RESUMEN
"Yerba mate" (YM), an aqueous extract of Ilex paraguariensis, has antioxidant, diuretic, cardio-protective and hypoglycaemic properties. Since its effect on the pancreatic islets remains unclear, we evaluated insulin sensitivity and glucose-stimulated insulin secretion (GSIS) in rats consuming YM or tap water (C) for 21 days. Glucose tolerance, glycemia, triglyceridemia, insulinemia, TBARS and FRAP serum levels were evaluated. GSIS and mRNA levels of insulin signaling pathway and inflammatory markers were measured in isolated pancreatic islets from both groups. In C rats, islets were incubated with YM extract or its phenolic components to measure GSIS. YM improved glucose tolerance, enhanced GSIS, increased FRAP plasma levels and islet mRNA levels of IRS-1 and PI3K (p110), and decreased TBARS plasma levels and islet gene expression of TNF-α and PAI-1. Islets from C rats incubated with 100 µg/mL dry YM extract, 1 µM chlorogenic acid, 0.1 and 1 µM rutin, 1 µM caffeic acid or 1 µM quercetin showed an increase in GSIS. Our results suggest that YM enhances glucose tolerance because of its positive effects on GSIS, oxidative stress rate and insulin sensitivity in rat islets, suggesting that long-term dietary supplementation with YM may improve glucose homeostasis in pre-diabetes or type 2 diabetes.
RESUMEN
The reason that determines the pathological deposition of human apolipoprotein A-I variants inducing organ failure has been under research since the early description of natural mutations in patients. To shed light into the events associated with protein aggregation, we studied the structural perturbations that may occur in the natural variant that shows a substitution of a Leucine by an Arginine in position 60 (L60R). Circular dichroism, intrinsic fluorescence measurements, and proteolysis analysis indicated that L60R was more unstable, more sensitive to cleavage and the N-terminus was more disorganized than the protein with the native sequence (Wt). A higher tendency to aggregate was also detected when L60R was incubated at physiological pH. In addition, the small structural rearrangement observed for the freshly folded variant led to the release of tumor necrosis factor-α and interleukin-1ß from a model of macrophages. However, the mutant preserved both its dimeric conformation and its lipid-binding capacity. Our results strongly suggest that the chronic disease may be a consequence of the native conformation loss which elicits the release of protein conformations that could be either cytotoxic or precursors of amyloid conformations.
Asunto(s)
Proteínas Amiloidogénicas/metabolismo , Apolipoproteína A-I/metabolismo , Proteínas Amiloidogénicas/química , Proteínas Amiloidogénicas/genética , Amiloidosis/etiología , Amiloidosis/genética , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Humanos , Mutación Puntual , Multimerización de Proteína , Estabilidad Proteica , Estructura Secundaria de ProteínaRESUMEN
BACKGROUND: Different protein conformations may be involved in the development of clinical manifestations associated with human amyloidosis. Although a fibrillar conformation is usually the signature of damage in the tissues of patients, it is not clear whether this species is per se the cause or the consequence of the disease. Hereditary amyloidosis due to variants of apolipoprotein A-I (apoA-I) with a substitution of a single amino acid is characterized by the presence of fibrillar protein within the lesions. Thus mutations result in increased protein aggregation. Here we set up to characterize the folding of a natural variant with a mutation leading to a deletion at position 107 (apoA-I Lys107-0). Patients carrying this variant show amyloidosis and severe atherosclerosis. METHODS: We oxidized this variant under controlled concentrations of hydrogen peroxide and analyzed the structure obtained after 30-day incubation by fluorescence, circular dichroism and microscopy approaches. Neutrophils activation was characterized by confocal microscopy. RESULTS: We obtained a high yield of well-defined stable fibrillar structures of apoA-I Lys107-0. In an in vitro neutrophils system, we were able to detect the induction of Neutrophils Extracellular Traps (NETs) when we incubated with oxidized apoA-I variants. This effect was exacerbated by the fibrillar structure of oxidized Lys 107-0. CONCLUSIONS: We conclude that a pro-inflammatory microenvironment could result in the formation of aggregation-prone species, which, in addition may induce a positive feed-back in the activation of an inflammatory response. GENERAL SIGNIFICANCE: These events may explain a close association between amyloidosis due to apoA-I Lys107-0 and atherosclerosis.
Asunto(s)
Amiloidosis Familiar/genética , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Aterosclerosis/genética , Mutación , Amiloidosis Familiar/metabolismo , Apolipoproteína A-I/metabolismo , Aterosclerosis/metabolismo , Humanos , Conformación ProteicaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Cocoa extracts rich in polyphenols are used as potential agent for treating diabetes. Cocoa polyphenols have been proved to ameliorate important hallmarks of type-2 diabetes (T2D). They can regulate glucose levels by increasing insulin secretion, promoting ß-cell proliferation and a reduction of insulin resistance. In addition, epidemiological evidence indicates that consumption of flavonoid decreases the incidence of T2D. AIM OF THE STUDY: T2D is preceded by a prediabetic state in which the endocrine-metabolic changes described in T2D are already present. Since epidemiological evidence indicates that consumption of flavonoid decreases its incidence, we evaluated possible preventive effects of polyphenol-enriched cocoa extract on a model of prediabetes induced by sucrose. MATERIALS AND METHODS: We determined circulating parameters and insulin sensitivity indexes, liver protein carbonyl groups and reduced glutathione, liver mRNA expression levels of lipogenic enzymes, expression of different pro-inflammatory mediators, fructokinase activity and liver glycogen content. For that, radioimmunoassay, real-time polymerase chain reaction, Western blot, spectrophotometry, and immunohistochemistry were used. RESULTS: We demonstrated that sucrose administration triggered hypertriglyceridemia, insulin-resistance, and liver increased oxidative stress and inflammation markers compared to control rats. Additionally, we found an increase in glycogen deposit, fructokinase activity, and lipogenic genes expression (SREBP-1c, FAS and GPAT) together with a decrease in P-Akt and P-eNOS protein content (Pâ¯<â¯0.05). Sucrose-induced insulin resistance, hepatic carbohydrate and lipid dysmetabolism, oxidative stress, and inflammation were effectively disrupted by polyphenol-enriched cocoa extract (PECE) co-administration (Pâ¯<â¯0.05). CONCLUSION: Dietary administration of cocoa flavanols may be an effective and complementary tool for preventing or reverting T2D at an early stage of its development (prediabetes).
Asunto(s)
Cacao/química , Diabetes Mellitus Tipo 2/prevención & control , Extractos Vegetales/farmacología , Polifenoles/farmacología , Estado Prediabético/tratamiento farmacológico , Animales , Diabetes Mellitus Tipo 2/metabolismo , Sacarosa en la Dieta/efectos adversos , Modelos Animales de Enfermedad , Humanos , Resistencia a la Insulina , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Estrés Oxidativo/efectos de los fármacos , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/uso terapéutico , Polifenoles/aislamiento & purificación , Polifenoles/uso terapéutico , Estado Prediabético/sangre , Estado Prediabético/etiología , Estado Prediabético/metabolismo , Ratas , Triglicéridos/sangre , Triglicéridos/metabolismoRESUMEN
PURPOSE: To determine the actions of isoespintanol (Isoesp) on post-ischemic myocardial and mitochondrial alterations. METHODS: Hearts removed from Wistar rats were perfused by 20 min. After this period, the coronary flow was interrupted by half an hour and re-established during 1 h. In the treated group, Isoesp was administered at the beginning of reperfusion. To assess the participation of ε isoform of protein kinase C (PKCε), protein kinase B (PKB/Akt), and nitric oxide synthase (NOS), hearts were treated with Isoesp plus the respective inhibitors (chelerythrine, wortmannin, and N-nitro-L-arginine methyl ester). Cell death was determined by triphenyl tetrazolium chloride staining technique. Post-ischemic recovery of contractility, oxidative stress, and content of phosphorylated forms of PKCε, Akt, and eNOS were also examined. Mitochondrial state was assessed through the measurement of calcium-mediated response, calcium retention capacity, and mitochondrial potential. RESULTS: Isoesp limited cell death, decreased post-ischemic dysfunction and oxidative stress, improved mitochondrial state, and increased the expression of PKCε, Akt, and eNOS phosphorylated. All these beneficial effects achieved by Isoesp were annulled by the inhibitors. CONCLUSION: These findings suggest that activation of Akt/eNOS and PKCε signaling pathways are involved in the development of Isoesp-induced cardiac and mitochondria tolerance to ischemia-reperfusion.
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Cardiotónicos/farmacología , Monoterpenos/farmacología , Infarto del Miocardio/tratamiento farmacológico , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Animales , Annonaceae , Corazón/efectos de los fármacos , Corazón/fisiología , Técnicas In Vitro , Masculino , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/fisiología , Monoterpenos/aislamiento & purificación , Contracción Miocárdica/efectos de los fármacos , Infarto del Miocardio/metabolismo , Infarto del Miocardio/fisiopatología , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/fisiopatología , Miocardio/metabolismo , Óxido Nítrico Sintasa de Tipo III , Proteína Quinasa C-epsilon , Proteínas Proto-Oncogénicas c-akt , Ratas WistarRESUMEN
Type-2 Diabetes (T2D) is a metabolic disease characterized by permanent hyperglycemia, whose development can be prevented or delayed by using therapeutic agents and implementing lifestyle changes. Some therapeutic alternatives include regulation of glycemia through modulation of different mediators and enzymes, such as AMP-activated protein kinase (AMPK), a highly relevant cellular energy sensor for metabolic homeostasis regulation, with particular relevance in the modulation of liver and muscle insulin sensitivity. This makes it a potential therapeutic target for antidiabetic drugs. In fact, some of them are standard drugs used for treatment of T2D, such as biguanides and thiazolidindiones. In this review, we compile the principal natural products that are activators of AMPK and their effect on glucose metabolism, which could make them candidates as future antidiabetic agents. Phenolics such as flavonoids and resveratrol, alkaloids such as berberine, and some saponins are potential natural activators of AMPK with a potential future as antidiabetic drugs.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Productos Biológicos/uso terapéutico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Plantas Medicinales/química , Proteínas Quinasas Activadas por AMP/química , Biguanidas/uso terapéutico , Productos Biológicos/química , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Glucosa/metabolismo , Humanos , Hipoglucemiantes/uso terapéutico , Plantas Medicinales/metabolismo , Tiazolidinedionas/química , Tiazolidinedionas/uso terapéuticoRESUMEN
Myocardial ischemia is the leading cause of death worldwide. Despite better outcomes with early coronary artery reperfusion strategies, morbidity and mortality remain significant. The principal myocardial hallmark of myocardial ischemia is cell death and the associated impairment of cardiac contractility. In this way, the use of extracts from medicinal plants versus synthetic drugs to mitigate post-ischemic damage constitutes an alternative. Despite their proven beneficial effects in cardiovascular disorders, the use of many plants is questioned. Our aim is to update the clinical and experimental studies about the actions of medicinal plants and polyphenol-enriched extracts against ischemia-reperfusion injury and the involved mechanisms. A review of the recent scientific literature (last ten years) on cardioprotective medicinal plants was developed using the following bibliographic databases: PubMed, Scopus, Web of Knowledge and Google Scholar. Herein, the clinical and experimental studies on medicinal plants and their phenolic compounds have been reviewed. The second part of this review was centered on the search for medicinal plant extracts and natural products isolated from them as potential cardioprotective agents. The botanical names of the cited plants have been authenticated by searching the Plant List and Royal Botanical Garden, Kew databases. The data collected show that treatment with natural products diminishes post-ischemic damage through an improvement of the mitochondrial functionality mainly mediated by enhanced nitric oxide bioavailability. Despite these results, further studies must be carried out to validate their use to prevent or mitigate ischemia-reperfusion injury in the clinical setting.
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Cardiotónicos/administración & dosificación , Isquemia Miocárdica/prevención & control , Extractos Vegetales/administración & dosificación , Polifenoles/administración & dosificación , Animales , Cardiotónicos/química , Ensayos Clínicos como Asunto , Humanos , Extractos Vegetales/química , Polifenoles/químicaRESUMEN
The cascade of molecular events leading to Human apolipoprotein A-I (apoA-I) amyloidosis is not completely understood, not even the pathways that determine clinical manifestations associated to systemic protein deposition in organs such as liver, kidney and heart. About twenty natural variants of apoA-I were described as inducing amyloidosis, but the mechanisms driving their aggregation and deposition are still unclear. We previously identified that the mutant Gly26Arg but not Lys107-0 induced the release of cytokines and reactive oxygen species from cultured RAW 264.7 murine macrophages, suggesting that part of the pathogenic pathway could elicit of an inflammatory signal. In this work we gained deep insight into this mechanism and determined that Gly26Arg induced a specific pro-inflammatory cascade involving activation of NF-κB and its translocation into the nucleus. These findings suggest that some but not all apoA-I natural variants might promote a pro-oxidant microenvironment which could in turn result in oxidative processing of the variants into a misfolded conformation.
RESUMEN
Our objective was to determine the effects of a polyphenol-enriched cocoa extract (PCE) on myocardial postischemic alterations in normotensive (Wistar rats, W) and spontaneously hypertensive rats (SHR). Isolated hearts were submitted to 110 min of perfusion or 20 min stabilization, 30 min global ischemia, and 60 min reperfusion (R). Other hearts were treated with PCE at the onset of R. Infarct size, the reduced glutathione (GSH), and the expression of phospho-Akt, P-GSK-3ß, and P-eNOS were assessed. In isolated mitochondria, the Ca(2+)-mediated response of mitochondrial permeability transition pore (mPTP), membrane potential (Δψm), and superoxide production were determined. PCE decreased infarct size, partly preserved GSH, increased the P-Akt, P-GSK-3ß, and P-eNOS contents, improved mPTP response to Ca(2+), decreased the superoxide production, and restored Δψm. These data show that PCE decreases the cardiac postischemic damage in W rats and SHR and suggest that Akt/GSK-3ß/eNOS dependent pathways are involved.
Asunto(s)
Cardiotónicos/administración & dosificación , Coca/química , Hipertensión/tratamiento farmacológico , Isquemia/complicaciones , Infarto del Miocardio/complicaciones , Extractos Vegetales/administración & dosificación , Polifenoles/administración & dosificación , Animales , Presión Sanguínea/efectos de los fármacos , Glutatión/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Corazón/efectos de los fármacos , Corazón/fisiopatología , Humanos , Hipertensión/etiología , Hipertensión/fisiopatología , Técnicas In Vitro , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Miocardio/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Wistar , Superóxidos/metabolismoRESUMEN
Tea made from Ilex paraguariensis (IP) dried and minced leaves is a beverage widely consumed by large populations in South America as a source of caffeine (stimulant action) and for its medicinal properties. However, there is little information about the action of IP on the myocardium in the ischemia-reperfusion condition. Therefore, the objective of this study was to examine the effects of an aqueous extract of IP on infarct size in a model of regional ischemia. Isolated rat hearts were perfused by the Langendorff technique and subjected to 40 min of coronary artery occlusion followed by 60 min of reperfusion (ischemic control hearts). Other hearts received IP 30 µg mL(-1) during the first 10 min of reperfusion in the absence or presence of l(G)-nitro-l-arginine methyl ester [l-NAME, a nitric oxide synthase (NOS) inhibitor]. The infarct size was determined by triphenyltetrazolium chloride (TTC) staining. Post-ischemic myocardial function and coronary perfusion were also assessed. Cardiac oxidative damage was evaluated by using the thiobarbituric acid reactive substance (TBARS) concentration and the reduced glutathione (GSH) content. To analyze the mechanisms involved, the expressions of phosphorylated forms of eNOS and Akt were measured. In isolated mitochondria the Ca(2+)-induced mitochondrial permeability transition pore (mPTP) opening was determined. IP significantly decreased the infarct size and improved post-ischemic myocardial function and coronary perfusion. TBARS decreased, GSH was partially preserved, the levels of P-eNOS and P-Akt increased and mPTP opening diminished after IP addition. These changes were abolished by l-NAME. Therefore, our data demonstrate that acute treatment with IP only during reperfusion was effective in reducing myocardial post-ischemic alterations. These actions would be mediated by a decrease of mitochondrial permeability through IP-activated Akt/eNOS-dependent pathways.
Asunto(s)
Corazón/efectos de los fármacos , Ilex paraguariensis/química , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/metabolismo , Extractos Vegetales/farmacología , Animales , Glutatión/metabolismo , Humanos , Técnicas In Vitro , Masculino , Infarto del Miocardio/genética , Miocardio/metabolismo , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Ratas , Ratas WistarRESUMEN
BACKGROUND: The mitochondrial permeability transition pore (mPTP) plays an important role in ischemia-reperfusion in normotensive animals. Our study aims to define their participation in the ischemic preconditioning (IP) in hypertrophied hearts and to assess the role played by NO and mitochondrial ATP-dependent K channels (mitoKATP). MATERIAL AND METHODS: Isolated hearts from spontaneously hypertensive rats (SHR) and age-matched normotensive rats Wistar Kyoto (WKY) were subjected to 35-min or 50-min global ischemia (GI) followed by 2-hour reperfusion (R). IP was induced by a single cycle of 5-min GI and 10-min R (IP1) or three cycles of 2-min GI and 5-min R (IP3) applied before to prolonged ischemia. L-NAME (NOS inhibitor) or 5-HD (mitoKATP blocker) to investigate the role played by NO and mitoKATP, respectively were administered. Infarct size (IS), myocardial function, reduced glutathione (GSH) - as marker of oxidative stress and MnSOD cytosolic activity - as an index of mPTP opening were determined. RESULTS: IP1 significantly decreased the IS in WKY hearts at both ischemia duration times. In SHR, IP1 decreased the IS observed in GI35 but it did not modify that detected at 50-min GI, which was limited by IP3. IP preserved GSH content and decreased MnSOD cytosolic activity in both rat strains. These protective effects were annulled by L-NAME and 5-HD for both ischemic periods in SHR, whereas in WKY they were only effective for 50-min GI. CONCLUSION: Our data demonstrate that the cardioprotection achieved by ischemic preconditioning in hearts from SHR hearts involves an attenuation of mPTP opening NO and mitoKATP-mediated.
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Cardiomegalia/metabolismo , Precondicionamiento Isquémico Miocárdico/métodos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Óxido Nítrico/fisiología , Canales de Potasio/fisiología , Animales , Cardiomegalia/patología , Masculino , Poro de Transición de la Permeabilidad Mitocondrial , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/antagonistas & inhibidores , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Resultado del TratamientoRESUMEN
Human apolipoprotein A-I (apoA-I)-derived amyloidosis can present with either wild-type (Wt) protein deposits in atherosclerotic plaques or as a hereditary form in which apoA-I variants deposit causing multiple organ failure. More than 15 single amino acid replacement amyloidogenic apoA-I variants have been described, but the molecular mechanisms involved in amyloid-associated pathology remain largely unknown. Here, we have investigated by fluorescence and biochemical approaches the stabilities and propensities to aggregate of two disease-associated apoA-I variants, apoA-IGly26Arg, associated with polyneuropathy and kidney dysfunction, and apoA-ILys107-0, implicated in amyloidosis in severe atherosclerosis. Results showed that both variants share common structural properties including decreased stability compared to Wt apoA-I and a more flexible structure that gives rise to formation of partially folded states. Interestingly, however, distinct features appear to determine their pathogenic mechanisms. ApoA-ILys107-0 has an increased propensity to aggregate at physiological pH and in a pro-inflammatory microenvironment than Wt apoA-I, whereas apoA-IGly26Arg elicited macrophage activation, thus stimulating local chronic inflammation. Our results strongly suggest that some natural mutations in apoA-I variants elicit protein tendency to aggregate, but in addition the specific interaction of different variants with macrophages may contribute to cellular stress and toxicity in hereditary amyloidosis.
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Sustitución de Aminoácidos , Proteínas Amiloidogénicas/química , Proteínas Amiloidogénicas/genética , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Animales , Línea Celular , Humanos , Macrófagos/metabolismo , Ratones , Pliegue de Proteína , Multimerización de Proteína , Estabilidad Proteica , Estructura Secundaria de ProteínaRESUMEN
Amyloidoses constitute a group of diseases in which soluble proteins aggregate and deposit extracellularly in tissues. Nonhereditary apolipoprotein A-I (apoA-I) amyloid is characterized by deposits of nonvariant protein in atherosclerotic arteries. Despite being common, little is known about the pathogenesis and significance of apoA-I deposition. In this work we investigated by fluorescence and biochemical approaches the impact of a cellular microenvironment associated with chronic inflammation on the folding and pro-amyloidogenic processing of apoA-I. Results showed that mildly acidic pH promotes misfolding, aggregation, and increased binding of apoA-I to extracellular matrix elements, thus favoring protein deposition as amyloid like-complexes. In addition, activated neutrophils and oxidative/proteolytic cleavage of the protein give rise to pro amyloidogenic products. We conclude that, even though apoA-I is not inherently amyloidogenic, it may produce non hereditary amyloidosis as a consequence of the pro-inflammatory microenvironment associated to atherogenesis.
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Amiloide/metabolismo , Apolipoproteína A-I/metabolismo , Aterosclerosis/metabolismo , Naftalenosulfonatos de Anilina/metabolismo , Animales , Apolipoproteína A-I/química , Apolipoproteína A-I/ultraestructura , Benzotiazoles , Células CHO , Colesterol/metabolismo , Cricetinae , Cricetulus , Heparina/metabolismo , Humanos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Ácido Hipocloroso/farmacología , Metaloproteinasa 12 de la Matriz/metabolismo , Activación Neutrófila/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Proteolisis/efectos de los fármacos , Solventes , Acetato de Tetradecanoilforbol/farmacología , Tiazoles/metabolismo , Triptófano/metabolismoRESUMEN
The possibility of a direct mitochondrial action of Na(+)/H(+) exchanger-1 (NHE-1) inhibitors decreasing reactive oxygen species (ROS) production was assessed in cat myocardium. Angiotensin II and endothelin-1 induced an NADPH oxidase (NOX)-dependent increase in anion superoxide (O(2)(-)) production detected by chemiluminescence. Three different NHE-1 inhibitors [cariporide, BIIB-723, and EMD-87580] with no ROS scavenger activity prevented this increase. The mitochondria appeared to be the source of the NOX-dependent ROS released by the "ROS-induced ROS release mechanism" that was blunted by the mitochondrial ATP-sensitive potassium channel blockers 5-hydroxydecanoate and glibenclamide, inhibition of complex I of the electron transport chain with rotenone, and inhibition of the permeability transition pore (MPTP) by cyclosporin A. Cariporide also prevented O(2)(-) production induced by the opening of mK(ATP) with diazoxide. Ca(2+)-induced swelling was evaluated in isolated mitochondria as an indicator of MPTP formation. Cariporide decreased mitochondrial swelling to the same extent as cyclosporin A and bongkrekic acid, confirming its direct mitochondrial action. Increased O(2)(-) production, as expected, stimulated ERK1/2 and p90 ribosomal S6 kinase phosphorylation. This was also prevented by cariporide, giving additional support to the existence of a direct mitochondrial action of NHE-1 inhibitors in preventing ROS release. In conclusion, we report a mitochondrial action of NHE-1 inhibitors that should lead us to revisit or reinterpret previous landmark observations about their beneficial effect in several cardiac diseases, such as ischemia-reperfusion injury and cardiac hypertrophy and failure. Further studies are needed to clarify the precise mechanism and site of action of these drugs in blunting MPTP formation and ROS release.
Asunto(s)
Mitocondrias Cardíacas/metabolismo , Miocardio/metabolismo , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Superóxidos/metabolismo , Angiotensina II/farmacología , Animales , Antiarrítmicos/farmacología , Cloruro de Calcio/farmacología , Gatos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Guanidinas/farmacología , Técnicas In Vitro , Mitocondrias Cardíacas/efectos de los fármacos , Dilatación Mitocondrial/efectos de los fármacos , NADPH Oxidasas/metabolismo , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Sulfonas/farmacologíaRESUMEN
Two flavonoids, gnaphaliin and tiliroside, isolated from Helichrysum italicum, were studied in vitro for their capacity to inhibit Cu(2+)-induced human low density lipoprotein (LDL) and diluted plasma oxidation. LDL oxidation was monitored by conjugated diene, thiobarbituric acid-reactive substances (TBARS) formation and electrophoretic mobility on agarose gel. Gnaphaliin and tiliroside increased the lag-phase for diene conjugate production in a dose-dependent manner. The reduction of TBARS production confirmed the antioxidant activity of gnaphaliin and tiliroside with 50% inhibitory concentration (IC(50)) values of 8.0+/-3.9 microM and 7.0+/-2.6 microM respectively. Furthermore, the flavonoids negated the Cu(2+)-induced increase in electrophoretic mobility of LDL. Antioxidant activity of gnaphaliin and tiliroside was significantly different when diluted plasma was oxidised by adding 1 mM CuSO(4). Although both flavonoids again reduced the TBARS production, tiliroside showed higher activity than gnaphaliin (IC(50)=10.6+/-2.5 microM vs. IC(50)>50 microM). In conclusion, tiliroside and gnaphaliin are antioxidants against in vitro Cu(2+)-induced LDL oxidation in the same order of magnitude compared to that of the reference drug, probucol.