We investigated the changes in redox state and protein expression in selected parts of the rat brain induced by a 4 week administration of morphine (10 mg/kg/day). We found a significant reduction in lipid peroxidation that mostly persisted for 1 week after morphine withdrawal. Morphine treatment led to a significant increase in complex II in the cerebral cortex (Crt), which was accompanied by increased protein carbonylation, in contrast to the other brain regions studied. Glutathione levels were altered differently in the different brain regions after morphine treatment. Using label-free quantitative proteomic analysis, we found some specific changes in protein expression profiles in the Crt, hippocampus, striatum, and cerebellum on the day after morphine withdrawal and 1 week later. A common feature was the upregulation of anti-apoptotic proteins and dysregulation of the extracellular matrix. Our results indicate that the tested protocol of morphine administration has no significant toxic effect on the rat brain. On the contrary, it led to a decrease in lipid peroxidation and activation of anti-apoptotic proteins. Furthermore, our data suggest that long-term treatment with morphine acts specifically on different brain regions and that a 1 week drug withdrawal is not sufficient to normalize cellular redox state and protein levels.

• Klíčová slova
• brain, morphine, oxidative stress, proteomics, withdrawal,
• Publikační typ
• časopisecké články MeSH

This work aimed to test the effect of 7-day exposure of rats to multifunctional enkephalin analogs LYS739 and LYS744 at doses of 3 mg/kg and 10 mg/kg on the protein composition of rat spleen lymphocytes, brain cortex, and hippocampus. Alterations of proteome induced by LYS739 and LYS744 were compared with those elicited by morphine. The changes in rat proteome profiles were analyzed by label-free quantification (MaxLFQ). Proteomic analysis indicated that the treatment with 3 mg/kg of LYS744 caused significant alterations in protein expression levels in spleen lymphocytes (45), rat brain cortex (31), and hippocampus (42). The identified proteins were primarily involved in RNA processing and the regulation of cytoskeletal dynamics. In spleen lymphocytes, the administration of the higher 10 mg/kg dose of both enkephalin analogs caused major, extensive modifications in protein expression levels: LYS739 (119) and LYS744 (182). Among these changes, the number of proteins associated with immune responses and apoptotic processes was increased. LYS739 treatment resulted in the highest number of alterations in the rat brain cortex (152) and hippocampus (45). The altered proteins were functionally related to the regulation of transcription and cytoskeletal reorganization, which plays an essential role in neuronal plasticity. Administration with LYS744 did not increase the number of altered proteins in the brain cortex (26) and hippocampus (26). Our findings demonstrate that the effect of κ-OR full antagonism of LYS744 is opposite in the central nervous system and the peripheral region (spleen lymphocytes).

• Klíčová slova
• chronic pain treatment, label-free quantification, morphine, multifunctional enkephalin analogs, proteomic analysis, rat brain, rat spleen lymphocytes,
• Publikační typ
• časopisecké články MeSH

The knowledge about proteome changes proceeding during protracted opioid withdrawal is lacking. Therefore, the aim of this work was to analyze the spectrum of altered proteins in the rat hippocampus in comparison with the forebrain cortex after 6-month morphine withdrawal. We utilized 2D electrophoretic workflow (Pro-Q® Diamond staining and Colloidal Coomassie Blue staining) which was preceded by label-free quantification (MaxLFQ). The phosphoproteomic analysis revealed six significantly altered hippocampal (Calm1, Ywhaz, Tuba1b, Stip1, Pgk1, and Aldoa) and three cortical proteins (Tubb2a, Tuba1a, and Actb). The impact of 6-month morphine withdrawal on the changes in the proteomic profiles was higher in the hippocampus-14 proteins, only three proteins were detected in the forebrain cortex. Gene Ontology (GO) enrichment analysis of differentially expressed hippocampal proteins revealed the most enriched terms related to metabolic changes, cytoskeleton organization and response to oxidative stress. There is increasing evidence that energy metabolism plays an important role in opioid addiction. However, the way how morphine treatment and withdrawal alter energy metabolism is not fully understood. Our results indicate that the rat hippocampus is more susceptible to changes in proteome and phosphoproteome profiles induced by 6-month morphine withdrawal than is the forebrain cortex.

• Klíčová slova
• energy metabolism, gel-based proteomics, nLC-MS/MS, oxidative stress, protracted morphine withdrawal, rat brain cortex, rat hippocampus,
• Publikační typ
• časopisecké články MeSH

The observation of the immunomodulatory effects of opioid drugs opened the discussion about possible mechanisms of action and led researchers to consider the presence of opioid receptors (OR) in cells of the immune system. To date, numerous studies analyzing the expression of OR subtypes in animal and human immune cells have been performed. Some of them confirmed the expression of OR at both the mRNA and protein level, while others did not detect the receptor mRNA either. Although this topic remains controversial, further studies are constantly being published. The most recent articles suggested that the expression level of OR in human peripheral blood lymphocytes could help to evaluate the success of methadone maintenance therapy in former opioid addicts, or could serve as a biomarker for chronic pain diagnosis. However, the applicability of these findings to clinical practice needs to be verified by further investigations.

• Klíčová slova
• addiction, chronic pain, immune cells, opioid drugs, opioid receptors, stem cells,
• MeSH
• biologické markery MeSH
• chronická bolest farmakoterapie   etiologie   metabolismus   MeSH
• imunitní systém účinky léků   imunologie   metabolismus   MeSH
• kmenové buňky účinky léků   metabolismus   MeSH
• lidé MeSH
• opioidní analgetika farmakologie   MeSH
• receptory opiátové genetika   metabolismus   MeSH
• regulace genové exprese * MeSH
• zánět komplikace   etiologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• biologické markery MeSH
• opioidní analgetika MeSH
• receptory opiátové MeSH

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 two-dimensional 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-3-phosphate 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.

• MeSH
• abstinenční syndrom patologie   MeSH
• časové faktory MeSH
• hipokampus účinky léků   patologie   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• morfin škodlivé účinky   MeSH
• mozková kůra účinky léků   patologie   MeSH
• opioidní analgetika škodlivé účinky   MeSH
• poruchy spojené s užíváním opiátů patologie   MeSH
• potkani Wistar MeSH
• proteomika MeSH
• regulace genové exprese účinky léků   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• morfin MeSH
• opioidní analgetika MeSH

β-Adrenergic signaling plays an important role in regulating diverse brain functions and alterations in this signaling have been observed in different neuropathological conditions. In this study, we investigated the effect of a 10-day treatment with high doses of morphine (10 mg/kg per day) on major components and functional state of the β-adrenergic receptor (β-AR) signaling system in the rat cerebral cortex. β-ARs were characterized by radioligand binding assays and amounts of various G protein subunits, adenylyl cyclase (AC) isoforms, G protein-coupled receptor kinases (GRKs), and β-arrestin were examined by Western blot analysis. AC activity was determined as a measure of functionality of the signaling system. We also assessed the partitioning of selected signaling proteins between the lipid raft and non-raft fractions prepared from cerebrocortical plasma membranes. Morphine treatment resulted in a significant upregulation of β-ARs, GRK3, and some AC isoforms (AC-I, -II, and -III). There was no change in quantity of G proteins and some other signaling molecules (AC-IV, AC-V/VI, GRK2, GRK5, GRK6, and β-arrestin) compared with controls. Interestingly, morphine exposure caused a partial redistribution of β-ARs, Gsα, Goα, and GRK2 between lipid rafts and bulk plasma membranes. Spatial localization of other signaling molecules within the plasma membrane was not changed. Basal as well as fluoride- and forskolin-stimulated AC activities were not significantly different in membrane preparations from control and morphine-treated animals. However, AC activity stimulated by the beta-AR agonist isoprenaline was markedly increased. This is the first study to demonstrate lipid raft association of key components of the cortical β-AR system and its sensitivity to morphine.

• Klíčová slova
• Adenylyl cyclase, Beta-adrenergic receptors, G proteins, Lipid rafts, Morphine, Rat brain cortex,
• MeSH
• adenylátcyklasy genetika   metabolismus   MeSH
• beta arrestiny genetika   metabolismus   MeSH
• beta-adrenergní receptory metabolismus   MeSH
• kinasa 3 receptorů spřažených s G-proteiny genetika   metabolismus   MeSH
• krysa rodu Rattus MeSH
• membránové mikrodomény metabolismus   MeSH
• morfin farmakologie   MeSH
• mozková kůra účinky léků   metabolismus   MeSH
• narkotika farmakologie   MeSH
• potkani Wistar MeSH
• signální transdukce * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenylátcyklasy MeSH
• beta arrestiny MeSH
• beta-adrenergní receptory MeSH
• Grk3 protein, rat MeSH Prohlížeč
• kinasa 3 receptorů spřažených s G-proteiny MeSH
• morfin MeSH
• narkotika MeSH

BACKGROUND: Chronic 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. METHODS: Rats 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. RESULTS: Significant 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. CONCLUSIONS: In 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 SIGNIFICANCE: Analysis of reversibility of morphine effect on mammalian brain.

• MeSH
• 2D gelová elektroforéza MeSH
• abstinenční syndrom * MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• krysa rodu Rattus MeSH
• morfin aplikace a dávkování   škodlivé účinky   MeSH
• potkani Wistar MeSH
• přední mozek metabolismus   MeSH
• receptory opiátové delta metabolismus   MeSH
• receptory opiátové kappa metabolismus   MeSH
• receptory opiátové mu metabolismus   MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• morfin MeSH
• receptory opiátové delta MeSH
• receptory opiátové kappa MeSH
• receptory opiátové mu MeSH

Decrease of cholesterol level in plasma membrane of living HEK293 cells transiently expressing FLAG-δ-OR by β-cyclodextrin (β-CDX) resulted in a slight internalization of δ-OR. Massive internalization of δ-OR induced by specific agonist DADLE was diminished in cholesterol-depleted cells. These results suggest that agonist-induced internalization of δ-OR, which has been traditionally attributed exclusively to clathrin-mediated pathway, proceeds at least partially via membrane domains. Identification of internalized pools of FLAG-δ-OR by colocalization studies with proteins of Rab family indicated the decreased presence of receptors in early endosomes (Rab5), late endosomes and lysosomes (Rab7) and fast recycling vesicles (Rab4). Slow type of recycling (Rab11) was unchanged by cholesterol depletion. As expected, agonist-induced internalization of oxytocin receptors was totally suppressed in β-CDX-treated cells. Determination of average fluorescence lifetime of TMA-DPH, the polar derivative of hydrophobic membrane probe diphenylhexatriene, in live cells by FLIM indicated a significant alteration of the overall PM structure which may be interpreted as an increased "water-accessible space" within PM area. Data obtained by studies of HEK293 cells transiently expressing FLAG-δ-OR by "antibody feeding" method were extended by analysis of the effect of cholesterol depletion on distribution of FLAG-δ-OR in sucrose density gradients prepared from HEK293 cells stably expressing FLAG-δ-OR. Major part of FLAG-δ-OR was co-localized with plasma membrane marker Na,K-ATPase and β-CDX treatment resulted in shift of PM fragments containing both FLAG-δ-OR and Na,K-ATPase to higher density. Thus, the decrease in content of the major lipid constituent of PM resulted in increased density of resulting PM fragments.

• Klíčová slova
• Cholesterol, Internalization, Plasma membrane, Rab proteins, δ-opioid receptor,
• MeSH
• buněčná membrána chemie   MeSH
• cholesterol metabolismus   MeSH
• HEK293 buňky MeSH
• intracelulární membrány chemie   MeSH
• lidé MeSH
• Rab proteiny vázající GTP metabolismus   MeSH
• receptory opiátové delta agonisté   metabolismus   MeSH
• struktury buněčné membrány chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cholesterol MeSH
• Rab proteiny vázající GTP MeSH
• receptory opiátové delta MeSH

BACKGROUND: Proteomic analysis was performed in post-nuclear supernatant (PNS) and Percoll-purified membranes (PM) prepared from fore brain cortex of rats exposed to increasing doses of morphine (10-50 mg/kg) for 10 days. RESULTS: In PNS, the 10 up (↑)- or down (↓)-regulated proteins exhibiting the largest morphine-induced change were selected, excised manually from the gel and identified by MALDI-TOF MS/MS: 1-(gi|148747414, Guanine deaminase), ↑2.5×; 2-(gi|17105370, Vacuolar-type proton ATP subunit B, brain isoform), ↑2.6×; 3-(gi|1352384, Protein disulfide-isomerase A3), ↑3.4×; 4-(gi|40254595, Dihydropyrimidinase-related protein 2), ↑3.6×; 5-(gi|149054470, N-ethylmaleimide sensitive fusion protein, isoform CRAa), ↑2.0×; 6-(gi|42476181, Malate dehydrogenase, mitochondrial precursor), ↑1.4×; 7-(gi|62653546, Glyceraldehyde-3-phosphate dehydrogenase), ↑1.6×; 8-(gi|202837, Aldolase A), ↑1.3×; 9-(gi|31542401, Creatine kinase B-type), ↓0.86×; 10-(gi|40538860, Aconitate hydratase, mitochondrial precursor), ↑1.3×. The identified proteins were of cytoplasmic (1, 4, 5, 7, 9), cell membrane (2), endoplasmic reticulum (3) and mitochondrial (6, 8, 10) origin and 9 of them were significantly increased, 1.3-3.6×. The 4 out of 9 up-regulated proteins (4, 6, 7, 10) were described as functionally related to oxidative stress; the 2 proteins participate in genesis of apoptotic cell death.In PM, the 18 up (↑)- or down (↓)-regulated proteins were identified by LC-MS/MS and were of plasma membrane [Brain acid soluble protein, ↓2.1×; trimeric Gβ subunit, ↓2.0x], myelin membrane [MBP, ↓2.5×], cytoplasmic [Internexin, ↑5.2×; DPYL2, ↑4.9×; Ubiquitin hydrolase, ↓2.0×; 60S ribosomal protein, ↑2.7×; KCRB, ↓2.6×; Sirtuin-2, ↑2.5×; Peroxiredoxin-2, ↑2.2×; Septin-11, ↑2.2×; TERA, ↑2.1×; SYUA, ↑2.0×; Coronin-1A, ↓5.4×] and mitochondrial [Glutamate dehydrogenase 1, ↑2.7×; SCOT1, ↑2.2×; Prohibitin, ↑2.2×; Aspartate aminotransferase, ↓2.2×] origin. Surprisingly, the immunoblot analysis of the same PM resolved by 2D-ELFO indicated that the "active", morphine-induced pool of Gβ subunits represented just a minor fraction of the total signal of Gβ which was decreased 1.2x only. The dominant signal of Gβ was unchanged. CONCLUSION: Brain cortex of rats exposed to increasing doses of morphine is far from being adapted. Significant up-regulation of proteins functionally related to oxidative stress and apoptosis suggests a major change of energy metabolism resulting in the state of severe brain cell "discomfort" or even death.

• Publikační typ
• časopisecké články MeSH