Pharmacological modulation of ion transport across wild-type and ΔF508 CFTR-expressing human bronchial epithelia

2000 ◽  
Vol 279 (2) ◽  
pp. C461-C479 ◽  
Author(s):  
Daniel C. Devor ◽  
Robert J. Bridges ◽  
Joseph M. Pilewski
Keyword(s):  
Cystic Fibrosis ◽  
Ion Transport ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Bronchial Epithelium ◽  
Disulfonic Acid ◽  
Wild Type ◽  
Pharmacological Agents ◽  
Transport Defect

Forskolin, UTP, 1-ethyl-2-benzimidazolinone (1-EBIO), NS004, 8-methoxypsoralen (Methoxsalen; 8-MOP), and genistein were evaluated for their effects on ion transport across primary cultures of human bronchial epithelium (HBE) expressing wild-type (wt HBE) and ΔF508 (ΔF-HBE) cystic fibrosis transmembrane conductance regulator. In wt HBE, the baseline short-circuit current ( I sc) averaged 27.0 ± 0.6 μA/cm2 ( n = 350). Amiloride reduced this I sc by 13.5 ± 0.5 μA/cm2 ( n = 317). In ΔF-HBE, baseline I sc was 33.8 ± 1.2 μA/cm2 ( n = 200), and amiloride reduced this by 29.6 ± 1.5 μA/cm2 ( n = 116), demonstrating the characteristic hyperabsorption of Na+ associated with cystic fibrosis (CF). In wt HBE, subsequent to amiloride, forskolin induced a sustained, bumetanide-sensitive I sc(Δ I sc = 8.4 ± 0.8 μA/cm2; n = 119). Addition of acetazolamide, 5-( N-ethyl- N-isopropyl)-amiloride, and serosal 4,4′-dinitrostilben-2,2′-disulfonic acid further reduced I sc, suggesting forskolin also stimulates HCO3 − secretion. This was confirmed by ion substitution studies. The forskolin-induced I scwas inhibited by 293B, Ba2+, clofilium, and quinine, whereas charybdotoxin was without effect. In ΔF-HBE the forskolin I sc response was reduced to 1.2 ± 0.3 μA/cm2 ( n = 30). In wt HBE, mucosal UTP induced a transient increase in I sc (Δ I sc = 15.5 ± 1.1 μA/cm2; n = 44) followed by a sustained plateau, whereas in ΔF-HBE the increase in I sc was reduced to 5.8 ± 0.7 μA/cm2 ( n = 13). In wt HBE, 1-EBIO, NS004, 8-MOP, and genistein increased I sc by 11.6 ± 0.9 ( n = 20), 10.8 ± 1.7 ( n = 18), 10.0 ± 1.6 ( n = 5), and 7.9 ± 0.8 μA/cm2( n = 17), respectively. In ΔF-HBE, 1-EBIO, NS004, and 8-MOP failed to stimulate Cl− secretion. However, addition of NS004 subsequent to forskolin induced a sustained Cl−secretory response (2.1 ± 0.3 μA/cm2, n = 21). In ΔF-HBE, genistein alone stimulated Cl− secretion (2.5 ± 0.5 μA/cm2, n = 11). After incubation of ΔF-HBE at 26°C for 24 h, the responses to 1-EBIO, NS004, and genistein were all potentiated. 1-EBIO and genistein increased Na+ absorption across ΔF-HBE, whereas NS004 and 8-MOP had no effect. Finally, Ca2+-, but not cAMP-mediated agonists, stimulated K+ secretion across both wt HBE and ΔF-HBE in a glibenclamide-dependent fashion. Our results demonstrate that pharmacological agents directed at both basolateral K+ and apical Cl− conductances directly modulate Cl−secretion across HBE, indicating they may be useful in ameliorating the ion transport defect associated with CF.

Role of CFTR in chloride secretion across human tracheal epithelium

1995 ◽  
Vol 269 (5) ◽  
pp. L561-L566 ◽  
Author(s):  
B. Q. Shen ◽  
R. J. Mrsny ◽  
W. E. Finkbeiner ◽  
J. H. Widdicombe
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Culture Conditions ◽  
Tracheal Epithelium ◽  
Disulfonic Acid ◽  
Acetoxymethyl Ester ◽  
Cl Channels

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.

Ion transport characteristics of the murine trachea and caecum

1992 ◽  
Vol 82 (6) ◽  
pp. 667-672 ◽  
Author(s):  
S. N. Smith ◽  
E. W. F. W. Alton ◽  
D. M. Geddes
Keyword(s):  
Cystic Fibrosis ◽  
Ion Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Open Circuit ◽  
Potential Difference ◽  
Cystic Fibrosis Gene ◽  
Tissue Type ◽  
Ussing Chambers

1. The basic defect in cystic fibrosis relates to abnormalities of ion transport in affected tissues, such as the respiratory and gastrointestinal tracts. The identification of the cystic fibrosis gene has enabled studies on the production of a cystic fibrosis transgenic mouse to be undertaken. Knowledge of normal ion transport will be necessary for the validation of any such animal model. We have therefore characterized selected responses of the murine trachea and caecum mounted in ‘mini’ Ussing chambers under open-circuit conditions. 2. Basal values for the trachea were: potential difference, 1.1 mV (sem 0.2; n=18); equivalent short-circuit current, 20.4 μA/cm2 (3.6); conductance, 18.2 mS/cm2 (1.7). Corresponding values for the caecum were: potential difference, 0.7 mV (0.1; n=18); equivalent short-circuit current, 11.0 μA/cm2 (1.6); conductance, 14.5 mS/cm2 (1.4). 3. Amiloride (10 μmol/l) produced a significant (P < 0.001) fall in potential difference of 43.0% (5.7) in the trachea, but had no significant effect in the caecum. 4. Subsequently, one of three protocols was used to assess the capacity of either tissue for chloride secretion. Addition of a combination of forskolin (1 μmol/l) and zardaverine (10 μmol/l) produced rises in the potential difference of 873% (509) in the trachea and 399% (202) in the caecum. Both A23187 (10 μmol/l) and phorbol dibutyrate (10 nmol/l) increased tracheal potential difference by 350% (182) and 147% (47), respectively. Neither had a significant effect in the caecum. 5. Subsequent addition of bumetanide caused a fall in the stimulated potential difference of between 39.8% and 71.7%, depending on secretagogue and tissue type. 6. When a homozygous transgenic cystic fibrosis mouse becomes available, these responses should allow such an animal to be distinguished from normal or heterozygous mice.

Regulation of Na-Cl absorption in rabbit proximal colon in vitro

1987 ◽  
Vol 252 (1) ◽  
pp. G45-G51 ◽  
Author(s):  
J. H. Sellin ◽  
R. De Soignie
Keyword(s):  
Ion Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Proximal Colon ◽  
Beta Agonist ◽  
Disulfonic Acid ◽  
Low Concentrations ◽  
Cotransport System ◽  
Net Flux

Ion transport in rabbit proximal colon (PC) in vitro is dominated by a Na-Cl cotransport system stimulated by epinephrine. To further characterize the regulation of Na-Cl transport, we tested the effects of specific adrenergic agonists on ion fluxes under short-circuit conditions. Additionally, we tested the effects of the transport inhibitors bumetanide, furosemide, and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). Basal Na and Cl absorption were essentially nil [Na net flux (JNanet) = 0.3 +/- 0.4, and Cl net flux (JClnet) = -0.5 +/- 0.5 mu eq X cm-2 X h-1, means +/- SE]. The alpha 2-agonist clonidine significantly increased net Na and Cl absorption (delta JNanet = 3.0 +/- 0.6 mu eq X cm-2 X h-1, delta JClnet = 2.0 +/- 0.4 mu eq X cm-2 X h-1) with a minimal change in short-circuit current (delta Isc = 0.1 +/- 0.1 mu eq X cm-2 X h-1). The alpha 1-agonist phenylephrine and the beta-agonist isoproterenol did not alter ion transport. The alpha 2-blocker yohimbine (YOH) had a complex, concentration-dependent effect. At low concentrations (10(-6)-10(-8) M) YOH effectively inhibited epinephrine-stimulated cotransport. Compared with 10(-8)M YOH, 10(-6) YOH blocked 90% of the epinephrine-induced increases in Na and Cl absorption.(ABSTRACT TRUNCATED AT 250 WORDS)

cAMP- but not Ca(2+)-regulated Cl- conductance in the oviduct is defective in mouse model of cystic fibrosis

1995 ◽  
Vol 268 (3) ◽  
pp. C708-C712 ◽  
Author(s):  
A. Y. Leung ◽  
P. Y. Wong ◽  
S. E. Gabriel ◽  
J. R. Yankaskas ◽  
R. C. Boucher
Keyword(s):  
Cystic Fibrosis ◽  
Secretory Pathway ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Functional Expression ◽  
Relative Magnitude ◽  
Cl Secretion ◽  
Oviductal Epithelium ◽  
High Level

Defective adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- transport in cystic fibrosis (CF) reflects defects in the cystic fibrosis transmembrane conductance regulator (CFTR). A moderate level of CFTR mRNA expression has been found in rodent and human oviductal epithelium, but unlike other CFTR-expressing tissues, the oviduct in CF patients is apparently normal. The present study was carried out to investigate the relative magnitude of the cAMP- and intracellular Ca2+ (Cai2+)-regulated Cl- secretion in primary cultures of the oviduct from normal and CF mice generated by targeted disruption of the murine CF gene. Normal oviductal epithelium exhibited a basal equivalent short-circuit current (Ieq) of 20.3 +/- 1.7 muA/cm2. CF oviduct exhibited a lower basal Ieq of 4.5 +/- 1.9 muA/cm2. In normal mice, forskolin (10(-5) M, apical) elicited a slowly developing sustained rise in Ieq, whereas ionomycin (5 x 10(-6) M, apical) and ATP (10(-4) M, apical) induced larger increases in Ieq consisting of a prompt, transient response followed by a slowly decreasing component. The Ieq response to forskolin was totally abolished in CF mouse oviducts, but the magnitudes of the peak Ieq responses to ionomycin and ATP were not different from normal. The time courses of the ionomycin- and ATP-evoked responses, however, were significantly more transient in CF than in normal oviducts. These results demonstrate that CF mouse oviduct exhibits defective cAMP- but not Cai(2+)-mediated Cl- secretion. The relatively high level of functional expression of the alternative Cai(2+)-activated Cl- secretory pathway in the mouse oviduct may contribute to the absence of major pathology in the CF oviduct.

Effects of carbon monoxide on ion transport across rat distal colon

2011 ◽  
Vol 300 (2) ◽  
pp. G207-G216 ◽  
Author(s):  
Julia Steidle ◽  
Martin Diener
Keyword(s):  
Carbon Monoxide ◽  
Ion Transport ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Disulfonic Acid ◽  
Maximal Response ◽  
Colonic Crypts ◽  
Acid Sodium Salt

The aim of the present study was to investigate whether carbon monoxide (CO) induces changes in ion transport across the distal colon of rats and to study the mechanisms involved. In Ussing chamber experiments, tricarbonyldichlororuthenium(II) dimer (CORM-2), a CO donor, evoked a concentration-dependent increase in short-circuit current ( Isc). A maximal response was achieved at a concentration of 2.5·10−4 mol/l. Repeated application of CORM-2 resulted in a pronounced desensitization of the tissue. Anion substitution experiments suggest that a secretion of Cl− and HCO3− underlie the CORM-2-induced current. Glibenclamide, a blocker of the apical cystic fibrosis transmembrane regulator channel, inhibited the Isc induced by the CO donor. Similarly, bumetanide, a blocker of the basolateral Na+-K+-2Cl− cotransporter, combined with 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid sodium salt, an inhibitor of the basolateral Cl−/HCO3− exchanger, inhibited the CORM-2-induced Isc. Membrane permeabilization experiments indicated an activation of basolateral K+ and apical Cl− channels by CORM-2. A partial inhibition by the neurotoxin, tetrodotoxin, suggests the involvement of secretomotor neurons in this response. In imaging experiments at fura-2-loaded colonic crypts, CORM-2 induced an increase of the cytosolic Ca2+ concentration. This increase depended on the influx of extracellular Ca2+, but not on the release of Ca2+ from intracellular stores. Both enzymes for CO production, heme oxygenase I and II, are expressed in the colon as observed immunohistochemically and by RT-PCR. Consequently, endogenous CO might be a physiological modulator of colonic ion transport.

Effect of nitric oxide on epithelial ion transports in noncystic fibrosis and cystic fibrosis human proximal and distal airways

2012 ◽  
Vol 303 (7) ◽  
pp. L617-L625 ◽  
Author(s):  
Sabine Blouquit-Laye ◽  
Luc Dannhoffer ◽  
Camille Braun ◽  
Anh-Tuan Dinh-Xuan ◽  
Edouard Sage ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cystic Fibrosis ◽  
Ion Transport ◽  
Soluble Guanylate Cyclase ◽  
Short Circuit ◽  
Primary Cultures ◽  
Airway Epithelia ◽  
Ussing Chambers ◽  
Intracellular Cgmp ◽  
Bronchial Cells

The airways of patients with cystic fibrosis (CF) exhibit decreased nitric oxide (NO) concentrations, which might affect airway function. The aim of this study was to determine the effects of NO on ion transport in human airway epithelia. Primary cultures of non-CF and CF bronchial and bronchiolar epithelial cells were exposed to the NO donor sodium nitroprusside (SNP), and bioelectric variables were measured in Ussing chambers. Amiloride was added to inhibit the Na+channel ENaC, and forskolin and ATP were added successively to stimulate cAMP- and Ca2+-dependent Cl−secretions, respectively. The involvement of cGMP was assessed by measuring the intracellular cGMP concentration in bronchial cells exposed to SNP and the ion transports in cultures exposed to 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, an inhibitor of the soluble guanylate cyclase (ODQ), or to 8Z, a cocktail of 8-bromo-cGMP and zaprinast (phosphodiesterase 5 inhibitor). SNP decreased the baseline short-circuit current ( Isc) and the changes in Iscinduced by amiloride, forskolin, and ATP in non-CF bronchial and bronchiolar cultures. The mechanism of this inhibition was studied in bronchial cells. SNP increased the intracellular cGMP concentration ([cGMP]i). The inhibitory effect of SNP was abolished by 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, an NO scavenger (PTIO) and ODQ and was partly mimicked by increasing [cGMP]i. In CF cultures, SNP did not significantly modify ion transport; in CF bronchial cells, 8Z had no effect; however, SNP increased the [cGMP]i. In conclusion, exogenous NO may reduce transepithelial Na+absorption and Cl−secretion in human non-CF airway epithelia through a cGMP-dependent pathway. In CF airways, the NO/cGMP pathway appears to exert no effect on transepithelial ion transport.

scholarly journals Evidence that Calu-3 human airway cells secrete bicarbonate

1998 ◽  
Vol 274 (3) ◽  
pp. L450-L453 ◽  
Author(s):  
Michael C. Lee ◽  
Christopher M. Penland ◽  
Jonathan H. Widdicombe ◽  
Jeffrey J. Wine
Keyword(s):  
Cystic Fibrosis ◽  
Previous Investigation ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Disulfonic Acid ◽  
Transmembrane Conductance Regulator ◽  
Submucosal Gland ◽  
Transmembrane Conductance ◽  
Air Interface ◽  
Cystic Fibrosis Transmembrane

The Calu-3 cell line is being investigated as a model for human submucosal gland serous cells. In a previous investigation of basal short-circuit current ( I sc) in Calu-3 cells, high levels of bumetanide-insensitive basal I sc (∼60 μA/cm2) were measured in cells grown at an air interface. Basal I sc was reduced only 7% by bumetanide, and the largest component of basal I sc required both Cl− and[Formula: see text] in the bathing solutions. Because I sc could be partially inhibited by basolateral 4,4′-dinitrostilbene-2,2′-disulfonic acid and because the only known apical exit pathway for anions is the cystic fibrosis transmembrane conductance regulator, which has a relatively poor conductance for [Formula: see text], it was concluded that most basal I sc is[Formula: see text]-dependent Cl− secretion [M. Singh, M. Krouse, S. Moon, and J. J. Wine. Am. J. Physiol. 272 ( Lung Cell. Mol. Physiol. 16): L690–L698, 1997]. We have now measured isotopic fluxes of36Cl−and22Na+across short-circuited Calu-3 cells and found that virtually none of the basal I sc is Cl− secretion or Na+ absorption. Thus, in contrast to the earlier report, we conclude that the major component of basal I sc is[Formula: see text] secretion. Stimulation recruits primarily Cl− secretion, as previously proposed.

Primary culture of duck salt gland. II. Neurohormonal stimulation of active transport

1985 ◽  
Vol 249 (1) ◽  
pp. C41-C47 ◽  
Author(s):  
R. J. Lowy ◽  
D. C. Dawson ◽  
S. A. Ernst
Keyword(s):  
Ion Transport ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Salt Gland ◽  
Hormonal Control ◽  
Regulatory Control ◽  
Secretory Cells ◽  
Active Ion Transport ◽  
Secretory Transport

Primary cultures of structurally polarized sheets of avian salt gland secretory cells were mounted in Lucite chambers for transmural electrophysiological analysis. Transmural resistance values increased during the first 3 days of culture to 293 +/- 35 omega X cm2 and then decreased slowly thereafter. There was little short-circuit current (Isc) in the absence of secretagogues. Serosal addition of either carbachol or epinephrine resulted in a Isc consistent with positive charge flow from mucosa to serosa, thus demonstrating that these cell layers were capable of active ion transport in response to either cholinergic or adrenergic neurohormonal stimulation. Serosal ouabain or furosemide abolished the response to either agonist, while theophylline enhanced the response. Receptor specificity for the electrical responses was shown by selective inhibition of carbachol- and epinephrine-induced Isc by atropine and propranolol, respectively. The results demonstrate that these primary epithelial cell cultures are capable of active ion transport and are sensitive to known inhibitors of secretory transport, and suggest that intracellular coupling mechanisms for hormonal control are retained in culture. These cultures should be useful for studying mechanisms of ion secretory transport and their regulatory control.

Transient permeabilization of airway epithelium by mucosal water

1996 ◽  
Vol 81 (1) ◽  
pp. 491-499 ◽  
Author(s):  
J. H. Widdicombe ◽  
F. Azizi ◽  
T. Kang ◽  
J. F. Pittet
Keyword(s):  
Lactate Dehydrogenase ◽  
Epithelial Cells ◽  
Airway Epithelium ◽  
Short Circuit ◽  
Primary Cultures ◽  
Physiological Saline ◽  
Short Circuit Current ◽  
Bronchial Epithelium ◽  
Mucosal Surface ◽  
Hyposmotic Shock

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.

Beta-adrenergic stimulation of ion transport in primary cultures of avian salt glands

1987 ◽  
Vol 252 (6) ◽  
pp. C670-C676 ◽  
Author(s):  
R. J. Lowy ◽  
S. A. Ernst
Keyword(s):  
Ion Transport ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Salt Gland ◽  
Adrenergic Stimulation ◽  
Salt Glands ◽  
Beta Adrenergic ◽  
Effector Pathway ◽  
Stimulation Of

Adrenergic stimulation of transmural ion transport was identified and characterized in primary cultures of avian salt gland. Adrenergic activation was mediated by beta-receptors since stimulation of the short-circuit current (Isc) was blocked by propranolol but not phentolamine. The Isc's elicited by isoproterenol, epinephrine, and norepinephrine were dose dependent, with respective EC50 values of 1.5 X 10(-8) M, 5.0 X 10(-6) M, and 1.1 X 10(-5) M. The apparent Ki for propranolol inhibition after isoproterenol stimulation was 7.5 X 10(-10) M. 8-Br cyclic AMP (8-Br cAMP) and forskolin-elicited Isc's that were insensitive to propranolol, were potentiated by theophylline, and inhibited by furosemide or ouabain. Isoproterenol also induced an increase in ouabain-sensitive respiration in acutely dispersed cells from salt-stressed juvenile or unstressed adult animals, but not in fully salt-stressed adults. The data indicate that, in addition to the well-established cholinergic receptors, beta-adrenergic receptors can control ion transport in these glands. Furthermore, the results suggest for the first time that an intracellular effector pathway involving cAMP is present.

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