Browsing by Author "Carvalho, Rui Albuquerque"
Now showing 1 - 4 of 4
Results Per Page
Sort Options
- Adrenaline and reactive oxygen species elicit proteome and energetic metabolism modifications in freshly isolated rat cardiomyocytesPublication . Costa, Vera Marisa; Silva, Renata; Tavares, Ludgero Canário; Vitorino, Rui; Amado, Francisco; Carvalho, Félix; Bastos, Maria de Lourdes; Carvalho, Márcia; Carvalho, Rui Albuquerque; Remião, FernandoThe sustained elevation of plasma and interstitial catecholamine levels, namely adrenaline (ADR), and the generation of reactive oxygen species (ROS) are well recognized hallmarks of several cardiopathologic conditions, like cardiac ischemia/reperfusion (I/R) and heart failure (HF). The present work aimed to investigate the proteomics and energetic metabolism of cardiomyocytes incubated with ADR and/or ROS. To mimic pathologic conditions, freshly isolated calcium-tolerant cardiomyocytes from adult rat were incubated with ADR alone or in the presence of a system capable of generating ROS [(xanthine with xanthine oxidase) (XXO)]. Two-dimensional electrophoresis with matrix-assisted laser desorption/ionization and time-of-flight mass spectrometer analysis were used to define protein spot alterations in the cardiomyocytes incubated with ADR and/or ROS. Moreover, the energetic metabolism and the activity of mitochondrial complexes were evaluated by nuclear magnetic resonance and spectrophotometric determinations, respectively. The protein extract was mainly constituted by cardiac mitochondrial proteins and the alterations found were included in five functional classes: (i) structural proteins, notably myosin light chain-2; (ii) redox regulation proteins, in particular superoxide dismutase (SOD); (iii) energetic metabolism proteins, encompassing ATP synthase alpha chain and dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex; (iv) stress response proteins, like the heat shock proteins; and (v) regulatory proteins, like cytochrome c and voltage-dependent anion channel 1. The XXO system elicited alterations in cardiac contractile proteins, as they showed high levels of cleavage, and also altered energetic metabolism, through increased lactate and alanine levels. The cardiomyocytes incubation with ADR resulted in an accentuated increase in mitochondrial complexes activity and the decrease in alanine/lactate ratio, thus reflecting a high cytosolic NADH/NAD(+) ratio. Furthermore, an increase in manganese SOD expression and total SOD activity occurred in the ADR group, as the increase in the mitochondrial complexes presumably led to higher 'electron leakage'. The modifications in proteins, enzymes activity, and energetic metabolism were indicative that different pathways are activated by catecholamines and ROS. These alterations altogether determine the I/R and HF specific features and contribute for the initiation or aggravation of those cardiopathologic conditions.
- Adrenaline in pro-oxidant conditions elicits intracellular survival pathways in isolated rat cardiomyocytesPublication . Costa, Vera Marisa; Silva, Renata; Ferreira, Rita; Amado, Francisco; Carvalho, Félix; Bastos, Maria de Lourdes; Carvalho, Rui Albuquerque; Carvalho, Márcia; Remião, FernandoIn several pathologic conditions, like cardiac ischemia/reperfusion, the sustained elevation of plasma and interstitial catecholamine levels, namely adrenaline (ADR), and the generation of reactive oxygen species (ROS) are hallmarks. The present work aimed to investigate in cardiomyocytes which intracellular signalling pathways are altered by ADR redox ability. To mimic pathologic conditions, freshly isolated calcium tolerant cardiomyocytes from adult rat were incubated with ADR alone or in the presence of a system capable of generating ROS [(xanthine with xanthine oxidase) (X/XO)]. ADR elicited a pro-oxidant signal with generation of reactive species, which was largely magnified by the ROS generating system. However, no change in cardiomyocytes viability was observed. The pro-oxidant signal promoted the translocation to the nucleus of the transcription factors, Heat shock factor-1 (HSF-1) and Nuclear factor-kappaB (NF-kappaB). In addition, proteasome activity was compromised in the experimental groups where the generation of reactive species occurred. The decrease in the proteasome activity of the ADR group resulted from its redox sensitivity, since the activity was recovered by adding the ROS scavenger, tiron. Proteasome inhibition seemed to elicit an increase in HSP70 levels. Furthermore, retention of mitochondrial cytochrome c and inhibition of caspase 3 activity were observed by X/XO incubation in presence or absence of ADR. In conclusion, in spite of all the insults inflicted to the cardiomyocytes, they were capable to activate intracellular responses that enabled their survival. These mechanisms, namely the pathways altered by catecholamine proteasome inhibition, should be further characterized, as they could be of relevance in the ischemia preconditioning and the reperfusion injury.
- Cross-functioning between the extraneuronal monoamine transporter and multidrug resistance protein 1 in the uptake of adrenaline and export of 5-(glutathion-S-yl) adrenaline in rat cardiomyocytesPublication . Costa, Vera Marisa; Ferreira, Luísa Maria; Branco, Paula Sério; Carvalho, Félix; Bastos, Maria de Lourdes; Carvalho, Rui Albuquerque; Carvalho, Márcia; Remião, FernandoIsolated heart cells are highly susceptible to the toxicity of catecholamine oxidation products, namely, to catecholamine-glutathione adducts. Although cellular uptake and/or efflux of these products may constitute a crucial step, the knowledge about the involvement of transporters is still very scarce. This work aimed to contribute to the characterization of membrane transport mechanisms, namely, extraneuronal monoamine transporter (EMT), the multidrug resistant protein 1 (MRP1), and P-glycoprotein (P-gp) in freshly isolated cardiomyocytes from adult rats. These transporters may be accountable for uptake and/or efflux of adrenaline and an adrenaline oxidation product, 5-(glutathion-S-yl)adrenaline, in cardiomyocyte suspensions. Our results showed that 5-(glutathion-S-yl)adrenaline efflux was mediated by MRP1. Additionally, we demonstrated that the adduct formation occurs within the cardiomyocytes, since EMT inhibition reduced the intracellular adduct levels. The classical uptake2 transport in rat myocardial cells was inhibited by the typical EMT inhibitor, corticosterone, and surprisingly was also inhibited by low concentrations of another drug, a well-known P-gp inhibitor, GF120918. The P-gp activity was absent in the cells since P-gp-mediated efflux of quinidine was not blocked by GF120918. In conclusion, this work showed that freshly isolated cardiomyocytes from adult rats constitute a good model for the study of catecholamines and catecholamines metabolites membrane transport. The cardiomyocytes maintain EMT and MRP1 fully active, and these transporters contribute to the formation and efflux of 5-(glutathion-S-yl)adrenaline. In the present experimental conditions, P-gp activity is absent in the isolated cardiomyocytes.
- Oxidation process of adrenaline in freshly isolated rat cardiomyocytes: formation of adrenochrome, quinoproteins, and GSH adductPublication . Costa, Vera Marisa; Silva, Renata; Ferreira, Luísa Maria; Branco, Paula Sério; Carvalho, Félix; Bastos, Maria de Lourdes; Carvalho, Rui Albuquerque; Carvalho, Márcia; Remião, FernandoHigh concentrations of circulating biogenic catecholamines often exist during the course of several cardiovascular disorders. Additionally, coronary dysfunctions are prominent and frequently related to the ischemic and reperfusion phenomenon (I/R) in the heart, which leads to the release of large amounts of catecholamines, namely adrenaline, and to a sustained generation of reactive oxygen species (ROS). Thus, this work aimed to study the toxicity of adrenaline either alone or in the presence of a system capable of generating ROS [xanthine with xanthine oxidase (X/XO)], in freshly isolated, calcium tolerant cardiomyocytes from adult rats. Studies were performed for 3 h, and cardiomyocyte viability, ATP level, lipid peroxidation, protein carbonylation content, and glutathione status were evaluated, in addition to the formation of adrenaline's oxidation products and quinoproteins. Intracellular GSH levels were time-dependently depleted with no GSSG formation when cardiomyocytes were exposed to adrenaline or to adrenaline with X/XO. Meanwhile, a time-dependent increase in the rate of formation of adrenochrome and quinoproteins was observed. Additionally, as a new outcome, 5-(glutathion- S-yl)adrenaline, an adrenaline adduct of glutathione, was identified and quantified. Noteworthy is the fact that the exposure to adrenaline alone promotes a higher rate of formation of quinoproteins and glutathione adduct, while adrenochrome formation is favored where ROS production is stimulated. This study shows that the redox status of the surrounding environment greatly influences adrenaline's oxidation pathway, which may trigger cellular changes responsible for cardiotoxicity.