RESUMEN
Although a great deal of emphasis has been placed on the vasculopathy that is associated with oxygen-induced retinopathy (OIR), our studies also revealed significant and irreversible structural (retinal histology) and functional (scotopic and photopic electroretinograms) impairments that were significantly more severe in pigmented Long-Evans rats compared to the more commonly used albino Sprague Dawley rats. In the following pages, we will highlight what we have learned about the retinal pathophysiological processes of OIR taking place in strains of both rats with the hope that this will trigger investigations into new therapeutic strategies to complement those geared at preventing the vasculopathy.
Asunto(s)
Modelos Animales de Enfermedad , Hiperoxia/fisiopatología , Retina/fisiopatología , Neovascularización Retiniana/fisiopatología , Retinopatía de la Prematuridad/fisiopatología , Animales , Animales Recién Nacidos , Electrorretinografía , Humanos , Recién Nacido , Oxígeno/toxicidad , Ratas , Vasos Retinianos/efectos de los fármacosRESUMEN
Vasopressin type 2 receptor (V2R) exhibits mostly important properties for hydroosmotic equilibrium and, to a lesser extent, on vasomotricity. Drugs currently acting on this receptor are analogs of the natural neuropeptide, arginine vasopressin (AVP), and hence are competitive ligands. Peptides that reproduce specific sequences of a given receptor have lately been reported to interfere with its action, and if such molecules arise from regions remote from the binding site they would be anticipated to exhibit noncompetitive antagonism, but this has yet to be shown for V2R. Six peptides reproducing juxtamembranous regions of V2R were designed and screened; the most effective peptide, cravky (labeled VRQ397), was characterized. VRQ397 was potent (IC(50) = 0.69 +/- 0.25 nM) and fully effective in inhibiting V2R-dependent physiological function, specifically desmopressin-L-desamino-8-arginine-vasopressin (DDAVP)-induced cremasteric vasorelaxation; this physiological functional assay was utilized to avoid overlooking interference of specific signaling events. A dose-response profile revealed a noncompetitive property of VRQ397; correspondingly, VRQ397 bound specifically to V2R-expressing cells could not displace its natural ligand, AVP, but modulated AVP binding kinetics (dissociation rate). Specificity of VRQ397 was further confirmed by its inability to bind to homologous V1 and oxytocin receptors and its inefficacy to alter responses to stimulation of these receptors. VRQ397 exhibited pharmacological permissiveness on V2R-induced signals, as it inhibited DDAVP-induced PGI(2) generation but not that of cAMP or recruitment of beta-arrestin2. Consistent with in vitro and ex vivo effects as a V2R antagonist, VRQ397 displayed anticipated in vivo aquaretic efficacy. We hereby describe the discovery of a first potent noncompetitive antagonist of V2R, which exhibits functional selectivity, in line with properties of a negative allosteric modulator.
Asunto(s)
Antagonistas de los Receptores de Hormonas Antidiuréticas , Antagonistas de Hormonas/farmacología , Músculo Liso/efectos de los fármacos , Miometrio/efectos de los fármacos , Oligopéptidos/farmacología , Vejiga Urinaria/efectos de los fármacos , 6-Cetoprostaglandina F1 alfa/metabolismo , Regulación Alostérica , Animales , Arginina Vasopresina/metabolismo , Línea Celular , AMP Cíclico/metabolismo , Desamino Arginina Vasopresina/metabolismo , Diuresis/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Antagonistas de Hormonas/metabolismo , Humanos , Técnicas In Vitro , Ligandos , Masculino , Ratones , Relajación Muscular/efectos de los fármacos , Músculo Liso/metabolismo , Miometrio/metabolismo , Oligopéptidos/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Vasopresinas/genética , Receptores de Vasopresinas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/efectos de los fármacos , Transfección , Vejiga Urinaria/metabolismoRESUMEN
The neonatal rat is born with its eyes closed and an immature visual system, that some say is equivalent to that of a human fetus at 26 weeks of gestation. From birth, the visual system of the newborn rat will gradually mature, the first manifestation of that being the opening of the eye which usually take place at postnatal day 14. Complete maturation of the retina and visual pathways is normally reached at the end of the first month of life. The neonatal rat model thus represents a unique paradigm to study the normal and abnormal maturation of the primary visual pathways that normally occurs in utero in human subjects. Our laboratory has, over the past decade, developed two animal models of postnatally induced retinopathy, namely the Oxygen-Induced Retinopathy (OIR) that share several common features with the human Retinopathy of Prematurity (ROP) and the Light-Induced Retinopathy that is viewed by some as a valid model of some forms of Retinitis Pigmentosa (RP). The following pages review what is known of the pathophysiological processes taking place and suggest possible therapeutic avenues that could be explored in order to halt the degenerative process.
Asunto(s)
Luz , Oxígeno , Retina/efectos de los fármacos , Retina/embriología , Vías Visuales/embriología , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Desarrollo Fetal , Humanos , Recién Nacido , Isoprostanos/fisiología , Factor de Activación Plaquetaria/fisiología , Ratas , Retinitis Pigmentosa/fisiopatología , Retinopatía de la Prematuridad/fisiopatología , Tromboxano A2/fisiologíaRESUMEN
By comparing mRNA profiles in cultured fibroblasts from patients affected with lysosomal storage diseases, we identified differentially expressed genes common to these conditions. These studies, confirmed by biochemical experiments, demonstrated that lysosomal storage is associated with downregulation of ubiquitin C-terminal hydrolase, UCH-L1 in the cells of eight different lysosomal disorders, as well as in the brain of a mouse model of Sandhoff disease. Induction of lysosomal storage by the cysteine protease inhibitor E-64 also reduced UCH-L1 mRNA, protein level and activity. All cells exhibiting lysosomal storage contained ubiquitinated protein aggregates and showed reduced levels of free ubiquitin and decreased proteasome activity. The caspase-mediated apoptosis in E-64-treated fibroblasts was reversed by transfection with a UCH-L1 plasmid, and increased after downregulation of UCH-L1 by siRNA, suggesting that UCH-L1 deficiency and impairment of the ubiquitin-dependent protein degradation pathway can contribute to the increased cell death observed in many lysosomal storage disorders.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Enfermedades por Almacenamiento Lisosomal/metabolismo , ARN/metabolismo , Transducción de Señal , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina/metabolismo , Animales , Apoptosis , Inhibidores de Cisteína Proteinasa/farmacología , Fibroblastos/enzimología , Fibroblastos/metabolismo , Humanos , Leucina/análogos & derivados , Leucina/farmacología , Enfermedades por Almacenamiento Lisosomal/enzimología , Enfermedades por Almacenamiento Lisosomal/genética , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/fisiología , ARN Interferente Pequeño , Piel/citología , Piel/enzimología , Piel/metabolismo , Ubiquitina Tiolesterasa/genéticaRESUMEN
Mechanisms for secondary sustained increase in cerebral blood flow (CBF) during prolonged hypercapnia are unknown. We show that induction of endothelial NO synthase (eNOS) by an increase in prostaglandins (PGs) contributes to the secondary CBF increase during hypercapnic acidosis. Ventilation of pigs with 6% CO(2) (PaCO(2 approximately)65 mm Hg; pH approximately 7.2) caused a approximately 2.5-fold increase in CBF at 30 minutes, which declined to basal values at 3 hours and gradually rose again at 6 and 8 hours; the latter increase was associated with PG elevation, nitrite formation, eNOS mRNA expression, and in situ NO synthase (NOS) reactivity (NADPH-diaphorase staining). Subjecting free-floating brain sections to acidotic conditions increased eNOS expression, the time course of which was similar to that of CBF increase. Treatment of pigs with the cyclooxygenase inhibitor diclofenac or the NOS inhibitor Nomega-nitro-L-arginine blunted the initial rise and prevented the secondary CBF increase during hypercapnic acidosis; neuronal NOS blockers 1-(2-trifluoromethylphenyl) imidazole and 3-bromo-7-nitroindazole were ineffective. Diclofenac abolished the hypercapnia-induced rise in cerebrovascular nitrite production, eNOS mRNA expression, and NADPH-diaphorase reactivity. Acidosis (pH approximately 7.15, PCO(2 approximately )40 mm Hg; 6 hours) produced similar increases in prostaglandin E(2) (PGE(2)) and eNOS mRNA levels in isolated brain microvessels and in NADPH-diaphorase reactivity of brain microvasculature; these changes were prevented by diclofenac, by the receptor-operated Ca(2+) channel blocker SK&F96365, and by the K(ATP) channel blocker glybenclamide. Acidosis increased Ca(2+) transients in brain endothelial cells, which were blocked by glybenclamide and SK&F96365 but not by diclofenac. Increased PG-related eNOS mRNA and NO-dependent vasorelaxation to substance P was detected as well in rat brain exposed to 6 hours of hypercapnia. PGE(2) was the only major prostanoid that modulated brain eNOS expression during acidosis. Thus, in prolonged hypercapnic acidosis, the secondary CBF rise is closely associated with induction of eNOS expression; this seems to be mediated by PGE(2) generated by a K(ATP) and Ca(2+) channel-dependent process.
Asunto(s)
Dinoprostona/metabolismo , Hipercapnia/complicaciones , Hiperemia/etiología , Óxido Nítrico Sintasa/biosíntesis , Canales de Potasio/metabolismo , Acidosis/metabolismo , Animales , Señalización del Calcio/fisiología , Dióxido de Carbono/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Endotelio Vascular/metabolismo , Inducción Enzimática , Técnicas In Vitro , NADPH Deshidrogenasa/metabolismo , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo III , Nitritos/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Flujo Sanguíneo Regional , Porcinos , Factores de TiempoRESUMEN
BACKGROUND: The ductus arteriosus (DA) of newborn infants exposed in utero to indomethacin is resistant to postnatal indomethacin; we hypothesized that this is due to ductus constriction in utero, with subsequent remodeling of the vessel. METHODS AND RESULTS: Infusion of fetal lambs with indomethacin for 48 hours constricted the DA and increased the thickness of the avascular zone of the DA, which in turn induced the expression of vascular endothelial growth factor, endothelial nitric oxide synthase (due to ingrowth of vasa vasorum), neointima formation, and loss of smooth muscle cells; moderate degrees of DA constriction in utero increased NO production, which inhibited DA contractility. Marked degrees of DA constriction decreased tissue distensibility and contractile capacity. CONCLUSIONS: DA patency is no longer controlled primarily by prostaglandins once it has been exposed to indomethacin in utero.
Asunto(s)
Conducto Arterial/anomalías , Conducto Arterial/efectos de los fármacos , Feto/anomalías , Indometacina/farmacología , Ovinos/embriología , 6-Cetoprostaglandina F1 alfa/biosíntesis , Animales , Western Blotting , Muerte Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Circulación Coronaria/efectos de los fármacos , Dinoprostona/biosíntesis , Dinoprostona/farmacología , Conducto Arterial/embriología , Conducto Arterial/metabolismo , Conducto Arterioso Permeable/inducido químicamente , Conducto Arterioso Permeable/enzimología , Conducto Arterioso Permeable/metabolismo , Conducto Arterioso Permeable/patología , Factores de Crecimiento Endotelial/biosíntesis , Feto/irrigación sanguínea , Feto/efectos de los fármacos , Feto/enzimología , Técnicas In Vitro , Linfocinas/biosíntesis , Modelos Biológicos , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo III , Nitroprusiato/farmacología , Presión , Ovinos/anomalías , Túnica Íntima/efectos de los fármacos , Túnica Íntima/patología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
BACKGROUND: Prostaglandin E(2) (PGE(2)) is important in ductus arteriosus (DA) patency, but the types of functional PGE(2) receptors (EP) in the developing DA are not known. We postulated that age-dependent alterations in EP and/or their subtypes may possibly contribute to the reduced responsiveness of the newborn DA to PGE(2). METHODS AND RESULTS: We determined PGE(2) receptor subtypes by competition binding and immunoblot studies on the DA of fetal ( approximately 75% and 90% gestation) and newborn (<45 minutes old) pigs. We studied the effects of EP receptor stimulation on cAMP signaling in vitro and on term newborn (<3 hours old) DA patency in vivo. Fetal pig DA expressed EP(2), EP(3), and EP(4) receptors equivalently, but not EP(1). In neonatal DA, EP(1), EP(3), and EP(4) were undetectable, whereas EP(2) density was similar in fetus and newborn. Prostaglandin-induced changes in cAMP mirrored binding data. 16,16-Dimethyl PGE(2) and 11-deoxy PGE(1) (EP(2)/EP(3)/EP(4) agonist) produced more cAMP in fetus than newborn, but butaprost (selective EP(2) agonist) caused similar cAMP increases in both; EP(3) and EP(4) ligands (M&B28767 and AH23848B, respectively) affected cAMP production only in fetus. After birth, administration of butaprost alone was as effective as 11-deoxy PGE(1) and 16,16-dimethyl PGE(2) in dilating DA in vivo. CONCLUSIONS: The data reveal fewer PGE(2) receptors in the DA of the newborn than in that of the fetus; this may contribute to the decreased responsiveness of the DA to PGE(2) in newborn. Because EP(2) receptors seem to mediate the effects of PGE(2) on the newborn DA, one may propose that a selective EP(2) agonist may be preferred as a pharmacological agent to maintain DA patency in infants with certain congenital heart diseases.
Asunto(s)
Dinoprostona/farmacología , Conducto Arterial/fisiología , Desarrollo Embrionario y Fetal/fisiología , Receptores de Prostaglandina E/clasificación , Receptores de Prostaglandina E/metabolismo , Animales , Animales Recién Nacidos , Unión Competitiva , AMP Cíclico/metabolismo , Dinoprostona/análogos & derivados , Dinoprostona/metabolismo , Conducto Arterial/embriología , Edad Gestacional , Cinética , Ensayo de Unión Radioligante , Transducción de Señal/efectos de los fármacos , Porcinos , Tritio , Grado de Desobstrucción VascularRESUMEN
BACKGROUND: Intravitreal neovascular diseases, as in ischemic retinopathies, are a major cause of blindness. Because inflammatory mechanisms influence vitreal neovascularization and cyclooxygenase (COX)-2 promotes tumor angiogenesis, we investigated the role of COX-2 in ischemic proliferative retinopathy. METHODS AND RESULTS: We describe here that COX-2 is induced in retinal astrocytes in human diabetic retinopathy, in the murine and rat model of ischemic proliferative retinopathy in vivo, and in hypoxic astrocytes in vitro. Specific COX-2 but not COX-1 inhibitors prevented intravitreal neovascularization, whereas prostaglandin E2, mainly via its prostaglandin E receptor 3 (EP3), exacerbated neovascularization. COX-2 inhibition induced an upregulation of thrombospondin-1 and its CD36 receptor, consistent with the observed antiangiogenic effects of COX-2 inhibition; EP3 stimulation reversed effects of COX-2 inhibitors on thrombospondin-1 and CD36. CONCLUSIONS: These findings point to an important role for COX-2 in ischemic proliferative retinopathy, as in diabetes.
Asunto(s)
Retinopatía Diabética/enzimología , Isquemia/enzimología , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Receptores Inmunológicos , Vitreorretinopatía Proliferativa/enzimología , Adulto , Anciano , Animales , Astrocitos/efectos de los fármacos , Astrocitos/enzimología , Astrocitos/patología , Antígenos CD36/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , Ciclooxigenasa 2 , Retinopatía Diabética/complicaciones , Retinopatía Diabética/tratamiento farmacológico , Retinopatía Diabética/patología , Dinoprostona/metabolismo , Modelos Animales de Enfermedad , Factores de Crecimiento Endotelial/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Isquemia/complicaciones , Isquemia/patología , Isoenzimas/antagonistas & inhibidores , Linfocinas/metabolismo , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/patología , Ratas , Ratas Sprague-Dawley , Receptores de Lipoproteína/metabolismo , Receptores de Prostaglandina E/efectos de los fármacos , Receptores de Prostaglandina E/metabolismo , Subtipo EP3 de Receptores de Prostaglandina E , Subtipo EP4 de Receptores de Prostaglandina E , Receptores Depuradores , Retina/efectos de los fármacos , Retina/enzimología , Retina/patología , Vasos Retinianos/efectos de los fármacos , Vasos Retinianos/patología , Trombospondina 1/metabolismo , Factor A de Crecimiento Endotelial Vascular , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Factores de Crecimiento Endotelial Vascular , Vitreorretinopatía Proliferativa/complicaciones , Vitreorretinopatía Proliferativa/tratamiento farmacológicoRESUMEN
In addition to its hemostatic functions, factor (F)VIIa exhibits cell proliferative properties as seen in angiogenesis and tumor growth. A role for tissue factor (TF) and protease-activated receptors (PAR)-1 and -2 in cell proliferation remain to be clarified. We tested the hypothesis that FVIIa induces cell proliferation by a mechanism involving TF and PAR-2. Human recombinant FVIIa induced cell proliferation of human BOSC23 cells transfected with plasmid containing human TF DNA sequence. Because DNA primase 1 (PRIM1) plays an essential role in cell proliferation, we used the cloned PRIM1 promoter upstream of the reporter gene chloramphenicol acetyl transferase (CAT) to elucidate the mode of action of FVIIa. FVIIa evoked a dose-dependent increase in cell proliferation and PRIM1 induction, which were markedly potentiated (4-5-fold) by the presence of TF and abrogated by TF antisense oligonucleotide. PRIM1 induction by FVIIa was also abolished by PAR-2 but not by PAR-1 antisense. In contrast, thrombin induced a small increase in CAT activity which was unaffected by TF, but was prevented only by PAR-1 antisense as well as the thrombin inhibitor hirudin. Proliferative properties of FVIIa were associated with a TF-dependent increase in intracellular calcium and were mediated by a concordant phosphorylation of p44/42 MAP kinase. In conclusion, data reveal that FVIIa induces PRIM1 and ensuing cellular proliferation via a TF- and of the PARs entirely PAR-2-dependent pathway, in distinction to that of thrombin which is PAR-1-dependent and TF-independent. We speculate that FVIIa-TF-PAR-2 inhibitors may be effective in suppressing cell proliferation.
Asunto(s)
Factor VIIa/metabolismo , Receptor PAR-2/metabolismo , Tromboplastina/fisiología , Northern Blotting , Western Blotting , Línea Celular , Proliferación Celular , Cloranfenicol O-Acetiltransferasa/metabolismo , Clonación Molecular , ADN/química , ADN Primasa/metabolismo , Relación Dosis-Respuesta a Droga , Genes Reporteros , Hirudinas/metabolismo , Humanos , Oligonucleótidos Antisentido/química , Fosforilación , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Receptor PAR-1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Trombina/metabolismo , Tromboplastina/metabolismo , Timidina/química , Factores de Tiempo , TransfecciónRESUMEN
The choroid is the main source of oxygen to the retina. In contrast to the adult, the absence of autoregulation of choroidal blood flow in the newborn leads to hyperoxygenation of the retina. In the immature retina which contains relatively low levels of antioxidants this hyperoxygenation favors peroxidation including the generation of biologically active isoprostanes, and results in vasoconstriction and vascular cytotoxicity leading to ischemia, which predisposes to the development of a vasoproliferative retinopathy, commonly termed retinopathy of prematurity. During frequently encountered oxidative stress to the perinate, the combined absence of vascular autoregulation and excessive oxygen delivery to the eyes of the developing subject is largely the result of a complex epigenetic and genetic interplay between prostanoids and nitric oxide (NO) systems on vasomotor regulation. The effects of certain prostaglandins are NO-dependent; conversely, those of NO have also been found to be largely prostaglandin I(2)-mediated in the eye; and NO synthase expression seems to be significantly regulated by other prostaglandins apparently through activation of functional perinuclear prostanoid receptors which affect gene transcription. The increased production of both prostaglandins and NO in the perinate augment ocular blood flow and as a result oxygen delivery to an immature retina partly devoid of antioxidant defenses. The ensuing peroxidation results in impaired circulation (partly thromboxane A(2)-dependent) and vascular integrity, leading to ischemia which predisposes to abnormal preretinal neovascularization, a major feature of ischemic retinopathy. Because tissue oxygenation is largely dependent upon circulation and critical in the generation of reactive oxygen species, and since the latter exert a major contribution in the pathogenesis of retinopathy of prematurity, it is important to understand the mechanisms that govern ocular blood flow. In this review we focus on the important and complex interaction between prostanoid, NO and peroxidation products on circulatory control of the immature retina.
Asunto(s)
Dinoprost/metabolismo , Peroxidación de Lípido , Óxido Nítrico/metabolismo , Estrés Oxidativo , Retina/metabolismo , Retinopatía de la Prematuridad/etiología , Coroides/irrigación sanguínea , Factores de Crecimiento Endotelial/metabolismo , Radicales Libres/metabolismo , Humanos , Recién Nacido , Recien Nacido Prematuro/metabolismo , Isquemia/metabolismo , Linfocinas/metabolismo , Neovascularización Patológica , Nitratos/metabolismo , Óxido Nítrico Sintasa/metabolismo , Oxígeno/metabolismo , Receptores de Prostaglandina E/metabolismo , Vasos Retinianos , Retinopatía de la Prematuridad/metabolismo , Retinopatía de la Prematuridad/fisiopatología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
The relationship between arachidonic acid (AA) mobilization and transcription of immediate-early genes, particularly of prostaglandin G/H synthase-2 (PGHS-2), in intestinal crypt epithelial cells was analyzed. PGHS-2 mRNA and protein synthesis were stimulated by its own substrate, AA; actinomycin D, a transcription inhibitor, prevented the AA-induced increase in PGHS-2 mRNA. Eicosatetraynoic acid, an inhibitor of AA utilization, significantly reduced PGHS-2 mRNA synthesis elicited by AA. Inhibitors of cytochrome P450 monoxygenases, ketoconazole and miconazole, also prevented PGHS-2 mRNA synthesis in a dose-dependent manner. Phenyl chalcone oxide, an epoxide hydrolase inhibitor, potentiated AA-induced PGHS-2 mRNA synthesis. Of the four regioisomers of arachidonic acid epoxides, only 14,15-epoxyeicosatrienoic acid elicited the expression of PGHS-2 in intestinal crypt epithelial cells. This is the first direct evidence of stimulation of an immediate-early gene product, specifically PGHS-2, by an AA epoxygenase metabolite, 14,15-epoxyeicosatrienoic acid, as well as of a heterologous regulation of PGHS-2 synthesis by these monoxygenase products.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Hidrocarburo de Aril Hidroxilasas , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Isoenzimas/biosíntesis , Oxigenasas de Función Mixta , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Transcripción Genética/efectos de los fármacos , Ácido 5,8,11,14-Eicosatetrainoico/farmacología , Ácido 8,11,14-Eicosatrienoico/farmacología , Animales , Línea Celular , Ciclooxigenasa 2 , Familia 2 del Citocromo P450 , Dactinomicina/farmacología , Mucosa Intestinal , Isomerismo , Cinética , Oxigenasas/metabolismo , ARN Mensajero/biosíntesis , RatasRESUMEN
Primary cultures of oligodendrocytes were used to study the toxic effects of cadmium chloride. Cell viability was evaluated by the mitochondrial dehydrogenase activity and confirmed by propidium iodide (PI) fluorescence staining. The expression of the 72 kDa stress protein, HSP72, was assayed by Western blot analysis. The results showed that Cd(2+)-induced toxicity was dependent on the time and dose of exposure, as well as on the developmental stage of the cultures. Oligodendrocyte progenitors were more vulnerable to Cd(2+) toxicity than were mature oligodendrocytes. Mature oligodendrocytes accumulated relatively higher levels of Cd(2+) than did progenitors, as determined by (109)CdCl(2) uptake; treatment with the metal ion caused a more pronounced reduction in intracellular glutathione levels and significantly higher free radical accumulation in progenitors. The latter could explain the observed differences in Cd(2+) susceptibility. HSP72 protein expression was increased both in progenitors and in mature cells exposed to Cd(2+). Pretreatment with N-acetylcysteine, a thiocompound with antioxidant activity and a precursor of glutathione, prevented Cd(2+)-induced (i) reduction in glutathione levels and (ii) induction of HSP72 and diminished (i) Cd(2+) uptake and (ii) Cd(2+)-evoked cell death. In contrast, buthionine sulfoximine, an inhibitor of gamma-glutamyl-cysteine synthetase, depleted glutathione, and potentiated the toxic effect of Cd(2+). These results strongly suggest that Cd(2+)-induced cytotoxicity in oligodendrocytes is mediated by reactive oxygen species and is modulated by glutathione levels.
Asunto(s)
Cadmio/toxicidad , Proteínas de Choque Térmico/biosíntesis , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Acetilcisteína/farmacología , Animales , Butionina Sulfoximina/farmacología , Muerte Celular/efectos de los fármacos , Diferenciación Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Radicales Libres/metabolismo , Glutatión/análogos & derivados , Glutatión/metabolismo , Glutatión/farmacología , Proteínas del Choque Térmico HSP72 , Oligodendroglía/citología , Estrés Oxidativo/efectos de los fármacos , RatasRESUMEN
The newborn retina is particularly sensitive and frequently subjected to peroxidative stresses that result in visual sequelae. We compared two iron chelators, deferoxamine and a newer compound, pyridoxal isonicotinoyl hydrazone (PIH), in protecting the retina of newborn pigs (1-3 d old) from asphyxia-reoxygenation insults. Animals were treated IV with either saline, deferoxamine 15.2 mumol/kg (10 mg/kg) or PIH 34.8 mumol/kg (10 mg/kg); n = 10 in each treatment group. Scotopic and photopic electroretinograms (ERG) were recorded before and 40 min after drug treatment as well as 45 min following a 5-min period of asphyxia by interrupting ventilation. In separate animals the indices of peroxidation, malondialdehyde (MDA: TBARS) and hydroperoxides, were measured in retina at the same times. In saline-treated animals, there was a marked increase in MDA and hydroperoxide concentrations in the retina following the asphyxia-reoxygenation period. This was associated with a decrease in the a- (photoreceptor generated) and b-wave (generated by Müller and bipolar cells) amplitudes measured under photopic (cone-mediated response) and scotopic (rod-mediated response) conditions, and an increase in their implicit times. PIH and deferoxamine prevented the postasphyxial increase in MDA and hydroperoxides. However, only PIH prevented the postasphyxial changes in a- and b-wave amplitudes and implicit times, whereas deferoxamine markedly altered the preasphyxial ERG and provided only partial postasphyxial protection simply to the retinal outer segment. Our findings indicate that the iron chelator PIH effectively inhibits peroxidation and retinal electrophysiological alterations secondary to asphyxia-reoxygenation-induced oxidative stresses to newborn animals, whereas deferoxamine adversely affects retinal function; hence, PIH may be a preferred alternative to deferoxamine.
Asunto(s)
Asfixia Neonatal/tratamiento farmacológico , Quelantes del Hierro/uso terapéutico , Isoniazida/análogos & derivados , Estrés Oxidativo/efectos de los fármacos , Piridoxal/análogos & derivados , Retina/efectos de los fármacos , Trastornos de la Visión/prevención & control , Animales , Animales Recién Nacidos , Asfixia Neonatal/complicaciones , Deferoxamina/uso terapéutico , Modelos Animales de Enfermedad , Electrorretinografía , Humanos , Peróxido de Hidrógeno/metabolismo , Recién Nacido , Isoniazida/uso terapéutico , Malondialdehído/metabolismo , Peróxidos/metabolismo , Piridoxal/uso terapéutico , Retina/metabolismo , PorcinosRESUMEN
Prostaglandin G/H synthase (PGHS) has been shown to generate peroxides to a significant extent in the retina and absorbs light at the lower end of the visible spectrum. We postulated that PGHS could be an important initial source of peroxidation in the retina exposed to light, which would in turn alter retinal function. Exposure of pig eyes (in vivo) to light (350 fc/3770 lx) caused after 3 h a 50% increase and by 5 h a 30% decrease in a- and b-wave amplitudes of the electroretinogram (ERG) which were comparable at 380-650 nm and 380-440 nm but were not observed at wavelengths > 450 nm. These effects of light were prevented by free radical scavengers (dimethylthiourea and high-dose allopurinol) and PGHS inhibitors (naproxen and diclofenac), but stable analogs of prostaglandins did not affect the ERG. Both increases and subsequent decreases in ERG wave amplitudes following light exposure in vivo were associated with increases in retinal prostaglandin and malondialdehyde (peroxidation product) levels, which were inhibited by the nonselective PGHS blockers, naproxen and diclofenac. Similar observations were made in vitro on isolated porcine eyecups as well as on retinal membranes exposed to light (250 fc/ 2700 lx) 380-650 nm and 380-440 nm but not at > 500 nm. Both PGHS-1 and PGHS-2 contributed equivalently to light-induced prostaglandin synthesis, as shown after selective PGHS-2 blockers, but mRNA expression of PGHS-1 and 2 was not affected by light. Finally, light stimulated activities of pure PGHS-1 and PGHS-2 isozymes, and these were also shown to produce superoxide radical (detected with fluorogenic spin trap, proxyl fluorescamine). Taken together, data suggest that PGHS- (1 and 2) is activated by short wavelength visible light, and in the retina is an important source of reactive oxygen species which in turn alter retinal electrophysiological function. PGHS thus seems a likely chromophore in setting forth photic-induced retinal injury. Findings provide an explanation for increased sensitivity of the retina to visible light predominantly at the far blue range of its spectrum.
Asunto(s)
Luz/efectos adversos , Peróxidos/metabolismo , Prostaglandina-Endoperóxido Sintasas/efectos de la radiación , Retina/enzimología , Retina/efectos de la radiación , Animales , Electrorretinografía/efectos de la radiación , Activación Enzimática/efectos de la radiación , Estabilidad de Enzimas/efectos de la radiación , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/efectos de la radiación , Peroxidación de Lípido/efectos de la radiación , Malondialdehído/metabolismo , Malondialdehído/efectos de la radiación , Membranas/enzimología , Membranas/efectos de la radiación , Peróxidos/efectos adversos , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandina-Endoperóxido Sintasas/metabolismo , Prostaglandinas/metabolismo , Prostaglandinas/efectos de la radiación , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Mensajero/efectos de la radiación , Retina/metabolismo , Superóxidos/metabolismo , Superóxidos/efectos de la radiación , PorcinosRESUMEN
To assess whether prostaglandins contribute to the control of basal retinal and choroidal hemodynamics, retinal (RBF) and choroidal blood flow (ChBF) were measured by a microsphere technique in 1- to 4-day-old pigs before and after (at 20 and 60 min) administration of indomethacin (0.3 mg/kg, n = 6 or 10 mg/kg, n = 5), ibuprofen (40 mg/kg, n = 7), naproxen (20 mg/kg, n = 5) or vehicle (n = 8). In 40 other animals, PGF2 alpha, PGE2, and 6-keto-PGF1 alpha were measured in the retina and choroid at times corresponding to blood flow measurements. Mean arterial blood pressure and blood gases and pH were not altered by any of the agents. Except for the lower dose of indomethacin (0.3 mg/kg), which did not change retinal and choroidal prostaglandin concentrations, the prostaglandin levels were decreased similarly (P < 0.01) by the three drugs. However, RBF and ChBF were not changed by ibuprofen and naproxen, but decreased to the same extent after low and high doses of indomethacin. The data suggest that the effects of indomethacin on RBF and ChBF cannot be simply attributed to prostaglandin synthesis inhibition, and that prostaglandins may not play a significant role in controlling basal blood flow to the retina and choroid.
Asunto(s)
Animales Recién Nacidos/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Coroides/metabolismo , Prostaglandinas/metabolismo , Retina/metabolismo , Vasos Retinianos/fisiología , Animales , Animales Recién Nacidos/sangre , Coroides/irrigación sanguínea , Ibuprofeno/farmacología , Indometacina/farmacología , Naproxeno/farmacología , Concentración Osmolar , Flujo Sanguíneo Regional/efectos de los fármacos , Vasos Retinianos/efectos de los fármacos , PorcinosRESUMEN
PURPOSE: Newborn rats exposed to hyperoxia during the first days of life have been shown to exhibit not only vasculopathy but also permanent changes in the structure and function of the retina. Given that the rat retina is immature at birth and that the maturation process continues until the opening of the eyes at 14 days of life, this study was conducted to investigate the susceptibility of the retina to oxygen toxicity as a function of the degree of retinal maturity reached at the time of oxygen exposure. METHODS: Newborn rats were exposed to hyperoxia during selected postnatal day intervals. Scotopic electroretinograms were recorded at 30 and 60 days of age, and retinal histology was obtained at the end of the study. RESULTS: There was a strong correlation between the duration of the hyperoxic event and the structural and functional consequences in the retina. However, the repercussions were significantly more profound when the exposure to oxygen occurred within the second week of life (6-14 days), compared with earlier (0-6 days) or later periods (14-28 days). CONCLUSIONS: The results strongly suggest that the structural and functional retinal changes secondary to postnatal hyperoxia are not only the direct consequence of exposure to high levels of oxygen (i.e., free radicals), but also are determined by the level of retinal maturity reached at the time of oxygen exposure. The results also indicate that the structural anomalies precede the functional impairments.
Asunto(s)
Hiperoxia/complicaciones , Retina/crecimiento & desarrollo , Retinopatía de la Prematuridad/etiología , Animales , Animales Recién Nacidos , Electrorretinografía , Humanos , Hiperoxia/patología , Recién Nacido , Oxígeno/toxicidad , Ratas , Ratas Sprague-Dawley , Retina/efectos de los fármacos , Retina/fisiopatología , Retinopatía de la Prematuridad/patología , Retinopatía de la Prematuridad/fisiopatología , Factores de TiempoRESUMEN
The role of prostanoids in setting the range of autoregulation of retinal blood flow (RBF) and choroidal blood flow (ChBF) in the newborn was assessed. The RBF, ChBF, and arterial and cerebral sinus concentrations of PGE, PGF2 alpha, 6-keto-PGF1 alpha and TXB2 were measured over a wide range of mean systemic blood pressure (blood pressure (BP): 17-117 mm Hg) in newborn piglets treated with ibuprofen (30 mg/kg iv) or its vehicle (n = 8, in each group). Hypertension and hypotension were induced 80 min apart on each animal, by inflating balloon-tipped catheters placed at the aortic isthmus and root, respectively. Blood flow and prostanoid concentrations were measured 20 min before (basal) and during the induced changes in BP. In vehicle-treated piglets, RBF did not change with BP between 50 and 90 mm Hg (r = 0.33, P = 0.27), but changed as a function of BP beyond this range (tau = 0.52, P less than 0.01); ChBF increased with BP throughout the range studied (17-117 mm Hg; tau = 0.89, P less than 0.001). The relationship between O2 delivery to the retina and choroid and BP (tau greater than 0.43, P less than 0.01) was similar to that seen between RBF and ChBF with BP. The concentration of all prostanoids increased when BP was reduced to less than 50 mm Hg. When BP was raised to more than 90 mm Hg, prostaglandin concentrations increased, and those of TXB2 did not change. Ibuprofen treatment reduced the basal concentrations of all prostanoids to nearly undetectable levels and prevented their changes during hypotension and hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Coroides/irrigación sanguínea , Homeostasis/efectos de los fármacos , Ibuprofeno/farmacología , Vasos Retinianos/fisiopatología , Animales , Animales Recién Nacidos , Presión Sanguínea , Coroides/metabolismo , Hipertensión/metabolismo , Hipertensión/fisiopatología , Hipotensión/metabolismo , Hipotensión/fisiopatología , Prostaglandinas/sangre , Flujo Sanguíneo Regional , Vasos Retinianos/metabolismo , Porcinos , Resistencia Vascular/efectos de los fármacosRESUMEN
PURPOSE: To study the role of free radicals in autoregulation of retinal blood flow (RBF) and choroidal blood flow (ChBF) and the contribution of the cyclooxygenase pathway in free radical formation during blood pressure changes in 1- to 3-day-old pigs. METHODS: Blood pressure was adjusted by inflating balloon-tipped catheters placed at the aortic isthmus and the aortic root to induce hypertension and hypotension, respectively. Blood flow was measured using the microsphere technique. Also, the effects of peroxides on retinal artery diameter were studied on eyecup preparations using time-frame photography processed by digital imaging. RESULTS: Blood gases and intraocular pressure (13 +/- 1 mm Hg) remained stable throughout the experiments. In control animals, RBF was constant only between 30 and 75 mm Hg of ocular perfusion pressure and ChBF increased as a function of ocular perfusion pressure (tau = 0.58, P < 0.01). Inhibition of peroxidation with the free radical scavenger 21-aminosteroid U74389F (2.5 mg/kg) widened the range of RBF and ChBF autoregulation (ocular perfusion pressure from 30 to 131 mm Hg). Hypertension caused an increase in the products of peroxidation, malondialdehyde, and hydroperoxides, as well as in prostaglandin E2, prostaglandin F2 alpha, and 6-keto-prostaglandin F1 alpha in the retina and choroid of control animals; these changes were inhibited by the free radical scavengers U74389F (2.5 mg/kg) and high-dose allopurinol (140 mg/kg) as well as by the cyclooxygenase inhibitors ibuprofen (40 mg/kg) and indomethacin (5 mg/kg). In isolated eyecup preparations, H2O2 and cumene hydroperoxide dilated retinal vessels, and this effect was completely blocked by indomethacin. CONCLUSIONS: These findings indicate that free radicals play a major role in setting the upper limit of RBF and ChBF autoregulation of the newborn animal. In addition, there exists a positive feedback interaction between free radicals and cyclooxygenase activity in ocular tissues, such that during hypertension the cyclooxygenase pathway is an important producer of free radicals and in turn is also activated by them.
Asunto(s)
Coroides/irrigación sanguínea , Homeostasis/fisiología , Prostaglandinas/metabolismo , Vasos Retinianos/fisiología , Animales , Antiinflamatorios no Esteroideos/farmacología , Antioxidantes/farmacología , Circulación Sanguínea/fisiología , Radicales Libres/metabolismo , Hipertensión Ocular/fisiopatología , Peróxidos/metabolismo , Pregnatrienos/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Flujo Sanguíneo Regional/fisiología , Porcinos , VasodilataciónRESUMEN
PURPOSE: To determine the relative contribution of cyclooxygenase (COX)-1 and COX-2 in regulating prostaglandin (PG) E2 and PGF2alpha receptors (EP and FP, respectively) densities and their functions in retinal vasculature of neonatal pigs. METHODS: Newborn pigs were treated intravenously every 8 hours for 48 hours with saline, 40 mg/kg nonselective COX inhibitor ibuprofen, 80 mg/kg COX-1 inhibitor valeryl salicylate, or 5 mg/kg DuP697 and 5 mg/kg NS398, COX-2 inhibitors. Retinal microvessel EP and FP receptor densities were measured by radioligand binding and receptor-coupled effects by determining second-messenger inositol 1,4,5-trisphosphate (IP3) and vasomotor responses. Retinal blood flow (RBF) response to incremental increases in blood pressure (BP) was measured by a microsphere technique. RESULTS: Valeryl salicylate, DuP697, and NS398 reduced retinal PGE2 and PGF2alpha concentrations in the newborn by approximately half, whereas ibuprofen caused further reduction to levels observed in adults. Retinal vessel EP1, EP3, and FP receptor densities increased approximately threefold after treatments with COX-1 or COX-2 inhibitors, and five- to sixfold after ibuprofen treatment. EP and FP receptor upregulation was associated with corresponding increases in IP3 production and retinal vasoconstriction in response to PGF2alpha, fenprostalene (an FP agonist), PGE2, 17-phenyl trinor PGE2 (an EP1 agonist), and M&B28,767 (an EP3 agonist) and with enhanced RBF autoregulation of high BP (> or =125 mm Hg). Conversely, EP2 receptor density and coupled functions were minimally affected by COX inhibition. CONCLUSIONS: Data suggest that increased COX-1- and COX-2-catalyzed prostaglandin synthesis contribute equivalently to the downregulation of retinovascular EP1, EP3, and FP receptors and their vasoconstrictor functions in newborn pigs; the EP2 receptor was not significantly influenced by ontogenic alterations in prostaglandin levels.