Interaction among mu-opioid receptors and alpha 2-adrenoceptors on SH-SY5Y human neuroblastoma cells.

The clonal human neuroblastoma cell line SK-N-SH-SY5Y was previously shown to express mu-opioid and alpha 2-adrenoceptors which are both negatively coupled to adenylyl cyclase. Because of the potential use of alpha 2-agonists in the treatment of narcotic dependence, we tested the interactions among he alpha 2-agonists, clonidine and norepinephrine, and morphine on AC in SH-SY5Y cells. Pretreatment with retinoic acid resulting in partial neuronal differentiation greatly enhanced the cells' sensitivity towards adenylyl cyclase stimulation by prostaglandin E1, and its inhibition by morphine and alpha 2-agonists. Norepinephrine (EC50 = 69 nM) maximally inhibited prostaglandin E1-stimulated cAMP accumulation (by approximately 83%), and the alpha 2-agonist yohimbine reversed these effects. Clonidine (EC50 = 32 nM) was a partial agonist, with 50 to 60% maximal inhibition. The combined effects of morphine (maximum approximately 70% inhibition) and norepinephrine exceeded the effect of either agent alone, yielding more than 90% inhibition of prostaglandin E1-stimulated cAMP accumulation. As previously reported for morphine, only a partial tolerance was observed for adenylyl cyclase inhibition by norepinephrine. Further, no cross-tolerance was observed between clonidine and morphine. The combined results indicate that mu-opioid receptors and an alpha 2-adrenoceptor subtype are colocalized on the same cells in SH-SY5Y culture, which hence serves as a model to study opioid-alpha 2-adrenergic interactions.     

Mu-opioid receptor binding in intact SH-SY5Y neuroblastoma cells

In order to determine affinities at the mu opioid receptor binding was conducted to intact SH-SY5Y neuroblastoma cells using the mu-selective ligand [3H][H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2] [( 3H]CTOP). Binding appeared to be a single receptor site, and a single state of the mu receptor. Under intact cell conditions, some but not all mu agonists display low affinity binding, while antagonists maintain high affinity for the mu receptor. These studies indicate the usefulness of [3H]CTOP for the determination of affinities at the mu receptor under physiological conditions.     

Organophosphorus compound-induced apoptosis in SH-SY5Y human neuroblastoma cells

Organophosphorus (OP) compounds have been shown to be cytotoxic to SH-SY5Y human neuroblastoma cell cultures. The mechanisms involved in OP compound-induced cell death (apoptosis versus necrosis) were assessed morphologically by looking at nuclear fragmentation and budding using the fluorescent stain Hoechst 33342 (10 microgram/ml). Hoechst staining revealed significant paraoxon (1 mM), parathion (1 mM), phenyl saligenin phosphate (PSP, 10 and 100 microM), tri-ortho-tolyl phosphate (TOTP, 100 microM and 1 mM), and triphenyl phosphite (TPPi, 1 mM) induced time-dependent increases in traditional apoptosis (p < 0.05). In many cells, PSP and TOTP (1 mM) also induced nuclear condensation with little fragmentation or budding. Pretreatment with cyclosporin A (500 nM, 30 h) decreased apoptosis following 1 mM parathion and TOTP exposures. Apoptotic nuclear changes were verified by DNA gel electrophoresis. Activation of caspase-3, a cysteine aspartate protease, was also monitored. OP compounds induced significant time-dependent increases in caspase-3 activation following paraoxon (1 mM), parathion (100 microM, 1 mM), PSP (10 microM, 100 microM, 1 mM), TOTP (100 microM, 1 mM), and TPPi (1 mM) exposure (p < 0.05). Pretreatment with cyclosporin A (500 nM, 30 h) significantly decreased caspase-3 activation during extended incubations with paraoxon, parathion, and TPPi (p < 0.05). In addition, pretreatment with the caspase-3 inhibitor Ac-DEVD-CHO and the caspase-8 inhibitor Ac-IETD-CHO (25 microM, 8 h) significantly decreased caspase-3 activation following exposure to 1 mM PSP and parathion (p < 0.05). Pretreatment with the serine protease inhibitor phenylmethyl sulfonyl fluoride (PMSF; 1 mM, 8 h) also significantly decreased caspase activation following 1 mM PSP and TOTP exposures (p < 0.05). Alteration of OP compound-induced nuclear fragmentation or caspase-3 activation by pretreatment with cyclosporin A, Ac-IETD-CHO, or PMSF suggested that OP compound-induced cytotoxicity may be modulated through multiple sites, including mitochondrial permeability pores, receptor-mediated caspase pathways, or serine proteases.     

Expression of CD38 in human neuroblastoma SH-SY5Y cells

Human CD38 antigen is a 42-45 kDa type II transmembrane glycoprotein with a short N-terminal cytoplasmic domain and a long C-terminal extracellular region. It is widely expressed in different cell types including thymocytes, activated T cells, and terminally differentiated B cells (plasma cells) and it is involved in cellular proliferation and adhesion. CD38 acts as an ectocyclase that converts NAD+ to the Ca2+ -releasing second messenger cyclic ADP-ribose (cADPR). It has been also demonstrated that increased extracellular levels of NAD+ and cADPR are involved in inflammatory diseases and in cellular damage, such as ischemia. In the present study, we have characterized the expression of CD38 in human neuroblastoma SH-SY5Y cell line. All-trans-retinoic acid (ATRA) treatment was used to induce cell differentiation. Our results indicate that: a) even if SH-SY5Y cells have a negative phenotype express CD38 at nuclear level, ATRA treatment does not influence this pattern; b) CD38 localizing to the nucleus may co-localize with p80-coilin positive nuclear-coiled bodies; c) purified nuclei, by Western blot determinations using anti-CD38 antibodies, display a band with a molecular mass of approximately 42 kDa; d) SH-SY5Y cells show nuclear ADP-ribosyl cyclase due to CD38 activity; e) the basal level of CD38 mRNA shows a time-dependent increase after treatment with ATRA. These results suggest that the presence of constitutive fully functional CD38 in the SH-SY5Y nucleus has some important implications for intracellular generation of cADP-ribose and subsequent nucleoplasmic calcium release.     

Effect of toluene diisocyanate on homeostasis of intracellular-free calcium in human neuroblastoma SH-SY5Y cells

The mechanisms of TDI (2,4-toluene diisocyanate)-induced occupational asthma are not fully established. Previous studies have indicated that TDI induces non-specific bronchial hyperreactivity to methacholine and induces contraction of smooth muscle tissue by activating 'capsaicin-sensitive' nerves resulting asthma. Cytosolic-free calcium ion concentrations ([Ca(2+)](c)) are elevated when either capsaicin acts at vanilloid receptors, or methacholine at muscarinic receptors. This study therefore investigated the effects of TDI on Ca(2+) mobilization in human neuroblastoma SH-SY5Y cells. TDI was found to elevate [Ca(2+)](c) by releasing Ca(2+) from the intracellular stores and extracellular Ca(2+) influx. 500 microM TDI induced a net [Ca(2+)](c) increase of 112+/-8 and 78+/-6 nM in the presence and absence of extracellular Ca(2+), respectively. In Ca(2+)-free buffer, TDI induced Ca(2+) release from internal stores to reduce their Ca(2+) content and this reduction was evidenced by a suppression occurring on the [Ca(2+)](c) rise induced by thapsigargin, ionomycin, and methacholine after TDI incubation. In the presence of extracellular Ca(2+), simultaneous exposure to TDI and methacholine led a higher level of [Ca(2+)](c) compared to single methacholine stimulation, that might explain that TDI induces bronchial hyperreactivity to methacholine. We conclude that TDI is capable of interfering the [Ca(2+)](c) homeostasis including releasing Ca(2+) from internal stores and inducing extracellular Ca(2+) influx. The interaction of this novel character and bronchial hyperreactivity need further investigation.     

Activation of phospholipase C in SH-SY5Y neuroblastoma cells by potassium-induced calcium entry.

1. We used SH-SY5Y human neuroblastoma cells to investigate whether depolarization with high K+ could stimulate inositol (1,4,5)trisphosphate (Ins(1,4,5)P3) formation and, if so, the mechanism involved. 2. Ins(1,4,5)P3 was measured by a specific radioreceptor mass assay, whilst [Ca2+]i was measured fluorimetrically with the Ca2+ indicator dye, Fura-2. 3. Depolarization with K+ caused a time- and dose-dependent increase in [Ca2+]i (peak at 27 s, EC50 of 50.0 +/- 9.0 mM) and Ins(1,4,5)P3 formation (peak at 30 s, EC50 of 47.4 +/- 1.1 mM). 4. Both the K(+)-induced Ins(1,4,5)P3 formation and increase in [Ca2+]i were inhibited dose-dependently by the L-type voltage-sensitive Ca2+ channel closer, (R+)-BayK8644, with IC50 values of 53.4 nM and 87.9 nM respectively. 5. These data show a close temporal and dose-response relationship between Ca2+ entry via L-type voltage-sensitive Ca2+ channels and Ins(1,4,5)P3 formation following depolarization with K+, indicating that Ca2+ influx can activate phospholipase C in SH-SY5Y cells.     

Honokiol and magnolol induce Ca2+ mobilization in rat cortical neurons and human neuroblastoma SH-SY5Y cells

We examined the intracellular Ca(2+) response in primary cultured rat cortical neurons and human neuroblastoma SH-SY5Y cells by Fluo 3 fluorescence imaging analysis. In these two kinds of neuronal cells, honokiol and magnolol increased cytoplasmic free Ca(2+) with a characteristic lag phase. The cytoplasmic free Ca(2+) increase was independent of extracellular Ca(2+), but dependent on activation of phospholipase C and inositol 1,4,5-triphosphate (IP(3)) receptors. These results suggest that honokiol and magnolol increase cytoplasmic free Ca(2+) through a phospholipase C-mediated pathway, and that the release of Ca(2+) from intracellular stores mainly contributes to the increase in cytoplasmic free Ca(2+). Thus, honokiol and magnolol may be involved in a new activation mechanism closely associated with intracellular Ca(2+) mobilization.     

Analysis of human Nav1.8 expressed in SH-SY5Y neuroblastoma cells

The tetrodotoxin-resistant voltage-gated sodium channel alpha-subunit Nav1.8 is expressed in nociceptors and has been implicated in chronic pain. Difficulties of heterologous expression have so far precluded analysis of the pharmacological properties of human Nav1.8. To address this we have introduced human Nav1.8 in neuroblastoma SH-SY5Y cells. Voltage-clamp analysis showed that human Nav1.8 generated an inward tetrodotoxin-resistant sodium current with an activating threshold around -50 mV, half maximal activation at -11+/-3 mV and a reversal potential of 67+/-4 mV. These properties closely match those of the endogenous rat tetrodotoxin-resistant sodium current in dorsal root ganglia suggesting that the expressed human channel is in a near physiological conformation. Human Nav1.8 was resistant to tetrodotoxin and activated by the pyrethroid toxin deltamethrin. Both voltage-activated and deltamethrin-activated human Nav1.8 were inhibited by the sodium channel blockers BIII 890 CL, NW-1029, and mexiletine. Inhibition of Nav1.8 by these compounds may underlie their known analgesic effects in animal models.     

Adiponectin 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 (ROS) level and apoptosis. Adiponectin, secreted from adipose tissue, mediates systemic insulin sensitivity with liver and muscle as target organs. In this study, we investigated the protective effects of adiponectin on acetaldehyde-induced apoptosis in human neuroblastoma SH-SY5Y cells and attempted to examine its mechanism. Acetaldehyde-induced apoptosis was moderately reversed by adiponectin treatment. Our results suggest that the protective effects of adiponectin 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 adiponectin may provide a useful therapeutic strategy for the prevention of progressive neurodegenerative disease such as Parkinson's disease.     

Suppression by phthalates of the calcium signaling of human nicotinic acetylcholine receptors in human neuroblastoma SH-SY5Y cells

Phthalates are widely used in industry and cause public concern since they have genomic estrogenic-like effects via estrogen receptors. We previously found that some phthalates have nongenomic effects, exerting inhibitory effects on the functional activities of nicotinic acetylcholine receptors (nAChRs) in bovine chromaffin cells. In this study, we investigated the effects of eight phthalates on the calcium signaling of human nAChR by using human neuroblastoma SH-SY5Y cells. All eight phthalates, with different potency, have inhibitory roles on the calcium signaling coupled with human nAChR, but not muscarinic acetylcholine receptors (mAChRs). For inhibition of human nAChR, the strongest to weakest potencies were observed as di-n-pentyl phthalate (DPP) → butyl benzyl phthalate (BBP) → di-n-butyl phthalate (DBP) → dicyclohexyl phthalate (DCHP) → di-n-hexyl phthalate (DHP) → di-(2-ethyl hexyl) phthalate (DEHP) → di-n-propyl phthalate (DPrP) → diethyl phthalate (DEP). The potencies of phthalates were associated with their structures such that the most effective ones had dialkyl group carbon numbers of C4 or C5, with shorter or longer numbers resulting in decreased potency. At as low as 0.1 μM, DPP, DBP, BBP, DCHP and DHP significantly inhibited the calcium signaling of human nAChR. The IC₅₀ of phthalates on human nAChR, ranging from 0.32 to 7.96 μM, were 10–50 lower than those for bovine nAChR. We suggest that some phthalates effectively inhibit the calcium signaling of human nAChR, and these nongenomic effects are cause for concern.     

Effects of dichlorobenzene on acetylcholine receptors in human neuroblastoma SH-SY5Y cells

para-Dichlorobenzene (DCB), a deodorant and an industrial chemical, is a highly volatile compound and is known to be an indoor air contaminant. Because of its widespread use and volatility, the toxicity of DCB presents a concern to industrial workers and public. Some toxic aspects of DCB have already been focused but its effects on neuronal signal transduction have been hitherto unknown. The effects of DCB on the cytosolic calcium homeostasis are investigated in human neuroblastoma SH-SY5Y cells in this study. DCB, above 200 microM, was found to induce a rise in cytosolic calcium concentration that could not be counteracted by nicotinic acetylcholine receptor (nAChR) and muscarinic acetylcholine receptor (mAChR) antagonists but was partially inhibited by thapsigargin. To understand the actions of DCB on the acetylcholine receptors, we investigated its effects on the changes of cytosolic calcium concentration following nicotinic AChR stimulation with epibatidine and muscarinic AChR stimulation with methacholine in human neuroblastoma SH-SY5Y cells. DCB inhibited the cytosolic calcium concentration rise induced by epibatidine and methacholine with respective IC(50)s of 34 and 294 microM. The inhibitions of DCB were not the same as thapsigargin's inhibition. In the electrophysiological observations, DCB blocked the influx currents induced by epibatidine. Our findings suggest that DCB interferes with the functional activities of AChR, including its coupling influx currents and cytosolic calcium elevations.     

Tribromophenol induces the differentiation of SH-SY5Y human neuroblastoma cells in vitro

Tribromophenol is a pesticide with fungicide activity, presently used as a replacement of pentachlorophenol as a wood preservative, and as a flame retardant in electronic and electrotechnical devices. Retinoic acid differentiated and non-differentiated SH-SY5Y human neuroblastoma cell cultures were exposed to a range of concentrations of tribromophenol for 24, 48 and 72 h and the effects evaluated at morphological, basal cytotoxicity and biochemical levels. Neuroblastoma cell number, evaluated by quantification of total protein content, was increasingly inhibited in accordance with the concentration of tribromophenol and the exposure time period. According to the mean effective concentrations, differentiated cultures were nearly three times more sensitive than naive cells. Lysosomal function evaluated by the neutral red uptake was stimulated, particularly in non-differentiated cells. MTS metabolization was stimulated by all the treatments, with more potency at 24 h for differentiated cells. Acetylcholinesterase activity increased with the time of exposure in non-differentiated cells, while in differentiated cells the activity was doubled at 24 h. Morphological alterations were evident from 12.5 μ , showing hydropic degeneration and reduction in cell number, and from that concentration, piknosis and apoptotic bodies were observed. In conclusion, the main effects detected for tribromophenol were the induction of neuroblastoma cell differentiation, as expressed by the inhibition of cell growth and the increase in acetylcholinesterase activity with a critical cell concentration of 0.1 μ . Apoptosis was observed at high concentrations. The induction of cell differentiation and the special sensitivity of differentiated cells can explain some mechanisms involved in the embryotoxic and foetotoxic potential of tribromophenol.     

Synergistic neurotoxic effects of arsenic and dopamine in human dopaminergic neuroblastoma SH-SY5Y cells.

Parkinson's disease is an environmentally influenced, neurodegenerative disease of unknown origin that is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta of the brain. Arsenic is an environmental contaminant found naturally in ground water, industrial waste, and fertilizers. The initial goal of the present study was to determine if a mixture of arsenite (As(+3)) and dopamine (DA) could cause enhanced degeneration of dopaminergic neuronal cells. Additional goals were to determine the mechanism (apoptosis or necrosis) of As- and DA-induced cell death and if death could be attenuated by antioxidants. The cell culture model employed was the SH-SY5Y neuroblastoma cell line that has been shown to possess differentiated characteristics of dopaminergic neurons. The results demonstrated that a mixture of As(+3) and DA was synergistic in producing the death of the SH-SY5Y cells when compared with exposure to either agent alone. A mixture of 10muM As(+3) and 100muM DA produced almost a complete loss of cell viability over a 24-h period of exposure, whereas, each agent alone had minimal toxicity. It was shown that necrosis, and not apoptosis, was the mechanism of cell death produced by exposure of the SH-SY5Y cells to the mixture of As(+3) and DA. It was also demonstrated that the antioxidants, N-acetylcysteine, and Sulforaphane, attenuated the toxicity of the mixture of As(+3) and DA to the SH-SY5Y cells. This study provides initial evidence that As(+3) and DA synergistically can cause enhanced toxicity in cultured neuronal cells possessing dopaminergic differentiation.     

d-Lysergic acid reduces microtubule-associated tau protein in SH-SY5Y human neuroblastoma cells

• 1.1. SH-SY5Y, an adrenergic human neuroblastoma cell line, was used to examine the hypothesis that d-lysergic acid (LSD) affects the metabolism of microtubule-associated tau protein, thus affecting microtubule assembly and the transport of neurotransmitters.
• 2.2. After 48 hr treatment LSD (10⁻⁵ and 10⁻⁷ M) decreased 50 kDa tau protein in the membrane (pellet) fraction. The drug (10⁻⁵ M) also decreased in the cytoplasmic (supernatant) fraction.
• 3.3. This reduction in tau protein was accompanied by a 65% increase (P < 0.05) in total protein after LSD (10⁻⁷ M) in the cytoplasmic fraction.

     

Tranexamic acid protects against rotenone-induced apoptosis in human neuroblastoma SH-SY5Y cells

Rotenone is a pesticide that has been shown to induce the pathological symptoms of Parkinson's disease (PD) in animal models. In this study, we investigated the protective effects of tranexamic acid (TA) on rotenone-induced apoptosis in human dopaminergic SH-SY5Y cells. TA blocked the rotenone-induced phosphorylation of JNK and P38, the downregulation of BCL2 and the upregulation of BAX. Furthermore, TA not only decreased the rotenone-induced cleavage of caspase 9, PARP, and caspase 3, but also increased caspase 3 enzymatic activity. Our findings indicate that TA is able to protect neuronal cells against apoptosis and suggest that TA might potentially serve as an agent for prevention or therapy of PD.     

Investigation of nanoplastic cytotoxicity using SH-SY5Y human neuroblastoma cells and polystyrene nanoparticles

Nanoplastics have spread widely throughout not only the oceans but also the atmosphere, and recently created great concern about human health relevant to ingestion and accumulation of the nanoparticles by aquatic organisms in the human food-chain. However, how the nanoplastics have an affect on actual human body remains largely unknown, and in particular, little knowledge about nanoplastic exposure to the nervous system in human has been obtained in vitro and still less vivo. Here, we evaluated how much concentration of nanoplastics had a direct impact on cells in the nervous system as the fundamental information. Specifically, the cytotoxicity was investigated by exposure of polystyrene nanoparticles (PS) to cultured neural cells, human neuroblastoma cells, SH-SY5Y. Our results demonstrated that the PS exposure induced the cytotoxicity in the cells promoted differentiation into neuronal phenotype, and the adverse effect was comparable to or exceed that of acrylamide, a well-recognized potent neurotoxin. Also, the cells under PS exposure exhibited shrinkage of neurite outgrowth, morphology alteration and swelling of the nuclei, and spilling of intracellular components. Moreover, our findings indicate that the concentration of nanoplastics caused the cytotoxicity on neuronal cells is likely to be much higher than those predicted from the marine environment.     

Induction of GADD153 expression by tributyltin in SH-SY5Y human neuroblastoma cells

The effects of tributyltin (TBT) exposure on the expression of growth arrest- and DNA damage-inducible gene 153 (GADD153), also called C/EBP homologous protein (CHOP), were examined in SH-SY5Y human neuroblastoma cells. In response to TBT exposure, the levels of both GADD153 mRNA and GADD153 protein increased significantly. This effect was preceded by phosphorylation of c-Jun NH(2)-terminal kinase (JNK). Treatment with the JNK inhibitor, SP600125, markedly suppressed TBT-induced GADD153 expression. TBT may induce the expression of GADD153, a gene highly responsive to endoplasmic reticulum (ER) stress, in a manner at least partially dependent upon the JNK pathway in SH-SY5Y cells.     

The bipyridyl herbicide paraquat induces proteasome dysfunction in human neuroblastoma SH-SY5Y cells

Paraquat (PQ) is suspected to be an environmental risk factor for Parkinson's disease (PD). A strong correlation between exposure to paraquat and the occurrence of PD was reported in Canada, Taiwan, and the United States. This correlation is supported by in vivo work showing that paraquat produces dopaminergic pathogenesis. In particular, paraquat forms abnormal protein aggregates in dopaminergic neurons of mice. However, it is not clear how paraquat produces this pathology. Given that proteasome dysfunction induces aberrant protein aggregation, it was hypothesized that paraquat induces proteasome dysfunction. To explore this possibility, proteasome activity and some factors possibly contributing to proteasome dysfunction were investigated in dopaminergic SY5Y cells treated with paraquat. Furthermore, levels of alpha-synuclein and ubiquitin-conjugated proteins were measured to test whether paraquat induces protein accumulation in SY5Y cells. Results showed that at a concentration of paraquat that reduced viability by about 60% at 48 h (0.5 mM) loss of proteasome activity occurred. In addition, the cells showed decreased ATP levels and reduced mitochondrial complex V activity. These changes were significant 24 h after treatment with paraquat. Furthermore, paraquat-treated cells showed decreased protein levels of proteasome 19S subunits, but not 20S alpha or beta subunits, suggesting that the effects observed were not the result of general cytotoxicity. Paraquat also increased levels of alpha-synuclein and ubiquitinated proteins, suggesting that paraquat-induced proteasome dysfunction leads to aberrant protein accumulation. Taken together, these findings support the hypothesis that paraquat impairs proteasome function in SY5Y cells.