Adenosine 3′,5′-monophosphate in cultured neuroblastoma cells: Effect of adenosine,phosphodiesterase inhibitors and benzazepines

Summary The effect of various neurohormones on intracellular levels of adenosine 3,5-monophosphate were evaluated in a neuroblastoma cell line both, in the presence and in the absence of the phosphodiesterase inhibitors isobutylmethylxanthine and papaverine. Without the phosphodiesterase inhibitors only prostaglandin E₁ increased intracellular adenosine 3,5-monophosphate levels. In the presence of isobutylmethylxanthine and/or papaverine, however, adenosine stimulated adenosine 3,5-monophosphate formation and the effect of prostaglandin E₁ was greatly potentiated. Treatment of the cells with dopamine, 5-hydroxytryptamine, noradrenaline, adrenaline, histamine and prostaglandin F₁ was without effect on adenosine 3,5-monophosphate levels either in the presence or absence of the phosphodiesterase inhibitors. The adenosine concentration for a half maximal effect was about 75 M. The effect of 0.1 mM adenosine was not antagonized by 1 mM theophylline. Several adenosine analogs were tested and found to have little or no effect on adenosine 3,5-monophosphate levels in neuroblastoma N4TG3. Diazepam and to a lesser extent chlordiazepoxide act like phosphodiesterase inhibitors when incubated together with prostaglandin E₁.Part of this work was done during a visit of the authors to NIH, U.S.A., J. S. being a fellow of the Deutsche Forschungsgemeinschaft and B. H. of the Max-Planck-Gesellschaft.     

Effects of adenosine analogues on basal, prostaglandin E1- and forskolin-stimulated cyclic AMP formation in intact neuroblastoma cells

We have examined the effects of R-phenylisopropyladenosine (R-PIA) and other adenosine analogues on basal, prostaglandin E1 (PGE1)- and forskolin-stimulated cyclic AMP (cAMP) formation in intact N1E-115 neuroblastoma cells, to determine whether the cells contain A1 adenosine receptors that are negatively coupled with adenylate cyclase. Basal levels of cAMP (68 +/- 7 pmol/mg protein; mean +/- SE, N = 15) were not altered by low concentrations of R-PIA. The apparent lack of inhibition was not due to increases in cAMP due to activation of a stimulatory A2 receptor by endogenously-synthesized adenosine. By comparison, low levels of R-PIA did reduce significantly (P less than 0.05) PGE1-dependent increases in cAMP formation (maximum response to PGE1, 972 +/- 77 pmol cAMP/mg protein; EC50 for PGE1, 0.2 microM). Inhibition was dose dependent, and resulted in a 30-50% maximum reduction in production stimulated by PGE1. Nanomolar concentrations of R-PIA elicited half-maximal inhibition; the inhibitory response was blocked by 8-phenyltheophylline (8-PT). The order of potencies of several adenosine analogues in eliciting this response suggested that inhibition was mediated by an A1 adenosine receptor. Examination of the effects of R-PIA on forskolin-stimulated cAMP formation yielded several interesting findings. First, stimulation by the diterpene by itself was blocked by both adenosine deaminase (ADA) and 8-PT (40 and 25% inhibition respectively). Low concentrations of R-PIA (less than 10(-6) M) had no effect on forskolin-stimulated cAMP production. At higher levels (greater than or equal to 10(-6) M) the analogues acted synergistically with the diterpene, to yield cAMP levels that were up to 3-fold higher than the additive effect of the two agents. Potentiation was stereospecific, Ca2+ dependent, and was blocked by 8-PT. The results of this study suggest that, in N1E-115 neuroblastoma cells, inhibitory A1 receptors are not stimulated in response to non-specific elevations in cAMP, but are associated with specific stimulatory receptors such as those activated by PGE1.     

Protein carboxyl-O-methyltransferase activity in cultured C-1300 neuroblastoma cells

Protein carboxylmethyltransferase (PCM) has been identified in a variety of tissues derived from neural crest anlage, including in vivo C-1300 murine neuroblastoma (MNB). These observations have stimulated interest in further defining the role of PCM as a potential modulator of neoplastic cell behavior. The subcellular distribution and kinetic behavior of PCM have been characterized in a tissue culture line derived from the C-1300 murine neuroblastoma (clone NB41A3). The specific and total activities of PCM in the presence and absence of exogenous substrate were determined in subcellular fractions of MNB cells prepared by differential centrifugation. In the presence of exogenous substrate (+ gelatin), 40% of the total PCM activity was present in the 100,000 g supernatant fraction and 41% in the 800 g paniculate fraction, whereas the higher specific activity of PCM was present in the 100,000 g supernatant fraction. Enzyme activity measured in the absence of gelatin, which reflects the concentration of endogenous methyl acceptor proteins in a cell fraction, was negligible. This activity represented less than 1.6 and 0.4% of the total PCM activity present in the 800 g paniculate and 100,000 g soluble fractions respectively. Cytosolic PCM had an apparent K_m of 13.9 × 10^?6 M for AdoMet and a V_max of 33 pmoles per min per mg protein. Cytoplasmic PCM was inhibited competitively by S-adenosylhomocysteine(K_i= 3.0 μM) and its analogues, sinefungin (K_i = 1.7 μM) and A-9145C (K_i = 0.2 μM). These data demonstrate that the specific activity of PCM was greatest in the soluble component of subcellular fractions prepared from cultured MNB cells. This distribution pattern of PCM is similar to that observed in the C-1300 MNB tumor grown in situ and in non-malignant neural tissues. In contrast to the latter tissues, cultured MNB cells exhibited low PCM activity when assayed in the absence of exogenous substrate.     

Interaction of tetanus toxin and toxoid with cultured neuroblastoma cells

Summary The primary interaction of tetanus toxin and toxoid with mouse neuroblastoma cells (C 1300, clone NB2A) in tissue culture was studied using direct immunofluorescence. Experiments were done in standard routine cultures and also those influenced by chemical modulators.There is a difference in the characteristic binding response between the growth culture cells (grown in presence of fetal calf serum) and differentiating culture cells (grown in absence of serum). Exposure to the toxin gives no visible effect on the cell division or viability in growth cultures; whereas in differentiating cells the processes are shortened and the adherence to the glass is diminished without involving significant cell death. The toxoid did not bind at all under the same experimental conditions.Since there was no biological effect in growth cultures we have called this binding ineffective, and in the case of the differentiating cells, effective binding. Stimulation of pinocytosis increases the uptake of toxin in both cultures. Presence of some surface bound toxin still remaining on the differentiating cells indicates the possibility of another sort of mechanism for internalization. Pre-treatment of the cells with neuraminidase of -galactosidase to alter the membrane gangliosides eliminates binding in growth cultures but not in differentiating cultures.From these results we suggest that even though the toxin may well bind to gangliosides, at least in the differentiating cultures they are not solely responsible for the fixation. The morphologically observed effective binding is probably that not related to gangliosides.This work was supported by a Medical Research Grant from World Health Organization to J.M.Z. A communication was presented at the Conference of the Swiss Society for Microbiology, Geneva, June 17–19, 1976     

The cytotoxicity of methylmercuric hydroxide and colchicine in cultured mouse neuroblastoma cells

After a 24-hr growth period in unsealed flasks, uncloned C1300 mouse neuroblastoma cells were exposed to either methylmercuric hydroxide (CH₃HgOH) (3 × 10^?9 –3 × 10^?6m) or colchicine (3 × 10^?10–1 × 10^?7m) in nutrient mixture F-12 (Ham) for 24–72 hr in sealed flasks. After 48 hr, CH₃HgOH (1 × 10^?6m) produced a 50% decrease in the number of morphologically differentiated cells and a significant increase in cell sloughing without inhibiting cell division or decreasing cell viability. However, after 48 hr, colchicine inhibited cell division and morphological differentiation at concentrations lower than those that caused increased cell sloughing or decreased cell viability. When CH₃HgOH (1 × 10^?6m) was replaced with control medium after 24 hr, most cytotoxic effects were reversed. The toxic effects were not as readily reversed after exposure to CH₃HgOH for 48 hr. Sloughed cells did not survive 48 hr exposure to CH₃HgOH when replaced in the control medium although they did survive after a 24-hr exposure. Recovery from the cytotoxic effects of CH₃HgOH was reduced in the presence of 3 × 10^?6m cycloheximide. It is concluded that CH₃HgOH exerts its toxicity at several sites within the neuroblastoma cell. Although the organomercurials have been shown to bind to neurotubles, the cytotoxicity of CH₃HgOH in neuroblastoma cells can not be ascribed solely to this mechanism. Recovery from the effects of short-term exposure may involve the synthesis of new protein.     

Adenosine and its analogs stimulate phosphoinositide hydrolysis in the kidney

Renal blood flow, glomerular filtration rate and sodium excretion are known to be affected by adenosine. The present studies were undertaken to investigate the actions of adenosine and its analogs (both agonists and antagonists) on phosphoinositide (PI) hydrolysis in the outer medullary slices. Adenosine was found to cause a dose-dependent stimulation of PI hydrolysis (ED50, 2.8 microM) in renal slices from outer medulla. The adenosine analogs 5'-(N-cyclopropyl)-carboxamidoadenosine (NCCA) and 5'-N-ethylcarboxamidoadenosine (NECA) also stimulated PI hydrolysis in renal medulla. Stimulation of PI hydrolysis was blocked by the adenosine antagonists: aminophylline, 1,3-dipropyl-7-methylxanthine (DMX) and 8-(p-sulfophenyl)-theophylline (8-SPT). Caffeine not only antagonized adenosine-stimulated PI hydrolysis but also increased PI hydrolysis independently. These results indicate that adenosine stimulates PI hydrolysis in renal medulla through a receptor-mediated mechanism.     

On the application of cultured neuroblastoma cells in chemical toxicity screening

The acute toxic action of a number of common chemicals was tested by their ability to cause detachment of cultured mouse neuroblastoma C1300 cells. A TD25 value was obtained by graphic estimation of the concentration needed to cause 25% of the total cell number to detach. These TD25 values were compared with LD50 values obtained from the literature, and they were found to correlate with a coefficient of 0.86. For six of the tested substances-diuron, butylated hydroxytoluene, benzidine, cyclophosphamide, Na2SeO3, and KCN-a very poor correlation was obtained. These diverging results could be ascribed to deficiencies in the neuroblastoma cell detachment test and emphasize the necessity for combined in vitro test procedures.     

1-Oleoyl-2-acetyl-glycerol and phorbol diester stimulate Ca2+ influx through Ca2+ channels in neuroblastoma x glioma hybrid NG108-15 cells

The effect of 1-oleoyl-2-acetyl-glycerol (OAG) and the phorbol diester 12-O-tetradecanoyl-phorbol-acetate (TPA) on the intracellular Ca2+ concentration ([Ca2+]i) in NG108-15 cells was studied using a Ca2+ indicator, quin 2. OAG and TPA induced an increase in [Ca2+]i from 100 +/- 19 to 187 +/- 24 nM and 192 +/- 15 nM, respectively, within 15 min. The increase in [Ca2+]i induced by activators of protein kinase C was dependent on the extracellular Ca2+ concentration [Ca2+]o) and was inhibited by the Ca2+ blockers, verapamil and nifedipine. These results indicate that the OAG- and TPA-induced [Ca2+]i increase is mediated by the influx of extracellular Ca2+ through voltage-sensitive Ca2+ channels.     

Membrane lesions in cultured mouse neuroblastoma cells exposed to metal compounds

Tritiated 2-deoxy-D-glucose (dGlc) was rapidly taken up into cultured mouse neuroblastoma C1300 cells (clone 41A₃). Upon perfusion the pre-loaded cultures slowly released radioactivity as [³H]2-deoxy-D-glucose-6-phosphate ([³H]dGlc-6-P) (rate const. = 0.017 min^?1) from a pool corresponding to 74% (t1/2 = 41 min) of the total radioactivity incorporated. Destruction of the plasma membrane of the cells by means of Triton X-100 (1.0%) resulted in a rapid and total release of the radioactivity. CH₃HgCl, HgCl, (C₂H₅)₃SnCl and K₂Cr₂O₇ all caused an increase in the passive cell membrane permeability to [³H]dGlc-6-P. A membrane toxic concentration (MTC) was defined as the concentration of the tested metal compound giving rise to an increase in the relative efflux from 1.0 to 1.2 during 60 min perfusion. Using this MTC-value, the membrane toxicity of the compounds could be ranked in the following order: CH₃HgCl (MTC = 9 × 10^?7 M >; HgCl (MTC = 6 × 10^?6 M) > (C₂H₅)₃SnCl (MTC = 3 × 10^?4 M) > K₂Cr₂O₇ (MTC = 7 × 10^?4 M. Since this differential toxicity is in accordance with other reports it is concluded that 2-deoxy-D-glucose (dGlc) may be used together with 41A₃ cells to screen metal compounds for their membrane toxicity.     

Cytotoxicity of cyclophosphamide and acrylamide in glioma and neuroblastoma cell lines cocultured with liver cells

A method for the cocultivation of nervous system cells with liver cells has been developed. When acrylamide was tested using the cocultivation procedure no significant liver metabolism-mediated changes in the cytotoxicity in C6 culture were detected. In neuroblastoma NIE115 cultures the toxicity was increased by induced chick hepatocytes at high acrylamide concentrations.     

Binding of radioactive methylmercuric chloride in subcellular fractions of neuroblastoma and glioma cells in culture

The binding of radioactive methylmercuric chloride ([²⁰³Hg] -CH₃HgCl) with various subcellular fractions of glioma (C-6) and neuroblastoma (NBP₂) cells was studied. Confluent cells were incubated in the presence of radioactive CH₃HgCl for a period of 5 h, and then various fractions were isolated. Most of the radioactivity was associated with the cytosol and particulate fractions. The cytosol and lipoprotein fractions of glioma cells incorporated more radioactive CH₃HgCl than those of NB cells. The specific radioactivities of the particulate, lipid and chromatin fractions were comparable in both cell types.     

Relationship between prostacyclin biosynthesis and cyclic AMP in cultured rabbit mesothelial cells

Calcium ionophore A23187 (10 microM) as well as thrombin (10 U/ml) stimulated the biosynthesis of prostacyclin in cultured rabbit mesothelial cells; in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (Mix, 1 mM) the cyclic AMP (cAMP) content was also elevated. Both effects were inhibited by indomethacin (28 microM). Exogenous prostacyclin elicited by itself a clear enhancement of intracellular cAMP. An increased cAMP content was also obtained with isoproterenol (10 microM), whose activity was antagonized by propranolol (10 microM). These two products however, had no effect on the prostacyclin release. In all these experiments, inhibition of phosphodiesterase with Mix, was necessary to obtain detectable cAMP levels. In the presence of Mix, the stimulation of prostacyclin production by A23187 and thrombin was significantly lower as compared to the stimulation in the absence of Mix. Our results suggest that increased prostacyclin biosynthesis results in adenylate cyclase stimulation. This rise in intracellular cAMP in the presence of Mix, is accompanied by a downward regulation of further prostacyclin production.     

Effect of methylmercuric chloride on gene expression in neuroblastoma and glioma cells after acute and chronic treatments

To understand the cellular and molecular mechanisms ofmethylmercuric chloride (CH₃HgCl)-induced damage to nerve tissue, monolayer cultures of glioma cells (C-6) and of neuroblastoma cells (NBP2) were used in this study. Chronic (6–8 weeks) and acute (5 days) treatment of glioma cells with low concentrations (0.05 to 0.1 μM) of CH₃HgCl produced marked increases and decreases in the amounts and net phosphorylation profiles of specific proteins. Chronic treatment of neuroblastoma cells (0.1 and 0.2 μM) did not produce any significant alterations in the amounts of specific proteins, but it caused marked changes in the phosphorylation levels of cellular proteins. The morphology and doubling time of chronically treated glioma and neuroblastoma cells did not change. Adenosine 3',5'-cyclic monophosphate (cyclic AMP)-stimulating agents produced morphological changes in chronically treated glioma and neuroblastoma cells similar to those produced in untreated cells.     

Hallucinogens antagonize histamine H1 receptors of cultured mouse neuroblastoma cells.

Hallucinogens were competitive antagonists of histamine at the H1-receptor of cultured mouse neuroblastoma cells. Their rank order of potency at this receptor was similar to that for their potency at eliciting subjective effects in vivo. Comparison with studies of other receptors, however, suggests that no single model of drug--receptor interaction adequately accounts for the known subjective effects of these compounds.     

尼卡地平抑制dbcAMP分化的神经母细胞瘤x神经胶质瘤杂种细胞（NG1 …

AIM: To investigate the possibility of dihydropyridine inhibition of N-type calcium channels. METHODS: Effects of nifedipine and nicardipine on the high K(+)-induced intracellular Ca2+ concentration ([Ca2+]i) increase were studied by measuring [Ca2+]i using the fluorescent indicator Fura-2. RESULTS: Pretreatment of cells with nifedipine 50 mumol.L-1 inhibited the high K(+)-induced [Ca2+]i transient by about 60% (n = 3); however, pretreatment of cells with nicardipine 10 mumol.L-1 completely prevented the high K(+)-evoked [Ca2+]i increase in dibutyryl cyclic AMP (dbcAMP)-differentiated NG 108-15 cells (n = 5). The high K(+)-induced [Ca2+]i increase was mediated by L- and N-type voltage-sensitive calcium channels (VSCC) in NG 108-15 cells. CONCLUSION: Nicardipine at micromolar range inhibited both L- and N-type VSCC in dbcAMP-differentiated NG 108-15 cells whereas nifedipine mainly inhibited L-type calcium channels.     

Multiple states of opioid receptors may modulate adenylate cyclase in intact neuroblastoma X glioma hybrid cells

Opioid receptor binding and opioid-mediated inhibition of cAMP accumulation were studied simultaneously in intact NG108-15 cells. The dose-response curves for the biological response were suggestive of positive cooperativity and systematically occurred at lower ligand concentrations than those for the binding of [3H] [D-Ala2, D-Leu5]enkephalin (DADLE), which were instead shallow and suggestive of a site heterogeneity or of a cooperative phenomenon. Computer modeling of the binding isotherms revealed that the data are best described assuming two binding sites with different affinities for the agonist; the mean ratio between the DADLE concentrations yielding half-maximal occupancy of the high affinity site and half-maximal response was 1.5, but it was 36 when the fractional occupancy of the sum of the two sites was considered. On examining several opioids, no direct correlation was found between high affinity site and biological response; however, several agonists displayed different affinities for the two sites, while the antagonist naloxone and the partial agonist diprenorphine bound to them with identical affinities. Furthermore, naloxone exhibited a good agreement between half-maximal receptor occupancy and Ki in blocking the agonist response. Thus, the binding heterogeneity detectable in intact cells is agonist-specific, and suggests rather that the sites are states of an identical receptor population. When [3H]diprenorphine was used to label the opioid receptors, the competition curves for DADLE were consistent with the existence of an additional, very low affinity state undetectable by direct binding with labeled agonist and, again, not discriminated by naloxone. Multiple affinity states of the opioid receptor in intact cells may reflect its interaction with the effector system in the plasma membrane.     

Cyclic AMP inhibits inositol polyphosphate production and calcium mobilization in neuroblastoma X glioma NG108-15 cells

In the neuroblastoma X glioma hybrid cell line NG108-15, bradykinin (BK) receptor stimulation induced a rapid and concentration-dependent rise in cytosolic free Ca2+ levels, as measured with the Ca2(+)-sensitive fluorescent dye fura-2. The Ca2+ transient was present in the absence of extracellular Ca2+ and was associated with a concentration-dependent production of inositol phosphates, particularly inositol trisphosphate (InsP3). Pretreatment of intact NG108-15 cells with forskolin or dibutyryl-cAMP plus isobutylmethylxanthine reduced BK-stimulated InsP3 production and the increase in cytosolic free Ca2+. Membranes prepared from forskolin- and [3H]inositol-pretreated NG108-15 cells also showed a diminished production of InsP3 elicited by guanosine 5'-[gamma-thio]triphosphate, NaF, or BK plus GTP. On the other hand, the Ca2+ sensitivity of membrane-associated phosphoinositide-specific phospholipase C (PI-PLC) was unaffected by forskolin pretreatment of intact NG108-15 cells. Collectively, these results suggest that A-kinase may inhibit receptor-mediated and postreceptor stimulation of PI-PLC in neuron-like cells, perhaps by impairing the coupling between a guanine nucleotide-binding protein and PI-PLC.     

Characterization of cyclic nucleotide phosphodiesterases from cultured bovine aortic endothelial cells

Experiments were carried out in order to isolate and characterize the cyclic nucleotide phosphodiesterase activities in primary and low passages of cultured bovine aortic endothelial cells. The subcellular characterization of the cyclic nucleotide hydrolytic activity showed that both cAMP and cGMP hydrolytic activities were predominant in the cytosolic rather than the particulate fraction of the endothelial cell homogenate. At a low substrate concentration (0.25 microM), the major hydrolytic activity was for cAMP while at a high concentration (20 microM) it was for both cAMP and cGMP. Both cAMP and cGMP hydrolytic activities were insensitive to calmodulin. Cytosolic cyclic nucleotide phosphodiesterase activity was resolved into two distinct phosphodiesterase forms using HPLC. The first eluted form was designated cGS-PDE: it hydrolysed both cAMP and cGMP and its cAMP hydrolytic activity was markedly enhanced by the presence of cGMP. The second form was designated cAMP-PDE: it selectively hydrolysed cAMP. The cytosolic cAMP-PDE was inhibited by micromolar concentrations of cAMP-PDE inhibitors such as trequinsin, rolipram, dipyridamole or papaverine. The cGS-PDE was inhibited by micromolar concentrations of trequinsin, dipyridamole and papaverine and was insensitive to rolipram, except for the hydrolysis of cAMP which was inhibited in the micromolar range. Both the cAMP-PDE and the cGS-PDE were relatively insensitive to the selective cGMP-PDE inhibitor, zaprinast which was about 750-fold less potent on endothelial PDEs than on smooth muscle cGMP-PDE. The identification of selective and specific PDE inhibitors of the different PDE forms may allow a better understanding of the regulation and the role of cyclic nucleotides in endothelial cells.     

Effects of toxic chemicals on the respiratory activity of cultured mouse neuroblastoma cells

Twenty common toxic chemicals were tested for their ability to inhibit respiratory activity in cultured mouse neuroblastoma C1300 cells, clone 41A3. Pentachlorophenol and hexachlorophene exhibited the properties of uncouplers of oxidative phosphorylation, whereas for KCN, pyridine, 2,5-hexandione, NaAsO2, K2Cr2O7, HgCl2, methylmercury and triethyltin more simple time-courses of inhibition were obtained. Ethanol, methanol, dimethyl sulphoxide, benzidine, nickel acetate, MnCl2, phenol, CoCl2, Na2SeO3 and CdCl2 did not cause any significant changes in respiratory activity. Among the effective compounds, those with well-known neurotoxic properties were the most potent in inhibiting respiration in 41A3 cells.