beta-endorphin and Glioma

The astrocytoma cell line rat C6 glioma has been used as a model system to study the mechanism of various opioid actions. Nevertheless, the type of opioid receptor(s) involved has not been established. Here we demonstrate the presence of high-affinity U69,593, endomorphin-1, morphine, and beta-endorphin binding in desipramine (DMI)-treated C6 cell membranes by performing homologous and heterologous binding assays with [3H]U69,593, [3H]morphine, or 125I-beta-endorphin. Naive C6 cell membranes displayed U69,593 but neither endomorphin-1, morphine, nor beta-endorphin binding. Cross-linking of 125I-beta-endorphin to C6 membranes gave labeled bands characteristic of opioid receptors. Moreover, RT-PCR analysis of opioid receptor expression in control and DMI-treated C6 cells indicate that both kappa- and mu-opioid receptors are expressed. There does not appear to be a significant difference in the level of mu nor kappa receptor expression in naive versus C6 cells treated with DMI over a 20-h period. Collectively, the data indicate that kappa- and mu-opioid receptors are present in C6 glioma cells.

Topics: Analgesics; Analgesics, Opioid; Animals; Benzeneacetamides; beta-Endorphin; Binding, Competitive; Desipramine; Glioma; Morphine; Polymerase Chain Reaction; Pyrrolidines; Rats; Receptors, Opioid, kappa; Receptors, Opioid, mu; Transcription, Genetic; Tumor Cells, Cultured

Opioid agonists inhibit DNA synthesis in C6 rat glioma cells that express opioid receptors, induced by desipramine (DMI). This inhibition was not observed in cells that were not treated with DMI, and thus did not express opioid-binding sites. Endothelin, a known mitogen, increased thymidine incorporation dose dependently (up to 1.7-fold) in DMI-treated C6 cells. This increase was reversed by an anti-idiotypic antibody to opioid receptors, Ab2AOR, which has opioid agonist properties. The opioid antagonist naltrexone blocked the inhibition caused by Ab2AOR. Endothelin also stimulated phosphoinositide (PI) turnover and this effect was inhibited by morphine (50%) or by Ab2AOR (72%) in DMI-treated but not in DMI-untreated C6 cells. These actions of morphine and Ab2AOR were reversed by naltrexone. The inhibition of PI turnover and of thymidine incorporation by Ab2AOR or morphine was insensitive to pertussis toxin (PTX). Since PI turnover is known to induce Ca2+ mobilization, it was of interest to examine the effects of the applied opioids on intracellular Ca2+ concentrations. Endothelin increased the concentration of cytosolic free Ca2+ in the cells while Ab2AOR, morphine, and beta-endorphin reversed the endothelin-induced Ca2+ mobilization in DMI-treated but not in DMI-untreated C6 cells. The effect of these agonists was also blocked by naltrexone. The results indicate that glial cells can be a target of an opioid receptor-mediated antimitogenic action and that an abatement in PI turnover and Ca2+ mobilization may be associated with this mechanism.

Topics: Animals; Antibodies, Anti-Idiotypic; beta-Endorphin; Calcium; Desipramine; DNA, Neoplasm; Dose-Response Relationship, Drug; Endothelins; Glioma; Morphine; Naltrexone; Phosphatidylinositols; Rats; Receptors, Opioid; Thymidine; Tumor Cells, Cultured

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.

Topics: Animals; Autoradiography; beta-Endorphin; Binding Sites; Binding, Competitive; Glioma; Mice; Neuroblastoma; Rats; Receptors, Opioid, delta; Tumor Cells, Cultured

The affinity cross-linking of the delta-opioid receptor in neuroblastoma x glioma NG108-15 cells was undertaken using (3-[125I]iodotyrosyl27)human-beta-endorphin ([125I]beta-endorphin) and disuccinimidyl suberate (DSS) or bis(sulfosuccinimidyl) suberate (BS3) in order to estimate molecular size. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, two radioactive bands were observed. Labeling of a major band of 29 kDa diminished in the presence of unlabeled selective delta-opioid agonist, [D-Pen2,D-Pen5]enkephalin (DPDPE), in a concentration-dependent manner, while labeling of a minor band of 58 kDa was hardly affected. The labeling intensity of the 29 kDa band decreased by addition of guanosine 5'-(3-o-thio)triphosphate (GTP gamma S) or by pretreatment of cells with pertussis toxin. These results, taking the molecular weight of covalently bound beta-endorphin (3.6 kDa) into consideration, suggest that the delta-opioid receptor in NG108-15 cell membrane is a 25 kDa protein which is coupled to pertussis toxin-sensitive guanosine triphosphate-binding proteins (G-proteins).

Topics: beta-Endorphin; Cell Membrane; Chemical Fractionation; Electrophoresis, Polyacrylamide Gel; Glioma; GTP-Binding Proteins; Humans; Molecular Weight; Neuroblastoma; Pertussis Toxin; Receptors, Opioid; Succinimides; Tumor Cells, Cultured; Virulence Factors, Bordetella

Regulation of [125I]beta h-endorphin binding by guanine nucleotides was investigated in membrane preparations from two opioid receptor-containing cell lines: NG108-15, which contains only delta opioid receptors, and SK-N-SH, which contains predominantly mu opioid receptors. In contrast to the binding of the delta-selective agonist [3H][D-penicillamine2,D-penicillamine5]enkephalin to NG108-15 cell membranes, and of the mu-selective agonist [3H][D-Ala2,MePhe4,Gly-ol5]enkephalin to SK-N-SH cell membranes, [125I]beta h-endorphin binding to NG108-15 and SK-N-SH cell membranes was not altered by guanosine triphosphate (GTP) or guanylyl-5'-imidodiphosphate (Gpp(NH)p) in the absence of cations. However, in the presence of NaCl, [125I]beta h-endorphin binding to both cell lines was inhibited by GTP and Gpp(NH)p in a concentration-dependent manner. In SK-N-SH cell membranes, the ability of sodium to promote regulation of [125I]beta h-endorphin binding by GTP was mimicked by the monovalent cations lithium and potassium, but not by the divalent cations magnesium, calcium, or manganese. In NG108-15 cell membranes, only sodium was effective in promoting inhibition of [125I]beta h-endorphin binding by GTP. The effect of GTP or Gpp(NH)p in the presence of sodium was also observed with guanosine diphosphate, but not guanosine monophosphate or any of the non-guanine nucleotides tested. These results indicate that the presence of monovalent cations is required for regulation of [125I]beta h-endorphin binding by guanine nucleotides, and that the specificity of this cation requirement differs between the mu and delta receptor-containing cell lines.

Topics: beta-Endorphin; Cations; Cell Membrane; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalins; Glioma; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Humans; Hybrid Cells; Neuroblastoma; Oligopeptides; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Sodium Chloride; Tumor Cells, Cultured

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.

Topics: Animals; beta-Endorphin; Binding, Competitive; Biotin; Cell Line; Glioma; Humans; Hybrid Cells; Kinetics; Neuroblastoma; Receptors, Opioid; Structure-Activity Relationship

The properties of [125I]beta h-endorphin-binding sites from rat brain membranes and membranes from the NG108-15 cell line were compared using a monoclonal antibody directed against the opioid receptor and opioid peptides as probes. The binding of [125I]beta h-endorphin to both rat brain and NG108-15 membranes yielded linear Scatchard plots with Kd values of 1.2 nM and 1.5 nM, respectively, and Bmax values of 865 fmol/mg rat brain membrane protein and 1077 fmol/mg NG108-15 membrane protein. A monoclonal antibody, OR-689.2.4, capable of inhibiting mu and delta binding but not kappa binding to rat brain membranes, noncompetitively inhibited the binding of 1 nM [125I]beta h-endorphin to rat brain and NG108-15 membranes with an IC50 value of 405 nM for rat brain membranes and 543 nM for NG108-15 membranes. The monoclonal antibody also inhibited the binding of 3 nM [3H] [D-penicillamine2, D-penicillamine5] enkephalin to NG108-15 membranes with an IC50 value of 370 nM. In addition to blocking the binding of [125I]beta h-endorphin to brain membranes, the antibody also displaced [125I]beta h-endorphin from membranes. Site-specific opioid peptides had large variations in their IC50 values depending on whether they were inhibiting [125I]beta h-endorphin binding to rat brain or the NG108-15 membranes. When the peptides were tested with the monoclonal antibody for their combined ability to inhibit [125I]beta h-endorphin binding to both membrane preparations, the peptides and antibody blocked binding as though they were acting at allosterically coupled sites, not two totally independent sites. These studies suggest that mu-, delta-, and beta-endorphin-binding sites share some sequence homology with the 35,000-dalton protein that the antibody is directed against.

Topics: Animals; Antibodies, Monoclonal; beta-Endorphin; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Glioma; Immunoglobulin M; In Vitro Techniques; Iodine Radioisotopes; Male; Molecular Weight; Neuroblastoma; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Tumor Cells, Cultured

The putative delta-opiate receptor complex has been identified by a new approach which employed an antibody that is directed against a peptide which binds gamma-endorphin and is specified by RNA that is complementary to that of gamma-endorphin mRNA. This antibody competes with beta-endorphin and naloxone for binding sites on the surface of neuroblastoma X glioma NG108-15 hybrid cells. The opiate receptor complex has an apparent molecular weight of 210,000 and is composed of four noncovalently associated subunits with apparent molecular weights of 68,000, 58,000, 45,000 and 30,000.

Topics: Antibodies; beta-Endorphin; Binding, Competitive; Cell Line; Endorphins; gamma-Endorphin; Glioma; Hybrid Cells; Immunologic Techniques; Molecular Weight; Naloxone; Neuroblastoma; Peptides; Receptors, Opioid; Receptors, Opioid, delta; RNA, Messenger

Pituitary carcinoma is defined as a malignant pituitary tumour associated with blood- or lymph-borne metastases. Cushing's disease is frequently present in patients with this condition. After adrenalectomy for Cushing's disease, a 37-year-old man developed Nelson's syndrome resulting from a pituitary carcinoma with metastases to the spinal cord, cauda equina, heart, liver, and pancreas. The primary tumour and its metastases showed immunocytochemical staining for ACTH, beta-lipotrophin, and variably for beta-endorphin and alpha-melanocyte stimulating hormone (alpha-MSH). A coincidental glioblastoma was also present. Nine cases of Cushing's disease associated with pituitary carcinoma, including the present patient, are documented in the literature. The case reported is only the second in which immunohistochemical staining of the primary pituitary tumour and its metastases was performed, and the first in which ACTH-related peptides, in addition to ACTH itself, were demonstrated in the carcinoma cells.

Topics: Adrenalectomy; Adrenocorticotropic Hormone; Adult; beta-Endorphin; beta-Lipotropin; Brain Neoplasms; Cushing Syndrome; Endorphins; Glioma; Humans; Male; Melanocyte-Stimulating Hormones; Nelson Syndrome; Neoplasms, Multiple Primary; Pituitary Neoplasms; Pro-Opiomelanocortin

Acid extracts from mouse Neuroblastoma x rat Glioma hybrid cells have been purified by means of Sep-Pak C-18 and fractionated by high performance liquid chromatography. Each fraction has been submitted to a sensitive beta-endorphin radioimmunoassay and an immunoreactivity peak at camel beta-endorphin retention time was found.

Topics: Animals; beta-Endorphin; Cell Line; Chromatography, High Pressure Liquid; Endorphins; Glioma; Hybrid Cells; Mice; Neuroblastoma; Radioimmunoassay; Rats; Spectrophotometry, Ultraviolet

Inhibition of binding of beta h-endorphin or Leu-enkephalin by beta h-endorphin analogs of various chain lengths in memmbrane preparations of the neuroblastoma x glioma NG108-15 cells has been investigated. The removal of even a single residue from the C-terminus results in the inability of the resulting peptide to completely displace beta h-endorphin. In addition, the proportion of nondisplaceable binding increases with decreasing chain length.

Topics: Animals; beta-Endorphin; Cell Membrane; Endorphins; Enkephalin, Leucine; Glioma; In Vitro Techniques; Kinetics; Neuroblastoma; Rats; Receptors, Opioid; Structure-Activity Relationship

The established human glioblastoma cell line SF126 was found to bind tritiated human beta-endorphin (beta h-EP) in a saturable fashion. From displacement studies, the ED50 was estimated to be about 2.5 nM. The Kd was estimated as 1.9 X 10(-9) M and Scatchard analysis showed a biphasic pattern with a predominant low-affinity component. Binding reached a maximum at about 90 min at 22 degrees C and was instantaneously reversible. Tritiated [D-Ala2,D-Leu5]enkephalin and tritiated dihydromorphine did not bind to the cells. Sodium at a concentration of 150 mM decreased the specific binding by 80%. The interaction with the cellular binding site appeared to be mediated by the COOH-terminal segment of beta h-EP, as beta h-EP-(6-31) retained a high potency for displacing tritiated beta h-EP, and beta h-EP-(1-27) has no activity. Camel beta-EP was only about 1% as active as the human hormone.

Topics: beta-Endorphin; Binding, Competitive; Cells, Cultured; Endorphins; Glioma; Humans; Receptors, Opioid; Structure-Activity Relationship

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.

Topics: beta-Endorphin; Binding, Competitive; Cell Line; Endorphins; Glioma; Humans; Hybrid Cells; Neuroblastoma; Neuroglia; Neurons; Receptors, Opioid; Tritium

The potency of a series of synthetic analogs of beta-endorphin in inhibiting binding of [3H2-Tyr27]-beta h-endorphin to either rat brain membranes or neuroblastoma x glioma hybrid cells (NG108-15) has been determined and compared with the previously determined analgesic potency. There is a very good correlation between inhibitory potency in membranes and cells, but the correlation between analgesic potency and inhibitory potency in either membranes or cells is not as good.

Topics: Animals; beta-Endorphin; Brain; Cell Membrane; Endorphins; Glioma; Hybrid Cells; Kinetics; Mice; Neuroblastoma; Rats; Receptors, Opioid; Structure-Activity Relationship

Topics: Amino Acid Sequence; Animals; beta-Endorphin; Cell Line; Endorphins; Female; Glioma; Humans; Hybrid Cells; Male; Mice; Neuroblastoma; Pregnancy; Rats; Receptors, Opioid; Tissue Distribution

Topics: Amino Acid Sequence; Animals; Behavior, Animal; beta-Endorphin; Cell Line; Endorphins; Glioma; Humans; Hybrid Cells; Mice; Neuroblastoma; Rats; Receptors, Opioid; Species Specificity