Mouse Neuroblastoma N18TG2 Cells Research Articles

Glycine N-acyltransferase-like 3 is responsible for long-chain N-acylglycine formation in N18TG2 cells

Long-chain fatty acid amides are signaling lipids found in mammals and other organisms; however, details of the metabolic pathways for the N-acylglycines and primary fatty acid amides (PFAMs) have remained elusive. Heavy-labeled precursor and subtraction lipidomic experiments in mouse neuroblastoma N18TG2 cells, a model cell line for the study of fatty acid amide metabolism, establish the biosynthetic pathways for the N-acylglycines and the PFAMs. We provide evidence that the N-acylglycines are formed by a long-chain specific glycine-conjugating enzyme, glycine N-acyltransferase-like 3 (GLYATL3). siRNA knockdown of GLYATL3 in the N18TG2 cells resulted in a decrease in the levels of the N-acylglycines and the PFAMs. This is the first report of an enzyme responsible for long-chain N-acylglycine production in cellula. The production of the PFAMs in N18TG2 cells was reported to occur by the oxidative cleavage of the N-acylglycines, as catalyzed by peptidylglycine α-amidating monooxygenase (PAM). siRNA knockdown of PAM resulted in an accumulation of [(13)C18]N-oleoylglycine and decreased levels of [(13)C18]oleamide when the N18TG2 cells were grown in the presence of [(13)C18]oleic acid. The addition of [1-(13)C]palmitate to the N18TG2 cell growth media led to the production of a family of [1-(13)C]palmitoylated fatty acid amides, consistent with the biosynthetic pathways detailed herein.

Synthesis and evaluation of a novel adenosine-ribose probe for global-scale profiling of nucleoside and nucleotide-binding proteins.

Proteomics is a powerful approach used for investigating the complex molecular mechanisms of disease pathogenesis and progression. An important challenge in modern protein profiling approaches involves targeting of specific protein activities in order to identify altered molecular processes associated with disease pathophysiology. Adenosine-binding proteins represent an important subset of the proteome where aberrant expression or activity changes of these proteins have been implicated in numerous human diseases. Herein, we describe an affinity-based approach for the enrichment of adenosine-binding proteins from a complex cell proteome. A novel N 6-biotinylated-8-azido-adenosine probe (AdoR probe) was synthesized, which contains a reactive group that forms a covalent bond with the target proteins, as well as a biotin tag for affinity enrichment using avidin chromatography. Probe specificity was confirmed with protein standards prior to further evaluation in a complex protein mixture consisting of a lysate derived from mouse neuroblastoma N18TG2 cells. Protein identification and relative quantitation using mass spectrometry allowed for the identification of small variations in abundance of nucleoside- and nucleotide-binding proteins in these samples where a significant enrichment of AdoR-binding proteins in the labeled proteome from the neuroblastoma cells was observed. The results from this study demonstrate the utility of this method to enrich for nucleoside- and nucleotide-binding proteins in a complex protein mixture, pointing towards a unique set of proteins that can be examined in the context of further understanding mechanisms of disease, or fundamental biological processes in general.

A novel fluorophosphonate inhibitor of the biosynthesis of the endocannabinoid 2‐arachidonoylglycerol with potential anti‐obesity effects

The development of potent and selective inhibitors of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) via DAG lipases (DAGL) α and β is just starting to be considered as a novel and promising source of pharmaceuticals for the treatment of disorders that might benefit from a reduction in endocannabinoid tone, such as hyperphagia in obese subjects. Three new fluorophosphonate compounds O-7458, O-7459 and O-7460 were synthesized and characterized in various enzymatic assays. The effects of O-7460 on high-fat diet intake were tested in mice. Of the new compounds, O-7460 exhibited the highest potency (IC₅₀ = 690 nM) against the human recombinant DAGLα, and selectivity (IC₅₀ > 10 μM) towards COS-7 cell and human monoacylglycerol lipase (MAGL), and rat brain fatty acid amide hydrolase. Competitive activity-based protein profiling confirmed that O-7460 inhibits mouse brain MAGL only at concentrations ≥ 10 μM, and showed that this compound has only one major 'off-target', that is, the serine hydrolase KIAA1363. O-7460 did not exhibit measurable affinity for human recombinant CB₁ or CB₂ cannabinoid receptors (Ki > 10 μM). In mouse neuroblastoma N18TG2 cells stimulated with ionomycin, O-7460 (10 μM) reduced 2-AG levels. When administered to mice, O-7460 dose-dependently (0-12 mg·kg⁻¹, i.p.) inhibited the intake of a high-fat diet over a 14 h observation period, and, subsequently, slightly but significantly reduced body weight. O-7460 might be considered a useful pharmacological tool to investigate further the role played by 2-AG both in vitro and in vivo under physiological as well as pathological conditions.

Primary fatty acid amide metabolism: conversion of fatty acids and an ethanolamine in N18TG2 and SCP cells

Primary fatty acid amides (PFAM) are important signaling molecules in the mammalian nervous system, binding to many drug receptors and demonstrating control over sleep, locomotion, angiogenesis, and many other processes. Oleamide is the best-studied of the primary fatty acid amides, whereas the other known PFAMs are significantly less studied. Herein, quantitative assays were used to examine the endogenous amounts of a panel of PFAMs, as well as the amounts produced after incubation of mouse neuroblastoma N(18)TG(2) and sheep choroid plexus (SCP) cells with the corresponding fatty acids or N-tridecanoylethanolamine. Although five endogenous primary amides were discovered in the N(18)TG(2) and SCP cells, a different pattern of relative amounts were found between the two cell lines. Higher amounts of primary amides were found in SCP cells, and the conversion of N-tridecanoylethanolamine to tridecanamide was observed in the two cell lines. The data reported here show that the N(18)TG(2) and SCP cells are excellent model systems for the study of PFAM metabolism. Furthermore, the data support a role for the N-acylethanolamines as precursors for the PFAMs and provide valuable new kinetic results useful in modeling the metabolic flux through the pathways for PFAM biosynthesis and degradation.

Uncovering the Biosynthetic Pathway of Oleamide

Oleamide belongs to a recently discovered class of signaling molecules called primary fatty acid amides (PFAMs). As of yet, the in vivo biosynthetic pathway for oleamide and the other cell-signalling PFAMs is unknown. The pathway we have proposed is shown in Fig. 1. Figure 1Open in figure viewerPowerPoint Hypothesized Pathway for PFAM Biosynthesis RT-PCR and Western analysis were employed to determine which enzymes from the pathway shown in Fig. 1 are expressed in mouse neuroblastoma N18TG2 cells (which are known to produce oleamide from oleic acid), choroid plexus cells, liver cells, and kidney cells. The expression of ACS and PAM in the N18TG2 cells supports the proposed biosynthetic pathway. None of the enzymes thought to catalyze reaction 2 in Fig. 1 (bile acid:amino acid transferase, acyl-CoA:glycine N-acyltransferase, or N-myristoyl transferase) were expressed in N18TG2 cells. This suggests that an undiscovered enzyme exists for the biosynthesis of the N-fatty acylglycine intermediates. Previous work from the Merkler laboratory has shown that addition of a PAM inhibitor to the N18TG2 cells culture medium results in accumulation of N-oleoylglycine. Future directions include identifying the enzyme responsible for production of the N-fatty acylglycines and using RNAi to target the PFAM biosynthetic enzymes to examine the accumulation of metabolic precursors.

Effects of AMP derivatives on cyclic AMP levels in NG108-15 cells.

1. In NG108-15 neuroblastomaxglioma hybrid cells, ATP stimulates intracellular cyclic AMP formation, which is inhibited by both adenosine (P(1)) and P2 receptor antagonists. In the present study, we examined the effects of several AMP derivatives in NG108-15 cells and mouse neuroblastoma N18TG-2 cells. 2. Adenosine 2'-monophosphate (A2P), adenosine 3'-monophosphate (A3P) and adenosine 5'-phosphosulphate (A5PS) increased cyclic AMP levels with similar concentration-dependencies in NG108-15 cells. 3. Increases in cyclic AMP by AMP derivatives were inhibited by the P2 receptor antagonist PPADS, but not by suramin. Effects of AMP derivatives were also inhibited by P(1) receptor antagonists ZM241385, XAC, DPCPX and partially by alloxazine. The ecto-nucleotidase inhibitor alpha, beta-methyleneADP was without effect. 4. In contrast, AMP derivatives did not change cyclic AMP levels in N18TG-2 cells. Accumulation of cyclic AMP in N18TG-2 cells was stimulated by adenosine A(2) receptor agonists CGS21680 and NECA, but not by ATP or beta, gamma-methyleneATP, agonists for cyclic AMP production in NG108-15 cells. 5. Reverse transcription-coupled polymerase chain reaction (RT - PCR) analyses revealed that N18TG-2 cells express both A(2A) and A(2B) receptors, while NG108-15 cells express mainly A(2A) receptors. 6. AMP derivatives did not affect the P2X and P2Y receptors expressed in NG108-15 cells. 7. These results suggest that A2P, A3P and A5PS act as agonists for cyclic AMP production and that these compounds are valuable tools for determinating the mechanism of ATP-stimulated cyclic AMP response in NG108-15 cells.

Morphological changes related to reconstituted acetylcholine release in a release-deficient cell line

The membrane changes accompanying Ca 2+-dependent acetylcholine release were investigated by comparing release-competent and release-incompetent clones of mouse neuroblastoma N18TG-2 cells. No release could be elicited in native N18 cells or in a N18–choline acetyltransferase clone in which acetylcholine synthesis was induced by transfection with the gene for rat choline acetyltransferase. However, acetylcholine release was operative in a To/9 clone which was co-transfected with complementary DNAs from rat choline acetyltransferase and Torpedo mediatophore 16,000 mol. wt subunit. In thin sections, the aspect of resting N18 and To/9 cells was identical: a very dense cytoplasm with practically no vesicle-like organelles. Cells were chemically fixed at different times during a stimulation using A-23187 and Ca 2+, and examined following both freeze-fracture and thin section. Stimulation of To/9 cells induced a marked change affecting the intramembrane particles. The number of medium-sized particles (9.9–12.38 nm) increased, while that of the small particles decreased. This change was not observed in control, release-incompetent cell lines. In the To/9 clone (but not in control clones), this was followed by occurrence of a large new population of pits which initially had a large diameter, but subsequently became smaller as their number decreased. Coated depressions and invaginations became abundant after stimulation, suggesting an endocytosis process. By considering the succession of events and by comparison with data from experiments performed on synapses in situ, it is proposed that a particle alteration was the counterpart of acetylcholine release in co-transfected To/9 cells; this was followed by a massive endocytosis.

Phosphatidic acid as the biosynthetic precursor of the endocannabinoid 2-arachidonoylglycerol in intact mouse neuroblastoma cells stimulated with ionomycin.

In mouse neuroblastoma N18TG2 cells prelabeled with [3H]arachidonic acid ([3H]AA) the biosynthesis of 2-arachidonoylglycerol (2-AG) is induced by ionomycin in a fashion sensitive to an inhibitor of diacylglycerol (DAG) lipase, RHC 80267, but not to four different phospholipase C (PLC) blockers. Pulse experiments with [3H]AA showed that ionomycin stimulation leads to the sequential formation of [3H]phosphatidic acid ([3H]PA), [3H]DAG, and [3H]2-AG. [3H]2-AG biosynthesis in N18TG2 cells prelabeled with [3H]AA was counteracted by propranolol and N-ethylmaleimide, two inhibitors of the Mg2+/Ca2(+)-dependent brain PA phosphohydrolase. Pretreatment of cells with exogenous phospholipase D (PLD) led to a strong potentiation of ionomycin-induced [3H]2-AG formation. These data indicate that DAG precursors for 2-AG in intact N18TG2 cells are obtained from the hydrolysis of PA and not through the activation of PLC. The presence of 2% ethanol during ionomycin stimulation failed to elicit the synthesis of [3H]phosphatidylethanol and did not counteract the formation of [3H]PA, thus arguing against the activation of PLD by the Ca2+ ionophore. Selective inhibitors of secretory phospholipase A2 and the acyl-CoA acylase inhibitor thimerosal significantly reduced [3H]2-AG biosynthesis. The implications of these latter findings, and of the PA-dependent pathways of 2-AG formation described here, are discussed.

CAMP-dependent induction of PDE5 expression in murine neuroblastoma cell differentiation

The present study demonstrates, in both hybrid NG108-15 and mouse neuroblastoma N18TG2 cells, the presence and regulation of PDE5 mRNA during cell differentiation. PDE5 cDNA probes in Northern blot analysis recognize a ∼9 kb transcript in bovine lung as well as in mouse neuroblastoma cells. Hybridization on total RNA extracted from dibutyryl-cAMP-treated NG108-15 cells shows a 5-fold increase of PDE5 9 kb mRNA: such an increase is not observed in N18TG2 although we observed a similar increase in the enzymatic activity of both cell lines. Our data demonstrate that PDE5 gene expression can be regulated by cAMP and suggest the existence of a complex regulatory system for PDE5 activity.

Arachidonoylserotonin and Other Novel Inhibitors of Fatty Acid Amide Hydrolase

Fatty acid amide hydrolase (FAAH) catalyzes the hydrolysis of bioactive fatty acid amides and esters such as the endogenous cannabinoid receptor ligands, anandamide (N-arachidonoyl-ethanolamine) and 2-arachidonoylglycerol, and the putative sleep inducing factorcis-9-octadecenoamide (oleamide). Most FAAH blockers developed to date also inhibit cytosolic phospholipase A2(cPLA2) and/or bind to the CB1 cannabinoid receptor subtype. Here we report the finding of four novel FAAH inhibitors, two of which, malhamensilipin A and grenadadiene, were screened out of a series of thirty-two different algal natural products, and two others, arachidonoylethylene glycol (AEG) and arachidonoyl-serotonin (AA-5-HT) were selected out of five artificially functionalized polyunsaturated fatty acids. When using FAAH preparations from mouse neuroblastoma N18TG2 cells and [14C]anandamide as a substrate, the IC50s for these compounds ranged from 12.0 to 26 μM, the most active compound being AA-5-HT. This substance was also active on FAAH from rat basophilic leukaemia (RBL-2H3) cells (IC50=5.6 μM), and inhibited [14C]anandamide hydrolysis by both N18TG2 and RBL-2H3 intact cells without affecting [14C]anandamide uptake. While AEG behaved as a competitive inhibitor and was hydrolyzed to arachidonic acid (AA) by FAAH preparations, AA-5-HT was resistant to FAAH-catalyzed hydrolysis and behaved as a tight-binding, albeit non-covalent, mixed inhibitor. AA-5-HT did not interfere with cPLA2-mediated, ionomycin or antigen-induced release of [3H]AA from RBL-2H3 cells, nor with cPLA2activity in cell-free experiments. Finally, AA-5-HT did not activate CB1 cannabinoid receptors since it acted as a very weak ligand in in vitro binding assays, and, at 10-15 mg/kg body weight, it was not active in the ‘open field’, ‘hot plate’ and rectal hypothermia tests carried out in mice. Conversely AEG behaved as a cannabimimetic substance in these tests as well as in the ‘ring’ immobility test where AA-5-HT was also active. AA-5-HT is the first FAAH inhibitor reported to date which is inactive both against cPLA2and at CB1 receptors, whereas AEG represents a new type of cannabinoid receptor agonist.

Heat shock protein 72 restores cyclic AMP accumulation after heat shock in N18TG2 cells

Although there are several reports on the alteration of intracellular signal transduction during heat shock in somatic cells, the long term effects of heat shock on neuronal cells remain unknown. In this report, we investigated cyclic AMP (cAMP) accumulation and the expression of heat shock proteins following heat shock in mouse neuroblastoma N18TG2 cells. Basal cAMP accumulation, or that stimulated by serotonin (10 μM), cholera toxin (1 μg/ml), and forskolin (1 μM) was suppressed at 0, 3, and 6 h following heat shock (45°C for 30 min). The cAMP levels were restored at 15 and 24 h after heat shock, corresponding with the expression of stress-induced heat shock protein 72 (HSP72). Quercetin, an inhibitor of HSP expression, decreased the expression of HSP72 and inhibited the recovery of cAMP levels 24 h after heat shock. Quercetin also decreased the basal expression of the constitutive heat shock cognate protein 70 (HSC70) and suppressed cAMP accumulation in non-heat shocked cells. These results suggest that stress-induced HSP72 restores cAMP accumulation to control levels following heat shock and that constitutive HSC70 is related to cAMP levels in non-stress conditions.

The Sleep Inducing Factor Oleamide Is Produced by Mouse Neuroblastoma Cells

Cis-9,10-octadecenoamide (oleamide) was isolated from the cerebrospinal fluid of sleep-deprived mammals and shown to induce sleep in rats. The enzyme catalyzing the hydrolysis of the amide bond of oleamide as well as of anandamide, the putative endogenous ligand of cannabinoid receptors, was purified from rat liver, cloned, shown to be expressed also in brain and named fatty acid amide hydrolase (FAAH). The enzymatic synthesis of oleamide from oleic acid and ammonia by rat brain microsomes has been also described. However, no evidence has been reported so far on the neuronal origin of oleamide, necessary in order to postulate for this compound a role as a neuromodulator. Here we show for the first time that oleamide is produced by a neuronal cell type and that its biosynthesis in intact neurons is not likely to occur through the direct condensation of oleic acid and ammonia. A lipid metabolite was extracted and purified from mouse neuroblastoma N18TG2cells through a sequence of chromatographic steps and characterized as oleamide by means of gas chromatography/electron impact mass spectrometry (GC/EIMS). The amount of oleamide, as estimated by GC analyses carried out in comparison with known amounts of synthetic oleamide, was 55.0±09.5 pmols/107cells, compared to less than 0.7 pmol/107cells for anandamide in the same cells. When N18TG2cells were prelabeled with [14C]oleic acid and the lipids extracted and purified, a radioactive component with the same chromatographic behavior as oleamide was found whose levels: (1) were not significantly influenced by stimulation with ionomycin; (2) were slightly increased by incubation with FAAH inhibitor phenyl-methyl-sulphonyl-fluoride (PMSF); (3) appeared to correlate with [14C]oleic acid incorporation into phospholipids but not with free [14C]oleic acid levels. N18TG2cell membranes were shown to contain an enzymatic activity catalyzing the synthesis of oleamide from oleic acid and ammonia. This activity was inhibited by FAAH selective inhibitors arachidonoyltrifluoromethylketone and methylarachidonoylfluorophosphonate, as well as by an excess of anandamide, and by PMSF at the same concentration which increased oleamide formation in intact cells. These data suggest that a FAAH-like enzyme working “in reverse” may be responsible for the formation of oleamide in cell-free preparations but not in whole cells.

Biosynthesis of 2-arachidonoyl-glycerol, a novel cannabimimetic eicosanoid, in mouse neuroblastoma cells.

Following the original isolation from porcine brain of the first endogenous ligand of cannabinoid receptors, anandamide (arachidonoyl-ethanolamide)1, another arachidonic acid (AA)-derived metabolite, 2-arachidonoyl-glycerol (2-AG), was found in canine gut2 and reported to share with anandamide and plant cannabinoids several pharmacological actions. 2-AG binds to either brain (CB1) or spleen (CB2) cannabinoid receptor subtypes with Ki values in the high nmolar-low μmolar range and inhibits forskolin-induced adenylate cyclase activation; in vivo, 2-AG reproduces the typical cannabimimetic effects of hypothermy, hypomotility and analgesia2. Moreover, 2-AG inhibits cell proliferation in lymphocytes that do not express the CB1 receptor subtype3, and, due to the inability of anandamide to functionally activate CB2 receptors4, has been suggested to act as the possible endogenous cannabinoid agonist in the immune system3, also on the basis of its recent finding in the spleen5. The isolation of mono-arachidonoyl-glycerol isomers from rat brain in levels 50–100 fold higher than those of anandamide6 and the recent report of a CB1 receptor-mediated stimulatory action by 2-AG on intracellular free Ca2+ in neuroblastoma x glioma hybrid cells7, however, raised the possibility that the monoglyceride might function as a possible ‘endocannabinoid’ also in the CNS. In this study we provide biochemical grounds to the hypothesis that 2-AG may be a neuronal mediator by describing: a) its Ca2+-dependent biosynthesis in mouse neuroblastoma N18TG2 cells, and b) the occurrence in the same cells of enzymatic activities responsible for 2-AG formation from diacylglycerols and (lyso)phosphatidylcholine.

Biosynthesis of anandamide and related acylethanolamides in mouse J774 macrophages and N18 neuroblastoma cells

Anandamide (arachidonoylethanolamide, AnNH) has been recently proposed as the endogenous ligand at the brain cannabinoid receptor CB1. Two alternative pathways have been suggested for the biosynthesis of this putative mediator in the central nervous system. Here we present data (1) substantiating further the mechanism by which AnNH is produced by phospholipase D (PLD)-catalysed hydrolysis of N-arachidonoylphosphatidylethanolamine in mouse neuroblastoma N18TG2 cells, and (2) suggesting for the first time that AnNH is biosynthesized via the same mechanism in a non-neuronal cell line, mouse J774 macrophages, together with other acylethanolamides and is possibly involved in the control of the immune/inflammatory response. Lipids from both neuroblastoma cells and J774 macrophages were shown to contain a family of N-acylphosphatidylethanolamines (N-aPEs), including the possible precursor of AnNH, N-arachidonoyl-PE. Treatment with exogenous PLD, but not with exogenous phospholipase A2 and ethanolamine, resulted in the production of a series of acylethanolamides (AEs), including AnNH, from both cell types. The formation of AEs was accompanied by a decrease in the levels of the corresponding N-aPEs. Enzymically active homogenates from either neuroblastoma cells or J774 macrophages were shown to convert synthetic N-[3H]arachidonoyl-PE into [3H]AnNH, thus suggesting that in both cells an enzyme is present which is capable of catalysing the hydrolysis of N-aPE(s) to the corresponding AE(s). Finally, as previously shown in central neurons, on stimulation with ionomycin, J774 macrophages also produced a mixture of AEs including AnNH and palmitoylethanolamide, which has been proposed as the preferential endogenous ligand at the peripheral cannabinoid receptor CB2 and, consequently, as a possible down-modulator of mast cells. On the basis of this as well as previous findings it is now possible to hypothesize for AnNH and palmitoylethanolamide, co-synthesized by macrophages, a role as peripheral mediators with multiple actions on blood cell function.

Evoked Acetylcholine Release Expressed in Neuroblastoma Cells by Transfection of Mediatophore cDNA

Transmitter release was elicited in two ways from cultured cells filled with acetylcholine: (a) in a biochemical assay by successive addition of a calcium ionophore and calcium and (b) electrophysiologically, by electrical stimulation of individual cells and real-time recording with an embryonic Xenopus myocyte. Glioma C6-Bu-1 cells were found to be competent for Ca(2+)-dependent and quantal release. In contrast, no release could be elicited from mouse neuroblastoma N18TG-2 cells. However, acetylcholine release could be restored when N18TG-2 cells were transfected with a plasmid coding for mediatophore. Mediatophore is a protein of nerve terminal membranes purified from the Torpedo electric organ on the basis of its acetylcholine-releasing capacity. The transfected N18TG-2 cells expressed Torpedo mediatophore in their plasma membrane. In response to an electrical stimulus, they generated in the myocyte evoked currents that were curare sensitive and calcium dependent and displayed, discrete amplitude levels, like in naturally occurring synapses.

Nucleotide sequence and promoter-specific effect of a negative regulatory region located upstream of the mouse proliferating cell nuclear antigen gene.

Different portions of the 5'-upstream region of the mouse proliferating cell-nuclear-antigen (PCNA) gene were combined with the bacterial chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in mouse neuroblastoma N18TG2 cells transfected with these recombinant plasmids and RNase protection analysis have revealed the existence of a negative regulatory region between nucleotides -1231 and -624 (+1 denotes the transcription initiation site). The CAT expression levels were gradually increased, depending on the extent of deletion from the 5'-terminus in this region, suggesting that the negative regulatory region consists of multiple elements with rather weak repressing activities. Significant sequence similarity was found between the negative regulatory region of the PCNA gene and those of the several reported genes. A 752-bp segment containing this negative regulatory region repressed the function of the PCNA gene promoter in an orientation-independent and position-independent manner. However, the negative regulatory region showed almost no repressing effect on the functions of the heterologous gene promoters such as the simian virus 40 enhancer promoter, the enhancer promoter in the Rous sarcoma virus long-terminal repeat and the mouse DNA polymerase beta gene promoter. These results suggest that the negative regulatory region of the mouse PCNA gene functions specifically to its own promoter. This unique property is discussed in comparison with that of the negative regulatory elements of the mouse DNA polymerase beta gene.

Characterization of 3':5' cyclic nucleotide phosphodiesterase activities of mouse neuroblastoma N18TG2 cells

Characterization of ‘low K m’ 3':5' cyclic nucleotide phosphodiesterase activities (PDE) expressed in mouse N 18TG2 neuroblastoma cells is reported. At least 3 peaks of activity were isolated by DEAE chromatography, none of which was calcium-calmodulin stimulated and cGMP stimulated or inhibited. A first peak elutes at 200 mM sodium acetate: it specifically hydrolyzes cGMP with a K m of 4.7 μM and shows sensitivity to zaprinast [M&B 22948] (1.8 μM). A second peak eluting at 410 mM sodium acetate hydrolyzes both cyclic nucleotides. A third peak. specific for cAMP hydrolysis, elutes at 580 mM sodium acetate, has a K m of 3.2 μM and is sensitive to RO 20 1724 (7.6 μM) and rolipram (2 μM). Hydrodynamic analysis showed for the first peak a Stokes radius of 5.3 nm with a sedimentation coefficient of 8.1 S, a frictional ratio ( f/ f 0) of 1.41 and a native molecular mass of 182 kDa. The same analysis for peak 3 showed a Stokes radius of 4.1 nm with a sedimentation coefficient of 3.2 S, a frictional ratio of 1.63 andanative molecular mass of 56kDa. The biochemical features reported for the enzyme eluting in the first peak, and its cGMP-binding activity stimulated by inhibitors of phosphodiesterase activity, demonstrate that it belongs to the PDE V subfamily; on the other hand the cAMP specific enzyme eluting in the third peak can be assigned to the ‘RO 20 1724 inhibited’ form. The significance of these findings is discussed in relation to the functional characteristics of the N18TG2 cell line.

Neurite outgrowth from N18TG2 neuroblastoma induced by H-7, a protein kinase inhibitor, in the presence of colchicine

Our previous studies have demonstrated that the protein kinase inhibitor H-7 promotes neurite outgrowth from mouse neuroblastoma N18TG2 cells as well as from primary cerebellar cells, and also that the neuntes induced by H-7 were more tolerant of colchicine (COL) than those induced by dibutyryl cAMP (dB-cAMP). In the present study, we tested the effects of H-7 and dB-cAMP on neurite growth from N18TG2 cells in the presence of COL. We found that only H-7 promoted neurite formation in the presence of COL. The percentage of cells with neurites induced by H-7 in the presence of COL (H-7+COL) was similar to that induced by H-7 alone. The neuntes induced by H-7+COL grew straight. They were very thin (less than 1 μm in diameter) and had round varicosities, as did the neurites induced by H-7 alone. By transmission electron microscopy, the neurites induced by H-7+COL were found to contain longitudinally arranged intermediate filaments (IF). Microtubules (MT) were not observed within the neurites. We also examined the effect of cytochalasin B (CB) on the neurites induced by H-7+COL and by H-7 alone. The neurites induced by H-7+COL were tolerant to CB, but those induced by H-7 were resorbed completely within 24 h after CB was applied. Neurites tolerant to CB contained longitudinally IF. Simultaneous application of CB with H-7+COL or with H-7 alone did not induce neurite formation. The present findings suggest that the neurite growth from N18TG2 cells induced by H-7 is independent of MT formation, although that induced by dB-cANP is closely related to MT. In addition, actin filaments play a crucial roles in H-7-induced neurite formation in N18TG2 cells, and IF enhanced the mechanical strength of the neurites.

Comparison of endothelin binding and calcium mobilization in C6-BU-1 rat glioma and N18TG2 mouse neuroblastoma cells

We have previously reported that endothelin (RT) receptor activation increases intracellular calcium concentrations ([Ca 2+] i) in NG108-15 cells, a hybrid of rat glioma C6-BU-1 and mouse neuroblastoma N18TG2 cells. This study was designed to further explore the origin of the ET receptor and [Ca 2+] i mobilization in the parent cell lines hybridized to form the NG108-15 cells. [ 125I]ET-1 bound to a single class of high affinity sites in C6-BU-1 cells with a K D value of 108pM and B max of 12400 sites/cell. ET-1, ET-2, ET-3 and big ET inhibited [ 125I]ET-1 binding to C6-BU-1 cells with K D values of 0.074, 0.167, 261 and 187 nM, respectively. All ETs produced a rapid increase in [Ca 2+] i in C6-Bu-1 cells. EC 50 values for ET-1, ET-2, ET-3 and big ET were 0.71, 1.14, 120 and 243 nM respectively. There was a significant correlation between the K D values obtained from competition binding experiments and the EC 50 values from [Ca 2+] i response curves in C6-BU-1 cells (r = 0.996, p < 0.004). Ten nM ET-1 produced about 85% of the maximal [Ca 2+] i increase in C6-BU-1 cells which was reduced by 96% in the absence of extracellular calcium. Furthermore, diltiazem (10 μM) and nifedipine (1 μM) failed to block ET-induced [Ca 2+] i mobilization. None of the ETs elevated [Ca 2+] i or displayed any specific [ 125I]ET-1 binding in N18TG2 cells. These data suggest that ET binds to a specific ET receptor in C6-BU-1 cells, and elevates [Ca 2+] i through dihydropyridine-insensitive, receptor-mediated calcium influx. Further, the ability of ETs to elevate [Ca 2+] i in NG108-15 hybrid cells is due to the ET receptor inherent to the C6-BU-1 glioma parent line.

Morphology of neurites from N18TG2 cell induced by protein kinase inhibitor H-7 and by cAMP

Protein kinase inhibitor H-7 and dibutyryl (dB)-cAMP were found to induce neuritic processes in mouse neuroblastoma N18TG2 cells (36). In the present study, morphological differences between the neurites induced by H-7 and those by dB-cAMP were examined using electron microscopy (TEM and SEM) and tubulin immunohistochemistry. It was observed that: 1. 1) The neurites induced by H-7 were relatively thin and frequently had varicosities. On the other hand, the neurites induced by dB-cAMP were thick but they had few varicosities. 2. 2) Centrioles were frequently observed in the cells treated with dB-cAMP but were not encountered in the H-7-treated cells. 3. 3) TEM and tubulin immunohistochemistry revealed that the main shafts of the neurites induced either by H-7 or dB-cAMP were filled with microtubules, but that the varicosities induced by H-7 contained a smaller amount of microtubules. 4. 4) The stability to colchicine was greater in the neurites induced by H-7 than in those by dB-cAMP. From these features of the neurites, it was inferred that neurite outgrowth induced by dB-cAMP is deeply related to the formation of microtubules and that the neurites induced by H-7 were involved in other processes probably including an adhesive property of cell surfaces.