levorphanol and Neuroblastoma

Topics: Animals; Cell Line; Cyclic AMP; Enkephalins; Glycolipids; Levorphanol; Mice; Models, Biological; Morphine; Neoplasms, Experimental; Neuroblastoma; Receptors, Opioid

Opioids induce dual (inhibitory and excitatory) regulation of depolarization-evoked [3H]dopamine release in SK-N-SH cells through either mu or delta receptors. The potentiation of dopamine release by opioid agonists is mediated by N-type voltage-dependent calcium channels and does not involve Gi/Go proteins. Removal of the excitatory opioid effect by blockade with omega-conotoxin, an N-channel antagonist, reveals the inhibitory effect of opioids on release, thus suggesting that both modulatory effects of opioids are exerted in parallel.

Topics: Brain Neoplasms; Calcium Channel Blockers; Calcium Channels; Dopamine; GTP-Binding Proteins; Humans; Levorphanol; Morphine; Narcotics; Neuroblastoma; Neurotransmitter Agents; Opioid Peptides; Tumor Cells, Cultured

Depolarization-evoked 3H-norepinephrine release from SK-N-SH cells was found to be regulated by opioid ligands. Opioids exerted either inhibition or augmentation of 3H-norepinephrine release. Both effects were mediated by opioid receptors. In addition, a nonopioid inhibitory effect of opiates on release was observed. The SK-N-SH cell-line provides a suitable model for studying the various mechanisms underlying the opioid regulatory pathways within single cells.

Topics: Cell Line; Dextrorphan; Drug Interactions; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Humans; Kinetics; Levorphanol; Morphine; Naloxone; Narcotics; Neuroblastoma; Norepinephrine; Receptors, Opioid; Tritium; Tumor Cells, Cultured

In differentiated SH-SY5Y human neuroblastoma cells, various opioids exhibited a wide range of potencies (Ki) in acutely inhibiting adenylate cyclase to different extents (Imax). After exposure of the cells to opioids for 24 hr, the initially reduced cAMP content of the cells recovered toward pre-exposure levels. Withdrawal of agonist from, or addition of antagonist to, the tolerant cells rapidly increased the cAMP content to 1.5 times the basal value. Long term treatment of the cells with agonists of high acute potency, such as Tyr-D-Ala-Gly-(Me)Phe-Gly-ol and levorphanol, decreased the Bmax of the antagonist [3H]naltrexone by 80-95%, increased the Ks for GTPase stimulation 10-14-fold, and increased the Ki for adenylate cyclase inhibition 2-3-fold. On the other hand, these parameters were only marginally affected by agonists of lower acute potency, such as profadol and morphiceptin, regardless of their Imax in inhibiting adenylate cyclase. The reduction in the level of receptor binding was experimentally not dissociable from effector desensitization. Tyr-D-Ala-Gly-(Me)Phe-Gly-ol retained the characteristics of a potent agonist in inducing tolerance even under conditions of submaximal signal, produced by lower concentrations of the peptide or by pretreatment with pertussis toxin. Alkylation of receptors by beta-chlornaltrexamine, although it reduced [3H]naltrexone binding by 50%, did not significantly alter the rank order of opioid agonists based on their ability to acutely inhibit adenylate cyclase. These results show that in opioid-tolerant SH-SY5Y cells the concurrently occurring down-regulation of receptor and shifts in the concentration dependence of effector response correlate with the potency of a given opioid in producing its acute effect but not with the maximum extent of that effect.

Topics: Adenylyl Cyclase Inhibitors; Amino Acid Sequence; Cyclic AMP; Down-Regulation; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; GTP Phosphohydrolases; Humans; Levorphanol; Molecular Sequence Data; Naltrexone; Narcotics; Neuroblastoma; Neurons; Receptors, Opioid; Signal Transduction; Tumor Cells, Cultured

Evidence is presented for linkage of opioid receptors directly to the stimulatory G protein (guanine nucleotide-binding protein), Gs, in addition to the generally accepted linkage to the inhibitory and "other" G proteins, gi and Go, in F-11 (neuroblastoma-dorsal root ganglion neuron) hybrid cells. Treatment of intact F-11 cells with cholera toxin decreased specific binding of the opioid agonist [D-Ala2,D-Leu5]enkephalin to F-11 cell membranes by 35%, with the remaining binding retaining high affinity for agonist. Under these conditions cholera toxin influenced the alpha subunit of Gs (Gs alpha) but had no effect on the alpha subunit of Gi/o (Gi/o alpha), based on ADP-ribosylation studies. Pertussis toxin treatment decreased high-affinity opioid agonist binding by about 50%; remaining binding was also of high affinity, even though pertussis toxin had inactivated Gi/o alpha selectively and essentially completely. Simultaneous treatment with both toxins had an additive effect, reducing specific binding by about 80%. While opioid agonists inhibited forskolin-stimulated adenylate cyclase activity of F-11 cells as expected, opioids also stimulated basal adenylate cyclase activity, indicative of interaction with Gs as well as Gi. Cholera toxin treatment attenuated opioid-stimulation of basal adenylate cyclase, whereas pertussis toxin treatment enhanced stimulation. In contrast, inhibition by opioid of forskolin-stimulated activity was attenuated by pertussis toxin but not by cholera toxin. It is concluded that a subset of opioid receptors may be linked directly to Gs and thereby mediate stimulation of adenylate cyclase. This Gs-adenylate cyclase interaction is postulated to be responsible for the novel excitatory electrophysiologic responses to opioids found in our previous studies of sensory neurons and F-11 cells.

Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Adenylyl Cyclases; Animals; Binding, Competitive; Cell Line; Cell Membrane; Cholera Toxin; Colforsin; Cricetinae; Cricetulus; Enkephalin, Leucine-2-Alanine; Ganglia, Spinal; GTP-Binding Proteins; Guanosine Triphosphate; Hybrid Cells; Kinetics; Levorphanol; Mice; Neuroblastoma; Neurons, Afferent; Pertussis Toxin; Receptors, Opioid; Sodium; Tumor Cells, Cultured; Virulence Factors, Bordetella

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

Mouse neuroblastoma cells in continuous culture, incubated for 1 to 2 days in the presence of 10--6 M levorphanol or morphine, were found to become tolerant to and dependent on those biologically active opiates. Examination of the interaction between levorphanol and the whole neuroblastoma cell suggested that levorphanol was binding to stereospecific opiate receptor sites. This binding was time and temperature dependent, and saturable at concentrations greater than 10--5 M levorphanol. Competition by other opiates for levorphanol sites correlated with their biological activity. This is the first evidence for saturable, stereospecific opiate binding in a homogeneous population of unhybridized cells in continuous culture.

Topics: Animals; Cell Division; Cells, Cultured; Hot Temperature; Levorphanol; Mice; Morphine; Neuroblastoma; Stereoisomerism; Time Factors

Topics: Animals; Cyclic AMP; Cyclic GMP; Dextrorphan; Dose-Response Relationship, Drug; Glioma; Hybrid Cells; Levorphanol; Mice; Morphine; Naloxone; Neuroblastoma; Neuroglia; Neurons; Rats; Receptors, Drug

Topics: Acetylcholinesterase; Animals; Carbon Isotopes; Cell Line; DNA, Neoplasm; Enzyme Induction; Levorphanol; Mice; Neoplasm Proteins; Neuroblastoma; RNA, Neoplasm; Thymidine; Time Factors; Tritium; Uridine