N6-methyladenosine (m6A) is one of the most prevalent methylated modifications of mRNA in eukaryotes. This reversible alteration can directly or indirectly influence biological functions, including RNA degradation, translation, and splicing. This study investigates the impact of chronic morphine administration and varying withdrawal durations (1 day, 1 week, 4 weeks, and 12 weeks) on the m6A modification levels in brain regions critical to addiction development and persistence. Our findings indicate that in the prefrontal cortex, the m6A levels and METTL3 expression decrease, accompanied by an increase in FTO and ALKBH5 expression, followed by fluctuating, but statistically insignificant changes in methylation-regulating enzymes over prolonged withdrawal. In the striatum, reductions in m6A levels and METTL3 expression are observed at 4 weeks of withdrawal, preceded by non-significant fluctuations in enzyme expression and the m6A modification levels. In contrast, no changes in the m6A modification levels or the expression of related enzymes are detected in the hippocampus and the cerebellum. Our data suggest that m6A modification and its regulatory enzymes undergo region-specific and time-dependent changes in response to chronic morphine exposure and subsequent withdrawal.

• Keywords
• FTO, N6-methyladenosine, morphine, morphine withdrawal, rat brain,
• MeSH
• Substance Withdrawal Syndrome * metabolism   genetics   MeSH
• Adenosine * analogs & derivatives   metabolism   MeSH
• AlkB Homolog 5, RNA Demethylase metabolism   genetics   MeSH
• Alpha-Ketoglutarate-Dependent Dioxygenase FTO metabolism   genetics   MeSH
• Rats MeSH
• RNA, Messenger metabolism   genetics   MeSH
• Methyltransferases metabolism   genetics   MeSH
• Methylation MeSH
• Morphine * adverse effects   pharmacology   administration & dosage   MeSH
• Brain * metabolism   drug effects   MeSH
• Rats, Sprague-Dawley MeSH
• Morphine Dependence metabolism   genetics   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenosine * MeSH
• AlkB Homolog 5, RNA Demethylase MeSH
• Alpha-Ketoglutarate-Dependent Dioxygenase FTO MeSH
• RNA, Messenger MeSH
• Methyltransferases MeSH
• Morphine * MeSH
• N-methyladenosine MeSH Browser

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.

• Keywords
• brain, morphine, oxidative stress, proteomics, withdrawal,
• Publication type
• Journal Article MeSH

Moderate cold acclimation (MCA) is a non-invasive intervention mitigating effects of various pathological conditions including myocardial infarction. We aim to determine the shortest cardioprotective regimen of MCA and the response of β1/2/3-adrenoceptors (β-AR), its downstream signaling, and inflammatory status, which play a role in cell-survival during myocardial infarction. Adult male Wistar rats were acclimated (9 °C, 1-3-10 days). Infarct size, echocardiography, western blotting, ELISA, mitochondrial respirometry, receptor binding assay, and quantitative immunofluorescence microscopy were carried out on left ventricular myocardium and brown adipose tissue (BAT). MultiPlex analysis of cytokines and chemokines in serum was accomplished. We found that short-term MCA reduced myocardial infarction, improved resistance of mitochondria to Ca2+-overload, and downregulated β1-ARs. The β2-ARs/protein kinase B/Akt were attenuated while β3-ARs translocated on the T-tubular system suggesting its activation. Protein kinase G (PKG) translocated to sarcoplasmic reticulum and phosphorylation of AMPKThr172 increased after 10 days. Principal component analysis revealed a significant shift in cytokine/chemokine serum levels on day 10 of acclimation, which corresponds to maturation of BAT. In conclusion, short-term MCA increases heart resilience to ischemia without any negative side effects such as hypertension or hypertrophy. Cold-elicited cardioprotection is accompanied by β1/2-AR desensitization, activation of the β3-AR/PKG/AMPK pathways, and an immunomodulatory effect.

• MeSH
• Adrenergic Agents * metabolism   MeSH
• Myocardial Infarction * pathology   MeSH
• Rats MeSH
• Myocardium metabolism   MeSH
• Rats, Wistar MeSH
• AMP-Activated Protein Kinases metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adrenergic Agents * MeSH
• AMP-Activated Protein Kinases MeSH

The β-adrenergic signaling pathways and antioxidant defence mechanisms play important roles in maintaining proper heart function. Here, we examined the effect of chronic normobaric hypoxia (CNH, 10% O2, 3 weeks) on myocardial β-adrenergic signaling and selected components of the antioxidant system in spontaneously hypertensive rats (SHR) and in a conplastic SHR-mtBN strain characterized by the selective replacement of the mitochondrial genome of SHR with that of the more ischemia-resistant Brown Norway strain. Our investigations revealed some intriguing differences between the two strains at the level of β-adrenergic receptors (β-ARs), activity of adenylyl cyclase (AC) and monoamine oxidase A (MAO-A), as well as distinct changes after CNH exposure. The β2-AR/β1-AR ratio was significantly higher in SHR-mtBN than in SHR, apparently due to increased expression of β2-ARs. Adaptation to hypoxia elevated β2-ARs in SHR and decreased the total number of β-ARs in SHR-mtBN. In parallel, the ability of isoprenaline to stimulate AC activity was found to be higher in SHR-mtBN than that in SHR. Interestingly, the activity of MAO-A was notably lower in SHR-mtBN than in SHR, and it was markedly elevated in both strains after exposure to hypoxia. In addition to that, CNH markedly enhanced the expression of catalase and aldehyde dehydrogenase-2 in both strains, and decreased the expression of Cu/Zn superoxide dismutase in SHR. Adaptation to CNH intensified oxidative stress to a similar extent in both strains and elevated the IL-10/TNF-α ratio in SHR-mtBN only. These data indicate that alterations in the mitochondrial genome can result in peculiar changes in myocardial β-adrenergic signaling, MAO-A activity and antioxidant defence and may, thus, affect the adaptive responses to hypoxia.

• Keywords
• Adenylyl cyclase, Antioxidant defence, Chronic hypoxia, Mitochondrial genome, Monoamine oxidase A, Myocardium, SHR, β-adrenergic receptors,
• MeSH
• Adenylyl Cyclases metabolism   MeSH
• Receptors, Adrenergic, beta metabolism   MeSH
• Hypoxia metabolism   MeSH
• Rats MeSH
• Malondialdehyde metabolism   MeSH
• Monoamine Oxidase metabolism   MeSH
• Myocardium metabolism   MeSH
• Rats, Inbred SHR MeSH
• Signal Transduction physiology   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenylyl Cyclases MeSH
• Receptors, Adrenergic, beta MeSH
• Malondialdehyde MeSH
• Monoamine Oxidase 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.

• Keywords
• Adenylyl cyclase, Beta-adrenergic receptors, G proteins, Lipid rafts, Morphine, Rat brain cortex,
• MeSH
• Adenylyl Cyclases genetics   metabolism   MeSH
• beta-Arrestins genetics   metabolism   MeSH
• Receptors, Adrenergic, beta metabolism   MeSH
• G-Protein-Coupled Receptor Kinase 3 genetics   metabolism   MeSH
• Rats MeSH
• Membrane Microdomains metabolism   MeSH
• Morphine pharmacology   MeSH
• Cerebral Cortex drug effects   metabolism   MeSH
• Narcotics pharmacology   MeSH
• Rats, Wistar MeSH
• Signal Transduction * MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenylyl Cyclases MeSH
• beta-Arrestins MeSH
• Receptors, Adrenergic, beta MeSH
• Grk3 protein, rat MeSH Browser
• G-Protein-Coupled Receptor Kinase 3 MeSH
• Morphine MeSH
• Narcotics MeSH