Asunto(s)
Analgesia/métodos , Dolor Postoperatorio/tratamiento farmacológico , Analgesia Epidural , Analgésicos/administración & dosificación , Analgésicos/uso terapéutico , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/uso terapéutico , Medicina Basada en la Evidencia , Humanos , Narcóticos/administración & dosificación , Narcóticos/uso terapéutico , Bloqueo Nervioso , Dimensión del Dolor , Dolor Postoperatorio/prevención & control , Planificación de Atención al PacienteRESUMEN
Peroxynitrite (PN), the product of the diffusion-limited reaction between nitric oxide (*NO) and superoxide (O*-(2)), represents a relevant mediator of oxidative modifications in low-density lipoprotein (LDL). This work shows for the first time the simultaneous action of low-controlled fluxes of PN and *NO on LDL oxidation in terms of lipid and protein modifications as well as oxidized lipid-protein adduct formation. Fluxes of PN (e.g., 1 microM min(-1)) initiated lipid oxidation in LDL as measured by conjugated dienes and cholesteryl ester hydroperoxides formation. Oxidized-LDL exhibited a characteristic fluorescent emission spectra (lambda(exc) = 365 nm, lambda(max) = 417 nm) in parallel with changes in both the free amino groups content and the relative electrophoretic mobility of the particle. Physiologically relevant fluxes of *NO (80-300 nM min(-1)) potently inhibited these PN-dependent oxidative processes. These results are consistent with PN-induced adduct formation between lipid oxidation products and free amino groups of LDL in a process prevented by the simultaneous presence of *NO. The balance between rates of PN and *NO production in the vascular wall will critically determine the final extent of LDL oxidative modifications leading or not to scavenger receptor-mediated LDL uptake and foam cell formation.
Asunto(s)
Metabolismo de los Lípidos , Lipoproteínas LDL/metabolismo , Óxido Nítrico/metabolismo , Ácido Peroxinitroso/metabolismo , Proteínas/metabolismo , Humanos , Liposomas/metabolismo , Modelos Químicos , Oxígeno/metabolismo , Unión Proteica , Factores de TiempoRESUMEN
Several novel semicarbazone derivatives were prepared from 5-nitro-2-furaldehyde or 5-nitrothiophene-2-carboxaldehyde and semicarbazides bearing a spermidine-mimetic moiety. All derivatives presented the E-configuration, as determined by NMR-NOE experiments. These compounds were tested in vitro as potential antitrypanosomal agents, and some of them, together with the parent compounds, 5-nitro-2-furaldehyde and 5-nitrothiophene-2-carboxaldehyde semicarbazone derivatives, were also evaluated in vivo using infected mice. Structure-activity relationship studies were carried out using voltammetric response and lipophilic-hydrophilic balance as parameters. Two of the compounds (1 and 3) displayed the highest in vivo activity. A correlation was found between lipophilic-hydrophilic properties and trypanocidal activity, high R(M) values being associated with low in vivo effects.
Asunto(s)
Aldehídos/síntesis química , Furaldehído/análogos & derivados , Compuestos de Sulfhidrilo/síntesis química , Tripanocidas/síntesis química , Aldehídos/farmacología , Animales , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Fenómenos Químicos , Química Física , Cromatografía en Capa Delgada , Electroquímica , Furaldehído/síntesis química , Furaldehído/farmacología , Lípidos/química , Espectroscopía de Resonancia Magnética , Masculino , Espectrometría de Masas , Ratones , Espectrofotometría Infrarroja , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/farmacología , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacosRESUMEN
Peroxynitrite, the reactive species formed in vivo by the reaction of nitric oxide with superoxide anion, is capable of diffusing across erythrocyte membranes via anion channels and passive diffusion (A. Denicola, J. M. Souza, and R. Radi, Proc. Natl. Acad. Sci. USA 95, 3566-3571, 1998). However, peroxynitrite diffusion could be limited by extracellular targets, with the reaction with CO(2) (k(2) = 4.6 x 10(4) at 37 degrees C and pH 7.4) the most relevant. Herein, we studied the influence of physiological concentrations of CO(2) on peroxynitrite diffusion across intact red blood cells. The presence of CO(2) inhibited the oxidation of intracellular oxyhemoglobin by externally added peroxynitrite. However, the inhibition by CO(2) decreased at increasing red blood cell densities. At 45% hematocrit, 1.3 mM CO(2) (in equilibrium with 24 mM bicarbonate, at pH 7.4 and 25 degrees C) only inhibited 30% of intracellular oxyhemoglobin oxidation. This partial inhibition was also observed in red blood cells pretreated with the anion exchanger inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, ruling out a competition between peroxynitrite and bicarbonate for the transport through the anion channel. A theoretical model was developed to estimate the diffusion distance and half-life of extracellular peroxynitrite before reacting with intracellular oxyhemoglobin, at different red blood cell densities, and in the presence or absence of CO(2). The theoretical model correlated well with the experimental data. Our results indicate that, even in the presence of CO(2), peroxynitrite is able to diffuse and reach the inside of the erythrocyte.
Asunto(s)
Dióxido de Carbono/farmacología , Membrana Eritrocítica/metabolismo , Nitratos/sangre , Transporte Biológico Activo , Dióxido de Carbono/sangre , Difusión , Membrana Eritrocítica/efectos de los fármacos , Hematócrito , Humanos , Técnicas In Vitro , Canales Iónicos/metabolismo , Modelos Biológicos , Oxidantes/sangre , Oxidación-Reducción , Oxihemoglobinas/metabolismoRESUMEN
The syntheses of a new series of derivatives of 1,2,5-oxadiazole N-oxide, benzo[1,2-c]1,2,5-oxadiazole N-oxide, and quinoxaline di-N-oxide are described. In vitro antitrypanosomal activity of these compounds was tested against epimastigote forms of Trypanosoma cruzi. For the most effective drugs, derivatives IIIe and IIIf, the 50% inhibitory dose (ID50) was determined as well as their cytotoxicity against mammalian fibroblasts. Electrochemical studies and ESR spectroscopy show that the highest activities observed are associated with the facile monoelectronation of the N-oxide moiety. Lipophilic-hydrophilic balance of the compounds could also play an important role in their effectiveness as antichagasic drugs.
Asunto(s)
Óxidos N-Cíclicos/síntesis química , Oxadiazoles/síntesis química , Tripanocidas/síntesis química , Animales , Línea Celular , Cricetinae , Cricetulus , Óxidos N-Cíclicos/química , Electroquímica , Espectroscopía de Resonancia por Spin del Electrón , Fibroblastos , Concentración 50 Inhibidora , Oxadiazoles/química , Relación Estructura-Actividad , Tripanocidas/química , Trypanosoma cruzi/efectos de los fármacosRESUMEN
Peroxynitrite anion (ONOO-) is a reactive species of increasingly recognized biological relevance that contributes to oxidative tissue damage. At present, however, there is limited knowledge about the mechanisms of peroxynitrite diffusion through biological compartments. In this work we have studied the diffusion of peroxynitrite across erythrocyte membranes. In solution, peroxynitrite rapidly reacts with oxyhemoglobin to yield methemoglobin, with k2 = (10.4 +/- 0.3) x 10(3) M-1.s-1 at pH 7.4 and 25 degrees C. Addition of peroxynitrite to intact erythrocytes caused oxidation of intracellular oxyhemoglobin to methemoglobin. Oxidation yields in red blood cells at pH 7.0 were approximately 40% of those obtained in solution, which results mostly from competition of other cytosolic components for peroxynitrite. Indeed, rather small differences were observed between oxidation yields in lysates compared with intact erythrocytes, in particular at acidic and neutral pH values, indicating that membrane was not precluding peroxynitrite diffusion. Incubation of erythrocytes at pH 7.0 with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a specific inhibitor of anion exchange, resulted in up to 50% inhibition of oxyhemoglobin oxidation by peroxynitrite. More protection by DIDS was achieved at alkaline pH, while no effect was observed at pH 5.5, where 95% of peroxynitrite is in the acidic form, ONOOH (pKa = 6.8). In addition, peroxynitrite caused nitration of intracellular hemoglobin, in a process that was enhanced in thiol-depleted erythrocytes. Our results indicate that peroxynitrite is able to cross the erythrocyte membrane by two different mechanisms: in the anionic form through the DIDS-inhibitable anion channel, and in the protonated form by passive diffusion.
Asunto(s)
Membrana Eritrocítica/metabolismo , Nitratos/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/metabolismo , Transporte Biológico , Humanos , Concentración de Iones de Hidrógeno , Oxidantes/metabolismo , Oxidación-ReducciónRESUMEN
Several novel semicarbazones derivatives were prepared from 5-nitro-2-furaldehyde or 5-nitrothiophene-2-carboxaldehyde, and tested in vitro as potential anti-trypanosomal agents. The compounds were prepared in good to excellent yields in 2-3 steps from readily available starting materials. Some derivatives were found to be active against Trypanosoma cruzi with an activity similar to that of Nifurtimox.
Asunto(s)
Semicarbazonas/síntesis química , Tripanocidas/síntesis química , Animales , Semicarbazonas/farmacología , Relación Estructura-Actividad , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacosRESUMEN
There has been a recent renewal of interest in the antioxidant properties of pyruvate which are usually attributed to its capacity to undergo oxidative decarboxylation in the presence of hydrogen peroxide. The interaction of pyruvate with other oxidizing biological intermediates, however, has been scarcely considered in the literature. Here we report that peroxynitrite, the oxidant produced by the reaction between superoxide anion and nitric oxide, reacts with pyruvate with an apparent second-order rate constant of 88 +/- 7 M-1 s-1 at pH 7.4 and 37 degrees C. Kinetic studies indicated that pyruvate reacts with peroxynitrite anion (k = 100 +/- 7 M-1 s-1, peroxynitrous acid (k = 49 +/- 7 M-1 s-1, and a highly oxidizing species derived from peroxynitrous acid. Pyruvate decarboxylation was proved by anion exchange chromatography detection of acetate in incubations of peroxynitrite and pyruvate at pH 7.4 and 5.5. Formation of carbon dioxide radical anion was ascertained by EPR spin-trapping studies in the presence of GSH and the spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The use of pyruvate labeled with 13C at the 1-position led to the detection of the labeled DMPO carbon dioxide radical anion adduct. In the absence of GSH, oxygen consumption studies confirmed that peroxynitrite mediates the decarboxylation of pyruvate to free radical intermediates. Comparing the yields of acetate and free radicals estimated from the oxygen uptake studies, it is concluded that pyruvate is oxidized by both one- and two-electron oxidation pathways, the latter being preponderant. Hydrogen peroxide-mediated pyruvate oxidation does not produce detectable levels of carbon dioxide radical anion except in the presence of iron(II)-ethylenediamine-N,N,N',N'-tetraacetate (EDTA). The apparent second-order rate constant of the reaction between pyruvate and hydrogen peroxide was determined to be 1 order of magnitude lower than that of the reaction between pyruvate and peroxynitrite. The latter process may contribute to the antioxidant properties of pyruvate.
Asunto(s)
Dióxido de Carbono/química , Nitratos/química , Oxidantes/química , Ácido Pirúvico/química , Aniones , Cromatografía por Intercambio Iónico , Descarboxilación , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres/química , Cinética , Consumo de OxígenoRESUMEN
Peroxynitrite is a strong oxidant produced in vivo as the reaction product of superoxide anion and nitric oxide (k approximately 5 x 10(9) M-1 s-1) and can be formed and mediate reactions in the extracellular environment. It has recently been reported that peroxynitrite and carbon dioxide react in a second-order process (S. V. Lymar and K. Hurst (1995) J. Am. Chem. Soc. 117, 8867-8868). Since one of the most abundant constituents of the extracellular milieu is bicarbonate anion (25 mM in plasma) which is in equilibrium with carbon dioxide (1.3 mM in plasma) we have further studied the kinetics of the reaction between peroxynitrite and carbon dioxide/ bicarbonate and the effect of bicarbonate on different peroxynitrite-mediated oxidations. The apparent second-order rate constant for the reaction is (2.3 +/- 0.1) x 10(3) M-1 s-1 at 37 degrees C and pH 7.4 and a pH-independent second-order rate constant of (5.8 +/- 0.2) x 10(4) M-1 s-1 at 37 degrees C was obtained considering peroxynitrite anion and carbon dioxide as the reacting species. The enthalpy and entropy of activation are delta H* = +10.7 +/- 0.8 kcal mol-1 and delta S* = -6.5 +/- 0.5 cal mol-1 K-1, respectively. The presence of bicarbonate had variable influence on peroxynitrite-mediated oxidations. While bicarbonate significantly enhanced peroxynitrite-mediated nitration of aromatics, it partially inhibited the oxidation of thiols, dimethylsulfoxide, oxyhemoglobin, and cytochrome c+2 and totally inhibited the hydroxylation of benzoate. Spontaneous chemiluminescence studies suggest the formation of bicarbonate radicals during the interactions of peroxynitrite with carbon dioxide/ bicarbonate. Our results support that peroxynitrite anion rapidly reacts with carbon dioxide to yield an adduct (ONOOCO2-) which can participate in oxidation and nitration processes, thus redirecting the primary reactivity of peroxynitrite.
Asunto(s)
Bicarbonatos/metabolismo , Dióxido de Carbono/metabolismo , Nitratos/metabolismo , Bicarbonatos/química , Dióxido de Carbono/química , Radicales Libres/química , Técnicas In Vitro , Cinética , Mediciones Luminiscentes , Nitratos/química , Oxidación-Reducción , TermodinámicaRESUMEN
Quenching of pyrene derivative fluorescence by nitric oxide was used to evaluate the apparent diffusion coefficients of nitric oxide in artificial and biological membranes. The apparent second-order quenching constants of nitric oxide were obtained from Stern-Volmer plots using methyl- and undecylpyrene derivatives incorporated into liposomes and erythrocyte plasma membranes in order to assess the ability of nitric oxide to interact with molecules located at different positions in the membrane. Diffusion coefficients were estimated from the determined second-order quenching constants and compared to that of oxygen obtained under the same conditions. Oxygen and nitric oxide presented similar diffusional behavior in agreement with their similarity in structures, with the differences observed attributable to the higher lipophilicity of oxygen compared to nitric oxide. In solution, both showed the same quenching efficiency while in liposomes and erythrocyte ghosts oxygen diffusion was twice that of nitric oxide (k(O2)/k(NO) = 2). Nitric oxide diffusion coefficients determined at 20 degrees C ranged from 1.3 x 10(-5) cm2 s-1 in liposomes to 0.4 x 10(-5) cm2 s-1 in surface erythrocyte plasma membranes. Both nitric oxide and oxygen had larger quenching constants for the undecyl derivative compared to the methylpyrene compound incorporated into erythrocyte plasma membranes, indicating an increased solubility of both gases toward the center of the membrane.
Asunto(s)
Membrana Eritrocítica/metabolismo , Liposomas/metabolismo , Óxido Nítrico/metabolismo , Oxígeno/metabolismo , Animales , Difusión , Colorantes Fluorescentes , Cinética , Pirenos , Ovinos , Espectrometría de FluorescenciaRESUMEN
Two Connecticut populations of the black-legged tick, Ixodes scapularis (Say), the vector of Lyme disease spirochetes, Borrelia bugrdorferi sensu stricto Johnson, Schmid, Hyde, Steigerwalt & Brenner, in the northeastern United States, are parasitized by the encyrtid wasp Ixodiphagus hookeri (Howard), formerly Hunterellus hookeri. The wasp was first detected in ticks from a forested site in Bridgeport in 1992. I. hookeri was reared from 18.6% of 148 host-seeking I. scapularis nymphs. In 1993 and 1994, this wasp was found to parasitize 26.0% of 192 engorged nymphs from Bridgeport and 21.8% of 101 nymphs from the Bluff Point Coastal Preserve in Groton. Each parasitized nymph produced an average of 6-8 wasps (range, 3-16) with a female to male sex ratio of 1.9-1. Both study sites are wooded, geographically isolated tracts (Bridgeport, 176 ha and Bluff Point, 326 ha) with high densities (51-72/km2) of white-tailed deer, Odocoileus virginianus (Zimmerman), and superabundant tick populations similar to that of Prudence Island, RI, where I. hookeri also has been reported. We found that I. hookeri emerged from 16.3% of 399 engorged nymphs and 13.7% of 1,081 engorged nymphs collected as unfed ticks from Prudence Island in 1990 and 1991, respectively. No wasps were obtained from nymphs collected in Stamford (n = 38) or Old Lyme, CT (n = 241). A high proportion (23.1% of 39) of engorged nymphs obtained from Bridgeport deer produced I. hookeri. However, only 2 nymphs (6.7%) recovered from white-footed mice, Peromyscus leucopus (Rafinesque), at Bridgeport were parasitized by the wasp. None of the engorged larvae recovered from deer or mice and fed as nymphs in the laboratory produced I. hookeri (n = 26 from deer and n = 384 from mice). The presence of this wasp in I. scapularis at these 2 insular-like sites on the Connecticut mainland supports the observation that high tick densities are required for the establishment and maintenance of I. hookeri and that the potential role of this wasp in the biological control of I. scapularis is limited.
Asunto(s)
Grupo Borrelia Burgdorferi/aislamiento & purificación , Ixodes/microbiología , Ixodes/parasitología , Infestaciones por Garrapatas/parasitología , Avispas , Animales , Connecticut , Ciervos , Femenino , Masculino , Ratones , Estaciones del Año , Infestaciones por Garrapatas/veterinariaRESUMEN
Peroxynitrite, the reaction product of nitric oxide and superoxide, is a potent and versatile oxidant that can attack a wide range of targets. In this work, we studied the oxidation of hydrogen peroxide by peroxynitrite, which led to oxygen evolution. Oxygen yields increased at alkaline pH with an apparent pKa of 7.05 +/- 0.04. The maximum yields were 16% and 32% of added peroxynitrite at pH 5.9 and 7.4, respectively, assuming that two molecules of peroxynitrite are needed to produce one of oxygen. Hydroxyl radical scavengers (dimethyl sulfoxide, mannitol, ethanol, formate, and acetate) inhibited oxygen evolution to a similar extent to that predicted from their rate constants with hydroxyl radical. The apparent rate constant of peroxynitrite decomposition was zero-order in hydrogen peroxide at acidic pH. At neutral and alkaline pH, the rate of peroxynitrite disappearance decreased in the presence of millimolar concentrations of hydrogen peroxide by up to 50%. The apparent activation enthalpy and entropy for peroxynitrite decomposition increased by 1.7 kcal mol-1 and 4.7 cal mol-1 K-1, respectively, in the presence of hydrogen peroxide. We propose that an activated intermediate of peroxynitrous acid is responsible for hydrogen peroxide oxidation at acidic pH, while at more alkaline pH the formation of a stabilizing complex between hydrogen peroxide and transperoxynitrite anion is involved.
Asunto(s)
Peróxido de Hidrógeno/química , Nitratos/química , Oxígeno/química , Concentración de Iones de Hidrógeno , Radical Hidroxilo/química , Oxidación-Reducción , TermodinámicaRESUMEN
Nitric oxide reacts with superoxide to form peroxynitrite, a strong oxidizing species. Peroxynitrite can either directly oxidize molecules such as thiols or protonate to peroxynitrous acid, which can yield an oxidant with a reactivity similar to that of hydroxyl radical in a transition metal-independent mechanism. This oxidative chemistry of peroxynitrite, however, is inhibited by the metal chelator desferrioxamine. Indeed, desferrioxamine, was a potent inhibitor of dimethylsulfoxide, hydrogen peroxide, 5,5-dimethyl-1-pyrroline-N-oxide, and luminol oxidation, whereas the metal chelator diethylenetriaminepentaacetic acid, and ferrioxamine, the iron complex of desferioxamine, were not. Two other hydroxamates, acetohydroxamate and salicylhydroxamate, were also effective inhibitors. Stopped-flow experiments showed that there is no direct reaction between peroxynitrite anion or cis-peroxynitrous acid with desferrioxamine. Electron paramagnetic resonance (EPR) studies showed the formation of the desferrioxamine nitroxide radical in incubations containing desferrioxamine, but not ferrioxamine, indicating that the hydroxamic group acts as a one-electron donor to peroxynitrite-derived oxidants. Taken together, our results led us to propose that desferrioxamine can inhibit the oxidative chemistry of peroxynitrite by reaction of the hydroxamic acid moieties with trans-peroxynitrous acid.
Asunto(s)
Deferoxamina/farmacología , Ácidos Hidroxámicos/química , Radical Hidroxilo/química , Nitratos/química , Óxidos N-Cíclicos/química , Dimetilsulfóxido/química , Espectroscopía de Resonancia por Spin del Electrón , Peróxido de Hidrógeno/química , Mediciones Luminiscentes , Luminol/química , Oxidación-Reducción , EspectrofotometríaRESUMEN
Ticks and blood samples were collected from white-tailed deer (Odocoileus virginianus) in forests located in an insular, urban area of Bridgeport, Conn., and in rural south central Connecticut during 1992 and 1993. Immature and adult Ixodes scapularis ticks were tested for Borrelia burgdorferi, the etiologic agent of Lyme borreliosis, by indirect fluorescent-antibody staining methods. Deer sera were analyzed for antibodies to this bacterium by an enzyme-linked immunosorbent assay. Infected ticks parasitized deer in Bridgeport from May through December; the prevalence of infection varied from 1.1% of 93 larvae to 28.1% of 114 adult females. The percentages of infected males (10.5% of 380 ticks) and females (13.7% of 328 ticks) were relatively lower in south central Connecticut. In antibody tests, the prevalence of seropositive specimens collected in Bridgeport (61% of 146 serum specimens) was more than twofold greater than that of specimens obtained in south central Connecticut (26.7% of 116 serum specimens). Foci for Lyme borreliosis can occur in forested, urban settings as well as in rural areas if there are ticks, rodents, birds, and large mammals present. Human exposure to ticks in such sites should be considered as a possible source of B. burgdorferi infection.
Asunto(s)
Vectores Arácnidos/microbiología , Grupo Borrelia Burgdorferi/aislamiento & purificación , Ciervos/microbiología , Garrapatas/microbiología , Animales , Anticuerpos Antibacterianos/sangre , Connecticut/epidemiología , Ciervos/sangre , Femenino , Enfermedad de Lyme/epidemiología , Enfermedad de Lyme/microbiología , Masculino , Prevalencia , Salud UrbanaRESUMEN
Activated macrophages release peroxynitrite anion (ONOO-), which has been recently shown to be highly cytotoxic against Trypanosoma cruzi epimastigotes. In this work, we report that two critical enzymes for the energetic metabolism of the parasite, succinate dehydrogenase and fumarate reductase, are inactivated by biologically relevant concentrations of peroxynitrite. Enzyme inactivation was accompanied by a significant inhibition of succinate-dependent respiration in intact cells as well as in the membrane-rich fraction. Peroxynitrite also inhibited NADH-dependent oxygen consumption which depends almost exclusively on fumarate reductase activity in T. cruzi epimastigotes. Direct reactions of peroxynitrite anion with critical sulfhydryl residues of the two enzymes were responsible for most of the observed inactivation as indicated by the protection afforded by peroxynitrite scavengers and the reactivation of the enzymes by dithiothreitol. We propose that peroxynitrite-mediated inactivation of succinate dehydrogenase and fumarate reductase may be a key mechanism of macrophage-mediated cytotoxicity to T. cruzi, through inhibition of the energetic metabolism of the parasite.
Asunto(s)
Metabolismo Energético/efectos de los fármacos , Nitratos/toxicidad , Succinato Deshidrogenasa/antagonistas & inhibidores , Trypanosoma cruzi/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Animales , Quelantes/farmacología , Etilmaleimida/farmacología , Humanos , Cinética , Pulmón/metabolismo , Mitocondrias Cardíacas/enzimología , NADH Deshidrogenasa/antagonistas & inhibidores , Succinato Deshidrogenasa/metabolismo , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/enzimología , alfa 1-Antitripsina/metabolismoRESUMEN
Macrophages produce and release superoxide anion (O2.-) and nitric oxide (.NO) as part of their microbicidal effector molecules. The simultaneous production of O2.- and .NO results in the rapid formation of peroxynitrite anion (ONOO-) by macrophages. Peroxynitrite is a strong oxidant with a half-life of less than 1 s in biological systems. There is solid experimental evidence implicating .NO and O2.- in macrophage-induced cytotoxicity against bacteria, parasites, and tumor cells. However, the cytotoxic role of peroxynitrite in these processes remains to be studied. In this work we demonstrate the parasiticidal activity of ONOO- against Trypanosoma cruzi. Peroxynitrite was highly trypanocidal, killing T. cruzi in a dose-dependent manner. Addition of 500 microM ONOO- as a single bolus resulted in 50% inhibition of cell proliferation as followed by growth curves. Fifty percent inhibition of [3H]thymidine incorporation measured at 6 h postaddition of ONOO- was obtained at 150 microM. Addition of ONOO- as a continuous infusion rather than a single bolus resulted in an even stronger inhibition in cell growth. Other cytotoxic effects of ONOO- included cellular swelling and inhibition of cell motility. Classical hydroxyl radical scavengers and metal chelators afforded minimal protection against ONOO(-)-mediated cytotoxicity, indicating that peroxynitrite anion itself, rather than the .OH-like oxidant derived from its proton-catalyzed decomposition, was the main damaging species. From literature data we estimated the production of ONOO- by activated macrophages inside phagolysosomes to be around 500 microM/min. Therefore, our results demonstrate that ONOO- may operate in vivo as a critical macrophage-derived reactive intermediate against T. cruzi.
Asunto(s)
Nitratos/toxicidad , Trypanosoma cruzi/efectos de los fármacos , Animales , Quelantes , ADN Protozoario/biosíntesis , Radicales Libres , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
Lower concentrations of immunoreactive (IR) beta-endorphin were present in the neurointermediate pituitary lobes of streptozocin-induced diabetic versus control animals at both 2 and 4 weeks after the onset of diabetes. The forms of beta-endorphin-like material present appeared to be similar in both groups when studied with cation-exchange chromatography. Insulin therapy via minipump for 2 weeks did not alter this finding of lowered beta-endorphin concentrations in diabetic animals, despite normalization of blood glucose levels and body weight gain. Lower IR beta-endorphin levels were also found in neurointermediate lobes of weight-restricted rats, but this group had increased plasma IR beta-endorphin concentrations compared to diabetic animals. Concentrations of IR beta-endorphin in microdissected brain regions and in anterior pituitaries of the diabetic animals failed to show consistent changes; in addition, ACTH concentrations in pituitary lobes and plasma did not differ among groups. Circadian rhythmicity of plasma insulin and corticosterone concentrations was absent in the diabetic animals, although food and water intake, while elevated, showed the normal nocturnal pattern of increased ingestion. Furthermore, adrenal hypertrophy was present in the diabetic animals and was accompanied by an elevation of mean plasma corticosterone levels. The present findings indicate that diabetes is associated with a decrease of neurointermediate pituitary lobe synthesis of beta-endorphin, while not affecting the processing of the peptide in this lobe, and confirm previous reports of altered adrenal function in diabetic animals.