Richard L. Lubman

Na+-K+-ATPase expression in alveolar epithelial cells: upregulation of active ion transport by KGF

We evaluated the effects of keratinocyte growth factor (KGF) on alveolar epithelial cell (AEC) active ion transport and on rat epithelial Na channel (rENaC) subunit and Na+-K+-adenosinetriphosphatase (ATPase) subunit isoform expression using monolayers of AEC grown in primary culture. Rat alveolar type II cells were plated on polycarbonate filters in serum-free medium, and KGF (10 ng/ml) was added to confluent AEC monolayers on day 4 in culture. Exposure of AEC monolayers to KGF on day 4 resulted in dose-dependent increases in short-circuit current ( I sc) compared with controls by day 5, with further increases occurring through day 8. Relative Na+-K+-ATPase α1-subunit mRNA abundance was increased by 41% on days 6 and 8 after exposure to KGF, whereas α2-subunit mRNA remained only marginally detectable in both the absence and presence of KGF. Levels of mRNA for the β1-subunit of Na+-K+-ATPase did not increase, whereas cellular α1- and β1-subunit protein increased 70 and 31%, respectively, on day 6. mRNA for α-, β-, and γ-rENaC all decreased in abundance after treatment with KGF. These results indicate that KGF upregulates active ion transport across AEC monolayers via a KGF-induced increase in Na pumps, primarily due to increased Na+-K+-ATPase α1-subunit mRNA expression. We conclude that KGF may enhance alveolar fluid clearance after acute lung injury by upregulating Na pump expression and transepithelial Na transport across the alveolar epithelium.We evaluated the effects of keratinocyte growth factor (KGF) on alveolar epithelial cell (AEC) active ion transport and on rat epithelial Na channel (rENaC) subunit and Na(+)-K(+)-adenosinetriphosphatase (ATPase) subunit isoform expression using monolayers of AEC grown in primary culture. Rat alveolar type II cells were plated on polycarbonate filters in serum-free medium, and KGF (10 ng/ml) was added to confluent AEC monolayers on day 4 in culture. Exposure of AEC monolayers to KGF on day 4 resulted in dose-dependent increases in short-circuit current (Isc) compared with controls by day 5, with further increases occurring through day 8. Relative Na(+)-K(+)-ATPase alpha 1-subunit mRNA abundance was increased by 41% on days 6 and 8 after exposure to KGF, whereas alpha 2-subunit mRNA remained only marginally detectable in both the absence and presence of KGF. Levels of mRNA for the beta 1-subunit of Na(+)-K(+)-ATPase did not increase, whereas cellular alpha 1- and beta 1-subunit protein increased 70 and 31%, respectively, on day 6. mRNA for alpha-, beta-, and gamma-rENaC all decreased in abundance after treatment with KGF. These results indicate that KGF upregulates active ion transport across AEC monolayers via a KGF-induced increase in Na pumps, primarily due to increased Na(+)-K(+)-ATPase alpha 1-subunit mRNA expression. We conclude that KGF may enhance alveolar fluid clearance after acute lung injury by upregulating Na pump expression and transepithelial Na transport across the alveolar epithelium.

Modulation of T1α expression with alveolar epithelial cell phenotype in vitro

T1α is a recently identified gene expressed in the adult rat lung by alveolar type I (AT1) epithelial cells but not by alveolar type II (AT2) epithelial cells. We evaluated the effects of modulating alveolar epithelial cell (AEC) phenotype in vitro on T1α expression using either soluble factors or changes in cell shape to influence phenotype. For studies on the effects of soluble factors on T1α expression, rat AT2 cells were grown on polycarbonate filters in serum-free medium (MDSF) or in MDSF supplemented with either bovine serum (BS, 10%), rat serum (RS, 5%), or keratinocyte growth factor (KGF, 10 ng/ml) from either day 0 or day 4 through day 8 in culture. For studies on the effects of cell shape on T1α expression, AT2 cells were plated on thick collagen gels in MDSF supplemented with BS. Gels were detached on either day 1(DG1) or day 4 (DG4) or were left attached until day 8. RNA and protein were harvested at intervals between days 1 and 8 in culture, and T1α expression was quantified by Northern and Western blotting, respectively. Expression of T1α progressively increases in AEC grown in MDSF ± BS between day 1 and day 8 in culture, consistent with transition toward an AT1 cell phenotype. Exposure to RS or KGF from day 0 prevents the increase in T1α expression on day 8, whereas addition of either factor from day 4 through day 8 reverses the increase. AEC cultured on attached gels express high levels of T1α on days 4 and 8. T1α expression is markedly inhibited in both DG1 and DG4 cultures, consistent with both inhibition and reversal of the transition toward the AT1 cell phenotype. These results demonstrate that both soluble factors and alterations in cell shape modulate T1α expression in parallel with AEC phenotype and provide further support for the concept that transdifferentiation between AT2 and AT1 cell phenotypes is at least partially reversible.

Mechanisms of EGF-induced stimulation of sodium reabsorption by alveolar epithelial cells

We investigated the effects of epidermal growth factor (EGF) on active Na+ absorption by alveolar epithelium. Rat alveolar epithelial cells (AEC) were isolated and cultivated in serum-free medium on tissue culture-treated polycarbonate filters. mRNA for rat epithelial Na+ channel (rENaC) alpha-, beta-, and gamma-subunits and Na+ pump alpha1- and beta1-subunits were detected in day 4 monolayers by Northern analysis and were unchanged in abundance in day 5 monolayers in the absence of EGF. Monolayers cultivated in the presence of EGF (20 ng/ml) for 24 h from day 4 to day 5 showed an increase in both alpha1 and beta1 Na+ pump subunit mRNA but no increase in rENaC subunit mRNA. EGF-treated monolayers showed parallel increases in Na+ pump alpha1- and beta1-subunit protein by immunoblot relative to untreated monolayers. Fixed AEC monolayers demonstrated predominantly membrane-associated immunofluorescent labeling with anti-Na+ pump alpha1- and beta1-subunit antibodies, with increased intensity of cell labeling for both subunits seen at 24 h following exposure to EGF. These changes in Na+ pump mRNA and protein preceded a delayed (>12 h) increase in short-current circuit (measure of active transepithelial Na+ transport) across monolayers treated with EGF compared with untreated monolayers. We conclude that EGF increases active Na+ resorption across AEC monolayers primarily via direct effects on Na+ pump subunit mRNA expression and protein synthesis, leading to increased numbers of functional Na+ pumps in the basolateral membranes.

Extracellular matrix-driven alveolar epithelial cell differentiation in vitro.

During homeostasis and in response to injury, alveolar type II (AT2) cells serve as progenitor cells to proliferate, migrate, differentiate, and reestablish both alveolar type I (AT1) and AT2 cells into a functional alveolar epithelium. To understand specific changes in cell differentiation, we monitored morphological characteristics and cell--specific protein markers over time for isolated rat AT2 cells cultured on combinations of collagen, fibronectin and/or laminin-5 (Ln5). For all matrices tested, cultured AT2 cells displayed reduced expression of AT2 cell--specific markers from days 1 to 4 and increased expression of AT1-specific markers by day 3, with continued expression until at least day 5. Over days 5 to 7 in culture, cells took on an AT1-like phenotype (on collagen/fibronectin; collagen alone; or Ln5 alone), an AT2-like phenotype (on collagen/fibronectin/Ln5; or collagen/Ln5), or both AT1-like and AT2-like phenotypes (on collagen/fibronectin matrix with a subsaturating amount of Ln5). Cells transferred between matrices at day 4 of culture retained the ability to alter day 7 phenotype. We conclude that in vitro, (1) AT2 cells exhibited phenotypic plasticity that included an intermediate cell type with both AT1 and AT2 cell characteristics independent of day 7 phenotype; (2) both collagen and Ln5 were needed to promote the development of an AT2-like phenotype at day 7; and (3) components of the extracellular matrix (ECM) contribute to phenotypic switching of alveolar cells in culture. The described tissue culture models provide accessible models for studying changes in alveolar epithelial cell physiology from AT2 cell progenitors to the establishment of alveolar epithelial monolayers that represent AT1-like, AT2-like, or a mix of AT1- and AT2-like cells.

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

To investigate the cell physiologic and biological properties of the alveolar epithelium, we studied rat alveolar epithelial cell monolayers grown on permeable supports in primary culture. Type II alveolar epithelial cells were disaggregated using elastase, and partially purified on a discontinuous metrizamide gradient. These isolated cells were plated onto tissue culture-treated Nuclepore membrane filters at 1.5×106 cells/cm2 and maintained in a humidified incubator (5% CO2 in air, 37° C). After 2 days in culture, the bathing media on both sides of the cell monolayers were changed to fresh culture medium, thus removing nonadherent cells (mostly leukocytes). These monolayers exhibit a high transmonolayer resistance (>2000 Ω-cm2) and actively transport ions. Radionuclide flux studies indicate that Na+ is the predominant ionic species absorbed actively under baseline conditions, accounting for about 80% of the total active ion transport. Cl− seems to be passively transported across the epithelium. However, when the epithelium is exposed to a beta-agonist (terbutaline), active absorption of Na+ is increased and active absorption of Cl− occurs. Although it is clear that both active Na+ and Cl− transport are dependent on Na+/K+-ATPase activity, and that Na+ enters cells predominantly through channels, the specific mechanisms by which Cl− enters and exits the alveolar epithelial cells remain unclear. The stimulated reabsorption of Na+ and Cl− may be important in helping to remove excess fluid from alveolar air spaces in the lung.

KGF prevents hyperoxia-induced reduction of active ion transport in alveolar epithelial cells.

We evaluated the effects of acute hyperoxic exposure on alveolar epithelial cell (AEC) active ion transport and on expression of Na+ pump (Na+-K+-ATPase) and rat epithelial Na+ channel subunits. Rat AEC were cultivated in minimal defined serum-free medium (MDSF) on polycarbonate filters. Beginning on day 5, confluent monolayers were exposed to either 95% air-5% CO2(normoxia) or 95% O2-5% CO2 (hyperoxia) for 48 h. Transepithelial resistance ( R t) and short-circuit current ( I sc) were determined before and after exposure. Na+ channel α-, β-, and γ-subunit and Na+-K+-ATPase α1- and β1-subunit mRNA levels were quantified by Northern analysis. Na+ pump α1- and β1-subunit protein abundance was quantified by Western blotting. After hyperoxic exposure, I sc across AEC monolayers decreased by ∼60% at 48 h relative to monolayers maintained under normoxic conditions. Na+ channel β-subunit mRNA expression was reduced by hyperoxia, whereas α- and γ-subunit mRNA expression was unchanged. Na+ pump α1-subunit mRNA was unchanged, whereas β1-subunit mRNA was decreased ∼80% by hyperoxia in parallel with a reduction in β1-subunit protein. Because keratinocyte growth factor (KGF) has recently been shown to upregulate AEC active ion transport and expression of Na+-K+-ATPase under normoxic conditions, we assessed the ability of KGF to prevent hyperoxia-induced changes in active ion transport by supplementing medium with KGF (10 ng/ml) from day 2. The presence of KGF prevented the effects of hyperoxia on ion transport (as measured by I sc) relative to normoxic controls. Levels of β1 mRNA and protein were relatively preserved in monolayers maintained in MDSF and KGF compared with those cultivated in MDSF alone. These results indicate that AEC net active ion transport is decreased after 48 h of hyperoxia, likely as a result of a decrease in the number of functional Na+ pumps per cell. KGF largely prevents this decrease in active ion transport, at least in part, by preserving Na+ pump expression.We evaluated the effects of acute hyperoxic exposure on alveolar epithelial cell (AEC) active ion transport and on expression of Na+ pump (Na+-K+-ATPase) and rat epithelial Na+ channel subunits. Rat AEC were cultivated in minimal defined serum-free medium (MDSF) on polycarbonate filters. Beginning on day 5, confluent monolayers were exposed to either 95% air-5% CO2 (normoxia) or 95% O2-5% CO2 (hyperoxia) for 48 h. Transepithelial resistance (Rt) and short-circuit current (Isc) were determined before and after exposure. Na+ channel alpha-, beta-, and gamma-subunit and Na+-K+-ATPase alpha1- and beta1-subunit mRNA levels were quantified by Northern analysis. Na+ pump alpha1- and beta1-subunit protein abundance was quantified by Western blotting. After hyperoxic exposure, Isc across AEC monolayers decreased by approximately 60% at 48 h relative to monolayers maintained under normoxic conditions. Na+ channel beta-subunit mRNA expression was reduced by hyperoxia, whereas alpha- and gamma-subunit mRNA expression was unchanged. Na+ pump alpha1-subunit mRNA was unchanged, whereas beta1-subunit mRNA was decreased approximately 80% by hyperoxia in parallel with a reduction in beta1-subunit protein. Because keratinocyte growth factor (KGF) has recently been shown to upregulate AEC active ion transport and expression of Na+-K+-ATPase under normoxic conditions, we assessed the ability of KGF to prevent hyperoxia-induced changes in active ion transport by supplementing medium with KGF (10 ng/ml) from day 2. The presence of KGF prevented the effects of hyperoxia on ion transport (as measured by Isc) relative to normoxic controls. Levels of beta1 mRNA and protein were relatively preserved in monolayers maintained in MDSF and KGF compared with those cultivated in MDSF alone. These results indicate that AEC net active ion transport is decreased after 48 h of hyperoxia, likely as a result of a decrease in the number of functional Na+ pumps per cell. KGF largely prevents this decrease in active ion transport, at least in part, by preserving Na+ pump expression.

Epidermal growth factor regulation in adult rat alveolar type II cells of amiloride-sensitive cation channels

Using the patch-clamp technique, we studied the effects of epidermal growth factor (EGF) on whole cell and single channel currents in adult rat alveolar epithelial type II cells in primary culture in the presence or absence of EGF for 48 h. In symmetrical sodium isethionate solutions, EGF exposure caused a significant increase in the type II cell whole cell conductance. Amiloride (10 μM) produced ∼20-30% inhibition of the whole cell conductance in both the presence and absence of EGF, such that EGF caused the magnitude of the amiloride-sensitive component to more than double. Northern analysis showed that α-, β- and γ-subunits of rat epithelial Na+ channel (rENaC) steady-state mRNA levels were all significantly decreased by EGF. At the single channel level, all active inside-out patches demonstrated only 25-pS channels that were amiloride sensitive and relatively nonselective for cations ([Formula: see text]/[Formula: see text]≈ 1.0:0.48). Although the biophysical characteristics (conductance, open-state probability, and selectivity) of the channels from EGF-treated and untreated cells were essentially identical, channel density was increased by EGF; the modal channel per patch was increased from 1 to 2. These findings indicate that EGF increases expression of nonselective, amiloride-sensitive cation channels in adult alveolar epithelial type II cells. The contribution of rENaC to the total EGF-dependent cation current under these conditions is quantitatively less important than that of the nonselective cation channels in these cells.

Basolateral localization of Na+-HCO3− cotransporter activity in alveolar epithelial cells

We investigated the polarized distribution of Na(+)- and HCO3(-)-dependent recovery from intracellular acidification in alveolar epithelial cell monolayers. Rat alveolar type II cells were grown in primary culture on detachable tissue culture-treated Nuclepore filters. Each filter was mounted in a cuvette containing two fluid compartments (apical and basolateral) separated by the monolayer. Cells were loaded with the pH-sensitive dye BCECF 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 = 1000 omega.cm2). After the cells were acidified by NH3 prepulse, pHi recovered to baseline when Na+ was present in the basolateral fluid, but did not recover when Na+ was present only in the apical fluid. This basolateral Na(+)-dependent pHi recovery in the presence of HCO3-/CO2 was reduced, but present, in experiments where dimethylamiloride (DMA, 100 microM) or the stilbene derivative DIDS (500 microM) was in basolateral fluid. However, recovery was completely inhibited when both DMA and DIDS were present basolaterally. pHi recovery was not inhibited under Cl(-)-free conditions, indicating that cytoplasmic realkalinization was not effected by Na(+)-dependent Cl-HCO3- exchange. These data indicate that alveolar epithelial cells express a basolateral Na(+)- and HCO3(-)-dependent, DIDS-sensitive, Cl(-)-independent pHi recovery process that probably represents Na(+)-HCO3(-)-cotransport (symport). Basolateral Na(+)-HCO3- cotransport modulates pHi in alveolar epithelial cells, may contribute to regulation of intracellular volume and osmolarity, and may participate in signal transduction by hormones and growth factors.

Heterocellular cultures of pulmonary alveolar epithelial cells grown on laminin-5 supplemented matrix.

SummaryThe pulmonary alveolar epithelium consists of alveolar type I (AT1) and alveolar type II (AT2) cells. Interactions between these two cell types are necessary for alveolar homeostasis and remodeling. These interactions have been difficult to study in vitro because current cell culture models of the alveolar epithelium do not provide a heterocellular population of AT1 and AT2 cells for an extended period of time in culture. In this study, a new method for obtaining heterocellular cultures of AT1- and AT2-like alveolar epithelial cells maintained for 7 d on a rat tail collagen-fibronectin matrix supplemented with laminin-5 is described. These cultures contain cells that appear by their morphology to be either AT1 cells (larger flattened cells without lamellar bodies) or AT2 cells (smaller cuboidal cells with lamellar bodies). AT1-like cells stain for the type I cell marker aquaporin-5, whereas AT2-like cells stain for the type II cell markers surfactant protein C or prosurfactant protein C. AT1/AT2 cell ratios, cell morphology, and cell phenotype-specific staining patterns seen in 7-d-old heterocellular cultures are similar to those seen in alveoli in situ. This culture system, in which a mixed population of phenotypically distinct alveolar epithelial cells are maintained, may facilitate in vitro studies that are more representative of AT1-AT2 cell interactions that occur in vivo.

Measurement of solute fluxes in isolated rat lungs

Most previous studies in isolated perfused lungs have utilized measurements of solute flow from alveolar to vascular space to characterize the barrier and transport properties of the alveolar epithelium. In this study, we measured flux of a series of nonionic hydrophilic solutes and sodium across the alveolar epithelium of the isolated rat lung from perfusate to airspace (P-->A), as well as from airspace to perfusate (A-->P). Apparent permeability-surface area products (PS) were calculated from the rates of isotope appearance downstream in either the airspace or the perfusate. Equivalent pore analysis of data for P-->A solute flow demonstrated a small pore population with radius 0.6 nm occupying 85% of the total pore area and a large pore population with radius 3.8 nm occupying 15% of the total area. Similar analysis of A-->P solute flux demonstrated a small pore population of 0.6 nm occupying 86% of the total pore area and a large pore population with radius 2.9 nm occupying 14% of total pore area. The ratio (R) of PSP-->A divided by PSA-->P was 0.8 for the nonionic hydrophilic solutes, while R for sodium was 0.5. In the presence of amiloride and ouabain, R for sucrose was unchanged while R for sodium increased to 0.8 due to a fall in PSA-->P. The difference between R for sodium and R for the passively transported solutes, and the reduction in this difference in the presence of sodium transport inhibitors, are consistent with active sodium reabsorption by the intact alveolar epithelium. Differences in measured unidirectional passive solute fluxes probably result from unequal effective surface areas for diffusion from vascular space to airspace and vice versa in the anatomically complex mammalian lung.