The Effect of a Common Daily Schedule on Human Circadian Rhythms During the Polar Day in Svalbard: A Field Study.

All Arctic visitors have to deal with extreme conditions, including a constant high light intensity during the summer season or constant darkness during winter. The light/dark cycle serves as the most potent synchronizing signal for the biological clock, and any Arctic visitor attending those regions during winter or summer would struggle with the absence of those entraining signals. However, the inner clock can be synchronized by other zeitgebers such as physical activity, food intake, or social interactions. Here, we investigated the effect of the polar day on the circadian clock of 10 researchers attending the polar base station in the Svalbard region during the summer season. The data collected in Svalbard was compared with data obtained just before leaving for the expedition (in the Czech Republic 49.8175°N, 15.4730°E). To determine the circadian functions, we monitored activity/rest rhythm with wrist actigraphy followed by sleep diaries, melatonin rhythm in saliva, and clock gene expression (Per1, Bmal1, and Nr1D1) in buccal mucosa samples. Our data shows that the two-week stay in Svalbard delayed melatonin onset but did not affect its rhythmic secretion, and delayed the activity/rest rhythm. Furthermore, the clock gene expression displayed a higher amplitude in Svalbard compared to the amplitude detected in the Czech Republic. We hypothesize that the common daily schedule at the Svalbard expedition strengthens circadian rhythmicity even in conditions of compromised light/dark cycles. To our knowledge, this is the first study to demonstrate peripheral clock gene expression during a polar expedition.

Circadian rhythms of melatonin and peripheral clock gene expression in idiopathic REM sleep behavior disorder.

OBJECTIVE: To evaluate changes in the expression of clock genes and melatonin levels in patients with idiopathic REM sleep behavior disorder (RBD) as a potential early stage of synucleinopathies. METHODS: We assessed the rhythmicity of circadian clock genes using real time-quantitative polymerase chain reaction and 24-h blood melatonin profiles using radio-immunoassay in 10 RBD patients and nine age-matched controls. RESULTS: The RBD patients did not show circadian rhythmicity for clock genes Per2, Bmal1, and Nr1d1 but the rhythmicity of Per 1 remained, and the amplitude of Per3 was diminished. The 24-h melatonin rhythm did not differ between RBD patients and healthy control subjects. Melatonin profile in RBD patients was delayed by 2 h compared to controls, the habitual sleep phases were phase delayed by about 1 h, however no phase shift occurred in any of the clock genes studied. The control group had stable acrophases of melatonin rhythms of approximately 5 h whereas the RBD patients had a more dispersed range over 11 h. CONCLUSIONS: Our results suggest that RBD could be associated with altered expression of clock genes and delayed melatonin secretion. Thus, we argue that circadian system dysregulation could play a role in RBD.

Protective Effect of Morphine Against the Oxidant-Induced Injury in H9c2 Cells.

There are some indications that morphine may exert myocardial protective effects under certain conditions. The aim of the present study was to investigate the effect of morphine on viability and oxidative state of H9c2 cells (rat cardiomyoblasts) influenced by oxidative stress that was elicited by exposure to tert-butyl hydroperoxide (t-BHP). Our experiments showed that pretreatment with morphine before the addition of t-BHP markedly improved cell viability. Morphine was able to increase total antioxidant capacity of H9c2 cells and to reduce the production of reactive oxygen species, protein carbonylation, and lipid peroxidation. Cellular damage caused by t-BHP was associated with low levels of p38 MAPK and GSK-3ß phosphorylation. Pretreatment with morphine augmented p38 phosphorylation, and the increased phospho-p38/p38 ratio was preserved even in the presence of t-BHP. Morphine did not change the level of GSK-3ß phosphorylation, but interestingly, the phospho-GSK-3ß/GSK-3ß ratio significantly increased after subsequent incubation with t-BHP. Furthermore, morphine exposure resulted in upregulation of the antioxidant enzyme catalase. The protective effect of morphine was abrogated by the addition of the PI3K inhibitor wortmannin and/or p38 MAPK inhibitor SB203580. It can be concluded that morphine may protect H9c2 cells against oxidative stress and that this protection is at least partially mediated through activation of the p38 MAPK and PI3K/GSK-3ß pathways.

Prolonged Morphine Treatment Alters Expression and Plasma Membrane Distribution of ß-Adrenergic Receptors and Some Other Components of Their Signaling System in Rat Cerebral Cortex.

ß-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 ß-adrenergic receptor (ß-AR) signaling system in the rat cerebral cortex. ß-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 ß-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 ß-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 ß-arrestin) compared with controls. Interestingly, morphine exposure caused a partial redistribution of ß-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 ß-AR system and its sensitivity to morphine.

Morphine as a Potential Oxidative Stress-Causing Agent.

Morphine exhibits important pharmacological effects for which it has been used in medical practice for quite a long time. However, it has a high addictive potential and can be abused. Long-term use of this drug can be connected with some pathological consequences including neurotoxicity and neuronal dysfunction, hepatotoxicity, kidney dysfunction, oxidative stress and apoptosis. Therefore, most studies examining the impact of morphine have been aimed at determining the effects induced by chronic morphine exposure in the brain, liver, cardiovascular system and macrophages. It appears that different tissues may respond to morphine diversely and are distinctly susceptible to oxidative stress and subsequent oxidative damage of biomolecules. Importantly, production of reactive oxygen/nitrogen species induced by morphine, which have been observed under different experimental conditions, can contribute to some pathological processes, degenerative diseases and organ dysfunctions occurring in morphine abusers or morphine-treated patients. This review attempts to provide insights into the possible relationship between morphine actions and oxidative stress.

Global changes in the rat heart proteome induced by prolonged morphine treatment and withdrawal.

Morphine belongs among the most commonly used opioids in medical practice due to its strong analgesic effects. However, sustained administration of morphine leads to the development of tolerance and dependence and may cause long-lasting alterations in nervous tissue. Although proteomic approaches enabled to reveal changes in multiple gene expression in the brain as a consequence of morphine treatment, there is lack of information about the effect of this drug on heart tissue. Here we studied the effect of 10-day morphine exposure and subsequent drug withdrawal (3 or 6 days) on the rat heart proteome. Using the iTRAQ technique, we identified 541 proteins in the cytosol, 595 proteins in the plasma membrane-enriched fraction and 538 proteins in the mitochondria-enriched fraction derived from the left ventricles. Altogether, the expression levels of 237 proteins were altered by morphine treatment or withdrawal. The majority of changes (58 proteins) occurred in the cytosol after a 3-day abstinence period. Significant alterations were found in the expression of heat shock proteins (HSP27, α-B crystallin, HSP70, HSP10 and HSP60), whose levels were markedly up-regulated after morphine treatment or withdrawal. Besides that morphine exposure up-regulated MAPK p38 (isoform CRA_b) which is a well-known up-stream mediator of phosphorylation and activation of HSP27 and α-B crystallin. Whereas there were no alterations in the levels of proteins involved in oxidative stress, several changes were determined in the levels of pro- and anti-apoptotic proteins. These data provide a complex view on quantitative changes in the cardiac proteome induced by morphine treatment or withdrawal and demonstrate great sensitivity of this organ to morphine.

Antiarrhythmic effect of prolonged morphine exposure is accompanied by altered myocardial adenylyl cyclase signaling in rats.

BACKGROUND: Morphine is often administered to patients for pain management, but it is also recommended to ameliorate some types of cardiovascular diseases. Nevertheless, there is a lack of information regarding the effect of prolonged morphine treatment on myocardial adenylyl cyclase (AC) signaling, which plays an important role in regulating heart function. METHOD: The present work has investigated the consequences of 10-day administration of high morphine doses (10 mg/kg per day) to adult Wistar rats for functioning of the G-protein-mediated AC signaling system. RESULTS: Morphine treatment appreciably affected neither the number of myocardial ß-adrenoceptors nor the content of selected subunits of trimeric G-proteins (G(s)α, G(i/o)α, G(z)α, G(q/11)α and Gß) but the amount of the dominant myocardial AC isoform V/VI almost doubled. These alterations were accompanied by a marked AC supersensitization: the enzyme activity stimulated by manganese, fluoride, forskolin or isoproterenol was considerably increased (by about 50-100%). In contrast, the ability of opioid agonists to inhibit forskolin-stimulated AC activity was slightly but significantly decreased in both groups. Besides that, morphine markedly decreased the incidence of ischemic ventricular arrhythmias induced by coronary artery occlusion, but did not significantly influence infarct size and arrhythmias occurring during reperfusion. CONCLUSION: Overall, these results indicate that prolonged treatment of rats with high doses of morphine substantially alters the function of myocardial G-protein-regulated AC signaling. These alterations are accompanied by a reduced susceptibility to ischemia-induced ventricular arrhythmias.

Prolonged morphine administration alters protein expression in the rat myocardium.

BACKGROUND: Morphine is used in clinical practice as a highly effective painkiller as well as the drug of choice for treatment of certain heart diseases. However, there is lack of information about its effect on protein expression in the heart. Therefore, here we aimed to identify the presumed alterations in rat myocardial protein levels after prolonged morphine treatment. METHODS: Morphine was administered to adult male Wistar rats in high doses (10 mg/kg per day) for 10 days. Proteins from the plasma membrane- and mitochondria-enriched fractions or cytosolic proteins isolated from left ventricles were run on 2D gel electrophoresis, scanned and quantified with specific software to reveal differentially expressed proteins. RESULTS: Nine proteins were found to show markedly altered expression levels in samples from morphine-treaded rats and these proteins were identified by mass spectrometric analysis. They belong to different cell pathways including signaling, cytoprotective, and structural elements. CONCLUSIONS: The present identification of several important myocardial proteins altered by prolonged morphine treatment points to global effects of this drug on heart tissue. These findings represent an initial step toward a more complex view on the action of morphine on the heart.