scholarly journals Autoregulation of apical membrane Na+ permeability of tight epithelia. Noise analysis with amiloride and CGS 4270.

1985 ◽  
Vol 85 (4) ◽  
pp. 555-582 ◽  
Author(s):  
F J Abramcheck ◽  
W Van Driessche ◽  
S I Helman
Keyword(s):  
Single Channel ◽  
Apical Membrane ◽  
Noise Analysis ◽  
Ringer Solution ◽  
Critical Examination ◽  
Na Channel ◽  
Channel Density ◽  
Na Permeability ◽  
Na Currents ◽  
Tight Epithelia

Noise analysis of the Na+ channels of the apical membranes of frog skin bathed symmetrically in a Cl-HCO3 Ringer solution was done with amiloride and CGS 4270. Tissues were studied in their control states and after inhibition of transepithelial Na+ transport (Isc) by addition of quinine or quinidine to the apical solution. A critical examination of the amiloride-induced noise indicated that the single channel Na+ currents (iNa) were decreased by quinine and quinidine, probably because of depolarization of apical membrane voltage. Despite considerable statistical uncertainty in the methods of estimation of the Na+ channel density with amiloride-induced noise (NA, see text), the striking observation was a large increase of NA with amiloride inhibition of the rate of Na+ entry into the cells. NA was increased to 406% of control, whereas Isc was inhibited to 8.6% of control by 6 microM amiloride. Studies were done also with the Na+ channel blocker CGS 4270. Noise analysis with this compound was advantageous, permitting iCGSNa and NCGS to be measured in individual tissues with a relatively small inhibition of Isc. As with amiloride, inhibition of Isc with CGS 4270 caused large increases of the Na+ channel density (approximately 200% at approximately 35% inhibition of the Isc). Quinine and quinidine caused an approximately 50% increase of Na+ channel density while inhibiting iNa by approximately 60-70%. As inhibition of Na+ entry leads to an increase of Na+ channel density, a mechanism of autoregulation appears to be a major factor in adjusting the apical membrane Na+ permeability of the cells.

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.

Current-noise analysis of Na absorption in the embryonic coprodeum: stimulation by aldosterone and thyroxine

1993 ◽  
Vol 265 (5) ◽  
pp. R1100-R1108 ◽  
Author(s):  
W. Clauss ◽  
B. Hoffmann ◽  
R. Krattenmacher ◽  
W. Van Driessche
Keyword(s):  
Sodium Transport ◽  
Single Channel ◽  
Short Circuit ◽  
Noise Analysis ◽  
Current Noise ◽  
Na Channel ◽  
Channel Density ◽  
Channel Current ◽  
Single Channel Current

The mechanism and regulation of sodium transport in the embryonic coprodeum of chicken were investigated with isolated epithelia in vitro by electrophysiological techniques. Electrogenic sodium transport (INa) was measured in Ussing chambers by the short-circuit current (Isc) technique and identified by the diuretic amiloride or by removal of sodium from the apical medium. Apical sodium channels and the kinetics of amiloride binding were investigated by current-noise analysis. Isc and INa were measured under control conditions and under the influence of in vitro incubation with aldosterone and thyroxine. At 20 days the embryonic coprodeum has an Isc of 12.6 +/- 1.4 microA/cm2 and a transepithelial resistance of 519 +/- 40 omega.cm2. Amiloride blocks 9.0 +/- 1.3 microA/cm2 of the Isc, which represents electrogenic Na+ absorption and can be inhibited by serosal ouabain. Aldosterone does not stimulate Isc or INa, whereas thyroxine increases Isc and INa about threefold. Aldosterone in combination with thyroxine increases Isc and INa further to about five- to sixfold. In both cases the hormonal stimulation can be totally blocked by spironolactone. Current-noise analysis of the apical Na+ entry step reveals amiloride-sensitive Na+ channels with a single-channel current of approximately 2.3 pA and a channel density of 9-16 million/cm2 under stimulated conditions. Half-maximal amiloride block occurs at 0.8-1 microM. The hormones stimulate Na+ absorption by increasing the Na+ channel density and not the single-channel current.(ABSTRACT TRUNCATED AT 250 WORDS)

Dual action of aldosterone on toad bladder: Na+ permeability and Na+ pump modulation

1984 ◽  
Vol 246 (4) ◽  
pp. F517-F525 ◽  
Author(s):  
C. S. Park ◽  
I. S. Edelman
Keyword(s):  
Urinary Bladder ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Electrical Conductance ◽  
Na Channel ◽  
Metabolic State ◽  
Na Pump ◽  
Na Permeability ◽  
Basolateral Side ◽  
Dual Action

The effects of aldosterone on the functional characteristics of the Na+ entry step across the apical membrane and on the Na+ exit step across the basolateral membrane of the urinary bladder of toads were examined using amiloride and ouabain as probes of the respective surfaces of the cell. Aldosterone stimulated Na+ transport with a concurrent increase in the transepithelial electrical conductance as did two other agents, vasopressin (ADH) and p-chloromercuriphenylsulfonate (PCMPS), primarily active on the apical membrane. Unlike the effects of ADH and PCMPS, however, the effect of aldosterone on Na+ conductance was blocked by actinomycin D and was associated with a decreased sensitivity of the apical Na+ channel to amiloride. In addition, aldosterone increased the sensitivity of the Na+ pump on the basolateral side to ouabain, an effect that was dependent on the metabolic state of the urinary bladder. These results support the inference of coordinate effects on Na+ permeability of the apical membrane and the Na+ pump of the basolateral membrane. Both effects of aldosterone appear to be dependent on the metabolic state of the transporting epithelium.

Brefeldin A inhibition of apical Na+ channels in epithelia

1996 ◽  
Vol 270 (1) ◽  
pp. C138-C147 ◽  
Author(s):  
R. S. Fisher ◽  
F. G. Grillo ◽  
S. Sariban-Sohraby
Keyword(s):  
Cultured Cells ◽  
Single Channel ◽  
Noise Analysis ◽  
Basolateral Membrane ◽  
Brefeldin A ◽  
Na Channel ◽  
Na Channels ◽  
Na Transport ◽  
Channel Current ◽  
Vacuolar System

Brefeldin A (BFA) is used to probe trafficking of proteins through the central vacuolar system (CVS) in a variety of cells. Transepithelial Na+ transport by high-resistance epithelia, such as A6 cultured cells, is inhibited by BFA. Apical Na+ channels, as well as basolateral pumps and K+ channels, are complex proteins that probably traverse the CVS for routing to the plasma membrane. BFA (5 micrograms/ml) decreases transepithelial Na+ current near zero and increases resistance reversibly after 4 h. Longer exposures are toxic. When tissues were treated for 20 h with 0.2 microgram/ml BFA, Na+ transport also was reversibly inhibited. Using noise analysis, we found that BFA drastically reduced apical Na+ channel density. The increase in single channel current was consistent with cell hyperpolarization. After apical permeabilization with nystatin, changes in transepithelial current reflect changes in basolateral membrane transport. Transport at this membrane was inhibited by ouabain and cycloheximide, but not by BFA. After BFA, aldosterone was ineffective, suggesting that an intact CVS is required for stimulation by this hormone. Thus BFA inhibition of Na+ transport is localized at the apical membrane. Implications for channel turnover as a mechanism for regulating the Na+ transport rate are discussed.

Quantification of K+ secretion through apical low-conductance K channels in the CCD

2005 ◽  
Vol 289 (1) ◽  
pp. F117-F126 ◽  
Author(s):  
Daniel A. Gray ◽  
Gustavo Frindt ◽  
Lawrence G. Palmer
Keyword(s):  
Single Channel ◽  
Collecting Duct ◽  
Reversal Potential ◽  
Inward Rectification ◽  
Na Channel ◽  
Sk Channels ◽  
Maximal Conductance ◽  
Channel Density ◽  
Sk Channel ◽  
High K

Outward and inward currents through single small-conductance K+ (SK) channels were measured in cell-attached patches of the apical membrane of principal cells of the rat cortical collecting duct (CCD). Currents showed mild inward rectification with high [K+] in the pipette (Kp+), which decreased as Kp+ was lowered. Inward conductances had a hyperbolic dependence on Kp+ with half-maximal conductance at ∼20 mM. Outward conductances, measured near the reversal potential, also increased with Kp+ from 15 pS (Kp+ = 0) to 50 pS (Kp+ = 134 mM). SK channel density was measured as the number of conducting channels per patch in cell-attached patches. As reported previously, channel density increased when animals were on a high-K diet for 7 days. Addition of 8-cpt-cAMP to the bath at least 5 min before making a seal increased SK channel density to an even greater extent, although this increase was not additive with the effect of a high-K diet. In contrast, increases in Na channel activity, assessed as the whole cell amiloride-sensitive current, due to K loading and 8-cpt-cAMP treatment were additive. Single-channel conductances and channel densities were used as inputs to a simple mathematical model of the CCD to predict rates of transepithelial Na+ and K+ transport as a function of apical Na+ permeability and K+ conductance, basolateral pump rates and K+ conductance, and the paracellular conductance. With measured values for these parameters, the model predicted transport rates that were in good agreement with values measured in isolated, perfused tubules. The number and properties of SK channels account for K+ transport by the CCD under all physiological conditions tested.

scholarly journals Endogenous Protease Activation of ENaC

2005 ◽  
Vol 126 (4) ◽  
pp. 339-352 ◽  
Author(s):  
Adedotun Adebamiro ◽  
Yi Cheng ◽  
John P. Johnson ◽  
Robert J. Bridges
Keyword(s):  
Single Channel ◽  
Apical Membrane ◽  
Open Channels ◽  
Na Channel ◽  
Fluctuation Analysis ◽  
Protease Inhibition ◽  
Open Probability ◽  
Channel Current ◽  
Single Channel Current ◽  
Channel Properties

Endogenous serine proteases have been reported to control the reabsorption of Na+ by kidney- and lung-derived epithelial cells via stimulation of electrogenic Na+ transport mediated by the epithelial Na+ channel (ENaC). In this study we investigated the effects of aprotinin on ENaC single channel properties using transepithelial fluctuation analysis in the amphibian kidney epithelium, A6. Aprotinin caused a time- and concentration-dependent inhibition (84 ± 10.5%) in the amiloride-sensitive sodium transport (INa) with a time constant of 18 min and half maximal inhibition constant of 1 μM. Analysis of amiloride analogue blocker–induced fluctuations in INa showed linear rate–concentration plots with identical blocker on and off rates in control and aprotinin-inhibited conditions. Verification of open-block kinetics allowed for the use of a pulse protocol method (Helman, S.I., X. Liu, K. Baldwin, B.L. Blazer-Yost, and W.J. Els. 1998. Am. J. Physiol. 274:C947–C957) to study the same cells under different conditions as well as the reversibility of the aprotinin effect on single channel properties. Aprotinin caused reversible changes in all three single channel properties but only the change in the number of open channels was consistent with the inhibition of INa. A 50% decrease in INa was accompanied by 50% increases in the single channel current and open probability but an 80% decrease in the number of open channels. Washout of aprotinin led to a time-dependent restoration of INa as well as the single channel properties to the control, pre-aprotinin, values. We conclude that protease regulation of INa is mediated by changes in the number of open channels in the apical membrane. The increase in the single channel current caused by protease inhibition can be explained by a hyperpolarization of the apical membrane potential as active Na+ channels are retrieved. The paradoxical increase in channel open probability caused by protease inhibition will require further investigation but does suggest a potential compensatory regulatory mechanism to maintain INa at some minimal threshold value.

Mechanism of sodium hyperabsorption in cultured cystic fibrosis nasal epithelium: a patch-clamp study

1994 ◽  
Vol 266 (4) ◽  
pp. C1061-C1068 ◽  
Author(s):  
T. C. Chinet ◽  
J. M. Fullton ◽  
J. R. Yankaskas ◽  
R. C. Boucher ◽  
M. J. Stutts
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Patch Clamp ◽  
Single Channel ◽  
Apical Membrane ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Probability ◽  
Nasal Epithelial Cells ◽  
Na Permeability

Transepithelial Na+ absorption is increased two to three times in cystic fibrosis (CF) compared with normal (NL) airway epithelia. This increase has been associated with a higher Na+ permeability of the apical membrane of airway epithelial cells. Because Na+ absorption is electrogenic and abolished by amiloride, Na+ channels are thought to dominate the apical membrane Na+ permeability. Three Na+ channel-related mechanisms may explain the increase in apical Na+ permeability in CF cells: increased number of channels, increased single-channel conductance, and increased single-channel open probability. We compared the properties of Na(+)-permeable channels in the apical membrane of confluent preparations of human NL and CF nasal epithelial cells cultured on permeable supports. Na(+)-permeable channels were studied using the patch-clamp technique in the excised inside-out and cell-attached configurations. The same types of Na(+)-permeable channels were recorded in CF and NL cells. In excised patches, nonselective (Na+/K+) cation channels were recorded, and no differences between CF and NL were found in the properties, incidence, single-channel conductance, and single-channel open probability. In cell-attached patches, channels with a higher Na+ vs. K+ selectivity dominated. There was no difference between CF and NL cells in the incidence (18.8 vs. 21.4%, respectively) and conductance (17.2 +/- 2.8 vs. 21.4 +/- 1.5 pS, respectively) of Na(+)-permeable channels. However, the open probability was higher in CF cells compared with NL cells (30.0 +/- 3.4%, n = 6, vs. 15.0 +/- 3.9%, n = 13; P < 0.05). We conclude that, in CF nasal epithelial cells, the increase in Na+ permeability of the apical membrane results from an increase in the open probability of Na(+)-permeable channels in the apical membrane.

Insulin activates single amiloride-blockable Na channels in a distal nephron cell line (A6)

1992 ◽  
Vol 263 (3) ◽  
pp. F392-F400 ◽  
Author(s):  
Y. Marunaka ◽  
N. Hagiwara ◽  
H. Tohda
Keyword(s):  
Cell Line ◽  
Single Channel ◽  
Apical Membrane ◽  
Na Channel ◽  
Na Channels ◽  
Distal Nephron ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Patch Clamp Technique ◽  
Open Probability

Using the patch-clamp technique, we studied the effect of insulin on an amiloride-blockable Na channel in the apical membrane of a distal nephron cell line (A6) cultured on permeable collagen films for 10-14 days. NPo (N, number of channels per patch membrane; Po, average value of open probability of individual channels in the patch) under baseline conditions was 0.88 +/- 0.12 (SE)(n = 17). After making cell-attached patches on the apical membrane which contained Na channels, insulin (1 mU/ml) was applied to the serosal bath. While maintaining the cell-attached patch, NPo significantly increased to 1.48 +/- 0.19 (n = 17; P less than 0.001) after 5-10 min of insulin application. The open probability of Na channels was 0.39 +/- 0.01 (n = 38) under baseline condition, and increased to 0.66 +/- 0.03 (n = 38, P less than 0.001) after addition of insulin. The baseline single-channel conductance was 4pS, and neither the single-channel conductance nor the current-voltage relationship was significantly changed by insulin. These results indicate that insulin increases Na absorption in the distal nephron by increasing the open probability of the amiloride-blockable Na channel.

scholarly journals Hormonal control of apical membrane Na transport in epithelia. Studies with fluctuation analysis.

1983 ◽  
Vol 82 (2) ◽  
pp. 201-220 ◽  
Author(s):  
S I Helman ◽  
T C Cox ◽  
W Van Driessche
Keyword(s):  
Single Channel ◽  
Apical Membrane ◽  
Ringer Solution ◽  
Hormonal Control ◽  
Fluctuation Analysis ◽  
Noise Power ◽  
Na Current ◽  
Na Transport ◽  
Electrically Conductive ◽  
Apical Membranes

To study the mechanisms by which antidiuretic hormone and prostaglandins regulate Na transport at the apical membranes of the cells of anuran tissues, studies were done with fluctuation analysis. Epithelia of frog skin (Rana pipiens) were treated with vasopressin alone, or treated with vasopressin after inhibition of Na transport by indomethacin. The tissues were bathed symmetrically with a Cl-HCO3 Ringer solution and short-circuited continuously. In this experimental circumstance, the amiloride-induced current noise power density spectra were of the Lorentzian type with little or no l/f noise, provided that "scraped" skins were used for study. Despite large changes of Na transport, especially in epithelia treated with indomethacin and vasopressin, the single-channel Na current remained essentially unchanged, whereas the density of amiloride-inhibitable, electrically conductive Na channels was increased by vasopressin and decreased by indomethacin.

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