scholarly journals cAMP Increases Density of ENaC Subunits in the Apical Membrane of MDCK Cells in Direct Proportion to Amiloride-sensitive Na+ Transport

2002 ◽  
Vol 120 (1) ◽  
pp. 71-85 ◽  
Author(s):  
Ryan G. Morris ◽  
James A. Schafer
Keyword(s):  
Single Channel ◽  
Apical Membrane ◽  
Mdck Cells ◽  
Specific Binding ◽  
Collecting Duct ◽  
Short Circuit ◽  
Na Channels ◽  
Direct Proportion ◽  
Na Transport ◽  
Open Probability

Antidiuretic hormone and/or cAMP increase Na+ transport in the rat renal collecting duct and similar epithelia, including Madin-Darby canine kidney (MDCK) cell monolayers grown in culture. This study was undertaken to determine if that increment in Na+ transport could be explained quantitatively by an increased density of ENaC Na+ channels in the apical membrane. MDCK cells with no endogenous ENaC expression were retrovirally transfected with rat α-, β-, and γENaC subunits, each of which were labeled with the FLAG epitope in their extracellular loop as described previously (Firsov, D., L. Schild, I. Gautschi, A.-M. Mérillat, E. Schneeberger, and B.C. Rossier. 1996. Proc. Natl. Acad. Sci. USA. 93:15370–15375). The density of ENaC subunits was quantified by specific binding of 125I-labeled anti-FLAG antibody (M2) to the apical membrane, which was found to be a saturable function of M2 concentration with half-maximal binding at 4–8 nM. Transepithelial Na+ transport was measured as the amiloride-sensitive short-circuit current (AS-Isc) across MDCK cells grown on permeable supports. Specific M2 binding was positively correlated with AS-Isc measured in the same experiments. Stimulation with cAMP (20 μM 8-p-chlorothio-cAMP plus 200 μM IBMX) significantly increased AS-Isc from 11.2 ± 1.3 to 18.1 ± 1.3 μA/cm2. M2 binding (at 1.7 nM M2) increased in direct proportion to AS-Isc from 0.62 ± 0.13 to 1.16 ± 0.18 fmol/cm2. Based on the concentration dependence of M2 binding, the quantity of Na+ channels per unit of AS-Isc was calculated to be the same in the presence and absence of cAMP, 0.23 ± 0.04 and 0.21 ±0.05 fmol/μA, respectively. These values would be consistent with a single channel conductance of ∼5 pS (typically reported for ENaC channels) only if the open probability is <0.02, i.e., less than one-tenth of the typical value. We interpret the proportional increases in binding and AS-Isc to indicate that the increased density of ENaC subunits in the apical membrane can account completely for the Isc increase produced by cAMP.

scholarly journals Amiloride-sensitive trypsinization of apical sodium channels. Analysis of hormonal regulation of sodium transport in toad bladder.

1983 ◽  
Vol 81 (6) ◽  
pp. 785-803 ◽  
Author(s):  
H Garty ◽  
I S Edelman
Keyword(s):  
Hormonal Regulation ◽  
Apical Membrane ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Toad Urinary Bladder ◽  
Apical Surface ◽  
Na Channels ◽  
Na Transport ◽  
Na Permeability ◽  
Trypsin Inhibition

Incubation of the mucosal surface of the toad urinary bladder with trypsin (1 mg/ml) irreversibly decreased the short-circuit current to 50% of the initial value. This decrease was accompanied by a proportionate decrease in apical Na permeability, estimated from the change in amiloride-sensitive resistance in depolarized preparations. In contrast, the paracellular resistance was unaffected by trypsinization. Amiloride, a specific blocker of the apical Na channels, prevented inactivation by trypsin. Inhibition of Na transport by substitution of mucosal Na, however, had no effect on the response to trypsin. Trypsinization of the apical membrane was also used to study regulation of Na transport by anti-diuretic hormone (ADH) and aldosterone. Prior exposure of the apical surface to trypsin did not reduce the response to ADH, which indicates that the ADH-induced Na channels were inaccessible to trypsin before addition of the hormone. On the other hand, stimulation of short-circuit current by aldosterone or pyruvate (added to substrate-depleted, aldosterone-repleted bladders) was substantially reduced by prior trypsinization of the apical surface. Thus, the increase in apical Na permeability elicited by aldosterone or substrate involves activation of Na channels that are continuously present in the apical membrane in nonconductive but trypsin-sensitive forms.

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.

Steroid hormone-dependent expression of blockersensitive ENaCs in apical membranes of A6 epithelia

1997 ◽  
Vol 273 (5) ◽  
pp. C1650-C1656 ◽  
Author(s):  
Lynn M. Baxendale-Cox ◽  
Randall L. Duncan ◽  
Xuehong Liu ◽  
Kieron Baldwin ◽  
Willem J. Els ◽  
...  
Keyword(s):  
Growth Medium ◽  
Single Channel ◽  
Apical Membrane ◽  
Short Circuit ◽  
Noise Analysis ◽  
Short Circuit Current ◽  
Growth Media ◽  
Open Probability ◽  
A6 Epithelia ◽  
Single Channel Currents

Weak channel blocker-induced noise analysis was used to determine the way in which the steroids aldosterone and corticosterone stimulated apical membrane Na+ entry into the cells of tissue-cultured A6 epithelia. Among groups of tissues grown on a variety of substrates, in a variety of growth media, and with cells at passages 73–112, the steroids stimulated both amiloride-sensitive and amiloride-insensitive Na+ transport as measured by short-circuit currents in chambers perfused with either growth medium or a Ringer solution. From baseline rates of blocker-sensitive short-circuit current between 2 and 7 μA/cm2, transport was stimulated about threefold in all groups of experiments. Single channel currents averaged near 0.3 pA (growth medium) and 0.5 pA (Ringer) and were decreased 6–20% from controls by steroid due to the expected decreases of fractional transcellular resistance. Irrespective of baseline transport rates, the steroids in all groups of tissues stimulated transport by increase of the density of blocker-sensitive epithelial Na+ channels (ENaCs). Channel open probability was the same in control and stimulated tissues, averaging ∼0.3 in all groups of tissues. Accordingly, steroid-mediated increases of open channel density responsible for stimulation of Na+ transport are due to increases of the apical membrane pool of functional channels and not their open probability.

Contrasting effects of cPLA2 on epithelial Na+ transport

2001 ◽  
Vol 281 (1) ◽  
pp. C147-C156 ◽  
Author(s):  
Roger T. Worrell ◽  
Hui-Fang Bao ◽  
Don D. Denson ◽  
Douglas C. Eaton
Keyword(s):  
Aristolochic Acid ◽  
Single Channel ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Fine Tuning ◽  
Prostaglandin E ◽  
Cortical Collecting Duct ◽  
Open Probability ◽  
A6 Cells ◽  
Metabolic Products

Activity of the epithelial Na+ channel (ENaC) is the limiting step for discretionary Na+reabsorption in the cortical collecting duct. Xenopus laeviskidney A6 cells were used to investigate the effects of cytosolic phospholipase A2 (cPLA2) activity on Na+ transport. Application of aristolochic acid, a cPLA2 inhibitor, to the apical membrane of monolayers produced a decrease in apical [3H]arachidonic acid (AA) release and led to an approximate twofold increase in transepithelial Na+ current. Increased current was abolished by the nonmetabolized AA analog 5,8,11,14-eicosatetraynoic acid (ETYA), suggesting that AA, rather than one of its metabolic products, affected current. In single channel studies, ETYA produced a decrease in ENaC open probability. This suggests that cPLA2 is tonically active in A6 cells and that the end effect of liberated AA at the apical membrane is to reduce Na+ transport via actions on ENaC. In contrast, aristolochic acid applied basolaterally inhibited current, and the effect was not reversed by ETYA. Basolateral application of the cyclooxygenase inhibitor ibuprofen also inhibited current. Both effects were reversed by prostaglandin E2(PGE2). This suggests that cPLA2 activity and free AA, which is metabolized to PGE2, are necessary to support transport. This study supports the fine-tuning of Na+ transport and reabsorption through the regulation of free AA and AA metabolism.

Stimulatory effects on Na+ transport in renal epithelia induced by extracts of Nigella arvensis are caused by adenosine

2002 ◽  
Vol 205 (23) ◽  
pp. 3729-3737 ◽  
Author(s):  
Fatima Atia ◽  
Irina Mountian ◽  
Jeannine Simaels ◽  
Etienne Waelkens ◽  
Willy Van Driessche
Keyword(s):  
Liquid Chromatography ◽  
System Analysis ◽  
Single Channel ◽  
Short Circuit ◽  
Noise Analysis ◽  
Gel Filtration ◽  
Maximal Effect ◽  
Na Transport ◽  
Open Probability ◽  
Stimulatory Action

SUMMARY Effects of the extract of Nigella arvensis (NA) seeds on transepithelial Na+ transport were studied in cultured A6 toad kidney cells by recording short-circuit current (Isc),transepithelial conductance (GT), transepithelial capacitance (CT) and fluctuation in Isc. Apical application of NA extract had merely a small stimulatory effect on Na+ transport, whereas basolateral administration markedly increased Isc, GT and CT. A maximal effect was obtained at 500 μl l-1 of lyophilized NA extract. The increase in CT suggests that the activation of Isc occurs through the insertion of transport sites in the apical membrane. In experiments performed in the absence of Na+transport [apical Na+ was replaced by N-methyl-D-glucamine(NMDG+)], basolateral NA extract did not affect Isc and GT, indicating that Cl- conductance was not influenced. Noise analysis of Isc using 6-chloro-3,5-diaminopyrazine-2-carboxamide(CDPC) showed that NA extract reduced single-channel current(iNa) and decreased channel open probability(Po) but evoked a threefold increase in channel density(NT), which confirms the insertion of Na+channels. The separation of the compounds in the crude extract of NAwas performed by fast protein liquid chromatography (FPLC) on a Superdex 200 gel-filtration column and by reverse-phase high-pressure liquid chromatography(RPHPLC) on an μRPC C2/C18 SC2.1/10 column connected to a SMART system. Analysis of the purified active fraction by mass spectrometry demonstrated the presence of adenosine as the single organic compound in the extract that had a stimulatory effect on Na+ transport. In a separate series of experiments, we confirmed that 1 μmol l-1 adenosine had similar effects on the parameters of Na+ transport as did the NAextract. The action of adenosine was further identified by experiments in which NA extract was added after adenosine. In these experiments, NA extract did not affect Isc, GT or CT. These results clearly demonstrate an essential role of adenosine in the stimulatory action of NA extract.

Apical membrane properties and amiloride binding kinetics of the human descending colon

1984 ◽  
Vol 247 (6) ◽  
pp. G749-G757 ◽  
Author(s):  
N. K. Wills ◽  
W. P. Alles ◽  
G. I. Sandle ◽  
H. J. Binder
Keyword(s):  
Single Channel ◽  
Apical Membrane ◽  
Short Circuit ◽  
Membrane Properties ◽  
Na Channel ◽  
Fluctuation Analysis ◽  
Bathing Solution ◽  
Na Channels ◽  
Descending Colon ◽  
Single Channel Currents

The apical membrane properties of the isolated human descending colon were characterized by use of current fluctuation analysis methods and microelectrode techniques. The Na+ channel blocker amiloride was used to evaluate apical membrane conductance and the transepithelial short-circuit current (Isc). Amiloride significantly reduced Isc and increased the membrane resistance ratio. At submaximal doses of amiloride in the mucosal bathing solution, fluctuation analysis of the Isc revealed a Lorentzian component in the power-density spectra. The dose-response relationship between amiloride and current noise parameters was consistent with a two-state mechanism of blocker interaction with the channel. The on and off rate constants for the blocker-receptor reactions, the single-channel currents, and the Na+ channel densitywere estimated and were similar to those from Na+ channels from other so-called tight epithelia. In addition, these studies revealed an amiloride-insensitive conductance in the apical membrane in parallel to the amiloride-blockable Na+ channels. This conductance may be due to potassium ions. If so, the apical membrane properties of the human descending colon may closely resemble those of the rabbit descending colon and rat distal colon.

scholarly journals Apical membrane of native OMCDi cells has nonselective cation channels

2001 ◽  
Vol 281 (1) ◽  
pp. F48-F55 ◽  
Author(s):  
Shen-Ling Xia ◽  
Seung-Hyun Noh ◽  
Jill W. Verlander ◽  
Craig H. Gelband ◽  
Charles S. Wingo
Keyword(s):  
Single Channel ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Reversal Potential ◽  
Cation Channels ◽  
Channel Conductance ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Potassium Gluconate ◽  
Medullary Collecting Duct

The purpose of this study was to examine cation channel activity in the apical membrane of the outer medullary collecting duct of the inner stripe (OMCDi) using the patch-clamp technique. In freshly isolated and lumen-opened rabbit OMCDi, we have observed a single channel conductance of 23.3 ± 0.6 pS ( n = 17) in cell-attached (c/a) patches with high KCl in the bath and in the pipette at room temperature. Channel open probability varied among patches from 0.06 ± 0.01 at −60 mV ( n = 5) to 0.31 ± 0.04 at 60 mV ( n = 6) and consistently increased upon membrane depolarization. In inside-out (i/o) patches with symmetrical KCl solutions, the channel conductance (22.8 ± 0.8 pS; n = 10) was similar as in the c/a configuration. Substitution of the majority of Cl− with gluconate from KCl solution in the pipette and bath did not significantly alter reversal potential ( E rev) or the channel conductance (19.7 ± 1.1 pS in asymmetrical potassium gluconate, n = 4; 21.4 ± 0.5 pS in symmetrical potassium gluconate, n = 3). Experiments with 10-fold lower KCl concentration in bath solution in i/o patches shifted E rev to near the E rev of K+. The estimated permeability of K+ vs. Cl− was over 10, and the conductance was 13.4 ± 0.1 pS ( n = 3). The channel did not discriminate between K+ and Na+, as evidenced by a lack of a shift in the E rev with different K+ and Na+ concentration solutions in i/o patches ( n = 3). The current studies demonstrate the presence of cation channels in the apical membrane of native OMCDicells that could participate in K+ secretion or Na+ absorption.

Apical potassium channels in the rat connecting tubule

2004 ◽  
Vol 287 (5) ◽  
pp. F1030-F1037 ◽  
Author(s):  
Gustavo Frindt ◽  
Lawrence G. Palmer
Keyword(s):  
Single Channel ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Membrane Voltage ◽  
Sk Channels ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
K Channels ◽  
Connecting Tubule ◽  
Channel Type

Apical membrane K channels in the rat connecting tubule (CNT) were studied using the patch-clamp technique. Tubules were isolated from the cortical labyrinth of the kidney and split open to provide access to the apical membrane. Cell-attached patches were formed on presumed principal and/or connecting tubule cells. The major channel type observed had a single-channel conductance of 52 pS, high open probability and kinetics that were only weakly dependent on voltage. These correspond closely to the “SK”-type channels in the cortical collecting duct, identified with the ROMK (Kir1.1) gene product. A second channel type, which was less frequently observed, mediated larger currents and was strongly activated by depolarization of the apical membrane voltage. These were identified as BK or maxi-K channels. The density of active SK channels revealed a high degree of clustering. Although heterogeneity of tubules or of cell types within a tubule could not be excluded, the major factor underlying the distribution appeared to be the presence of channel clusters on the membrane of individual cells. The overall density of channels was higher than that previously found in the cortical collecting tubule (CCT). In contrast to results in the CCT, we did not detect an increase in the overall density of SK channels in the apical membrane after feeding the animals a high-K diet. However, the activity of amiloride-sensitive Na channels was undetectable under control conditions but was increased after both 1 day (90 ± 24 pA/cell) or 7 days (385 ± 82 pA/cell) of K loading. Thus one important factor leading to an increased K secretion in the CNT in response to increased dietary K is an increased apical Na conductance, leading to depolarization of the apical membrane voltage and an increased driving force for K movement out into the tubular lumen.

scholarly journals Gating of Na channels in the rat cortical collecting tubule: effects of voltage and membrane stretch.

1996 ◽  
Vol 107 (1) ◽  
pp. 35-45 ◽  
Author(s):  
L G Palmer ◽  
G Frindt
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Apical Membrane ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Time ◽  
Collecting Tubule ◽  
Excised Patches ◽  
The Mean ◽  
Cortical Collecting Tubule

The gating kinetics of apical membrane Na channels in the rat cortical collecting tubule were assessed in cell-attached and inside-out excised patches from split-open tubules using the patch-clamp technique. In patches containing a single channel the open probability (Po) was variable, ranging from 0.05 to 0.9. The average Po was 0.5. However, the individual values were not distributed normally, but were mainly < or = 0.25 or > or = 0.75. Mean open times and mean closed times were correlated directly and inversely, respectively, with Po. In patches where a sufficient number of events could be recorded, two time constants were required to describe the open-time and closed-time distributions. In most patches in which basal Po was < 0.3 the channels could be activated by hyperpolarization of the apical membrane. In five such patches containing a single channel hyperpolarization by 40 mV increased Po by 10-fold, from 0.055 +/- 0.023 to 0.58 +/- 0.07. This change reflected an increase in the mean open time of the channels from 52 +/- 17 to 494 +/- 175 ms and a decrease in the mean closed time from 1,940 +/- 350 to 336 +/- 100 ms. These responses, however, could not be described by a simple voltage dependence of the opening and closing rates. In many cases significant delays in both the activation by hyperpolarization and deactivation by depolarization were observed. These delays ranged from several seconds to several tens of seconds. Similar effects of voltage were seen in cell-attached and excised patches, arguing against a voltage-dependent chemical modification of the channel, such as a phosphorylation. Rather, the channels appeared to switch between gating modes. These switches could be spontaneous but were strongly influenced by changes in membrane voltage. Voltage dependence of channel gating was also observed under whole-cell clamp conditions. To see if mechanical perturbations could also influence channel kinetics or gating mode, negative pressures of 10-60 mm Hg were applied to the patch pipette. In most cases (15 out of 22), this maneuver had no significant effect on channel behavior. In 6 out of 22 patches, however, there was a rapid and reversible increase in Po when the pressure was applied. In one patch, there was a reversible decrease. While no consistent effects of pressure could be documented, membrane deformation could contribute to the variation in Po under some conditions.

scholarly journals Blocker-related changes of channel density. Analysis of a three-state model for apical Na channels of frog skin.

1990 ◽  
Vol 95 (4) ◽  
pp. 647-678 ◽  
Author(s):  
S I Helman ◽  
L M Baxendale
Keyword(s):  
Frog Skin ◽  
Noise Analysis ◽  
Kinetic Scheme ◽  
State Model ◽  
Na Channel ◽  
Na Channels ◽  
Na Transport ◽  
Open Probability ◽  
Wide Range ◽  
Three State Model

Blocker-induced noise analysis of apical membrane Na channels of epithelia of frog skin was carried out with the electroneutral blocker (CDPC, 6-chloro-3,5-diamino-pyrazine-2-carboxamide) that permitted determination of the changes of single-channel Na currents and channel densities with minimal inhibition of the macroscopic rates of Na transport (Baxendale, L. M., and S. I. Helman. 1986. Biophys. J. 49:160a). Experiments were designed to resolve changes of channel densities due to mass law action (and hence the kinetic scheme of blocker interaction with the Na channel) and to autoregulation of Na channel densities that occur as a consequence of inhibition of Na transport. Mass law action changes of channel densities conformed to a kinetic scheme of closed, open, and blocked states where blocker interacts predominantly if not solely with open channels. Such behavior was best observed in "pulse" protocol experiments that minimized the time of exposure to blocker and thus minimized the contribution of much longer time constant autoregulatory influences on channel densities. Analysis of data derived from pulse, staircase, and other experimental protocols using both CDPC and amiloride as noise-inducing blockers and interpreted within the context of a three-state model revealed that Na channel open probability in the absence of blocker averaged near 0.5 with a wide range among tissues between 0.1 and 0.9.

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