Ontogeny of CLCN3 chloride channel gene expression in human pulmonary epithelium.

Human fetal bronchopulmonary epithelia secrete liquid, and this chloride (Cl)-dependent process is important for normal lung growth. At the time of birth there is a maturational transition from a secretory to an absorptive phenotype. The pathways for Cl exit from the apical membrane which are required for fetal lung liquid secretion are unknown but are thought to be independent of the cystic fibrosis transmembrane conductance regulator. We determined the ontogeny of expression of the CLCN family of voltage-dependent Cl channel genes (CLCN2 through 6, K(a) and K(b)) in the human lung to identify potential pathways for pulmonary liquid secretion. Only CLCN3 and CLCN6 messenger RNA were detected by Northern analysis of fetal whole lung tissue. Ribonuclease protection assays confirmed the expression of CLCN3 and also revealed expression of CLCN2. The ontogeny of expression of these two channels was similar, peaking in midgestation and declining postnatally. In situ hybridization localized the CLCN2 and CLCN3 messages to airway and distal pulmonary epithelia and to pulmonary blood vessels. We conclude that CLCN3 is expressed in human airway epithelia and expression is developmentally regulated. The contribution of these channels to pulmonary epithelial liquid transport and lung development remains to be determined.

KGF and FGF-10 stimulate liquid secretion in human fetal lung.

During fetal life, the pulmonary epithelium secretes liquid that distends the airways and is important for normal lung growth and development. The factors regulating human fetal lung liquid secretion are poorly understood; however, recent studies in murine models show that keratinocyte growth factor (KGF, FGF-7) and fibroblast growth factor 10 (FGF-10) stimulate liquid secretion. We asked whether KGF and FGF-10 stimulate liquid secretion in human fetal lung. First trimester fetal lung explants developed dose-dependent increases in intraluminal volume in response to KGF and FGF-10. Although there were no acute changes in explant transepithelial potential difference in response to KGF (0.1-1000 ng/mL), exposure to 5-50 ng/mL KGF over 60 h depolarized transepithelial potential difference compared with controls. We used ribonuclease protection assays to quantitate the ontogeny and regulation of mRNA expression for KGF and its receptor. Both mRNA were expressed in fetal and postnatal lung. Because the promoter region of the human KGF gene contains cAMP and IL-6 response elements, we asked whether cAMP or IL-6 stimulated expression of KGF or its receptor. We have previously shown that cAMP stimulates liquid secretion in this model. Both cAMP and IL-6 significantly increased expression of KGF but not KGF receptor during a 48-h experiment. Thus, stimulation of liquid secretion in explant models by cAMP may be mediated in part by induction of KGF expression. KGF and FGF-10 may be important paracrine factors regulating liquid secretion in human fetal lung.

Keratinocyte growth factor stimulates CFTR-independent fluid secretion in the fetal lung in vitro.

Keratinocyte growth factor (KGF) caused cystic dilation of mouse fetal lung explants in vitro, markedly increasing the luminal volume of lung buds and disrupting branching morphogenesis. Effects of KGF were dose dependent, were detected within 4 h of treatment, and were blocked by cycloheximide but not by actinomycin D, indicating that de novo protein synthesis mediated the response. Effects of KGF were inhibited by bumetanide, an inhibitor of the Na(+)-K(+)-Cl- cotransporter, and ouabain, an inhibitor of the Na(+)-K(+)-ATPase. KGF stimulated fluid secretion equally in lung buds from cystic fibrosis transmembrane conductance regulators (CFTR) -/- and wild-type embryos, indicating that the effects were mediated by CFTR-independent Cl- transport. Microelectrode studies demonstrated that, whereas KGF did not acutely alter the transepithelial potential difference (PD) across the respiratory epithelium, the PD decreased while luminal volume increased during chronic exposure. KGF inhibited expression of alpha-subunit of epithelial Na+ channel (alpha-ENaC) mRNA, suggesting that KGF may inhibit Na+ absorption, which may contribute to KGF-induced fluid accumulation. KGF-induced fluid accumulation is driven by CFTR-independent Cl- transport and associated with decreased expression of alpha-ENaC.

Superoxide production by wound neutrophils. Evidence for increased activity of the NADPH oxidase.

Oxygen radical secretion by neutrophils is potentiated or "primed" by extravascular migration into wounds. To define this change in responsiveness more precisely we measured superoxide production by blood and wound neutrophils from rabbits using formylmethionyl-leucyl-phenylalanine and phorbol myristate acetate as agonists. In all experiments, the time- and dose-dependency of superoxide secretion were the same for blood and wound neutrophils. However, wound neutrophils produced significantly more superoxide. Furthermore, the cytochrome b component of the NADPH oxidase was found in greater quantities within wound neutrophils. We conclude that priming does little to alter the requirements for activating the NADPH oxidase but does significantly increase the velocity of superoxide generation. The data suggest that alterations in the assembly and function of the NADPH oxidase may contribute to enhanced superoxide secretion by wound neutrophils.

Biologic priming of neutrophils in subcutaneous wounds.

Migration of neutrophils from blood into tissue is a complex response by circulating cells to chemotactic stimulation. Previous studies of the functional changes induced by this process have produced variable results. We compared neutrophils isolated from blood and from subcutaneous wounds in rabbits using established assays for adherence, chemotaxis, superoxide anion production, and hydrogen peroxide production. No differences in adherence to biologic surfaces or chemotaxis toward activated plasma were found. However, our results confirm the observation that wound neutrophils are "primed" for increased production of oxygen radicals. Primed responses were observed for both soluble (formyl methionyl leucylphenylalanine, phorbol myristate acetate) and particulate (opsonized zymosan) stimulants. Priming was also observed for peritoneal exudate neutrophils. The data suggest that the process of extravascular migration includes priming of the superoxide generating system.

Oral filament proteins and their regulation in Tetrahymena pyriformis.

Two proteins from the Triton X-100-insoluble fraction of Tetrahymena pyriformis have been isolated and shown by immunological methods to be major components of a pervasive system of filaments localized within the oral apparatus. These proteins, OF-1 and OF-2, have apparent molecular weights (MWapp) in polyacrylamide gels of 87,000 and 80,000 D, respectively. Peptide maps obtained and the absence of immunological cross-reactivity suggest that these proteins are not closely related to each other. Indirect immunofluorescence studies on dividing cells have shown that the oral filament system forms late in the cell cycle. The filaments appeared first after the basal bodies in the oral primordium had organized into groups and the fission furrow had begun to form. Dedifferentiation of the oral filament system in the anterior (old) oral apparatus was also observed at this point in the cell cycle. Following this, the oral filament systems in both old and new oral apparatuses completed development synchronously. Proteins showing antigenic similarity to OF-1 were found in a number of other cell types. Tests with heterologous antisera failed to demonstrate a relationship between vertebrate cytoskeletal proteins and the oral filament proteins of Tetrahymena.