C6BU-1 Cells Research Articles

Homologous Desensitization of Serotonin 5‐HT2 Receptor‐Stimulated Intracellular Calcium Mobilization in C6BU‐1 Glioma Cells via a Mechanism Involving a Calmodulin Pathway

Serotonin 5-HT2 receptor-mediated intracellular Ca2+ mobilization was investigated in rat glioma C6BU-1 cells. The receptors became desensitized after previous exposure to 5-HT in a time- and concentration-dependent manner. The desensitization of 5-HT2 receptor-mediated intracellular signaling appeared to be homologous because previous exposure to 5-HT did not alter the response to other transmitters such as thrombin or isoproterenol and because previous exposure to thrombin or isoproterenol did not diminish the response to 5-HT. The desensitization induced by pretreatment with 5-HT was potently prevented by the naphthalenesulfonamide derivative W-7, a calmodulin antagonist, when it was cosupplied with 5-HT. Furthermore, the preventive effect of W-7 was greater than that of W-5, a weak analogue of W-7, and than that of H-7, a nonselective inhibitor of protein kinases. These results suggest that 5-HT2 receptor-mediated Ca2+ mobilization can be desensitized homologously after prolonged exposure to 5-HT in a calmodulin-dependent manner in rat glioma C6BU-1 cells.

338 Serotonin-induced desensitization of the serotonin-1A receptor expressed in rat glioma C6BU-1 cells

338 Serotonin-induced desensitization of the serotonin-1A receptor expressed in rat glioma C6BU-1 cells

Characterization of beta-125I-endorphin cross-linked proteins in NG108-15 cell membranes. A 25-kilodalton protein with properties of delta-opioid-binding site.

Cross-linking of beta-125I-endorphin to NG108-15 cell membranes labeled bands with molecular masses of 55, 35, and 25 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We applied several criteria to evaluate the relevance of these cross-linked bands to delta-opioid receptors, including selectivity, stereospecificity, affinity, G-protein coupling, down-regulation, and correlation with opioid receptor level in different well-characterized cell lines. Only the 25 kDa protein adequately fulfilled all these criteria. Thus, cross-linking to the 25-kDa band was selectively inhibited by ligands with delta-opioid affinity, but not by mu-opioid, kappa-opioid, or optically inactive opioid ligands or by non-opioid ligands. Based on inhibition of cross-linking, we calculated an affinity of [D-Ala2,D-Leu5]enkephalin binding to the 25-kDa and (Kd = 6 nM) that is similar to that reported for [D-Ala2,D-Leu5]enkephalin binding to NG108-15 membranes; this affinity decreased approximately 10-fold in the presence of Na+/guanyl-5'-yl imidodiphosphate. Chronic agonist treatment of NG108-15 cells reduced cross-linking to the 25-kDa band, but not to others, in a manner parallel to down-regulation of opioid receptors. Finally, the amount of the 25-kDa band was roughly proportional to the level of opioid receptors present in N18TG2, NS20Y, ST7-3, and ST8-4 cells. The 25-kDa band was absent in PC12h, NIH3T3, and C6BU1 cells as well as in liver, all of which had no detectable opioid binding.

Effect of Dexamethasone and Antidepressants on 5-HT-2 Receptor-Mediated Intracellular Calcium Mobilization in Rat Glioma C6BU-1 Cells

Effect of Dexamethasone and Antidepressants on 5-HT-2 Receptor-Mediated Intracellular Calcium Mobilization in Rat Glioma C6BU-1 Cells

Effects of NMDA receptor antagonists on 5-HT-induced intracellular calcium mobilization in C6BU-1 cells

Effects of NMDA receptor antagonists on 5-HT-induced intracellular calcium mobilization in C6BU-1 cells

Feedback regulation of phospholipase C-beta by protein kinase C.

Treatment of a variety of cells and tissues with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C (PKC) results in the inhibition of receptor-coupled inositol phospholipid-specific phospholipase C (PLC) activity. To determine whether or not the targets of TPA-activated PKC include one or more isozymes of PLC, studies were carried out with PC12, C6Bu1, and NIH 3T3 cells, which contain at least three PLC isozymes, PLC-beta, PLC-gamma, and PLC-delta. Treatment of the cells with TPA stimulated the phosphorylation of serine residues in PLC-beta, but the phosphorylation state of PLC-gamma and PLC-delta was not changed significantly. Phosphorylation of bovine brain PLC-beta by PKC in vitro resulted in a stoichiometric incorporation of phosphate at serine 887, without any concomitant effect on PLC-beta activity. We propose, therefore, that rather than having a direct effect on enzyme activity, the phosphorylation of PLC-beta by PKC may alter its interaction with a putative guanine nucleotide-binding regulatory protein and thereby prevent its activation.

Chronic exposure of rat glioma C6 cells to cholera toxin induces loss of the α-subunit of the stimulatory guanine nucleotide-binding protein (G s)

Rat glioma C6 BU1 cells were treated in tissue culture with cholera toxin. Incubation of membranes derived from these cells with fresh cholera toxin and [ 32P]NAD + failed to promote incorporation of radioactivity into polypeptides corresponding to forms of G sα. This is generally assumed to reflect prior ADP ribosylation of these polypeptides in vivo using endogenouss NAD + as substrate. However, immunological studies with anti-peptide antisera which identify all forms of G sα demonstrated that concentrations of this polypeptide were now substantially reduced in the membranes. This effect was specific for G sα as neither the α-subunits of the pertussis toxin-sensitive G-proteins G i2 and G i3, nor the β subunit common to the various G-proteins were lost in parallel. Pertussis toxin-catalysed ADP ribosylation did not cause the downregulation of G sα nor of the α-subunits of G i2 or G i3 although it did cause ADP ribosylation of the entire complement of both G i2 and G i3 in the membranes. Despite the reduction in levels of immunoreactive G sα from the membranes of cholera toxin-treated cells, no alterations in levels of MRNA corresponding to this G-protein were noted.

Phosphorylation of Phospholipase C-γ by cAMP-dependent Protein Kinase

The mechanism by which cAMP modulates the activity of phosphoinositide-specific phospholipase C (PLC) was studied. Elevation of cAMP inhibited both basal and norepinephrine-stimulated phosphoinositide breakdown in C6Bu1 cells which contain at least three PLC isozymes, PLC-beta, PLC-gamma, and PLC-delta. Treatment of C6Bu1 cells with cAMP-elevating agents (cholera toxin, isobutylmethylxanthine, forskolin, and 8-bromo-cAMP) increased serine phosphate in PLC-gamma, but the phosphate contents in PLC-beta and PLC-delta were not changed. In addition, cAMP-dependent protein kinase selectively phosphorylated purified PLC-gamma among the three isozymes and added a single phosphate at serine. The serine phosphorylation, nevertheless, did not affect the activity of PLC-gamma in vitro. We propose, therefore, that the phosphorylation of PLC-gamma by cAMP-dependent protein kinase alters its interaction with putative modulatory proteins and leads to its inhibition.

Foetal calf serum enhances cholera toxin-catalysed ADP-ribosylation of the pertussis toxin-sensitive guanine nucleotide binding protein, G i2, in rat glioma C6BU1 cells

The major G-protein of rat glioma C6BU1 cells corresponds immunologically to G i2. In the absence of guanine nucleotides, this protein is shown to be a substrate for ADP-ribosylation catalysed by both cholera and pertussis toxins. Under these conditions, a receptor for a growth factor, which has previously been shown to be activated by foetal calf serum, modulated the effects of both cholera and pertussis toxins on the G-protein. These ligand-mediated alterations of cholera and pertussis toxin-catalysed ADP ribosylation demonstrated that, in this system, the growth factor receptor interacts functionally with G i2.

Persistent activation of the α subunit of Gs promotes its removal from the plasma membrane

As assessed both by cholera-toxin-catalysed ADP-ribosylation and by immunoblotting with an anti-peptide antiserum raised against the C-terminal decapeptide of forms of Gs alpha (the alpha subunit of the stimulatory guanine nucleotide-binding protein), rat glioma C6 BU1 cells express two forms of Gs alpha: a major 44 kDa form and a much less prevalent 42 kDa form. We examined the effects of guanine nucleotides on the interaction of the 44 kDa form with the plasma membrane. Incubation of membranes of C6 BU1 cells with poorly hydrolysed analogues of GTP, but not with analogues of either ATP or GDP, caused the release of this Gs alpha from the membrane fraction. Release of Gs alpha was observed within 5 min, and continued throughout the incubation period. After treatment with guanosine 5'-[beta gamma-imido]triphosphate for 60 min, some 75% of this polypeptide had been released from its site of membrane attachment. These experiments demonstrate that Gs alpha need not remain associated invariantly with the plasma membrane.

GTP analogues promote release of the alpha subunit of the guanine nucleotide binding protein, Gi2, from membranes of rat glioma C6 BU1 cells.

The major pertussis-toxin-sensitive guanine nucleotide-binding protein of rat glioma C6 BU1 cells corresponded immunologically to Gi2. Antibodies which recognize the alpha subunit of this protein indicated that it has an apparent molecular mass of 40 kDa and a pI of 5.7. Incubation of membranes of these cells with guanosine 5'-[beta gamma-imido]triphosphate, or other analogues of GTP, caused release of this polypeptide from the membrane in a time-dependent manner. Analogues of GDP or of ATP did not mimic this effect. The GTP analogues similarly caused release of the alpha subunit of Gi2 from membranes of C6 cells in which this G-protein had been inactivated by pretreatment with pertussis toxin. The beta subunit was not released from the membrane under any of these conditions, indicating that the release process was a specific response to the dissociation of the G-protein after binding of the GTP analogue. Similar nucleotide profiles for release of the alpha subunits of forms of Gi were noted for membranes of both the neuroblastoma x glioma hybrid cell line NG108-15 and of human platelets. These data provide evidence that: (1) pertussis-toxin-sensitive G-proteins, in native membranes, do indeed dissociate into alpha and beta gamma subunits upon activation; (2) the alpha subunit of 'Gi-like' proteins need not always remain in intimate association with the plasma membrane; and (3) the alpha subunit of Gi2 can still dissociate from the beta/gamma subunits after pertussis-toxin-catalysed ADP-ribosylation.

Alteration in morphology and induction of glutamine synthetase in rat glioma C6BU-1 cells cultured with prostaglandin D2-supplemented media

We evaluated the effects of prostaglandins (PGs) on rat glioma C6BU-1 cells by supplementing the culture media with PGs. In the medium containing PGD(2) (15 or 20 ?M), the glial cells showed altered morphology from an elongated fibroblastic form to a spreading multipolar one within 24 h, and their growth rate was suppressed to half of that of control cultures. In these cultures, the specific activity of glutamine synthetase (GS) increased approximately twofold within 48 h in comparison to the value for vehicle-treated controls. Simultaneous treatment with actinomycin D or cycloheximide completely blocked the PGD(2)-elicited increase in GS specific activity, suggesting that the increase was due to de novo synthesis of the enzyme. PGD(2)-like prostanoids such as PGD(1) and 9-deoxy-?(9), ?(12)-13,14-dihydro-PGD(2) (?(12)-PGJ(2)), when added to the culture medium, mimicked the actions of PGD(2) on the C6BU-I cells, though their effective concentrations were not necessarily identical. PGs of the E- and F-series had almost no discernible effect on the glioma. These results might imply a possibility that PGD(2) plays a regulatory effect in growth and/or differentiation of rat glioma C6BU-1 cells.

Expression of β-nerve growth factor mRNA in rat glioma cells and astrocytes from rat brain

A 50-base synthetic oligodeoxynucleotide complementary to a portion of mouse nerve growth factor (NGF) mRNA was used as a probe for analysis of the expression of NGF gene. Northern blot analysis showed the presence of a major 1.3 kb transcript, which was identical in size to mouse NGF mRNA, in both C6Bu1 cells and rat astrocytes cultured from newborn rat brain. Further, the rearrangement of DNA sequence in and around the NGF gene locus of C6Bu1 cells was not detected by Southern blot analysis. These results indicate the expression of NGF mRNA in both C6Bu1 cells and astrocytes from rat brain, suggesting that astrocytes may produce NGF protein in the rat brain, especially in developing rat brain.

Foetal-calf serum stimulates a pertussis-toxin-sensitive high-affinity GTPase activity in rat glioma C6 BU1 cells.

Cellular proliferation of rat glioma C6 BU1 cells in tissue culture is dependent on the presence of either calf or foetal-calf serum in the medium. Foetal-calf serum stimulated a high-affinity GTPase in membranes derived from C6 BU1 cells. Pretreatment of the cells with pertussis toxin decreased the high-affinity GTPase activity substantially, and attenuated the foetal-calf-serum-stimulated increase in this GTPase activity. Cholera toxin, in contrast, did not modulate the response to foetal-calf serum. Foetal-calf serum did not inhibit adenylate cyclase activity in membranes of these cells, indicating that the G-protein that was stimulated by foetal-calf serum was not Gi (the inhibitory one). Although the nature of the specific component of foetal-calf serum responsible for this pertussis-toxin-sensitive receptor-mediated stimulation of high-affinity GTPase activity has not been identified, it was mimicked neither by bombesin, which can stimulate inositol phospholipid turnover via a guanine nucleotide binding protein, nor by platelet-derived growth factor, which is present in substantial concentrations in foetal-calf serum. This report represents the first demonstration of a pertussis-toxin-substrate-mediated response in this cell line and provides further evidence that G-proteins other than Gi can be functionally inactivated by pertussis toxin.

Guanine nucleotide regulation of the pertussis and cholera toxin substrates of rat glioma C6 BU1 cells

Rat glioma C6 BU1 cells contain a pertussis toxin substrate of 40 kDa which does not appear to be identical with G i,G o or transducin. The GTP analogue, GTP[γS], inhibited the rate of pertussis toxin-catalysed ADPribosylation of this protein, while the GDP analogue GDP[βS] stimulated this reaction. A protein of the same kDa value was ADPribosylated by cholera toxin in the absence of added guanine nucleotides. It is suggested that this 40 kDa protein can be a substrate for both cholera and pertussis toxins under appropriate conditions.

Cytosolic calcium elevation and cGMP production induced by serotonin in a clonal cell of glial origin

It has been shown recently that astroglial cells of the mammalian CNS possess receptors for neurotransmitters. In order to analyze what sequences of cellular events occur upon activation of these glial receptors, we utilized a 5-HT receptor in a rat clonal cell of glial origin as a model system. When the C6BU-1 glioma cells were exposed to 5-HT, the cytosolic Ca2+ concentration ([Ca2+]i) was elevated and the cellular content of cGMP was increased in a dose-dependent manner. 5-HT receptor antagonists and a Ca2+ entry blocker suppressed the increases in both [Ca2+]i and cGMP. The magnitude of the cGMP increment depended on the environmental Ca2+ concentration and was totally blocked by Ca2+ depletion. Application of a Ca2+ ionophore increased [Ca2+]i and cGMP. There was a tendency for extremely high [Ca2+]i to suppress the cGMP increment. On the contrary, membrane-permeable cyclic nucleotide analogs failed to increase [Ca2+]i. These results suggest that the following sequence of events occurs in 5-HT-induced C6BU-1 cells: activation of 5-HT receptors, Ca2+ influx, a rise in [Ca2+]i, activation of guanylate cyclase, and, finally, activation of cyclic nucleotide phosphodiesterase.

Forskolin induction of S-100 protein in glioma and hybrid cells.

The S-100 protein level in mouse neuroblastoma (N18TG-2 and NIE-115), rat glioma (C6, C6BU-1, and C6V-1), and hybrid (NG108-15, 140-3, 141-B, NBr10A, NBr20A, NCB20, and NX3IT) cells was determined with a sensitive enzyme immunoassay system that uses a rabbit antibody to bovine brain S-100 protein. S-100 protein was detected in glioma but not in neuroblastoma cells. All seven hybrid cells derived from neuroblastoma and glioma or other types of cells were found to possess a very little or undetectable S-100 protein. The induction of S-100 protein level in prestationary phase cultures of glioma C6BU-1 cells was examined by forskolin, which was a highly specific activator of adenylate cyclase of the cells and produced morphological differentiation. After incubation with 10 microM forskolin for 48 hr, the S-100 protein level increased 2-2.5-fold in C6BU-1 glioma cells whose mean control level was 60 +/- 26 ng/mg protein (+/- SD). The forskolin induction of S-100 protein in the cells was dose dependent, and the concentration of forskolin required for 50% activation of S-100 protein was about 0.6 microM. The increase by forskolin was initiated from 10-15 hr after incubation with it and was inhibited with cycloheximide and actinomycin D. In NG108-15 hybrid cells the induction of S-100 protein was also observed by forskolin as well as prostaglandin (PG) E1 plus theophylline which are known to activate adenylate cyclase of the cells. The results indicate that S-100 protein biosynthesis is genetically controlled in these clonal cells, and that S-100 protein can be regulated in a cAMP-dependent fashion in prestationary cultures.

Isolation and characterization of intraspecific cybrids: Effect of mitochondrial DNA on their cellular properties

Cybrid clones were obtained by fusing whole cells of rat glioma C6BU-1, resistant to 5-bromodeoxyuridine (BrdU), with cytoplasts of embryonic rat 3Y1CAP cells, resistant to chloramphenicol (CAP), in selective medium with BrdU and CAP. The clones resistant to BrdU and CAP were confirmed to be cybrids by chromosome and mtDNA analyses. More than half the mtDNA of all the cybrid clones was from the 3Y1CAP cells. After cultivation of a cybrid clone Y22 for 3 months in the absence of CAP, subclones were isolated. One subclone Y22-22 contained predominantly mitochondrial DNA (mtDNA) from the 3Y1CAP cells. Using this subclone, the effects of the mitochondrial genome on cellular properties were examined. The growth patterns, expression of glioma-specific β-adrenergic receptor, and composition of the major proteins of C6BU-1 cells were not affected by transmitted mtDNA from the 3Y1CAP cells. This procedure for isolating cells containing predominantly foreign mtDNA will be useful in studies on the interaction between genomes of the mitochondria and nucleus.

A quantitative analysis of intramembranous particles during the development of neuroblastoma × glioma hybrid cells

The process of neural differentiation of neuroblastoma × glioma hybrid cells (NG108-15 cells) was studied by freeze-fracture electron microscopy. The distribution of intramembranous particles (IMPs) was random and their density increased in both P-, and E-face of the plasmalemma when the membrane excitability developed. Undifferentiated cells grown in growth medium for 2–6 days had an average of 1300–1500 IMPs/μm 2 in the P-face and 250–300/μ 2 in the E-face. After the cells were differentiated with dBcAMP for 1 week, the density of IMPs was 2600/μm 2 in the P-face and 540/μm 2 in the E-face. The analysis of size distribution showed the increase of the number of medium (7.5–10 nm) and large (> 10 nm) particles. The slight increase of large particles was observed in the case of differentiated N18TG2 (the parental neuroblastoma clone) cells. Treatment with dBcAMP did not affect the number and the size distribution of IMPs in the C6BU1 (the parental glioma clone) cells.

Modulation in the levels and localization of plasminogen activator in differentiating neuroblastoma cells

Variations in the specific activity and localization of plasminogen activator (PA) were examined prior to and during the differentiation of mouse neuroblastoma and rat glioma cells in culture, in an attempt to reveal whether PA is actively involved in neurite extension and development in these cells. Following differentiation induced by dibutyryl cyclic AMP, the level of PA increased by 10 to 20-fold in neuroblastoma N18TG-2 cells and in the hybrid cell line NG108-15, obtained by fusion of mouse neuroblastoma N18TG-2 and rat glioma C6BU-1 cells, but was very low in the glioma parental cells throughout differentiation. Plasminogen-dependent proteolysis was found not to be an obligatory requirement for the differentiation of the hybrid cells in culture, since the extension of neurites, the appearance of toxin-activated Rb +-efflux and the eletrical properties were largely unaffected by eliminating and blocking this activity. Confluent cells from several clones derived from mouse neuroblastoma C1300 cells displayed significant intracellular and secreted PA activities, and the axon-minus neuroblastoma NIA-103 cells were found to secrete the highest amounts of PA. Upon gel electrophoresis, all mouse neuroblastoma cell lines examined displayed a major molecular form of active PA with an apparent molecular weight of 80,000 and a minor one of 50,000. Only the larger enzyme form was expressed by the rat glioma or the hybrid cells, and the electrophoretic migration pattern of the active enzyme species remained unchanged upon differentiation of the various cell lines. Distribution of PA activity on the surface of differentiating neuroblastoma N1E-115 cells and hybrid NG108-15 cells was visualized by high resolution autoradiography of [ 125I]fibrin degradation products, cross-linked to the cell surface. Particularly intense labeling was observed along membranes of cell bodies in both developing and mature cells, around branching zones, along individual neurite extensions and in part, but not all, of the growth cones. These findings suggest that PA plays a role in the metabolism of mature cells of neuronal origin, and imply its involvement in the development and/or the functions of specific neuronal processes and growth cones.