scholarly journals Electrophysiological measurement of transepithelial ion transport: short-circuit current (Ussing chamber) technique.

2004 ◽  
Vol 123 (3) ◽  
pp. 211-218 ◽  
Author(s):  
Shin-ichiro KARAKI ◽  
Atsukazu KUWAHARA
Keyword(s):  
Ion Transport ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Electrophysiological Measurement ◽  
Chamber Technique

Basolateral Cl− uptake mechanisms in Xenopus laevis lung epithelium

2010 ◽  
Vol 299 (1) ◽  
pp. R92-R100 ◽  
Author(s):  
Jens Berger ◽  
Martin Hardt ◽  
Wolfgang G. Clauss ◽  
Martin Fronius
Keyword(s):  
Xenopus Laevis ◽  
Ion Transport ◽  
Anion Exchanger ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Transport Processes ◽  
Lung Epithelium ◽  
Active Ion Transport ◽  
Uptake Mechanisms

A thin liquid layer covers the lungs of air-breathing vertebrates. Active ion transport processes via the pulmonary epithelial cells regulate the maintenance of this layer. This study focuses on basolateral Cl− uptake mechanisms in native lungs of Xenopus laevis and the involvement of the Na+/K+/2 Cl− cotransporter (NKCC) and HCO3−/Cl− anion exchanger (AE), in particular. Western blot analysis and immunofluorescence staining revealed the expression of the NKCC protein in the Xenopus lung. Ussing chamber experiments demonstrated that the NKCC inhibitors (bumetanide and furosemide) were ineffective at blocking the cotransporter under basal conditions, as well as under pharmacologically stimulated Cl−-secreting conditions (forskolin and chlorzoxazone application). However, functional evidence for the NKCC was detected by generating a transepithelial Cl− gradient. Further, we were interested in the involvement of the HCO3−/Cl− anion exchanger to transepithelial ion transport processes. Basolateral application of DIDS, an inhibitor of the AE, resulted in a significantly decreased the short-circuit current (ISC). The effect of DIDS was diminished by acetazolamide and reduced by increased external HCO3− concentrations. Cl− secretion induced by forskolin was decreased by DIDS, but this effect was abolished in the presence of HCO3−. These experiments indicate that the AE at least partially contributes to Cl− secretion. Taken together, our data show that in Xenopus lung epithelia, the AE, rather than the NKCC, is involved in basolateral Cl− uptake, which contrasts with the common model for Cl− secretion in pulmonary epithelia.

scholarly journals Enterohemorrhagic E. coli (EHEC)—Secreted Serine Protease EspP Stimulates Electrogenic Ion Transport in Human Colonoid Monolayers

Toxins ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 351 ◽  
Author(s):  
C. Tse ◽  
Julie In ◽  
Jianyi Yin ◽  
Mark Donowitz ◽  
Michele Doucet ◽  
...  
Keyword(s):  
Ion Transport ◽  
Serine Protease ◽  
Shiga Toxin ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Adult Stem Cell ◽  
Short Circuit Current ◽  
Watery Diarrhea ◽  
E Coli ◽  
Electrogenic Ion Transport

One of the characteristic manifestations of Shiga-toxin-producing Escherichia coli (E. coli) infection in humans, including EHEC and Enteroaggregative E. coli O104:H4, is watery diarrhea. However, neither Shiga toxin nor numerous components of the type-3 secretion system have been found to independently elicit fluid secretion. We used the adult stem-cell-derived human colonoid monolayers (HCM) to test whether EHEC-secreted extracellular serine protease P (EspP), a member of the serine protease family broadly expressed by diarrheagenic E. coli can act as an enterotoxin. We applied the Ussing chamber/voltage clamp technique to determine whether EspP stimulates electrogenic ion transport indicated by a change in short-circuit current (Isc). EspP stimulates Isc in HCM. The EspP-stimulated Isc does not require protease activity, is not cystic fibrosis transmembrane conductance regulator (CFTR)-mediated, but is partially Ca2+-dependent. EspP neutralization with a specific antibody reduces its potency in stimulating Isc. Serine Protease A, secreted by Enteroaggregative E. coli, also stimulates Isc in HCM, but this current is CFTR-dependent. In conclusion, EspP stimulates colonic CFTR-independent active ion transport and may be involved in the pathophysiology of EHEC diarrhea. Serine protease toxins from E. coli pathogens appear to serve as enterotoxins, potentially significantly contributing to watery diarrhea.

Dantrolene and basal ileal sodium and chloride transport: involvement of calcium stores

1983 ◽  
Vol 245 (6) ◽  
pp. G780-G785
Author(s):  
M. Donowitz ◽  
S. Cusolito ◽  
L. Battisti ◽  
G. W. Sharp
Keyword(s):  
Ion Transport ◽  
Intracellular Calcium ◽  
Chloride Transport ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Calcium Stores ◽  
Ionophore A23187 ◽  
Rabbit Ileum ◽  
Intracellular Calcium Stores

The effect of dantrolene on active ion transport in rabbit ileum was determined using the Ussing chamber short-circuiting technique. Dantrolene prevents the release of calcium from intracellular stores in skeletal muscle and was used to probe the role of intracellular calcium stores in intestinal ion transport. A saturated solution of dantrolene (approx 25 microM) decreased ileal short-circuit current and potential difference, increased conductance and mucosal-to-serosal and net Na and Cl fluxes, but did not alter serosal-to-mucosal Na and Cl fluxes. The dantrolene stimulation of active Na and Cl absorption was specific since it did not alter glucose-dependent Na absorption, transport changes caused by Ca2+ ionophore A23187, or the increase in short-circuit current caused by dibutyryl cAMP or theophylline. These effects were associated with an increase in total ileal calcium content and a decreased rate of 45Ca2+ efflux without any change in 45Ca2+ influx from the serosal or mucosal surfaces. These findings are consistent with an effect of dantrolene to stimulate active ileal Na and Cl absorption by a mechanism involving lowered cytosol Ca2+ levels and compatible with trapping calcium in intracellular stores. It thus appears as if intracellular calcium stores have an important role in the control of basal ion transport in the intestine.

Mast cells in the regulation of intestinal electrolyte transport

1986 ◽  
Vol 251 (2) ◽  
pp. G253-G262 ◽  
Author(s):  
D. A. Russell
Keyword(s):  
Mast Cells ◽  
Ion Transport ◽  
Trichinella Spiralis ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Antigen Challenge ◽  
Intracellular Camp ◽  
Camp Content ◽  
Ionic Basis

Experiments were performed to determine the ionic basis and physiological messengers of transepithelial ion transport alterations (short-circuit current, Isc) measured during the induction of intestinal anaphylaxis in an Ussing chamber. Antigens derived from Trichinella spiralis, an intestinal parasite, were used to challenge jejunal tissue from guinea pigs immunized by infection with the parasite. Histamine (10(-4) M) caused an increased in Isc that was similar to that induced by antigen. Diphenhydramine (10(-5) M) inhibited the epithelial electrical responses to histamine by 100% and to antigen by 60-70%. Indomethacin (10(-5) M), in combination with diphenhydramine, completely inhibited the antigen-induced rise in Isc. Furosemide (10(-4) M) caused 50-60% inhibition of the increase in Isc induced by antigen and histamine. Antigen challenge of isolated enterocytes did not alter intracellular cAMP content. However, antigen challenge of jejunal segments in which epithelial cells were in contact with sensitized mast cells increased mucosal cAMP content. These results suggest that electrogenic Cl- secretion, mediated in part by cAMP, contributes to antigen-induced jejunal ion transport changes and that histamine and prostaglandins are involved in eliciting these epithelial responses.

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.

scholarly journals Segment-specific effects of epinephrine on ion transport in the colon of the rat

1998 ◽  
Vol 275 (6) ◽  
pp. G1367-G1376 ◽  
Author(s):  
Silke Hörger ◽  
Gerhard Schultheiß ◽  
Martin Diener
Keyword(s):  
Ion Transport ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Proximal Colon ◽  
Rat Colon ◽  
Second Phase ◽  
Stimulatory Action ◽  
Β Receptor

The effect of epinephrine on transport of K+, Na+, Cl−, and[Formula: see text] across the rat colon was studied using the Ussing chamber technique. Epinephrine (5 × 10−6mol/l) induced a biphasic change in short-circuit current ( Isc) in distal and proximal colon: a transient increase followed by a long-lasting decay. The first phase of the Iscresponse was abolished in Cl−-poor solution or after bumetanide administration, indicating a transient induction of Cl−secretion. The second phase of the response to epinephrine was suppressed by apical administration of the K+channel blocker, quinine, and was concomitant with an increase in serosal-to-mucosal Rb+flux, indicating that epinephrine induced K+secretion, although this response was much smaller than the change in Isc. In addition, the distal colon displayed a decrease in mucosal-to-serosal and serosal-to-mucosal Cl−fluxes when treated with epinephrine. In the distal colon, indomethacin abolished the first phase of the epinephrine effect, whereas the second phase was suppressed by TTX. In the proximal colon, indomethacin and TTX were ineffective. The neuronally mediated response to epinephrine in the distal colon was suppressed by the nonselective β-receptor blocker, propranolol, and by the β2-selective blocker, ICI-118551, whereas the epithelial response in the proximal colon was suppressed by the nonselective α-blocker, phentolamine, and by the selective α2-blocker, yohimbine. These results indicate a segment-specific action of epinephrine on ion transport: a direct stimulatory action on epithelial α2-receptors in the proximal colon and an indirect action on secretomotoneurons via β2-receptors in the distal colon.

Interleukin-13 induces a hypersecretory ion transport phenotype in human bronchial epithelial cells

2002 ◽  
Vol 282 (2) ◽  
pp. L226-L236 ◽  
Author(s):  
Henry Danahay ◽  
Hazel Atherton ◽  
Gareth Jones ◽  
Robert J. Bridges ◽  
Christopher T. Poll
Keyword(s):  
Epithelial Cells ◽  
Ion Transport ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Fluid Secretion ◽  
Bronchial Epithelial Cells ◽  
Human Bronchial Epithelial Cells ◽  
Bronchial Epithelial ◽  
Anion Conductance

Interleukin (IL)-13 has been associated with asthma, allergic rhinitis, and chronic sinusitis, all conditions where an imbalance in epithelial fluid secretion and absorption could impact upon the disease. We have investigated the effects of IL-13 on the ion transport characteristics of human bronchial epithelial cells cultured at an apical-air interface. Ussing chamber studies indicated that 48 h pretreatment with IL-13 or IL-4 significantly reduced the basal short-circuit current ( I sc) and inhibited the amiloride-sensitive current by >98%. Furthermore, the I scresponses were increased by more than six- and twofold over control values when stimulated with UTP or forskolin, respectively, after cytokine treatment. The IL-13-enhanced response to UTP/ionomycin was sensitive to bumetanide and DIDS and was reduced in a low-chloride, bicarbonate-free solution. Membrane permeablization studies indicated that IL-13 induced the functional expression of an apical Ca2+-activated anion conductance and that changes in apical or basolateral K+ conductances could not account for the increased I sc responses to UTP or ionomycin. The results indicate that IL-13 converts the human bronchial epithelium from an absorptive to a secretory phenotype that is the result of loss of amiloride-sensitive current and an increase in a DIDS-sensitive apical anion conductance.

Hyperosmoregulation in the red freshwater crab Dilocarcinus pagei (Brachyura, Trichodactylidae): structural and functional asymmetries of the posterior gills

2002 ◽  
Vol 205 (2) ◽  
pp. 167-175 ◽  
Author(s):  
Horst Onken ◽  
John Campbell McNamara
Keyword(s):  
Ion Transport ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Freshwater Crab ◽  
Pillar Cell ◽  
Na Substitution ◽  
Apical Vesicles ◽  
Gill Lamellae ◽  
Dilocarcinus Pagei

SUMMARY The osmotic and ionic status of the haemolymph and the structural and ion-transport characteristics of the posterior gills of Dilocarcinus pagei, a hololimnetic crab, were investigated. Haemolymph osmolality was 386±18 mosmol kg–1, while [Na+] and [Cl–] were 190±13 and 206±12 mmol l–1, respectively; [K+], [Ca2+] and [Mg2+] were 9.7±0.7, 10.2±0.5 and 2.8±0.4 mmol l–1, respectively (means ± s.e.m., N=12–17). The gill lamellae possess a central, osmiophilic area, which exhibits a marked structural asymmetry. The thick (18–20 μm) proximal epithelium is characterised by basal invaginations and a few apical vesicles, while the thin (3–10 μm) distal epithelium consists of apical pillar cell flanges populated by vesicles and membrane invaginations. Isolated gills, bathed and perfused with NaCl saline, spontaneously generate a negative transbranchial potential difference (Vte), which stabilises at positive or negative values. Ouabain shifts Vte to more positive values. When mounted in an Ussing chamber, distal split lamellae generate a negative, Cl–-dependent short-circuit current (Isc). Na+ substitution leads to more negative values of Isc. Internal ouabain is without effect, while diphenylamine-2-carboxylate and acetazolamide abolish Isc. Proximal split lamellae show a positive, Na+-dependent Isc, which decreases after internal application of ouabain. These data suggest that the thin epithelium actively absorbs Cl–, while the thick epithelium actively absorbs Na+.

scholarly journals Enterotoxigenicity of Mature 45-Kilodalton and Processed 35-Kilodalton Forms of Hemagglutinin Protease Purified from a Cholera Toxin Gene-Negative Vibrio cholerae Non-O1, Non-O139 Strain

2006 ◽  
Vol 74 (5) ◽  
pp. 2937-2946 ◽  
Author(s):  
A. Ghosh ◽  
D. R. Saha ◽  
K. M. Hoque ◽  
M. Asakuna ◽  
S. Yamasaki ◽  
...  
Keyword(s):  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Hela Cells ◽  
Histopathological Examination ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Toxin Gene ◽  
Dependent Manner ◽  
A Cell

ABSTRACT Cholera toxin gene-negative Vibrio cholerae non-O1, non-O139 strain PL-21 is the etiologic agent of cholera-like syndrome. Hemagglutinin protease (HAP) is one of the major secretory proteins of PL-21. The mature 45-kDa and processed 35-kDa forms of HAP were purified in the presence and absence of EDTA from culture supernatants of PL-21. Enterotoxigenicities of both forms of HAP were tested in rabbit ileal loop (RIL), Ussing chamber, and tissue culture assays. The 35-kDa HAP showed hemorrhagic fluid response in a dose-dependent manner in the RIL assay. Histopathological examination of 20 μg of purified protease-treated rabbit ileum showed the presence of erythrocytes and neutrophils in the upper part of the villous lamina propria. Treatment with 40 μg of protease resulted in gross damage of the villous epithelium with inflammation, hemorrhage, and necrosis. The 35-kDa form of HAP, when added to the lumenal surface of rat ileum loaded in an Ussing chamber, showed a decrease in the intestinal short-circuit current and a cell rounding effect on HeLa cells. The mature 45-kDa form of HAP showed an increase in intestinal short-circuit current in an Ussing chamber and a cell distending effect on HeLa cells. These results show that HAP may play a role in the pathogenesis of PL-21.

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