Fetal Alveolar Epithelial Cells Research Articles

The Rho Pathway Mediates Transition to an Alveolar Type I Cell Phenotype During Static Stretch of Alveolar Type II Cells

Stretch is an essential mechanism for lung growth and development. Animal models in which fetal lungs have been chronically over or underdistended demonstrate a disrupted mix of type II and type I cells, with static overdistention typically promoting a type I cell phenotype. The Rho GTPase family, key regulators of cytoskeletal signaling, are known to mediate cellular differentiation in response to stretch in other organs. Using a well-described model of alveolar epithelial cell differentiation and a validated stretch device, we investigated the effects of supraphysiologic stretch on human fetal lung alveolar epithelial cell phenotype. Static stretch applied to epithelial cells suppressed type II cell markers (SP-B and Pepsinogen C, PGC), and induced type I cell markers (Caveolin-1, Claudin 7 and Plasminogen Activator Inhibitor-1, PAI-1) as predicted. Static stretch was also associated with Rho A activation. Furthermore, the Rho kinase inhibitor Y27632 decreased Rho A activation and blunted the stretch-induced changes in alveolar epithelial cell marker expression. Together these data provide further evidence that mechanical stimulation of the cytoskeleton and Rho activation are key upstream events in mechanotransduction-associated alveolar epithelial cell differentiation.

Pulmonary effects of keratinocyte growth factor in newborn rats exposed to hyperoxia

Acute lung injury and compromised alveolar development characterize bronchopulmonary dysplasia (BPD) of the premature neonate. High levels of keratinocyte growth factor (KGF), a cell-cell mediator with pleiotrophic lung effects, are associated with low BPD risk. KGF decreases mortality in hyperoxia-exposed newborn rodents, a classic model of injury-induced impaired alveolarization, although the pulmonary mechanisms of this protection are poorly defined. These were explored through in vitro and in vivo approaches in the rat. Hyperoxia decreased by 30% the rate of wound closure of a monolayer of fetal alveolar epithelial cells, due to cell death, which was overcome by recombinant human KGF (100 ng/ml). In rat pups exposed to >95% O2 from birth, increased viability induced by intraperitoneal injection of KGF (2 microg/g body wt) every other day was associated with prevention of neutrophil influx in bronchoalveolar lavage (BAL), prevention of decreases in whole lung DNA content and cell proliferation rate, partial prevention of apoptosis increase, and a markedly increased proportion of surfactant protein B-immunoreactive cells in lung parenchyma. Increased lung antioxidant capacity is likely to be due in part to enhanced CAAT/enhancer binding protein alpha expression. By contrast, KGF neither corrected changes induced by hyperoxia in parameters of lung morphometry that clearly indicated impaired alveolarization nor had any significant effect on tissue or BAL surfactant phospholipids. These findings evidence KGF alveolar epithelial cell protection, enhancing effects on alveolar repair capacity, and anti-inflammatory effects in the injured neonatal lung that may account, at least in part, for its ability to reduce mortality. They argue in favor of a therapeutic potential of KGF in the injured neonatal lung.

Pregnancy complicated by diffuse chorioamniotic hemosiderosis: Obstetric features and influence on respiratory diseases of the infant

To clarify the clinical features of pregnancy and neonatal respiratory problems associated with diffuse chorioamniotic hemosiderosis (DCH). Sixteen singleton cases of DCH without chorioamnionitis (CAM) were retrospectively analyzed and compared with gestation- and birthweight-matched controls (32 cases of CAM and 32 cases of non-DCH-non-CAM). Maternal symptoms and respiratory problems of the infants were investigated. All 16 cases with DCH resulted in preterm delivery from 23 to 35 weeks' gestation. The presence of subchorionic hematoma in the first trimester (P < 0.001), recurrent vaginal bleeding (P < 0.001), brownish amniotic fluid (P < 0.001) and amniotic necrosis or degeneration (P < 0.001) were significantly more frequent in the DCH group compared to the CAM and non-DCH-non-CAM groups. The incidence of dry lung syndrome and persistent pulmonary hypertension of the newborn (PPHN) was significantly higher in the DCH group than in the CAM (P < 0.001) and non-DCH-non-CAM (P < 0.001) groups. Long-term exposure to degenerating red blood cells is supposed to damage amnion, fetal alveolar epithelial cells and fetal pulmonary arteries, and may lead to dry lung syndrome and PPHN in the infant complicated by DCH.

In Vitro Transdifferentiation of Human Fetal Type II Cells Toward a Type I–like Cell

For alveolar type I cells, phenotype plasticity and physiology other than gas exchange await further clarification due to in vitro study difficulties in isolating and maintaining type I cells in primary culture. Using an established in vitro model of human fetal type II cells, in which the type II phenotype is induced and maintained by adding hormones, we assessed for transdifferentiation in culture toward a type I-like cell with hormone removal for up to 144 h, followed by electron microscopy, permeability studies, and RNA and protein analysis. Hormone withdrawal resulted in diminished type II cell characteristics, including decreased microvilli, lamellar bodies, and type II cell marker RNA and protein. There was a simultaneous increase in type I characteristics, including increased epithelial cell barrier function indicative of a tight monolayer and increased type I cell marker RNA and protein. Our results indicate that hormone removal from cultured human fetal type II cells results in transdifferentiation toward a type I-like cell. This model will be useful for continued in vitro studies of human fetal alveolar epithelial cell differentiation and phenotype plasticity.

Rat alveolar epithelial type I cells express CFTR

CFTR, the cystic fibrosis transmembrane regulator, is a Cl− channel present in fetal alveolar epithelial cells where it contributes to Cl−, and thus fluid, secretion into the developing airspaces. At birth, the fetal lung converts from fluid secretion to fluid absorption. As the lung matures, CFTR expression decreases in the alveolar epithelium but remains prominent in the bronchiolar epithelium. Recently, CFTR has been shown to play an important role in fluid absorption in the distal airspaces of adult lung. The alveolar epithelium, the major site of fluid clearance in the lung, is composed of alveolar type I (TI) and type II (TII) cells, both of which contain sodium channels and Na+, K+-ATPase, by which active Na+ transport occurs. However, the routes for anion movement were unclear. CFTR has been demonstrated in adult TII cells, providing a means for Cl− transport. It is not known if CFTR is also present in TI cells, which cover >95% of the internal surface area of the lung. Hypothesis Alveolar TI cells contain CFTR, which may facilitate Cl− transport. Methods Immunohistochemistry and confocal microscopy were used to localize CFTR to specific alveolar epithelial cells in 2μ sections of adult rat lung and in cytocentrifuged preparations of isolated lung cells. Total RNA was extracted from isolated adult rat TI and TII cells and transcribed to cDNA. Quantitative PCR was performed using probes for CFTR and results were normalized to 18S. Results CFTR colocalizes to TI cells using immunohistochemistry. By QPCR analysis, both TI and TII cells contain mRNA for CFTR. Conclusion Alveolar TI cells contain CFTR, further implicating a role for TI cells in modulating lung fluid balance. Supported by RWJ Foundation and NIH Grants HL57426 and HL24075.

VDUP1: a potential mediator of expansion-induced lung growth and epithelial cell differentiation in the ovine fetus

The degree of fetal lung expansion is a critical determinant of fetal lung growth and alveolar epithelial cell (AEC) differentiation, although the mechanisms involved are unknown. As VDUP1 (vitamin D3-upregulated protein 1) can modulate cell proliferation, can induce cell differentiation, and is highly expressed in the lung, we have investigated the effects of fetal lung expansion on VDUP1 expression and its relationship to expansion-induced fetal lung growth and AEC differentiation in fetal sheep. Alterations in fetal lung expansion caused profound changes in VDUP1 mRNA levels in lung tissue. Increased fetal lung expansion significantly reduced VDUP1 mRNA levels from 100+/-8% in control fetuses to 37+/-4, 46+/-4, and 45+/-9% of control values at 2, 4, and 10 days of increased fetal lung expansion, respectively. Reduced fetal lung expansion increased VDUP1 mRNA levels from 100+/-16% in control fetuses to 162+/-16% of control values after 7 days. VDUP1 was localized to airway epithelium in small bronchioles, AECs, and some mesenchymal cells. Its expression was inversely correlated with cell proliferation during normal lung development (R2=0.972, P<0.002) as well as in response to alterations in fetal lung expansion (R2=0.956, P<0.001) and was positively correlated with SP-B expression during normal lung development (R2=0.803, P<0.0001) and following altered lung expansion (R2=0.817, P<0.001). We suggest that VDUP1 may be an important mediator of expansion-induced lung cell proliferation and AEC differentiation in the developing lung.

Developmental regulation of claudin localization by fetal alveolar epithelial cells.

Tight junction proteins in the claudin family regulate epithelial barrier function. We examined claudin expression by human fetal lung (HFL) alveolar epithelial cells cultured in medium containing dexamethasone, 8-bromo-cAMP, and isobutylmethylxanthanine (DCI), which promotes alveolar epithelial cell differentiation to a type II phenotype. At the protein level, HFL cells expressed claudin-1, claudin-3, claudin-4, claudin-5, claudin-7, and claudin-18, where levels of expression varied with culture conditions. DCI-treated differentiated HFL cells cultured on permeable supports formed tight transepithelial barriers, with transepithelial resistance (TER) >1,700 ohm/cm(2). In contrast, HFL cells cultured in control medium without DCI did not form tight barriers (TER <250 ohm/cm(2)). Consistent with this difference in barrier function, claudins expressed by HFL cells cultured in DCI medium were tightly localized to the plasma membrane; however, claudins expressed by HFL cells cultured in control medium accumulated in an intracellular compartment and showed discontinuities in claudin plasma membrane localization. In contrast to claudins, localization of other tight junction proteins, zonula occludens (ZO)-1, ZO-2, and occludin, was not sensitive to HFL cell phenotype. Intracellular claudins expressed by undifferentiated HFL cells were localized to a compartment containing early endosome antigen-1, and treatment of HFL cells with the endocytosis inhibitor monodansylcadaverine increased barrier function. This suggests that during differentiation to a type II cell phenotype, fetal alveolar epithelial cells use differential claudin expression and localization to the plasma membrane to help regulate tight junction permeability.

Dexamethasone responsive element in the rat Na, K-ATPase β 1 gene coding region

The Na, K-ATPase plays an essential role in active alveolar epithelial fluid resorption. In fetal and adult alveolar epithelial cells, glucocorticoids (GC) increase Na, K-ATPase activity, mRNA levels, and transcription rate of the β 1 subunit. In this study, we describe a glucocorticoid responsive element (GRE) in the coding region of the rat Na, K-ATPase β 1 gene in a rat lung epithelial cell line. Transient transfection experiments with the β 1 subunit coding region with or without the 5′ and 3′ untranslated regions demonstrated responsiveness to dexamethasone induction and also identified a GRE at +434 in exon IV. The +434 GRE conferred dexamethasone responsiveness in a heterologous thymidine kinase promoter irrespective of its orientation to the β 1 promoter. Transcriptional upregulation by dexamethasone was abolished in +434 mutants. Electrophoretic mobility shift assays (EMSA) demonstrated specific binding of nuclear proteins to the +434 GRE and the presence of the GC receptor. This specific binding was inhibited by a GRE previously described in the rat Na, K-ATPase β 1 gene at −631. In conclusion, we identified a GRE at +434 in the exon IV of the rat Na, K-ATPase β 1 gene.

Mechanical Stretch Induces Fetal Type II Cell Differentiation Via an Epidermal Growth Factor Receptor–Extracellular-Regulated Protein Kinase Signaling Pathway

Mechanical forces are important for fetal alveolar epithelial cell differentiation. However, the signal transduction pathways regulating this process remain largely unknown. Based on the importance of the extracellular-regulated protein kinase (ERK) pathway in cell differentiation, we hypothesized that this cascade mediates stretch-induced fetal type II cell differentiation. We demonstrate that ERK1/2 was maximally activated (> 3-fold) after 15 min of cyclic stretch. Blockage of the ERK pathway with U0126 (a selective MEK1/2 inhibitor) significantly decreased stretch-inducible surfactant protein-C (SP-C) mRNA expression. We examined upstream activators of ERK1/2 and found that stretch induced phosphorylation of Raf-1 and activation of Ras. Moreover, GW5074, a selective c-Raf-1 inhibitor, decreased stretch-inducible SP-C mRNA accumulation. Mechanical stretch also stimulated epidermal growth factor receptor (EGFR) phosphorylation. Finally, blockage of the EGFR, either with tyrphostin AG1478 or neutralizing antibody, decreased stretch-inducible SP-C mRNA expression. We conclude that stretch, at least in part, induces differentiation of fetal epithelial cells via EGFR activation of the ERK pathway. These results suggest that EGFR may be a mechanosensor during fetal lung development. These findings may have significant implications for the design of strategies to accelerate lung maturation.

Dexamethasone stimulates transcription of the Na+-K+-ATPase beta1 gene in adult rat lung epithelial cells.

Na+-K+-ATPase plays an essential role in active alveolar epithelial fluid resorption. In fetal and adult alveolar epithelial cells, glucocorticoids (GC) increase Na+-K+-ATPase activity and mRNA levels. We sought to define the mechanism of Na+-K+-ATPase gene upregulation by GC. In a rat alveolar epithelial cell line (RLE), dexamethasone (Dex) increased beta1-subunit Na+-K+-ATPase mRNA expression two- to threefold within 3 h after exposure to the GC. The increased gene expression was due to increased transcription as demonstrated by nuclear run-on assays, whereas mRNA stability remained unchanged. Transient transfection of 5' deletion mutants of a beta1 promoter-reporter construct demonstrated a 1.5- to 2.2-fold increase in promoter activity by Dex. All of the 5' deletion constructs contained partial or palindromic GC regulatory elements (GRE) and responded to GC. The increased expression of promoter reporter was inhibited by RU-486, a GC receptor (GR) antagonist, suggesting the involvement of GR. The palindromic GRE at -631 demonstrated Dex induction in a heterologous promoter construct. Gel mobility shift assays using RLE nuclear extracts demonstrated specific binding to this site and the presence of GR. We conclude that GC directly stimulate transcription of Na+-K+-ATPase beta1 gene expression in adult rat lung epithelial cells through a GR-dependent mechanism that can act at multiple sites.

Hypercapnia induces injury to alveolar epithelial cells via a nitric oxide-dependent pathway.

Ventilator strategies allowing for increases in carbon dioxide (CO(2)) tensions (hypercapnia) are being emphasized to ameliorate the consequences of inflammatory-mediated lung injury. Inflammatory responses lead to the generation of reactive species including superoxide (O(2)(-)), nitric oxide (.NO), and their product peroxynitrite (ONOO(-)). The reaction of CO(2) and ONOO(-) can yield the nitrosoperoxocarbonate adduct ONOOCO(2)(-), a more potent nitrating species than ONOO(-). Based on these premises, monolayers of fetal rat alveolar epithelial cells were utilized to investigate whether hypercapnia would modify pathways of.NO production and reactivity that impact pulmonary metabolism and function. Stimulated cells exposed to 15% CO(2) (hypercapnia) revealed a significant increase in.NO production and nitric oxide synthase (NOS) activity. Cell 3-nitrotyrosine content as measured by both HPLC and immunofluorescence staining also increased when exposed to these same conditions. Hypercapnia significantly enhanced cell injury as evidenced by impairment of monolayer barrier function and increased induction of apoptosis. These results were attenuated by the NOS inhibitor N-monomethyl-L-arginine. Our studies reveal that hypercapnia modifies.NO-dependent pathways to amplify cell injury. These results affirm the underlying role of.NO in tissue inflammatory reactions and reveal the impact of hypercapnia on inflammatory reactions and its potential detrimental influences.

Repulsive axon guidance molecule Sema3A inhibits branching morphogenesis of fetal mouse lung

Semaphorin III/collapsin-1 (Sema3A) guides a specific subset of neuronal growth cones as a repulsive molecule. In this study, we have investigated a possible role of non-neuronal Sema3A in lung morphogenesis. Expression of mRNAs of Sema3A and neuropilin-1 (NP-1), a Sema3A receptor, was detected in fetal and adult lungs. Sema3A-immunoreactive cells were found in airway and alveolar epithelial cells of the fetal and adult lungs. Immunoreactivity for NP-1 was seen in fetal and adult alveolar epithelial cells as well as endothelial cells. Immunoreactivity of collapsin response mediator protein CRMP (CRMP-2), an intracellular protein mediating Sema3A signaling, was localized in alveolar epithelial cells, nerve tissue and airway neuroendocrine cells. The expression of CRMP-2 increased during the fetal, neonate and adult periods, and this pattern paralleled that of NP-1. In a two-day culture of lung explants from fetal mouse lung (E11.5), with exogenous Sema3A at a dose comparable to that which induces growth cone collapse of dorsal root ganglia neurons, the number of terminal buds was reduced in a dose-dependent manner when compared with control or untreated lung explants. This decrease was not accompanied with any alteration of the bromodeoxyuridine-positive DNA-synthesizing fraction. A soluble NP-1 lacking the transmembrane and intracellular region, neutralized the inhibitory effect of Sema3A. The fetal lung explants from neuropilin-1 homozygous null mice grew normally in vitro regardless of Sema3A treatment. These results provide evidence that Sema3A inhibits branching morphogenesis in lung bud organ cultures via NP-1 as a receptor or a component of a possible multimeric Sema3A receptor complex.

Expression and Activity of Epidermal Growth Factor Receptor in Late Fetal Rat Lung Is Cell- and Sex-Specific

Epidermal growth factor (EGF) augments late fetal lung maturation by advancing the ontogeny of fetal lung development and by stimulating surfactant synthesis. Previous studies have indicated that fibroblast-alveolar epithelial cell communications mediate surfactant synthesis in the fetal lung and EGF acts through such a mechanism. We investigated the hypothesis that differential activity and expression of the epidermal growth factor receptor (EGF-R) in fetal lung fibroblasts during the canalicular stage of lung development mediates EGF effects. To test this hypothesis, we examined fetal rat lung fibroblasts (FLFs) and type II cells of late gestation (canalicular and saccular stages; 17–22 days) by EGF-R binding techniques, SDS–PAGE, and Western blot analysis. Specific EGF binding increased 181% in day 18 female FLFs, with male FLFs exhibiting a similar increase on day 19. In contrast, specific EGF binding was low in type II cells, did not increase during late gestation, and there were no sex-specific differences. SDS–PAGE and Western blot analysis revealed a predominant 170-kDa EGF-R band in fibroblasts that increased with gestation (peak = 19 days), and was stronger in females. Immunoprecipitation of EGF-treated cells demonstrated the tyrosine kinase activity of the identified receptor. In contrast, type II cells showed minimal signal that did not increase until day 21 of gestation. We also examined whole fetal lung sections by immunohistochemistry to determine cell-specific expression of the EGF-Rin vivo.Immunohistochemistry revealed specific EGF-R staining in columnar and cuboidal epithelia of small conducting airways and in mesenchyme of epithelial-mesenchymal borders (including subepithelial mesenchyme). In contrast, alveolar epithelia showed minimal staining, while subalveolar mesenchyme EGF-R staining peaked at day 19 of gestation. We conclude that cell-specific and sex-specific differences in EGF-R binding and EGF-R immunolocalization appears in the fetal lung at a developmental stage that is critical for alveolar epithelial cell differentiation. The results suggest a role for EGF-R activation in late fetal alveolar epithelial cell maturation, which is mediated through mesenchymal–epithelial cell communication.

Intercellular adhesion molecule I (ICAM-1) expression by rat fetal alveolar epithelial cells in vitro. • 1784

ICAM-1 is an important molecule in immune and inflammatory interactions that is expressed abundantly on normal type I cells in the adult lung. ICAM-1 expression by adult alveolar epithelial cells in vitro is a function of cell differentiation that is greatly influenced by cell shape. Previously we found that there is low level expression of ICAM-1 on primitive cuboidal epithelial cells in the developing rat lung, and that this expression increases dramatically in neonatal life during alveolarization, suggesting that ICAM-1 expression on maturing alveolar epithelial cells (AEC) is a developmentally regulated process. To further investigate the mechanisms involved in this process, AEC were isolated from 21 day fetal rats (term=22 day) by enzymatic cell dispersion and differential adherence. Epithelial cell purity was consistently >90%. Adult type II AEC were used for comparison. After 2 days, adult AEC that had spread in culture expressed abundant ICAM-1 that remained stable through 7 days in culture. Although both adult and fetal cells had assumed a similar flat, squamous shape at 2 days, fetal AEC expressed dramatically less ICAM-1 by Western blot analysis and immunofluorescence microscopy. However, by 4 days in culture, ICAM-1 expression had increased markedly in the fetal AEC, and was similar to that in adult cells. Thus, fetal AEC recapitulate the differentiation-related induction of ICAM-1 expression by isolated cells in vitro found with cells from adult animals. However, ICAM-1 expression in fetal AEC is determined by additional AEC features beyond cell shape and cell-cell interactions.

Ion transport properties of fetal sheep alveolar epithelial cells in monolayer culture.

Alveolar type II cells were isolated from late-term fetal sheep to investigate ion transport across fetal distal lung epithelium. In Ussing chambers, basal transepithelial potential difference (PD; reference apical side), equivalent short-circuit current (Ieq), and resistance were -0.10 +/- 0.05 mV, 0.10 +/- 0.08 microA/cm2, and 821.5 +/- 38.8 omega .cm2, respectively. Epinephrine (100 nM) increased PD from -0.13 +/- 0.19 to -1.37 +/- 0.20 mV and Ieq from 0.18 +/- 0.26 to 1.47 +/- 0.28 microA/cm2. Propranolol (100 nM) inhibited responses to epinephrine. Forskolin (10 microM) increased PD to -0.81 +/- 0.08 mV and Ieq to 1.02 +/- 0.12 microA/cm2. Mucosal amiloride (200 microM) and serosal bumetanide (10 microM) decreased the forskolin-stimulated PD by 23.42 +/- 4.73 and 25.57 +/- 3.9%, respectively. We conclude that in fetal sheep distal lung epithelium amiloride-inhibitable sodium absorption and bumetanide-sensitive chloride secretion are stimulated by forskolin and that epinephrine effects on ion transport are mediated by beta-adrenergic receptors.

Nitric oxide regulation of superoxide-dependent lung injury: Oxidant-protective actions of endogenously produced and exogenously administered nitric oxide

The influence of endogenous cell ·NO production and ·NO derived from exogenous sources on oxidant injury to cultured fetal rat lung alveolar epithelium and an animal model of pulmonary oxidant injury was examined. Confluent fetal rat alveolar epithelial cell monolayers were stimulated to produce ·NO after treatment with a combination of cytokines (IL-1β, TNF-α, IFN-γ), LPS and zymosan-activated serum (CZ). Cell injury, assessed by 14C-adenine release, was significantly increased compared to basal and CZ-induced cells after inhibition of ·NO synthesis by L-NMMA. Cell monolayer macromolecule barrier function was determined by the rate of diffusion of 125I-albumin from the apical to basolateral side of monolayers. Following exposure to CZ and/or O 2 - generated by xanthine oxidase + lumazine (XO), endogenous cell ·NO production and exogenously administered ·NO (from ·NO donors S-nitrosyl-glutathione and S-nitroso-N-acetylpenicillamine) significantly inhibited the increased monolayer permeability induced by exposure to reactive oxygen species. Furthermore, inhalation of 5–10 ppm of ·NO significantly reduced the toxicity of > 95% oxygen to adult rats. We conclude that when cultured pulmonary epithelial cells and lung tissue in vivo are subjected to inflammatory mediators or acute oxidative stress, ·NO can play a protective role by inhibiting O 2 ·− --dependent toxicity.

Expression of the tyrosine phosphatase LAR-PTP2 is developmentally regulated in lung epithelia

The role of tyrosine kinases in regulating cell proliferation, differentiation, and development has been well documented. In contrast, little is known about the role of protein tyrosine phosphatases (PTPs) in mammalian development. To identify PTPs that may be involved in lung development, we have isolated (by polymerase chain reaction) from rat fetal alveolar epithelial cells a cDNA fragment which was identified as the recently cloned tyrosine phosphatase LAR-PTP2. Analysis of tissue expression of LAR-PTP2 identified a approximately 7.5-kb message in the lung, which is also expressed weakly in brain, and an alternatively spliced approximately 6.0-kb message (LAR-PTP2B) expressed in brain. In the fetal lung, LAR-PTP2 was preferentially expressed in lung epithelial (but not fibroblast) cells grown briefly in primary culture, and its expression was tightly regulated during lung development, peaking at 20 days of gestational age (term = 22 days), when mature alveolar type II epithelium first appears. Accordingly, immunoblot analysis revealed high expression of endogenous LAR-PTP2 protein in alveolar epithelial cells from 21-day gestation fetuses. LAR-PTP2 was also expressed in lungs of newborn rats, but transcripts (and protein) were barely detectable in adult lungs and in the nonproliferating adult alveolar type II cells. Interestingly, expression was restored in the transformed adult type II-like A549 cells. These results suggest that LAR-PTP2 may play a role in the proliferation and/or differentiation of epithelial cells during lung development.

Vitamin D stimulates DNA synthesis in alveolar type-II cells

Alveolar type-II cells are responsible for alveolar epithelial cell proliferation during growth and development and in response to lung injury. Based on the observation of abnormal lung development in rachitic rat pups and the expression of receptors for vitamin D by fetal alveolar epithelial cells, the present study examined the influence of 1,25-dihydroxy vitamin D (DHD) on the proliferation of primary cultures of fetal, neonatal and adult alveolar epithelial cells. The ontogony of vitamin D responsiveness was examined, using fetal ( days 18, 19 and 22 = term), neonatal (days 7 and 18) alveolar epithelial cells as well as adult alveolar type-II cells. Maximal stimulation of [ 3H]thymidine incorporation occurred in neonatal d18 cells: (250 ± 4.8%, n = 4, P < 0.05). Incubation of adult type-II cells, in the presence of 10 −9 M DHD increased thymidine incorporation into DNA (149.1 ± 33.2%, mean ± S.E., n = 3, P < 0.001) compared to control cells maintained in basal medium. Exposure to DHD also increased thymidine incorporation after stimulation with a mixture of conventional progression factors (insulin (10 μg/ml) (I), cholera toxin (10 μg/ml) (C) and EGF (20 ng/ml) (E)) (349.4 ± 42.9% vs. 213.5 ± 23.6%, n = 6, P < 0.005). Autoradiographic labeling indices of adult type-II cells increased from 3.1 ± 0.6% for cells cultured in basal medium to 7.2 ± 1.7% in cells exposed to DHD from the time of plating and I, C, E from 20–68 h in culture ( n = 4, P < 0.05). Although no increase in the number of adult type-II cells was observed in these experiments, flow cytometric analysis of nuclear DNA content revealed an increased proportion of cells in the S and G2 phases of the cell cycle (basal: S = 2.6%, G2/M = 3.0%, DHD + GF: S = 4.7%, G2/M = 5.6%, P < 0.05 for each comparison). These data demonstrate that vitamin D3 is a growth factor for alveolar type-II cells and suggest the possibility that local elaboration of vitamin D may provide a novel mechanism of modulation of epithelial proliferation in the context of lung development and repair.

A Nonselective Cation Channel in Adult Alveolar Epithelial Cells

OBJECTIVE: Efficient gas exchange across the alveolar membrane requires a dry air space. At birth, active Na+transport provides the major driving force for absorption of fetal lung liquid, and during adult life, similar transport mechanisms balance fluid exchange across the alveolarcapillary membrane. A cation channel, previously identified in fetal alveolar epithe lium, may represent one of several pathways which regulate apical-basal transepithelial Na+flux. The purpose of this study was to determine if cation channels are also present in adult alveolar epithelial cells.DESIGN: Standard inside-out patch-clamp recording techniques were used to study channels present in patches of membrane from adult rat alveolar epithelium.POPULATION: Alveolar epithelial cells were obtained from adult male Sprague-Dawley rats and studied in primary cell culture.MAIN RESULTS: A 23 pS nonselective cation channel was identified in 68% of patches voltage clamped to -60 mV. The probability of channel opening was not influenced by changes in membrane potential. Amiloride ( I05M) applied to the extracellular membrane induced channel flickering and reduced the apparent mean channel open time without affecting channel conductance.CONCLUSIONS: These results provide direct evidence for a cation channel in the apical membrane of adult alveolar epithelial cells. The channel is similar to a nonselective cation channel in fetal alveolar epithelial cells and may play an important role in Na+-coupled fluid absorption and/or K+secretion.

Expression of CFTR and a cAMP-stimulated chloride secretory current in cultured human fetal alveolar epithelial cells.

During development the fetal lung secretes fluid that is osmotically linked to chloride (Cl-) transport. One possible pathway for Cl- secretion across the fetal pulmonary epithelium is through the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is expressed in epithelia and functions as a Cl- channel regulated by cyclic adenosine monophosphate (cAMP)-dependent protein kinase and intracellular ATP. Previous studies have shown that CFTR mRNA is expressed throughout the human fetal pulmonary epithelium and CFTR protein can be immunoprecipitated from human fetal lung homogenates. In cultured fetal lung tissue explants, CFTR mRNA was localized to alveolar epithelial cells. To test the hypothesis that fetal alveolar epithelial cells express functional CFTR, we immunolocalized CFTR in human fetal lung and looked for evidence of Cl- secretion in cultured alveolar epithelial cell monolayers. Monoclonal anti-CFTR antibodies localized CFTR in cultured lung explants to the epithelial cells, predominantly at the apical surface. Bioelectric properties of cultured monolayers of midgestation fetal alveolar epithelial cells were measured in modified Ussing chambers. In unstimulated monolayers, transepithelial electrical potential difference (psi t) = -1.1 +/- 0.1 mV, transepithelial resistance (Rt) = 768 +/- 58 omega.cm2, and short-circuit current (Isc) = 1.9 +/- 0.2 microA/cm2 (mean +/- SE, n = 17). Addition of amiloride to the apical surface significantly decreased basal Isc. Apical diphenylamine-2-carboxylate (DPC), a Cl- channel inhibitor, caused no significant change in basal Isc. In the presence of apical amiloride, isoproterenol significantly increased Isc, a response that was inhibited by apical DPC and submucosal bumetanide. The cAMP agonists forskolin and IBMX also stimulated Isc.(ABSTRACT TRUNCATED AT 250 WORDS)