Opioid addiction is recognized as a chronic relapsing brain disease resulting from repeated exposure to opioid drugs. Cellular and molecular mechanisms underlying the ability of organism to return back to the physiological norm after cessation of drug supply are not fully understood. The aim of this work was to extend our previous studies of morphine-induced alteration of rat forebrain cortex protein composition to the hippocampus. Rats were exposed to morphine for 10 days and sacrificed 24 h (groups +M10 and −M10) or 20 days after the last dose of morphine (groups +M10/−M20 and −M10/−M20). The six altered proteins (�2-fold) were identified in group (+M10) when compared with group (−M10) by twodimensional fluorescence difference gel electrophoresis (2D-DIGE). The number of differentially expressed proteins was increased to thirteen after 20 days of the drug withdrawal. Noticeably, the altered level of α-synuclein, β-synuclein, α-enolase and glyceraldehyde-3phosphate dehydrogenase (GAPDH) was also determined in both (±M10) and (±M10/ −M20) samples of hippocampus. Immunoblot analysis of 2D gels by specific antibodies oriented against α/β-synucleins and GAPDH confirmed the data obtained by 2D-DIGE analysis. Label-free quantification identified nineteen differentially expressed proteins in group (+M10) when compared with group (−M10). After 20 days of morphine withdrawal (±M10/−-M20), the number of altered proteins was increased to twenty. We conclude that the morphine-induced alteration of protein composition in rat hippocampus after cessation of drug supply proceeds in a different manner when compared with the forebrain cortex. In forebrain cortex, the total number of altered proteins was decreased after 20 days without morphine, whilst in hippocampus, it was increased.

Morphine is an analgesic drug therapeutically administered to relieve pain. However, this drug has numerous side effects, which include impaired healing and regeneration after injuries or tissue damages. It suggests negative effects of morphine on stem cells which are responsible for tissue regeneration. Therefore, we studied the impact of morphine on the properties and functional characteristics of human bone marrow-derived mesenchymal stem cells (MSCs). The presence of μ-, δ- and κ-opioid receptors (OR) in untreated MSCs, and the enhanced expression of OR in MSCs pretreated with proinflammatory cytokines, was demonstrated using immunoblotting and by flow cytometry. Morphine modified in a dose-dependent manner the MSC phenotype, inhibited MSC proliferation and altered the ability of MSCs to differentiate into adipocytes or osteoblasts. Furthermore, morphine rather enhanced the expression of genes for various immunoregulatory molecules in activated MSCs, but significantly inhibited the production of the vascular endothelial growth factor, hepatocyte growth factor or leukemia inhibitory factor. All of these observations are underlying the selective impact of morphine on stem cells, and offer an explanation for the mechanisms of the negative effects of opioid drugs on stem cells and regenerative processes after morphine administration or in opioid addicts.

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Opioid receptors (ORs) have been observed as homo- and heterodimers, but it is unclear if the dimers are stable under physiological conditions, and whether monomers or dimers comprise the predominant fraction in a cell. Here, we use three live-cell imaging approaches to assess dimerization of ORs at expression levels that are 10–100 × smaller than in classical biochemical assays. At membrane densities around 25/µm2, a split-GFP assay reveals that κOR dimerizes, while µOR and δOR stay monomeric. At receptor densities < 5/µm2, single-molecule imaging showed no κOR dimers, supporting the concept that dimer formation depends on receptor membrane density. To directly observe the transition from monomers to dimers, we used a single-molecule assay to assess membrane protein interactions at densities up to 100 × higher than conventional single-molecule imaging. We observe that κOR is monomeric at densities < 10/µm2 and forms dimers at densities that are considered physiological. In contrast, µOR and δOR stay monomeric even at the highest densities covered by our approach. The observation of long-lasting co-localization of red and green κOR spots suggests that it is a specific effect based on OR dimerization and not an artefact of coincidental encounters.

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BackgroundChronic exposure of mammalian organism to morphine results in adaption to persistent high opioid tone through homeostatic adjustments. Our previous results indicated that in the frontal brain cortex (FBC) of rats exposed to morphine for 10 days, such a compensatory adjustment was detected as large up-regulation of adenylylcyclases I (8-fold) and II (2.5–fold). The other isoforms of AC (III-IX) were unchanged. Importantly, the increase of ACI and ACII was reversible as it disappeared after 20 days of morphine withdrawal. Changes of down-stream signaling molecules such as G proteins and adenylylcyclases should respond to and be preceded by primary changes proceeding at receptor level. Therefore in our present work, we addressed the problem of reversibility of the long-term morphine effects on μ-, δ- and κ-OR protein levels in FBC.MethodsRats were exposed to increasing doses of morphine (10–40 mg/kg) for 10 days and sacrificed either 24 h (group +M10) or 20 days (group +M10/−M20) after the last dose of morphine in parallel with control animals (groups −M10 and −M10/−M20). Post-nuclear supernatant (PNS) fraction was prepared from forebrain cortex, resolved by 1D-SDS-PAGE under non-dissociated (−DTT) and dissociated (+DTT) conditions, and analyzed for the content of μ-, δ- and κ-OR by immunoblotting with C- and N-terminus oriented antibodies.ResultsSignificant down-regulation of δ-OR form exhibiting Mw ≈ 60 kDa was detected in PNS prepared from both (+M10) and (+M10/−M20) rats. However, the total immunoblot signals of μ-, δ- and κ-OR, respectively, were unchanged. Plasma membrane marker Na, K-ATPase, actin and GAPDH were unaffected by morphine in both types of PNS. Membrane-domain marker caveolin-1 and cholesterol level increased in (+M10) rats and this increase was reversed back to control level in (+M10/−M20) rats.ConclusionsIn FBC, prolonged exposure of rats to morphine results in minor (δ-OR) or no change (μ- and κ-OR) of opioid receptor content. The reversible increases of caveolin-1 and cholesterol levels suggest participation of membrane domains in compensatory responses during opioid withdrawal.General significanceAnalysis of reversibility of morphine effect on mammalian brain.

Opioid receptors (ORs) have been observed as homo-and heterodimers, but it is unclear if the dimers are stable under physiological conditions. Here we use three live-cell imaging approaches to assess dimerization of ORs at different expression levels. At high membrane densities, a split GFP assay reveals that OR dimerizes, while OR and OR stay monomeric. In contrast, singlemolecule imaging showed no OR dimers at low receptor densities. To reconcile our seemingly contradictory results, we used a high-density single-molecule assay to assess membrane protein interactions at densities 100x higher than conventional single-molecule imaging. We observe that OR is monomeric at low densities and forms dimers at densities that are considered physiological. In contrast, OR and OR stay monomeric even at the highest densities covered. The observation of long-lasting OR dimers but not higher order aggregates suggests that OR dimerization is a specific effect and not a result of increasing expression. IntroductionORs are G protein-coupled receptors (GPCRs) from class A with three genes coding for the OR, OR, and OR. Based on pharmacological profiles, more subtypes were proposed, which can be explained by the existence of splicing variants, posttranslational modifications and/or direct interactions between receptors. Dimerization of ORs has been covered in multiple studies, yet the conclusions were contradictory, as for many other GPCRs, likely due to the use of differing methodological approaches and experimental conditions [1].So far, the major techniques to assess dimerization of ORs were co-immunoprecipitation followed by Western blotting, and bioluminescence resonance energy transfer (BRET) [2][3][4][5][6]. Both are bulk techniques, where the signal is obtained from a large population of cells. Not only is the major part of the signal caused by a small, highly expressing fraction of the cells, but in addition, these few cells express the receptors at the highest density. Therefore, the signal mainly reflects the receptor's behavior at a membrane density that is far above the physiological range, but cannot capture the state of the receptors at low membrane densities, as they prevail in many cells in vivo.In this work, we quantitatively measured OR dimerization using different microscopy approaches that work at different densities. Our starting point was a split GFP fluorescence complementation assay in cells with expression levels of 50-100 / µm 2 . This assay gave us a first indication that the OR has a tendency to dimerize, while the OR and OR resembled the monomeric control, which was the PDGFR transmembrane domain (PDGFRTM). However, with conventional dualcolor single-molecule imaging at densities below 5 / µm 2 , we did not observe a significantly higher dimerization for OR than for the other ORs. Therefore, we turned to a recently developed technique called PhotoGate that allows tracking of single molecules in an originally crowded environment by controlling the density of fluorescent molecules in a re...

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