naltrindole and Neuroblastoma

To investigate the dual effects by the delta opioid receptor agonists DPDPE on the delayed rectified potassium channels in NG108-15 cells.. A series of outward currents were evoked in NG108-15 cells by depolarizing voltage from -50 mV to +80 mV at holding potential of -90 mV. These currents were delayed rectified potassium currents. Relatively selected delta opioid receptor agonists DPDPE of higher and lower concentrations were used to modulate the delayed rectified K+ current in NG108-15 cells. Opioid receptor antagonist Naloxone (NAL) and relatively selected delta opioid receptor antagonist Naltrindole (NTI) were used in the present experiments for the characterization of the actions of opioid receptors.. The relatively higher concentrations of delta opioid receptor agonist DPDPE (> or = 10(-6) mol/L) significantly increased the amplitude of the delayed rectified K+ current. On the contrary, the relatively lower concentrations of DPDPE (< or = 10(-12) mol/L) decreased the amplitude of the delayed rectified K+ current (P < 0.05). Furthermore both the increase and decrease were time-dependent.. delta opioid receptor agonist has dual regulatory effects on the delayed rectified potassium channels in NG108-15 cells.

Topics: Animals; Cell Membrane; Enkephalin, D-Penicillamine (2,5)-; Glioma; Hybrid Cells; Mice; Naloxone; Naltrexone; Neuroblastoma; Patch-Clamp Techniques; Potassium Channels, Inwardly Rectifying; Rats; Receptors, Opioid, delta; Tumor Cells, Cultured

Opioids play an important role in the regulation of lutenizing hormone releasing hormone (LHRH). In the present study, we attempted to find out the subtype of opioid receptors involved in the inhibitory effect of morphine on LHRH. Experiments were conducted on SK-N-SH neuroblastoma cells that express both micro and delta opioid receptors, LHRH mRNA, and release the LHRH peptide. Enzyme-linked immunosorbent assay (ELISA) was employed to measure the levels of LHRH. LHRH level was decreased by 1000 microM of morphine regardless of the duration of exposure or differentiation status of the SK-N-SH cells and was not reversed by naloxone. Selective antagonism of micro opioid receptors, but not delta opioid receptors, allowed lower concentrations (1-100 microM) of morphine to inhibit LHRH. The results of this study imply that (1) delta opioid receptors may mediate the inhibitory effect of lower concentrations of morphine on LHRH levels in SK-N-SH cells, and (2) inhibition of LHRH level by high concentrations of morphine may involve systems other than opioid receptors.

Topics: Analgesics, Opioid; Cell Line, Tumor; Dose-Response Relationship, Drug; Gonadotropin-Releasing Hormone; Humans; Morphine; Naloxone; Naltrexone; Narcotic Antagonists; Neuroblastoma; Receptors, Opioid, delta; Somatostatin

Gi/Go proteins are uncoupled from receptors by ADP-ribosylation with pertussis toxin (PTX). However, PTX treatment of delta opioid receptor-containing NG108-15 cells reduces, but does not eliminate, opioid inhibition of adenylyl cyclase. The present study explored potential mechanisms of this residual inhibition. Overnight treatment of NG108-15 cells with 100 ng/ml PTX eliminated both PTX-catalyzed [adenylyl-32P]NAD+-labeling of G proteins and agonist stimulation of low Km GTPase in membranes. Although PTX-treatment decreased the maximal opioid inhibition of adenylyl cyclase by 50-65%, the inhibition that remained was concentration-dependent and antagonist-reversible. This inhibition persisted in the absence of GTP (even though opioid inhibition of adenylyl cyclase in untreated membranes was GTP-dependent), but was eliminated by hydrolysis-resistant guanine nucleotide analogs, indicating that G-proteins were still involved in the coupling mechanism. However, assays of agonist-stimulated [35S]GTPgammaS binding in the presence of excess GDP indicated that PTX pretreatment eliminated stimulation of guanine nucleotide exchange by opioid agonists. These results suggest that in membranes from PTX-treated NG108-15 cells, a subpopulation of G proteins may transduce an inhibitory signal from agonist-bound opioid receptors without involvement of guanine nucleotide exchange.

Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Animals; Catalysis; Cell Membrane; Enzyme Activation; GTP Phosphohydrolases; GTP-Binding Proteins; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Hydrolysis; Kinetics; Mice; Naltrexone; Narcotics; Neuroblastoma; Pertussis Toxin; Tumor Cells, Cultured; Virulence Factors, Bordetella

Neuroblastoma x glioma NG108-15 hybrid cells have been examined for the expression of opioid receptor-like receptor (ORL1). [3H]Nociceptin/orphanin FQ (OFQ) bound to the cell membrane specifically (Kd = 3.6 +/- 0.6 nM) and inhibited forskolin-stimulated cAMP accumulation (EC50 = 0.72 +/- 0.3 nM). The responsiveness of NG108-15 cells to nociceptin/OFQ was blocked by pertussis toxin but not by naltrindole. The inhibitory activity of nociceptin/OFQ was significantly reduced after a prechallenge with the same peptide but was not influenced by DPDPE pretreatment, indicating acute and homologous desensitization of ORL1 receptors. Naltrindole caused the overshoot of cAMP in DPDPE-pretreated cells but not in nociceptin/OFQ-pretreated cells. The results indicate that ORL1 is functionally expressed and does not cross-interact with specific ligands of the delta opioid receptor in NG108-15 cells.

Topics: Colforsin; Cyclic AMP; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Glioma; GTP-Binding Proteins; Hybrid Cells; Naltrexone; Narcotic Antagonists; Neuroblastoma; Nociceptin; Nociceptin Receptor; Opioid Peptides; Pertussis Toxin; Receptors, Opioid; Receptors, Opioid, delta; Virulence Factors, Bordetella

To compare activation of G proteins by opioid receptors, opioid agonist-stimulated guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTP gamma S) binding in the presence of excess GDP was assayed in membranes from NG108-15 (delta) and SK-N-SH (primarily mu) cells. Basal [35S]GTP gamma S binding consisted of a single class of low-affinity sites (KD 400-500 nM). Addition of agonists produced a high-affinity site 100-300-fold higher in affinity than the basal site. The receptor/transducer amplification factor (ratio of activated G protein Bmax to opioid receptor Bmax) was 10-fold higher for SK-N-SH mu receptors than for NG108-15 delta receptors. Chronic delta agonist ([D-Ser2]-Leu-enkephalin-Thr; DSLET) treatment of NG108-15 cells resulted in an 80% loss of DSLET-stimulated [35S]-GTP gamma S binding within 1 h. Morphine treatment of SK-N-SH cells decreased mu agonist ([D-Ala2, N-Me-Phe4,Gly5-ol]-enkephalin; DAMGO)-stimulated [35S]GTP gamma S binding by 45% after 16 h, with no effect after 1 h. Loss of agonist response was due to a decrease in the Bmax of activated G proteins with no change in the KD. These results provide a quantitative description of G protein activation occurring on acute and chronic exposure to opioid agonists.

Topics: Analgesics; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalins; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Hybrid Cells; Membrane Proteins; Mice; Naloxone; Naltrexone; Narcotic Antagonists; Narcotics; Neuroblastoma; Protein Binding; Rats; Receptors, Opioid, delta; Receptors, Opioid, mu; Signal Transduction; Sulfur Radioisotopes; Time Factors