Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027819 (neuroblastoma)
 27,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Stimulation of muscarinic receptors expressed in SH-SY5Y human neuroblastoma cells resulted in a complex profile of inositol 1,4,5-trisphosphate (InsP3) accumulation, with a dramatic increase (six- to eightfold) over the first 10 s (the "peak" phase) and subsequently from approximately 60 s onward, maintained at a lower but sustained level (the "plateau" phase). Chelation of extracellular Ca2+ with EGTA or inhibition of Ca2+ channels with Ni2+ showed that the plateau phase was dependent upon Ca2+ entry. Furthermore, use of thapsigargin and EGTA to discharge and sequester Ca2+ from intracellular stores revealed that Ca2+ from this source was capable of supporting the peak phase of the InsP3 response. Carbachol-stimulated phosphoinositidase C activity in permeabilized SH-SY5Y cells was also shown to be highly dependent on free Ca2+ concentration (20-100 nM) and suggests that under normal conditions, InsP3 formation is enhanced by increases in cytosolic free Ca2+ concentration that accompany muscarinic receptor activation. Measurement of carbachol-stimulated total inositol phosphate accumulation in the presence of Li+ indicated that the initial rate of phosphoinositide hydrolysis (from 0 to 30 s) was about fivefold greater than that from 30 to 300 s. This rapid but partial desensitization of receptor-mediated phosphoinositide hydrolysis provides strong evidence for the mechanism underlying the changes in InsP3 accumulation over this time. Because very similar data were obtained in Chinese hamster ovary cells transfected with human m3 receptor cDNA, we suggest that although increases in cytosolic free CA2+ concentration amplify InsP3 formation during stimulation of m3 muscarinic receptors, the primary factor that governs the profile of InsP3 accumulation is rapid, but partial, desensitization.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Muscarinic receptor-mediated inositol 1,4,5-trisphosphate formation in SH-SY5Y neuroblastoma cells is regulated acutely by cytosolic Ca2+ and by rapid desensitization. 820 26

The psychotherapeutic action of Li+ in brain has been proposed to result from the depletion of cellular inositol secondary to its block of inositol monophosphatase. This action is thought to slow phosphoinositide resynthesis, thereby attenuating stimulated phosphoinositidase-mediated signal transduction in affected cells. In the present study, the effect of Li+ on muscarinic receptor-stimulated formation of the immediate precursor of phosphatidylinositol, CDP-diacylglycerol (CDP-DAG), has been examined in human SK-N-SH neuroblastoma cells that have been cultured under conditions that alter the cellular content of myo-inositol. Resting neuroblastoma cells, like brain cells in vivo, were found to concentrate inositol from the culture medium, achieving an intracellular level of 60.0 +/- 4 nmol/mg of protein. The addition of carbachol to [3H]cytidine-prelabeled cells elicited a four- to fivefold increase in the accumulation of labeled CDP-DAG. This stimulated formation of [3H]CDP-DAG was completely blocked by the addition of 10 microM atropine, was not dependent on the presence of Li+, nor was it affected by co-incubation with myo-inositol. This result was in sharp contrast to findings in rat brain slices, in which carbachol-stimulated formation of [3H]CDP-DAG was potentiated approximately 10-fold by Li+ and substantially reduced by coincubation with inositol. The formation of [3H]CDP-DAG in labeled SK-N-SH cells by carbachol was both concentration and time dependent. The order of efficacy of muscarinic ligands in stimulating [3H]-CDP-DAG accumulation paralleled that established in these cells for inositol phosphate accumulation, i.e., carbachol > or = oxotremorine-M > bethanecol > or = arecoline > oxotremorine > pilocarpine.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Lithium enhances muscarinic receptor-stimulated CDP-diacylglycerol formation in inositol-depleted SK-N-SH neuroblastoma cells. 845 27

The human neuroblastoma line SH-SY5Y expresses three muscarinic receptor genes (m1, m2, and m3). In this study, we have investigated the effect of agonist exposure on the steady state levels of each muscarinic receptor transcript, using a comparative polymerase chain reaction (PCR) assay that allows changes in levels of very rare transcripts to be monitored. Northern blot analysis of cellular RNA revealed the presence of m3 mRNA, whereas PCR amplification of SH-SY5Y cDNA additionally revealed the presence of m1 and m2 transcripts. Cell surface muscarinic receptor number, as assessed by N-[3H]methylscopolamine binding to whole cells, rapidly decreased to 42% of control levels within 1 hr of exposure to 100 microM carbachol; this was followed by a slower decline to 6% of control levels after 48 hr. Total receptor number, measured by binding of [3H]quinuclidinyl benzilate, showed a much slower decline to 21% of control levels after 48 hr of treatment. Comparative PCR analysis showed that each muscarinic transcript was differentially regulated. The level of transcript encoding the major receptor population, the m3 mRNA, was rapidly elevated within 1 hr of agonist challenge and subsequently decreased to about 30% of prestimulation levels within 9 hr; this decrease was sustained for the time course of the experiment. m2 mRNA levels showed a transient increase followed by a decrease to 30% of prestimulation levels after 6 hr but, in contrast to the m3 transcripts, this depression was followed by a transient rise to 270% of prestimulation levels after 24 hr before declining to normal levels by 72 hr after stimulation. Exposure of cells to agonist clearly instigates a complex pattern of changes in levels of receptor and receptor mRNA; comparison of the relative time courses of these changes indicates that the decline in m3 transcripts precedes the loss of muscarinic receptor binding sites.
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PMID:Differential regulation of muscarinic receptor mRNA levels in neuroblastoma cells by chronic agonist exposure: a comparative polymerase chain reaction study. 850 26

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.
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PMID:Muscarinic receptor sequestration in SH-SY5Y neuroblastoma cells is inhibited when clathrin distribution is perturbed. 852 52

Cholinergic muscarinic receptor genes are members of the G-protein receptor gene superfamily. In this study we describe the structure of the gene and promoter of the rat m4 muscarinic receptor gene. A rat cosmid clone containing the coding region for the m4 gene and 25 kilobases of upstream sequence was isolated. This clone directed expression of the rat m4 gene when introduced in IMR32 cells, a human neuroblastoma that expresses m4, but did not drive expression when introduced into Chinese hamster ovary cells, a line that does not express the m4 gene. S1 nuclease, modified 5'-rapid amplification of cDNA ends and polymerase chain reaction analysis of rat cosmid DNA and cDNA showed that the gene consists of a 2.6-kilobase coding exon, extending 34 base pairs (bp) upstream from the initiating ATG, separated from a 460-493 bp noncoding exon by a 4.8-kilobase intron. DNA sequence analysis shows that the non-coding exon is GC-rich and that the promoter does not contain a TATA or CAAT box and has several consensus sequences for enhancer elements including five Sp-1 binding sites, one AP-2 site, one AP-3 binding site and two E-boxes within the proximal 600 bp. A reporter construct consisting of 1440 bp of flanking DNA and 80 bp of the first exon cloned into a luciferase reporter plasmid, drove cell specific expression in transient transfection assays. Removal of 1088 bp of the 5' end of this construct resulted in expression in non-m4 expressing cell lines suggesting there is a repressor element in this region.
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PMID:Structure of the m4 cholinergic muscarinic receptor gene and its promoter. 853 49

The muscarinic receptor-stimulated mobilisation of calcium ions in SH-SY5Y neuroblastoma cells was measured as function of the concentration of seven muscarinic receptor agonists and partial agonists: carbamoylcholine, acetylcholine, propionylcholine, butyrylcholine, acetylthiocholine, methylfurmethide and tetrametylammonium. The dose-response curves reached a clear maximum followed by a downturn of the curve. The concentration interval where the activatory and inhibitory effects occurred depended on the structure of the ligand. The bell-shaped dose-response curves were analysed assuming that the drugs interact with two sites, which are responsible for agonistic and antagonistic effects, on the muscarinic receptors. The results indicate that full vs. partial agonism is at least in part determined by relative affinities of these two sites.
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PMID:Dual effect of muscarinic receptor agonists on Ca2+ mobilization in SH-SY5Y neuroblastoma cells. 854 46

The present study was designed to determine to determine the in vitro affinity profile of (R)-(-)-dimethindene and (S)-(+)-dimethindene at muscarinic receptor subtypes using both functional and binding assays. In addition, the racemate was investigated in functional studies. The functional muscarinic receptors studied were putative M1 receptors in rabbit vas deferens and rat duodenum, M2 receptors in guinea-pig left atria and rabbit vas deferens, as well as M3 receptors in guinea-pig ileum and trachea. Furthermore, the histamine H1 antagonism by (R)-(-)- and (S)-(+)-dimethindene has been examined in guinea-pig ileum. Muscarinic binding selectivity was assessed in homogenates from human neuroblastoma NB-OF 1 cells (M1), rat heart (M2), pancreas (3) and striatum (M4). The results demonstrate that (S)-(+)-dimethindene is a potent M2-selective muscarinic receptor antagonist (pA2 = 7.86/7.74; pKi = 7.78) with lower affinities for the muscarinic M1 (pA2 = 6.83/6.36; pKi = 7.08), the M3 (pA2 = 6.92/6.96; pKi = 6.70) and the M4 receptors (pKi = 7.00), respectively. The (S)-(+)-enantiomer was more potent (up to 41-fold) than the (R)-(-)-enantiomer in all muscarinic assays. In contrast, the stereoselectivity was inverse at histamine H1 receptors, the (R)-(-)-enantiomer being the eutomer (pA2 = 9.42; pA2/(S)-isomer = 7.48). In conclusion, (S)-(+)-dimethindene is a useful tool to investigate muscarinic receptor heterogeneity. In addition, this lipophilic compound might become the starting point for the development of M2-selective muscarinic receptor antagonists useful as diagnostic tools for quantifying muscarinic M2 receptors in the central nervous system with positron emission tomography imaging, and to test the hypothesis that muscarinic M2 receptor antagonists show beneficial effects in the treatment of cognitive disorders.
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PMID:The (S)-(+)-enantiomer of dimethindene: a novel M2-selective muscarinic receptor antagonist. 860 84

Agonist-induced decrease of surface muscarinic receptor number occurs in a number of cell lines. Recent work has suggested that some muscarinic receptor subtypes undergo internalization, whereas others do not. We investigated the agonist-induced trafficking of various muscarinic receptor subtypes transfected into CHO cells and compared it with the trafficking of receptors expressed natively in neuronal cells, fibroblasts, or epithelial cells. SH-SY5Y neuroblastoma cells, which express predominantly the m3 receptor subtype, show qualitatively similar changes in surface receptor number in response to agonist stimulation to those occurring in NG108-15 cells, which express predominantly the m4 subtype. The rate constants for internalization, however, were considerably different, indicating that receptors in SH-SY5Y cells show a much faster turnover than those in NG108-15 cells. In the transfected cells, the muscarinic receptor subtypes m1 and m3, which are coupled to second messenger systems via Gq/11, showed little agonist-induced loss of surface receptors. In contrast, the muscarinic receptor subtypes m2 and m4, which are coupled via Gi or G(o), showed a substantial loss of surface receptors after treatment with agonist. An interesting implication of this result is that agonist-induced receptor trafficking can still occur efficiently, even at very high receptor densities. Significant agonist-induced internalization also occurs in a fibroblast line (HeLa) and an epithelial cell line (HT29), both of which express predominantly m3 receptors. Our results suggest that the extent and rate of the loss of receptors from the cell surface in response to agonist stimulation are governed by both the receptor subtype and the cell type in which it is expressed.
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PMID:Intracellular trafficking of the muscarinic acetylcholine receptor: importance of subtype and cell type. 863 69

The effects of protein kinase C (PKC) activation on muscarinic receptor-mediated phosphoinositide and Ca2+ signalling were examined in the human neuroblastoma, SH-SY5Y. Carbachol evoked rapid transient elevations of Ins(1,4,5)P3 and intracellular [Ca2+] followed by lower sustained elevations. Phorbol 12,13-dibutyrate (PDBu) preferentially attenuated transient phases. Removal of the transplasmalemmal Ca2+ gradient coupled with depletion of intracellular Ca2+ stores with thapsigargin also reduced carbachol-mediated Ins(1,4,5)P3 accumulation. Under these conditions, PDBu virtually abolished Ins(1,4,5)P3 responses to carbachol thereby implicating both Ca(2+)- and PKC-sensitive components. PDBu also reduced agonist-mediated accumulation of inositol phosphates and depletion of lipids, thereby eliminating an effect of PKC on Ins(1,4,5)P3 metabolism or phosphoinositide synthesis. In electroporated cells, PDBu inhibited Ins(1,4,5)P3 accumulation mediated by carbachol or guanosine 5'-[gamma-thio]-triphosphate, the latter indicating that some PDBu-sensitive elements were downstream of the receptor. The PKC inhibitor, Ro-318220, protected against PDBu but did not enhance responses to maximal concentrations of carbachol, indicating no feedback inhibition by agonist-activated PKC. Muscarinic antagonist activity of Ro-318220 complicated such assessment at low agonist concentrations. Carbachol or PDBu induced cytosol to membrane translocation of PKC alpha. This was faster and possibly greater with PDBu, which may explain the lack of feedback by agonist-activated PKC. These results indicate that, in SH-SY5Y cells, PDBu activation of PKC preferentially inhibits rapid muscarinic receptor-mediated phosphoinositide and Ca2+ responses via suppression of PtdIns(4,5)P2 hydrolysis. This is at least partially through inhibition of Gq-protein/phosphoinositidase C coupling. However, at least at high agonist concentrations, a major agonist-mediated PKC feedback is not present in these cells.
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PMID:Contrasting effects of phorbol ester and agonist-mediated activation of protein kinase C on phosphoinositide and Ca2+ signalling in a human neuroblastoma. 867 Jan 70

We have examined the effects of two intravenous anaesthetic induction agents, propofol and thiopentone, on K+ and carbachol evoked [3H]noradrenaline release from a human neuroblastoma cell line, SH-SY5Y. In this model, we have previously demonstrated that K+ evoked [3H]noradrenaline release was dependent on Ca2+ entry and carbachol evoked release was extracellular Ca(2+)- independent. Propofol inhibited K+ (100 mM)-evoked (IC50 of 42 +/- 11 microM), but not carbachol (1 mM)-evoked, [3H]noradrenaline release. Thiopentone inhibited both K+- and carbachol-evoked release with IC50 values of 116 +/- 15 microM and 169 +/- 39 microM, respectively. These inhibitory effects were not due to changes in the release dynamics, as assessed using perfused cells. Furthermore, thiopentone inhibition of carbachol-evoked release was not due to muscarinic receptor antagonism. Both propofol and thiopentone caused noncompetitive inhibition of K+-stimulated Ca2+ influx, with IC50 values of 127 +/- 7 microM and 121 +/- 10 microM, respectively. These effects were not due to interaction with GABAA receptors, but suggest that both compounds block voltage-sensitive Ca2+ channels. Thiopentone, but not propofol, inhibited carbachol-stimulated increased intracellular Ca2+ concentrations in the presence and absence of extracellular Ca2+. However, thiopentone had no effect on carbachol-stimulated inositol (1,4,5)-triphosphate formation, suggesting that thiopentone may directly inhibit Ca2+ release from intracellular stores.
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PMID:Effects of propofol and thiopentone on potassium- and carbachol-evoked [3H]noradrenaline release and increased [Ca2+]i from SH-SY5Y human neuroblastoma cells. 868 76


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