Enhanced Acetylcholine Release from Cells that Have More 15-kDa Proteolipid in Their Membrane, a Constituent V-ATPase, and Mediatophore

Abstract: Mediatophore is a protein that translocates acetylcholine (ACh) on calcium action. It is a homopolymer of a 15-kDa proteolipid that is also a constituent of the membrane sector of vacuolar H⁺-adenosine trisphosphatase (V-ATPase; vacuolar proton pump). Experiments on neuroblastoma cell lines (N18TG-2) that are deficient for ACh release and on cells that are competent for release, such as the glioma C6BU-1 or the N18TG-2/C6BU-1 fusion product NG108-15, show that there is a correlation between ACh release and the 15-kDa proteolipid content of the cell membrane. In another cell line, L-M(TK⁻), it has been possible to up-regulate ACh release and the membrane proteolipid content after treating the cells with dibutyryl-cyclic AMP or dexamethasone. As mediatophore translocates ACh and as V-ATPase may help vesicular ACh storage, it was interesting to determine the respective role of the two proteins in the observed correlation between release and proteolipid content. After blocking vesicular loading with vesamicol, we did not affect release from these cells, suggesting that the observed correlation may be attributed to mediatophore. The acquisition of an ACh release mechanism would then depend on the process that guides the proteolipid to the plasma membrane of the cell.     

Rapid Degradation of Neurotensin by Intact Murine Neuroblastoma Cells (Clone N1E-115)

Murine neuroblastoma clone N1E-115, which possesses receptors for neurotensin mediating the formation of intracellular cyclic GMP and the stimulation of inositol phospholipid hydrolysis, exhibited only partial desensitization to neurotensin. This result led to the observation that neurotensin was very rapidly degraded by intact N1E-115 cells. In experiments measuring the time course of [³H]neurotensin degradation, a minimum of six major tritiated products were found, with the breakdown peptides formed and the degree of proteolysis of [³H]neurotensin being dependent upon the length of incubation and the concentration of cells. Clone N1E-115 degraded [³H]neurotensin in an apparently sequential fashion; the primary initial cleavage of intact neurotensin was at the peptide bond between residues Arg⁸ and Arg⁹. Initial degradation peptides from the active carboxyl-terminal portion of neurotensin were more rapidly degraded, after formation, than were the peptides from the inactive amino-terminal half of neurotensin. The final two degradation products found were tyrosine, from the carboxyl-terminal portion of neurotensin, and an as yet unidentified peptide from the amino-terminal half of neurotensin. [³H]Neurotensin(8–13) was more rapidly hydrolyzed under identical conditions than was [³H]neurotensin itself. A combination of the protease inhibitors 1, 10-phenanthroline and Z-Pro-Prolinal was able to inhibit almost completely the degradation of neurotensin by clone N1E-115.     

Evoked Acetylcholine Release by Immortalized Brain Endothelial Cells Genetically Modified to Express Choline Acetyltransferase and/or the Vesicular Acetylcholine Transporter

Immortalized rat brain endothelial RBE4 cells do not express choline acetyltransferase (ChAT), but they do express an endogenous machinery that enables them to release specifically acetylcholine (ACh) on calcium entry when they have been passively loaded with the neurotransmitter. Indeed, we have previously reported that these cells do not release glutamate or GABA after loading with these transmitters. The present study was set up to engineer stable cell lines producing ACh by transfecting them with an expression vector construct containing the rat ChAT. ChAT transfectants expressed a high level of ChAT activity and accumulated endogenous ACh. We examined evoked ACh release from RBE4 cells using two parallel approaches. First, Ca2+-dependent ACh release induced by a calcium ionophore was followed with a chemiluminescent procedure. We showed that ChAT-transfected cells released the transmitter they had synthesized and accumulated in the presence of an esterase inhibitor. Second, ACh released on an electrical depolarization was detected in real time by a whole-cell voltage-clamped Xenopus myocyte in contact with the cell. Whether cells synthesized ACh or whether they were passively loaded with ACh, electrical stimulation elicited the release of ACh quanta detected as inward synaptic-like currents in the myocyte. Repetitive stimulation elicited a continuous train of responses of decreasing amplitudes, with rare failures. Amplitude analysis showed that the currents peaked at preferential levels, as if they were multiples of an elementary component. Furthermore, we selected an RBE4 transgenic clone exhibiting a high level of ChAT activity to introduce the Torpedo vesicular ACh transporter (VAChT) gene. However, as the expression of ChAT was inactivated in stable VAChT transfectants, the potential influence of VAChT on evoked ACh release could only be studied on cells passively loaded with ACh. VAChT expression modified the pattern of ACh delivery on repetitive electrical stimulation. Stimulation trains evoked several groups of responses interrupted by many failures. The total amount of released ACh and the mean quantal size were not modified. As brain endothelial cells are known as suitable cellular vectors for delivering gene products to the brain, the present results suggest that RBE4 cells genetically modified to produce ACh and intrinsically able to support evoked ACh release may provide a useful tool for improving altered cholinergic function in the CNS.     

Muscarinic Acetylcholine Receptors in Neuroblastoma Cells: Lack of Effect of Veratrum Alkaloids on Receptor Number

The effect of compounds that activate sodium channels on the number of muscarinic acetylcholine receptors in neuroblastoma NIE 115 cells has been investigated. The cells were used in electrically unexcitable ("control" cells) and excitable ("differentiated" cells) states. Although receptor assays using a single concentration of the radioligand [3H]scopolamine methyl chloride indicated a loss of receptors after a 6-h incubation of cells with veratrine, no true loss of receptors was seen with any of the compounds tested (veratridine, veratrine, aconitine) when full saturation analyses were performed in either control or differentiated cells. The apparent receptor loss seen with veratrine was due to a muscarinic receptor-active component of veratrine (not veratridine) occluded by the cells and released into the binding assays upon cell breakage. Veratridine and aconitine have a very low affinity for muscarinic acetylcholine receptors, and the binding of carbamoylcholine to the receptors is unaffected by tetrodotoxin, so that there is no evidence in this system for interaction between muscarinic receptors and sodium channels.     

Biochemical Characterization of Two Distinct Angiotensin AT2 Receptor Populations in Murine Neuroblastoma N1E-115 Cells

Abstract: The murine neuroblastoma N1E-115 cell line possesses a high density of angiotensin II (Angll) receptors that can be solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. These solubilized binding sites exhibited high affinity for CGP-42112A and not Losartan, indicating that they were of the AT₂ subtype. However, displacement of ¹²⁵I-Angll with the AT₂ nonpeptide antagonist PD-123319 resulted in a biphasic curve, suggesting heterogeneity of the AT₂ receptor population in N1E-115 cells. In support of this view, separation of two receptor populations was accomplished with heparin-Sepharose chromatography. More specifically, three distinct protein peaks eluted from the heparin-Sepharose column, two of which bound ¹²⁵I-Angll with high affinity and saturability. One of these binding peaks (peak I) eluted rapidly and represented ～80% of the total binding activity, whereas the remaining binding activity was contained within a second peak (peak III) that required the addition of 1.5 M NaCI for its complete elution. Pharmacological analysis revealed that both peaks of binding activity were exclusively AT₂ receptors insofar as they exhibited high affinity for CGP-42112A and little or no affinity for the AT₁-selective antagonist Losartan. However, whereas the nonpeptidic AT₂-selective antagonist PD-123319 completely displaced the binding of ¹²⁶I-Angll from peak I in a monophasic fashion (IC₅₀= 9.1 ± 4.1 nM; mean ± SEM; n = 3), PD-123319 was much less effective in displacing ¹²⁵I-Angll from peak III (IC₅₀= 196 β 27 nM; mean β SEM; n = 3). Treatment of individual peaks with the reducing agent dithiothreitol caused a large increase in ¹²⁵I-Angll specific binding in peak III, whereas a decrease in binding was observed in peak I. Moreover, GTPγS significantly reduced high-affinity agonist binding in peak I but not peak III, further suggesting heterogeneity in the AT₂ receptor family. Finally, immunoblotting studies with polyclonal antisera raised against peak I specifically detected two proteins of 110 and 66 kDa, as is true in crude solubilized membranes, whereas no immunospecific proteins were detected in peak III. These same antisera immunoprecipitated ¹²⁵I-Angll binding activity in peak I but were ineffective in peak III. Collectively, these results suggest that heparin-Sepharose chromatography can efficiently separate two pharmacologically, biochemically and immunologically distinct populations of AT₂ receptors.     

Increase in Neurite Formation and Acetylene line Release by Transfection of Growth-Associated Protein-43 cDNA into NG108–15 Cells

Abstract: We previously reported that growth-associated protein-43 (GAP-43) could be involved in the maintenance of elongated neurites and that a decline in protein kinase C activity may be involved in accumulation of GAP-43. In the present study, to clarify the functional significance of GAP-43 for neurite maintenance and acetylcholine (ACh) release, we prepared NG-G11 cells by transfection of GAP-43 cDNA into NG108-15 cells. NG-G11 cells expressed GAP-43 mRNA at levels approximately twice that in nontransfected or vector-transfected cells under control conditions and after treatment with dibutyryl cyclic AMP (diBu-cAMP) or 12-O-tetradecanoylphorbol 13-acetate (TPA) plus diBu-cAMP. Neurite outgrowth after addition of diBu-cAMP was greater in NG-G11 than in control cells. In NG-G11 cells, neurites elongated by treatment with diBu-cAMP for 72 h were maintained after removal of the drug. Treatment with TPA plus diBu-cAMP for 24 h induced neurite outgrowth in NG-G11 cells, although control cells required 72 h. Depolarization by 50 m M KCI induced ACh release in both NG-G11 and control cells treated with diBu-cAMP or TPA/diBu-cAMP. Although removal of the drugs following diBu-cAMP treatment reversed ACh release to nontreated levels in control cells, a high-K⁺-induced level of ACh release remained in NG-G11 cells after removal of diBu-cAMP. ACh release induced by TPA plus diBu-cAMP for 24 h was further enhanced after removal of the drugs in NG-G11 cells, but it was not seen in control cells. These results suggest that levels of GAP-43 mRNA are correlated with neurite maintenance and the level of ACh release. Thus, GAP-43 may be involved in neuronal differentiation in NG108-15 cells.     

Differential Expression of MARCKS and Other Calmodulin-Binding Protein Kinase C Substrates in Cultured Neuroblastoma and Glioma Cells

Abstract: Expression of the protein kinase C substrate MARCKS and other heat-stable myristoylated proteins have been studied in four cultured neural cell lines. Amounts of MARCKS protein, measured by [³H]myristate labeling and western blotting, were severalfold higher in rat C6 glioma and human HTB-11 (SK-N-SH) neuroblastoma cells than in HTB-10 (SK-N-MC) or mouse N1E-115 neuroblastoma cells. Higher levels of MARCKS mRNA were also detected in the former cell lines by S1 nuclease protection assay. At least two additional ³H-myristoylated proteins of 50 and 40–45 kDa were observed in cell extracts heated to >80°C or treated with perchloric acid. The 50-kDa protein, which bound to calmodulin in the presence of Ca²⁺, was more prominent in cells (N1E-115 and HTB-10) with less MARCKS, whereas neuromodulin (GAP-43) was detected in N1E-115 and HTB-11 cells only. Heating resulted in a fourfold increase in the detection of MARCKS by western blotting; this was not paralleled by a similar increase in [³H]myristate-labeled MARCKS and may be due to a conformational change affecting the C-terminal epitope or enhanced retention of the protein on nitrocellulose. Addition of β-12- O -tetradecanoylphorbol 13-acetate resulted in three- to fourfold increased phosphorylation of MARCKS in HTB-11 cells, with little increase noted in HTB-10 cells. These results indicate that MARCKS, neuromodulin, and other calmodulin-binding protein kinase C substrates exhibit distinct levels of expression in cultured neurotumor cell lines. Of these proteins, only MARCKS appears to be correlated with phorbol ester stimulation of phosphatidylcholine turnover in these cells.     

Promotion of Neuritogenesis in Mouse Neuroblastoma Cells by Exogenous Gangliosides. Relationship Between the Effect and the Cell Association of Ganglioside GM1

Ganglioside GM1 promoted neuritogenesis of neuroblastoma cells, neuro-2a clone, in monolayer culture. GM1 bound to neuro-2a cells in three distinct forms, one removable by treatment with serum-containing solutions, one serum-resistant and labile to trypsin treatment, and one resistant to serum and trypsin treatments. The proportions among the three forms of cell-associated GM1 varied in relation to duration of exposure to ganglioside, ganglioside concentration in the medium, and number of cells in culture. The form removable by serum was predominant at the initial stages of association and at the highest ganglioside concentrations (over 10(-6)M); the trypsin-labile and -stable forms tended to increase with increasing cell number and decreasing ganglioside concentration. The neuritogenic effect of GM1 was higher when neuro-2a cells were incubated for 24 h in the presence of GM1 and fetal calf serum. Under this condition the percentage of neurite-bearing cells increased from 11% of control to 62% at the optimal ganglioside concentration of 10-4M. The effect was still present, although to a lower extent (from 11% to 28% of neurite-bearing cells), when cells were first exposed for only 2 h to GM1, then washed and incubated for 24 h in the presence of fetal calf serum. The trypsin-labile and -stable forms of cell-associated GM1 had a fundamental role in the effect, whereas the form removable by serum was not involved. The preparation of GM1 used was extremely pure (99%) and, in particular, had a peptide contamination, if any, less than 1:20,000-1:50,000.(ABSTRACT TRUNCATED AT 250 WORDS)     

瞬时转染金属硫蛋白-3基因后人神经母细胞瘤细胞系SH—SY5Y细胞的蛋白质差异表达

金属硫蛋白 3(MT 3) ,又称神经生长抑制因子 ,主要表达于中枢神经系统。它属于金属硫蛋白家族 ,但具有几项其他家族蛋白质如MT 1/ 2等所不具有的独特性质 ,是一种多功能蛋白质 ,可在中枢神经系统中发挥重要的神经调节和神经保护作用 ,但是具体发挥机制还很不清楚。实验以人神经母细胞瘤细胞系SH SY5Y为模型 ,运用最近发展起来的比较蛋白质组学研究方法对MT 3基因瞬时转染引起的SH SY5Y细胞蛋白质的整体变化进行了系统的研究。经考马斯亮蓝染色 ,结果表明 ,MT 3转基因后平均每块胶上可检测到约 75 0个蛋白质点。利用ImageMaster 2DElite软件对胶上的蛋白质点进行半定量分析 ,发现共有 17个蛋白质点呈显著的变化 :和对照组比较 ,在这 17个点中 ,有 12个表达明显上调 ,有 5个表达水平明显下降 ,实验结果具有可重复性。结合pI值和分子量 ,应用基质辅助激光解吸 /电离飞行时间质谱对这 17个点进行分析 ,鉴定了其中 10个点 ,包括类锌指蛋白 ,谷氨酸转运蛋白和增强蛋白等。这些蛋白质都可在神经系统功能的调节中发挥作用。实验结果表明MT 3可能是通过调节和 /或协同这些蛋白质来发挥它的多种功能的。     

NGX6基因转染对鼻咽癌细胞基因表达谱的影响

鼻咽癌是我国南方的常见肿瘤 .NGX6是新近克隆的定位于 9p2 1 2 2 ,在鼻咽癌组织中表达下调的基因 .初步的研究结果显示 ,NGX6基因转染鼻咽癌细胞HNE1能够延缓其生长速度 ,使肿瘤细胞更多地停留在G0 G1期 .为探索NGX6基因在鼻咽癌发病机制中的作用 ,建立了高表达NGX6的鼻咽癌细胞系 .利用包含 14 0 0 0个基因的cDNA微阵列分析了NGX6基因转染对HNE1细胞基因表达谱的影响 ,发现NGX6基因的转染能够上调p2 9、APC7、NEU1、RNasek6、αE catenin和TFⅡEα等基因的表达 ;同时也下调properdinP因子、G0S2、BAZ2B、ZHX1,OS4和PBX3等基因的表达 .研究结果提示 ,NGX6基因对鼻咽癌细胞的生物学行为的影响可能与它对一些细胞周期调控因子和转录调控因子的影响相关 .作为一种高通量的分析技术 ,cDNA微阵列为新基因的功能研究提供了重要的线索     

Thiamine Deficiency-Induced Partial Necrosis and Mitochondrial Uncoupling in Neuroblastoma Cells Are Rapidly Reversed by Addition of Thiamine

Abstract: Culture of neuroblastoma cells in a medium of low-thiamine concentration (6 n M ) and in the presence of the transport inhibitor amprolium leads to the appearance of overt signs of necrosis; i.e., the chromatin condenses in dark patches, the oxygen consumption decreases, mitochondria are uncoupled, and their cristae are disorganized. Glutamate formed from glutamine is no longer oxidized and accumulates, suggesting that the thiamine diphosphate-dependent α-ketoglutarate dehydrogenase activity is impaired. When thiamine (10 µ M ) is added to the cells, the O₂ consumption increases, respiratory control is restored, and normal cell and mitochondrial morphology is recovered within 1 h. Succinate, which is oxidized via the thiamine diphosphate-independent succinate dehydrogenase, is also able to restore a normal O₂ consumption (with respiratory control) in digitonin-permeabilized thiamine-deficient cells. Our results therefore suggest that the slowing of the citric acid cycle is the main cause of the biochemical lesion induced by thiamine deficiency as observed in Wernicke's encephalopathy.     

Dissociation of Phosphorylation and Translocation of a Myristoylated Protein Kinase C Substrate (MARCKS Protein) in C6 Glioma and N1E-115 Neuroblastoma Cells

Abstract: An 80-kDa protein labeled with [³H]myristic acid in C6 glioma and N1E-115 neuroblastoma cells has been identified as the myristoylated alanine-rich C kinase substrate (MARCKS protein) on the basis of its calmodulin-binding, acidic nature, heat stability, and immunochemical properties. When C6 cells preincubated with [³H]myristate were treated with 200 n M 4β-12- O -tetradecanoylphorbol 13-acetate (β-TPA), labeled MARCKS was rapidly increased in the soluble digitonin fraction (maximal, fivefold at 10 min) with a concomitant decrease in the Triton X-100–soluble membrane fraction. However, phosphorylation of this protein was increased in the presence of β-TPA to a similar extent in both fractions (maximal, fourfold at 30 min). In contrast, β-TPA–stimulated phosphorylation of MARCKS in N1E-115 cells was confined to the membrane fraction only and no change in the distribution of the myristoylated protein was noted relative to α-TPA controls. These results indicate that although phosphorylation of MARCKS by protein kinase C occurs in both cell lines, it is not directly associated with translocation from membrane to cytosol, which occurs in C6 cells only. The cell-specific translocation of MARCKS appears to correlate with previously demonstrated differential effects of phorbol esters on stimulation of phosphatidylcholine turnover in these two cell lines.     

Stimulation of Neurite Outgrowth in Neuroblastoma Cells by Neuraminidase: Putative Role of GM1 Ganglioside in Differentiation

Treatment of three neuroblastoma cell types in culture with neuraminidase resulted in enhanced neurite outgrowth. These included the mouse Neuro-2A and rat B104 and B50 lines. The morphological changes depended on the presence of exogenous Ca2+ and were accompanied by modest but statistically significant increases in 45Ca2+ influx. Neuraminidase-stimulated neuritogenesis was blocked by the B subunit of cholera toxin (cholera B) and anti-GM1 antibody, a finding suggesting the effect was due to an increased amount of GM1 on the cell surface. Cholera B also blocked the increase in 45Ca2+ influx. The mouse N1A-103 line, previously characterized as "neurite minus," did not respond to neuraminidase with either neurite outgrowth or enhanced Ca2+ influx. These results point to an influence of GM1 on neuritogenesis in cells with differentiation potential and suggest a mechanism involving modulation of Ca2+ flux.     

Stimulation of Phospholipase D Activity in Human Neuroblastoma (LA-N-2) Cells by Activation of Muscarinic Acetylcholine Receptors or by Phorbol Esters: Relationship to Phosphoinositide Turnover

We have investigated the coupling of muscarinic acetylcholine receptors (mAChR) to phospholipid hydrolysis in a human neuroblastoma cell line, LA-N-2, by measuring the formation of 3H-inositol phosphates (3H-IP) and of [3H]phosphatidylethanol ([3H]PEt) in cells prelabeled with [3H]inositol and [3H]oleic acid. The muscarinic agonist carbachol (CCh) stimulated the phospholipase C (PLC)-mediated formation of 3H-IP in a time- and dose-dependent manner (EC50 = 40-55 microM). In addition, in the presence of ethanol (170-300 mM), CCh elevated levels of [3H]PEt [which is regarded as a specific indicator of phospholipase D (PLD) activity] by three- to sixfold. The effect of CCh on PEt formation also was dose dependent (EC50 = 50 microM). Both effects of CCh were antagonized by atropine, indicating that they were mediated by mAChR. Incubation of LA-N-2 cells with the phorbol ester phorbol 12-myristate 13-acetate (PMA, 0.1 microM; 10 min) increased [3H]PEt levels by up to 10-fold. This effect was inhibited by the protein kinase C (PKC) inhibitor staurosporine (1 microM) or by pretreatment for 24 h with 0.1 microM PMA, by 74% and 65%, respectively. In contrast, the effect of CCh on PEt accumulation was attenuated by only 28% in the presence of staurosporine (1 microM). In summary, these results suggest that, in LA-N-2 neuroblastoma cells, mAChR are coupled both to phosphoinositide-specific PLC and to PLD. PKC is capable of stimulating PLD activity in these cells; however, it is not required for stimulation of the enzyme by mAChR activation.     

LPS诱导肝细胞L02释放HMGB1蛋白的研究

以人单核巨噬细胞系U937细胞为对照,探讨肝细胞L02分泌晚期炎症介质高迁移率族蛋白HMGB1过程的特点.400μg/L LPS作用于人U937细胞和L02细胞,MTT和荧光TUNEL结果显示0～24 h,两种细胞均未见明显坏死和凋亡.RT-PCR结果表明L02细胞HMGB1 mRNA表达水平高于相应对照组的时相(20 h)晚于U937细胞(16 h).Western blot显示L02细胞上清中检测到HMGB1蛋白的时相(16 h)晚于U937细胞(8h);两者上清HMGBl蛋白水平呈时间依赖性,但U937细胞分泌HMGB1蛋白的量占有绝对优势.细胞免疫荧光观察到两者均有HMGB1从细胞核向胞浆移位的现象,但L02细胞晚于U937细胞.由此可见,LPS诱导肝细胞分泌晚期炎症介质HMGB1具有时相晚、量少的特点,且其分泌HMGB1的过程与HMGB1蛋白移位有关,而并不依赖于细胞坏死与凋亡.     

Stimulation of Inositol Phosphate Production by Neurotensin in Neuroblastoma N1E115 Cells: Implication of GTP-Binding Proteins and Relationship with the Cyclic GMP Response

The association of neurotensin to its receptor in differentiated neuroblastoma N1E115 cells led to a fast and transitory increase of the intracellular concentration in inositol triphosphate and inositol biphosphate, followed by a slower and more stable increase inositol monophosphate. The action of inositol 1,4,5-triphosphate on digitonin-permeabilized N1E115 cells resulted in a stimulation of cyclic GMP levels that mimicked that induced by neurotensin. Therefore, the cyclic GMP stimulation is probably a consequence of the initial inositol triphosphate formation triggered by neurotensin. Fluoroaluminate ions and pertussis toxin had the capacity to modulate positively and negatively, respectively, the formation of inositol triphosphate induced by neurotensin, indicating that GTP-binding proteins are involved in the regulation of inositol phosphate levels by neurotensin receptors.     

Heterogeneity of Nucleotide Receptors in NG108-15 Neuroblastoma and C6 Glioma Cells for Mediating Phosphoinositide Turnover

Abstract: We have compared the characteristics of receptors for nucleotide analogues and the involvement of phospholipase C (PLC) in the effector mechanism in NG108-15 neuroblastoma and C6 glioma cells. The relative potency of these analogues to stimulate inositol phosphate (IP) formation is UTP > UDP ? 2-methylthio-ATP (2-MeSATP), GTP > ATP, CTP > ADP > UMP in NG108-15 cells and ATP > UTP > ADP > GTP > UDP ? 2Me-SATP, CTP, UMP in C6 glioma cells. α,β-Methylene-ATP, β,γ-methylene-ATP, AMP, and adenosine had little or no effect in both types of cells. The EC₅₀ values were 3 and 106 µM for UTP in NG108-15 and C6 glioma cells, respectively. The EC₅₀ value for ATP in C6 glioma cells was 43 µM. 2-MeSATP was threefold more potent than ATP in NG108-15 cells but had little effect in C6 glioma cells at 1 mM. In NCB-20 cells, a similar rank order of potency to that found in NG108-15 cells, i.e., UTP ? GTP > ATP > CTP, was observed. In both NG108-15 and C6 glioma cells, preincubation with ATP or UTP caused a pronounced cross-desensitization of subsequent nucleotide-stimulated IP production. ATP and UTP displayed no additivity in terms of IP formation at maximally effective concentrations. In contrast, endothelin-1, bradykinin, and NaF interacted in an additive manner with either nucleotide in stimulating PI hydrolysis. Pretreatment with pertussis toxin did not affect ATP-, UTP-, and GTP-stimulated IP generation in these cells, indicating that nucleotide receptors coupled to PLC by a pertussis toxin-resistant G protein in both cell types. Short-term treatment of the cells with protein kinase C (PKC) activators [phorbol 12-myristate 13-acetate (PMA) and octylindolactam V] produced a dose-dependent inhibition of ATP- and UTP-induced IP formation with a greater extent and higher susceptibility in C6 glioma cells than in NG108-15 cells. Furthermore, a 24-h exposure of the cells to PMA resulted in an obvious attenuation of nucleotide-induced IP formation in C6 glioma cells but failed to change the response in NG108-15 cells. These results suggest that distinct nucleotide receptors that respond to ATP and UTP with different selectivity exist in NG108-15 and C6 glioma cells. These heterogeneous nucleotide receptors coupled to PLC undergo discriminative modulation by PKC. NG108-15 and NCB-20 neuroblastoma are two cell lines that showed the highest specificity to extracellular UTP rather than ATP among the nucleotide receptors so far studied in various cells, suggesting the presence of a pyrimidine receptor in these cells.     

Products of Cultured Neuroglial Cells. III. Release of an 85,000 Molecular Weight Glycoprotein by C6 Glioma Cells In Vitro

Abstract: With [³H]fucose as a marker, C6 glioma cells in culture released an 85,000 molecular weight molecule into the medium as the major extracellular glycoprotein. The quantity and extracellularkytoplasmic ratio of this glycoprotein suggest that its cellular processing is different from that of five other released glycoproteins of molecular weights 55,000, 115,000, 130,000, 150,000, and 170,000. Nearly 40% of newly synthesized glycoproteins in the cells was released into the culture medium. Major glycoproteins retained by the cells migrated electrophoretically to molecular weight positions of 82,000, 110,000, 120,000, 140,000, and 160,000, and approximately one-third of these retained glycoproteins were labile to trypsinization. Both synthesis and release of these macromolecules were inhibited more than 95% with cycloheximide treatment, demonstrating that nearly all fucosylation was linked to protein synthesis. Since 40% of all glycoproteins was released under conditions of more than 99% cellular viability, it is likely that these extracellular glycoproteins are physiological products of membrane turnover and secretion, but not of cell lysis. The results provide a basis for the further study of glial differentiation and of shed glioma antigens.     

Activation of Phospholipase A2 by the Nociceptin Receptor Expressed in Chinese Hamster Ovary Cells

Abstract: To gain insight into the molecular mechanism for nociceptin function, functional coupling of the nociceptin receptor expressed in Chinese hamster ovary (CHO) cells with phospholipase A₂ (PLA₂) was examined. In the presence of A23187, a calcium ionophore, activation of the nociceptin receptor induced time- and dose-dependent release of arachidonate, which was abolished by pretreatment of the cells with pertussis toxin (PTX). Immunoblot analysis using anti-Ca²⁺-dependent cytosolic PLA₂ (cPLA₂) monoclonal antibody demonstrates that activation of the nociceptin receptor induces a time- and dose-dependent electrophoretic mobility shift of cPLA₂, suggesting that phosphorylation of cPLA₂ is induced by the nociceptin receptor. Pretreatment of the cells with PD98059, a specific mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 inhibitor, or staurosporine, a potent inhibitor of serine/threonine protein kinases and tyrosine protein kinases, partially inhibited the nociceptin-induced cPLA₂ phosphorylation and arachidonate release. These results indicate that the nociceptin receptor expressed in CHO cells couples with cPLA₂ through the action of PTX-sensitive G proteins and suggest that cPLA₂ is activated by phosphorylation induced by the nociceptin receptor via mechanisms partially dependent on p44 and p42 mitogen-activated protein kinases.