Intestinal ion transport in NKCC1-deficient mice

2000 ◽  
Vol 279 (4) ◽  
pp. G707-G718 ◽  
Author(s):  
B. R. Grubb ◽  
E. Lee ◽  
A. J. Pace ◽  
B. H. Koller ◽  
R. C. Boucher
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Targeted Mutagenesis ◽  
Intestinal Epithelia ◽  
Entry Pathway ◽  
Intestinal Pathology ◽  
Ion Substitution ◽  
Deficient Mice ◽  
Intestinal Ion Transport

The Na+-K+-2Cl− cotransporter (NKCC1) located on the basolateral membrane of intestinal epithelia has been postulated to be the major basolateral Cl− entry pathway. With targeted mutagenesis, mice deficient in the NKCC1 protein were generated. The basal short-circuit current did not differ between normal and NKCC1 −/− jejuna. In the −/− jejuna, the forskolin response (22 μA/cm2; bumetanide insensitive) was significantly attenuated compared with the bumetanide-sensitive response (52 μA/cm2) in normal tissue. Ion-replacement studies demonstrated that the forskolin response in the NKCC1 −/− jejuna was HCO3 − dependent, whereas in the normal jejuna it was independent of the HCO3 − concentration in the buffer. NKCC1 −/− ceca exhibited a forskolin response that did not differ significantly from that of normal ceca, but unlike that of normal ceca, was bumetanide insensitive. Ion-substitution studies suggested that basolateral HCO3 − as well as Cl− entry (via non-NKCC1) paths played a role in the NKCC1 −/− secretory response. In contrast to cystic fibrosis mice, which lack both basal and stimulated Cl− secretion and exhibit severe intestinal pathology, the absence of intestinal pathology in NKCC1 −/− mice likely reflects the ability of the intestine to secrete HCO3 − and Cl− by basolateral entry mechanisms independent of NKCC1.

scholarly journals Molecular mechanisms for intestinal HCO3- secretion and its regulation by guanylin in seawater-acclimated eels

2019 ◽  
Author(s):  
Yoshio Takei ◽  
Marty K.S. Wong ◽  
Masaaki Ando
Keyword(s):  
Hormonal Regulation ◽  
Time Course ◽  
Molecular Mechanisms ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Disulfonic Acid ◽  
Intestinal Epithelia ◽  
Mucosal Side

AbstractThe intestine of marine teleosts secretes HCO3- into the lumen and precipitates Ca2+ and Mg2+ in the imbibed seawater as carbonates to decrease luminal fluid osmolality and facilitate water absorption. However, reports on studies on the hormonal regulation of HCO3- secretion are just emerging. Here, we showed that guanylin (GN) applied to the mucosal side of intestinal epithelia increased HCO3- secretion in seawater-acclimated eels. The effect of GN on HCO3- secretion was slower than that on the short-circuit current, and the time-course of the GN effect was similar to that of bumetanide. Mucosal bumetanide and serosal 4,4’-dinitrostilbene-2,2’-disulfonic acid (DNDS) inhibited the GN effect, suggesting an involvement of apical Na+-K+-2Cl- cotransporter (NKCC2) and basolateral Cl-/HCO3- exchanger (AE)/Na+-HCO3- cotransporter (NBC) in the GN effect. However, mucosal DNDS and diphenylamine-2-carboxylic acid (DPC) failed to inhibit the GN effect, showing that apical AE and Cl- channel are not involved. To identify molecular species of possible transporters involved in the GN effect, we performed RNA-seq analyses followed by quantitative real-time PCR after transfer of eels to seawater. Among the genes upregulated after seawater transfer, those of Slc26a3a, b (DRAa, b) and Slc26a6a, c (Pat-1a, c) on the apical membrane of the intestinal epithelial cells, and those of Sls4a4a (NBCe1a), Slc4a7 (NBCn1), Slc4a10a (NBCn2a) and Slc26a1 (Sat-1) on the basolateral membrane were candidate transporters involved in HCO3- secretion. Judging from the slow effect of GN, we suggest that GN inhibits NKCC2b on the apical membrane and decreases cytosolic Cl- and Na+, which then activates apical DNDS-insensitive DRAa, b and basolateral DNDS-sensitive NBCela, n1, n2a to enhance transcellular HCO3- flux across the intestinal epithelia of seawater-acclimated eels.

Alterations in airway ion transport in NKCC1-deficient mice

2001 ◽  
Vol 281 (2) ◽  
pp. C615-C623 ◽  
Author(s):  
B. R. Grubb ◽  
A. J. Pace ◽  
E. Lee ◽  
B. H. Koller ◽  
R. C. Boucher
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Airway Disease ◽  
Treatment Protocols ◽  
Anion Secretion ◽  
Ussing Chambers ◽  
Adult Mice ◽  
Ion Substitution ◽  
Deficient Mice

Airways of Na+-K+-2Cl−(NKCC1)-deficient mice (−/−) were studied in Ussing chambers to determine the role of the basolateral NKCC1 in transepithelial anion secretion. The basal short-circuit current ( I sc) of tracheae and bronchi from adult mice did not differ between NKCC1−/− and normal mice, whereas NKCC1−/− tracheae from neonatal mice exhibited a significantly reduced basal I sc. In normal mouse tracheae, sensitivity to the NKCC1 inhibitor bumetanide correlated inversely with the age of the mouse. In contrast, tracheae from NKCC1−/− mice at all ages were insensitive to bumetanide. The anion secretory response to forskolin did not differ between normal and NKCC1−/− tissues. However, when larger anion secretory responses were induced with UTP, airways from the NKCC1−/− mice exhibited an attenuated response. Ion substitution and drug treatment protocols suggested that HCO[Formula: see text]secretion compensated for reduced Cl− secretion in NKCC1−/− airway epithelia. The absence of spontaneous airway disease or pathology in airways from the NKCC1−/− mice suggests that the NKCC1 mutant mice are able to compensate adequately for absence of the NKCC1 protein.

Nystatin studies of the skin of larval Rana catesbeiana

1983 ◽  
Vol 244 (1) ◽  
pp. R58-R65
Author(s):  
T. C. Cox ◽  
R. H. Alvarado
Keyword(s):  
Rana Catesbeiana ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Electrical Potential ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Electrical Characteristics ◽  
Shunt Resistance ◽  
Selective Permeability ◽  
Ion Substitution

Transport and electrical characteristics of the isolated skin of larval Rana catesbeiana were analyzed using ion substitution and nystatin. When the inner (IBS) and outer (OBS) bathing solutions contained Na Ringer solution the electrical potential (TEP), short-circuit current (SCC), and resistance (R2) were 23.5 +/- 7.0 mV, 2.8 +/- 0.7 microA . cm-2, and 8.00 +/- 0.74 k omega. cm2, respectively (n = 4). When K was substituted for Na in the OBS these values were not changed significantly. When nystatin (120 U.cm-3), a drug that increases the permeability of membranes to small cations, was added to the OBS (Na Ringer) there was a striking increase in the TEP to 52.8 +/- 3.1 mV, SCC to 14.8 +/- 2.0 microA . cm-2, and drop in R2 to 3.75 +/- 0.52 k omega . cm2. The response to nystatin was similar with Na or K Ringer solution in the OBS (Na Ringer in the IBS). With Na Ringer in the OBS and IBS, the increase in SCC induced by low doses of nystatin equaled net Na flux measured isotopically. Plots of transepithelial conductance against SCC after nystatin were linear and provided estimates of shunt resistance (R*sh = 14.6 +/- 1.3 k omega . cm2) and electromotive driving force for ions (E*A = 76 +/- 3 mV). Similar curves were obtained with K Ringer in the OBS. In the presence of nystatin, characteristics of the basolateral membrane were evaluated. It displayed selective permeability to K relative to Na or Tris.

Electrogenic bicarbonate secretion by prairie dog gallbladder

2007 ◽  
Vol 292 (6) ◽  
pp. G1683-G1694 ◽  
Author(s):  
A. James Moser ◽  
A. Gangopadhyay ◽  
N. A. Bradbury ◽  
K. W. Peters ◽  
R. A. Frizzell ◽  
...  
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Impedance Analysis ◽  
Membrane Resistance ◽  
Disulfonic Acid ◽  
Prairie Dog ◽  
Anion Secretion ◽  
Ion Substitution

Pathological rates of gallbladder salt and water transport may promote the formation of cholesterol gallstones. Because prairie dogs are widely used as a model of this event, we characterized gallbladder ion transport in animals fed control chow by using electrophysiology, ion substitution, pharmacology, isotopic fluxes, impedance analysis, and molecular biology. In contrast to the electroneutral properties of rabbit and Necturus gallbladders, prairie dog gallbladders generated significant short-circuit current ( Isc; 171 ± 21 μA/cm2) and lumen-negative potential difference (−10.1 ± 1.2 mV) under basal conditions. Unidirectional radioisotopic fluxes demonstrated electroneutral NaCl absorption, whereas the residual net ion flux corresponded to Isc. In response to 2 μM forskolin, Isc exceeded 270 μA/cm2, and impedance estimates of the apical membrane resistance decreased from 200 Ω·cm2 to 13 Ω·cm2. The forskolin-induced Isc was dependent on extracellular HCO3− and was blocked by serosal 4,4′-dinitrostilben-2,2′-disulfonic acid (DNDS) and acetazolamide, whereas serosal bumetanide and Cl− ion substitution had little effect. Serosal trans-6-cyano-4-( N-ethylsulfonyl- N-methylamino)-3-hydroxy-2,2-dimethyl-chroman and Ba2+ reduced Isc, consistent with the inhibition of cAMP-dependent K+ channels. Immunoprecipitation and confocal microscopy localized cystic fibrosis transmembrane conductance regulator protein (CFTR) to the apical membrane and subapical vesicles. Consistent with serosal DNDS sensitivity, pancreatic sodium-bicarbonate cotransporter protein pNBC1 expression was localized to the basolateral membrane. We conclude that prairie dog gallbladders secrete bicarbonate through cAMP-dependent apical CFTR anion channels. Basolateral HCO3− entry is mediated by DNDS-sensitive pNBC1, and the driving force for apical anion secretion is provided by K+ channel activation.

scholarly journals Interferon-γ Down-regulates Adenosine 2b Receptor-mediated Signaling and Short Circuit Current in the Intestinal Epithelia by Inhibiting the Expression of Adenylate Cyclase

2004 ◽  
Vol 280 (6) ◽  
pp. 4048-4057 ◽  
Author(s):  
Vasantha Kolachala ◽  
Vivian Asamoah ◽  
Lixin Wang ◽  
Shanthi Srinivasan ◽  
Didier Merlin ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Interferon Γ ◽  
Intestinal Epithelia

Suppression of gastric acid secretion by furosemide in isolated gastric mucosa of guinea pig

1980 ◽  
Vol 239 (6) ◽  
pp. G532-G535 ◽  
Author(s):  
A. Ayalon ◽  
A. Corcia ◽  
G. Klemperer ◽  
S. R. Caplan
Keyword(s):  
Guinea Pig ◽  
Acid Secretion ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Electrical Conductance ◽  
Short Circuit Current ◽  
Cl Transport ◽  
Consequent Reduction ◽  
Net Flux ◽  
Mucosal Side

The effect of furosemide on acid secretion and Cl- transport was studied in isolated fundic mucosa of the guinea pig. Furosemide (10(-3) M), applied to the serosal side produced an immediate effect on the short-circuit current (Isc), lowering it by 47 +/- 2%. Potential difference decreased by 29 +/- 3%, electrical conductance by 18 +/- 4%, acid secretion by 38 +/- 1%, and net flux of Cl- from serosal-to-mucosal side by 37%. Application of the drug to the mucosal side produced similar effects on acid secretion and on the electrical parameters. It is suggested that furosemide blocks the entrance of Cl-, by the Na+--Cl- cotransport mechanism, through the basolateral membrane of the secreting cell. The consequent reduction in electrogenic Cl- transport would cause Isc and acid secretion to decrease. A reduction of Cl- conductance of the apical membrane, upon mucosal application of the drug, would cause similar effects on acid secretion and Cl- transport.

Active electrolyte transport in mammalian buccal mucosa

1988 ◽  
Vol 255 (3) ◽  
pp. G286-G291 ◽  
Author(s):  
R. C. Orlando ◽  
N. A. Tobey ◽  
V. J. Schreiner ◽  
R. D. Readling
Keyword(s):  
Buccal Mucosa ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Electrical Potential ◽  
Short Circuit Current ◽  
Electrolyte Transport ◽  
Electrical Potential Difference ◽  
Ussing Chambers ◽  
Net Fluxes

The transmural electrical potential difference (PD) was measured in vivo across the buccal mucosa of humans and experimental animals. Mean PD was -31 +/- 2 mV in humans, -34 +/- 2 mV in dogs, -39 +/- 2 mV in rabbits, and -18 +/- 1 mV in hamsters. The mechanisms responsible for this PD were explored in Ussing chambers using dog buccal mucosa. After equilibration, mean PD was -16 +/- 2 mV, short-circuit current (Isc) was 15 +/- 1 microA/cm2, and resistance was 1,090 +/- 100 omega.cm2, the latter indicating an electrically "tight" tissue. Fluxes of [14C]mannitol, a marker of paracellular permeability, varied directly with tissue conductance. The net fluxes of 22Na and 36Cl were +0.21 +/- 0.05 and -0.04 +/- 0.02 mueq/h.cm2, respectively, but only the Na+ flux differed significantly from zero. Isc was reduced by luminal amiloride, serosal ouabain, or by reducing luminal Na+ below 20 mM. This indicated that the Isc was determined primarily by active Na+ absorption and that Na+ traverses the apical membrane at least partly through amiloride-sensitive channels and exits across the basolateral membrane through Na+-K+-ATPase activity. We conclude that buccal mucosa is capable of active electrolyte transport and that this capacity contributes to generation of the buccal PD in vivo.

scholarly journals Zinc inhibits cAMP-stimulated Cl secretion via basolateral K-channel blockade in rat ileum

2005 ◽  
Vol 288 (5) ◽  
pp. G956-G963 ◽  
Author(s):  
Kazi Mirajul Hoque ◽  
Vazhaikkurichi M. Rajendran ◽  
Henry J. Binder
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
K Channel ◽  
Isolated Cells ◽  
Rat Ileum ◽  
Anion Secretion ◽  
Cl Secretion ◽  
Ion Absorption ◽  
Isotonic Buffer

Zn, an essential micronutrient and second most abundant trace element in cell and tissues, reduces stool output when administered to children with acute diarrhea. The mechanism by which Zn improves diarrhea is not known but could result from stimulating Na absorption and/or inhibiting anion secretion. The aim of this study was to investigate the direct effect of Zn on intestinal epithelial ion absorption and secretion. Rat ileum was partially stripped of serosal and muscle layers, and the mucosa was mounted in lucite chambers. Potential difference and short-circuit current were measured by conventional current-voltage clamp method.86Rb efflux and uptake were assessed for serosal K channel and Na-K-2Cl cotransport activity, respectively. Efflux experiments were performed in isolated cells preloaded with86Rb in the presence of ouabain and bumetanide, whereas uptake experiments were performed in low-Cl isotonic buffer containing Ba and ouabain. Neither mucosal nor serosal Zn affected glucose-stimulated Na absorption. In contrast, forskolin-induced Cl secretion was markedly reduced by serosal but not mucosal addition of Zn. Zn also substantially reversed the increase in Cl secretion induced by 8-bromoadenosine 3′,5′-cyclic monophosphate (8-BrcAMP) with half-maximal inhibitory concentration of 0.43 mM. In contrast, serosal Zn did not alter Cl secretion stimulated by carbachol, a Ca-dependent agonist. Zn inhibited 8-BrcAMP-stimulated86Rb efflux but not carbachol-stimulated86Rb efflux. Zn had no effect on bumetanide-sensitive86Rb uptake, Na-K-ATPase, or CFTR. We conclude from these studies that Zn inhibits cAMP-induced Cl secretion by blocking basolateral membrane K channels.

Mechanisms of sodium and chloride transport across equine tracheal epithelium

1990 ◽  
Vol 259 (6) ◽  
pp. L459-L467 ◽  
Author(s):  
G. J. Tessier ◽  
T. R. Traynor ◽  
M. S. Kannan ◽  
S. M. O3'Grady
Keyword(s):  
Chloride Transport ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Tracheal Epithelium ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Cotransport System ◽  
Ethanesulfonic Acid

Equine tracheal epithelium, stripped of serosal muscle, mounted in Ussing chambers, and bathed in plasmalike Ringer solution generates a serosa-positive transepithelial potential of 10–22 mV and a short-circuit current (Isc) of 70–200 microA/cm2. Mucosal amiloride (10 microM) causes a 40–60% decrease in Isc and inhibits the net transepithelial Na flux by 95%. Substitution of Cl with gluconate resulted in a 30% decrease in basal Isc. Bicarbonate substitution with 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid decreased the Isc by 21%. The Cl-dependent Isc was inhibited by serosal addition of 1 mM amiloride. Bicarbonate replacement or serosal amiloride (1 mM) inhibits the net Cl flux by 72 and 69%, respectively. Bicarbonate replacement significantly reduces the effects of serosal amiloride (1 mM) on Isc, indicating its effect is HCO3 dependent. Addition of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP; 100 microM) causes a 40% increase in Isc. This effect is inhibited by subsequent addition of 10 microM serosal bumetanide. Bumetanide (10 microM) reduces net Cl secretion following stimulation with 8-BrcAMP (100 microM). Serosal addition of BaCl2 (1 mM) causes a reduction in Isc equal to that following Cl replacement in the presence or absence of 100 microM cAMP. These results suggest that 1) Na absorption depends on amiloride-inhibitable Na channels in the apical membrane, 2) Cl influx across the basolateral membrane occurs by both a Na-H/Cl-HCO3 parallel exchange mechanism under basal conditions and by a bumetanide-sensitive Na-(K?)-Cl cotransport system under cAMP-stimulated conditions, and 3) basal and cAMP-stimulated Cl secretion depends on Ba-sensitive K channels in the basolateral membrane.

ENaC- and CFTR-dependent ion and fluid transport in mammary epithelia

2001 ◽  
Vol 281 (2) ◽  
pp. C633-C648 ◽  
Author(s):  
Sasha Blaug ◽  
Kevin Hybiske ◽  
Jonathan Cohn ◽  
Gary L. Firestone ◽  
Terry E. Machen ◽  
...  
Keyword(s):  
Tight Junctions ◽  
Fluid Transport ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Fluid Secretion ◽  
Equivalent Circuit Analysis ◽  
Mean Values ◽  
Apical Side

Mammary epithelial 31EG4 cells (MEC) were grown as monolayers on filters to analyze the apical membrane mechanisms that help mediate ion and fluid transport across the epithelium. RT-PCR showed the presence of cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial Na+ channel (ENaC) message, and immunomicroscopy showed apical membrane staining for both proteins. CFTR was also localized to the apical membrane of native human mammary duct epithelium. In control conditions, mean values of transepithelial potential (apical-side negative) and resistance ( R T) are −5.9 mV and 829 Ω · cm2, respectively. The apical membrane potential ( V A) is −40.7 mV, and the mean ratio of apical to basolateral membrane resistance ( R A/ R B) is 2.8. Apical amiloride hyperpolarized V A by 19.7 mV and tripled R A/ R B. A cAMP-elevating cocktail depolarized V A by 17.6 mV, decreased R A/ R B by 60%, increased short-circuit current by 6 μA/cm2, decreased R T by 155 Ω · cm2, and largely eliminated responses to amiloride. Whole cell patch-clamp measurements demonstrated amiloride-inhibited Na+ currents [linear current-voltage ( I-V) relation] and forskolin-stimulated Cl−currents (linear I-V relation). A capacitance probe method showed that in the control state, MEC monolayers either absorbed or secreted fluid (2–4 μl · cm−2 · h−1). Fluid secretion was stimulated either by activating CFTR (cAMP) or blocking ENaC (amiloride). These data plus equivalent circuit analysis showed that 1) fluid absorption across MEC is mediated by Na+ transport via apical membrane ENaC, and fluid secretion is mediated, in part, by Cl− transport via apical CFTR; 2) in both cases, appropriate counterions move through tight junctions to maintain electroneutrality; and 3) interactions among CFTR, ENaC, and tight junctions allow MEC to either absorb or secrete fluid and, in situ, may help control luminal [Na+] and [Cl−].

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