RESUMEN
As understanding of the mechanisms driving and regulating insulin secretion from pancreatic beta cells grows, there is increasing and compelling evidence that nitric oxide (â¢NO) and other closely-related reactive nitrogen species (RNS) play important roles in this exocytic process. â¢NO and associated RNS, in particular peroxynitrite, possess the capability to effect signals across both intracellular and extracellular compartments in rapid fashion, affording extraordinary signaling potential. It is well established that nitric oxide signals through activation of guanylate cyclase-mediated production of cyclic GMP. The intricate intracellular redox environment, however, lends credence to the possibility that â¢NO and peroxynitrite could interact with a wider variety of biological targets, with two leading mechanisms involving 1) Snitrosylation of cysteine, and 2) nitration of tyrosine residues comprised within a variety of proteins. Efforts aimed at delineating the specific roles of â¢NO and peroxynitrite in regulated insulin secretion indicate that a highly-complex and nuanced system exists, with evidence that â¢NO and peroxynitrite can contribute in both positive and negative regulatory ways in beta cells. Furthermore, the ultimate biochemical outcome within beta cells, whether to compensate and recover from a given stress, or not, is likely a summation of contributory signals and redox status. Such seeming regulatory dichotomy provides ample opportunity for these mechanisms to serve both physiological and pathophysiologic roles in onset and progression of diabetes. This review focuses attention upon recent accumulating evidence pointing to roles for nitric oxide induced post-translational modifications in the normal regulation as well as the dysfunction of beta cell insulin exocytosis.
Asunto(s)
Cisteína/metabolismo , Diabetes Mellitus/metabolismo , Exocitosis/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Óxido Nítrico/metabolismo , Ácido Peroxinitroso/metabolismo , Tirosina/metabolismo , Apoptosis , GMP Cíclico/metabolismo , Cisteína/farmacología , Femenino , Humanos , Masculino , Oxidación-Reducción , Ácido Peroxinitroso/farmacología , Procesamiento Proteico-Postraduccional , Transducción de Señal , Tirosina/farmacologíaRESUMEN
The plasma membrane soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein syntaxin (Syn)4 is required for biphasic insulin secretion, although how it regulates each phase remains unclear. In a screen to identify new Syn4-interacting factors, the calcium-activated F-actin-severing protein gelsolin was revealed. Gelsolin has been previously implicated as a positive effector of insulin secretion, although a molecular mechanism to underlie this function is lacking. Toward this, our in vitro binding studies showed the Syn4-gelsolin interaction to be direct and mediated by the N-terminal Ha domain (amino acid residues 39-70) of Syn4. Syn4-gelsolin complexes formed under basal conditions and dissociated upon acute glucose or KCl stimulation; nifedipine blocked dissociation. The dissociating action of secretagogues could be mimicked by expression of the N-terminal Ha domain of Syn4 fused to green fluorescent protein (GFP) (GFP-39-70). Furthermore, GFP-39-70 expression in isolated mouse islet and clonal MIN6 ß-cells initiated insulin release in the absence of appropriate stimuli. Consistent with this, the inhibitory GFP-39-70 peptide also initiated Syn4 activation in the absence of stimuli. Moreover, although MIN6 ß-cells expressing the GFP-39-70 peptide maintained normal calcium influx in response to KCl, KCl-stimulated insulin secretion and the triggering pathway of insulin secretion were significantly impaired. Taken together, these data support a mechanistic model for gelsolin's role in insulin exocytosis: gelsolin clamps unsolicited soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE)-regulated exocytosis through direct association with Syn4 in the absence of appropriate stimuli, which is relieved upon stimulus-induced calcium influx to activate gelsolin and induce its dissociation from Syn4 to facilitate insulin exocytosis.
Asunto(s)
Exocitosis , Gelsolina/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas Qa-SNARE/metabolismo , Vesículas Secretoras/metabolismo , Animales , Transporte Biológico , Calcio/metabolismo , Línea Celular , Membrana Celular/metabolismo , Glucosa/farmacología , Proteínas Fluorescentes Verdes , Secreción de Insulina , Ratones , Nifedipino/farmacología , Cloruro de Potasio/farmacología , Proteínas SNARE/metabolismoRESUMEN
Glucose-stimulated insulin release from pancreatic islet ß-cells involves increased levels of reactive oxygen and nitrogen species. Although this is normal, under pathophysiological conditions such as chronic hyperglycemia and inflammation, insulin exocytosis fails, and yet the mechanistic reason for failure is unclear. Hypothesizing that exocytotic proteins might be targets of S-nitrosylation, with their dysfunction under conditions of nitrosative stress serving as a mechanistic basis for insulin secretory dysfunction, we identified the t-SNARE protein Syntaxin 4 as a target of modification by S-nitrosylation. The cellular content of S-nitrosylated Syntaxin 4 peaked acutely, within 5 min of glucose stimulation in both human islets and MIN6 ß-cells, corresponding to the time at which Syntaxin 4 activation was detectable. S-Nitrosylation was mapped to Syntaxin 4 residue Cys(141), located within the Hc domain predicted to increase accessibility for v-SNARE interaction. A C141S-Syntaxin 4 mutant resisted S-nitrosylation induced in vitro by the nitric oxide donor compound S-nitroso-L-glutathione, failed to exhibit glucose-induced activation and VAMP2 binding, and failed to potentiate insulin release akin to that of wild-type Syntaxin 4. Strikingly, S-nitrosylation of Syntaxin 4 could be induced by acute treatment with inflammatory cytokines (TNFα, IL-1ß, and IFNγ), coordinate with inappropriate Syntaxin 4 activation and insulin release in the absence of the glucose stimulus, consistent with nitrosative stress and dysfunctional exocytosis, preceding the cell dysfunction and death associated with more chronic stimulation (24 h). Taken together, these data indicate a significant role for reactive nitrogen species in the insulin exocytosis mechanism in ß-cells and expose a potential pathophysiological exploitation of this mechanism to underlie dysfunctional exocytosis.
Asunto(s)
Exocitosis , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Qa-SNARE/metabolismo , Vesículas Secretoras/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Línea Celular , Glucosa/farmacología , Humanos , Insulina/genética , Secreción de Insulina , Células Secretoras de Insulina/patología , Ratones , Estructura Terciaria de Proteína , Proteínas Qa-SNARE/genética , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Vesículas Secretoras/genética , Vesículas Secretoras/patología , Edulcorantes/farmacología , Proteína 2 de Membrana Asociada a Vesículas/genética , Proteína 2 de Membrana Asociada a Vesículas/metabolismoRESUMEN
Evidence suggests that estrogen mediates rapid endothelial nitric oxide synthase (eNOS) activation via estrogen receptor-a (ERalpha) within the plasma membrane of endothelial cells (EC). ERalpha is known to colocalize with caveolin 1, the major structural protein of caveolae, and caveolin 1 stimulates the translocation of ERalpha to the plasma membrane. However, the role played by caveolin 1 in regulating 17beta-estradiol-mediated NO signaling in EC has not been adequately resolved. Thus, the purpose of this study was to explore how 17beta-estradiol stimulates eNOS activity and the role of caveolin 1 in this process. Our data demonstrate that modulation of caveolin 1 expression using small interfering RNA or adenoviral gene delivery alters ERalpha localization to the plasma membrane in EC. Further, before estrogen stimulation ERalpha associates with caveolin 1, whereas stimulation promotes a pp60(Src)-mediated phosphorylation of caveolin 1 at tyrosine 14, increasing ERalpha-PI3 kinase interactions and disrupting caveolin 1-ERalpha interactions. Adenoviral mediated overexpression of a phosphorylation-deficient mutant of caveolin (Y14FCav) attenuated the ERalpha/PI3 kinase interaction and prevented Akt-mediated eNOS activation. Furthermore, Y14FCav overexpression reduced eNOS phosphorylation at serine1177 and decreased NO generation after estrogen exposure. Using a library of overlapping peptides we identified residues 62-73 of caveolin 1 as the ERalpha-binding site. Delivery of a synthetic peptide based on this sequence decreased ERalpha plasma membrane translocation and reduced estrogen-mediated activation of eNOS. In conclusion, caveolin 1 stimulates 17beta-estradiol-induced NO production by promoting ERalpha to the plasma membrane, which facilitates the activation of the PI3 kinase pathway, leading to eNOS activation and NO generation.
Asunto(s)
Caveolina 1/metabolismo , Estradiol/farmacología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Animales , Caveolina 1/genética , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Receptor alfa de Estrógeno/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/metabolismo , Ovinos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
To understand how microtubules contribute to the dynamic reorganization of the endothelial cell (EC) cytoskeleton, we established an EC model expressing EB3-GFP, a protein that marks microtubule plus-ends. Using this model, we were able to measure microtubule growth rate at the centrosome region and near the cell periphery of a single human EC and in the EC monolayer. We demonstrate that the majority of microtubules in EC are dynamic, the growth rate of their plus-ends is highest in the internal cytoplasm, in the region of the centrosome. Growth rate of microtubule plus-ends decreases from the cell center toward the periphery. Our data suggest the existing mechanism(s) of local regulation of microtubule plus-ends growth in EC. Microtubule growth rate in the internal cytoplasm of EC in the monolayer is lower than that of single EC suggesting the regulatory effect of cell-cell contacts. Centrosomal microtubule growth rate distribution in single EC indicated the presence of two subpopulations of microtubules with "normal" (similar to those in monolayer EC) and "fast" (three times as much) growth rates. Our results indicate functional interactions between cell-cell contacts and microtubules.
Asunto(s)
Células Endoteliales/fisiología , Microtúbulos/fisiología , Modelos Biológicos , Línea Celular , Centrosoma/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía Fluorescente , Microscopía por Video , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Acute partial compression of the fetal ductus arteriosus (DA) results in an initial abrupt increase in pulmonary blood flow (PBF), which is followed by a significant reduction in PBF to baseline values over the ensuing 2-4 h. We have previously demonstrated that this potent vasoconstricting response is due, in part, to an endothelin-1 (ET-1)-mediated decrease in nitric oxide synthase (NOS) activity. In addition, in vitro data demonstrate that ET-1 increases superoxide levels in pulmonary arterial smooth muscle cells and that oxidative stress alters NOS activity. Therefore, the objectives of this study were to determine the potential role of superoxide in the alterations of hemodynamics and NOS activity following acute ductal constriction in the late-gestation fetal lamb. Eighteen anesthetized near-term fetal lambs were instrumented, and a lung biopsy was performed. After a 48-h recovery, acute constriction of the DA was performed by inflating a vascular occluder. Polyethylene glycol-superoxide dismutase (PEG-SOD; 1,000-1,500 units/kg, n = 7) or PEG-alone (vehicle control group, n = 5) was injected into the pulmonary artery before ductal constriction. Six animals had a sham operation. In PEG-alone-treated lambs, acute ductal constriction rapidly decreased pulmonary vascular resistance (PVR) by 88%. However, by 4 h, PVR returned to preconstriction baseline. This vasoconstriction was associated with an increase in lung superoxide levels (82%), a decrease in total NOS activity (50%), and an increase in P-eNOS-Thr495 (52%) (P < 0.05). PEG-SOD prevented the increase of superoxide after ductal constriction, attenuated the vasoconstriction, preserved NOS activity, and increased P-eNOS Ser1177 (307%, P < 0.05). Sham procedure induced no changes. These data suggest that an acute decrease in NOS activity that is mediated, in part, by increased superoxide levels, and alterations in the phosphorylation status of the endothelial NOS isoform, underlie the pulmonary vascular response to acute ductal constriction.
Asunto(s)
Conducto Arterial/fisiología , Pulmón/irrigación sanguínea , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico Sintasa/metabolismo , Superóxidos/metabolismo , Vasoconstricción , Animales , Feto/metabolismo , Pulmón/embriología , Ovinos , Resistencia Vascular/efectos de los fármacosRESUMEN
Although alterations in ROS generating systems are well described in several vascular disorders, there is very limited information on the perinatal regulation of these systems in the lung both during normal development and in pulmonary hypertension. Thus, this study was undertaken to explore how the two predominant superoxide generating systems, nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) and xanthine oxidase (XO), are developmentally regulated in control lambs and in our established lamb model of increased pulmonary blood flow (Shunt) over the first 2months of life. We found that the levels of p47(phox), p67(phox), and Rac1 subunits of NADPH oxidase complex were altered. During the first two months of life there was no change in p47(phox) protein levels in either normal or Shunt lambs. However, both p67(phox) and Rac1 protein levels decreased over time. In addition, p47(phox) protein levels were significantly increased in shunt lambs at 2- and 4-, but not 8-weeks of age compared to age-matched controls while levels of the p67(phox) subunit were decreased at 8-weeks of age in the Shunts but unchanged at other time periods. Furthermore, Rac1 protein expression was significantly increased in the Shunts only at 4weeks of age. These data correlated with a significant increase in NADPH oxidase dependent superoxide generation at 2- and 4-, but not 8-weeks of age in the Shunts. During normal development XO levels significantly increased over time in normal lambs but significantly decreased in the Shunts. In addition, XO protein levels were significantly increased in the Shunt at 2- and 4-weeks of age but significantly decreased at 8-weeks. Again this correlated with a significant increase in XO dependent superoxide generation at 2- and 4-, but not 8-weeks of age in the Shunts. Collectively, our findings suggest that NADPH oxidase and XO are major contributors to superoxide generation both during the normal development and during the development of pulmonary hypertension.
Asunto(s)
Pulmón/metabolismo , NADPH Oxidasas/metabolismo , Superóxidos/metabolismo , Xantina Oxidasa/metabolismo , Factores de Edad , Animales , Femenino , Hipertensión Pulmonar/enzimología , Pulmón/enzimología , Pulmón/crecimiento & desarrollo , Embarazo , Circulación Pulmonar , Ovinos , Factores de Tiempo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Previous studies have shown that acute increases in oxidative stress induced by the addition of hydrogen peroxide (H(2)O(2)) can increase endothelial nitric oxide synthase (eNOS) catalytic activity via an increase in the phosphorylation of eNOS at serine 1177. However, it is unclear how increased H(2)O(2) affects nitric oxide (NO) signaling when endothelial cells are exposed to biomechanical forces. Thus, the purpose of this study was to evaluate the acute effects of H(2)O(2) on NO signaling in the presence or absence of laminar shear stress. We found that acute sustained increases in cellular H(2)O(2) levels in bovine aortic endothelial cells did not alter basal NO generation but the NO produced in response to shear stress was significantly increased. This amplification in NO signaling was found to correlate with an H(2)O(2)-induced increase in eNOS localized to the plasma membrane and an increase in total caveolin-1 protein levels. We further demonstrated that overexpressing caveolin-1 increased eNOS localized to the plasma membrane again without altering total eNOS protein levels. We also found that caveolin-1 overexpression increased NO generation in response to shear stress but only in the presence of H(2)O(2). Conversely, depleting caveolin-1 with an siRNA decreased eNOS localized to the plasma membrane and abolished the enhanced NO generation. Finally, we found that expressing a caveolin-1 binding-site deletion mutant of eNOS in COS-7 cells decreased its plasma membrane localization and resulted in attenuated NO production in response to calcium activation. In conclusion, we have identified a new role for caveolin-1 in enhancing eNOS trafficking to the plasma membrane that seems to be involved in priming eNOS for flow-mediated activation under conditions of oxidative stress. To our knowledge, this is the first report that H(2)O(2) modulates eNOS activity by altering its subcellular location and that caveolin-1 can play a stimulatory role in NO signaling.
Asunto(s)
Caveolina 1/metabolismo , Endotelio Vascular/efectos de los fármacos , Activación Enzimática , Peróxido de Hidrógeno/farmacología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Resistencia al Corte , Animales , Sitios de Unión/genética , Células COS , Bovinos , Caveolina 1/genética , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Chlorocebus aethiops , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Activación Enzimática/efectos de los fármacos , Óxido Nítrico/genética , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , ARN Interferente Pequeño/genética , Eliminación de Secuencia/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transgenes/genéticaRESUMEN
Zinc is the second-most abundant transition metal within cells and an essential micronutrient. Although adequate zinc is essential for cellular function, intracellular free zinc (Zn(2+)) is tightly controlled, as sustained increases in free Zn(2+) levels can directly contribute to apoptotic endothelial cell death. Moreover, exposure of endothelial cells to acute nitrosative and/or oxidative stress induces a rapid rise of Zn(2+) with mitochondrial dysfunction and the initiation of apoptosis. This apoptotic induction can be mimicked through addition of exogenous ZnCl(2) and mitigated by zinc-chelation strategies, indicating Zn(2+)-dependent mechanisms in this process. However, the molecular mechanisms of Zn(2+)-mediated mitochondrial dysfunction are unknown. Here we report that free Zn(2+) disrupts cellular redox status through inhibition of glutathione reductase, and induces apoptosis by redox-mediated inhibition of the mitochondrial adenine nucleotide transporter (ANT). Inhibition of ANT causes increased mitochondrial oxidation, loss of ADP uptake, mitochondrial translocation of bax, and apoptosis. Interestingly, pre-incubation with glutathione ethyl ester protects endothelial cells from these observed effects. We conclude that key mechanisms of Zn(2+)-mediated apoptotic induction include disruption of cellular glutathione homeostasis leading to ANT inhibition and decreases in mitochondrial ATP synthesis. These pathways could represent novel therapeutic targets during acute oxidative or nitrosative stress in cells and tissues.
Asunto(s)
Apoptosis/efectos de los fármacos , Cloruros/farmacología , Células Endoteliales/efectos de los fármacos , Glutatión/antagonistas & inhibidores , Translocasas Mitocondriales de ADP y ATP/metabolismo , Compuestos de Zinc/farmacología , Adenosina Difosfato/metabolismo , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Endoteliales/citología , Células Endoteliales/metabolismo , Glutatión/metabolismo , Ovinos , Relación Estructura-ActividadRESUMEN
Previous studies demonstrate impaired nitric oxide (NO) signaling in children and animal models with congenital heart defects and increased pulmonary blood flow. However, the molecular mechanisms underlying these alterations remain incompletely understood. The purpose of this study was to determine if early changes in arginine metabolic pathways could play a role in the reduced NO signaling demonstrated in our lamb model of congenital heart disease with increased pulmonary blood flow (Shunt lambs). The activities of the arginine recycling enzymes, argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) were both decreased in lung tissues of Shunt lambs while arginase activity was increased. Associated with these alterations, lung L-arginine levels were decreased. These changes correlated with an increase in NO synthase-derived reactive oxygen species (ROS) generation. This study provides further insights into the molecular mechanisms leading to decreased NO signaling in Shunt lambs and suggests that altered arginine metabolism may play a role in the development of the endothelial dysfunction associated with pulmonary hypertension secondary to increased pulmonary blood flow.
Asunto(s)
Arginina/metabolismo , Hipertensión Pulmonar/metabolismo , Pulmón/metabolismo , Circulación Pulmonar , Secuencia de Aminoácidos , Aminoácidos/sangre , Animales , Arginasa/metabolismo , Presión Sanguínea , Femenino , Datos de Secuencia Molecular , Óxido Nítrico/fisiología , Embarazo , OvinosRESUMEN
We have previously shown that endothelial nitric oxide synthase (eNOS) promoter activity is decreased in endothelial cells in response to the addition of hydrogen peroxide (H(2)O(2)), and this involves, at least in part, the inhibition of AP-1 activity. Thus, the objective of this study was to determine if other cis-element(s) and transcription factor(s) are involved in the oxidant-mediated downregulation of eNOS. Our initial experiments indicated that although H(2)O(2) treatment increased eNOS mRNA levels in ovine pulmonary arterial endothelial cells (OPAECs), there was a significant decrease in the promoter activity of an eNOS promoter construct containing 840 bp of upstream sequence. However, a truncated promoter construct that lacked the AP-1 element (650 bp) was also inhibited by H(2)O(2). A similar effect was observed when the 650 bp human eNOS promoter construct was transfected into human PAECs. We also found that although exposure of the cells to PEG-catalase prevented the inhibitory effect on eNOS promoter activity, the hydroxyl radical scavenger, deferoxamine myslate, did not. Nor could we identify an increase in hydroxyl radical levels in cells exposed to H(2)O(2). Exposure of PAECs caused a significant increase in labile zinc levels in response to H(2)O(2). As the eNOS promoter has a cis-element for Sp1 binding, we evaluated the role of Sp1 in response to H(2)O(2). As previously reported, mutation of the Sp1 consensus lead to the complete loss of eNOS promoter activity, confirming the key role of Sp1 in regulating basal eNOS promoter activity. In addition, we found, using electrophoretic mobility and supershift assays, that H(2)O(2) decreased Sp1 binding. Finally, using chromatin immunoprecipitation analysis, we found a significant decrease in Sp1 binding to the eNOS promoter in vivo in response to treatment with H(2)O(2). Together, these data suggest that the inhibition of Sp1 activity, possibly through loss of zinc in the protein, plays a role in the H(2)O(2)-induced inhibition of eNOS promoter activity.
Asunto(s)
Peróxido de Hidrógeno/farmacología , Óxido Nítrico Sintasa de Tipo III/genética , Factor de Transcripción Sp1/genética , Factor de Transcripción AP-1/genética , Zinc/metabolismo , Animales , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Radical Hidroxilo/metabolismo , Óxido Nítrico Sintasa de Tipo III/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Regiones Promotoras Genéticas , Arteria Pulmonar/citología , Arteria Pulmonar/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Ovinos , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción AP-1/metabolismoRESUMEN
Cardiac defects associated with increased pulmonary blood flow result in pulmonary vascular dysfunction that may relate to a decrease in bioavailable nitric oxide (NO). An 8-mm graft (shunt) was placed between the aorta and pulmonary artery in 30 late gestation fetal lambs; 27 fetal lambs underwent a sham procedure. Hemodynamic responses to ACh (1 microg/kg) and inhaled NO (40 ppm) were assessed at 2, 4, and 8 wk of age. Lung tissue nitric oxide synthase (NOS) activity, endothelial NOS (eNOS), neuronal NOS (nNOS), inducible NOS (iNOS), and heat shock protein 90 (HSP90), lung tissue and plasma nitrate and nitrite (NO(x)), and lung tissue superoxide anion and nitrated eNOS levels were determined. In shunted lambs, ACh decreased pulmonary artery pressure at 2 wk (P < 0.05) but not at 4 and 8 wk. Inhaled NO decreased pulmonary artery pressure at each age (P < 0.05). In control lambs, ACh and inhaled NO decreased pulmonary artery pressure at each age (P < 0.05). Total NOS activity did not change from 2 to 8 wk in control lambs but increased in shunted lambs (ANOVA, P < 0.05). Conversely, NO(x) levels relative to NOS activity were lower in shunted lambs than controls at 4 and 8 wk (P < 0.05). eNOS protein levels were greater in shunted lambs than controls at 4 wk of age (P < 0.05). Superoxide levels increased from 2 to 8 wk in control and shunted lambs (ANOVA, P < 0.05) and were greater in shunted lambs than controls at all ages (P < 0.05). Nitrated eNOS levels were greater in shunted lambs than controls at each age (P < 0.05). We conclude that increased pulmonary blood flow results in progressive impairment of basal and agonist-induced NOS function, in part secondary to oxidative stress that decreases bioavailable NO.
Asunto(s)
Óxido Nítrico Sintasa/metabolismo , Estrés Oxidativo , Circulación Pulmonar/fisiología , Acetilcolina/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Enfermedad Crónica , Modelos Animales de Enfermedad , Femenino , Proteínas HSP90 de Choque Térmico/metabolismo , Pulmón/enzimología , Pulmón/patología , Pulmón/fisiopatología , Nitratos/sangre , Óxido Nítrico Sintasa de Tipo III/metabolismo , Nitritos/sangre , Estrés Oxidativo/efectos de los fármacos , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/enzimología , Arteria Pulmonar/patología , Arteria Pulmonar/fisiopatología , Circulación Pulmonar/efectos de los fármacos , Ovinos , Superóxidos/metabolismoRESUMEN
Increased asymmetric dimethylarginine (ADMA) levels have been implicated in the pathogenesis of a number of conditions affecting the cardiovascular system. However, the mechanism(s) by which ADMA exerts its effect has not been adequately elucidated. Thus the purpose of this study was to determine the effect of increased ADMA on nitric oxide (NO) signaling and to begin to elucidate the mechanism by which ADMA acts. Our initial data demonstrated that ADMA increased NO synthase (NOS) uncoupling in both recombinant human endothelial NO synthase (eNOS) and pulmonary arterial endothelial cells (PAEC). Furthermore, we found that this endothelial NOS (eNOS) uncoupling increased 3-nitrotyrosine levels preferentially in the mitochondria of PAEC due to a redistribution of eNOS from the plasma membrane to the mitochondria. This increase in nitration in the mitochondria was found to induce mitochondrial dysfunction as determined by increased mitochondrial-derived reactive oxygen species and decreased generation of ATP. Finally, we found that the decrease in ATP resulted in a reduction in the chaperone activity of HSP90 resulting in a decrease in its interaction with eNOS. In conclusion increased levels of ADMA causes mitochondrial dysfunction and a loss of heat shock protein-90 chaperone activity secondary to an uncoupling of eNOS. Mitochondrial dysfunction may be an understudied component of the endothelial dysfunction associated with various cardiovascular disease states.
Asunto(s)
Arginina/análogos & derivados , Proteínas HSP90 de Choque Térmico/metabolismo , Mitocondrias/metabolismo , Óxido Nítrico/metabolismo , Arteria Pulmonar/metabolismo , Transducción de Señal , Adenosina Trifosfato/metabolismo , Animales , Arginina/metabolismo , Células Cultivadas , Humanos , Canales Iónicos/metabolismo , Proteínas Mitocondriales/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Estrés Oxidativo , Ácido Peroxinitroso/metabolismo , Unión Proteica , Transporte de Proteínas , Arteria Pulmonar/embriología , Arteria Pulmonar/fisiopatología , Proteínas Recombinantes/metabolismo , Ovinos , Superóxidos/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo , Proteína Desacopladora 2RESUMEN
Utilizing aortopulmonary vascular graft placement in the fetal lamb, we have developed a model (shunt) of pulmonary hypertension that mimics congenital heart disease with increased pulmonary blood flow. Our previous studies have identified a progressive development of endothelial dysfunction in shunt lambs that is dependent, at least in part, on decreased nitric oxide (NO) signaling. The purpose of this study was to evaluate the possible role of a disruption in carnitine metabolism in shunt lambs and to determine the effect on NO signaling. Our data indicate that at 2 wk of age, shunt lambs have significantly reduced expression (P < 0.05) of the key enzymes in carnitine metabolism: carnitine palmitoyltransferases 1 and 2 as well as carnitine acetyltransferase (CrAT). In addition, we found that CrAT activity was inhibited due to increased nitration. Furthermore, free carnitine levels were significantly decreased whereas acylcarnitine levels were significantly higher in shunt lambs (P < 0.05). We also found that alterations in carnitine metabolism resulted in mitochondrial dysfunction, since shunt lambs had significantly decreased pyruvate, increased lactate, and a reduced pyruvate/lactate ratio. In pulmonary arterial endothelial cells cultured from juvenile lambs, we found that mild uncoupling of the mitochondria led to a decrease in cellular ATP levels and a reduction in both endothelial NO synthase-heat shock protein 90 (eNOS-HSP90) interactions and NO signaling. Similarly, in shunt lambs we found a loss of eNOS-HSP90 interactions that correlated with a progressive decrease in NO signaling. Our data suggest that mitochondrial dysfunction may play a role in the development of endothelial dysfunction and pulmonary hypertension and increased pulmonary blood flow.
Asunto(s)
Carnitina/metabolismo , Hipertensión Pulmonar/fisiopatología , Mitocondrias/fisiología , Óxido Nítrico/fisiología , Animales , Animales Recién Nacidos , Carnitina O-Acetiltransferasa/metabolismo , Carnitina O-Palmitoiltransferasa/metabolismo , Parto Obstétrico , Modelos Animales de Enfermedad , Femenino , Proteínas HSP90 de Choque Térmico/fisiología , Homeostasis , Hipertensión Pulmonar/enzimología , Óxido Nítrico Sintasa/metabolismo , Embarazo , Circulación Pulmonar/fisiología , Flujo Sanguíneo Regional , Ovinos , Transducción de Señal/fisiologíaRESUMEN
Previously, we have shown that pulmonary arterial endothelial cells (PAECs) isolated from fetal lambs produce significant levels of nitric oxide (NO) but minimal superoxide upon stimulation, whereas PAECs isolated from 4-wk-old lambs produce significant amounts of both NO and superoxide. These data indicated that a certain degree of uncoupling of endothelial NO synthase (eNOS) occurs in PAECs during postnatal development. In this study, we sought to extend these studies by investigating the potential role of heat shock protein 90 (HSP90) in eNOS coupling. Western blot analyses revealed higher HSP90 expression in PAECs isolated from fetal compared with 4-wk-old lambs, whereas the analysis of recombinant human eNOS activation in vitro in the presence of HSP90 indicated that HSP90 significantly augmented NO production while inhibiting superoxide generation from eNOS. To further investigate whether HSP90 could be involved in uncoupling of eNOS in PAECs isolated from 4-wk-old lambs, we utilized an adenovirus to overexpress HSP90. We found that overexpression of HSP90 significantly increased the shear-stimulated association of HSP90 with eNOS and led to significant increases in NO production and reduced NOS-dependent superoxide generation. Conversely, the exposure of PAECs isolated from fetal lambs to the HSP90 inhibitor radicicol led to significant decreases in eNOS-HSP90 interactions, decreased shear-stimulated NO generation, and increased NOS-dependent superoxide production indicative of eNOS uncoupling. Finally, we examined eNOS-HSP90 interactions in our lamb model of pulmonary hypertension associated with increased pulmonary blood flow (shunt). Our data indicate that HSP90-eNOS interactions were decreased in shunt lambs and that this was associated with decreased NO generation and an increase in eNOS-dependent generation of superoxide. Together, our data support a significant role for HSP90 in promoting NO generation and inhibiting superoxide generation by eNOS and indicate that the disruption of this interaction may be involved in the endothelial dysfunction associated with pulmonary hypertension.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/biosíntesis , Superóxidos/metabolismo , Envejecimiento , Animales , Separación Celular , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Feto/citología , Feto/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Pulmón/citología , Pulmón/efectos de los fármacos , Pulmón/enzimología , Macrólidos/farmacología , Unión Proteica/efectos de los fármacos , Arteria Pulmonar/citología , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/enzimología , Resistencia al Corte , OvinosRESUMEN
Increasing data suggest that oxidative stress, due to an increased production of reactive oxygen species and/or a decrease in antioxidants, is involved in the pathophysiology of pulmonary hypertension. Several antioxidant systems regulate the presence of oxidant species in vivo, and of primary interest are the superoxide dismutases (SOD) and catalase. However, little is known about the expression of antioxidant enzymes during the development of pulmonary hypertension. This study uses our lamb model of increased postnatal pulmonary blood flow, secondary to in utero aortopulmonary graft placement (shunt lambs), to investigate the expression patterns as well as activities of antioxidant enzymes during the early development of pulmonary hypertension. Protein levels of catalase, SOD1, SOD2, and SOD3 were evaluated by Western blot, and the activities of catalase and SOD were also quantified. In control lambs, protein expression and activities of catalase and SOD2 increased postnatally (P < 0.05). However, SOD1 and SOD3 protein levels did not change. In shunt lambs, catalase, SOD1, and SOD2 protein levels all increased over the first 8 wk of life (P < 0.05). However, SOD3 did not change. This was associated with an increase in the activities of catalase and SOD2 (P < 0.05). Compared with control lambs, catalase and SOD2 protein levels were decreased in 2-wk-old shunt lambs and this was associated with increased levels of hydrogen peroxide (H(2)O(2)) and superoxide (P < 0.05). Developmentally superoxide but not H(2)O(2) levels significantly increased in both shunt and control lambs with levels being significantly higher in shunt compared with control lambs at 2 and 4 but not 8 wk. These data suggest that the antioxidant enzyme systems are dynamically regulated postnatally, and this regulation is altered during the development of pulmonary hypertension secondary to increased pulmonary blood flow. An increased understanding of these alterations may have important therapeutic implications for the treatment of pulmonary hypertension secondary to increased pulmonary blood flow.
Asunto(s)
Envejecimiento/metabolismo , Pulmón/enzimología , Pulmón/fisiopatología , Oxidorreductasas/metabolismo , Circulación Pulmonar , Animales , Catalasa/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Peróxido de Hidrógeno/metabolismo , Inmunohistoquímica , Pulmón/crecimiento & desarrollo , Pulmón/metabolismo , Arteria Pulmonar/enzimología , Ovinos , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1 , Superóxidos/metabolismo , Distribución TisularRESUMEN
Oxidative stress has been associated with multiple pathologies and disease states, including those involving the cardiovascular system. Previously, we showed that pulmonary artery endothelial cells (PAECs) undergo apoptosis after acute exposure to H(2)O(2). However, the underlying mechanisms regulating this process remain unclear. Because of the prevalence of H(2)O(2) in normal physiological processes and apparent loss of regulation in disease states, the purpose of this study was to develop a more complete understanding of H(2)O(2)-mediated adverse effects on endothelial cell survival. Acute exposure of PAECs to H(2)O(2) caused a dose-dependent increase in cellular release of lactate dehydrogenase and a significant increase in production of superoxide ions, which appear to be generated within the mitochondria, as well as a significant loss of mitochondrial membrane potential and activity. Subsequent to the loss of mitochondrial membrane potential, PAECs exhibited significant caspase activation and apoptotic nuclei. We also observed a significant increase in intracellular free Zn(2+) after bolus exposure to H(2)O(2). To determine whether this increase in Zn(2+) was involved in the apoptotic pathway induced by acute H(2)O(2) exposure, we developed an adenoviral construct for overexpression of the Zn(2+)-binding protein metallothionein-1. Our data indicate that chelating Zn(2+), either pharmacologically with N,N,N',N-tetrakis(2-pyridylmethyl)ethylene diamine or by overexpression of the Zn(2+)-binding protein metallothionein-1, in PAECs conferred significant protection from induction of apoptosis and cell death associated with the effects of acute H(2)O(2) exposure. Our results show that the acute toxicity profile of H(2)O(2) can be attributed, at least in part, to liberation of Zn(2+) within PAECs. We speculate that regulation of Zn(2+) levels may represent a potential therapeutic target for cardiovascular disease associated with acute oxidative stress.
Asunto(s)
Células Endoteliales/metabolismo , Células Endoteliales/patología , Peróxido de Hidrógeno/toxicidad , Zinc/metabolismo , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Quelantes/farmacología , Células Endoteliales/efectos de los fármacos , Etilenodiaminas/farmacología , Homeostasis , Metalotioneína/genética , Metalotioneína/metabolismo , Estrés Oxidativo , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ovinos , Transducción de Señal/efectos de los fármacosRESUMEN
Pancreatic cancer, the fourth leading cause of cancer-associated mortality in the United States, usually presents in an advanced stage and is generally refractory to chemotherapy. As such, there is a great need for novel therapies for this disease. The naturally derived isoprenoids perillyl alcohol, farnesol, and geraniol have chemotherapeutic potential in pancreatic and other tumor types. However, their mechanisms of action in these systems are not completely defined. In this study, we investigated isoprenoid effects on the cell cycle and observed a similar antiproliferative mechanism of action among the three compounds. First, when given in combination, the isoprenoids exhibited an additive antiproliferative effect against MIA PaCa-2 human pancreatic cancer cells. Furthermore, all three compounds induced a G(0)/G(1) cell cycle arrest that coincided with an increase in the expression of the cyclin kinase inhibitor proteins p21(Cip1) and p27(Kip1) and a reduction in cyclin A, cyclin B1, and cyclin-dependent kinase (Cdk) 2 protein levels. Immunoprecipitation studies demonstrated increased association of both p21(Cip1) and p27(Kip1) with Cdk2 as well as diminished Cdk2 kinase activity after isoprenoid exposure, indicating a cell cycle-inhibitory role for p21(Cip1) and p27(Kip1) in pancreatic adenocarcinoma cells. When siRNA was used to inhibit expression of p21(Cip1) and p27(Kip1) proteins in MIA PaCa-2 cells, conditional resistance to all three isoprenoid compounds was evident. Given similar findings in this cell line and in BxPC-3 human pancreatic adenocarcinoma cells, we conclude that the chemotherapeutic isoprenoid compounds perillyl alcohol, farnesol, and geraniol invoke a p21(Cip1)- and p27(Kip1)-dependent antiproliferative mechanism in human pancreatic adenocarcinoma cells.
Asunto(s)
Ciclo Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Farnesol/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Monoterpenos/farmacología , Terpenos/farmacología , Monoterpenos Acíclicos , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Fase G1/efectos de los fármacos , Humanos , Inmunoprecipitación , Páncreas , Interferencia de ARN , Fase de Descanso del Ciclo Celular/efectos de los fármacosRESUMEN
While nitric oxide (NO)-mediated biological interactions have been intensively studied, the underlying mechanisms of nitrosative stress with resulting pathology remain unclear. Previous studies have demonstrated that NO exposure increases free zinc ions (Zn(2+)) within cells. However, the resulting effects on endothelial cell survival have not been adequately resolved. Thus the purpose of this study was to investigate the role of altered zinc homeostasis on endothelial cell survival. Initially, we confirmed the previously observed significant increase in free Zn(2+) with a subsequent induction of apoptosis in our pulmonary artery endothelial cells (PAECs) exposed to the NO donor N-[2-aminoethyl]-N-[2-hydroxy-2-nitrosohydrazino]-1,2-ethylenediamine. However, NO has many effects upon cell function and we wanted to specifically evaluate the effects mediated by zinc. To accomplish this we utilized the direct addition of zinc chloride (ZnCl(2)) to PAEC. We observed that Zn(2+)-exposed PAECs exhibited a dose-dependent increase in superoxide (O(2)(-).) generation that was localized to the mitochondria. Furthermore, we found Zn(2+)-exposed PAECs exhibited a significant reduction in mitochondrial membrane potential, loss of cardiolipin from the inner leaflet, caspase activation, and significant increases in TdT-mediated dUTP nick end labeling-positive cells. Furthermore, using an adenoviral construct for the overexpression of the Zn(2+)-binding protein, metallothionein-1 (MT-1), we found either MT-1 overexpression or coincubation with a Zn(2+)-selective chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylene-diamide, in PAECs significantly protected the mitochondria from both NO and Zn(2+)-mediated disruption and induction of apoptosis and cell death. In summary, our results indicate that a loss of Zn(2+) homeostasis produces mitochondrial dysfunction, increased oxidative stress, and apoptotic cell death. We propose that regulation of Zn(2+) levels may represent a potential therapeutic target for disease associated with both nitrosative and oxidative stress.