Binding of tetanus toxin to somatic neural hybrid cells with varying ganglioside composition

125I-labelled tetanus toxin interaction with several somatic hybrid cell lines was investigated. Binding of toxin is most effective in NCB-20, followed by NBr-10A, NG108-C15, and SB21-B1 cells. Specific binding of toxin to NCB-20 and SB21-B1 cells is 7- and 60-fold lower, respectively, in comparison to enriched rat cerebral neuron cultures. The NCB-20, NBr-10A, and NG108-C15 clones display a complex ganglioside pattern, including the presence of [N-acetyl-neuraminyl]-galactosyl-N-acetylgalactosaminyl[ N-acetylneuraminyl]-galactosylglucosyl-ceramide (GD1a) and two unidentified [14C]galactose-labelled lipid-soluble compounds, while the SB21-B1 is most abundant in [N-acetyl-neuraminyl]-galactosylglucosyl-ceramide (GM3) and N-acetyl-galactosaminyl-[N-acetyl-neuraminyl]-galactosylglucosyl-c eramide (GM2) gangliosides. None of the cells tested contain measurable levels of [14C]galactose-labelled or resorcinol-positive bands of galactosyl-N-acetyl-galactosaminyl-[ N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosyl-ceramide (GD1b) and [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[ N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosyl-ceramide (GT1b) gangliosides. After 2 h at 37 degrees C a near plateau of toxin association with NCB-20 cells is seen. Binding in low-ionic-strength medium is 1.35-fold higher at 37 degrees C than at 4 degrees C, but is reduced by 21 and 51% at 4 degrees C and 37 degrees C, respectively, in physiologic medium. Treatment of NCB-20 cells with neuraminidase causes a partial loss (29%) of toxin-binding sites. Binding to the hybrid cells is significantly different from that of cerebral cultures with respect to temperature, salt effect, and sensitivity to neuraminidase, suggesting perhaps a different class of receptors for the toxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of tetanus toxin binding to rat brain membranes. Evidence for a high-affinity proteinase-sensitive receptor.

Binding of 125I-labelled tetanus toxin to rat brain membranes in 25 mM-Tris/acetate, pH 6.0, was saturable and there was a single class of high-affinity site (KD 0.26-1.14 nM) present in high abundance (Bmax. 0.9-1.89 nmol/mg). The sites were largely resistant to proteolysis and heating but were markedly sensitive to neuraminidase. Trisialogangliosides were effective inhibitors of toxin binding (IC50 10 nM) and trisialogangliosides inserted into membranes lacking a toxin receptor were able to bind toxin with high affinity (KD 2.6 nM). The results are consistent with previous studies and the hypothesis that di- and trisialogangliosides act as the primary receptor for tetanus toxin under these conditions. In contrast, when toxin binding was assayed in Krebs-Ringer buffer, pH 7.4, binding was greatly reduced, was non-saturable and competition binding studies showed evidence for a small number of high-affinity sites (KD 0.42 nM, Bmax. 0.90 pmol/mg) and a larger number of low-affinity sites (KD 146 nM, Bmax. 179 pmol/mg). Treatment of membranes with proteinases, heat, and neuraminidase markedly reduced binding. Trisialogangliosides were poor inhibitors of toxin binding (IC50 11.0 microM), and trisialogangliosides inserted into membranes bound toxin with low affinity. The results suggest that in physiological buffers tetanus toxin binds with high affinity to a protein receptor, and that gangliosides represent only a low-affinity site.     

125I-beta-endorphin binding to neuroblastoma X glioma NG108-15 cells: distribution of delta opioid receptors.

The mouse neuroblastoma x rat glioma hybrid NG108-15 was previously shown to express delta opioid receptors. Because neuroblastoma cells display different phenotypes and cloned cell lines are heterogenous, we studied the characteristics and distribution of human 125I-beta-endorphin (125I-beta E) binding sites in cultures of NG108-15 cells with the use of micro-autoradiography and light microscopy. 125I-beta E labeled delta sites in NG108-15 in the presence of the non-opioid blocking peptide, beta-endorphin (6-31) (beta E (6-31)). Silver grains resulting from 125I-beta E binding to the opioid sites occurred in diffuse patches over several cells, with preferential location in dense cell patches. Pretreatment of NG108-15 with the delta agonist DADLE, previously shown to decrease beta E binding to delta sites on intact cells, also reduced silver grain density; however, some cells located in dense cell clusters were resistant to substantial agonist induced loss of labeling. These results suggest that delta opioid binding has a heterogenous cellular distribution in NG108.     

Identification and characterization of the beta-adrenergic receptor on neuroblastoma × glioma hybrid NG108-15 cells

1. Using [3H]DHA and unlabeled L-alprenolol, a substantial amount of over 64% specific binding of beta-adrenergic receptor has been identified on the neuroblastoma x glioma hybrid NG108-15 cell, which has been proven to display numerous functional characteristics of intact neurons. 2. Beta-adrenergic receptor binding on intact NG108-15 cells does not change significantly upon morphological differentiation, induced by 1 mM dibutyryl cyclic AMP (dBcAMP). 3. The [3H]DHA binding on intact NG108-15 cells is rapid, saturable, and reversible, having a t1/2 of 1.0 min for association and 3.5 min for dissociation. 4. The affinity constant (Kd) and maximum binding capacity (Bmax) for binding of [3H]DHA to beta-adrenergic receptors on NG108-15 cells have been estimated by Scatchard plot analysis to be 2.5 and 0.23 nM, respectively. Further analysis indicates a single class of receptors for [3HDHA binding on NG108-15 cells. 5. Studies on kinetic properties have revealed on-rate (K + 1) and off-rate (K - 1) constants of 0.7 X 10(-9) M min-1 and 0.19 min-1, respectively. Further, the IC50 value and inhibition constant (Ki) for unlabeled L-alprenolol to inhibit [3HDHA binding on NG108-15 cells have been estimated to be 10(-5) and 8.9 X 10(-6) M, respectively. 6. The rank-order potency of catecholamine agonists, (-)ISO greater than (+)ISO greater than EPI greater than NE, reveals the presence of type 2 receptor for the beta-adrenergic binding on both differentiated and undifferentiated NG108-15 cells. 7. The present study indicates that the clonal neuroblastoma x glioma hybrid NG108-15 cell line possesses substantial amounts of beta-adrenergic receptors with characteristics similar to those on neuronal cells.     

Action of tetanus toxin on cholinergic neuroblastoma X glioma hybrid cells: selective blockade of Ca spikes

We examined the effect of tetanus toxin on clonal neuroblastoma X glioma hybrid cells, NG108-15, by intracellular microelectrode studies of passive membrane electrical properties and action potentials generated under various conditions. Binding of tetanus toxin to the surface of the cells was demonstrated by indirect immunofluorescent staining but no morphological alteration was observed in tetanus toxin-treated cells under a phase contrast microscope. These is no significant difference between the tetanus toxin-treated and untreated cells in their passive electrical membrane properties, i.e. resting membrane potentials, input resistances, time constants and input capacities. Cells in 120 mM Na+, 2 mM Ca2+ salt solution showed Na spikes, and cells in high Ca2+ (30 mM), Na+-free salt solution showed Ca spikes in response to depolarizing current pulses. While the Na spike was not affected by tetanus toxin, the Ca spike was blocked by the toxin. The minimum dose of tetanus toxin for maximum suppression of the peak potential level of the Ca spike was 250 ng/ml. Addition of tetraethyl ammonium (TEA) to extracellular fluid enhanced the Ca spike in untreated cells. In toxin-treated cells, TEA did not alter the effect of tetanus toxin on the Ca spike. Blockade of the Ca spike by tetanus toxin could be detected even at low extracellular Ca2+ concentration (10 mM) by adding TEA to the extracellular fluid and adjusting the membrane potential to a steady hyperpolarized level (-80 mV) to ensure optimal and uniform electrical responses. The usefulness of NG108-15 hybrid cells for in vitro investigations on the mechanism of action of tetanus toxin was discussed.     

G Protein Modulation of ω-Conotoxin Binding Sites in Neuroblastoma X Glioma NG 108-15 Hybrid Cells

Electrophysiological evidence shows that voltage-dependent calcium channel (VDCC) activity can be regulated by a large number of neurotransmitters. In particular, guanine nucleotide binding regulatory protein (G protein)-mediated inhibitory modulation of the channel activity has been deduced from evidence that GTP analogues and purified G proteins are able to mimic this effect. The G proteins involved are pertussis toxin (PTx) sensitive. The purpose of the present study was to investigate, using biochemical techniques, whether G protein activation modulates the recognition site for omega-conotoxin GVIA (CgTx), a peptide neurotoxin that selectively labels a population of high-threshold VDCC. Undifferentiated and differentiated (1 mM dibutyryl cyclic AMP, 4 days) NG 108-15 cells were used. In both crude cellular extracts specific binding of 125I-CgTx was characterized. Differentiation induced a sixfold increase in the number of binding sites and doubled the KD value. The in vitro addition of guanylylimidodiphosphate (GMP-PNP; a nonhydrolyzable analogue of GTP) to extracts prepared from differentiated cells reduced the 125I-CgTx binding by 48%. This effect, observed in undifferentiated cells as well, was also caused by other triphosphate guanine nucleotides, such as GTP, but not by guanosine 5'-O-(2-thiodiphosphate) or adenine nucleotides. Treatment of the cells with PTx prevented the GMP-PNP effect. Moreover, the results obtained after preincubation with specific antisera raised against the alpha subunits of Gi1-2 and Go suggest that Go is the G protein responsible for the observed effect.     

Characterization by [3H]Dihydroergocryptine Binding of Alpha-Adrenergic Receptors in Neuroblastoma × Glioma Hybrid Cells

[3H]Dihydroergocryptine ([3H]DHE) was shown to bind to sites in membranes from neuroblastoma X glioma hybrid cells (NG 108-15) that had the characteristics expected of alpha-adrenergic receptors. The binding was saturable with 0.3 pmol [3H]DHE bound per mg of protein and of high affinity, with an apparent dissociation constant (KD) of 1.8 nM. The specificity of the binding site for various ligands was more similar to that of alpha 2 receptors than to that of alpha 1. No specific binding of [3H]WB-4101 was found in the membranes derived from NG 108 cells. This finding also indicated that the [3H]DHE binding site in the cell is the alpha 2 receptor. GTP lowered the affinity of agonists for the [3H]DHE binding site, although the nucleotide hardly affected the affinity of antagonists including [3H]DHE.     

Glycosylation-Dependent Regulation of Opiate (Enkephalin) Receptors in Neurotumor Cells

Abstract: Electron inactivation analysis revealed that the opiate (enkephalin) binding site in neurotumor cell lines NG108-15 and NCB-20 had an apparent target size of 200,000 daltons. Expression of functional opiate receptors in neurotumor cells appeared to require glycosylation, as treatment of such cells with tunicamycin (TM; under conditions where de novo glycosylation of asparagine residues in protein was reduced by 80%, but overall protein and DNA synthesis were inhibited by <10%) resulted in the loss of 50% of the opiate binding sites. The loss of binding sites could not be prevented by addition of protease inhibitors to cell cultures, but binding sites were partially restored 48–60 h after removal of the TM. In addition, the number of enkephalin binding sites in TM-treated cells was also restored to near-normal levels by addition of physiological concentrations (1-10 mM) of manganese ions to the in vitro receptor binding incubation mixture. TM treatment resulted in receptor supersensitivity to manganese ions for both opiate agonists and antagonists, no change in the sodium effect for either agonists or antagonists, and subsensitivity to GTP for both agonists and antagonists. However, opiate binding to cell membranes was not substantially inhibited by either neuraminidase treatment or short-term incubation with lectins such as wheat germ agglutinin, ricin, or concanavalin A. Thus, the data suggest that oligosaccharide units are not directly involved in opiate receptor-ligand interactions, but protein glycosylation is required for functional expression of receptors.     

Adaptive increase in adenylyl cyclase activity in NG108-15 and S49 cells induced by chronic treatment with inhibitory drugs is not due to a decrease in cyclic AMP concentrations.

NG108-15 neuroblastoma x glioma hybrid cells and S49 lymphoma cells exhibit an enhancement in adenylyl cyclase activity after chronic treatment with receptor agonists that acutely inhibit the enzyme. Using agonists that activate five distinct inhibitory receptors in NG108-15 cells, we have found that there is a correlation between the extent of acute inhibition of prostaglandin E1 (PGE1)-stimulated cAMP accumulation and efficacy for induction of enhanced PGE1 stimulation of cAMP accumulation after chronic treatment and withdrawal. Chronic treatment with dideoxyadenosine, which acutely inhibits adenylyl cyclase activity by a mechanism independent or cell surface receptors or pertussis toxin-sensitive G proteins, did not induce enhanced PGE1 stimulation of cAMP accumulation in NG108-15 cells or forskolin stimulation of cAMP accumulation in S49 cells. While control basal cAMP concentrations were acutely decreased by carbachol in NG108-15 cells and by somatostatin in S49 cells, when the cAMP concentrations were maintained above the control basal values with a phosphodiesterase inhibitor, chronic treatment with these inhibitory drugs nonetheless resulted in enhanced cAMP responses in both NG108-15 and S49 cells. These results provide evidence that the initial decrement in cAMP concentrations caused by inhibitory drug is not the requisite signal for inducing the subsequent sensitization of adenylyl cyclase in NG108-15 and S49 cells but that activation of a pertussis toxin-sensitive G protein is involved in the development of this important adaptation.     

Preparation of affinity-purified,biotinylated tetanus toxin,and characterization and localization of cell surface binding sites on nerve growth factor-treated PC12 cells

Biotinylated derivatives of tetanus toxin were prepared and isolated by chromatofocusing and ganglioside-affinity chromatography. Biotinylation was monitored by the appearance of a 210,00 dalton complex upon SDS-polyacrylamide gel electrophoresis in the presence of avidin, and by selective binding to an avidin-Sepharose gel. At molar biotin:toxin ratios from 11 to 201 only biotinylated derivatives with low toxicity were obtained; these derivatives, however, retained 60–80% of their specific binding affinity for brain synaptosomes. A biotinylated tetanus toxin derivative purified by ganglioside-affinity chromatography was used to identify and localize tetanus toxin binding sites on PC12 cells. Electron microscopic analysis with streptavidin-gold revealed very low levels of tetanus toxin binding sites on the surface of untreated cells, and the appearance of such binding sites during the second week of nerve growth factor-induced differentiation. Examination of micrographs of the differentiated cells indicated that the tetanus toxin binding sites sites are concentrated on the neurites, with relatively few appearing on the cell bodies. Cognate studies using¹²⁵I-labeled, affinity-purified tetanus toxin revealed an increase in PC12 binding capacity from about 0.07 nmol/mg protein in untreated cells to 0.8 nmoles/mg protein in cells treated for 14 days with nerve growth factor. Cells treated in suspension for 2–3 weeks with nerve growth factor do not express tetanus toxin binding sites; upon plating, these cells required one week for the appearance of binding sites, although neurites grew much more rapidly from these primed cells. The high binding capacity of these tetanus toxin sites, as well as their sensitivity to neuraminidase, is indicative of a polysialoganglioside structure. The advantages of biotinylated tetanus toxin derivatives are discussed and the significance of nerve growth factor-differentiated PC12 cells grown as monolayers as a model for the study of the development, localization, and function of neuraminidase-sensitive tetanus toxin binding sites is presented.Abbreviations PBS phosphate-buffered saline - STS sucrose-Tris-serum solution - NGF nerve growth factor - C collagen - PL polylysine - BBG bovine brain ganglioside mixture - GM₁ gafactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GD_1a [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GT_1a [N-aceylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GD_1b galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosyl ceramide - GT_1b [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl] galactosylglucosyl ceramide - NANA N-acetylneuraminic acid     

Desensitization of PGE1 receptors in neuroblastoma-glioma hybrid cells

Prostaglandin E1 receptor sites were measured in homogenates of NG108-15 neuroblastoma-glioma hybrid cells after exposure of intact cells to PGE1. Scatchard analysis of competitive binding studies showed that incubation of NG108-15 cells in the presence of 2.5 microM PGE1 for 16 h resulted in a loss of PGE1 receptors and an increase in the dissociation constant of the remaining receptors. Thus, cells challenged with PGE1 not only lose adenylate cyclase activity, but also lose PGE1 receptors and decreased the affinity of the remaining receptors for PGE1.     

Binding of Aminoalkylindoles to Noncannabinoid Binding Sites in NG108-15 Cells

1. Aminoalkylindoles, typified by WIN 55212-2, bind to G protein-coupled cannabinoid receptors in brain. Although cannabinoids inhibit adenylyl cyclase in NG108-15 neuroblastoma × glioma hybrid cells, cannabinoid receptor binding in these cells has not been described previously. This study compares pharamcological characteristics of [³H]WIN 55212-2 binding sites in rat cerebellar membranes and in NG108-15 membranes.2. Although the K _D of specifid [³H]WIN 55212-2 binding was similar in brain and NG108-15 membranes, the B _max was 10 times lower in NG108-15 than in cerebellar membranes. In both brain and NG108-15 membranes, aminoalkylindole analogues were relatively potent in displacing [³H]WIN 55212-2 binding.However, IC₅₀ values for more traditional cannabinoids were significantly higher in NG108-15 membranes than in brain, e.g., the K _i values for CP55,940 were1.2nM in brain and >5000nM in NG108-15 membranes. Moreover, sodium and GTP--S decreased [³H]WIN 55212-2 binding in brain but not in NG108-15membranes.3. These data suggest that WIN 55212-2 does not label traditional cannabinoid receptors in NG108-15 cells and that these novel aminoalkylindolebinding sites are not coupled to G proteins.     

Insulin-like growth factor-1 inhibits STS-induced cell death and increases functional recovery of in vitro differentiated neurons

NG108-15 cells differentiate into neurons by 1 mM sodium butyrate (NaB) treatment. Differentiated cells resulted more resistant to staurosporine (STS) than proliferating cells. In particular, STS treatment decreased Bcl-2 and Bcl-xL content in mitochondria of proliferating cells, but not in mitochondria of differentiated cells. Bad was phosphorylated and down-regulated only in differentiated cells. Bax accumulated in the mitochondria of proliferating but not differentiated cells. Mitochondrial release of cytochrome c was observed in proliferating cells, whereas mitochondria of differentiated cells retained cytochrome c. Proliferating cells treated with STS accumulated Endo G and AIF in the nucleus. By contrast, differentiated cells did not show such nuclear accumulation. Treatment of differentiated cells with Insulin-like Growth Factor-1 (IGF-1) and STS resulted in a 17,1% increase of cell viability. The survival role of IGF-1 was demonstrated by treating differentiated cells with an anti-IGF-1 neutralizing antibody. Such treatment significantly increased STS-induced cell death. Electrophysiology studies showed that in STS-treated cells membrane potential oscillations were reduced in amplitude without giving rise to spontaneous action potentials (APs). However, the percentage of cells yielding overshooting APs returned to 100% after STS removal. It is concluded that neuronal differentiation of NG108-15 cells induces resistance to apoptotic cell death and that IGF-1 plays a central role in sustaining this mechanism.     

Sodium regulation of opioid agonist binding is potentiated by pertussis toxin

Pretreatment of intact NG108-15 cells with pertussis toxin suppresses opioid inhibition of cyclic AMP accumulation mediated by the inhibitory guanine nucleotide-binding regulatory protein, Ni, which apparently also mediates the inhibitory nucleotide effects on opioid against binding. The toxin treatment had no effect on opioid agonist binding measured in NG108-15 cell membranes without sodium present. However, the toxin potentiated the inhibitory effect of sodium on agonist binding, leading to an agonist-specific reduction of opioid receptor affinity in the presence of sodium in the binding reaction. The potency of the stable GTP analog, GTP gamma S, to reduce agonist binding in the presence of sodium was little changed in membranes prepared from pertussis toxin-treated cells compared to control membranes, whereas the potency of the stable GDP analog, GDP beta S, was magnified. The data indicate that ADP-ribosylation of Ni by pertussis toxin potentiates sodium regulation of opioid agonist binding and that the communication between Ni and opioid receptors is not lost by the covalent modification of Ni.     

Temperature-Mediated Interaction of Tetanus Toxin with Cerebral Neuron Cultures: Characterization of a Neuraminidase-Insensitive Toxin-Receptor Complex

Abstract: Energy-dependent internalization of ¹²⁵I-labeled tetanus toxin into cultured neural cells is shown to follow an energy-independent binding process. A three-step model, involving receptor-mediated binding followed by sequestration and internalization is proposed. In the first step, binding of toxin is enhanced in appearance under low ionic strength medium, at 0–4°C; it is suppressed, however, with increasing incubation temperature under physiological salt concentrations. Cell-bound toxin is displaced by approximately 35.5% when high-salt medium (physiological concentrations) is added to cells at 0–4°C; the effect is further amplified at 37°C. Addition of disialoganglioside G_D1b (1–5 μg/ml) also lowers the amount of cell-associated toxin. The fraction of ¹²⁵I-labeled toxin retained by the cells after exposure to high-salt medium at 0–4°C or after addition of G_D1b is operationally defined as sequestered toxin. This second step, characterized by a stable association of the toxin with the neural cells, is affected by both physiological salt and by 37°C conditions. Lastly, an energy-dependent phenomenon of firm association of tetanus toxin with neural cells, compatible with internalization, is described. The toxin residing in this fraction is bioactive and cannot be removed by salts, gangliosides, or by treatment with protease or neuraminidase. Binding, sequestration, and internalization are mutually dependent, as they are all blocked by pretreatment of cells with neuraminidase and by an enhanced energy-independent sequestration event, which results in enhanced tetanus toxin internalization by an energy-dependent process.     

Effect of angiotensin II and III on inositol polyphosphate production in differentiated NG108-15 hybrid cells

Neuroblastoma x glioma hybrid cells (NG108-15), differentiated by treatment with 1.5% dimethyl sulfoxide (DMSO) and 0.5% fetal bovine serum, were used to measure the effect of angiotensin II and III (ANG II and ANG III) on the generation of inositol polyphosphates. ANG II increased the synthesis of inositol monophosphates (IP1), inositol diphosphates (IP2), and inositol trisphosphates (IP3) with maximal responses observed at 300, 120, and 30 sec, respectively. The percent increases above basal values at the maximal responses were 140% +/- 9% (IP1), 142% +/- 4% (IP2), and 132% +/- 4% (IP3). This effect was not attenuated by pretreatment of the cells with pertussis toxin. Furthermore, both ANG II and ANG III increased the production of inositol polyphosphates in a dose-dependent manner with ED50 values of 145 nM and 11 nM, respectively. We conclude that differentiated NG108-15 cells express an ANG III selective receptor that mediates phosphatidylinositol breakdown through a pertussis toxin insensitive G-protein.     

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

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

Comparison of the butyrate effects on neurotransmitter receptors in neurohybrids NG108-15 and NCB-20 cells

Our previous study demonstrated that long term treatment of NCB-20 cells with sodium butyrate resulted in a marked increase in the density of delta-opioid receptors with a much lesser effect on muscarinic cholinergic and no effect on alpha 2-adrenergic receptors. In the present study we investigated the effect of sodium butyrate on these three types of receptors in NG108-15 cells whose neuroblastoma parent is the same as that of NCB-20 cells. Long term treatment of NG108-15 cells with sodium butyrate (0.5 mM) induced a 2-fold increase in the density of the specific binding of 3H-clonidine. A comparable increase in the number of binding sites was detected when 3H-yohimbine was used as the receptor ligand. The butyrate-induced increase in the alpha 2-adrenergic receptor binding could be totally abolished by treatment with a protein synthesis inhibitor, cycloheximide, suggesting that synthesis of receptor protein is involved. The same butyrate treatment had no significant effect on opioid and muscarinic cholinergic receptor bindings. Thus, butyrate effects on the expression of these three types of receptors in NG108-15 and NCB-20 cells are dramatically different. These data suggest that induction by butyrate of neurotransmitter receptors requires concerted action of genetic factors of both parents of the neurohybrids.     

Protein kinase C activator phorbol 12, 13-dibutyrate inhibits platelet activating factor-stimulated Ca2+ mobilization and phosphoinositide turnover in neurohybrid NG108-15 cells

The protein kinase C (PKC) activator, phorbol 12, 13-dibutyrate (PDBa) dose-dependently inhibited platelet-activating factor (PAF)-induced [Ca²⁺]_i elevation and inositol monophosphate (IP₁) accumulation in neurohybrid NG108-15 cells with IC₅₀ values of 162 nM and 35 nM, respectively. Pretreatment of NG108-15 cells with PKC inhibitor H-7 partially prevented the inhibitory effect of PDBu on PAF-induced [Ca²⁺]_i elevation as well as PI metabolism in NG108-15 cells. Pretreatment of the cells with pertussis toxin (PTX) resulted in a dose-dependent inhibition of PAF-induced IP₁ and IP₃ accumulation but only slightly affected PAF-induced [Ca²⁺]_i elevation in NG108-15 cells. The results reveal that PAF receptor-mediated Ca²⁺ mobilization and PI metabolism in NG108-15 cells are regulated by PKC while a PTX-sensitive G protein is coupled to PAF receptor for inducing activation of phospholipase C.     

Enkephalins and Opiate Antagonists Control Calmodulin Distribution in Neuroblastoma-Glioma Cells

The calcium binding protein calmodulin and the opiate receptor binding sites are unevenly distributed in various subcellular fractions of neuroblastoma-glioma NG108-15 cells. The crude mitochondrial-membrane fraction of these cells contains two membrane fractions that are separable by sucrose gradient centrifugation. These two differ in the content of both calmodulin and opiate receptors. Leucine enkephalin and D-Ala2-methionine enkephalinamide decrease the amount of membrane-bound calmodulin in the NC108-15 cells in a time- and dose-dependent manner, whereas the opiate antagonists naloxone and levallorphan have an opposite effect. Naloxone blocks the effect of leucine enkephalin and dextrallorphan has no significant effect. The opiate alkaloids entorphine and phenazocine induce changes similar to that of the enkephalins whereas morphine is inactive even at high concentrations. The alteration in the amount of membrane-bound calmodulin after a short incubation (15 min) with the enkephalins or with naloxone is reflected as an opposite change in the amount of calmodulin in the cell cytosol. Naloxone and levallorphan also increase the number of opiate receptors in NG108-15 cells but dextrallorphan has no such effect. Modulation of the intracellular distribution of calmodulin by opioid peptides and alkaloids may control the activity of various membrane-bound and cytosolic systems that are calmodulin- and/or calcium-dependent.