Neuroblastoma Neuro2A cells stably expressing a cloned μ-opioid receptor: a specific cellular model to study acute and chronic effects of morphine

doi:10.1016/0169-328X(95)00014-J

Molecular Brain Research

Volume 30, Issue 2, June 1995, Pages 269-278

https://doi.org/10.1016/0169-328X(95)00014-J Get rights and content

Abstract

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 μ-opioid receptors, the receptors at which morphine and related opioids act, has been lacking. To approach this problem, the μ-opioid receptor (MOR-1) was stably expressed in murine neuroblastoma Neuro_2A 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 μ-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 μ-opioid receptors, providing an ideal cellular model to study the molecular mechanisms underlying chronic; morphine treatment.

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Cited by (53)

Modulating μ-opioid receptor phosphorylation switches agonist-dependent signaling as reflected in PKCε activation and dendritic spine stability
2011, Journal of Biological Chemistry
Citation Excerpt :
Morphine induces maximum ERK phosphorylation and adenylyl cyclase inhibition at 1 μm (20). Considering the relative affinity of the four agonists for OPRM1 and their abilities to induce maximum ERK phosphorylation and adenylyl cyclase inhibition (4, 22), DAMGO was used at 1 μm, whereas etorphine and fentanyl were used at 10 nm, to achieved equivalent concentrations. At these concentrations, the agonists induce ERK phosphorylation and adenylyl cyclase inhibition to similar levels.
A new role of G protein-coupled receptor (GPCR) phosphorylation was demonstrated in the current studies by using the μ-opioid receptor (OPRM1) as a model. Morphine induces a low level of receptor phosphorylation and uses the PKCϵ pathway to induce ERK phosphorylation and receptor desensitization, whereas etorphine, fentanyl, and [d-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAMGO) induce extensive receptor phosphorylation and use the β-arrestin2 pathway. Blocking OPRM1 phosphorylation (by mutating Ser³⁶³, Thr³⁷⁰ and Ser³⁷⁵ to Ala) enabled etorphine, fentanyl, and DAMGO to use the PKCϵ pathway. This was not due to the decreased recruitment of β-arrestin2 to the receptor signaling complex, because these agonists were unable to use the PKCϵ pathway when β-arrestin2 was absent. In addition, overexpressing G protein-coupled receptor kinase 2 (GRK2) decreased the ability of morphine to activate PKCϵ, whereas overexpressing dominant-negative GRK2 enabled etorphine, fentanyl, and DAMGO to activate PKCϵ. Furthermore, by overexpressing wild-type OPRM1 and a phosphorylation-deficient mutant in primary cultures of hippocampal neurons, we demonstrated that receptor phosphorylation contributes to the differential effects of agonists on dendritic spine stability. Phosphorylation blockage made etorphine, fentanyl, and DAMGO function as morphine in the primary cultures. Therefore, agonist-dependent phosphorylation of GPCR regulates the activation of the PKC pathway and the subsequent responses.
Chronic morphine treatment up-regulates mu opioid receptor binding in cells lacking filamin A
2007, Brain Research
Citation Excerpt :
Extensive studies in many different cell lines demonstrated MOP down-regulation by different agonists (Baumhaker et al., 1993; Chakrabarti et al., 1995; Kato et al., 1998; Yabaluri and Medzihradsky, 1997). Chronic morphine-induced MOP down-regulation has been well investigated and characterized in a various cell culture systems, such as SH-SY5Y (Zadina et al., 1993), HEK (Onoprishvili et al., 1999), CHO (Kato et al., 1998), C6 (Yabaluri and Medzihradsky, 1997), Neuro2a (Chakrabarti et al., 1995), 7315c (only 20%) (Puttfarcken and Cox, 1989) and SK-N-SH (Baumhaker et al., 1993). Opioid receptor up-regulation by antagonist treatment is a well-established phenomenon in animals and cell cultures.
We investigated the effects of morphine and other agonists on the human mu opioid receptor (MOP) expressed in M2 melanoma cells, lacking the actin cytoskeleton protein filamin A and in A7, a subclone of the M2 melanoma cells, stably transfected with filamin A cDNA. The results of binding experiments showed that after chronic morphine treatment (24 h) of A7 cells, MOP-binding sites were down-regulated to 63% of control, whereas, unexpectedly, in M2 cells, MOP binding was up-regulated to 188% of control naive cells. Similar up-regulation was observed with the agonists methadone and levorphanol. The presence of antagonists (naloxone or CTAP) during chronic morphine treatment inhibited MOP down-regulation in A7 cells. In contrast, morphine-induced up-regulation of MOP in M2 cells was further increased by these antagonists. Chronic morphine desensitized MOP in A7 cells, i.e., it decreased DAMGO-induced stimulation of GTPγS binding. In M2 cells DAMGO stimulation of GTPγS binding was significantly greater than in A7 cells and was not desensitized by chronic morphine. Pertussis toxin treatment abolished morphine-induced receptor up-regulation in M2 cells, whereas it had no effect on morphine-induced down-regulation in A7 cells. These results indicate that, in the absence of filamin A, chronic treatment with morphine, methadone or levorphanol leads to up-regulation of MOP, to our knowledge, the first instance of opioid receptor up-regulation by agonists in cell culture.
Post-opioid receptor adaptations to chronic morphine; Altered functionality and associations of signaling molecules
2006, Life Sciences
Opioid desensitization/tolerance mechanisms have largely focused on adaptations that occur on the level of the μ-opioid receptor (MOR) itself. These include opioid receptor phosphorylation and ensuing trafficking events. Recent research, however, has revealed additional adaptations that occur downstream from the opioid receptor, which involve covalent modification of signaling molecules and altered associations among them. These include augmented isoform-specific synthesis of adenylyl cyclase (AC) and their phosphorylation as well as augmented phosphorylation of the G_β subunit of G_βγ. The aggregate effect of these changes is to shift μ-opioid receptor-coupled signaling from predominantly G_iα inhibitory to (G_i-derived) G_βγ stimulatory AC signaling. Most recently, chronic morphine has been shown to enhance the association (interaction) between MOR and G_s, which should provide an additional avenue for offsetting inhibitory MOR signaling sequelae. The unfolding complexity of chronic morphine-induced sequelae demands an evolving broader and more encompassing perspective on opioid tolerance-producing mechanisms. This should facilitate understanding tolerance within the context of physiological plasticity that is activated by chronic exposure to drugs of abuse. Additional research is required to integrate the various tolerance-producing adaptations that have been elucidated to date. Specifically, the relative contribution to opioid tolerance of identified adaptations is still unknown as is the extent to which they vary among different regions of the central nervous system.
Opioid abuse and brain gene expression
2004, European Journal of Pharmacology
Opiate addiction is a central nervous system disorder of unknown mechanism. Neuronal basis of positive reinforcement, which is essential to the action of opioids, relies on activation of dopaminergic neurons resulting in an increased dopamine release in the mesolimbic brain structures. Certain aspects of opioid dependence and withdrawal syndrome are also related to the activity of noradrenergic and serotonergic systems, as well as to both excitatory and inhibitory amino acid and peptidergic systems. The latter pathways have been recently proven to be involved both in the development of dependence and in counteracting the states related to relapse. An important role in neurochemical mechanisms of opioid reward, dependence and vulnerability to addiction has been ascribed to endogenous opioid peptides, particularly those acting via the mu- and kappa-opioid receptors. Opiate abuse leads to adaptive reactions in the nervous system which occur at the cellular and molecular levels. Recent research indicates that intracellular mechanisms of signal transmission—from the receptor, through G proteins, cyclic AMP, MAP kinases to transcription factors—also play an important role in opioid tolerance and dependence. The latter link in this chain of reactions may modify synthesis of target genes and in this manner, it may be responsible for opiate-induced long-lasting neural plasticity.
Insights into the receptor transcription and signaling: Implications in opioid tolerance and dependence
2004, Neuropharmacology
Drug addiction has great social and economical implications. In order to resolve this problem, the molecular and cellular basis for drug addiction must be elucidated. For the past three decades, our research has focused on elucidating the molecular mechanisms behind morphine tolerance and dependence. Although there are many working hypotheses, it is our premise that cellular modulation of the receptor signaling, either via transcriptional or post-translational control of the receptor, is the basis for morphine tolerance and dependence. Thus, in the current review, we will summarize our recent work on the transcriptional and post-translational control of the opioid receptor, with special emphasis on the μ-opioid receptor, which is demonstrated to mediate the in vivo functions of morphine.
Turnover of μ-opioid receptors in neuroblastoma cells
2002, Molecular Brain Research
Citation Excerpt :
N2A cells were cultured in DMEM supplemented with 10% fetal calf serum, 100 μg/ml streptomycin 100 IU/ml penicillin in a humidified atmosphere with 90% air and 10% CO2. N2A cells were transfected with constructs of MOR as previously described [5]. In order to facilitate the identification of the MOR with monoclonal antibody, a hemagglutinin (HA) epitope tag (YPYDVPDYA) recognized by the monoclonal antibody was spliced to the N-terminus immediately after the initial methionine codon as described earlier [2].
This study investigated the turnover of μ-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 μ-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 μg/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.

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Research reportNeuroblastoma Neuro2A cells stably expressing a cloned μ-opioid receptor: a specific cellular model to study acute and chronic effects of morphine

Abstract

Life Sci.

Life Sci.

J. Biol. Chem.

J. Biol. Chem.

Anal. Biochem.

Biochem. Pharmacol.

Life Sci.

Eur. J. Pharmacol.

Brain. Res.

Brain. Res.

Life Sci.

Anal. Biochem.

5-Hydroxytryptamine, receptor density and mRNA levels in choroid plexus epithelial cells after treatment with mianserin and (−)-1-(4-bromo-2,5-di-methoxyphenyl)-2-aminopropane

Mol. Pharmacol.

Receptor mechanisms of opioid tolerance in SHSY5Y human neural cells

Mol. Pharmacol.

Opioid peptides induce reduction of enkephalin receptors in cultured neuroblastoma cells

Nature

Calcium phosphate-mediate gene transfer: a high efficient transfection system for stable transforming cells with plasmid DNA

Mol. Cell. Biol.

Research report
Neuroblastoma Neuro_2A cells stably expressing a cloned μ-opioid receptor: a specific cellular model to study acute and chronic effects of morphine