Evidence for κ‐ and μ‐Opioid Receptor Expression in C6 Glioma Cells

Bohn, Laura M.; Belcheva, Mariana M.; Coscia, Carmine J.

doi:10.1046/j.1471-4159.1998.70051819.x

Cited by 23 publications

(23 citation statements)

References 43 publications

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“…In the current in vitro experiments, we used the C6 glioma cell line that expresses both EAAC1 (27,28), a neuronal glutamate transporter, and opioid receptors (29). Although C6 glioma cells have been extensively used for studying the biosynthesis, metabolism, and regulatory mechanisms of EAAC1 glutamate transporter (30 -32), C6 glioma cells are tumorbased cell lines with mixed neuronal and astroglial properties.…”

Section: Discussionmentioning

confidence: 99%

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Morphine Induces Ubiquitin-Proteasome Activity and Glutamate Transporter Degradation

Yang

Wang

Sung

et al. 2008

Journal of Biological Chemistry

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Glutamate transporters play a crucial role in physiological glutamate homeostasis, neurotoxicity, and glutamatergic regulation of opioid tolerance. However, how the glutamate transporter turnover is regulated remains poorly understood. Here we show that chronic morphine exposure induced posttranscriptional downregulation of the glutamate transporter EAAC1 in C6 glioma cells with a concurrent decrease in glutamate uptake and increase in proteasome activity, which were blocked by the selective proteasome inhibitor MG-132 or lactacystin but not the lysosomal inhibitor chloroquin. At the cellular level, chronic morphine induced the PTEN (phosphatase and tensin homolog deleted on chromosome Ten)-mediated up-regulation of the ubiquitin E3 ligase Nedd4 via cAMP/protein kinase A signaling, leading to EAAC1 ubiquitination and proteasomal degradation. Either Nedd4 or PTEN knockdown with small interfering RNA prevented the morphine-induced EAAC1 degradation and decreased glutamate uptake. These data indicate that cAMP/protein kinase A signaling serves as an intracellular regulator upstream to the activation of the PTEN/ Nedd4-mediated ubiquitin-proteasome system activity that is critical for glutamate transporter turnover. Under an in vivo condition, chronic morphine exposure also induced posttranscriptional down-regulation of the glutamate transporter EAAC1, which was prevented by MG-132, and transcriptional up-regulation of PTEN and Nedd4 within the spinal cord dorsal horn. Thus, inhibition of the ubiquitin-proteasome-mediated glutamate transporter degradation may be an important mechanism for preventing glutamate overexcitation and may offer a new strategy for treating certain neurological disorders and improving opioid therapy in chronic pain management.Glutamate transporters play a crucial role in physiological glutamate homeostasis, neurotoxicity, and glutamatergic regulation of opioid tolerance (1-5). However, how the glutamate transporter degradation is regulated remains unclear (6 -8). The ubiquitin-proteasome system (UPS) 2 is a major non-lysosomal proteolytic pathway that degrades cellular proteins including those with important roles in the regulation of cell growth and function (9 -11). In addition, activation of UPS has been shown to regulate the PSD-95 degradation and ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor surface expression (12), suggesting a possible relationship between UPS and glutamatergic activities. Ubiquitination is a process involving three enzymes: E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin ligase) (13,14). Interactions between an E3 ligase and its target molecule are considered a key step in determining the selectivity of UPS for a target molecule and its subsequent proteasomal degradation, a process that is subject to intracellular modulation by various upstream regulators (14). PTEN (phosphatase and tensin homolog deleted on chromosome Ten) is a tumor suppressor and lipid phosphatase, which has been shown to regulate cell surviva...

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Section: Discussionmentioning

confidence: 99%

“…28) and opioid receptors (29). This cell line has been extensively used to examine the biosynthesis, metabolism, and regulatory mechanisms of the EAAC1 glutamate transporter (30 -32).…”

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confidence: 99%

Morphine Induces Ubiquitin-Proteasome Activity and Glutamate Transporter Degradation

Yang

Wang

Sung

et al. 2008

Journal of Biological Chemistry

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“…(33). Similarly, there are endogenous MOR-expressing cells from different species, e.g., SY-SH5Y and NMB as human neuroblastoma cell lines and the MOR-positive C6 glioma cells from the rat (5). In addition, regulation of the MOR gene has been studied in various physiological and neuronal states in several species.…”

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confidence: 99%

Evidence of Endogenous Mu Opioid Receptor Regulation by Epigenetic Control of the Promoters

Hwang

Song

Kim

et al. 2007

Molecular and Cellular Biology

145

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The pharmacological effect of morphine as a painkiller is mediated mainly via the mu opioid receptor (MOR) and is dependent on the number of MORs in the cell surface membrane. While several studies have reported that the MOR gene is regulated by various cis-and trans-acting factors, many questions remain unanswered regarding in vivo regulation. The present study shows that epigenetic silencing and activation of the MOR gene are achieved through coordinated regulation at both the histone and DNA levels. In P19 mouse embryonal carcinoma cells, expression of the MOR was greatly increased after neuronal differentiation. MOR expression could also be induced by a demethylating agent (5-aza-2-deoxycytidine) or histone deacetylase inhibitors in the P19 cells, suggesting involvement of DNA methylation and histone deacetylation for MOR gene silencing. Analysis of CpG DNA methylation revealed that the proximal promoter region was unmethylated in differentiated cells compared to its hypermethylation in undifferentiated cells. In contrast, the methylation of other regions was not changed in either cell type. Similar methylation patterns were observed in the mouse brain. In vitro methylation of the MOR promoters suppressed promoter activity in the reporter assay. Upon differentiation, the in vivo interaction of MeCP2 was reduced in the MOR promoter region, coincident with histone modifications that are relevant to active transcription. When MeCP2 was disrupted using MeCP2 small interfering RNA, the endogenous MOR gene was increased. These data suggest that DNA methylation is closely linked to the MeCP2-mediated chromatin structure of the MOR gene. Here, we propose that an epigenetic mechanism consisting of DNA methylation and chromatin modification underlies the cell stage-specific mechanism of MOR gene expression.Opioids exert their pharmacological and physiological effects through binding to their endogenous receptors. Three types of opioid receptors, mu (), delta (␦), and kappa (), all belonging to the G-protein-coupled receptor superfamily, have been cloned. Upon agonist binding, these receptors couple to G proteins and affect several signal transduction pathways thought to mediate a broad range of functions and pharmacological effects of endogenous and exogenous opioids (51). Previous studies suggested that the opioid receptor (MOR) plays a key role in mediating the major clinical effects of analgesics, such as morphine, as well as the development of tolerance and physical dependence upon prolonged administration (39). MOR is mainly expressed in the central nervous system, with densities varying greatly in different regions, which can display different functional roles (55). During mouse embryonic development, the MOR message was specifically observed as early as embryonic day 8.5 (E8.5) using the reverse transcription (RT)-PCR method (44). In contrast, MOR transcripts were detected only beginning at E12 using the radioligand binding method (70) and at E10.5 by in situ hybridization (85). Transcript levels gradually incr...

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“…However, growing evidence indicates modulatory effects of opioids on these cells. Indeed, classical opioid receptors are present in glial cells, while a new class of receptors termed ''toll-like'' receptor (TLR) has been demonstrated in the spinal cord and brain (Bohn et al, 1998;Song and Zhao, 2001;Hutchinson et al, 2007).…”

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Effects of morphine on testosterone levels in rat C6 glioma cells: Modulation by anastrozole

Ceccarelli

Rossi

Maddalena

et al. 2009

Journal Cellular Physiology

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Rat C6 glioma cells are commonly used to investigate the functions of glial cells. To evaluate the presence of testosterone and its metabolism in rat C6 glioma cells, we cultured them in media with or without the addition of testosterone propionate and anastrozole, a blocker of aromatase, the enzyme needed to transform testosterone into estradiol. The same procedure was repeated with morphine (10 and 100 microM), known to decrease testosterone levels in the brain (in rats) and plasma (in rats and humans). Confluent cells were exposed to the test media for 48 h and then collected. Cell pellets were used to determine testosterone by radioimmunoassay. The C6 cells contained detectable levels of testosterone and the levels increased with the addition of testosterone to the medium. Aromatase blockage by anastrozole increased cellular levels of testosterone regardless of the addition of exogenous testosterone. Both concentrations of morphine dose-dependently decreased testosterone levels in the C6 cells; this effect was also present with the contemporary administration of anastrozole. Our findings show that testosterone is present in rat C6 glioma cells and can be metabolized by aromatase. Moreover, the presence of morphine in the culture medium strongly decreased testosterone, demonstrating that the glia would be a target of the morphine-induced hypogonadal effect.

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Evidence for κ‐ and μ‐Opioid Receptor Expression in C6 Glioma Cells

Cited by 23 publications

References 43 publications

Morphine Induces Ubiquitin-Proteasome Activity and Glutamate Transporter Degradation

Morphine Induces Ubiquitin-Proteasome Activity and Glutamate Transporter Degradation

Evidence of Endogenous Mu Opioid Receptor Regulation by Epigenetic Control of the Promoters

Effects of morphine on testosterone levels in rat C6 glioma cells: Modulation by anastrozole

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