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BACKGROUND: Healthcare organisations and teams perform improvement activities to facilitate high-quality healthcare. The use of an improvement coach who provides support and guidance to the healthcare team may facilitate improvement activities; however, no systematic review exists on the facilitators and barriers to implementing an improvement coach. AIMS: We conducted a qualitative evidence synthesis to examine the facilitators and barriers to the implementation of improvement coaching. METHODS: We searched MEDLINE® , Embase and CINAHL. The final search was in March 2021. The screening eligibility criteria included the following: interdisciplinary team receiving the coaching, improvement coaching, designs with a qualitative component and primary purpose of evaluating practice facilitation in OECD countries. An ecologically-informed consolidated framework for implementation research (CFIR) served as the framework for coding. Patterns of barriers and facilitators across domains were identified through matrix analysis. Risk of bias was assessed using Critical Appraisal Skills Program. PRISMA reporting guidelines served as a guide for reporting this review. RESULTS: Nineteen studies with a qualitative component met the inclusion criteria. Four themes of barriers and facilitators crossed multiple CFIR domains: adaptability (e.g. making adjustments to the project; process, or approach); knowledge and skills (e.g. understanding of content and process for the project); engagement (e.g. willingness to be involved in the process) and resources (e.g. assets required to complete the improvement process). CONCLUSION: Improvement coaching is a complex intervention that influences the context, healthcare team being coached and improvement activities. Improvement coaches should understand how to minimise barriers and promote facilitators that are unique to each improvement project across the domains. Limitations of the study are related to the nature of the intervention including potential publication bias given quality improvement focus; the variety of terms similar to improvement coaching or selection of framework.
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Atención a la Salud , Tutoría , Humanos , Grupo de Atención al Paciente , Investigación CualitativaRESUMEN
BACKGROUND: A culture of improvement is an important feature of high-quality health care systems. However, health care teams often need support to translate quality improvement (QI) activities into practice. One method of support is consultation from a QI coach. The literature suggests that coaching interventions have a positive impact on clinical outcomes. However, the impact of coaching on specific process outcomes, like adoption of clinical care activities, is unknown. Identifying the process outcomes for which QI coaching is most effective could provide specific guidance on when to employ this strategy. METHODS: We searched multiple databases from inception through July 2021. Studies that addressed the effects of QI coaching on process of care outcomes were included. Two reviewers independently extracted study characteristics and assessed risk of bias. Certainty of evidence was assessed using GRADE. RESULTS: We identified 1983 articles, of which 23 cluster-randomized trials met eligibility criteria. All but two took place in a primary care setting. Overall, interventions typically targeted multiple simultaneous processes of care activities. We found that coaching probably has a beneficial effect on composite process of care outcomes (n = 9) and ordering of labs and vital signs (n = 6), and possibly has a beneficial effect on changes in organizational process of care (n = 5), appropriate documentation (n = 5), and delivery of appropriate counseling (n = 3). We did not perform meta-analyses because of conceptual heterogeneity around intervention design and outcomes; rather, we synthesized the data narratively. Due to imprecision, inconsistency, and high risk of bias of the included studies, we judged the certainty of these results as low or very low. CONCLUSION: QI coaching interventions may affect certain processes of care activities such as ordering of labs and vital signs. Future research that advances the identification of when QI coaching is most beneficial for health care teams seeking to implement improvement processes in pursuit of high-quality care will support efficient use of QI resources. PROTOCOL REGISTRATION: This study was registered and followed a published protocol (PROSPERO: CRD42020165069).
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Tutoría , Mejoramiento de la Calidad , Atención a la Salud , Servicios de Salud , Humanos , Calidad de la Atención de SaludRESUMEN
INTRODUCTION: Military and civil aviation have documented physiological episodes among aircrews. Therefore, continued efforts are being made to improve the internal environment. Studies have shown that exposures to many organic compounds present in emissions are known to cause a variety of physiological symptoms. We hypothesize that these compounds may reversibly inhibit acetylcholinesterase, which may disrupt synaptic signaling. As a result, neural proteins leak through the damaged blood-brain barrier into the blood and in some, elicit an autoimmune response. MATERIALS AND METHODS: Neural-specific autoantibodies of immunoglobulin-G (IgG) class were estimated by the Western blotting technique in the sera of 26 aircrew members and compared with the sera of 19 normal healthy nonaircrew members, used as controls. RESULTS: We found significantly elevated levels of circulating IgG-class autoantibodies to neurofilament triplet proteins, tubulin, microtubule-associated tau proteins (Tau), microtubule-associated protein-2, myelin basic protein, and glial fibrillary acidic protein, but not S100 calcium-binding protein B compared to healthy controls. CONCLUSION: Repetitive physiological episodes may initiate cellular injury, leading to neuronal degeneration in selected individuals. Diagnosis and intervention should occur at early postinjury periods. Use of blood-based biomarkers to assess subclinical brain injury would help in both diagnosis and treatment.
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Personal Militar/estadística & datos numéricos , Fenómenos Fisiológicos/fisiología , Medicina Aeroespacial/métodos , Medicina Aeroespacial/estadística & datos numéricos , Aeronaves , Autoanticuerpos/análisis , Autoanticuerpos/sangre , Biomarcadores/análisis , Biomarcadores/sangre , Western Blotting/métodos , Proteína Ácida Fibrilar de la Glía/análisis , Proteína Ácida Fibrilar de la Glía/sangre , Humanos , Inmunoglobulina G/análisis , Inmunoglobulina G/sangre , Proteínas Asociadas a Microtúbulos/análisis , Proteínas Asociadas a Microtúbulos/sangre , Proteína Básica de Mielina/análisis , Proteína Básica de Mielina/sangre , Proteínas de Neurofilamentos/análisis , Proteínas de Neurofilamentos/sangre , Proteínas S100/análisis , Proteínas S100/sangre , Tubulina (Proteína)/análisis , Tubulina (Proteína)/sangreRESUMEN
INTRODUCTION: Posttraumatic stress disorder (PTSD) can develop during the aftermath of traumatic events. Although many are impacted by several stressors, nearly 3.6% suffer from PTSD in the United States with higher incidence reported in military service personnel. Any injury to the blood-brain barrier can ignite an array of biological signaling molecules in the immune-privileged brain parenchyma, which can disrupt the synaptic neural network, resulting in altered behavior. MATERIALS AND METHODS: In this preliminary study, we compared 20 PTSD veterans with age-matched healthy veterans to identify plasma levels of brain-specific protein markers using enzyme-linked immunosorbent assay/immunofluorometric sandwich assay for neurotrophic factors and neuropoietic cytokines, and catalytic activity of matrix metalloproteinase (MMP) by zymography. RESULTS: We observed an increased level of glial fibrillary acidic protein, tumor necrosis factor-alpha, interleukin 6, and MMP2 and MMP9 but decreased level of brain-derived neurotrophic factor, nerve growth factor-beta, and negligible difference in astroglial marker S100 calcium-binding protein B compared to controls. CONCLUSION: Identification of neural biomarkers is essential to understand the subclinical symptoms for the diagnosis PTSD, which may not be visible by magnetic resonance imaging (MRI/fMRI) and may take years to clinically manifest.
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Trastornos por Estrés Postraumático/etiología , Adulto , Síntomas Conductuales/etiología , Síntomas Conductuales/psicología , Factor Neurotrófico Derivado del Encéfalo/análisis , Factor Neurotrófico Derivado del Encéfalo/sangre , Femenino , Proteína Ácida Fibrilar de la Glía/análisis , Proteína Ácida Fibrilar de la Glía/sangre , Humanos , Interleucina-6/análisis , Interleucina-6/sangre , Imagen por Resonancia Magnética/métodos , Masculino , Persona de Mediana Edad , Factor de Crecimiento Nervioso/análisis , Factor de Crecimiento Nervioso/sangre , Subunidad beta de la Proteína de Unión al Calcio S100/análisis , Subunidad beta de la Proteína de Unión al Calcio S100/sangre , Trastornos por Estrés Postraumático/diagnóstico , Trastornos por Estrés Postraumático/psicología , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/sangre , Estados UnidosRESUMEN
OBJECTIVES: Serum repositories are foundations for seroepidemiological data, revealing targeted information about morbidities and existing heterogeneity in human populations. With the recent technological advances, we can perform high-throughput screening at an affordable cost using minimal plasma. Monitoring brain health after an injury is critical since mild Traumatic Brain Injury (mTBI) and other neurological symptoms are under-diagnosed. Our objective in this study is to present our preliminary serological data from one of our ongoing studies on mTBI. METHODS: In this retrospective study, we used stored plasma samples to understand biomarkers of mTBI. We compared plasma samples from five patients with mTBI following their first concussive episode to five gender and age-matched healthy controls. We assessed multiple biomarkers to show the importance of biorepositories. RESULTS: Most of the estimated plasma factors in mTBI subjects at baseline were comparable to normal healthy individuals except for the astroglial markers S100B and glial fibrillary acidic protein. Fluctuations of these biomarkers can affect the homeostasis of brain parenchyma by altering the neural network signaling, which in turn may result in intermittent behavioral symptoms. CONCLUSION: Biorepositories are powerful resources for understanding the spectrum of morbidity. Biomarkers serve as a valuable diagnostic and therapeutic tool.
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Biomarcadores/análisis , Conmoción Encefálica/sangre , Guerra , Adulto , Biomarcadores/sangre , Conmoción Encefálica/fisiopatología , Factor Neurotrófico Derivado del Encéfalo/análisis , Factor Neurotrófico Derivado del Encéfalo/sangre , Estudios de Cohortes , Complemento C3/análisis , Proteína 1 de la Respuesta de Crecimiento Precoz/análisis , Proteína 1 de la Respuesta de Crecimiento Precoz/sangre , Femenino , Proteína Ácida Fibrilar de la Glía/análisis , Proteína Ácida Fibrilar de la Glía/sangre , Humanos , Interleucina-6/análisis , Interleucina-6/sangre , Estudios Longitudinales , Masculino , Factor de Activación Plaquetaria/análisis , Estudios Retrospectivos , Subunidad beta de la Proteína de Unión al Calcio S100/análisis , Subunidad beta de la Proteína de Unión al Calcio S100/sangre , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/sangreRESUMEN
This paper has been retracted at the request of the authors.
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Bronchopulmonary dysplasia (BPD) is an inflammatory lung disorder common in premature infants who undergo mechanical ventilation with supplemental oxygen. Inhaled nitric oxide (iNO) has been used to prevent experimental and clinical BPD. Earlier studies showed that NO effects in alveolar epithelial cells (AEC) are mediated by S-nitrosothiol uptake via L-type amino acid transporter-1 (LAT1). Because LAT1 expression could influence the efficacy of iNO therapy, we sought to determine whether pulmonary LAT1 expression is altered in preterm baboons with experimental BPD and in human newborns susceptible to developing BPD. Using fixed lung obtained from 125 d to 140 d gestation baboon models of BPD, LAT1 immunostaining was measured in control and BPD animals. In adult controls and in 140 d gestational controls (GC), LAT1 was expressed in both type I and type II AECs. In 140 d BPD lungs, LAT1 expression density in alveolar tissue was decreased. In 125 d GC baboons, LAT1 immunostaining was largely confined to cuboidal AECs, whereas animals given 14 d of mechanical ventilation exhibited diminished alveolar septal LAT1 Labeling. The pattern in adult human donor lung was comparable to that observed in adult baboons. LAT1 was expressed in lungs obtained from some but not all very premature newborns at autopsy. In human and baboon lung, adult and newborn, pulmonary vascular cells expressed LAT1. In summary, LAT1 is expressed in AECs and pulmonary vascular cells in baboons and humans. Experimental BPD in premature baboons decreases pulmonary LAT1 expression and alters its spatial localization. Heterogeneity of functional LAT1 could affect S-nitrosothiol importation, which could impair iNO therapy. Pediatr Pulmonol. 2016;51:1048-1056. © 2016 Wiley Periodicals, Inc.
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Displasia Broncopulmonar/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Pulmón/metabolismo , Adulto , Animales , Humanos , Recién Nacido , Recien Nacido Prematuro , Recién Nacido de muy Bajo Peso , Óxido Nítrico/metabolismo , Oxígeno/metabolismo , Papio , Respiración ArtificialRESUMEN
AIMS: The aims of this study were to determine hyperoxia effects on S-nitrosothiol (SNO) accumulation and L-type amino acid transporter 1 (LAT1) expression/function in alveolar epithelium and to determine whether hyperoxia impairs exogenous nitric oxide (NO) treatment effects in alveolar epithelium through effects on LAT1 expression and/or function. RESULTS: SNO accumulation in vitro and in vivo after NO treatment was dependent on the LAT1 system transport. Hyperoxia (60% or 90%) impaired NO effects on SNO accumulation and soluble guanylyl cyclase activation in proportion to the magnitude of hyperoxia and the duration of exposure, up to 12 h, in type I-like (R3/1) and type II-like (L2) rat and human (A549) alveolar epithelial cells. LAT function, determined by sodium-independent (3)H-leucine uptake, was impaired in a parallel manner. Hyperoxia impaired LAT1 expression in alveolar epithelial cells, determined by immunoblots and immunofluorescence, and in newborn rats exposed to 60% O2 for 4 days, determined by immunohistochemistry. INNOVATION: Despite significant preclinical evidence, inhaled NO has shown disappointing limitations in clinical applications. Our studies suggest an important explanation: oxidative stress, a common feature of diseases in which therapeutic NO would be considered, impairs LAT1 expression and function, blocking a major route for inhaled NO (iNO) action, that is, the uptake of S-nitrosocysteine via LAT1. CONCLUSIONS: SNO uptake after NO treatment is dependent on LAT1. Hyperoxia impairs SNO uptake and NO effects during NO exposure and impairs LAT system function and LAT1 expression. Effects on SNO formation and transport must be considered for rational optimization of NO-based therapeutics.
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Células Epiteliales/metabolismo , Hiperoxia/patología , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Óxido Nítrico/metabolismo , Alveolos Pulmonares/metabolismo , Animales , Transporte Biológico/fisiología , Línea Celular Tumoral , Cisteína/análogos & derivados , Cisteína/metabolismo , Células Epiteliales/patología , Guanilato Ciclasa/metabolismo , Humanos , Hiperoxia/metabolismo , Leucina/metabolismo , Masculino , Estrés Oxidativo/fisiología , Alveolos Pulmonares/patología , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/metabolismo , S-Nitrosotioles/metabolismo , Guanilil Ciclasa SolubleRESUMEN
BACKGROUND: We previously showed that intra-amniotic lipopolysaccharide (LPS) amplifies alveolar hypoplasia induced by postnatal hyperoxia. We determined whether the priming effect of intra-amniotic LPS amplifies hyperoxia-induced airway hyperreactivity (AHR). METHODS: LPS or normal saline was injected into the amniotic cavities of pregnant rats at the 20th day of gestation. After birth, rat pups were exposed to 60% O2 or air for 14 d. On postnatal day 14, rat pups underwent forced oscillometry, which included a challenge with nebulized methacholine, and the lungs were harvested for morphological studies. RESULTS: Hyperoxia significantly increased airway reactivity and decreased compliance. Intra-amniotic LPS further increased hyperoxia-induced AHR but did not further impair respiratory system compliance. Hyperoxia-induced changes in lung parenchymal and small airway morphology were not further altered by intra-amniotic LPS. However, combined exposure to intra-amniotic LPS and hyperoxia increased the proportion of degranulating mast cells in the hilar airways. CONCLUSION: Intra-amniotic LPS amplified postnatal hyperoxia-induced AHR. This was associated with increased airway mast cell degranulation, which has previously been linked with hyperoxia-induced AHR. There were no morphologic changes of parenchyma or airways that would account for the LPS augmentation of hyperoxia-induced AHR.
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Hiperreactividad Bronquial/inducido químicamente , Hiperoxia/fisiopatología , Lipopolisacáridos/administración & dosificación , Animales , Animales Recién Nacidos , Femenino , Hiperoxia/inducido químicamente , Embarazo , Ratas , Ratas Sprague-DawleyRESUMEN
The pharmacological effects of nitric oxide (NO) administered as a gas are dependent on the conversion to S-nitrosocysteine, and as such are largely mediated by the L-type amino-acid transporters (LATs) in several cell types. The dipeptide transporter PEPT2 has been proposed as a second route for S-nitrosothiol (SNO) transport, but this has never been demonstrated. Because NO governs important immune functions in alveolar macrophages, we exposed rat alveolar macrophages (primary and NR8383 cells) to NO gas at the air-liquid interface ± LPS stimulation in the presence of PEPT2 substrate Cys-Gly (or the LAT substrate L-Cys) ± transporter competitors. We found that SNO uptake and NO-dependent actions, such as the activation of soluble guanylyl cyclase (sGC), the augmentation of sGC-dependent filamentous actin (F-actin) polymerization, phagocytosis, and the inhibition of NF-κB activation, were significantly augmented by the addition of Cys-Gly in a manner dependent on PEPT2 transport. We found parallel (and greater) effects that were dependent on LAT transport. The contribution of cystine/cysteine shuttling via system x cystine transporter (xCT) to SNO uptake was relatively minor. The observed effects were unaffected by NO synthase inhibition. The NO gas treatment of alveolar macrophages increased SNO uptake, the activation of sGC, F-actin polymerization, and phagocytosis, and inhibited NF-κB activation, in a manner dependent on SNO transport via PEPT2, as well as via LAT.
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Macrófagos Alveolares/efectos de los fármacos , Óxido Nítrico/farmacología , S-Nitrosotioles/metabolismo , Simportadores/fisiología , Animales , Transporte Biológico , Células Cultivadas , Macrófagos Alveolares/metabolismo , RatasRESUMEN
Inhaled nitric oxide (iNO) is used to treat pulmonary hypertension and is being investigated for prevention of bronchopulmonary dysplasia in neonates. Extrapulmonary effects of iNO are widely recognized, but the underlying chemistry and pharmacology are poorly understood. Growing evidence suggests that, in addition to acting via diffusion, NO can be converted into nitrosants capable of reacting with endogenous L-cysteine (L-Cys) in the alveolar lining fluid, forming S-nitrosothiol (SNO)-L-cysteine (CSNO). CSNO can then enter cells via the type L amino acid transporter (LAT). To determine the influence of LAT and supplemental L-Cys on the functional activity of iNO and transpulmonary movement of SNOs or other related species, we exposed C57Bl6 mice to nebulized L-Cys or D-cysteine (D-Cys) and/or LAT competitors. Isolated lungs were then perfused with physiologic buffer while effluent was collected to assay perfusate SNOs. Nebulized L-Cys, but not D-Cys, augmented the iNO-induced increase in circulating SNOs in the effluent without altering iNO-induced pulmonary vasodilation. Addition to the perfusate of either L-leucine (L-Leu) or 2-amino-2-norborane carboxylic acid, two distinct LAT competitors, inhibited appearance in the perfusate of SNOs in L-Cys-exposed lungs; a higher concentration of L-Leu significantly inhibited the iNO-induced pulmonary vasodilation as well as SNO accumulation. We conclude that iNO-induced pulmonary vasodilation and the transpulmonary movement of iNO-derived SNOs are mediated in part by formation of extracellular CSNO, uptake by alveolar epithelial LAT, and/or export by LAT from the pulmonary endothelium into the circulation. Therapies that exploit and optimize LAT-dependent SNO transport might improve the efficacy of and clinical outcomes with NO-based therapy by improving systemic SNO delivery.
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Pulmón/irrigación sanguínea , Pulmón/metabolismo , Óxido Nítrico/farmacología , S-Nitrosotioles/metabolismo , Vasodilatación , Administración por Inhalación , Aminoácidos Cíclicos/farmacología , Animales , Transporte Biológico Activo , Cisteína/administración & dosificación , Cisteína/farmacología , Femenino , Hipertensión Pulmonar/tratamiento farmacológico , Leucina/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/administración & dosificación , Óxido Nítrico/uso terapéutico , Vasodilatación/efectos de los fármacosRESUMEN
Inducible nitric oxide synthase (NOS2) expression is increased in the airway epithelium in acute inflammatory disorders although the physiological impact remains unclear. We have previously shown that NOS2 inhibits NF-κB (p50-p65) activation in respiratory epithelial cells by inducing S-nitrosylation of the p65 monomer (SNO-p65). In addition, we have demonstrated that mouse lung SNO-p65 levels are acutely depleted in a lipopolysaccharide (LPS) model of lung injury and that augmenting SNO-p65 levels before LPS treatment results in decreased airway epithelial NF-κB activation, airway inflammation, and lung injury. We now show that aerosolized LPS induces NOS2 expression in the respiratory epithelium concomitant with an increase in lung SNO-p65 levels and a decrease in airway NF-κB activity. Genetic deletion of NOS2 results in an absence of SNO-p65 formation, persistent NF-κB activity in the respiratory epithelium, and prolonged airway inflammation. These results indicate that a primary function of LPS-induced NOS2 expression in the respiratory epithelium is to modulate the inflammatory response through deactivation of NF-κB via S-nitrosylation of p65, thereby counteracting the initial stimulus-coupled denitrosylation.
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Óxido Nítrico Sintasa de Tipo II/fisiología , Mucosa Respiratoria/metabolismo , Factor de Transcripción ReIA/metabolismo , Animales , Líquido del Lavado Bronquioalveolar/química , Inflamación/inducido químicamente , Lipopolisacáridos , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/biosíntesisRESUMEN
Spontaneous diastolic depolarization in the sinoatrial (SA) node enables it to serve as pacemaker of the heart. The variable cell morphology within the SA node predicts that ion channel expression would be heterogeneous and different from that in the atrium. To evaluate ion channel heterogeneity within the SA node, we used fluorescent in situ hybridization to examine ion channel expression in the ferret SA node region and atrial appendage. SA nodal cells were distinguished from surrounding cardiac myocytes by expression of the slow (SA node) and cardiac (surrounding tissue) forms of troponin I. Nerve cells in the sections were identified by detection of GAP-43 and cytoskeletal middle neurofilament. Transcript expression was characterized for the 4 hyperpolarization-activated cation channels, 6 voltage-gated Na(+) channels, 3 voltage-gated Ca(2+) channels, 24 voltage-gated K(+) channel α-subunits, and 3 ancillary subunits. To ensure that transcript expression was representative of protein expression, immunofluorescence was used to verify localization patterns of voltage-dependent K(+) channels. Colocalizations were performed to observe any preferential patterns. Some overlapping and nonoverlapping binding patterns were observed. Measurement of different cation channel transcripts showed heterogeneous expression with many different patterns of expression, attesting to the complexity of electrical activity in the SA node. This study provides insight into the possible role ion channel heterogeneity plays in SA node pacemaker activity.
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Hurones/genética , Canales Iónicos/genética , Canales Iónicos/metabolismo , Nodo Sinoatrial/metabolismo , Animales , Biomarcadores/metabolismo , Hurones/anatomía & histología , Hurones/metabolismo , Regulación de la Expresión Génica , Hibridación Fluorescente in Situ , Técnicas In Vitro , Activación del Canal Iónico/genética , Masculino , Neuronas/citología , Neuronas/metabolismo , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Nodo Sinoatrial/citologíaRESUMEN
The pathway by which inhaled NO gas enters pulmonary alveolar epithelial cells has not been directly tested. Although the expected mechanism is diffusion, another route is the formation of S-nitroso-L-cysteine, which then enters the cell through the L-type amino acid transporter (LAT). To determine if NO gas also enters alveolar epithelium this way, we exposed alveolar epithelial-rat type I, type II, L2, R3/1, and human A549-cells to NO gas at the air liquid interface in the presence of L- and D-cysteine+/-LAT competitors. NO gas exposure concentration dependently increased intracellular NO and S-nitrosothiol levels in the presence of L- but not D-cysteine, which was inhibited by LAT competitors, and was inversely proportional to diffusion distance. The effect of L-cysteine on NO uptake was also concentration dependent. Without preincubation with L-cysteine, NO uptake was significantly reduced. We found similar effects using ethyl nitrite gas in place of NO. Exposure to either gas induced activation of soluble guanylyl cylase in a parallel manner, consistent with LAT dependence. We conclude that NO gas uptake by alveolar epithelium achieves NO-based signaling predominantly by forming extracellular S-nitroso-L-cysteine that is taken up through LAT, rather than by diffusion. Augmenting extracellular S-nitroso-L-cysteine formation may augment pharmacological actions of inhaled NO gas.
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Sistema de Transporte de Aminoácidos y+L/metabolismo , Transporte Biológico/efectos de los fármacos , Epitelio/metabolismo , Guanilato Ciclasa/metabolismo , Proteínas de Neoplasias/metabolismo , Óxido Nítrico/metabolismo , Sistema de Transporte de Aminoácidos y+L/antagonistas & inhibidores , Animales , Unión Competitiva , Cisteína/análogos & derivados , Cisteína/metabolismo , Cisteína/farmacología , Difusión , Epitelio/patología , Guanilato Ciclasa/genética , Humanos , Leucina/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Óxido Nítrico/farmacología , Alveolos Pulmonares/patología , Ratas , S-Nitrosotioles/metabolismo , EstereoisomerismoRESUMEN
Nitric oxide (NO) effects are often mediated via S-nitrosothiol (SNO) formation; SNO uptake has recently been shown to be mediated in some cell types via system L-type amino acid transporters (LAT-1, 2). Inhaled NO therapy may exert some biological effects via SNO formation. We therefore sought to determine if pulmonary epithelial SNO uptake depended on LAT or peptide transporter 2 (PEPT2). Both LAT-1 and PEPT2 proteins were detected by immunoblot and immunocytochemistry in L2 cells and rat lung. We tested SNO uptake through the transporters by exposing rat alveolar epithelial cells (L2 and type II) to RSNOs: S-nitrosoglutathione, S-nitrosocysteinylglycine (SNO-Cys-Gly), S-nitrosocysteine (CSNO), and to NO donor diethylamine NONOate (DEA-NONOate). SNO was detected in cell lysates by ozone chemiluminescence. NO uptake was detected by fluorescence in alveolar epithelial cells loaded with 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) diacetate cultured in submersion and exposed to RSNOs and DEA NONOate. Addition of L-Cys but not D-Cys to RSNOs or DEA NONOate increased SNO and DAF-FM signal that was inhibited by coincubation with LAT competitors. Incubation of cells with PEPT2 substrate SNO-Cys-Gly showed no increase in SNO or DAF-FM signal unless incubated with L-Cys. This was unaffected by PEPT2 inhibition. We conclude that RSNOs (thionitrites, S-nitrosothiols) and NO enter alveolar epithelial cells predominantly by S-nitrosation of L-Cys, which is then imported through LAT.
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Sistema de Transporte de Aminoácidos y+/metabolismo , Cisteína/metabolismo , Dipéptidos/metabolismo , Células Epiteliales/metabolismo , Cadenas Ligeras de la Proteína-1 Reguladora de Fusión/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Óxido Nítrico/metabolismo , Compuestos Nitrosos/metabolismo , Alveolos Pulmonares/metabolismo , Animales , Transporte Biológico , Línea Celular , Cisteína/análogos & derivados , Cisteína/farmacología , Dipéptidos/farmacología , Células Epiteliales/citología , Fluoresceínas/farmacología , Hidrazinas/farmacología , Óxido Nítrico/farmacología , Donantes de Óxido Nítrico/farmacología , Compuestos Nitrosos/farmacología , Alveolos Pulmonares/citología , Ratas , Ratas Sprague-Dawley , Simportadores/metabolismoRESUMEN
Nitrogen oxides exert significant but diverse regulatory effects on cardiac myocytes. Many of these effects are due to modulation of voltage-sensitive ion channel function. The redox-status of NO-related compounds is a critical factor in determining whether indirect (cGMP-dependent) versus direct (cGMP-independent) effects are dominant. However, molecular mechanisms by which different cardiac myocyte types, and associated different ion channel types expressed within them, could achieve selectivity between NO-related indirect versus direct effects are unclear We have previously demonstrated heterogeneous expression gradients of Type III NO synthase (eNOS) and sarcolemmal superoxide dismutase (ECSOD) in ferret and human ventricle, with both enzymes being highly expressed in right ventricle and left ventricular subepicardium but markedly reduced in left ventricular subendocardium. In this study we extend this previous analysis by analyzing NO-activated soluble guanylyl cyclase (sGC) expression in the heart (ferret and human). We demonstrate that, at both tissue and single myocyte levels, sGC protein expression is heterogeneous, being high in sinoatrial node, right atrium, right ventricle and left ventricular subepicardium, but markedly reduced to absent in left atrium and left ventricular subendocardium. Thus, there is a significant overlap in expression gradients of sGC, eNOS, and ECSOD among distinct cardiac tissue and myocyte types. These gradients positively correlate with both: (i) experimentally measured basal NO production levels; and (ii) expression gradients of specific voltage-gated ion channels (particularly Kv1 and Kv4 channels). Our results provide the first demonstration in the heart of an expressed coupled multienzymatic system for selective regulation of indirect (sGC-dependent) versus direct (sGC-independent) NO- and redox-related modulation of voltage-gated ion channel function in different myocyte types. Our results also have functional implications for NO(*)/redox-related modulation of ion channels expressed in other cell types, including neurons, skeletal muscle and smooth muscle.