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1.
Moody M Pennington C Schultz C Caldwell R Dinkel C Rossi MW McNamara S Widdicombe J Gabriel S Traynor-Kaplan AE 《American journal of physiology. Cell physiology》2005,289(3):C512-C520
Amiloride-sensitive, epithelial Na+ channel (ENaC)-mediated, active absorption of Na+ is elevated in the airway epithelium of cystic fibrosis (CF) patients, resulting in excess fluid removal from the airway lumen. This excess fluid/volume absorption corresponds to CF transmembrane regulator-linked defects in ENaC regulation, resulting in the reduced mucociliary clearance found in CF airways. Herein we show that INO-4995, a synthetic analog of the intracellular signaling molecule, D-myo-inositol 3,4,5,6-tetrakisphosphate, inhibits Na+ and fluid absorption across CF airway epithelia, thus alleviating this critical pathology. This conclusion was based on electrophysiological studies, fluid absorption, and 22Na+ flux measurements in CF airway epithelia, contrasted with normal epithelia, and on electrophysiological studies in Madin-Darby canine kidney cells and 3T3 cells overexpressing ENaC. The effects of INO-4995 were long-lasting, dose-dependent, and more pronounced in epithelia from CF patients vs. controls. These findings support preclinical development of INO-4995 for CF treatment and demonstrate for the first time the therapeutic potential of inositol polyphosphate derivatives. epithelial Na+ channels; fluid absorption 相似文献
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Airway surface fluid volume and Cl content in cystic fibrosis and normal bronchial xenografts 总被引:1,自引:0,他引:1
We describe theuse of an in vivo human bronchial xenograft model of cystic fibrosis(CF) and non-CF airways to investigate pathophysiological alterationsin airway surface fluid (ASF) volume (Vs) and Cl content.Vs was calculated based on thedilution of an impermeable marker,[3H]inulin, duringharvesting of ASF from xenografts with an isosmotic Cl-free solution.These calculations demonstrated thatVs in CF xenographs (28 ± 3.0 µl/cm2;n = 17) was significantly less thanthat of non-CF xenografts (35 ± 2.4 µl/cm2;n = 30). The Cl concentration of ASF([Cl]s) wasdetermined using a solid-state AgCl electrode and adjusted for dilutionduring harvesting using the impermeable[3H]inulin marker.Cumulative results demonstrate small but significant elevations(P < 0.045) in[Cl]s in CF (125 ± 4 mM; n = 27) compared with non-CF(114 ± 4 mM; n = 48) xenografts.To investigate potential mechanisms by which CF airways may facilitatea higher level of fluid absorption yet retain slightly elevated levelsof Cl, we sought to evaluate the capacity of CF and non-CF airways toabsorb both 22Na and36Cl. Two consistent findings wereevident from these studies. First, in both CF and non-CF xenografts,22Na and36Cl were always absorbed in anequal molar ratio. Second, CF xenografts hyperabsorbed (~1.5-foldhigher) both 22Na and36Cl compared with non-CFxenografts. These results substantiate previously documented findingsof elevated Na absorption in CF airways and also suggest that theslightly elevated[Cl]s found in thisstudy of CF xenograft epithelia does not occur through a mechanism ofdecreased apical permeability to Cl. 相似文献
3.
Chen JH Stoltz DA Karp PH Ernst SE Pezzulo AA Moninger TO Rector MV Reznikov LR Launspach JL Chaloner K Zabner J Welsh MJ 《Cell》2010,143(6):911-923
Defective transepithelial electrolyte transport is thought to initiate cystic fibrosis (CF) lung disease. Yet, how loss of CFTR affects electrolyte transport remains uncertain. CFTR?(/)? pigs spontaneously develop lung disease resembling human CF. At birth, their airways exhibit a bacterial host defense defect, but are not inflamed. Therefore, we studied ion transport in newborn nasal and tracheal/bronchial epithelia in tissues, cultures, and in vivo. CFTR?(/)? epithelia showed markedly reduced Cl? and HCO?? transport. However, in contrast to a widely held view, lack of CFTR did not increase transepithelial Na(+) or liquid absorption or reduce periciliary liquid depth. Like human CF, CFTR?(/)? pigs showed increased amiloride-sensitive voltage and current, but lack of apical Cl? conductance caused the change, not increased Na(+) transport. These results indicate that CFTR provides the predominant transcellular pathway for Cl? and HCO?? in porcine airway epithelia, and reduced anion permeability may initiate CF airway disease. 相似文献
4.
Abnormal localization of cystic fibrosis transmembrane conductance regulator in primary cultures of cystic fibrosis airway epithelia
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Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a membrane glycoprotein that forms Cl- channels. Previous work has shown that when some CF-associated mutants of CFTR are expressed in heterologous cells, their glycosylation is incomplete. That observation led to the hypothesis that such mutants are not delivered to the plasma membrane where they can mediate Cl- transport. Testing this hypothesis requires localization of CFTR in nonrecombinant cells and a specific determination of whether CFTR is in the apical membrane of normal and CF epithelia. To test the hypothesis, we used primary cultures of airway epithelia grown on permeable supports because they polarize and express the CF defect in apical Cl- permeability. Moreover, their dysfunction contributes to disease. We developed a semiquantitative assay, using nonpermeabilized epithelia, an antibody directed against an extracellular epitope of CFTR, and large (1 microns) fluorescent beads which bound to secondary antibodies. We observed specific binding to airway epithelia from non-CF subjects, indicating that CFTR is located in the apical membrane. In contrast, there was no specific binding to the apical membrane of CF airway epithelia. These data were supported by qualitative studies using confocal microscopy: the most prominent immunostaining was in the apical region of non-CF cells and in cytoplasmic regions of CF cells. The results indicate that CFTR is either missing from the apical membrane of these CF cells or it is present at a much reduced level. The data support the proposed defective delivery of some CF-associated mutants to the plasma membrane and explain the lack of apical Cl- permeability in most CF airway epithelia. 相似文献
5.
Polarized signaling via purinoceptors in normal and cystic fibrosis airway epithelia 总被引:8,自引:0,他引:8
Airway epithelia are confronted with distinct signals emanating from the luminal and/or serosal environments. This study tested whether airway epithelia exhibit polarized intracellular free calcium (Ca(2+)(i)) and anion secretory responses to 5' triphosphate nucleotides (ATP/UTP), which may be released across both barriers of these epithelia. In both normal and cystic fibrosis (CF) airway epithelia, mucosal exposure to ATP/UTP increased Ca(2+)(i) and anion secretion, but both responses were greater in magnitude for CF epithelia. In CF epithelia, the mucosal nucleotide-induced response was mediated exclusively via Ca(2+)(i) interacting with a Ca(2+)-activated Cl(-) channel (CaCC). In normal airway epithelia (but not CF), nucleotides stimulated a component of anion secretion via a chelerythrine-sensitive, Ca(2+)-independent PKC activation of cystic fibrosis transmembrane conductance regulator. In normal and CF airway epithelia, serosally applied ATP or UTP were equally effective in mobilizing Ca(2+)(i). However, serosally applied nucleotides failed to induce anion transport in CF epithelia, whereas a PKC-regulated anion secretory response was detected in normal airway epithelia. We conclude that (1) in normal nasal epithelium, apical/basolateral purinergic receptor activation by ATP/UTP regulates separate Ca(2+)-sensitive and Ca(2+)-insensitive (PKC-mediated) anion conductances; (2) in CF airway epithelia, the mucosal ATP/UTP-dependent anion secretory response is mediated exclusively via Ca(2+)(i); and (3) Ca(2+)(i) regulation of the Ca(2+)-sensitive anion conductance (via CaCC) is compartmentalized in both CF and normal airway epithelia, with basolaterally released Ca(2+)(i) failing to activate CaCC in both epithelia. 相似文献
6.
Gao L Broughman JR Iwamoto T Tomich JM Venglarik CJ Forman HJ 《American journal of physiology. Lung cellular and molecular physiology》2001,281(1):L24-L30
Cystic fibrosis (CF), an inherited disease characterized by defective epithelial Cl- transport, damages lungs via chronic inflammation and oxidative stress. Glutathione, a major antioxidant in the epithelial lung lining fluid, is decreased in the apical fluid of CF airway epithelia due to reduced glutathione efflux (Gao L, Kim KJ, Yankaskas JR, and Forman HJ. Am J Physiol Lung Cell Mol Physiol 277: L113-L118, 1999). The present study examined the question of whether restoration of chloride transport would also restore glutathione secretion. We found that a Cl- channel-forming peptide (N-K4-M2GlyR) and a K+ channel activator (chlorzoxazone) increased Cl- secretion, measured as bumetanide-sensitive short-circuit current, and glutathione efflux, measured by high-performance liquid chromatography, in a human CF airway epithelial cell line (CFT1). Addition of the peptide alone increased glutathione secretion (181 +/- 8% of the control value), whereas chlorzoxazone alone did not significantly affect glutathione efflux; however, chlorzoxazone potentiated the effect of the peptide on glutathione release (359 +/- 16% of the control value). These studies demonstrate that glutathione efflux is associated with apical chloride secretion, not with the CF transmembrane conductance regulator per se, and the defect of glutathione efflux in CF can be overcome pharmacologically. 相似文献
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Pseudomonas aeruginosa induces changes in fluid transport across airway surface epithelia 总被引:3,自引:0,他引:3
Evans D. J.; Matsumoto P. S.; Widdicombe J. H.; Li-Yun C.; Maminishkis A. A.; Miller S. S. 《American journal of physiology. Cell physiology》1998,275(5):C1284
Fluid transport across cultures of bovine tracheal epitheliumwas measured with a capacitance probe technique. Baseline fluid absorption (Jv)across bovine cells of 3.2 µl · cm2 · h1was inhibited by ~78% after 1 h of exposure to suspensions of Pseudomonas aeruginosa, with aconcomitant decrease in transepithelial potential (TEP) and increase intransepithelial resistance(Rt). Effectsof P. aeruginosa were blocked byamiloride, which decreased Jv by 112% frombaseline of 2.35 ± 1.25 µl · cm2 · h1,increased Rt by101% from baseline of 610 ± 257 · cm2, anddecreased TEP by 91% from baseline of 55 ± 18.5 mV.Microelectrode studies suggested that effects of P. aeruginosa on amiloride-sensitive Na absorption weredue in part to a block of basolateral membrane K channels. In thepresence of Cl transport inhibitors[5-nitro-2-(3-phenylpropylamino)-benzoic acid,H2-DIDS, and bumetanide],P. aeruginosa induced a fluid secretion of ~2.5 ± 0.4 µl · cm2 · h1and decreased Rtwithout changing TEP. However, these changes were abolished when thetransport inhibitors were used in a medium in which Cl was replaced byan impermeant organic anion. Filtrates of P. aeruginosa suspensions had no effect onJv, TEP, orRt. Mutantslacking exotoxin A or rhamnolipids or with defective lipopolysaccharide still inhibited fluid absorption and altered bioelectrical properties. By contrast, mutations in the rpoN gene encodinga factor of RNA polymerase abolished actions of P. aeruginosa. In vivo, changes in transepithelial saltand water transport induced by P. aeruginosa may alter viscosity and ionic composition ofairway secretions so as to foster further bacterial colonization. 相似文献
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We haveinvestigated the role in vivo of mineralocorticoid and glucocorticoidhormones in regulating the rate of electrogenic amiloride-sensitiveNa+ absorption across murineairway tissue studied in vivo (nasal potential difference) and in vitro(Ussing chambers). We found that elevating the plasma aldosteroneconcentration 10-fold (low-Na+diet) had no significant effect on amiloride-sensitiveNa+ absorption across tracheal ornasal epithelia. High doses of dexamethasone for 1 wk likewise did notchange the rate of amiloride-sensitive Na+ absorption across airwayepithelia. In contrast, both hormonal manipulations elevated the rateof colonic Na+ absorption.Furthermore, adrenalectomy (both normal and cystic fibrosis mice) alsofailed to alter Na+ absorptionacross airway epithelia. We conclude that, in vivo, neither themineralocorticoid nor the glucocorticoid hormones significantlyregulate the rates of amiloride-sensitive electrogenic Na+ absorption across airwayepithelia in the adult mouse. 相似文献
12.
Miller TJ Davis PB 《American journal of physiology. Lung cellular and molecular physiology》2008,294(4):L654-L664
FXYD5, also known as dysadherin, belongs to a family of tissue-specific regulators of the Na(+)-K(+)-ATPase. We determined the kinetic effects of FXYD5 on Na(+)-K(+)-ATPase pump activity in stably transfected Madin-Darby canine kidney cells. FXYD5 significantly increased the apparent affinity for Na(+) twofold and decreased the apparent affinity for K(+) by 60% with a twofold increase in V(max) of K(+), a pattern that would increase activity and Na(+) removal from the cell. To test the effect of increased Na(+) uptake on FXYD5 expression, we analyzed Madin-Darby canine kidney cells stably transfected with an inducible vector expressing all three subunits of the epithelial Na(+) channel (ENaC). Na(+)-K(+)-ATPase activity increased sixfold after 48-h ENaC induction, but FXYD5 expression decreased 75%. FXYD5 expression was also decreased in lung epithelia from mice that overexpress ENaC, suggesting that chronic Na(+) absorption by itself downregulates epithelial FXYD5 expression. Patients with cystic fibrosis (CF) display ENaC-mediated hyperabsorption of Na(+) in the airways, accompanied by increased Na(+)-K(+)-ATPase activity. However, FXYD5 was significantly increased in the lungs and nasal epithelium of CF mice as assessed by RT-PCR, immunohistochemistry, and immunoblot analysis (P < 0.001). FXYD5 was also upregulated in nasal scrapings from human CF patients compared with controls (P < 0.02). Treatment of human tracheal epithelial cells with a CFTR inhibitor (I-172) confirmed that loss of CFTR function correlated with increased FXYD5 expression (P < 0.001), which was abrogated by an inhibitor of NF-kappaB. Thus FXYD5 is upregulated in CF epithelia, and this change may exacerbate the Na(+) hyperabsorption and surface liquid dehydration observed in CF airway epithelia. 相似文献
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J H Widdicombe 《Biorheology》1987,24(6):551-555
Airway epithelia possess transepithelial ion transport processes which may help to regulate the fluid content of airway secretions. Chloride secretion promotes fluid movement from blood to airway lumen. Active absorption of Na favours fluid movement in the opposite direction. The balance between these two processes can be altered by a number of agents which stimulate Cl secretion. The importance of ion transport for normal mucociliary clearance is suggested by the finding that airway epithelia in patients with cystic fibrosis are unable to secrete chloride. This defect may cause the characteristically sticky and tenacious mucous secretions which are the major cause of death in this disease. 相似文献
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Trinh NT Privé A Maillé E Noël J Brochiero E 《American journal of physiology. Lung cellular and molecular physiology》2008,295(5):L866-L880
Severe lesions of airway epithelia are observed in cystic fibrosis (CF) patients. The regulatory mechanisms of cell migration and proliferation processes, involved in the repair of injured epithelia, then need to be better understood. A model of mechanical wounding of non-CF (NuLi) and CF (CuFi) bronchial monolayers was employed to study the repair mechanisms. We first observed that wound repair, under paracrine and autocrine EGF control, was slower (up to 33%) in CuFi than in NuLi. Furthermore, EGF receptor (EGFR) activation, following wounding, was lower in CuFi than in NuLi monolayers. Cell proliferation and migration assays indicated a similar rate of proliferation in both cell lines but with reduced (by 25%) CuFi cell migration. In addition, cell migration experiments performed in the presence of conditioned medium, collected from NuLi and CuFi wounded bronchial monolayers, suggested a defect in EGF/EGFR signaling in CF cells. We (49) recently demonstrated coupling between the EGF response and K(+) channel function, which is crucial for EGF-stimulated alveolar repair. In CuFi cells, lower EGF/EGFR signaling was accompanied by a 40-70% reduction in K(+) currents and KvLQT1, ATP-sensitive potassium (K(ATP)), and Ca(2+)-activated K(+) (KCa3.1) channel expression. In addition, EGF-stimulated bronchial wound healing, cell migration, and proliferation were severely decreased by K(+) channel inhibitors. Finally, acute CFTR inhibition failed to reduce wound healing, EGF secretion, and K(+) channel expression in NuLi. In summary, the delay in CuFi wound healing could be due to diminished EGFR signaling coupled with lower K(+) channel function, which play a crucial role in bronchial repair. 相似文献
15.
Ribeiro CM Paradiso AM Schwab U Perez-Vilar J Jones L O'neal W Boucher RC 《The Journal of biological chemistry》2005,280(18):17798-17806
Hyperinflammatory responses to infection have been postulated as a component of cystic fibrosis (CF) lung disease. Studies have linked intracellular calcium (Ca(2+)(i)) mobilization with inflammatory responses in several systems. We have reported that the pro-inflammatory mediator bradykinin (BK) promotes larger Ca(2+)(i) signals in CF compared with normal bronchial epithelia, a response that reflects endoplasmic reticulum (ER)/Ca(2+) store expansion induced by chronic luminal airway infection/inflammation. The present study investigated whether CF airway epithelia were hyperinflammatory and, if so, whether the hyperinflammatory CF phenotype was linked to larger Ca(2+) stores in the ER. We found that DeltaF508 CF bronchial epithelia were hyperinflammatory as defined by an increased basal and mucosal BK-induced interleukin (IL)-8 secretion. However, the CF hyperinflammation expressed in short-term (6-11-day-old) primary cultures of DeltaF508 bronchial epithelia was lost in long-term (30-40-day-old) primary cultures of DeltaF508 bronchial epithelia, indicating this response was independent of mutant cystic fibrosis transmembrane conductance regulator. Exposure of 30-40-day-old cultures of normal airway epithelia to supernatant from mucopurulent material (SMM) from CF airways reproduced the increased basal and mucosal BK-stimulated IL-8 secretion of short-term CF cultures. The BK-triggered increased IL-8 secretion in SMM-treated cultures was mediated by an increased Ca(2+)(i) mobilization consequent to an ER expansion associated with increases in protein synthesis (total, cytokines, and antimicrobial factors). The increased ER-dependent, Ca(2+)(i)-mediated hyperinflammatory epithelial response may represent a general beneficial airway epithelial adaptation to transient luminal infection. However, in CF airways, the Ca(2+)(i)-mediated hyperinflammation may be ineffective in promoting the eradication of infection in thickened mucus and, consequently, may have adverse effects in the lung. 相似文献
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Soluble mediators, not cilia, determine airway surface liquid volume in normal and cystic fibrosis superficial airway epithelia
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A key aspect of the lung's innate defense system is the ability of the superficial epithelium to regulate airway surface liquid (ASL) volume to maintain a 7-mum periciliary liquid layer (PCL), which is required for cilia to beat and produce mucus flow. The mechanisms whereby airway epithelia regulate ASL height to >or=7 microm are poorly understood. Using bumetanide as an inhibitor of Cl- secretion, and nystatin as an activator of Na+ absorption, we found that a coordinated "blending" of both Cl- secretion and Na+ absorption must occur to effect ASL volume homeostasis. We then investigated how ASL volume status is regulated by the underlying epithelia. Cilia were not critical to this process as (a) ASL volume was normal in cultures from patients with primary ciliary dyskinesia with immotile cilia, and (b) in normal cultures that had not yet undergone ciliogenesis. However, we found that maneuvers that mimic deposition of excess ASL onto the proximal airways, which occurs during mucociliary clearance and after glandular secretion, acutely stimulated Na+ absorption, suggesting that volume regulation was sensitive to changes in concentrations of soluble mediators in the ASL rather than alterations in ciliary beating. To investigate this hypothesis further, we added potential "soluble mediators" to the ASL. ASL volume regulation was sensitive to a channel-activating protein (CAP; trypsin) and a CAP inhibitor (aprotinin), which regulated Na+ absorption via changes in epithelial Na+ channel (ENaC) activity in both normal and cystic fibrosis cultures. ATP was also found to acutely regulate ASL volume by inducing secretion in normal and cystic fibrosis (CF) cultures, while its metabolite adenosine (ADO) evoked secretion in normal cultures but stimulated absorption in CF cultures. Interestingly, the amount of ASL/Cl- secretion elicited by ATP/ADO was influenced by the level of CAP-induced Na+ absorption, suggesting that there are important interactions between the soluble regulators which finely tune ASL volume. 相似文献
18.
Puchelle E 《Journal de la Société de Biologie》2002,196(1):29-35
Cystic fibrosis (CF) is the most common genetic autosomal recessive disease in caucasian north-american and european populations. The CF gene codes for a transmembrane glycoprotein called CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), a chloride channel which regulates the luminal secretion of chloride and the active ion and water transport in the airway epithelial cells. Mutations of the CF gene lead to a dysregulation of chloride and sodium channel associated to airway mucus dehydration, neutrophil-dominated airway inflammation and chronic infection responsible for the morbidity and mortality of CF patients. Although a high number of studies has been devoted to the CFTR pleiotropic functions, the chronology of the physiopathological events leading to the airway inflammation linked to mutations of the CF gene is still an open question. The issue of whether airway inflammation takes place before infection or is a consequence of infection during CF pathogenesis is still controversial. It has been recently reported that in broncho-alveolar lavages collected in CF infants, there is an increased level of interleukin IL-8 and abnormal low level of IL-10. The decreased IL-10 production has been confirmed in peripheral blood monocytes as well as in airway cell lines. Under basal conditions, the increased expression of the pro-inflammatory IL-8 cytokine has also been recently observed in the airway liquid secreted by CF na?ve humanized airway xenografts and in the supernatant culture of CF human airway epithelial cells. These results suggest that CFTR dysfunction may result in a constitutive pro-inflammatory vs anti-inflammatory imbalance in CF disease. Recent data from the literature suggest that the failure of chloride transport, the maturation defect and mistraffricking of mutated CFTR, lead to its accumulation in the endoplasmic reticulum and activation of NF-kappa B, responsible for the imbalance in the CF airway cell cytokine production. 相似文献
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Arniges M Vázquez E Fernández-Fernández JM Valverde MA 《The Journal of biological chemistry》2004,279(52):54062-54068
The vertebrate transient receptor potential cationic channel TRPV4 has been proposed as an osmo- and mechanosensor channel. Studies using knock-out animal models have further emphasized the relevance of the TRPV4 channel in the maintenance of the internal osmotic equilibrium and mechanosensation. However, at the cellular level, there is still one important question to answer: does the TRPV4 channel generate the Ca(2+) signal in those cells undergoing a Ca(2+)-dependent regulatory volume decrease (RVD) response? RVD in human airway epithelia requires the generation of a Ca(2+) signal to activate Ca(2+)-dependent K(+) channels. The RVD response is lost in airway epithelia affected with cystic fibrosis (CF), a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator channel. We have previously shown that the defective RVD in CF epithelia is linked to the lack of swelling-dependent activation of Ca(2+)-dependent K(+) channels. In the present study, we show the expression of TRPV4 in normal human airway epithelia, where it functions as the Ca(2+) entry pathway that triggers the RVD response after hypotonic stress, as demonstrated by TRPV4 antisense experiments. However, cell swelling failed to trigger Ca(2+) entry via TRPV4 channels in CF airway epithelia, although the channel's response to a specific synthetic activator, 4 alpha-phorbol 12,13-didecanoate, was maintained. Furthermore, RVD was recovered in CF airway epithelia treated with 4 alpha-phorbol 12,13-didecanoate. Together, these results suggest that defective RVD in CF airway epithelia might be caused by the absence of a TRPV4-mediated Ca(2+) signal and the subsequent activation of Ca(2+)-dependent K(+) channels. 相似文献