Coupling of muscarinic receptors to the mobilization of intracellular Ca2+ stores in permeabilized SH-SY5Y human neuroblastoma cells.
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ABSTRACT:
SUBMITTER: Wojcikiewicz RJ
PROVIDER: S-EPMC1149690 | biostudies-other | 1990 Nov
REPOSITORIES: biostudies-other
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Project description:This study reports increased intracellular Ca2+ and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in response to muscarinic-cholinergic stimulation of human neuroblastoma (SH-SY5Y) cells. Carbachol stimulation leads to a rapid increase in intracellular Ca2+ and Ins(1,4,5)P3 mass, both reaching a peak at around 10 s and then declining to a new maintained phase significantly above basal. Dose-response analysis of peak and plateau phases of intracellular Ca2+ shows different agonist potencies for both phases, carbachol being more potent for the plateau phase. The plateau-phase intracellular Ca2+ was dependent on extracellular Ca2+, which is admitted to the cell through a non-voltage-sensitive Ni2(+)-blockable Ca2+ channel. Using a Mn2+ quench protocol, we have shown that Ca2+ entry occurs early during the discharge of the internal stores. The plateau phase (Ca2(+)-channel opening) is dependent on the continued presence of agonist, since addition of atropine closes the Ca2+ channel and intracellular Ca2+ declines rapidly back to basal. We also failed to detect a refilling transient when we added back Ca2+ after intracellular Ca2+ had reached a peak and then declined in Ca2(+)-free conditions. These data strongly suggest that muscarinic stimulation of SH-SY5Y cells leads to a rapid release of Ca2+ from an Ins(1,4,5)P3-sensitive internal store and a parallel early entry of Ca2+ across the plasma membrane.
| S-EPMC1136919 | biostudies-other
Project description:Inositol-polyphosphate-induced Ca2+ mobilization was investigated in saponin-permeabilized SH-SY5Y human neuroblastoma cells. Ins(1,4,5)P3 induced a dose-related release from intracellular Ca2+ stores with an EC50 (concn. giving half-maximal effect) of 0.1 microM and a maximal release of 70%. Ins(1,3,4)P3, DL-Ins(1,4,5,6)P4 and Ins(1,3,4,5,6)P5 did not evoke Ca2+ mobilization in these cells when used at concentrations up to 10 microM. However, Ins(1,3,4,5)P4 was found to release Ca2+ in a dose-related manner, but the response was dependent on the source of Ins(1,3,4,5)P4 used. When commercially available D-Ins(1,3,4,5)P4 was used, the EC50 and maximal response values were 1 microM and 50% respectively, compared with values for chemically synthesized DL-Ins(1,3,4,5)P4 of 2 microM and 25%. The enhanced maximal response of commercial D-Ins(1,3,4,5)P4 was decreased by pretreatment with rat brain crude Ins(1,4,5)P3 3-kinase and was therefore concluded to be indicative of initial Ins(1,4,5)P3 contamination of the Ins(1,3,4,5)P4 preparation. When metabolism of DL-Ins(1,3,4,5)P4 (10 microM) in these cells at 25 degrees C was investigated by h.p.l.c., substantial amounts of Ins(1,4,5)P3 (0.2 microM) and Ins(1,3,4)P3 (0.8 microM) were found to be produced within 3 min. Analysis of DL-Ins(1,3,4,5)P4 incubation with cells at 4 degrees C, however, indicated that metabolism had been arrested ([3H]Ins(1,4,5)P3 detection limits were estimated to be approx. 0.01 microM). When chemically synthesized DL-Ins(1,3,4,5)P4 and incubation conditions of low temperature were used, the Ca2(+)-releasing properties of this compound were established to be 1 microM and 19% for the EC50 and maximal response values respectively. The results obtained strongly suggest that Ins(1,3,4,5)P4 alone has the ability to release intracellular Ca2+. However, in the presence of sub-maximal concentrations of Ins(1,4,5)P3, Ca2+ release appears to be synergistic with Ins(1,3,4,5)P4, but at supramaximal concentrations not even additive effects are observed.
| S-EPMC1149731 | biostudies-other
Project description:The potential Ca2(+)-releasing activity of the inositol tetrakisphosphates Ins(1,3,4,6)P4 and DL-Ins(1,4,5,6)P4 and the inositol pentakisphosphate Ins(1,3,4,5,6)P5 and their effect on Ins(1,4,5)P3- and DL-Ins (1,3,4,5)P4-mediated Ca2+ release were examined in permeabilized SH-SY5Y human neuroblastoma cells. Neither DL-Ins(1,4,5,6)P4 nor Ins(1,3,4,5,6)P5 exhibit Ca2(+)-releasing activity at concentrations up to 10 microM, but Ins(1,3,4,6)P4 releases Ca2+ dose-dependently, with an EC50 value (conen, giving half-maximal effect) of 5.92 +/- 0.47 microM. Maximal response by this tetrakisphosphate (49 +/- 2.5%) is significantly less than that seen with Ins(1,4,5)P3 (60 +/- 3%) and is achieved at a concentration of 30 microM. In the presence of this concentration of Ins(1,3,4,6)P4 the EC50 value for Ins(1,4,5)P3-mediated Ca2+ release increases from 0.12 +/- 0.02 microM to 2.11 +/- 0.51 microM, providing evidence that this naturally occurring inositol tetrakisphosphate may recognize and exhibit its Ca2(+)-releasing activity via the Ins(1,4,5)P3 receptor. DL-Ins(1,3,4,5)P4, however, at its maximally effective concentration (10 microM) does not significantly affect Ins(1,4,5)P3-mediated Ca2+ release, and therefore appears to mediate its Ca2(+)-mobilizing action through a receptor distinct from that for Ins(1,4,5)P3.
| S-EPMC1151159 | biostudies-other
Project description:Short-term pretreatment (9 min) with the phorbol ester 12-myristate 13-acetate (PMA) alone had no effect on the basal release of [3H]noradrenaline ([3H]NA), but enhanced K+ (100 mM)-, acetylcholine (0.1 mM)-, carbachol (1 mM)-, muscarine (1 mM)- and arecoline (1 mM)-evoked release by 2.3-, 6.4-, 3.0-, 2.0- and 2.0-fold respectively in SH-SY5Y cells. Maximum effects of PMA were observed after a 10 min preincubation at a concentration of 0.1 microM. There was a 4-fold decrease in the EC50 values (concentration required for 50% of maximal stimulation) observed for carbachol- and acetylcholine-evoked release of [3H]NA in the presence of PMA. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate did not alter the K(+)- or carbachol-evoked release of [3H]NA. The enhancement of release in the presence of PMA was more potently inhibited by the protein kinase C inhibitors RO 31-7549 [concentration required for 50% inhibition (IC50) = 0.18 microM/bd and RO 31-8220 (IC50 = 0.56 microM) than by either polymyxin-B or H-7. Furthermore, in the absence of PMA, both K(+)- and carbachol-evoked release was inhibited by these antagonists. Atropine, hexahydro-sila-difenidol and pirenzepine antagonized the PMA-enhanced carbachol-evoked release of [3H]NA, with Ki values of 2.75 +/- 0.25 nM, 2.6 +/- 0.64 nM and 294 +/- 17 nM respectively. These values were consistent with the coupling of an M3 muscarinic receptor to the release of [3H]NA in SH-SY5Y cells. Whereas pretreatment with PMA (5 min) enhanced M3-evoked release of [3H]NA, it decreased the muscarinic-agonist-evoked initial peak (greater than 85%) and plateau phase in intracellular Ca2+. These results suggest that noradrenaline release evoked by muscarinic agonists was triggered not only by relatively small changes in Ca2+ but also by activation of protein kinase C.
| S-EPMC1130836 | biostudies-other
Project description:Extracellular application of lysophosphatidic acid (LPA) elevated intracellular Ca(2+) concentration ([Ca(2+)](i)) in human SH-SY5Y neuroblastoma cells. The maximal response to LPA occurred between 0. 1 and 1 microM, at which point [Ca(2+)](i) was increased by approx. 500 nM. This increase was of similar magnitude to that caused by the muscarinic acetylcholine receptor agonist methacholine (MCh), although the initial rate of release by LPA was slower. Both LPA and MCh released Ca(2+) from intracellular stores, as assessed by inhibition of their effects by thapsigargin, a blocker of endoplasmic reticular Ca(2+) uptake, and by the persistence of their action in nominally Ca(2+)-free extracellular medium. Similarly, both agonists appeared to stimulate store-refilling Ca(2+) entry. MCh produced a marked elevation in cellular Ins(1,4,5)P(3) and stimulated [(3)H]InsP accumulation in the presence of Li(+). In contrast, LPA failed to stimulate detectable phosphoinositide turnover. Chronic down-regulation of Ins(1,4,5)P(3) receptor (InsP(3)R) proteins with MCh did not affect Ca(2+) responses to LPA. In addition, heparin, a competitive antagonist of InsP(3)Rs, blocked Ca(2+)-mobilization in permeabilized SH-SY5Y cells in response to MCh or exogenously added Ins(1,4,5)P(3), but failed to inhibit Ca(2+)-release induced by LPA. Elevation of [Ca(2+)](i) elicited by LPA was blocked by guanosine 5'-[beta-thio]-diphosphate, indicating that this agonist acts via a G-protein-coupled receptor. However, pertussis toxin was without effect on LPA-evoked [Ca(2+)](i) responses, suggesting that G(i/o)-proteins were not involved. In the absence of extracellular Ca(2+), N,N-dimethylsphingosine (DMS, 30 microM), a competitive inhibitor of sphingosine kinase, blocked LPA-induced Ca(2+) responses by almost 90%. In addition, MCh-induced Ca(2+) responses were also diminished by the addition of DMS, although to a lesser extent than with LPA. We conclude that LPA mobilizes intracellular Ca(2+)-stores in SH-SY5Y cells independently of the generation and action of Ins(1,4,5)P(3). Furthermore, the Ca(2+)-response to LPA appears to be dependent on sphingosine kinase activation and the potential generation of the putative second messenger sphingosine 1-phosphate.
| S-EPMC1220522 | biostudies-other
Project description:Stimulation of M3 muscarinic receptors expressed by SH-SY5Y cells induced a dose- and time-related increase in the mass of Ins(1,4,5)P3 (basal 38.3 +/- 5.8 pmol/mg of protein) and Ins(1,3,4,5)P4 (basal 6.1 +/- 1.2 pmol/mg of protein). Comparison of radioreceptor mass assays with [3H]inositol labelling showed higher-fold stimulations with the former protocol. The later accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 mass was dependent upon extracellular Ca2+.
| S-EPMC1149832 | biostudies-other
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: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