Biophysical and molecular properties of amiloride-inhibitable Na+ channels in alveolar epithelial cells

The recent immunopurification and cloning of various lung Na+ channel proteins has provided the necessary tools to study Na+ transport at a fundamental level across a number of epithelial tissues. Various macroscopic measurements of Na+ transport have shown that Na+ ions enter the cytoplasm of alveolar cells mainly through amiloride-inhibitable Na+ channels. Molecular biology studies have shown the existence of three Na+ channel subunit mRNAs (alpha-, beta-, and gamma-rENaC) in mature fetal (FDLE) and adult alveolar type II (ATII) cells. Patch-clamp studies have demonstrated the existence of various types of amiloride-inhibitable Na+ channels, located in the apical membranes of FDLE and ATII cells. beta-Agonists and agents that enhance intracellular adenosine 3',5'-cyclic monophosphate levels increase the open probability of these channels, leading to increased Na+ transport across the alveolar epithelium in vivo. Immunopurification of a putative channel protein from adult ATII cells showed that it contains an amiloride-binding subunit with a molecular mass of 150 kDa. When this protein was reconstituted in planar lipid bilayers, it exhibited single channels with a conductance of 25 pS, which were moderately selective for Na+ over K+. The open probability of these channels was increased by the addition of protein kinase A (PKA) and ATP, and was decreased to the same extent by addition of [N-ethyl-N-isopropyl]-2'-4'-amiloride (EIPA) and amiloride (1 microM each) in the apical side of the bilayer, in agreement with the results of patch-clamp studies in ATII cells. Exposure of rats to sublethal hyperoxia increased alpha-rENaC mRNA and the functional expression of Na+ channels in alveolar epithelial cells and limited alveolar edema. These findings indicate that alveolar epithelial channels contain at least one family of amiloride-sensitive Na+ channel proteins, which displays a number of unique properties, including sensitivity to EIPA.

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

The Journal of General Physiology ◽

10.1085/jgp.95.4.647 ◽

1990 ◽

Vol 95 (4) ◽

pp. 647-678 ◽

Cited By ~ 33

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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Mechanisms and sequelae of increased alveolar fluid clearance in hyperoxic rats

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1997.272.3.l407 ◽

1997 ◽

Vol 272 (3) ◽

pp. L407-L412 ◽

Cited By ~ 35

Author(s):

G. Yue ◽

S. Matalon

Keyword(s):

Alveolar Epithelial Cells ◽

Na Channels ◽

Alveolar Fluid Clearance ◽

Na Transport ◽

Alveolar Epithelial ◽

Lung Fluid ◽

Isotonic Fluid ◽

Epithelial Na Channels ◽

Alveolar Edema

We instilled 4 ml isotonic fluid containing trace amounts of fluorescently labeled dextran (molecular mass 150 kDa) in the lungs of rats exposed to either 85% O(2) for 7 days or to 85% O(2) for 7 days and 100% O(2) for 3 days. We withdrew the fluid every hour for a 3-h period and calculated alveolar fluid clearance (AFC) from changes in dextran concentration. Postinstillation (3 h), AFC values in the control and the two hyperoxic groups were 51 +/- 1, 63 +/- 2, and 62 +/- 3 (SE), respectively (%instilled volume; n > or = 5; P < 0.05). Addition of either 1 mM amiloride or N-ethyl-N-isopropyl amiloride (EIPA) in the instillate decreased the AFC values in all groups 3 h later to approximately 30% of instilled volume. Instillation of phenamil, an irreversible blocker of epithelial Na+ channels into the lungs of rats exposed to 85% O(2) for 7 days and 100% O(2) for 2 days, resulted in a significant increase of their extravascular lung fluid volumes 24 h later. These results demonstrate the existence of EIPA-inhibitable Na+ channels in alveolar epithelial cells in vivo and indicate that an increase in Na+ transport plays an important role in limiting the amount of alveolar edema in O(2)-damaged lungs.

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Cl-channel activation is necessary for stimulation of Na transport in adult alveolar epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2000.278.2.l239 ◽

2000 ◽

Vol 278 (2) ◽

pp. L239-L244 ◽

Cited By ~ 34

Author(s):

Scott M. O'Grady ◽

Xinpo Jiang ◽

David H. Ingbar

Keyword(s):

Epithelial Cells ◽

Adrenergic Receptor ◽

Apical Membrane ◽

Receptor Activation ◽

Alveolar Epithelial Cells ◽

Na Channels ◽

Channel Activation ◽

Alveolar Epithelial ◽

Cl Channel ◽

Stimulation Of

In this review, we discuss evidence that supports the hypothesis that adrenergic stimulation of transepithelial Na absorption across the alveolar epithelium occurs indirectly by activation of apical Cl channels, resulting in hyperpolarization and an increased driving force for Na uptake through amiloride-sensitive Na channels. This hypothesis differs from the prevailing idea that adrenergic-receptor activation increases the open probability of Na channels, leading to an increase in apical membrane Na permeability and an increase in Na and fluid uptake from the alveolar space. We review results from cultured alveolar epithelial cell monolayer experiments that show increases in apical membrane Cl conductance in the absence of any change in Na conductance after stimulation by selective β-adrenergic-receptor agonists. We also discuss possible reasons for differences in Na-channel regulation in cells grown in monolayer culture compared with that in dissociated alveolar epithelial cells. Finally, we describe some preliminary in vivo data that suggest a role for Cl-channel activation in the process of amiloride-sensitive alveolar fluid absorption.

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Mechanism Of Epithelial Na+ Channel (ENaC) Inhibition By Hypoxia In Alveolar Epithelial Cells

10.1164/ajrccm-conference.2012.185.1_meetingabstracts.a3534 ◽

2012 ◽

Author(s):

Thomas Gille ◽

Nadia Randrianarison ◽

Arnaud Goolaerts ◽

Yurdagül Uzunhan ◽

Evelyne Ferrary ◽

...

Keyword(s):

Epithelial Cells ◽

Alveolar Epithelial Cells ◽

Na Channel ◽

Alveolar Epithelial ◽

Epithelial Na Channel

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Chapter 13 Inhibition of Vectorial Na+ Transport across Alveolar Epithelial Cells by Nitrogen-Oxygen Reactive Species

Current Topics in Membranes - Amiloride-Sensitive Sodium Channels - Physiology and Functional Diversity ◽

10.1016/s0070-2161(08)60961-2 ◽

1999 ◽

pp. 219-238

Author(s):

Sadis Matalon ◽

Ahmed Lazrak ◽

Michael D. DuVall

Keyword(s):

Epithelial Cells ◽

Reactive Species ◽

Alveolar Epithelial Cells ◽

Na Transport ◽

Alveolar Epithelial ◽

Oxygen Reactive Species

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Long-term stimulation of alveolar epithelial cells by β-adrenergic agonists: increased Na+ transport and modulation of cell growth?

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00166.2003 ◽

2003 ◽

Vol 285 (4) ◽

pp. L798-L801 ◽

Cited By ~ 1

Author(s):

Yves Berthiaume

Keyword(s):

Cell Growth ◽

Epithelial Cells ◽

Alveolar Epithelial Cells ◽

Adrenergic Agonists ◽

Na Transport ◽

Alveolar Epithelial ◽

Stimulation Of

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Evidence for active Na+ transport by cultured monolayers of pulmonary alveolar epithelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.1983.245.1.c78 ◽

1983 ◽

Vol 245 (1) ◽

pp. C78-C83 ◽

Cited By ~ 82

Author(s):

B. E. Goodman ◽

R. S. Fleischer ◽

E. D. Crandall

Keyword(s):

Epithelial Cells ◽

Active Transport ◽

Alveolar Epithelial Cells ◽

Metabolic Inhibitors ◽

Normal Lung ◽

Type Ii ◽

Na Transport ◽

Alveolar Epithelial ◽

Cl Transport ◽

Transport Inhibitors

Primary cultured type II alveolar epithelial cells grown to confluence on nonporous surfaces form many small fluid-filled hemicysts or domes. These domes are generally thought to result from active transport of solutes from the medium above the cell monolayer to the substratum, with water following passively. We have investigated the characteristics of active transport by primary cultured monolayers of type II alveolar epithelial cells from rat lungs. Changes in dome density were measured after exposure to metabolic inhibitors, Na+ or Cl- transport inhibitors, and low-Na+ or low-Cl- culture media. Metabolic and Na+ transport inhibitors, and low-Na+ medium, lead to disappearance of domes, whereas Cl- transport inhibitors and low-Cl- medium seem to have no effect on dome density. These results suggest the presence of a Na+-dependent active transepithelial transport process across the monolayer, which is responsible for the formation of domes. This finding implies that absorption of fluid by mammalian alveolar epithelium in vivo may be important in the maintenance of normal lung fluid balance.

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G protein-regulated large-conductance chloride channels in freshly isolated fetal type II alveolar epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1993.265.4.l323 ◽

1993 ◽

Vol 265 (4) ◽

pp. L323-L329 ◽

Cited By ~ 6

Author(s):

P. J. Kemp ◽

G. G. MacGregor ◽

R. E. Olver

Keyword(s):

Epithelial Cells ◽

G Protein ◽

Alveolar Epithelial Cells ◽

Disulfonic Acid ◽

Single Channels ◽

Type Ii ◽

Channel Activity ◽

Patch Clamp Technique ◽

Alveolar Epithelial ◽

Cl Channel

Using the patch-clamp technique, we have recorded single channels in cell-attached and inside-out excised patches from the plasma membrane of type II alveolar epithelial cells freshly isolated from fetal guinea pig lung by elastase digestion and differential filtration. In cell-free patches the channels were highly selective for Cl- (PCl:Pcat = 9:1), had a large unitary conductance (375 pS +/- 23 pS), and current reversal of 0 mV in either symmetrical Na(+)-rich solutions or when the inner membrane leaflet was bathed in a K(+)-rich solution. The large-conductance Cl- channel exhibited little or no voltage inactivation at positive potentials, remained open for a significant amount of time at potentials negative to -40 mV, and was blocked at all potentials by 0.1 mM 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid. Channel activity was independent of intracellular calcium concentration. Bath addition of the nonmetabolizable analogue of GTP, GTP gamma S (0.1 mM), caused a voltage-dependent inhibition of channel activity [open probability (Po) plot was shifted by at least +25 mV]. Smaller channels (25 +/- 3 pS) were recorded in the cell-attached configuration with a current-voltage (I-V) relationship which was compatible with a Cl- conductance. On excision, the patches previously containing small-conductance channels exhibited only large-conductance Cl- channel behavior. These large-conductance, G protein-regulatable Cl- channels may provide a route for alveolar cell Cl- exit and as such may be an integral part of the mechanism responsible for secretion of fetal lung fluid.

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Na transport proteins are expressed by rat alveolar epithelial type I cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00130.2000 ◽

2002 ◽

Vol 282 (4) ◽

pp. L599-L608 ◽

Cited By ~ 98

Author(s):

Zea Borok ◽

Janice M. Liebler ◽

Richard L. Lubman ◽

Martha J. Foster ◽

Beiyun Zhou ◽

...

Keyword(s):

Alveolar Epithelial Cells ◽

Na Channel ◽

Type I ◽

Rt Pcr ◽

Na Transport ◽

Double Labeling ◽

Α Subunit ◽

Alveolar Epithelial ◽

Na Pump

Despite a presumptive role for type I (AT1) cells in alveolar epithelial transport, specific Na transporters have not previously been localized to these cells. To evaluate expression of Na transporters in AT1 cells, double labeling immunofluorescence microscopy was utilized in whole lung and in cytocentrifuged preparations of partially purified alveolar epithelial cells (AEC). Expression of Na pump subunit isoforms and the α-subunit of the rat (r) epithelial Na channel (α-ENaC) was evaluated in isolated AT1 cells identified by their immunoreactivity with AT1 cell-specific antibody markers (VIIIB2 and/or anti-aquaporin-5) and lack of reactivity with antibodies specific for AT2 cells (anti-surfactant protein A) or leukocytes (anti-leukocyte common antigen). Expression of the Na pump α1-subunit in AEC was assessed in situ. Na pump subunit isoform and α-rENaC expression was also evaluated by RT-PCR in highly purified (∼95%) AT1 cell preparations. Labeling of isolated AT1 cells with anti-α1 and anti-β1 Na pump subunit and anti-α-rENaC antibodies was detected, while reactivity with anti-α2 Na pump subunit antibody was absent. AT1 cells in situ were reactive with anti-α1 Na pump subunit antibody. Na pump α1- and β1- (but not α2-) subunits and α-rENaC were detected in highly purified AT1 cells by RT-PCR. These data demonstrate that AT1 cells express Na pump and Na channel proteins, supporting a role for AT1 cells in active transalveolar epithelial Na transport.

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