RESUMEN
RNA polymerase III (Pol III) transcribes medium-sized non-coding RNAs (collectively termed Pol III genes). Emerging diverse roles of Pol III genes suggest that individual Pol III genes are exquisitely regulated by transcription and epigenetic factors. Here we report global Pol III expression/methylation profiles and molecular mechanisms of Pol III regulation that have not been as extensively studied, using nc886 as a representative Pol III gene. In a human mammary epithelial cell system that recapitulates early breast tumorigenesis, the fraction of actively transcribed Pol III genes increases reaching a plateau during immortalization. Hyper-methylation of Pol III genes inhibits Pol III binding to DNA via inducing repressed chromatin and is a determinant for the Pol III repertoire. When Pol III genes are hypo-methylated, MYC amplifies their transcription, regardless of its recognition DNA motif. Thus, Pol III expression during tumorigenesis is delineated by methylation and magnified by MYC.
Asunto(s)
Mama/metabolismo , Transformación Celular Neoplásica/genética , Epigénesis Genética , ARN Polimerasa III/metabolismo , Transcripción Genética , Mama/citología , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , ADN/genética , ADN/metabolismo , Metilación de ADN , Células Epiteliales/metabolismo , Humanos , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/genética , ARN no Traducido/genéticaRESUMEN
Nitric oxide (NO)-derived species could potentially react with arachidonic acid to generate novel vasoactive metabolites. We studied the reaction of arachidonic acid with nitrogen dioxide (NO2), a free radical that originates from NO oxidation. The reaction mixture contained lipid products that relaxed endothelium-removed bovine coronary arteries. Relaxation to the lipid mixture was inhibited approximately 20% by indomethacin and approximately 70% by a soluble guanylate cyclase (sGC) inhibitor (ODQ). Thus, novel lipid products, which activate sGC presumably through a mechanism involving NO, appeared to have contributed to the observed vasorelaxation. Lipids that eluted at 9 to 12 min during high-performance liquid chromatography fractionation accounted for about one-half of the vasodilator activity in the reaction mixture, which was inhibited by ODQ. Lipid products in fractions 9 to 12 were identified by electrospray tandem mass spectrometry to be eight isomers having molecular weight of 367 and a fragmentation pattern indicative of arachidonic acid derivatives containing nitro and hydroxy groups and consistent with the structures of vicinal nitrohydroxyeicosatrienoic acids. These lipids spontaneously released NO (183 +/- 12 nmol NO/15 min/micromol) as detected by head space/chemiluminescence analysis. Mild alkaline hydrolysis of total lipids extracted from bovine cardiac muscle followed by isotopic dilution gas chromatography/mass spectrometry analysis detected basal levels of nitrohydroxyeicosatrienoic acids (6.8 +/- 2.6 ng/g tissue; n = 4). Thus, the oxidation product of NO, NO2, reacts with arachidonic acid to generate biologically active vicinal nitrohydroxyeicosatrienoic acids, which may be important endogenous mediators of vascular relaxation and sGC activation.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido Araquidónico/química , Lípidos/síntesis química , Lípidos/farmacología , Donantes de Óxido Nítrico/síntesis química , Donantes de Óxido Nítrico/farmacología , Dióxido de Nitrógeno/química , Nitroparafinas/síntesis química , Nitroparafinas/farmacología , Vasodilatadores/síntesis química , Vasodilatadores/farmacología , Ácido 8,11,14-Eicosatrienoico/síntesis química , Ácido 8,11,14-Eicosatrienoico/farmacología , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Bovinos , Cromatografía Líquida de Alta Presión , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/metabolismo , Técnicas In Vitro , Metabolismo de los Lípidos , Mediciones Luminiscentes , Masculino , Espectrometría de Masas , Contracción Muscular/efectos de los fármacos , Ratas , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Heme oxygenase (HO-1, encoded by Hmox1) is an inducible protein activated in systemic inflammatory conditions by oxidant stress. Vascular injury is characterized by a local reparative process with inflammatory components, indicating a potential protective role for HO-1 in arterial wound repair. Here we report that HO-1 directly reduces vasoconstriction and inhibits cell proliferation during vascular injury. Expression of HO-1 in arteries stimulated vascular relaxation, mediated by guanylate cyclase and cGMP, independent of nitric oxide. The unexpected effects of HO-1 on vascular smooth muscle cell growth were mediated by cell-cycle arrest involving p21Cip1. HO-1 reduced the proliferative response to vascular injury in vivo; expression of HO-1 in pig arteries inhibited lesion formation and Hmox1-/- mice produced hyperplastic arteries compared with controls. Induction of the HO-1 pathway moderates the severity of vascular injury by at least two adaptive mechanisms independent of nitric oxide, and is a potential therapeutic target for diseases of the vasculature.
Asunto(s)
Arterias/fisiología , Hemo Oxigenasa (Desciclizante)/metabolismo , Músculo Liso Vascular/citología , Vasoconstricción , Animales , Arterias/enzimología , Arterias/lesiones , Ciclo Celular/fisiología , Células Cultivadas , Medio de Cultivo Libre de Suero , GMP Cíclico/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Ciclinas/metabolismo , Inducción Enzimática , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Eliminación de Gen , Hemo Oxigenasa (Desciclizante)/antagonistas & inhibidores , Hemo-Oxigenasa 1 , Proteínas de la Membrana , Ratones , Músculo Liso Vascular/fisiología , Protoporfirinas/farmacología , Porcinos , Transfección , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacosRESUMEN
Acute myocardial infarct (MI) results in ischemia distal to lesions which puts heart muscle at risk for reperfusion injury (RI). Neutrophils, platelets and complement are putative mediators of RI. Recent advances in filtration technology provide integrated neutrophil and platelet removal together with complement-attenuating properties in a single blood-conditioning device. The present study characterizes the properties of a blood-conditioning filter and describes its clinical effect when used in conjunction with active hemoperfusion for acute MI. The filter reduces leukocytes by 99.9998 +/- 0.0002% (p<0.0001) and platelets by 99.9934 +/- 0.0069% (p<0.0001). Human plasma, derived from heparinized blood that was 'conditioned' by filtration, was studied using the Langendorff isolated rabbit heart preparation. The deposition of membrane attack complex and the resultant functional myocardial impairments [reflected in hemodynamic and biochemical measurements, including developed pressure, coronary blood flow, lymph-derived myocardial creatine kinase (CK)] are significantly attenuated by blood conditioning. Integration of the blood-conditioning filter into an active hemoperfusion system during primary percutaneous transluminal coronary angioplasty (PTCA) for acute MI (n=8) did not delay the procedure or cause any complications. Reperfusion of occluded coronary arteries with 300 cm3 of conditioned blood led to significant improvement in echocardiographic global wall motion scores (in standard deviations) following treatment (-1.64 +/- 0.18 to -1.45 +/- 0.15, p=0.02). Initial reperfusion of totally occluded coronary arteries with conditioned blood leads to acutely improved ventricular function. Collectively, these data provide a strong indication for continued investigation of conditioned blood reperfusion in angioplasty following acute MI for the long-term effect upon recovery of salvagable myocardium.
Asunto(s)
Angioplastia Coronaria con Balón/métodos , Infarto del Miocardio/cirugía , Reperfusión/métodos , Adulto , Anciano , Anciano de 80 o más Años , Animales , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/farmacología , Electrocardiografía , Femenino , Filtración/métodos , Humanos , Técnicas In Vitro , Leucaféresis , Masculino , Persona de Mediana Edad , Infarto del Miocardio/sangre , Infarto del Miocardio/fisiopatología , Proyectos Piloto , Plaquetoferesis , Estudios Prospectivos , Conejos , Daño por Reperfusión/etiología , Daño por Reperfusión/prevención & control , Volumen Sistólico , Función Ventricular IzquierdaRESUMEN
Reperfusion of the ischemic myocardium results in irreversible tissue injury and cell necrosis, leading to decreased cardiac performance. While early reperfusion of the heart is essential in preventing further tissue damage due to ischemia, reintroduction of blood flow can expedite the death of vulnerable, but still viable, myocardial tissue, by initiating a series of events involving both intracellular and extracellular mechanisms. In the last decade, extensive efforts have focused on the role of cytotoxic reactive oxygen species, complement activation, neutrophil adhesion, and the interactions between complement and neutrophils during myocardial reperfusion injury. Without reperfusion, myocardial cell death evolves slowly over the course of hours. In contrast, reperfusion after an ischemic insult of sufficient duration initiates an inflammatory response, beginning with complement activation, followed by the recruitment and accumulation of neutrophils into the reperfused myocardium. Modulation of the inflammatory response, therefore, constitutes a potential pharmacological target to protect the heart from reperfusion injury. Recognition of the initiating factor(s) involved in myocardial reperfusion injury should aid in development of pharmacological interventions to selectively or collectively attenuate the sequence of events that mediate extension of tissue injury beyond that caused by the ischemic insult.
Asunto(s)
Daño por Reperfusión Miocárdica/fisiopatología , Animales , Supervivencia Celular/fisiología , Activación de Complemento/fisiología , Humanos , Contracción Miocárdica/fisiología , Activación Neutrófila/fisiología , Especies Reactivas de Oxígeno/metabolismo , Síndrome de Respuesta Inflamatoria Sistémica/fisiopatologíaRESUMEN
We compared the effects of the D1/D5 receptor antagonist SCH-23390 with the beta-adrenergic receptor antagonist propranolol on the persistence of long-term potentiation in the CA1 and dentate gyrus subregions of the hippocampus. In slices, SCH-23390 but not propranolol reduced the persistence of long-term potentiation in area CA1 without affecting its induction. The drugs exerted reverse effects in the dentate gyrus, although in this case the induction of long-term potentiation was also affected by propranolol. The lack of effect of SCH-23390 on the induction and maintenance of long-term potentiation in the dentate gyrus was confirmed in awake animals. The drug also had little or no effect on the expression of inducible transcription factors. In area CA1 of awake animals, SCH-23390 blocked persistence of long-term potentiation beyond 3 h, confirming the results in slices. To rule out a differential release of catecholamines induced by our stimulation protocols between brain areas, we compared the effects of the D1/D5 agonist SKF-38393 with the beta-adrenergic agonist isoproterenol on the persistence of a weakly induced, decremental long-term potentiation in CA1 slices. SKF-38393 but not isoproterenol promoted greater persistence of long-term potentiation over a 2-h period. In contrast, isoproterenol but not SKF-38392 facilitated the induction of long-term potentiation. These data demonstrate that there is a double dissociation of the catecholamine modulation of long-term potentiation between CA1 and the dentate gyrus, suggesting that long-term potentiation in these brain areas may be differentially consolidated according to the animal's behavioural state.
Asunto(s)
Antagonistas Adrenérgicos beta/farmacología , Benzazepinas/farmacología , Antagonistas de Dopamina/farmacología , Hipocampo/efectos de los fármacos , Potenciación a Largo Plazo/efectos de los fármacos , Propranolol/farmacología , Animales , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Hipocampo/fisiología , Potenciación a Largo Plazo/fisiología , Masculino , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos beta/efectos de los fármacos , Receptores Adrenérgicos beta/fisiología , Receptores de Dopamina D1/efectos de los fármacos , Receptores de Dopamina D1/fisiología , Receptores de Dopamina D5RESUMEN
Activation of the complement system contributes to the tissue destruction associated with myocardial ischemia/reperfusion. Pentosan polysulfate (PPS), a negatively charged sulfated glycosaminoglycan (GAG) and an effective inhibitor of complement activation, was studied for its potential to decrease infarct size in an experimental model of myocardial ischemia/reperfusion injury. Open-chest rabbits were subjected to 30-min occlusion of the left coronary artery followed by 5 h of reperfusion. Vehicle (saline) or PPS (30 mg/kg/h) was administered intravenously immediately before the onset of reperfusion and every hour during the reperfusion period. Treatment with PPS significantly (p < 0.05) reduced infarct size as compared with vehicle-treated animals (27.5+/-2.9% vs. 13.34+/-2.6%). Analysis of tissue demonstrated decreased deposition of membrane-attack complex and neutrophil accumulation in the area at risk. The results indicate that, like heparin and related GAGs, PPS possesses the ability to decrease infarct size after an acute period of myocardial ischemia and reperfusion. The observations are consistent with the suggestion that PPS may mediate its cytoprotective effect through modulation of the complement cascade.
Asunto(s)
Quimiotaxis/efectos de los fármacos , Infarto del Miocardio/patología , Poliéster Pentosan Sulfúrico/uso terapéutico , Daño por Reperfusión/tratamiento farmacológico , Animales , Anticuerpos/inmunología , Plaquetas/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proteínas Inactivadoras de Complemento/farmacología , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Vasos Coronarios/fisiología , Inhibidores Enzimáticos/farmacología , Eritrocitos/efectos de los fármacos , Glicosaminoglicanos/uso terapéutico , Hemodinámica/efectos de los fármacos , Técnicas In Vitro , Ligadura , Neutrófilos/fisiología , Conejos , OvinosRESUMEN
2-(4-Nitrophenyl)sulfonylethoxycarbonyl (Nsc) is an alternative base-labile N(alpha)-protecting group to 9-fluorenylmethoxycarbonyl (Fmoc) for amino acids. The UV spectrum of the Nsc group exhibits moderate absorption at 380 nm which is excellent for real-time monitoring of the deprotection process. It also decreases the rearrangement of X-Asp, which can be a serious problem in SPPS.
Asunto(s)
Aminoácidos/química , Técnicas de Química Analítica/métodos , Fluorenos/química , Biosíntesis de Péptidos , Proteínas Bacterianas/síntesis química , Cromatografía Líquida de Alta Presión , Endotelina-1 , Endotelinas/síntesis química , Humanos , Nitrobencenos/química , Precursores de Proteínas/síntesis química , Sustancia P/síntesis química , Factores de TiempoRESUMEN
OBJECTIVE: To determine if dehydroepiandrosterone (DHEA) is beneficial in severe systemic lupus erythematosus (SLE). METHODS: A double-blinded, placebo-controlled, randomized clinical trial in 21 patients with severe and active SLE, manifestated primarily by nephritis, serositis or hematological abnormalities. In addition to conventional treatment with corticosteroids +/- immunosuppressives, patients received DHEA 200 mg/d vs. placebo for 6 months, followed by a 6-month open label period. The primary outcome was a prospectively defined responder analysis, based on a quantitatively specified improvement of the principal severe lupus manifestation at 6 months. RESULTS: Nineteen patients were available for evaluation at 6 months. Baseline imbalance between the groups was noted, with the DHEA group having greater disease activity at baseline (P<0.05 by physician's global assessment). Eleven patients were responders: 7/9 patients on DHEA vs. 4/10 patients on placebo (P<0.10). Of the secondary outcomes, mean improvement in SLE disease activity index (SLE-DAI) score was greater in the DHEA group (-10.3+/-3.1 vs. -3.9+/-1.4. P<0.07). Bone mineral density at the lumbo-sacral spine showed significant reduction in the placebo group, but was maintained in the DHEA group. CONCLUSION: DHEA therapy, when added to conventional treatment for severe SLE, may at most have a small added benefit with respect to lupus outcomes, but baseline imbalances in the study population limit the generalizability of the results. DHEA appears to have a protective effect with respect to corticosteroid-induced osteopenia in such patients.
Asunto(s)
Deshidroepiandrosterona/uso terapéutico , Lupus Eritematoso Sistémico/tratamiento farmacológico , Corticoesteroides/administración & dosificación , Adulto , Densidad Ósea/efectos de los fármacos , Deshidroepiandrosterona/administración & dosificación , Deshidroepiandrosterona/efectos adversos , Método Doble Ciego , Quimioterapia Combinada , Femenino , Humanos , Inmunosupresores/administración & dosificación , Lupus Eritematoso Sistémico/metabolismo , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Resultado del TratamientoRESUMEN
The ability of the heparin derivative, N-acetylheparin (NHEP) to protect the heart from regional ischemia/reperfusion injury was examined in vivo. NHEP (2 mg/kg i.v.) or vehicle was administered 2 h before occlusion of the left circumflex coronary (LCX) artery. Open-chest, anesthetized rabbits were subjected to 30 min of regional myocardial ischemia followed by 5 h of reperfusion. Myocardial myeloperoxidase activity, membrane attack complex (MAC) deposition and IL-8 generation were assessed in supernatant samples from the area at risk. Infarct size in rabbits pretreated with NHEP (32.5 +/- 3.8%, n = 10) decreased by 41% compared to infarct size in rabbits that received vehicle (55.3 +/- 4.9%, n = 10; p = 0.002). Accumulation of neutrophils within the ischemic region, as assessed by myeloperoxidase activity, declined by 45% (p < 0.05) in AAR from NHEP-treated animals compared to AAR from vehicle-treated animals. Levels of MAC and IL-8 obtained from AAR were less in NHEP-pretreated animals compared to controls. These results suggest that NHEP may protect the myocardium by inhibiting complement activation and subsequent neutrophil infiltration.
Asunto(s)
Heparina/análogos & derivados , Infarto del Miocardio/tratamiento farmacológico , Daño por Reperfusión Miocárdica/prevención & control , Animales , Calcio/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Hemodinámica/efectos de los fármacos , Heparina/farmacología , Interleucina-8/metabolismo , Recuento de Leucocitos , Masculino , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Miocardio/enzimología , Miocardio/patología , Activación Neutrófila/efectos de los fármacos , Peroxidasa/metabolismo , ConejosRESUMEN
Pentosan polysulfate (PPS) is a highly sulfated semisynthetic polysaccharide possessing a higher negative charge density and degree of sulfation than heparin. Like other glycosaminoglycans, the structural and chemical properties of PPS promote binding of the drug to the endothelium. Glycosaminoglycans, including heparin, inhibit complement activation independent of an action on the coagulation system. This ability provides a compelling argument for the implementation of this class of compounds in experimental models of cellular injury mediated by complement. The objective of this study was to examine whether PPS could reduce myocardial injury resulting from activation of the complement system. We used the rabbit isolated heart perfused with 4% normal human plasma as a source of complement. Hemodynamic variables were obtained before addition of PPS (0.03 01 mg/ml) and every 10 min after the addition of human plasma. Compared with vehicle-treated hearts, left ventricular end-diastolic pressure was improved at the conclusion of the 60-min protocol in hearts treated with PPS (58.9 +/- 13.6 vs. 15. 2 +/- 4.8 mm Hg). Further evidence as to the protective effects of PPS was demonstrated by decreased creatine kinase release compared with vehicle (86.5 +/- 28.5 U/l vs. 631.0 +/- 124.8 U/l). An enzyme-linked immunosorbent assay for the presence of the membrane attack complex in lymph and tissue samples demonstrated decreased membrane attack complex formation in PPS-treated hearts, which suggests inhibition of complement activation. This conclusion was supported further by the ability of PPS to inhibit complement-mediated red blood cell lysis in vitro. The results of this study indicate that PPS can reduce tissue injury and preserve organ function that otherwise would be compromised during activation of the human complement cascade.
Asunto(s)
Anticoagulantes/farmacología , Activación de Complemento/efectos de los fármacos , Corazón/efectos de los fármacos , Daño por Reperfusión Miocárdica/prevención & control , Poliéster Pentosan Sulfúrico/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Proteínas del Sistema Complemento/análisis , Proteínas del Sistema Complemento/efectos de los fármacos , Creatina Quinasa/metabolismo , Relación Dosis-Respuesta a Droga , Glicoproteínas/análisis , Glicoproteínas/efectos de los fármacos , Corazón/fisiología , Humanos , Masculino , Daño por Reperfusión Miocárdica/inmunología , Conejos , Función Ventricular Izquierda/efectos de los fármacosRESUMEN
Neutrophil accumulation and activation of the complement system with subsequent deposition of the cytolytic membrane attack complex (MAC) have been implicated in the pathogenesis of myocardial ischemia/reperfusion injury. The MAC, when present in high concentrations, promotes target cell lysis. However, relatively little is known about the potential modulatory role of sublytic concentrations of the MAC on nucleated cell function in vivo. In vitro studies demonstrated that the MAC regulates cell function by promoting the expression of pro-inflammatory mediators, including adhesion molecules and pro-inflammatory cytokines. We examined, using C6-deficient and C6-sufficient rabbits, the regulatory role of the MAC in mediating IL-8 expression and subsequent neutrophil recruitment in the setting of myocardial ischemia/reperfusion injury. C6-deficient and C6-sufficient rabbits were subjected to 30 min of regional myocardial ischemia followed by a period of reperfusion. In addition to a significant reduction in myocardial infarct size in C6-deficient animals, analysis of myocardial tissue demonstrated a decrease in neutrophil influx into the infarcted region. The reduction in neutrophil influx correlated with the decreased expression of the neutrophil chemotactic cytokine IL-8, as determined by ELISA and immunohistochemical analysis. The results derived from this study provide evidence that the MAC has an important function in mediating the recruitment of neutrophils to the reperfused myocardium through the local induction of IL-8.
Asunto(s)
Complemento C6/fisiología , Complejo de Ataque a Membrana del Sistema Complemento/fisiología , Interleucina-8/metabolismo , Isquemia Miocárdica/inmunología , Daño por Reperfusión Miocárdica/inmunología , Animales , Movimiento Celular , Recuento de Leucocitos , Infarto del Miocardio , Isquemia Miocárdica/sangre , Daño por Reperfusión Miocárdica/sangre , Daño por Reperfusión Miocárdica/metabolismo , Miocardio/metabolismo , Neutrófilos/citología , Neutrófilos/enzimología , Peroxidasa/metabolismo , ConejosRESUMEN
One of the foremost mechanisms involved in the pathogenesis of myocardial reperfusion injury is the adhesion of neutrophils within the myocardium. The initial neutrophil-endothelial cell interactions are mediated by the selectin family of adhesion molecules. Blockade of this group of adhesion molecules, through the use of synthetic carbohydrate analogs to the selectin ligand sialyl Lewisx and glycomimetics, has been beneficial in reducing neutrophil influx and infarct size. In the present study, glycyrrhizin (GM1292), a natural structural glycomimetic, was analyzed for the ability to decrease myocardial infarct size after regional myocardial ischemia/reperfusion. To determine the structural requirements for optimal cardioprotective activity, two additional compounds related to glycyrrhizin, GM3290 and GM1658 (glycyrrhetinic acid), were studied. The molecular structures of the latter two compounds differ in the number of glucuronic acid residues in their respective molecules. Open-chest, anesthetized rabbits were subjected to 30 min occlusion of the left coronary artery followed by 5 hr of reperfusion. Vehicle or glycomimetic (10 mg/kg/hr) was administered intravenously immediately before the onset of reperfusion and every hour during the reperfusion period. Myocardial infarct size in rabbits treated with GM1292 (two glucuronic acid residues) and GM3290 (one glucuronic acid residue) was reduced significantly when compared with vehicle-treated animals (P < .05). GM1658, which lacks glucuronic acid residues, did not provide a protective effect in vivo. The data suggest that GM1292 and GM3290, which contain carbohydrate moieties, are effective in reducing the degree of myocardial injury after an acute period of ischemia/reperfusion.
Asunto(s)
Ácido Glicirrínico/uso terapéutico , Infarto del Miocardio/tratamiento farmacológico , Animales , Adhesión Celular/efectos de los fármacos , Humanos , Inmunohistoquímica , Recuento de Leucocitos , Neutrófilos/efectos de los fármacos , Selectina-P/análisis , Peroxidasa/metabolismo , ConejosRESUMEN
The cytoprotective action of reviparin-sodium (LU-47311: Clivarin), a low-molecular-weight heparin, was examined in an ex vivo model of complement-mediated myocardial injury. The effective concentration of reviparin was determined by using an in vitro rabbit erythrocyte-lysis assay using 4% normal human plasma. Reviparin (0.01-2.73 mg/ml) reduced erythrocyte lysis in a concentration-dependent manner. Subsequently, 0.91 mg/ml of reviparin was evaluated in an ex vivo rabbit isolated-heart model of human complement-mediated injury. Hearts perfused in the presence of 0.91 mg/ml of reviparin (n = 10) demonstrated significant preservation of ventricular function compared with vehicle-treated hearts (n = 10), as evidenced by coronary artery perfusion pressure, left ventricular developed pressure, and left ventricular end-diastolic pressure. A reduction in myocyte creatine kinase release was observed in reviparin-treated hearts compared with controls. Myocardial injury in vehicle-treated hearts was associated with an increased assembly of the membrane-attack complex, as determined by immunohistochemical localization of C5b-9 neoantigen. Reviparin decreased fluid-phase Bb formation detected in the lymphatic drainage of plasma-perfused hearts. The results of this study demonstrate that reviparin inhibits complement-mediated myocardial injury as assessed in an ex vivo experimental model of complement activation.
Asunto(s)
Anticoagulantes/farmacología , Activación de Complemento/efectos de los fármacos , Complejo de Ataque a Membrana del Sistema Complemento/análisis , Fibrinolíticos/farmacología , Corazón/efectos de los fármacos , Heparina de Bajo-Peso-Molecular/farmacología , Miocardio/patología , Animales , Cardiomiopatías/tratamiento farmacológico , C3 Convertasa de la Vía Alternativa del Complemento , Complemento C3b/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/antagonistas & inhibidores , Creatina Quinasa/metabolismo , Humanos , Técnicas para Inmunoenzimas , Inmunohistoquímica , Técnicas In Vitro , Miocardio/enzimología , Miocardio/inmunología , Fragmentos de Péptidos/metabolismo , ConejosRESUMEN
Evidence is presented that treatment with a highly sulfated low-molecular-weight heparin fraction, LU 51198, protects the ex vivo perfused rabbit heart from human complement-mediated injury. Hearts from male New Zealand White rabbits were perfused under constant flow in the Langendorff mode. After equilibration, normal human plasma was added to the perfusate as a source of complement. Either control (n = 8) or LU 51198 (0.6 mg/ml; n = 7) was added to the perfusion medium 10 min before the addition of human plasma. Hemodynamic variables were obtained for both groups before treatment of human plasma. Hemodynamic variables were obtained for both groups before treatment (baseline), 10 min after treatment (zero) and after the addition of human plasma. Compared to control-treated hearts, variables recorded during perfusion with human plasma, including coronary perfusion pressure, left ventricular developed pressure, and left ventricular end-diastolic pressure, along with a reduction of creatine kinase and potassium efflux, were significantly improved in hearts treated with LU 51198 (P < .05). ELISA assays were used to analyze lymphatic effluent for the presence of iC3b, Bb and SC5b-9 proteins derived from the activation of human complement. The increased presence of the Bb fragment in the effluent obtained from LU 51198-perfused hearts suggests an accelerated dissociation of the convertases responsible for complement amplification, an observation that coincided with protection from complement-mediated damage in the presence of the glycosaminoglycan. The lysis of rabbit red blood cells upon exposure to human plasma was inhibited by LU 51198, which is evidence of the drug's ability to modulate complement reactivity. The results of this study indicate that a highly sulfated heparin fraction, LU 51198, can reduce tissue injury and preserve discordant organ function that otherwise would be compromised during activation of the human complement cascade.
Asunto(s)
Proteínas del Sistema Complemento/toxicidad , Corazón/efectos de los fármacos , Heparina/análogos & derivados , Heparina/química , Miocardio/patología , Animales , Proteínas Inactivadoras de Complemento/farmacología , Creatina Quinasa/metabolismo , Corazón/fisiopatología , Hemodinámica/efectos de los fármacos , Heparina/farmacología , Humanos , Masculino , Peso Molecular , Miocardio/enzimología , Miocardio/metabolismo , Potasio/metabolismo , ConejosRESUMEN
We determined if heat stress induction of heat shock protein (HSP) 70 modulates complement activation in an experimental model of xenograft rejection. Male New Zealand White rabbits were heat stressed (core body temperature to 42 degrees C for 15 min; n = 9). Control rabbits (n = 13) were not exposed to heat stress. Hearts were removed 18 h later and perfused by the Langendorff method. After equilibration, human plasma (source of human complement) was added to the perfusion medium. Hemodynamic variables recorded during perfusion with human plasma were improved in hearts from heat-stressed animals compared with control hearts. Assembly of the soluble membrane attack complex was reduced in the interstitial fluid effluent from the heat-stressed hearts. Electron microscopic evidence of ultrastructural changes was attenuated in the hearts from heat-stressed rabbits. Myocardial tissue from heat-stressed animals exhibited an increase in inducible HSP 70 that was virtually absent in the hearts of control rabbits. Previous whole body hyperthermia protects the rabbit heart against the detrimental effects of heterologous plasma, suggesting that heat-stress induction of HSP 70 limits the extent of complement activation by a discordant vascularized tissue (xenograft). Induction of heat stress proteins by the donor organ might be an important mechanism affecting the outcome of xenograft transplants.
Asunto(s)
Proteínas del Sistema Complemento/fisiología , Corazón/fisiopatología , Trastornos de Estrés por Calor/fisiopatología , Animales , Western Blotting , Activación de Complemento/fisiología , Complejo de Ataque a Membrana del Sistema Complemento , Proteínas del Sistema Complemento/metabolismo , Glicoproteínas/metabolismo , Proteínas HSP70 de Choque Térmico/fisiología , Hemodinámica , Humanos , Masculino , Microscopía Electrónica , Miocardio/patología , Perfusión , ConejosRESUMEN
1. The direct cardiac electrophysiological and antifibrillatory actions of tedisamil (KC-8857) were studied in rabbit isolated hearts. 2. Tedisamil (1, 3, and 10 microM), prolonged the ventricular effective refractory period (VRP) from 120 +/- 18 ms (baseline) to 155 +/- 19, 171 +/- 20, and 205 +/- 14 ms, respectively. Three groups of isolated hearts (n = 6 each) were used to test the antifibrillatory action of tedisamil. Hearts were perfused with 1.25 microM pinacidil, a KATP channel activator. Hearts were subjected to hypoxia for 12 min followed by 40 min of reoxygenation. Ventricular fibrillation (VF) developed during hypoxia and reoxygenation in both the control and 1 microM tedisamil-treated groups (5/6 and 4/6, respectively). Tedisamil (3 microM) reduced the incidence of VF (0/6, P = 0.007 vs. control). 3. In a separate group of hearts, VF was initiated by electrical stimulation. The administration of 0.3 ml of 10 mM tedisamil, via the aortic cannula, terminated VF in all hearts, converting them to normal sinus rhythm. 4. Tedisamil (3 microM) reversed pinacidil-induced negative inotropic effects in rabbit isolated atrial muscle which were equilibrated under normoxia, as well as in atrial muscle subjected to hypoxia and reoxygenation. 5. The results demonstrate a direct antifibrillatory action of tedisamil in vitro. The mechanism responsible for the observed effects may involve modulation by tedisamil of the cardiac ATP-regulated potassium channel, in addition to its antagonism of IK and Ito.
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Antiarrítmicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Ciclopropanos/farmacología , Ciclopropanos/uso terapéutico , Electrocardiografía/efectos de los fármacos , Corazón/efectos de los fármacos , Animales , Antiarrítmicos/uso terapéutico , Relación Dosis-Respuesta a Droga , Guanidinas/farmacología , Corazón/fisiología , Técnicas In Vitro , Pinacidilo , Conejos , Estereoisomerismo , Vasodilatadores/farmacología , Fibrilación Ventricular/tratamiento farmacológicoRESUMEN
The murine cDNA, encoding the purine catabolic enzyme, ecto-5'-nucleotidase (NT), was cloned and the tissue-specific distribution of both the mRNA and enzyme activity was examined. Starting with kidney RNA and primers based on the known rat sequence, reverse transcriptase-polymerase chain reaction (RT-PCR) was utilized to obtain the complete sequence for the translated portion of the murine cDNA. Murine NT is 94% identical to human NT at the amino acid (aa) level and 86% identical at the nucleotide (nt) level. NT enzyme assays revealed greater than tenfold more NT activity in mature vs. immature murine T- and B-lymphocytes. A similar increase in NT activity was also found when the pre-B-cell line, 70Z/3, was induced to produce surface kappa light chains with lipopolysaccharide (LPS) and gamma-interferon (gamma-IFN). Thus, culture systems in which murine lymphocytes mature may be useful for examining the mechanisms of NT gene regulation, as well as the function of NT in the immune system. In tissues, enzyme activity varied over 30-fold, from the lowest levels in skeletal muscle, thymus and spleen to highest in placenta, kidney and forestomach. Levels of mRNA, as determined by RNase protection assay, showed increased NT expression in the early gestation site, as compared to non-pregnant uterus, and in day-19.5 placenta, as compared to day-13 chorioallantoic placenta. Messenger RNA levels were in general proportional to enzyme activity, except in the lung and glandular stomach where mRNA levels were higher than expected, based on enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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5'-Nucleotidasa/genética , 5'-Nucleotidasa/metabolismo , Envejecimiento/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN , Humanos , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Reacción en Cadena de la Polimerasa , Ribonucleasas , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
Single fibers of rabbit fast-twitch tibialis anterior (TA) muscles were analyzed after continuous low-frequency stimulation for up to 8 wk. After 2-5 wk, every fiber showed higher levels of citrate synthase, hexokinase, and 3-oxoacid CoA-transferase than any control fiber; in some cases these levels were 2-10 times higher (well above any found even in the control soleus, a slow-twitch muscle). Average levels of malate dehydrogenase and alanine transaminase also rose dramatically, but peak single fiber levels were not much above the highest in controls. These differential effects confirm at the single fiber level that chronic stimulation can alter mitochondrial composition. Lactate dehydrogenase, fructose-bisphosphatase, and adenylate kinase declined to levels far below those of any control TA fiber, and, in the case of fructose-bisphosphatase, to within the activity range of control soleus fibers. According to their staining reaction for myofibrillar ATPase, TA fibers were initially 23% type IIA, and 74% type IIB, but by 5 wk these had been converted to a mixture of type I, IIA, and IIC fibers. At 5 wk, levels of lactate dehydrogenase, adenylate kinase, and malate dehydrogenase were characteristic of their (new) ATPase type, but 3-oxoacid CoA transferase had increased to levels 6-15 times higher than in control fibers of the same type.
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Adaptación Fisiológica , Coenzima A Transferasas , Músculos/enzimología , Adenosina Trifosfatasas/metabolismo , Alanina Transaminasa/metabolismo , Animales , Citrato (si)-Sintasa/metabolismo , Estimulación Eléctrica , Femenino , Fructosa-Bifosfatasa/metabolismo , Hexoquinasa/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Malato Deshidrogenasa/metabolismo , Masculino , Músculos/anatomía & histología , Miofibrillas/enzimología , Conejos , Sulfurtransferasas/metabolismo , Factores de TiempoRESUMEN
Activities of choline acetyltransferase and acetylcholinesterase were measured for the acetylcholinesterase-positive fiber bundles containing axons projecting from the brainstem to the labyrinth of the rat. These activities were compared to those of a well-established cholinergic tract: the facial motor root. The choline acetyltransferase activities were roughly similar between the tracts, consistent with a conclusion that the centrifugal labyrinthine fibers are all cholinergic. The acetylcholinesterase activities were much higher in the centrifugal labyrinthine bundle than in the facial motor root, probably relating to the smaller diameters of the labyrinthine fibers. Transection of the centrifugal labyrinthine bundle led to virtually total loss of its choline acetyltransferase activity lateral to the cut, consistent with a centrifugal direction of all the fibers, but loss of only half its acetylcholinesterase activity, even after 34 days. These results agree with those for well-established cholinergic pathways, including the facial motor root in the present study, and with previous suggestions that a component of the acetylcholinesterase in cholinergic tracts might be synthesized by cells other than the neurons in the tract.