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J H Widdicombe

Publications and source records attributed to J H Widdicombe.

At least 37 records · Page 2Linked to original sources
PubMed1997

Cultured lung epithelium: A cellular model for lung preservation.

Cellular models have helped with the development of conditions needed for hypothermic preservation of kidney, liver, and heart. Recently, highly differentiated cultured lung epithelial cell lines grown with basolateral side feeding technique have become available that can mimic airspace, epithelium, and interstitium of lung parenchyma. Cultured lung epithelium coupled with Ussing's short-circuit current technique was used as a cellular model system for lung preservation. A parametric study was conducted to correlate the effects of luminal fluid composition (University of Wisconsin (UW) solution and phosphate-buffered saline) and storage gas (air vs nitrogen) at 4 degrees C for 24 h on postischemic electrogenic properties (transepithelial ion transport and resistance). The results showed that cells were better preserved with the UW solution on both sides as measured by their transepithelial resistance, an indicator of tight junction integrity (Rte approximately 65% of control values approximately 135 Omega cm2). In addition, they responded better to mediators that stimulate chloride secretion than cells preserved with other conditions. Cells preserved with no additional fluid on the apical side had substantially lowered Rte (<20%) than those preserved with an additional thin layer of fluid ( approximately 35-65%). This cellular model system is a realistic representation of lung epithelium and can provide an accurate assessment of preservation quality through the measurements of tight junction integrity and active ion transport.

Adenosine↗
PubMed1997

Hepatocyte growth factor stimulates the differentiation of human tracheal epithelia in vitro.

Hepatocyte growth factor (HGF) can influence epithelial cell growth and differentiation. We examined the actions of recombinant human HGF (rhHGF) on the differentiation of human primary tracheal epithelial (HTE) cells cultured in vitro for up to 96 h. Basolateral, but not apical, treatment of confluent HTE cell sheets for 48 h with rhHGF led to increases in cell height, cell volume, cilia, and total protein content. Basolateral rhHGF treatment produced a decrease in HGF receptor (c-met) expression but had no effect on c-met mRNA levels. HTE cell sheets treated with rhHGF for 48 h showed a significant increase in mediator-induced Cl- secretion and a decrease in amiloride-sensitive sodium absorption. No effect on transepithelial resistance was observed with rhHGF treatment. The enhancement of short-circuit responses by basolateral rhHGF was dose dependent. Our results demonstrate that rhHGF has hypertrophic actions on, and can influence the differentiation of, human airway epithelia in vitro, presumably through the activation of c-met at the basolateral surface of these cells.

Amiloride↗
PubMed1997

Regulation of depth and composition of airway surface liquid.

The depth and composition of human airway surface liquid (ASL) may depend on secretion from airway glands, ion transport across the surface epithelium, goblet cell discharge, transepithelial gradients in hydrostatic pressure, and surface tension. Published values for the frequency of airway glands and for the secretory rates of individual glands suggest that total gland secretion in human trachea can amount to approximately 60 microL x cm(-2) x h(-1). Volume absorption directly measured across cultures of surface epithelium from human trachea is approximately 5 microL x cm(-2) x h(-1). These flows should alter the depth of ASL at +10 and -1 microm x min(-1). We have looked for changes in ASL depth of this magnitude using low-temperature scanning electron microscopy (LT-SEM) of rapidly frozen specimens of bovine trachea. Stimulation of gland secretion with methacholine led to an initial increase in depth of approximately 25 microm x min(-1) followed by a decline at approximately 1.5 microm x min(-1). Whereas the initial increase in depth was probably due to transient gland secretion, the later decline reflected active absorption of liquid across the surface epithelium. Finally, we present preliminary data showing that LT-SEM can be combined with X-ray microanalysis to determine the elemental composition of ASL.

Adult↗
PubMed1996

Intraluminal water increases expression of plasmid DNA in rat lung.

Effective gene delivery to specific organs is a major goal for human gene therapy. The lung's structure allows instillation of agents into the airspaces, directly adjacent to the lung epithelium. We hypothesized that the airspace instillation of hypotonic solutions would increase the permeability of the lung epithelium and increase DNA uptake. This hypothesis was tested by instilling plasmid DNA (p4241) encoding the luciferase gene in isotonic and hypotonic solutions. The highest luciferase expression in the lung was achieved after the instillation of this plasmid DNA in distilled water. Aerosolization of water just before the instillation of the plasmid DNA also enhanced the expression level of luciferase in the lung. In addition, an intralobar instillation of the plasmid DNA in water significantly increased the luciferase expression, suggesting that the instillation of the plasmid over a smaller surface area increased expression. Levels of expression could be measured for 3 days. Water increases the permeability of lung epithelial cells transiently and/or enhances gene expression and can be used to achieve gene expression in the lung airspaces for short intervals without toxicity.

Animals↗
PubMed1996

Ultrastructure of tracheal surface liquid: low-temperature scanning electron microscopy.

A layer of liquid lines the airways in the lung. Previous microscopic studies have suggested that it is in two phases, with a mucous gel lying above a periciliary sol. However, shrinkage artifacts due to chemical fixation, dehydration, and drying have prevented reliable estimates of the depth of these layers. To avoid such problems, we have studied the surface liquid of bovine trachea by low-temperature scanning electron microscopy (LTSEM). A polished copper probe cooled to liquid nitrogen temperature was applied to the mucosal surface of sheets of excised tracheal epithelium to effect rapid freezing of surface liquid. Tissue sheets were then mounted in an LTSEM (AMRay 1000A with Biochamber) which maintains samples at -180 degrees C with a Joule-Thompson refrigerator built into the stage. Tissues were fractured at right angles to the epithelial surface, coated with gold, and viewed, all at 10(-5) to 10(-6) torr without transfer through air. The sample was stable under the electron beam at accelerating voltages up to 20 kV. Epithelial features (nuclei, cilia, microvilli, mucous granules) were well preserved. The mucosal surface of the cells was covered with material on the order of 8 microns in depth. The mucous gel and periciliary sol could be seen as distinct layers and could be distinguished by the size and pattern of ice crystal voids generated by radiant-etching of the fractured surface of the sample.

Animals↗
PubMed1996

Transient permeabilization of airway epithelium by mucosal water.

We describe a simple hyposmotic shock procedure whereby the apical membrane of airway epithelium can be made transiently leaky to proteins and other macromolecules. Bovine or human tracheal epithelial cells were grown as confluent polarized cell sheets on porous inserts. While physiological saline was maintained on the basolateral surface, the mucosal surface was exposed to water. This led to marked increases in the uptake of [14C]mannitol across both apical and basolateral membranes. On restoring saline to the mucosal surface, the [14C]mannitol permeability returned to preexposure levels with a half-life of approximately 5 min. Mucosal water also increased efflux of lactate dehydrogenase and the uptakes of fluorescent albumin and dextran (2,000 kDa). Water-induced increases in mannitol permeability were similar at 4 and 37 degrees C, suggesting that pinocytosis was not the mechanism. Detailed time courses of the uptake of dextran and the loss of lactate dehydrogenase and 36Cl showed that the bulk of the permeability increase occurred during the first 2- to 4-min exposure to water. Transepithelial resistance was reversibly decreased by exposure to water, but short-circuit current responses to transport blockers and secretagogues remained qualitatively normal. The hyposmotic shock procedure also successfully permeabilized apical membranes of primary cultures of nasal epithelial cells from a patient with cystic fibrosis (CF) and of JME/CF 15 cells, a cell line derived from CF bronchial epithelium. This simple and efficient procedure may prove useful in studies on the cell and molecular biology of airway and other epithelia.

Animals↗
PubMed1995

Effects of lovastatin on trafficking of cystic fibrosis transmembrane conductance regulator in human tracheal epithelium.

Genetic defects in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel, cause cystic fibrosis. Most defective forms of CFTR show improper intracellular trafficking. Because isoprenylated, small GTP-binding proteins are involved in the vesicular trafficking of other integral membrane proteins, we have investigated the role of isoprenylation in the trafficking of CFTR to the apical membranes of primary cultures of human airway epithelium and of Calu-3 cells, a human lung carcinoma cell line. CFTR function was measured as short circuit current, 125I efflux, and conductance of cell sheets with permeabilized basolateral membranes. Lovastatin, an inhibitor of isoprenyl lipid biosynthesis, markedly inhibited all measures of CFTR function. The lovastatin-induced declines in CFTR function were corrected by the simultaneous addition of mevalonate or the isoprenyl lipids geranylgeranyl and farnesyl but not cholesterol. Lovastatin reduced total cellular CFTR as assessed by immunoprecipitation. Mevalonate or isoprenyl lipids protected CFTR levels from the actions of lovastatin. Together, these results suggest a role for isoprenyl lipids, presumably through the actions of small GTP-binding proteins, in the trafficking of CFTR to the apical membrane of human airway epithelium.

Biological Transport↗
PubMed1995

Regulation of human airway surface liquid.

Human airways are lined with a film of liquid from 5-100 microns in depth, consisting of a periciliary sol around and a mucous gel above the cilia. Microscopical studies have shown the sol to be invariably the same depth as the length of the cilia, and we discuss possible reasons for this. The composition and sources of the airway surface liquid are also described. In addition the forces regulating its volume are analyzed. Several airway diseases are characterised by dramatic changes in the volume and composition of airway liquid. We review recent research suggesting that the accumulation of airway mucous secretions in cystic fibrosis is caused by alterations in active transport of ions and water across both the surface and gland epithelia.

Animals↗
PubMed1995

cAMP-independent activation of CFTR Cl channels by the tyrosine kinase inhibitor genistein.

Genistein, a protein tyrosine kinase inhibitor, activates the cystic fibrosis transmembrane conductance regulator (CFTR) in transfected NIH-3T3 fibroblasts that express the CFTR (3T3-CFTR). CFTR activity was assayed by 125I efflux and by patch clamping in the cell-attached mode. Both forskolin and genistein stimulated 125I efflux and activated a 9-10 pS anion channel in 3T3-CFTR cells but failed to activate 125I efflux in mock-transfected NIH-3T3 cells. Genistein, unlike forskolin and 3-isobutyl-1-methylxanthine, did not increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) above control levels. This demonstrates that genistein-dependent activation does not involve inhibition of phosphodiesterase activity and suggests that stimulation does not involve a direct activation of protein kinase A. Genistein stimulated 125I efflux to approximately 50% of the maximal rate with forskolin. Genistein did not increase 125I efflux at saturating forskolin but decreased the concentration of forskolin required for half-maximal stimulation. Orthovanadate (VO4), a phosphotyrosine phosphatase inhibitor, inhibited genistein-induced channel activation with an inhibition constant of approximately 20 microM. These effects suggest that, in addition to activation by protein kinase A, the CFTR is regulated by a tyrosine kinase-dependent pathway.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Culture and transformation of human airway epithelial cells.

The culture of human airway epithelial cells has played an important role in advancing our understanding of the metabolic and molecular mechanisms underlying normal function and disease pathology of airway epithelial cells. Recent advances in culturing primary epithelial cells and the development of transformed airway epithelial cell lines have been particularly important in enhancing our understanding of the pathology associated with cystic fibrosis and lung cancer. The establishment of conditions that enhance the proliferative capacity of airway epithelial cells in primary culture was the first technical hurdle overcome in the development of in vitro culture systems. Research is now being geared toward the development of cell culture conditions that facilitate the expression in culture of the differentiated characteristics found in the native epithelium. Aside from the advances that have been made in defining the growth media and extracellular matrixes that enhance the expression of differentiated features, the use of an air-liquid interface has been a significant advance in the culture of airway epithelial cells. The implementation of the in vitro cell culture systems that have now been established and the research into optimizing the conditions for the growth of airway epithelial cells have been and will continue to be essential in the development of therapies for airway disease.

Animals↗
PubMed1995

Role of CFTR in chloride secretion across human tracheal epithelium.

We have tested two hypotheses: 1) the cystic fibrosis transmembrane conductance regulator (CFTR) represents the predominant Cl conductance in the apical membrane of human tracheal epithelium, and 2) CFTR in this tissue is close to maximally activated under baseline conditions. In support of the first hypothesis, we found 1) when the level of differentiation of cultures was varied by varying the culture conditions, there was a significant positive correlation between the levels of CFTR and the magnitude of mediator-induced Cl secretion. 2) Amiloride-insensitive baseline short-circuit current (Isc) and mediator-induced increases in Isc were inhibited by diphenylamine-2-carboxylic acid (DPAC) but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a pharmacology consistent with passage of apical membrane Cl current through CFTR; Ca-activated Cl channels are inhibited by DIDS but not by DPAC. 3) Raising temperature from 22 degrees to 37 degrees C increased 125I efflux, and this increase was inhibited by DPAC and blockers of protein kinase A, but not by DIDS or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester. In support of the second hypothesis, we have earlier shown [M. Yamaya, W.E. Finkbeiner, S.Y. Chun, and J.H. Widdicombe. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L713-L724, 1992] that adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents are essentially without effect on Isc across primary cultures of human tracheal epithelium. Here, we further show that these agents are also usually without effect on 125I efflux; the mean increase in efflux in response to elevating cAMP was approximately 20% that of raising temperature from 22 degrees to 37 degrees C.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1995

A method for measuring Cl efflux from dispersed cells of airway epithelium.

This paper describes a method for measuring the increase in halide permeability of isolated airway epithelial cells induced by adenosine 3',5'-cyclic monophosphate (cAMP). Suspensions of isolated cells, known to contain the cystic fibrosis transmembrane conductance regulator (CFTR), were placed in the upper part of a Swinnex filter holder containing a filter with pores of 0.65 micron diameter. Medium was perfused over the cells at room temperature and collected at minute intervals following its passage through the filter. Experiments were performed on Calu-3 and T84 cells (human lung and colonic epithelial cell lines), primary cultures of dog and human tracheal epithelium, and Swiss 3T3 fibroblasts stably transfected with CFTR. In all cell types, addition of agents that elevate cAMP led to increases in the rates of loss of 36Cl and 125I. However, in human tracheal epithelial cells, warming the medium from room temperature to 37 degrees C was a more effective way of stimulating tracer efflux. Increases in efflux in response to either temperature or cAMP-elevating agents were inhibited by diphenylamine-2-carboxylate, a blocker of CFTR. Reproducible increases in tracer efflux were seen with as few as 10(6) cells. Cells that had been trypsinized off their culture dishes responded better than cells that had been scraped off, although treatment of scraped cells with trypsin enhanced their responsiveness to cAMP-elevating agents. Cystic fibrosis is characterized by the lack of a cAMP-activated Cl conductance in the apical membrane of airway epithlia.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Radiotracer studies of cystic fibrosis transmembrane conductance regulator expressed in Xenopus oocytes.

We measured fluxes of radiotracers in Xenopus oocytes expressing the cystic fibrosis transmembrane conductance regulator (CFTR). Addition of adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents [forskolin and 3-isobutyl-1-methylxanthine (I/F)] led to large increases in uptake of 36Cl, 125I, and 82Br into oocytes expressing wild-type CFTR or delta F508 CFTR but not sham-injected oocytes. I/F also stimulated halide efflux from CFTR and delta F508 oocytes in the sequence Cl > Br > I. cAMP-induced increases in 36Cl efflux from delta F508 oocytes were approximately 20% of those in CFTR oocytes. Increases in halide efflux were blocked by diphenylamine-2-carboxylic acid but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The phorbol ester, phorbol 12-myristate 13-acetate, also stimulated 36Cl efflux from CFTR oocytes. ATP uptakes into CFTR and sham oocytes were similar, and both were reduced by I/F. However, ATP uptake into I/F-treated CFTR oocytes was slightly greater (approximately 40%) than into I/F-treated sham oocytes. Urea uptake into CFTR and sham oocytes was similar and in both cases was increased by I/F. However, the I/F-induced increase in urea uptake into CFTR oocytes was significantly greater than for sham oocytes. I/F stimulated formate uptake into CFTR oocytes but not into sham oocytes. Fluxes of 22Na, 86Rb, 35SO4, 32PO4, and mannitol were unaltered by expression and activation of CFTR.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Calu-3: a human airway epithelial cell line that shows cAMP-dependent Cl- secretion.

Of 12 cell lines derived from human lung cancers, only Calu-3 cells showed high transepithelial resistance (Rte) and increases in short-circuit current (Isc) in response to mediators. Calu-3 cells formed polarized monolayers with tight junctions and Rte of approximately 100 omega.cm2. Baseline Isc was approximately 35 microA/cm2 and was increased by approximately 75 microA/cm2 on elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) by isoproterenol. Flux studies showed that the increase in Isc was due to Cl- secretion. Forskolin and permeant analogues of cAMP also increased Isc. Consistent with the presence of cAMP-dependent Cl- secretion, immunoprecipitation demonstrated the presence of the cystic fibrosis transmembrane conductance regulator (CFTR). Bradykinin, methacholine, trypsin, and histamine all transiently (15-30 s) elevated Isc, probably by increasing intracellular Ca concentration. Experiments in which the basolateral membrane was permeabilized with nystatin indicated that CFTR was substantially activated under baseline conditions and that Ca-activated Cl- channels were absent from the apical membrane. We anticipate that Calu-3 cells will prove useful in the study of Cl- secretion and other functions of human airway epithelial cells.

Bradykinin↗
PubMed1994

CFTR in Calu-3 human airway cells: channel properties and role in cAMP-activated Cl- conductance.

Calu-3, a cell line derived from a lung adenocarcinoma, forms tight junctions, expresses cystic fibrosis transmembrane conductance regulator (CFTR), and secretes Cl- in response to adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents. Anion conductance of Calu-3 cells was assessed with isotopic flux and patch-clamp methods at 22 degrees C. Iodide efflux was increased by cAMP-elevating agents and brief trypsin treatment. A 7.1 +/- 0.4-pS voltage-independent Cl- channel with linear current-voltage relation was the most common channel observed in cell-attached recordings and was identified as CFTR on the basis of shared features with recombinant CFTR. In unstimulated cells, the mean minimum number of active CFTR channels per patch was 1 +/- 1 (n = 12), increasing to 6 +/- 8 (n = 40) after stimulation with cAMP-elevating agents or after brief trypsin treatment. Channel closure after excision was biexponential with tau 1 approximately 4 s and tau 2 approximately 79 s; typically channels were open continuously until closing permanently. In 11 of 12 excised patches, channels were reactivated by exposure to cAMP-dependent protein kinase (PKA) plus ATP. Efficacy of reactivation was inversely related to the duration from excision to addition of PKA. Channels were blocked by 20-40 microM 5-nitro-2-(3-phenylpropylamino)benzoate on cytosolic but not external side. Active CFTR channels were recorded in 83% of total patches. Other types of Cl- channels were observed in 5 of 52 (10%) cell-attached patches and in 17 of 34 (50%) excised patches, including an outwardly rectifying channel in 2 patches. CFTR channels are the predominant pathway for cAMP-stimulated Cl- conductance in Calu-3 cells; the long open times in the absence of ATP are not explained by present models of CFTR activation.

Adenosine Triphosphate↗
PubMed1994

Chloride secretion and function of serous and mucous cells of human airway glands.

Cells from the acini of human tracheal glands were grown in culture to produce confluent cell sheets of mucous or mixed seromucous phenotype. Levels of mediator-induced Cl secretion in mucous cells were 2-18% those of seromucous cells. Levels of the cystic fibrosis transmembrane conductance regulator (an apical membrane Cl channel) were also much less in mucous than in seromucous cells. These results suggest that serous cells are more important than mucous cells in providing the fluid component of gland secretions.

Biological Transport↗