Opioids Alleviate Oxidative Stress via the Nrf2/HO-1 Pathway in LPS-Stimulated Microglia.

Opioids are known to have antioxidant effects and to modulate microglial function under certain conditions. It has been previously shown that opioid ligands can effectively inhibit the release of proinflammatory cytokines when stimulated with lipopolysaccharide (LPS) and convert microglia to an anti-inflammatory polarization state. Here, we used C8-B4 cells, the mouse microglial cell line activated by LPS as a model to investigate the anti-inflammatory/antioxidant potential of selected opioid receptor agonists (DAMGO, DADLE, and U-50488). We found that all of these ligands could exert cytoprotective effects through the mechanism affecting LPS-induced ROS production, NADPH synthesis, and glucose uptake. Interestingly, opioids elevated the level of reduced glutathione, increased ATP content, and enhanced mitochondrial respiration in microglial cells exposed to LPS. These beneficial effects were associated with the upregulation of the Nrf2/HO-1 pathway. The present results indicate that activation of opioid signaling supports the preservation of mitochondrial function with concomitant elimination of ROS in microglia and suggest that an Nrf2/HO-1 signaling pathway-dependent mechanism is involved in the antioxidant efficacy of opioids. Opioid receptor agonists may therefore be considered as agents to suppress oxidative stress and inflammatory responses of microglia.

Excess ischemic tachyarrhythmias trigger protection against myocardial infarction in hypertensive rats.

Increased level of C-reactive protein (CRP) is a risk factor for cardiovascular diseases, including myocardial infarction and hypertension. Here, we analyzed the effects of CRP overexpression on cardiac susceptibility to ischemia/reperfusion (I/R) injury in adult spontaneously hypertensive rats (SHR) expressing human CRP transgene (SHR-CRP). Using an in vivo model of coronary artery occlusion, we found that transgenic expression of CRP predisposed SHR-CRP to repeated and prolonged ventricular tachyarrhythmias. Excessive ischemic arrhythmias in SHR-CRP led to a significant reduction in infarct size (IS) compared with SHR. The proarrhythmic phenotype in SHR-CRP was associated with altered heart and plasma eicosanoids, myocardial composition of fatty acids (FAs) in phospholipids, and autonomic nervous system imbalance before ischemia. To explain unexpected IS-limiting effect in SHR-CRP, we performed metabolomic analysis of plasma before and after ischemia. We also determined cardiac ischemic tolerance in hearts subjected to remote ischemic perconditioning (RIPer) and in hearts ex vivo. Acute ischemia in SHR-CRP markedly increased plasma levels of multiple potent cardioprotective molecules that could reduce IS at reperfusion. RIPer provided IS-limiting effect in SHR that was comparable with myocardial infarction observed in naïve SHR-CRP. In hearts ex vivo, IS did not differ between the strains, suggesting that extra-cardiac factors play a crucial role in protection. Our study shows that transgenic expression of human CRP predisposes SHR-CRP to excess ischemic ventricular tachyarrhythmias associated with a drop of pump function that triggers myocardial salvage against lethal I/R injury likely mediated by protective substances released to blood from hypoxic organs and tissue at reperfusion.

Renal Sympathetic Denervation Attenuates Congestive Heart Failure in Angiotensin II-Dependent Hypertension: Studies with Ren-2 Transgenic Hypertensive Rats with Aortocaval Fistula.

OBJECTIVE: We examined if renal denervation (RDN) attenuates the progression of aortocaval fistula (ACF)-induced heart failure or improves renal hemodynamics in Ren-2 transgenic rats (TGR), a model of angiotensin II (ANG II)-dependent hypertension. METHODS: Bilateral RDN was performed 1 week after creation of ACF. The animals studied were ACF TGR and sham-operated controls, and both groups were subjected to RDN or sham denervation. In separate groups, renal artery blood flow (RBF) responses were determined to intrarenal ANG II (2 and 8 ng), norepinephrine (NE) (20 and 40 ng) and acetylcholine (Ach) (10 and 40 ng) 3 weeks after ACF creation. RESULTS: In nondenervated ACF TGR, the final survival rate was 10 versus 50% in RDN rats. RBF was significantly lower in ACF TGR than in sham-operated TGR (6.2 ± 0.3 vs. 9.7 ± 0.5 mL min-1 g-1, p < 0.05), the levels unaffected by RDN. Both doses of ANG II decreased RBF more in ACF TGR than in sham-operated TGR (-19 ± 3 vs. -9 ± 2% and -47 ± 3 vs. -22 ± 2%, p < 0.05 in both cases). RDN did not alter RBF responses to the lower dose, but increased it to the higher dose of ANG II in sham-operated as well as in ACF TGR. NE comparably decreased RBF in ACF TGR and sham-operated TGR, and RDN increased RBF responsiveness. Intrarenal Ach increased RBF significantly more in ACF TGR than in sham-operated TGR (29 ± 3 vs. 17 ± 3%, p < 0.05), the changes unaffected by RDN. ACF creation induced marked bilateral cardiac hypertrophy and lung congestion, both attenuated by RDN. In sham-operated but not in ACF TGR, RDN significantly decreased mean arterial pressure. CONCLUSION: The results show that RDN significantly improved survival rate in ACF TGR; however, this beneficial effect was not associated with improvement of reduced RBF or with attenuation of exaggerated renal vascular responsiveness to ANG II.

Naloxone Is a Potential Binding Ligand and Activator of the Capsaicin Receptor TRPV1.

The receptor channel transient receptor potential vanilloid 1 (TRPV1) functions as a sensor of noxious heat and various chemicals. There is increasing evidence for a crosstalk between TRPV1 and opioid receptors. Here we investigated the effect of the prototypical TRPV1 agonist capsaicin and selected opioid ligands on TRPV1 movement in the plasma membrane and intracellular calcium levels in HEK293 cells expressing TRPV1 tagged with cyan fluorescent protein (CFP). We observed that lateral mobility of TRPV1 increased after treatment of cells with capsaicin or naloxone (a nonselective opioid receptor antagonist) but not with DAMGO (a µ-opioid receptor agonist). Interestingly, both capsaicin and naloxone, unlike DAMGO, elicited intracellular calcium responses. The increased TRPV1 movement and calcium influx induced by capsaicin and naloxone were blocked by the TRPV1 antagonist capsazepine. The ability of naloxone to directly interact with TRPV1 was further corroborated by [3H]-naloxone binding. In conclusion, our data suggest that besides acting as an opioid receptor antagonist, naloxone may function as a potential TRPV1 agonist.

ß-Arrestin 2 and ERK1/2 Are Important Mediators Engaged in Close Cooperation between TRPV1 and µ-Opioid Receptors in the Plasma Membrane.

The interactions between TRPV1 and µ-opioid receptors (MOR) have recently attracted much attention because these two receptors play important roles in pain pathways and can apparently modulate each other's functioning. However, the knowledge about signaling interactions and crosstalk between these two receptors is still limited. In this study, we investigated the mutual interactions between MOR and TRPV1 shortly after their activation in HEK293 cells expressing these two receptors. After activation of one receptor we observed significant changes in the other receptor's lateral mobility and vice versa. However, the changes in receptor movement within the plasma membrane were not connected with activation of the other receptor. We also observed that plasma membrane ß-arrestin 2 levels were altered after treatment with agonists of both these receptors. Knockdown of ß-arrestin 2 blocked all changes in the lateral mobility of both receptors. Furthermore, we found that ß-arrestin 2 can play an important role in modulating the effectiveness of ERK1/2 phosphorylation after activation of MOR in the presence of TRPV1. These data suggest that ß-arrestin 2 and ERK1/2 are important mediators between these two receptors and their signaling pathways. Collectively, MOR and TRPV1 can mutually affect each other's behavior and ß-arrestin 2 apparently plays a key role in the bidirectional crosstalk between these two receptors in the plasma membrane.

Long-term administration of morphine specifically alters the level of protein expression in different brain regions and affects the redox state.

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.

Heart rate changes mediate the embryotoxic effect of antiarrhythmic drugs in the chick embryo.

A significant increase in cardiovascular medication use during pregnancy occurred in recent years. Only limited evidence on safety profiles is available, and little is known about the mechanisms of adverse effect on the fetus. We hypothesized that drug-induced bradycardia is the leading mechanism of developmental toxicity. Embryotoxicity was tested in ovo after administration of various doses of metoprolol, carvedilol, or ivabradine. Embryonic day (ED) 4 and 8 chick embryos were studied by video microscopy and ultrasound biomicroscopy ex ovo after intraamniotic injection of the drug for a period of 30 min. Stroke volume was calculated by the Simpson method and prolate ellipsoid formula. Significant dose-dependent mortality was achieved in embryos injected with carvedilol and ivabradine. In ED4 embryos, metoprolol, carvedilol, and ivabradine reduced the heart rate by 33%, 27%, and 55%, respectively, compared with controls (6%). In ED8 embryos this effect was more pronounced with a heart rate reduction by 71%, 54%, and 53%, respectively (controls, 36%). Cardiac output decreased in all tested groups but only proved significant in the metoprolol group in ED8 embryos. The number of ß-adrenergic receptors showed a downward tendency during embryonic development. A negative chronotropic effect of metoprolol, carvedilol, and ivabradine was increasingly pronounced with embryonic maturity despite a downward trend in the number of ß-adrenergic receptors. This effect was associated with reduced cardiac output in chick embryos, probably leading to premature death. Although standard doses of these drugs appear relatively safe, high doses have a potentially adverse effect on the fetus through reduced heart rate.

Protracted morphine withdrawal induces upregulation of peroxiredoxin II and reduces 14-3-3 protein levels in the rat brain cortex and hippocampus.

Protracted opioid withdrawal is considered to be a traumatic event with many adverse effects. However, little attention is paid to its consequences on the protein expression in the rat brain. A better understanding of the changes at the molecular level is essential for designing future innovative drug therapies. Our previous proteomic data indicated that long-term morphine withdrawal is associated with altered proteins functionally involved in energy metabolism, cytoskeletal changes, oxidative stress, apoptosis, or signal transduction. In this study, we selected peroxiredoxin II (PRX II) as a marker of oxidative stress, 14-3-3 proteins as adaptors, and creatine kinase-B (CK-B) as a marker of energy metabolism to detect their amounts in the brain cortex and hippocampus isolated from rats after 3-month (3 MW) and 6-month morphine withdrawal (6 MW). Methodically, our work was based on immunoblotting accompanied by 2D resolution of PRX II and 14-3-3 proteins. Our results demonstrate significant upregulation of PRX II in the rat brain cortex (3-fold) and hippocampus (1.3-fold) after 3-month morphine abstinence, which returned to the baseline six months since the drug was withdrawn. Interestingly, the level of 14-3-3 proteins was downregulated in both brain areas in 3 MW samples and remained decreased only in the brain cortex of 6 MW. Our findings suggest that the rat brain cortex and hippocampus exhibit the oxidative stress-induced vulnerability represented by compensatory upregulation of PRX II after three months of morphine withdrawal.

Ghrelin/GHS-R1A antagonism in memory test and its effects on central molecular signaling involved in addiction in rats.

Central ghrelin signaling seems to play important role in addiction as well as memory processing. Antagonism of the growth hormone secretagogue receptor (GHS-R1A) has been recently proposed as a promising tool for the unsatisfactory drug addiction therapy. However, molecular aspects of GHS-R1A involvement in specific brain regions remain unclear. The present study demonstrated for the first time that acute as well as subchronic (4 days) administration of the experimental GHS-R1A antagonist JMV2959 in usual intraperitoneal doses including 3 mg/kg, had no influence on memory functions tested in the Morris Water Maze in rats as well as no significant effects on the molecular markers linked with memory processing in selected brain areas in rats, specifically on the ß-actin, c-Fos, two forms of the calcium/calmodulin-dependent protein kinase II (CaMKII, p-CaMKII) and the cAMP-response element binding protein (CREB, p-CREB), within the medial prefrontal cortex (mPFC), nucleus accumbens (NAc), dorsal striatum, and hippocampus (HIPP). Furthermore, following the methamphetamine intravenous self-administration in rats, the 3 mg/kg JMV2959 pretreatment significantly reduced or prevented the methamphetamine-induced significant decrease of hippocampal ß-actin and c-Fos as well as it prevented the significant decrease of CREB in the NAC and mPFC. These results imply, that the GHS-R1A antagonist/JMV2959 might reduce/prevent some of the memory-linked molecular changes elicited by methamphetamine addiction within brain structures associated with memory (HIPP), reward (NAc), and motivation (mPFC), which may contribute to the previously observed significant JMV2959-induced reduction of the methamphetamine self-administration and drug-seeking behavior in the same animals. Further research is necessary to corroborate these results.

Continuous short-term acclimation to moderate cold elicits cardioprotection in rats, and alters ß-adrenergic signaling and immune status.

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 ß1/2/3-adrenoceptors (ß-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 ß1-ARs. The ß2-ARs/protein kinase B/Akt were attenuated while ß3-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 ß1/2-AR desensitization, activation of the ß3-AR/PKG/AMPK pathways, and an immunomodulatory effect.

Proteomic Analysis Unveils Expressional Changes in Cytoskeleton- and Synaptic Plasticity-Associated Proteins in Rat Brain Six Months after Withdrawal from Morphine.

Drug withdrawal is associated with abstinence symptoms including deficits in cognitive functions that may persist even after prolonged discontinuation of drug intake. Cognitive deficits are, at least partially, caused by alterations in synaptic plasticity but the precise molecular mechanisms have not yet been fully identified. In the present study, changes in proteomic and phosphoproteomic profiles of selected brain regions (cortex, hippocampus, striatum, and cerebellum) from rats abstaining for six months after cessation of chronic treatment with morphine were determined by label-free quantitative (LFQ) proteomic analysis. Interestingly, prolonged morphine withdrawal was found to be associated especially with alterations in protein phosphorylation and to a lesser extent in protein expression. Gene ontology (GO) term analysis revealed enrichment in biological processes related to synaptic plasticity, cytoskeleton organization, and GTPase activity. More specifically, significant changes were observed in proteins localized in synaptic vesicles (e.g., synapsin-1, SV2a, Rab3a), in the active zone of the presynaptic nerve terminal (e.g., Bassoon, Piccolo, Rims1), and in the postsynaptic density (e.g., cadherin 13, catenins, Arhgap35, Shank3, Arhgef7). Other differentially phosphorylated proteins were associated with microtubule dynamics (microtubule-associated proteins, Tppp, collapsin response mediator proteins) and the actin-spectrin network (e.g., spectrins, adducins, band 4.1-like protein 1). Taken together, a six-month morphine withdrawal was manifested by significant alterations in the phosphorylation of synaptic proteins. The altered phosphorylation patterns modulating the function of synaptic proteins may contribute to long-term neuroadaptations induced by drug use and withdrawal.

Alterations in the Proteome and Phosphoproteome Profiles of Rat Hippocampus after Six Months of Morphine Withdrawal: Comparison with the Forebrain Cortex.

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.

ß-Arrestin 1 and 2 similarly influence µ-opioid receptor mobility and distinctly modulate adenylyl cyclase activity.

ß-Arrestins are known to play a crucial role in GPCR-mediated transmembrane signaling processes. However, there are still many unanswered questions, especially those concerning the presumed similarities and differences of ß-arrestin isoforms. Here, we examined the roles of ß-arrestin 1 and ß-arrestin 2 at different levels of µ-opioid receptor (MOR)-regulated signaling, including MOR mobility, internalization of MORs, and adenylyl cyclase (AC) activity. For this purpose, naïve HEK293 cells or HEK293 cells stably expressing YFP-tagged MOR were transfected with appropriate siRNAs to block in a specific way the expression of ß-arrestin 1 or ß-arrestin 2. We did not find any significant differences in the ability of ß-arrestin isoforms to influence the lateral mobility of MORs in the plasma membrane. Using FRAP and line-scan FCS, we observed that knockdown of both ß-arrestins similarly increased MOR lateral mobility and diminished the ability of DAMGO and endomorphin-2, respectively, to enhance and slow down receptor diffusion kinetics. However, ß-arrestin 1 and ß-arrestin 2 diversely affected the process of agonist-induced MOR endocytosis and exhibited distinct modulatory effects on AC function. Knockdown of ß-arrestin 1, in contrast to ß-arrestin 2, more effectively suppressed forskolin-stimulated AC activity and prevented the ability of activated-MORs to inhibit the enzyme activity. Moreover, we have demonstrated for the first time that ß-arrestin 1, and partially ß-arrestin 2, may somehow interact with AC and that this interaction is strongly supported by the enzyme activation. These data provide new insights into the functioning of ß-arrestin isoforms and their distinct roles in GPCR-mediated signaling.

Effects of Renal Denervation on the Enhanced Renal Vascular Responsiveness to Angiotensin II in High-Output Heart Failure: Angiotensin II Receptor Binding Assessment and Functional Studies in Ren-2 Transgenic Hypertensive Rats.

Detailed mechanism(s) of the beneficial effects of renal denervation (RDN) on the course of heart failure (HF) remain unclear. The study aimed to evaluate renal vascular responsiveness to angiotensin II (ANG II) and to characterize ANG II type 1 (AT1) and type 2 (AT2) receptors in the kidney of Ren-2 transgenic rats (TGR), a model of ANG II-dependent hypertension. HF was induced by volume overload using aorto-caval fistula (ACF). The studies were performed two weeks after RDN (three weeks after the creation of ACF), i.e., when non-denervated ACF TGR enter the decompensation phase of HF whereas those after RDN are still in the compensation phase. We found that ACF TGR showed lower renal blood flow (RBF) and its exaggerated response to intrarenal ANG II (8 ng); RDN further augmented this responsiveness. We found that all ANG II receptors in the kidney cortex were of the AT1 subtype. ANG II receptor binding characteristics in the renal cortex did not significantly differ between experimental groups, hence AT1 alterations are not responsible for renal vascular hyperresponsiveness to ANG II in ACF TGR, denervated or not. In conclusion, maintained renal AT1 receptor binding combined with elevated ANG II levels and renal vascular hyperresponsiveness to ANG II in ACF TGR influence renal hemodynamics and tubular reabsorption and lead to renal dysfunction in the high-output HF model. Since RDN did not attenuate the RBF decrease and enhanced renal vascular responsiveness to ANG II, the beneficial actions of RDN on HF-related mortality are probably not dominantly mediated by renal mechanism(s).

Impact of three-month morphine withdrawal on rat brain cortex, hippocampus, striatum and cerebellum: proteomic and phosphoproteomic studies.

Opioid addiction is characterized by compulsive drug seeking and taking behavior, which is thought to result from persistent neuroadaptations. However, there is a lack of information about the changes at both the cellular and molecular levels occurring after cessation of drug administration. The aim of our study was to determine alterations of both phosphoproteome and proteome in selected brain regions of the rats (brain cortex, hippocampus, striatum, and cerebellum) 3 months after cessation of 10-day morphine treatment. Phosphoproteome profiling was performed by Pro-Q® Diamond staining. The gel-based proteomic approach accompanied by label-free quantification (MaxLFQ) was used for characterization of proteome changes. The phosphoproteomic analysis revealed the largest change in the hippocampus (14); only few altered proteins were detected in the forebrain cortex (5), striatum (4), and cerebellum (3). The change of total protein composition, determined by 2D electrophoresis followed by LFQ analysis, identified 22 proteins with significantly altered expression levels in the forebrain cortex, 19 proteins in the hippocampus, 12 in the striatum and 10 in the cerebellum. The majority of altered proteins were functionally related to energy metabolism and cytoskeleton reorganization. As the most important change we regard down-regulation of 14-3-3 proteins in rat cortex and hippocampus. Our findings indicate that i) different parts of the brain respond in a distinct manner to the protracted morphine withdrawal, ii) characterize changes of protein composition in these brain parts, and iii) enlarge the scope of evidence for adaptability and distinct neuroplasticity proceeding in the brain of drug-addicted organism.

The cardioprotective effect persisting during recovery from cold acclimation is mediated by the ß2-adrenoceptor pathway and Akt activation.

The infarct size-limiting effect elicited by cold acclimation (CA) is accompanied by increased mitochondrial resistance and unaltered ß1-adrenergic receptor (AR) signaling persisting for 2 wk at room temperature. As the mechanism of CA-elicited cardioprotection is not fully understood, we examined the role of the salvage ß2-AR/Gi/Akt pathway. Male Wistar rats were exposed to CA (8°C, 5 wk), whereas the recovery group (CAR) was kept at 24°C for additional 2 wk. We show that the total number of myocardial ß-ARs in the left ventricular myocardium did not change after CA but decreased after CAR. We confirmed the infarct size-limiting effect in both CA and CAR groups. Acute administration of ß2-AR inhibitor ICI-118551 abolished the protective effect in the CAR group but had no effect in the control and CA groups. The inhibitory Giα1/2 and Giα3 proteins increased in the membrane fraction of the CAR group, and the phospho-Akt (Ser473)-to-Akt ratio also increased. Expression, phosphorylation, and mitochondrial location of the Akt target glycogen synthase kinase (GSK-3ß) were affected neither by CA nor by CAR. However, GSK-3ß translocated from the Z-disk to the H-zone after CA, and acquired its original location after CAR. Our data indicate that the cardioprotection observed after CAR is mediated by the ß2-AR/Gi pathway and Akt activation. Further studies are needed to unravel downstream targets of the central regulators of the CA process and the downstream targets of the Akt protein after CAR.NEW & NOTEWORTHY Cardioprotective effect of cold acclimation and that persisting for 2 wk after recovery engage in different mechanisms. The ß2-adrenoceptor/Gi pathway and Akt are involved only in the mechanism of infarct size-limiting effect occurring during the recovery phase. GSK-3ß translocated from the Z-line to the H-zone of sarcomeres by cold acclimation returns back to the original position after the recovery phase. The results provide new insights potentially useful for the development of cardiac therapies.

Protein alterations induced by long-term agonist treatment of HEK293 cells expressing thyrotropin-releasing hormone receptor and G11alpha protein.

This study aimed to determine whether sustained stimulation with thyrotropin-releasing hormone (TRH), a peptide with important physiological functions, can possibly affect expression of plasma membrane proteins in HEK293 cells expressing high levels of TRH receptor and G(11)alpha protein. Our previous experiments using silver-stained two-dimensional polyacrylamide gel electrophoretograms did not reveal any significant changes in an overall composition of membrane microdomain proteins after long-term treatment with TRH of these cells (Matousek et al. 2005 Cell Biochem Biophys 42: 21-40). Here we used a purified plasma membrane fraction prepared by Percoll gradient centrifugation and proteins resolved by 2D electrophoresis were stained with SYPRO Ruby gel stain. The high enrichment in plasma membrane proteins of this preparation was confirmed by a multifold increase in the number of TRH receptors and agonist stimulated G-protein activity, compared to postnuclear supernatant. By a combination of these approaches we were able to determine a number of clearly discernible protein changes in the plasma membrane-enriched fraction isolated from cells treated with TRH (1 x 10(-5) M, 16 h): 4 proteins disappeared, the level of 18 proteins decreased and the level of 39 proteins increased. Our concomitant immunochemical determinations also indicated a clear down-regulation of G(q/11)alpha proteins in preparations from hormone-treated cells. In parallel, we observed decrease in caspase 3 and alterations in some other apoptotic marker proteins, which were in line with the presumed antiapoptotic effect of TRH.

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.

Biased µ-opioid receptor agonists diversely regulate lateral mobility and functional coupling of the receptor to its cognate G proteins.

There are some indications that biased µ-opioid ligands may diversely affect µ-opioid receptor (MOR) properties. Here, we used confocal fluorescence recovery after photobleaching (FRAP) to study the regulation by different MOR agonists of receptor movement within the plasma membrane of HEK293 cells stably expressing a functional yellow fluorescent protein (YFP)-tagged µ-opioid receptor (MOR-YFP). We found that the lateral mobility of MOR-YFP was increased by (D-Ala2,N-MePhe4,Gly5-ol)-enkephalin (DAMGO) and to a lesser extent also by morphine but decreased by endomorphin-2. Interestingly, cholesterol depletion strongly enhanced the ability of morphine to elevate receptor mobility but significantly reduced or even eliminated the effect of DAMGO and endomorphin-2, respectively. Moreover, the ability of DAMGO and endomorphin-2 to influence MOR-YFP movement was diminished by pertussis toxin treatment. The results obtained by agonist-stimulated [35S]GTPγS binding assays indicated that DAMGO exhibited higher efficacy than morphine and endomorphin-2 did and that the efficacy of DAMGO, contrary to the latter agonists, was enhanced by cholesterol depletion. Overall, our study provides clear evidence that biased MOR agonists diversely affect receptor mobility in plasma membranes as well as MOR/G protein coupling and that the regulatory effect of different ligands depends on the membrane cholesterol content. These findings help to delineate the fundamental properties of MOR regarding their interaction with biased MOR ligands and cognate G proteins.