Mu-opioids activate phospholipase C in SH-SY5Y human neuroblastoma cells via calcium-channel opening.
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ABSTRACT:
SUBMITTER: Smart D
PROVIDER: S-EPMC1136401 | biostudies-other | 1995 Jan
REPOSITORIES: biostudies-other
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Project description:1. There is evidence for interactions between mu and delta opioid systems both in vitro and in vivo. This work examines the hypothesis that interaction between these two receptors can occur intracellularly at the level of G protein in human neuroblastoma SH-SY5Y cells. 2. The [(35)S]GTP gamma S binding assay was used to measure G protein activation following agonist occupation of opioid receptors. The agonists DAMGO (EC(50), 45 nM) and SNC80 (EC(50), 32 nM) were found to be completely selective for stimulation of [(35)S]-GTP gamma S binding through mu and delta opioid receptors respectively. Maximal stimulation of [(35)S]-GTP gamma S binding produced by SNC80 was 57% of that seen with DAMGO. When combined with a maximally effective concentration of DAMGO, SNC80 caused no additional [(35)S]-GTP gamma S binding. This effect was also seen when measured at the level of adenylyl cyclase. 3. Receptor activation increased the dissociation of pre-bound [(35)S]-GTP gamma S. In addition, the delta agonist SNC80 promoted the dissociation of [(35)S]-GTP gamma S from G proteins initially labelled using the mu agonist DAMGO. Conversely, DAMGO promoted the dissociation of [(35)S]-GTP gamma S from G proteins initially labelled using SNC80. 4. Tolerance to DAMGO and SNC80 in membranes from cells exposed to agonist for 18 h was homologous and there was no evidence for alteration in G protein activity. 5. The findings support the hypothesis that mu- and delta-opioid receptors share a common G protein pool, possibly through a close organization of the two receptors and G protein at the plasma membrane.
| S-EPMC1573101 | biostudies-other
Project description:Western-blot analysis of human neuroblastoma SH-SY5Y cells (mu- and delta-receptors) revealed the presence of the following G-protein subunits: Gi alpha 1, Gi alpha 2, Gs alpha, G(o) alpha, Gz alpha, and G beta, a pattern resembling that observed in central nervous tissue. Chronic treatment of differentiated [all-trans-retinoic acid (10 microM; 6 days)] SH-SY5Y cells with D(-)-morphine (10 microM; 3 days) significantly increased the abundance of all G-protein subunits identified. Co-incubation of morphine-exposed cells together with naloxone (10 microM; 3 days) or the mu-selective opioid antagonist CTOP (10 microM; 3 days), but not with the delta-selective antagonist ICI-174,864 (10 microM; 3 days), completely abolished this effect, suggesting that the increase in G-protein abundance is specifically mediated by mu-receptors. Moreover, the biologically inactive enantiomer L(+)-morphine (10 microM; 3 days) failed to produce a similar effect. G-protein up-regulation developed in a time- and dose-dependent manner and is most likely due to enhanced protein synthesis de novo, since concomitant treatment of the cells with cycloheximide (100 micrograms/ml; 3 days) prevented this effect. Chronic treatment with the low-efficacy mu-selective opioid peptide morphiceptin (10 microM; 3 days), but not with the highly potent mu-agonist DAGO (0.1 microM; 3 days) produced a comparable increase in G-protein abundance. Coincident with quantitative effects on G-protein levels in morphine-tolerant/dependent SH-SY5Y cells, we found elevated levels of basal, forskolin (1 microM)- and prostaglandin-E1 (1 microM)-stimulated adenylate cyclase activities. Reconstitution experiments using S49 cyc- lymphoma-cell membranes suggest that this increase is most likely due to elevated levels of functionally intact Gs. Chronic treatment with both morphine and DAGO induces high degrees of tolerance in this cell line. However, the intrinsic activity of G1 was unchanged, as assessed in functional studies with low-nanomolar concentrations of guanosine 5'-[beta gamma- imido]triphosphate. Our data demonstrate that chronic treatment of SH-SY5Y cells with low-efficacy mu-opioids increases G-protein abundance, a phenomenon which might contribute to the biochemical mechanisms underlying opioid tolerance/dependence.
| S-EPMC1134848 | biostudies-other
Project description:Estrogens exert extensive influences on the nervous system besides their well-known roles in regulation of reproduction and metabolism. Estrogens act via the nuclear receptor ER? and ER? to regulate gene transcription (classical genomic effects). In addition, estrogens are also known to cause rapid non-genomic effects on neuronal functions including inducing fast changes in cytosolic calcium level and rapidly desensitizing the ? type opioid receptor (MOR). The receptors responsible for the rapid actions of estrogens remain uncertain, but recent evidence points to the G protein-coupled estrogen receptor (GPER), which has been shown to be expressed widely in the nervous system. In the current study, we test the hypothesis that activation of GPER may mediate rapid calcium signaling, which may promote phosphorylation of MOR through the calcium-dependent protein kinases in neuronal cells. By qPCR and immunocytochemistry, we found that the human neuroblastoma SH-SY5Y cells endogenously express GPER and MOR. Activation of GPER by 17?-estradiol (E2) and G-1 (GPER selective agonist) evoked a rapid calcium rise in a concentration-dependent manner, which was due to store release rather than calcium entry. The GPER antagonist G15, the PLC inhibitor U73122 and the IP3 receptor inhibitor 2-APB each virtually abolished the calcium responses to E2 or G-1. Activation of GPER stimulated translocation of PKC isoforms (? and ?) to the plasma membrane, which led to MOR phosphorylation. Additionally, E2 and G-1 stimulated c-Fos expression in SH-SY5Y cells in a PLC/IP3-dependent manner. In conclusion, the present study has revealed a novel GPER-mediated estrogenic signaling in neuroblastoma cells in which activation of GPER is followed by rapid calcium mobilization, PKC activation and MOR phosphorylation. GPER-mediated rapid calcium signal may also be transmitted to the nucleus to impact on gene transcription. Such signaling cascade may play important roles in the regulation of opioid signaling in the brain.
| S-EPMC6928052 | biostudies-literature
Project description:Manganese (Mn)-associated neurotoxicity has been well recognized. However, Mn is also an essential nutrient to maintain physiological function. Our previous study of human neuroblastoma SH-SY5Y cells showed that Mn treatment comparable to physiological and toxicological concentrations in human brain resulted in different mitochondrial responses, yet cellular metabolic responses associated with such different outcomes remain uncharacterized. Herein, SH-SY5Y cells were examined for metabolic responses discriminated by physiological and toxicological levels of Mn using high-resolution metabolomics (HRM). Before performing HRM, we examined Mn dose (from 0 to100 ?M) and time effects on cell death. Although we did not observe any immediate cell death after 5?h exposure to any of the Mn concentrations assessed (0-100??M), cell loss was present after a 24-h recovery period in cultures treated with Mn???50??M. Exposure to Mn for 5?h resulted in a wide range of changes in cellular metabolism including amino acids (AA), neurotransmitters, energy, and fatty acids metabolism. Adaptive responses at 10??M showed increases in neuroprotective AA metabolites (creatine, phosphocreatine, phosphoserine). A 5-h exposure to 100?µM Mn, a time before any cell death occurred, resulted in decreases in energy and fatty acid metabolites (hexose-1,6 bisphosphate, acyl carnitines). The results show that adjustments in AA metabolism occur in response to Mn that does not cause cell death while disruption in energy and fatty acid metabolism occur in response to Mn that results in subsequent cell death. The present study establishes utility for metabolomics analyses to discriminate adaptive and toxic molecular responses in a human in vitro cellular model that could be exploited in evaluation of Mn toxicity.
| S-EPMC6484887 | biostudies-literature
Project description:The increasing appearance of engineered nanomaterials in broad biomedical and industrial sectors poses an escalating health concern from unintended exposure with unknown consequences. Routine in vitro assessments of nanomaterial toxicity are a vital component to addressing these mounting health concerns; however, despite the known role of cell-cell and cell-matrix contacts in governing cell survival, these physical interactions are generally ignored. Herein, we demonstrate that exposure to amorphous silica particles destabilizes mitochondrial membrane potential, stimulates reactive oxygen species (ROS) production and promotes cytotoxicity in SH-SY5Y human neuroblastoma through mechanisms that are potently matrix dependent, with SH-SY5Y cells plated on the softest matrix displaying a near complete recovery in viability compared to dose-matched cells plated on tissue-culture plastic. Cells on the softest matrix (3 kPa) further displayed a 50% reduction in ROS production and preserved mitochondrial membrane potential. The actin cytoskeleton is mechanosensitive and closely related to ROS production. SH-SY5Y cells exposed to a 100 μg/mL dose of 50 nm silica particles displayed distinct cytoskeletal aberrations and a 70% increase in cell stiffness. Overall, this study establishes that the mechanical environment can significantly impact silica nanoparticle toxicity in SH-SY5Y cells. The mechanobiochemical mechanisms behind this regulation, which are initiated at the cell-matrix interface to adjust cytoskeletal structure and intracellular tension, demand specific attention for a comprehensive understanding of nanotoxicity.
| S-EPMC7877221 | biostudies-literature
Project description:Compounds of natural origin may constitute an interesting tool for the treatment of neuroblastoma, the most prevalent extracranial solid tumor in children. PRES is a commercially available food supplement, composed of a 13:2 (w/w) extracts mix of Olea europaea L. leaves (OE) and Hibiscus sabdariffa L. flowers (HS). Its potential towards neuroblastoma is still unexplored and was thus investigated in human neuroblastoma SH-SY5Y cells. PRES decreased the viability of cells in a concentration-dependent fashion (24 h IC50 247.2 ± 31.8 µg/mL). Cytotoxicity was accompanied by an increase in early and late apoptotic cells (AV-PI assay) and sub G0/G1 cells (cell cycle analysis), ROS formation, reduction in mitochondrial membrane potential, and caspases activities. The ROS scavenger N-acetyl-L-cysteine reverted the cytotoxic effects of PRES, suggesting a key role played by ROS in PRES-mediated SH-SY5Y cell death. Finally, the effects of OE and HS extracts were singularly tested and compared to those of the corresponding mixture. OE- or HS-mediated cytotoxicity was always significantly lower than that caused by PRES, suggesting a synergic effect. In conclusion, the present findings highlight the potential of PRES for the treatment of neuroblastoma and offers the basis for a further characterization of the mechanisms underlying its effects.
| S-EPMC8750347 | biostudies-literature
Project description:BackgroundMaleic acid is a multi-functional chemical widely used in the field of industrial chemistry for producing food additives and food contact materials. As maleic acid may contaminate food by the release from food packages or intentional addition, it raises the concern about the effects of excessive dietary exposure to maleic acid on human health. However, the influence of maleic acid on human health has not been thoroughly studied. In silico toxicogenomics approaches have found the association between maleic acid and nervous system disease in human. The aim of this study is to experimentally explore the effects of maleic acid on human neuronal cells.MethodsA microarray-based transcriptome profiling was performed to offer a better understanding of the effects of maleic acid on human health. Gene expression profiles of human neuroblastoma SH-SY5Y cells exposed to three concentrations of maleic acid (10, 50, and 100 μM) for 24 h were analyzed. Genes which were differentially expressed in dose-dependent manners were identified and further analyzed with an enrichment analysis. The expression profile of selected genes related to the inferred functional changes was validated using quantitative polymerase chain reaction (qPCR). Specific fluorescence probes were applied to observe the inferred functional changes in maleic acid-treated neuronal cells.ResultsA total of 316 differentially expressed genes (141 upregulated and 175 downregulated) were identified in response to the treatment of maleic acid. The enrichment analysis showed that DNA binding and metal ion binding were the significant molecular functions (MFs) of the neuronal cells affected by maleic acid. Maleic acid exposure decreased the expression of genes associated with calcium and thiol levels of the cells in a dose-dependent manner. The levels of intracellular calcium and thiol levels were also affected by maleic acid dose-dependent.DiscussionThe exposure to maleic acid is found to decrease the cellular calcium and thiol levels in human neuronal cells at both transcriptional and functional levels. This study reported the first transcriptomic profiling of human neuronal cells treated with maleic acid. It is also the first experimental validation of chemical effects predicted by in silico toxicogenomics approaches. The proposed approach may be useful in understanding the potential effects of other poorly characterized chemicals on human health.
| S-EPMC5384566 | biostudies-literature
Project description:OBJECTIVE:Isatin has gained attention in recent years owing to its anticancer properties and is thought to offer medical benefits. Isatin is an endogenous oxidized indole with various behavioral and metabolic properties and is commonly found in mammalian tissues and fluids. It has several plausible applications in biomedical research and has also been investigated as a potential anticancer agent. However, its effects on neuroblastoma (NB) cells are unclear. Here, we evaluate the effects of isatin on neuroblastoma cell metastasis and invasion and reveal the underlying mechanism. METHODS:NB cell viability was evaluated with the cell counting kit (CCK)-8 assay. NB cell invasion and migration abilities were tested with transwell and wound healing experiments. The relative mRNA expression of associated molecules was detected with real-time polymerase chain reaction (RT-PCR) and quantitative PCR. The expression level of related proteins was detected with western blotting. RESULTS:Isatin inhibited the proliferation, invasion, and migration of neuroblastoma cells in a dose-dependent manner. Isatin increased the expression level of H3K4m1 and phosphatase and tensin homolog (PTEN) and decreased the phosphorylation level of PTEN downstream proteins phosphoinositide 3-kinase, protein kinase B, mammalian target of rapamycin, focal adhesion kinase, and SHC. Together, these results support the potential anti-metastatic effects of isatin on NB cells.
| S-EPMC6949577 | biostudies-literature
Project description:The beta-adrenoceptor blockers exhibit a well-characterized anti-apoptotic property in the heart and kidney while less is known about the effect of this class of drugs on neuronal apoptosis. We studied the effects of three beta-adrenoceptor blockers propranolol (1-(isoproplyamino)-3-(naphthalene-1-yloxy)propan-2-ol), atenolol (2-[4-[2-hydroxy-3-(1-methylethylamino)propoxyl]phenyl]ehanamide), and ICI 118551 (1-[2,3-(dihydro-7-methyl-1H-iden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol), against staurosporine-induced apoptosis in SH-SY5Y human neuroblastoma cells. Staurosporine increased caspase 3-like activity, DNA fragmentation, PARP cleavage, and the number of TUNEL positive cells consistent with the induction of apoptosis. Propranolol and ICI 118551, but not atenolol, demonstrated a concentration-dependent inhibition of caspase 3-like activity. Propranolol and ICI 118551 directly inhibited the enzymatic activity of recombinant caspase 9 while atenolol did not; however, none of the beta-adrenoceptor blockers that were examined directly blocked caspases 2 or 3 activity. In isolated mitochondria, propranolol and ICI 118551 inhibited staurosporine-induced cytochrome c release while atenolol did not. We conclude that propranolol and ICI 118551 protect SH-SY5Y cells against staurosporine-induced apoptosis through a dual action on the mitochondria and on caspase 9 in a cell type and an apoptotic paradigm where the conventional inhibitors of mitochondrial permeability transition such as cyclosporin A and bongkrekic acid demonstrate no protection.
| S-EPMC2529477 | biostudies-literature
Project description:Parkinson's disease (PD) is characterized by progressive dopaminergic neuron loss or dysfunction and is the second most prevalent neurodegenerative disorder after Alzheimer's disease. However, current therapeutic strategies for PD are limited to treating the outcomes of this disease rather than preventing it. Sinapic acid (SA) is a phenolic compound with potential antioxidant properties, which reportedly acts as a therapeutic agent against many diseases including cancer, as well as cardiac and liver diseases. However, little is known about the effects of SA against neurodegenerative disorders. Therefore, our study sought to evaluate the neuroprotective effects of non-cytotoxic concentrations of SA against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in SH-SY5Y human neuroblastoma cells, which we used as an in vitro PD model. SA increased cell viability and rescued the cells from 6-OHDA-induced apoptotic cell death. Additionally, oxidative stress responses were significantly blocked by SA, including reactive oxygen species (ROS) overproduction and decreased expression levels of antioxidant proteins. Notably, SA also attenuated mitochondrial dysfunction and endoplasmic reticulum (ER) stress. Moreover, SA dramatically inhibited the activation of mitogen-activated protein kinase (MAPK) proteins. Taken together, our findings highlight the potential PD prevention effects of SA, as well as its underlying mechanisms, making this compound a promising prevention and treatment agent for PD.
| S-EPMC8000777 | biostudies-literature