Stimulation of choline release from NG108-15 cells by 12-O-tetradecanoylphorbol 13-acetate.

The effects of the potent tumour-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on phosphatidylcholine (PtdCho) metabolism were investigated in the neuroblastoma X glioma hybrid cell line NG108-15. TPA (100 nM) stimulated by 150-200% the release into the medium of 3H radioactivity from cells that had been pre-labelled with [3H]choline. H.p.l.c. analysis of the medium revealed that TPA stimulated the release of only free [3H]choline (212 +/- 11% of control), without affecting such other labelled metabolites as [3H]phosphocholine and [3H]glycerophosphocholine. This effect was concentration-dependent, with a half-maximal effect obtained at 27.5 +/- 6.8 nM, and was observable as early as 5-10 min after exposure to TPA. The TPA-induced release of [3H]choline into the medium was accompanied by a small and variable decrease in cellular [3H]PtdCho (to 93 +/- 4% of control). However, the radioactivity associated with water-soluble cellular choline metabolites (mainly [3H]phosphocholine and [3H]glycerophosphocholine) remained unchanged. TPA also stimulated the release of [3H]choline derived from [3H]PtdCho that had been produced via the methylation pathway from [3H]methionine. These data suggest that phosphatidylcholine may serve as the source of free choline released from the cells in response to TPA. The possible enzymic mechanisms underlying this response are discussed.     

Calcium-dependent proliferation of NG108-15 neuroblastoma cells

While there is increasing evidence that Ca2+ plays an important role in regulating cell proliferation, the precise mechanisms have not been clearly elucidated so far. In order to gain insight into how Ca2+ controls cell division, the rate of proliferation, cell volume, viability and attachment to the culture support were measured in NG108-15 neuroblastoma cells in the presence of various extracellular Ca2+ concentrations ([Ca2+]o). Culture medium [Ca2+]o was decreased from 1.8 mmol/l to various values down to 1 micromol/l with EGTA. The rate of cell proliferation was almost independent of [Ca2+]o between 1.8 mmol/l and 45 micromol/l. It was decreased by about 50% at 12 umol/l Ca2+ and was almost zero in the presence of 1 micromol/l Ca2+. As we hawe shown previously (Rouzaire-Dubois and Dubois 1998) long-term hypertonicity increased the cell volume and decreased the rate of proliferation. The effects of hypertonicity and decrease in [Ca2+]o on cell proliferation were synergistic and can be described by cell size-dependent and independent mechanisms, respectively. Relative to control conditions (1.8 mmol/l Ca2+), decreases in [Ca2+]o to 12 and 1 micromol/l decreased the cell viability to 76 and 52% and the cell adhesion to dishes to 16 and 3%, respectively. Altogether, these results indicate that the effects of alteration in [Ca2+]o and cell size on neuroblastoma cell proliferation are independent and act on different signalling pathways controlling cell division.     

Specific reduction of delta-opioid receptor binding in transfected NG108-15 cells.

We have recently identified and sequenced the cDNA for an opioid-binding protein with homologies to cell adhesion molecules (OBCAM) (Schofield, P. R., McFarlard, K. C., Hayflick, J. S., Wilcox, J. N., Cho, T. M., Roy, S., Lee, N. M., Loh, H. H., and Seeburg, P. H. (1989) EMBO J. 8, 489-495). Several lines of evidence using antibodies suggest that OBCAM may play a functional role in NG108-15 neuroblastoma x glioma cells, a useful model system that contains a homogeneous population of delta-opioid receptors. A logical extension of this research is to further test this hypothesis. As part of this study, NG108-15 cells were stably transfected with either sense or antisense sequences of a portion of pROM, the rat cDNA for OBCAM. [3H] Diprenorphine binding was greatly reduced in antisense-transfected cells relative to non-transfected cells. Binding to alpha 2-adrenergic, muscarinic, and insulin receptors was unaffected. These results further support the notion that OBCAM or its analogue is part (or a subunit) of an opioid receptor. Furthermore, our observation of an apparently specific reduction in opioid binding in these transfected cells suggests that they may provide a novel genetic approach for studying regulation of the opioid receptor in this defined cell line.     

Cholera toxin differentially decreases membrane levels of alpha and beta subunits of G proteins in NG108-15 cells

Treatment of NG108-15 neuroblastoma x glioma cells (24 h) with cholera toxin (0.1-10 micrograms/ml) resulted in a concentration-dependent reduction of the membrane levels of subunits of GTP-binding regulatory proteins (G proteins), as determined by quantitative immunoblot procedures. The extent of reduction differed for different types of subunits: the levels of Go alpha and G beta 1 were reduced by 40-50%, whereas those of G alpha common immunoreactivity and Gi2 alpha were only reduced by 10-20% following treatment with 10 micrograms/ml cholera toxin. This effect of the toxin could not be mimicked by incubation with the resolved B oligomer of cholera toxin, nor by exposure of cells to agents able to raise the intracellular levels of cAMP. Basal adenylate cyclase was stimulated in a biphasic manner by cholera toxin, being stimulated at low concentrations (0.01-10 ng/ml) and then decreased at high (0.1-10 micrograms/ml) concentrations. Thus, the down regulation of G-protein subunits produced by cholera toxin requires its (ADP-ribosyl)transferase activity but does not result from a cAMP-mediated mechanism. The toxin-mediated decrease of Go alpha in the membrane was correlated with a diminution of opioid-receptor-mediated stimulation of high-affinity GTPase activity, suggesting that opioid receptors interact with Go in native membranes of NG108-15 cells. Northern-blot analysis of cytoplasmic RNA prepared from cells treated with cholera toxin showed that the levels of mRNA coding for G beta 1 did not change. Thus, the cholera-toxin-induced decrease of G-protein subunits may not result from an alteration in mRNA levels, but may involve a direct effect of the toxin on the process of insertion and/or clearance of G proteins into and/or from the membrane. These data indicate that cholera toxin, besides catalyzing the ADP-ribosylation of Gs and Gi/Go types of G proteins, can also reduce the steady state levels of Go alpha and G beta 1 subunits in the membrane and thus alter by an additional mechanism the function of inhibitory receptor systems.     

Cross-dependence to opioid and alpha 2-adrenergic receptor agonists in NG108-15 cells

Clonidine, a partial alpha 2-agonist, has been used empirically to alleviate opiate withdrawal symptoms, but the mechanism of its effects is not completely understood. We studied the interactions of opioid and adrenergic receptor agonists in the NG108-15 cells, which are a model of opiate dependence. We determined that in these cells the adenylate cyclase (AC) [EC 4.6.1.1; ATP pyrophosphate-lyase (cyclizing) overshoot response to opioid or alpha 2-agonist withdrawal can be significantly attenuated or suppressed by the other agonist. Subsequently, the AC overshoot response can be triggered with the antagonist to the second agonist to which the cells were not dependent. These results demonstrate that convergent dependence to morphine and alpha 2 agonists can occur in a homogeneous cell population without neuronal loops. Therefore, the basic mechanisms that can account for convergent dependence in this model take place at the level of intracellular regulatory pathways that do not require neuronal networks.     

Trachynilysin,a neurosecretory protein isolated from stonefish (Synanceia trachynis) venom,forms nonselective pores in the membrane of NG108-15 cells

Trachynilysin, a protein toxin isolated from the venom of the stonefish Synanceia trachynis, has been reported to elicit massive acetylcholine release from motor nerve endings of isolated neuromuscular preparations and to increase both cytosolic Ca2+ and catecholamine release from chromaffin cells. In the present study, we used the patch clamp technique to investigate the effect of trachynilysin on the cytoplasmic membrane of differentiated NG108-15 cells in culture. Trachynilysin increased membrane conductance the most when the negativity of the cell holding membrane potential was reduced. The trachynilysin-induced current was carried by cations and reversed at about -3 mV in standard physiological solutions, which led to strong membrane depolarization and Ca2+ influx. La3+ blocked the trachynilysin current in a dose-, voltage-, and time-dependent manner, and antibodies raised against the toxin antagonized its effect on the cell membrane. The inside-out configuration of the patch clamp technique allowed the recording of single channel activity from which various multiples of 22 pS elementary conductance were resolved. These results indicate that trachynilysin forms pores in the NG108-15 cell membrane, and they advance our understanding of the toxin's mode of action on motor nerve endings and neurosecretory cells.     

Bradykinin stimulates GTP hydrolysis in NG108-15 membranes by a high-affinity, pertussis toxin-insensitive GTPase

In membranes of neuroblastoma x glioma hybrid (NG108-15) cells, bradykinin (EC50 approximately equal to 5 nM) stimulates GTP hydrolysis by a high-affinity GTPase (Km approximately equal to 0.2 microM). The octapeptide, des-Arg9-bradykinin, was inactive. Stimulation of GTP hydrolysis by bradykinin and an opioid agonist was partially additive. Treatment of NG108-15 cells with pertussis toxin, which inactivates Ni, eliminated GTPase stimulation by the opioid agonist but not by bradykinin. The data suggest that bradykinin activates in NG108-15 membranes a guanine nucleotide-binding protein which is not sensitive to pertussis toxin and which may be involved in bradykinin-induced stimulation of phosphoinositide metabolism in these cells.     

The alpha 2B adrenergic receptor of undifferentiated neuroblastoma x glioma hybrid NG108-15 cells, interacts directly with the guanine nucleotide binding protein, Gi2

In membranes of undifferentiated neuroblastoma x glioma hybrid cell line NG108-15, the apparent specific binding of [3H]yohimbine measured in the presence of 1 microM noradrenaline, was increased substantially by the presence of the poorly hydrolysed analogue of GTP, guanylyl-imidodiphosphate (Gpp[NH]p) or by preincubation of membranes with antibodies against the C-terminal decapeptide of the alpha subunit of the G-protein Gi2. Such an effect was not produced by antibodies against the equivalent region of Go alpha Gi3 alpha or Gs alpha or from non-immune serum. By contrast, total specific binding of [3H]yohimbine was not modified by co-incubation with Gpp[NH]p or by preincubation with the antibodies from any of the anti-G protein antisera. These results demonstrate a direct interaction of the alpha 2B adrenergic receptor of NG108-15 cells with Gi2.     

Opiate binding to membrane preparations of neuroblastoma x glioma hybrid cells NG108-15: effects of ions and nucleotides.

Interaction of a number of opiate agonists with the opiate receptors in NG108-15 cell membranes is influenced by ions, as well as certain nucleotides. Steady state binding of [³H]leu-enkephalin is increased by Mg⁺⁺ and decreased by Na⁺, GMP-P(NH)P, GTP, GDP, ITP and IMP-P(NH)P. Half-maximal inhibition produced by GMP-P(NH)P occurred at 4.6 μM. The dissociation of [³H]leu- and [³H]met-enkephalin, as well as [³H]etorphine, from these opiate receptors was also shown to be altered by both ions and nucleotides.     

Different modes of growth cone collapse in NG 108-15 cells

In the fundamental process of neuronal path-finding, a growth cone at the tip of every neurite detects and follows multiple guidance cues regulating outgrowth and initiating directional changes. While the main focus of research lies on the cytoskeletal dynamics underlying growth cone advancement, we investigated collapse and retraction mechanisms in NG108-15 growth cones transiently transfected with mCherry-LifeAct and pCS2+/EMTB-3XGFP for filamentous actin and microtubules, respectively. Using fluorescence time lapse microscopy we could identify two distinct modes of growth cone collapse leading either to neurite retraction or to a controlled halt of neurite extension. In the latter case, lateral movement and folding of actin bundles (filopodia) confine microtubule extension and limit microtubule-based expansion processes without the necessity of a constantly engaged actin turnover machinery. We term this previously unreported second type fold collapse and suggest that it marks an intermediate-term mode of growth regulation closing the gap between full retraction and small scale fluctuations.     

Muscarinic receptor regulation of NG108-15 adenylate cyclase: requirement for Na+ and GTP

Cholinergic agonists inhibit the basal and PGE1-activated adenylate cyclase activity in membranes isolated from the mouse neuroblastoma x glioma hybrid cell NG108-15. Inhibition is observed with acetylcholine, acetyl-beta-methylcholine and carbachol and is blocked by two specific muscarinic antagonists, atropine and quinuclydinylbenzilate. Inhibition of basal and PGE1-activated activity is only partial. Carbachol-directed inhibition has an apparent Km of 6 microM in the presence or absence of PGE1. Both the guanine nucleotide GTP and the monovalent cation Na+ are required for this muscarinic inhibition of basal and PGE1-activated NG108-15 adenylate cyclase. The selectivity observed for monovalent cations (all chloride salts) in this process is Na+ congruent to Li+ greater than K+ greater than Choline+ with the ED50 for Na+ congruent 40 microM. Of the nucleotides tested, only IT (and not ATP, UTP or CTP) replaces GTP in this process. GTP at 10 microM represents a saturating nucleotide concentration. Opiate-directed inhibition of NG108-15 adenylate cyclase has recently been shown to exhibit a similar requirement for GTP and Na+ [Blume, A. J., Lichtshtein, D. and Boone, G. (1979) Proc. National Academy of Sciences, USA, in press]. The data presented here therefore support the hypothesis that the general transfer of inhibitory information from membrane receptors to adenylate cyclase involves both a Na+ and GTP-sensitive process.     

Pertussis toxin inhibits enkephalin stimulation of GTPase of NG108-15 cells

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.     

Flavonoids from the leaves of Diospyros kaki reduce hydrogen peroxide-induced injury of NG108-15 cells

Flavonoids from the leaves of Diospyros kaki L (FLDK-P70) are main therapeutic components of NaoXingQing, which is a novel and patented Traditional Chinese Medicine drug used for the treatment of syndrome of apoplexy for years in China. The present study investigated the effects of FLDK-P70 on hydrogen peroxide (H2O2)-induced apoptosis-like damage of NG108-15 cells. Pretreatment of cells with FLDK-P70 alleviated H2O2-induced cell injury and apoptosis by upregulating Bcl-2 expression and improving redox imbalance as indicated by the alleviation of the decline in the intracellular endogenous antioxidants: glutathione and glutathione peroxidase as well as catalase, with the decrease of the leak of lactate dehydrogenase and the reduction of the accumulation of malondialdehyde. These results indicate that FLDK-P70 may be potentially used in the prevention and treatment of ischemia/reperfusion injury and other neurodegenerative disease. Upregulating bcl-2 and improving cellular redox state by FLDK-P70 may play critical roles in attenuating oxidative injury.     

Neurosteroids alter glutamate-induced changes in neurite morphology of NG108-15 cells

Activation of the NMDA receptor leads to increased intracellular Ca2+ levels ([Ca2+]i) which induces outgrowth of and morphologic changes in the neurites of the NG108-15 cell line. This effect can be blocked by antagonists for this glutamate receptor subtype (e.g. ifenprodil or AP5). We have previously shown that nanomolar concentrations of various neurosteroids modulate ifenprodil binding to the NMDA receptor. To investigate whether this interaction affects the functioning of the receptor, we studied the effect of 24 and 48 h of pregnenolone sulphate (PS) or pregnanolone sulphate (3alpha5betaS) on glutamate-stimulated NG108-15 cells. Unexpectedly, the neurosteroids themselves had an inhibitory effect on glutamate-induced changes in neurite patterns. This effect was comparable to that of ifenprodil or AP5. Moreover, the effect of combined treatment with 3alpha5betaS and ifenprodil on neurite morphology indicated a functional interaction between the substances. Interestingly, PS induced cell detachment over time, an effect that was further enhanced by ifenprodil. Cell detachment was also seen after 48 h of treatment with 3alpha5betaS; however, the effect was blocked by ifenprodil and weaker than that of PS. The interaction with the NR2B-selective antagonist ifenprodil indicates that this NMDA receptor subunit may be involved in neurosteroid-induced NG108-15 cell detachment.