Regulation of Cyclic AMP by the μ‐Opioid Receptor in Human Neuroblastoma SH‐SY5Y Cells

SUMMARYIn C 6 glial cells stably expressing rat -opioid receptor, opioid agonist activation is negatively coupled to adenylyl cyclase through pertussis toxin-sensitive G proteins. H]DAMGO binding in membranes with the rank order of etorphine Ͼ DAMGO ϭ ␤-endorphin Ͼ morphine Ͼ butorphanol, and the affinity of DAMGO in alkaloid-but not peptide-treated membranes was significantly lower in comparison with control. Pertussis toxin treatment of the cells before agonist treatment did not prevent the down-regulation by full agonists; DAMGO and etorphine exhibited ϳ80% internalization, whereas the ability of partial agonists was greatly impaired. In addition to establishing this cell line as a good model for further studies on the mechanisms of opioid tolerance, these results indicate important differences in the inactivation pathways of receptor triggered by full and partial agonists.Opioid receptors are activated by endogenous opioid peptides and alkaloids, which cause a multitude of important physiological functions. Recent cloning of -, ␦-, and -opioid receptors showed that these proteins contain seven transmembrane domains and belong to the family of GPCRs (1). The -opioid receptor is the molecular target for potent analgesics such as morphine and fentanyl, which are indispensable in the management of pain despite their abuse potential (2). The biochemical mechanisms of tolerance have been studied in many systems, including cell lines containing ␦-opioid receptors such as N4TG1 (3) and NG108 -15 (4) cells. Although studies conducted in the central nervous system often led to inconsistent results due to the heterogeneity of the system, experiments carried out in a single brain region, such as locus ceruleus, demonstrated the physiological relevance of the cellular model originally proposed by Sharma et al. (5) in NG108 -15 cells based on the alterations in the opioid/AC system. Subsequent studies using 7315c (6) and SH-SY5Y (7, 8) cells examined altered properties of -opioid receptor/effector components during tolerance; however, the exact mechanisms involved in this process are largely unknown. To study the molecular mechanisms of -opioid receptor selectively, we transfected C 6 glial cells that express many other receptors, but not opioid receptors (9), with the rat receptor cDNA. Transfected receptor in these cells is coupled to AC through PTX-sensitive G proteins (10). We characterized opioid agonist efficacies (11) and showed that this cell line exhibits sodium regulation of receptor in much the same fashion as SH-SY5Y cells (12).The major goal of the current study was to investigate the molecular changes involved in the development of tolerance by different agonists of varying efficacies. In the C 6 cell line stably expressing high levels of receptor (ϳ8 pmol/mg), tolerance to peptides and alkaloids was induced, and alterations were examined at every step of the signal transduction pathway (i.e., ligand/receptor interactions, G protein and effector functions). The diminished receptor activation of G protein, ...

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“…2). In SH-SY5Y cells, a 4-fold shift was produced by prolonged morphine exposure (15). In addition, as depicted in Fig.…”

Section: Resultsmentioning

confidence: 78%

Down-regulation of μ-Opioid Receptor by Full but Not Partial Agonists Is Independent of G Protein Coupling

Yabaluri

Medzihradsky

1997

Mol Pharmacol

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“…Desensitization of receptors, observed in systems including CHO cells, Xenopus oocytes, SHSY5Y cells, and locus coeruleus neurons (1,(25)(26)(27)(28), appears unlikely to require a kinase of limited distribution. It is conceivable that a widely distributed member of the G-linked receptor kinase family, or another broadly-distributed kinase, could be responsible for agonist-induced receptor phosphorylation.…”

Section: Figmentioning

confidence: 99%

μ Opioid Receptor Phosphorylation, Desensitization, and Ligand Efficacy

Zhang

Yin

et al. 1997

Journal of Biological Chemistry

201

142

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Opioid receptors are G-protein coupled receptors that mediate the potent analgesic actions and addictive properties of morphine-derived compounds. Under physiological conditions, these receptors interact with endogenous opioid peptides to modulate pain-controlling pathways and circuits that modulate behaviors including mood and reward (2). opioid receptors interact with rapidly acting opioid drugs, such as heroin, to produce marked euphoria and behavioral reward. They are also primary targets of the slower and longer acting opioids, such as methadone and LAAM, 1 that represent the best current substitution therapeutics for opiate addiction (3).receptors desensitize after repeated stimulation by opioid agonists, in fashions that display similarities to desensitizing events noted for other G-protein coupled receptors. Agonistinduced receptor desensitization can be correlated with receptor phosphorylation. receptors display naloxone-reversible phosphorylation and desensitize after morphine or DAMGO treatments (1). Both of these agonist-induced events are insensitive to pretreatments with the protein kinase C inhibitor staurosporine, which inhibits phorbol ester-induced receptor phosphorylation and desensitization.Many opiates and opioid ligands can recognize receptors with high affinities. Plant-derived alkaloids, synthetic compounds of several classes, and endogenous opioid peptides can function as agonists, partial agonists, or antagonists with a range of abilities to induce or block induction of analgesia and euphoria. However, mutagenesis studies and studies with receptor chimeras support the idea that different receptor features could be involved in recognition of these different ligand classes (4 -6). Mutations that change naloxone from a full antagonist to a partial agonist can leave the intrinsic activity of opioid peptides unchanged, for example see Claude et al. (7). These differences raise the possibility that receptor occupancies by opioid drugs of different classes could alter the conformation of the receptor in distinct fashions. Some of these differences could render the receptor an improved or a worse substrate for kinases and phosphatases and thus directly confer different susceptibilities to phosphorylation or dephosphorylation. Alternatively, selective activation of different G-protein classes by different agonists could trigger different receptor phosphorylation and desensitization events.The current study thus investigates the effects of opioid ligands of various classes and varying intrinsic activities on receptor activation, phosphorylation, and desensitization using human receptors expressed in CHO cells (hCHO), and the receptors coexpressed in Xenopus oocytes with a G-protein linked K ϩ channel. The results support striking parallels between opioid efficacies in opening ion channels and inhibiting

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“…Chronic activation of opioid receptors in NG108-15 neuroblastoma x glioma cell line (containing g-receptors), as well as in the human neuroblastoma SH-SY5Y (containing mainly preceptors and some g-receptors), followed by the withdrawal of the opioid agonist, was shown to lead to AC supersensitivity ('overshoot') [4][5][6][7][8]. It has been suggested that this phenomenon represents a possible cellular adaptation mechanism associated with chronic opioid exposure.…”

Section: Introductionmentioning

confidence: 99%

κ‐Opioid receptor‐transfected cell lines: modulation of adenylyl cyclase activity following acute and chronic opioid treatments

et al. 1995

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Regulation of Cyclic AMP by the μ‐Opioid Receptor in Human Neuroblastoma SH‐SY5Y Cells

Cited by 95 publications

References 15 publications

Down-regulation of μ-Opioid Receptor by Full but Not Partial Agonists Is Independent of G Protein Coupling

Down-regulation of μ-Opioid Receptor by Full but Not Partial Agonists Is Independent of G Protein Coupling

μ Opioid Receptor Phosphorylation, Desensitization, and Ligand Efficacy

κ‐Opioid receptor‐transfected cell lines: modulation of adenylyl cyclase activity following acute and chronic opioid treatments

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