scholarly journals ION TRANSPORT IN THE INTESTINE OF ANGUILLA ANGUILLA: GRADIENTS AND TRANSLOCATORS

1994 ◽  
Vol 193 (1) ◽  
pp. 97-117 ◽  
Author(s):  
P Marvão ◽  
M G Emílio ◽  
K Gil Ferreira ◽  
P L Fernandes ◽  
H Gil Ferreira
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Specific Inhibitor ◽  
Anguilla Anguilla ◽  
Short Circuit Current ◽  
Inhibitory Effect ◽  
Serosal Side ◽  
Sodium Dependent ◽  
Transport Inhibitors ◽  
Experimental Strategies

The transport of Na+, K+ and Cl- across the isolated epithelium of the eel intestine was studied using a combination of four experimental strategies: short-circuiting, measurements of intracellular potentials and ion concentrations, application of a variety of transport inhibitors and measurement of unidirectional fluxes with radioactive tracers. When short-circuited, the system performs a net transport of Cl- and Na+ towards the blood side, with a stoichiometry approaching 2, and a much smaller net transport of K+ towards the lumen. The system is totally driven by the sodium pump located in the basolateral barrier and the main coupling between the fluxes of the three ions is through the operation of a furosemide-sensitive transporter in the apical barrier, probably a 2Cl-/Na+/K+ symporter. The inhibitory effect of DIDS and picrylsulphonic acid on the short-circuit current, when added to the serosal side, suggests the presence of a sodium-dependent anionic shuttle located in the basolateral membrane. The short-circuit current is inhibited by H25, a non-specific inhibitor of the K+/Cl- symport, added to the serosal side. This effect occurs after a delay of at least 5 min and may result from the diffusion of the drug to the apical barrier, where it blocks the 2Cl-/Na+/K+ symport with much higher affinity.

Active ammonia excretion across the gills of the green shore crabCarcinus maenas: participation of Na+/K+-ATPase, V-type H+-ATPase and functional microtubules

2002 ◽  
Vol 205 (18) ◽  
pp. 2765-2775 ◽  
Author(s):  
Dirk Weihrauch ◽  
Andreas Ziegler ◽  
Dietrich Siebers ◽  
David W. Towle
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Carcinus Maenas ◽  
Specific Inhibitor ◽  
Ammonia Excretion ◽  
Short Circuit Current ◽  
Shore Crab ◽  
Dependent Manner ◽  
Microtubule Network ◽  
Experimental Conditions

SUMMARYAlthough aquatic animals are generally believed to export nitrogenous waste by diffusion of NH3 or NH4+ across external epithelia, evidence for active ammonia excretion has been found in a number of species. In the euryhaline green shore crab Carcinus maenas, active excretion of ammonia across isolated gills is reduced by inhibitors of the Na+/K+-ATPase and vacuolar-type H+-ATPase. In addition, a functional dynamic microtubule network is necessary, since application of colchicine, taxol or thiabendazole leads to almost complete blockage of active and gradient-driven ammonia excretion. Actin filaments seem not to play a role in the excretory process. The NH4+-dependent short-circuit current and the conductance of the isolated cuticle were reduced in a dose-dependent manner by amiloride,a non-specific inhibitor of the Na+/H+ exchanger and Na+ channels. Combined with an analysis of gill morphology, the strong intracellular but weak apical abundance of V-type H+-ATPase and the fact that ammonia flux rates are equal under buffered and unbuffered experimental conditions, our observations suggest a hypothetical model of transepithelial ammonia movement that features active uptake across the basolateral membrane, sequestration in acidified vesicles, vesicle transport via microtubules and exocytosis at the apical membrane.

scholarly journals A Ba2+-resistant, acid-sensitive K+ conductance in Na+-absorbing H441 human airway epithelial cells

2007 ◽  
Vol 292 (5) ◽  
pp. L1304-L1312 ◽  
Author(s):  
Sarah K. Inglis ◽  
Sean G. Brown ◽  
Maree J. Constable ◽  
Niall McTavish ◽  
Richard E. Olver ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Inhibitory Effect ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Human Airway ◽  
K2p Channels ◽  
Human Airway Epithelial Cells

By analysis of whole cell membrane currents in Na+-absorbing H441 human airway epithelial cells, we have identified a K+ conductance ( GK) resistant to Ba2+ but sensitive to bupivacaine or extracellular acidification. In polarized H441 monolayers, we have demonstrated that bupivacaine, lidocaine, and quinidine inhibit basolateral membrane K+ current ( IBl) whereas Ba2+ has only a weak inhibitory effect. IBl was also inhibited by basolateral acidification, and, although subsequent addition of bupivacaine caused a further fall in IBl, acidification had no effect after bupivacaine, demonstrating that cells grown under these conditions express at least two different bupivacaine-sensitive K+ channels, only one of which is acid sensitive. Basolateral acidification also inhibited short-circuit current ( ISC), and basolateral bupivacaine, lidocaine, quinidine, and Ba2+ inhibited ISC at concentrations similar to those needed to inhibit IBl, suggesting that the K+ channels underlying IBl are part of the absorptive mechanism. Analyses using RT-PCR showed that mRNA encoding several two-pore domain K+ (K2P) channels was detected in cells grown under standard conditions (TWIK-1, TREK-1, TASK-2, TWIK-2, KCNK-7, TASK-3, TREK-2, THIK-1, and TALK-2). We therefore suggest that K2P channels underlie GK in unstimulated cells and so maintain the driving force for Na+ absorption. Since this ion transport process is vital to lung function, K2P channels thus play an important but previously undocumented role in pulmonary physiology.

Stimulation by cGMP of apical Na channels and cation channels in toad urinary bladder

1991 ◽  
Vol 260 (2) ◽  
pp. C234-C241 ◽  
Author(s):  
S. Das ◽  
M. Garepapaghi ◽  
L. G. Palmer
Keyword(s):  
Urinary Bladder ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Toad Urinary Bladder ◽  
Na Channels ◽  
Serosal Side ◽  
Cyclic Monophosphate ◽  
Stimulation Of

The effects of 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) on apical membrane cation conductances in the toad urinary bladder were investigated. 8-BrcGMP (1 mM) added to the serosal solution increased the amiloride-sensitive short-circuit current (INa) after a delay of 5 min to a steady-state value 1.8 times that of controls achieved after 30 min. Similar effects were seen when the bladders were bathed on the serosal side with a normal NaCl Ringer solution and with a high-K sucrose solution to depolarize the basolateral membrane. Under the latter conditions, the amiloride-sensitive transepithelial conductance increased in parallel with the short-circuit current, indicating stimulation of apical membrane Na channels. The threshold concentration for observing the stimulation of INa was 100 microM, 10-100 times larger than the concentration of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) required to elicit an increase in INa. Currents through an outwardly rectifying Ca-sensitive cation conductance (Iout) were also increased by 1.8-fold relative to controls. This stimulatory effect occurred after a delay of 15 min and reached maximal levels 90-120 min after addition of the nucleotide. The effects of cGMP on INa were not additive with those of 8-BrcAMP or with antidiuretic hormone, an agent known to act by increasing cAMP within the cell. Addition of 1 mM 3-isobutyl-1-methylxanthine to the serosal side of the bladders stimulated INa by 1.3-fold and Iout by 2.4-fold. In both cases, subsequent addition of cGMP produced no further activation of either conductance.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of ENaC by intracellular Cl− in an MDCK clone with high ENaC expression

2004 ◽  
Vol 287 (4) ◽  
pp. F722-F731 ◽  
Author(s):  
Yi Xie ◽  
James A. Schafer
Keyword(s):  
Apical Membrane ◽  
Mdck Cells ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Inhibitory Effect ◽  
High Rate ◽  
Na Channel ◽  
Transmembrane Conductance Regulator ◽  
Average Current

We examined the effects of intracellular Cl− concentration ([Cl−]i) on the epithelial Na channel (ENaC) in a line of Madin-Darby canine kidney (MDCK) cells (FL-MDCK) with a high rate of Na+ transport produced by stable retroviral transfection with rENaC subunits (Morris RG and Schafer JA. J Gen Physiol 120: 71–85, 2002). Treatment with cAMP (100 μM 8-cpt-cAMP plus 100 μM IBMX) stimulated ENaC-mediated Na+ absorption as well as Cl− secretion via cystic fibrosis transmembrane conductance regulator, which was characterized in α-toxin-permeabilized monolayers to have the anion selectivity sequence NO3− > Br− > Cl− > I−. With the use of FL-MDCK monolayers in which the basolateral membrane was permeabilized by nystatin, the ENaC conductance of the apical membrane [determined from the amiloride-sensitive short-circuit current (AS- Isc) driven by an apical-to-basolateral Na+ concentration gradient] was progressively inhibited by increasing the [Cl−] in the basolateral solution (and hence in the cytosol), but it was insensitive to the [Cl−] in the apical solution. This inhibitory effect of [Cl−]i occurred regardless of the presence or absence of net Cl− transport. However, from fluorometric measurements using the Cl−-sensitive dye 6-methoxy- N-(3-sulfopropyl)-quinolinium in intact FL-MDCK monolayers on permeable supports, cAMP, which activates both Na+ absorption and Cl− secretion, produced a decrease of [Cl−]i from 76 ± 14 to 36 ± 8 mM ( P = 0.03). Thus it might be expected that activation of Cl− secretion by cAMP would lead to stimulation rather than inhibition of ENaC. In the nystatin-treated monolayers, an increase in [Cl−]i from 15 to 145 mM decreased AS- Isc from 24.5 ± 1.0 to 10.2 ± 1.6 μA/cm2. This inhibition of ENaC could be attributed to nearly proportional decreases in the density of ENaC in the apical membrane from 1.91 ± 0.16 to 1.32 ± 0.17 fmol/cm2 and in the intrinsic channel activity (the average current per ENaC subunit) from 13.3 ± 1.2 to 8.2 ± 1.4 μA/fmol.

UTP inhibits Na+ absorption in wild-type and ΔF508 CFTR-expressing human bronchial epithelia

1999 ◽  
Vol 276 (4) ◽  
pp. C827-C837 ◽  
Author(s):  
Daniel C. Devor ◽  
Joseph M. Pilewski
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Receptor Occupancy ◽  
Short Circuit Current ◽  
Inhibitory Effect ◽  
Initial Increase ◽  
Bronchial Epithelia ◽  
Sustained Inhibition

Ca2+-mediated agonists, including UTP, are being developed for therapeutic use in cystic fibrosis (CF) based on their ability to modulate alternative Cl− conductances. As CF is also characterized by hyperabsorption of Na+, we determined the effect of mucosal UTP on transepithelial Na+transport in primary cultures of human bronchial epithelia (HBE). In symmetrical NaCl, UTP induced an initial increase in short-circuit current ( I sc) followed by a sustained inhibition. To differentiate between effects on Na+ absorption and Cl− secretion, I sc was measured in the absence of mucosal and serosal Cl−( I Na). Again, mucosal UTP induced an initial increase and then a sustained decrease that reduced amiloride-sensitive I Na by 73%. The Ca2+-dependent agonists histamine, bradykinin, serosal UTP, and thapsigargin similarly induced sustained inhibition (62–84%) of I Na. Mucosal UTP induced similar sustained inhibition (half-maximal inhibitory concentration 296 nM) of I Na in primary cultures of human CF airway homozygous for the ΔF508 mutation. BAPTA-AM blunted UTP-dependent inhibition of I Na, but inhibitors of protein kinase C (PKC) and phospholipase A2 had no effect. Indeed, direct activation of PKC by phorbol 12-myristate 13-acetate failed to inhibit Na+ absorption. Apyrase, a tri- and diphosphatase, did not reverse inhibitory effects of UTP on I Na, suggesting a long-term inhibitory effect of UTP that is independent of receptor occupancy. After establishment of a mucosa-to-serosa K+ concentration gradient and permeabilization of the mucosal membrane with nystatin, mucosal UTP induced an initial increase in K+current followed by a sustained inhibition. We conclude that increasing cellular Ca2+ induces a long-term inhibition of transepithelial Na+transport across normal and CF HBE at least partly due to downregulation of a basolateral membrane K+ conductance. Thus UTP may have a dual therapeutic effect in CF airway: 1) stimulation of a Cl− secretory response and 2) inhibition of Na+ transport.

Electrical properties of monolayers cultured from cells of human tracheal mucosa

1985 ◽  
Vol 58 (5) ◽  
pp. 1729-1735 ◽  
Author(s):  
J. H. Widdicombe ◽  
D. L. Coleman ◽  
W. E. Finkbeiner ◽  
I. K. Tuet
Keyword(s):  
Loop Diuretic ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Serosal Side ◽  
Tracheal Mucosa ◽  
Isolated Cells ◽  
Trypan Blue Exclusion ◽  
Mucosal Side

Dispersed isolated cells were obtained from human tracheal mucosa by digestion with collagenase. Up to 1.5 X 10(8) cells were obtained per trachea and showed up to 95% viability, as judged by trypan blue exclusion. When grown in culture, the cells formed monolayers after approximately 4 days. Electron microscopy of the monolayers revealed a polarized structure. An apical membrane, containing microvilli and a pronounced glycocalyx, was separated from a relatively unspecialized basolateral membrane by typical tight junctions. Monolayers grown on nucleopore filters showed resistances of 44–1,800 omega. cm2 and transepithelial potential differences of 0.1–7.6 mV. Short-circuit current (Isc) was increased by isoproterenol, prostaglandins E2 and F2 alpha, and bradykinin. The loop diuretic, bumetanide, reduced Isc when added to the basolateral (serosal) side but had no effect from the apical (mucosal) side of the monolayers. Furosemide and MK-196 also inhibited Isc. Mucosal amiloride inhibited Isc. Serosal amiloride or mucosal ouabain had no effect on Isc. Serosal ouabain brought Isc to zero after approximately 15 min.

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.

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