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)

Calmodulin (CaM) mediates the Ca(2+)-dependent activation of many enzyme systems in accordance with its cellular localization. We have described previously a muscarinic receptor-mediated translocation of CaM from membranes into the cytosol of SK-N-SH human neuroblastoma cells. To explore the potential targets (CaM-binding proteins, CaMBP) for CaM upon translocation, a photoreactive CaM derivative was introduced into living SK-N-SH cells using a scrape-loading technique. Scrape-loading incorporated rhodamine isothiocyanate-labeled CaM with an efficiency of 38%. CaM-diazopyruvamide (CaM-DAP), a Ca(2+)-dependent and CaM-specific probe, was also introduced into the cells. The muscarinic agonist carbachol stimulated a translocation of CaM from membranes into cytosol in CaM-DAP-loaded SK-N-SH cells. Upon photochemical cross-linking, cross-linked adducts of CaM-CaMBP were detected by immunoblotting with anti-CaM antibody. Carbachol stimulated increased photoaffinity labeling of three proteins with relative adduct molecular masses of 70, 120, and 180 kDa. The time course of labeling for the 70- and 120-kDa adducts showed maximal increased by 15-30 min. The 180-kDa adduct displayed a slower time course of maximal labeling, with increases maintained for 2-4 h. Subtracting the molecular mass of CaM, carbachol stimulated binding to CaMBPs of 55, 105, and 163 kDa. Predominant cellular CaMBP were identified using a biotinylated CaM overlay procedure. Western blot analysis indicated the expression of specific CaM-dependent enzymes such as calcineurin, phosphodiesterase, the beta-isoform (rat brain) of CaM kinase II, and Ca(2+)-ATPase. Numerous cytoskeletal CaMBP were expressed such as microtubule-associated protein-2, spectrin, tubulin, caldesmon, adducin, and neuromodulin. Of the CaMBP expressed, phosphodiesterase, calcineurin, caldesmon, and adducin cross-linked with CaM-DAP in the loaded SK-N-SH cells. Carbachol stimulated the time-dependent CaM-DAP labeling of calcineurin and adducin. This study demonstrates the novel incorporation of a photoreactive CaM derivative into living cells, as well as muscarinic receptor-activated CaM-DAP interaction with several cellular CaMBP. We postulate that carbachol-stimulated CaM translocation in SK-N-SH cells may affect the activity of CaM-dependent enzymes and may alter aspects of cytoskeletal function.
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PMID:Carbachol stimulates binding of a photoreactive calmodulin derivative to calmodulin-binding proteins in intact SK-N-SH human neuroblastoma cells. 155 1

1. We compared the binding properties of 4-diphenyl-acetoxy-N-methyl-piperidine methiodide (4-DAMP) and nine analogues of this compound on muscarinic receptors of human neuroblastoma NB-OK1 cells (M1 subtype), rat heart (M2 subtype), rat pancreas (M3 subtype) and to the putative M4 subtype in striatum. 2. The requirements for high affinity binding were somewhat different for the four receptor subtypes. In general, the requirements of M3 receptors were more stringent than for M1, M2 or putative M4 receptors. 3. The abilities of the compounds to discriminate muscarinic receptor subtypes were not correlated with their affinities at any subtype. 4. The temperature-dependence of binding of 4-DAMP analogues to M2 receptors varied with the drug structure. In particular, the increased affinity of the alpha-methyl derivative of 4-DAMP could be ascribed to van der Waals interactions. 5. The affinities of most 4-DAMP analogues for M2 and M3 receptors were similar to their pharmacological potencies on atrial and ileum preparations, respectively. 6. At concentrations above 1 microM, all 4-DAMP analogues as well as atropine, reduced the [3H]-N-methyl scopolamine ([3H]-NMS) dissociation rate from cardiac muscarinic receptors, with no obvious structure-activity relationship.
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PMID:Binding properties of nine 4-diphenyl-acetoxy-N-methyl-piperidine (4-DAMP) analogues to M1, M2, M3 and putative M4 muscarinic receptor subtypes. 159 94

Muscarinic receptors in N1E 115 mouse neuroblastoma cells were characterized by competition binding experiments using three agonists and five antagonists, including 4-DAMP and AF-DX 116, and by studying the effect of agonist stimulation on the cellular cAMP and cGMP content. The results of the binding studies with the antagonists suggest that only one single homogeneous binding site of the M1 muscarinic receptor subtype is present. For the binding with the agonists, two binding sites were detected, one with high affinity for the ligand (between 53 and 77% of the total binding sites depending on the agonist) and one with low affinity. In contrast to the results obtained with the binding experiments using antagonists, the study of the cellular cyclic nucleotide response upon carbachol stimulation suggested the presence of both the M1 and M2 subtypes as there was an increase in cyclic GMP concentration while at the same time, the prostaglandin-stimulated synthesis of cyclic AMP was inhibited. Considering both binding and functional data we suggest that in N1E 115 cells a majority of M1 and a minority of M2 muscarinic receptors are present; there is no evidence for the presence of M3 muscarinic receptors.
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PMID:Study of the muscarinic receptor subtypes in N1E 115 mouse neuroblastoma cells. 164 47

Quinacrine altered the equilibrium binding of the muscarinic antagonist quinuclidinyl benzilate (QNB) to the muscarinic receptor in membranes from N18TG2 neuroblastoma cells. At 1 microM quinacrine, the apparent Kd for [3H]QNB binding was shifted from 48 to 210 pM without a decrease in the maximum binding. The competition binding profile of quinacrine indicates that it displaced [3H]QNB binding with an IC50 of 460 nM in a manner consistent with competitive inhibition. Quinacrine blocked the muscarinic inhibition of cyclic AMP accumulation by carbachol in cultured N18TG2 neuroblastoma cells without affecting arachidonic acid release or Gi coupling to the adenylate cyclase. The disruption of muscarinic receptor function by quinacrine may provide an explanation for its blockade of the muscarinic response in cells.
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PMID:Muscarinic receptor binding is inhibited by quinacrine. 165 14

The mechanisms of muscarinic receptor-linked increase in cAMP accumulation in SH-SY5Y human neuroblastoma cells has been investigated. The dose-response relations of carbachol-induced cAMP synthesis and carbachol-induced rise in intracellular free Ca2+ were similar. The stimulated cAMP synthesis was inhibited by about 50% when cells were entrapped with the Ca2+ chelator BAPTA or in the presence of the protein kinase C (PKC) inhibitor staurosporine. Production of cAMP could be induced also by the Ca2+ ionophore, ionomycin and by TPA, an activator of PKC. When added together TPA and ionomycin had a synergistic effect. When cAMP synthesis was activated with cholera toxin, PGE1 or PGE1 + pertussis toxin carbachol stimulated cAMP production to the same extent as in control cells. Ca2+ and protein kinase C thus seem to be the mediators of muscarinic-receptor linked cAMP synthesis by a direct action on adenylate cyclase.
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PMID:Muscarinic receptor-linked elevation of cAMP in SH-SY5Y neuroblastoma cells is mediated by Ca2+ and protein kinase C. 165 8

The relationship between muscarinic receptor activation of phosphoinositide hydrolysis and the sequestration of cell surface muscarinic receptors has been examined for both intact and digitonin-permeabilized human SK-N-SH neuroblastoma cells. Addition of the aminosteroid 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1H-pyrrole-2,5-dione (U-73122) to intact cells resulted in the inhibition of oxotremorine-M-stimulated inositol phosphate release and of Ca2+ signaling by greater than 75%. In contrast, when phospholipase C was directly activated by the addition of the calcium ionophore ionomycin, inclusion of U-73122 had little inhibitory effect. Addition of U-73122 to intact cells also inhibited the agonist-induced sequestration of cell surface muscarinic receptors and their subsequent down-regulation with an IC50 value (4.1 microM) similar to that observed for inhibition of inositol phosphate release (3.7 microM). In contrast, when oxotremorine-M-stimulated phosphoinositide hydrolysis was inhibited by depletion of extracellular Ca2+, no reduction in the extent of receptor sequestration was observed. When introduced into digitonin-permeabilized cells, U-73122 more markedly inhibited inositol phosphate release elicited by either oxotremorine-M or guanosine-5'-O-(3-thiotriphosphate) than that induced by added Ca2+. Addition of oxotremorine-M to permeabilized cells resulted in muscarinic receptor sequestration and down-regulation. Both the loss of muscarinic acetylcholine receptors and activation of phosphoinositide hydrolysis in permeabilized cells were inhibited by the inclusion of guanosine-5'-O-(2-thiodiphosphate). The results indicate that the agonist-induced sequestration of muscarinic acetylcholine receptor in SK-N-SH cells requires the involvement of a GTP-binding protein but not the production of phosphoinositide-derived second messenger molecules.
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PMID:The aminosteroid U-73122 inhibits muscarinic receptor sequestration and phosphoinositide hydrolysis in SK-N-SH neuroblastoma cells. A role for Gp in receptor compartmentation. 166 Aug 86

The receptor-mediated generation of an endothelial-derived relaxing factor (EDRF)-free radical intermediate in a neuronal cell line detected by spin trapping techniques has been reported. Here we report the time course of the appearance of the 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) spin adduct and cyclic GMP formation following addition of carbamylcholine to suspensions of cultured mouse neuroblastoma cells (clone N1E-115). The time course of the appearance of the DBNBS spin adduct shows that spin adduct formation decreases possibly reaching a minimum approximately between 35 and 40 s. This is inversely proportional to cGMP formation which reaches a maximum at approximately 40 s after carbamylcholine activation. In addition, the inhibitory effect of NG-monomethyl-L-arginine (NMMA), potassium ferricyanide, K3Fe(CN)6 and methylene blue in cytosol preparation was investigated. A mechanism is proposed that essentially accounts for the combined results observed by spin trapping/electron paramagnetic resonance (EPR) study providing direct evidence for the muscarinic receptor-mediated formation of a labile, diffusible precursor of nitric oxide (NO.) derived from L-arginine that activates soluble guanylate cyclase.
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PMID:Activation of cyclic GMP formation in mouse neuroblastoma cells by a labile nitroxyl radical. An electron paramagnetic resonance/spin trapping study. 168 65

Dopamine or agonists with D1 receptor potency stimulated cyclic AMP (cAMP) accumulation in whole cell preparations of NS20Y neuroblastoma cells. The accumulation of cAMP after D1 stimulation was rapid and linear for 3 min. Both dopamine and the novel D1 receptor agonist dihydrexidine stimulated cAMP accumulation two- to three-fold over baseline. The pseudo-Km for dopamine was approximately 2 microM, whereas for dihydrexidine it was approximately 30 nM. The effects of both drugs were blocked by either the D1-selective antagonist SCH23390 (Ki, 0.3 nM) or the nonselective antagonist (+)-butaclamol (Ki, 5 nM). Both (-)-butaclamol and the D2-selective antagonist (-)-sulpiride were ineffective (Ki greater than 3 microM). Forskolin (10 microM), prostaglandin E1 (1 microM), and adenosine (10 microM) also stimulated cAMP accumulation, but none were antagonized by SCH23390 (1 microM). Finally, muscarinic receptor stimulation (100 microM carbachol) inhibited both D1- and forskolin-stimulated increases in cAMP accumulation by 80%. The present results indicate that NS20Y neuroblastoma cells have D1 receptors that are coupled to adenylate cyclase, and that these receptors have a pharmacological profile similar to that of the D1 receptor(s) found in rat striatum.
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PMID:D1 dopamine receptors of NS20Y neuroblastoma cells are functionally similar to rat striatal D1 receptors. 171 49

Quantitative changes in the total mass and the molecular species of 1,2-diacyl-sn-glycerol (DAG) and phosphatidic acid (PA) formed upon muscarinic receptor activation were studied in cultured human SK-N-SH neuroblastoma cells. DAG was isolated from the total lipid extracts of carbachol (CCh)-stimulated and unstimulated cells and after benzoylation, was subjected to reverse phase high performance liquid chromatography to separate the component species. The molecular species of DAG were identified by analyzing the fatty acid composition of each separated fraction by gas chromatography, and their total and individual masses were quantified from the known amount of an internal standard, 1,2-distearoyl-sn-glycerol, added during the extraction of the lipid. Relatively high basal levels of DAG (1.5 nmol/mg protein) are present in these cells, and addition of CCh elicited a 50-60% increase in the total amounts of DAG within 5 min. The increase was biphasic: an initial major peak at 5 min was followed by a sustained increase that persisted for at least 30 min. An increase in DAG was elicited by both full and partial muscarinic agonists and was blocked by atropine. The presence of extracellular Ca2+ was necessary for muscarinic receptor-activated formation of DAG. To determine the source of the DAG, the molecular species of the major phospholipids present in SK-N-SH cells were also analyzed. The phospholipids were first enzymatically hydrolyzed to DAGs which were then analyzed as described above. A number of unusual fatty acids, the major one being 20:3 (n-9), were present in these lipids especially in the phosphoinositides and also in the DAG formed after CCh stimulation. Within 5 s of CCh stimulation there were transient increases in the DAG species representative of phosphoinositides. By 5 min the newly formed molecular species of DAG resembled a mixture of phosphoinositides and phosphatidylcholine (PC). Quantitative comparison of the molecular species compositions of phosphoinositides, PC, and newly formed DAGs indicated that at time periods up to 10 min, approximately 30% of the DAG originated from the phosphoinositides and the rest from PC. At longer intervals (greater than 20 min), most (85%) of DAGs originated from PC. Activation of muscarinic receptors in SK-N-SH cells also elicited an increase in PA (200% in 5 min). A quantitative molecular species analysis, using 1,2-distearoyl-sn-glycerol-3-P as internal standard, was performed by enzymatic (alkaline phosphatase) hydrolysis of PA to DAG and subsequent analysis.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Quantitative analysis of molecular species of diacylglycerol and phosphatidate formed upon muscarinic receptor activation of human SK-N-SH neuroblastoma cells. 174 76

IMR-32 and SK-N-MC cells were found to contain [3H]quinuclidinyl benzilate specific binding sites inhibited by pirenzepine in a manner suggesting the presence of both M1-type and M2-type muscarinic receptor recognition sites. Neither cell had detectable [3H]8-OH-DPAT binding sites. Carbachol stimulated the rate of inositol phospholipid breakdown in IMR-32 and SK-N-MC human neuroblastoma cells with an EC50 value of about 50 microM in both cases. Pirenzepine inhibited the carbachol (100 microM)-stimulated inositol phospholipid breakdown in both cells with Hill slopes of unity and IC50 values of 15 nM (IMR-32) and 12 nM (SK-N-MC). The 5-HT1A receptor agonist 8-OH-DPAT competitively inhibited carbachol-stimulated inositol phospholipid breakdown with pA2 values of 5.78 (IMR-32) and 5.61 (SK-N-MC). These values are consistent with the inhibitory potency of 8-OH-DPAT towards [3H]quinuclidinyl benzilate binding in these cells. The 5-HT agonists 5-MeODMT and buspirone at micromolar concentrations inhibited carbachol-stimulated breakdown in IMR-32 cells. The inhibition by 8-OH-DPAT and 5-MeODMT was not affected by preincubation with (-)alprenolol. 5-HT (10-100 microM) was without effect on either basal or carbachol-stimulated breakdown. It is concluded that IMR-32 and SK-N-MC neuroblastoma cells express muscarinic M1-type but not serotoninergic receptors coupled to phosphoinositide-specific phospholipase C. 8-OH-DPAT acts as a weak antagonist at these muscarinic receptors.
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PMID:Antagonism by 8-hydroxy-2(di-n-propylamino)tetraline and other serotonin agonists of muscarinic M1-type receptors coupled to inositol phospholipid breakdown in human IMR-32 and SK-N-MC neuroblastoma cells. 182 86


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