Studies on the mechanisms of active ion fluxes across alveolar epithelial cell monolayers

Kim, Kwang‐Jin; Suh, Duk-Joon; Lubman, Richard L.; Danto, Spencer I.; Borok, Zea; Crandall, Edward D.

doi:10.1007/bf01409010

Cited by 27 publications

(22 citation statements)

References 45 publications

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“…Type II pneumocytes isolated enzymatically from male Sprague-Dawley rats were further enriched by IgG panning and plated onto tissue culture-treated polycarbonate filters (0.45-m pores, 12-mm-diameter Transwells; Costar-Corning, Cambridge, MA) at 1.2 ϫ 10 6 cells/cm 2 (2,21). Cells were maintained for 48 h at 37°C in a humidified atmosphere of 5% CO2/95% air, with culture medium containing 10% newborn bovine serum in minimal defined serum-free medium (MDSF), a 1:1 mixture of DME/ F-12 medium (Sigma, St. Louis, MO) supplemented with 1% nonessential amino acids, 2 mM glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, and 0.1% bovine serum albumin.…”

Section: Methodsmentioning

confidence: 99%

“…In this regard, a simplified model of the alveolar epithelial barrier [primary cultured rat alveolar epithelial cell monolayers (RAECM) (16,21)] has been utilized widely to study transport mechanisms. The monolayers exhibit morphologic and phenotypic characteristics of in vivo type I pneumocytes (4), develop high barrier resistance (Ͼ2,000 ⍀cm 2 ), and actively reabsorb Na ϩ (ϳ0.2 eq⅐cm Ϫ2 ⅐h Ϫ1 ) from apical fluid (17).…”

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confidence: 99%

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Net absorption of IgG via FcRn-mediated transcytosis across rat alveolar epithelial cell monolayers

Kim¹,

Fandy²,

Lee³

et al. 2004

American Journal of Physiology-Lung Cellular and Molecular Phys

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We characterized immunoglobulin G (IgG) transport across rat alveolar epithelial cell monolayers cultured on permeable supports. Unidirectional fluxes of biotin-labeled rat IgG (biot-rIgG) were measured in the apical-to-basolateral (ab) and opposite (ba) directions as functions of [rIgG] in upstream fluids at 37 and 4°C. We explored specificity of IgG transport by measuring fluxes in the presence of excess Fc, Fab, F(abЈ)2, or chicken Ig (IgY). Expression of the IgG receptor FcRn and the effects of dexamethasone on FcRn expression and biot-rIgG fluxes were determined. Results show that ab flux of biot-rIgG is about fivefold greater than ba flux at an upstream concentration of 25 nM biot-rIgG at 37°C. Both ab and ba fluxes of rIgG saturate, resulting in net absorption with half-maximal concentration and maximal flow of 7.1 nM and 1.3 fmol ⅐ cm Ϫ2 ⅐ h Ϫ1 . At 4°C, both ab and ba fluxes significantly decrease, nearly collapsing net absorption. The presence of excess unlabeled Fc [but not Fab, F(abЈ)2, or IgY] significantly reduces biot-rIgG fluxes. RT-PCR demonstrates expression of ␣-and ␤-subunits of rat FcRn. Northern analysis further confirms the presence of ␣-subunit of rat FcRn mRNA of ϳ1.6 kb. Dexamethasone exposure for 72 h decreases the steady-state level of mRNA for rat FcRn ␣-subunit and the ab (but not ba) flux of biot-rIgG. These data indicate that IgG transport across alveolar epithelium takes place via regulable FcRn-mediated transcytosis, which may play an important role in alveolar homeostasis in health and disease. receptor mediated; saturable transcytosis; net IgG absorption; lung defense; pulmonary immune system ALVEOLAR EPITHELIUM LINES the distal air spaces of the lung and provides high resistance to the leak of solutes and fluid from the surrounding interstitial and vascular spaces (18). Various serum proteins (e.g., albumin, transferrin, and IgG) are known to be present in alveolar fluid lining distal air spaces, although the underlying transport mechanisms that account for their presence are not well delineated (10,12,19). Alveolar protein clearance is essential for resolution of both hydrostatic and (especially) high permeability pulmonary edema. Understanding the mechanisms of alveolar protein clearance may be useful in the management of patients with alveolar pulmonary edema and in providing new insights into transpulmonary delivery of exogenous protein drugs.Proteins 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 (10,12,19,20). The relative contributions of each of these three pathways to total clearance of proteins from the air spaces are not known. Previous reports suggest the possibility of transcellular mechanism(s) [e.g., receptor-mediated or adsorptive transcytosis (24)] for transport of macromolecules across alveolar epithelium.Protein transport studies utilizing intact lung are not ideal for inferring mechanistic information bec...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Net absorption of IgG via FcRn-mediated transcytosis across rat alveolar epithelial cell monolayers

Kim¹,

Fandy²,

Lee³

et al. 2004

American Journal of Physiology-Lung Cellular and Molecular Phys

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“…Primary cultures of rat alveolar epithelial cells, which establish polarized monolayers with tight junctions (Ն1,500 ⍀/cm 2 ), were prepared as described in detail elsewhere (16,17). Briefly, the lungs of pathogen-free male Sprague-Dawley rats (125 to 150 g) were perfused via the pulmonary artery, lavaged with Ca 2ϩ -and Mg 2ϩ -free Ringer's solution, and exteriorized.…”

Section: Iodination Of Proteinmentioning

confidence: 99%

Inhalational Poisoning by Botulinum Toxin and Inhalation Vaccination with Its Heavy-Chain Component

Park¹,

Simpson

2003

Infect Immun

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Botulinum toxin is the etiologic agent responsible for the disease botulism, which is characterized by peripheral neuromuscular blockade. Botulism is ordinarily encountered as a form of oral poisoning. The toxin is absorbed from the lumen of the gut to reach the general circulation and is then distributed to peripheral cholinergic nerve endings. However, there is a widespread presumption that botulinum toxin can also act as an inhalation poison, which would require that it be absorbed from the airway. Experiments have been done to show that both pure toxin and progenitor toxin (a complex with auxiliary proteins) are inhalation poisons. Interestingly, the data indicate that auxiliary proteins are not necessary to protect the toxin or to facilitate its absorption. When studied on rat primary alveolar epithelial cells or on immortalized human pulmonary adenocarcinoma (Calu-3) cells, botulinum toxin displayed both specific binding and transcytosis. The rate of transport was greater in the apical-to-basolateral direction than in the basolateral-to-apical direction. Transcytosis was energy dependent, and it was blocked by serotype-specific antibody. The results demonstrated that the holotoxin was not essential for the process of binding and transcytosis. Both in vivo and in vitro experiments showed that the heavy-chain component of the toxin was transported across epithelial monolayers, which indicates that the structural determinants governing binding and transcytosis are found in this fragment. The heavy chain was not toxic, and therefore it was tested for utility as an inhalation vaccine against the parent molecule. This fragment was shown to evoke complete protection against toxin doses of at least 10 4 times the 50% lethal dose.

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“…We have previously reported the routine generation of tight monolayers of rat alveolar epithelial cells in primary culture (6,22,25,26). Briefly, lungs of male specific pathogen-free Sprague-Dawley rats (ϳ150 g) were perfused, lavaged, treated with porcine pancreatic elastase (2.5 U/ml; Worthington, Lakewood, NJ), and chopped into ϳ1-mm 3 tissue blocks (devoid of large airways).…”

Section: Primary Culture Of Rat Alveolar Epithelial Cell Monolayersmentioning

confidence: 99%

“…In this study, we investigated albumin transport properties of the alveolar epithelium, utilizing an in vitro model of primary cultured rat pneumocyte monolayers grown on tissue culture-treated polycarbonate filters (6,22,25,26). These monolayers comprise alveolar epithelial type I-like cells (5,8), develop high barrier resistance (Ͼ2,000 ⍀-cm 2 ), and actively reabsorb Na ϩ (ϳ0.2 eq ⅐ cm Ϫ2 ⅐ h…”

mentioning

confidence: 99%

Absorption of intact albumin across rat alveolar epithelial cell monolayers

Kim¹,

Matsukawa²,

Yamahara³

et al. 2003

American Journal of Physiology-Lung Cellular and Molecular Phys

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Transport characteristics of intact albumin were investigated using primary cultured rat alveolar epithelial cell monolayers. The apical-to-basolateral ( ab) flux of intact fluorescein isothiocyanate (FITC)-labeled albumin (F-Alb) is greater than basolateral-to-apical ( ba) flux at the same upstream [F-Alb]. Net absorption of intact F-Alb occurs with half-maximal concentration of ∼1.6 μM and maximal transport rate of ∼0.15 fmol · cm−2 · s−1. At 15 and 4°C, both ab and ba F-Alb fluxes are not different from zero, collapsing net absorption. The presence of excess unlabeled albumin (but not other macromolecule species) in either the apical or basolateral fluid significantly reduces both ab and ba unidirectional F-Alb fluxes. Photoaffinity labeling of apical cell membranes revealed an ∼60-kDa protein that exhibits specificity for albumin. These data indicate that net absorption of intact albumin takes place via saturable receptor-mediated transcellular endocytotic processes recognizing albumin, but not other macromolecules, that may play an important role in alveolar homeostasis in the mammalian lung.

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Studies on the mechanisms of active ion fluxes across alveolar epithelial cell monolayers

Cited by 27 publications

References 45 publications

Net absorption of IgG via FcRn-mediated transcytosis across rat alveolar epithelial cell monolayers

Net absorption of IgG via FcRn-mediated transcytosis across rat alveolar epithelial cell monolayers

Inhalational Poisoning by Botulinum Toxin and Inhalation Vaccination with Its Heavy-Chain Component

Absorption of intact albumin across rat alveolar epithelial cell monolayers

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