morphinans and Neuroblastoma

The p73 gene codes for various different protein isoforms. They include proteins expressed under the control of the P1 promoter that contain a transactivation domain and are similar in function to p53 (TAp73 isoforms), as well as proteins regulated by the P2 promoter that lack this domain and function as dominant negative inhibitors of TAp73 and p53 (DeltaNp73 isoforms). Whereas TAp73 functions as a tumor suppressor with pro-apoptotic function, DeltaNp73 is likely to prevent the induction of apoptosis in tumor cells and to participate in oncogenesis. Here we used a loss-of-function strategy to assess the role of DeltaNp73 in SH-SY5Y neuroblastoma cells. An antisense oligonucleotide designed to target DeltaNp73 mRNA, but not TAp73, was used to effectively downregulate this transcript. DeltaNp73 downregulation was accompanied by increased levels of the pro-apoptotic BH3 family member PUMA at the mRNA and protein level, and by conformational activation of BAX which translocated to mitochondria. These DeltaNp73 antisense-mediated alterations led to the induction of apoptosis as detected by decreased cell viability, augmented DNA fragmentation and increased caspase-3 activity in cell lysates. Our results demonstrate the cytoprotective role of DeltaNp73 in neuroblastoma and suggest its use as a target for molecular intervention therapy.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Brain Neoplasms; Cell Line, Tumor; DNA-Binding Proteins; Down-Regulation; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Tumor Suppressor; Humans; Morphinans; Neuroblastoma; Nuclear Proteins; Oligonucleotides, Antisense; Protein Isoforms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Delta-opiate receptors have been solubilized with the non-ionic bile salt detergent digitonin from NG108-15 cell membranes and reconstituted into lipid vesicles. Specific opiate binding was restored to soluble receptor preparations after supplementation with a brain lipid extract, and dilution below the effective detergent concentration. Saturable and specific opiate binding was measured for both membrane and vesicle preparations; dissociation constants (Kd) obtained from saturation isotherms of [3H]bremazocine binding were 1.3 and 4.2 nM, respectively. Relative affinity (IC50) values of ligand binding measured for subtype-selective agonists confirmed that a delta-opiate binding site interaction was recovered in vesicle preparations. Changes in agonist binding affinity noted for these experiments were explained by dissociation of the GTP-binding protein Gi from the receptor in detergent. The recovery of solubilized opiate receptors was nearly quantitative, and strictly dependent upon the total brain lipid preparation used in the reconstitution. Ligand binding was incompletely recovered after substituting pure, vesicle-forming phospholipid preparations. [3H]Bremazocine binding was also reconstituted after lectin affinity chromatography of solubilized receptor preparations, using conditions which likely effect the removal of endogenous lipid cofactors. A photoaffinity cross-linking methodology was employed to verify recovery of the delta-opiate receptor after its solubilization from membranes and reconstitution. Two membrane-associated proteins (50 and 70 kDa) were covalently tagged with an azido analog of beta-endorphin(Leu5) in cell membranes and subsequently identified by immunoblotting with antisera directed against this opioid. Labeling of the 50-kDa polypeptide was prevented by coincubating assay samples with a relative excess of (D-Pen2,5)enkephalin. This opioid binding polypeptide was also present in solubilized/reconstituted receptor preparations.

Topics: Animals; Benzomorphans; Binding Sites; Cell Line; Cell Membrane; Chromatography, Affinity; Glioma; Kinetics; Liposomes; Morphinans; Neuroblastoma; Phospholipids; Receptors, Opioid; Receptors, Opioid, delta; Solubility

The neuroblastoma-glioma NG108 cell line has been shown to contain both a delta-opiate receptor and enkephalin peptides. In this paper, the presence of authentic proenkephalin mRNA and proenkephalin-derived peptides are demonstrated. Growth of the cells in the presence of etorphine for 5-7 days resulted in a 3-fold increase of proenkephalin mRNA, which was accompanied by comparable increases in proenkephalin peptides and free enkephalin. The effect was mimicked by either morphine or [D-Ala2,D-Met5]enkephalinamide, and was blocked by naloxone. The EC50 for the effect of etorphine was 10(-9) M. The cyclic AMP content of cells grown for 5 days in the presence of etorphine was the same as that of control cells. Forskolin treatment also increased the proenkephalin mRNA content of the cells: the effect was not additive with that of etorphine, suggesting that the effect of opiate agonists was not occurring through their inhibition of adenylate cyclase. The results suggest that proenkephalin synthesis in NG108 cells can be regulated by two different mechanisms, one involving cyclic AMP while the other, regulated by the opiate receptor, is yet to be determined.

Topics: Animals; Cell Line; Colforsin; Enkephalin, Methionine; Enkephalins; Etorphine; Genes; Glioma; Hybrid Cells; Morphinans; Morphine; Naloxone; Neuroblastoma; Protein Precursors; RNA, Messenger; Transcription, Genetic

Chronic treatment of neuroblastoma X glioma NG108-15 hybrid cells with opiate agonist resulted in loss of the acute opiate inhibition of adenylate cyclase activity with a concomitant increase in the enzymatic activity observable on addition of the antagonist naloxone. The role of membrane lipids in the cellular expression of these chronic opiate effects was investigated by the hydrolysis of phospholipids with various lipases. Treatment with phospholipase C from Clostridium welchii produced an enzyme concentration-dependent decrease of prostaglandin E1-stimulated adenylate cyclase activity in control or etorphine-treated (1 microM for 4 h) hybrid cells. In addition, incubation of hybrid cells with phospholipase C concentrations of greater than or equal to 0.5 U/ml completely abolished the compensatory increase in adenylate cyclase activity after chronic opiate treatment. This attenuation of the increase in adenylate cyclase activity by phospholipase C could be prevented by inclusion of phosphatidylcholine but not of phosphatidic acid during the enzymatic incubations. The specificity of the phospholipids involved in expression of the chronic opiate effect could be demonstrated further by the absence of effect exhibited by phospholipase C from Bacillus cereus and phospholipase D. Hydrolysis of the acyl side chains of phospholipids with phospholipase A2 did not alter the chronic opiate effect after removal of lysophosphatides with bovine serum albumin. Because the guanylylimidodiphosphate- and NaF-sensitive adenylate cyclase activities were not affected by these phospholipase treatments, the expression of the compensatory increase in adenylate cyclase activity is mediated via an increase in the coupling between hormonal receptor and adenylate cyclase with the participation of the polar head groups of the phospholipids and not the hydrophobic side chains.

Topics: Adenylyl Cyclase Inhibitors; Alprostadil; Cell Line; Cyclic AMP; Etorphine; Glioma; Hybrid Cells; Membrane Lipids; Morphinans; Neuroblastoma; Phospholipase D; Phospholipases; Phospholipases A; Phospholipases A2; Phospholipids; Type C Phospholipases

The adenylate cyclase (AC) of the neuroblastoma-glioma hybrid cells (NG108-15), is generally considered to be a model for the study of the biochemical correlates of opiate tolerance and dependence. However, the naloxone-induced rebound response of adenylate cyclase, described in some recent reports, is much smaller than that originally described by Sharma, Klee and Nirenberg (1975). Possible explanations for these discrepancies are: (1) a marked down-regulation of opioid receptors and tolerance produced by the use of delta agonists or (2) the use of etorphine, a relatively hydrophobic drug which has slower dissociation rates than morphine. To test these possibilities, neuroblastoma-glioma hybrid cells were treated cells with morphine, etorphine, [D-Ala2,D-Leu5]enkephalin (DADLE), [D-Ala2]Leu-enkephalinamide (DALAMID) or vehicle. In addition, some of the cells treated with etorphine were washed with DADLE to replace the etorphine without producing the rebound response of adenylate cyclase prior to the addition of naloxone. The cells treated with morphine, DADLE and DALAMID, and incubated with prostaglandin E1 (PGE1) and naloxone showed a significant rebound of adenylate cyclase when compared with control groups and opiate-treated cells, incubated only with PGE1. In contrast, naloxone did not induce any significant rebound response in cells treated with etorphine unless they were previously washed with DADLE. These results demonstrate that the lack of a rebound response in cells treated with etorphine was due to the slow dissociation rates of the opiate and not to tolerance or to down-regulation of opioid receptors produced by agonists of high intrinsic activity.

Topics: Adenylyl Cyclases; Alprostadil; Cell Line; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalin, Methionine; Etorphine; Glioma; Morphinans; Morphine; Naloxone; Neuroblastoma; Receptors, Opioid

Neuroblastoma X glioma NG108-15 hybrid cells cultured in a chemically defined medium within 3 cell passages, exhibited viability, growth rate and morphology similar to those of cells grown in medium supplemented with 5% fetal calf serum. Hybrid cells cultured in the chemically defined medium within these periods of time also did not exhibit a difference in basal adenylate cyclase activity nor in the enzymatic activities stimulated by adenosine, forskolin, NaF, GppNHp or Mn2+. Furthermore, opiate receptor density in chemically defined medium cultured cells remained identical to that in cells cultured in 5% fetal calf serum. The acute and chronic effects of opiates on adenylate cyclase were similar for cells grown under either set of conditions.

Topics: Adenylyl Cyclases; Culture Media; Etorphine; Glioma; Hybrid Cells; Levorphanol; Morphinans; Neuroblastoma; Receptors, Opioid

Chronic etorphine treatment of neuroblastoma X glioma NG108-15 cells results in both an increase in adenylate cyclase activity (upon addition of the opiate antagonist naloxone) as well as an homologous desensitization of the opiate receptor. The continued ability of opiate agonists to regulate adenylate cyclase activity following opiate receptor desensitization can be understood by proposing that the catalytic subunit of adenylate cyclase in NG108-15 cells is under tonic regulation by both guanine nucleotide regulatory (Ni) and stimulatory (NS) components. Inactivation of Ni by pertussis toxin (PT) treatment resulted in elevated adenylate cyclase activities comparable to those observed in control cells following chronic opiate treatment. This increased enzymatic activity could not be further induced by PT treatment of cells exposed to opiate previously. In addition, procedures that prevented receptor-mediated activation of NS, i.e., treatment with NaF or desensitization of the stimulatory receptors (prostaglandin E1, adenosine) eliminated the increase in adenylate cyclase activity induced by naloxone following chronic opiate exposure. Hence, the increase in enzymatic activity observed following chronic opiate treatment may be due to a loss in tonic inhibitory regulation of adenylate cyclase mediated through Ni resulting in the unimpeded expression of NS activity. This tonic inhibition of adenylate cyclase activity is one of the multiple mechanisms by which Ni regulates adenylate cyclase in this cell line.

Topics: Adenosine; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Alprostadil; Animals; Cell Line; Colforsin; Etorphine; Glioma; GTP-Binding Proteins; Hybrid Cells; Kinetics; Mice; Morphinans; Neuroblastoma; Rats; Sodium Fluoride

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.

Topics: Animals; Bacterial Toxins; Cell Line; Diprenorphine; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Glioma; Guanine Nucleotides; Hybrid Cells; Mice; Morphinans; Neuroblastoma; Pertussis Toxin; Rats; Receptors, Opioid; Sodium; Virulence Factors, Bordetella

The benozomorphan derivative (-)-2-[2-(p-bromoacetamidophenyl)ethyl]-5,9 alpha-dimethyl-2'-hydroxy-6,7-benzomorphan (BAB), capable of reacting with nucleophilic groups, acts on neuroblastoma X glioma hybrid cells as a potent, irreversible opiate agonist. Its potency in inhibiting the increase in cellular cyclic AMP, evoked by prostaglandin E1, is comparable to that of Leu-enkephalin. This also applies to its capacity to compete with [3H]D-Ala2-Met-enkephalinamide ([3H]DAEA) in binding on cell membrane preparations. The comparatively lower potency of (-)-2-[2-(p-acetamidophenyl)-ethyl]-5,9 alpha-dimethly-2'-hydroxy-5,7-benzomorphan (AB), which differs from BAB in the substitution of the bromoacetamido group by an acetamido group, is of the same order of magnitude as that of morphine. The covalent interaction of BAB with the opiate receptors is deduced from the observations that (1) it is not possible to wash away this compound from the receptors, (2) the potency of BAB in inhibiting the specific binding of [3H]DAEA increases with prolonged preincubation time, and (3) AB behaves as a reversible agonist.

Topics: Affinity Labels; Alprostadil; Animals; Benzomorphans; Cells, Cultured; Cyclic AMP; Dose-Response Relationship, Drug; Enkephalin, Methionine; Glioma; Hybrid Cells; Morphinans; Neuroblastoma; Prostaglandins E; Receptors, Opioid

Nine new compounds have been synthesized as potential affinity ligands for specific opioid receptors. The biochemical properties of three of these compounds were examined in detail and one of them, N-cyclopropylmethyl-7 alpha-methylfumaroylamido-6, 14-endoethenotetrahydronororipavine (NIH 10236), was found to be a potent irreversible ligand for the delta opioid receptor. It had the properties of a narcotic antagonist, as determined by its effect on adenylate cyclase activity of NG108-15 neuroblastoma-glioma cell homogenates. It is, thus, the first delta specific alkylating ligand known which is a narcotic antagonist. A second compound, the N-cyclopropylmethyl-7 alpha-isothiocyanato-6, 14-endoethenotetrahydronororipavine (NIH 10235) was found to be a mu specific alkylating ligand in brain and a reversible antagonist in the NG108-15 cells.

Topics: Animals; Binding, Competitive; Brain; Cell Line; Cell Membrane; Glioma; Hybrid Cells; Ligands; Mice; Morphinans; Neuroblastoma; Rats; Receptors, Opioid; Thebaine

A soluble cytosolic factor from neuroblastoma x glioma NG108-15 hybrid cells stimulates adenylate cyclase activity in isolated membrane preparations. This cytosolic component is heat stable, pronase insensitive, has a molecular weight less than 350 daltons and an absorbance peak at 260 nm. The stimulation is immediate, independent of Ca++ and exhibits a sigmoidal concentration dependency curve. The cytosolic factor stimulated adenylate cyclase activity in etorphine treated cells (100 nM etorphine, 16 hrs) to a greater extent than in control cells. In addition, cytosolic factor derived from etorphine treated cells, as compared to control cells, displayed an increased capacity to stimulate adenylate cyclase. It is suggested that the observed cytosolic factor may be adenosine and that cells chronically treated with an opiate exhibit an increase in both concentration and sensitivity to this agent.

Topics: Adenylyl Cyclases; Animals; Cell Line; Cell Membrane; Cytosol; Etorphine; Glioma; Hybrid Cells; Kinetics; Mice; Morphinans; Neuroblastoma; Rats

In NG108-15 hybrid cells ascorbate suppresses the delayed etorphine-induced compensatory increase in the levels of cyclic AMP. It has, however, no effect on the early response of the cells to etorphine, as manifested in a transient decrease in the levels of cyclic AMP.

Topics: Ascorbic Acid; Cyclic AMP; Etorphine; Glioma; Hybrid Cells; Morphinans; Neuroblastoma; Receptors, Opioid

The high-affinity binding of benzomorphan drugs (ethylketocyclazocine and N-allylnorcyclazocine) and [DAla2,DLeu5] enkephalin was examined in a mouse neuroblastoma--Chinese hamster brain clonal hybrid cell line (NCB-20). Scatchard analysis of saturation binding isotherms indicated the presence of a single binding site for 3H-labeled [DAla2,DLeu5]enkephalin (Kd = 3 nM) and multiple binding sites for [3H]ethylketocyclazocine (Kd = 4 and 20 nM) and N-[3H]allylnorcyclazocine (Kd = 0.5 and 15 nM). Both ethylketocyclazocine and N-allylnorcyclazocine competed (Ki = 10 and 30 nM, respectively) with [3H][DAla2,DLeu5]enkephalin binding in NCB-20 cells but neither [DAla2,DLeu5]enkephalin nor morphine could completely inhibit the specific binding of [3H]ethylketocyclazocine (7 nM) or N-[3H]allylnorcyclazocine (3 nM). Furthermore, not all benzomorphan drugs (e.g., ethylketocyclazocine) were totally efficacious in displacing 3 nM N-[3H]allylnorcyclazocine binding in the presence or absence of high concentrations of [DAla2,DLeu5]enkephalin. The data presented suggest that benzomorphan drugs interact with three distinct high-affinity binding sites: (i) a site that binds enkephalin and morphine in addition to ethylketocyclazocine and N-allylnorcyclazocine; (ii) a site that binds both ethylketocyclazocine and N-allylnorcyclazocine but not enkephalin and morphine; and (iii) a site that binds N-allylnorcyclazocine but not enkephalin, morphine, or ethylketocyclazocine. The first of these sites was comparable to the delta opiate receptor expressed in NG108-15 and N4TG1 cell lines based on the potency series obtained for various opiates and benzomorphan drugs in competition studies with [3H][DAla2,DLeu5]-enkephalin. However, the specific high-affinity benzomorphan binding sites thus far are unique and may represent biochemical correlates of kappa and sigma opiate receptors which have been proposed to exist on the basis of physiological studies.

Topics: Analgesics, Opioid; Animals; Benzomorphans; Binding Sites; Binding, Competitive; Brain; Cell Line; Cyclazocine; Enkephalin, Leucine-2-Alanine; Enkephalins; Ethylketocyclazocine; Hybrid Cells; Kinetics; Morphinans; Neuroblastoma; Phenazocine; Rats; Receptors, Opioid

The guanine nucleotides GDP, GTP, and guanosine-5'-(beta, gamma-imido)triphosphate inhibit binding of opiates and opioid peptides to receptors solubilized from membranes of neuroblastoma X glioma NG108-15 hybrid cells. The inhibition reflects decreased affinity of receptors for opioid ligands. Whereas in membranes, only opioid agonist binding is sensitive to guanine nucleotide inhibition, both agonist and antagonist binding is reduced in the case of soluble receptors. Furthermore, soluble receptors are more sensitive to the effects of guanine nucleotides than are membrane-bound receptors. These observations are consistent with the suggestion that solubilized receptors may be complexes of an opiate binding protein and a guanine nucleotide-sensitive regulatory component.

Topics: Animals; Cell Line; Cell Membrane; Diprenorphine; Etorphine; Glioma; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Hybrid Cells; Kinetics; Mice; Morphinans; Neuroblastoma; Rats; Receptors, Opioid

The binding of many opiates and enkephalins to enkephalin (delta) and morphine (mu) receptors was compared by using three different binding assays: (i) 125I-labeled[D-Ala2, D-Leu5]enkephalin or 125I-labeled[D-Ala2,N-Me-Phe4,Met(O)5ol]-enkephalin to brain membranes; (ii) [3H]ethylketocyclazocine to brain membranes; and (iii) [3H]diprenorphine and [3H]naloxone to neuroblastoma cell and brain membranes, respectively. According to their relative binding potencies and the effects of Na+ and GTP on the binding to these two receptors, opiates and enkephalins can be classified into seven classes: (i) morphine-type mu agonists; (ii) enkephalin-type delta agonists; (iii) mixed agonists-antagonists; (iv) putative kappa agonists; (v) putative sigma agonists; (vi) nalorphine-type antagonists; and (vii) opiate antagonists. Studies with [3H]ethylketocyclazocine do not reveal specific kappa receptors distinct from those already described that bind morphine and enkephalins. The benzomorphan analogs ketocyclazocine and ethylketocyclazocine (putative kappa agonists) and N-allylnormetazocine (putative sigma agonist) bind to morphine (mu) and enkephalin (delta) receptors with similarly high affinities. The potency of putative kappa agonists, measured by competition with binding of the 3H-labeled antagonist, is greatly reduced by the presence of Na+ and GTP; the "Na+ and GTP ratios" are similar to those of morphine and enkephalins. However, Na+ and GTP greatly decrease the potency of binding of putative sigma agonists to enkephalin receptors but only slightly decrease the binding to morphine receptors. These data suggest that putative kappa agonists have agonistic activity toward both receptors, whereas putative sigma agonists behave as agonists for enkephalin receptors but have antagonist activity for morphine receptors. Mixed agonist-antagonists also show smaller difference in affinity to both receptors. These findings may have important implications for understanding the differences in the pharmacological effects of these drugs.

Topics: Animals; Benzomorphans; Brain; Cyclazocine; Diprenorphine; Endorphins; Enkephalins; Ethylketocyclazocine; Guanosine Triphosphate; Morphinans; Morphine; Naloxone; Neuroblastoma; Rats; Receptors, Opioid; Sodium; Synaptic Membranes

The reaction of N-2,4,5-tribenzyloxyphenyl)ethyl methanesulfonate, prepared in a seven-step sequence, with normetazocine followed by hydrogenolysis of the benzyloxy-protecting groups, gave N-(2,4,5-trihydroxyphenethyl)normetazocine. This compound was prepared to study the effect of a narcotic analgesic containing a functional group which could be activated in situ to a moiety potentially capable of reacting irreversibly with the narcotic receptor. This 6-hydroxydopamin derivative of normetazocine did not prove to be a useful affinity label. Its low toxicity could indicate the necessity for the formation of an aminochrome system for the expression of toxicity by 6-hydroxydopamine.

Topics: Adenylyl Cyclases; Analgesics, Opioid; Animals; Benzomorphans; Brain; Cyclazocine; Depression, Chemical; In Vitro Techniques; Male; Mice; Morphinans; Neoplasms, Experimental; Neuroblastoma; Norepinephrine; Protein Binding; Rats; Receptors, Opioid

Reactions mediated by the opiate receptors that inhibit adenylate cyclase (EC 4.6.1.1) are closely coupled to subsequent reactions that gradually increase adenylate cyclase activity of neuroblastoma X glioma NG108-15 hybrid cells. Opiate-treated cells have higher basal-, prostaglandin E1-, and 2-chloroadenosine-stimulated activities than do control cells. However, NaF or guanosine 5'-(beta, gamma-imido)triphosphate abolishes most of the differences in adenylate cyclase activity observed with homogenates from control and opiate-treated cells. Cycloheximide blocked some, but not all, of the opiate-dependent increase in adenylate cyclase activity. These results suggest that the opiate-dependent increase in adenylate cyclase is due to conversion of adenylate cyclase to a form with altered activity. Protein synthesis also is required for part of the opiate effect. We propose that activity of adenylate cyclase determines the rate of conversion of the enzyme from one form to the other and that opiates, by inhibiting adenylate cyclase, alter the relative abundance of low- and high-activity forms of the enzyme.

Topics: Adenosine; Adenylyl Cyclases; Cell Line; Cycloheximide; Enzyme Activation; Etorphine; Fluorides; Glioma; Guanylyl Imidodiphosphate; Hybrid Cells; Kinetics; Morphinans; Morphine; Naloxone; Neuroblastoma; Prostaglandins E