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.

Acid-base effects on intestinal Na+ absorption and vesicular trafficking

2002 ◽  
Vol 283 (3) ◽  
pp. C971-C979 ◽  
Author(s):  
Alan N. Charney ◽  
Richard W. Egnor ◽  
Jesline Alexander-Chacko ◽  
Nicholas Cassai ◽  
Gurdip S. Sidhu
Keyword(s):  
Electron Microscopy ◽  
Apical Membrane ◽  
Ringer Solution ◽  
Vesicular Trafficking ◽  
Acid Base ◽  
Western Blots ◽  
Ph Values ◽  
Transmission Electron ◽  
Apical Membranes ◽  
Nhe Isoforms

We examined for vesicular trafficking of the Na+/H+ exchanger (NHE) in pH-stimulated ileal and CO2-stimulated colonic Na+absorption. Subapical vesicles in rat distal ileum were quantified by transmission electron microscopy at ×27,500 magnification. Internalization of ileal apical membranes labeled with FITC-phytohemagglutinin was assessed using confocal microscopy, and pH-stimulated ileal Na+ absorption was measured after exposure to wortmannin. Apical membrane protein biotinylation of ileal and colonic segments and Western blots of recovered proteins were performed. In ileal epithelial cells incubated in HCO[Formula: see text]/Ringer or HEPES/Ringer solution, the number of subapical vesicles, the relative quantity of apical membrane NHE isoforms 2 and 3 (NHE2 and NHE3, respectively), and apical membrane fluorescence under the confocal microscope were not affected by pH values between 7.1 and 7.6. Wortmannin did not inhibit pH-stimulated ileal Na+ absorption. In colonic epithelial apical membranes, NHE3 protein content was greater at a Pco 2 value of 70 than 21 mmHg, was internalized when Pco 2 was reduced, and was exocytosed when Pco 2 was increased. We conclude that vesicle trafficking plays no part in pH-stimulated ileal Na+absorption but is important in CO2-stimulated colonic Na+ absorption.

Cholinergic modulation of apical Na+ channels in turtle colon: analysis of CDPC-induced fluctuations

1990 ◽  
Vol 259 (4) ◽  
pp. C668-C674 ◽  
Author(s):  
D. J. Wilkinson ◽  
D. C. Dawson
Keyword(s):  
Single Channel ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Rate Coefficients ◽  
Corner Frequency ◽  
Na Channel ◽  
Fluctuation Analysis ◽  
Na Channels ◽  
Na Current

Current fluctuation analysis was used to investigate the properties of apical Na+ channels during muscarinic inhibition of active Na+ absorption. A reversible Na+ channel blocker, 6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC), was used to induce fluctuations in the short-circuit current (I(sc)). Power density spectra of the CDPC-induced fluctuations exhibited a clearly discernible Lorentzian component, characterized by a corner frequency that was linearly related to CDPC concentration between 20 and 100 microM. The on (k'on) and off (k(off)) rate coefficients for the CDPC blocking reaction were k'on = 11.1 +/- 0.8 rad.s-1.microM-1 and k(off) = 744 +/- 53 rad/s, and the microscopic inhibition constant was 67 microM (n = 11). CDPC blocking kinetics were not significantly different after inhibition of Isc by 5 microM serosal carbachol. Single-channel Na+ current (iNa) and the density of open and blocked Na+ channels (N(ob)) were estimated from the fluctuations induced by 40 microM CDPC. Under control conditions, iNa was 0.43 +/- 0.05 pA and N(ob) was 251 +/- 42 X 10(6)/cm2 (n = 10). After exposure to serosal carbachol (2-10 microM) for 60 min, Na+ current and N(ob) were reduced by approximately 50%, but iNa was not changed significantly. These results indicate that muscarinic inhibition of electrogenic Na+ absorption was associated with a reduction in the number of open Na+ channels in the apical membrane. They also suggest that this downregulation of transport involved a coordinated decrease in both apical and basolateral membrane conductances.

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.

scholarly journals Na+ and K+ transport at basolateral membranes of epithelial cells. I. Stoichiometry of the Na,K-ATPase.

1986 ◽  
Vol 87 (3) ◽  
pp. 467-483 ◽  
Author(s):  
T C Cox ◽  
S I Helman
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Na Transport ◽  
Basolateral Membranes ◽  
Dependent Component ◽  
Epithelial Sheets ◽  
Apical Membranes ◽  
K Transport

The stoichiometry of pump-mediated Na/K exchange was studied in isolated epithelial sheets of frog skin. 42K influx across basolateral membranes was measured with tissues in a steady state and incubated in either beakers or in chambers. The short-circuit current provided estimates of Na+ influx at the apical membranes of the cells. 42K influx of tissues bathed in Cl- or SO4-Ringer solution averaged approximately 8 microA/cm2. Ouabain inhibited 94% of the 42K influx. Furosemide was without effect on pre-ouabain-treated tissues but inhibited a ouabain-induced and Cl--dependent component of 42K influx. After taking into account the contribution of the Na+ load to the pump by way of basolateral membrane recycling of Na+, the stoichiometry was found to increase from approximately 2 to 6 as the pump-mediated Na+ transport rate increased from 10 to 70 microA/cm2. Extrapolation of the data to low rates of Na+ transport (less than 10 microA/cm2) indicated that the stoichiometry would be in the vicinity of 3:2. As pump-mediated K+ influx saturates with increasing rates of Na+ transport, Na+ efflux cannot be obligatorily coupled to K+ influx at all rates of transepithelial Na+ transport. These results are similar to those of Mullins and Brinley (1969. Journal of General Physiology. 53:504-740) in studies of the squid axon.

cAMP-induced changes of apical membrane potentials of confluent H441 monolayers

2003 ◽  
Vol 285 (2) ◽  
pp. L443-L450 ◽  
Author(s):  
Ahmed Lazrak ◽  
Sadis Matalon
Keyword(s):  
Apical Membrane ◽  
Ringer Solution ◽  
Membrane Potentials ◽  
Open Probability ◽  
Open Time ◽  
Current Clamp Mode ◽  
Apical Membranes ◽  
Induced Changes ◽  
Human Lung Cell Line ◽  
Lung Cell Line

We recorded apical membrane potentials ( Va) of H441 cells [a human lung cell line exhibiting both epithelial Na+ (ENaC) and CFTR-type channels] grown as confluent monolayers, using the microelectrode technique in current-clamp mode before, during, and after perfusion of the apical membranes with 10 μM forskolin. When perfused with normal Ringer solution, the cells had a Va of -43 ± 10 mV (means ± SD; n = 31). Perfusion with forskolin resulted in sustained depolarization by 25.0 ± 3.5 mV (means ± SD; n = 23) and increased the number, open time, and the open probability of a 4.2-pS ENaC. In contrast to a previous report (Jiang J, Song C, Koller BH, Matthay MA, and Verkman AS. Am J Physiol Cell Physiol 275: C1610–C1620, 1998), no transient hyperpolarization was observed. The forskolin-induced depolarization of Va was almost totally prevented by pretreatment of monolayers with 10 μM amiloride or by substitution of Na+ ions in the bath solution with N-methyl-d-glucamine. These findings indicate that cAMP stimulation of Na+ influx across H441 confluent monolayers results from activation of an amiloride-sensitive apical Na+ conductance and not from Va hyperpolarization due to Cl- influx through CFTR-type channels.

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.

Apical membrane chloride channels in a colonic cell line activated by secretory agonists

1988 ◽  
Vol 254 (4) ◽  
pp. C505-C511 ◽  
Author(s):  
D. R. Halm ◽  
G. R. Rechkemmer ◽  
R. A. Schoumacher ◽  
R. A. Frizzell
Keyword(s):  
Cell Line ◽  
Chloride Channels ◽  
Single Channel ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Anion Channel ◽  
Cell Types ◽  
Cl Channel ◽  
Evoked Activity ◽  
Apical Membranes

We characterized the anion channel responsible for the increase in apical membrane Cl secretion using a model salt-secreting epithelium, the T84 colonic cell line. The adenosine 3',5'-cyclic monophosphate (cAMP)-mediated secretagogues, prostaglandin E2, forskolin, and 8-bromo-cAMP, evoked activity of an outwardly rectifying Cl channel in previously quiet cell-attached membrane patches. The channel remained active in excised, inside-out membranes, where its single-channel conductance was 40-45 pS at 0 mV with 160 mM NaCl in pipette and bath. Selectivities were PCl/PNa = 50 and for halides I(1.8)/Br(1.4)/Cl(1.0)/F(0.4). This halide sequence illustrates that the ability of various anions to undergo transepithelial secretion is determined by the selectivity of the basolateral membrane Cl entry step rather than by the apical Cl channel. Open-channel probability increased with depolarization, an effect that would adjust the rate of Cl exit across secretory cell apical membranes with agonist-induced changes in apical membrane potential. Comparison with the properties of Cl channels detected in other cell types suggests that this cAMP-stimulated Cl channel is uniquely present in the apical membranes of salt-secreting epithelial cells.

Single-channel currents in renal tubules

1984 ◽  
Vol 247 (2) ◽  
pp. F380-F384
Author(s):  
B. M. Koeppen ◽  
K. W. Beyenbach ◽  
S. I. Helman
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Ringer Solution ◽  
Na Channel ◽  
Renal Tubules ◽  
Cell Type ◽  
Channel Current ◽  
Excised Patches ◽  
Single Channel Currents ◽  
Apical Membranes

Patch-clamp techniques were used to study isolated renal cortical collecting ducts of rabbits. Gigaohm seals of the native apical membranes of the principal cells were obtained from tissues superfused with a Ringer solution. No enzymatic or other pretreatment of the tissues was required. The patches studied were primarily of the on-cell type, although excised patches could be obtained. Unitary currents in a range of tenths of picoamperes were observed at holding voltages between +/- 100 mV. Since the apparent reversal potential was at a holding voltage at or near 0 eatment of the tissues was required. The patches studied were primarily of the on-cell type, although excised patches could be obtained. Unitary currents in a range of tenths of picoamperes were observed at holding voltages between +/- 100 mV. Since the apparent reversal potential was at a holding voltage at or near 0 eatment of the tissues was required. The patches studied were primarily of the on-cell type, although excised patches could be obtained. Unitary currents in a range of tenths of picoamperes were observed at holding voltages between +/- 100 mV. Since the apparent reversal potential was at a holding voltage at or near 0 mV and since the current-voltage relationship was markedly nonlinear, the unitary currents are most likely due to K+ . Na+-channel current fluctuations, if present, could not be uniquely identified in the presence or absence of amiloride.

Intrinsic regulation of apical sodium entry in epithelia

1991 ◽  
Vol 71 (2) ◽  
pp. 429-445 ◽  
Author(s):  
K. Turnheim
Keyword(s):  
Steady State ◽  
Cell Function ◽  
Single Channel ◽  
Apical Membrane ◽  
Feedback Inhibition ◽  
Cell Models ◽  
Na Transport ◽  
Membrane Area ◽  
Na Permeability ◽  
Regulatory Systems

In the past 30 years the basic features of Na+ absorption by epithelia have been unraveled and generally accepted cell models have been established. However, these cell models of transepithelial Na+ transport represent, for the most part, a static view of cell function, i.e., all transport parameters are assumed to be in a steady state. Today the focus is on the dynamic properties of epithelia, the non-steady-state condition, and the adaptation to environmental or transport changes. This review deals with mechanisms intrinsic to the epithelium that regulate apical membrane Na+ permeability in response to changes in transport load and ambient conditions. Together with parallel autoregulatory events concerning the basolateral K+ conductance, the described mechanisms controlling apical membrane Na+ permeability serve to maintain the intracellular ionic composition within the limits that are compatible with cell function and survival. Extraepithelial factors that influence epithelial Na+ transport such as mineralocorticoids and glucocorticoids, ADH, catecholamines, and other neurotransmitters are discussed elsewhere. Apical membrane Na+ permeability appears to be determined by several intrinsic or autoregulatory mechanisms. The PmNa of epithelia with channel-mediated apical Na+ entry is downregulated by increases in the Na+ concentration of the apical bathing solution (self-inhibition) and by procedures that inhibit basolateral Na+ extrusion (feedback inhibition). The underlying mechanisms of both regulatory systems are unclear. With the use of current-noise (fluctuation) analysis, on the one hand, and single-channel recordings, on the other hand, conflicting results were obtained concerning the saturability of single-channel conductance with increasing external Na+ concentrations. Results from Na(+)-uptake studies in apical membrane vesicles from amiloride-sensitive epithelia render it unlikely that cell Na+ itself is the mediator of feedback inhibition. Both self-inhibition and feedback inhibition of PmNa are prevented by titrating superficial sulfhydryl groups in the apical membrane. Elevations of cell Ca2+ decrease apical Na+ entry, possibly via an indirect mechanism involving protein kinase C. The PmNa is markedly dependent on cell metabolism and pHc; inhibition of ATP supply and lowering cell pH reduce PmNa. Additionally, PmNa may be altered by exocytotic expansion and endocytotic retrieval of the apical membrane area or by insertion of channel proteins into the apical membrane without increasing the apical membrane area. The diversity of regulatory systems may insure the high degree of flexibility and plasticity of epithelia in their response to environmental changes.

Functional groups of the Na+ channel: role of carboxyl and histidyl groups

1983 ◽  
Vol 245 (6) ◽  
pp. F716-F725 ◽  
Author(s):  
C. S. Park ◽  
D. D. Fanestil
Keyword(s):  
Urinary Bladder ◽  
Apical Membrane ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Ringer Solution ◽  
Acid Dissociation ◽  
Na Channel ◽  
Toad Urinary Bladder ◽  
Carboxyl Group ◽  
Na Transport

Two titratable groups, with effect on Na+ transport and with apparent acid dissociation constants (pKaS) of 4.2 and 6.7, were found in the apical membrane of toad urinary bladder and are tentatively identified as a carboxyl and an imidazole. N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), a reagent selective for carboxyl residues, inhibits Na+ transport in the urinary bladder of toads. The underlying chemical reaction whereby EEDQ produces inhibition through potential modification of carboxyl residues was studied. The inhibitory action of EEDQ on Na+ transport was dependent on pH of reaction media and availability of nucleophile, indicating that formation of a covalent acyl-nucleophile bond is probably involved in the irreversible inhibition of Na+ transport. The kinetics of the inhibition showed a stoichiometry of formation of one acyl-nucleophile bond per closure of one Na+ transport site, presumably the Na+ channel. The nucleophile that appears to be involved in the formation of the acyl-nucleophile bond was tentatively identified as having an apparent pKa of 6.7. Amiloride and two analogues of amiloride added to the mucosal Ringer solution (but not serosal amiloride) protected against inhibition of Na+ transport by EEDQ--a finding consistent with the hypothesis that the EEDQ-activated carboxyl group undergoes reaction with a nucleophile at or near the site of specific binding of amiloride onto the apical membrane, most likely at the Na+ channel. Our findings led us to postulate that amiloride must interact with at least two sites on the Na+ channel in order to block the channel. One of the two sites appears to be an ionic interaction between the anionic carboxyl group at the Na+ channel and the cationic guanidinium group of amiloride.

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