RESUMO
BACKGROUND: High doses of doxorubicin put cancer patients at risk for developing dilated cardiomyopathy. Previously, we showed that doxorubicin treatment decreases SIRT3 (sirtuin 3), the main mitochondrial deacetylase and increases protein acetylation in rat cardiomyocytes. Here, we hypothesize that SIRT3 expression can attenuate doxorubicin induced dilated cardiomyopathy in vivo by preventing the acetylation of mitochondrial proteins. METHODS: Nontransgenic, M3-SIRT3 (truncated SIRT3; short isoform), and M1-SIRT3 (full-length SIRT3; mitochondrial localized) transgenic mice were treated with doxorubicin for 4 weeks (8 mg/kg body weight per week). Echocardiography was performed to assess cardiac structure and function and validated by immunohistochemistry and immunofluorescence (n=4-10). Mass spectrometry was performed on cardiac mitochondrial peptides in saline (n=6) and doxorubicin (n=5) treated hearts. Validation was performed in doxorubicin treated primary rat and human induced stem cell derived cardiomyocytes transduced with adenoviruses for M3-SIRT3 and M1-SIRT3 and deacetylase deficient mutants (n=4-10). RESULTS: Echocardiography revealed that M3-SIRT3 transgenic mice were partially resistant to doxorubicin induced changes to cardiac structure and function whereas M1-SIRT3 expression prevented cardiac remodeling and dysfunction. In doxorubicin hearts, 37 unique acetylation sites on mitochondrial proteins were altered. Pathway analysis revealed these proteins are involved in energy production, fatty acid metabolism, and oxidative stress resistance. Increased M1-SIRT3 expression in primary rat and human cardiomyocytes attenuated doxorubicin-induced superoxide formation, whereas deacetylase deficient mutants were unable to prevent oxidative stress. CONCLUSIONS: Doxorubicin reduced SIRT3 expression and markedly affected the cardiac mitochondrial acetylome. Increased M1-SIRT3 expression in vivo prevented doxorubicin-induced cardiac dysfunction, suggesting that SIRT3 could be a potential therapeutic target for mitigating doxorubicin-induced dilated cardiomyopathy.
Assuntos
Cardiomiopatia Dilatada , Doxorrubicina , Estresse Oxidativo , Sirtuína 3 , Acetilação/efeitos dos fármacos , Animais , Cardiomiopatia Dilatada/induzido quimicamente , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Cardiomiopatia Dilatada/prevenção & controle , Doxorrubicina/efeitos adversos , Doxorrubicina/farmacologia , Insuficiência Cardíaca/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Estresse Oxidativo/efeitos dos fármacos , Ratos , Sirtuína 3/genética , Sirtuína 3/metabolismoRESUMO
The dominant corticosteroid in elasmobranchs, 1α-hydroxycorticosterone (1α-OHB), has a described role in mineral regulation but a presumptive role in energy balance. Energy demand in vertebrates following exposure to a stressor typically involves an immediate but transient release of glucocorticoids as a means of mobilizing available energy stores, usually in the form of glucose. Although a glucocorticoid role for 1α-OHB would be expected, direct glucocorticoid function of this steroid has yet to be reported in any elasmobranch. In addition, elasmobranchs also utilize the metabolite ß-hydroxybutyrate (ß-HB), which is thought to replace the role fatty acids play in most vertebrates as a predominant fuel source in extrahepatic tissues. To determine the mobilization of metabolites and corticosteroids during a stress event, North Pacific spiny dogfish, Squalus acanthias suckleyi, were cannulated and held in a darkened isolation box to recover (24-48 h) before being subjected to an acute air exposure or corticosterone injection. Dogfish were then serially blood sampled at nine timepoints over 48 h. Glucose, ß-HB, 1α-OHB, corticosterone, as well as lactate, pH, and osmolality were quantified in plasma samples. All measured variables increased in control and treatment groups within 48 h from the start of experimentation, and ß-HB and 1α-OHB remained elevated for the duration of the experiment. There was no linear correlation between glucose and 1α-OHB, but there was a weak (R2 = 0.230) although significant (p = 0.001), positive correlation between ß-HB and 1α-OHB. Interestingly, there were also significant correlations between increasing circulating glucose and corticosterone (R2 = 0.349; p < 0.001), and decreasing ß-HB and corticosterone concentrations (R2 = 0.180; p = 0.008). Our data suggest that following successive stressors of capture, surgery, and confinement, 1α-OHB was not correlated with circulating glucose, only weakly correlated with circulating ß-HB concentrations (R2 = 0.230; p = 0.001), and that corticosterone may also serve a role in energy mobilization in this species.
Assuntos
Elasmobrânquios , Squalus acanthias , Squalus , Animais , Corticosterona/metabolismo , Cação (Peixe) , Squalus/metabolismoRESUMO
RNA sequencing is an effective approach for studying aquatic species yielding both physiological and genomic data. However, its population genetic applications are not well-characterized. We investigate this possible role for RNA sequencing for population genomics in Lake Winnipeg, Manitoba, Canada, walleye (Sander vitreus). Lake Winnipeg walleye represent the largest component of the second-largest freshwater fishery in Canada. In the present study, large female walleye were sampled via nonlethal gill biopsy over two years at three spawning sites representing a latitudinal gradient in the lake. Genetic variation from sequenced mRNA was analyzed for neutral and adaptive markers to investigate population structure and possible adaptive variation. We find low population divergence (F ST = 0.0095), possible northward gene flow, and outlier loci that vary latitudinally in transcripts associated with cell membrane proteins and cytoskeletal function. These results indicate that Lake Winnipeg walleye may be effectively managed as a single demographically connected metapopulation with contributing subpopulations and suggest genomic differences possibly underlying observed phenotypic differences. Despite its high cost relative to other genotyping methods, RNA sequencing data can yield physiological in addition to genetic information discussed here. We therefore argue that it is useful for addressing diverse molecular questions in the conservation of freshwater species.
RESUMO
Age is an important parameter for a variety of ecological applications, including population viability analyses, contaminants monitoring and targeting of individuals for conservation. While many organisms can be aged by annual rings, dentition and other techniques (i.e., fish otoliths, clam growth rings, mammal tooth wear), there are no minimally invasive biomarkers for accurately aging birds in the wild. For the past century, banding has been the only way to identify a bird of known age, which requires continuous effort on a large scale with possibly low return rates. Recent studies have identified pentosidine as a potential biomarker of chronological aging in several bird species. To test this idea in four species of long-lived seabirds, we collected skin biopsies from the foot webs of previously banded, known-age seabirds: black-legged kittiwakes (Rissa tridactyla; 0-19 y old), Atlantic puffins (Fratercula arctica; 5-26 y old), razorbills (Alca torda; 0-15 d old) and thick-billed murres (Uria lomvia; 0-35 y old). Foot web samples were specifically chosen because this was the least invasive site for substantial skin biopsy. Samples were analysed with high performance liquid chromatography to quantify pentosidine levels. Collagen levels were estimated through hydroxyproline assays to normalize pentosidine content across individuals. Kittiwakes displayed a weak correlation (r2 = 0.20) between age and pentosidine/collagen. Puffins (adults only, r2 = 0.02), razorbills (chicks only, r2 = 0.08), and murres (adults, r2 = 0.04) did not show any associations with age. We concluded that pentosidine content in the foot web does not appear to be a reliable method for aging seabirds in the wild. An absence of change in pentosidine in the foot web with age is further evidence that long-lived seabirds may maintain physiological performance into old age.
Assuntos
Envelhecimento/fisiologia , Arginina/análogos & derivados , Charadriiformes/fisiologia , Lisina/análogos & derivados , Animais , Arginina/análise , Arginina/metabolismo , Biomarcadores , Aves/fisiologia , Lisina/análise , Lisina/metabolismoRESUMO
Dietary methionine restriction (MR) where methionine is the sole source of sulfur amino acid increases lifespan in diverse species. Methionine restricted rodents experience a decrease in glutathione (GSH), a major antioxidant, in several tissues, which is paradoxical to longevity interventions because tissues with low GSH might experience more oxidative damage. Liver plays a key role in GSH synthesis and here we examined how MR influences GSH metabolism in the liver. We also hypothesised that low GSH might be subsidized by compensatory pathway(s) in the liver. To investigate GSH synthesis and antioxidant responses, Fischer-344 rats were given either a MR diet or a control diet for 8â¯weeks. Based on γ-glutamylcysteine synthetase activity, GSH synthetic capacity did not respond to low dietary methionine availability. Tissue level protein and lipid oxidation markers do not support elevated oxidative damage, despite low GSH availability. Whole tissue and mitochondrial level responses to MR differed. Specifically, the activity of glutathione reductase and thioredoxin reductase increase in whole liver tissue which might offset the effects of declined GSH availability whereas mitochondrial GSH levels were unperturbed by MR. Moreover, enhanced proton leak in liver mitochondria by MR (4â¯week) presumably diminishes ROS production. Taken together, we suggest that the effect of low GSH in liver tissue is subsidized, at least in part, by increased antioxidant activity and possibly by enhanced mitochondrial proton leak.
Assuntos
Antioxidantes/fisiologia , Glutationa/metabolismo , Metionina/deficiência , Mitocôndrias Hepáticas/metabolismo , Animais , Respiração Celular/fisiologia , Dipeptídeos/metabolismo , Glutamato-Cisteína Ligase/metabolismo , Glutationa/biossíntese , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Peróxido de Hidrogênio/metabolismo , Fígado/metabolismo , Masculino , Malondialdeído/metabolismo , Estresse Oxidativo/fisiologia , Consumo de Oxigênio/fisiologia , Carbonilação Proteica/fisiologia , Distribuição Aleatória , Ratos Endogâmicos F344 , Espécies Reativas de Oxigênio/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismoRESUMO
Naked mole-rats (NMRs) are mouse-sized mammals that exhibit an exceptionally long lifespan (>30 vs. <4 years for mice), and resist aging-related pathologies such as cardiovascular and pulmonary diseases, cancer, and neurodegeneration. However, the mechanisms underlying this exceptional longevity and disease resistance remain poorly understood. The oxidative stress theory of aging posits that (a) senescence results from the accumulation of oxidative damage inflicted by reactive oxygen species (ROS) of mitochondrial origin, and (b) mitochondria of long-lived species produce less ROS than do mitochondria of short-lived species. However, comparative studies over the past 28 years have produced equivocal results supporting this latter prediction. We hypothesized that, rather than differences in ROS generation, the capacity of mitochondria to consume ROS might distinguish long-lived species from short-lived species. To test this hypothesis, we compared mitochondrial production and consumption of hydrogen peroxide (H2 O2 ; as a proxy of overall ROS metabolism) between NMR and mouse skeletal muscle and heart. We found that the two species had comparable rates of mitochondrial H2 O2 generation in both tissues; however, the capacity of mitochondria to consume ROS was markedly greater in NMRs. Specifically, maximal observed consumption rates were approximately two and fivefold greater in NMRs than in mice, for skeletal muscle and heart, respectively. Our results indicate that differences in matrix ROS detoxification capacity between species may contribute to their divergence in lifespan.
Assuntos
Antioxidantes/metabolismo , Longevidade , Mitocôndrias/metabolismo , Animais , Peróxido de Hidrogênio/antagonistas & inibidores , Peróxido de Hidrogênio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos-Toupeira , Espécies Reativas de Oxigênio/metabolismoRESUMO
Dietary methionine restriction (MR) increases lifespan in several animal models. Despite low dietary intake of sulphur amino acids, rodents on MR develop hyperhomocysteinemia. On the contrary, MR has been reported to increase H2S production in mice. Enzymes involved in homocysteine metabolism also take part in H2S production and hence, in this study, the impact of MR on hyperhomocysteinemia and H2S production capacity were investigated using Fischer-344 rats assigned either a control or a MR diet for 8 weeks. The MR animals showed elevated plasma homocysteine accompanied with a reduction in liver cysteine content and methylation potential. It was further found that MR decreased cystathionine-ß-synthase (CBS) activity in the liver, however, MR increased hepatic cystathionine-γ-lyase (CGL) activity which is the second enzyme in the transsulfuration pathway and also participates in regulating H2S production. The relative contribution of CGL in H2S production increased concomitantly with the increased CGL activity. Additionally, hepatic mercaptopyruvate-sulphur-transferase (MPST) activity also increased in response to MR. Taken together, our results suggest that reduced CBS activity and S-Adenosylmethionine availability contributes to hyperhomocysteinimia in MR animals. Elevated CGL and MPST activities may provide a compensatory mechanism for maintaining hepatic H2S production capacity in response to the decreased CBS activity.
Assuntos
Alimentos Formulados , Sulfeto de Hidrogênio/metabolismo , Hipercolesterolemia/metabolismo , Fígado/metabolismo , Metionina/deficiência , Animais , Hipercolesterolemia/etiologia , Hipercolesterolemia/patologia , Fígado/patologia , Masculino , Ratos , Ratos Endogâmicos F344RESUMO
Many studies have investigated ammonia excretion and acid-base regulation in aquatic arthropods, yet current knowledge of marine chelicerates is non-existent. In American horseshoe crabs (Limulus polyphemus), book gills bear physiologically distinct regions: dorsal and ventral half-lamellae, a central mitochondria-rich area (CMRA) and peripheral mitochondria-poor areas (PMPAs). In the present study, the CMRA and ventral half-lamella exhibited characteristics important for ammonia excretion and/or acid-base regulation, as supported by high expression levels of Rhesus-protein 1 (LpRh-1), cytoplasmic carbonic anhydrase (CA-2) and hyperpolarization-activated cyclic nucleotide-gated K+ channel (HCN) compared with the PMPA and dorsal half-lamella. The half-lamellae displayed remarkable differences; the ventral epithelium was ion-leaky whereas the dorsal counterpart possessed an exceptionally tight epithelium. LpRh-1 was more abundant than Rhesus-protein 2 (LpRh-2) in all investigated tissues, but LpRh-2 was more prevalent in the PMPA than in the CMRA. Ammonia influx associated with high ambient ammonia (HAA) treatment was counteracted by intact animals and complemented by upregulation of branchial CA-2, V-type H+-ATPase (HAT), HCN and LpRh-1 mRNA expression. The dorsal epithelium demonstrated characteristics of active ammonia excretion. However, an influx was observed across the ventral epithelium as a result of the tissue's high ion conductance, although the influx rate was not proportionately high considering the â¼3-fold inwardly directed ammonia gradient. These novel findings suggest a role for the coxal gland in excretion and in the maintenance of hemolymph ammonia regulation under HAA. Hypercapnic exposure induced compensatory respiratory acidosis and partial metabolic depression. Functional differences between the two halves of a branchial lamella may be physiologically beneficial in reducing the backflow of waste products into adjacent lamellae, especially in fluctuating environments where ammonia levels can increase.
Assuntos
Equilíbrio Ácido-Base , Amônia/metabolismo , Proteínas de Artrópodes/metabolismo , Caranguejos Ferradura/metabolismo , Animais , Brânquias/enzimologia , Brânquias/metabolismo , Brânquias/ultraestrutura , Caranguejos Ferradura/enzimologia , MasculinoRESUMO
The most common methods of measuring mitochondrial hydrogen peroxide production are based on the extramitochondrial oxidation of a fluorescent probe such as amplex ultra red (AUR) by horseradish peroxidase (HRP). These traditional HRP-based assays only detect H2O2 that has escaped the matrix, raising the potential for substantial underestimation of production if H2O2 is consumed by matrix antioxidant pathways. To measure this underestimation, we characterized matrix consumers of H2O2 in rat skeletal muscle mitochondria, and developed specific means to inhibit these consumers. Mitochondria removed exogenously added H2O2 (2.5µM) at rates of 4.7 and 5.0nmol min(-1) mg protein(-1) when respiring on glutamate+malate and succinate+rotenone, respectively. In the absence of respiratory substrate, or after disrupting membranes by cycles of freeze-thaw, rates of H2O2 consumption were negligible. We concluded that matrix consumers are respiration-dependent (requiring respiratory substrates), suggesting the involvement of either the thioredoxin (Trx) and/or glutathione (GSH)-dependent enzymatic pathways. The Trx-reductase inhibitor auranofin (2µM), and a pre-treatment of mitochondria with 35µM of 1-chloro-2,4-dintrobenzene (CDNB) to deplete GSH specifically compromise these two consumption pathways. These inhibition approaches presented no undesirable "off-target" effects during extensive preliminary tests. These inhibition approaches independently and additively decreased the rate of consumption of H2O2 exogenously added to the medium (2.5µM). During traditional HRP-based H2O2 efflux assays, these inhibition approaches independently and additively increased apparent efflux rates. When used in combination (double inhibition), these inhibition approaches allowed accumulation of (endogenously produced) H2O2 in the medium at a comparable rate whether it was measured with an end point assay where 2.5µM H2O2 is initially added to the medium or with traditional HRP-based efflux assays. This finding confirms that a high degree of inhibition of all matrix consumers is attained with the double inhibition. Importantly, this double inhibition of the matrix consumers allowed revealing that a large part of the H2O2 produced in muscle mitochondria is consumed before escaping the matrix during traditional HRP-based efflux assays. The degree of this underestimation was substrate dependent, reaching >80% with malate, which complicates comparisons of substrates for their capacity to generate H2O2 in normal conditions i.e. when matrix consumers are active. Our results also urge caution in interpreting changes in H2O2 efflux in response to a treatment; when HRP-based assays are used, large changes in apparent H2O2 efflux may come from altered capacity of the matrix consumers.
Assuntos
Glutationa/metabolismo , Peróxido de Hidrogênio/metabolismo , Mitocôndrias Musculares/metabolismo , Tiorredoxinas/metabolismo , Animais , Antioxidantes/metabolismo , Auranofina/administração & dosagem , Dinitroclorobenzeno/administração & dosagem , Humanos , Estresse Oxidativo/genética , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
At its core mitochondrial function relies on redox reactions. Electrons stripped from nutrients are used to form NADH and NADPH, electron carriers that are similar in structure but support different functions. NADH supports ATP production but also generates reactive oxygen species (ROS), superoxide (O2(·-)) and hydrogen peroxide (H2O2). NADH-driven ROS production is counterbalanced by NADPH which maintains antioxidants in an active state. Mitochondria rely on a redox buffering network composed of reduced glutathione (GSH) and peroxiredoxins (Prx) to quench ROS generated by nutrient metabolism. As H2O2 is quenched, NADPH is expended to reactivate antioxidant networks and reset the redox environment. Thus, the mitochondrial redox environment is in a constant state of flux reflecting changes in nutrient and ROS metabolism. Changes in redox environment can modulate protein function through oxidation of protein cysteine thiols. Typically cysteine oxidation is considered to be mediated by H2O2 which oxidizes protein thiols (SH) forming sulfenic acid (SOH). However, problems begin to emerge when one critically evaluates the regulatory function of SOH. Indeed SOH formation is slow, non-specific, and once formed SOH reacts rapidly with a variety of molecules. By contrast, protein S-glutathionylation (PGlu) reactions involve the conjugation and removal of glutathione moieties from modifiable cysteine residues. PGlu reactions are driven by fluctuations in the availability of GSH and oxidized glutathione (GSSG) and thus should be exquisitely sensitive to changes ROS flux due to shifts in the glutathione pool in response to varying H2O2 availability. Here, we propose that energy metabolism-linked redox signals originating from mitochondria are mediated indirectly by H2O2 through the GSH redox buffering network in and outside mitochondria. This proposal is based on several observations that have shown that unlike other redox modifications PGlu reactions fulfill the requisite criteria to serve as an effective posttranslational modification that controls protein function.
Assuntos
Metabolismo Energético , Mitocôndrias/metabolismo , Oxirredução , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Animais , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , NADP/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Remarkably little is known about nitrogenous excretion in freshwater invertebrates. In the current study, the nitrogen excretion mechanism in the carnivorous ribbon leech, Nephelopsis obscura, was investigated. Excretion experiments showed that the ribbon leech is ammonotelic, excreting 166.0 ± 8.6 nmol·grams fresh weight (gFW)(-1)·h(-1) ammonia and 14.7 ± 1.9 nmol·gFW(-1)·h(-1) urea. Exposure to high and low pH hampered and enhanced, respectively, ammonia excretion rates, indicating an acid-linked ammonia trapping mechanism across the skin epithelia. Accordingly, compared with body tissues, the skin exhibited elevated mRNA expression levels of a newly identified Rhesus protein and at least in tendency the Na(+)/K(+)-ATPase. Pharmacological experiments and enzyme assays suggested an ammonia excretion mechanism that involves the V-ATPase, Na(+)/K(+)-ATPase, and carbonic anhydrase, but not necessarily a functional microtubule system. Most importantly, functional expression studies of the identified Rh protein cloned from leech skin tissue revealed an ammonia transport capability of this protein when expressed in yeast. The leech Rh-ammonia transporter (NoRhp) is a member of the primitive Rh protein family, which is a sister group to the common ancestor of vertebrate ammonia-transporting Rh proteins. Exposure to high environmental ammonia (HEA) caused a new adjustment of body ammonia, accompanied with a decrease in NoRhp and Na(+)/K(+)-ATPase mRNA levels, but unaltered ammonia excretion rates. To our knowledge, this is only the second comprehensive study regarding the ammonia excretion mechanisms in a freshwater invertebrate, but our results show that basic processes of ammonia excretion appear to also be comparable to those found in freshwater fish, suggesting an early evolution of ionoregulatory mechanisms in freshwater organisms.
Assuntos
Amônia/metabolismo , Proteínas de Transporte/metabolismo , Sanguessugas/metabolismo , Animais , Proteínas de Transporte/genética , Ingestão de Alimentos/fisiologia , Meio Ambiente , Água Doce , Concentração de Íons de Hidrogênio , Saccharomyces cerevisiae/metabolismo , Pele/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Ureia/metabolismoRESUMO
The soil-dwelling nematode Caenorhabditis elegans is a bacteriovorous animal, excreting the vast majority of its nitrogenous waste as ammonia (25.3±1.2â µmol gFW(-1) day(-1)) and very little urea (0.21±0.004â µmol gFW(-1) day(-1)). Although these roundworms have been used for decades as genetic model systems, very little is known about their strategy to eliminate the toxic waste product ammonia from their bodies into the environment. The current study provides evidence that ammonia is at least partially excreted via the hypodermis. Starvation reduced the ammonia excretion rates by more than half, whereas mRNA expression levels of the Rhesus protein CeRhr-2, V-type H(+)-ATPase (subunit A) and Na(+)/K(+)-ATPase (α-subunit) decreased correspondingly. Moreover, ammonia excretion rates were enhanced in media buffered to pH 5 and decreased at pH 9.5. Inhibitor experiments, combined with enzyme activity measurements and mRNA expression analyses, further suggested that the excretion mechanism involves the participation of the V-type H(+)-ATPase, carbonic anhydrase, Na(+)/K(+)-ATPase, and a functional microtubule network. These findings indicate that ammonia is excreted, not only by apical ammonia trapping, but also via vesicular transport and exocytosis. Exposure to 1â mmol l(-1) NH4Cl caused a 10-fold increase in body ammonia and a tripling of ammonia excretion rates. Gene expression levels of CeRhr-1 and CeRhr-2, V-ATPase and Na(+)/K(+)-ATPase also increased significantly in response to 1â mmol l(-1) NH4Cl. Importantly, a functional expression analysis showed, for the first time, ammonia transport capabilities for CeRhr-1 in a phylogenetically ancient invertebrate system, identifying these proteins as potential functional precursors to the vertebrate ammonia-transporting Rh-glycoproteins.
Assuntos
Amônia/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Amônia/farmacologia , Animais , Anidrases Carbônicas/metabolismo , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Transporte Proteico , ATPase Trocadora de Sódio-Potássio/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Ammonia is a highly toxic molecule and often introduced in considerable amounts into aquatic environments due to anthropogenic activities. Many aquatic and semi-aquatic amphibians utilize, in addition to their kidneys, the skin for osmoregulation and nitrogen excretion. In the present study the effects of prolonged (7-21 days) exposure to high environmental ammonia (HEA, 1 mmol l(-1) NH4Cl) on cutaneous nitrogen excretion and gene expression of key-transporters involved in nitrogen excretion and acid-base regulation were investigated in the fully aquatic African clawed frog, Xenopus laevis. The study revealed that X. laevis excretes predominately ammonia of which approximately 50% is excreted via the skin. Both the ventral and dorsal skin were capable to generate a net ammonia efflux, which was significantly activated by 10 mmol l(-1) of the phosphodiesterase blocker theophylline. The obtained data further suggest that the ammonia efflux was promoted by an acidification of the unstirred boundary layer, likely generated by an apical localized V-ATPase, with NH3 being transported via cutaneous expressed ammonia transporters, Rhbg and Rhcg. Prolonged HEA exposure did significantly reduce the net-flux rates over the ventral skin with Vmax changing from 256 nmol cm(-2) h(-1) in control frogs to 196 nmol cm(-2) h(-1) in HEA exposed animals. Further, prolonged HEA exposure caused a decrease in mRNA expression levels of the ammonia transporter Rhbg, Na(+)/K(+)-ATPase (α-subunit) and V-ATPase (subunit H) in the ventral and dorsal skin and the kidney. In contrast, Rhcg expression levels were unaffected by HEA in skin tissues.
Assuntos
Amônia/análise , Exposição Ambiental , Nitrogênio/metabolismo , Pele/metabolismo , Xenopus laevis/fisiologia , Análise de Variância , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Primers do DNA/genética , Masculino , Modelos Biológicos , Reação em Cadeia da Polimerase em Tempo Real , ATPase Trocadora de Sódio-Potássio/metabolismo , Teofilina/farmacologia , Ureia/metabolismoRESUMO
The production of H(2)O(2) by isolated mitochondria is frequently used as a measure of mitochondrial superoxide formation. Matrix superoxide dismutase quantitatively converts matrix superoxide to H(2)O(2). However, matrix enzymes such as the glutathione peroxidases can consume H(2)O(2) and compete with efflux of H(2)O(2), causing an underestimation of superoxide production. To assess this underestimate, we depleted matrix glutathione in rat skeletal muscle mitochondria by more than 90% as a consequence of pretreatment with 1-chloro-2,4-dintrobenzene (CDNB). The pretreatment protocol strongly diminished the mitochondrial capacity to consume exogenous H(2)O(2), consistent with decreased peroxidase capacity, but avoided direct stimulation of superoxide production from complex I. It elevated the observed rates of H(2)O(2) formation from matrix-directed superoxide by up to two-fold from several sites of production, as defined by substrates and electron transport inhibitors, over a wide range of control rates, from 0.2-2.5 nmol H(2)O(2) min(-1) mg protein(-1). Similar results were obtained when glutathione was depleted using monochlorobimane or when soluble matrix peroxidase activity was removed by preparation of submitochondrial particles. The data indicate that the increased H(2)O(2) efflux observed with CDNB pretreatment was a result of glutathione depletion and compromised peroxidase activity. A hyperbolic correction curve was constructed, making H(2)O(2) efflux a more quantitative measure of matrix superoxide production. For rat muscle mitochondria, the correction equation was: CDNB-pretreated rate = control rate + [1.43 x (control rate)]/(0.55 + control rate). These results have significant ramifications for the rates and topology of superoxide production by isolated mitochondria.
Assuntos
Glutationa/deficiência , Peróxido de Hidrogênio/metabolismo , Mitocôndrias Musculares/metabolismo , Superóxidos/metabolismo , Animais , Feminino , Glutationa/metabolismo , Mitocôndrias Musculares/química , Mitocôndrias Musculares/enzimologia , Ratos , Ratos Wistar , Superóxido Dismutase/metabolismoRESUMO
The unusual energy metabolism of elasmobranchs is characterized by limited or absent fatty acid oxidation in cardiac and skeletal muscle and a great reliance on ketone bodies and amino acids as oxidative fuels in these tissues. Other extrahepatic tissues in elasmobranchs rely on ketone bodies and amino acids for aerobic energy production but, unlike muscle, also appear to possess a significant capacity to oxidize fatty acids. This organization of energy metabolism is reflected by relatively low plasma levels of non-esterified fatty acids (NEFA) and by plasma levels of the ketone body ss-hydroxybutyrate that are as high as those seen in fasted mammals. The preference for ketone body oxidation rather than fatty acid oxidation in muscle of elasmobranchs under routine conditions is opposite to the situation in teleosts and mammals. Carbohydrates appear to be utilized as a fuel source in elasmobranchs, similar to other vertebrates. Amino acid- and lipid-fueled ketogenesis in the liver, the lipid storage site in elasmobranchs, sustains the demand for ketone bodies as oxidative fuels. The liver also appears to export NEFA and serves a buoyancy role. The regulation of energy metabolism in elasmobranchs and the effects of environmental factors remain poorly understood. The metabolic organization of elasmobranchs was likely present in the common ancestor of the Chondrichthyes ca. 400million years ago and, speculatively, it may reflect the ancestral metabolism of jawed vertebrates. We assess hypotheses for the evolution of the unusual energy metabolism of elasmobranchs and propose that the need to synthesize urea has influenced the utilization of ketone bodies and amino acids as oxidative fuels.
Assuntos
Elasmobrânquios/metabolismo , Metabolismo Energético , Fígado/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Adaptação Fisiológica , Aminoácidos/metabolismo , Animais , Evolução Biológica , Metabolismo dos Carboidratos , Meio Ambiente , Ácidos Graxos/metabolismo , Doenças dos Peixes/metabolismo , Homeostase , Corpos Cetônicos/metabolismo , Neoplasias/metabolismo , OxirreduçãoRESUMO
The time course of betaine accumulation and activities of enzymes involved in betaine metabolism were studied in developing rats. In study 1, pups weaned on a nonpurified diet had a transient increase in liver and kidney betaine content followed by a decline after approximately 42-56 d. In study 2, dams and, following weaning, pups were fed an AIN-93G (betaine-free) or an AIN-93G betaine-supplemented diet (0.3%) to determine the source of the transient increase in betaine levels previously observed. In study 2, only rats fed betaine had an increase in plasma betaine concentration. Similarly, liver and kidney betaine contents increased postweaning; however, betaine levels returned to that found in rats fed a betaine-free diet by 49 d of age. The dietary content of betaine fed to dams did not affect pup betaine. The activities of choline dehydrogenase, an enzyme of betaine synthesis, and betaine:homocysteine methyltransferase (BHMT), which is the only known betaine-consuming enzyme in mammals, were also measured in study 2. Liver BHMT activity decreased after weaning, whereas liver and kidney choline dehydrogenase activity increased with age, possibly reaching a plateau by 42 d of age. We conclude that the transient increase in betaine reflects high dietary betaine and not a change in endogenous betaine synthesis.
Assuntos
Betaína/metabolismo , Dieta , Envelhecimento , Animais , Betaína/administração & dosagem , Suplementos Nutricionais , Relação Dose-Resposta a Droga , Feminino , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
The present study investigated aspects of betaine metabolism in an elasmobranch fish, the winter skate (Leucoraja ocellata). Based on the level of choline dehydrogenase (ChoDH) activity, the liver and kidney appear to be the major sites of betaine synthesis and the mitochondrial localization of ChoDH and betaine aldehyde dehydrogenase (BADH) indicates that the metabolic organization of betaine synthesis in winter skate is similar to other vertebrates. Food deprivation did not affect white muscle betaine content, and prolonged starvation (70 days) appeared to decrease the total hepatic betaine synthetic capacity. There was no decrease in ChoDH or BADH activity at the mitochondrial level with starvation, suggesting any decrease is due to catabolism of hepatic reserves rather than downregulation of betaine synthesis. Skates fed a high betaine diet (frozen squid approximately 55 micromol g(-1)) had elevated white muscle betaine content compared to those fed a low betaine diet (frozen herring <2 micromol g(-1)); however, high dietary betaine intake did not affect the activity of betaine synthesizing enzymes in liver. Acclimation to elevated salinity (120 and 130% seawater) did not result in an increase in white muscle betaine content. Taken as a whole, the present data suggest that diet is a major determinant of muscle betaine in the winter skate and that betaine is of marginal importance as an intracellular osmolyte in this species.
Assuntos
Betaína/metabolismo , Rajidae/metabolismo , Animais , Biomarcadores , Tamanho Corporal/efeitos dos fármacos , Colina Desidrogenase/metabolismo , Decapodiformes , Privação de Alimentos/fisiologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Metilaminas/análise , Músculos/química , Músculos/efeitos dos fármacos , Pressão Osmótica/efeitos dos fármacos , Cloreto de Sódio/farmacologia , Frações Subcelulares/efeitos dos fármacos , Fatores de Tempo , Distribuição Tecidual/efeitos dos fármacosRESUMO
The contribution of alterations in mitochondrial K(ATP) channel activity and the sarcoplasmic reticulum (SR) to anaerobic cardiac function in the anoxia tolerant armored catfish Liposarcus pardalis were assessed. K(ATP) channels contribute to hypoxic cardioprotection in mammals, but little is known of their action in more hypoxia tolerant animals. Anoxia resulted in a decrease in force in isometrically contracting ventricle strips to approximately 40% of the pre-anoxic level. This was maintained for at least 2 h. Upon reoxygenation, hearts recovered to the same level as control preparations. Treatment with 5-hydroxydecanoic acid (5HD), a specific mitochondrial K(ATP) blocker significantly increased force in preparations during anoxia and caused hypercontracture at reoxygenation. Ryanodine, a specific inhibitor of SR function, significantly increased force loss in ventricle preparations under anoxia. Results show that mitochondrial K(ATP) channel activity and SR function are important in anaerobic and post-anaerobic contractility in armored catfish heart.