Gangliosides Inhibit Platelet-Derived Growth Factor-Stimulated Receptor Dimerization in Human Glioma U-1242MG and Swiss 3T3 Cells

Abstract: We previously showed that gangliosides inhibit DNA synthesis in Swiss 3T3 cells stimulated with platelet-derived growth factor (PDGF) in a dose-responsive manner. This correlated with the inhibitory effects of several gangliosides (except GM3) on tyrosine phosphorylation of the PDGF receptor (PDGFR). [³⁵S]Methionine-labeled Swiss 3T3 cells were incubated either with or without gangliosides and stimulated with PDGF, and proteins were cross-linked with bis(sulfosuccinimidyl) suberate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that two protein bands (170 and 350 kDa) were specifically immunoprecipitated with an anti-PDGFR antibody. Using both Swiss 3T3 and human glioma U-1242MG cells, western blots with anti-PDGFR and anti-phosphotyrosine antibodies confirmed that these bands were the PDGFR monomer and dimer, respectively, and that phosphotyrosine was present in these bands only after cells were stimulated with PDGF. Of the gangliosides tested, GM1, GM2, GD1a, GD1b, GD3, and GT1b, but not GM3, inhibited the formation of the 350-kDa band. These results demonstrate that all gangliosides tested, except GM3, probably inhibit PDGF-mediated growth by preventing dimerization of PDGFR monomers. Loss of more complex gangliosides in human gliomas would permit unregulated activation of the PDGFR, contributing to uncontrolled growth stimulation. We propose that ganglioside inhibition of receptor dimerization is a novel mechanism for regulating and coordinating several trophic factor-mediated cell functions.     

Suramin Induces Phosphorylation of the High-Affinity Nerve Growth Factor Receptor in PC12 Cells and Dorsal Root Ganglion Neurons

Abstract: Suramin is a polysulfonated naphthylurea with demonstrated antineoplastic activity. Toxicity includes adrenal insufficiency and peripheral neuropathy. Although the mechanism of antitumor activity is unknown, inhibition of binding of growth factors to their receptors has been suggested. Growth factors inhibited by suramin include platelet-derived growth factor, fibroblast growth factor, transforming growth factor, epidermal growth factor, insulin-like growth factor, and nerve growth factor (NGF). In these studies, suramin was shown to be cytotoxic to PC12 cells in a dose-dependent manner. At lower doses and in surviving cells, we observed the induction of neurite outgrowth. To determine the mechanism of suramin-induced neurite outgrowth, PC12 cells were exposed to suramin and/or NGF for various time periods and treated cells were analyzed, by western blot analysis, for expression of tyrosine phosphoproteins. There was a similarity in the pattern of tyrosine-phosphorylated proteins in PC12 cells stimulated with suramin or NGF. Of particular interest was the rapid phosphorylation (by 1 min) of the high-affinity NGF (TrkA) receptor. Activation of other members of the signal-transduction cascade (Shc, p21 ^ras , Raf-1, ERK-1) revealed similar phosphorylation levels induced by suramin and NGF. Parallel studies were performed in rat dorsal root ganglion cultures; suramin potentiated neurite outgrowth and activated the NGF receptor on these cells. This finding of specific patterns of tyrosine phosphorylation of cellular proteins in response to suramin treatment demonstrated that suramin is a partial agonist for the NGF receptor in both PC12 cells and dorsal root ganglion neurons.     

K-252a Induces Tyrosine Phosphorylation of the Focal Adhesion Kinase and Neurite Outgrowth in Human Neuroblastoma SH-SY5Y Cells

Abstract: The protein kinase inhibitor K-252a has been shown to promote cholinergic activity in cultures of rat spinal cord and neuronal survival in chick dorsal root ganglion cultures. To determine the mechanism by which K-252a acts as a neurotrophic factor, we examined the effects of this molecule on a human neuroblastoma cell line, SH-SY5Y. K-252a induced neurite outgrowth in a dose-dependent manner. Coincident with neurite outgrowth was the early tyrosine phosphorylation of 125- and 140-kDa proteins. The phosphorylation events were independent of protein kinase C inhibition because down-regulation of protein kinase C by long-term treatment with phorbol ester did not prevent K-252a-induced tyrosine phosphorylation. Similarly, the protein kinase C inhibitors H7, GF-109203X, and calphostin C did not induce the phosphorylation. We have identified one of the phosphosubstrates as the pp125 focal adhesion protein tyrosine kinase (Fak). Induction of phosphorylation coincided with increased Fak activity and appeared to be independent of ligand/integrin interaction. The induction of Fak phosphorylation by K-252a was also observed in LA-N-5 cells and primary cultures of rat embryonic striatal cells but not in PC12 cells. The protein kinase C-independent induction of tyrosine phosphorylation and the identification of Fak as a substrate of K-252a-induced tyrosine kinase activity suggest that this compound mediates neurotrophic effects through a novel signaling pathway.     

Density-Dependent Changes in Gangliosides and Sialidase Activity of Murine Neuroblastoma Cells

Abstract: Density-dependent changes in ganglioside composition, Vibrio cholerae neuraminidase (VCN)-susceptible sialyl residues, and membrane- associated sialidase activity were determined for the cholinergic murine neuroblastoma cell line S20Y. A decrease in total ganglioside sialic acid and VCN-releasable sialic acid was observed with increasing cell density. G^M3 was the major ganglioside component of preconfluent S20Y cells, whereas G_DIA was predominant in postconfluent cells. Sialidase activity increased in confluent and postconfluent cells and may account for the reduction in total ganglioside sialic acid observed with increasing cell density. In contrast, while adrenergic N115 cells showed a decrease in VCN-susceptible sialic acid residues with increasing cell density, there was no significant change in ganglioside composition or ganglioside sialic acid levels.     

Distinct Mechanisms of Differentiation of SH-SY5Y Neuroblastoma Cells by Protein Kinase C Activators and Inhibitors

Certain biological actions of phorbol esters cannot be duplicated by diacylglycerol (DAG). Thus, the human neuroblastoma cell line SH-SY5Y differentiates when exposed to 12-tetradecanoyl-13-acetyl-beta-phorbol (TPA) and protein kinase C (PKC) inhibitors, but not when exposed to DAG. To investigate the specific features of the phorbol diester molecule that might be responsible for these effects, we examined the extension of neurites, expression of neuron-specific enolase, and appearance and localization of phosphorylated high molecular weight neurofilament subunits (NF-H). TPA, 12-deoxy-13-tetradecanoyl-beta-phorbol, and staurosporine, but not DAG or 4-O-methyl-TPA, caused neurite outgrowth. Neuron-specific enolase was expressed in cells treated with TPA and 12-deoxy-13-tetradecanoyl-beta-phorbol but not with DAG, staurosporine, or 4-O-methyl-TPA. NF-H increased in the perikarya of cells treated with DAG and 4-O-methyl-TPA, in processes and to varying degrees in perikarya of TPA- and 12-deoxy-13-tetradecanoyl-beta-phorbol-treated cells, but much more in the processes than in the perikarya of staurosporine-differentiated cells. These findings and additional differences between the differentiation induced by TPA (a PKC activator) and staurosporine (a PKC inhibitor), including distinct morphology of the cell body and processes and time of appearance of the morphological phenotype, suggest that activators and inhibitors of PKC induce differentiation of SH-SY5Y cells by different mechanisms, and that the five-membered/seven-membered terpene ring region present in TPA must be intact for the induction of morphological differentiation.     

Promotion of Neuritogenesis in Mouse Neuroblastoma Cells by Exogenous Gangliosides. Relationship Between the Effect and the Cell Association of Ganglioside GM1

Ganglioside GM1 promoted neuritogenesis of neuroblastoma cells, neuro-2a clone, in monolayer culture. GM1 bound to neuro-2a cells in three distinct forms, one removable by treatment with serum-containing solutions, one serum-resistant and labile to trypsin treatment, and one resistant to serum and trypsin treatments. The proportions among the three forms of cell-associated GM1 varied in relation to duration of exposure to ganglioside, ganglioside concentration in the medium, and number of cells in culture. The form removable by serum was predominant at the initial stages of association and at the highest ganglioside concentrations (over 10(-6)M); the trypsin-labile and -stable forms tended to increase with increasing cell number and decreasing ganglioside concentration. The neuritogenic effect of GM1 was higher when neuro-2a cells were incubated for 24 h in the presence of GM1 and fetal calf serum. Under this condition the percentage of neurite-bearing cells increased from 11% of control to 62% at the optimal ganglioside concentration of 10-4M. The effect was still present, although to a lower extent (from 11% to 28% of neurite-bearing cells), when cells were first exposed for only 2 h to GM1, then washed and incubated for 24 h in the presence of fetal calf serum. The trypsin-labile and -stable forms of cell-associated GM1 had a fundamental role in the effect, whereas the form removable by serum was not involved. The preparation of GM1 used was extremely pure (99%) and, in particular, had a peptide contamination, if any, less than 1:20,000-1:50,000.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of NMDA and Dopamine D2L Receptors in Human Neuroblastoma SH-SY5Y Cells

Abstract: To understand the mechanism of interaction of the dopamine D_2L receptors with NMDA receptors, we have developed a model by transfecting human neuroblastoma SH-SY5Y cells with the human dopamine D_2L receptor gene. In vitro blockade of NMDA receptors by the specific antagonists MK-801 and (±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) on human neuroblastoma SH-SY5Y cells expressing human dopamine D_2L receptors resulted in a significant increase in the density of D_2L receptors without a significant change in receptor affinity. Moreover, the dopamine receptor mRNA level increased by ∼50% by the blockade of NMDA with MK-801. These results suggest a possible interaction of NMDA and dopamine D_2L receptors in neuroblastoma SH-SY5Y cells. This system would serve as an excellent model to study the molecular mechanisms involved in the interaction of these two receptors.     

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.     

Muscarinic Receptor Sequestration in SH-SY5Y Neuroblastoma Cells Is Inhibited when Clathrin Distribution Is Perturbed

Abstract: The possibility that clathrin plays a role in the agonist-mediated sequestration of muscarinic cholinergic receptors in human SH-SY5Y neuroblastoma cells has been investigated by the application of experimental paradigms previously established to perturb clathrin distribution and receptor cycling events. Preincubation of SH-SY5Y cells under hypertonic conditions resulted in a pronounced inhibition of agonist-induced muscarinic receptor sequestration (70–80% at 550 mOsm), which was reversed when cells were returned to isotonic medium. Depletion of intracellular K⁺ or acidification of the cytosol also resulted in >80% inhibition of muscarinic receptor sequestration. Under conditions of hypertonicity, depletion of intracellular K⁺, or acidification of cytosol, muscarinic receptor-stimulated phosphoinositide hydrolysis and Ca²⁺ signaling events were either unaffected or markedly less inhibited than receptor sequestration. That these same experimental conditions did perturb clathrin distribution was verified by immunofluorescence studies. Hypertonicity and depletion of intracellular K⁺ resulted in a pronounced accumulation of clathrin in the perinuclear region, whereas acidification of the cytosol resulted in the appearance of microaggregates of clathrin throughout the cytoplasm and at the plasma membrane. The results are consistent with the possibility that muscarinic receptors in SH-SY5Y cells are endocytosed via a clathrin-dependent mechanism.     

Postinjury Changes in Platelet-Derived Growth Factor-Like Activity in Fish and Rat Optic Nerves

The poor regenerative ability of the CNS of mammals has been attributed, at least in part, to the presence of mature oligodendrocytes, which have been shown to inhibit axonal growth. Proliferation of oligodendrocyte progenitor cells in the rat optic nerve during development, and thereby the timing of oligodendrocyte differentiation, has been shown to depend on a factor derived from type 1 astrocytes, later characterized as platelet-derived growth factor (PDGF). In the present study we examine whether injury to the optic nerve induces changes in the levels of PDGF in spontaneously regenerating systems, compared with nonregenerating systems. Soluble substances, derived from nonneuronal cells surrounding injured fish and rat optic nerves, were prepared and examined for the presence of PDGF immunoreactivity and biological mitogenic activity on PDGF-responsive cells. The results suggest that PDGF-like mitogenic activity and immunoreactivity are present in both fish and rat optic nerves. However, in the rat optic nerve PDGF levels increased after axonal injury, whereas in the fish optic nerve injury was accompanied by an apparent decrease in PDGF-like levels. The results are discussed with respect to the possible role of PDGF in regeneration.     

Inositol 1,4,5-Trisphosphate Receptor Immunoreactivity in SH-SY5Y Human Neuroblastoma Cells Is Reduced by Chronic Muscarinic Receptor Activation

Inositol 1,4,5-trisphosphate (InsP3) receptor immunoreactivity in SH-SY5Y human neuroblastoma cells was monitored with a monoclonal antibody raised against the mouse cerebellar InsP3 receptor. Recognition of a protein corresponding to the InsP3 receptor (molecular mass, approximately 275 kDa) was inhibited markedly following exposure of cells to 0.1 mM carbachol. This effect was half-maximal and maximal at approximately 2 and approximately 6 h, respectively; was blocked by atropine; but was not mimicked by thapsigargin, K+, or phorbol 12-myristate 13-acetate. However, the decrease in immunoreactivity following exposure of cells to carbachol for 5 h was blocked if the extracellular Ca2+ concentration was reduced from 1.3 mM to 200 nM. This manipulation also reduced markedly carbachol-induced increases in InsP3 concentration at 5 h. These data indicate that chronic muscarinic stimulation of phosphoinositide hydrolysis reduces InsP3 receptor concentration in SH-SY5Y cells, perhaps via a mechanism that involves prolonged elevation of InsP3 levels.     

不同能量的培养条件对人神经母细胞瘤株SH-SY5Y细胞生长的影响

目的建立热量限制的体外模型,观察不同能量培养条件下对人神经母细胞瘤细胞株SH-SY5Y细胞生长代谢的影响。方法将人神经母细胞瘤细胞株SH-SY5Y细胞分别采用含有低浓度（2 g/L）、正常浓度（3.15g/L）或高浓度（4.5 g/L）葡萄糖的培养基进行常规传代培养,利用MTT代谢率、细胞生长曲线及LDH漏出率等指标观察各组细胞生长情况。结果与正常葡萄糖浓度培养条件下培养的对照组相比,高糖组细胞突起缩短,细胞胞体皱缩,MTT代谢率稍低（0.573±0.001）,LDH漏出率高,细胞生长状态差;与对照组相比,低糖组细胞突起伸展,MTT代谢率较低（0.428±0.003）,LDH漏出率低,细胞生长速度缓慢,但是形态良好。结论高糖培养对细胞有损伤作用,细胞代谢加速,更容易衰老死亡;而低糖培养起到保护作用,在热量限制允许范围内降低培养液的含糖量,不但不会对细胞造成损伤,反而对细胞的代谢及生长起到保护作用,延长细胞的总体寿命。     

Binding of Erythroagglutinating Phytohemagglutinin Lectin from Phaseolus vulgaris to the Epidermal Growth Factor Receptor Inhibits Receptor Function in the Human Glioma Cell Line, U373 MG

Abstract: Little is known about the role of the N -linked oligosaccharides in the function of the epidermal growth factor (EGF) receptor (EGF-R). In a human glioma cell line, U373 MG, EGF-Rs contain the bisecting N -linked oligosaccharide sequence recognized by erythroagglutinating phytohemagglutinin lectin from Phaseolus vulgaris (E-PHA). Incubation of E-PHA with cultured U373 MG cells results in inhibition of EGF binding to its receptor and consequently inhibition of EGF-induced autophosphorylation of the receptor. Consistent with the inhibitory effects on the EGF-R, phenotypic events that depend on EGF-R signaling, such as cell spreading and proliferation, were also found to be modified. The effect of this lectin seems to be specific because leukoagglutinating phytohemagglutinin lectin from P. vulgaris (L-PHA), an isolectin of E-PHA, had no effect on EGF-R activity or the biological functions of these cells even though L-PHA was able to bind to the EGF-R. These findings suggest the presence of an important bisecting N -linked oligosaccharide structure in close proximity to the EGF binding site on the receptor. Furthermore, these results suggest the possibility that E-PHA lectin binding may provide an additional approach to blocking EGF-dependent glioma cell growth.     

CDK-5-Mediated Neurofilament Phosphorylation in SHSY5Y Human Neuroblastoma Cells

Cyclin-dependent kinase-5 (CDK-5) has been shown to play important roles in neuronal development and neurogenesis. In vitro studies indicate a role of CDK-5 in phosphorylation of neurofilaments (NFs). In this study, we have chosen the human neuroblastoma cell line SHSY5Y as a model system to study the in vivo phosphorylation of NF proteins by CDK-5. Upon differentiation of SHSY5Y cells with retinoic acid, we found that the phosphorylation of high molecular mass (NF-H) and medium molecular mass (NF-M) NFs increased, whereas the CDK-5 protein level and kinase activity were unaffected. The role of CDK-5 in the phosphorylation of cytoskeletal proteins was studied by using antisense oligonucleotides (ONs) to inhibit the expression of the CDK-5 gene. We found that inhibition of CDK-5 levels by antisense ON treatment resulted in a decrease in phosphorylation of NF-H that correlated with a decline in neurite outgrowth. These results demonstrate that CDK-5 is a major proline-directed kinase phosphorylating the human NF-H tail domain.     

Selective Inhibition of Nerve Growth Factor-Stimulated Protein Kinases by K-252a and 5''-S-Methyladenosine in PC12 Cells

K-252a, a protein kinase inhibitor isolated from the culture broth of Nocardiopsis sp., inhibits the nerve growth factor (NGF)-stimulated phosphorylation of microtubule-associated protein 2 (MAP2) and Kemptide (synthetic Leu-Arg-Arg-Ala-Ser-Leu-Gly) by blocking the activation of two independent kinases in PC12 cells: MAP2/pp250 kinase and Kemptide kinase. The NGF-stimulated activation of these kinases is inhibited in a dose-dependent manner following treatment of the cells with K-252a. Although these kinases also are activated by epidermal growth factor (EGF) and 12-O-tetradecanoyl-phorbol 13-acetate, K-252a has no inhibitory effect when these agents are used. Half-maximal inhibition of the activation of both kinases was observed at 10-30 nM K-252a. K-252a was shown to directly inhibit the activity of MAP2/pp250 kinase and Kemptide kinase when added to the phosphorylation reaction mixture in vitro; however, half-maximal inhibition under these conditions was observed at greater than or equal to 50 nM K-252a. These data suggest that K-252a exerts its effects at a step early in the cascade of events following NGF binding. The effects of K-252a are similar to those reported for 5'-S-methyladenosine (MTA) and other methyltransferase inhibitors. Treatment of PC12 cells with MTA inhibited NGF-, but not EGF-mediated activation of MAP2/pp250-kinase (Ki greater than 500 microM). MTA, when added to the phosphorylation reaction mixture in vitro, directly inhibited kinase activity (Ki = 50 microM), suggesting that the effects of MTA may be the result of its action on protein kinases rather than methyltransferases.     

Regulation of Cyclic AMP by the μ-Opioid Receptor in Human Neuroblastoma SH-SY5Y Cells

The human neuroblastoma clonal cell line SH-SY5Y expresses both mu- and delta-opioid receptors (ratio approximately 4.5:1). Differentiation with retinoic acid (RA) was previously shown to enhance the inhibition of adenylyl cyclase (AC) by mu-opioid agonists. We tested here the inhibition of cyclic AMP (cAMP) accumulation by morphine under a variety of conditions: after stimulation with prostaglandin E1 (PGE1), forskolin, and vasoactive intestinal peptide (VIP), both in the presence and in the absence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Morphine inhibition of the forskolin cAMP response (approximately 65%) was largely unaffected by the presence of IBMX. In contrast, deletion of IBMX enhanced morphine's inhibition of the PGE1 and VIP cAMP response from approximately 50 to approximately 80%. The use of highly mu- and delta-selective agents confirmed previous results that inhibition of cAMP accumulation by opioids is mostly mu, and not delta, receptor mediated in SH-SY5Y cells, regardless of the presence or absence of IBMX. Because of the large morphine inhibition and the high cAMP levels even in the absence of IBMX, PGE1-stimulated, RA-differentiated SH-SY5Y cells were subsequently used to study narcotic analgesic tolerance and dependence in vitro. Upon pretreatment with morphine over greater than or equal to 12 h, a fourfold shift of the PGE1-morphine dose-response curve was observed, whether or not IBMX was added. However, mu-opioid receptor number and affinity to the mu-selective [D-Ala2, N-Me-Phe4, Gly5-ol]enkephalin were largely unaffected, and Na(+)- and guanyl nucleotide-induced shifts of morphine-[3H]naloxone competition curves were unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Degradative Pathway of Gangliosides GM1 and GM2 in Neuro2a Cells by Sialidase

Abstract: Gangliosides GM1 [³H-labeled at the sphingosine (Sph) moiety] and GM2 [³H-labeled at the Sph or N -acetylgalactosamine (GalNAc) moiety] were administered to cultured Neuro2a cells for varying pulse (1–4 h) and chase (up to 4 h) periods, and their metabolic processing was followed. The main and earliest formed ³H-metabolites of [ Sph -³H]GM1 were GM2, asialo-GM1, asialo-GM2, and lactose-ceramide, and those of [ Sph -³H]GM2 were asialo-GM2 and lactose-ceramide. The asialo-GM1 and asialo-GM2 formed were isolated and chemically characterized. [³H]Asialo-GM2 was produced in identical amounts after treatment with equimolar [ Sph -³H]GM2 and [ GalNAc -³H]GM2. At low temperature or in the presence of chloroquine, the formation of all ³H-metabolites, including asialo-GM2 and asialo-GM1, was undetectable, indicating that ganglioside metabolic processing was an endocytosis- and lysosome-dependent process. These results demonstrate that in Neuro2a cells exogenous GM1 (and GM2) is mainly degraded through the pathway GM1 → GM2 → asialo-GM2 →→ Sph, with a minor fraction of GM1 undergoing degradation with the sequence GM1 → asialo-GM1 → asialo-GM2 →→ Sph. These findings are consistent with the hypothesis that Neuro2a cells contain a sialidase (likely of lysosomal nature) affecting ganglioside GM1 and GM2. The sialidase-mediated degradative pathway of GM1 and GM2 in Neuro2a cells might be related to the tumoral nature of these cells.     

Regulation of Nicotinic Receptor Subtypes Following Chronic Nicotinic Agonist Exposure in M10 and SH-SY5Y Neuroblastoma Cells

Abstract: The present study further investigated whether nicotinic acetylcholine receptor (nAChR) subtypes differ in their ability to up-regulate following chronic exposure to nicotinic agonists. Seven nicotinic agonists were studied for their ability to influence the number of chick α4β2 nAChR binding sites stably transfected in fibroblasts (M10 cells) following 3 days of exposure. The result showed a positive correlation between the K _i values for binding inhibition and EC₅₀ values for agonist-induced α4β2 nAChR up-regulation. The effects of epibatidine and nicotine were further investigated in human neuroblastoma SH-SY5Y cells (expressing α3, α5, β2, and β4 nAChR subunits). Nicotine exhibited a 14 times lower affinity for the nAChRs in SH-SY5Y cells as compared with M10 cells, whereas epibatidine showed similar affinities for the nAChRs expressed in the two cell lines. The nicotine-induced up-regulation of nAChR binding sites in SH-SY5Y cells was shifted to the right by two orders of magnitude as compared with that in M10 cells. The epibatidine-induced up-regulation of nAChR binding sites in SH-SY5Y cells was one-fourth that in M10 cells. The levels of mRNA of the various nAChR subunits were measured following the nicotinic agonist exposure. In summary, the various nAChR subtypes show different properties in their response to chronic stimulation.     

MAPK磷酸酯酶-1通过抑制JNK和p38的磷酸化调节SH-SY5Y神经母细胞瘤的凋亡

目的研究MKP-1在SH-SY5Y神经母细胞瘤中的抗凋亡。方法建立稳定表达MKP-1的SH-SY5Y细胞,用H2O2诱导细胞凋亡,并通过Western blotting比较分析MKP-1的表达对JNK和p38磷酸化的调节。结果①H2O2诱导SH-SY5Y细胞表达MKP-1,同时导致JNK和p38的去磷酸化;②在稳定表达MKP-1的SH-SY5Y细胞中,MKP-1可以抑制JNK和p38的磷酸化。③稳定表达MKP-1的SH-SY5Y细胞抵抗H2O2诱导细胞凋亡的能力比对照细胞提高了1倍左右。结论MKP-1对神经细胞的凋亡具有重要的调节作用,提示MKP-1作为调节ERK、JNK和p38蛋白激酶信号途径的重要分子,可能对退行性神经系统疾病的发病机制和治疗有重要的作用。     

The Growth Response to Beta-Hydroxybutyrate in SH-SY5Y Neuroblastoma Cells is Suppressed by Glucose and Pyruvate Supplementation

Neurochemical Research - Neuroblastoma (NB) is a childhood malignancy of the sympathetic nervous system and is commonly studied using the SH-SY5Y cell line. Its neoplastic and neurodevelopmental...