RESUMEN
The heme oxygenase isozymes, HO-1 and HO-2, oxidatively cleave the heme molecule to produce biliverdin and the gaseous messenger, CO. The cleavage results in the release of iron, a regulator of transferrin, ferritin, and nitric oxide (NO) synthase gene expression. Biliverdin reductase (BVR) then catalyzes the reduction of biliverdin, generating the potent intracellular antioxidant, bilirubin. We report an age-related decrease in HO-1 and HO-2 expression present in select brain regions including the hippocampus and the substantia nigra, that are involved in the high order cognitive processes of learning and memory. The age-related loss of monoxide-producing potential in select regions of the brain was not specific to the HO system but was also observed in neuronal NO-generating system. Furthermore, compared to 2-month old rats, the ability of aged brain tissue to respond to hypoxic/hyperthermia was compromised at both the protein and the transcription levels as judged by attenuated induction of HO-1 immunoreactive protein and its 1.8 Kb transcript. Neotrofin (AIT), a cognitive-enhancing and neuroprotective drug, caused a robust increase in HO-1 immunoreactive protein in select neuronal regions and increased the expression of HO-2 transcripts. The potential interplay between regulation of HO-2 gene expression and the serum levels of the adrenal steroids is discussed. We suggest the search for therapeutic agents that reverse the decline and aberrant stress response of HO enzymes may lead to effective treatment regimens for age-associated neuronal deficits.
Asunto(s)
Envejecimiento , Encéfalo/enzimología , Hemo Oxigenasa (Desciclizante)/biosíntesis , Aminobenzoatos/farmacología , Animales , Northern Blotting , Encéfalo/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Expresión Génica/efectos de los fármacos , Hemo Oxigenasa (Desciclizante)/efectos de los fármacos , Hipertermia Inducida , Hipoxantinas/farmacología , Inmunohistoquímica , Isoenzimas/biosíntesis , Isoenzimas/efectos de los fármacos , Aprendizaje/efectos de los fármacos , Aprendizaje/fisiología , Memoria/efectos de los fármacos , Memoria/fisiología , NADPH Deshidrogenasa/metabolismo , Fármacos Neuroprotectores/farmacología , Óxido Nítrico Sintasa/biosíntesis , ARN Mensajero/análisis , RatasAsunto(s)
Taponamiento Cardíaco/diagnóstico , Rotura Cardíaca/diagnóstico , Infarto del Miocardio/tratamiento farmacológico , Derrame Pericárdico/inducido químicamente , Terapia Trombolítica/efectos adversos , Activador de Tejido Plasminógeno/efectos adversos , Anticoagulantes/efectos adversos , Anticoagulantes/uso terapéutico , Aspirina/efectos adversos , Aspirina/uso terapéutico , Taponamiento Cardíaco/inducido químicamente , Taponamiento Cardíaco/cirugía , Diagnóstico Diferencial , Femenino , Humanos , Persona de Mediana Edad , Infarto del Miocardio/complicaciones , Nitroglicerina/uso terapéutico , Derrame Pericárdico/complicaciones , Pericardiocentesis , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/uso terapéutico , Activador de Tejido Plasminógeno/uso terapéuticoRESUMEN
Heme oxygenase-2 (HO-2) degrades heme [Fe-protoporphyrin IX (Fe-PP)] to CO and bilirubin. The enzyme is a hemoprotein and interacts with nitric oxide. HO-2 has two copies of heme regulatory motif (HRM) with a conserved core of Cys264-Pro265 and Cys281-Pro282. We examined interaction of HO-2 HRMs with Fe-PP, Zn-protoporphyrin IX (Zn-PP; HO-2 inhibitor), and protoporphyrin IX (PP IX). Spectral analyses, using 1:4 or 1:1 molar ratio of the heme to 10-residue peptides, corresponding to HRM containing HO-2 sequences, revealed specific interactions as indicated by a shift in the absorption spectrum of heme. Five residue peptides qualitatively produced similar results. Substitution of cysteine with alanine in either peptide eliminated interactions, and substitution of proline with alanine reduced the peptides' affinity for heme. Neither Zn-PP nor PP IX absorption spectrum was affected by HRM peptides. The circular dichroism spectra confirmed heme-HRM peptides interactions. An astounding 4,000-6,000-fold higher concentrations of KCN were required at pH 7.5 to displace HRM peptides from heme. Data suggest (a) each HRM can contribute to HO-2-heme interaction, (b) heme iron interacts with cysteine thiol, (c) charged residues upstream of Cys264-Pro265 result in its high-affinity heme binding, and (d) inhibition of HO-2 activity by synthetic metalloporphyrins does not involve HRMs. We suggest that heme bound to HRMs may serve as a binding site/reservoir for gaseous signal molecules.
Asunto(s)
Hemo Oxigenasa (Desciclizante)/metabolismo , Metaloporfirinas/metabolismo , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Dominio Catalítico , Dicroismo Circular , Cisteína/química , Hemo/química , Hemo Oxigenasa (Desciclizante)/química , Humanos , Concentración de Iones de Hidrógeno , Fragmentos de Péptidos/química , Prolina/química , Unión Proteica , Protoporfirinas/metabolismo , Espectrofotometría , Espectrofotometría Ultravioleta , Reactivos de Sulfhidrilo/químicaRESUMEN
OBJECTIVES: We sought to evaluate if angiographic dye videointensity of the risk area during percutaneous transluminal coronary angioplasty (PTCA) of the infarct-related artery (IRA) relates to remodeling. BACKGROUND: Poor reflow after myocardial infarction (MI) predicts worse ventricular remodeling. METHODS: Fifty-three patients with a first anterior MI and isolated disease of the left anterior descending (LAD), who underwent "primary" (n = 14), "rescue" (n = 7) or "late" (after 10 +/- 4 days, n = 32) PTCA, were retrospectively selected. In 10 patients prospectively collected, we assessed Doppler flow velocities and Doppler flow reserve (DFR), relating them to the videointensity technique. Coronary stenosis and TIMI flow were determined, and echocardiographic volumes (end-diastolic and end-systolic volume indexes) and regional asynergy were computed before hospital discharge (baseline) and at six months. Assuming higher peak videointensity reflects greater myocardial blood volume, a 1- to 5-point (poor-optimal) perfusion scale was devised. RESULTS: The correlation of Doppler peak velocity and DFR with videointensity was significant (r = 0.58, p = 0.007 and r = 0.71, p < 0.001, respectively). Patients were subdivided into group A (increased videointensity post-PTCA > or = 1.5 points, n = 29) and group B (unchanged videointensity, n = 24). Analysis of variance showed a time-group interaction for end-diastolic volume index (-4.6 +/- 23% vs. +22 +/- 22%, p = 0.003) and end-systolic volume index (-3.05 +/- 11.1% vs. +4.1 +/- 12.5%, p = 0.027). There was no interaction for changes in LAD stenosis (p = 0.39) and TIMI flow after PTCA (p = 0.27), or regional asynergy at six months (p = 0.31). CONCLUSIONS: Angiographic dye videointensity in the risk area correlates with Doppler peak velocity and DFR, and its increase after PTCA of IRA has a limiting effect on ventricular volumes, independent of coronary stenosis resolution, changes in Thrombolysis In Myocardial Infarction (TIMI) flow or extent of regional asynergy.
Asunto(s)
Volumen Sanguíneo/fisiología , Medios de Contraste/farmacocinética , Angiografía Coronaria , Circulación Coronaria/fisiología , Ecocardiografía Doppler , Yopamidol , Infarto del Miocardio/terapia , Volumen Sistólico/fisiología , Remodelación Ventricular/fisiología , Adulto , Anciano , Velocidad del Flujo Sanguíneo/fisiología , Terapia Combinada , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Yopamidol/farmacocinética , Masculino , Persona de Mediana Edad , Infarto del Miocardio/fisiopatología , Miocardio/metabolismo , Factores de Riesgo , Terapia TrombolíticaRESUMEN
Biliverdin reductase (BVR) reduces heme oxygenase (HO) activity product, biliverdin, to bilirubin. BVR is unique in having dual pH/dual cofactor requirements. Using Escherichia coli-expressed human BVR and COS cells, we show that BVR is autophosphorylated and that phosphorylation is required for its activity. An "in blot" autophosphorylation assay showed that BVR is a renaturable phosphoprotein. Controls for the experiments were HO-1 and HO-2; both are phosphoproteins but are not autophosphorylated. Autophosphorylation was pH-dependent, with activity at pH 8.7 being most prominent. In addition, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate fluorescence titration of BVR gave a lower K(d) at pH 8.7 than at pH 7.4 (15.5 versus 28.0 micrometer). Mn(2+) was required for binding of the ATP analogue and for autophosphorylation; the autokinase activity was lost when treated at 60 degrees C for 10 min. The loss of transferred phosphates by alkaline treatment suggested that BVR is a serine/threonine kinase. Potato acid phosphatase treatment reversibly inactivated the enzyme. The enzyme was also inactivated by treatment with the serine/threonine phosphatase, protein phosphatase 2A; okadaic acid attenuated the inhibition. Titration of protein phosphatase 2A-released phosphates indicated a 1:6 molar ratio of BVR to phosphate. The BVR immunoprecipitated from COS cell lysates was a phosphoprotein, and its activity and phosphorylation levels increased in response to H(2)O(2). The results define a previously unknown mechanism for regulation of BVR activity and are discussed in the context of their relevance to heme metabolism.
Asunto(s)
Bilirrubina/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/metabolismo , Animales , Células COS , Humanos , Concentración de Iones de Hidrógeno , Fosforilación , Especificidad por SustratoRESUMEN
The heme oxygenase (HO) isozymes catalyze oxidation of the heme molecule to biliverdin and carbon monoxide (CO) with the release of chelated iron. Presently, we have defined, for the first time, propensity for site of injury-directed induction of isozymes--the stress-inducible isozyme, HO-1, responds distal (below) and the glucocorticoid (GC)-inducible HO-2 responds proximal (above) to the site of injury. We have also shown that reactive iron (Fe3+) and cGMP staining spatially resemble that of HO-1; which, in turn, colocalizes in motor neurons with transcription factors: Fas-associated protein containing death domain (FADD), tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and p53. Spinal cord injury (SCI) was inflicted by clip compression for 30 min, and analyses were carried out after 4 h or 16 h. When compared with spinal cord segments proximal to the site of injury, northern blot analysis showed remarkably higher levels of HO-1 mRNA distal (below) to the site of injury at both time points. In contrast, HO-2 mRNA levels were elevated proximal (above) to the site of injury and more prominently at 16 h post SCI. Immunohistochemical analyses were carried out using 2 x 5 mm segments above and below the compression site. When compared with segments above the site of injury, the intensity of HO-1 immunostaining and the number of HO-1 positive neurons in the ventral horn motor neurons were prominently increased in segments below the injury. Western blot analysis confirmed the observations. HO-2 protein was mapped to the ventral horn motor neurons, oligodendrocytes, the Clarke's nucleus neurons and the ependymal cells. When compared with segments below the site of injury, neuronal HO-2 staining intensity was increased above the site of injury, and most notably at 16 h. These observations were also confirmed by western blotting and HO activity measurements. Tissue Fe3+ and cGMP staining were increased and prominently mapped below the site of injury, where cGMP colocalized with HO-1 in the nucleus of the motor neurons. Also, a site of injury-directed pattern of induction of FADD, TRAIL, and p53 immunoreactivity, and a widespread colocalization of the oncogenes with HO-1 protein, were found within motor neurons below the level of injury. We forward the hypothesis that HO-1 and HO-2 have different roles in the defense mechanisms of the injured nervous system. We hypothesize that HO-1 protects against further damage by contributing to controlled cell death through their intrinsic suicide program, while HO-2 is involved in suppression of inflammatory response by NO derived radicals.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Hemo Oxigenasa (Desciclizante)/metabolismo , Neuronas/enzimología , Traumatismos de la Médula Espinal/enzimología , Médula Espinal/enzimología , Animales , Proteínas Reguladoras de la Apoptosis , Proteínas Portadoras/metabolismo , GMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Inducción Enzimática , Proteína de Dominio de Muerte Asociada a Fas , Hemo-Oxigenasa 1 , Hierro/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana , Ratones , Neuronas/patología , Médula Espinal/química , Médula Espinal/patología , Traumatismos de la Médula Espinal/patología , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Biliverdin reductase catalyzes the reduction of biliverdin, the product of heme oxygenase (HO) activity, to bilirubin. The reductase is unique among all enzymes characterized to date in being dual pH/cofactor-dependent. Until now the enzyme was assumed to be a noninducible cytosolic protein. This report, for the first time, demonstrates induction and nuclear localization of reductase in rat kidney in response to HO-1 inducers: bacterial lipopolysaccharide (LPS) and bromobenzene. The study also demonstrates that nuclear localization requires an intact nuclear localization signal and is responsive to cGMP. Specifically 16 h after treatment of rats (i.p.) with LPS (5 mg/kg), there was an increase in nuclear biliverdin reductase as determined by immunostaining, Western blotting, and activity analysis. Induction and nuclear localization of the reductase in kidney was also observed in bromobenzene-treated rats (2 mmol/kg, s.c., 24 h). The reductase message levels, however, were not increased in response to either treatment, suggesting post-transcriptional activation of the reductase by LPS and bromobenzene. The mechanism of nuclear transport of the reductase was examined using HeLa cells transfected with the hemagglutinin-tagged reductase construct. When cells were treated with 8-Br-cGMP the protein translocated into the nucleus. Mutation of the putative nuclear localization signal domain of the reductase blocked nuclear transport of the protein. We suggest the significance of nuclear localization of the reductase may relate to: 1) chain-breaking antioxidant activity of bilirubin; 2) inhibition of superoxide formation by bilirubin; and 3) modulation of the signal transduction pathways.
Asunto(s)
Núcleo Celular/efectos de los fármacos , Hemo Oxigenasa (Desciclizante)/biosíntesis , Riñón/efectos de los fármacos , Lipopolisacáridos/farmacología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/metabolismo , Animales , Bilirrubina/fisiología , Bromobencenos/farmacología , Núcleo Celular/enzimología , Inducción Enzimática , Hemo-Oxigenasa 1 , Inmunohistoquímica , Riñón/enzimología , Riñón/patología , Masculino , Mutagénesis , Oxidorreductasas/efectos de los fármacos , Oxidorreductasas/genética , Ratas , Ratas Sprague-Dawley , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/enzimologíaRESUMEN
Heme oxygenase 1 and 2 activities are responsible for initiating most of the degradation of heme, although other enzyme pathways play a role as well. The degradation pathway also includes biliverdin reductase, the activity of which is coupled to oxidation of NADH and NADPH. This overview discusses the pathways and enzymes involved in heme degradation.
Asunto(s)
Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo-Oxigenasa 1/metabolismo , Hemo/metabolismo , Animales , Bilirrubina/metabolismo , Biliverdina/metabolismo , Monóxido de Carbono/metabolismo , Hemo/química , Hemo Oxigenasa (Desciclizante)/genética , Hemo-Oxigenasa 1/genética , Humanos , Hierro/metabolismo , Estructura Molecular , Oxidación-ReducciónRESUMEN
Heme oxygenase isozymes, HO-1, HO-2 and HO-3, are HSP32 protein cognates, with a known function of catalyzing the isomer specific oxidation of the heme molecule, including that of NO synthase. Unknown until recent years was that the system is a central component of the cellular defense mechanisms; this can be attributed to a combination of many factors. In biological systems HO activity is responsible for production of equimolar amounts of CO, biliverdin and free Fe. The serine/threonine kinase, biliverdin reductase, catalyzes reduction of biliverdin to bilirubin. Bilirubin is a potent antioxidant and CO is a signal molecule. Although both active HO isozymes catalyze the same reaction, HO-1 and HO-2 may function in a rather distinct fashion in protection against tissue injury. HO-1 is the stress responsive cognate that is rapidly induced by free and stable radicals as well as by hypoxia. Supra induction of HO-1 completely protects ischemic kidney against tissue pathology. This involves rapid inactivation of the pro-oxidant heme of denatured hemoproteins and converting it to bilirubin and CO. In the case of severe tissue injury, such as compression injury, HO-1 is induced and colocalizes with cGMP and pro-apoptotic oncogenes. HO-2, which is the constitutive form, in addition to maintaining cell heme homeostasis, inactivates NO derived radicals. The isozyme binds the free radical at its "heme regulatory motifs" and is "suicide" inactivated at the protein and transcript levels. Data are shown that provide evidence for role of the HO system in the cellular defense mechanism against free radical-mediated tissue damage, and are consistent with the forwarded concept that HO isozymes have common, as well as distinct, roles in cellular defense mechanisms.
Asunto(s)
Hemo Oxigenasa (Desciclizante)/fisiología , Animales , Óxidos N-Cíclicos , Ferritinas/metabolismo , Depuradores de Radicales Libres/farmacología , Expresión Génica , Hemo Oxigenasa (Desciclizante)/genética , Hemo-Oxigenasa 1 , Hierro/metabolismo , Riñón/enzimología , Reperfusión Miocárdica , Miocardio/enzimología , Óxidos de Nitrógeno/farmacología , Ratas , ReperfusiónRESUMEN
Reproducible animal models of stroke are indispensable for investigation of pathogenesis and treatment of ischemic brain injury. Defined location and size of infarction as well as consistent production of neurological deficits make it possible to evaluate therapeutic potential of neuroprotective agents as well as to assess the impact of gene deletion [Nat. Med. 3 (1997) 1089; Science 265 (1994) 1883; Nat. Med. 4 (1998) 228] or overexpression [J. Neurochem. 72 (1999) 1187; J. Neurosci. 17 (1997) 7655] on neuroprotection in genetically altered mice. Ischemic stroke in mice can be reliably replicated by means of an open craniectomy exposure followed by permanent occlusion of the trunk and branches of the middle cerebral artery (MCA). Open craniectomy model is known to be statistically robust, yielding a coefficient of variation of <10%, and requiring minimal number of animals to validate the concept of statistical power. In the past, this model as well as some of its variants had been used in pivotal scientific studies to demonstrate impact of therapeutic genes on the course of ischemic neuronal injury [Neuron 13 (1994) 1017], as well as identification of 'culprit genes' responsible for progression of ischemic injury [J. Cerebr. Blood Flow Metab. 14 (1994) 887; Science 265 (1994) 1883; J. Neurosci. 17 (1997) 7655] through continuous recruitment of marginally ischemic penumbra into ischemic core [Trends Neurosci. 22 (1999) 391]. This protocol describes mapping of the ischemic penumbra using NADPH diaphorase staining as well as assessment of penumbral endogenous antioxidant reserves by detection of cellular biliverdin reductase mRNA and protein levels using immunocytochemistry and in situ hybridization histological techniques.
Asunto(s)
Arteriopatías Oclusivas/enzimología , Arterias Cerebrales , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/metabolismo , Animales , Inducción Enzimática , Femenino , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratones , NADPH Deshidrogenasa/metabolismo , Oxidorreductasas/genética , ARN Mensajero/metabolismo , Coloración y EtiquetadoRESUMEN
AIM: To compare the predictive value of thallium-201 single photon emission computed tomography (SPECT) scintigraphy (Sci) and low-dose dobutamine echocardiography (Dob) in predicting late recovery of dysfunctioning myocardium in patients with recent, uncomplicated myocardial infarction (MI). METHODS AND RESULTS: 19 patients (18 male, aged 58+/-8 years) with recent MI and ejection fraction <50% (35.5+/-8.3%) underwent 5-15 microg/kg per min Dob, rest-redistribution Sci and coronary angiography, respectively, 14+/-6, 16+/-7 and 17+/-5 days after MI. On an eleven-segment ventricular model devised to compare Dob and Sci segment by segment, each dysfunctioning ventricular segment was considered viable if it showed recovery of mechanical function at the echocardiographic follow-up, performed 6.3+/-1.5 months after revascularization (five PTCA, five GABG) or medical therapy. Among the 104 dysfunctioning segments, of which 26 (25%) showed recovery at follow-up, Dob and Sci gave a concordant response in 50 (48%, k = 0.13), correctly predicting the recovery (or not) of function in 42. Forty-two of 54 discordant responses were due to segments judged viable only by Sci and which had no recovery at follow-up (of these 37 were akinetic or severely hypokinetic at baseline). At the segment-by-segment analysis, the sensitivity, specificity, and accuracy in predicting recovery of function at follow-up were, respectively, 69, 88 and 84% for Dob as against 88, 36 and 49% for Sci (P<0.001 for both specificity and accuracy, P=NS for sensitivity). CONCLUSION: In patients with recent MI, the specificity of Dob in the detection of myocardium capable of late mechanical recovery is significantly higher with respect to Sci, whereas sensitivity is slightly, not significantly higher for the latter. It is conceivable that Sci detects viable myocardium even if it is transmurally limited to epicardial layers in segments with severely impaired mechanical function in which viability will not affect late recovery of function.
Asunto(s)
Cardiotónicos , Dobutamina , Infarto del Miocardio/diagnóstico , Radioisótopos de Talio , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Infarto del Miocardio/diagnóstico por imagen , Valor Predictivo de las Pruebas , Cintigrafía , Sensibilidad y Especificidad , UltrasonografíaRESUMEN
The heme oxygenase (HO) system was identified in the early 1970s as a distinct microsomal enzyme system that catalyzes formation of bile pigments (Maines and Kappas, 1974). Up to the early 1990s the system was considered only as a "molecular wrecking ball" (Lane, 1998) for degradation of the heme molecule and production of toxic waste products, CO and bile pigments. For those years, the HO system remained relatively unknown to the research community. In a rather short span of the past 10 years following the discovery of high levels of a second form of the enzyme, HO-2, in the brain, suggesting that "heme oxygenase in the brain has functions aside from heme degradation" (Sun et al., 1990); concomitant with finding that another toxic gas, NO, is a signal molecule for generation of cGMP (Ignarro et al., 1982), the system was propelled into main stream research. This propulsion was fueled by the realization of the multiple and diverse functions of heme degradation products. Heme oxygenase has now found relevance in all kinds of human pathophysiology ranging from stroke, cancer, multiple sclerosis, and malaria to transplantation and immune response. As it turns out, its potential benefits are mesmerizing investigators in diverse fields (Lane, 1998). The most recent findings with HO-2 being a hemoprotein and potentially an intracellular "sink" for NO (McCoubrey et al., 1997a; Ding et al., 1999), together with the discovery of the third form of the enzyme, HO-3 (McCoubrey et al., 1997b), are likely to insure the widespread interest in the enzyme system in the coming years. The present review is intended to highlight molecular properties of HO isozymes and their likely functions in the brain. Extended reviews of the system are found in Maines (1992, 1997).
Asunto(s)
Encéfalo/enzimología , Hemo Oxigenasa (Desciclizante)/fisiología , Animales , Regulación de la Expresión Génica , Hemo Oxigenasa (Desciclizante)/clasificación , Hemo Oxigenasa (Desciclizante)/genética , Hemo Oxigenasa (Desciclizante)/metabolismo , Humanos , Líquido Intracelular/metabolismo , Isoenzimas/clasificación , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/fisiología , Óxido Nítrico/metabolismoRESUMEN
Heme oxygenase-1 (HO), the heat shock/stress cognate of the heat shock protein 32 (HSP32) family of proteins, is postulated to be a component of cellular defense mechanisms against oxidative stress-mediated injury. Nitric oxide (NO) is among the extensive array of stimuli that induce HO-1. The cellular signaling mechanisms that regulate the induction of HO-1 by NO are not understood. In the present study, we have demonstrated that exposure of HeLa cells to the NO donor, sodium nitroprusside (SNP), results in concentration and time-dependent increase in HO-1 mRNA and activation of MAPKs: ERK (ERK1 and ERK2) and p38 pathways, but not SAPK/JNK pathway. Pre-treatment of the cells with PD98059, a selective ERK pathway inhibitor, and SB203580, a p38 MAPK inhibitor, blocked the induction of HO-1 by the NO donor in a dose-dependent manner. In addition, an increase in HO-1 mRNA level that was detected as early as 2 hrs.following SNP treatment preceded c-jun and c-fos induction. These transcription factors are downstream of SAPK/JNK pathway, and their increased expression was detected at 3hr. and 6hr. after SNP treatment. Similarly, AP-1 DNA binding activity was not increased when measured 6 hrs. after SNP treatment. ERK and p38 inhibitors also suppressed induction of HO-1 by SNAP and GSNO. The increase in HO-1 mRNA was inhibited by actinomycin D and cycloheximide, but not by NAC, and was not mimicked by the lipophilic cGMP analogue, 8-bromo-cGMP, suggesting that NO-mediated induction required de novo RNA and protein synthesis and was unrelated to cGMP and redox signaling. Collectively, the findings suggest that MAP kinase ERK and p38 pathways are involved in the NO-mediated induction of HO-1 and that SAPK/JNK pathway and increased DNA binding of AP-1 transcription factor are not involved in HO-1 gene activation by NO. A plausible mechanism by which the NO donors cause HO-1 induction may involve HO-1 gene regulation by its substrate, heme.
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Hemo Oxigenasa (Desciclizante)/biosíntesis , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Óxido Nítrico/metabolismo , Activación Enzimática , Inducción Enzimática , Expresión Génica/efectos de los fármacos , Células HeLa , Hemo Oxigenasa (Desciclizante)/genética , Hemo-Oxigenasa 1 , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas de la Membrana , Proteína Quinasa 3 Activada por Mitógenos , ARN Mensajero , Transducción de Señal , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
This is the first report on the protective effect of heme oxygenase-1 (HO-1) overexpression against oxidative stress-mediated neuronal cell death and demonstration of a decreased production of oxygen free radicals when HO-1 levels are increased. HO-1 is the heat shock/stress cognate of the heat shock protein 32 family of proteins. A known function of these proteins is alpha-meso bridge-specific cleavage of the heme molecule. For the present study, we used cerebellar granular neurons (CGNs) isolated from homozygous transgenic (Tg) mice that overexpress HO-1 under neuron-specific enolase control and nontransgenic (Ntg) littermates. The Tg mouse CGNs were characterized by increased levels of HO-1 mRNA and protein, a lower resting intracellular calcium concentration, and a reduced HO-1 transcriptional response to glutamate-mediated oxidative stress. Compared with the Ntg neurons, when exposed to glutamate (30 microM or 3 mM), the magnitude of cell viability was increased and the number of cells exhibiting membrane permeability and chromatin condensation were significantly decreased in the Tg CGN cultures. The population of neurons surviving glutamate toxicity decreased when HO-1 activity was inhibited by a peptide inhibitor. The neuroprotective effect by HO-1 was extended to H(2)O(2)-induced cell death. The mechanism of protection may involve in part a reduced production of reactive oxygen species upon exposure to glutamate. We suggest that induction of HO-1 by pharmacological means may be a novel approach to amelioration of oxidative insults to neurons.
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Muerte Celular , Expresión Génica , Hemo Oxigenasa (Desciclizante)/genética , Neuronas/enzimología , Estrés Oxidativo , Animales , Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Cerebelo/citología , Inhibidores Enzimáticos/farmacología , Ácido Glutámico/farmacología , Hemo Oxigenasa (Desciclizante)/antagonistas & inhibidores , Hemo Oxigenasa (Desciclizante)/fisiología , Hemo-Oxigenasa 1 , Peróxido de Hidrógeno/farmacología , Proteínas de la Membrana , Ratones , Ratones Transgénicos , Fármacos Neuroprotectores , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Heme oxygenase (HO)-2, the constitutive cognate of oxidative stress inducible HO-1 (HSP32), degrades heme to biliverdin, carbon monoxide, and iron. The highest levels of HO-2 are found in the testis. Previously we identified multiple HO-2 homologous transcripts that differ in size and use three different 5' UTRs that form the untranslated first exon of the gene (referred to as rHO-2, rHO-2-1 and rHO-2-2) and two poly(A) signals. Also, we have characterized a functional glucocorticoid response element (GRE) in the promoter region of rHO-2. In this study, we have examined the structural basis for size heterogeneity of HO-2 transcripts and whether expression of HO-2 at mRNA and protein levels is subject to regulation by corticosterone. Age and tissue-dependence of transcript expression were examined as well. Our data indicate that the remarkable increase in HO-2 mRNA in adult rat testis is due primarily to generation of two HO-2 homologous transcripts of approx. 2.1kb and approx. 1.45kb size that use rHO-2 and are unique to this tissue, and that rHO-2 is not used within other organs. These transcripts are not present in the brain, kidney, thymus, heart, spleen, liver, or in prepubertal 14day old rat testis. The testis-specific transcripts contain all of the coding region exons present in the approx. 1.3kb and approx. 1.9kb transcripts that are common to all organs, including the adult and prepubertal rat testis. Differential use of the poly(A) signals accounts for the difference in size of these two transcripts. Treatment of newborn rats with corticosterone for 5days, starting on day 2 after birth, induced HO-2 protein expression in the testis as detected by Western blotting. In adult rat testis, corticosterone treatment, however, was not an effective regulator of HO-2 transcript populations or levels. The findings suggest that HO-2 levels in the testis are controlled by glucocorticoids; and that developmental and tissue-specific factor(s) determine generation of transcripts unique to the organ. The apparent exclusive use of rHO-2 by the mature testis is consistent with the possibility that HO-2 may play a role in male reproduction.
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Corticosterona/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Hemo Oxigenasa (Desciclizante)/genética , Testículo/embriología , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Animales , Animales Recién Nacidos , Secuencia de Bases , Northern Blotting , Western Blotting , Exones , Hemo Oxigenasa (Desciclizante)/biosíntesis , Masculino , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Ratas , Ribonucleasa H/metabolismo , Testículo/enzimología , Factores de Tiempo , Distribución TisularRESUMEN
In mammals the rate-limiting step in heme catabolism is the heme oxygenase (HO) system. Two isozymes, HO-1 and HO-2, oxidatively cleave the substrate to form biliverdin, and the potential cellular messenger, CO; the chelated iron is released as the result of the tetrapyrrole ring opening. Biliverdin is subsequently reduced to bilirubin, an antioxidant, by biliverdin reductase. The aim of the present study was to investigate the involvement of HO-1, a heat shock/stress protein, in protection offered by the spin trap agent, N-tert-butyl-alpha-phenyl-nitrone (PBN), against kidney ischemia/reperfusion injury. For this, HO-1 expression and assessment of the parameters associated with tissue-oxidative injury were compared in the presence or absence of PBN pretreatment of rats (100 mg/kg i.p., 30 min) before the onset of 30-min ischemia. Twenty-four hours after reperfusion, Northern blot analysis showed an unprecedented approximately 37-fold increase in 1.8-kb HO-1 mRNA in PBN pretreated rat kidney; HO-2 mRNA levels did not increase. At 48 h, the levels of HO-1 mRNA remained nearly 14-fold higher than the control value. In the absence of PBN, the levels measured approximately 5- and 2-fold higher than control values at the 24- and 48-h intervals, respectively. PBN pretreatment also resulted in a most impressive increase in the levels of HO-1 protein as judged by Western blot analysis and measurement of enzyme activity at the 24-h time point. As detected by immunohistochemical analysis, PBN pretreatment caused an increase in HO-1 and biliverdin reductase-immunoreactive proteins in the cortex and in the outer stripe of the outer medulla. In the absence of PBN pretreatment, there was an intense immunostaining for HO-1 in the medullary rays, which corresponded with iron and lipid peroxidation staining of the region; these observations were not made with PBN-pretreated kidneys. Collectively, the findings are consistent with the likelihood that suprainduction of HO-1 gene expression protects the kidney from free radical-mediated injury by increasing the capacity to produce the potent cellular antioxidant bilirubin. We also suggest spin trap-mediated protection against ischemia/reperfusion injury is likely due to a sustained elevation of HO-1 gene expression by formation of stable radicals.
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Hemo Oxigenasa (Desciclizante)/genética , Riñón/fisiología , Óxidos de Nitrógeno/farmacología , Estrés Oxidativo/fisiología , ARN Mensajero/metabolismo , Daño por Reperfusión/patología , Animales , Anticuerpos/inmunología , Northern Blotting , Western Blotting , Óxidos N-Cíclicos , Modelos Animales de Enfermedad , Hemo-Oxigenasa 1 , Inmunohistoquímica , Hierro/metabolismo , Isoenzimas , Peroxidación de Lípido/efectos de los fármacos , Oxigenasas/fisiología , Ratas , Detección de Spin , Factores de TiempoRESUMEN
Heme oxygenase-2 (HO-2) is the constitutive cognate of the heat-shock protein-32 family of proteins. These proteins catalyze oxidative cleavage of heme to CO and biliverdin, and release Fe. HO-2 is a hemoprotein and binds heme at heme regulatory motifs (HRMs) with a conserved Cys-Pro pair; two copies of HRM are present in HO-2 (Cys264 and Cys281). The HO-2 HRMs are not present in HO-1 and are not involved in HO-2 catalytic activity. Optical CD, and spectral and activity analyses were used to examine reactivity of HO isozymes with NO species produced by NO donors. Purified Escherichia coli-expressed HO preparations, wild-type HO-2, Cys264/Cys281 --> Ala/Ala HO-2-mutant (HO-2-mut) and HO-1 preparations were used. A type II change (red shift) of the Soret band (405 nm --> 413-419 nm) was observed when wild-type HO-2 was treated with sodium nitroprusside (SNP), S-nitroglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholinosydnonimine (SIN-1); the NO scavenger, hydroxocobalamin (HCB) prevented the shift. Only SIN-1, which produces peroxynitrite by generating both NO and superoxide anion, decreased the Soret region absorption and the pyridine hemochromogen spectrum of HO-2; superoxide dismutase (SOD) blocked the decrease. Binding of heme to HO-2 protein was required for shift and/or decrease in absorption of the Soret band. NO donors significantly inhibited HO-2 activity, with SNP being the most potent inhibitor (> 40%). Again, trapping NO with HCB blocked HO-2 inactivation. HO-1 and HO-2-mut were not inactivated by NO donors. CD data suggest that the decrease in HO-2 activity was not related to change by NO species of the secondary structure of HO-2. Western blot analysis suggests that NO donors did not cause HO-1 protein loss and Northern blot analysis of HeLa cells treated with SIN-1 and SNP indicates that, unlike HO-1 mRNA, which is remarkably responsive to the treatments, HO-2 mRNA levels were modestly increased ( approximately two to threefold) by NO donors. The data are consistent with the possibility that NO interaction with HO-2-bound heme effects electronic interactions of residues involved in substrate binding and/or oxygen activation. The findings permit the hypothesis that HO-2 and NO are trans-inhibitors, whereby biological activity of NO is attenuated by interaction with HO-2, serving as an intracellular 'sink' for the heme ligand, and NO inhibits HO-2 catalytic activity. As such, the cellular level of both signaling molecules, CO and NO would be moderated.
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Hemo Oxigenasa (Desciclizante)/metabolismo , Donantes de Óxido Nítrico/metabolismo , Óxido Nítrico/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Cartilla de ADN/genética , Escherichia coli/genética , Células HeLa , Hemo/metabolismo , Hemo Oxigenasa (Desciclizante)/genética , Humanos , Técnicas In Vitro , Ligandos , Molsidomina/análogos & derivados , Molsidomina/metabolismo , Molsidomina/farmacología , Mutagénesis Sitio-Dirigida , Donantes de Óxido Nítrico/farmacología , Nitroprusiato/metabolismo , Nitroprusiato/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
PURPOSE: Biliverdin reductase is an oxidoreductase unique among all enzymes characterized to date in having dual pH/dual cofactor requirement - NADH and NADPH at 6.7 and 8.7, respectively. The protein shows extensive microheterogeneity that is caused by post-translational modification. The reductase converts the heme degradation product, biliverdin, to bilirubin. Bilirubin has been shown to inhibit responses of human lymphocytes, including phytohemagglutinin-induced proliferation, interleukin-2 production, and antibody dependent and independent cell mediated cytotoxicity. In addition to acting as an antioxidant, it inhibits protein phosphorylation and activity of enzymes such as protein kinase C and NADPH oxidase. This research was to evaluate whether renal cell carcinoma differs from normal tissue in regard to the expression and activity of the reductase. MATERIALS AND METHODS: Kidney tissue with or without visible renal carcinoma and normal kidney tissue from a brain dead patient were frozen at -80C shortly after removal. Ten microm. tissue sections were used for immunostaining of biliverdin reductase, pooled isolated tumors and surrounding tissue that did not contain visible tumor were used for Northern blot analysis of mRNA and Western blot analysis of protein. Enzyme activity was also measured in these preparations at pH 6.7 with NADH, and at pH 8.7 with NADPH. Ten additional formalin fixed specimens of renal cell carcinoma were also used for immunostaining. RESULTS: There was a striking increase in the reductase protein levels, as visualized by immunostaining in tumor tissue cells. The increase was also evident by Western blotting, and involved in increased transcription of biliverdin reductase as suggested by Northern blot analysis. The protein would also be detected in the infiltrating monocytes, macrophages, T cells and neutrophils as well as in circulating lymphocytes. The enzyme activity was nearly doubled in the tumor tissue, but selectively with NADH as the cofactor. CONCLUSION: Increases in biliverdin reductase expression and activity only with NADH is found in renal cell carcinoma. The net effects of this change are uncertain at present but several pathways, which could be affected by the reductase, may alter local physiology. Biliverdin reductase as a zinc metalloprotein may directly interact with other regulatory proteins, generation of increased bilirubin may alter immune function and increased enzyme activity may deplete NADH with contrasting consequence of blocking free radical formation and depleting cellular ATP. To the benefit of the host, the latter could culminate in tumor cell death.
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Carcinoma de Células Renales/enzimología , Neoplasias Renales/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/metabolismo , Carcinoma de Células Renales/química , Activación Enzimática , Humanos , Concentración de Iones de Hidrógeno , Neoplasias Renales/química , NAD/metabolismo , NADP/metabolismo , Oxidorreductasas/análisisRESUMEN
Heme oxygenase-1 (HO-1, HSP32) is an early gene that is responsive to an array of pathological conditions including, but not limited to, hypoxia and cerebral ischemia. HO-1 cleaves the heme molecule and produces carbon monoxide (CO) and biliverdin (an antioxidant) and is essential for iron homeostasis. The purpose of this study was to investigate, using transgenic (Tg) mice, whether overexpression of HO-1 in the brain augments or attenuates cellular injury caused by ischemic stroke. Homozygous HO-1 Tg mice that overexpress HO-1 under the control of the neuron-specific enolase promoter (characterized previously) were used. Under halothane anesthesia and normothermic conditions, wild-type nontransgenic (nTg; n = 22) and HO-1 Tg (n = 24) mice were subjected to middle cerebral artery occlusion (MCAo). Six hours after induction of ischemia, Tg and nTg mice developed infarcts that were 39 +/- 6 and 63 +/- 9 mm3, respectively (p < 0.01). No significant difference between the two strains was observed in the values of brain edema (11.3 +/- 4% in Tg vs. 14.6 +/- 5% in nTg; p < 0.1). At 24 h after MCAo, Tg mice exhibited significant neuroprotection as determined by the stroke volumes (41 +/- 2 mm3 in Tg vs. 74 +/- 5 mm3 in nTg; p < 0.01) and values of ischemic cerebral edema (21 +/- 6% in Tg vs. 35 +/- 11% in nTg; p < 0.01). Data suggest that neuroprotection in Tg mice was, at least in part, related to the following findings: (a) constitutively up-regulated cyclic GMP and bcl-2 levels in neurons; (b) inhibition of nuclear localization of p53 protein; and (c) antioxidant action of HO-1, as detected by postischemic neuronal expression of ferritin, and decreases in iron staining and tissue lipid peroxidation. We suggest that pharmacological stimulation of HO-1 activity may constitute a novel therapeutic approach in the amelioration of ischemic injury during the acute period of stroke.
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Arteriopatías Oclusivas/patología , Isquemia Encefálica/patología , Enfermedades Arteriales Cerebrales/patología , Regulación Enzimológica de la Expresión Génica/fisiología , Hemo Oxigenasa (Desciclizante)/genética , Animales , Arteriopatías Oclusivas/complicaciones , Arteriopatías Oclusivas/enzimología , Arteriopatías Oclusivas/genética , Conducta Animal , Northern Blotting , Edema Encefálico/patología , Isquemia Encefálica/enzimología , Isquemia Encefálica/etiología , Isquemia Encefálica/genética , Enfermedades Arteriales Cerebrales/complicaciones , Enfermedades Arteriales Cerebrales/enzimología , Enfermedades Arteriales Cerebrales/genética , Circulación Cerebrovascular , GMP Cíclico/metabolismo , Ferritinas/biosíntesis , Hemo Oxigenasa (Desciclizante)/biosíntesis , Hemo-Oxigenasa 1 , Inmunohistoquímica , Peroxidación de Lípido/fisiología , Proteínas de la Membrana , Ratones , Ratones Transgénicos , NADPH Deshidrogenasa , Neuronas/enzimología , Neuronas/patología , Neuronas/ultraestructura , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Volumen Sistólico , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
This is the first report on increased neuronal levels of biliverdin reductase (BVR) in response to ischemic brain injury. BVR is an oxidoreductase, and is unique among all enzymes characterized to date in having dual pH/dual cofactor requirements--NADH and NADPH at 6.7 and 8.7, respectively. BVR catalyses the final step in the heme metabolic pathway and reduces the heme degradation product, biliverdin, to bilirubin. Bilirubin can be both a neurotoxicant and an antioxidant depending on its ratio to protein and concentration. Bilirubin also has immunomodulatory activity. Other biologically active heme degradation products are iron and CO. This study assessed time-dependent changes in the level of BVR, following permanent middle cerebral artery occlusion (MCAo). It also examined correlation of the change in BVR expression with display of indices of ischemic tissue injury. Under halothane anesthesia and normothermic conditions, 72 DNX inbred mice were subjected to MCAo. A time-dependent enlargement of an ischemic lesion over the course of 24 h was observed and measured 55 +/- 5 mm3 at 6 h, 63 +/- 6.7 mm3 at 12 h, and 73 +/- 5 mm3 at 24 h. Six hours after MCAo, increased immunoreactivity for BVR was noted in neurons in the peri-ischemic areas, intraischemic cortical layers 3 and 5, as well as in neurons in regions distant from the borders of vascular distribution of the MCA, such as those in substantia nigra, in the Purkinje layer of the cerebellum and in the central nucleus of inferior colliculus. Twenty-four hours after MCAo, immunoreactivity for BVR remained increased in the peri-ischemia areas. At all time points staining for BVR was decreased in the ischemic core. At the 24 h time point there was an increase in Fe staining in the perimeter of the lesion and an increase in Schiff's staining for lipid peroxidation at the rim of the lesion. In situ hybridization analysis demonstrated a time dependent increase in BVR mRNA labeling in neurons of the peri-ischemic area. In the ischemic hemisphere, when compared with the contralateral hemisphere, neither measurable decreases in BVR mRNA or total protein levels nor a decrease in NADH-dependent BVR activity at pH 6.7 were observed. As judged by Northern and Western blots and activity analysis, despite the apparent loss of BVR from the ischemic core, and its increase in the peri-ischemic region, when compared with the contralateral hemisphere, the overall capacity of the ischemic hemisphere to catalyze the reduction of biliverdin was unchanged throughout the experiment. Should, in the case of ischemia, the conditions favor the antioxidant activity of bilirubin, then we suggest that increase in BVR expression in ischemic penumbra may present a cellular defense mechanism against free radical-mediated neuronal damage. Furthermore, we interpret the apparent tightly regulated expression of BVR in the ischemic hemisphere as an important factor in protection against bilirubin neurotoxicity. Data suggest that pharmacological modulation of BVR expression is a possible new direction for protecting neurons against ischemic injury and oxidative stress.