NG108-15 Neuroblastoma-glioma Hybrid Research Articles

New G Protein Interactions

Two groups have uncovered new wrinkles in reactions involving heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins). Ligand binding to G protein-coupled receptors (GPCRs) activates G proteins by catalyzing guanine nucleotide exchange and dissociation of the GTP-bound α subunit (Gα) from the βγ heterodimer (Gβγ). Both subunits are then free to activate various effectors. Ribas et al. partially purified a ligand-independent G-protein activator, NG108-15 G-protein activator (NG-GPA), from the neuroblastoma-glioma hybrid NG108-15. NG-GPA stimulated guanosine 5'-O-thiotriphosphate (GTPγS) binding to purified brain G protein, as well as to recombinant Gαo and Gαi, and inhibited adenylyl cyclase activity in DDT1-MF-2 cell membranes. NG-GPA's stimulation of GTPγS binding, unlike GPCR-stimulated GTPγS binding, was unaffected by treatment with pertussis toxin, which prevents agonist-induced receptor interactions with the Gi family of G proteins, suggesting that NG-GPA interacts with the G protein through a previously unidentified regulatory domain. Dell et al. used a yeast two-hybrid screen to identify new Gβγ effectors and isolated three proteins containing WD40 motifs (which Gβγ also contains) that bound to Gβγ. Gβγ interaction with two of these proteins, receptor for activated C kinase 1 (RACK1) and dynein intermediate chain (DIC), was confirmed by Western analysis of immunoprecipitates. A RACK1 fusion protein bound to both Gβ1γ1 and the heterotrimeric Gαtβ1γ1 transducin heterotrimer but not to Gαt alone; Gαtβ1γ1 had a higher binding affinity than Gβ1γ1. RACK1 interaction with Gαtβ1γ1 was surprising, because few proteins other than GPCRs interact with the heterotrimer. Indeed, all known Gβγ effectors bind to the Gα-interacting region, suggesting a novel mechanism for RACK1-G protein interaction.C. Ribas, A. Takesono, M. Sato, J. D. Hildebrandt, S. M. Lanier, Pertussis toxin-insensitive activation of the heterotrimeric G-proteins Gi/Go by the NG108-15 G-protein activator. J. Biol. Chem. 277, 50223-50225 (2002). [Abstract] [Full Text]E. J. Dell, J. Connor, S. Chen, E. G. Stebbins, N. P. Skiba, D. Mochly-Rosen, H. E. Hamm, The βγ subunit of heterotrimeric G proteins interacts with RACK1 and two other WD repeat proteins. J. Biol. Chem. 277, 49888-49895 (2002). [Abstract] [Full Text]

New G Protein Interactions

Two groups have uncovered new wrinkles in reactions involving heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins). Ligand binding to G protein-coupled receptors (GPCRs) activates G proteins by catalyzing guanine nucleotide exchange and dissociation of the GTP-bound α subunit (Gα) from the βγ heterodimer (Gβγ). Both subunits are then free to activate various effectors. Ribas et al. partially purified a ligand-independent G-protein activator, NG108-15 G-protein activator (NG-GPA), from the neuroblastoma-glioma hybrid NG108-15. NG-GPA stimulated guanosine 5'- O -thiotriphosphate (GTPγS) binding to purified brain G protein, as well as to recombinant Gα o and Gα i , and inhibited adenylyl cyclase activity in DDT 1 -MF-2 cell membranes. NG-GPA's stimulation of GTPγS binding, unlike GPCR-stimulated GTPγS binding, was unaffected by treatment with pertussis toxin, which prevents agonist-induced receptor interactions with the G i family of G proteins, suggesting that NG-GPA interacts with the G protein through a previously unidentified regulatory domain. Dell et al. used a yeast two-hybrid screen to identify new Gβγ effectors and isolated three proteins containing WD40 motifs (which Gβγ also contains) that bound to Gβγ. Gβγ interaction with two of these proteins, receptor for activated C kinase 1 (RACK1) and dynein intermediate chain (DIC), was confirmed by Western analysis of immunoprecipitates. A RACK1 fusion protein bound to both Gβ 1 γ 1 and the heterotrimeric Gα t β 1 γ 1 transducin heterotrimer but not to Gα t alone; Gα t β 1 γ 1 had a higher binding affinity than Gβ 1 γ 1 . RACK1 interaction with Gα t β 1 γ 1 was surprising, because few proteins other than GPCRs interact with the heterotrimer. Indeed, all known Gβγ effectors bind to the Gα-interacting region, suggesting a novel mechanism for RACK1-G protein interaction. C. Ribas, A. Takesono, M. Sato, J. D. Hildebrandt, S. M. Lanier, Pertussis toxin-insensitive activation of the heterotrimeric G-proteins G i /G o by the NG108-15 G-protein activator. J. Biol. Chem. 277 , 50223-50225 (2002). [Abstract] [Full Text] E. J. Dell, J. Connor, S. Chen, E. G. Stebbins, N. P. Skiba, D. Mochly-Rosen, H. E. Hamm, The βγ subunit of heterotrimeric G proteins interacts with RACK1 and two other WD repeat proteins. J. Biol. Chem. 277 , 49888-49895 (2002). [Abstract] [Full Text]

Muscarinic acetylcholine m1–m4 receptors overexpressed in neuroblastoma-glioma hybrid NG108-15 cells as a cholinergic model

Muscarinic acetylcholine m1–m4 receptors overexpressed in neuroblastoma-glioma hybrid NG108-15 cells as a cholinergic model

Inhibition of bioenergetics alters intracellular calcium, membrane composition, and fluidity in a neuronal cell line

The effect of inhibited bioenergetics and ATP depletion on membrane composition and fluidity was examined in cultured neuroblastoma-glioma hybrid NG108-15 cells. Sodium cyanide (CN) and 2-deoxyglucose (2-DG) were used to block, oxidative phosphorylation and anaerobic glycolysis, respectively. Endoplasmic reticulum (ER) Ca(2+)-pump activity measured by 45Ca2+ uptake was > 92% inhibited in intact cells incubated with CN (1 mM) and 2-DG (20 mM) for 30 min. In addition, exposure of cells to CN and 2-DG caused a 134% increased release of isotopically labeled arachidonic acid (3H-AA) or arachidonate-derived metabolites from membranes. Removal of Ca2+ from the incubation medium ablated the CN/2-DG induced release of 3H-AA or its metabolites. Membrane fluidity of intact cells was measured by electron spin resonance spectroscopy using the spin label 12-doxyl stearic acid. The mean rotational correlation time (tau c) of the spin label increased 49% in CN/2-DG exposed cells compared to controls, indicating a decrease in membrane fluidity. These results show that depletion of cellular ATP results in inhibition of the ER Ca(2+)-pump, loss of AA from membranes, and decreased membrane fluidity. We propose that impaired bioenergetics can increase intracellular Ca2+ as a result of Ca(2+)-pump inhibition and thereby activate Ca(2+)-dependent phospholipases causing membrane effects. Since neurons derive energy predominantly from oxidative metabolism, ATP depletion during brain hypoxia may initiate a similar cytotoxic mechanism.

Agonist regulation of cellular levels of the stimulatory guanine nucleotide-binding protein, Gs, in wild type and transfected neuroblastoma-glioma hybrid NG108-15 cells.

Conference Article| May 01 1993 Agonist regulation of cellular levels of the stimulatory guanine nucleotide-binding protein, Gs, in wild type and transfected neuroblastoma-glioma hybrid NG108-15 cells Elaine J. Adie; Elaine J. Adie 1Molecular Pharmacology Group, Department of Biochemistry and Pharmacology, University of Glasgow, Glasgow GI2 8QQ, Scotland, U.K. Search for other works by this author on: This Site PubMed Google Scholar Graeme Milligan Graeme Milligan * *To whom correspondence should be addressed Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1993) 21 (2): 432–435. https://doi.org/10.1042/bst0210432 Article history Received: December 09 1992 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation Elaine J. Adie, Graeme Milligan; Agonist regulation of cellular levels of the stimulatory guanine nucleotide-binding protein, Gs, in wild type and transfected neuroblastoma-glioma hybrid NG108-15 cells. Biochem Soc Trans 1 May 1993; 21 (2): 432–435. doi: https://doi.org/10.1042/bst0210432 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: NECA, 5′-(N-ethyl)carboxamido-adenosine This content is only available as a PDF. © 1993 Biochemical Society1993 Article PDF first page preview Close Modal You do not currently have access to this content.

O-336 - Bradykinin-evoked multiphasic conductance-change in differentiated and non-differrentiated neuroblastoma-glioma hybrid NG108-15 cells

O-336 - Bradykinin-evoked multiphasic conductance-change in differentiated and non-differrentiated neuroblastoma-glioma hybrid NG108-15 cells

Regeneration of acetylcholinesterase in clonal neuroblastoma-glioma hybrid NG108-15 cells after soman inhibition: effect of glycyl-l-glutamine

Acetylcholinesterase (AChE) in the clonal NG108-15 cell line has been previously characterized. This cell line represents an in vitro system to study AChE regulation and effects of chemical compounds that may alter AChE activity. Recently, glycyl-L-glutamine (GLG) was demonstrated to function as a neurotrophic factor for maintenance of AChE content in cat denervated superior cervical ganglion cells. In the present study, regeneration of AChE activity in cultures of undifferentiated NG108-15 cells after soman inhibition was investigated in the presence and absence of GLG. Cells were treated with soman (5.5 x 10(-6) M) for 15 min and then washed to remove excess soman. Culture medium containing either GLG (10(-6), 10(-5), or 10(-4) M) or glycyl-L-glutamic acid (10(-6) M) was added to cultures after soman treatment and remained in the medium until cell harvest. Cells were physically detached at various times after soman treatment and specific AChE activity was determined. After soman, AChE activity dramatically decreased to less than 1% of untreated cellular activity at 1 hr. AChE activity gradually increased after 5 hr, while untreated cell AChE activity was regained 20 hr after soman. The t1/2 for AChE regeneration was approximately 10 hr. GLG did not increase the rate of AChE regeneration after soman inhibition. These results indicate that GLG is not a directly acting neurotrophic factor for AChE synthesis in NG108-15 cells after chemical AChE inactivation.

Prevention of muscarinic acetylcholine receptor down-regulation by chloroquine: antilysosomal or antimuscarinic mechanisms.

The effect of the antimalarial drug chloroquine on the carbachol-induced down-regulation of muscarinic acetylcholine receptors (mAChRs) was studied in the neuroblastoma-glioma hybrid NG108-15 cells. Chloroquine, which is proposed to have both antilysosomal and antimuscarinic effects (4,11), blocked the loss of both cell surface and total mAChRs as monitored by [3H]N-methyl-scopolamine (NMS) and [3H] quinuclidinyl benzilate (QNB) bindings respectively. To the contrary, NH4Cl, only an antilysosomal agent, had no effect on the loss of surface receptors, but blocked degradation of internalized receptors following the effect of carbachol. These findings demonstrate that chloroquine prevents the agonist-induced mAChR down-regulation in NG108-15 cells by both its antilysosomal and antimuscarinic effects.

Specific soman-hydrolyzing enzyme activity in a clonal neuronal cell culture

An enzymatic activity that specifically hydrolyzes the highly toxic organophosphorus anticholinesterase compound soman (pinacolyl methylphosphonofluoridate) has been identified and partially characterized in the clonal neuronal neuroblastoma-glioma hybrid NG108-15 cell line. Using the whole cell homogenate as the enzyme source and 1 mM substrate, the relative rate of hydrolysis of two other toxic anticholinesterase compounds sarin (isopropyl methylphosphonofluoridate) and tabun (ethyl- N-dimethyl phosphoramidocyanidate) is approximatelt one-tenth the rate of hydrolysis of soman, while DFP (diisopropyl phosphorofluoridate), paraoxon ( p-nitrophenyl diethylphosphate), and a phosphinate PNMPP ( p-nitrophenyl methyl (phenyl) phosphinate) are not hydrolyzed. Analysis of the kinetics of soman hydrolysis reveals two components of the enzyme activity with different affinities and reaction rates. Unlike previously reported enzymes of this type, this enzyme lacks chiral specificity and thus hydrolyzes both toxic and non-toxic soman stereoisomers at equal rates. The enzyme activity is stable at low temperature, found almost exclusively in the soluble fraction of these cells, and enhanced significantly by Mn 2+ and by chemical differentiation of these cells in culture. The results suggest possible application of this enzyme for soman detection and/or detoxication, and use of the NG108-15 cell line to study the natural function(s) of enzymes of this type.

Block of calcium channels by enkephalin and somatostatin in neuroblastoma-glioma hybrid NG108-15 cells.

Leucine-enkephalin, methionine-enkephalin, and morphine caused a reversible block of Ca2+ channel currents in neuroblastoma-glioma hybrid cells (NG108-15). The long-lasting (type 2) component of the Ca2+ channel current was blocked by leucine-enkephalin, while the transient (type 1) component was not affected. The enkephalin-induced blocking action was antagonized by naloxone and appears to be mediated by delta-opiate receptors. Two different aspects of the blocking effect were detected, a resting block and a recovery from block during prolonged depolarizing pulses. Recovery from block was more complete, and its time course was more rapid, with depolarization to more positive potentials. The dose dependence of the type 2 channel block at rest indicated a one-to-one binding stoichiometry, with an apparent dissociation constant of 8.8 nM. Somatostatin exerted a similar selective blocking action on the type 2 Ca2+ channel. The time- and voltage-dependent block of type 2 Ca2+ channels may provide a mechanism underlying the enkephalinergic presynaptic inhibition of transmitter release and the somatostatin block of pituitary growth hormone release.

Specific Muscarinic‐Cholinergic Desensitization in the Neuroblastoma‐Glioma Hybrid NG108‐15

The cholinergic agonist carbachol, epinephrine, and the opiate morphine all inhibit prostaglandin E1 (PGE1)-stimulated adenylate cyclase in homogenates from the neuroblastoma-glioma hybrid NG108-15. Pretreatment of the hybrid with 100 microM carbachol resulted in the rapid loss (desensitization) of the carbachol inhibition of adenylate cyclase (t1/2 less than 3 min). The desensitization of the carbachol inhibition was blocked by 0.1 microM atropine. Pretreatment with carbachol (1-24 h) did not significantly affect the inhibition of adenylate cyclase by either epinephrine or morphine, nor did it alter the PGE1-stimulated activity, that is, no supersensitization was observed. Cholate extracts of the particulate fraction from either carbachol-desensitized or of control NG108-15 were able to reconstitute adenylate cyclase activities of the coupling proteins (G/F)-deficient cyc- lymphoma cell membranes with equal efficacy. These results suggested that the coupling proteins of the adenylate cyclase were not altered by the carbachol pretreatment and that desensitization occurs at the receptor or at a receptor-associated level. However, the possibility remained that specific domains of the G/F, which interact only with muscarinic receptors, were altered.

Hyperpolarization of neuroblastoma-glioma hybrid NG108-15 by vanadium ions.

Vanadate hyperpolarizes mouse neuroblastoma-glioma hybrid NG108-15 by 20-30 mV. These changes in membrane potential (delta psi) are observed by monitoring the equilibrium distribution (intracellular/extracellular) of the lipophilic cation [3H]tetraphenylphosphonium (TTP+) and by directly measuring delta psi with intracellular microelectrodes. In physiological media (i.e., 135 mM NaCl/5 mM KCl), the half-maximal effective concentrations of sodium orthovanadate (Na3VO4) and sodium metavanadate (NaVO3) are 35 and 160 microM, respectively. The maximal effects for both these ions are quantitatively indistinguishable. The hyperpolarizing responses to vanadate occur without any observable lag, have t1/2 less than or equal to 30 sec, and are always accompanied by simultaneous decreases in membrane resistance. Neither ouabain nor media containing high K (i.e., 120 mM) devoid of Na and K (isotonicity maintained by choline) prevent the change in delta psi induced by vanadate. Therefore, vanadate produces a unique hyperpolarization which does not depend upon Na, K, or the Na/K pump. Furthermore, the accompanying decreases in membrane resistance indicate that vanadate must increase the permeability of the membrane to some ion. Our data are consistent with it being an anion, such as chloride or vanadate itself. Finally, vanadate hyperpolarizes many different types of cultured cells, only some of which are of neuronal origin. This indicates that a hyperpolarization of delta psi must be considered in any assessment of the physiological actions of the vanadates.

Mechanism of monensin-induced hyperpolarization of neuroblastoma-glioma hybrid NG108-15.

Addition of the ionophore monensin to mouse neuroblastoma-rat glioma hybrid NG108-15 cells leads to a 20 to 30-mV increase in the electrical potential across the plasma membrane as shown by direct intracellular recording techniques and by distribution studies with the lipophilic cation [3H]-tetraphenylphosphonium+ (TPP+) [Lichtshtein, D., Kaback, H.R. & Blume, A.J. (1979) Proc. Natl. Acad. Sci. USA 76, 650-654]. The effect is not observed with cells suspended in high K+ medium, is dependent upon the presence of Na+ externally, and the concentration of monensin that induces half-maximal stimulation of TPP+ accumulation is approximately 1 microM. The ionophore also causes rapid influx of Na+, a transient increase in intracellular pH, and a decrease in extracellular pH, all of which are consistent with the known ability of monensin to catalyze the transmembrane exchange of H+ for Na+. Although ouabain has no immediate effect on the membrane potential, the cardiac glycoside completely blocks the increase in TPP+ accumulation observed in the presence of monensin. Thus, the hyperpolarizing effect of monensin is mediated apparently by an increase in intracellular Na+ that acts to stimulate the electrogenic activity of the Na+,K+-ATPase. Because monensin stimulates TPP+ accumulation in a number of other cultured cell lines in addition to NG108-15, the techniques described may be of general use for studying the Na+,K+ pump and its regulation in situ.