cAMP and protein kinase A contribute to the downregulation of spinal glutamate transporters after chronic morphine

doi:10.1016/j.neulet.2004.11.016

Neuroscience Letters

Volume 376, Issue 1, 7 March 2005, Pages 9-13

https://doi.org/10.1016/j.neulet.2004.11.016 Get rights and content

Abstract

Our previous study has shown that spinal glutamate transporters (GTs) are downregulated following chronic morphine administration; however, how spinal GTs are regulated in this process remains unclear. Here we show that the downregulation of spinal GTs (EAAC1 and GLT-1) induced by a 6-day intrathecal morphine (10 μg, twice daily) treatment regimen was prevented by co-administration of morphine with 2′,5′-dideoxyadenosine (ddA, 1 μg, a broad adenylyl cyclase inhibitor) or H89 (10 μg, a selective protein kinase A inhibitor). When co-administered with morphine, ddA or H89 also effectively attenuated the development of morphine tolerance in the same rats, while ddA or H89 alone did not affect the baseline nociceptive response. These results indicate that the downregulation of spinal GTs following chronic morphine is at least in part mediated through the intracellular cyclic AMP and protein kinase A pathway, suggesting that this cellular mechanism of GT regulation may be contributory to the development of morphine tolerance in rats.

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Acknowledgements

This work was supported by PHS Grants RO1 DA08835, NS42661, and NS45681 to J.M.

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Notably, chronic morphine treatment decreases EAAT3 expression through stimulation of opioid receptors in both rats, mice and cell lines (Mao et al., 2002). This was dependent on ubiquitination by NEDD4, an E3 ubiquitin ligase, which in turn was dependent on the phosphatase PTEN (Lim et al., 2005; Yang et al., 2008), though also EAAT3 mRNA decreases (Guo et al., 2015). The expression of EAAT3 was time-dependent, and after 12 h of withdrawal, the levels returned to baseline (Guo et al., 2015).
The neuronal excitatory amino acid transporter 3 (EAAT3) is fairly ubiquitously expressed in the brain, though it does not necessarily maintain the same function everywhere. It is important in maintaining low local concentrations of glutamate, where its predominant post-synaptic localization can buffer nearby glutamate receptors and modulate excitatory neurotransmission and synaptic plasticity. It is also the main neuronal cysteine uptake system acting as the rate-limiting factor for the synthesis of glutathione, a potent antioxidant, in EAAT3 expressing neurons, while on GABAergic neurons, it is important in supplying glutamate as a precursor for GABA synthesis. Several diseases implicate EAAT3, and modulation of this transporter could prove a useful therapeutic approach. Regulation of EAAT3 could be targeted at several points for functional modulation, including the level of transcription, trafficking and direct pharmacological modulation, and indeed, compounds and experimental treatments have been identified that regulate EAAT3 function at different stages, which together with observations of EAAT3 regulation in patients is giving us insight into the endogenous function of this transporter, as well as the consequences of altered function. This review summarizes work done on elucidating the role and regulation of EAAT3.
Chronic exposure to morphine decreases the expression of EAAT3 via opioid receptors in hippocampal neurons
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Alterations in glutamate transporter expression are closely related to opiate addition behavior, but the role of opioid receptors is unclear. In this study, we used primary cultures of hippocampal neurons from neonatal rats to study the effects of chronic exposure to morphine on excitatory amino acid transporter 3 (EAAT3) expression and the roles of µ opioid receptor (MOR), δ opioid receptor (DOR), and κ opioid receptor (KOR) in the morphine-dependent alterations in EAAT3 expression. The results showed that the EAAT3 protein and mRNA expression levels decreased significantly after chronic exposure to morphine (10 μmol/L) for 48 h, whereas the concentration of extracellular glutamate increased. In addition, we found that both the MOR inhibitor CTOP and the DOR inhibitor naltrindole could reverse the decreased expression of EAAT3 after exposure to morphine, whereas the MOR activator DAMGO and the DOR activator DPDPE significantly decreased EAAT3 expression. The KOR inhibitor had no effect on the expression of EAAT3, whereas its activator increased EAAT3 expression. These results suggest that the down-regulation of morphine-dependent EAAT3 expression in primary rat hippocampal cultures may be mediated by MOR and DOR and that KOR may not contribute significantly to this effect.
This article is part of a Special Issue entitled SI:Addiction circuits.
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l-Glutamate is the predominant excitatory neurotransmitter in the central nervous system (CNS) and is directly and indirectly involved in a variety of brain functions. Glutamate is released in the synaptic cleft at a particular concentration that further activates the various glutaminergic receptors. This concentration of glutamate in the synapse is maintained by either glutamine synthetase or excitatory amino acid proteins which reuptake the excessive glutamate from the synapse and named as excitatory amino acid transporters (EAATs). Out of all the subtypes GLT-1 (glutamate transporter 1) is abundantly distributed in the CNS. Down-regulation of GLT-1 is reported in various neurological diseases such as, epilepsy, stroke, Alzheimer's disease and movement disorders. Therefore, positive modulators of GLT-1 which up-regulate the GLT-1 expression can serve as a potential target for the treatment of neurological disorders. GLT-1 translational activators such as ceftriaxone are found to have significant protective effects in ALS and epilepsy animal models, suggesting that this translational activation approach works well in rodents and that these compounds are worth further pursuit for various neurological disorders. This drug is currently in human clinical trials for ALS. In addition, a thorough understanding of the mechanisms underlying translational regulation of GLT-1, such as identifying the molecular targets of the compounds, signaling pathways involved in the regulation, and translational activation processes, is very important for this novel drug-development effort. This review mainly emphasizes the role of glutamate and its transporter, GLT-1 subtype in excitotoxicity. Further, recent reports on GLT-1 transporters for the treatment of various neurological diseases, including a summary of the presumed physiologic mechanisms behind the pharmacology of these disorders are also explained.
Effects of the altered activity of δ-opioid receptor on the expression of glutamate transporter type 3 induced by chronic exposure to morphine
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Citation Excerpt :
The MOR agonist morphine was the focus of this study. Chronic morphine treatment has been shown to result in the down-regulation of Glu transporters (EAAT3 and EAAT2) in the spinal cord in rats, which is mediated (at least in part) by adenylate cyclase and PKA [11]. Yang et al. found that down-regulation of EAAT3 expression and decreased function caused by chronic morphine exposure could be blocked in C6 cells by the protease inhibitor MG-132, suggesting that the ubiquitin–proteasome system (UPS) is involved in this process.
Altered δ-opioid receptor (DOR) activity can affect the activity and function of excitatory amino acid transporter 3 (EAAT3), but the effects of DOR on EAAT3 expression in morphine relapse remain unknown. In this study, a C6δ cell line and SD rats in a conditioned place preference (CPP) reinstatement model were used. Here, we show that EAAT3 protein levels in C6δ cells decreased significantly after chronic exposure to morphine (10 μM) for 48 h and returned to normal 12 h after drug withdrawal. When C6δ cells were re-exposed to 5 μM morphine for 4 h, EAAT3 protein levels again decreased significantly. The selective μ opioid receptor (MOR) specific agonist DAMGO had a similar effect as morphine, and CTOP, a specific MOR blocker, reversed the declined expression of EAAT3 protein triggered by morphine exposure. The selective DOR agonist [d-pen2, 5] enkephalin (DPDPE) significantly increased EAAT3 expression in C6δ cells and even reversed the decreased EAAT3 expression caused by chronic morphine exposure. The non specific antagonist naloxone, but not the DOR inhibitor Naltrindole (NTI), reversed the decreased EAAT3 expression in C6δ cells caused by chronic morphine exposure. In vivo, EAAT3 levels in the prefrontal cortex of rats with morphine-induced CPP reinstatement significantly decreased. Naloxone completely suppressed reinstatement and reversed the decrease in EAAT3 expression induced by morphine re-exposure. In contrast, NTI only weakened CPP reinstatement and exerted no influence on EAAT3 expression. These findings suggest that DOR can affect the expression of EAAT3. However, the morphine-induced down-regulation of EAAT3 in C6δ cells and in the prefrontal cortex of rats may not be mediated by DOR.
Role of neuroinflammation in morphine tolerance: Effect of tumor necrosis factor-α
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Citation Excerpt :
Glutamatergic receptors, in particular NMDA receptors, are critically involved in chronic opioid-induced neuronal adaptations, such as opioid tolerance, dependence, and withdrawal,10,48,49 and in chronic pain-associated hyperalgesia.50–52 Emerging evidence suggests that opioid tolerance and abnormal pain sensitivity, the opioid-induced hyperalgesia, may share common cellular mechanisms and mediate, at least in part, through NMDA receptors12,53 and GT system.54 Our previous studies have demonstrated that coinfusion of the NMDA antagonist MK-801 with morphine shifted the AD50 (analgesic dose) of morphine to 11.2 μg compared to 83.8 μg in morphine tolerant-rats.55
Opioids have been used as potent analgesics in clinics for decades; however, their long-term administration leads to tolerance. Two possible mechanisms for drug tolerance are postulated as within-system and between-systems adaptation. The within-system tolerance is involved in the signal transduction of opioid receptors, including downregulation of opioid receptors, uncoupling of G-protein from opioid receptors, and β-arrestin recruitment to opioid receptors, which causes receptor desensitization and internalization/endocytosis. The between-systems tolerance comprehends the glutamatergic receptor system and glial activation with the release of proinflammatory cytokines, and thus the analgesic effect of morphine is reduced. Tumor necrosis factor-α (TNF-α) is a vital proinflammatory cytokine and exerts either a neurotoxic or neuroprotective effect on different diseases of the central nervous system. TNF-α has also been demonstrated to correlate with neuronal plasticity via activation of spinal glial cells and enhancement of glutamatergic transmission. Previous studies had revealed an increased expression of TNF-α in morphine tolerance. This review article focuses on the role of TNF-α in neuroinflammation and the glutamatergic receptor system in morphine tolerance. It may provide another adjuvant therapy for morphine tolerance, which extends the effectiveness of opioids in clinical pain management.

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cAMP and protein kinase A contribute to the downregulation of spinal glutamate transporters after chronic morphine

Abstract

Section snippets

Acknowledgements

Prog. Neurol.

Pain

Neuron

Cell

Pain

Pain

Eur. J. Pharmacol.

J. Biol. Chem.

Physiol. Behav.

Different mechanisms mediate development and expression of tolerance and dependence for peripheral-opioid antinociception in rat

J. Neurosci.

Mu-opioid receptor desensitization by beta-arrestin-2 determines morphine tolerance but not dependence

Nature

Enhanced morphine analgesia in mice lacking beta-arrestin-2

Science

A method for determining loss of pain sensation

J. Pharmacol. Exp. Ther.

Regional deafferentation downregulates subtypes of glutamate transporter protein

J. Neurochem.

Opioid tolerance and the emergence of new opioid receptro-coupled signaling

Mol. Neurobiol.

Chronic morphine increases GABA tone on serotonergic neurons of the dorsal raphe nucleus: association with an up-regulation of the cyclic AMP pathway

Neuroscience

Characterization of striatal lesions produced by glutamate uptake alteration: cell death, reactive gliosis, and changes in GLT?1 and GADD45 mRNA expression

Glia