Normalization of ion transport in murine cystic fibrosis nasal epithelium using gene transfer

1997 ◽  
Vol 273 (2) ◽  
pp. C734-C740 ◽  
Author(s):  
L. J. MacVinish ◽  
C. Goddard ◽  
W. H. Colledge ◽  
C. F. Higgins ◽  
M. J. Evans ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Channels ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Nasal Epithelium ◽  
Sodium Absorption ◽  
Calcium Dependent ◽  
Cf Transmembrane Conductance Regulator ◽  
Therapy Studies

The murine nasal epithelium was investigated by the short-circuit current (SCC) technique. Electrogenic sodium absorption was revealed by addition of amiloride and calcium-dependent chloride secretion by the addition of amiloride and calcium-dependent chloride secretion by the addition of 2,5-di-(tert-butyl)-1,4-benzohydroquinone (TBHQ)/ionomycin. In the presence of these agents a further increase in SCC was obtained by addition of forskolin. Epithelia from both cystic fibrosis (CF) null (Cftrtm1Cam) and CF delta F508 (Cftrtm2Cam) mice had enhanced sodium absorption compared with controls, whereas only delta F508 epithelia had increased calcium-dependent chloride secretion. Both strains gave nasal epithelia that showed significantly reduced responses to forskolin, due to the absence of CF transmembrane conductance regulator (CFTR) chloride channels. In Cftrtm2Cam nasal epithelia the forskolin responses were not significantly different from zero. Transfection of these mice with the plasmid pTRIAL10-CFTR2 complexed with cationic liposomes normalized the transporting activity in the nasal epithelium. Basal SCC and calcium-dependent chloride secretion were significantly reduced, whereas CFTR-dependent chloride secretion was increased to normal values. Amiloride-sensitive SCC was reduced by transfection but failed to reach significance. The similarity of murine CF nasal epithelium to that in human CF airways makes the model valuable for gene therapy studies.

The primary and final effector mechanisms required for kinin-induced epithelial chloride secretion

1998 ◽  
Vol 274 (3) ◽  
pp. G578-G583
Author(s):  
Alan W. Cuthbert ◽  
Clare Huxley
Keyword(s):  
Chloride Channels ◽  
Short Circuit ◽  
Receptor Gene ◽  
Secretory Activity ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Secretory Process ◽  
Mouse Intestine ◽  
Cf Transmembrane Conductance Regulator ◽  
Effector Mechanisms

The short-circuit current technique was used to examine the effects of N 2-l-lysylbradykinin (LBK) on chloride secretion in the mucosae of the mouse intestine. It was found to be a potent chloride secretagogue in the mucosa lining the colon, jejunum, and cecum, as it is in most mammals, with 2 nM being sufficient to cause half-maximal secretion. The extent of the responses was in the order cecum > colon > jejunum. In cystic fibrosis (CF) null mice, with no CF transmembrane conductance regulator (CFTR) chloride channels, LBK caused no chloride secretion, but transporting activities for other ions were revealed. Introduction of the human CF gene into the genome of CF null mice at the zygote stage restored the chloride secretory activity of LBK, with only minor differences in potency. In mice in which the kinin B2 receptor gene had been disrupted, LBK had no effect, whereas the responses to forskolin were unchanged. Thus the acute effects of kinins on chloride secretion depend uniquely on kinin B2receptors and CFTR chloride channels, which form the primary and final effector mechanisms of the secretory process.

scholarly journals Nobiletin Stimulates Chloride Secretion in Human Bronchial Epithelia via a cAMP/PKA-Dependent Pathway

2015 ◽  
Vol 37 (1) ◽  
pp. 306-320 ◽  
Author(s):  
Yuan Hao ◽  
Cindy S.T. Cheung ◽  
Wallace C.Y. Yip ◽  
Wing-hung Ko
Keyword(s):  
Cystic Fibrosis ◽  
Adenylate Cyclase ◽  
Cell Line ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Epithelial Cell Line ◽  
Electrical Measurements ◽  
Cl Secretion ◽  
Bronchial Epithelia

Background/Aims: Nobiletin, a citrus flavonoid isolated from tangerines, alters ion transport functions in intestinal epithelia, and has antagonistic effects on eosinophilic airway inflammation of asthmatic rats. The present study examined the effects of nobiletin on basal short-circuit current (ISC) in a human bronchial epithelial cell line (16HBE14o-), and characterized the signal transduction pathways that allowed nobiletin to regulate electrolyte transport. Methods: The ISC measurement technique was used for transepithelial electrical measurements. Intracellular calcium ([Ca2+]i) and cAMP were also quantified. Results: Nobiletin stimulated a concentration-dependent increase in ISC, which was due to Cl- secretion. The increase in ISC was inhibited by a cystic fibrosis transmembrane conductance regulator inhibitor (CFTRinh-172), but not by 4,4'-diisothiocyano-stilbene-2,2'-disulphonic acid (DIDS), Chromanol 293B, clotrimazole, or TRAM-34. Nobiletin-stimulated ISC was also sensitive to a protein kinase A (PKA) inhibitor, H89, and an adenylate cyclase inhibitor, MDL-12330A. Nobiletin could not stimulate any increase in ISC in a cystic fibrosis (CF) cell line, CFBE41o-, which lacked a functional CFTR. Nobiletin stimulated a real-time increase in cAMP, but not [Ca2+]i. Conclusion: Nobiletin stimulated transepithelial Cl- secretion across human bronchial epithelia. The mechanisms involved activation of adenylate cyclase- and cAMP/PKA-dependent pathways, leading to activation of apical CFTR Cl- channels.

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.

Intestinal ion transport in rats with spontaneous arterial hypertension

1988 ◽  
Vol 75 (2) ◽  
pp. 127-133 ◽  
Author(s):  
Ralf Lübcke ◽  
Gilbert O. Barbezat
Keyword(s):  
Ion Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Membrane Resistance ◽  
Chloride Secretion ◽  
Sodium Absorption ◽  
Spontaneously Hypertensive ◽  
Wistar Kyoto ◽  
Intestinal Ion Transport

1. Ion balance, intestinal ion transport in vivo with luminal Ringer, and direct voltage clamping in vivo with luminal Ringer and sodium-free choline-Ringer were studied in young (40 days old) and adult (120 days old) spontaneously hypertensive rats (SHR) and age-matched normotensive controls (Wistar–Kyoto rats, WKY). 2. Faecal sodium output was significantly higher in SHR compared with WKY in both young (+ 67%) and adult (+ 43%) rats. 3. Small-intestinal sodium absorption was equal in young SHR and WKY, but significantly greater net sodium absorption was found in the ileum of adult SHR. In contrast, net sodium absorption was reduced from the colon of both young and adult SHR. 4. In adult SHR, the colonic transepithelial short-circuit current (Isc) and the transepithelial potential difference (PD) were significantly higher, whereas the transepithelial membrane resistance (Rm) was significantly lower than in WKY. There was an identical drop in Isc in both strains when luminal sodium was replaced by choline. These data cannot be explained by increased electrogenic cation (sodium) absorption in the SHR, but would favour chloride secretion. 5. It is suggested that in SHR membrane electrolyte transport abnormalities may also be present in the epithelial cells of the small and large intestine, as have been demonstrated already in blood cells by several investigators. The SHR may become an interesting experimental animal model for the study of generalized ion transport disorders.

Calcium-dependent chloride secretion across cultures of human tracheal surface epithelium and glands

1993 ◽  
Vol 265 (2) ◽  
pp. L170-L177 ◽  
Author(s):  
M. Yamaya ◽  
T. Ohrui ◽  
W. E. Finkbeiner ◽  
J. H. Widdicombe
Keyword(s):  
Time Course ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Cell Types ◽  
Chloride Secretion ◽  
Surface Epithelium ◽  
Acetoxymethyl Ester ◽  
Calcium Dependent ◽  
Gland Cells ◽  
Active Ion Transport

Surface epithelium and gland cells from human trachea were cultured on porous-bottom inserts and loaded with fura 2 to permit measurement of the intracellular calcium concentration ([Ca2+]i). Short-circuit current (Isc), an index of transepithelial active ion transport, was measured on cells from the same cultures. Surface epithelial [Ca2+]i of 82 +/- 15 nM was increased transiently by isoproterenol, histamine, and bradykinin with maximal increases of 88 +/- 17, 480 +/- 149, and 978 +/- 214 nM (n = 15), respectively. Baseline [Ca2+]i in cultured gland cells of 68 +/- 11 nM was increased transiently by isoproterenol, histamine, methacholine, and bradykinin with maximal increases of 105 +/- 19, 233 +/- 47, 327 +/- 121, and 634 +/- 151 nM (n = 17-21), respectively. In both cell types, mediators that increased [Ca2+]i also increased Isc with a time course identical to the increase in [Ca2+]i. Pretreatment with the calcium chelator, 1,2-bis-(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid, acetoxymethyl ester (BAPTA-AM), had no effect on basal Isc or transepithelial resistance but markedly inhibited both the Isc and [Ca2+]i responses to agonists. Forskolin (10(-5) M), 3-isobutyl-1-methylxanthine (10(-3) M), dibutyryl adenosine 3',5'-cyclic monophosphate (10(-3) M), and 8-(4-chlorophenylthio)-cAMP (10(-3) M) had no or only trivial effects on Isc and [Ca2+]i. We suggest that mediators increase Isc across human airway epithelium by activating Ca-dependent basolateral K channels, resulting in hyperpolarization and an increased driving force for Cl exit through apical membrane Cl channels.

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.

Phospholipase C controls chloride-dependent short-circuit current in human bronchial epithelial cells

2020 ◽  
Author(s):  
Chloé Grebert ◽  
Frederic Becq ◽  
Clarisse Vandebrouck
Keyword(s):  
Epithelial Cells ◽  
Phospholipase C ◽  
Chloride Channels ◽  
Short Circuit ◽  
Ion Homeostasis ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Bronchial Epithelial Cells ◽  
Airway Epithelial ◽  
Bronchial Epithelial

Chloride secretion by airway epithelial cells is primordial for water and ion homeostasis and airways surface prevention of infections. This secretion is impaired in several human diseases, including cystic fibrosis, a genetic pathology due to CFTR gene mutations leading to chloride channel defects. A potential therapeutic approach is aiming at increasing chloride secretion either by correcting the mutated CFTR itself or by stimulating non-CFTR chloride channels at the plasma membrane. Here we studied the role of phospholipase C in regulating the transepithelial chloride secretion in human airway epithelial 16HBE14o- and CFBE cells over expressing WT‑ or F508del‑CFTR. Western blot analysis shows expression of the three endogenous PLC isoforms PLCd1, PLCg1 and PLCb3 in 16HBE14o‑ cells. In 16HBE14o‑ cells we performed Ussing chamber experiments after silencing each of these PLC isoforms or using the PLC inhibitor U73122 or its inactive analogue U73343. Our results show the involvement of PLCb3 and PLCg1 in CFTR-dependent short-circuit current activated by forskolin, but not PLCd1. In CFBE-WT CFTR and corrected CFBE‑F508del CFTR cells, PLCb3 silencing also inhibits CFTR‑dependent current activated by forskolin and UTP-activated calcium-dependent chloride channels (CaCC). Our study supports the importance of PLC in maintaining CFTR‑dependent chloride secretion over time, getting maximal CFTR-dependent current and increasing CaCC activation in bronchial epithelial cells.

Synthetic chloride channel restores glutathione secretion in cystic fibrosis airway epithelia

2001 ◽  
Vol 281 (1) ◽  
pp. L24-L30 ◽  
Author(s):  
Lin Gao ◽  
James R. Broughman ◽  
Takeo Iwamoto ◽  
John M. Tomich ◽  
Charles J. Venglarik ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
High Performance ◽  
Chloride Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Epithelial Cell Line ◽  
Inherited Disease ◽  
Airway Epithelia ◽  
Control Value

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.

Effects of ion transport inhibitors on MCh-mediated secretion from porcine airway submucosal glands

2002 ◽  
Vol 93 (3) ◽  
pp. 873-881 ◽  
Author(s):  
Jonathan E. Phillips ◽  
John A. Hey ◽  
Michel R. Corboz
Keyword(s):  
Ion Transport ◽  
Channel Blocker ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Gland Secretion ◽  
Sodium Absorption ◽  
Airway Function ◽  
Submucosal Glands ◽  
Chloride Channel Blocker

Submucosal glands secrete macromolecules and liquid that are essential for normal airway function. To determine the mechanisms responsible for airway gland secretion and the interaction between gland secretion and epithelial ion transport, studies were performed in porcine tracheal epithelia by using the hillocks and Ussing techniques. No significant baseline gland fluid flux ( J G) was measured by the hillocks technique after 3 min, and the epithelia had an average potential difference of 7.5 ± 0.5 mV (lumen negative) with a short-circuit current of 73 ± 4 μA/cm2, as measured by the Ussing technique. The secretagogue methacholine induced concentration-dependent increases in J G after 3 min from 0.003 μl · min−1 · cm−2 at 0.1 μM to 0.41 ± 0.04 μl · min−1 · cm−2 at 1,000 μM, with a 0.9 ± 0.1 mV hyperpolarization of the epithelium at 1,000 μM. When the epithelium was pretreated for 3 min with the sodium channel blocker amiloride, the methacholine (1,000 μM)-induced J G increased to 0.67 ± 0.09 μl · min−1 · cm−2, and the hyperpolarization increased to 2.2 ± 0.5 mV over the amiloride-pretreated level. When pretreated for 3 min with the chloride channel blocker diphenylamine-2-carboxylic acid, the methacholine (1,000 μM)-induced J G was inhibited to 0.20 ± 0.06 μl · min−1 · cm−2, and the methacholine-induced hyperpolarization was abolished. These data indicate that, in porcine airways, methacholine-induced J G may be increased by inhibition of sodium absorption and decreased by inhibition of chloride secretion.

Role of calcium in the regulation of colonic secretion in the rat

1983 ◽  
Vol 244 (5) ◽  
pp. G552-G560 ◽  
Author(s):  
T. W. Zimmerman ◽  
J. W. Dobbins ◽  
H. J. Binder
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Extracellular Calcium ◽  
Electrolyte Transport ◽  
Rat Colon ◽  
Sodium Absorption ◽  
Chloride Absorption

In vitro experiments were performed in rat colon to define the role of calcium in the regulation of electrolyte transport. Neither basal net sodium absorption (JNanet) nor JClnet was affected by varying serosal calcium from 0 to 3.0 mM, but both were decreased by 4.8 mM calcium. Removal of serosal calcium completely inhibited the effect of bethanechol, a muscarinic cholinergic agonist, which inhibits neutral sodium-chloride absorption in 1.2 mM calcium. In contrast, theophylline significantly decreased JNanet and JClnet both in the presence and absence of calcium, but the effects of theophylline were significantly less in calcium-free media. In 3.0 mM calcium bethanechol inhibited JCLnet significantly greater than JNanet and in 4.8 mM calcium bethanechol decreased JClnet equivalent to the increase in short-circuit current without significantly altering JNanet. We conclude that 1) high [Ca2+] inhibits net sodium and net chloride absorption; 2) the alteration of electrolyte transport by bethanechol is dependent on extracellular calcium, and the alteration of electrolyte transport by theophylline is not dependent on extracellular calcium but may be dependent on intracellular calcium; and 3) in addition to inhibition of neutral NaCl absorption, bethanechol stimulates chloride secretion.

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