Proenkephalin deletion in hematopoietic cells induces intestinal barrier failure resulting in clinical feature similarities with irritable bowel syndrome in mice.

Opioid-dependent immune-mediated analgesic effects have been broadly reported upon inflammation. In preclinical mouse models of intestinal inflammatory diseases, the local release of enkephalins (endogenous opioids) by colitogenic T lymphocytes alleviate inflammation-induced pain by down-modulating gut-innervating nociceptor activation in periphery. In this study, we wondered whether this immune cell-derived enkephalin-mediated regulation of the nociceptor activity also operates under steady state conditions. Here, we show that chimeric mice engrafted with enkephalin-deficient bone marrow cells exhibit not only visceral hypersensitivity but also an increase in both epithelial paracellular and transcellular permeability, an alteration of the microbial topography resulting in increased bacteria-epithelium interactions and a higher frequency of IgA-producing plasma cells in Peyer's patches. All these alterations of the intestinal homeostasis are associated with an anxiety-like behavior despite the absence of an overt inflammation as observed in patients with irritable bowel syndrome. Thus, our results show that immune cell-derived enkephalins play a pivotal role in maintaining gut homeostasis and normal behavior in mice. Because a defect in the mucosal opioid system remarkably mimics some major clinical symptoms of the irritable bowel syndrome, its identification might help to stratify subgroups of patients.

Human PSEN1 Mutant Glia Improve Spatial Learning and Memory in Aged Mice.

The PSEN1 ΔE9 mutation causes a familial form of Alzheimer's disease (AD) by shifting the processing of amyloid precursor protein (APP) towards the generation of highly amyloidogenic Aß42 peptide. We have previously shown that the PSEN1 ΔE9 mutation in human-induced pluripotent stem cell (iPSC)-derived astrocytes increases Aß42 production and impairs cellular responses. Here, we injected PSEN1 ΔE9 mutant astrosphere-derived glial progenitors into newborn mice and investigated mouse behavior at the ages of 8, 12, and 16 months. While we did not find significant behavioral changes in younger mice, spatial learning and memory were paradoxically improved in 16-month-old PSEN1 ΔE9 glia-transplanted male mice as compared to age-matched isogenic control-transplanted animals. Memory improvement was associated with lower levels of soluble, but not insoluble, human Aß42 in the mouse brain. We also found a decreased engraftment of PSEN1 ΔE9 mutant cells in the cingulate cortex and significant transcriptional changes in both human and mouse genes in the hippocampus, including the extracellular matrix-related genes. Overall, the presence of PSEN1 ΔE9 mutant glia exerted a more beneficial effect on aged mouse brain than the isogenic control human cells likely as a combination of several factors.

Efficacy of multimodal analgesic treatment of severe traumatic acute pain in mice pretreated with chronic high dose of buprenorphine inducing mechanical allodynia.

Managing severe acute nociceptive pain in buprenorphine-maintained individuals for opioid use disorder management is challenging owing to the high affinity and very slow dissociation of buprenorphine from µ-opioid receptors that hinders the use of full agonist opioid analgesics. In a translational approach, the aim of this study was to use an animal setting to investigate the effects of a chronic high dose of buprenorphine treatment on nociceptive thresholds before and after applying a severe acute nociceptive traumatic surgery stimulus and to screen postoperative pharmacological analgesic strategies. A chronic treatment of mice with a high dose of buprenorphine (BUP HD, 2 × 200 µg/kg/day; i.p.) revealed significant mechanical allodynia. One and two days after having discontinued buprenorphine administration and having induced a severe nociceptive acute pain by a closed tibial fracture, acute administration of morphine at a dose which has analgesic effects in absence of pretreatment (4.5 mg/kg; i.p.), was ineffective to reduce pain in the BUP HD group. However, mimicking multimodal analgesia strategy used in human postoperative context, the combination of morphine (administered at the same dose) with a NMDA receptor antagonist (ketamine) or an NSAID (ketoprofen) produced antinociceptive responses in these animals. The mouse model of closed tibial fracture could be useful to identify analgesic strategies of postoperative pain for patients with chronic exposure to opioids and suffering from hyperalgesia.

Tibial post fracture pain is reduced in kinin receptors deficient mice and blunted by kinin receptor antagonists.

BACKGROUND: Tibial fracture is associated with inflammatory reaction leading to severe pain syndrome. Bradykinin receptor activation is involved in inflammatory reactions, but has never been investigated in fracture pain. METHODS: This study aims at defining the role of B1 and B2-kinin receptors (B1R and B2R) in a closed tibial fracture pain model by using knockout mice for B1R (B1KO) or B2R (B2KO) and wild-type (WT) mice treated with antagonists for B1R (SSR 240612 and R954) and B2R (HOE140) or vehicle. A cyclooxygenase (COX) inhibitor (ketoprofen) and an antagonist (SB366791) of Transient Receptor Potential Vaniloid1 (TRPV1) were also investigated since these pathways are associated with BK-induced pain in other models. The impact on mechanical and thermal hyperalgesia and locomotion was assessed by behavior tests. Gene expression of B1R and B2R and spinal cord expression of c-Fos were measured by RT-PCR and immunohistochemistry, respectively. RESULTS: B1KO and B2KO mice demonstrated a reduction in post-fracture pain sensitivity compared to WT mice that was associated with decreased c-Fos expression in the ipsilateral spinal dorsal horn in B2KO. B1R and B2R mRNA and protein levels were markedly enhanced at the fracture site. B1R and B2R antagonists and inhibition of COX and TRPV1 pathways reduced pain in WT. However, the analgesic effect of the COX-1/COX-2 inhibitor disappeared in B1KO and B2KO. In contrast, the analgesic effect of the TRPV1 antagonist persisted after gene deletion of either receptor. CONCLUSIONS: It is suggested that B1R and B2R activation contributes significantly to tibial fracture pain through COX. Hence, B1R and B2R antagonists appear potential therapeutic agents to manage post fracture pain.

Development and characterization of sphingosine 1-phosphate receptor 1 monoclonal antibody suitable for cell imaging and biochemical studies of endogenous receptors.

Although sphingosine-1-phosphate receptor 1 (S1P1) has been shown to trigger several S1P targeted functions such as immune cell trafficking, cell proliferation, migration, or angiogenesis, tools that allow the accurate detection of endogenous S1P1 localization and trafficking remain to be obtained and validated. In this study, we developed and characterized a novel monoclonal S1P1 antibody. Mice were immunized with S1P1 produced in the yeast Pichia pastoris and nine hybridoma clones producing monoclonal antibodies were created. Using different technical approaches including Western blot, immunoprecipitation and immunocytochemistry, we show that a selected clone, hereinafter referred to as 2B9, recognizes human and mouse S1P1 in various cell lineages. The interaction between 2B9 and S1P1 is specific over receptor subtypes, as the antibody does not binds to S1P2 or S1P5 receptors. Using cell-imaging methods, we demonstrate that 2B9 binds to an epitope located at the intracellular domain of S1P1; reveals cytosolic and membrane localization of the endogenous S1P1; and receptor internalization upon S1P or FTY720-P stimulation. Finally, loss of 2B9 signal upon knockdown of endogenous S1P1 by specific small interference RNAs further confirms its specificity. 2B9 was also able to detect S1P1 in human kidney and spinal cord tissue by immunohistochemistry. Altogether, our results suggest that 2B9 could be a useful tool to detect, quantify or localize low amounts of endogenous S1P1 in various physiological and pathological processes.

Activation of nociceptin/orphanin FQ receptors inhibits contextual fear memory reconsolidation.

Several neuropeptidergic systems act as modulators of cognitive performances. Among them, nociceptin, an opioid-like peptide also known as orphanin FQ (N/OFQ), has recently gained attention. Stimulation of its receptor, the N/OFQ opioid receptor (NOP), which is expressed in brain regions involved in emotion, memory and stress response, has inhibitory effects on the acquisition and/or consolidation of spatial and emotional memory in rodents. Recently, N/OFQ was also proposed to be linked to the pathogenesis of Post-Traumatic Stress Disorder in humans. However, until now the effect of the activation of the N/OFQ-NOP system on already consolidated memory, such as during retrieval and reconsolidation phases, has never been explored. In the present study, we investigated the consequences of systemic injection of NOP agonists or i.c.v. injection of the N/OFQ peptide on the retrieval and the reconsolidation of contextual fear memory in mice. We demonstrate that the activation of the N/OFQ system impairs the reconsolidation of context-dependent but not cue-dependent aversive memories. We also show that this amnestic effect is associated with decreased c-Fos expression in the hippocampus and amygdala. Our data thus provide the first evidence that the NOP receptor could be targeted during the reconsolidation process to weaken maladaptive memories. The N/OFQ-NOP system might constitute in the future an interesting pharmacological target for interfering with so-called "pathological memories", in particular those involving maladaptive contextual memories.

Beneficial effects of levobupivacaine regional anaesthesia on postoperative opioid induced hyperalgesia in diabetic mice.

BACKGROUND: Diabetic neuropathy is one of the most common complications of diabetes and causes various problems in daily life. The aim of this study was to assess the effect of regional anaesthesia on post surgery opioid induced hyperalgesia in diabetic and non-diabetic mice. METHODS: Diabetic and non-diabetic mice underwent plantar surgery. Levobupivacaine and sufentanil were used before surgery, for sciatic nerve block (regional anaesthesia) and analgesia, respectively. Diabetic and non-diabetic groups were each randomly assigned to three subgroups: control, no sufentanil and no levobupivacaine; sufentanil and no levobupivacaine; sufentanil and levobupivacaine. Three tests were used to assess pain behaviour: mechanical nociception; thermal nociception and guarding behaviours using a pain scale. RESULTS: Sufentanil, alone or in combination with levobupivacaine, produced antinociceptive effects shortly after administration. Subsequently, sufentanil induced hyperalgesia in diabetic and non-diabetic mice. Opioid-induced hyperalgesia was enhanced in diabetic mice. Levobupivacaine associated to sufentanil completely prevented hyperalgesia in both groups of mice. CONCLUSION: The results suggest that regional anaesthesia can decrease opioid-induced hyperalgesia in diabetic as well as in non-diabetic mice. These observations may be clinically relevant for the management of diabetic patients.

Identification and functional characterization of the phosphorylation sites of the neuropeptide FF2 receptor.

The neuropeptide FF2 (NPFF2) receptor belongs to the rhodopsin family of G protein-coupled receptors and mediates the effects of several related RFamide neuropeptides. One of the main pharmacological interests of this system resides in its ability to regulate endogenous opioid systems, making it a potential target to reduce the negative effects of chronic opioid use. Phosphorylation of intracellular residues is the most extensively studied post-translational modification regulating G protein-coupled receptor activity. However, until now, no information concerning NPFF2 receptor phosphorylation is available. In this study, we combined mass spectrometric analysis and site-directed mutagenesis to analyze for the first time the phosphorylation pattern of the NPFF2 receptor and the role of the various phosphorylation sites in receptor signaling, desensitization, and trafficking in a SH-SY5Y model cell line. We identified the major, likely GRK-dependent, phosphorylation cluster responsible for acute desensitization, (412)TNST(415) at the end of the C terminus of the receptor, and additional sites involved in desensitization ((372)TS(373)) and internalization (Ser(395)). We thus demonstrate the key role played by phosphorylation in the regulation of NPFF2 receptor activity and trafficking. Our data also provide additional evidence supporting the concept that desensitization and internalization are partially independent processes relying on distinct phosphorylation patterns.

Heterologous regulation of Mu-opioid (MOP) receptor mobility in the membrane of SH-SY5Y cells.

The dynamic organization of G protein-coupled receptors in the plasma membrane is suspected of playing a role in their function. The regulation of the diffusion mode of the mu-opioid (MOP) receptor was previously shown to be agonist-specific. Here we investigate the regulation of MOP receptor diffusion by heterologous activation of other G protein-coupled receptors and characterize the dynamic properties of the MOP receptor within the heterodimer MOP/neuropeptide FF (NPFF2) receptor. The data show that the dynamics and signaling of the MOP receptor in SH-SY5Y cells are modified by the activation of α2-adrenergic and NPFF2 receptors, but not by the activation of receptors not described to interact with the opioid receptor. By combining, for the first time, fluorescence recovery after photobleaching at variable radius experiments with bimolecular fluorescence complementation, we show that the MOP/NPFF2 heterodimer adopts a specific diffusion behavior that corresponds to a mix of the dynamic properties of both MOP and NPFF2 receptors. Altogether, the data suggest that heterologous regulation is accompanied by a specific organization of receptors in the membrane.

Solubilization and reconstitution of the mu-opioid receptor expressed in human neuronal SH-SY5Y and CHO cells.

The zwitterionic detergent CHAPS was used to solubilize the human mu-opioid receptor (hMOR) from SH-SY5Y neuroblastoma cells and recombinant hMOR-CHO (CHO-T7-hMOR) and hMOR-SH-SY5Y (SH-SY5Y-T7-hMOR) cell membranes. Agonist stimulation and G-protein activation by the mu-selective opioid agonist DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin) were recovered after removing of CHAPS after polyethylene glycol (PEG) precipitation. Binding assays show that hMOR solubilized and reconstituted this way was functional and able to interact with both agonist peptides and with G-protein. The effective solubilization and reconstitution of hMOR from mammalian cells, without truncation and extensive modification, represent an essential step toward the purification of a receptor bearing important post-translational modifications.

A high-affinity, radioiodinatable neuropeptide FF analogue incorporating a photolabile p-(4-hydroxybenzoyl)phenylalanine.

A new radioiodinated photoaffinity compound, [(125)I]YE(Bpa)WSLAAPQRFNH2, derived from a peptide present in the rat neuropeptide FF (NPFF) precursor was synthesized, and its binding characteristics were investigated on a neuroblastoma clone, SH-SY5Y, stably expressing rat NPFF2 receptors tagged with the T7 epitope. The binding of the probe was saturable and revealed a high-affinity interaction (KD=0.24nM) with a single class of binding sites. It was also able to affinity label NPFF2 receptor in a specific and efficient manner given that 38% of the bound radioligand at saturating concentration formed a wash-resistant binding after ultraviolet (UV) irradiation. Photoaffinity labeling with [(125)I]YE(Bpa)WSLAAPQRFamide showed two molecular forms of NPFF2 receptor with apparent molecular weights of 140 and 95kDa in a 2:1 ratio. The comparison of the results between photoaffinity labeling and Western blot analysis suggests that all receptor forms bind the probe irreversibly with the same efficiency. On membranes of mouse olfactory bulb, only the high molecular weight form of NPFF2 receptor is observed. [(125)I]YE(Bpa)WSLAAPQRFamide is an excellent radioiodinated peptidic ligand for direct and selective labeling of NPFF2 receptors in vitro.

GRK2 protein-mediated transphosphorylation contributes to loss of function of µ-opioid receptors induced by neuropeptide FF (NPFF2) receptors.

Neuropeptide FF (NPFF) interacts with specific receptors to modulate opioid functions in the central nervous system. On dissociated neurons and neuroblastoma cells (SH-SY5Y) transfected with NPFF receptors, NPFF acts as a functional antagonist of µ-opioid (MOP) receptors by attenuating the opioid-induced inhibition of calcium conductance. In the SH-SY5Y model, MOP and NPFF(2) receptors have been shown to heteromerize. To understand the molecular mechanism involved in the anti-opioid activity of NPFF, we have investigated the phosphorylation status of the MOP receptor using phospho-specific antibody and mass spectrometry. Similarly to direct opioid receptor stimulation, activation of the NPFF(2) receptor by [D-Tyr-1-(NMe)Phe-3]NPFF (1DMe), an analog of NPFF, induced the phosphorylation of Ser-377 of the human MOP receptor. This heterologous phosphorylation was unaffected by inhibition of second messenger-dependent kinases and, contrarily to homologous phosphorylation, was prevented by inactivation of G(i/o) proteins by pertussis toxin. Using siRNA knockdown we could demonstrate that 1DMe-induced Ser-377 cross-phosphorylation and MOP receptor loss of function were mediated by the G protein receptor kinase GRK2. In addition, mass spectrometric analysis revealed that the phosphorylation pattern of MOP receptors was qualitatively similar after treatment with the MOP agonist Tyr-D-Ala-Gly (NMe)-Phe-Gly-ol (DAMGO) or after treatment with the NPFF agonist 1DMe, but the level of multiple phosphorylation was more intense after DAMGO. Finally, NPFF(2) receptor activation was sufficient to recruit ß-arrestin2 to the MOP receptor but not to induce its internalization. These data show that NPFF-induced heterologous desensitization of MOP receptor signaling is mediated by GRK2 and could involve transphosphorylation within the heteromeric receptor complex.

Modulation by neuropeptide FF of the interaction of mu-opioid (MOP) receptor with G-proteins.

The Neuropeptide FF (NPFF) system is known to modulate the effects of opioids in vivo and in vitro. In the present study, we have investigated the effect of NPFF agonists on the coupling of the Mu-opioid (MOP) receptor to G-proteins in a model of SH-SY5Y cells transfected with NPFF(2) receptor, in which the neuronal anti-opioid activity of NPFF was previously reproduced. Activation of G-proteins was monitored by [(35)S]GTPgammaS binding assay and analysis of G-protein subunits associated with MOP receptors was performed by Western blotting after immunoprecipitation of the receptor. The results demonstrate that concentrations of NPFF agonists that produce a cellular anti-opioid effect, did not affect the ability of the opioid agonist DAMGO to activate G-proteins. However, at saturating concentration of agonist or when expression of receptor was high, opioid and NPFF agonists did not stimulate [(35)S]GTPgammaS binding in an additive manner, indicating that both receptors share a common fraction of a G-protein pool. In addition, stimulation of NPFF receptors in living cells modified the G-protein environment of MOP receptor by favoring its interaction with alpha(s), alpha(i2) and beta subunits. This change in G-protein coupling to MOP receptor might participate in the mechanism by which NPFF agonists reduce the inhibitory activity of opioids.

Modulation of basal and morphine-induced neuronal activity by a NPFF(2) selective agonist measured by c-Fos mapping of the mouse brain.

Neuropeptide FF (NPFF) is a neurotransmitter known to modulate opioid-induced analgesia, sensitization, and reward. The expression of the immediate early gene c-Fos was analyzed to map the distribution of neurons whose activity is regulated by central administration of the NPFF(2)-selective agonist dNPA in naive mice and in animals who had received a systemic injection of morphine. The number of c-Fos positive nuclei was quantified in 28 brain regions. Intracerebro-ventricular injection of 1 nmol dNPA alone produced an overall inhibition of basal c-Fos expression in the brain with a statistically significant decrease in the lateral ventral part of the bed nucleus of the stria terminalis, the medial preoptic area, and the medial parvicellular part of the paraventricular nucleus of the hypothalamus. In contrast, intraperitoneal injection of morphine 5 mg.kg(-1) induced a statistically significant increase in c-Fos expression in the prelimbic cortex, the nucleus accumbens core and shell, the ventral pallidum, the lateral hypothalamus, and the nucleus of the tractus solitarius. dNPA counteracted morphine effect only in the nucleus accumbens shell and the ventral pallidum. The inhibitory effects of a low dose of dNPA in the hypothalamus and its afferents suggest that NPFF(2) receptors negatively regulate the hypothalamic-pituitary-adrenal axis in mouse. Moreover, our study identified the nucleus accumbens shell and ventral pallidum as putative sites of interaction between NPFF and opioid systems in relation with the modulation of acute morphine rewarding and locomotor effects.

Pharmacological characterization of the mouse NPFF2 receptor.

This study presents the binding and functional properties of the mouse NPFF(2) (mNPFF(2)) receptor, in comparison with its human counterpart (hNPFF(2)). Binding experiments were performed by using the NPFF(2) selective radioligand [(3)H]-EYF in membranes from CHO cells transfected with mouse and human NPFF(2) receptors and compared to membranes from mouse olfactory bulb, the brain region expressing the highest density of NPFF(2) receptors in mouse. mNPFF(2) receptors exhibited a high affinity (Kd=0.2-0.4 nM) for [(3)H]-EYF, comparable to that of hNPFF(2) receptors. Also, the binding selectivity profile of mNPFF(2) receptors was comparable to that of hNPFF(2) receptors, except for three ligands (NPSF, NPVF, RF9) that were about tenfold more potent and active on mouse receptors than on human receptors. In particular, compared to hNPFF(2) receptors, mNPFF(2) receptors were less discriminative towards the proNPFF(B)-derived peptide. This suggests some species-related differences in the binding properties of NPFF(2) receptors that could have repercussion when evaluating the pharmacological properties of drugs in vivo.

Description of the low-affinity interaction between nociceptin and the second extracellular loop of its receptor by fluorescence and NMR spectroscopies.

The second extracellular loop (ECL2) of the Noc receptor has been proposed to be involved in ligand binding and selectivity. The interaction of Noc with a constrained cyclic synthetic peptide, mimicking the ECL2, has been studied using fluorescence and NMR spectroscopies. Selective binding was shown with a dissociation constant of approximately 10 microM (observed with the constrained cyclic loop and not with the open chain), and residues involved in ligand binding and selectivity have been identified. This bimolecular complex is stabilized by (i) ionic interactions between the two Noc basic motives and the ECL2 acidic residues; (ii) hydrophobic contacts involving Noc FGGF N-terminal sequence and an ECL2 tryptophane residue. Our data confirm that Noc receptor's ECL2 contributes actively to ligand binding and selectivity by providing the peptidic ligand with a low affinity-binding site.

Protein degradation, as with protein synthesis, is required during not only long-term spatial memory consolidation but also reconsolidation.

The formation of long-term memory requires protein synthesis, particularly during initial memory consolidation. This process also seems to be dependant upon protein degradation, particularly degradation by the ubiquitin-proteasome system. The aim of this study was to investigate the temporal requirement of protein synthesis and degradation during the initial consolidation of allocentric spatial learning. As memory returns to a labile state during reactivation, we also focus on the role of protein synthesis and degradation during memory reconsolidation of this spatial learning. Male CD1 mice were submitted to massed training in the spatial version of the Morris water maze. At various time intervals after initial acquisition or after a reactivation trial taking place 24 h after acquisition, mice received an injection of either the protein synthesis inhibitor anisomycin or the protein degradation inhibitor lactacystin. This injection was performed into the hippocampal CA3 region, which is specifically implicated in the processing of spatial information. Results show that, in the CA3 hippocampal region, consolidation of an allocentric spatial learning task requires two waves of protein synthesis taking place immediately and 4 h after acquisition, whereas reconsolidation requires only the first wave. However, for protein degradation, both consolidation and reconsolidation require only one wave, taking place immediately after acquisition or reactivation, respectively. These findings suggest that protein degradation is a key step for memory reconsolidation, as for consolidation. Moreover, as protein synthesis-dependent reconsolidation occurred faster than consolidation, reconsolidation did not consist of a simple repetition of the initial consolidation.

Neuropeptide FF-sensitive confinement of mu opioid receptor does not involve lipid rafts in SH-SY5Y cells.

Mu opioid (MOP) receptor activation can be functionally modulated by stimulation of Neuropeptide FF 2 (NPFF(2)) G protein-coupled receptors. Fluorescence recovery after photobleaching experiments have shown that activation of the NPFF(2) receptor dramatically reduces the fraction of MOP receptors confined in microdomains of the plasma membrane of SH-SY5Y neuroblastoma cells. The aim of the present work was to assess if the direct observation of receptor compartmentation by fluorescence techniques in living cells could be related to indirect estimation of receptor partitioning in lipid rafts after biochemical fractionation of the cell. Our results show that MOP receptor distribution in lipid rafts is highly dependent upon the method of purification, questioning the interpretation of previous data regarding MOP receptor compartmentation. Moreover, the NPFF analogue 1DMe does not modify the distribution profile of MOP receptors, clearly demonstrating that membrane fractionation data do not correlate with direct measurement of receptor compartmentation in living cells.

Effect of long-term exposure of SH-SY5Y cells to morphine: a whole cell proteomic analysis.

BACKGROUND: Opiate addiction reflects plastic changes that endurably alter synaptic transmission within relevant neuronal circuits. The biochemical mechanisms of these adaptations remain largely unknown and proteomics-based approaches could lead to a broad characterization of the molecular events underlying adaptations to chronic drug exposure. RESULTS: Thus, we have started proteomic analyses of the effects of chronic morphine exposure in a recombinant human neuroblastoma SH-SY5Y clone that stably overexpresses the mu-opioid receptor. Cells were treated with morphine for 6, 24 and 72 hours, the proteins were separated by 2-D gel electrophoresis and stained with Coomassie blue, and the protein map was compared with that obtained from untreated cells. Spots showing a statistically significant variation were selected for identification using mass spectrometric analyses. CONCLUSION: A total of 45 proteins were identified, including proteins involved in cellular metabolism, cytoskeleton organization, vesicular trafficking, transcriptional and translational regulation, and cell signaling.

Long-term morphine treatment enhances proteasome-dependent degradation of G beta in human neuroblastoma SH-SY5Y cells: correlation with onset of adenylate cyclase sensitization.

The initial aim of this study was to identify protein changes associated with long-term morphine treatment in a recombinant human neuroblastoma SH-SY5Y clone (sc2) stably overexpressing the human mu-opioid (MOP) receptor. In MOP receptor-overexpressing sc2 cells, short-term morphine exposure was found to be much more potent and efficacious in inhibiting forskolin-elicited production of cAMP, and long-term morphine exposure was shown to induce a substantially higher degree of opiate dependence, as reflected by adenylate cyclase sensitization, than it did in wild-type neuroblastoma cells. Differential proteomic analysis of detergent-resistant membrane rafts isolated from untreated and chronically morphine-treated sc2 cells revealed long-term morphine exposure to have reliably induced a 30 to 40% decrease in the abundance of five proteins, subsequently identified by mass spectrometry as G protein subunits alphai(2), alphai(3), beta(1), and beta(2), and prohibitin. Quantitative Western blot analyses of whole-cell extracts showed that long-term morphine treatment-induced down-regulation of Gbeta but not of the other proteins is highly correlated (r(2) = 0.96) with sensitization of adenylate cyclase. Down-regulation of Gbeta and adenylate cyclase sensitization elicited by long-term morphine treatment were suppressed in the presence of carbobenzoxy-l-leucyl-l-leucyl-l-norvalinal (MG-115) or lactacystin. Thus, sustained activation of the MOP receptor by morphine in sc2 cells seems to promote proteasomal degradation of Gbeta to sensitize adenylate cyclase. Together, our data suggest that the long-term administration of opiates may elicit dependence by altering the neuronal balance of heterotrimeric G proteins and adenylate cyclases, with the ubiquitin-proteasome pathway playing a pivotal role.