RESUMEN

This study examines if heme biosynthesis-associated iron metabolism is regulated in pulmonary arteries by endothelin-1 (ET1) potentially through modulating cartilage oligomeric matrix protein (COMP) availability. Our studies in organoid-cultured endothelium-rubbed bovine pulmonary arteries (BPAs) observed COMP depletion by siRNA or hypoxia increases NOX2 and superoxide and depletes mitochondrial SOD2. ET1 also increases superoxide in a manner that potentially impairs mitochondrial heme biosynthesis. In this study, organoid culture of BPA with ET1 (10 nM) increases superoxide in the mitochondrial matrix and extramitochondrial regions associated with COMP depletion, and COMP (0.5 µM) inhibited these superoxide increases. As mitochondrial matrix superoxide could impair heme biosynthesis from protoporphyrin IX (PpIX) by decreasing Fe2+ availability and/or ferrochelatase (FECH), we studied ET1, COMP, and COMP siRNA effects on the expression of FECH, transferrin receptor-1 (TfR1, an indicator of iron availability) and soluble guanylate cyclase (sGC, a key heme-dependent protein), and on measurements of PpIX (HPLC) and heme content. ET1 decreased FECH, heme, and sGC, and increased TfR1 and iron. COMP reversed these effects of ET1, and COMP decreased PpIX and increased heme in the absence of ET1. COMP siRNA increased PpIX detection and TfR1 expression and decreased the expression of FECH and sGC. Nitric oxide (spermine NONOate) relaxation of BPA was inhibited by ET1, and this was attenuated by COMP during exposure to ET1. Thus, COMP depletion by ET1 or siRNA modulates pulmonary artery iron metabolism, which results in loss of heme biosynthesis and heme-dependent cGMP mechanisms.

Asunto(s)

Arteria Pulmonar , Superóxidos , Animales , Proteína de la Matriz Oligomérica del Cartílago/genética , Bovinos , Endotelina-1/metabolismo , Ferroquelatasa/metabolismo , Ferroquelatasa/farmacología , Hemo/metabolismo , Hierro/metabolismo , Óxido Nítrico/metabolismo , Arteria Pulmonar/metabolismo , ARN Interferente Pequeño/metabolismo , Receptores de Transferrina/metabolismo , Guanilil Ciclasa Soluble/metabolismo , Superóxidos/metabolismo

RESUMEN

Erythropoietic protoporphyria is an inherited disorder of heme biosynthesis caused by partial ferrochelatase deficiency, resulting in protoporphyrin (PP) overproduction by erythrocytes. In humans, it is responsible for painful skin photosensitivity and, occasionally, liver failure due to accumulation of PP in the liver. The ferrochelatase deficiency mouse mutation is the best animal model available for human erythropoietic protoporphyria. The original description, based on mice with a BALB/cByJCrl genetic background, reported a disease resembling the severe form of the human disease, with anemia, jaundice, and liver failure. Using congenic strains, we investigated the effect of genetic background on the severity of the phenotype. Compared with BALB/cByJCrl, C57BL/6JCrl mice developed moderate but increasing anemia and intense liver accumulation of PP with severe hepatocyte damage and loss. Bile excretory function was not affected, and bilirubin remained low. Despite the highest PP concentration in erythrocytes, anemia was mild and there were few PP deposits in the liver in SJL/JOrlCrl homozygotes. Discriminant analysis using six hematologic and biochemical parameters showed that homozygotes of the three genetic backgrounds could be clustered in three well-separated groups. These three congenic strains provide strong evidence for independent genetic control of bone marrow contribution of PP overproduction to development of liver disease and biliary PP excretion. They provide a tool to investigate the physiological mechanisms involved in these phenotypic differences and to identify modifying genes.

Asunto(s)

Anemia/etiología , Anemia/genética , Médula Ósea/fisiología , Modelos Animales de Enfermedad , Ferroquelatasa/genética , Fallo Hepático/genética , Fallo Hepático/fisiopatología , Protoporfiria Eritropoyética/complicaciones , Protoporfiria Eritropoyética/genética , Protoporfirinas/biosíntesis , Animales , Animales Congénicos , Femenino , Ferroquelatasa/farmacología , Humanos , Ictericia/etiología , Ictericia/veterinaria , Fallo Hepático/veterinaria , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mutación , Fenotipo , Protoporfiria Eritropoyética/veterinaria , Índice de Severidad de la Enfermedad

RESUMEN

In order to study effects of environmental contamination, a suite of biomarkers were measured over the period 1996 to 1999 in livers of flounder (Platichthys flesus) from two urban embayments and one non-urban reference site of the Gulf of Finland in the vicinity of Tallinn, Estonia. Total cytochrome P450 (CYP) level, aryl hydrocarbon hydroxylase (AHH), 5-aminolevulinic acid synthetase (ALA-S), and heme synthetase (HEM-S) activities were quantified by means of spectrophotometry. These data were compared to results obtained in 1994 for the same biomarkers at one of the urban embayments and the non-urban site, as measured by the same protocols. For the flounder collected from the non-urban site, changes occurred in AHH activity and the total CYP level, which were significantly lower in 1996 and 1999 compared with 1994 (p < 0.05). Activity of ALA-S decreased slightly over this same period. The activity of HEM-S increased between 1996 and 1999. In the urban site first investigated in 1994, the activities of AHH and ALA-S, as well as the total level of CYP in flounder liver were significantly higher compared with 1999 (p < 0.05). HEM-S activities did not show any significant changes over this time period. AHH activities of flounder collected in another urban site decreased slightly between 1996 and 1999, in contrast to data on the total CYP level which diminished drastically over these years (p < 0.05). Activities of HEM-S increased significantly (p < 0.05) during the period investigated, while activities of ALA-S remained unchanged. These findings suggest that contamination of the marine environments by PAHs has gone down everywhere in the Tallinn area during the last 3 to 5 years. However, the results indicate that the area is still contaminated, as indicated by elevated heme synthesis enzymes and the total CYP content, and that monitoring of contaminants and their effects should be continued in this region.

Asunto(s)

5-Aminolevulinato Sintetasa/farmacología , Hidrocarburo de Aril Hidroxilasas/farmacología , Biomarcadores/análisis , Carcinógenos/análisis , Sistema Enzimático del Citocromo P-450/farmacología , Exposición a Riesgos Ambientales , Ferroquelatasa/farmacología , Lenguado/fisiología , Contaminantes Químicos del Agua/análisis , 5-Aminolevulinato Sintetasa/análisis , Animales , Hidrocarburo de Aril Hidroxilasas/análisis , Carcinógenos/efectos adversos , Ciudades , Sistema Enzimático del Citocromo P-450/análisis , Ferroquelatasa/análisis , Hígado/enzimología , Contaminantes Químicos del Agua/efectos adversos

RESUMEN

Erythropoietic protoporphyria (EPP) is an inherited disorder of heme synthesis caused by deficiency of the mitochondrial enzyme ferrochelatase. EPP in humans is associated with liver disease, hypertriglyceridemia, and a low level of high density lipoprotein (HDL) cholesterol. To explore consequences of ferrochelatase deficiency in lipid metabolism, we have analyzed hepatic lipid content and plasma lipoprotein levels in chow-fed BALB/c mice homozygous ( fch/fch) or heterozygous ( fch/1) for a point mutation in the ferrochelatase gene and in wild-type controls (1/1). Livers of fch/fch mice show bile duct proliferation and biliary fibrosis, but bile formation is not impaired. The free cholesterol content of fch/fch livers is significantly increased when compared with fch/1 and 1/1 livers. Plasma cholesterol in fch/fch mice (9.9 +/- 6.4 mM) is elevated when compared with fch/1 and 1/1 mice (2.9 +/- 0.2 and 2.5 +/- 0.3 mM, respectively), because of an increased cholesterol content in the very low density lipoprotein-sized fractions, whereas HDL cholesterol is reduced. The ratio of cholesteryl ester to free cholesterol is 4.3 +/- 0.6, 3.3 +/- 0.3, and 0.3 +/- 0.1 in the plasma of 1/1, fch/1, and fch/fch mice, respectively. The latter is not due to reduced lecithin:cholesterol acyltransferase activity in plasma of fch/fch mice but to the presence of lipoprotein-X (Lp-X), a particle composed of bile-type lipids usually seen only in cholestatic conditions. Expression of mdr2, essential for biliary phospholipid/cholesterol secretion, is increased in fch/fch livers. In spite of this, biliary phospholipid/cholesterol secretion is reduced relative to that of bile salts. It is postulated that an inability of bile salts to stimulate lipid secretion adequately leads to formation of Lp-X in this noncholestatic condition. Distinct atherosclerotic lesions were found in aged fch/fch mice.Thus, ferrochelatase deficiency in mice leads to liver disease associated with altered hepatic lipid metabolism, a characteristic hyperlipidemia, and development of atherosclerosis.-Bloks, V. W., T. Plösch, H. van Goor, H. Roelofsen, J. Baller, R. Havinga, H. J. Verkade, A. van Tol, P. L. M. Jansen, and F. Kuipers. Hyperlipidemia and atherosclerosis associated with liver disease in ferrochelatase-deficient mice. J. Lipid Res. 2001. 42: 41;-50.

Asunto(s)

Arteriosclerosis/enzimología , Ferroquelatasa/farmacología , Hiperlipidemias/enzimología , Hepatopatías/enzimología , Animales , Arteriosclerosis/etiología , Arteriosclerosis/metabolismo , Hígado Graso/enzimología , Hígado Graso/etiología , Hígado Graso/metabolismo , Ferroquelatasa/genética , Hiperlipidemias/etiología , Hiperlipidemias/metabolismo , Metabolismo de los Lípidos , Lípidos/análisis , Lípidos/sangre , Lipoproteína X/sangre , Lipoproteína X/metabolismo , Lipoproteínas HDL/sangre , Lipoproteínas HDL/efectos de los fármacos , Lipoproteínas HDL/metabolismo , Hígado/metabolismo , Hígado/patología , Hepatopatías/etiología , Hepatopatías/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Mutantes , Protoporfiria Eritropoyética

RESUMEN

A 33-nucleotide, guanine-rich DNA oligomer, PS5.ST1, has been reported to catalyze the metallation of mesoporphyrin IX (MPIX) by copper and zinc ions. In this paper we report a thorough investigation of the properties of this DNAzyme. We have established that a 24-nucleotide sequence (PS5.M), from within PS5.ST1, is both the minimal and most optimal catalytic unit. We have found that three related porphyrins are acceptable as substrates by this DNAzyme, of which protoporphyrin IX is preferred as a substrate over the expected substrate, MPIX. We have determined that it is unlikely that a strong, catalytically relevant binding site for copper ions exists in the DNAzyme and that high concentrations of copper destroy the active DNAzyme. This enzyme, whose folded structure likely contains guanine quartets, requires potassium ions for activity; we have shown that as little as 1 mM potassium is sufficient for its catalytic robustness, whereas as much as 0.5 M sodium still will not support catalysis. In determining the pH, temperature, and salt optima for the catalyzed reaction, we have found an unexpected stabilizing role for Tris buffer in both the catalyzed and background metallation reactions. As a consequence of various steps of optimization, we now have a vastly improved DNAzyme, one whose enzymatic parameters compare well both with those of natural ferrochelatases, as well as with those of artificially derived chelatases, composed of protein (a catalytic antibody) and RNA. The existence of this array of biocatalysts for porphyrin metallations allows one-to-one comparisons of the ways in which different biopolymers solve a given catalytic problem.

Asunto(s)

Oligodesoxirribonucleótidos/metabolismo , Porfirinas/metabolismo , Sitios de Unión , Tampones (Química) , Catálisis/efectos de los fármacos , Cobre/metabolismo , Detergentes/farmacología , Dimetilsulfóxido/farmacología , Ferroquelatasa/farmacología , Concentración de Iones de Hidrógeno , Cinética , Mesoporfirinas/metabolismo , Oligodesoxirribonucleótidos/química , Potasio/farmacología , Protoporfirinas/metabolismo , Sodio/farmacología , Especificidad por Sustrato

RESUMEN

Protoporphyria is generally an autosomal dominant disease characterized genetically by mutations in the ferrochelatase gene. The interaction between the wild-type and mutant ferrochelatase protein is unknown. The aim of this study was to evaluate the ability to correct the enzymatic and biochemical defects in cells from patients with protoporphyria, using a replication-defective human adenovirus for gene transfer. Overexpression of ferrochelatase was accomplished by construction of a vector in which expression of the wild-type ferrochelatase cDNA was driven by the constitutive cytomegalovirus (CMV) promoter, introduction and packaging of the cDNA into human adenovirus dl309, and transduction of normal and protoporphyric fibroblasts. Fibroblasts from controls and patients were infected with the ferrochelatase adenovirus or a control adenovirus and assayed for ferrochelatase activity and the accumulation of protoporphyrin upon challenge with the precursor delta-aminolevulinic acid (ALA). At a multiplicity of infection (moi) of 10, greater than 85% of both the wild-type and protoporphyric fibroblasts were infected. The recombinant adenovirus increased the ferrochelatase protein content and activity in the wild-type and protoporphyric fibroblasts with equal efficiency. Therefore, the presence of the mutant ferrochelatase protein did not inhibit the ferrochelatase activity expressed by the transgene.

Asunto(s)

Adenoviridae/genética , Ferroquelatasa/genética , Ferroquelatasa/metabolismo , Porfiria Hepatoeritropoyética/terapia , Adenoviridae/química , Adenoviridae/patogenicidad , Ácido Aminolevulínico/metabolismo , Células Cultivadas , Citomegalovirus/genética , ADN Complementario , Deuteroporfirinas/metabolismo , Ferroquelatasa/farmacología , Fibroblastos/virología , Células HeLa/metabolismo , Células HeLa/virología , Humanos , Immunoblotting , Porfiria Hepatoeritropoyética/genética , Porfiria Hepatoeritropoyética/patología , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Transfección , beta-Galactosidasa/genética