Ovine male genital duct epithelial cells differentiate in vitro and express functional CFTR and ENaC

2000 ◽  
Vol 278 (5) ◽  
pp. C885-C894 ◽  
Author(s):  
Marko Bertog ◽  
David J. Smith ◽  
Andreas Bielfeld-Ackermann ◽  
John Bassett ◽  
David J. P. Ferguson ◽  
...  
Keyword(s):  
Vas Deferens ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Transmembrane Conductance Regulator ◽  
Genital Duct ◽  
Inhibitor Concentration ◽  
Transmembrane Conductance ◽  
Established Cell ◽  
Male Genital

To investigate the biology of the male genital duct epithelium, we have established cell cultures from the ovine vas deferens and epididymis epithelium. These cells develop tight junctions, high transepithelial electrical resistance, and a lumen-negative transepithelial potential difference as a sign of active transepithelial ion transport. In epididymis cultures the equivalent short-circuit current ( I sc) averaged 20.8 ± 0.7 μA/cm2 ( n = 150) and was partially inhibited by apical application of amiloride with an inhibitor concentration of 0.64 μM. In vas deferens cultures, I sc averaged 14.4 ± 1.1 μA/cm2 ( n = 18) and was also inhibited by apical application of amiloride with a half-maximal inhibitor concentration ( K i) of 0.68 μM. The remaining amiloride-insensitive I sc component in epididymis and vas deferens cells was partially inhibited by apical application of the Cl− channel blocker diphenylamine-2-carboxylic acid (1 mM). It was largely dependent on extracellular Cl− and, to a lesser extent, on extracellular[Formula: see text]. It was further stimulated by basolateral application of forskolin (10− 5 M), which increased I sc by 3.1 ± 0.3 μA/cm2 ( n=65) in epididymis and 0.9 ± 0.1 μA/cm2 ( n = 11) in vas deferens. These findings suggest that cultured ovine vas deferens and epididymis cells absorb Na+ via amiloride-sensitive epithelial Na+ channels (ENaC) and secrete Cl− and [Formula: see text]via apical cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels. This interpretation is supported by RT-PCR data showing that vas deferens and epididymis cells express CFTR and ENaC mRNA.

CFTR Function and Clinical Response to Modulators Parallel Nasal Epithelial Organoid Swelling

2021 ◽  
Author(s):  
Justin D Anderson ◽  
Zhongyu Liu ◽  
L. Victoria Odom ◽  
Latona Kersh ◽  
Jennifer S Guimbellot
Keyword(s):  
Ion Channel ◽  
Clinical Response ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Transmembrane Conductance Regulator ◽  
Channel Activity ◽  
Clinical Tool ◽  
Transmembrane Conductance ◽  
Novel Treatments

Rationale. In vitro biomarkers to assess Cystic Fibrosis Transmembrane Conductance Regulator activity are desirable for precision modulator selection and as a tool for clinical trials. Objectives. We describe an organoid swelling assay derived from human nasal epithelia using commercially available reagents and equipment and an automated imaging process. Methods. Cells were collected in nasal brush biopsies, expanded in vitro, and cultured as spherical organoids or as monolayers. Organoids were used in a functional swelling assay with automated measurements and analysis, while monolayers were used for short-circuit current measurements to assess ion channel activity. Clinical data was collected from patients on modulators. Relationships between swelling data and short-circuit current, as well as between swelling data and clinical outcome measures, were assessed. Main results. The organoid assay measurements correlate with short-circuit current measurements for ion channel activity. The functional organoid assay distinguished individual responses as well as differences between groups. The organoid assay distinguished incremental drug responses to modulator monotherapy with ivacaftor and combination therapy with ivacaftor, tezacaftor, and elexacaftor. The swelling activity paralleled clinical response. Conclusions. An in vitro biomarker derived from patients' cells can be used to predict responses to drugs and is likely to be useful as a pre-clinical tool to aid in development of novel treatments, and as a clinical trial outcome measure for a variety of applications, including gene therapy or editing.

scholarly journals Huqi San-Evoked Rat Colonic Anion Secretion through Increasing CFTR Expression

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Xiaowei Xue ◽  
Zhengming Shi ◽  
Wen Wang ◽  
Xiaotong Yu ◽  
Ping Feng ◽  
...  
Keyword(s):  
Protein Content ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Anion Secretion ◽  
Mrna Transcripts ◽  
And Function ◽  
Cystic Fibrosis Transmembrane

Huqi San (HQS) is a Chinese herbal preparation of eight medicinal herbs that promote diuresis, detoxification, blood circulation, and cholestasis. Defects in transporter expression and function can cause cholestasis and jaundice. However, the mechanism of the cholestasis underlying HQS effects, especially on the gastrointestinal tract ion secretion, has not been elucidated. Real-time RT-PCR and Western blotting were used to study the expression and localization of cystic fibrosis transmembrane conductance regulator (CFTR) andα-ENaC in rat alimentary tract, and then the effect of HQS on the ion transport in rat distal colon mucosa was investigated using the short-circuit current (ISC) technique. The results showed that pretreatment with HQS significantly enhanced mRNA transcripts and protein content of CFTR in liver and distal colon but notα-ENaC in alimentary organs. HQS increasesISCand decreases the transepithelial resistance. Pretreatment with epithelial Na+channel blocker did not affect theISCresponses elicited by HQS, but removal of extracellular Cl−or pretreatment with Cl−channel or Na+-K+-2Cl−cotransporter blocker inhibited HQS-elicitedISCresponses. These findings demonstrated that HQS, RA, and RP can stimulate Cl−secretion in the distal colon by increasing the mRNA transcripts and protein content of CFTR in liver and distal colon.

CFTR disruption impairs cAMP-dependent Cl−secretion in primary cultures of mouse cortical collecting ducts

2001 ◽  
Vol 281 (3) ◽  
pp. F434-F442 ◽  
Author(s):  
Marcelle Bens ◽  
Jean-Paul Duong Van Huyen ◽  
Françoise Cluzeaud ◽  
Jacques Teulon ◽  
Alain Vandewalle
Keyword(s):  
Collecting Duct ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Similar Degree ◽  
Transmembrane Conductance Regulator ◽  
Cortical Collecting Duct ◽  
Transmembrane Conductance ◽  
Nacl Transport ◽  
Cystic Fibrosis Transmembrane

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in the renal cortical collecting duct (CCD) has not yet been fully elucidated. Here, we investigated the effects of deamino-8-d-arginine vasopressin (dDAVP) and isoproterenol (ISO) on NaCl transport in primary cultured CCDs microdissected from normal [CFTR(+/+)] and CFTR-knockout [CFTR(−/−)] mice. dDAVP stimulated the benzamyl amiloride (BAm)-sensitive transport of Na+ assessed by the short-circuit current ( I sc) method in both CFTR(+/+) and CFTR(−/−) CCDs to a very similar degree. Apical addition of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) or glibenclamide partially inhibited the rise in I sc induced by dDAVP and ISO in BAm-treated CFTR(+/+) CCDs, whereas dDAVP, ISO, and NPPB did not alter I sc in BAm-treated CFTR(−/−) CCDs. dDAVP stimulated the apical-to-basal flux and, to a lesser extent, the basal-to-apical flux of 36Cl− in CFTR(+/+) CCDs. dDAVP also increased the apical-to-basal36Cl− flux in CFTR(−/−) CCDs but not the basal-to-apical 36Cl− flux. These results demonstrate that CFTR mediates the cAMP-stimulated component of secreted Cl− in mouse CCD.

Enhanced colonic Na+ absorption in cystic fibrosis mice versus normal mice

1997 ◽  
Vol 272 (2) ◽  
pp. G393-G400 ◽  
Author(s):  
B. R. Grubb ◽  
R. C. Boucher
Keyword(s):  
Cystic Fibrosis ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Proximal Colon ◽  
Na Channel ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Constant Dose ◽  
Cf Transmembrane Conductance Regulator

Because there are reports that electrogenic Na+ absorption is increased in colonic epithelia of cystic fibrosis (CF) subjects, we tested whether amiloride-sensitive Na+ absorption was increased in the colonic epithelia of CF mice compared with normal mice on high- or low-Na+ diets. When mice consumed a diet high in Na+, none of the colonic regions (distal colon, proximal colon, or cecum) from either group of mice exhibited an amiloride-sensitive short-circuit current (Isc). However, when mice were placed on a low-Na+ diet for 2 wk, all three intestinal regions from the CF mice exhibited a significant response to amiloride (P < or = 0.05). In contrast, normal mice on the low-Na+ diet exhibited an amiloride-sensitive Isc that was smaller and only significant in the cecum and distal colon. Measurement of plasma aldosterone levels revealed that the CF mice on the low-Na+ diet had significantly greater aldosterone levels than similarly treated controls [8,906 +/- 1,039 (n = 14) vs. 5,243 +/- 1,410 pg/ml (n = 14), respectively]. When mice were infused with a constant dose of aldosterone (1 microg x g(-1) x day(-1)) for 7 days, the distal colon of the CF mice still had a significantly greater amiloride-sensitive Isc than did the normal distal colon. If the presence of CF transmembrane conductance regulator (CFTR) down-regulates Na+ absorption in the colonic tissue from normal mice, our data suggest that at least some CFTR may be colocalized with the Na+ channel. Alternatively, other factors may be involved.

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.

Mechanisms of guanylin action on water and ion absorption at different regions of seawater eel intestine

2014 ◽  
Vol 307 (6) ◽  
pp. R653-R663 ◽  
Author(s):  
Masaaki Ando ◽  
Marty K. S. Wong ◽  
Yoshio Takei
Keyword(s):  
Water Absorption ◽  
Transport System ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Pcr Analysis ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Ion Absorption ◽  
Seawater Acclimation ◽  
Cystic Fibrosis Transmembrane

Guanylin (GN) inhibited water absorption and short-circuit current ( Isc) in seawater eel intestine. Similar inhibition was observed after bumetanide, and the effect of bumetanide was abolished by GN or vice versa, suggesting that both act on the same target, Na+-K+-2Cl− cotransporter (NKCC), which is a key player for the Na+-K+-Cl− transport system responsible for water absorption in marine teleost intestine. However, effect of GN was always greater than that of bumetanide: 10% greater in middle intestine (MI) and 40% in posterior intestine (PI) for Isc, and 25% greater in MI and 34% in PI for water absorption. After treatment with GN, Isc decreased to zero, but 20–30% water absorption still remained. The remainder may be due to the Cl−/HCO3− exchanger and Na+-Cl− cotransporter (NCC), since inhibitors for these transporters almost nullified the remaining water absorption. Quantitative PCR analysis revealed the presence of major proteins involved in water absorption; the NKCC2β and AQP1 genes whose expression was markedly upregulated after seawater acclimation. The SLC26A6 (anion exchanger) and NCCβ genes were also expressed in small amounts. Consistent with the inhibitors' effect, expression of NKCC2β was MI > PI, and that of NCCβ was MI << PI. The present study showed that GN not only inhibits the bumetanide-sensitive Na+-K+-Cl− transport system governed by NKCC2β, but also regulates unknown ion transporters different from GN-insensitive SLC26A6 and NCC. A candidate is cystic fibrosis transmembrane conductance regulator Cl− channel, as demonstrated in mammals, but its expression is low in eel intestine, and its role may be minor, as indicated by the small effect of its inhibitors.

scholarly journals Effect of TongXie-YaoFang on Cl−andHCO3-Transport in Diarrhea-Predominant Irritable Bowel Syndrome Rats

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Xiaofang Lu ◽  
Shengsheng Zhang ◽  
Cheng Yang ◽  
Zhengfang Wang ◽  
Luqing Zhao ◽  
...  
Keyword(s):  
Irritable Bowel Syndrome ◽  
Maternal Separation ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Formula Group ◽  
Neonatal Maternal Separation ◽  
Combined Action ◽  
Irritable Bowel

TongXie-YaoFang (TXYF) can effectively alleviate the symptoms of diarrhea-predominant irritable bowel syndrome (D-IBS) patients. However, the curative mechanism has not been fully clarified. The study was designed to investigate the effect of TXYF on the colonic ion transport induced by serotonin (5-HT) in D-IBS rats. A method of multiple stress (neonatal maternal separation (NMS) combined with restraint stress (RS)) was used to induce the D-IBS model. The model rats were randomly divided into two groups: NMS + RS group and TXYF-formula group, and the normal control (no handling) rats were classified as NH group. In the NMS + RS group, the change of short-circuit current (ΔIsc) induced by 5-HT was lower than that in the NH and TXYF-formula groups. After removing of the extracellular Cl−orHCO3-or basolateral Na+or blocking the cystic fibrosis transmembrane conductance regulator (CFTR), Na+-K+-2Cl−cotransporter (NKCC),Na+-HCO3-cotransporter,Cl-/HCO3-exchanger, K+channel, or Na+/K+-ATPase, respectively, there was no difference in 5-HT-inducedΔIscamong the three groups. These data suggest that TXYF can regulate 5-HT-induced Cl−andHCO3-secretion, possibly mediated by the combined action of CFTR, NKCC, Na+-HCO3-cotransporter,Cl-/HCO3-exchanger, K+channel, and Na+/K+-ATPase.

Stimulation of chloride secretion by baicalein in isolated rat distal colon

2002 ◽  
Vol 282 (3) ◽  
pp. G508-G518 ◽  
Author(s):  
W. H. Ko ◽  
V. W. Y. Law ◽  
W. C. Y. Yip ◽  
G. G. L. Yue ◽  
C. W. Lau ◽  
...  
Keyword(s):  
Colonic Mucosa ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Chloride Secretion ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Ussing Chambers ◽  
Rat Distal Colon ◽  
Cystic Fibrosis Transmembrane

The effect of baicalein on mucosal ion transport in the rat distal colon was investigated in Ussing chambers. Mucosal addition of baicalein (1–100 μM) elicited a concentration-dependent short-circuit current ( I sc) response. The increase in I sc was mainly due to Cl−secretion. The presence of mucosal indomethacin (10 μM) significantly reduced both the basal and subsequent baicalein-evoked I sc responses. The baicalein-induced I sc were inhibited by mucosal application of diphenylamine-2-carboxylic acid (100 μM) and glibenclamide (500 μM) and basolateral application of chromanol 293B (30 μM), a blocker of KvLQT1 channels and Ba2+ ions (5 mM). Treatment of the colonic mucosa with baicalein elicited a threefold increase in cAMP production. Pretreating the colonic mucosa with carbachol (100 μM, serosal) but not thapsigargin (1 μM, both sides) abolished the baicalein-induced I sc. Addition of baicalein subsequent to forskolin induced a further increase in I sc. These results indicate that the baicalein evoked Cl− secretion across rat colonic mucosa, possibly via a cAMP-dependent pathway. However, the action of baicalein cannot be solely explained by its cAMP-elevating effect. Baicalein may stimulate Cl− secretion via a cAMP-independent pathway or have a direct effect on cystic fibrosis transmembrane conductance regulator.

Ion transport across the jejunum in normal and cystic fibrosis mice

1995 ◽  
Vol 268 (3) ◽  
pp. G505-G513 ◽  
Author(s):  
B. R. Grubb
Keyword(s):  
Cystic Fibrosis ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Fluid Secretion ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Targeted Disruption ◽  
Cl Secretion ◽  
Ion Substitution ◽  
Cystic Fibrosis Transmembrane

Cystic fibrosis (CF) mice created by targeted disruption of the murine cystic fibrosis transmembrane conductance regulator gene lack adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion and exhibit marked intestinal complications secondary to inadequate fluid secretion. The basal short-circuit current (Isc) in the normal murine jejuna [43.2 +/- 5.9 microA.cm-2, n = 10 (mean +/- SE)] exhibits marked spontaneous n = 10 (mean +/- SE)] exhibits marked spontaneous oscillations (amplitude = 47.9 microA.cm-2, n = 18), which were completely absent in the CF jejunum. Treatment of normal jejuna with the neuronal blocker tetrodotoxin completely eliminated the oscillations and decreased the Isc to levels not significantly different from the low basal Isc (5.4 +/- 2.8 microA.cm-2, n = 16) exhibited by CF tissue. Ion substitution studies revealed basal Isc in normal jejuna to be due primarily to Cl- secretion but these tissues appeared to be capable of HCO3- secretion as well. In contrast, CF jejuna spontaneously secreted neither Cl- nor HCO3-, which may indicate that CF jejuna have a defect in the ability to secrete both of these anions. Apical glucose elicited an electrogenic absorption of Na+ of identical magnitude in normal and CF jejuna. Without apical glucose, CF jejuna exhibited a very small Isc response to forskolin (delta 2.2 +/- 0.67 microA.cm-2, n = 10). However, in the presence of apical glucose, forskolin elicited an eightfold greater Isc response in the CF tissue (delta 17.2 +/- 4.8 microA.cm-2, n = 9).(ABSTRACT TRUNCATED AT 250 WORDS)

Genistein activates CFTR-mediated Cl−secretion in the murine trachea and colon

2000 ◽  
Vol 279 (2) ◽  
pp. C383-C392 ◽  
Author(s):  
Catharine A. Goddard ◽  
Martin J. Evans ◽  
William H. Colledge
Keyword(s):  
Cystic Fibrosis ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Cell Systems ◽  
Cl Secretion ◽  
Cystic Fibrosis Transmembrane

The action of the isoflavone genistein on the cystic fibrosis transmembrane conductance regulator (CFTR) has been studied in many cell systems but not in intact murine tissues. We have investigated the action of genistein on murine tissues from normal and cystic fibrosis (CF) mice. Genistein increased the short-circuit current ( I sc) in tracheal (16.4 ± 2.8 μA/cm2) and colonic (40.0 ± 4.4 μA/cm2) epithelia of wild-type mice. This increase was inhibited by furosemide, diphenylamine-2-carboxylate, and glibenclamide, but not by DIDS. In contrast, genistein produced no significant change in the I sc of the tracheal epithelium (0.9 ± 1.1 μA/cm2) and decreased the I sc of colons from CF null (−13.1 ± 2.3 μA/cm2) and ΔF508 mice (−10.3 ± 1.3 μA/cm2). Delivery of a human CFTRcDNA-liposome complex to the airways of CF null mice restored the genistein response in the tracheas to wild-type levels. Tracheas from ΔF508 mice were also studied: 46% of trachea showed no response to genistein, whereas 54% gave an increase in I scsimilar to that in wild type. We conclude that genistein activates CFTR-mediated Cl− secretion in the murine trachea and distal colon.

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