scholarly journals Amiloride analog stimulation of short-circuit current in larval frog skin epithelium.

1997 ◽  
Vol 200 (23) ◽  
pp. 3055-3065
Author(s):  
T Cox
Keyword(s):  
Rana Catesbeiana ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Membrane Resistance ◽  
Similar Time ◽  
Peak Response ◽  
Ussing Chambers ◽  
Apical Side ◽  
Basolateral Side

The skin of the bullfrog Rana catesbeiana tadpole contains an apical non-selective cation channel that is activated by amiloride. This is in contrast to the adult skin, which has a highly Na+-selective channel that is blocked by amiloride. The purpose of the present study was to characterize further the nature of the tadpole channel using amiloride and its analogs benzamil, dimethyl amiloride (DMA), 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and methyl isobutyl amiloride (MIBA). Tadpole skins were mounted in modified Ussing chambers with Ca2+-free KCl or NaCl Ringer on the apical side and standard NaCl Ringer (containing 2 mmol l-1 Ca2+) on the basolateral side. Drugs were added to the apical solution at concentrations between 0.1 and 1000 micromol l-1. Amiloride caused the short-circuit current (Isc) to increase rapidly from near zero to a peak of approximately 30-50 microA and then to decline back towards zero over several seconds. The peak response was largest at 100 micromol l-1. The rate of decline was noticeably faster at the higher concentrations. Benzamil and DMA had similar time courses to amiloride, but with smaller effects on Isc. The largest peak responses occurred at 5-50 micromol l-1. EIPA and MIBA gave small responses at 1-10 micromol l-1 and, at higher concentrations (50-500 micromol l-1), the responses consisted of rapid, small increases in Isc followed by rapid decreases. The largest peak response occurred at 10 micromol l-1 for both drugs. After apical membrane resistance had been reduced by nystatin, addition of analogs to the apical solution caused no change in Isc or transepithelial resistance. This suggests that the decline in Isc after amiloride analog treatment was not due to increases in the resistance of the basolateral membrane. N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) blocked stimulation by all of the analogs. These data are consistent with amiloride analogs acting as both activators and inhibitors of short-circuit current in tadpole skin and extend the list of ligands that activate these channels.

Cultured monolayers of the dog jejunum with the structural and functional properties resembling the normal epithelium

2005 ◽  
Vol 288 (4) ◽  
pp. G705-G717 ◽  
Author(s):  
Xing-He Weng ◽  
Klaus W. Beyenbach ◽  
Andrea Quaroni
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Immunofluorescent Staining ◽  
Normal Epithelium ◽  
Ussing Chambers ◽  
Apical Side ◽  
Structural And Functional Properties

The development of a culture of the normal mammalian jejunum motivated this work. Isolated crypt cells of the dog jejunum were induced to form primary cultures on Snapwell filters. Up to seven subcultures were studied under the electron microscope and in Ussing chambers. Epithelial markers were identified by RT-PCR, Western blot, and immunofluorescent staining. Confluent monolayers exhibit a dense apical brush border, basolateral membrane infoldings, desmosomes, and tight junctions expressing zonula occludens-1, occludin-1, and claudin-3 and -4. In OptiMEM medium fortified with epidermal growth factor, hydrocortisone, and insulin, monolayer transepithelial voltage was −6.8 mV (apical side), transepithelial resistance was 1,050 Ω·cm2, and short-circuit current ( Isc) was 8.1 μA/cm2. Transcellular and paracellular resistances were estimated as 14.8 and 1.1 kΩ·cm2, respectively. Serosal ouabain reduced voltage and current toward zero, as did apical amiloride. The presence of mRNA of α-epithelial Na+ channel (ENaC) was confirmed. Na-d-glucose cotransport was identified with an antibody to Na+-glucose cotransporter (SGLT) 1. The unidirectional mucosa-to-serosa Na+ flux (19 nmol·min−1·cm−2) was two times as large as the reverse flux, and net transepithelial Na+ flux was nearly double the amiloride-sensitive Isc. In plain Ringer solution, the amiloride-sensitive Isc went toward zero. Under these conditions plus mucosal amiloride, serosal dibutyryl-cAMP elicited a Cl−-dependent Isc consistent with the stimulation of transepithelial Cl− secretion. In conclusion, primary cultures and subcultures of the normal mammalian jejunum form polarized epithelial monolayers with 1) the properties of a leaky epithelium, 2) claudins specific to the jejunal tight junction, 3) transepithelial Na+ absorption mediated in part by SGLT1 and ENaC, and 4) electrogenic Cl− secretion activated by cAMP.

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.

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−].

Forskolin-induced HCO3- current across apical membrane of the frog corneal epithelium

1990 ◽  
Vol 259 (2) ◽  
pp. C215-C223 ◽  
Author(s):  
O. A. Candia
Keyword(s):  
Corneal Epithelium ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Pump Current ◽  
Gas Composition ◽  
Apical Side ◽  
Disulfonic Stilbene ◽  
Stimulation Of

Forskolin (and other Cl- secretagogues) does not affect the very small Na(+)-originated short-circuit current (Isc) across frog corneal epithelium bathed in Cl- free solutions. However, forskolin in combination with increased PCO2 bubbling of the solutions (5-20% CO2) stimulated Isc proportionally to PCO2 to a maximum of approximately 8 microA/cm2. This current could be eliminated and reinstated by sequentially changing the gas composition of the bubbling to 100% air and 20% CO2-80% air. The same effects were observed when PCO2 changes were limited to the apical-side solution. Stroma-to-tear HCO3- movement was deemed unlikely, since the increase in Isc was observed with a HCO3(-)-free solution on the stromal side and CO2 gassing limited to the tear side. From the effects of ouabain and tryptamine, at least 80% of the Isc across the basolateral membrane can be accounted for by the Na+ pump current plus K+ movement from cell to bath. Methazolamide also inhibited Isc. Current across the apical membrane cannot be attributed to an electronegative Na(+)-HCO3- symport given the insensitivity of Isc to a disulfonic stilbene and the fact that stroma-to-tear Na+ fluxes did not increase on stimulation of Isc. The tear-to-stroma Na+ flux also remained unaltered, negating an increased apical bath-to-cell Na+ flow. The forskolin-20% CO2 manipulation produced a depolarization of the intracellular potential, a reduction in the apical-to-basolateral resistance ratio, and a decrease in transepithelial resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of K+ conductance in basolateral membrane of toad urinary bladder by oxytocin and cAMP

1988 ◽  
Vol 254 (6) ◽  
pp. C816-C821 ◽  
Author(s):  
W. Van Driessche ◽  
D. Erlij
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Potassium Conductance ◽  
Short Circuit Current ◽  
Membrane Properties ◽  
Toad Urinary Bladder ◽  
Electrical Behavior ◽  
Apical Side ◽  
Camp Analogue

We incubated toad urinary bladders with Na+-free, isotonic K+ solutions on the apical side and increased the cationic conductance of the apical membrane with nystatin (150 U/ml). Under these conditions, the short-circuit current is mostly carried by K+ flowing from mucosa to serosa. Impedance measurements showed that in nystatin-treated preparations, the electrical behavior of the tissue is dominated by the basolateral membrane properties. Oxytocin (0.1 U/ml) produced an increase of the current and the conductance of the basolateral membrane. Both the resting and the oxytocin-stimulated current were rapidly and reversibly blocked by serosal Ba2+. Addition of the adenosine 3',5'-cyclic monophosphate (cAMP) analogue [8-(4-chloropheylthio)-cAMP] to the basolateral solution mimicked the effects of oxytocin. These results show that oxytocin and cAMP stimulate a potassium conductance in the basolateral membrane and that the stimulation is not related to an increase in sodium entry through the apical membrane. Addition of ouabain (10(-3) M) to the serosal solution did not modify the stimulation by oxytocin, indicating that the activated pathway is not linked to the rate of turnover of the Na+ pump.

Effect of chloride on pH microclimate and electrogenic Na+ absorption across the rumen epithelium of goat and sheep

2006 ◽  
Vol 291 (2) ◽  
pp. G246-G252 ◽  
Author(s):  
S. Leonhard-Marek ◽  
G. Breves ◽  
R. Busche
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Epithelial Surface ◽  
Ussing Chambers ◽  
Rumen Epithelium ◽  
Microclimate Ph ◽  
Cation Conductance ◽  
Sheep Rumen

Active Na+ absorption across rumen epithelium comprises Na+/H+ exchange and a nonselective cation conductance (NSCC). Luminal chloride is able to stimulate Na+ absorption, which has been attributed to an interaction between Cl−/HCO3− and Na+/H+ exchangers. However, isolated rumen epithelial cells also express a Cl− conductance. We investigated whether Cl− has an additional effect on electrogenic Na+ absorption via NSCC. NSCC was estimated from short-circuit current ( Isc) across epithelia of goat and sheep rumen in Ussing chambers. Epithelial surface pH (pHs) was measured with 5- N-hexadecanoyl-aminofluorescence. Membrane potentials were measured with microelelectrodes. Luminal, but not serosal, Cl− stimulated the Ca2+ and Mg2+ sensitive Isc. This effect was independent of the replacing anion (gluconate or acetate) and of the presence of bicarbonate. The mean pHs of rumen epithelium amounted to 7.47 ± 0.03 in a low-Cl− solution. It was increased by 0.21 pH units when luminal Cl− was increased from 10 to 68 mM. Increasing mucosal pH from 7.5 to 8.0 also increased the Ca2+ and Mg2+ sensitive Isc and transepithelial conductance and reduced the fractional resistance of the apical membrane. Luminal Cl− depolarized the apical membrane of rumen epithelium. 5-Nitro-2-(3-phenylpropylamino)-benzoate reduced the divalent cation sensitive Isc, but only in low-Cl− solutions. The results show that luminal Cl− can increase the microclimate pH via apical Cl−/HCO3− or Cl−/OH− exchangers. Electrogenic Na+ absorption via NSCC increases with pH, explaining part of the Cl− effects on Na+ absorption. The data further show that the Cl− conductance of rumen epithelium must be located at the basolateral membrane.

Apical adenosine activates an amiloride-sensitive conductance in human intestinal cell line HT29cl.19A

1997 ◽  
Vol 272 (3) ◽  
pp. C931-C936 ◽  
Author(s):  
H. Bouritius ◽  
J. A. Groot
Keyword(s):  
Adenosine Receptor ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Membrane Resistance ◽  
Intestinal Cell ◽  
Cation Conductance ◽  
Intestinal Cell Line ◽  
Stimulation Of

We studied the effects of stimulation of the apical adenosine receptor on ion transport by HT29cl.19A cells with the conventional microelectrode technique. Adenosine (100 microM) caused an increase in the transepithelial potential (3.6 +/- 0.4 mV) and equivalent short-circuit current (I(sc), 21 +/- 3 microA/cm2), a transient depolarization of the apical membrane potential (14 +/- 2 mV), and a decrease in the apical membrane resistance. The increase in I(sc) was additive to the effect of forskolin or basolateral addition of a maximal concentration of adenosine. Bumetanide, applied after adenosine, caused a further depolarization (7 +/- 2 mV) concomitant with a decrease in I(sc) (-13 +/- 2 microA/cm2) and an increase in the basolateral membrane resistance. Substitution of Cl- with gluconate or Na+ with N-methylglucamine reduced the response to adenosine by >60%. The response was also reduced by a low concentration of amiloride. We conclude that stimulation of the apical adenosine receptor activated a cation conductance in the apical membrane.

On the natriuretic effect of verapamil: inhibition of ENaC and transepithelial sodium transport

2002 ◽  
Vol 283 (4) ◽  
pp. F765-F770 ◽  
Author(s):  
Alan S. Segal ◽  
John P. Hayslett ◽  
Gary V. Desir
Keyword(s):  
Short Circuit ◽  
Ussing Chamber ◽  
Surrogate Marker ◽  
Distal Tubule ◽  
Short Circuit Current ◽  
Channel Blockers ◽  
A6 Cells ◽  
Apical Side ◽  
Basolateral Side ◽  
Natriuretic Effect

The natriuretic effect of Ca2+ channel blockers has been attributed to hemodynamic changes and to poorly defined direct tubular effects. To test the possibility that verapamil may inhibit Na+ reabsorption at the distal tubule, its effect on transepithelial Na+transport in aldosterone-stimulated A6 cells was determined. Cells were grown on permeable supports, and short-circuit current ( I sc) measured in an Ussing chamber was used as a surrogate marker for transepithelial Na+ transport. Application of 300 μM verapamil to the apical side inhibited I sc by 77% and was nearly as potent as 100 μM amiloride, which inhibited I sc by 87%. Verapamil-induced inhibition of I sc was accompanied by a significant increase in transepithelial resistance, suggesting blockade of an apical conductance. Its action on transepithelial Na+ transport does not appear to occur through inhibition of L-type Ca2+ channels, since I sc was unaffected by removal of extracellular Ca2+. Verapamil also does not appear to inhibit I sc by modulating intracellular Ca2+stores, since it fails to inhibit transepithelial Na+transport when added to the basolateral side. The effect on Na+ transport is specific for verapamil, since nifedipine, Ba2+, 4-aminopyridine, and charybdotoxin do not significantly affect I sc. A direct effect of verapamil on the epithelial Na+ channel (ENaC) was tested using oocytes injected with the α-, β-, and γ-subunits. We conclude that verapamil inhibits transepithelial Na+transport in A6 cells by blocking ENaC and that the natriuresis observed with administration of verapamil may be due in part to its action on ENaC.

Electrophysiology and noise analysis of K+-depolarized epithelia of frog skin

1985 ◽  
Vol 249 (5) ◽  
pp. C421-C429 ◽  
Author(s):  
J. Tang ◽  
F. J. Abramcheck ◽  
W. Van Driessche ◽  
S. I. Helman
Keyword(s):  
Frog Skin ◽  
Single Channel ◽  
Apical Membrane ◽  
Short Circuit ◽  
Noise Analysis ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Membrane Resistance ◽  
Na Channel ◽  
Channel Density

Epithelia of frog skin bathed either symmetrically with a sulfate-Ringer solution or bathed asymmetrically and depolarized with a 112 mM K+ basolateral solution (Kb+) were studied with intracellular microelectrode techniques. Kb+ depolarization caused an initial decrease of the short-circuit current (Isc) with a subsequent return of the Isc toward control values in 60-90 min. Whereas basolateral membrane resistance (Rb) and voltage were decreased markedly by high [Kb+], apical membrane electrical resistance (Ra) was decreased also. After 60 min, intracellular voltage averaged -27.3 mV, transcellular fractional resistance (fRa) was 86.8%, and Ra and Rb were decreased to 36.1 and 13.0%, of their control values, respectively. Amiloride-induced noise analysis of the apical membrane Na+ channels revealed that Na+ channel density was increased approximately 72% while single-channel Na+ current was decreased to 39.9% of control, roughly proportional to the decrease of apical membrane voltage (34.0% of control). In control and Kb+-depolarized epithelia, the Na+ channel density exhibited a phenomenon of autoregulation. Inhibition of Na+ entry (by amiloride) caused large increases of Na+ channel density toward saturating values of approximately 520 X 10(6) channels/cm2 in Kb+-depolarized tissues.

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