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The Journal of pediatrics 11/2009; 155(5):603-4.e1. · 4.02 Impact Factor
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ABSTRACT: Proteinases contribute to the pathogenesis of various lung diseases, partly through activating cell surface receptors by limited proteolytic cleavage. The authors provide evidence that in primary cultures of distal lung epithelia, basolateral protease-activated receptor 1 activation rapidly reduces transepithelial resistance but does not alter paracellular permeability to small uncharged solutes. Changes in transepithelial resistance were partially blocked by ion transport inhibitors and were completely blocked by placing cells in low chloride buffer. In vivo studies did not reveal enhanced lung permeability in response to pulmonary or intravenous administration of protease-activated receptor 1 activators. This information is relevant as strategies to inhibit protease-activated receptor 1 signaling are considered in order to preserve lung epithelial barrier function.
Experimental Lung Research 04/2009; 35(2):136-54. · 1.22 Impact Factor
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ABSTRACT: The neuroendocrine system is most active at birth and may play a role in the transition from fetal to postnatal life, in particular in the lungs' transition from fluid secretion to fluid absorption. Pulmonary neuroendocrine cells do release dopamine (DA), serotonin, and gastrin-releasing peptide but their effects on lung ion and fluid transport are poorly understood. Therefore, we studied their effects on fetal distal lung explants and primary cultures of fetal distal lung epithelium (FDLE). We show that DA, but neither serotonin nor gastrin-releasing peptide, alters ion and fluid transport, in a dose-dependent manner. DAs effects were abrogated by D1/D2 receptor blockers in FDLE but not in explants. Propranolol abrogated DAs effects in both models. DA increased intracellular cAMP levels in FDLE. Terbutaline, forskolin, and isobutylmethylxanthine did not increase short circuit current (Isc) in DA-treated cells, despite a further increase in cAMP. We conclude that at least one, but not all mediators released by pulmonary neuroendocrine cells alter distal lung epithelial ion transport.
Pediatric Research 01/2009; 65(3):274-8. · 2.70 Impact Factor
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ABSTRACT: REDD1 (Regulated in Development and DNA Damage-1) is a stress-response gene that represses mammalian target of rapamycin (mTOR) thus decreasing protein synthesis. In contrast to studies using cell lines and adult alveolar type II (ATII) cells, we find that REDD1 mRNA levels did not increase in rat fetal distal lung epithelia (FDLE) or fetal lung fibroblasts grown in primary cultures and then exposed to 3% O2. REDD1 mRNA expression was repressed by dexamethasone (DEX) in FDLE and ATII, but induced by DEX in fibroblasts. Lung epithelial cell lines, A549 and MLE-15, showed increases in REDD1 mRNA in response to hypoxia and DEX. The effect of DEX on REDD1 mRNA and protein in FDLE and fibroblasts was dose- and time-dependent. Inhibitor studies support repression of REDD1 mRNA by DEX in FDLE was mediated via glucocorticoid receptor and not by nongenomic effects of glucocorticoids via MAPK pathways. The half-life of REDD1 mRNA was shorter in DEX-exposed FDLE compared with hormone-free media suggesting that DEX reduced REDD1 mRNA stability in FDLE. These studies indicate that REDD1 expression in response to hypoxia and DEX is cell-type specific and that physiologically appropriate levels of PO2 should be used when investigating fetal lung development.
Pediatric Research 01/2009; 65(5):514-9. · 2.70 Impact Factor
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ABSTRACT: During the peripartum period, the lung must respond to dramatic changes in circulating hormones, nutritional factors, and physiologic signals during its transition to becoming the organ of gas exchange. Protein synthesis consumes a significant proportion of metabolic resources and is inhibited by many environmental stresses. We hypothesized that translational control mechanisms play a role in the perinatal lung. Immunoblots of late-gestation (Fetal Day [FD] 17-22) rat lung extracts revealed gradual decreases in phosphorylated forms of the mammalian target of rapamycin effectors, eukaryotic initiation factor (eIF) 4E-binding protein, p70 S6 kinase, and ribosomal protein S6, followed by sharp increases on Postnatal Day 1 (P1). Immunohistochemistry showed phospho-S6 staining was most prominent in epithelial cells of the large and small airways. m(7)GTP-sepharose pulldown experiments showed a decrease in association of translation initiation factor, eIF4E, with its inhibitor, eIF4E-binding protein, and a concomitant increase in eIF4E association with eIF4G immediately after birth, and polysome profiles confirmed a decrease in abundance of large polysomes between FD19 and FD22, which was reversed on P1. Microarray analysis of polysomal versus total RNA from FD19, FD22, and P1 lungs was used to identify specific genes, the association of which with large polysomes changed either pre- or postnatally. RT-PCR and Northern blotting were used to confirm translational changes in selected candidate genes, including a prenatal increase in IL-18 and a postnatal decrease in regulatory subunit 2 of protein phosphatase 1. Translational regulation of IL-18 and protein phosphatase 1 regulatory (inhibitor) subunit 2 is gene-specific, as these changes contrast with the corresponding global changes in polysome abundance.
American Journal of Respiratory Cell and Molecular Biology 11/2008; 40(5):555-67. · 5.13 Impact Factor
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ABSTRACT: Current indirect measurements of alveolar fluid clearance (AFC) suggest that the rate of fluid clearance correlates with morbidity and mortality in patients with pulmonary edema. In a traditional AFC-measurement, fluid laced with a tracer macromolecule is instilled into the lung and thereafter repeated samples of the instilled fluid are extracted from the lung's fluid-filled airspaces. The change in concentration of the tracer molecule indicates the AFC-rate. In this work, a new MRI technique was developed to image lung water clearance by adding Gadolinium-DTPA to the instilled fluid. As fluid is absorbed by the animal, the concentration of gadolinium will increase, reducing the T(1) relaxation time. By repeatedly measuring the T(1) relaxation time, the AFC can be tracked over time with high spatial resolution. The new technique was tested both in phantoms and 10 Yorkshire piglets.
Magnetic Resonance in Medicine 08/2008; 60(1):230-5. · 2.96 Impact Factor
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ABSTRACT: The ability of the distal lung epithelia to actively transport Na+, with Cl- and water following, from the alveolar spaces inversely correlates with morbidity and mortality of infants, children, and adults with alveolar pulmonary edema. It is now recognized, in contrast to many other Na+ transporting epithelia, that at least half of this active transport is not sensitive to amiloride, which inhibits the epithelial Na+ channel. This paper reviews amiloride-insensitive Na+ and fluid transport in the mammalian distal lung unit under basal conditions and speculates on potential explanations for this amiloride-insensitive transport. It also provides new information, using primary cultures of rat fetal distal lung epithelia and alveolar type II cells grown under submersion and air-liquid interface culture conditions, regarding putative blockers of this transport.
AJP Lung Cellular and Molecular Physiology 04/2008; 294(3):L401-8. · 3.66 Impact Factor
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ABSTRACT: Fetal distal lung epithelium (FDLE) must increase amiloride-sensitive epithelial Na(+) channel (ENaC) activity during the perinatal period to increase Na(+) transport and fluid clearance. Glucocorticosteroid (GC) levels increase, there is a 7-fold increase in Po(2) at birth, and we have previously shown that dexamethasone (DEX)-induced alpha-ENaC mRNA is efficiently translated only under postnatal (21%) O(2) (Otulakowski et al., AJRCMB 2006;34:204-212). Translation of mRNAs with long GC-rich 5'UTRs, such as alpha-ENaC mRNA, are sensitive to the amount of eIF4F, the mRNA 5'-cap binding complex composed of eIF4E and eIF4G. We now show, by Western blotting and m(7)GTP-Sepharose pull-down experiments, that in FDLE cultured under 3% O(2), DEX decreases formation of eIF4F and increases association of eIF4E with its inhibitor 4E-BP by changing 4E-BP phosphorylation. Conversely, FDLE cultured at 21% O(2) expressed lower levels of 4E-BP and maintained eIF4E-eIF4G association independent of DEX. Phosphorylation of 4E-BP is regulated by the kinase mTOR. Under 3% O(2), DEX decreased abundance of phosphorylated forms of the mTOR effectors, S6 kinase and ribosomal protein S6. Neither effect was associated with changes in REDD1, an upstream regulator of mTOR. When mTOR was inhibited (3 nM rapamycin) there was reduced 4E-BP phosphorylation, fewer ribosomes on alpha-ENaC mRNA, and decreased amiloride-sensitive short-circuit current, but no change in ribosomal loading onto any of beta- or gamma-ENaC or cytokeratin 18 mRNAs. We speculate that at birth increased Po(2) acts with GC through an mTOR-related pathway to increase alpha-ENaC protein synthesis, thereby promoting lung fluid absorption.
American Journal of Respiratory Cell and Molecular Biology 11/2007; 37(4):457-66. · 5.13 Impact Factor
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Shephali G Gandhi,
Bijan Rafii,
Michael S Harris,
Alexandra Garces,
Don Mahuran,
Xi-Juan Chen,
Hui-Fang Bao,
Lucky Jain,
Douglas C Eaton,
Gail Otulakowski, Hugh O'Brodovich
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ABSTRACT: We have previously shown that cardiogenic pulmonary edema fluid (EF) increases Na(+) and fluid transport by fetal distal lung epithelia (FDLE) (Rafii B, Gillie DJ, Sulowski C, Hannam V, Cheung T, Otulakowski G, Barker PM and O'Brodovich H. J Physiol 544: 537-548, 2002). We now report the effect of EF on Na(+) and fluid transport by the adult lung. We first studied primary cultures of adult type II (ATII) epithelium and found that overnight exposure to EF increased Na(+) transport, and this effect was mainly due to factors other than catecholamines. Plasma did not stimulate Na(+) transport in ATII. Purification of EF demonstrated that at least some agent(s) responsible for the amiloride-insensitive component resided within the globulin fraction. ATII exposed to globulins demonstrated a conversion of amiloride-sensitive short-circuit current (I(sc)) to amiloride-insensitive I(sc) with no increase in total I(sc). Patch-clamp studies showed that ATII exposed to EF for 18 h had increased the number of highly selective Na(+) channels in their apical membrane. In situ acute exposure to EF increased the open probability of Na(+)-permeant ion channels in ATII within rat lung slices. EF did increase, by amiloride-sensitive pathways, the alveolar fluid clearance from the lungs of adult rats. We conclude that cardiogenic EF increases Na(+) transport by adult lung epithelia in primary cell culture, in situ and in vivo.
AJP Lung Cellular and Molecular Physiology 10/2007; 293(3):L651-9. · 3.66 Impact Factor
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ABSTRACT: Edema fluid (EF) increases epithelial Na(+) transport by rat fetal distal lung epithelia (FDLE) and induces net lung fluid absorption in fetal mouse lung explants [Rafii B, Gillie DJ, Sulowski C, Hannam V, Cheung T, Otulakowski G, Barker PM, O'Brodovich H. J Physiol (Lond) 544: 537-548, 2002]. We now show that EF increases fluid absorption across monolayers of rat FDLE in a dose-dependent manner. To study the role of subunits of the epithelial Na(+) channel (ENaC) in the phenomena, we cultured explants from the distal lungs of 16-day gestational age wild-type (WT) or alpha-, beta-, or gamma-ENaC knockout or heterozygote (HT) mice. WT explants cultured in media continuously expanded over time as a result of net fluid secretion. In contrast, when explants were exposed to EF for 24 h, net fluid absorption occurred. EF-exposed explants had normal histology, but marked changes were seen after Triton X-100 or staurosporine exposure. Transmission electron microscopy showed EF promoted lamellar body formation and abundant surfactant in the explants' lumens. EF-induced changes in explant size were similar in alpha-ENaC knockout, WT, and HT littermate fetal lung explants (P > 0.05). In contrast, EF's effect was attenuated in beta- and gamma-ENaC knockouts (P < 0.05) vs. WT and HT littermate fetal lung explants. EF exposure slightly decreased or had no effect on mRNA levels for alpha-ENaC in various mouse genotypes but decreased expression of beta- and gamma-ENaC subunit mRNAs (P < 0.01) across all genotype groups. We conclude that beta- and gamma-, but not alpha-, ENaC subunits are essential for EF to exert its maximal effect on net fluid absorption by distal lung epithelia.
AJP Lung Cellular and Molecular Physiology 09/2007; 293(3):L537-45. · 3.66 Impact Factor
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ABSTRACT: The academic physicians of our department developed a novel Career Development and Compensation Program to outline job expectations, enhance career development, and provide a peer-review process to assess performance. The Career Development and Compensation Program was founded on the principle that sustained achievement in education, clinical care, or research should be valued, supported, and rewarded in an equivalent manner and that reward for clinical work should not be limited by the focus of the university on research and education. The objective of this study was to determine whether the principles of the Career Development and Compensation Program were sustained during the initial 7 years of its implementation.
The outcome of the 7 triennial reviews that occurred from 1999 to 2005 was evaluated. For the purposes of some analyses, physicians were classified as predominately clinical (clinician-specialists and clinician-teachers), predominately education (clinician-educators), or predominately research (clinician-investigators and clinician-scientists).
Each of the job profiles had a similar probability to increase a level within the Career Development and Compensation Program at the time of triennial review. Similarly, all 5 job profiles had a similar rate of increase in their level in relation to the total number of years of experience at an academic health science center. Neither the university academic rank nor gender of the physician affected the probability of increasing a level at the time of the triennial review.
The peer-reviewed Career Development and Compensation Program recognizes sustained achievement in each area of education, clinical care, and research in an equivalent manner with no detectable effect of academic rank or gender.
PEDIATRICS 05/2007; 119(4):e791-7. · 4.47 Impact Factor
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AJP Lung Cellular and Molecular Physiology 02/2007; 292(1):L4-5. · 3.66 Impact Factor
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ABSTRACT: There are no accepted and practical measures of the relative clinical and educational activities of pediatricians who work in an academic health science center. Such measures are necessary for justification of existing and future human resource plans and evaluation of the activities and performance of physicians. The limited literature on the measurement of physician workload usually focuses on a specific subspecialty group and does not account for such issues as indirect patient care, such as telephone calls or e-mail consultations; variables that affect the delivery of clinical care, including patient acuity and complexity; and the presence of students during the patient care activities. After completing a pilot study that assessed the educational workload of faculty members, we adapted existing personal digital assistant technology and software to document clinical and educational activities.
Twenty full-time physicians from 4 subspecialty pediatric divisions participated in a 2-week evaluation project in May through June 2005. Clinical activities, with and without trainees, and educational activities were collected with the use of personal digital assistants. Software allowed an individualized division-specific drop-down menu. Information that was collected included clinical (location of activity, diagnosis, and time requirement) and educational activities. After completion of a 2-week data collection period, each physician was asked to complete a 5-question evaluation form.
The project was completed successfully with capture of additional clinical and educational activities. A 5-question evaluation form was completed by 70% of the participants at the end of the 2-week data collection. Data on clinical and educational activities were analyzed qualitatively and graphed.
This method of workload data collection added significant information in capturing activities that are not measured in traditional workload evaluations for either clinical activities, such as e-mail, telephone, and patient information review, or educational endeavors, including mentoring and educational lectures and presentations.
PEDIATRICS 11/2006; 118(4):e985-91. · 4.47 Impact Factor
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ABSTRACT: Glucocorticoid hormones play an important role in fetal lung maturation. It is unknown how they interact with changes in O2 tension, which play an important role in converting the lung from a fluid-secreting to a fluid-absorbing organ at birth. Airspace fluid absorption arises from active transepithelial Na+ transport with the amiloride-sensitive epithelial Na channel (ENaC), consisting of alpha, beta, and gamma subunits, representing the rate-limiting step under nonpathologic conditions. We investigated the individual and combined effects of dexamethasone (DEX) and PO2 on alphaENaC mRNA levels, rate of alphaENaC protein synthesis, and amiloride-sensitive short-circuit current in primary cultures of rat fetal distal lung epithelial cells. DEX significantly induced alphaENaC mRNA in fetal (3%) and postnatal (21%) O2, but increases in alphaENaC protein synthesis and function occurred only when epithelia were grown under a postnatal PO2. Sucrose density gradient analyses showed that DEX treatment of cells cultured at 3% O2 decreased the association of alphaENaC mRNA with large polysomes and enhanced the association with small polysomes. Conversely, incubation of DEX-treated cells in 21% O2 restored alphaENaC mRNA association with large polysomes. No significant changes were seen in the overall polyribosome profiles or in the distribution of mRNAs encoding beta and gamma subunits of ENaC or cytokeratin 18, indicating specific modulation of alphaENaC mRNA translation. These data suggest that postnatal O2 exposure may be important for efficient translation of the alphaENaC mRNA.
American Journal of Respiratory Cell and Molecular Biology 03/2006; 34(2):204-12. · 5.13 Impact Factor
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Hugh O'Brodovich
Paediatric respiratory reviews 02/2006; 7 Suppl 1:S62-3. · 2.20 Impact Factor
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ABSTRACT: Determining fitness to fly is a difficult task for physicians, especially for those caring for children with respiratory disorders, as the available information for guidance is scarce. This case describes the use of a flight simulation in a decompression chamber in order to assess fitness to fly in an 18-mo-old infant with congenital lobar emphysema (CLE). The case discussion focuses on the need for an understanding of flight physiology in order for physicians to determine the most appropriate method to assess fitness to fly in children with medical concerns.
Aviation Space and Environmental Medicine 11/2005; 76(10):989-91. · 0.88 Impact Factor
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Hugh O'Brodovich
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ABSTRACT: To provide an overview of the pathogenesis of pulmonary edema and describe recent discoveries related to the clearance of airspace fluid and potential new therapies for this life-threatening disorder.
It is clinically important to determine the mechanisms responsible for the clearance of fluid from the airspaces. At birth inadequate clearance of fetal lung liquid is one of the two mechanisms leading to respiratory distress syndrome in the premature infant. Adults with heart failure or adult respiratory distress syndrome survive when they had active absorption of airspace fluid, yet have greater morbidity or die when they show no evidence of active fluid clearance. Humans who are susceptible to high-altitude pulmonary edema have less ability to actively transport fluid across their respiratory epithelium.
New approaches to increase the active clearance of fluid from the airspaces, combined with further improvements in the intensive care and monitoring of patients with serious illnesses, will lead to improved care for patients with lung diseases characterized by increased lung water content.
Current Opinion in Pediatrics 07/2005; 17(3):381-4. · 2.83 Impact Factor
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Journal of Pediatrics 11/2004; 145(4):425-6. · 4.11 Impact Factor
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ABSTRACT: In preparation for birth, lung epithelia must switch from net fluid secretion, required for lung development, to net absorption, which prepares the lungs for postnatal gas exchange. The apical membrane amiloride-sensitive epithelial Na channel (ENaC) is the rate-limiting step for Na+ and fluid absorption. Expression of alpha-ENaC mRNA has been detected in human lung as early as the embryonic stage of development. However, humans express multiple transcripts for alpha-ENaC, containing differing 5'-untranslated regions (UTR) with unknown effects on protein translation, and different ontogenies for individual transcripts could provide a novel mechanism for developmental regulation of ENaC function. To assess the relative expression of the two most abundant alpha-ENaC transcripts (alpha-ENaC1 and alpha-ENaC2) during lung development, we performed nonradioactive in situ hybridization using probes specific to the alternative 5'-UTRs. Both transcripts were expressed throughout intrauterine lung development (8 to 40 wk gestation), and expression was localized to the surface epithelial cells of the conductive and respiratory airways in both ciliated cells and nonciliated Clara cells. alpha-ENaC mRNA expression was also identified in the serous cells of the submucosal glands surrounding the proximal airways. In the mature prenatal lung, subsets of alveolar type II (ATII) cells expressed one or both of the alpha-ENaC transcripts. Our observations demonstrate that a developmentally regulated switch between alpha-ENaC 5'-UTR variants is not the trigger by which the developing human lung becomes a fluid-absorbing organ at birth, that individual ATII cells express neither, one, or both of the alpha-ENaC transcripts, and that the overall expression is linked to epithelial cell differentiation and lung maturation.
AJP Lung Cellular and Molecular Physiology 10/2004; 287(3):L608-15. · 3.66 Impact Factor
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ABSTRACT: The amiloride-sensitive epithelial Na(+) channel (ENaC), the rate-limiting step in epithelial Na(+) transport, consists of three subunits: alpha, beta, and gamma. The abundance of mRNA encoding the alpha-subunit far surpasses the amount for other subunits, and considerably exceeds the predicted subunit protein stoichiometry. We evaluated 5'-untranslated region (UTR) expression and found that fetal rat lung uses alternative 5'UTRs for alpha-ENaC during development. Sucrose density gradient analysis of postnuclear supernatants from fetal rat lung homogenates demonstrated that all three ENaC subunits were associated with high molecular weight polysomes, indicating active translation of the mRNAs, but translational efficiency was much lower for the alpha-subunit. Sucrose density gradient distributions were comparable for the endogenously expressed alpha-ENaC 5'UTRs in rat lung at Fetal Day 20 or Postnatal Day 1 using Northern analysis. Although birth resulted in a global decrease in lung mRNA translation, the loading of ribosomes on ENaC subunit mRNAs was largely unaffected. Evaluation of cytokeratin 18 and vimentin mRNAs in these gradients suggested a cell-specific effect. We conclude that there are different translational efficiencies for ENaC subunits and that perinatal processes globally modulate lung mRNA translation.
American Journal of Respiratory Cell and Molecular Biology 07/2004; 30(6):862-70. · 5.13 Impact Factor
