Opioid Signal Transduction in Intact and Fragmented SH‐SY5Y Neural Cells

Carter, Bruce D.; Medzihradsky, Fedor

doi:10.1111/j.1471-4159.1992.tb10032.x

Cited by 11 publications

(18 citation statements)

References 31 publications

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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: 97%

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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Section: Figmentioning

confidence: 97%

μ Opioid Receptor Phosphorylation, Desensitization, and Ligand Efficacy

Zhang

Yin

et al. 1997

Journal of Biological Chemistry

201

142

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“…The results provide direct evidence for an additional function ofGo in cAMP-dependent signal transduction. A preliminary account of these findings has been presented (28).…”

mentioning

confidence: 94%

“…Recently, we have quantified the functional responses of G protein and adenylyl cyclase in normal (26) and opioid-tolerant (27) SH-SY5Y neural cells. In the present study, we identify G-protein subtypes that modulate the coupling of ,u and 8 opioid receptors to adenylyl cyclase.…”

mentioning

confidence: 99%

“…The surface-growing cells were rinsed with phosphate-buffered saline and lifted by incubation with a Ca2+-and Mg2+-free modified Puck's solution for 10 min, and the membranes were isolated as described (26). The striatal region from rat brain was dissected at 4°C.…”

mentioning

confidence: 99%

“…The striatal region from rat brain was dissected at 4°C. The tissue was homogenized and further treated as described for the SH-SY5Y cells (26). The membranes from both sources were used immediately after isolation.…”

mentioning

confidence: 99%

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Go mediates the coupling of the mu opioid receptor to adenylyl cyclase in cloned neural cells and brain.

Carter

Medzihradsky

1993

Proc. Natl. Acad. Sci. U.S.A.

104

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In membranes from SH-SY5Y human neuroblastoma cells differentiated with retinoic acid, the ,u-selective agonist Tyr-D-Ala-Gly-N-Me,-Phe-Gly-ol (DAMGO) inhibited cAMP formation with an IC5o of 26 nM. Two separate antibodies raised against distinct regions of the Go. sequence attenuated the effect of DAMGO by 50-60%, whereas antibodies to Gial,2 or G1a3 reduced the ,u-opioid signal insignificantly or to a lesser extent. In contrast, inhibition of adenylyl cyclase by the 8-opioid agonist Tyr-D-Pen-Gly-Phe-D-Pen-OH (DPDPE; Pen = peniciliamine) was very sensitive to the Gial,2 antibody. In membranes from rat brain striatum, coupling of the 1t opioid receptor to adenylyl cyclase was also maximally blocked by antibodies to G... After long-term treatment of the cells with DAMGO, the content of Goa was reduced by 26%, whereas the levels of G1.1,2, G1.3, and Gsa were unaltered.

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Modulation of opioid signal transduction in SH-SY5Y neural cells by differentiating agents: Concurrent mu receptor upregulation and effector desensitization by phorbol ester

Lin

Carter

Haas

et al. 1994

Regulatory Peptides

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Long-term exposure of SH-SY5Y human neural cells to retinoic acid (RA) increased the binding of [3H] DAMGO and [3H]DPDPE by up to 3-fold compared to membranes of untreated cells. In contrast, incubation of the cells with phorbol ester (TPA) selectively up-regulated the binding of [3H]DAMGO. RA enhanced the maximal inhibition of cAMP formation in SH-SY5Y cells by both DAMGO and DPDPE from 20% for both opioids in control cells to 75% and 50%, respectively. On the other hand, the effect of TPA was limited to a marginal increase in the inhibition of cAMP formation by DAMGO. Parameters of GTP3PsS binding as well as low-Km GTPase activity revealed no deterioration in the structure or function of total G protein in the TPA-treated cells, and initial Western blots showed no difference in the cell content of Go. Ongoing experiments are focusing on the covalent modification of specific G protein subtypes involved in # and ~ opioid signal transduction in SH-SY5Y cells differentiated by TPA. METHODS:Human neuroblastoma cells SH-SY5Y were cultured and differentiated with RA or TPA for 9-11 days as described (1). Ligand binding was resolved and quantified by computer fitting using the statistical program SYSTAT, and the activities of adenylyl cyclase and GTPase were determined by a radioligand binding assay and measurement of released 32p, respectively (2). Binding of GTP3Pss (3), Western blotting (4), and the determination of protein (3) were implemented as described previously.

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Opioid Signal Transduction in Intact and Fragmented SH‐SY5Y Neural Cells

Cited by 11 publications

References 31 publications

μ Opioid Receptor Phosphorylation, Desensitization, and Ligand Efficacy

μ Opioid Receptor Phosphorylation, Desensitization, and Ligand Efficacy

Go mediates the coupling of the mu opioid receptor to adenylyl cyclase in cloned neural cells and brain.

Modulation of opioid signal transduction in SH-SY5Y neural cells by differentiating agents: Concurrent mu receptor upregulation and effector desensitization by phorbol ester

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