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)

NG108-15 neuroblastoma x glioma somatic hybrid cells were permeabilized in the presence of [32P]NAD+ and then cultured for 18 h. Resolution of the cell proteins on polyacrylamide gels revealed [32P]ADP-ribosylation of five major protein species with molecular mass values of 52 kDa, 44 kDa, 35 kDa, 30 kDa and 25 kDa. A similar pattern of labelling was also seen when NG108-15 cell membranes were incubated with [32P]NAD+ and hydrolysis of the product revealed mono(ADP-ribosyl)ation. Immunoprecipitation of these products with anti-Gs alpha antiserum revealed a single band identical to cholera toxin substrate. Culture of [32P]NAD(+)-loaded cells for 18 h in the presence of 50 mM-nicotinamide inhibited the eukaryotic mono(ADP-ribosyl)transferase activity. Inhibition of the eukaryotic enzyme was also accompanied by an increase in the abundance of Gs alpha, whether measured by Western blotting with anti-Gs alpha antibody (two separate antisera) or by cholera toxin-dependent [32P]ADP-ribosylation. There was no accompanying change in the abundance of G beta. The increase in Gs alpha abundance in nicotinamide-treated NG108-15 cells was accompanied by a 2-fold increase in basal adenylate cyclase activity (measured in the presence of GTP), and by a smaller but significant increase in iloprost-dependent activation of adenylate cyclase. Receptor number or affinity was not affected by nicotinamide, since this treatment did not alter the binding parameters of [3H]iloprost to NG108-15 cell membranes. Short-term exposure of cells to nicotinamide for 1 h revealed no significant difference in either basal or agonist-stimulated adenylate cyclase activity. These results reveal that mono(ADP-ribosyl)ation of Gs alpha by eukaryotic ADP-ribosyltransferase modifies the abundance and activity of Gs alpha in NG108-15 cells, and hence may play a role in the hormonal regulation of cell function.
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PMID:Gs alpha is a substrate for mono(ADP-ribosyl)transferase of NG108-15 cells. ADP-ribosylation regulates Gs alpha activity and abundance. 128 Jan 14

Desensitization of the responsiveness to hormones or drugs is often mediated by down-regulation of receptors. The stimulatory coupling protein (Ns) of adenylate cyclase has been shown to be involved in the down-regulation of stimulatory beta-adrenergic receptors. Whether the inhibitory coupling protein (Ni) is involved in the down-regulation of receptors that inhibit adenylate cyclase is not known. We wished to determine whether down-regulation of inhibitory muscarinic cholinergic and alpha 2-adrenergic receptors occurs in neuroblastoma X glioma hybrid cells after the ability of Ni to inhibit adenylate cyclase is inactivated by pertussis toxin. After treatment of cells with pertussis toxin, the ability of carbachol or epinephrine to inhibit prostaglandin E1-stimulated cAMP accumulation in intact cells was either completely prevented or markedly attenuated, respectively, indicating functional inactivation of Ni. Furthermore, pertussis toxin treatment of membrane fragments from these cells did not result in labeling of the 41,000-dalton alpha-subunit of Ni with ADP ribose from [32P] NAD, indicating maximal ADP ribosylation of Ni by prior treatment of cells with pertussis toxin. Carbachol treatment of cells resulted in down-regulation of muscarinic cholinergic receptors to 45.7 +/- 12.5% and 52.5 +/- 13.5% of control values for toxin-untreated and toxin-treated cells, respectively. Epinephrine treatment of cells caused homologous desensitization of alpha 2-receptor-mediated inhibition of cAMP accumulation and down-regulation of alpha 2-adrenergic receptors to 42.9 +/- 11.4% and 53.2 +/- 5.3% of control values for toxin-untreated and toxin-treated cells, respectively. Down-regulation of muscarinic cholinergic receptors by carbachol and of alpha 2-adrenergic receptors by epinephrine was not due to the effect of retained agonist and was agonist specific, since it could be prevented by the antagonists atropine and yohimbine, respectively. We conclude that agonist-mediated down-regulation of both the muscarinic cholinergic receptor and the alpha 2-adrenergic receptor does not require functional inhibitory coupling.
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PMID:Agonist-induced down-regulation of muscarinic cholinergic and alpha 2-adrenergic receptors after inactivation of Ni by pertussis toxin. 242 98

Mouse wild-type neuroblastoma cells (NB cells) were stepwise selected for 10,000-fold increased resistance to mycophenolic acid (NB-Myco cells), an inhibitor of IMP dehydrogenase (IMP:NAD+ oxidoreductase, EC 1.1.1.205). IMP dehydrogenase activity was increased 25-fold, from 3.1 to 75 nmol/min.mg of protein; and a 56.7-kDa peptide was increased in abundance 200-500-fold in NB-Myco as compared to NB cells. Purification and sequence analysis confirmed that the abundant protein was IMP dehydrogenase. The stepwise selection, increased activity and protein abundance, and unstable phenotype are indirect evidence for a process of gene amplification. Kinetic findings consistent with an Ordered Bi Bi mechanism were indicative of IMP dehydrogenase having undergone mutation. The Michaelis constants were unchanged for IMP (14 and 13 microM) and increased 4-fold for NAD from 25 to 94 microM for NB and NB-Myco cells, respectively. The Ki for mycophenolic acid was increased 2400-fold from 1.4 nM to 3.4 microM for the enzyme from NB versus NB-Myco cells, and the Ki for XMP was increased 4-fold from 78 to 336 microM. Mycophenolic acid exhibited uncompetitive inhibition with IMP, consistent with the formation of a dead end E-XMP-inhibitor complex. The cellular GTP concentration was increased 2-fold in resistant cells and, upon removal of mycophenolic acid, further increased to 4.5-fold that of NB cells.
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PMID:Increased activity, amount, and altered kinetic properties of IMP dehydrogenase from mycophenolic acid-resistant neuroblastoma cells. 257 89

Activation of muscarinic cholinergic receptors of 1321N1 human astrocytoma cells attenuates cyclic AMP accumulation. This effect results from an activation of phosphodiesterase with no direct inhibition of adenylate cyclase activity. In spite of this lack of coupling of muscarinic receptors to adenylate cyclase, guanine nucleotides reduce the apparent binding affinity of the agonist carbachol in a washed membrane preparation of 1321N1 cells. The order of potency for this effect is guanosine 5'-O-(3-thiotriphosphate) greater than 5'-guanylyl-imidodiphosphate = GTP = GDP; ATP has no effect. The occurrence of a Mr = 41,000 protein labeled in the presence of [32P]NAD and pertussis toxin as well as the occurrence of guanine nucleotide-mediated inhibition of forskolin-stimulated adenylate cyclase activity indicate that the functional inhibitory guanine nucleotide regulatory component of adenylate cyclase (Ni) is present in 1321N1 cells. Pertussis toxin pretreatment of NG108-15 neuroblastoma X glioma cells, which express muscarinic receptors that link through Ni to inhibit adenylate cyclase, blocked the GTP-sensitive, high affinity binding of carbachol. In contrast, pretreatment of 1321N1 cells with a concentration of pertussis toxin that blocked [32P]ADP ribosylation of the Mr = 41,000 substrate and GTP-mediated inhibition of forskolin-stimulated adenylate cyclase activity had no effect on GTP-sensitive high affinity binding of carbachol. These results suggest that muscarinic cholinergic receptors of 1321N1 cells couple to a guanine nucleotide regulatory protein that is distinct from Ni.
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PMID:Guanine nucleotide-sensitive, high affinity binding of carbachol to muscarinic cholinergic receptors of 1321N1 astrocytoma cells is insensitive to pertussis toxin. 298

As noted previously, in N1E-115 neuroblastoma cells, carbamylcholine, a muscarinic cholinergic agonist, increased cGMP over 15-fold and decreased basal and prostaglandin E1 (PGE1)-stimulated cAMP content. In contrast to the stimulatory effects of PGE1 on cAMP, which were immediate, the carbamylcholine-induced decrease in basal and PGE1-stimulated cAMP exhibited a delay. The delay in carbamylcholine inhibition was independent of the extent of adenylate cyclase activation. Although basal cAMP content was suppressed within 30 sec after addition of carbamylcholine, inhibition was not maximal for at least 2 min following agonist addition; the delay was similar in cells exposed to PGE1 for 10 min prior to carbamylcholine but could be eliminated by incubation of the cells with muscarinic cholinergic agonist for 5 min prior to addition of prostaglandin. N1E-115 neuroblastoma cells possess a 41,000-Da membrane protein believed to be a component of the inhibitory GTP-binding protein of adenylate cyclase that is ADP ribosylated by pertussis toxin. Incubation of the cells with pertussis toxin prior to the addition of carbamylcholine reduced the maximal extent of inhibition of cAMP content and prevented the [32P]ADP-ribosylation of a 41,000-Da protein by toxin and [32P]NAD in membrane preparations from these cells. Incubation of cells with pertussis toxin, however, did not significantly alter the dose-response curve for carbamylcholine effects on cGMP. Even high concentrations of carbamylcholine, effective in stimulating cGMP, had minimal effects on cAMP content in toxin-treated cells; thus, ADP-ribosylation of Gi converts the adenylate cyclase but not the guanylate cyclase system to an agonist-insensitive state.
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PMID:Effects of pertussis toxin on cAMP and cGMP responses to carbamylcholine in N1E-115 neuroblastoma cells. 299 40

Previous electron spin resonance studies have demonstrated that the decay of ascorbyl plus semiquinone radicals, produced in an aqueous mixture of ascorbate and 2,6-dimethoxy-p-quinone, is accelerated by ascites cells. This effect was concluded to involve a sulfhydryl-containing NAD(P)H-enzyme, and work on cultured cell lines showed that on neoplastic transformation the activity against the radicals was increased. We show here that at least three disulfide-oxidoreductases are able to quench the radicals in a similar way to that of viable cells. Glutathione reductase (EC 1.6.4.2) in the presence of NADPH and oxidised glutathione, and dihydrolipoamide dehydrogenase (EC 1.8.1.4) with NADH and lipoamide, are found to accelerate the radical decay by reducing the quinone or semiquinone. DT-diaphorase (EC 1.6.99.2) in the presence of NAD(P)H can also achieve this by reducing the quinone directly. Lipoamide dehydrogenase and glutathione reductase are also capable of reducing nitroxide spin labels, a finding considered of relevance to the reported reduction of such spin labels by neuroblastoma cells.
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PMID:Electron spin resonance studies of the interaction of oxidoreductases with 2,6-dimethoxy-p-quinone and semiquinone. 302 90

It has been proposed elsewhere [Meeker, R.B. & Harden, T. K. (1982) Mol. Pharmacol. 22, 310-319] that muscarinic cholinergic receptor-mediated attenuation of cAMP accumulation occurs through activation of phosphodiesterase in 1321N1 human astrocytoma cells. Pertussis toxin, which ADP-ribosylates the guanine nucleotide regulatory protein involved in receptor-mediated inhibition of adenylate cyclase (Ni), has been utilized to further differentiate between the mechanism of cholinergic regulation of cAMP metabolism in 1321N1 cells and the mechanism involving inhibition of adenylate cyclase in other tissues. Muscarinic receptor-mediated regulation of cAMP accumulation in NG108-15 neuroblastoma-glioma cells occurs through inhibition of adenylate cyclase. Pretreatment of these cells with pertussis toxin completely blocked the capacity of carbachol to attenuate cAMP accumulation. In contrast, concentrations of pertussis toxin two to three orders of magnitude higher than those effective in NG108-15 cells had no effect on muscarinic receptor-mediated attentuation of cAMP accumulation in 1321N1 cells. In addition, no effect of pertussis toxin was observed either on the control rate or the carbachol-stimulated rate of cAMP degradation measured directly in intact 1321N1 cells. A 41,000 Mr protein previously proposed to be the alpha subunit of Ni was labeled during incubation of a plasma membrane fraction from 1321N1 cells with [32P]NAD and pertussis toxin. Pertussis toxin is apparently active in 1321N1 cells, since this protein substrate was not labeled in plasma membrane preparations from cells previously incubated with toxin. Functional activity of Ni was demonstrated by the observation that guanosine 5'-[gamma-thio]triphosphate- and GTP-mediated inhibition of forskolin-stimulated adenylate cyclase activity occurred in cell-free preparations from 1321N1 cells. The inhibitory activity of these guanine nucleotides was lost in membrane preparations from pertussis toxin-treated cells. The data suggest that adenylate cyclase is not involved in cholinergic action in 1321N1 cells and, furthermore, Ni is not involved in muscarinic receptor-mediated activation of phosphodiesterase in these cells. Thus, pertussis toxin can be used to differentiate between two mechanisms of cholinergic regulation of cAMP metabolism.
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PMID:Pertussis toxin differentiates between two mechanisms of attenuation of cyclic AMP accumulation by muscarinic cholinergic receptors. 609 Nov 3

In neuroblastoma-glioma (NG108-15) hybrid cells, opiates inhibit adenylate cyclase and stimulate a low Km GTPase. It has been postulated that the stimulation of GTPase plays a role in opiate inhibition of adenylate cyclase (Koski, G., and Klee, W. A. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4185-4189). Treatment of NG108-15 cells with pertussis toxin attenuates receptor-mediated inhibition of adenylate cyclase. The toxin acts by catalyzing the ADP-ribosylation of a 41,000-dalton substrate believed to be a part of the receptor-adenylate cyclase complex. We have found that toxin treatment of NG108-15 results in inhibition of the opiate-stimulated GTPase. The concentration of toxin required for inhibition of this GTPase was similar to that needed for both attenuation of opiate inhibition of adenylate cyclase and ADP ribosylation of the 41,000-dalton substrate. Inhibition of the opiate-induced GTPase by pertussis toxin in isolated membranes required NAD, consistent with the hypothesis that this effect of the toxin resulted from ADP ribosylation of a protein component of the system. Since the opiate-stimulated GTPase is believed to play a role in the receptor-mediated decrease in adenylate cyclase activity, inhibition of this GTPase may be an important part of the mechanism by which the toxin interferes with opiate action on adenylate cyclase.
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PMID:Pertussis toxin inhibits enkephalin stimulation of GTPase of NG108-15 cells. 613 91

Pertussis toxin (islet-activating protein) activates adenylate cyclase in susceptible cells by ADP-ribosylating an inhibitory component of the cyclase system. This toxin, assayed in a cell-free system in the presence of high concentrations of thiol, catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide. This NAD glycohydrolase activity co-chromatographed on Sephacryl G-200 in 6.5 M urea, pH 3.2, 0.1 M glycine with the ADP-ribosyltransferase activity of the toxin, as monitored by the transfer of [32P]ADP-ribose from [32P]NAD to a 41,000-Da protein in NG108-15 neuroblastoma X glioma hybrid cells. In the absence of thiol, the native holotoxin was enzymatically inactive. Following addition of 250 mM dithiothreitol to the assay, maximal enzymatic activity was evident after a delay of approximately 1 h; with 20 mM thiol, the delay was longer. The Km for NAD with the fully activated enzyme was 25 microM; the Km did not appear to vary with the extent of activation. Thiol was necessary in a cell-free system to demonstrate NAD glycohydrolase activity. When extensively washed membranes were used as a source of 41,000-Da substrate, thiol was necessary to observe ADP-ribosylation in some cases (human erythrocytes) and significantly stimulated activity in others (NG108-15 cells). In contrast to the bacterial toxins choleragen and Escherichia coli heat-labile enterotoxin that ADP-ribosylate stimulatory components of the cyclase system, pertussis toxin did not transfer ADP-ribose to low molecular weight guanidino compounds, such as arginine or agmatine.
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PMID:Activation by thiol of the latent NAD glycohydrolase and ADP-ribosyltransferase activities of Bordetella pertussis toxin (islet-activating protein). 631 27

Incubation of purified rat brain tubulin with cholera toxin and radiolabeled [32P] or [8-3H]NAD results in the labeling of both alpha and beta subunits as revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of these protein bands with snake venom phosphodiesterase resulted in quantitative release of labeled 5'-AMP, respectively labeled with the corresponding isotope. Two-dimensional separation by isoelectric focusing and SDS-PAGE of labeled and native tubulin revealed that labeling occurs at least in four different isotubulins. The isoelectric point of the labeled isotubulins was slightly lower than that of native purified tubulin. This shift in mobility is probably due to additional negative charges involved with the incorporation of ADP-ribosyl residues into the tubulin subunits. SDS-PAGE of peptides derived from [32P]ADP-ribosylated alpha and beta tubulin subunits by Staphylococcus aureus protease cleavage showed a peptide pattern identical with that of native tubulin. Microtubule-associated proteins (MAP1 and MAP2) of high molecular weight were also shown to undergo ADP-ribosylation. Incubation of permeated rat neuroblastoma cells in the presence of [32P]NAD and cholera toxin results in the labeling of only a few cell proteins of which tubulin is one of the major substrates.
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PMID:ADP-ribosylation of microtubule proteins as catalyzed by cholera toxin. 676 71


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