RESUMEN
Hephaestin (Heph), a membrane-bound multicopper ferroxidase (FOX) expressed in duodenal enterocytes, is required for optimal iron absorption. However, sex-linked anemia (sla) mice harboring a 194-amino acid deletion in the Heph protein are able to absorb dietary iron despite reduced expression and mislocalization of the mutant protein. Thus Heph may not be essential, and mice are able to compensate for the loss of its activity. The current studies were undertaken to search for undiscovered FOXs in rodent enterocytes. An experimental approach was developed to investigate intestinal FOXs in which separate membrane and cytosolic fractions were prepared and FOX activity was measured by a spectrophotometric transferrin-coupled assay. Unexpectedly, FOX activity was noted in membrane and cytosolic fractions of rat enterocytes. Different experimental approaches demonstrated that cytosolic FOX activity was not caused by contamination with membrane Heph or a method-induced artifact. Cytosolic FOX activity was abolished by SDS and heat (78 °C), suggesting protein-mediated iron oxidation, and was also sensitive to Triton X-100. Furthermore, cytosolic FOX activity increased â¼30% in iron-deficient rats (compared with controls) but was unchanged in copper-deficient rats (in contrast to the reported dramatic reduction of Heph expression and activity during copper deficiency). Additional studies done in sla, Heph-knockout, and ceruloplasmin-knockout mice proved that cytosolic FOX activity could not be fully explained by Heph or ceruloplasmin. Therefore rodent enterocytes contain a previously undescribed soluble cytosolic FOX that may function in transepithelial iron transport and complement membrane-bound Heph.
Asunto(s)
Ceruloplasmina/aislamiento & purificación , Enterocitos/enzimología , Anemia Ferropénica/genética , Animales , Fraccionamiento Celular , Membrana Celular/enzimología , Ceruloplasmina/deficiencia , Ceruloplasmina/metabolismo , Citosol/enzimología , Duodeno/citología , Duodeno/enzimología , Ferrozina/análisis , Deficiencias de Hierro , Trastornos del Metabolismo del Hierro/metabolismo , Yeyuno/citología , Yeyuno/enzimología , Masculino , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedades Neurodegenerativas/metabolismo , Oxidación-Reducción , Ratas , Ratas Sprague-Dawley , SolubilidadRESUMEN
Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is a natural compound with antiproliferative properties. Recent studies suggest that these properties might be due to the ability of curcumin to induce apoptosis in tumor cells by increasing the permeability of the mitochondrial membrane. In the present study, we confirm these observations and provide a molecular mechanism for the action of curcumin in rat liver mitochondria. Curcumin induced mitochondrial swelling, the collapse of Deltapsi, and the release of cytochrome C, events associated with the opening of the permeability transition pore (PTP). Experiments were performed with chemically substituted curcumin derivatives. Some derivatives were obtained by modification of groups on the terminal aromatic rings, and others were obtained by substitution of the diketone function with the cyclohexanone function. They demonstrated that phenol and methoxy groups were essential to promote PTP opening. Curcumin and curcumin derivatives that open the PTP were able to oxidize thiol groups. In addition, PTP opening was abolished in medium devoid of O2 and decreased in the presence of catalase, ferrozine, o-phenanthroline, mannitol, or N-ethylmaleimide. These data suggest that the mechanism by which curcumin promotes PTP opening involves the reduction of Fe3+ to Fe2+, inducing hydroxyl radical (HO*) production and oxidation of thiol groups in the membrane, leading to pore opening.
Asunto(s)
Curcumina/análogos & derivados , Curcumina/farmacología , Canales Iónicos/metabolismo , Mitocondrias Hepáticas/efectos de los fármacos , Animales , Citocromos c/metabolismo , Ferrozina/análisis , Ferrozina/química , Hierro/química , Hierro/metabolismo , Masculino , Mitocondrias Hepáticas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial , Poro de Transición de la Permeabilidad Mitocondrial , Dilatación Mitocondrial/efectos de los fármacos , Estructura Molecular , Oxidación-Reducción/efectos de los fármacos , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Compuestos de Sulfhidrilo/análisis , Compuestos de Sulfhidrilo/metabolismoRESUMEN
59Fe uptake rates by mouse duodenal fragments incubated in vitro were markedly reduced by non-permeable reagents, ferricyanide (oxidising agent) and ferrozine (Fe2+ chelator), in the medium; ferrocyanide had no effect. Reduction of Fe3+, as reflected by an increase in ferrozine-(Fe2+)-chelatable iron, was observed in the presence of the tissue fragments. The generation of Fe2+ occurred linearly with time, was independent of the medium ferrozine concentration, and was not due to release of reducing factors from the duodenal fragments. Fe(3+)-reducing activity was mainly present on the mucosal surface and was localised primarily to the proximal region of the small intestine. Changes in Fe3+ reduction rates closely parallelled the changes in duodenal 59Fe uptake, when metabolic inhibitors or modulators of membrane potential were included in the medium. The enhancement in duodenal mucosal 59Fe uptake in chronic hypoxic and iron-deficient mice parallelled the changes in the tissue reduction of medium Fe3+. Moreover, the rates of reduction were quantitatively similar to rates of uptake. These observations indicate that a sequential reduction and uptake process operates for Fe3+ uptake in mouse duodenum.