³⁺) on carbachol-induced inositol 1,4,5-trisphosphate (lnsP₃) production arid Ca²⁺ mobilisation were assessed in electropermeabilised human SH-SY5Y neuroblastoma cells. Al³⁺ had no effect on lnsP₃-induced Ca²⁺ release but appreciably reduced carbachol-induced Ca²⁺ release (lC₅₀ of ∼90 μ M ). Aβ³⁺ also inhibited lnsP₃ production (lC₆₀ of ∼15 μ M ). Dimethyl hydroxypyridin-4-one, a potent Al³⁺ chelator (K₅= 31), at 100 μ M was able to abort and reverse the effects of Al³⁺ on both Ca²⁺ release and lnsP₃ production. These data suggest that, in permeabilised cells, the effect of Al³⁺ on the phosphoinositide-mediated signalling pathway is at the level of phosphatidylinositol 4,5-bisphosphate hydrolysis. This may reflect interference with receptor-G protein-phospholipase C coupling or an interaction with phosphatidylinositol 4,5-bisphosphate.     

PXR激活在拮抗SH-SY5Y细胞凋亡中的作用

人神经母细胞瘤细胞SH-SY5Y细胞可以表达神经元特异性的酪氨酸羟化酶、多巴胺-β-羟化酶以及多巴胺转运体等,因此可用于建立帕金森病的体外模型。虽然帕金森综合症发病的确切机制至今尚不清楚,但众多的病理学资料证实该病患者存在中脑黑质多巴胺能神经元的凋亡。自由基、兴奋性     

Regulation of Muscarinic Agonist-Induced Activation of Phosphoinositidase C in Electrically Permeabilized SH-SY5Y Human Neuroblastoma Cells by Guanine Nucleotides

myo-[3H]Inositol-labelled SH-SY5Y cells were permeabilized with electrical discharges. 3H-Inositol phosphate formation in cells shown to be fully permeable was stimulated by the muscarinic agonist carbachol, by guanosine 5'-(gamma-thio)triphosphate [GTP(S)], and by guanosine 5'-(beta gamma-imido)diphosphate (GppNHp). Synergism was observed on coincubation of these GTP analogues with carbachol. GTP was also stimulatory and guanosine 5'-(beta-thio)diphosphate was inhibitory in the presence of agonist. Atropine blocked the effects of carbachol. Stimulation by GTP(S) (0.1 mM) occurred after a 1-2-min lag, whereas Ca2+ (0.5 mM), carbachol (1 mM), and carbachol plus GTP(S) stimulated without delay. The effects of carbachol plus GTP(S) but not those of Ca2+ were inhibited by spermine (4 mM). Accumulation of 3H-inositol phosphates was enhanced by Li+ (4 mM) only in intact cells. In intact or permeabilized cells, the "partial" agonist arecoline was maximally 40-50% as efficacious as carbachol. In permeabilized cells, the maximal effects of carbachol and arecoline were enhanced 2.8- and 5.3-fold, respectively, by 0.1 mM GTP(S), but only the EC50 for carbachol was substantially reduced. The binding affinity of carbachol but not that of arecoline in permeabilized cells was significantly reduced by 0.1 mM GppNHp. These data indicate that a guanine nucleotide-binding regulatory protein is involved in coupling muscarinic receptors to phosphoinositidase C in SH-SY5Y cells and that the activity of this protein influences the relationship between receptor occupation and phosphoinositide response.     

Involvement of Matrix Metalloproteinases on the Inhibition of Cells Invasion and Migration by Emodin in Human Neuroblastoma SH-SY5Y Cells

Emodin (1,3,8-trihydroxy-6-methylanthaquinone), an active component present in the root and rhizome of Rheum palmatum L. (Polygonaceae) has anti-bacterial, anti-tumor, diuretic and vasorelaxant effects. However, its mechanism of action on the cell migration and invasion of human neuroblastoma cancer SH-SY5Y cells is not fully understood. In this study, firstly, the effects of emodin on the percentage of viable cells were examined by using MTT assay and it was found that emodin induced dose-and time-dependent inhibition in human neuroblastoma SH-SY5Y cells. Second, the effects of emodin on the migration and invasion of SH-SY5Y cells were examined by using wound assay and matrigel counting and the results showed that emodin suppressed the migration and invasion of SH-SY5Y cells. Third, we examined the effect of emodin on the levels of associated proteins by using Western blotting and the results indicated that emodin inhibited the levels of GRB2, RhoA, HIF-1α, VEGF, FAK, iNOS, COX2, p-p38, p-c-jun, MMP2, MMP9 and MMP7 but promoted the levels of PKC, PI3K, MEKK3 and NF-κB p65 that led to the inhibition of migration and invasion of SH-SY5Y cells in vitro.     

胡椒碱及其衍生物对SH-SY5Y细胞的保护作用研究

对胡椒碱进行结构改造,合成了4个胡椒碱衍生物。以高浓度皮质酮(corticosterone)模拟抑郁及焦虑症神经细胞损伤状态。对包括胡椒碱在内的5个化合物进行了SH-SY5Y细胞保护作用的研究。结果表明:胡椒碱、5-(3,4-methylenedioxy phenyl)-2E,4E-pentadienoic acid n-propylamine amide(2)以及5-(3,4-methylenedioxyphenyl)-2E,4E-pentadienoic acid N-methyl piperazine amide(4)对SH-SY5Y细胞有较好的保护作用。其中化合物2对SH-SY5Y细胞具有明显和持久的保护作用。     

Carbachol Inhibits Insulin-Stimulated Phosphatidylinositol 3-Kinase Activity in SH-SY5Y Neuroblastoma Cells

Abstract: SH-SY5Y human neuroblastoma cells express muscarinic M₃ receptors as well as insulin receptors, thus offering the opportunity to investigate possible cross-talk following activation of two distinct intracellular signal transduction pathways that convert the precursor phosphatidylinositol (PI) to its 3′ phosphate or its 4′ phosphate, respectively. In this study, the effect of carbachol on insulin-stimulated PI 3-kinase (PI3K) activity was examined in SH-SY5Y cells. Insulin addition to the cell medium induced a 10–26-fold increase in anti-phosphotyrosine-immunoprecipitable PI3K activity. Preincubation with 1 mM carbachol inhibited the insulin-stimulated PI3K activity in a time-dependent manner, with half-maximal and maximal inhibition times of 4 and 15 min, respectively. Atropine blocked the inhibitory effect of carbachol. Although carbachol did not change the amount of 85-kDa subunit protein regulatory unit associated with tyrosine-phosphorylated proteins, either in control or in insulin-stimulated cells, it appears to decrease the amount of associated 110-kDa catalytic subunit protein in the latter instance. Because PI3K activity from SH-SY5Y cells has been shown to be inhibited in vitro in the presence of cytidine diphosphodiacylglycerol (CDP-DAG) or phosphatidate (PA), we examined the presence of these lipids in SH-SY5Y cells that had been treated with carbachol. Formation of both lipids was increased in a time-dependent manner following carbachol addition, and their increased levels are proposed to account for the observed in vivo inhibition of PI3K. Addition of the cell-permeable homologue didecanoyl-CDP-DAG to intact cells inhibited insulin-stimulated PI3K activity up to 75%, with an IC₅₀ of 0.5 µM, a result that further supports a proposed lipid-mediated inhibition of PI3K. Exogenously added didecanoyl-PA, however, did not affect PI3K activity. The possibility that stimulation of the PI 4-kinase-mediated signal transduction pathway leads to down-regulation of the PI3K-mediated signal transduction pathway in vivo, via inhibition of PI3K by CDP-DAG or by other consequences of phosphoinositidase C-linked receptor activation, is discussed.     

Characterization of CPP32-Like Protease Activity Following Apoptotic Challenge in SH-SY5Y Neuroblastoma Cells

Abstract: We characterized the activation of interleukin-1β-converting enzyme (ICE)-like proteases (caspases) in human neuroblastoma cells (SH-SY5Y) following challenge with staurosporine, an established agent known to induce apoptosis. Time course analyses of lactate dehydrogenase release detected a significant increase in cell death as early as 6 h that continued at least until 24 h following staurosporine treatment. Western blot analyses using anti-poly(ADP-ribose) polymerase (anti-PARP) and anti-CPP32 antibodies revealed proteolytic processing of CPP32 (an ICE homologue) as well as fragmentation of PARP as early as 3 h following staurosporine challenge. Furthermore, the hydrolysis of the CPP32 substrate acetyl-DEVD-7-amido-4-methylcoumarin was detected as early as 3 h and became maximal at 6 h after staurosporine challenge, suggesting a delayed and sustained period of CPP32-like activation. In addition, we used the first immunohistochemical examination of CPP32 and PARP in cells following an apoptotic challenge. The localization of CPP32 in untreated SH-SY5Y cells was exclusively restricted to the cytoplasm. Following staurosporine challenge there was a condensing of CPP32 immunofluorescence from the cytoplasm to a region adjacent to the plasma membrane. In contrast, PARP immunofluorescence was evenly distributed in the nucleus in untreated SH-SY5Y cells and on staurosporine challenge was found to be associated with condensed chromatin. It is important that a pan ICE inhibitor [carbobenzoxy-Asp-CH₂OC(O)-2,6-dichlorobenzene] was able to attenuate lactate dehydrogenase release and PARP and CPP32 cleavage and altered immunohistochemical staining patterns for PARP and CPP32 following staurosporine challenge.     

Role of 1,25-Dihydroxyvitamin D3 and Extracellular Calcium in the Regulation of Proliferation in Cultured SH-SY5Y Human Neuroblastoma Cells

This study examines the effects of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] on SH-SY5Y human neuroblastoma cells cultured in the presence of medium containing varying concentrations of calcium (0.1, 0.9, 1.4, 1.8 mM). Pyruvate kinase activity was assayed in SH-SY5Y cells incubated in variable calcium medium with or without 1, 10 or 100 nM 1,25(OH)₂D₃ for 48 h. The enzyme levels showed a significant increase in comparison with control, when the cells were incubated with 100 nM hormone in the presence of 0.1 mM calcium, while pyruvate kinase activity decreased, when the cells were treated with 100 nM 1,25(OH)₂D₃ in the presence of 1.8 mM calcium. The proliferative activity of SH-SY5Y was dependent on the extracellular concentration of calcium, being the highest at 1.8 mM calcium and completely absent at 0.1 mM calcium. In the presence of 1,25(OH)₂D₃, at the three concentrations used and after 48 h incubation, a significant decrease in cell number was always observed, without a direct correlation between 1,25(OH)₂D₃ effect and calcium concentration in the medium. [³H]Thymidine incorporation in SH-SY5Y cells significantly increased in comparison with control, when the 48 h incubation with 1, 10 or 100 nM 1,25(OH)₂D₃ was carried out in the presence of 0.1 mM calcium, while, at the other calcium concentrations, the hormone did not cause any significant change in this parameter. The treatment of SH-SY5Y cells with 1 nM 1,25(OH)₂D₃ for 48 h did not affect cell morphology, when 0.1 mM calcium was present, while, in the medium containing 1.8 mM calcium, the treated cells showed a slight trend to differentiation. The differentiating effect of 10 M all-trans retinoic acid, even if incomplete after 48 h treatment, was only observed in the cultures grown in 1.8 mM calcium, in comparison with those maintained in 0.1 mM calcium.     

Potassium- and Carbachol-Evoked Release of [3H]Noradrenaline from Human Neuroblastoma Cells, SH-SY5Y

The human neuroblastoma clone SH-SY5Y expresses potassium-, carbachol-, and calcium ionophore A23187-evoked, calcium-dependent release of [3H]noradrenaline. Release in response to carbachol and potassium was greater than additive. Atropine (Ki = 0.33 nM), hexahydrosiladifenidol (Ki = 18 nM), and pirenzepine (Ki = 1,183 nM) completely inhibited the carbachol-evoked noradrenaline release, an order of potency suggesting that an M3 receptor was linked to release. In contrast, noradrenaline release was only partially inhibited by the M2-selective antagonists methoctramine (10(-4) M) and AFDX-116 (10(-4) M), by approximately 14 and 46%, respectively. The nicotinic antagonist d-tubocurarine (10(-4) M) resulted in a partial inhibition of release, a finding suggesting that a nicotinic receptor may also be involved. SH-SY5Y provides a suitable cell line in which to study the biochemical mechanisms underlying the cholinergic receptor regulation of noradrenaline release.     

Neuroprotective Effects of Ugonin K on Hydrogen Peroxide-Induced Cell Death in Human Neuroblastoma SH-SY5Y Cells

Oxidative stress plays an important role in the pathological processes of various neurodegenerative diseases. Ugonin K, a flavonoid isolated from the rhizomes of Helminthostachys zeylanica, possesses potent antioxidant property. In this study, we investigate the neuroprotective effects of ugonin K on hydrogen peroxide (H₂O₂)-induced apoptosis in SH-SY5Y cells. Incubation of SH-SY5Y cells with H₂O₂ for 24 h induced cell death measured with MTT assay. Hoechst 33258 staining confirmed that the reduced cell viability by H₂O₂ was due to apoptosis. In addition, H₂O₂ increased the expression of 17-kDa cleaved fragment of caspase-3 which could be reversed by pretreatment with ugonin K. Pretreatment with ugonin K attenuated H₂O₂-induced cell death in a dose-dependent manner. Neuroprotective effect of ugonin K was abolished by ERK and PI3K inhibitors. Pretreatment with JNK kinase and p38 MAPK inhibitors had no effect on ugonin K-mediated protection against H₂O₂-induced apoptosis. Western blotting with anti-phospho-ERK1/2 and anti-phospho-Akt (pS473) antibodies showed that ugonin K increased both ERK1/2 and Akt phosphorylation. These results suggest that ugonin K by activation of ERK1/2 and PI3K/Akt signal pathways protects SH-SY5Y cells from H₂O₂-induced apoptosis.     

Ascorbate Transport and Recycling by SH-SY5Y Neuroblastoma Cells: Response to Glutamate Toxicity

Neurons maintain relatively high intracellular concentrations of vitamin C, or ascorbic acid. In this work we studied the mechanisms by which neuronal cells in culture transport and maintain ascorbate, as well as how this system responds to oxidant stress induced by glutamate. Cultured SH-SY5Y neuroblastoma cells took up ascorbate, achieving steady-state intracellular concentrations of 6 mM and higher at extracellular concentrations of 200 μM and greater. This gradient was generated by relatively high affinity sodium-dependent ascorbate transport (K _m of 113 μM). Ascorbate was also recycled from dehydroascorbate, the reduction of which was dependent on GSH, but not on d-glucose. Glutamate in concentrations up to 2 mM caused an acute concentration-dependent efflux of ascorbate from the cells, which was prevented by the anion channel blocker 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid. Intracellular ascorbate did not affect radiolabeled glutamate uptake, showing absence of heteroexchange.     

Occurrence of Two Types of Secretory Vesicles in the Human Neuroblastoma SH-SY5Y

Abstract: Western blot analysis showed that the human neuroblastoma SH-SY5Y expresses the proteins synaptotagmin I, synaptobrevin, synapsin I, rab3a, syntaxin, SNAP-25, NSF, α-SNAP, and munc-18, which have been implicated in the movement, docking, and fusion of vesicles during exocytosis from other neuroendocrine cells. The subcellular localization of secretogranins I and II, synaptotagmin I, neuropeptide Y, rab3a, synaptobrevin, synaptophysin, and syntaxin was investigated by immunofluorescence microscopy and revealed punctate staining patterns characteristic of secretory vesicles. The comigration of noradrenaline, secretogranin II, and dopamine-β-hydroxylase on sucrose-D₂O gradient fractions indicates the presence of a population of noradrenaline-containing large dense-cored vesicles (LDCVs). In addition, a lighter vesicle population is also present that does not appear to be noradrenergic and contains a 48-kDa synaptophysin antigen absent from the large dense-cored vesicles. Immunocytochemical experiments show that not all of the vesicles that express synaptotagmin I contain secretogranin II. Thus, our studies suggest that two types of vesicle are present in SH-SY5Y cells, one of which, the LDCVs, contains noradrenaline. These findings confirm our previous studies suggesting that depolarization-evoked release of noradrenaline from SH-SY5Y occurs by LDCV exocytosis. This enhances the value of SH-SY5Y as a cell line in which to study the mechanism by which noradrenaline release is regulated.     

Reduction of Muscarinic Receptor Density and of Guanine Nucleotide-Stimulated Phosphoinositide Hydrolysis in Human SH-SY5Y Neuroblastoma Cells Following Long-Term Treatment with 12-O-Tetradecanoylphorbol 13-Acetate or Mezerein

The actions of tumor promoters on the coupling of muscarinic receptors to the hydrolysis of inositol lipids and the generation of Ca2+ signals were examined in the human neuroblastoma SH-SY5Y cell line. Pretreatment of SH-SY5Y cells with 50 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) for 5 days resulted in neuronal differentiation, a 28% decrease in both N-[3H]methylscopolamine and [3H]-scopolamine binding, and a significantly larger reduction (48%) in agonist-stimulated 3H-inositol phosphate generation. Whereas mezerein could mimic the effects produced by TPA, the biologically inactive 4 alpha-phorbol 12,13-didecanoate was without effect on both antagonist binding and agonist-stimulated phosphoinositide (PPI) turnover. A decline (approximately 50%) in the agonist-mediated rise in cytoplasmic Ca2+ and a substantial loss of protein kinase C activity also were observed following pretreatment with TPA or mezerein. The ability of fluoride, an agent capable of direct activation of guanine nucleotide binding proteins, to stimulate 3H-inositol phosphate release was significantly reduced in SH-SY5Y cells treated with these agents. Furthermore, pretreatment of SH-SY5Y neuroblastoma cells with TPA or mezerein impaired 3H-inositol phosphate formation induced by the addition of either guanosine 5'-O-(3-thiotriphosphate) or carbamylcholine to digitonin-permeabilized cells, but not that elicited by the addition of 2 mM CaCl2. Although cells cultured in the presence of serum-free media also exhibited neuronal differentiation, no significant alteration in either muscarinic receptor number or agonist-stimulated PPI hydrolysis was observed. The results suggest that TPA and mezerein decrease agonist-stimulated PPI hydrolysis and Ca2+ signaling in SH-SY5Y cells not only by a reduction in muscarinic receptor number but also through an inhibition of guanine nucleotide-stimulated PPI turnover.     

热量限制对SH-SY5Y细胞氧化损伤的影响

目的观察热量限制培养条件下,SH-SY5Y细胞抗氧化应激损伤的能力。方法建立过氧化氢诱导的SH-SY5Y细胞损伤模型。体外培养SH-SY5Y细胞,分为对照组、损伤组（50、100、250、500、1 000μmol/L H2O2）、低糖组（2 g/L）、低糖＋损伤组,进行细胞形态观察、测定各组细胞的噻唑蓝（MTT）代谢率、乳酸脱氢酶（LDH）漏出率。结果与对照组比较,（50、100、250、500、1 000）μmol/L H2O2损伤1 h后MTT代谢率测定细胞活力,50μmol/L组与对照组比较差异无统计学意义（P〉0.05）;其他组与对照组比较,随着H2O2浓度的增加,细胞活力呈递减趋势,差异具有显著性（P〈0.01）;选定250μmol/L H2O2组为损伤应激源。用低糖预处理细胞24 h,给与250μmol/L H2O2损伤1 h后测定MTT代谢率显示,与对照组比较,损伤组活力明显下降,低糖组活力上升（P〈0.01）;与损伤组比较,低糖＋损伤组活力明显上升（p〈0.01）;继续培养至7 h发现,与对照组比较,低糖组活力上升（P〈0.01）;与损伤组比较,低糖＋损伤组活力明显上升（P〈0.01）。进一步检测LDH漏出率显示,损伤1 h后结果显示,与对照组比较,损伤组漏出率明显增加（P〈0.05）,低糖组漏出率稍有减少（P〉0.05）;与损伤组比较,低糖＋损伤组漏出率明显减少（P〈0.01）;继续培养7h显示,低糖7h组与低糖1 h组比较,漏出稍有增多（P〉0.05）,低糖＋损伤组7 h组与低糖＋损伤组1 h比较漏出率稍有增加（P〈0.05）;细胞形态学观察显示,未加损伤之前,低糖组的细胞形态,与对照组比较无明显改变。加入损伤药物1h后的细胞形态与对照组比较无明显改变。加入损伤药物7 h后的细胞形态,低糖组和对照组细胞突起伸展良好细长,损伤组可见细胞数目明显减少,死细胞多,突起回缩,细胞明显变圆,贴壁性不好,透光性差。结论热量限制能提高神经细胞的抗氧化应激能力,增加细胞生存率,降低死亡率。     

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.