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.     

Covalent labeling of opioid receptors with radioiodinated human beta-endorphin. Identification of binding site subunit

125I-beta-Endorphin (human) binds with high affinity, specificity, and saturability to rat brain and neuroblastoma X glioma hybrid cell (NG 108-15) membranes. Dissociation constants and binding capacities were obtained from Scatchard plots and are 2 nM and 0.62 pmol/mg of protein for rat whole brain and 6 nM and 0.8 pmol/mg of protein for NG 108-15 cells. Results from competition experiments also indicate that this ligand interacts with high affinity with both mu and delta opioid binding sites, with a slight preference for mu sites, while exhibiting low affinity at kappa sites. We have demonstrated that human 125I-beta-endorphin is a useful probe for the investigation of the subunit structure of opioid receptors. The specific cross-linking of this ligand has revealed the presence of four reproducible bands or areas after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography at 65, 53, 38, and 25 kDa. All labeled bands seem to be opioid receptor related since they are eliminated when binding is carried out in an excess of various opiates. The evidence we have obtained using rat whole brain (delta congruent to mu), rat thalamus (largely mu), bovine frontal cortex (delta:mu congruent to 2:1), and NG 108-15 cells (delta) demonstrates that different labeling patterns are obtained when mu and delta binding sites are cross-linked. The pattern obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from cross-linked mu sites contains a major (heavily labeled) component of 65 kDa and a minor component of 38 kDa, while patterns from delta sites contain a major labeled component of 53 kDa. This 53-kDa band appears clearly in extracts from NG 108-15 cells and bovine frontal cortex, while in rat whole brain a diffusely labeled region is present between 55 and 41 kDa. In addition, NG 108-15 cells also display a minor labeled component at 25 kDa. The relationship of the minor bands to the major bands is not clear.     

A human neuroblastoma cell line expresses mu and delta opioid receptor sites

A series of neuroblastoma cell lines were screened for the presence of opioid receptor sites with the tracers [3H]diprenorphine (mu, delta, kappa ligand) and [3H]naloxone (mu-selective ligand). One human neuroblastoma cell line, SK-N-SH, displayed avid binding for both tracers. Binding experiments with multiple tracers revealed the presence of both mu and delta sites. These sites were stereospecific, saturable, and proteinaceous in character. Saturation binding experiments provided an estimate of 50,000 mu and 10,000 delta sites/cell. NaCl (100 mM) and guanine nucleotide, guanylyl imidodiphosphate (50 microM), reduced opioid agonist but not antagonist binding to these sites. Etorphine at 1 nM inhibited prostaglandin E1-stimulated cyclic AMP production by approximately 20%, which was reversible by naloxone. The opioid-binding sites on SK-N-SH cells closely resemble the previously reported mu and delta sites in human and rodent brain. Therefore, the SK-N-SH neuroblastoma cell line represents a useful tool to study the molecular functions of opioid receptors.     

Monoclonal antiidiotypic antibodies against delta opioid receptors as an electron microscopy probe.

Four different rat monoclonal antibodies were produced against delta opioid receptor using an antiidiotypic approach in which antibodies directed against the opioid agonist DADLE were used as immunogen. In the first step, seven hybridomas were selected on the basis of their ability to inhibit the DADLE-anti-DADLE antibody interaction. After purification from ascitic fluids, these monoclonal antibodies were characterized. Four antiidiotypic antibodies, named 5, 11, 16, and 51, directed toward different epitopes, recognized the delta opioid receptor: (i) they bound directly to the NG108-15 cells, (ii) they inhibited the [3H]DADLE binding on the NG108-15 cells, (iii) they immunoprecipitated a 52,500 dalton protein present on the surface of the NG108-15 cells. The four monoclonal antiidiotypic anti-opioid receptor antibodies were used to immunocytologically detect the opioid receptors under light and electron microscopy in the rat spinal cord. The regional distribution of the immunoreactivity corresponded to layers known to be rich delta opioid receptor subtype. Moreover, at the ultrastructural level, the labeling was located mainly on plasma membranes, especially on non-synaptic zones. Our results show that monoclonal antiidiotypic antibodies constitute a valuable tool for visualizing cell surface receptors.     

Co-localization of μ and δ opioid receptors on SK-N-SH cells detected by fluorescence microscopy using labeled anti-idiotypic antibodies

Selective fluorescence labeling of opioid receptor subclasses on SK-N-SH cultured cells has been accomplished using labeled polyclonal anti-idiotypic antibodies along with subclass-selective opioid agonists (DPDPE, δ-selective; DAMGO, μ-selective) as blocking reagents. Labeling of the cells was examined using conventional fluorescence microscopy. Co-localization of μ- and δ- opioid receptors on SK-N-SH cells has been studied by double labeling fluorescence experiments. In agreement with our own, and other workers', previous observations on NG108-15 cells, a subpopulation of viable cells in asynchronous cultures are labeled. Amon those SK-N-SH cells that are labeled, both subclasses of receptors are seen. On the basis of sequential blocking experiments we interpret our combined results to be consistent with a model where μ- and δ- binding sites reside on different subunits of a multimeric complex.     

Dimeric pentapeptide enkephalin: a novel probe of delta opiate receptors

A dimeric pentapeptide enkephalin (DPE2) consisting of two molecules of [D-Ala 2, Leu 5] enkephalin linked at C-terminal leucine with ethylenediamine, (H-Tyr-D-Ala-Gly-Phe-Leu-NH-Ch2)2 is a bivalent ligand for the delta enkephalin receptors of rat brain and neuroblastoma-glioma hybrid (NG108-15) cells. This new enkephalin analog shows dramatically increased affinity in radioligand assays using whole brain membranes when delta but not mu specific radioligands are employed. When membranes from NG108-15 cells are used, the dimer shows greatly increased activity irrespective of the mu or delta specificity of the tracer. The dimer DPE2 shows a four-fold, "sodium shift" in its IC50 for competition with [3H]naloxone, suggestive of agonist behavior. Agonist activity was confirmed by demonstrating that DPE2 inhibits cyclic AMP production in prostaglandin E1 stimulated NG108-15 cells, and by demonstrating very high potency in the mouse vas deferens bioassay. DPE2 binds to the same delta sites as the delta-selective monomer [D-Ala2, D-Leu5] enkephalin, since the two ligands show complete crossdisplacement. Radiolabeled 3H-DPE2 shows a five-fold higher affinity constant, a 2.5-fold higher association rate constant, and a two-fold lower dissociation rate than the monomer. These results are consistent with the hypothesis that the dimeric pentapeptide enkephalin can bridge two delta receptors. This enkephalin dimer provides a valuable new probe of opiate receptors and their organization in cell membranes.     

Biotinylated human beta-endorphins as probes for the opioid receptor

The reaction of human beta-endorphin and biotinyl N-hydroxysuccinimide with or without spacer arm, afforded a series of products that were separated by high performance liquid chromatography (HPLC). Liquid secondary ion mass spectrometry of the biotinylated products and their tryptic digests produced abundant protonated molecular ions (MH+), which specified the number and location of biotinylation. Between 1 and 4 biotinyl residues were incorporated per human beta-endorphin molecule, at Lys-9, -19, -24, -28, and -29, but not at the amino-terminal Tyr-1. Three HPLC fractions were isolated for receptor binding studies with monobiotinylation of Lys-9 (B1 beta and B1X beta; X = C6 spacer arm), Lys-19 (B1 gamma), and a mixture of Lys-24, Lys-28, and Lys-29 derivatives (B1 alpha, BX1 alpha). All derivatives displayed tight binding to avidin, and no dissociation from avidin was detectable over several hours at 0 degrees C for the derivatives (BX1 alpha) tested. IC50 values for binding to mu and delta opioid receptor sites were 3-8 times higher for monobiotinylated derivatives than for the parent human beta-endorphin (IC50,mu = 1.5 nM, IC50,delta = 1.3 nM). Association with avidin decreased opioid receptor affinities for the C6 spacer derivative biotinylated at position Lys-9, which is close to the (1-5) enkephalin receptor region. In contrast, avidin did not affect or even increased apparent affinities to mu and delta sites for derivatives biotinylated at the alpha-helical part of the molecule (Lys-19, -24, -28, and -29). Thus, when bound to avidin, the biotinylated human beta-endorphin derivatives with spacer arm (BX1 alpha), substituted near the carboxyl terminal (Lys-24, -28, and -29), displayed mu binding affinities equal to and delta binding affinities only four times lower than underivatized human beta-endorphin. Biotinylated human beta-endorphins also bound to low affinity nonopioid binding sites on NG-108-15 cells; however, affinities to these sites were considerably reduced when derivatives were bound to avidin. The ability of biotinylated human beta-endorphin to cross-link the mu and delta opioid receptors to avidin allows application of the biotin-avidin system as a molecular probe of the opioid receptor.     

Analgesic effect of interferon-alpha via mu opioid receptor in the rat

Using the tail-flick induced by electro-stimulation as a pain marker, it was found that pain threshold (PT) was significantly increased after injecting interferon-alpha (IFN alpha) into the lateral ventricle of rats. This effect was dosage-dependent and abolished by monoclonal antibody (McAb) to IFN alpha. Naloxone could inhibit the analgesic effect of IFN alpha, suggesting that the analgesic effect of IFN alpha be related to the opioid receptors. Beta-funaltrexamine (beta-FNA), the mu specific receptor antagonist could completely block the analgesic effect of IFN alpha. The selective delta-opioid receptor antagonist, ICI174,864 and the kappa-opioid receptor antagonist, nor-BNI both failed to prevent the analgesic effect of IFN alpha. IFN alpha could significantly inhibit the production of the cAMP stimulated by forskolin in SK-N-SH cells expressing the mu-opioid receptor, not in NG108-15 cells expressing the delta-opioid receptor uniformly. The results obtained provide further evidence for opioid activity of IFN alpha and suggest that this effect is mediated by central opioid receptors of the mu subtype. The evidence is consistent with the hypothesis that multiple actions of cytokines, such as immunoregulatory and neuroregulatory effects, might be mediated by distinct domains of cytokines interacting with different receptors.     

Different molecular weight forms of opioid receptors revealed by polyclonal antibodies

Polyclonal antibodies were raised against a purified opioid receptor from bovine brain (Cho, et. al., 1986), and shown to inhibit 3H-diprenorphine binding to this receptor in a dose-dependent fashion. These antibodies were then used to characterize opioid-binding material present in rat brain and in NG108-15 neuroblastoma-glioma hybrid cells. Western blot analysis revealed that the antibodies reacted with a single species of 58,000 molecular weight in rat brain membranes; this closely corresponds in size to the bovine opioid receptor used to raise the antibodies. In contrast, the polyclonal antibodies reacted with a 45,000 molecular weight species in NG108-15 neuroblastoma-glioma hybrid cells; moreover, this band was specifically reduced in NG108-15 cells in which opioid receptors had been down-regulated by incubation with D-ala2-D-leu5-enkephalin for 24 hours. Thus at least two distinct opioid receptor molecules have been identified, which have antigenic similarities.     

Only Pyrimidinoceptors Are Functionally Expressed in Mouse Neuroblastoma Cell Lines

The ability of UTP, UDP, ATP, and ADP to influence inositol phospholipid hydrolysis in neuroblastoma origin cell lines was assessed. The mouse neuroblastoma lines N1E 115, Neuro 2a, and NB4 1A3 and the rat glioma/mouse neuroblastoma hybrid line NG108-15 gave robust responses to both UTP and UDP, which were essentially equipotent. Thus a range of cell lines of mouse neuroblastoma origin express a pyrimidine-selective P2Y receptor. The NG108-15 cells were the only cell type tested at which ATP and ADP displayed activity with EC₅₀ values of greater than 100 μM, compared with values of 0.58 and 1.25 μM for UTP and UDP, respectively. In contrast to the cell lines derived from mouse neuroblastoma, the human neuroblastoma lines SH-SY5Y and SK-N-SH did not respond to any nucleotides, although both responded well to carbachol.     

Target size analysis of opioid receptors. No difference between receptor types, but discrimination between two receptor states

Target size analysis of opioid receptor is complicated by the presence of multi-exponential inactivation curves. Irradiation of intact frozen tissue proved essential to eliminate such artifacts, due to indirect irradiation effects. Upon irradiation condition, opioid binding activity was inactivated in a single mono-exponential manner. Identical inactivation curves were obtained for mu, delta and kappa binding activities in brain membranes from rat, guinea-pig and frog and in NG 108-15 cells: the molecular mass obtained was 98 +/- 2 kDa. However, when opioid binding was assayed in the presence of Na+, Mg2+ and GTP, the molecular mass was found to be only 56 +/- 4.4 kDa. We suggest that the opioid recognition site comprises a unit of 56 kDa and that in the absence of Na+, Mg2+ and GTP an additional membrane component of 40-44 kDa is necessary for high-affinity opioid binding.     

The regulation of steroidogenesis by opioid peptides in porcine theca cells

The present study was designed to investigate basal and LH-induced steroidogenesis in porcine theca cells from large follicles in response to various concentrations (1-1000 nM) of mu opioid receptor agonists (beta-endorphin, DAMGO, FK 33-824), delta receptor agonists (met-enkephalin, leu-enkephalin, DPLPE) and kappa receptor agonists (dynorphin A, dynorphin B, U 50488). Agonists of mu opioid receptors suppressed basal androstenedione (A4), testosterone (T) and oestradiol-17beta (E2) secretion and enhanced LH-induced A4 and T release by theca cells. The inhibitory effect of the agonists on E2 secretion was abolished in the presence of LH. All delta receptor agonists depressed basal progesterone (P4) output. However, the influence of these agents on LH-treated cells was negligible. Among delta receptor agonist used only leu-enkephalin and DPLPE at the lowest concentrations inhibited basal A4 release. The presence of LH in culture media changed the influence of these opioids from inhibitory to stimulatory. Similarly, DPLPE reduced T secretion by non-stimulated theca cells and enhanced T secretion of stimulated cells. All of delta agonists inhibited basal E2 secretion and unaffected its release from LH-treated theca cells. Agonists of kappa receptors inhibited basal, non-stimulated, P4 secretion and two of them (dynorphin B, U 50488) potentiated LH-induced P4 output. Basal A4 and T release remained unaffected by kappa agonist treatment, but the cells cultured in the presence of LH generally increased both androgen production in response to these opioids. Basal secretion of E2 was also suppressed by kappa agonists. This inhibitory effect was not observed when the cells were additionally treated with LH. In view of these findings we suggest that opioid peptides derived from three major opioid precursors may directly participate in the regulation of porcine theca cell steroidogenesis.     

beta-Endorphin: evidence for the existence of opioid and non-opioid binding components for the tritiated human hormone in NG108-15 cells

Human beta-endorphin (beta h-EP) binding on neuroblastoma X glioma hybrid NG108-15 cells using tritiated human beta endorphin (3H-beta h-EP) as a primary ligand was found to have a component which was not displacable with [D-Ser2 )-Leu-enkephalin-Thr6 (DSLET). The beta h-EP binding on these cells after saturation of the delta opiate sites with 200 nM DSLET was further characterized with synthetic beta h-EP analogs. The nonopioid binding site appears to recognize beta h-EP-(6-31), beta h-EP-(21-31) and beta h-EP-(28-31). Under these conditions, these COOH-terminal segments fully displace the tritiated beta h-EP. However, beta h-EP-(1-27) does not further displace 3H-beta h-EP in the presence of DSLET. The fact that a combination of DSLET and beta h-EP-(6-31) results in a full displacement of 3H-beta h-EP provides direct evidence for the existence of two binding sites for beta h-EP in NG108-15 cells, one recognizing the NH2-terminal enkephalin sequence and the other the non-opioid COOH-terminal segment.     

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.     

Attenuation of Enkephalin Activity in Neuroblastoma × Glioma NG108—15 Hybrid Cells by Phospholipases

The role of membrane phospholipids in enkephalin receptor-mediated inhibition of adenylate cyclase (EC 4.6.1.1) activity in neuroblastoma X glioma NG108-15 hybrids was studied by selective hydrolysis of lipids with phospholipases. When NG108-15 cells were treated with phospholipase C from Clostridium welchii at 37 degrees C, an enzyme concentration--dependent decrease in adenylate cyclase activity was observed. The basal and prostaglandin E1 (PGE1)-stimulated adenylate cyclase activities were more sensitive to phospholipase C (EC 3.1.4.3) treatment than were the NaF-5'-guanylylimidodiphosphate (Gpp(NH)p)-sensitive adenylate cyclase activities. Further, Leu5-enkephalin inhibition of basal or PGE1-stimulated adenylate cyclase activity was attenuated by phospholipase C treatment, characterized by a decrease of enkephalin potency and of maximal inhibitory level. [3H]D-Ala2-Met5-enkephalinamide binding revealed a decrease in receptor affinity with no measurable reduction in number of binding sites after phospholipase C treatment. Although opiate receptor was still under the regulation of guanine nucleotide after phospholipase C treatment, adenylate cyclase activity was more sensitive to the stimulation of Gpp(NH)p. Thus, the reduction of opiate agonist affinity was not due to the uncoupling of opiate receptor from N-component. Further, treatment of NG108-15 hybrid cell membrane with phospholipase C at 24 degrees C produced analogous attenuation of enkephalin potency and efficacy without alteration in receptor binding. The reduction in enkephalin potency could be reversed by treating NG108-15 membrane with phosphatidylcholine, but not with phosphatidylserine, phosphatidylinositol, or cerebroside sulfate. The enkephalin activity in NG108-15 cells was not altered by treating the cells with phospholipase A2 o phospholipase C from Bacillus cereus. Hence, apparently, there was a specific lipid dependency in enkephalin inhibition of adenylate cyclase activity.     

Synthesis and binding properties to DNA and to opioid receptors of enkephalin-ellipticinium conjugates

In order to specifically direct cytotoxic agents against tumor cells bearing delta opioid receptors, the DNA intercalating agents ellipticine and 9-OH-ellipticine were coupled by quaternarization of the pyridine nitrogen to an enkephalin modified pentapeptide through a short chemical linker. The ellipticine ring of these conjugates was shown to intercalate into DNA, with DNA affinity constants close to those of the non-conjugated ellipticines. Despite the addition of a polycyclic ring to the C-terminal amino acid, the D-Ala2-D-Leu5-enkephalin-ellipticine conjugates bind to the opioid receptor from rat brain and NG 108-15 cells with an affinity constant close to 10(8) M-1. Other derivatives were synthesized as a control using a tripeptide which does not bind to the opioid receptor.     

Neuronal Cell Surface Molecules Mediate Specific Binding to Rabies Virus Glycoprotein Expressed by a Recombinant Baculovirus on the Surfaces of Lepidopteran Cells

The existence of specific rabies virus (RV) glycoprotein (G) binding sites on the surfaces of neuroblastoma cells is demonstrated. Spodoptera frugiperda (Sf21) cells expressing G of the RV strain CVS (Gcvs-Sf21 cells) bind specifically to neuroblastoma cells of different species but not to any other cell type (fibroblast, myoblast, epithelial, or glioma). Attachment to mouse neuroblastoma NG108-15 cells is abolished by previous treatment of Gcvs-Sf21 cells with anti-G antibody. Substitutions for lysine at position 330 and for arginine at position 333 in RV G greatly reduce interaction between Gcvs-Sf21 cells and NG108-15 cells. These data are consistent with in vivo results: an avirulent RV mutant bearing the same double mutation is not able to infect sensory neurons or motoneurons (P. Coulon, J.-P. Ternaux, A. Flamand, and C. Tuffereau, J. Virol. 72:273–278, 1998) after intramuscular inoculation into a mouse. Furthermore, infection of NG108-15 cells by RV but not by vesicular stomatitis virus leads to a reduction of the number of binding sites at the neuronal-cell surface. Our data strongly suggest that these specific attachment sites on neuroblastoma cells represent a neuronal receptor(s) used by RV to infect certain types of neurons in vivo.     

Opioid tolerance/dependence in neuroblastoma x glioma (NG108-15) hybrid cells is associated with a reduction in spontaneous stimulatory receptor activity

Chronic opioid regulation of stimulatory receptor activity was investigated in neuroblastoma x glioma (NG108-15) hybrid cells stably transfected to express the human beta(2)-adrenoceptor (beta(2)-AR). Expressed beta(2)-ARs are functionally coupled to G proteins and display ligand-independent signalling activity, as demonstrated by the ability of an inverse agonist to attenuate basal adenylyl cyclase (AC) activity. Despite the relative increase in basal AC activity due to the development of tolerance/dependence, chronic morphine treatment was found to completely abolish spontaneous beta(2)-AR activity by reducing basal receptor/G protein precoupling. A similar chronic opioid effect was observed in transiently transfected COS-7 cells. These results indicate that during the state of opioid tolerance/dependence basal levels of AC activity are no longer under the control of spontaneously active stimulatory receptors.