scholarly journals Cholera toxin enhances Na+absorption across MCF10A human mammary epithelia

2014 ◽  
Vol 306 (5) ◽  
pp. C471-C484 ◽  
Author(s):  
Qian Wang ◽  
Bruce D. Schultz
Keyword(s):  
Ion Transport ◽  
Cholera Toxin ◽  
Short Circuit ◽  
Mammary Epithelium ◽  
Brefeldin A ◽  
Short Circuit Current ◽  
The Body ◽  
Cellular Mechanisms ◽  
Mammary Epithelia ◽  
Mcf10a Cells

Cellular mechanisms to account for the low Na+concentration in human milk are poorly defined. MCF10A cells, which were derived from human mammary epithelium and grown on permeable supports, exhibit amiloride- and benzamil-sensitive short-circuit current ( Isc; a sensitive indicator of net ion transport), suggesting activity of the epithelial Na+channel ENaC. When cultured in the presence of cholera toxin (Ctx), MCF10A cells exhibit greater amiloride-sensitive Iscat all time points tested (2 h to 7 days), an effect that is not reduced with Ctx washout for 12 h. Amiloride-sensitive Iscremains elevated by Ctx in the presence of inhibitors for PKA (H-89, Rp-cAMP), PI3K (LY294002), and protein trafficking (brefeldin A). Additionally, the Ctx B subunit, alone, does not replicate these effects. RT-PCR and Western blot analyses indicate no significant increase in either the mRNA or protein expression for α-, β-, or, γ-ENaC subunits. Ctx increases the abundance of both β- and γ-ENaC in the apical membrane. Additionally, Ctx increases both phosphorylated and nonphosphorylated Nedd4-2 expression. These results demonstrate that human mammary epithelia express ENaC, which can account for the low Na+concentration in milk. Importantly, the results suggest that Ctx increases the expression but reduces the activity of the E3 ubiquitin ligase Nedd4-2, which would tend to reduce the ENaC retrieval and increase steady-state membrane residency. The results reveal a novel mechanism in human mammary gland epithelia by which Ctx regulates ENaC-mediated Na+transport, which may have inferences for epithelial ion transport regulation in other tissues throughout the body.

Glucocorticoids stimulate ENaC upregulation in bovine mammary epithelium

2007 ◽  
Vol 292 (5) ◽  
pp. C1739-C1745 ◽  
Author(s):  
Rebecca R. Quesnell ◽  
Xiaobin Han ◽  
Bruce D. Schultz
Keyword(s):  
Glucocorticoid Receptor ◽  
Ion Transport ◽  
Receptor Antagonist ◽  
Short Circuit ◽  
Ionic Composition ◽  
Mammary Epithelium ◽  
Short Circuit Current ◽  
Mineralocorticoid Receptor Antagonist ◽  
Bovine Mammary Epithelial Cells ◽  
Mammary Epithelia

Mammary epithelia produce an isotonic, low-Na+ fluid that is rich in nutrients. Mechanisms that account for the low electrolyte concentration have not been elucidated, although amiloride-sensitive ion transport has been reported in some situations. We hypothesized that corticosteroid exposure modulates epithelial Na+ channel (ENaC) expression and/or activity in bovine mammary epithelial cells. BME-UV cells were grown to confluent monolayers on permeable supports with a standard basolateral medium and apical medium of low-electrolyte, high-lactose composition that resembles the ionic composition of milk. Ion transport was assessed in modified Ussing flux chambers. Exposure to glucocorticoids (dexamethasone, cortisol, or prednisolone), but not aldosterone, increased short-circuit current ( Isc), a sensitive measure of net ion transport, whereas apical exposure to amiloride or benzamil reduced corticosteroid-induced Isc close to basal levels. Quantitative RT-PCR indicated a glucocorticoid-induced increase in mRNA for β- and γ-ENaC, whereas α-ENaC mRNA expression was only mildly affected. Exposure to mifepristone (a glucocorticoid receptor antagonist), but not spironolactone (a mineralocorticoid receptor antagonist), precluded both the corticosteroid-induced elevation in amiloride-sensitive Isc and the induced changes in β- and γ-ENaC mRNA. We conclude that Na+ movement across mammary epithelia is modulated by corticosteroids via a glucocorticoid receptor-mediated mechanism that regulates the expression of the β- and γ-subunits of ENaC. ENaC expression and activity could account for the low Na+ concentration that is typical of milk.

Ileal mucosal cyclic AMP and Cl secretion: serosal vs. mucosal addition of cholera toxin.

1977 ◽  
Vol 232 (2) ◽  
pp. E210 ◽  
Author(s):  
H E Sheerin ◽  
M Field
Keyword(s):  
Cyclic Amp ◽  
Ion Transport ◽  
Cholera Toxin ◽  
Short Circuit ◽  
Electrical Conductance ◽  
Short Circuit Current ◽  
Serosal Side ◽  
Ileal Mucosa ◽  
Camp Content ◽  
Mucosal Side

Changes in ion transport and cyclic AMP (cAMP) concentration produced by addition of cholera toxin to the serosal side of isolated rabbit ileal mucosa (CTs) were compared to the changes produced by addition to the mucosal side (CTm). CTs increased short-circuit current (SCC) as did CTm but it did so more slowly. CTs, unlike CTm, did not significantly decrease electrical conductance. Inhibition of the SCC response to theophylline, a measure of preexisting secretion, was almost complete 180 min after CTm but was not yet significant 180 min after CTs. Longer (280 min) after CTs, the SCC response to theophylline was reduced by 59%, a significant reduction but less than that caused by CTm. A statistically significant change in net Cl flux could not be demonstrated after CTs, although at 280 min the measured flux was halfway between the fluxes for control and CTm tissues. Cyclic AMP concentrations were determined at 190 min, 10 min after addition of theophylline. CTs, despite little or no effect on ion transport, increased cAMP to the same level as did CTm, and the effect on cAMP of adding toxin to both sides was additive. We conclude that 1) active secretion is probably stimulated by cholera toxin added on the serosal side, although more slowly than after addition to the mucosal side and 2) much of the toxin-stimulated cAMP content of the mucosa is not coupled to secretion.

A regulated apical Na+ conductance in dexamethasone-treated H441 airway epithelial cells

2004 ◽  
Vol 287 (2) ◽  
pp. L411-L419 ◽  
Author(s):  
S. J. Ramminger ◽  
K. Richard ◽  
S. K. Inglis ◽  
S. C. Land ◽  
R. E. Olver ◽  
...  
Keyword(s):  
Ion Transport ◽  
Rank Order ◽  
Short Circuit ◽  
Brefeldin A ◽  
Short Circuit Current ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Ionic Selectivity ◽  
Camp Content ◽  
Permeabilized Cells

Treating H441 cells with dexamethasone raised the abundance of mRNA encoding the epithelial Na+ channel α- and β-subunits and increased transepithelial ion transport (measured as short-circuit current, Isc) from <4 μA·cm−2 to 10–20 μA·cm−2. This dexamethasone-stimulated ion transport was blocked by amiloride analogs with a rank order of potency of benzamil ≥ amiloride > EIPA and can thus be attributed to active Na+ absorption. Studies of apically permeabilized cells showed that this increased transport activity did not reflect a rise in Na+ pump capacity, whereas studies of basolateral permeabilized cells demonstrated that dexamethasone increased apical Na+ conductance ( GNa) from a negligible value to 100–200 μS·cm−2. Experiments that explored the ionic selectivity of this dexamethasone-induced conductance showed that it was equally permeable to Na+ and Li+ and that the permeability to these cations was approximately fourfold greater than to K+. There was also a small permeability to N-methyl-d-glucammonium, a nominally impermeant cation. Forskolin, an agent that increases cellular cAMP content, caused an ∼60% increase in Isc, and measurements made after these cells had been basolaterally permeabilized demonstrated that this response was associated with a rise in GNa. This cAMP-dependent control over GNa was disrupted by brefeldin A, an inhibitor of vesicular trafficking. Dexamethasone thus stimulates Na+ transport in H441 cells by evoking expression of an amiloride-sensitive apical conductance that displays moderate ionic selectivity and is subject to acute control via a cAMP-dependent pathway.

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.

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.

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.

A primary cation transport by a V-type ATPase of low specificity

1996 ◽  
Vol 199 (6) ◽  
pp. 1327-1334 ◽  
Author(s):  
J Küppers ◽  
I Bunse
Keyword(s):  
Ion Transport ◽  
Proton Transport ◽  
Short Circuit ◽  
Membrane Conductance ◽  
Short Circuit Current ◽  
Potential Difference ◽  
Lepidopteran Larvae ◽  
The Family ◽  
K Transport

The enzyme involved in outward K+ transport in insect epithelia belongs to the family of V-ATPases. Evidence has been reported relating the generation of the K+ gradient to a primary electrogenic proton transport via a distinct electrophoretic nH+/K+ antiport. The subject of this paper is the transport of K+ at a thread hair sensillum of the cockroach in situ. We recorded changes in the voltage and resistance of the ion-transporting membrane and of shifts in pH caused by inhibition of energy metabolism and by putative inhibitors of a proton/cation exchanger. The results are supplemented by previous determinations of the K+ activities in the same preparation. 1. In cockroach hair sensilla, the ion transport generates a membrane voltage of 105 mV. We found that the transport rendered the positive output compartment alkaline with respect to the cytoplasm by 1.0 pH unit compared with the pH at equilibrium distribution, and we infer that proton transport cannot be the process that energizes the generation of the K+ gradient. 2. The ion transport created an electrochemical potential difference for protons, DeltaetaH, of approximately 4.5 kJ mol-1, while the potential difference for K+, DeltaetaK, amounted to approximately 11 kJ mol-1. Both potential differences are directed to the cytosol. It follows from DeltaetaK/DeltaetaH that an antiport would have to be electrophoretic to drive K+ by DeltaetaH and it should, therefore, contribute to the membrane conductance. Amiloride and harmaline did not significantly change the pH in the adjacent spaces and did not affect the voltage or the resistance of the transporting membrane. Previous determinations of the impedance have shown that the ATP-independent conductance of this membrane is small, supporting the conclusion that it lacks an electrophoretic antiport. From these results, we deduce that K+ transport in cockroach sensilla is not secondary to a proton transport and an electrochemical proton gradient. The phenomena observed match the performance of a primary, electrogenic, cation-translocating ATPase of the type deduced from analyses of the short-circuit current at the midgut epithelium of lepidopteran larvae. The validity of the H+ transport/antiport hypothesis is discussed.

Avian cecum: role of glucose and volatile fatty acids in transepithelial ion transport

1991 ◽  
Vol 260 (5) ◽  
pp. G703-G710 ◽  
Author(s):  
B. R. Grubb
Keyword(s):  
Fatty Acids ◽  
Ion Transport ◽  
Volatile Fatty Acids ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Serosal Side ◽  
Na Transport ◽  
Cl Secretion ◽  
Mucosal Side

In the fowl cecum in vitro, the influence of glucose and the three most prevalent naturally occurring volatile fatty acids (acetate, propionate, butyrate) on short-circuit current (Isc), electrical resistance, and transport of Na and Cl was determined. When glucose, acetate, or butyrate was present, ion transport was characterized by electrogenic Na absorption, greater than 65% of which was amiloride inhibitable, and Cl secretion, which also was electrogenic. Isc could be completely accounted for by net fluxes of Na and Cl. When glucose, acetate, or butyrate (10 mM both sides) was included in the incubation medium, cecal tissue maintained its Isc and a constant rate of net Na absorption and Cl secretion for a 5-h period. When no substrate was present or propionate was included in the medium, a marked fall in Isc and net Na and Cl fluxes was seen. Glucose caused an increase in Isc when added only to the serosal side. As 3-O-methylglucose (not metabolized) was not effective in stimulating Isc of the cecum (serosal or mucosal addition), it appeared that glucose increased Isc by acting as an energy substrate for active Na transport. Acetate and butyrate appeared to be equally effective in stimulating Na transport and Isc when placed on either side of the membrane. When the preparation was supplied with glucose (serosal side) and acetate was added to the mucosal side, no further stimulation of Isc occurred. Thus it appeared that acetate and butyrate were acting as substrates for active Na transport rather than stimulating Na transport by some other mechanism such as a cotransport with Na.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of calcium on ion transport and electrical properties of tracheal epithelium

1978 ◽  
Vol 44 (6) ◽  
pp. 900-904 ◽  
Author(s):  
M. G. Marin ◽  
M. M. Zaremba
Keyword(s):  
Electrical Properties ◽  
Ion Transport ◽  
Calcium Concentration ◽  
Short Circuit ◽  
Electrical Potential ◽  
Short Circuit Current ◽  
Tracheal Epithelium ◽  
Electrical Potential Difference ◽  
Tracheal Lumen

Active transport of Cl- toward the tracheal lumen and Na+ away from the lumen creates an electrical potential difference across dog tracheal epithelium. This study examined in vitro the effect of varying calcium concentration in the bathing media on the ion transport and electrical properties of dog tracheal epithelium. In six pairs of epithelia, changing calcium concentration from 1.9 to 0 mM resulted in a significant decrease in electrical resistance, from 318 +/- 36 to 214 +/- 24 omega.cm2. Short-circuit current and net Cl- and Na+ fluxes measured under short-circuit conditions were not changed significantly. Changing calcium concentration from 1.9 to 10 mM resulted in no significant change from control in the electrical properties nor in net Cl- and Na+ fluxes (short-circuit conditions). Histamine (10(-4) M) produced a significantly smaller increase in short-circuit current in 0 than in 1.9 mM Ca2+ (+5 +/- 2 vs. +12 +/- 2 microamperemeter/cm2). However, electrical changes were not significantly different in 1 or 10 mM Ca2+. These results indicate that calcium lack increased permeability of tracheal epithelium and that the increase in short-circuit current due to histamine depended in part on calcium.

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