Fetal Rabbit Lung Research Articles

Effect of lung surfactant liposomes on the rabbit fetal lung type II cell antioxidant enzymes following prenatal dexamethasone treatment.

Prematurely born infants are at a high risk of developing neonatal respiratory distress syndrome (RDS), and it is believed that besides an insufficient surfactant (SF) system the initial breathing of O2-enriched air may in some way be responsible for this syndrome. Hyperventilation is a dominant stimulator for SF secretion but it may lead to oxidant-mediated cellular injury to type II cells. Since type II cells are the sole source of SF synthesis, the effect of ventilation support on these cells becomes important. In the present study, the changes in the antioxidant enzymes (AOEs) such as superoxide dismutase (SOD), catalase and glutathione peroxidase (GPX) in the type II cells of fetal rabbit lungs resulting from exogenous SF liposomes and steroid intervention have been investigated. The specific activities of the AOEs were found to be higher in the presence of SF liposomes in prematurely born pups whose mothers were not steroid treated (control), while such an increase was more pronounced in dexamethasone-treated groups. The results indicate that the exogenous surfactant and steroid intervention may favourably alter the AOE defences in the type II cells of fetal rabbits.

NADPH-oxidase and a hydrogen peroxide-sensitive K+ channel may function as an oxygen sensor complex in airway chemoreceptors and small cell lung carcinoma cell lines.

Pulmonary neuroepithelial bodies (NEB) are widely distributed throughout the airway mucosa of human and animal lungs. Based on the observation that NEB cells have a candidate oxygen sensor enzyme complex (NADPH oxidase) and an oxygen-sensitive K+ current, it has been suggested that NEB may function as airway chemoreceptors. Here we report that mRNAs for both the hydrogen peroxide sensitive voltage gated potassium channel subunit (KH2O2) KV3.3a and membrane components of NADPH oxidase (gp91phox and p22phox) are coexpressed in the NEB cells of fetal rabbit and neonatal human lungs. Using a microfluorometry and dihydrorhodamine 123 as a probe to assess H2O2 generation, NEB cells exhibited oxidase activity under basal conditions. The oxidase in NEB cells was significantly stimulated by exposure to phorbol esther (0.1 microM) and inhibited by diphenyliodonium (5 microM). Studies using whole-cell voltage clamp showed that the K+ current of cultured fetal rabbit NEB cells exhibited inactivating properties similar to KV3.3a transcripts expressed in Xenopus oocyte model. Exposure of NEB cells to hydrogen peroxide (H2O2, the dismuted by-product of the oxidase) under normoxia resulted in an increase of the outward K+ current indicating that H2O2 could be the transmitter modulating the O2-sensitive K+ channel. Expressed mRNAs or corresponding protein products for the NADPH oxidase membrane cytochrome b as well as mRNA encoding KV3.3a were identified in small cell lung carcinoma cell lines. The studies presented here provide strong evidence for an oxidase-O2 sensitive potassium channel molecular complex operating as an O2 sensor in NEB cells, which function as chemoreceptors in airways and in NEB related tumors. Such a complex may represent an evolutionary conserved biochemical link for a membrane bound O2-signaling mechanism proposed for other cells and life forms.

Isolation of type II epithelial cells from rabbit fetal lungs by adherence on an IgG-coated surface.

The lung is comprised of about 40 different cell types, of which only 15% are type II cells. These are the major, if not the sole, source of synthesis and secretion of lung surfactant. To date a large number of methods have been described for the isolation of pure populations of type II cells using a wide variety of techniques, but most of these have employed differential centrifugation methods and have used adult rodents. The present study reports the isolation of type II cells from fetal rabbit lungs by the immunoglobin G plating method. Pure populations of fetal type II cells in high yield and with good viability were obtained by the procedure for the first time from rabbit fetal tissue.

Regulation of the β2 subunit chain of laminin in developing rabbit fetal lung tissue

Laminins are a family of basement membrane-associated heterotrimeric proteins that are important in mediating the growth, migration, and differentiation of a variety of cell types. The β2 subunit chain is a component of several laminin isoforms, e.g., laminin-3, laminin-4, laminin-7, and possibly other, as yet uncharacterized laminin isoforms. Utilizing monoclonal antibodies directed against the β2 subunit chain of laminin, we detected this protein in fetal, neonatal, and adult lung tissues. The relative amount of laminin β2 subunit chain in fetal lung tissue increased as gestation proceeded, reaching its peak around the time of alveolar type II cell differentiation in the rabbit. The laminin β2 subunit chain was localized in early gestational age rabbit fetal lung tissue primarily in basement membranes of prealveolar ducts, airways, and smooth muscle cells of airways and arterial blood vessels. A rabbit laminin β2 cDNA was generated using RT-PCR and utilized as a probe in northern blot analysis to characterize the levels of laminin β2 mRNA in developing rabbit lung tissue. Similar to the pattern of laminin β2 protein induction observed in fetal lung tissue, laminin β2 mRNA levels were maximal late in gestation. Utilizing a laminin β2 chain cRNA probe and in situ hybridization, we detected laminin β2 mRNA in the epithelial cells of prealveolar ducts, the alveolar wall, and airways, as well as in connective tissue cells, and the smooth muscle cells of airways and blood vessels in fetal and adult lung tissues. In addition, using an in vitro explant model, we determined that alveolar type II cells are capable of synthesizing laminin β2 subunit mRNA and depositing this laminin subunit chain in the basement membrane beneath type II cells. The results of this study are suggestive that the laminin β2 chain may be involved in alveolar epithelial cell differentiation.

Cytokines and production of surfactant components

The production of pulmonary surfactant, a complex of lipids and proteins that reduces surface tension at the alveolar air-liquid interface, is developmentally regulated. Several hormones, most notably glucocorticoids, are known to accelerate maturation of the surfactant system. Cytokines are polypeptides that act mostly in a paracrine fashion and possess a wide spectrum of activities on multiple types of cells. Many cytokines are produced by different lung cells a various stages of fetal development or under pathological conditions affecting the fetus. In addition, cytokines present in amniotic fluid or in the blood stream may reach the fetal lungs. Some cytokines, including epidermal growth factor, transforming growth factor-alpha, and interferon-gamma have been shown to stimulate the production of surfactant components. On the other hand, tumor necrosis factor and transforming growth factor-beta downregulate the production of surfactant lipids and proteins. We have recently shown that the proinflammatory cytokine interleukin-1 (IL-I) enhances the expression of surfactant protein A (SP-A) in fetal rabbit lung explants. In addition, injection of IL-I into the amniotic fluid of fetal rabbits enhances the expression of surfactant proteins and improves the lung compliance of preterm animals. Preterm delivery is often associated with subclinical intraamniotic infection. In these cases, amniotic fluid concentrations of IL-I are often elevated. We propose that this cytokine accelerates maturation of the surfactant system in fetal lungs and thus prepares the fetus for extrauterine life.

Characterization of the cyclic adenosine 3',5'-monophosphate response element of the rabbit surfactant protein-A gene: evidence for transactivators distinct from CREB/ATF family members.

Surfactant protein-A (SP-A) gene transcription in fetal lung explants is stimulated by factors that increase intracellular cAMP. In transfected type II cells, expression of fusion genes containing 991 bp of 5'-flanking DNA from the rabbit SP-A gene linked to the human GH gene as reporter is stimulated more than 20-fold by cAMP. Mutagenesis of a putative cAMP responsive element (CRE) located -261 bp upstream of the SP-A transcription initiation site to a sequence known not to bind the transcription factor CRE-binding protein (CREB) caused a marked decrease in basal and cAMP-inducible reporter gene expression. This element, termed CREsp-a (TGACCTCA), differs by one nucleotide from a palindromic CRE (CREpal, TGACGTCA), which is known to bind CREB as a homodimer. In the present study, we found that mutagenesis of CREsp-a to CREpal also caused a marked decrease in basal and cAMP-induced fusion gene expression. The findings of competitive electrophoretic mobility shift assays (EMSA) using fetal rabbit lung nuclear extracts suggest that different protein complexes bind CREsp-a and CREpal. By UV cross-linking analysis, an approximately 43-kilodalton protein complex was found to interact both with CREsp-a and CREpal; however, purified CREB was ineffective in binding CREsp-a but did bind CREpal. In EMSA using fetal rabbit lung nuclear proteins, antibodies directed against CREB, CRE modulator (CREM), and activating transcription factor-1 (ATF-1) failed to supershift the complex of proteins bound to CREsp-a; whereas, a supershift was evident using CREpal as a probe. Moreover, in competition EMSA using radiolabeled CREsp-a and fetal rabbit lung nuclear proteins, a purified basic leucine zipper (bLZ) polypeptide failed to compete for binding. By contrast, the bLZ polypeptide competed effectively with CREpal for lung nuclear protein binding. This finding suggests that leucine zipper transcription factors do not bind CREsp-a. Additionally, expression of a CREsp-a:HIS3 fusion gene in yeast was unaffected either by CREB or bLZ polypeptides fused to the GAL4 activation domain. By contrast, HIS3 expression was markedly induced both by CREB and bLZ fusion proteins in a CREpal:HIS3 yeast strain. By competition EMSA using mutagenized CREsp-a oligonucleotides, the critical protein-binding nucleotides in CREsp-a were found to constitute a hexameric element, TGACCT, which corresponds to a binding site for members of the steroid receptor superfamily. Since the TGACCT motif is present in the SP-A gene as a single site, we propose that a unique orphan member of the steroid receptor superfamily may bind to this element.

Ca +2-phosphatidylserine-dependent protein kinase C activfty in fetal, neonatal and adult rabbit lung and isolated lamellar bodies

Evidence indicates that Ca +2-phosphatidylserine-dependent protein kinase C (PKC) is involved in regulation of surfactant secretion. This study was done to examine PKC activity in lung as surfactant synthesis and secretion is initiated, and at birth and to compare these enzyme levels with those in the adult lung. NZW rabbits were used. Fetal and adult lungs were fractionated into subcellular compartments including a lamellar body fraction, which represents intracellular surfactant. The time course for microsomal enzyme activity was compared between 24th gestational day and adult rabbit lung. The reactivity appeared similar in both fractions. PKC specific activity displayed a prominent peak between the 27th and 30th gestational days in total homogenate and lamellar bodies. Specific activity was also high in nuclear, mitochondrial and microsomal fractions the day prior to birth. Adult levels were similar or higher. Total PKC activity was high during late gestation but declined sharply the day prior to birth. A marked increase was present on the first postnatal day. In contrast lamellar bodies displayed a peak in activity between the 27th and 30th gestational days followed by a decline to adult levels. Delipidation of lamellar body fraction indicated that the high enzyme activity in this fraction on the 27th gestational day was not artifactual. The changes observed in PKC in fetal, neonatal and adult lung indicate this enzyme activity changes in lung during the period of onset of surfactant synthesis and secretion during late gestation and may be associated with lamellar bodies, in 27th gestational day fetal lung.

Characterization of alpha 1, beta 1, and gamma 1 laminin subunits during rabbit fetal lung development.

Laminin-1 is an extracellular matrix protein composed of three polypeptide chains that are designated alpha 1, beta 1, and gamma 1. We investigated the expression of laminin alpha 1, beta 1, and gamma 1 subunit chains during several stages of rabbit fetal lung development. Utilizing polyclonal antibodies directed against human placental laminin and immunoblot analysis, we found that the highest levels of laminin alpha 1, beta 1, and gamma 1 subunit chains in the fetal lung were present on day 26 of gestation (term = 31 days), coincident with the initiation of alveolar epithelial cell differentiation. Levels of the laminin chains were approximately five times higher in fetal lung at day 26 of gestation than in adult lung tissue. Different temporal patterns of laminin alpha 1, beta 1, and gamma 1 subunit chain expression were observed, data suggestive that the chains are independently regulated during lung development. Laminin was localized to the basement membranes of bronchi, bronchioles, prealveolar ducts, and blood vessels in fetal lung tissue, as shown by immunostaining with polyclonal laminin antibodies. A similar staining pattern was observed in adult lung tissue, but the alveolar wall was also stained. Laminin was also observed surrounding a few mesenchymal cells in fetal lung on day 19 of gestation; the number of positive mesenchymal cells increased with lung development. Laminin alpha 1 subunit chains, detected using a monoclonal antibody, were present in the basement membranes of bronchi, bronchioles, prealveolar ducts, and blood vessels in fetal lung tissue. No laminin alpha 1 chain staining was observed in the mesenchyme of early fetal lung tissue. Using a monoclonal antibody, laminin beta 1 subunit chains were immunolocalized in the basement membranes of bronchi, bronchioles, in prealveolar ducts, and surrounding some mesenchymal cells in fetal lung tissue. Laminin alpha 1 and beta 1 subunit chains in adult lung tissue were present in basement membranes of airways, blood vessels, and alveoli. Thus, changes in the localization and accumulation of laminin near the time of alveolar type I and type II epithelial cell differentiation suggest that laminin may play a role in mediating the differentiation of these cell types during rabbit fetal lung development.

Transcription and mRNA stability regulate developmental and hormonal expression of rabbit surfactant protein B gene

Surfactant protein B (SP-B), a hydrophobic protein of pulmonary surfactant, is essential for the surface tension-reducing properties of surfactant. In the present study, we isolated and characterized cDNAs encoding rabbit SP-B, and used transcription run-on assays and Northern blot analysis to investigate the role of transcriptional and posttranscriptional mechanisms in the developmental and cAMP and dexamethasone induction of SP-B mRNA. We found two forms of SP-B cDNAs that differed by an insertion of 69 nucleotides in the 3' untranslated regions. We found that transcription across the SP-B gene is nonequimolar and the 3' end of the gene has high levels of antisense transcription. SP-B gene transcription and SP-B mRNA levels increased during fetal lung development. However, increased SP-B mRNA levels could not be accounted for primarily on the basis of increased transcription. These results suggested that enhanced SP-B gene transcription and enhanced SP-B mRNA stability mediate developmental induction of SP-B gene. In rabbit fetal lung in vitro, both dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) and dexamethasone increased SP-B mRNA levels. DBcAMP-dependent increase in SP-B mRNA levels resulted from increased SP-B gene transcription, whereas dexamethasone-dependent increase resulted from combined effects on increased SP-B gene transcription and SP-B mRNA stability. In tissues treated with dexamethasone the half-life (t1/2) of SP-B mRNA increased > 2.5-fold (t1/2 control = 9 h; t1/2 dex-treated = 25 h). These data show that both transcription and mRNA stability regulate induction of SP-B gene expression during fetal lung development and by cAMP and dexamethasone in fetal lung in vitro.

Cell-Specific Expression of Fibronectin in Adult and Developing Rabbit Lung

Fibronectin (FN), a glycoprotein component of the extracellular matrix, plays a role in tissue morphogenesis and tissue-specific differentiation through its effects on cell adhesion, cell shape, and cytoskeletal organization. Immunohistochemistry has been used to show that during lung development FN deposition changes, yet the cell-specific sites of pulmonary FN synthesis have not been determined. Because cellular FN synthesis is reflected by FN mRNA abundance, we performed in situ hybridizations to identify pulmonary tissue with the capacity to synthesize FN. Both in situ mRNA hybridization and immunohistochemical staining were performed on tissue sections from lungs of adults and late gestation fetal and neonatal rabbits. In adults, FN transcripts and immunostaining were clearly seen in endothelial cells, smooth muscle cells, and chondrocytes. During lung development, FN transcripts were virtually ubiquitous except in airway epithelium. There was a gradual decrease in FN mRNA abundance with advancing fetal age, but low levels of FN mRNA persisted in neonatal and adult lungs. In contrast, parenchymal immunostaining increased throughout fetal development and remained elevated in the newborn. FN immunostaining was lower in adult lung. In all tissues examined, airway epithelial cells contained no FN transcripts above background. However, immunostaining was detected in airway basement membrane zones and on luminal surfaces of some epithelial cells. The lack of FN transcripts in airway epithelial cells suggests that FN synthesis does not normally occur in this cell type and that its associated FN immunostaining is from another source. The colocalization of FN mRNA and protein in pulmonary endothelial cells, smooth muscle cells, and chondrocytes in adults strongly suggests that these cells are sites of FN synthesis.

Thyrotrophin-releasing hormone (TRH) and lung maturation.

Clinical trials of thyrotrophin-releasing hormone (TRH) in conjunction with antepartum glucocorticoid treatment in the prevention of respiratory distress syndrome is based on experimental evidence that fetal lung maturation is accelerated by exposure to raised concentrations of triiodothyronine (T3) in fetal plasma. Studies of fetal rat and rabbit lung in vitro show an inconsistent increase in surfactant synthesis in response to T3 and potentiation of the response to corticosteroid. Experiments with fetal rodents in vivo are difficult to interpret because of confounding effects of the procedures and the responses to T3 are variable. In fetal sheep, very high concentrations of T3 are without effect on lung maturation. These observations suggest that the action of TRH on the lung may be mediated at least in part by one of the numerous, non-hormonal pathways known to be stimulated by TRH, particularly the autonomic nervous system. Experiments in rats and sheep lend support to this possibility. It is concluded that available evidence is inadequate to determine the mechanism of action of TRH.

Immunocharacterization and developmental regulation of rabbit lung calcium-dependent phospholipid-binding proteins

The purpose of this work was to use the immunoblotting methods to study the 36 kDa calcium-dependent phospholipid-binding protein (PLBP) in the adult and fetal rabbit lungs to gain insight into the significance of this protein in lung development. The identity of the 36 kDa PLBP and the antigen specificity of the antiserum raised against this protein in the guinea pig were tested against known annexins and antibodies to the annexins. Our results showed that the rabbit lung 36 kDa PLBP contained only one protein which cross-reacted with antibodies against annexin l. However, the 36 kDa PLBP was slightly smaller (36 vs. 37 kDa) and more acidic (pI 6.0 vs. 6.9) than the recombinant human annexin 1. The guinea pig antiserum only reacted with annexin 1, not with any of the other annexins tested. In the cytosolic fractions of the lung and the alveolar epithelial type II cells, and in the lung lavage fluid, the 36 kDa PLBP was by far the most prominent protein with minor presence of a 33 kDa protein recognized by the guinea pig antiserum. The amount of the 36 kDa PLBP of type II cells was 55% higher than that in the lung tissue and 2.6-times higher than that in the lung lavage (9.3 ± 0.62, 6.0 ± 0.31 and 3.6 ± 0.04 μg/ mg protein, respectively). The 36 kDa PLBP appeared in the fetal rabbit lungs as early as at 21 days gestation and increased 2-fold to reach the adult level at 27 days gestation (term 31 days). The high content of PLBP in type II cells and the rapid increase in this protein in the fetal lungs at late gestations suggest an important role of the 36 kDa PLBP in lung development and surfactant biogenesis.

Influence of protein kinase C activation by 4β-phorbol ester or 1-oleoyl-2-acetylglycerol on disaturated phosphatidylcholine synthesis and secretion, and protein phosphorylation in differentiating fetal rabbit type II alveolar cells

Undifferentiated type II alveolar cells were isolated from the fetal rabbit lung on the 24th gestational day, grown in vitro for 2–3 days, and used to test the hypothesis that activation of protein kinase C by 4β-phorbol ester (TPA) or the diacylglycerol analogue, sn-1-oleoyl-2-acetylglycerol (OAG), stimulates disaturated phosphatidylcholine (DSPC) synthesis and secretion. To measure secretion, cells were prelabelled with [ 3H]choline in serum-free medium or medium with 10% carbon-stripped fetal bovine serum for 24 h. The radiolabel was removed and TPA (10 −6–10 −9 M) or OAG (125, 250 or 500 μM) was incubated with the cells for 2 h. The medium was removed and filtered. Fresh medium with the same compound was added for an additional 16 h. To measure synthesis, cells were incubated with [ 3H]choline and concurrently TPA or OAG was added. Cells were removed at 2 or 18 h. After 2 h at concentrations of 10 −8 M, TPA augmented the release of 3H-labelled DSPC, the major component of the surfactant, by cells incubated in serum-free medium. In the presence of carbon-stripped fetal bovine serum, TPA (10 −7 and 10 −6 M) induced release of DSPC. The incorporation of [ 3H]choline into intracellular DSPC was increased after 2 or 18 h in fetal alveolar cells exposed to TPA at 10 −9 M or higher. OAG also significantly stimulated the release of labelled DSPC after 2 h at all concentrations tested. In contrast, OAG-exposed cells displayed a reduction of [ 3H]choline incorporation into cellular DSPC. Characterization of radioactive material released by prelabelled fetal type II cells showed that phorbol ester stimulation increased the release of material which co-migrated with adult rabbit lung lamellar bodies on a sucrose gradient. Electrophoretic examination of [γ- 32P]ATP phosphorylation sites in fetal type II cells showed that TPA and OAG induced an increase in phosphorylation of a group of proteins with apparent molecular masses of 45, 50 and 55 kDa. Addition of phosphatidylserine to the incubations produced substantial increases in the phosphorylation of these proteins, particularly in the presence of TPA. Fetal type II cells also displayed a phosphorylation product with an apparent molecular mass of 97 kDa. This protein as well as two high-molecular-mass products appeared to be particular to cells incubated with TPA plus phosphatidylserine and may in part account for the different action of TPA compared to OAG with regard to synthesis and secretion of DSPC by the fetal type II cells.

Oxygen sensing in airway chemoreceptors.

Pulmonary neuroepithelial bodies, composed of innervated clusters of amine- and peptide-containing cells, are widely distributed throughout the airway mucosa of human and animal lungs. Structurally, neuroepithelial bodies resemble chemoreceptors (such as carotid body, taste buds) and are thought to function as hypoxia sensitive airway sensors. Evidence for this is indirect, however, and the mechanism of oxygen sensing by these cells is unknown. Here we culture neuroepithelial bodies isolated from rabbit fetal lungs and identify voltage-activated potassium, calcium and sodium currents using the whole-cell patch clamp technique. Upon exposure to hypoxia there is a reversible reduction (25-30%) in the outward potassium current, with no change in inward currents. In addition, we demonstrate the expression of an oxygen-binding protein (b-cytochrome, NADPH oxidase) on the plasma membrane of these cells. The identification of an oxygen-sensing mechanism (namely the presence of an O2-sensitive potassium channel coupled to an O2 sensor protein) in the cells of pulmonary neuroepithelial bodies indicates that they are transducers of the hypoxia stimulus and hence may function as airway chemoreceptors in the regulation of respiration.

Exposure of rabbit fetal lung to glucocorticoids in vitro dose not enhance transcription of the gene encoding pulmonary surfactant-associated protein-B (SP-B)

We have investigated the ontogeny and hormonal regulation of both synthesis rates and cellular accumulation of the mRNA for surfactant-associated protein B (SP-B) in rabbit fetal lung. The developmental pattern for SP-B mRNA synthesis increased as a function of gestational age and paralleled that for SP-B mRNA levels except on days 22–26 of gestation where relatively higher levels of gene transcription were observed. Time-course studies with explants from 26- and 30-day fetal lung maintained in culture revealed a gradual increase in mRNA levels and a much smaller increase in gene transcription relative to adult values. Within 48 h of exposure of 26-day expiants to dexamethasone at 10 −8 M there was a rapid increase in SP-B mRNA levels to 7-fold adult levels. A similar overall although somewhat slower and attenuated pattern was observed with 30-day explants. Dexamethasone at 10 −8 M had no effect on SP-B gene transcription with explants of either gestational age. We conclude that the major effect of dexamethasone treatment in vitro on SP-B mRNA levels appears to be post-transcriptional and there are small but distinct differences in the effects of glucocorticoids on SP-B mRNA levels with explant cultures from early and late stages of fetal lung maturation.

Genomic elements involved in transcriptional regulation of the rabbit surfactant protein-A gene.

Expression of the surfactant protein-A (SP-A) gene is lung specific and is developmentally and hormonally regulated in fetal lung tissue. Cyclic AMP analogs and glucocorticoids stimulate transcriptional activity of the SP-A gene in fetal rabbit lung tissue in culture; an additive effect is observed when the agents are added in combination. To analyze the genomic regions that regulate SP-A promoter activity, fusion genes comprised of -1766, -991, -378, and -47 basepairs (bp) of DNA flanking the 5'-end of the SP-A gene, the transcription initiation site, and 20 bp of exon I linked to the human GH (hGH) structural gene were subcloned into a replication-defective human adenovirus vector and transfected into differentiated rat type II cells in primary culture. SP-A promoter activity was analyzed by RIA of hGH protein in the culture medium. In type II cells transfected with SP-A-1766:hGH and SP-A-991:hGH fusion genes, hGH production was induced 30- to 40-fold by (Bu)2AMP (Bt2cAMP; 1 mM). When type II cells were transfected with the SP-A-378:hGH fusion gene, basal levels of expression were reduced by more than 50%; however, Bt2cAMP caused an 11-fold increase in hGH production. In type II cells transfected with the SP-A-47:hGH fusion gene, basal levels of hGH production were essentially undetectable, and no stimulatory effect of Bt2cAMP was apparent. Cyclic AMP stimulation of expression of the SP-A-1766:hGH, SP-A-991:hGH, and SP-A-378:hGH fusion genes was limited to type II pneumonocytes in primary culture and was absent in two lung adenocarcinoma cell lines (NCl-H358 and A549), which do not express SP-A, and in cAMP-responsive adrenal Y1 cells. Mutations of a putative cAMP-responsive element (TGACCTCA) at -261 bp revealed its functional importance in mediating cAMP regulation of SP-A gene expression. Unexpectedly, dexamethasone (Dex; 10(-7) M) antagonized the stimulatory effect of Bt2cAMP on expression of SP-A:hGH fusion genes containing from -378 to -1766 bp of 5'-flanking DNA as well as that of a fusion gene construct containing -991 bp of 5'-flanking DNA, the first exon, the first intron, and 20 bp of the second exon (SP-A-991+670:hGH). The inhibitory effect of Dex was dose dependent, with half-maximal inhibition occurring at a Dex concentration of 8 x 10(-10) M. The inhibitory effect of Dex was prevented by the glucocorticoid receptor antagonist RU486.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of a novel serum and growth factor-inducible gene in vascular smooth muscle cells

We have used subtraction cloning to isolate a cDNA (PS4) that identified a serum-inducible mRNA of 1.9 kilobases in rabbit vascular smooth muscle cells. DNA sequence analysis revealed one major open reading frame encoding a 9,442 M(r) protein. Comparison of the DNA as well as the putative protein sequence with various data bases revealed no homology with other sequences. In vitro translation of synthesized PS4 mRNA generated a major polypeptide of 12 kDa. Serum stimulation of quiescent smooth muscle cells in culture induced a rapid increase in the level of PS4 mRNA. Expression of this message was detected by 1 h, peaked at approximately 4 h, and became undetectable by 12 h. The induction of PS4 by serum was completely blocked by cycloheximide, indicating its expression required prior protein synthesis. Epidermal growth factor, acidic fibroblast growth factor, and transforming growth factor-beta 1 also induced a strong increase in PS4 expression. By contrast, platelet-derived growth factor-BB was only able to mildly stimulate the level of PS4 mRNA and insulin-like growth factor-I was unable to enhance PS4 expression. There was a high level of PS4 mRNA in rabbit fetal muscle, esophagus, kidney, and lung, a low level in fetal aorta and heart, and an undetectable level in fetal liver, brain, as well as, in the placenta. The expression of PS4 in the corresponding adult tissues was low or undetectable. Our analysis indicate that PS4 expression is developmentally regulated and tightly controlled by growth factors, suggesting this novel gene has a role in cell growth and differentiation.

Influence of Strain on [3H]thymidine Incorporation, Surfactant-related Phospholipid Synthesis, and cAMP Levels in Fetal Type II Alveolar Cells

Episodic fetal breathing movements occur in utero; however, their purpose is not known. While some evidence indicates these movements produce distention of the fetal lung, the effects of eliminating the movements on lung development are not clear. Using preparations of the surfactant-producing fetal rabbit lung type II cells, we have examined the hypothesis that distention is involved in maturation of these cells as determined by replication rate and phospholipid synthesis. Differentiating type II cells were isolated from fetal rabbit lungs on gestational day 24. After allowing 24 h for the cells to attach, distention (10%) was applied using a Flexercell apparatus at a rate of 3 s of strain and 57 s of rest for 24 or 48 h. Cells were concurrently incubated with [3H]thymidine. Autoradiography showed that [3H]thymidine-labeled nuclei of fetal type II cells and fibroblasts could be identified. Strain significantly increased incorporation of the radioisotope into DNA. In the remaining studies, cells were grown to confluence prior to use. Cell viability after straining (3 or 50 cycles per minute [cpm] for 48 h) was assessed by incubating with diacetyl fluorescein/ethidium bromide and viewing by fluorescence microscopy. Straining did not alter cellular viability. Lactate dehydrogenase was also assayed in the culture medium. Straining did not cause release of lactate dehydrogenase. Ultrastructural examination showed typical cellular appearance in the strained cells. Extracellular lamellar body material was also observed. The incorporation of [3H]choline (3 or 50 cpm) into cellular phospholipids was measured under static conditions or under distention. [3H]choline-labeling of phosphatidylcholine and disaturated phosphatidylcholine was significantly increased by distention at either 3 or 50 cpm.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgen regulation of epidermal growth factor receptor binding activity during fetal rabbit lung development.

Fetal lung development progresses in a sex-specific manner with male fetuses exhibiting delayed maturation. Androgens, both exogenous and endogenous, inhibit while epidermal growth factor (EGF) enhances fetal lung development. We hypothesized that one mechanism responsible for the delay in male fetal lung development is an androgen-induced delay in EGF receptor binding activity. We measured EGF binding in sex-specific fetal rabbit lung plasma membranes isolated from control fetuses (days 21, 23, 25, 27, 29, and 30 of gestation) and from androgen-treated fetuses (days 21, 23, and 27 of gestation) that had been continuously exposed in vivo to exogenous 5 alpha-dihydrotestosterone from day 12 through 27 of gestation. Specific binding of EGF was significantly lower in male than in female fetal lung tissue isolated from controls at day 21 of gestation. Scatchard analysis revealed that this decrease in EGF binding was associated with decreased EGF receptor density without any significant change in affinity. Prenatal exogenous androgen treatment led to decreased EGF binding in fetal rabbit lung tissue from both sexes secondary to a decrease in EGF receptor density. These findings suggest that one mechanism responsible for the delay in male fetal lung maturation is an androgen-induced delay in EGF receptor binding activity during fetal lung development.

Cystic Fibrosis Gene and Protein Expression during Fetal Lung Development

The cystic fibrosis transmembrane conductance regulator (CFTR) transports Cl- in the apical membrane of secretory epithelial cells. Normal lung development is critically dependent on active Cl- secretion by developing airway epithelia. In this study, the polymerase chain reaction (PCR) was used to detect CFTR mRNA expression in rabbit lung at different stages of development. Using nested amplification in exons 7 and 11, a 494-bp fragment was isolated from cDNA prepared from lungs obtained at gestational days 22 (pseudoglandular) and 29 (terminal sac). Nucleotide sequencing verified the identification of rabbit CFTR sequences. At day 22 of gestation, CFTR was detected in fetal liver, kidney, heart, intestine, and brain. CFTR could not be amplified from adult rabbit brain. CFTR protein was detected using a polyclonal antibody raised in chickens against a synthetic peptide from the R domain conjugated to thyroglobulin. Rabbit CFTR migrated as a 165-kD protein on 5% sodium dodecyl sulfate polyacrylamide gels when detected either by chicken anti-R IgG or a previously characterized rabbit anti-R antibody. In frozen sections of fetal rabbit lung at day 22 of gestation, CFTR was localized to the membrane regions of primitive tubular epithelial cells and absent from surrounding connective tissue. At day 29, CFTR was concentrated in the apical region of bronchiolar epithelial cells and absent from alveoli and vessels. These results suggest that CFTR gene expression begins very early in lung development and, with differentiation of the airways, becomes confined to differentiated bronchiolar epithelium, the presumed site for cAMP-mediated Cl- secretion.