enkephalin--ala(2)-mephe(4)-gly(5)- and Neuroblastoma

Hyperalgesia often occurs in opioid-induced withdrawal syndrome. In the present study, we found that three hourly injections of DAMGO (a μ-opioid receptor agonist) followed by naloxone administration at the fourth hour significantly decreased rat paw nociceptive threshold, indicating the induction of withdrawal hyperalgesia. Application of NaHS (a hydrogen sulfide donor) together with each injection of DAMGO attenuated naloxone-precipitated withdrawal hyperalgesia. RT-PCR and Western blot analysis showed that NaHS significantly reversed the gene and protein expression of up-regulated spinal calcitonin gene-related peptide (CGRP) in naloxone-treated animals. NaHS also inhibited naloxone-induced cAMP rebound and cAMP response element-binding protein (CREB) phosphorylation in rat spinal cord. In SH-SY5Y neuronal cells, NaHS inhibited forskolin-stimulated cAMP production and adenylate cyclase (AC) activity. Moreover, NaHS pre-treatment suppressed naloxone-stimulated activation of protein kinase C (PKC) α, Raf-1, and extracellular signal-regulated kinase (ERK) 1/2 in rat spinal cord. Our data suggest that H2S prevents the development of opioid withdrawal-induced hyperalgesia via suppression of synthesis of CGRP in spine through inhibition of AC/cAMP and PKC/Raf-1/ERK pathways.

Topics: Adenylyl Cyclases; Analgesics, Opioid; Animals; Calcitonin Gene-Related Peptide; Cell Line, Tumor; CREB-Binding Protein; Down-Regulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Humans; Hydrogen Sulfide; Hyperalgesia; Male; MAP Kinase Signaling System; Naloxone; Neuroblastoma; Pain Threshold; Rats; Rats, Sprague-Dawley; Spine; Substance Withdrawal Syndrome

Neuropeptide FF (NPFF) interacts with specific receptors to modulate opioid functions in the central nervous system. On dissociated neurons and neuroblastoma cells (SH-SY5Y) transfected with NPFF receptors, NPFF acts as a functional antagonist of μ-opioid (MOP) receptors by attenuating the opioid-induced inhibition of calcium conductance. In the SH-SY5Y model, MOP and NPFF(2) receptors have been shown to heteromerize. To understand the molecular mechanism involved in the anti-opioid activity of NPFF, we have investigated the phosphorylation status of the MOP receptor using phospho-specific antibody and mass spectrometry. Similarly to direct opioid receptor stimulation, activation of the NPFF(2) receptor by [D-Tyr-1-(NMe)Phe-3]NPFF (1DMe), an analog of NPFF, induced the phosphorylation of Ser-377 of the human MOP receptor. This heterologous phosphorylation was unaffected by inhibition of second messenger-dependent kinases and, contrarily to homologous phosphorylation, was prevented by inactivation of G(i/o) proteins by pertussis toxin. Using siRNA knockdown we could demonstrate that 1DMe-induced Ser-377 cross-phosphorylation and MOP receptor loss of function were mediated by the G protein receptor kinase GRK2. In addition, mass spectrometric analysis revealed that the phosphorylation pattern of MOP receptors was qualitatively similar after treatment with the MOP agonist Tyr-D-Ala-Gly (NMe)-Phe-Gly-ol (DAMGO) or after treatment with the NPFF agonist 1DMe, but the level of multiple phosphorylation was more intense after DAMGO. Finally, NPFF(2) receptor activation was sufficient to recruit β-arrestin2 to the MOP receptor but not to induce its internalization. These data show that NPFF-induced heterologous desensitization of MOP receptor signaling is mediated by GRK2 and could involve transphosphorylation within the heteromeric receptor complex.

Topics: Amino Acid Sequence; Analgesics, Opioid; Arrestins; beta-Arrestins; Cell Line, Tumor; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; G-Protein-Coupled Receptor Kinase 2; Gene Knockdown Techniques; Heterotrimeric GTP-Binding Proteins; Humans; Molecular Sequence Data; Neuroblastoma; Phosphorylation; Receptors, G-Protein-Coupled; Receptors, Neuropeptide; Receptors, Opioid, mu; Second Messenger Systems; Serine; Signal Transduction

Organization of G protein-coupled receptors and cognate signaling partners at the plasma membrane has been proposed to occur via multiple mechanisms, including membrane microdomains, receptor oligomerization, and protein scaffolding. Here, we investigate the organization of six types of Gi/o-coupled receptors endogenously expressed in SH-SY5Y cells. The most abundant receptor in these cells was the μ-opioid receptor (MOR), the activation of which occluded acute inhibition of adenylyl cyclase (AC) by agonists to δ-opioid (DOR), nociceptin/orphanin FQ peptide (NOPr), α2-adrenergic (α2AR), cannabinoid 1, and serotonin 1A receptors. We further demonstrate that all receptor pairs share a common pool of AC. The MOR agonist [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) also occluded the ability of DOR agonist to stimulate G proteins. However, at lower agonist concentrations and at shorter incubation times when G proteins were not limiting, the relationship between MOR and DOR agonists was additive. The additive relationship was confirmed by isobolographic analysis. Long-term coadministration of MOR and DOR agonists caused cAMP overshoot that was not additive, suggesting that sensitization of AC mediated by these two receptors occurs by a common pathway. Furthermore, heterologous inhibition of AC by agonists to DOR, NOPr, and α2AR reduced the expression of cAMP overshoot in DAMGO-dependent cells. However, this cross-talk did not lead to heterologous tolerance. These results indicate that multiple receptors could be tethered into complexes with cognate signaling proteins and that access to shared AC by multiple receptor types may provide a means to prevent opioid withdrawal.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Analgesics, Opioid; Blotting, Western; Cell Line, Tumor; Cyclic AMP; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Protein alpha Subunits, Gi-Go; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Neuroblastoma; Radioligand Assay; Receptor, Cannabinoid, CB1; Receptor, Serotonin, 5-HT1A; Receptors, Opioid, delta; Receptors, Opioid, mu; Signal Transduction

Techniques for analyzing the membrane diffusion of molecules are the most promising methods for investigating the compartmentalization of G-protein-coupled receptors, particularly as relevant to receptor signaling processes. Here, we report fluorescence recovery after photobleaching (FRAP) measurements performed at variable spot radius for human mu opioid (hMOP) receptors on SH-SY5Y neuroblastoma cells in the presence of ligands. Although an antagonist did not affect the behavior of the receptors compared with the basal state, two different agonists, DAMGO and morphine, caused markedly different changes to receptor diffusion. Like receptors in the absence of ligand, receptors bound to morphine exhibited diffusion confined to joined semipermeable domains, but with smaller domain size and diffusion coefficient. This effect was inhibited by pertussis toxin, strongly suggesting that this dynamic behavior is associated with early steps of signaling. In the presence of DAMGO, half of the receptors displayed free long-range diffusion and the other half were confined to smaller isolated domains. Hypertonic sucrose buffer suppressed this effect, which we attribute to receptor entry into clathrin-coated pits. It is likely that the observation of distinct receptor dynamics in the presence of DAMGO and morphine involves the agonist-selective phosphorylation of the receptor.

Topics: Analgesics, Opioid; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Fluorescence Recovery After Photobleaching; Green Fluorescent Proteins; Humans; Morphine; Neuroblastoma; Pertussis Toxin; Phosphorylation; Receptors, Opioid, mu; Tumor Cells, Cultured

Prolonged (12h) exposure of SH-SY5Y neuroblastoma cells to the mu-opioid receptor (MOPr) agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) causes homologous desensitization as well as heterologous desensitization of the extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation induced by insulin-like growth factor (IGF)-I. Brief (15 min) but not prolonged exposure to DAMGO transregulates the insulin-like growth factor-I (IGF-I) receptor, as evidenced by its phosphorylation in the absence of IGF-I. Silencing of beta-arrestin 2 uncouples the crosstalk between the two receptors, thus maintaining IGF-I-mediated receptor phosphorylation and ERK 1/2 activation even after prolonged DAMGO exposure. Furthermore, MOPr-induced activation of IGF-I receptor requires the tyrosine kinase c-Src.

Topics: Arrestins; beta-Arrestin 2; beta-Arrestins; Cell Line, Tumor; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neuroblastoma; Phosphorylation; Receptor Cross-Talk; Receptor, IGF Type 1; Receptors, Opioid, mu; RNA, Small Interfering; Signal Transduction; Transcriptional Activation

mu-Opioid receptors (MORs) are G-protein-coupled receptors (GPCRs) that mediate the physiological effects of endogenous opioid neuropeptides and opiate drugs such as morphine. MORs are coexpressed with neurokinin 1 receptors (NK1Rs) in several regions of the CNS that control opioid dependence and reward. NK1R activation affects opioid reward specifically, however, and the cellular basis for this specificity is unknown. We found that ligand-induced activation of NK1Rs produces a cell-autonomous and nonreciprocal inhibition of MOR endocytosis induced by diverse opioids. Studies using epitope-tagged receptors expressed in cultured striatal neurons and a neuroblastoma cell model indicated that this heterologous regulation is mediated by NK1R-dependent sequestration of arrestins on endosome membranes. First, endocytic inhibition mediated by wild-type NK1Rs was overcome in cells overexpressing beta-arrestin2, a major arrestin isoform expressed in striatum. Second, NK1R activation promoted sequestration of beta-arrestin2 on endosomes, whereas MOR activation did not. Third, heterologous inhibition of MOR endocytosis was prevented by mutational disruption of beta-arrestin2 sequestration by NK1Rs. NK1R-mediated regulation of MOR trafficking was associated with reduced opioid-induced desensitization of adenylyl cyclase signaling in striatal neurons. Furthermore, heterologous regulation of MOR trafficking was observed in both amygdala and locus ceruleus neurons that naturally coexpress these receptors. These results identify a cell-autonomous mechanism that may underlie the highly specific effects of NK1R on opioid signaling and suggest, more generally, that receptor-specific trafficking of arrestins may represent a fundamental mechanism for coordinating distinct GPCR-mediated signals at the level of individual CNS neurons.

Topics: Analgesics, Opioid; Animals; Biotinylation; Cell Line, Tumor; Corpus Striatum; Cyclic AMP; Dose-Response Relationship, Drug; Embryo, Mammalian; Endocytosis; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Methionine; Female; Locus Coeruleus; Mice; Morphine; Mutation; Neuroblastoma; Neurons; Pregnancy; Rats; Rats, Sprague-Dawley; Receptors, Neurokinin-1; Receptors, Opioid, mu; Substance P; Transfection

Regulator of G-protein signaling (RGS) proteins are a family of molecules that control the duration of G protein signaling. A variety of RGS proteins have been reported to modulate opioid receptor signaling. Here we show that RGS4 is abundantly expressed in human neuroblastoma SH-SY5Y cells that endogenously express mu- and delta-opioid receptors and test the hypothesis that the activity of opioids in these cells is modulated by RGS4. Endogenous RGS4 protein was reduced by approximately 90% in SH-SY5Y cells stably expressing short hairpin RNA specifically targeted to RGS4. In these cells, the potency and maximal effect of delta-opioid receptor agonist (SNC80)-mediated inhibition of forskolin-stimulated cAMP accumulation was increased compared with control cells. This effect was reversed by transient transfection of a stable RGS4 mutant (HA-RGS4C2S). Furthermore, MAPK activation by SNC80 was increased in cells with knockdown of RGS4. In contrast, there was no change in the mu-opioid (morphine) response at adenylyl cyclase or MAPK. FLAG-tagged opioid receptors and HA-RGS4C2S were transiently expressed in HEK293T cells, and co-immunoprecipitation experiments showed that the delta-opioid receptor but not the mu-opioid receptor could be precipitated together with the stable RGS4. Using chimeras of the delta- and mu-opioid receptors, the C-tail and third intracellular domain of the delta-opioid receptor were suggested to be the sites of interaction with RGS4. The findings demonstrate a role for endogenous RGS4 protein in modulating delta-opioid receptor signaling in SH-SY5Y cells and provide evidence for a receptor-specific effect of RGS4.

Topics: Analgesics, Opioid; Animals; Benzamides; Cell Line, Tumor; Colforsin; Cyclic AMP; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Glioma; Glycosylation; Humans; Kidney; Mice; Mitogen-Activated Protein Kinases; Morphine; Neuroblastoma; Piperazines; Rats; Receptors, Opioid, delta; Receptors, Opioid, mu; Recombinant Fusion Proteins; RGS Proteins; RNA Interference; Signal Transduction; Transfection

In the present study we investigated the signal transduction pathways leading to the activation of extracellular signal-regulated kinase (ERK) by opioid or cannabinoid drugs, when their receptors are coexpressed in the same cell-type. In N18TG2 neuroblastoma cells, the opioid agonist etorphine and the cannabinoid agonist CP-55940 induced the phosphorylation of ERK by a similar mechanism that involved activation of delta-opioid receptors or CB1 cannabinoid receptors coupled to Gi/Go proteins, matrix metalloproteases, vascular endothelial growth factor (VEGF) receptors and MAPK/ERK kinase (MEK). In HEK-293 cells, these two drugs induced the phosphorylation of ERK by separate mechanisms. While CP-55940 activated ERK by transactivation of VEGFRs, similar to its effect in N18TG2 cells, the opioid agonist etorphine activated ERK by a mechanism that did not involve transactivation of a receptor tyrosine kinase. Interestingly, the activation of ERK by etorphine was resistant to the inhibition of MEK, suggesting the possible existence of a novel, undescribed yet mechanism for the activation of ERK by opioids. This mechanism was found to be specific to etorphine, as activation of ERK by the micro-opioid receptor (MOR) agonist DAMGO ([D-Ala(2), N-Me-Phe(4), Gly(5)-ol] enkephalin) was mediated by MEK in these cells, suggesting that etorphine and DAMGO activate distinct, ligand-specific, conformations of MOR. The characterization of cannabinoid- and opioid-induced ERK activation in these two cell-lines enables future studies into possible interactions between these two groups of drugs at the level of MAPK signaling.

Topics: Analgesics; Analgesics, Opioid; Animals; Cell Line; Cell Line, Tumor; Central Nervous System; Cyclohexanols; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Activation; Etorphine; Extracellular Signal-Regulated MAP Kinases; Humans; MAP Kinase Kinase 1; Mice; Neuroblastoma; Neurons; Rats; Receptor, Cannabinoid, CB1; Receptors, Cannabinoid; Receptors, G-Protein-Coupled; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Vascular Endothelial Growth Factor Receptor-1

Activation of the NPFF(2) receptor reduces the inhibitory effect of opioids on the N-type Ca(2+) channel. Although this anti-opioid effect is specific for opioid receptors in neurons and tissues, it also affects NPY Y2 and alpha(2)-adrenoreceptors in undifferentiated SH-SY5Y cells stably expressing the NPFF(2) receptor. To test whether this difference could be due to the immaturity of these cells, they were differentiated to a noradrenergic neuronal phenotype with staurosporine. The differentiated cells ceased to divide and grew long, thin neurites. The inhibition of the depolarization-triggered Ca(2+) transient by activation of G(i)-coupled receptors was either unaffected (micro-opioid), increased (NPY), reduced (NPFF(2)) or lost (alpha(2)-adrenoreceptors). Following a 20 min incubation with 1DMe, the effect of DAMGO was reduced, as in undifferentiated cells, but the effect of NPY was no longer affected. Staurosporine differentiation did not modify the coupling of the micro-opioid and NPFF(2) receptors to the G(i/o) proteins. We suggest that the specificity of the effect of NPFF may not reside in the molecular mechanism of its anti-opioid activity itself but in the organization of receptors within the membrane.

Topics: Calcium; Cell Differentiation; Cell Line, Tumor; Cell Membrane; Clonidine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Humans; Neuroblastoma; Neuropeptide Y; Oligopeptides; Receptors, Neuropeptide; Receptors, Opioid; Staurosporine

(1) This study investigated the functional genomics of glucocorticoid and opioid receptor stimulation in cellular adaptations using a cultured neuronal cell model. (2) Human SH-SY5Y neuroblastoma cells grown in hormone-depleted serum were treated for 2-days with the glucocorticoid receptor-II agonist dexamethasone (30 nM); the mu-opioid receptor agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate (DAMGO; 1 nM); or dexamethasone (30 nM) plus DAMGO (1 nM). RNA was extracted; purified, reverse transcribed, and labeled cDNA was hybridized to a 10,000-oliogonucleotide-array human gene chip. Gene expression changes that were significantly different between treatment groups and were of interest due to biological function were verified by real-time reverse transcription polymerase chain reaction (RT-PCR). Five relevant genes were identified for which the combination of dexamethasone plus DAMGO, but neither one alone, significantly up-regulated gene expression (ANOVA, P < 0.05). (3) Proteins coded by the identified genes: FRS2 (fibroblast growth factor receptor substrate-2; CTNNB1 (beta1-catenin); PRCP (prolyl-carboxypeptidase); MPHOSPH9 (M-phase phosphoprotein 9); and ZFP95 (zinc finger protein 95) serve important neuronal functions in signal transduction, synapse formation, neuronal growth and development, or transcription regulation. Neither opioid, glucocorticoid nor combined treatments significantly altered the cell growth rate determined by cell counts and protein. (4) We conclude that sustained mu-opioid receptor stimulation accompanied by glucocorticoids can synergistically regulate genes that influence neuronal function. Future studies are warranted to determine if combined influences of glucocorticoid fluctuations and opioid receptor stimulation in vivo can orchestrate exagerated neuroadaptation to reinforcing drugs under chronic mild stress conditions.

Topics: Analgesics, Opioid; Cell Division; Cell Line, Tumor; Dexamethasone; Drug Synergism; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Gene Expression Regulation; Genomics; Glucocorticoids; Humans; Neuroblastoma; Neuronal Plasticity; Neurons; Oligonucleotide Array Sequence Analysis; Receptors, Opioid, mu; Reverse Transcriptase Polymerase Chain Reaction; Up-Regulation

In order to elucidate the mechanisms of the neuronal anti-opioid activity of Neuropeptide FF, we have transfected the SH-SY5Y neuroblastoma cell line, which expresses mu- and delta-opioid receptors, with the human NPFF1 receptor. The SH1-C7 clone expresses high affinity NPFF1 receptors in the same range order of density as opioid receptors. Similarly to the opioids, acute stimulation with the NPFF1 agonist NPVF inhibits adenylyl cyclase activity and voltage-gated (N-type) Ca2+ currents and enhances the intracellular Ca2+ release triggered by muscarinic receptors activation. In contrast, preincubation of cells with NPVF decreases the response to opioids on both calcium signaling, thus reproducing the cellular anti-opioid activity described in neurons. SH1-C7 cells are therefore a suitable model to investigate the interactions between NPFF and opioid receptors.

Topics: Calcium Channels, N-Type; Carbachol; Cell Line, Tumor; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Humans; Narcotic Antagonists; Neuroblastoma; Neuropeptides; Oligopeptides; Pertussis Toxin; Receptors, Muscarinic; Receptors, Neuropeptide; Receptors, Opioid, mu; Signal Transduction; Transfection

Oxidative stress contributes to changes in neurosensory processing, including pain, that occur during aging and neurodegeneration. The effects of neuronal oxidation on the opioid system are poorly understood. In this in vitro study, oxidative stress was induced by 3-nitroproprionic acid (3-NPA) in opioid-responsive differentiated SK-N-SH cells. Changes in the inhibitory effects of opioid receptor agonists on intracellular cAMP were used as a marker of the function of mu and delta opioid receptors (MOR and DOR, respectively). Cells were treated with morphine and selective MOR and DOR agonists and antagonists to characterize the function of each receptor subtype. Cyclic AMP (cAMP) was measured by enzyme immunoassay. Levels of reactive oxygen species (ROS) were assessed using the 2',7'-dichlorofluorescin diacetate assay. Exposure of cells to 3-NPA resulted in an increase in ROS. After 3-NPA exposure, there was a significant attenuation of the inhibitory effect of morphine and DAMGO but not of DPDPE on cAMP. In cells pretreated with CTOP, 3-NPA did not change the inhibitory effect on cAMP. These findings demonstrate for the first time that under conditions of mitochondrial damage, the function of MOR is significantly decreased, while the function of DOR does not change, suggesting that the effect of 3-NPA on opioid receptors is subtype-specific.

Topics: Cell Line, Tumor; Energy Metabolism; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Humans; Mitochondria; Neuroblastoma; Neurons; Nitro Compounds; Propionates; Reactive Oxygen Species; Receptors, Opioid, delta; Receptors, Opioid, mu

Opioid receptors are involved in regulating neuronal survival. Here we demonstrate that activation of the mu-opioid receptor in human neuroblastoma SH-SY5Y cells led to the phosphorylations of IkappaB kinase (IKK) and p65, denoting the stimulation of the nuclear factor-kappaB (NFkappaB) transcription factor. This response was mediated through pertussis toxin-sensitive G proteins. The mu-opioid-induced IKK phosphorylation required extracellular signal-regulated protein kinase, phosphatidylinositol 3-kinase and c-Src. Moreover, c-Jun N-terminal kinase and calmodulin-dependent kinase II also participated in the IKK activation, despite the lack of involvement of phospholipase Cbeta and protein kinase C. These data suggest that the mu-opioid receptor is capable of simulating NFkappaB signaling via the phosphorylation of IKK and p65 in human neuroblastoma SH-SY5Y cells.

Topics: Analgesics, Opioid; Analysis of Variance; Blotting, Western; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Interactions; eIF-2 Kinase; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Inhibitors; Humans; Mitogen-Activated Protein Kinases; Naloxone; Narcotic Antagonists; Neuroblastoma; Pertussis Toxin; Phosphorylation; Receptors, Opioid, mu; Somatostatin; Time Factors

Activity-regulated cytoskeleton-associated protein (Arc) is an effector immediate early gene product implicated in long-term potentiation and other forms of neuroplasticity. Earlier studies demonstrated Arc induction in discrete brain regions by several psychoactive substances, including drugs of abuse. In the present experiments, the influence of morphine on Arc expression was assessed by quantitative reverse transcription real-time PCR and Western blotting in vivo in the mouse striatum/nucleus accumbens and, in vitro, in the mouse Neuro2A MOR1A cell line, expressing mu-opioid receptor. An acute administration of morphine produced a marked increase in Arc mRNA and protein level in the mouse striatum/nucleus accumbens complex. After prolonged opiate treatment, tolerance to the stimulatory effect of morphine on Arc expression developed. No changes in the striatal Arc mRNA levels were observed during spontaneous or opioid antagonist-precipitated morphine withdrawal. In Neuro2A MOR1A cells, acute, but not prolonged, morphine treatment elevated Arc mRNA level by activation of mu-opioid receptor. This was accompanied by a corresponding increase in Arc protein level. Inhibition experiments revealed that morphine induced Arc expression in Neuro2A MOR1A cells via intracellular signaling pathways involving mitogen-activated protein (MAP) kinases and protein kinase C. These results lend further support to the notion that stimulation of opioid receptors may exert an activating influence on some intracellular pathways and leads to induction of immediate early genes. They also demonstrate that Arc is induced in the brain in vivo after morphine administration and thus may play a role in neuroadaptations produced by the drug.

Topics: AIDS-Related Complex; Analysis of Variance; Animals; Blotting, Western; Cell Line, Tumor; Corpus Striatum; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Interactions; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Inhibitors; Flavonoids; Gene Expression; Gene Expression Regulation; Indoles; Male; Maleimides; Mice; Mice, Inbred C57BL; Morphine; Narcotics; Neuroblastoma; Receptors, Opioid, mu; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Somatostatin; Time Factors

Cultured cells from the rat brainstem were used to study opioid receptor (OpR) expression during late fetal and early postnatal development. Mu and delta opioid receptor (MOR and DOR) expression was investigated from embryonic day 16 (E16) to 6 days postnatal (P6). Postnatal neurons showed more intense MOR immunoreactivity (IR) than neurons cultured from fetal brainstem (P < 0.006). DOR IR showed a similar pattern, but the differences between fetal and neonatal animals were not statistically significant. Using confocal microscopy, MOR and DOR IR were shown to be present on both the cell membrane and within the cytoplasm, in a similar pattern to the IR seen in SH-SY5Y neuroblastoma cells that endogenously express both MOR and DOR. Double-labeling experiments demonstrated colocalization of MOR and DOR in the same brainstem neurons; however, not all MOR IR regions of a single neuron were also positively stained for DOR, and not all DOR IR regions were also positive for MOR. MOR was down-regulated after a 1- or 2-h treatment with 1 microM DAMGO, a potent mu opioid agonist, in both non-transfected and MOR-transfected SH-SY5Y cells and in primary cell cultures. It was concluded that many brainstem neurons express functional MOR or DOR or coexpress both receptors, although intracellular distributions of the receptors are unique for each receptor type.

Topics: Analgesics, Opioid; Animals; Animals, Newborn; Astrocytes; Blotting, Western; Brain Stem; Cell Count; Cells, Cultured; Cloning, Molecular; Embryo, Mammalian; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Gene Expression Regulation, Developmental; Humans; Immunohistochemistry; Kidney; Microtubule-Associated Proteins; Neuroblastoma; Neurons; Rats; Receptors, Opioid, delta; Receptors, Opioid, mu; Swine; Time Factors; Transfection

RGS proteins are a recently described class of regulators that influence G-protein-mediated signaling pathways. We have shown previously that chronic morphine results in functional uncoupling of the mu opioid receptor from its G protein in CHO cells expressing cloned human mu opioid receptors. In the present study, we examined the effects of morphine treatment (1 microM, 20 h) on DAMGO-stimulated high-affinity [35S]GTP-gamma-S binding and DAMGO-mediated inhibition of forskolin-stimulated cAMP accumulation in HN9.10 cells stably expressing the cloned rat mu opioid receptor, in the absence and presence of the RGS9 protein knock-down condition (confirmed by Western blot analysis). RGS9 protein expression was reduced by blocking its mRNA with an antisense oligodeoxynucleotide (AS-114). Binding surface analysis resolved two [35S]GTP-gamma-S binding sites (high affinity and low affinity sites). In sense-treated control cells, DAMGO-stimulated [35S]GTP-gamma-S binding by increasing the B(max) of the high-affinity site. In sense-treated morphine-treated cells, DAMGO-stimulated [35S]GTP-gamma-S binding by decreasing the high-affinity Kd without changing the B(max). AS-114 significantly inhibited chronic morphine-induced upregulation of adenylate cyclase activity and partially reversed chronic morphine effects as measured by DAMGO-stimulated [35S]GTP-gamma-S binding. Morphine treatment increased the EC50 (6.2-fold) for DAMGO-mediated inhibition of forskolin-stimulated cAMP activity in control cells but not in cells treated with AS-114 to knock-down RGS9. These results provide additional evidence for involvement of RGS9 protein in modulating opioid signaling, which may contribute to the development of morphine tolerance and dependence.

Topics: Adenylyl Cyclases; Analgesics, Opioid; Animals; Blotting, Western; Brain Neoplasms; Cell Line, Tumor; Cell Membrane; Cloning, Molecular; Colforsin; Cyclic AMP; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Guanosine 5'-O-(3-Thiotriphosphate); Mice; Morphine; Neuroblastoma; Oligonucleotides, Antisense; Receptors, Opioid, mu; RGS Proteins; Signal Transduction

Addiction to opiates depend on drug-induced neuroplastic changes and are underlain by alterations of gene expression. Transcription factors Ca2+/cAMP responsive element binding protein (CREB) and activator protein 1 (AP-1) may constitute a direct link between the opioid-regulated signal transduction pathways and modulation of gene expression. Acute treatment of Neuro2a MOR neuroblastoma cells with opioids stimulated CREB activity; prolonged treatment normalized it, while withdrawal from the drug again elicited an increase in phosphorylated CREB levels. Protein kinase C was responsible for the activation of transcription following acute opioid administration whereas the cAMP pathway activated similar mechanisms during withdrawal, making CREB a kind of 'a trigger' reacting to the presence or withdrawal of the opioid signal. Apart from the elevated CREB phosphorylation, CRE binding activity and expression of luciferase reporter gene regulated by CRE elements were increased after single administration and during withdrawal from the prolonged opioid treatment. Along with CREB, AP-1 binding activity and AP-1-directed transcription were stimulated after single administration and during withdrawal from the opioid. These results provide evidence that both single opioid administration and opioid withdrawal activate CREB and CRE-dependent transcriptional mechanisms via distinct intracellular signaling pathways.

Topics: Analgesics, Opioid; Animals; Cell Line; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; DNA; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Gene Expression; Genes, Reporter; Mice; Morphine; Neuroblastoma; Phosphorylation; Protein Kinase C; Receptors, Opioid, mu; Transcription Factor AP-1

The human neuroblastoma cell line, SH-SY5Y, was used to examine the effects of morphine and the endogenous opioid peptides, endomorphin-1 (EM-1) and endomorphin-2 (EM-2), on mu opioid receptor (MOR) internalization and down-regulation. Treatment for 24 h with EM-1, EM-2 or morphine at 100 nM, 1 microM and 10 microM resulted in a dose-dependent down-regulation of mu receptors. Exposure of cells to 10 microM EM-1 for 2.5, 5 and 24 h resulted in a time-dependent down-regulation of mu receptors. Down-regulation of mu receptors by morphine and EM-1 was blocked by treatment with hypertonic sucrose, consistent with an endocytosis-dependent mechanism. Sensitive cell-surface binding studies with a radiolabeled mu antagonist revealed that morphine was able to induce internalization of mu receptors naturally expressed in SH-SY5Y cells. EM-1 produced a more rapid internalization of mu receptors than morphine, but hypertonic sucrose blocked the internalization induced by each of these agonists. This study demonstrates that, like morphine, the endomorphins down-regulate mu opioid receptors in a dose- and time-dependent manner. This study also demonstrates that morphine, as well as EM-1, can induce rapid, endocytosis-dependent internalization of mu opioid receptors in SH-SY5Y cells. These results may help elucidate the ability of mu agonists to regulate the number and responsiveness of their receptors.

Topics: Analgesics, Opioid; Binding Sites; Cell Differentiation; Cell Line, Tumor; Cell Membrane; Dose-Response Relationship, Drug; Down-Regulation; Drug Interactions; Endocytosis; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Humans; Hypotonic Solutions; Morphine; Narcotic Antagonists; Neuroblastoma; Oligopeptides; Peptides; Radioligand Assay; Receptors, Opioid, mu; Sucrose; Time Factors; Tritium

The differential ability of various mu-opioid receptor (MOP) agonists to induce rapid receptor desensitization and endocytosis of MOP could arise simply from differences in their efficacy to activate G proteins or, alternatively, be due to differential capacity for activation of other signaling processes. We used AtT20 cells stably expressing a low density of FLAG-tagged MOP to compare the efficacies of a range of agonists to 1) activate G proteins using inhibition of calcium channel currents (ICa) as a reporter before and after inactivation of a fraction of receptors by beta-chlornaltrexamine, 2) produce rapid, homologous desensitization of ICa inhibition, and 3) internalize receptors. Relative efficacies determined for G protein coupling were [Tyr-D-Ala-Gly-MePhe-Glyol]enkephalin (DAMGO) (1) > or = methadone (0.98) > morphine (0.58) > pentazocine (0.15). The same rank order of efficacies for rapid desensitization of MOP was observed, but greater concentrations of agonist were required than for G protein activation. By contrast, relative efficacies for promoting endocytosis of MOP were DAMGO (1) > methadone (0.59) >> morphine (0.07) > or = pentazocine (0.03). These results indicate that the efficacy of opioids to produce activation of G proteins and rapid desensitization is distinct from their capacity to internalize mu-opioid receptors but that, contrary to some previous reports, morphine can produce rapid, homologous desensitization of MOP.

Topics: Animals; Calcium Channel Blockers; Calcium Channels; Electric Conductivity; Endocytosis; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Proteins; Methadone; Mice; Morphine; Naltrexone; Narcotic Antagonists; Narcotics; Neuroblastoma; Pentazocine; Pituitary Neoplasms; Receptors, Opioid, mu; Transfection; Tumor Cells, Cultured

Morphine tolerance in vivo is reduced following blockade of the orphanin FQ/nociceptin (OFQ/N)/opioid receptor-like 1 (ORL1) receptor system, suggesting that OFQ/N contributes to the development of morphine tolerance. We previously reported that a 60-min activation of ORL1 receptors natively expressed in BE(2)-C cells desensitized both mu and ORL1 receptor-mediated inhibition of cAMP. Investigating the mechanism(s) of OFQ/N-mediated mu and ORL1 receptor cross-talk, we found that pretreatment with the protein kinase C inhibitor, chelerythrine chloride (1 microM), blocked OFQ/N-mediated homologous desensitization of ORL1 and heterologous desensitization of mu opioid receptors. Furthermore, depletion of PKC by 12-O-tetradecanoylphorbol-13-acetate exposure (48 h, 1 microM) also prevented OFQ/N-mediated mu and ORL1 desensitization. OFQ/N pretreatment resulted in translocation of PKC-alpha, G protein-coupled receptor kinase 2 (GRK2) and GRK3 from the cytosol to the membrane, and this translocation was also blocked by chelerythrine. Reduction of GRK2 and GRK3 levels by antisense, but not sense DNA treatment blocks ORL1 and mu receptor desensitization. This suggests that PKC-alpha is required for GRK2 and GRK3 translocation to the membrane, where GRK can inactivate ORL1 and mu opioid receptors upon rechallenge with the appropriate agonist. Our results demonstrate for the first time the involvement of conventional PKC isozymes in OFQ/N-induced mu-ORL1 cross-talk, and represent a possible mechanism for OFQ/N-induced anti-opioid actions.

Topics: Alkaloids; Benzophenanthridines; beta-Adrenergic Receptor Kinases; Cell Membrane; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Inhibitors; G-Protein-Coupled Receptor Kinase 3; Humans; Neuroblastoma; Nociceptin; Nociceptin Receptor; Oligodeoxyribonucleotides, Antisense; Opioid Peptides; Phenanthridines; Protein Kinase C; Protein Serine-Threonine Kinases; Receptor Cross-Talk; Receptors, Opioid; Receptors, Opioid, mu; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

This study investigated the turnover of mu-opioid receptors (MOR) in neuroblastoma (N2A) cells under basal and agonist-stimulated opioid receptor down-regulation. Cells were labeled with [35S]methionine for 24 h and MOR degradation was quantified by immunoprecipitation using monoclonal anti (MOR) antibody followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. Treatment of N2A cells with the selective mu-opioid ligand (DAMGO) increased the rate of MOR degradation. The radiolabeled immunoprecipitable receptor was lost from the cells with a half-life (t(1/2)) of 12 and 7 h in the absence and presence of DAMGO, respectively. On the other hand, the protein synthesis inhibitor cycloheximide (10 microg/ml) produced a decrease in the rate of receptor degradation, t(1/2)=22 h indicated that the rate of MOR turnover was attenuated almost 2-fold following the inhibition of protein synthesis. Furthermore, when N2A cells were exposed to a combination of DAMGO and cycloheximide, the t(1/2) was 9.7 h. These data provided the first evidence that MOR is down-regulated during agonist stimulation and that the turnover rate of MOR is sum of both accelerated receptor degradation and decreased receptor biosynthesis.

Topics: Analgesics, Opioid; Animals; Cycloheximide; Down-Regulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Methionine; Mice; Neuroblastoma; Neurons; Protein Synthesis Inhibitors; Receptors, Opioid, mu; Sulfur Radioisotopes; Tumor Cells, Cultured

The molecular mechanism(s) underlying cross-tolerance between mu and opioid receptor-like 1 (ORL1) receptor agonists were investigated using two human neuroblastoma cell lines endogenously expressing these receptors and G protein-coupled receptor kinases (GRKs). Prolonged (24 h) activation of the mu receptor desensitized both mu and ORL1 receptor-mediated inhibition of forskolin-stimulated cAMP accumulation and upregulated GRK2 levels in SH-SY5Y and BE(2)-C cells. Prolonged ORL1 activation increased GRK2 levels and desensitized both receptors in SH-SY5Y cells. Upregulation of GRK2 correlated with increases in levels of transcription factors Sp1 or AP-2. PD98059, an upstream inhibitor of extracellular signal-regulated kinases 1 and 2 (ERK1/2), reversed all these events. Pretreatment with orphanin FQ/nociceptin (OFQ/N) also upregulated GRK3 levels in both cell lines, and desensitized both receptors in BE(2)-C cells. Protein kinase C (PKC), but not ERK1/2, inhibition blocked OFQ/N-mediated GRK3 induction and mu and ORL1 receptor desensitization in BE(2)-C cells. Antisense DNA treatment confirmed the involvement of GRK2/3 in mu and ORL1 desensitization. Here, we demonstrate for the first time a role for ERK1/2-mediated GRK2 induction in the development of tolerance to mu agonists, as well as cross-tolerance to OFQ/N. We also demonstrate that chronic OFQ/N-mediated desensitization of ORL1 and mu receptors occurs via cell-specific pathways, involving ERK1/2-dependent GRK2, or PKC-dependent and ERK1/2-independent GRK3 induction.

Topics: Analysis of Variance; beta-Adrenergic Receptor Kinases; Cell Membrane; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Drug Interactions; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Inhibitors; Flavonoids; G-Protein-Coupled Receptor Kinase 3; Humans; Mitogen-Activated Protein Kinases; Morphine; Neuroblastoma; Nociceptin; Nociceptin Receptor; Oligodeoxyribonucleotides, Antisense; Opioid Peptides; Protein Serine-Threonine Kinases; Receptor Cross-Talk; Receptors, Opioid; Receptors, Opioid, mu; Transcription Factors; Tumor Cells, Cultured

1. There is evidence for interactions between mu and delta opioid systems both in vitro and in vivo. This work examines the hypothesis that interaction between these two receptors can occur intracellularly at the level of G protein in human neuroblastoma SH-SY5Y cells. 2. The [(35)S]GTP gamma S binding assay was used to measure G protein activation following agonist occupation of opioid receptors. The agonists DAMGO (EC(50), 45 nM) and SNC80 (EC(50), 32 nM) were found to be completely selective for stimulation of [(35)S]-GTP gamma S binding through mu and delta opioid receptors respectively. Maximal stimulation of [(35)S]-GTP gamma S binding produced by SNC80 was 57% of that seen with DAMGO. When combined with a maximally effective concentration of DAMGO, SNC80 caused no additional [(35)S]-GTP gamma S binding. This effect was also seen when measured at the level of adenylyl cyclase. 3. Receptor activation increased the dissociation of pre-bound [(35)S]-GTP gamma S. In addition, the delta agonist SNC80 promoted the dissociation of [(35)S]-GTP gamma S from G proteins initially labelled using the mu agonist DAMGO. Conversely, DAMGO promoted the dissociation of [(35)S]-GTP gamma S from G proteins initially labelled using SNC80. 4. Tolerance to DAMGO and SNC80 in membranes from cells exposed to agonist for 18 h was homologous and there was no evidence for alteration in G protein activity. 5. The findings support the hypothesis that mu- and delta-opioid receptors share a common G protein pool, possibly through a close organization of the two receptors and G protein at the plasma membrane.

Topics: Adenylyl Cyclases; Analgesics, Opioid; Benzamides; Cyclic AMP; Dose-Response Relationship, Drug; Drug Interactions; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Ligands; Neuroblastoma; Piperazines; Receptors, Opioid, delta; Receptors, Opioid, mu; Sulfur Radioisotopes; Tumor Cells, Cultured

Recent studies have demonstrated the analgesic synergy between mu- and delta-opioid receptor, but evidence obtained at the cellular level is scanty. This work was designed to find the evidence of synergy between the actions of D-Ala2-Mephe4-glyol5 enkephalin (DAMGO) and D-Phe2, D-Phe5 enkephalin (DPDPE) on undifferentiated SH-SY5Y cells. Microphysiometer was used to detect the functional activity of cells by measuring the real-time extracellular acidification rate (ECAR). The results demonstrate the unequivocal synergy between DAMGO and DPDPE at least within certain ratios. In addition, combined administration of the two drugs in the synergistic ratios attenuates receptor desensitization. These data indicate that DAMGO and DPDPE have a synergistic effect at cellular level.

Topics: Analgesics, Opioid; Animals; Cell Differentiation; CHO Cells; Cricetinae; Dose-Response Relationship, Drug; Drug Synergism; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Humans; Neuroblastoma; Neurons; Receptors, Opioid, delta; Receptors, Opioid, mu; Time Factors; Tumor Cells, Cultured

Human SK-N-SH neuroblastoma cells expressed sigma-1 and sigma-2 receptors with similar pharmacological profiles to those of rodent-derived tissues, although sigma-2 receptors exhibited some affinity differences that might suggest heterogeneity or species differences. Structurally diverse sigma ligands produced two types of increases in intracellular (cytosolic) Ca(2+) concentration ([Ca(2+)](i)) in these cells. CB-64D, CB-64L, JL-II-147, BD737, LR172, BD1008, haloperidol, reduced haloperidol, and ibogaine all produced an immediate, dose-dependent, and transient rise in [Ca(2+)](i). Sigma-inactive compounds structurally similar to the most active sigma ligands and ligands for several neurotransmitter receptors produced little or no effect. The high activity of CB-64D and ibogaine (sigma-2-selective ligands) compared with the low activity of (+)-pentazocine and other (+)-benzomorphans (sigma-1-selective ligands), in addition to enantioselectivity for CB-64D over CB-64L, strongly indicated mediation by sigma-2 receptors. The effect of CB-64D and BD737 was blocked by the sigma antagonists BD1047 and BD1063, further confirming specificity as a receptor-mediated event. The transient rise in [Ca(2+)](i) occurred in the absence of extracellular Ca(2+) and was completely eliminated by pretreatment of cells with thapsigargin. Thus, sigma-2 receptors stimulate a transient release of Ca(2+) from the endoplasmic reticulum. Prolonged exposure of cells to sigma-receptor ligands resulted in a latent and sustained rise in [Ca(2+)](i), with a pharmacological profile identical to that of the transient rise. This sustained rise in [Ca(2+)](i) was affected by neither the removal of extracellular Ca(2+) nor thapsigargin pretreatment, suggesting latent sigma-2 receptor-induced release from thapsigargin-insensitive intracellular Ca(2+) stores. Sigma-2 receptors may use Ca(2+) signals in producing cellular effects.

Topics: Calcium; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Humans; Inositol 1,4,5-Trisphosphate; Morphine; Neuroblastoma; Receptors, sigma; Sigma-1 Receptor; Tumor Cells, Cultured

1. Opioid (mu, delta, kappa) and cannabinoid (CB1, CB2) receptors are coupled mainly to Gi/Go GTP-binding proteins. The goal of the present study was to determine whether different subtypes of opioid and cannabinoid receptors, when coexpressed in the same cell, share a common reservoir, or utilize different pools, of G proteins. 2. The stimulation of [35S]GTPgammaS binding by selective opioid and cannabinoid agonists was tested in transiently transfected COS-7 cells, as well as in neuroblastoma cell lines. In COS-7 cells, cotransfection of mu- and delta-opioid receptors led to stimulation of [35S]GTPgammaS binding by either mu-selective (DAMGO) or delta-selective (DPDPE) agonists. The combined effect of the two agonists was similar to the effect of either DAMGO or DPDPE alone, suggesting the activation of a common G-protein reservoir by the two receptor subtypes. 3. The same phenomenon was observed when COS-7 cells were cotransfected with CB1 cannabinoid receptors and either mu- or delta-opioid receptors. 4. On the other hand, in N18TG2 neuroblastoma cells, which endogenously coexpress CB1 and delta-opioid receptors, as well as in SK-N-SH neuroblastoma cells, which coexpress mu- and delta-opioid receptors, the combined effects of the various agonists (the selective cannabinoid DALN and the selective opioids DPDPE and DAMGO) were additive, implying the activation of different pools of G proteins by each receptor subtype. 5. These results suggest a fundamental difference between native and artificially transfected cells regarding the compartmentalization of receptors and GTP-binding proteins.

Topics: Analgesics; Analgesics, Opioid; Animals; COS Cells; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Etorphine; Gene Expression; GTP-Binding Protein alpha Subunits, Gi-Go; Guanosine 5'-O-(3-Thiotriphosphate); Heterotrimeric GTP-Binding Proteins; Neuroblastoma; Phenanthridines; Radioligand Assay; Receptors, Cannabinoid; Receptors, Drug; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Signal Transduction; Sulfur Radioisotopes; Transfection; Tumor Cells, Cultured

Cross-linking experiments using the (125)I-beta-endorphin revealed the presence of several receptor-related species in cell lines expressing endogenous opioid receptors, including a small molecular mass protein (approximately 22 kDa). Previous reports have suggested that this 22-kDa (125)I-beta-endorphin cross-linked protein could be the degradative product from a higher molecular mass species, i.e., a fragment of the receptor. To determine if this protein is indeed a degraded receptor fragment, (125)I-beta-endorphin was cross-linked to the (His)(6) epitope-tagged mu-opioid receptor (His-mu) stably expressed in the murine neuroblastoma Neuro(2A) cells. Similar to earlier reports with cell lines expressing endogenous receptors, two major bands of 72- and 25-kDa proteins were specifically cross-linked. Initial cross-linking experiments indicated the absolute requirement of the high-affinity (125)I-beta-endorphin binding to the mu-opioid receptor prior to the appearance of the low molecular weight species, suggesting that the 22-kDa protein could be a degraded fragment of the receptor. However, variations in the ratios of these protein bands being cross-linked by several homo- or heterobifunctional cross-linking agents were observed. Although neither the carboxyl terminus mu-opioid receptor-specific antibodies nor the antibodies against the epitope at the amino terminus of the receptor could recognize the 22-kDa protein, this (125)I-beta-endorphin cross-linked species could be coimmunoprecipitated with the receptor antibodies or could be isolated with a nickel resin affinity chromatography. The direct physical association of the 22-kDa protein with the receptor was demonstrated also by the observation that the 22-kDa protein could not bind to the nickel resin alone, but that its binding to the nickel resin was restored in the presence of the His-mu. Taken together, these results suggest that the 22-kDa protein cross-linked by (125)I-beta-endorphin is not a degradative product, but a protein located within the proximity of the mu-opioid receptor, and that it is tightly associated with the receptor.

Topics: Animals; beta-Endorphin; Cross-Linking Reagents; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Histidine; Iodine Radioisotopes; Mice; Molecular Weight; Morphine; Neuroblastoma; Proteins; Receptors, Opioid, mu; Somatostatin; Tumor Cells, Cultured

The potential interactions of natively expressed mu-opioid and opioid receptor-like (ORL1) receptors were studied by exposing intact BE(2)-C cells to agonists or antagonists for 1 h. Pretreatment with the mu-opioid receptor agonist, [D-Ala(2), N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), or the ORL1 receptor agonist, orphanin FQ/nociceptin desensitized both mu-opioid and ORL1 receptor responses. beta-Funaltrexamine (beta-FNA) pretreatment also blocked both mu-opioid and ORL1 receptor responses, but only mu-opioid receptor binding was reduced. Moreover, beta-FNA (1 microM) failed to inhibit specific ORL1 receptor binding.

Topics: Brain Neoplasms; Cyclic AMP; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Humans; Morphine; Naltrexone; Narcotic Antagonists; Narcotics; Neuroblastoma; Nociceptin; Nociceptin Receptor; Opioid Peptides; Receptors, Opioid; Receptors, Opioid, mu; Tumor Cells, Cultured

Phosphorylation of specific amino acid residues is believed to be crucial for the agonist-induced regulation of several G protein-coupled receptors. This is especially true for the three types of opioid receptors (mu, delta, and kappa), which contain consensus sites for phosphorylation by numerous protein kinases. Protein kinase C (PKC) has been shown to catalyze the in vitro phosphorylation of mu- and delta-opioid receptors and to potentiate agonist-induced receptor desensitization. In this series of experiments, we continue our investigation of how opioid-activated PKC contributes to homologous receptor down-regulation and then expand our focus to include the exploration of the mechanism(s) by which mu-opioids produce PKC translocation in SH-SY5Y neuroblastoma cells. [D-Ala2,N-Me-Phe4,Gly-ol]enkephalin (DAMGO)-induced PKC translocation follows a time-dependent and biphasic pattern beginning 2 h after opioid addition, when a pronounced translocation of PKC to the plasma membrane occurs. When opioid exposure is lengthened to >12 h, both cytosolic and particulate PKC levels drop significantly below those of control-treated cells in a process we termed "reverse translocation." The opioid receptor antagonist naloxone, the PKC inhibitor chelerythrine, and the L-type calcium channel antagonist nimodipine attenuated opioid-mediated effects on PKC and mu-receptor down-regulation, suggesting that this is a process partially regulated by Ca2+-dependent PKC isoforms. However, chronic exposure to phorbol ester, which depletes the cells of diacylglycerol (DAG) and Ca2+-sensitive PKC isoforms, before DAMGO exposure, had no effect on opioid receptor down-regulation. In addition to expressing conventional (PKC-alpha) and novel (PKC-epsilon) isoforms, SH-SY5Y cells also contain a DAG- and Ca2+-independent, atypical PKC isozyme (PKC-zeta), which does not decrease in expression after prolonged DAMGO or phorbol ester treatment. This led us to investigate whether PKC-zeta is similarly sensitive to activation by mu-opioids. PKC-zeta translocates from the cytosol to the membrane with kinetics similar to those of PKC-alpha and epsilon in response to DAMGO but does not undergo reverse translocation after longer exposure times. Our evidence suggests that direct PKC activation by mu-opioid agonists is involved in the processes that result in mu-receptor down-regulation in human neuroblastoma cells and that conventional, novel, and atypical PKC isozymes are involved.

Topics: Analgesics; Analgesics, Opioid; Biological Transport; Carcinogens; Diprenorphine; Down-Regulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalins; Enzyme Activation; Humans; Immunoblotting; Isoenzymes; Narcotic Antagonists; Neuroblastoma; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; Protein Kinase C-alpha; Protein Kinase C-epsilon; Receptors, Opioid, mu; Second Messenger Systems; Tritium; Tumor Cells, Cultured

Agonist-induced down-regulation of opioid receptors appears to require the phosphorylation of the receptor protein. However, the identities of the specific protein kinases that perform this task remain uncertain. Protein kinase C (PKC) has been shown to catalyze the phosphorylation of several G protein-coupled receptors and potentiate their desensitization toward agonists. However, it is unknown whether opioid receptor agonists induce PKC activation under physiological conditions. Using cultured SH-SY5Y neuroblastoma cells, which naturally express mu- and delta-opioid receptors, we investigated whether mu-opioid receptor agonists can activate PKC by measuring enzyme translocation to the membrane fraction. PKC translocation and opioid receptor densities were simultaneously measured by 3H-phorbol ester and [3H]diprenorphine binding, respectively, to correlate alterations in PKC localization with changes in receptor binding sites. We observed that mu-opioid agonists have a dual effect on membrane PKC density depending on the period of drug exposure. Exposure for 2-6 h to [D-Ala2,N-Me-Phe4,Gly-ol]enkephalin or morphine promotes the translocation of PKC from the cytosol to the plasma membrane. Longer periods of opioid exposure (>12 h) produce a decrease in membrane-bound PKC density to a level well below basal. A significant decrease in [3H]diprenorphine binding sites is first observed at 2 h and continues to decline through the last time point measured (48 h). The opioid receptor antagonist naloxone attenuated both opioid-mediated PKC translocation and receptor down-regulation. These results demonstrate that opioids are capable of activating PKC, as evidenced by enhanced translocation of the enzyme to the cell membrane, and this finding suggests that PKC may have a physiological role in opioid receptor plasticity.

Topics: Analgesics; Analgesics, Opioid; Binding, Competitive; Biological Transport; Carcinogens; Diprenorphine; Down-Regulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalins; Humans; Kinetics; Membrane Proteins; Morphine; Naloxone; Narcotic Antagonists; Neuroblastoma; Phorbol 12,13-Dibutyrate; Protein Kinase C; Receptors, Opioid, mu; Second Messenger Systems; Time Factors; Tritium; Tumor Cells, Cultured

The influence of membrane microviscosity on mu-opioid agonist and antagonist binding, as well as agonist efficacy, was examined in membranes prepared from SH-SY5Y cells and from a C6 glioma cell line stably expressing the rat mu-opioid receptor (C6mu). Addition of cholesteryl hemisuccinate (CHS) to cell membranes increased membrane microviscosity and reduced the inhibitory effect of sodium and guanine nucleotides on the affinity of the full agonists sufentanil and [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAMGO) for the mu-opioid receptor. Binding of the antagonists [3H]naltrexone and [3H]diprenorphine and the partial agonist nalbuphine was unaffected by CHS. The effect of CHS on agonist binding was reversed by subsequent addition of cis-vaccenic acid, suggesting that the effect of CHS is the result of increased membrane microviscosity and not a specific sterol-receptor interaction. CHS addition increased the potency of DAMGO to stimulate guanosine-5'-O-(3-[35S]thio)triphosphate binding by fourfold, whereas the potency of nalbuphine was unaffected. However, nalbuphine efficacy relative to that of the full agonist DAMGO was strongly increased in CHS-treated membranes compared with that in control membranes. Membrane rigidification also resulted in an increased efficacy for the partial agonists meperidine, profadol, and butorphanol relative to that of DAMGO as measured by agonist-stimulated GTPase activity in control and CHS-modified membranes. These findings support a regulatory role for membrane microviscosity in receptor-mediated G protein activation.

Topics: Animals; Cell Line; Cell Membrane; Cholesterol Esters; Diprenorphine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Glioma; Guanosine 5'-O-(3-Thiotriphosphate); Membrane Fluidity; Nalbuphine; Naltrexone; Narcotic Antagonists; Narcotics; Neuroblastoma; Protein Conformation; Rats; Receptors, Opioid, mu; Sodium Chloride; Sufentanil; Tumor Cells, Cultured; Viscosity

Monoclonal antibodies generated against multiple antigenic peptides of the N-terminal sequence (3LVPSARAELQSSPLV17) of the cloned delta opioid receptor immunoprecipitated a 58 kDa protein from CHAPS-solubilized NG108-15 membranes. The immunoprecipitates bound [3H]DPDPE--but not [3H]DAMGO--with a Kd of 6.4 nM and a Bmax of 75 pM. Western blot analysis revealed a distinct band of 58 kDa. The antibodies inhibited basal and PGE1-stimulated cAMP levels, and mimicked the effect of agonists manifest in a compensatory increase in cAMP formation. The antibody will be potentially useful in the analysis of functional epitopes on the delta opioid receptor.

Topics: Adenylyl Cyclases; Alprostadil; Amino Acid Sequence; Animals; Antibodies, Monoclonal; Blotting, Western; Cyclic AMP; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Epitopes; Fluorescent Antibody Technique; Glioma; Molecular Sequence Data; Neuroblastoma; Precipitin Tests; Receptors, Opioid, delta; Tumor Cells, Cultured

Opioid agonists either potentiate or suppress basal cAMP production in SK-N-SH cells. The inhibitory effect is mediated by PTX-sensitive GTP-binding proteins, while the stimulatory effect involves Ca++ entry and calmodulin activation. Both pathways can be activated simultaneously by opioid agonists. Low (nM) concentrations of either mu (DAMGO) or delta (DPDPE) selective opioids potentiate cAMP formation. At higher (100 nM) concentrations, however, a net suppression takes over; this suppression can be eliminated by PTX, and the underlying stimulatory effect is disclosed. Micromolar concentrations of either mu or delta selective agonists cross-activate the other (delta or mu) receptors, and augment the stimulatory pathway. The overall outcome (either stimulation or inhibition of cAMP production) is dependent on the balance between the two overlapping pathways, and can be modified by blocking either of the two opposing mechanisms.

Topics: Calcium; Cyclic AMP; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; GTP-Binding Proteins; Humans; Kinetics; Neuroblastoma; Opioid Peptides; Receptors, Opioid, delta; Receptors, Opioid, mu; Tumor Cells, Cultured; Virulence Factors, Bordetella

Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2), peptides recently isolated from bovine and human brain, have high affinity and selectivity for mu opiate receptors. They share sequence similarity with the endogenous opiate-modulating peptide Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2). The efficacies of these endogenous peptides and of the enkephalin analog DAMGO were compared by measuring their effects on the binding of guanosine-5'-O-(-gamma-[35S]thio)triphosphate ([35S]GTPgammaS) to G-proteins in membranes from SH-SYSY human neuroblastoma cells. DAMGO, endomorphin-1, and endomorphin-2 stimulated [35S]GTPgammaS binding dose dependently, with maximal effects of 60 +/- 9%, 47 +/- 9%, and 43 +/- 6% stimulation above basal and ED50 of 49 +/- 8 nM, 38 +/- 8 nM, and 64 +/- 13 nM, respectively. Tyr-W-MIF-1 showed only a small stimulation of binding (5% stimulation above basal, ED50 = 2 microM). When given in combination with the other opioids, however, Tyr-W-MIF-1 attenuated their ability to activate G-proteins. Thus, the endogenous opioids endomorphin-1 and endomorphin-2 activate G-proteins similarly to the synthetic agonist DAMGO, but the structurally similar peptide Tyr-W-MIF-1 produces only minimal stimulation of G-proteins.

Topics: Cell Membrane; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; MSH Release-Inhibiting Hormone; Narcotic Antagonists; Neuroblastoma; Oligopeptides; Receptors, Opioid; Tumor Cells, Cultured

The present study demonstrates a conditional, agonist-dependent phosphorylation of the mu-opioid receptor (MOR-1) by cyclic AMP-dependent protein kinase (PKA) in membrane preparations of MOR-1-transfected neuroblastoma Neuro2A cells. Opioid agonist-dependent phosphorylation occurs in a time- and concentration-dependent manner (EC50 approximately 40 nM) and can be abolished by the receptor antagonist naloxone. Stoichiometric analysis indicates incorporation of a maximum of 6 mol of phosphate/mol of receptor in the presence of 1 microM morphine and 6 nM PKA. Although morphine and related alkaloids as well as some peptide agonists (PLO17 and beta-endorphin) stimulated phosphorylation of MOR-1 by PKA, the potent mu-opioid-selective peptide [D-Ala2,N-MePhe4,Gly-ol5]-enkephalin (DAMGO) or other enkephalin analogues such as [D-Ala2]-Met5-enkephalinamide (DALA), [D-Ala2,D-Leu5]-enkephalin (DADLE), and Met5-enkephalin had no effect. The lack of the effect of DAMGO on MOR-1 phosphorylation state was evident also after chronic pretreatment. These results suggest the existence of different agonist-dependent conformations of MOR-1. Furthermore, phosphorylation may be a useful parameter with which to identify different agonist-receptor conformations.

Topics: Analgesics, Opioid; Antibody Specificity; Carbazoles; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine-2-Alanine; Enkephalin, Methionine; Enkephalins; Enzyme Inhibitors; Indoles; Morphine; Neuroblastoma; Neurons; Phosphorylation; Precipitin Tests; Pyrroles; Receptors, Opioid, mu; Tumor Cells, Cultured

Ca2+ channel modulation by the mu opioid agonist [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAGO) and the delta opiate agonists [d-Pen2, d-Pen5]-enkephalin (DPDPE) and [d-Ala2, d-Leu5]-enkephalin (DADLE) in cultured human neuroblastoma SH-SY5Y cells was investigated using the whole-cell variant of the patch-clamp technique. In SH-SY5Y cells, differentiated in vitro with retinoic acid, all agonists reversibly decreased high-voltage-activated, omega-conotoxin-sensitive Ba2+ currents in a concentration-dependent way. Inhibition was maximal with a 1 microM concentration of opiate agonists (76% with DAGO and 63% with delta agonists, when measured at 0 mV) and was characterized by a clear slow down of Ba2+ current activation at low test potentials. Both inhibition and slow down of activation were attenuated at more positive potentials, and could be partially relieved by strong conditioning depolarizations. Current suppression operated by both mu and delta agonists was prevented by pre-treatment of the cells with pertussis toxin. No sign of additivity was observed when delta agonists were applied to cells that were maximally activated by DAGO, suggesting that a common mechanism, involving the same type of modulating molecule, is responsible for Ca2+ channel inhibition promoted by activation of mu and delta opioid receptors in SH-SY5Y cells.

Topics: Barium; Calcium Channels; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Neuroblastoma; Receptors, Opioid, delta; Receptors, Opioid, mu; Tumor Cells, Cultured

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

The present study examines the stimulatory effect of opioids on adenosine 3':5'-cyclic monophosphate (cyclic AMP) production in the human neuroblastoma cell line SK-N-SH, and its dependence on calcium. We show that, in this culture, the mu-opioid selective agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin stimulates cyclic AMP production by 30% in a naloxone-reversible manner. This stimulation is completely dependent on calcium and involves the activation of calcium/calmodulin since it is abolished in the presence of EGTA, calcium channel blockers or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7). The results suggest that the activation of calcium/calmodulin dependent adenylyl cyclases by opioids in SK-N-SH cells is secondary to the induction of calcium influx and the consequent elevation of intracellular calcium level.

Topics: Calcium; Calcium Channel Blockers; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cell Line; Cyclic AMP; Egtazic Acid; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Humans; Naloxone; Narcotic Antagonists; Neuroblastoma; Opioid Peptides; Sulfonamides

1. The agonist action of morphine on membranes prepared from human neuroblastoma SH-SY5Y cells was measured by an increase in the binding of the GTP analogue [35S]-GTPgammaS. Morphine increased the binding of [35S]-GTPgammaS to SH-SY5Y cell membranes by 30 fmol mg(-1) protein with an EC50 value of 76 +/- 10 nM. 2. Incubation of SH-SY5Y cells with 10 microM morphine for 48 h caused a tolerance to morphine manifested by a 2.5 fold shift to the right in the EC50 value with a 31 +/- 6% decrease in the maximum stimulation of [35S]-GTPgammaS binding. The response caused by the partial agonist pentazocine was reduced to a greater extent. 3. Chronic treatment of the cells with the more efficacious mu-ligand [D-Ala2, MePhe4, Gly-ol5]enkephalin (DAMGO, 10 microM) for 48 h afforded a greater effect than treatment with morphine. The maximal agonist effect of morphine was reduced to 58.9 +/- 6% of that seen in control cells while the maximal effect of DAMGO was reduced to 62.8 +/- 4%. There was a complete loss of agonist activity for pentazocine. 4. The development of tolerance was complete within 24 h and was blocked by naloxone and by the nonselective protein kinase inhibitor H7, but not by the putative beta-adrenoceptor kinase (beta-ARK) inhibitor suramin. 5. The observed tolerance effect was accompanied by a down-regulation of mu-opioid receptors determined by a decrease in the maximal binding capacity for the opioid antagonist [3H]-diprenorphine of 66 +/- 4%, but with no change in binding affinity. Binding of the agonist [3H]-DAMGO was similarly reduced. 6. The modulation of [35S]-GTPgammaS binding in SH-SY5Y cell membranes by opioids provides a simple method for the study of opioid tolerance at a site early in the signal transduction cascade.

Topics: Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Morphine; Neuroblastoma; Receptors, Opioid, mu; Tumor Cells, Cultured

Whole-cell patch-clamp recordings were used to study Ba2+ currents through voltage-dependent Ca2+ channels in dorsal root ganglion x mouse neuroblastoma hybrid (F-11) cells. Opioid agonists selective for either mu (Tyr-D-Ala-Gly-Mephe-Gly-ol; DAMGO) or delta (Tyr-D-Pen-Gly-Phe-D-Pen-OH; DPDPE) receptors inhibited high-threshold Ba2+ currents. The inhibition was reversible, naloxone-sensitive, and dose-dependent. The inhibitory effects of both DAMGO and DPDPE were blocked by pretreatment of the cells with pertussis toxin (PTX) as well as by brief exposure to the sulfhydryl alkylating agent, N-ethylmaleimide (NEM). The N-type Ca2+ channel antagonist omega-conotoxin GVIA (omega-CTX GVIA) irreversibly inhibited high threshold Ba2+ currents by 66% and blocked the inhibitory effect of DAMGO or DPDPE. In contrast, the L-type Ca2+ channel blocker nifedipine inhibited high threshold Ba2+ currents by 15% and failed to block the inhibitory effect of DAMGO or DPDPE. These results demonstrate that mu and delta opioid receptors are negatively coupled to N-type Ca2+ channels via PTX- and NEM-sensitive GTP-binding proteins in F-11 cells.

Topics: Analgesics; Analgesics, Opioid; Animals; Barium; Calcium; Calcium Channel Blockers; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Ethylmaleimide; Gene Expression Regulation; Hybrid Cells; Ion Channel Gating; Mice; Neuroblastoma; omega-Conotoxin GVIA; Patch-Clamp Techniques; Peptides; Pertussis Toxin; Rats; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; RNA, Messenger; Sulfhydryl Reagents; Virulence Factors, Bordetella

Radioligand binding assays and functional experiments revealed that the SK-N-BE neuroblastoma cell line expresses a similar ratio of mu- and delta-opioid receptors, both negatively coupled to adenylyl cyclase through pertussis toxin-sensitive G proteins. Our findings also indicate that some functional interaction occurred between the two opioid subtypes; in fact, long-term exposure to [D-Ala2-N-methyl-Phe4-Gly-ol5]enkephalin (DAMGO), a mu-selective agonist, sensitized the functional response of the delta-selective agonist but not vice versa. It is interesting that in acute interaction experiments, we observed a shift to the right of the concentration-effect curve of either DAMGO or [D-Pen2,5]enkephalin (DPDPE), a delta-selective agonist, as a result of DPDPE or DAMGO administration, respectively. In addition, low doses of naloxone, an antagonist selective for mu receptors, increased the inhibitory effect [D-Ala2-D-Met5]enkephalinamide (DAME), a mixed mu/delta agonist, on adenylyl cyclase activity. Taken overall, these data support the hypothesis of the existence of a cross talk between mu and delta receptors in the SK-N-BE cell line.

Topics: Adenylyl Cyclases; Analgesics; Binding, Competitive; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Methionine; Enkephalins; Humans; Membrane Proteins; Neuroblastoma; Receptors, Opioid, delta; Receptors, Opioid, mu; Time Factors; Tumor Cells, Cultured

The present study showed that the glucocorticoid/progesterone antagonists, 17 beta-hydroxy-1 1 beta-(4-dimethylamino-phenyl-1)-17-(prop-1-ynyl)estra-4,9-dien+ ++-3-one (RU486) and 17 beta-hydroxy-11 beta-(4-dimethylamino-phenyl-1)-17-(propan-3-ol)estra-4,9-dien-3-o ne (ZK 98299), inhibit the binding of labeled dihydromorphine to mu-opioid receptors present on membrane preparations derived from rat and mouse brain, as well as from human neuroblastoma cells. The inhibitory effect of RU486 was dose-dependent and linked to a decrease of the affinity of labeled dihydromorphine to the mu-opioid receptors. Kinetic experiments have shown that RU486 induces a decrease of the association rate constant (k + 1) of dihydromorphine. RU486 also proved able to dissociate the dihydromorphine-mu-opioid receptor complex, although at a rate slower than that exhibited by unlabeled dihydromorphine. Finally, the addition of NaCl (100 mM) to the incubation buffer induced a 50% decrease of the inhibitory effect of RU486. A 6-day treatment of neuroblastoma cells with RU486 eliminated the inhibitory effect morphine exerts on the intracellular accumulation of cyclic AMP induced by prostaglandin E1. These results indicate that RU-486 may interact with brain mu-opioid receptors in vitro, by decreasing the affinity of opioid ligands.

Topics: Analgesics; Animals; Dihydromorphine; Diprenorphine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Female; Gonanes; Hormone Antagonists; Humans; In Vitro Techniques; Male; Membranes; Mice; Mifepristone; Narcotic Antagonists; Narcotics; Nerve Tissue; Nervous System Neoplasms; Neuroblastoma; Progestins; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Sodium; Tumor Cells, Cultured

gamma-Hydroxybutyrate (GHB) and morphine induce a number of similar effects. Moreover, the effects they elicit can be reversed by the opiate antagonist naloxone (NX), suggesting that GHB may produce at least some of its central effects by acting as an opiate agonist. The present study considered this possibility by examining the effect of GHB on mu, delta, and kappa-opioid receptor binding in concentrations of 1 nM-0.1 mM. GHB was inactive in each instance, at every dose examined. GHB is consequently not a direct opiate receptor agonist. It is also unlikely to be an indirect (enkephalin or dynorphin release-stimulating) agonist. The mechanism of action involved whereby NX can reverse the effects of GHB must therefore not involve opioid mechanisms; at least not directly.

Topics: Adjuvants, Anesthesia; Analgesics; Animals; Diprenorphine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Etorphine; Glioma; Guinea Pigs; Naloxone; Narcotic Antagonists; Narcotics; Neuroblastoma; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Sodium Oxybate; Tritium; Tumor Cells, Cultured

Incubation of SH-SY5Y neural cells with mycophenolic acid (MPA), an inhibitor of inosine monophosphate dehydrogenase (the key enzyme in purine nucleotide biosynthesis), reduced the cellular content of GTP by 94% relative to its concentration in control cells (43 nmol/mg protein) without altering the level of GDP. Although in GTP-depleted intact cells the receptor binding parameters (Kd and Bmax) of the opioid antagonist [3H]naltrexone were unchanged from those in untreated cells, the binding affinity of the mu-selective opioid agonist [3H]Tyr-D-Ala-Gly-(Me)- Phe-Gly-ol ([3H]DAMGO) was enhanced 2-fold. Furthermore, the kinetics of ligand/receptor interaction revealed that in the nucleotide-depleted cells, the dissociation rate constant for [3H]DAMGO was reduced by 44%. Initial exposure of SH-SY5Y cells to pertussis toxin reduced high-affinity ligand binding by 95% and abolished the effect of MPA treatment. Renewed incubation of the GTP-depleted cells with guanosine restored the original GTP levels and agonist binding. Neither MPA nor guanosine treatment changed the Bmax of [3H]DAMGO binding. Forskolin- and prostaglandin E1-stimulated adenylyl cyclase activities were decreased significantly in GTP-depleted cells. DAMGO and [D-Pen2,D-Pen5]enkephalin inhibitions of adenylyl cyclase were also affected with MPA treatment. Maximal inhibition of forskolin-stimulated adenylyl cyclase activity by both of the agonists was reduced, whereas MPA caused a 2-fold reduction in potency for DAMGO. The results show that reduction in endogenous GTP levels leads to noticeable changes in agonist, receptor, and G protein interactions, as measured by agonist binding, and to subsequent diminution of the signal transduction, as reflected by the cAMP levels.

Topics: Adenylyl Cyclases; Amino Acid Sequence; Analgesics; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Kinetics; Molecular Sequence Data; Mycophenolic Acid; Naltrexone; Narcotic Antagonists; Neuroblastoma; Neurons; Receptors, Opioid, mu; Tritium; Tumor Cells, Cultured

Several cellular systems display desensitization and downregulation of opioid receptors upon chronic treatment, suggesting that they could be used as a model system to understand opioid tolerance/dependence. However, a model system containing a homogeneous population of mu-opioid receptors, the receptors at which morphine and related opioids act, has been lacking. To approach this problem, the mu-opioid receptor (MOR-1) was stably expressed in murine neuroblastoma Neuro2A cells after transfection. The expressed receptor was negatively coupled to adenylyl cyclase through Gi/Go proteins, displayed high affinity ligand binding, and was expressed in high number (2.06 pmol/mg of [3H]diprenorphine binding sites). In addition, loss of ability of mu-opioids to acutely inhibit forskolin-stimulated cAMP formation was observed after 4-24 h of chronic exposure to these agonists with concentrations as low as 300-500 nM. The effects of chronic morphine or [D-Ala2,N-MePhe4,Gly-ol]enkephalin (DAMGO) administration were found to be time- and concentration-dependent. Cross 'tolerance' was also observed. Thus the IC50 value of DAMGO to inhibit adenylyl cyclase was increased by 27-fold from 4.3 nM in control cells to 117 nM in cells pretreated with 300 nM morphine; there was no effect on the inhibition of adenylyl cyclase mediated by muscarinic receptors. Further, receptor downregulation accompanied the desensitization process. However, different time-dependence for these two processes suggests, in line with other studies, that these are entirely different cellular adaptation processes. In addition, the opioid antagonist naloxone induced an acute increase in intracellular cAMP level (2-3 times above the control level) following chronic agonist exposure. This process was also concentration-dependent. Overall, these results suggest that the cell line utilized in this study has a homogeneous population of mu-opioid receptors, providing an ideal cellular model to study the molecular mechanisms underlying chronic morphine treatment.

Topics: Analgesics; Animals; Binding, Competitive; Cell Line; Colforsin; Dose-Response Relationship, Drug; Down-Regulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Mice; Models, Biological; Morphine; Neuroblastoma; Receptors, Opioid, mu; Time Factors

Voltage-dependent Ca2+ currents were measured in NG108-15 neuroblastoma x glioma hybrid cells transformed to express the rat mu-opioid receptor by the whole-cell configuration of the patch-clamp technique with Ba2+ as charge carrier. A mu-opioid receptor-selective agonist, [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin caused significant inhibition of voltage-dependent Ca2+ currents in mu-receptor-transformed NG108-15 cells but not in nontransfected or vector-transformed control cells. On the other hand, a delta-opioid receptor-selective agonist, [D-penicillamine2,D-penicillamine5]enkephalin, induced inhibition of voltage-dependent Ca2+ currents in both control and mu-receptor-transformed cells, which is mediated by the delta-opioid receptor expressed endogenously in NG108-15 cells. The inhibition of voltage-dependent Ca2+ currents induced by [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin [D-penicillamine2,D-penicillamine5]enkephalin was reduced by pretreatment of the cells with pertussis toxin or omega-contoxin GVIA. These results indicate that the mu-opioid receptor expressed from cDNA functionally couples with omega-contoxin-sensitive N-type Ca2+ channels through the action of pertussis toxin-sensitive G proteins in NG108-15 cells.

Topics: Animals; Calcium Channels; Cloning, Molecular; DNA, Complementary; Electric Conductivity; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Gene Expression; Glioma; GTP-Binding Proteins; Hybrid Cells; Neuroblastoma; omega-Conotoxin GVIA; Peptides; Pertussis Toxin; Rats; Receptors, Opioid, mu; Transfection; Tumor Cells, Cultured; Virulence Factors, Bordetella

The ability of mu-opioid agonists to activate G proteins has been demonstrated by studying the binding of the GTP analogue guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTP gamma S) to membranes from the human neuroblastoma SH-SY5Y cell line. The potent opioid agonist fentanyl caused an approximate doubling of basal [35S]GTP gamma S binding in a naloxone-sensitive manner, confirming this to be an opioid receptor-mediated process. The presence of GDP was necessary to observe this effect. Pretreatment of the cells with pertussis toxin (100 ng/ml, for 24 hr) completely prevented the fentanyl-stimulated increase in [35S]GTP gamma S binding and lowered the basal binding of [35S]GTP gamma S. These latter data suggest an involvement of Gi and/or Go proteins and their activation by added membrane-bound receptors even in the absence of agonist. The order of potency of a series of opioid agonists in stimulating the binding of [35S]GTP gamma S was buprenorphine > cyclazocine = levallorphan > nalorphine > [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAMGO) > fentanyl > morphine > pentazocine. DAMGO, fentanyl, and morphine were full agonists but the remaining compounds showed decreasing levels of intrinsic activity in the order buprenorphine > pentazocine > cyclazocine = nalorphine > levallorphan. The opioid antagonist naloxone was without effect. Under the conditions of the [35S]GTP gamma S assay, binding of agonists was to a high affinity site, indicating that a high agonist affinity state of the mu-opioid receptor is responsible for the observed stimulation of [35S]GTP gamma S binding. The level of [35S]GTP gamma S binding (597 fmol/mg of protein) stimulated by DAMGO was 2-fold greater than the maximal number of mu-opioid agonist binding sites (Bmax) determined using [3H]DAMGO (254 fmol/mg of protein). The opioid agonist-mediated stimulation of [35S]GTP gamma S binding in SH-SY5Y cell membranes thus provides a "functional" measure of agonist occupation of mu-opioid receptors and offers a simple method for the determination of efficacy and intrinsic activity of mu-opioid agonists.

Topics: Diprenorphine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Fentanyl; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Humans; Narcotics; Neuroblastoma; Receptors, Opioid, mu; Tumor Cells, Cultured

Topics: Adenylyl Cyclase Inhibitors; Cannabinoids; Colforsin; Cyclic AMP; Dronabinol; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Humans; Neuroblastoma; Potassium; Receptors, Cannabinoid; Receptors, Drug; Receptors, Opioid; Tumor Cells, Cultured

The site of interaction of opioids and inhalation anaesthetic agents is unknown, but may be at the level of the opioid receptor. In this study we have used SH-SY5Y human neuroblastoma cells, which express both mu and delta receptors, to examine the effects of halothane on the receptor binding profiles of [3H]diprenorphine (DPN), an opioid receptor antagonist, and [3H] [D-Ala2,MePhe4, Gly(ol)5]enkephalin (DAMGO), a mu receptor selective agonist. Binding of [3H]DPN and [3H]DAMGO was performed at 37 degrees C for 60 min in the presence of air, nitrous oxide (75%) or air containing halothane (0.5-5.0% v/v). Compared with air controls, neither 75% nitrous oxide nor 0.5, 1.0, 2.0 and 5.0% halothane influenced DPN binding variables. Binding of [3H]DAMGO was unaffected by 1.0% halothane, but 5.0% halothane reduced the affinity, with a modest increase in Kd (1.15 (0.16) to 1.7 (0.2) nmol litre-1) without effect on Bmax. Our data suggest that the site of opioid and volatile anaesthetic interaction is not at the opioid receptor.

Topics: Analgesics; Anesthetics, Inhalation; Diprenorphine; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Halothane; Humans; Neuroblastoma; Nitrous Oxide; Receptors, Opioid; Tumor Cells, Cultured

Evidence is presented for the occurrence of a unique opiate alkaloid-selective, opioid peptide-insensitive binding site in N18TG2 mouse neuroblastoma cells and in late passage hybrid F-11 cells, derived from N18TG2 neuroblastoma cells and rat dorsal root ganglion cells. Those cells lacked classical opioid peptide-sensitive receptor subtypes, but contained [3H]morphine and [3H]diprenorphine binding sites with affinity for certain opiate alkaloids but not for any endogenously occurring opioid peptide or peptide analog tested, including D-ala2-D-leu5-enkephalin (DADLE), D-Ala2,N-Me-Phe4,Gly5-ol (DAGO) and dynorphin A(1-17). The binding site differed from hitherto described mu, delta and kappa neuronal opioid receptors not only on the basis of peptide insensitivity, but also on the basis of selectivity and affinities of alkaloids. Saturation experiments with [3H]morphine indicated the presence of a single site with Kd = 49 nM and Bmax = 1510 fmol/mg protein. This novel binding site was not present in F-11 hybrid cells at early passage. Instead the hybrid cells contained conventional opioid receptors (predominantly delta and also mu) capable of binding DADLE and other peptides as well as opiate alkaloids. With additional passage (cell divisions) of the hybrid cells, during which a limited change occurred in mouse chromosome number, the peptide-insensitive binding appeared and the opioid peptide-binding (delta and mu) receptors were lost reciprocally. Thus, expression of the peptide-insensitive binding normally may be repressed when conventional opioid receptors are expressed. The peptide-insensitive opiate binding site described here appears to correspond to the mu 3 receptor subtype, recently identified pharmacologically and functionally in several cell types of the immune system.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Binding, Competitive; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Male; Mice; Morphine; NAD; Neuroblastoma; Opioid Peptides; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Tumor Cells, Cultured

The irreversible opioid receptor antagonists naloxonazine and beta-funaltrexamine have been used to determine whether multiple mu-opioid receptors exist on undifferentiated SH-SY5Y human neuroblastoma cells. Naloxonazine binds irreversibly to the mu 1-opioid receptor subtype and reversibly to the mu 2-opioid receptor subtype. On SH-SY5Y cells naloxonazine afforded a Ki of 3.4 +/- 0.7 nM, and was fully reversible, indicating the mu-opioid receptor population on SH-SY5Y cells was solely of the mu 2-opioid receptor subtype. The alkylating agent beta-funaltrexamine was maximally able to alkylate only 60% of the mu-opioid receptor sites on SH-SY5Y cells, labelled with [3H]diprenorphine or [3H][D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAMGO). The reversible binding of naloxonazine and the insensitivity of a percentage of the mu-opioid receptor sites to alkylation by beta-funaltrexamine suggests that differences do exist in the mu 2-opioid receptor population on undifferentiated SH-SY5Y cells. This may indicate further heterogeneity or the inability of beta-funaltrexamine to alkylate all relevant nucleophilic groups in a single population of receptors.

Topics: Amino Acid Sequence; Animals; Brain Neoplasms; Cerebral Cortex; Diprenorphine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Humans; Ligands; Molecular Sequence Data; Naloxone; Naltrexone; Neuroblastoma; Rats; Receptors, Opioid, mu; Somatostatin; Tumor Cells, Cultured

In this work, we have characterized the opioid receptor expressed by the human neuroblastoma cell line SK-N-BE and compared its hydrodynamic behaviour with those of well known opioid receptors: mu-opioid receptor of rabbit cerebellum and delta-opioid receptor of the hybrid cell line NG 108-15. Human neuroblastoma cell line SK-N-BE expresses a substantial amount of opioid receptors (200-300 fmoles/mg of protein). Pharmacological characterization suggests an heterogenous population of receptors and the presence of two delta subtypes which are, at least partially, negatively coupled with adenylate cyclase via a Gi protein. These receptors exist under two different molecular forms and, in this respect, strikingly contrast with the archetypic delta receptors of NG 108-15 hybrid cell line which show only a high molecular weight form and appear more tightly coupled with the G protein. Hydrodynamic behaviour of SK-N-BE opioid receptors is reminiscent of the profile observed with the rabbit cerebellum mu-opioid receptor. This observation is consistent with the presence of two delta-opioid receptors subtypes, one of which exhibiting properties close to those of mu opioid receptors. Taken overall, our results suggest that different types and subtypes of opioid receptors, even if they are coupled to the same inhibitory G protein, are more or less tightly coupled with their transduction proteins and that closely related opioid receptors can form allosterically interacting complexes.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Adenylyl Cyclases; Amino Acid Sequence; Animals; Binding, Competitive; Cerebellum; Colforsin; Diprenorphine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Etorphine; GTP-Binding Proteins; Guinea Pigs; Humans; Hybrid Cells; Molecular Sequence Data; Morphine; Naloxone; Neoplasm Proteins; Nerve Tissue Proteins; Neuroblastoma; Oligopeptides; Pyrrolidines; Rabbits; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; 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

We have examined the effect of fentanyl on [3H]noradrenaline release in a human neuroblastoma cell preparation, SH-SY5Y. Fentanyl produced a significant, concentration-dependent inhibition of [3H]noradrenaline release with IC50 values of 5.5 x 10(-6) mol litre-1 and 15.5 x 10(-6) mol litre-1 for carbachol- and potassium-evoked release, respectively. The small difference in IC50 between the two evoking stimuli may be explained by the weak binding affinity of fentanyl to muscarinic receptors (Ki = 570 nmol litre-1). The minimum concentrations at which a significant effect was observed were 0.3 x 0.10(-6) mol litre-1 and 10.0 x 10(-6) mol litre-1 for carbachol- and potassium-evoked release, respectively; these values are considerably in excess of the serum concentration of fentanyl required to produce analgesia. Naloxone failed to antagonize the fentanyl inhibition and, furthermore, morphine and an enkephalin had no effect on evoked release, implying a non-opioid receptor mediated effect.

Topics: Atropine; Carbachol; Cell Differentiation; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Fentanyl; Humans; Morphine; Neuroblastoma; Norepinephrine; Potassium; Receptors, Muscarinic; Tumor Cells, Cultured

The cellular mechanisms underlying opioid action remain to be fully determined, although there is now growing indirect evidence that some opioid receptors may be coupled to phospholipase C. Using SH-SY5Y human neuroblastoma cells (expressing both mu- and delta-opioid receptors), we demonstrated that fentanyl, a mu-preferring opioid, caused a dose-dependent (EC50 = 16 nM) monophasic increase in inositol (1,4,5)trisphosphate mass formation that peaked at 15 s and returned to basal within 1-2 min. This response was of similar magnitude (25.4 +/- 0.8 pmol/mg of protein for 0.1 microM fentanyl) to that found in the plateau phase (5 min) following stimulation with 1 mM carbachol (18.3 +/- 1.4 pmol/mg of protein), and was naloxone-, but not naltrindole- (a delta antagonist), reversible. Further studies using [D-Ala2, MePhe4, Gly(ol)5]enkephalin and [D-Pen2,5]enkephalin confirmed that the response was specific for the mu receptor. Incubation with Ni2+ (2.5 mM) or in Ca(2+)-free buffer abolished the response, as did pretreatment (100 ng/ml for 24 h) with pertussis toxin (control plus 0.1 microM fentanyl, 26.9 +/- 1.5 pmol/mg of protein; pertussis-treated plus 0.1 microM fentanyl, 5.1 +/- 1.3 pmol/mg of protein). In summary, we have demonstrated a mu-opioid receptor-mediated activation of phospholipase C, via a pertussis toxin-sensitive G protein, that is Ca(2+)-dependent. This stimulatory effect of opioids on phospholipase C, and the potential inositol (1,4,5)trisphosphate-mediated rises in intracellular Ca2+, could play a part in the cellular mechanisms of opioid action.

Topics: Calcium; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Fentanyl; GTP-Binding Proteins; Humans; Inositol 1,4,5-Trisphosphate; Morphine; Narcotic Antagonists; Neuroblastoma; Pertussis Toxin; Receptors, Opioid, mu; Tumor Cells, Cultured; Virulence Factors, Bordetella

Opioids elicit an increase in the intracellular free Ca2+ concentration ([Ca2+]i) in neuroblastoma x glioma hybrid NG108-15 cells, which, depending upon growth conditions, results from either Ca2+ influx in differentiated cells or Ca2+ release from internal stores in undifferentiated cells (Jin et al., 1992). In this report we describe fura-2-based digital imaging studies that demonstrate that opioid-evoked Ca2+ release in these cells results from the activation of phospholipase C (PLC) and subsequent mobilization of the inositol 1,4,5-trisphosphate (IP3)-sensitive store. D-Ala2-D-Leu5-enkephalin (DA-DLE) evoked concentration-dependent increases in [Ca2+]i (EC50 approximately equal to 4 nM). The response was blocked by naloxone (1 microM). In single cells, sequential application of selective opioid agonists (10 nM) evoked responses of the rank order DADLE = D-Pen2, D-Pen5-enkephalin (DPDPE) > trans-(+/-) 3,4-dichloro-N-methyl-N-(2-[1- pyrrolidinyl]cyclohexyl) benzeneacetamide (U50488) > D-ala2, N-Me-Phe4, Gly5-ol-enkephalin (DAMGO), consistent with activation of a delta-opioid receptor. Forty percent (n = 198) of the cells responded to 100 nM DADLE with a net [Ca2+]i increase of 483 +/- 40 nM. Bradykinin (100 nM) elicited a response in 91% of the cells with a mean net amplitude of 707 +/- 36 nM. The DADLE-evoked responses were not blocked by removal of extracellular Ca2+; instead, they were abolished by treatment with 10 nM thapsigargin, an agent that depletes and prevents refilling of IP3-sensitive Ca2+ stores. A 1 microM concentration of U73122, an aminosteroid inhibitor of PLC, completely blocked the DADLE-evoked [Ca2+]i increase, while an inactive analog, U73433, was without effect. To explore the possible role of G-proteins in mediating opioid-induced [Ca2+]i increases in NG108-15 cells, we pretreated cells with pertussis or cholera toxin; pertussis toxin blocked the opioid-induced response while cholera toxin was without effect, consistent with a Gi- or Go-mediated effect. Activation of the opioid inhibitory pathway previously described for these cells appears to stimulate the phosphoinositide (PI) cascade as well. Including the PI cascade among the multiple second messenger systems modulated by opioids may be key to understanding the biochemical events that underlie acute and chronic opioid action.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Calcium; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Fluorescent Dyes; Fura-2; Glioma; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Kinetics; Mice; Microscopy, Fluorescence; Naloxone; Narcotics; Neuroblastoma; Pyrrolidines; Rats; Tumor Cells, Cultured

Functional delta opioid receptors were expressed in Xenopus oocytes following injection of poly(A+)RNA isolated from the mouse neuroblastoma X rat glioma hybrid cell line, NG108-15. Oocytes coinjected with in vitro transcribed beta 2-adrenergic receptor mRNA and NG108-15 cell mRNA had 3-fold elevated cAMP levels following isoproterenol treatment. Application of delta opioids to these oocytes caused dose-dependent inhibition of isoproterenol-induced increase of cAMP, which was antagonized by naltrexone. This is the first demonstration of the expression of opioid receptors in Xenopus oocytes. The ability to assay expression of delta opioid receptors functionally coupled to the inhibition of adenylate cyclase in Xenopus oocytes following injection of exogenous mRNA will provide a system to investigate the relationship of molecular structure to function of opioid receptors.

Topics: Adenylyl Cyclases; Analgesics; Analysis of Variance; Animals; Cyclic AMP; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Female; Glioma; Hybrid Cells; Isoproterenol; Mice; Naltrexone; Neuroblastoma; Oocytes; Poly A; Rats; Receptors, Opioid, delta; RNA; RNA, Messenger; Xenopus laevis

Human neuroblastoma cells (SH-SY5Y) differentiated by retinoic acid exhibited high threshold-activated N-type Ca2+ currents, which were largely inhibited by the two enkephalins, DAGO and DPDPE, as well as by dopamine and somatostatin. The inhibitory effects were fully abolished after pretreatment of cells with pertussis toxin. After washing out the toxin, the inhibitory effects re-established with a time constant of about 16 h. The recovery of Ca2+ current inhibition was similar for all tested agonists. Unexpectedly, we observed a neurotransmitter-induced stimulation of Ca2+ currents in approximately 10% of all investigated cells during the recovery phase. Such a stimulatory effect by otherwise inhibitory receptors was never seen in control cells. It did also not occur when exogenous purified G-proteins of the Gi family were reconstituted via the patch pipette, suggesting that additional mechanisms may play a role in the appearance of stimulatory effects during the recovery phase after pertussis toxin pretreatment.

Topics: Calcium; Cell Differentiation; Dopamine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Humans; Membrane Potentials; Neuroblastoma; Pertussis Toxin; Somatostatin; Tretinoin; Tumor Cells, Cultured; Virulence Factors, Bordetella

In membranes from SH-SY5Y human neuroblastoma cells differentiated with retinoic acid, the mu-selective agonist Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO) inhibited cAMP formation with an IC50 of 26 nM. Two separate antibodies raised against distinct regions of the Go alpha sequence attenuated the effect of DAMGO by 50-60%, whereas antibodies to Gi alpha 1,2 or Gi alpha 3 reduced the mu-opioid signal insignificantly or to a lesser extent. In contrast, inhibition of adenylyl cyclase by the delta-opioid agonist Tyr-D-Pen-Gly-Phe-D-Pen-OH (DPDPE; Pen = penicillamine) was very sensitive to the Gi alpha 1,2 antibody. In membranes from rat brain striatum, coupling of the mu opioid receptor to adenylyl cyclase was also maximally blocked by antibodies to Go alpha. After long-term treatment of the cells with DAMGO, the content of Go alpha was reduced by 26%, whereas the levels of Gi alpha 1,2, Gi alpha 3, and Gs alpha were unaltered. Addition of Go, purified from bovine brain, to membranes from pertussis toxin-treated SH-SY5Y cells restored the inhibition of adenylyl cyclase by DAMGO to 70% of that in toxin-untreated cells. To comparably restore the effect of DPDPE, much higher concentrations of Go were required. By demonstrating mediation of cAMP-dependent signal transduction by Go, these results describe (i) an additional role for this G protein present at a high concentration in brain, (ii) preferential, although not exclusive, interaction of mu and delta opioid receptors with different G protein subtypes in coupling to adenylyl cyclase, and (iii) reduced levels of Go following chronic opioid treatment of SH-SY5Y cells with mu opioids.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclases; Analgesics; Animals; Antibodies; Cell Membrane; Clone Cells; Corpus Striatum; Cyclic AMP; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; GTP-Binding Proteins; Humans; Kinetics; Membrane Proteins; Neuroblastoma; Pertussis Toxin; Rats; Receptors, Opioid, mu; Somatostatin; Tumor Cells, Cultured; Virulence Factors, Bordetella

Topics: Analgesics; Cell Line; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Humans; Kinetics; Naloxone; Naltrexone; Narcotic Antagonists; Neuroblastoma; Receptors, Opioid, mu; Tumor Cells, Cultured

Western-blot analysis of human neuroblastoma SH-SY5Y cells (mu- and delta-receptors) revealed the presence of the following G-protein subunits: Gi alpha 1, Gi alpha 2, Gs alpha, G(o) alpha, Gz alpha, and G beta, a pattern resembling that observed in central nervous tissue. Chronic treatment of differentiated [all-trans-retinoic acid (10 microM; 6 days)] SH-SY5Y cells with D(-)-morphine (10 microM; 3 days) significantly increased the abundance of all G-protein subunits identified. Co-incubation of morphine-exposed cells together with naloxone (10 microM; 3 days) or the mu-selective opioid antagonist CTOP (10 microM; 3 days), but not with the delta-selective antagonist ICI-174,864 (10 microM; 3 days), completely abolished this effect, suggesting that the increase in G-protein abundance is specifically mediated by mu-receptors. Moreover, the biologically inactive enantiomer L(+)-morphine (10 microM; 3 days) failed to produce a similar effect. G-protein up-regulation developed in a time- and dose-dependent manner and is most likely due to enhanced protein synthesis de novo, since concomitant treatment of the cells with cycloheximide (100 micrograms/ml; 3 days) prevented this effect. Chronic treatment with the low-efficacy mu-selective opioid peptide morphiceptin (10 microM; 3 days), but not with the highly potent mu-agonist DAGO (0.1 microM; 3 days) produced a comparable increase in G-protein abundance. Coincident with quantitative effects on G-protein levels in morphine-tolerant/dependent SH-SY5Y cells, we found elevated levels of basal, forskolin (1 microM)- and prostaglandin-E1 (1 microM)-stimulated adenylate cyclase activities. Reconstitution experiments using S49 cyc- lymphoma-cell membranes suggest that this increase is most likely due to elevated levels of functionally intact Gs. Chronic treatment with both morphine and DAGO induces high degrees of tolerance in this cell line. However, the intrinsic activity of G1 was unchanged, as assessed in functional studies with low-nanomolar concentrations of guanosine 5'-[beta gamma- imido]triphosphate. Our data demonstrate that chronic treatment of SH-SY5Y cells with low-efficacy mu-opioids increases G-protein abundance, a phenomenon which might contribute to the biochemical mechanisms underlying opioid tolerance/dependence.

Topics: Adenylyl Cyclases; Amino Acid Sequence; Analgesics; Cell Differentiation; Cell Membrane; Cycloheximide; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; GTP-Binding Proteins; Humans; Molecular Sequence Data; Morphine; Narcotics; Neuroblastoma; Time Factors; Tumor Cells, Cultured; Up-Regulation

Opioids are regarded to act via receptors interacting with heterotrimeric pertussis toxin (PTX)-sensitive G proteins. In membranes of SH-SY5Y cells, the mu-selective agonist [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAGO) and the delta-selective agonist [D-Pen2,Pen5]-enkephalin (DPDPE) stimulated incorporation of the photoreactive GTP analog [alpha-32P]GTP azidoanilide into proteins comigrating with the alpha subunits of G(i1), G(i2), G(i3), G(o1), and another form of G(o), presumably G(o2). In membranes of PTX-treated cells, both agonists were ineffective. Subtype-specific immunoprecipitation of G protein alpha subunits photolabeled in the absence or presence of agonists revealed profound differences between mu and delta opioid receptors in coupling to PTX-sensitive G proteins. Whereas activated delta opioid receptors preferentially coupled to G(i1), activated mu opioid receptors more effectively coupled to G(i3). Additionally, we provide evidence that G(o) subtypes are also differentially activated by the two receptors. Thus, mu and delta opioid receptors appear to discriminate between PTX-sensitive G proteins and lead to activation of distinct G protein subtypes.

Topics: Affinity Labels; Azides; Cell Membrane; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Immunosorbent Techniques; Neuroblastoma; Pertussis Toxin; Photochemistry; Receptors, Opioid, delta; Receptors, Opioid, mu; Tumor Cells, Cultured; Virulence Factors, Bordetella

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

In a previous study, we showed that microM concentrations of mu or delta opioid agonists increase voltage-dependent outward K+ currents in neuroblastoma x DRG neuron hybrid F11 cells via pertussis toxin-sensitive receptors. The present study demonstrates that much lower concentrations (fM to nM) of these opioids (DAGO and DPDPE) decreased voltage-dependent outward K+ currents during step depolarization. The opioid antagonist, naloxone (3 nM) prevented these decreases in K+ current as did the cholera toxin subunits A or B (ca. 1 nM). Furthermore, the specific mu opioid receptor antagonist, beta-funaltrexamine (5 nM) blocked the decrease by DAGO and the specific delta antagonist, naltrindole (1 nM) blocked that by DPDPE. Acute GM1 ganglioside (1 microM) treatment markedly enhanced the efficacy of opioid-induced decrease in K+ current. After treating the cells with pertussis toxin (1 microgram/ml) for 2 days or more, these opioids decreased the K+ current even when tested at concentrations as high as 1 microM. These results indicate that the decrease in K+ current elicited in F11 cells by low concentrations of mu and delta opioid agonists resembles the opioid-induced prolongation of the action potential duration and decrease in voltage-dependent K+ conductance that occur in DRG neurons in primary cultures. The F11 cell line provides therefore a valuable model system for correlative pharmacologic, electrophysiologic and biochemical analyses of Gs-coupled, GM1 ganglioside-regulated excitatory opioid receptor functions, in addition to G(i)/G(o)-coupled inhibitory receptor functions, in sensory neurons.

Topics: Animals; Cholera Toxin; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Ganglia, Spinal; Hybrid Cells; Membrane Potentials; Neuroblastoma; Neurons; Pertussis Toxin; Potassium Channels; Rats; Receptors, Opioid, delta; Receptors, Opioid, mu; Virulence Factors, Bordetella

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, delta-selective; DAMGO, mu-selective) as blocking reagents. Labeling of the cells was examined using conventional fluorescence microscopy. Co-localization of mu- and delta-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. Among 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 mu- and delta- binding sites reside on different subunits of a multimeric complex.

Topics: Antibodies, Anti-Idiotypic; Binding, Competitive; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Humans; Microscopy, Fluorescence; Neuroblastoma; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Tumor Cells, Cultured

Human neuroblastoma cells were tested for the presence of opioid receptors. [3H]Diprenorphine binds to NMB cell membranes with a KD value of 0.46 +/- 0.13 nM and Bmax of 534 +/- 22 fmol/mg protein. The presence of mu, delta, and kappa opioid receptors was tested by displacing [3H]diprenorphine specific binding by the selective agonists DAMGO, DPDPE, and U50,488H, respectively. Using this procedure, the data suggest that the NMB neuroblastoma cells express the three opioid receptor types with the abundance of delta receptors (about 60%) and minor, yet substantial populations of mu and kappa receptors (about 20% each).

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Binding, Competitive; Diprenorphine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Humans; Kinetics; Neuroblastoma; Pyrrolidines; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Sensitivity and Specificity; Tritium; Tumor Cells, Cultured

Human neuroblastoma SK-N-SH cells, which contain both mu- and delta-opioid receptors, were grown under conditions that provided a mu:delta ratio of 1.5:1. Both receptors were down-regulated after 72 hr of exposure to 100 nM etorphine. Selective down-regulation was demonstrated using selective opioid agonists; the mu agonist Tyr-D-Ala2-Gly-(Me)Phe4-Gly-ol down-regulated mu- but not delta-opioid receptors, whereas prolonged exposure to the selective delta agonist D-Pen2,D-Pen5-enkephalin resulted in delta- but not mu-opioid receptor down-regulation. Morphine, which binds mu- as well as delta-opioid receptors, down-regulated both receptor subtypes. NG108-15 cells, which contain delta receptors exclusively, were also tested. NG108-15 cells did not exhibit delta-opioid receptor down-regulation when exposed to morphine. The discrepancy between the effect of chronic morphine treatment on delta receptors in SK-N-SH cells and in NG108-15 cells raised the question of whether the coexistence of mu receptors in the former allowed morphine to down-regulate delta receptors. The role of mu-opioid receptors in morphine-induced delta receptor down-regulation was studied by using the irreversible mu antagonist beta-funaltrexamine. Pretreatment of SK-N-SH cells with beta-funaltrexamine prevented down-regulation of delta receptors in response to chronic exposure to morphine but did not affect down-regulation of delta receptors in response to D-Pen2,D-Pen5-enkephalin. The experimental data indicate that morphine-induced delta-opioid receptor down-regulation is dependent on the presence of functional mu receptors in the same cell.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Down-Regulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Humans; Morphine; Naltrexone; Neuroblastoma; Pyrrolidines; Receptors, Opioid, delta; Receptors, Opioid, mu; Tumor Cells, Cultured

The F11 cell line is a fusion product of cells of mouse neuroblastoma cell line N18TG-2 with embryonic rat dorsal-root ganglion (DRG) neurons. Previous biochemical results suggest that they express mu- and delta-opioid receptors that are negatively coupled to adenylate cyclase. The present study provides direct agonist-binding and electrophysiologic evidence of mu and delta, but not kappa, receptor expression in F11 cells. Radioligand binding assays show that F11 cell membranes bind the mu- and delta-opioid receptor agonists, DAGO and DPDPE with Kd = 4.5 and 4.9 nM and Bmax = 111 and 195 fmol/mg, respectively. Tight-seal patch-clamp recordings of F11 cells after several days in a differentiating culture medium (low serum, cyclic AMP and nerve growth factor) showed that: (i) the outward K+ current during pulsed depolarization in most of these cells was increased by either DAGO or DPDPE, but none were responsive to both opioids or to the kappa-opioid receptor agonist, U-50,488H. The response was blocked by relevant receptor antagonists, naloxone, beta-funaltrexamine or naltrindole; (ii) cells without processes responded neither to DAGO nor to DPDPE; (iii) treatment with pertussis toxin blocked all opioid-induced increases in outward K+ current. The opioid-induced increase in voltage-dependent membrane K+ current in F11 cells resembles the inhibitory effect elicited by mu- and delta-opioid agonists in primary cultures of mouse DRG neurons.

Topics: Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Hybrid Cells; Mice; Neuroblastoma; Neurons; Potassium Channels; Radioligand Assay; Rats; Receptors, Opioid, delta; Receptors, Opioid, mu; Spinal Cord

The intracellular free calcium concentration ([Ca2+]i) was measured in single NG108-15 cells using indo-1-based microfluorimetry. In cells differentiated for 6-14 days in serum-free, forskolin (5 microM)-supplemented medium, application of micromolar concentrations of [D-Ala2,D-Leu5]-enkephalin (DADLE) inhibited Ca2+ influx mediated by voltage-gated Ca2+ channels. DADLE, at concentrations ranging from 1 nM to 1 microM, also produced rapid transient increases in [Ca2+]i (EC50 = 10 nM). The [Ca2+]i increases elicited by DADLE did not correlate with the inhibitory effects of the peptide. DADLE-induced [Ca2+]i increases were blocked by naloxone. In single cells, sequential application of selective opioid agonists (30 nM) evoked responses of the rank order DADLE = [D-Pen2,D-Pen5]-enkephalin > (trans)-(+-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl) benzeneacetamide > [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, consistent with activation of a delta-opioid receptor. The response was completely blocked by removal of extracellular Ca2+ or application of 1 microM nitrendipine, indicating that the increase in [Ca2+]i results from Ca2+ influx via dihydropyridine-sensitive, voltage-gated Ca2+ channels. Substitution of N-methyl-D-glucamine for extracellular Na+ or application of 1 microM tetrodotoxin greatly reduced, and in some cases blocked, the DADLE-induced [Ca2+]i increase, consistent with amplification of the response by voltage-gated Na+ channels. The [Ca2+]i increase was mimicked by both dibutyryl-cAMP and phorbol 12,13-dibutyrate. These findings are consistent with a delta-opioid-induced depolarization, possibly mediated by a second messenger, that subsequently recruits voltage-sensitive Ca2+ channels. In contrast to differentiated cells, undifferentiated cells responded to DADLE with a modest [Ca2+]i increase that was not sensitive to nitrendipine. In these cells, activation of the same second messenger system may elevate [Ca2+]i by mobilization from intracellular stores rather than influx. In addition to previously described inhibitory coupling to adenylyl cyclase and Ca2+ channels in NG108-15 cells, these results suggest that a novel, excitatory, effector system may also couple to opioid receptors.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Bucladesine; Calcium; Calcium Channels; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Glioma; Hybrid Cells; Ion Channel Gating; Narcotics; Neuroblastoma; Phorbol 12,13-Dibutyrate; Pyrrolidines; Receptors, Opioid, delta; Tumor Cells, Cultured

In SK-N-SH human neuroblastoma cells, the muscarinic agonist carbachol promotes polyphosphoinositide (PPI) hydrolysis via M3 receptors and increases cyclic AMP levels through an unidentified mechanism. Activation of PPI hydrolysis by carbachol elicits a robust translocation of CaM from membranes into cytosol which was previously shown to be mimicked by the addition of the calcium ionophore ionomycin and the phorbol ester TPA28. The effect of agonist-stimulated second messenger production on CaM localization was determined by activating receptors that increase and decrease adenylyl cyclase activity on SK-N-SH cells. VIP (10 microM), prostaglandin E1 (30 microM) and forskolin (10 microM) all increased adenylyl cyclase activity 8- to 10-fold above the activity with 1 microM GTP. Carbachol (100 microM) did not stimulate adenylyl cyclase activity. The alpha 2-adrenergic agonist UK 14,304 (0.1 microM) and the delta and mu opioid DPDPE (10 microM) and DAMGO (10 microM) inhibited forskolin-stimulated cyclic AMP formation by 27-32%. CaM did not stimulate adenylyl cyclase activity. Incubation of cells with vasoactive intestinal polypeptide (VIP), dibutyryl cyclic AMP and forskolin, resulted in 30% decrease in membrane CaM and an increase in cytosolic CaM of 40-50%. The CaM translocation with the combination of an agent that elevates cyclic AMP levels and a low dose of carbachol was not different from that observed with either agent alone. UK 14,304, DPDPE and DAMGO potentiated carbachol-stimulated increases in cytosolic CaM. Upon the addition of carbachol, a 5-fold increase in intracellular calcium concentration measured with fura-2 fluorescence was observed. VIP and UK 14,304 elevated intracellular calcium concentrations 2 to 3 fold, while forskolin (10 microM) had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenylyl Cyclases; Adrenergic alpha-Agonists; Alprostadil; Analgesics; Brimonidine Tartrate; Bucladesine; Calcium; Calmodulin; Carbachol; Cell Line; Colforsin; Cyclic AMP; Cytosol; Edetic Acid; Egtazic Acid; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Humans; Kinetics; Neuroblastoma; Quinoxalines; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

We have measured mu and delta opioid receptor sites on intact SK-N-SH and NG108-15 neuroblastoma cells, respectively, in culture. Use of 125I-beta-endorphin (beta E) as a tracer, together with beta E(6-31) to block high-affinity non-opioid binding in both cell lines, permitted the measurement of cell surface mu and delta opioid receptor sites. Labeling was at delta sites in NG108-15 cells and predominantly at mu sites in SK-N-SH cells. Pretreatment with the mu and delta agonist, DADLE, caused a rapid loss of cell surface delta receptor sites in NG108-15 cells, but failed to reduce significantly mu receptor density in SK-N-SH cells.

Topics: Analgesics; Animals; beta-Endorphin; Binding Sites; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Humans; Mice; Neuroblastoma; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Tumor Cells, Cultured

The relationship between muscarinic receptor activation of phosphoinositide hydrolysis and the sequestration of cell surface muscarinic receptors has been examined for both intact and digitonin-permeabilized human SK-N-SH neuroblastoma cells. Addition of the aminosteroid 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1H-pyrrole-2,5-dione (U-73122) to intact cells resulted in the inhibition of oxotremorine-M-stimulated inositol phosphate release and of Ca2+ signaling by greater than 75%. In contrast, when phospholipase C was directly activated by the addition of the calcium ionophore ionomycin, inclusion of U-73122 had little inhibitory effect. Addition of U-73122 to intact cells also inhibited the agonist-induced sequestration of cell surface muscarinic receptors and their subsequent down-regulation with an IC50 value (4.1 microM) similar to that observed for inhibition of inositol phosphate release (3.7 microM). In contrast, when oxotremorine-M-stimulated phosphoinositide hydrolysis was inhibited by depletion of extracellular Ca2+, no reduction in the extent of receptor sequestration was observed. When introduced into digitonin-permeabilized cells, U-73122 more markedly inhibited inositol phosphate release elicited by either oxotremorine-M or guanosine-5'-O-(3-thiotriphosphate) than that induced by added Ca2+. Addition of oxotremorine-M to permeabilized cells resulted in muscarinic receptor sequestration and down-regulation. Both the loss of muscarinic acetylcholine receptors and activation of phosphoinositide hydrolysis in permeabilized cells were inhibited by the inclusion of guanosine-5'-O-(2-thiodiphosphate). The results indicate that the agonist-induced sequestration of muscarinic acetylcholine receptor in SK-N-SH cells requires the involvement of a GTP-binding protein but not the production of phosphoinositide-derived second messenger molecules.

Topics: Alprostadil; Cell Line; Colforsin; Cyclic AMP; Dose-Response Relationship, Drug; Down-Regulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Estrenes; GTP-Binding Proteins; Humans; Inositol; Kinetics; Neuroblastoma; Oxotremorine; Phosphatidylinositols; Pyrrolidinones; Receptors, Muscarinic; Scopolamine; Type C Phospholipases

The human neuroblastoma clonal cell line SH-SY5Y expresses both mu- and delta-opioid receptors (ratio approximately 4.5:1). Differentiation with retinoic acid (RA) was previously shown to enhance the inhibition of adenylyl cyclase (AC) by mu-opioid agonists. We tested here the inhibition of cyclic AMP (cAMP) accumulation by morphine under a variety of conditions: after stimulation with prostaglandin E1 (PGE1), forskolin, and vasoactive intestinal peptide (VIP), both in the presence and in the absence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Morphine inhibition of the forskolin cAMP response (approximately 65%) was largely unaffected by the presence of IBMX. In contrast, deletion of IBMX enhanced morphine's inhibition of the PGE1 and VIP cAMP response from approximately 50 to approximately 80%. The use of highly mu- and delta-selective agents confirmed previous results that inhibition of cAMP accumulation by opioids is mostly mu, and not delta, receptor mediated in SH-SY5Y cells, regardless of the presence or absence of IBMX. Because of the large morphine inhibition and the high cAMP levels even in the absence of IBMX, PGE1-stimulated, RA-differentiated SH-SY5Y cells were subsequently used to study narcotic analgesic tolerance and dependence in vitro. Upon pretreatment with morphine over greater than or equal to 12 h, a fourfold shift of the PGE1-morphine dose-response curve was observed, whether or not IBMX was added. However, mu-opioid receptor number and affinity to the mu-selective [D-Ala2, N-Me-Phe4, Gly5-ol]enkephalin were largely unaffected, and Na(+)- and guanyl nucleotide-induced shifts of morphine-[3H]naloxone competition curves were unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 1-Methyl-3-isobutylxanthine; Alprostadil; Cell Line; Colforsin; Cyclic AMP; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Humans; Kinetics; Morphine; Neuroblastoma; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Theophylline; Vasoactive Intestinal Peptide

The endogenous opioids and their receptors are known to play a major role in neoplasia. In the present study, naltrexone (NTX), a potent opioid antagonist, was utilized to explore the interactions of opioids and opioid receptors in mice with transplanted neuroblastoma (S20Y). Tumors from mice subjected to either intermittent (4-6h/day; 0.1 mg/kg NTX) or complete (24 h/day; 10 mg/kg NTX) opioid receptor blockade exhibited an up-regulation of DADLE and Met-enkephalin binding sites, as well as tissue levels of beta-endorphin and Met-enkephalin. Binding affinity to [D-Ala2,D-Leu5]enkephalin (DADLE) or ethylketocyclazocine (EKC), the levels of plasma beta-endorphin, and the anatomical location and quantity of Met- and Leu-enkephalin and cytoskeletal components (i.e. tubulin, actin, brain spectrin (240/235) were similar in NTX and control tumor-bearing animals. Tissue viability of the 0.1 NTX group was increased compared to controls. Both mitotic and labeling indexes were increased during the period of opioid receptor blockade, but decreased in the period subsequent to receptor blockade. NTX treatment produced a 2-fold increased in sensitivity to opioids. Met-enkephalin (10 mg/kg) produced a depression in both mitotic and labeling indexes in tumor-bearing mice that could be reversed by naloxone (10 mg/kg) administration. Thus, the endogenous opioids are trophic agents that inhibit growth by suppressing cell proliferation. The duration of receptor blockade by opioid antagonists modulates these actions, affecting both tumor incidence and survival time. Complete opioid receptor block prevents the interaction of increased levels of putative growth-related peptides with a greater number of opioid receptors, thereby increasing cell proliferation and accelerating tumor growth. With intermittent blockade, an enhanced opioid-receptor interaction occurs during the interval when the opioid antagonist is no longer present, producing an exaggerated inhibitory action on cell proliferation and the repression of tumorigenic events.

Topics: Animals; Cell Division; Cell Line; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Male; Mice; Naltrexone; Neoplasms, Experimental; Neuroblastoma; Receptors, Opioid

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 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 binding of [3H] [D-Ala2, MePhe4, Gly-ol5]enkephalin ([3H]DAGO), [3H]D-Ala2,D-Leu5]enkephalin ([3H]DADLE) and (+/-)-[3H]ethylketocyclazocine ([3H]EKC) to neurotumor tissues derived from S20Y neuroblastoma cells transplanted into A/Jax mice was examined. Specific and saturable binding to [3H]DADLE and [3H]EKC was detected, and the data fit a single homogeneous binding site for each ligand. Scatchard analysis for [3H]DADLE and [3H]EKC yielded Kd values of 0.65 and 0.45 nM, respectively, and Bmax values of 9.2 and 116 fmol/mg protein. Binding was dependent on time, temperature, and pH, and was sensitive to Na+ and guanine nucleotides. Pretreatment of the tumor homogenates with trypsin markedly reduced binding to both ligands, suggesting that the binding sites were proteinaceous in character. Displacement experiments indicated that delta (delta) receptor related compounds (e.g. DPDPE, ICI 174,864) avidly displaced [3H]DADLE, whereas kappa (kappa) related compounds (e.g. U50,488, dynorphin) markedly competed with [3H]EKC. Mu (mu) receptor drugs (e.g. DAGO, beta-FNA, morphine) were not potent in displacing either [3H]DADLE or [3H]EKC. These results are the first to characterize opioid binding sites in tumor tissue. The function of these sites is unclear, but previous evidence as to the growth regulatory properties of endogenous opioid systems may suggest that either one, or both, binding sites may be involved in carcinogenic events.

Topics: Analgesics, Opioid; Animals; Binding, Competitive; Cell Line; Clone Cells; Cyclazocine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Ethylketocyclazocine; Kinetics; Ligands; Mice; Neuroblastoma; Receptors, Opioid

The characteristics of mu and delta opioid receptor sites present in human neuroblastoma SH-SY5Y cells were investigated using [D-Ala2-N-methyl-Phe4-Gly-(01)5]enkephalin (DAGO) and [2-D-penicillamine, 5-D-penicillamine]enkephalin (DPDPE), which are the most selective radioligands available for mu and delta sites, respectively. Scatchard analysis of the saturation isotherms revealed high affinity binding to a single class of sites for both [3H]DAGO (mu) and [3H]DPDPE (delta). [3H]DAGO labeled twice the number of sites compared to the binding capacity of [3H]DPDPE, yielding a mu/delta ratio of 2:1. Selective suppression of [3H]diprenorphine binding by specific opioid "blocking" ligands also showed a predominance of mu receptors, representing 65-70% of the total opioid sites. Competition binding studies carried out with a series of opiates and opioid peptides displayed higher potencies of mu- and delta-selective ligands in displacing the specific binding of [3H]DAGO and [3H]DPDPE, respectively. The [3H]diprenorphine/agonist competition curves were biphasic, indicating the high and low affinity states of mu and delta receptor sites in SH-SY5Y cells. Guanine nucleotide and sodium had differential effects on the agonist affinity and the proportion of high affinity states of mu and delta receptors. The mu and delta receptor sites were shown to be functionally coupled to adenylate cyclase. All of these data support the independent existence of mu and delta receptor types in human neuroblastoma cells. SH-SY5Y cells, therefore, represent a suitable model for investigating opioid-mediated responses in nerve cell populations.

Topics: Binding, Competitive; Cell Line; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Humans; Kinetics; Narcotics; Neuroblastoma; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu

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.

Topics: Bone Neoplasms; Cell Line; Child, Preschool; Diprenorphine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Etorphine; Female; Guanylyl Imidodiphosphate; Humans; Kinetics; Naloxone; Neuroblastoma; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Sodium Chloride

The effects of varying the sodium concentration (at constant ionic strength) on opioid binding at mu- and delta-opioid receptors in 7315c and NG108-15 cells has been examined. The binding of [3H]etorphine to mu-receptors on 7315c cells was increased by replacing the sodium in the incubation medium with potassium or N-methyl-D-glucamine. This effect was shown to be attributable to an increase in affinity, with no change in the maximum number of binding sites, both in cell membrane suspensions and in intact 7315c cells. Replacement of sodium with potassium or N-methyl-D-glucamine in NG108-15 membrane or intact cell suspensions also resulted in an increase in [3H]etorphine binding, but in these cells the effect was associated with an increase in the number of binding sites measurable under these experimental conditions. The effects of sodium on opioid inhibition of adenylate cyclase in membrane preparations from 7315c and NG108-15 cells also differed. Sodium reduced apparent agonist affinity in 7315c membranes. In NG108-15 cell membranes, sodium was essential for the demonstration of opioid inhibition of cyclase activity. Increasing the sodium concentration above 0.5 mM resulted in an increase in the fraction of total enzyme activity inhibited by opioid, but the opioid IC50 did not change. In the companion paper, it is shown that the effects of sodium removal on mu- and delta-receptor binding in guinea pig brain neural membranes were similar to those observed in the cell preparations. An increase in intracellular sodium concentration without change in extracellular concentration was effected by incubation of 7315c and NG108-15 cells with the sodium-selective ionophore, monensin. When sodium was present in the extracellular medium, monensin reduced [3H]etorphine binding by 50% or more, both at mu-receptors in 7315c cells and at delta-receptors in NG108-15 cells. In the absence of sodium, however, monensin treatment produced only a small inhibition of binding. These results suggest that sodium acts at an intracellular site to regulate opioid agonist binding at both mu- and delta-receptors, but that the mode of regulation is not identical at each site. Since a reduction in intracellular sodium concentration by removal of extracellular sodium increases agonist binding, and an increase in intracellular sodium following monensin treatment reduces agonist binding, it is probable that the intracellular sodium concentration is a critical regulator of opioid agonist b

Topics: Adenylyl Cyclase Inhibitors; Animals; Cell Line; Cell Membrane; Cyclazocine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Ethylketocyclazocine; Etorphine; Glioma; Guinea Pigs; Monensin; Naloxone; Neuroblastoma; Pituitary Neoplasms; Rats; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Sodium