bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytokine signaling in lung: transforming growth factor-beta secretion by lung fibroblasts
Authors: Kelley J. , Fabisiak J. P. , Hawes K. , Absher M.
Published: December 2017

Control of growth and phenotypic expression of interstitial fibroblasts is a critical determinant of lung architecture and physiology during processes of growth and remodeling. We examined the ability of lung fibroblasts to produce transforming growth factor-beta (TGF-beta), a cytokine that is known to modulate proliferation and phenotypic expression of mesenchymal cells. Cultures of fibroblasts isolated from rat lungs spontaneously secrete TGF-beta as measured in the standard bioassay of anchorage-independent growth of normal rat kidney (NRK) cells in soft agar. Rat lung fibroblasts secrete TGF-beta in an inactive precursor form. Fibroblasts cultured from adult and fetal rat lungs produced comparable amounts of TGF-beta. The ability of lung fibroblast supernatant fluids to induce colony formation in soft agar could be completely neutralized by preincubation of samples with anti-TGF-beta immunoglobulin (Ig). Anti-platelet-derived growth factor IgG had no effect on anchorage-independent growth of NRK cells driven by rat fibroblast culture supernatant samples. These results indicate that TGF-beta does not require the presence of and interaction with secondary cytokines for its activity. In contrast to the results obtained with rat cells, neither human fetal nor adult lung fibroblasts secreted detectable amount of active TGF-beta or its inactive precursor. This was not due to the presence of TGF-beta inhibitors in fibroblast culture media, because the addition of purified porcine TGF-beta to conditioned medium from human lung fibroblast cultures yielded the expected increase in NRK cell growth in soft agar. These results point to differing cytokine control patterns in the lungs of the two species.
The molecular biology of mammalian arachidonic acid metabolism
Authors: Sigal E.
Published: December 2017

The metabolism of arachidonic acid by cyclooxygenase and lipoxygenase enzymes results in a wide range of oxidized products with potent biological activities. These metabolites, which include the prostaglandins and leukotrienes, have been implicated in the pathogenesis of a variety of inflammatory diseases. Research over the last decade has focused primarily on the elucidation of the chemical structure of the metabolites and their biological effects in vitro and in vivo. Recently, research on the enzymes that produce these bioactive metabolites through oxidization of arachidonic acid has intensified. Recombinant DNA techniques have enabled investigators to determine the nucleotide sequences for several of the enzymes in the arachidonic acid cascade. The resulting cDNAs are now being used to further investigate the biochemical and biological features of arachidonic acid metabolism. The purpose of this paper is to review how the cDNAs for these enzymes were obtained, what information they convey, and how they are being applied in current research.
Protein kinase C activation allows pulmonary artery smooth muscle cells to proliferate to hypoxia
Authors: Dempsey E. C. , McMurtry I. F. , O'Brien R. F.
Published: December 2017

Pulmonary artery (PA) smooth muscle cell (SMC) proliferation occurs with hypoxic pulmonary hypertension in vivo. However, proliferation of cultured PA SMC to hypoxia has not been demonstrated, and thus the mechanism by which these cells respond to hypoxia is unknown. Because protein kinase C (PKC) plays a role in intracellular transduction of proliferative signals, we asked whether PKC activation 1) causes proliferation of bovine PA SMC and 2) is important in PA SMC proliferative response to hypoxia. By measuring [3H]thymidine incorporation and cell counts, we found that quiescent PA SMC from four different cows proliferated with the PKC activator, phorbol 12-myristate 13-acetate (PMA), in a concentration-dependent manner. The proliferation was blocked with a PKC inhibitor, dihydrosphingosine, or by downregulating SMC PKC. We tested whether “priming“ PA SMC by PKC activation was required for in vitro SMC proliferative response to hypoxia. Each SMC population was treated with PMA and then exposed for 24 h to 20, 10, 7, 3 or 0% O2. These cells proliferated with hypoxia reaching a peak response at 3% O2. The magnitude of the response to PMA and hypoxia was different for each cell population tested. No hypoxic proliferation occurred in control cells (no PMA). Dihydrosphingosine blocked the hypoxic response to the same extent that it inhibited the initial PMA conditioning stimulus. PKC-downregulated PA SMC did not proliferate to PMA or to subsequent hypoxia. The hypoxic response was not due to a reduction in O2 radical-mediated antiproliferative effect; rather, the PMA-primed cells seemed to “acquire” the ability to directly sense hypoxia and proliferate. In summary, PKC activation caused proliferation of PA SMC in vitro and allowed an additional proliferative response to hypoxia. Activation of PKC may be a requisite step for PA SMC to respond directly to hypoxia.
Dexamethasone in vivo raises surfactant protein B mRNA in alveolar and bronchiolar epithelium
Authors: Phelps D. S. , Floros J.
Published: December 2017

Glucocorticoids enhance the rate of maturation of the fetal lung, in part, by increasing production of surfactant by type II cells. We have examined the effect of in vivo dexamethasone treatment on mRNA levels for the hydrophobic surfactant protein, SP-B, in rat lung and compared the data to similar measurements of SP-A mRNA. Rats of known gestational and postnatal ages were injected intraperitoneally with dexamethasone (2 mg/kg) and killed after 24 h. SP-B mRNA levels in the lungs were determined by RNA blotting using rat SP-A and SP-B cDNA probes. SP-B mRNA levels increased 5.2-fold when 163 pairs of animals of all ages were examined. SP-B mRNA increases in fetuses (107 pairs) and postnatal animals (56 pairs) were 8.4- and 2.1-fold, respectively (P less than 0.0001). The high levels of stimulation in fetal lungs were largely due to high increases on gestational days 18 and 19 when the control levels are very low. There were no detectable gender differences in the population as a whole. Tissue in situ hybridization showed that type II cells, as well as some bronchiolar cells, responded to dexamethasone treatment throughout life. The significance and the reasons for the presence and hormonal responsiveness of SP-B in the bronchiolar epithelium are intriguing and warrant further investigation.
Surfactant apoprotein in adult rat lung compartments is increased by dexamethasone
Authors: Young S. L. , Ho Y. S. , Silbajoris R. A.
Published: December 2017

The distribution of the major surfactant apoprotein (SP-A) in adult rat lung was determined in order to gain insight into its metabolism, including packaging of SP-A into lamellar bodies. The effect of glucocorticoid treatment on surfactant apoprotein was studied to test whether regulation of surfactant apoprotein genes, which has been described for the fetal lung, can be demonstrated in the adult animal. We measured the amounts of immunoreactive SP-A in several lung tissue compartments and lavage fractions from control animals and from the lungs of rats given dexamethasone for 1 wk. Protein and phospholipids were measured, SP-A was quantitated with a noncompetitive enzyme-linked immunoabsorbent assay (ELISA) and SP-A, SP-B, and SP-C mRNAs were estimated by Northern blotting. We found an 85-fold concentration of SP-A in a lamellar body-rich fraction compared with lung tissue homogenate and we calculated that as much as one-half of all the tissue SP-A might be accounted for by a lamellar body pool. After 1 wk of dexamethasone treatment, there was an increase in adult rat lung SP-A, SP-B, and SP-C mRNA and a substantial increase in tissue and lavage fluid immunoreactive SP-A pools. Lamellar body fraction SP-A content per lung was 1.4-fold higher after dexamethasone, and there was a fivefold increase in the lavage SP-A pool, much of which was inseparable from the alveolar macrophages. We conclude that SP-A is concentrated in the lamellar bodies of type II cells, that dexamethasone treatment increased all surfactant mRNAs, and that it increased SP-A content in adult rat lung.
Quantitative approaches for studying gene expression
Authors: Wiesner R. J. , Zak R.
Published: December 2017

The methods currently available for measuring mRNAs and proteins are reviewed, with a special emphasis on their application to physiological questions. The article focuses on the quantitative determination of cellular contents, but also on assessment of rates of synthesis and degradation, and turnover.
Inflammation and cell-cell interactions in airway hyperresponsiveness
Authors: Leff A. R. , Hamann K. J. , Wegner C. D.
Published: December 2017

Airway hyperresponsiveness results from the conversion of normally reactive airways to a state of augmented responsiveness to constrictor stimuli. Although the mechanism accounting for the induction of airway hyperresponsiveness remains elusive, recent investigations have suggested that inflammation may be a sine qua non for human asthma. Numerous experimental models have demonstrated the necessity of circulating granulocytes as mediators of augmented bronchoconstriction during immune challenge. It is not known how granulocytes are targeted for selective migration to the conducting airways of the lung during hyperresponsive states; however, recent evidence implicates the upregulation of granulocyte adhesion molecules on both the endothelial and epithelial surfaces of the airway. There is evidence that during migration diapedesis, granulocytes interact with epithelial and endothelial cells to produce regionally secreted mediators that upregulate the responsiveness of adjacent airway smooth muscle and/or cause lumenal edema, thus augmenting the effect of constrictor stimuli. Most evidence suggests that the eosinophil is the most important granulocyte in these responses and that eosinophilic infiltration and activation may account for the unique, spasmodic, and cyclic nature of hyperreactive airways. The molecular biology of the eosinophil granule proteins has characterized four distinct substances, each of which exerts potential cytotoxic effects on airway epithelium by different mechanism. In addition, at least one of these proteins, the major basic protein, appears to cause direct, noncytotoxic stimulation of epithelial secretion that upregulates nonspecifically the response of airway smooth muscle to contractile stimuli. The recognition of inflammation as the essential component to airway hyperresponsiveness provides a fresh approach to a difficult problem and suggests a host of novel therapies for human asthma.
Neuroepithelial bodies stimulate proliferation of airway epithelium in fetal hamster lung
Authors: Hoyt R. F. , McNelly N. A. , McDowell E. M. , Sorokin S. P.
Published: December 2017

Autoradiographs were prepared from lungs of a newborn Syrian golden hamster exposed continuously to [3H]thymidine throughout the final 4.5 days of gestation. Silver grains were counted over nuclei of 1,145 nonendocrine airway epithelial cells adjacent to 28 mature neuroepithelial bodies (NEBs). Generally, accumulated label was greater in cells nearer a NEB than in those further away. Diminution of label with increasing distance from the closet NEB was confirmed statistically. In 24 of 28 instances, both rank-order correlation and linear regression were significant (P less than 0.001-0.05); in two others, only one test was significant; in another two, neither test was significant. The pattern was consistent and widespread. Rank-order correlations and linear regressions were significant (P less than 0.001) in populations pooled separately from left lung, right upper, and right lower lobes, and the three regression lines were superimposable. Confirmation was obtained in another animal by labeling S-phase cells with a 2-h transplacental pulse of bromodeoxyuridine (BrdU) on fetal day 15. Of 322 BrdU-positive cells counted in 270,204 microns 2 of bronchial epithelium, 174 (54%) lay within 20 microns of a neuroepithelial body. This concentration of dividing cells was significant by chi-square test: chi 2[1] = 101.62; P less than 0.001. We conclude that established NEBs promote growth of the developing airway by stimulating proliferation of local endoderm. A few daughter cells may enter NEBs; most move away to join the expanding nonendocrine airway lining.
Inhibition of surfactant subtype convertase in radiation model of adult respiratory distress syndrome
Authors: Gross N. J.
Published: December 2017

The accompanying paper [Am. J. Physiol. 260 (Lung Cell. Mol. Physiol. 4): L302-L310, 1991] showed that in the radiation pneumonitis model of adult respiratory distress syndrome (ARDS) there was an excess of the proximate, higher buoyant density subtypes of alveolar surfactant, and a decrease in the light buoyant density form. Because the surfactant subtypes normally evolve from the former to the latter a delay in the alveolar metabolism of surfactant could explain this disproportion. Three possible mechanisms of a delay in surfactant metabolism in radiation pneumonitis were explored using an in vitro model of surfactant subtype metabolism called “cycling”. The first was that the surfactant of mice with radiation pneumonitis was intrinsically less capable of conversion to the light subtype. It was found, however, that the proximate forms of surfactant of mice with radiation pneumonitis were as capable of generating light subtype as those of control mice. The second was that there was a deficit in the serine protease activity, called “convertase”, that mediates the conversion. But it was found that lungs of mice with radiation pneumonitis released convertase activity to the same extent as control lungs. The third was that an inhibitor of convertase activity was present in the alveoli. It was found that the alveolar lavage fluid of mice with radiation pneumonitis inhibited the conversion of exogenous surfactant by exogenous convertase. Moreover, it contained an 18-fold excess of antiprotease activity. The present data are interpreted as suggesting that an inhibitor in the alveolar space is responsible for the delay in surfactant subtype metabolism in radiation pneumonitis, resulting in the disproportion of surfactant subtypes in radiation pneumonitis.(ABSTRACT TRUNCATED AT 250 WORDS)
Immunological characterization of deglycosylated human and swine trachea and Cowper's gland mucin glycoproteins
Authors: Sangadala S. , Brewer J. M. , Mendicino J.
Published: December 2017

Antibodies were raised in rabbits against purified swine and human trachea and Cowper's gland mucin glycoproteins and their deglycosylated polypeptide chains. Three monospecific antibody fractions that recognize the carbohydrate, the deglycosylated or unglycosylated regions of the polypeptide chains in these glycoproteins, were isolated by immunoaffinity chromatography. The human and swine trachea mucin glycoproteins showed extensive immunological homology in both their carbohydrate and polypeptide chains. The carbohydrate chains and unglycosylated region of the polypeptide chain in Cowper's gland mucin glycoprotein showed little or no cross-reaction with comparable regions in respiratory mucin glycoproteins. However, the polypeptide chains in the deglycosylated regions of all three mucin glycoproteins showed extensive homology. Five bands with molecular masses ranging from 40 to 46 kDa that differed by a constant molecular mass of approximately 1.5 kDa were detected in hydrolysates of all of the polypeptide chains after treatment with S. aureus V8 protease. Monospecific antibodies to the deglycosylated region of these chains reacted with the peptides, whereas those directed against the unglycosylated region did not. The results suggest that these chains contain tandem repeating sequences of amino acids.
Synthesis of eicosanoids by isolated guinea pig tracheocytes
Authors: Prie S. , Maghni K. , Laporte J. , Borgeat P. , Sirois P.
Published: December 2017

Epithelial cells from the guinea pig trachea (tracheocytes) have been separated from the lamina propria by incubation with EDTA. Contaminating eosinophils were removed by plating on guinea pig immunoglobulin G (IgG)-coated plates. Purity of the cell suspension was assessed by electron microscopy. Enzyme immunoassay analyses of supernatants of guinea pig tracheocytes incubated with 10 microns arachidonic acid (AA) in the presence or absence of 2 microns ionophore A23187 showed that the cells released up to eight times more thromboxane, prostaglandin E2, and 6-ketoprostaglandin F1 alpha than control cells. Ionophore A23187 by itself stimulated to a smaller extent the release of these eicosanoids and did not potentiate the effect of AA. The release of cyclooxygenase products by these cells was highly modified by cell densities in the incubation media. The release of leukotrienes (LT) by stimulated epithelial cells was also investigated using reverse-phase high-performance liquid chromatography. Our results showed that the cells could not release LT on incubation with ionophore A23187 and/or AA. However, the cells released LTB4 in the medium on incubation with 2 microns LTA4. Peptido-leukotrienes were not detected. These results indicated that guinea pig tracheocytes have the cyclooxygenase and the LTA4 hydrolase but not the 5-lipoxygenase. It is postulated that tracheocytes may participate in transcellular metabolism of LTA4 generated by other cell types.
Growth and differentiation factors in tracheobronchial epithelium
Authors: Jetten A. M.
Published: December 2017

The normal tracheobronchial epithelium is continuously renewing itself: cells are lost and replaced by the proliferation and differentiation of stem cells. The proliferation and differentiation of these cells have to be tightly controlled in order to maintain the normal structure of the epithelium. A variety of biological and biochemical processes are involved in controlling the proliferation and differentiation of the tracheobronchial epithelium. Since the trachea and bronchus are comprised of a heterogeneous cell population, interactions between the different cell types are of crucial importance not only in controlling the normal maintenance of this tissue but also in the regulation of repair processes following injury and morphogenesis during lung development. A variety of factors, including several polypeptide growth factors and cytokines, have been identified that regulate positively or negatively the growth and differentiation of tracheobronchial epithelial cells by autocrine or paracrine mechanisms. Retinoids are another group of regulatory factors that appear to play a crucial role in controlling cell proliferation and differentiation in the tracheobronchial epithelium. Recently, many advances have been made in understanding the action of these agents in these cells. Alterations in the balance between growth and differentiation regulatory factors appear to play an important role in several pathophysiological changes such as hyperplasia, fibrosis, and neoplasia.
Ectopeptidases of alveolar epithelium: candidates for roles in alveolar regulatory mechanisms
Authors: Funkhouser J. D. , Tangada S. D. , Peterson R. D.
Published: December 2017

This commentary discusses the implications of recent observations indicating that at least one, and probably three, ectopeptidases are exhibited on the surface of alveolar epithelial cells. The unique biological feature of the ectopeptidases is that they are expressed on the surface of only selected cell types and at specific stages in the differentiation of those cells. Consequently they are positioned to exert their enzymatic activity in a very restricted locale. Drawing from information derived from studies of this activity on other cells, we develop the proposition that the ectopeptidases on alveolar epithelial cells regulate the biological activity of peptides encountered at this site. These may be peptides produced by the alveolar epithelial cells or cytokines that have the potential of influencing the alveolar cells. Awareness that these ectopeptidases are expressed on the luminal surface of these cells thus opens heretofore unrecognized avenues of investigation.
Inheritance of phenotype in mammalian cells: genetic vs. epigenetic mechanisms
Authors: Gruenert D. C. , Cozens A. L.
Published: December 2017

Inherited phenotypic changes in cultured cells, as observed during differentiation and transformation, reflect alterations in gene expression and have both a genetic and epigenetic basis. The causes of specific changes are often difficult to define especially when observing phenomenological end points. Although such observations are an important step in defining the phenotypic changes that endure for multiple generations, it is necessary to analyze cells at the molecular level to characterize the pathways leading to changes in phenotype. Gene expression can be regulated at multiple levels, i.e., DNA structure, gene transcription, and/or posttranscriptional modifications. Four genetic mechanisms (DNA point mutations, deletion, rearrangement, and amplification) and two epigenetic mechanisms (DNA methylation and the preservation of DNA-protein complexes) can account for the majority of enduring changes observed in cultured cells. Genetic alterations in DNA sequence appear to be largely responsible for altered growth regulation associated with transformation, but there is also evidence to suggest that epigenetic mechanisms play a role in transformation. Differentiation of cultured cells is often associated with lack of growth, and has been ascribed in part to epigenetic mechanisms. However, differentiation and transformation are not mutually exclusive but may be regulated by parallel multistep pathways. Analysis of the causes underlying transformation has been complicated by the use of aneuploid cells, but it is clear that the tools for overcoming the ambiguities associated with phenomenological analysis are available.
Rat lung antioxidant enzyme induction by ozone
Authors: Rahman I. , Clerch L. B. , Massaro D.
Published: December 2017

We exposed rats of different ages (weights approximately 45-300 g) to 0.7 ppm O3 for 1-5 days. At 5 days lungs of O3-exposed rats had higher activity of Cu,Zn superoxide dismutase (SOD), Mn SOD, catalase, and glutathione peroxidase than air-breathing rats; this greater activity was not due to blood-associated enzyme activity. The greater enzyme activity occurred with a higher concentration of the mRNA for each enzyme (Mn SOD not measured) without altered stability of these mRNAs. In adult rats the concentrations of these mRNAs were measured after 1, 3, and 5 days exposure to O3 and were elevated by day 3. The intergroup differences (air vs. O3) among antioxidant enzymes (AOEs) were unequal, and the intergroup differences in concentration of the specific AOE mRNA were greater than the differences in activity of their AOE. We conclude exposure to O3 led to greater expression of AOE genes; the increased expression was mediated pretranslationally probably at the level of transcription.
Mechanisms of action of endothelin 1 in maturing rabbit airway smooth muscle
Authors: Grunstein M. M. , Rosenberg S. M. , Schramm C. M. , Pawlowski N. A.
Published: December 2017

Maturational differences in the effects and mechanisms of action of endothelin 1 (ET-1) on airway contractility were investigated in tracheal smooth muscle (TSM) segments isolated from 2-wk-old and adult rabbits. In TSM under passive tension, ET-1 elicited dose-dependent contractions, with a potency of action that was significantly greater (P less than 0.001) in the 2-wk-old vs. adult tissues (i.e., mean +/- SE - log 50% of maximal response values: 8.59 +/- 0.17 vs. 7.79 +/- 0.15 - log M, respectively). In TSM half-maximally contracted with acetylcholine (ACh), however, ET-1 elicited dual and opposing dose-dependent effects. At lower doses (less than or equal to 10(-9) M), ET-1 induced TSM relaxation that was significantly greater in the adult vs. 2-wk-old TSM segments (i.e., approximately 100 vs. 26.5% decrease in active tension, respectively). The relaxant responses were associated with significantly enhanced (P less than 0.001) ET-1-induced release of prostaglandins E2 and I2 in the adult tissues. At higher doses (greater than 10(-9) M), ET-1 induced TSM contractions that were 1) attenuated to a relatively greater extent by the Ca2+ channel blocker, nifedipine (10(-5) M) in the 2-wk-old tissues and 2) associated with significantly (P less than 0.001) enhanced ET-1-stimulated accumulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in the immature TSM. Moreover, the TSM contractions were inhibited by the protein kinase C (PKC) antagonist, H-7, and the latter effect was more potent in the immature TSM. Collectively, these findings demonstrate that ET-1 exerts a potent duality of action in rabbit TSM which varies significantly with maturation, wherein 1) age-dependent differences in airway relaxation are associated with changes in the evoked release of bronchodilatory prostaglandins and 2) maturational differences in airway contraction are associated with changes in Ins(1,4,5)P3 accumulation and extracellular Ca2+ mobilization, coupled to differences in PKC activation.
One hour in 1 ATA oxygen enhances rat alveolar macrophage chemiluminescence and fungal cytotoxicity
Authors: Smith R. M. , Mohideen P.
Published: December 2017

The purpose of this study was to determine if 100% O2 would enhance rat pulmonary alveolar macrophage (PAM) oxidative killing of conidia of the fungus Neurospora crassa. First, we found that incubation in 100% O2 had no effect on conidia viability in the absence of PAM. We obtained resident PAM from nonpretreated anesthetized male Sprague-Dawley rats by bronchoalveolar lavage. Compared with similar air exposures we found that 1 h in vitro exposure of PAM to 100% O2 (1.0 atmosphere absolute) increased their killing of opsonized phagocytized conidia by 52%, without altering phagocytosis. A reactive oxygen killing mechanism is suggested because 1) 100% O2 increased PAM chemiluminescence (CL) by 60% both at rest and during 1 h of phagocytosis, and 2) PAM in 100% O2 killed albino conidia (lacking free radical-quenching carotenoids) 2.9 times more readily than they killed wild-type conidia. Compared with air, 100% O2 delayed the PAM respiratory burst time to peak by 18% but did not alter the burst maximal acceleration. The latter and the similar 60% increase in PAM CL in 100% O2 both at rest and during 1 h of phagocytosis suggests a nonenzymatic O2 enhancement of the respiratory burst. Changed timing of burst events in 100% O2 suggests early O2 toxicity. We conclude that 1 h in 100% O2 increases PAM free radical production and fungicidal activity.
A bronchial epithelial cell-derived factor in asthma that promotes eosinophil activation and survival as GM-CSF
Authors: Soloperto M. , Mattoso V. L. , Fasoli A. , Mattoli S.
Published: December 2017

We have examined the in vitro interaction between bronchial epithelial cells and eosinophils derived from five asthmatics by determining the effect of epithelial cell-conditioned medium on the survival and activation of peripheral blood eosinophils. The supernatants of epithelial cells from six normal donors were used as control. The asthmatic epithelial cell-conditioned medium significantly increased the survival of eosinophils cultured for 3 (P less than 0.025) and 6 (P less than 0.05) days. The incubation of eosinophils with the supernatants of asthmatic epithelial cells for 1 h also increased the generation of superoxide anion and the release of leukotriene C4, triggered by phorbol myristate acetate and calcium ionophore, by more than twofold. The preincubation of asthmatic epithelial cell-conditioned media with saturating concentrations of a mono-specific antiserum against granulocyte-macrophage colony-stimulating factor (GM-CSF) completely abolished their activity, whereas the addition of recombinant human GM-CSF restored it. The supernatants of asthmatic epithelial cells contained 0.88 +/- 0.09 (SD) ng/5 X 10(5) cells immunoreactive GM-CSF, and this amount was significantly greater than that measured in the supernatants of normal epithelial cells (0.21 +/- 0.105, P less than 0.025). Bronchial epithelial cells from asthmatics also expressed increased levels of GM-CSF mRNA when compared with normal epithelial cells. Thus both the synthesis and release of GM-CSF by bronchial epithelial cells are upregulated in asthma, and this may contribute to the persistence of eosinophil infiltration and activation in asthmatic airways.
Na(+)-HCO3- symport modulates intracellular pH in alveolar epithelial cells
Authors: Lubman R. L. , Crandall E. D.
Published: December 2017

We investigated Na(+)-HCO3- cotransport as a mechanism for regulation of intracellular pH (pHi) in rat alveolar pneumocytes grown in primary culture. pHi was monitored using the fluorescent pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Cells incubated in 6 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) medium at pH 7.4 were subjected to rapid acidification by CO2 pulse. pHi recovered in the presence of Na+ with an initial rate (dpHi/dt) of 0.15 min-1, which was reduced by 67% when Na+ was replaced by choline, unaffected by substitution of gluconate for Cl-, reduced 40% in the presence of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 500 microM), and unchanged by amiloride (1 mM). In parallel experiments, cells were incubated at pH 7.4 with 20 mM HCO3- and pHi acutely lowered by NH3 prepulse. dpHi/dt in these experiments was 0.14 min-1 in the presence of Na+ and HCO3-, and reduced 79% under Na(+)-free conditions. These data indicate the presence of a Na(+)-dependent, Cl(-)-independent, DIDS-sensitive and amiloride-insensitive mechanism of recovery from acute intracellular acidification in alveolar pneumocytes, most consistent with Na(+)-HCO3- cotransport (symport) effecting acid extrusion under these experimental conditions. This ion transport mechanism may contribute to regulation of pHi in alveolar pneumocytes, transepithelial transport of acid-base equivalents across the alveolar epithelium, and modulation of pH of alveolar fluid in adult mammalian lungs.
Ontogeny of rat lung type II cells correlated with surfactant lipid and surfactant apoprotein expression
Authors: Randell S. H. , Silbajoris R. , Young S. L.
Published: December 2017

During the last stages of intrauterine growth, remarkable changes occur in the alveolar epithelium that include cellular differentiation and increased production of surfactant lipid and apoprotein. We made morphometric measurements of type II cell characteristics from rats aged gestational day 20 to 14 days postnatal. We also measured the amounts of disaturated phosphatidylcholine (DSPC) and surfactant apoprotein (SP-A) in lung tissue, bronchoalveolar lavage, and a lamellar body-rich fraction, and we estimated the lung content of mRNAs for SP-A, SP-B, and SP-C. Lavage and lamellar body surfactant lipid and apoprotein content per lung showed a pattern of a sharp rise in the early postnatal period, then a substantial decline, and a second increase by day 14. When normalized for dry lung weight, the highest DSPC values were found on postnatal day 1 in all compartments. The fraction of whole lung DSPC found in lamellar body or lavage was greatest in the 48-h period surrounding birth. Lamellar body SP-A was greater than lavage SP-A on gestational day 22, but a day later the lavage SP-A was 16 times greater than the lamellar body SP-A. The lung tissue content of all three apoprotein mRNAs increased sharply before birth, fell during the 1st postnatal wk, and then rose again to adult levels. Type II cell number and lamellar body number per milligram of dry lung tissue was highest on post-natal day 1 and fell by one-half during the 1st postnatal wk. The amount of DSPC per unit of lamellar body volume rose to its greatest value on postnatal day 1 and then decreased more than threefold. These findings indicate a pattern of expansion of surfactant cellular and biochemical pools at the time of birth in the rat.
Simple technique for culture of highly differentiated cells from dog tracheal epithelium
Authors: Kondo M. , Finkbeiner W. E. , Widdicombe J. H.
Published: December 2017

Cultures of dog tracheal epithelium have proved very useful in studies of ion transport. Their short-circuit current (Isc), however, is usually much less than the original tissue. We have tested a variety of conditions in an attempt to produce large numbers of cells with electrical properties comparable with the original tissue. Of several growth supports, human placental collagen (HPC) gave the best results. When plated at 2.5 x 10(5) cells/cm2 onto HPC, cells grown in serum-free, growth factor-supplemented medium (GF medium) showed increases in cells per unit area, thickness of cell sheet, numbers of domes, numbers of apical microvilli, and degree of basolateral membrane interdigitation compared with cells grown in medium containing 5% fetal calf serum (FCS medium). Transepithelial resistance (Rte) and the increases in Isc and intracellular Ca in response to isoproterenol were also increased. However, baseline Isc and adenosine 3',5'-cyclic monophosphate levels were not changed. The improved electrical properties were maintained for up to 4 mo. GF medium combined with an air interface produced further increases in Rte, Isc, and changes in Isc in response to amiloride and isoproterenol. Ultrastructural features such as the presence of cilia, greater thickness of the cell sheet, and increased amplification of apical and basolateral membranes also indicated improved differentiation. Our results show that GF medium and an air interface can be combined with a simple growth support and a relatively low-plating density to allow the easy production of greater than 500 cm2 of cultured cells from a single trachea, with a level of differentiation similar to that of the original tissue.
Lung surfactant protein SP-C from human, bovine, and canine sources contains palmityl cysteine thioester linkages
Authors: Stults J. T. , Griffin P. R. , Lesikar D. D. , Naidu A. , Moffat B. , Benson B. J.
Published: December 2017

Lung surfactant is a complex mixture of lipids and proteins that coats the alveoli to reduce surface tension and prevent airspace collapse. One of the principal protein constituents, surfactant protein C (SP-C), has been characterized following isolation from human, canine, and bovine sources. In each species, this highly hydrophobic protein is composed of 33-35 amino acids, the differences being due to NH2-terminal heterogeneity. A COOH-terminal leucine is conserved throughout. The cysteines in each species were found by fast atom bombardment mass spectrometry to be present as thioesters of palmitic acid. Acylation of recombinant SP-C with palmityl coenzyme A, followed by characterization before and after release of the acyl group with 1,4-dithiothreitol, provided corroborating evidence for the native structure.
Biphasic contractile response of pulmonary artery to hypoxia
Authors: Bennie R. E. , Packer C. S. , Powell D. R. , Jin N. , Rhoades R. A.
Published: December 2017

Isolated perfused lungs exposed to low O2 exhibit a hypoxic pulmonary vasoconstriction response that is transient in nature. The purpose of this study was to determine whether the isolated pulmonary artery behaves similarly in response to hypoxia. Rat pulmonary arterial rings were placed in tissue baths (37 degrees C, air-5% CO2, pH = 7.4) and attached to force transducers. Maximum contractile responses (Po) to high K+ were elicited. After washout, arterial rings were submaximally contracted and made hypoxic (PO2 = 33.7 +/- 1.3, pH = 7.38 +/- 0.01). Aortic rings were used to obtain comparative data. The isolated pulmonary arterial hypoxic response was biphasic, displaying an initial rapid contraction of short duration (phase 1) then, before complete relaxation of this first response, a second slow but sustained contraction occurred (phase 2). Aortic rings did not exhibit a biphasic response, but showed only an initial short contraction followed by complete relaxation. The contractile response of the pulmonary artery was diminished when the endothelium was rendered nonfunctional. However, the phase 2 response was not endothelium dependent. Neither inhibitors of the lipoxygenase or cyclooxygenase pathways nor scavengers of extracellular reactive oxygen species had any effect on the biphasic hypoxic response. Pulmonary arterial hypoxic contractions were blunted when glucose was absent and appear to be dependent on glycolytic ATP. Results of this study show that hypoxia causes a biphasic contractile response of pulmonary arterial muscle and that two different mechanisms appear to be involved, since the transient phase 1 response is endothelium dependent, whereas the sustained contraction of phase 2 is endothelium independent.
Effects of perinatal undernutrition on elimination of immature myosin isoforms in the rat diaphragm
Authors: Brozanski B. S. , Daood M. J. , LaFramboise W. A. , Watchko J. F. , Foley T. P. , Butler-Browne G. S. , Whalen R. G. , Guthrie R. D. , Ontell M.
Published: December 2017

The effect of perinatal undernutrition on the postnatal elimination of immature myosin isoforms in rat diaphragm muscle was examined using electrophoretic and immunocytochemical techniques. Electrophoresis of native myosin showed that neonatal bands were present in diaphragm muscles of both control and undernourished rats on day 4. By day 21, the neonatal bands were diminished in the control diaphragm compared with the diaphragm of the undernourished rats. Neonatal bands persisted on postnatal day 30 in the diaphragm of the undernourished rats but not in the diaphragm of control rats. No significant difference in the time course of elimination of embryonic myosin light chain (LCemb) was observed between the diaphragm muscles of control and undernourished rats with two-dimensional gel electrophoresis. Immunocytochemical analysis demonstrated embryonic myosin heavy chain (MHCemb) in all myofibers of the diaphragm muscle of both groups at day 4, but this isoform was not detected in either group by day 14. Reactivity with anti-neonatal myosin heavy chain (MHCneo) indicated that rate of elimination of the MHCneo was delayed in the undernourished state as compared with the normal rats (P less than 0.001). Serum triiodothyronine levels were measured at 14, 21, and 30 days and were significantly lower in the undernourished rats compared with age-matched controls. These data demonstrate that the normal postnatal decrease in MHCneo, but not MHCemb or LCemb, is affected by the nutritional state of the animal. We speculate that these alterations in myosin isoform transitions are induced by hypothyroidism associated with undernutrition.
Hydrogen peroxide production by alveolar type II cells, alveolar macrophages, and endothelial cells
Authors: Kinnula V. L. , Everitt J. I. , Whorton A. R. , Crapo J. D.
Published: December 2017

Extracellular H2O2 release and intracellular H2O2 production were determined in rat lung alveolar macrophages, rat alveolar type II cells, and cultured bovine aortic endothelial cells. Isolated macrophages (5 h ex vivo) released 3.1 +/- 0.09 nmol H2O2.min-1.mg cell protein-1, freshly isolated (5 h ex vivo) type II cells released 0.7 +/- 0.07 nmol H2O2.min-1.mg protein-1, and cultured endothelial cells released 0.06 +/- 0.005 nmol H2O2.min-1.mg protein-1. The rate of extracellular H2O2 release decreased rapidly over time in both fresh macrophages and freshly isolated type II cells. When the measurements were repeated at different times ex vivo, the decrease was greater than 20%/h, and H2O2 release was almost undetectable 12 h ex vivo. The decrease occurred while lactate dehydrogenase release, catalase activity, and intracellular H2O2 production remained unchanged. Catalase activity was 59.3 +/- 4.9 nmol O2 produced.min-1.mg protein-1 in type II cells, 13.2 +/- 1.8 in macrophages, and 11.4 +/- 2.7 in endothelial cells. Aminotriazole is a compound that inhibits catalase in the presence of H2O2 at a rate that is proportional to the rate of intracellular H2O2 production in or near peroxisomes. Incubation of the cells with aminotriazole led to a rapid inhibition of catalase. In 15 min the reduction of catalase activity was 69% in type II cells, 53% in macrophages, and 37% in endothelial cells. When freshly isolated type II cells were exposed to hyperoxia (95% O2) for 30 min, no changes in the rate of either intracellular H2O2 production or extracellular H2O2 release were seen.
Sustained production of secretory component by human tracheal epithelial cells in primary culture
Authors: Fiedler M. A. , Kaetzel C. S. , Davis P. B.
Published: December 2017

Secretory immunoglobulin A (sIgA) is an important initial defense against environmental agents in the airway. The purposes of our study were to determine whether human tracheal epithelial (HTE) cells produce secretory component (SC), the receptor for dimeric IgA (dIgA), to determine whether HTE cells in primary culture continue to produce SC, and if so, to develop a model for studying SC metabolism in the airway. Immunoperoxidase staining of the human trachea using antibody raised against human SC reveals that many surface epithelial cells and the cells of the submucosal glands express SC but basal cells do not. HTE cells, obtained from tracheal specimens at necropsy, contain 10-51 ng of SC/10(5) cells, at the time of isolation. However, when these cells are placed in culture on plastic, SC release diminishes with time (from 19.6 ng/10(5) cells on day 2 to 6.4 on day 8) despite continued cell proliferation. In contrast, HTE cells cultured on floating collagen gels increase SC release over the same period (26.2 ng/10(5) cells on day 2 and 193.9 on day 8). HTE cells cultured on collagen-coated and uncoated nitrocellulose filters also produced SC at least through day 8 (collagen coated, 21.5 ng/10(5); uncoated, 6.3). Furthermore, SC was released preferentially to the apical surface (4:1 ratio) under both conditions. This system will allow us to study the production, processing, and release of SC by HTE cells and further understand the transport and function of secretory IgA in the airway.
Potassium dependence of Na-Cl cotransport in dog tracheal epithelium
Authors: Fong P. , Chao A. C. , Widdicombe J. H.
Published: December 2017

In confluent primary cultures of dog tracheal epithelium, we tested whether Cl entry across the basolateral membrane is by cotransport with K. Two approaches were taken. First, we measured the inhibition of short-circuit current (Isc) by the K channel inhibitor, Ba2+. Consistent with Na-K-2Cl cotransport, maximal doses of Ba2+ inhibited five-sixths of Isc in tissues previously stimulated to secrete Cl; only two-thirds of Isc should be sensitive to Ba2+ if NaCl cotransport is the entry mechanism. Second, we measured basolateral 86Rb uptake and demonstrated inhibition by bumetanide, an inhibitor of Na-K-2Cl cotransport in other tissues. The degree of inhibition by bumetanide was consistent with the levels of Cl secretion measured as Isc. Uptake of 86Rb was also reduced by removal of Na or Cl, and under these conditions Rb uptake was not further inhibited by bumetanide. These results suggest that the process responsible for Cl entry across the basolateral membrane of tracheal epithelium during Cl secretion is Na-K-2Cl rather than Na-Cl cotransport.
DNA distribution analysis of type II pneumocytes by laser flow cytometry: technical considerations
Authors: Uhal B. D. , Rannels D. E.
Published: December 2017

Optimal conditions were established for determination of cell cycle phase fractions of freshly isolated or cultured adult rat type II pneumocytes (T2P). Propidium iodide staining of ethanol-fixed cells treated with ribonuclease (RNase) consistently yielded histograms with low coefficients of variation. Contaminating cells and cell clumps were eliminated during data acquisition through electronic gating based on anti-vimentin immunofluorescence and peak red fluorescence, respectively. Failure to delete contaminants, clumps or RNA resulted in overestimation of S or G2/M phase fractions by as much as 20-fold. When T2P were cultured on plastic at an initial density of 2.5 x 10(5)/cm2, the S phase fraction did not change over a culture interval in which thymidine incorporation rates increased almost 10-fold. In contrast, a significant increase in the G2/M phase fraction by day 2 of culture occurred with no significant increase in cell number. These results support the hypothesis that adult rat T2P, when subjected to customary conditions of primary culture, undergo cell cycle block in G2/M phases. The data also indicate that under these in vitro conditions, net thymidine incorporation by T2P may vary independently of the S phase fraction. The methods described in this report address basic considerations crucial to future applications of flow cytokinetics to the study of T2P proliferation and differentiation.
Phosphocreatine pathway for energy transport: ADP diffusion and cardiomyopathy
Authors: Saks V. A. , Belikova Y. O. , Kuznetsov A. V. , Khuchua Z. A. , Branishte T. H. , Semenovsky M. L. , Naumov V. G.
Published: December 2017

Chemically skinned (by treatment with saponin, 40 μg/ml) isolated cardiomyocytes were used to study the intracellular diffusion of ADP and creatine (Cr). Stimulation of respiration was studied in these cardiomyocytes without intact sarcolemma and in isolated heart mitochondrial by addition of ADP and Cr in the presence of 0.2 mM ATP (via mitochondrial creatine kinase reaction: Cr + MgATP = MgADP + PCr). The Michaelis constant (Km) for Cr was similar in both cases, 5.67 ± 0.11 (SD) mM in skinned myocytes and 6.9 ± 0.2 mM in mitochondria, showing that there is no significant restriction to the diffusion of this substrate. However, the apparent Km for external ADP increased from 17.6 ± 1.0 μM for mitochondria to 250 ± 38 μM for skinned cardiomyocytes, showing decreased diffusivity of ADP as a result of binding to cellular structures. In the presence of 25 mM Cr, the Km for ADP for myocytes decreased to 35.6 ± 5.6 μM due to the coupling of the creatine kinase and oxidative phosphorylation reactions. Provision of substrate for the creatine kinase reaction amplified the weak ADP signal in the regulation of respiration. The activity of the mitochondrial creatine kinase was decreased by a factor of two in cardiomyopathic hamsters and human hearts and was associated with a twofold decrease in creatine-stimulated respiration. These data show a potentially key role of mitochondrial creatine kinase in the regulation of cellular respiration and the possible importance of changes in its activity for the functional disturbances of the cardiomyopathic heart.

cellular respiration; creatine kinase; oxidative phosphorylati
Colchicine has opposite effects on interleukin-1 beta and tumor necrosis factor-alpha production
Authors: Allen J. N. , Herzyk D. J. , Wewers M. D.
Published: December 2017

To study the role of microtubules in cytokine production, the effect of the microtubule depolymerizing agent colchicine on lipopolysaccharide endotoxin (LPS)-induced interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) release by blood monocytes and alveolar macrophages were examined. Immunofluorescence microscopy demonstrated that LPS resulted in the appearance of microtubule-containing cytoplasmic appendages and that colchicine, which resulted in microtubule disruption in monocytes, blocked appendage formation. Colchicine resulted in approximately 50% increase in LPS-induced IL-1 beta release and a 50% decrease in LPS-induced TNF-alpha release by human monocytes at all doses of LPS tested. Although colchicine resulted in a statistically significant increase in LPS-stimulated human alveolar macrophage IL-1 beta release, the increase was not as great as that observed with monocytes. Northern blot analysis suggested that the colchicine effect occurs pretranslationally because colchicine caused an increase in LPS-stimulated IL-1 beta mRNA levels and a decrease in TNF-alpha mRNA levels. These results suggest that microtubules contribute to the regulation of endotoxin-stimulated mononuclear phagocyte cytokine production and that this regulation differs significantly between IL-1 beta and TNF-alpha.