Fetal Lung Organ Culture Research Articles

MiR-124 regulates fetal pulmonary epithelial cell maturation.

MicroRNAs are a family of small noncoding RNAs that regulate the expression of their target proteins at the posttranscriptional level. Their functions cover almost every aspect of cell physiology. However, the roles of microRNAs in fetal lung development are not completely understood. The objective of this study is to investigate the regulation and molecular mechanisms of alveolar epithelial cell maturation during fetal lung development by miR-124. We discovered that miR-124 was downregulated during rat fetal lung development and predominantly expressed in the epithelial cells at late stage of the lung development. Overexpression of miR-124 with an adenovirus vector led to the inhibition of epithelial maturation in rat fetal lung organ cultures and fetal alveolar epithelial type II cells, as demonstrated by a decrease in the type II cell marker expression and an increase in glycogen content. We further demonstrated by luciferase reporter assays that miR-124 inhibited the NF-κB, cAMP/PKA, and MAPK/ERK pathways. In addition, nuclear factor I/B (NFIB), a critical protein in fetal lung maturation, was validated as a direct target of miR-124. Furthermore, miR-124 expression was induced by the Wnt/β-catenin signaling pathway through a direct interaction of LEF1 and the miR-124 promoter region. We concluded that miR-124 downregulation is critical to fetal lung epithelial maturation and miR-124 inhibits this maturation process at least partially through the inhibition of NFIB.

Pleiotrophin Regulates Lung Epithelial Cell Proliferation and Differentiation during Fetal Lung Development via β-Catenin and Dlk1

The role of pleiotrophin in fetal lung development was investigated. We found that pleiotrophin and its receptor, protein-tyrosine phosphatase receptor beta/zeta, were highly expressed in mesenchymal and epithelial cells of the fetal lungs, respectively. Using isolated fetal alveolar epithelial type II cells, we demonstrated that pleiotrophin promoted fetal type II cell proliferation and arrested type II cell trans-differentiation into alveolar epithelial type I cells. Pleiotrophin also increased wound healing of injured type II cell monolayer. Knockdown of pleiotrophin influenced lung branching morphogenesis in a fetal lung organ culture model. Pleiotrophin increased the tyrosine phosphorylation of beta-catenin, promoted beta-catenin translocation into the nucleus, and activated T cell factor/lymphoid enhancer factor transcription factors. Dlk1, a membrane ligand that initiates the Notch signaling pathway, was identified as a downstream target of the pleiotrophin/beta-catenin pathway by endogenous dlk1 expression, promoter assay, and chromatin immunoprecipitation. These results provide evidence that pleiotrophin regulates fetal type II cell proliferation and differentiation via integration of multiple signaling pathways including pleiotrophin, beta-catenin, and Notch pathways.

MicroRNA-127 modulates fetal lung development

MicroRNAs (miRNAs) are small endogenous RNAs and are widely regarded as one of the most important regulators of gene expression in both plants and animals. To define the roles of miRNAs in fetal lung development, we profiled the miRNA expression pattern during lung development with a miRNA microarray. We identified 21 miRNAs that showed significant changes in expression during lung development. These miRNAs were grouped into four distinct clusters based on their expression pattern. Cluster 1 contained miRNAs whose expression increased as development progressed, while clusters 2 and 3 showed the opposite trend of expression. miRNAs in cluster 4 including miRNA-127 (miR-127) had the highest expression at the late stage of fetal lung development. Quantitative real-time PCR validated the microarray results of six selected miRNAs. In situ hybridization demonstrated that miR-127 expression gradually shifted from mesenchymal cells to epithelial cells as development progressed. Overexpression of miR-127 in fetal lung organ culture significantly decreased the terminal bud count, increased terminal and internal bud sizes, and caused unevenness in bud sizes, indicating improper development. These findings suggest that miR-127 may have an important role in fetal lung development.

GABAA receptors play a role in fluid secretion in rat fetal lung

Fluid secretion in the fetal lung is driven by Cl−secretion and is essential for lung development. However, the mechanism of Cl− and fluid secretion in the fetal lung is not completely clear. GABAA receptors are ligand-gated Cl−channels. We show that native GABAA receptors in fetal distal lung epithelial cells are composed of α3-, α4-, β1-, β2-, and π-subunits by co-immunoprecipitation. GABA increased lumen size and Cl− concentration on fetal lung organ culture while GABA receptor antagonists, picrotoxin and biccuculine had an opposite effect. The knock-down of GABAA receptor π subunit by RNA interference also inhibited the fluid secretion. In vitro phosphorylation studies denoted that only β1-subunit of GABAA receptors was phosphorylated on serine residue and this phosphorylation was enhanced in the fetal distal lung epithelial cells stimulated with PMA. Inhibitors of protein kinase C, phospholipase C and P2Y receptor all reduced lumen size in the presence of GABA and inhibited the phosphorylation of the β1-subunit on fetal lung organ culture. Taken together, our results suggest that fetal distal lung epithelial cells contain functional ionotropic GABAA receptors. These receptors could participate in fluid secretion in the fetal lung and this process is regulated by protein phosphorylation (supported by NIH HL-052146, HL-071628 and March of Dimes to LL).

Pleiotrophin Promotes Alveolar Epithelial Cell Proliferation and Migration

Pleiotrophin (PTN) is a heparin-binding cytokine highly expressed during the late stage of embryogenesis and in postnatal central nerve system. It plays important roles in cell proliferation, mitogenesis, angiogenesis, and mesenchymal-epithelial interaction. Our previous DNA microarray and real-time PCR analyses have shown that PTN was highly expressed at the late stage of fetal lung development. Immunostainning revealed that PTN was localized in the mesenchyme adjacent to the developing epithelium, suggesting a role in mesenchymal-epithelial interaction. In the isolated alveolar epithelial cells, PTN significantly increased cell migration and proliferation in a dose-dependent manner. In fetal lung organ culture, PTN influenced lung branching morphogenesis, increased cell proliferation, alveolar epithelial type II cell differentiation, and decreased apoptosis. Our results demonstrate for the first time that PTN may play an important role in fetal lung development. (Supported by NIH R01 HL-52146 and R01 HL-071628 to LL).

Oxidation–Reduction (Redox) Controls Fetal Hypoplastic Lung Growth

Introduction. The persistent morbidity and mortality of congenital diaphragmatic hernia are largely due to associated pulmonary hypoplasia. We have shown previously that three antioxidants (vitamin C, glutathione, and vitamin E) could accelerate the growth of fetal hypoplastic lungs grown in culture. We hypothesize that this occurs via a reductant mechanism. Methods. Timed-pregnant rats were gavage-fed nitrofen (100 mg) on day 9.5 of gestation (term = day 22). Fetal lungs were harvested on day 13.5 and placed in organ culture containing serum-free BGJb medium with antibiotics. After randomization, the lung organ cultures were divided into a control group ( n = 31) and an experimental group that received the antioxidant N-acetylcysteine (NAC, 100 μM, n = 31). The fetal lung organ cultures were grown for 4 days at 37°C with 5% CO 2. Computer-assisted digital tracings of the airways were performed daily on live, unstained specimens, and lung bud count, perimeter, and area were measured. After 4 days, lungs were pooled, homogenized, and assayed for reduced and oxidized glutathione, normalized to protein, as an estimate of the tissue redox potential. Data were expressed as means ± SEM, and statistical comparisons were performed using Student's unpaired t test, with P < 0.05 considered significant. Results. Area, perimeter, lung bud count, and complexity (as measured by the perimeter/square root of area) were all significantly increased with NAC treatment from day 2 onward. Reduced glutathione levels were significantly increased following NAC administration (67.1 ± 5.8 versus 37.5 ± 4.2 μmol/mg, P = 0.0004). The ratio of reduced to oxidized glutathione was 2.23. Conclusions. N-Acetylcysteine stimulates nitrofen-induced hypoplastic fetal lung growth in organ culture and increases the ratio of reduced to oxidized glutathione. These data support the concept that oxidation–reduction (redox) may be an important control mechanism for fetal lung growth.

Differential release of prostaglandins by organ cultures of human fetal trachea and lung.

Human fetal lung at 16-19 weeks gestation has a partially differentiated epithelium, and in organ culture, distal airsacs dilate and the epithelium autodifferentiates to type I and II pneumatocytes, processes regulated by endogenous prostaglandin PGE2. Human fetal trachea, at the same gestation, has a terminally differentiated mucociliary epithelium but after 4-6 d in organ culture, develops squamous metaplasia. Tracheal cultures restricted to 3 d have normal phase-contrast and light microscopy appearances and immunohistochemical reactivities (epithelium: cytokeratin 7,8,18; glutathione S-transferase pi-isozyme; epithelial membrane antigen and mesenchyme; desmin; vimentin). In human fetal trachea organ cultures, the predominant prostaglandins released are 6-keto-PGF1 alpha, PGF2 alpha, and PGE2, a pattern similar to that previously described for human adult trachea and lung. In fetal lung cultures, 13,14-dihydro-15-keto-PGF2 alpha is the major prostaglandin released with lesser amounts of 13,14-dihydro-15-keto-PFG2 alpha,PGF2 alpha,PGE2, and 6-keto-PGF1 alpha. Human fetal lung in vitro has the competence to self-differentiate, as early as 12 weeks gestation and presence of high levels in fetal lung of the inactive metabolite 13,14-dihydro-15-keto-PGE2 relative to PGE2 suggests that active prostaglandin catabolism may be one of the mechanisms to retard this stage of maturation in vivo by limiting PGE2 availability. Surprisingly, the profile of prostaglandins released from fetal lung organ culture does not change to that of a mature lung with terminal differentiation of the epithelium, and this may indicate differences in the expression of key prostaglandin-metabolizing enzymes in developing human fetal lung in culture and with in utero ontogeny.

Development of human fetal lung in organ culture compared with in utero ontogeny

In utero, at around 23 wk gestation, the progenitor epithelium of distal airway differentiates into type I and type II pneumatocytes. Human fetal lung organ cultures, as early as 12 wk gestation, have the competence to self-differentiate. Distal airway epithelial immunoreactivity to cytokeratins CK 7, 8, and 18 decreases with differentiation both in utero and in organ culture, whereas reactivity to epithelial membrane antigen remains constant in both. As distal airways dilate, the mean percentage airspace of fetal lungs in organ culture increases to 58%, equivalent to lung of gestation 26.0 +/- 7.3 wk. In organ culture, capillary blood vessels, visualized by vimentin immunoreactivity, remodel and more closely approximate the epithelium but without direct invasion. In utero, at 23 wk gestation, elastin appears as condensation around airways and forms a basis for secondary crests which, by 29 wk gestation, evolve into alveolar septae. In organ culture, no elastin is deposited, no secondary or alveolar crests form, and the lung retains a simple saccular structure. Differentiation of the terminal airway epithelium and mesodermal maturational events to facilitate gas exchange, such as capillary invasion or secondary-alveolar crest formation, are almost synchronous in human lung in utero but clearly dissociate in organ culture.

Prostaglandin production and metabolism in self-differentiating human fetal lung organ culture.

PGE2 and PGF2 alpha are released into the media of human fetal lung organ cultures in decreasing amounts with time. This decline in PGs is not due to culture failure or loss of synthetic capacity, which can be stimulated by fetal bovine serum, nor is it due to increased catabolism of PGE2 to 13,14-dihydro-15-keto-PGE2 (PGEM) or of PGF2 alpha to 13,14-dihydro-15-keto-PGF2 alpha (PGFM). Immunohistochemically reactive PGs are not retained within lung cells. Antisera against methyl-moximated derivatives of PGEM or PGFM and preceded by derivatization on tissue sections of PGs by methyl-moximation not only demonstrate the localization of PGEM and PGFM in epithelial cells and blood vessels, but also show an overall decline in immunoreactivity with time. In addition electron microscopy of uncultured fetal lung removed directly after termination reveals various degrees of mitochondrial damage and in some cases plasma membrane blebs which resolve during the period in culture and as fetal lung self-differentiates. It is proposed that oxidative and mechanical stresses, occurring during termination of pregnancy or tissue preparation, result in cell damage and increased lung prostaglandin production, which, although decreasing during culture as cells recover, is sufficient to trigger terminal self-differentiation.

CD10/neutral endopeptidase 24.11 in developing human fetal lung. Patterns of expression and modulation of peptide-mediated proliferation.

The cell membrane-associated enzyme CD10/neutral endopeptidase 24.11 (CD10/NEP) functions in multiple organ systems to downregulate responses to peptide hormones. Recently, CD10/NEP was found to hydrolyze bombesin-like peptides (BLP), which are mitogens for normal bronchial epithelial cells and small cell lung carcinomas. Growth of BLP-responsive small cell lung carcinomas was potentiated by CD10/NEP inhibition, implicating CD10/NEP in regulation of BLP-mediated tumor growth. BLP are also likely to participate in normal lung development because high BLP levels are found in fetal lung, and bombesin induces proliferation and maturation of human fetal lung in organ cultures and murine fetal lung in utero. To explore potential roles for CD10/NEP in regulating peptide-mediated human fetal lung development, we have characterized temporal and cellular patterns of CD10/NEP expression and effects of CD10/NEP inhibition in organ cultures. Peak CD10/NEP transcript levels are identified at 11-13 wk gestation by Northern blots and localized to epithelial cells and mesenchyme of developing airways by in situ hybridization. CD10/NEP immunostaining is most intense in undifferentiated airway epithelium. In human fetal lung organ cultures, inhibition of CD10/NEP with either phosphoramidon or SCH32615 increases thymidine incorporation by 166-182% (P < 0.025). The specific BLP receptor antagonist, [Leu13-psi(CH2NH)Leu14]bombesin abolishes these effects on fetal lung growth, suggesting that CD10/NEP modulates BLP-mediated proliferation. CD10/NEP expression in the growing front of airway epithelium and the effects of CD10/NEP inhibitors in lung explants implicate the enzyme in the regulation of peptide-mediated fetal lung growth.

A novel system for the culture of human lung: lung development and the response to injury.

Existing methods of fetal lung organ culture are complicated and require special skills. With the use of a polyester-based plastic sheet, we have developed a simpler human fetal lung organ culture that is viable for 6 wk. This novel method permits the study of growth and differentiation, pulmonary surfactant secretion, and the response of human lung tissue to injury in vitro. Lung tissues, obtained from human fetuses ranging in gestational age from 14 to 18 wk, were cultured on the polyester sheet in Dulbecco's modified Eagle medium supplemented with 15% fetal calf serum and gentamicin. Microscopic study of the fetal lung before culturing revealed round epithelial tubules, lined by glycogen-rich columnar cells and a thick cellular interstitium. After 1 wk in culture, morphological examination showed the development and expansion of alveolar saccules and thinning of the interstitium; type I and II pneumocytes as well as fibroblasts and myofibroblasts were present. Lipid analysis of the tissues, 2 wk after the initiation of the culture, demonstrated a high percentage of dipalmitoyl phosphatidylcholine characteristic of pulmonary surfactant. Treatment of the organ culture with asbestos fibers induced type II cell hyperplasia, increased numbers of collagen fiber bundles within the interstitium, and the accumulation of multi-lamellated surfactant material within the alveolar lumens. We conclude that this organ culture system is suitable for studying lung growth, development, and injury in human tissue.

Genetic element from human surfactant protein SP-C gene confers bronchiolar-alveolar cell specificity in transgenic mice

Transgenic mice bearing chimeric genes consisting of 5'-sequences derived from the human surfactant protein C (SP-C) gene and the bacterial chloramphenicol acetyltransferase (CAT) gene were generated. Analysis of CAT activity was utilized to demonstrate tissue-specific and developmental expression of chimeric genes containing 3.7 kb of sequences from the human SP-C gene. Lung-specific expression of the 3.7 SP-C-CAT transgene was observed in eight distinct transgenic mouse lines. Expression of the 3.7 SP-C-CAT transgene was first detected in fetal lung on day 11 of gestation and increased dramatically with advancing gestational age, reaching adult levels of activity before birth. In situ hybridization demonstrated that expression of 3.7 SP-C-CAT mRNA was confined to the distal respiratory epithelium. Antisense CAT hybridization was detected in bronchiolar and type II epithelial cells in the adult lung of the 3.7 SP-C-CAT transgenic mice. In situ hybridization of four distinct 3.7 SP-C-CAT transgenic mouse lines demonstrated bronchiolar-alveolar expression of the chimeric CAT gene, although the relative intensity of expression at each site varied within the lines studied. Glucocorticoids increased murine SP-C mRNA in fetal lung organ culture. Likewise, expression of 3.7 SP-C-CAT transgene increased during fetal lung organ or explant culture and was further enhanced by glucocorticoid in vitro. The 5'-regions of human SP-C conferred developmental, lung epithelial, and glucocorticoid-enhanced expression of bacterial CAT in transgenic mice. The increased expression of SP-C accompanying prenatal lung development and exposure to glucocorticoid is mediated, at least in part, at the transcriptional level, being influenced by cis-active elements contained within the 5'-flanking region of the human SP-C gene.

Self-differentiation of human fetal lung organ culture: The role of prostaglandins PGE 2 and PGF 2α

Addition of PGE 2, but not PGF 2α, to fetal lung organ cultures accelerates the process of self-differentiation with increased dilatation of terminal airsacs and differentiation of the epithelial lining. Indomethacin reduces the endogenous production by organ cultures of PGE 2, PGF 2α, 13,14-dihydro-15-keto-PGE 2, and 13,14-dihydro-15-keto-PGF 2α and retards the process of self-differentiation. Prolonged exposure of cultures to indomethacin results in cell necrosis. Indomethacin inhibition of self-differentiation can be reversed and accelerated by the addition of PGE 2. Addition of PGF 2α in the presence of indomethacin prevents indomethacin-associated cell necrosis but does not accelerate dilatation or differentiation beyond that of cultures in serafree media without additions. We propose that the endogenous production of PGE 2 is a key process in the mechanism of self-differentiation of human fetal lung in organ culture.

Bombesin Increases Fetal Lung Growth and Maturation In Utero and in Organ Culture

Pulmonary neuroendocrine cells (PNECs) in fetuses synthesize gastrin-releasing peptide (GRP, or mammalian bombesin) at high levels, but the role of this hormone in lung development has been obscure. The present study demonstrates that bombesin administered for 2 to 4 d toward the end of gestation in utero led to increased DNA (days 17 and 18) and saturated phosphatidylcholine (SPC) synthesis (day 18) in a dose-dependent fashion in fetal lung. These kinetics coincide with the timing of endogenous GRP gene activation in untreated fetal mouse lung, where GRP mRNA is detectable on day 16 and peaks at day 18. Electron microscopy on in vivo bombesin-treated fetal lung showed an increase in the number of cells containing lamellar bodies on both days 17 and 18, consistent with increased growth and/or maturation of type II cells. In mouse fetal lung organ cultures, the addition of bombesin led to accelerated uptake of [3H]thymidine into DNA, [3H]leucine into protein, and [3H]choline into SPC, indicating that increased growth and maturation may be direct effects. Extending these observations to another species, bombesin was found to induce growth and maturation of human fetal lung in organ culture. A monoclonal antibody to bombesin (2A11) prevented bombesin-induced increases in choline and thymidine incorporation in lung organ cultures and also blocked baseline automaturation of control lung organ cultures in serum-free medium. These data suggest that bombesin, and thus PNECs, play a role in normal lung development.

The α and π isoenzymes of glutathione S-transferase in human fetal lung: in utero ontogeny compared with differentiation in lung organ culture

Polyclonal antisera to the α and π isoenzymes of glutathione S-transferase have been used in immunohistochemical studies of developing human lung. In utero expression of the π set was down-regulated in distal airway cells and the first appearance of π-negative cells coincided with phenotypic differentiation. In contrast, in the early phase of fetal lung organ culture π isoenzyme was detected in all differentiated epithelial cells and only as culture progressed did focal negativity develop. The α set showed no developmental changes in utero or in organ culture.

Glucocorticoid enhances surfactant proteolipid Phe and pVal synthesis and RNA in fetal lung.

Two newly described surfactant proteolipids (SPL), Phe and pVal, are produced by proteolytic processing of distinct precursors of Mr = 40,000 and 22,000, respectively. These proteins are structurally related and intimately associated with surfactant phospholipids. We now demonstrate the expression of both SPL(Phe) and SPL(pVal) in explants of human fetal lung from 16-24 weeks of gestation. Content, synthesis, and mRNA for the proteolipids were low prior to organ culture of fetal lung. Induction of synthesis of the proteolipids occurred rapidly in explant culture in the absence of exogenous hormones and was enhanced by addition of dexamethasone. Increased synthesis of the proteolipids was detected by enzyme-linked immunosorbent assay and by [35S]methionine incorporation into the glycosylated Mr = 40,000-43,000 SPL (Phe) precursor. The response to dexamethasone occurred rapidly and contrasted with effects of dexamethasone on the expression of surfactant-associated protein- (SAP) 35, a distinct surfactant glycoprotein. 8-Br-cAMP did not significantly increase proteolipid content but markedly increased synthesis of SAP-35 in identical cultures. Increased proteolipid content was associated with increased mRNA for each protein as determined by the Northern blot analysis. Proteolipid RNA was also increased by 8-Br-cAMP, however, not to the extent observed with the glucocorticoid. Immunohistochemical analysis of fetal lung with anti-proteolipid antiserum confirmed that the dexamethasone-enhanced synthesis of the proteins by Type II epithelial cells. The time and hormone dependence of the regulation of expression of both SPL(Phe) and SPL(pVal) precursors were distinct from that of SAP-35. Expression of the surfactant proteolipids increased during explant culture of human fetal lung and was further enhanced by glucocorticoid. Developmental and hormonal regulation of the surfactant proteolipids may be important factors in surfactant function at birth.

SYNTHESIS AND PROCESSING OF PULMONARY SURFACTANT PROTEOLIPID SPL(Phe) IN FETAL LUNG

Surfactant proteolipid, SPL(Phe), a small molecular weight hydrophobic protein isolated from pulmonary surfactant, has been associated with enhanced surface properties of surfactant phospholipids from various species. We have isolated cDNA's encoding SPL(Phe) and studies the induction of SPL(Phe) RNA and protein synthesis in human fetal lung tissue during explant culture usng the labelled SPL(Phe) cDNA to probe SPL(Phe) RNA. SPL(Phe) synthesis was not detected after [35S]methionine labelling and immunoprecipitation in fetal lung from 18-23 weeks gestation but increased markedly after 1-5 days in organ culture. Primary in vitro translation product of SPL(Phe) was a charge train of Mr=38-39,000, pI 5.0-5.4 processed in vitro to larger proteins of Mr=40,000. Hybrid selected and arrested translation of human lung RNA confirmed the relationship of the Mr=40,000 precursors to SPL(Phe). Synthesis of SPL(Phe) preprotein, Mr=40-41,000, pI 5.0-5.4, containing N-linked oligosaccharide, was enhanced between 0-4 days of fetal lung explant culture; Northern blot analysis of RNA of low abundance demonstrated a single 2.0 kilobase mRNA (in fetal lung), prior to explant culture and increased dramatically during 1-4 days of culture. Proteolytic processing of the SPL(Phe) to smaller peptides of Mr=6-14,000, which co-migrated with the peptides isolated from human surfactant was demonstrated in the fetal lung explants after organ culture. SPL(Phe) is a Type II cell surfactant-associated hydrophobic protein whose synthesis and RNA increased dramatically during organ culture of fetal lung in association with the morphologic saturation of the Type II epithelial cell.

Optimization of fetal lung organ culture for surfactant biosynthesis.

Lung organ culture has been a widely used system for studying differentiation and maturation of alveolar epithelium through various culture conditions. The purpose of this work was to carefully characterize in vitro lung biochemical differentiation through isolation of surfactant fraction from tissue and to search for optimal culture conditions. Fetal rat lung was explanted on the 18th gestational day for studying glycogen storage, and on the 20th gestational day for studying surfactant accretion, and cultivated for 48 h. Morphologic differentiation was studied by electron microscopy on tissue explanted on the 17th or 18th gestational days and cultivated for various times. Glycogen storage was greater on fluid medium, although less than occurring in vivo. Cellular integrity and surfactant accumulation were maximal on a semisolid medium containing 0.5% agar. Use of O2-CO2 instead of air-CO2 for gassing the explants slightly decreased phospholipid accumulation. Among media used in previous lung culture studies, Waymouth MB 752/1 was the only one to allow net glycogen accumulation in vitro. The most favorable media for surfactant phospholipid accretion were Waymouth MB 752/1, Eagle's minimum essential and its Dulbecco's modification, CMRL 1066, and NCTC 109. They allowed a 12- to 14-fold increase of surfactant fraction phospholipids in vitro, which is similar to the increase occurring in vivo during the same period. Ham's F10 and F12 media allowed a six fold increase. RPMI 1640 and medium 199 (M199) allowed only a three fold increase. Phospholipid concentration in nonsurfactant fraction only doubled during culture, and differences between various media were much less marked. DNA concentration changed little during culture. Morphologic differentiation of epithelial cells was advanced as compared with in vivo timing in a medium allowing maximal surfactant accretion (Waymouth MB 752/1) but not in a medium allowing low surfactant increase (RPMI 1640). The possible role of compositional differences between media is discussed.

Dexamethasone increases superoxide dismutase activity in serum-free rat fetal lung organ cultures.

Dexamethasone (Dex) injected intraperitoneally to dams on gestational days 19 through 21 significantly enhances the normal late gestational rise of rat pulmonary superoxide dismutase activity. To study if Dex could act directly on lung cells to increase the activity of superoxide dismutase, rat fetal lung organ cultures were established from 21- or 22-day-old pups and maintained in serum free Waymouth 752/1 medium in 95% O2 for 72 h with and without 10 nM Dex in the medium. The cultures increased spontaneously in total superoxide dismutase activity from 17.5 +/- 3.1 to 33.5 +/- 6.2 U/mg DNA during this interval (+90%). The presence of 10 nM Dex caused an increase in enzyme activity to 40.1 +/- 9.3 U/mg DNA (+130%) demonstrating this hormone can act directly on the lung independent of the systemic metabolic consequences of corticosteroid administration. Dex decreased the rate of Cu,Zn superoxide dismutase synthesis (13.5 +/- 3.4 nmol Phe incorporated/mg DNA/h control vs 7.2 +/- 1.6, Dex) and seemed to also decrease the rate of enzyme degradation.