scholarly journals Characteristics of Passive Solute Transport across Primary Rat Alveolar Epithelial Cell Monolayers

Membranes ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 331
Author(s):  
Yong Ho Kim ◽  
Kwang-Jin Kim ◽  
David Z. D’Argenio ◽  
Edward D. Crandall
Keyword(s):  
Epithelial Cell ◽  
Tight Junctions ◽  
Pore Radius ◽  
Alveolar Epithelial Cell ◽  
Alveolar Epithelial Cells ◽  
Type I ◽  
Cell Monolayers ◽  
Alveolar Epithelial ◽  
Epithelial Cell Monolayers ◽  
Hydrophilic Solutes

Primary rat alveolar epithelial cell monolayers (RAECM) were grown without (type I cell-like phenotype, RAECM-I) or with (type II cell-like phenotype, RAECM-II) keratinocyte growth factor to assess passive transport of 11 hydrophilic solutes. We estimated apparent permeability (Papp) in the absence/presence of calcium chelator EGTA to determine the effects of perturbing tight junctions on “equivalent” pores. Papp across RAECM-I and -II in the absence of EGTA are similar and decrease as solute size increases. We modeled Papp of the hydrophilic solutes across RAECM-I/-II as taking place via heterogeneous populations of equivalent pores comprised of small (0.41/0.32 nm radius) and large (9.88/11.56 nm radius) pores, respectively. Total equivalent pore area is dominated by small equivalent pores (99.92–99.97%). The number of small and large equivalent pores in RAECM-I was 8.55 and 1.29 times greater, respectively, than those in RAECM-II. With EGTA, the large pore radius in RAECM-I/-II increased by 1.58/4.34 times and the small equivalent pore radius increased by 1.84/1.90 times, respectively. These results indicate that passive diffusion of hydrophilic solutes across an alveolar epithelium occurs via small and large equivalent pores, reflecting interactions of transmembrane proteins expressed in intercellular tight junctions of alveolar epithelial cells.

Modulation of ion conductance and active transport by TGF-β1 in alveolar epithelial cell monolayers

2003 ◽  
Vol 285 (6) ◽  
pp. L1192-L1200 ◽  
Author(s):  
Brigham C. Willis ◽  
Kwang-Jin Kim ◽  
Xian Li ◽  
Janice Liebler ◽  
Edward D. Crandall ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Active Transport ◽  
Alveolar Epithelial Cell ◽  
Alveolar Type ◽  
Type I ◽  
Ion Conductance ◽  
Cell Monolayers ◽  
Alveolar Epithelial ◽  
Epithelial Cell Monolayers ◽  
Tgf Β1

Transforming growth factor-β1 (TGF-β1) may be a critical mediator of lung injury and subsequent remodeling during recovery. We evaluated the effects of TGF-β1 on the permeability and active ion transport properties of alveolar epithelial cell monolayers. Rat alveolar type II cells plated on polycarbonate filters in defined serum-free medium form confluent monolayers and acquire the phenotypic characteristics of alveolar type I cells. Exposure to TGF-β1 (0.1-100 pM) from day 0 resulted in a concentration- and time-dependent decrease in transepithelial resistance ( Rt) and increase in short-circuit current ( Isc). Apical amiloride or basolateral ouabain on day 6 inhibited Isc by 80 and 100%, respectively. Concurrent increases in expression of Na+-K+-ATPase α1- and β1-subunits were observed in TGF-β1-treated monolayers. No change in the α-subunit of the rat epithelial sodium channel (α-rENaC) was seen. Exposure of confluent monolayers to TGF-β1 from day 4 resulted in an initial decrease in Rt within 6 h, followed by an increase in Isc over 72-96 h. These results demonstrate that TGF-β1 modulates ion conductance and active transport characteristics of the alveolar epithelium, associated with increased Na+-K+-ATPase, but without a change in α-rENaC.

Polarized distribution of Na(+)-H+ antiport activity in rat alveolar epithelial cells

1994 ◽  
Vol 266 (2) ◽  
pp. L138-L147 ◽  
Author(s):  
R. L. Lubman ◽  
E. D. Crandall
Keyword(s):  
Epithelial Cell ◽  
Alveolar Epithelial Cell ◽  
Alveolar Epithelium ◽  
Alveolar Epithelial Cells ◽  
Alveolar Type ◽  
Cell Monolayers ◽  
Alveolar Epithelial ◽  
Tissue Resistance ◽  
Recovery From Acidification ◽  
Epithelial Cell Monolayers

In this study, we investigated the polarized distribution of Na(+)-H+ antiport activity in alveolar epithelial cell monolayers. Rat alveolar type II pneumocytes were grown on detachable tissue culture-treated Nuclepore filters. The membrane filters, with their adherent intact alveolar epithelial cell monolayers, were mounted in a cuvette designed to contain two fluid compartments separated by the monolayer. Cells were loaded with the pH-sensitive dye 2',7'-biscarboxyethyl-5,6-carboxylfluorescein and intracellular pH (pHi) measured spectrofluorometrically. Monolayers were studied at ambient temperature on days 3–4 in culture, coincident with the development of high tissue resistance (RT > or = 2000 omega.cm2). Cells were incubated in HCO(3-)-free Na+ buffer [(in mM) 140 NaCl, 6 HEPES, pH 7.4] and acidified by NH3 prepulse. Rates of realkalinization (JH+) were calculated as the product of the initial rate of recovery (dpHi/dt) and the intracellular buffer capacity (beta i). Under control conditions, recovery occurred with an initial JH+ of 28.4 mM/min. When 100 microM dimethylamiloride (DMA), an amiloride analogue with enhanced specificity for inhibiting the Na(+)-H+ antiporter, was present in the basolateral fluid, recovery was inhibited by > 90%. Conversely, when the monolayers were acidified in Na+ buffer containing DMA (100 microM) in the apical fluid, acidification and recovery were identical to control. Recovery from acidification was inhibited by basolateral DMA with a one-half maximal inhibitory concentration (IC50) of 100 nm and by basolateral amiloride with an IC50 of 10 microns. Recovery was completely inhibited by omission of Na+ from the basolateral fluid, but omission of Na+ from apical fluid had no effect. We conclude that Na(+)-H+ antiport activity is located exclusively on the basolateral surface of these alveolar epithelial cell monolayers, where it most likely represents the high-amiloride affinity isoform of the Na(+)-H+ antiporter, NHE-1. The Na(+)-H+ antiporter, asymmetrically distributed to the basolateral surface of the polarized alveolar epithelium, contributes to intracellular homeostasis in alveolar pneumocytes and may also play a role in signal transduction in these cells.

Estimation of paracellular conductance of primary rat alveolar epithelial cell monolayers

2005 ◽  
Vol 98 (1) ◽  
pp. 138-143 ◽  
Author(s):  
Kwang-Jin Kim ◽  
Zea Borok ◽  
Carsten Ehrhardt ◽  
Brigham C. Willis ◽  
Claus-Michael Lehr ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Bovine Serum ◽  
Alveolar Epithelial Cell ◽  
Ringer Solution ◽  
Type I ◽  
Ion Conductance ◽  
Cell Monolayers ◽  
Alveolar Epithelial ◽  
Newborn Bovine Serum ◽  
Epithelial Cell Monolayers

Freshly isolated rat type II pneumocytes, when grown on permeable tissue culture-treated polycarbonate filters, form confluent alveolar epithelial cell monolayers (RAECM). Cells in RAECM undergo transdifferentiation, exhibiting over time morphological and phenotypic characteristics of type I pneumocytes in vivo. We recently reported that transforming growth factor-β1 (TGF-β1) decreases overall monolayer resistance ( Rte) and stimulates short-circuit current in a dose-dependent manner. In this study, we investigated the effects of TGF-β1 (50 pM) or 10% newborn bovine serum (NBS) on modulation of paracellular passive ion conductance and its contribution to total passive ion conductance across RAECM. On days 5–7 in culture, tight-junctional resistance ( Rtj, kΩcm2) of RAECM, cultured in minimally defined serum-free medium (MDSF) with or without TGF-β1 or NBS, was estimated from the relationship between observed transmonolayer voltage and resistance after addition of gramicidin D to apical potassium isethionate Ringer solution under open-circuit conditions. NaCl Ringer solution bathed the basolateral side throughout the experimental period. Results showed that transmonolayer conductance (1/ Rte) and tight-junctional conductance (1/ Rtj) are 0.59 and 0.14 mS/cm2 for control monolayers in MDSF, 1.59 and 0.38 mS/cm2 for monolayers exposed to TGF-β1, and 0.38 and 0.18 mS/cm2 for monolayers grown in the presence of NBS. The contributions to total transepithelial ion conductance by the paracellular pathway are estimated to be 23, 23, and 47% for control, TGF-β1-exposed, and newborn bovine serum (NBS)-treated RAECM, respectively.

Hypoxia decreases active Na transport across primary rat alveolar epithelial cell monolayers

2002 ◽  
Vol 282 (4) ◽  
pp. L659-L665 ◽  
Author(s):  
Heimo Mairbäurl ◽  
Katrin Mayer ◽  
Kwang-Jin Kim ◽  
Zea Borok ◽  
Peter Bärtsch ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Alveolar Epithelial Cell ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Alveolar Epithelial Cells ◽  
Apical Plasma Membrane ◽  
Na Transport ◽  
Cell Monolayers ◽  
Alveolar Epithelial ◽  
Epithelial Cell Monolayers

L665, 2002. First published December 14, 2001; 10.1152/ajplung.00355.2001.—Hypoxia has been reported to inhibit activity and expression of ion transporters of alveolar epithelial cells. This study extended those observations by investigating the mechanisms underlying inhibition of active Na transport across primary cultured adult rat alveolar epithelial cell monolayers grown on polycarbonate filters. Cell monolayers were exposed to normoxia and hypoxia (1.5% and 5% O2, 5% CO2), and resultant changes in bioelectric properties [i.e., short-circuit current ( I sc) and transepithelial resistance ( R t)] were measured in Ussing chambers. Results showed that I sc decreased with duration of exposure to hypoxia, while relatively little change was observed for R t. In normoxia, amiloride inhibited ∼70% of I sc. The amiloride-sensitive portion of I sc decreased over time of exposure to hypoxia, whereas the magnitude of the amiloride-insensitive portion of I sc was not affected. Na pump capacity measured after permeabilization of the apical plasma membrane with amphotericin B decreased in monolayers exposed to 1.5% O2 for 24 h, as did the capacity of amiloride-sensitive Na uptake measured after imposing an apical to basolateral Na gradient and permeabilization of the basolateral membrane. These results demonstrate that exposure to hypoxia inhibits alveolar epithelial Na reabsorption by reducing the rates of both apical amiloride-sensitive Na entry and basolateral Na extrusion.

Mechanisms of liquid flux across pulmonary alveolar epithelial cell monolayers

1997 ◽  
Vol 33 (3) ◽  
pp. 195-200 ◽  
Author(s):  
Gerasimos S. Filippatos ◽  
W. Frank Hughes ◽  
Renli Qiao ◽  
J. Iasha Sznajder ◽  
Bruce D. Uhal
Keyword(s):  
Epithelial Cell ◽  
Alveolar Epithelial Cell ◽  
Cell Monolayers ◽  
Alveolar Epithelial ◽  
Liquid Flux ◽  
Epithelial Cell Monolayers

Rates of Protein Transport Across Rat Alveolar Epithelial Cell Monolayers

1999 ◽  
Vol 7 (5) ◽  
pp. 335-342 ◽  
Author(s):  
Yasuhisa Matsukawa ◽  
Hiroshi Yamahara ◽  
Fumiyoshi Yamashita ◽  
Vincent H.L. Lee ◽  
Edward D. Crandall ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Protein Transport ◽  
Alveolar Epithelial Cell ◽  
Cell Monolayers ◽  
Alveolar Epithelial ◽  
Epithelial Cell Monolayers

Regulation of transport across pulmonary alveolar epithelial cell monolayers

1984 ◽  
Vol 57 (3) ◽  
pp. 703-710 ◽  
Author(s):  
B. E. Goodman ◽  
S. E. Brown ◽  
E. D. Crandall
Keyword(s):  
Epithelial Cell ◽  
Alveolar Epithelial Cell ◽  
Phosphodiesterase Inhibitors ◽  
Beta Agonist ◽  
Type Ii ◽  
Dome Formation ◽  
Cell Monolayers ◽  
Cyclic Monophosphate ◽  
Alveolar Epithelial ◽  
Epithelial Cell Monolayers

Domes are formed in large numbers by primary cultured monolayers of type II alveolar epithelial cells from rat lungs. These fluid-filled structures are formed by active transport of solute from medium to substratum, with water following passively. In the present study, we used dome-forming monolayers to study the regulation of alveolar epithelial transport processes by determining the effects on dome formation of adenosine 3',5'-cyclic monophosphate (cAMP) analogues, phosphodiesterase inhibitors, neurotransmitters, and vasopressin (antidiuretic hormone, ADH). The cAMP analogues (dibutyryl cAMP and 8-bromo-cAMP) and phosphodiesterase inhibitors (theophylline, papaverine, and isobutylmethylxanthine) caused large increases in dome formation by 24 h. ADH and beta-adrenergic agonists (epinephrine, terbutaline, and isoproterenol) also caused significant increases in dome density. The beta-agonist response was completely eliminated in the presence of the beta-blocker propranolol. Dibutyryl guanosine 3',5'-cyclic monophosphate and acetylcholine (cholinergic agonist) had no effect on dome formation, whereas the alpha-adrenergic agonist methoxamine caused a small but significant decrease in dome formation. These findings suggest that the active solute flux resulting in dome formation by type II alveolar epithelial cell monolayers is increased by substances expected to elevate intracellular cAMP (or analogue) concentrations. An attractive speculation having major implications for lung fluid balance is that transepithelial fluxes can be modulated by endogenous, and perhaps exogenous, chemical agents in adult mammalian alveolar epithelium in vivo.

Effect of stretch on structural integrity and micromechanics of human alveolar epithelial cell monolayers exposed to thrombin

2006 ◽  
Vol 290 (6) ◽  
pp. L1104-L1110 ◽  
Author(s):  
Xavier Trepat ◽  
Ferranda Puig ◽  
Nuria Gavara ◽  
Jeffrey J. Fredberg ◽  
Ramon Farre ◽  
...  
Keyword(s):  
Mechanical Ventilation ◽  
Epithelial Cell ◽  
Structural Integrity ◽  
Alveolar Epithelial Cell ◽  
Compressive Strain ◽  
Cell Junctions ◽  
Cell Monolayers ◽  
Alveolar Epithelial ◽  
Epithelial Cell Monolayers ◽  
Human Alveolar Epithelial Cell

Alveolar epithelial cells in patients with acute lung injury subjected to mechanical ventilation are exposed to increased procoagulant activity and mechanical strain. Thrombin induces epithelial cell stiffening, contraction, and cytoskeletal remodeling, potentially compromising the balance of forces at the alveolar epithelium during cell stretching. This balance can be further compromised by the loss of integrity of cell-cell junctions in the injured epithelium. The aim of this work was to study the effect of stretch on the structural integrity and micromechanics of human alveolar epithelial cell monolayers exposed to thrombin. Confluent and subconfluent cells (A549) were cultured on collagen-coated elastic substrates. After exposure to thrombin (0.5 U/ml), a stepwise cell stretch (20%) was applied with a vacuum-driven system mounted on an inverted microscope. The structural integrity of the cell monolayers was assessed by comparing intercellular and intracellular strains within the monolayer. Strain was measured by tracking beads tightly bound to the cell surface. Simultaneously, cell viscoelasticity was measured using optical magnetic twisting cytometry. In confluent cells, thrombin did not induce significant changes in transmission of strain from the substrate to overlying cells. By contrast, thrombin dramatically impaired the ability of subconfluent cells to follow imposed substrate deformation. Upon substrate unstretching, thrombin-treated subconfluent cells exhibited compressive strain (9%). Stretch increased stiffness (56–62%) and decreased cell hysteresivity (13–22%) of vehicle cells. By contrast, stretch did not increase stiffness of thrombin-treated cells, suggesting disruption of cytoskeletal structures. Our findings suggest that thrombin could exacerbate epithelial barrier dysfunction in injured lungs subjected to mechanical ventilation.

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