scholarly journals β-Adrenergic agonists differentially regulate highly selective and nonselective epithelial sodium channels to promote alveolar fluid clearance in vivo

2012 ◽  
Vol 302 (11) ◽  
pp. L1167-L1178 ◽  
Author(s):  
Charles A. Downs ◽  
Lisa H. Kriener ◽  
Ling Yu ◽  
Douglas C. Eaton ◽  
Lucky Jain ◽  
...  
Keyword(s):  
Single Channel ◽  
Selective Inhibition ◽  
Alveolar Epithelial Cells ◽  
Alveolar Type ◽  
Channel Measurements ◽  
Open Probability ◽  
Alveolar Epithelial ◽  
Lung Fluid ◽  
T1 And T2

β-Adrenergic receptors (β-AR) increase epithelial sodium channel (ENaC) activity to promote lung fluid clearance. However, the effect of selective β-AR agonist on highly selective cation (HSC) channels or nonselective cation (NSC) channels in alveolar type 1 (T1) and type 2 (T2) cells is unknown. We hypothesized that stimulation with β1-AR agonist (denopamine) or β2-AR agonist (terbutaline) would increase HSC and/or NSC channel activity in alveolar epithelial cells. We performed single-channel measurements from T1 and T2 cells accessed from rat lung slices. Terbutaline (20 μM) increased HSC ENaC activity (open probability, NPo) in T1 (from 0.96 ± 0.61 to 1.25 ± 0.71, n = 5, P <0.05) and T2 cells (from 0.28 ± 0.14 to 1.0 ± 0.30, n = 8, P = 0.02). Denopamine (20 μM) increased NSC NPo in T1 cells (from 0.34 ± 0.09 to 0.63 ± 0.14, n = 7, P = 0.02) and in T2 cells (from 0.47 ± 0.09 to 0.68 ± 0.10, P = 0.004). In vivo X-ray imaging of lung fluid clearance and ICI 118,551 selective inhibition of β2-ARs confirmed patch-clamp findings. cAMP concentrations increased following treatment with denopamine or terbutaline ( n = 3, P < 0.002). The effects of systemic (intraperitoneal, IP) and local (intratracheal, IT) modes of delivery on lung fluid clearance were assessed. IT delivery of denopamine promoted alveolar flooding, whereas IP delivery promoted delayed fluid clearance. In summary, β-AR agonists differentially regulate HSC and NSC in T1 and T2 cells to promote lung fluid clearance in vivo, and the mode of drug delivery is critical for maximizing β-AR agonist efficacy.

Dopamine activates amiloride-sensitive sodium channels in alveolar type I cells in lung slice preparations

2006 ◽  
Vol 291 (4) ◽  
pp. L610-L618 ◽  
Author(s):  
My N. Helms ◽  
Julie Self ◽  
Hui Fang Bao ◽  
Lauren C. Job ◽  
Lucky Jain ◽  
...  
Keyword(s):  
Receptor Antagonist ◽  
Single Channel ◽  
Sch 23390 ◽  
Alveolar Epithelial Cells ◽  
Alveolar Type ◽  
Type I ◽  
Channel Activity ◽  
Open Probability ◽  
Single Channel Recordings ◽  
Alveolar Epithelial

Active Na+ reabsorption by alveolar epithelial cells generates the driving force used to clear fluids from the air space. Using single-channel methods, we examined epithelial Na+ channel (ENaC) activity of alveolar type I (AT1) cells from live 250- to 300-μm sections of lung tissue, circumventing concerns that protracted cell isolation procedures might compromise the innate transport properties of native lung cells. We used fluorescein-labeled Erythrina crystagalli lectin to positively identify AT1 cells for single-channel patch-clamp analysis. We demonstrated, for the first time, single-channel recordings of highly selective and nonselective amiloride-sensitive ENaC channels (HSC and NSC, respectively) from AT1 cells in situ, with mean conductances of 8.2 ± 2.5 and 22 ± 3.2 pS, respectively. Additionally, 25 nM amiloride in the patch electrode blocked Na+ channel activity in AT1 cells. Immunohistochemical studies demonstrated the presence of dopamine D1 and D2 receptors on the surface of AT1 cells, and single-channel recordings showed that 10 μM dopamine increased Na+ channel activity [product of the number of channels and single-channel open probability ( NPo)] from 0.31 ± 0.19 to 0.60 ± 0.21 ( P < 0.001). The D1 receptor antagonist SCH-23390 (10 μM) blocked the stimulatory effect of dopamine on AT1 cells, but the D2 receptor antagonist sulpiride did not.

scholarly journals Oxidized glutathione (GSSG) inhibits epithelial sodium channel activity in primary alveolar epithelial cells

2015 ◽  
Vol 308 (9) ◽  
pp. L943-L952 ◽  
Author(s):  
Charles A. Downs ◽  
Lisa Kreiner ◽  
Xing-Ming Zhao ◽  
Phi Trac ◽  
Nicholle M. Johnson ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Single Channel ◽  
Alveolar Epithelial Cells ◽  
Alveolar Fluid Clearance ◽  
Open Probability ◽  
Alveolar Epithelial ◽  
Biochemical Assays ◽  
Glutathione Redox ◽  
Back Filling

Amiloride-sensitive epithelial Na+ channels (ENaC) regulate fluid balance in the alveoli and are regulated by oxidative stress. Since glutathione (GSH) is the predominant antioxidant in the lungs, we proposed that changes in glutathione redox potential (Eh) would alter cell signaling and have an effect on ENaC open probability ( Po). In the present study, we used single channel patch-clamp recordings to examine the effect of oxidative stress, via direct application of glutathione disulfide (GSSG), on ENaC activity. We found a linear decrease in ENaC activity as the GSH/GSSG Eh became less negative ( n = 21; P < 0.05). Treatment of 400 μM GSSG to the cell bath significantly decreased ENaC Po from 0.39 ± 0.06 to 0.13 ± 0.05 ( n = 8; P < 0.05). Likewise, back-filling recording electrodes with 400 μM GSSG reduced ENaC Po from 0.32 ± 0.08 to 0.17 ± 0.05 ( n = 10; P < 0.05), thus implicating GSSG as an important regulatory factor. Biochemical assays indicated that oxidizing potentials promote S-glutathionylation of ENaC and irreversible oxidation of cysteine residues with N-ethylmaleimide blocked the effects of GSSG on ENaC Po. Additionally, real-time imaging studies showed that GSSG impairs alveolar fluid clearance in vivo as opposed to GSH, which did not impair clearance. Taken together, these data show that glutathione Eh is an important determinant of alveolar fluid clearance in vivo.

Mechanisms and sequelae of increased alveolar fluid clearance in hyperoxic rats

1997 ◽  
Vol 272 (3) ◽  
pp. L407-L412 ◽  
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.

Impairment of cation transport in A549 cells and rat alveolar epithelial cells by hypoxia

1997 ◽  
Vol 273 (4) ◽  
pp. L797-L806 ◽  
Author(s):  
Heimo Mairbäurl ◽  
Ralf Wodopia ◽  
Sigrid Eckes ◽  
Susanne Schulz ◽  
Peter Bärtsch
Keyword(s):  
Epithelial Cells ◽  
A549 Cells ◽  
Alveolar Epithelium ◽  
Cell Types ◽  
Cation Transport ◽  
Alveolar Epithelial Cells ◽  
Alveolar Type ◽  
Water Reabsorption ◽  
Alveolar Epithelial

A reduced cation reabsorption across the alveolar epithelium decreases water reabsorption from the alveoli and could diminish clearing accumulated fluid. To test whether hypoxia restricts cation transport in alveolar epithelial cells, cation uptake was measured in rat lung alveolar type II pneumocytes (AII cells) in primary culture and in A549 cells exposed to normoxia and hypoxia. In AII and A549 cells, hypoxia caused a[Formula: see text]-dependent inhibition of the Na-K pump, of Na-K-2Cl cotransport, and of total and amiloride-sensitive22Na uptake. Nifedipine failed to prevent hypoxia-induced transport inhibition in both cell types. In A549 cells, the inhibition of the Na-K pump and Na-K-2Cl cotransport occurred within ∼30 min of hypoxia, was stable >20 h, and was reversed by 2 h of reoxygenation. There was also a reduction in cell membrane-associated Na-K-ATPase and a decrease in Na-K-2Cl cotransport flux after full activation with calyculin A, indicating a decreased transport capacity. [14C]serine incorporation into cell proteins was reduced in hypoxic A549 cells, but inhibition of protein synthesis with cycloheximide did not reduce ion transport. In AII and A549 cells, ATP levels decreased slightly, and ADP and the ATP-to-ADP ratio were unchanged after 4 h of hypoxia. In A549 cells, lactate, intracellular Na, and intracellular K were unchanged. These results indicate that hypoxia inhibits apical Na entry pathways and the basolateral Na-K pump in A549 cells and rat AII pneumocytes in culture, indicating a hypoxia-induced reduction of transepithelial Na transport and water reabsorption by alveolar epithelium. If similar changes occur in vivo, the impaired cation transport across alveolar epithelial cells might contribute to the formation of hypoxic pulmonary edema.

Effect of endocytosis inhibitors on alveolar clearance of albumin, immunoglobulin G, and SP-A in rabbits

1994 ◽  
Vol 266 (5) ◽  
pp. L544-L552 ◽  
Author(s):  
R. H. Hastings ◽  
J. R. Wright ◽  
K. H. Albertine ◽  
R. Ciriales ◽  
M. A. Matthay
Keyword(s):  
Immunoglobulin G ◽  
Protein Transport ◽  
Protein A ◽  
Alveolar Epithelium ◽  
Alveolar Epithelial Cells ◽  
Capillary Endothelium ◽  
Alveolar Type ◽  
Protein Uptake ◽  
Alveolar Epithelial

Protein in the alveolar space may be cleared by endocytosis and degradation inside alveolar epithelial cells, by transcytosis across the alveolar epithelium, or by restricted diffusion through the epithelium. The relative contributions of these three pathways to clearance of large quantities of protein from the air spaces is not known. This study investigated the effects of monensin and nocodazole, agents which inhibit endocytosis in cell culture, on alveolar epithelial protein transport in anesthetized rabbits. There was evidence that monensin and nocodazole inhibited endocytosis by the alveolar epithelium in vivo. Nocodazole increased the number of vesicles in the alveolar epithelium and capillary endothelium. Monensin increased vesicle density in the endothelium. These results suggested that the inhibitors disrupted microtubules or interrupted cellular membrane traffic in the lung. Both inhibitors decreased lung parenchymal uptake of immunoreactive human albumin from the air spaces. Monensin and nocodazole inhibited albumin uptake in cultured alveolar type II cells. Monensin increased the amount of 125I-labeled surfactant protein A associated with the lungs, compared with the quantity remaining in the air space 2 h after instillation. Although the drugs decreased alveolar epithelial protein uptake, they did not decrease alveolar clearance of 125I-labeled immunoglobulin G or 131I-labeled albumin in anesthetized rabbits. Thus monensin- and nocodazole-sensitive protein-uptake pathways do not account for most alveolar protein clearance when the distal air spaces are filled with a protein solution.

Low expression of the β-ENaC subunit impairs lung fluid clearance in the mouse

2008 ◽  
Vol 294 (3) ◽  
pp. L409-L416 ◽  
Author(s):  
Nadia Randrianarison ◽  
Christine Clerici ◽  
Chrystophe Ferreira ◽  
Alexandre Fontayne ◽  
Sylvain Pradervand ◽  
...  
Keyword(s):  
Alveolar Epithelial Cells ◽  
Mouse Lung ◽  
Mrna Levels ◽  
Alveolar Epithelial ◽  
Lung Fluid ◽  
Low Levels ◽  
Low Expression ◽  
Epithelial Sodium Channel Enac

Transepithelial alveolar sodium (Na+) transport mediated by the amiloride-sensitive epithelial sodium channel (ENaC) constitutes the driving force for removal of fluid from the alveolar space. To define the role of the β-ENaC subunit in vivo in the mature lung, we studied a previously established mouse strain harboring a disruption of the β-ENaC gene locus resulting in low levels of β-ENaC mRNA expression. Real-time RT-PCR experiments confirmed that β-ENaC mRNA levels were decreased by >90% in alveolar epithelial cells from homozygous mutant (m/m) mice. β-ENaC protein was undetected in lung homogenates from m/m mice by Western blotting, but α- and γ-ENaC proteins were increased by 83% and 45%, respectively, compared with wild-type (WT) mice. At baseline, Na+-driven alveolar fluid clearance (AFC) was significantly reduced by 32% in m/m mice. Amiloride at the concentration 1 mM inhibited AFC by 75% and 34% in WT and m/m mice, respectively, whereas a higher concentration (5 mM) induced a 75% inhibition of AFC in both groups. The β2-agonist terbutaline significantly increased AFC in WT but not in m/m mice. These results show that despite the compensatory increase in α- and γ-ENaC protein expression observed in mutant mouse lung, low expression of β-ENaC results in a moderate impairment of baseline AFC and in decreased AFC sensitivity to amiloride, suggesting a possible change in the stoichiometry of ENaC channels. Finally, adequate β-ENaC expression appears to be required for AFC stimulation by β2-agonists.

Dexamethasone prevents transport inhibition by hypoxia in rat lung and alveolar epithelial cells by stimulating activity and expression of Na+-K+-ATPase and epithelial Na+ channels

2007 ◽  
Vol 293 (5) ◽  
pp. L1332-L1338 ◽  
Author(s):  
Şevin Güney ◽  
Akelei Schuler ◽  
Alexandra Ott ◽  
Sabine Höschele ◽  
Stefanie Zügel ◽  
...  
Keyword(s):  
Short Circuit ◽  
A549 Cells ◽  
Alveolar Epithelial Cells ◽  
Hypoxic Exposure ◽  
Alveolar Type ◽  
Rat Lung ◽  
Fluid Reabsorption ◽  
Alveolar Epithelial ◽  
Lung Fluid ◽  
Transport Inhibition

Hypoxia inhibits Na and lung fluid reabsorption, which contributes to the formation of pulmonary edema. We tested whether dexamethasone prevents hypoxia-induced inhibition of reabsorption by stimulation of alveolar Na transport. Fluid reabsorption, transport activity, and expression of Na transporters were measured in hypoxia-exposed rats and in primary alveolar type II (ATII) cells. Rats were treated with dexamethasone (DEX; 2 mg/kg) on 3 consecutive days and exposed to 10% O2 on the 2nd and 3rd day of treatment to measure hypoxia effects on reabsorption of fluid instilled into lungs. ATII cells were treated with DEX (1 μM) for 3 days before exposure to hypoxia (1.5% O2). In normoxic rats, DEX induced a twofold increase in alveolar fluid clearance. Hypoxia decreased reabsorption (−30%) by decreasing its amiloride-sensitive component; pretreatment with DEX prevented the hypoxia-induced inhibition. DEX increased short-circuit currents (ISC) of ATII monolayers in normoxia and blunted hypoxic transport inhibition by increasing the capacity of Na+-K+-ATPase and epithelial Na+ channels (ENaC) and amiloride-sensitive ISC. DEX slightly increased the mRNA of α- and γ-ENaC in whole rat lung. In ATII cells from DEX-treated rats, mRNA of α1-Na+-K+-ATPase and α-ENaC increased in normoxia and hypoxia, and γ-ENaC was increased in normoxia only. DEX stimulated the mRNA expression of α1-Na+-K+-ATPase and α-, β-, and γ-ENaC of A549 cells in normoxia and hypoxia (1.5% O2) when DEX treatment was begun before or during hypoxic exposure. These results indicate that DEX prevents inhibition of alveolar reabsorption by hypoxia and stimulates the expression of Na transporters even when it is applied in hypoxia.

Hypoxia decreases proteins involved in epithelial electrolyte transport in A549 cells and rat lung

2000 ◽  
Vol 279 (6) ◽  
pp. L1110-L1119 ◽  
Author(s):  
Ralf Wodopia ◽  
Hyun Soo Ko ◽  
Javiera Billian ◽  
Rudolf Wiesner ◽  
Peter Bärtsch ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Lung Tissue ◽  
A549 Cells ◽  
Alveolar Epithelial Cells ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Alveolar Type Ii Cells ◽  
Alveolar Epithelial

Fluid reabsorption from alveolar space is driven by active Na reabsorption via epithelial Na channels (ENaCs) and Na-K-ATPase. Both are inhibited by hypoxia. Here we tested whether hypoxia decreases Na transport by decreasing the number of copies of transporters in alveolar epithelial cells and in lungs of hypoxic rats. Membrane fractions were prepared from A549 cells exposed to hypoxia (3% O2) as well as from whole lung tissue and alveolar type II cells from rats exposed to hypoxia. Transport proteins were measured by Western blot analysis. In A549 cells, α1- and β1-Na-K-ATPase, Na/K/2Cl cotransport, and ENaC proteins decreased during hypoxia. In whole lung tissue, α1-Na-K-ATPase and Na/K/2Cl cotransport decreased. α- and β-ENaC mRNAs also decreased in hypoxic lungs. Similar results were seen in alveolar type II cells from hypoxic rats. These results indicate a slow decrease in the amount of Na-transporting proteins in alveolar epithelial cells during exposure to hypoxia that also occurs in vivo in lungs from hypoxic animals. The reduced number of transporters might account for the decreased transport activity and impaired edema clearance in hypoxic lungs.

scholarly journals Alveolar nonselective channels are ASIC1a/α-ENaC channels and contribute to AFC

2017 ◽  
Vol 312 (6) ◽  
pp. L797-L811 ◽  
Author(s):  
Phi T. Trac ◽  
Tiffany L. Thai ◽  
Valerie Linck ◽  
Li Zou ◽  
Megan Greenlee ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Single Channel ◽  
Fluid Layer ◽  
Alveolar Epithelial Cells ◽  
Percentage Increase ◽  
Channel Measurements ◽  
Water Reabsorption ◽  
Alveolar Epithelial ◽  
Fluid Uptake ◽  
Nonselective Cation Channels

A thin fluid layer in alveoli is normal and results from a balance of fluid entry and fluid uptake by transepithelial salt and water reabsorption. Conventional wisdom suggests the reabsorption is via epithelial Na+ channels (ENaC), but if all Na+ reabsorption were via ENaC, then amiloride, an ENaC inhibitor, should block alveolar fluid clearance (AFC). However, amiloride blocks only half of AFC. The reason for failure to block is clear from single-channel measurements from alveolar epithelial cells: ENaC channels are observed, but another channel is present at the same frequency that is nonselective for Na+ over K+, has a larger conductance, and has shorter open and closed times. These two channel types are known as highly selective channels (HSC) and nonselective cation channels (NSC). HSC channels are made up of three ENaC subunits since knocking down any of the subunits reduces HSC number. NSC channels contain α-ENaC since knocking down α-ENaC reduces the number of NSC (knocking down β- or γ-ENaC has no effect on NSC, but the molecular composition of NSC channels remains unclear). We show that NSC channels consist of at least one α-ENaC and one or more acid-sensing ion channel 1a (ASIC1a) proteins. Knocking down either α-ENaC or ASIC1a reduces both NSC and HSC number, and no NSC channels are observable in single-channel patches on lung slices from ASIC1a knockout mice. AFC is reduced in knockout mice, and wet wt-to-dry wt ratio is increased, but the percentage increase in wet wt-to-dry wt ratio is larger than expected based on the reduction in AFC.

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