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)

NS20Y neuroblastoma cells expressing a homogeneous population of D1-dopamine receptors were used in the present study as a model system to investigate the mechanisms of agonist-induced stimulation and desensitization of D1 receptor-coupled adenylyl cyclase activity. Membrane prepared from NS20Y cells showed a pharmacologically specific, dose-dependent increase in cAMP production in response to various dopaminergic agonists. Dopamine exhibited an EC50 of 5 microM, and at 100 microM a maximal stimulation of 3-4-fold over basal enzyme activity was observed, which could be selectively antagonized by the active stereoisomers of SCH-23390 and butaclamol. Preincubation of NS20Y cells with dopamine induced homologous desensitization of D1 receptor-coupled adenylyl cyclase activity, decreasing dopamine- but not prostaglandin-, adenosine-, or forskolin-stimulated cAMP production. Desensitization did not affect the EC50 for dopamine but resulted in an 85-90% reduction in the maximal response. Dopamine-induced desensitization of adenylyl cyclase activity was found to be both dose and time dependent. As early as 5 min after preincubation with dopamine, cAMP production was decreased by 45-50%, with maximal desensitization occurring by 90 min. Preincubation of NS20Y cells with dopamine also induced a decrease in D1 receptor ligand binding activity, as assessed with the radiolabeled antagonist [3H]SCH-23390. This decrease in binding activity occurred more slowly than the loss of enzyme activity, not achieving maximal levels until after 3 hr. [3H]SCH-23390 saturation binding isotherms in control and maximally desensitized NS20Y cell membranes revealed no change in affinity (KD); however, a 65-70% decrease in receptor number (Bmax) was observed. Because the maximal and temporal decrease in D1 receptors does not correlated with the decrease in dopamine-stimulated enzyme activity, the desensitization may involve a functional uncoupling of the D1 receptor in addition to receptor down-regulation. This is further suggested by a loss in high affinity agonist binding observed in agonist/[3H]SCH-23390 competition experiments after desensitization. Removal of dopamine after maximal desensitization/down-regulation results in recovery to control values by 24 hr. This recovery is mostly, but not completely, blocked by protein synthesis inhibitors, suggesting an involvement of receptor degradation in the desensitization process.
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PMID:Agonist-induced desensitization of D1-dopamine receptors linked to adenylyl cyclase activity in cultured NS20Y neuroblastoma cells. 197 40

Dopamine stimulated human neuroblastoma SK-N-MC cells to accumulated cyclic AMP. The D1 agonist SKF (R)-38393 also stimulated cyclic AMP production whereas the response to dopamine was inhibited by the D1 antagonist SCH (R)-23390. Membranes from SK-N-MC cells bound the D1 ligand [125I]SCH 23982 with a Kd of 2.1 nM and a Bmax of 102 fmol/mg protein. Binding was displaced by dopamine, SKF 38393, and SCH 23390. Up to 40% of the receptors were in an agonist high affinity, guanine nucleotide-sensitive state, compared to only 6% in rat striatum. A D1 photoaffinity probe labeled a 72 kDa protein in both SK-N-MC and rat striatal membranes. Thus, SK-N-MC human neuroblastoma cells contain D1 dopamine receptors which are similar to those found in mammalian striatum, but which are more tightly coupled to adenylate cyclase. SK-N-MC cells may be a useful model to investigate the properties and regulation of D1 dopamine receptors.
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PMID:Identification and characterization of functional D1 dopamine receptors in a human neuroblastoma cell line. 215 12

In order to clone the D1 dopamine receptor linked to adenylyl cyclase activation, the polymerase chain reaction was used with highly degenerate primers to selectively amplify a cDNA sequence from NS20Y neuroblastoma cell mRNA. This amplification produced a cDNA fragment exhibiting considerable sequence homology to guanine nucleotide-binding (G)-protein-coupled receptors that have been cloned previously. To characterize this cDNA further, a full-length clone was isolated from a rat striatal library by using the cDNA fragment as a probe. Sequence analysis of this cDNA clone indicated that it is indeed a member of the G-protein-coupled receptor family and exhibits greatest homology with the previously cloned catecholamine receptors. Northern blot analysis of various neural tissues revealed a transcript of approximately 4 kb that was predominantly located in the striatum with lesser amounts in the cortex and retina. In contrast, no mRNA was detected in the cerebellum, hippocampus, olfactory bulb, mesencephalon, or pituitary. In situ hybridization analysis also revealed a high abundance of mRNA in the striatum as well as in the olfactory tubercle. To establish the identity of this cDNA, we performed transient expression experiments in COS-7 cells. [3H]SCH-23390, a D1-selective radioligand, exhibited specific, saturable binding only in cells that were transfected with this cDNA. Competition binding analysis with a variety of dopaminergic ligands demonstrated a D1 dopaminergic pharmacology. In addition, dopamine as well as other D1-selective agonists stimulated cAMP accumulation in transfected COS-7 cells. We conclude that we have cloned a cDNA encoding the D1 dopamine receptor linked to the activation of adenylyl cyclase activity.
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PMID:Molecular cloning and expression of a D1 dopamine receptor linked to adenylyl cyclase activation. 216 56

The recent availability of high specific activity radiolabeled dopaminergic antagonists with specificity for dopamine receptor subtypes has allowed us to screen a wide variety of cultured mammalian cell lines for the presence of D1 and D2 dopamine receptors. Specific receptor binding of the D1 selective antagonists [3H]SCH 23390 and [125I]SCH 23982 was detected in membranes prepared from NS20Y cells, a clonal cell line derived from the C1300 murine neuroblastoma. Saturation analysis of [3H]SCH 23390 binding revealed the presence of saturable, high affinity binding sites with a dissociation constant (Kd) of 575 pM and a receptor density of 138 fmol/mg protein (approximately 9000 receptors/cell). Inhibition of [3H]SCH 23390 binding by a series of dopaminergic agonists and antagonists exhibited appropriate stereoselectivity and pharmacological specificity, verifying the D1 nature of this site. Dopamine inhibition of [3H]SCH 23390 binding revealed the presence of high and low affinity agonist binding sites which were converted to a homogeneous low affinity state by the addition of GppNHp. In membranes prepared from the WERI 27 human retinoblastoma cell line, specific receptor binding of the D2 antagonists [3H]methylspiperone and [125I]NAPS was observed. Saturation analysis of [3H]methylspiperone binding revealed the presence of a single class of high affinity, saturable binding sites with a Kd of 140 pM and a Bmax of 223 fmol/mg protein (approximately 2500 receptor sites/cell). Inhibition of [3H]methylspiperone binding by dopaminergic antagonists exhibited a rank order of potency consistent with the identification of a D2 dopamine receptor subtype. In addition, dopamine inhibition of [3H]methylspiperone binding exhibited both high and low affinity agonist binding sites which were converted to low affinity by the addition of GppNHp. These results represent the first direct demonstration of D1 and D2 dopamine receptors in cultured mammalian clonal cell lines. These cells should provide powerful model systems for investigating the molecular mechanisms involved in dopamine receptor/effector coupling and regulation.
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PMID:Identification and characterization of D1 and D2 dopamine receptors in cultured neuroblastoma and retinoblastoma clonal cell lines. 266 4

Through the technical advances in molecular biology during the past decade, important new insights into the fundamental chromosomal changes associated with brain tumors have been gained. The pace of such research is accelerating, and most of the published reports have appeared outside the neurosurgical literature. Furthermore, many neurosurgeons may not be sufficiently familiar with the terminology and techniques involved to remain abreast of the field. In this review, we discuss through specific examples of recent work on brain tumors the basic techniques of molecular biology, including the Southern and Northern blots, restriction enzyme digestion of DNA, molecular cloning of genes, and mapping of chromosomal deletions. Gene amplification and rearrangements are discussed through review of recent work on the N-myc gene in neuroblastoma and the epidermal growth factor receptor (EGFR) gene in glioblastoma. The molecular cloning of the gli gene from a glioblastoma illustrates the powerful analytic nature of these laboratory techniques and the investigative potential of a cloned gene. The concept of the "recessive oncogene" is discussed through a summary of recent work analyzing restriction fragment length polymorphisms (RFLPs) in families of patients with meningioma, acoustic neurinoma, and bilateral acoustic neurofibromatosis (BANF; NF-2). Throughout this article, emphasis is placed on ways in which molecular biology may soon affect clinical practice.
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PMID:Molecular biology of brain tumors. 305 15

[3H]ICS 205-930 recognition sites were analyzed in membranes prepared from murine neuroblastoma N1E-115 cells. [3H]ICS 205-930 bound rapidly, reversibly, and stereoselectively to a homogeneous population of high affinity recognition sites: Bmax = 40 +/- 5 fmol/mg of protein, pKD = 9.20 +/- 0.05 (n = 11). Nonlinear regression and Scatchard analysis of saturation data suggested the existence of a single class of [3H]ICS 205-930 recognition sites on N1E-115 cells. The affinity of [3H]ICS 205-930 determined in kinetic studies was in agreement with that obtained under equilibrium conditions. Competition studies carried out with a large variety of agonists and antagonists also suggested the presence of a homogeneous population of [3H]ICS 205-930 recognition sites. [3H]ICS 205-930-binding sites displayed the pharmacological profile of a 5-HT3 receptor. Potent 5-HT3 receptor antagonists showed nM affinities for [3H]ICS 205-930-binding sites with the following rank order of potency: SDZ 206-830 greater than SDZ 206-792 greater than ICS 205-930 greater than BRL 43694 greater than quipazine greater than BRL 24924 greater than MDL 72222 greater than GR 38032F. Methiothepine, mCPP, and metoclopramide showed sub-microM affinity. The rank order of potency of agonists was: 5-HT greater than phenylbiguanide = 2-methyl-5-HT much greater than 5-methoxytryptamine = 5-carboxamidotryptamine. All antagonist competition curves were steep (pseudo-Hill coefficients not lower than 1), monophasic, and best fit for a one-site model; 5-HT and 2-methyl-5-HT produced pseudo-Hill coefficients of 1.2-1.4. Drugs acting at 5-HT1, 5-HT2, alpha- and beta-adrenergic, dopaminergic, and histaminergic receptors (methysergide, ketanserin, propranolol, phentolamine, sulpiride, SCH 23390, cimetidine) were essentially inactive at 10 mumol/liter. The binding of [3H]ICS 205-930 was not affected by guanine and adenine nucleotides (GTP, GppNHp, and ATP) at 1 mmol/liter. These nucleotides did not affect the binding of agonists, suggesting that 5-HT3 recognition sites are not coupled to G-proteins. The interactions of agonists and antagonists with [3H]ICS 205-930 recognition sites were competitive in nature, as demonstrated by saturation experiments carried out with [3H]ICS 205-930 in the presence and the absence of unlabeled compounds: apparent Bmax values were not reduced, whereas apparent KD values were increased in the presence of competing ligands.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Identification of serotonin 5-HT3 recognition sites in membranes of N1E-115 neuroblastoma cells by radioligand binding. 335 95

The aim of this study was to investigate the pharmacological characteristics of the 5-hydroxytryptamine-(5-HT)-induced electrical response in cultured neuroblastoma N1E-115 cells of the mouse. In these cells 5-HT induces a transient membrane depolarization, which is associated with a transient inward current, that has been recorded in voltage clamp experiments on whole cells. The peak amplitude of the inward current depends on the concentration of 5-HT applied. Maximum peak inward current was evoked by 10 microM 5-HT and half maximum effect by 2 microM. Responses to 5-HT were blocked by nanomolar concentrations of selective 5-HT3-receptor antagonists, whereas the selective agonist 2-methyl-5-HT mimicked the membrane depolarization induced by 5-HT. A number of agonists and antagonists, which are known to act on 5-HT1-like, 5-HT2, dopaminergic and adrenergic receptors failed to affect the response to 5-HT in neuroblastoma cells. Observed antagonistic effects of SCH 23390 [(R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepi n-7-ol hemimaleate] and haloperidol are discussed. The inhibitory effect of the 5-HT3 receptor antagonist, ICS 205-930 [(3 alpha-tropanyl)-1H-indole-3-carboxylic acid ester] has been demonstrated. When cells were exposed to 0.1 nM ICS 205-930 the maximum evoked response was reduced by about 50%, but a surmountable shift of the concentration-response curve of 5-HT was not observed. The kinetics of the 5-HT-induced inward current remained unchanged in the presence of ICS 205-930. Recovery from the block by ICS 205-930 was very slow.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Pharmacological characterization of serotonin 5-HT3 receptor-mediated electrical response in cultured mouse neuroblastoma cells. 337 70

1. The effects of chronic in vitro administration of amitriptyline, a tricyclic antidepressant, on 5-hydroxytryptamine (5-HT) receptor-mediated adenylyl cyclase activity was studied in the neuroblastoma x glioma hybrid cell line, NG 108-15. 2. Treatment of NG 108-15 cells with 8 microM amitriptyline for 3 days increased forskolin-stimulated (0.1 microM) adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation. Addition of 5-HT (0.1-100 microM) increased forskolin-stimulated cyclic AMP accumulation in amitriptyline-treated cells in a concentration-dependent manner. However, 5-HT did not affect forskolin-stimulated cyclic AMP accumulation in untreated cells. 3. The 5-HT4 receptor agonist, 5-methoxytryptamine, significantly enhanced forskolin-stimulated cyclic AMP accumulation in amitriptyline-treated cells. In contrast, amitriptyline treatment failed to modify 8-hydroxy-2-(di-n-propylamine) tetralin-induced inhibition of forskolin-stimulated cyclic AMP accumulation. 4. Pretreatment of cells with pertussis toxin did not affect the 5-HT-induced enhancement of cyclic AMP accumulation. 5. The 5-HT-induced enhancement of cyclic AMP accumulation in amitriptyline-treated cells was attenuated by the 5-HT4 receptor antagonists, GR 113808 and ICS 205-930, with relatively low potency. However, spiperone, SCH 23390, and pindolol were completely ineffective against this 5-HT-induced enhancement. 6. Chronic treatment with amitriptyline did not modify the cyclic AMP production stimulated by prostaglandin E1 or cholera toxin. This treatment also had no effect on GTP gamma S-, NaF-, and Mn(2+)-stimulated cyclic AMP accumulation in isolated cell membranes. 7. Chronic treatment with the 5-HT receptor antagonists, pindolol or ICS 205-930, did not inhibit the 5-HT-induced enhancement of cyclic AMP accumulation.8. Chronic treatment with other antidepressant drugs, imipramine, mianserin or paroxetine, elicited the 5-HT-induced enhancement of cyclic AMP accumulation.9. Taken together, these results suggest that chronic amitriptyline treatment of NG 108-15 cells causes 5-HT to enhance forskolin-stimulated cyclic AMP accumulation by enhancing 5-HT receptor-mediated stimulation of adenylyl cyclase and not by reducing 5-HT-mediated inhibition of adenylyl cyclase. The 5-HT-induced enhancement of cyclic AMP accumulation in amitriptyline-treated cells may result from changes at the level of the 5-HT receptor rather than at the level of G, proteins or adenylyl cyclase. It is unlikely that this enhancement of cyclic AMP accumulation is caused by long-term antagonism of the 5-HT receptor by amitriptyline.
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PMID:Enhancement of cyclic AMP accumulation mediated by 5-HT after chronic amitriptyline treatment in NG 108-15 cells. 762 Jul 19

Cyclic AMP-mediated desensitization of D1 dopamine receptor-coupled adenylyl cyclase was investigated using NS20Y neuroblastoma cells. Pretreatment of the cells for 24 h with 8-(4-chlorophenylthio)-adenosine-3':5'-cyclic monophosphate (CPT-cAMP), a membrane-permeable analog of cAMP, resulted in an approximately 90% reduction of the maximum dopamine-stimulated adenylyl cyclase activity. In addition, there was a twofold reduction in the potency of dopamine for stimulating cAMP production that was not dependent on the concentration of Mg2+ in the assay. These effects of CPT-cAMP pretreatment were time dependent, showing a t1/2 of about 3 h and a maximum reduction after about 8 h. Receptor-binding activity, as measured using the D1-selective antagonist [3H]SCH-23390, also declined following CPT-cAMP pretreatment with a t1/2 of about 5 h and a maximum reduction of about 70% after 20 h. Saturation analysis indicated that the loss in radioligand binding was due to a reduction in maximum binding capacity (Bmax) with no alteration in receptor affinity (KD). The EC50 of CPT-cAMP for producing enzyme desensitization and D1 receptor downregulation was determined to be about 30 microM with a maximal response occurring at 1 mM. These regulatory effects of CPT-cAMP were pharmacologically specific as other analogs of cAMP, such as dibutryl-cAMP, 8-bromo-cAMP, and Sp-cAMPS, were capable of inducing D1 receptor desensitization and downregulation, whereas treatment of the cells with the cAMP antagonist Rp-cAMPS had no effect. Conversely, Rp-cAMPS was capable of blocking the regulatory effects of CPT-cAMP but was apparently without effect in blocking dopamine-induced desensitization.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cyclic AMP-mediated desensitization of D1 dopamine receptor-coupled adenylyl cyclase in NS20Y neuroblastoma cells. 770 30

The effect of long-term (72 h) treatment with dopamine D1 receptor agonists, SKF 38393 and dopamine on D1 dopamine receptor and G-protein (Gs alpha) was investigated in SK-N-MC neuroblastoma cells. The prolonged treatment of cells with 10 microM SKF 38393 or 10 microM dopamine resulted in a decrease in dopamine D1 receptor by 41 and 81%, respectively, as measured by specific antagonist [3H]SCH 23390 binding. Similarly, the prolonged treatment of SK-N-MC cells with 10 microM SKF 38393 or 10 microM dopamine resulted in a reduction of the level of Gs alpha subunit of G-protein. The results indicate that agonist-induced down-regulation of D1 dopamine receptor may also modulate G-proteins.
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PMID:Desensitization of D1 dopamine receptors down-regulates the Gs alpha subunit of G protein in SK-N-MC neuroblastoma cells. 810 56


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