bluuebunny/crossref_metadata_2025 huggingface dataset:
Dihydropyridine-sensitive single calcium channels in airway smooth muscle cells
Authors: Worley J. F. , Kotlikoff M. I.
Published: December 2017

We have identified and characterized single voltage-dependent calcium channels in both acutely dissociated rat bronchial and cultured human tracheobronchial smooth muscle cells using the patch-clamp technique. In both cell types, on-cell membrane patches displayed unitary currents selective for barium ions and exhibited one conductance level (21–26 pS), and the open state probability was increased by membrane depolarization. Unitary barium currents were enhanced by the calcium channel selective agonist, BAY R 5417, and inhibited by the dihydropyridine calcium channel antagonist, nisoldipine (apparent inhibition constant less than 100 nM). Moreover, the degree of nisoldipine inhibition of the rat bronchial smooth muscle channels was increased with membrane depolarization in a manner consistent with the drug interacting with highest affinity to the inactivated channel state. In addition, the sensitivity to BAY R 5417 augmentation and nisoldipine inhibition of depolarization-induced tonic force of intact rat bronchial ring segments was in close agreement to the single channel results. Thus these data suggest that activation of voltage-dependent calcium channels can influence airway contraction and that dihydropyridines may be effective modulators of depolarization-induced increases in bronchial tone. We conclude that both rat and human airway smooth muscle cells have high-conductance voltage-dependent calcium channels that interact in a predictable manner with dihydropyridines and are similar to voltage-dependent calcium channels observed in other smooth muscle cells.
Okadaic acid, a phosphatase inhibitor, decreases macrophage motility
Authors: Wilson A. K. , Takai A. , Ruegg J. C. , de Lanerolle P.
Published: December 2017

Cellular locomotion results from a series of spatially and temporally integrated reactions. The coordinated regulation of these reactions requires sensitive intracellular signaling mechanisms. Because protein phosphorylation reactions represent important signaling mechanisms in mammalian cells, we investigated the effect of okadaic acid, a phosphoprotein phosphatase inhibitor, on protein phosphorylation and macrophage motility. Okadaic acid was applied to rat alveolar macrophages, and motility was quantitated by a directed chemotaxis assay. Okadaic acid inhibits macrophage motility in a dose-dependent fashion; the concentrations for 50 and 100% inhibition were 3 and 25 microM, respectively. Protein phosphorylation studies demonstrated a 2.5-fold increase in total protein phosphorylation in macrophages treated with 25 microM okadaic acid. These experiments also demonstrated a dose-dependent increase in the phosphorylation of the 20-kDa light chain of myosin. Moreover, 25 microM okadaic acid 1) maximally increased myosin light chain phosphorylation by 6.6-fold, 2) raised the level of myosin associated with the cytoskeleton from a basal level of 47.0 to 96.7% of the total myosin, and 3) induced profound morphological changes as visualized by scanning electron microscopy. These data correlate an increase in protein phosphorylation with a decrease in macrophage motility. Furthermore, they suggest that phosphoprotein phosphatase inhibition may prevent motility by uncoupling coordinated processes, such as cytoskeletal reorganization, that are essential for macrophage motility.
Secretory granule calcium loss after isolation of rat alveolar type II cells
Authors: Eckenhoff R. G. , Rannels S. R. , Fisher A. B.
Published: December 2017

Morphological change and lamellar body loss suggests that alveolar type II cells rapidly de- or redifferentiate after several days of primary culture. To determine whether type II cells or lamellar body compositional changes precede these obvious morphological changes, we examined the in situ elemental composition of lamellar bodies and type II cells from intact lung and at different times after isolation using electron probe microanalysis (EPMA). Isolated cells were prepared by standard methods and plated on either tissue culture plastic or kept in suspension with stirrer flasks. Cell pellets obtained at 0, 3, 24, and 48 h after isolation were rapidly frozen, and thin freeze-dried cryosections were prepared and examined cold in a transmission electron microscope equipped for EPMA. Eight to ten type II cells from each of three to four different preparations for each time period were analyzed. A rapid, progressive, and sustained fall in lamellar body calcium and sulfur content occurred by 48 h of primary culture, suggesting rapid alteration in calcium and protein metabolism by type II cells and/or lamellar bodies after isolation. Also, marked changes in type II cell cytoplasmic Na and K occurred in freshly isolated cells, with incomplete normalization by 48 h. Culture on laminin-enriched Matrigel for 1 wk increased both lamellar body calcium or sulfur content, but 100 nM dexamethasone had no effect. Lamellar body calcium accumulation appears to be a very sensitive index of differentiated type II cell function.
Acetaldehyde-mediated cilia dysfunction in bovine bronchial epithelial cells
Authors: Sisson J. H. , Tuma D. J. , Rennard S. I.
Published: December 2017

Acetaldehyde, which is present in significant concentrations in cigarette smoke and is elevated during alcohol ingestion, has been demonstrated to impair mucociliary clearance of the lung. Acetaldehyde is also known to impair protein function through the formation of acetaldehyde-protein adducts. We hypothesized that acetaldehyde impairs bronchial epithelial cilia motion by inhibiting cilia dynein adenosinetriphosphatase (ATPase) activity through the formation of acetaldehyde adducts with cilia proteins. Acetaldehyde induced concentration- and time-dependent slowing of cilia beating and cilia-derived dynein ATPase activity in primary cultures and isolated axonemes of bovine airway epithelial cells. Cilia slowing and ATPase inhibitory effects were also observed with related aldehydes but not with ethanol. Acetaldehyde binding, assessed by gel electrophoresis using [14C] acetaldehyde, was demonstrated to occur with the dynein heavy chains and with tubulin and closely paralleled ATPase inhibition. We conclude that acetaldehyde directly impairs bronchial cilia function causing slowing of cilia beating, inhibits cilia dynein ATPase activity, and binds to cilia proteins critical for motion including dynein and tubulin. These data suggest that acetaldehyde-induced cilia dysfunction may be related to direct cilia ATPase inactivation and adduct formation with cilia dynein and tubulin. This may be an important mechanism by which airway host defenses are impaired in clinical settings where acetaldehyde exposure occurs, e.g., with cigarette smoking and alcohol ingestion.
Glucocorticoid enhances pulmonary surfactant protein B gene transcription
Authors: O'Reilly M. A. , Clark J. C. , Whitsett J. A.
Published: December 2017

The effect of glucocorticoid on the regulation of pulmonary surfactant protein B (SP-B) synthesis was studied in a human pulmonary adenocarcinoma cell line. Northern blot analysis demonstrated a marked increase in SP-B mRNA expression after treatment with dexamethasone for 48 h. Actinomycin D, puromycin, or cycloheximide blocked the induction of SP-B mRNA by glucocorticoid. Nuclear run-on experiments demonstrated that the effects of dexamethasone on SP-B mRNA were due in part to increased transcription of the SP-B gene. However, during this time period, there was a discrepancy between SP-B gene transcription, which was increased only 2- to 4-fold, and SP-B mRNA, which increased 60- to 150-fold after treatment with dexamethasone. In the presence of actinomycin D, SP-B mRNA was relatively stable, decreasing slowly in the presence or absence of glucocorticoid. In contrast to the relative stability of SP-B mRNA in the presence of actinomycin D, SP-B mRNA was markedly decreased after exposure to puromycin, supporting the premise that continued protein synthesis, rather than transcription alone, is required for maintenance of SP-B mRNA levels. Induction of SP-B expression by glucocorticoids was dependent on enhanced SP-B gene transcription and was also dependent on continued protein synthesis. The discrepancy between the relative enhancement of SP-B transcription and SP-B mRNA suggests that posttranscriptional factors influence SP-B expression in this cell line.
Evidence that fatty acid chain length is a type II cell lipid-sorting signal
Authors: Longmuir K. J. , Haynes S.
Published: December 2017

This study was undertaken to determine those structural features of phospholipid molecules which influence their enrichment in type II cell lamellar body material. Cultured fetal rabbit lung tissue was labeled with [1-14C]acetate, type II cells were isolated, and extracellular lamellar body and microsomal fractions were prepared. Radiolabeled molecular species of phosphatidylcholine (PC) and phosphatidylethanolamine were analyzed by high-performance liquid chromatography (HPLC), followed by silver nitrate thin-layer chromatography of HPLC peak fractions that overlapped. Compared with microsomes, lamellar body PC was selectively enriched with molecular species containing 14- and 16-carbon fatty acids and depleted of species containing 18-carbon fatty acids. Palmitoleic acid and an ether linkage positively influenced the enrichment of PC molecular species in the lamellar body material, but these structural features were secondary to the predominant influence of fatty acid chain length. In vivo, lung tissue normally contains low levels of palmitoleic acid; hence most unsaturated fatty acids are 18-carbons or longer. A cellular lipid-sorting mechanism that selects PCs by recognition of 14- and 16-carbon fatty acid chains (and not by recognition of fatty acid saturation) should serve to enrich the resulting pulmonary surfactant with disaturated molecular species of PC.
Adenosine A2-receptor-mediated phosphatidylcholine secretion in type II pneumocytes
Authors: Griese M. , Gobran L. I. , Douglas J. S. , Rooney S. A.
Published: December 2017

Adenosine and its analogues stimulate phosphatidylcholine secretion in cultured rat type II pneumocytes. There is evidence that this effect is mediated by A2 receptors. We have now employed the radioligand 5'(N-ethylcarboxyamido)adenosine (NECA) in an attempt to study the adenosine receptor in a membrane fraction of type II cells. Specific binding of [3H]-NECA was saturable with a total binding capacity (Bmax) of 4.91 pmol/mg protein and a dissociation constant (Kd) of 240 nM. The Kd was similar to the concentration of NECA which half-maximally stimulated phosphatidylcholine secretion (EC50, 150 nM). Although the relative potency of adenosine analogues in displacing bound [3H]NECA was consistent with that expected at an A2 receptor, there were discrepancies between binding and function with respect to some agonists as well as the antagonist xanthine amine congener (XAC). This together with the unusually high Bmax suggests that the NECA binding site is not the adenosine receptor mediating phosphatidylcholine secretion. The receptor was further characterized functionally by measuring its antagonist dissociation constant (Kb). KbS for XAC inhibition of the effects of NECA, N6-(L-2-phenylisopropyl)adenosine, and adenosine on phosphatidylcholine secretion were 19 +/- 8, 24 +/- 11, and 6 +/- 12 nM, respectively, suggesting that all three agonists act at the same receptor. The Kb value allowed comparison of the receptor in type II cells with that in other tissues and revealed that it was similar to the A2 receptor previously described in human platelets. These functional data further characterize and are consistent with a physiological role for the adenosine A2 receptor in the regulation of lung surfactant secretion.
Effects of P. aeruginosa-derived bacterial products on tracheal ciliary function: role of O2 radicals
Authors: Jackowski J. T. , Szepfalusi Z. , Wanner D. A. , Seybold Z. , Sielczak M. W. , Lauredo I. T. , Adams T. , Abraham W. M. , Wanner A.
Published: December 2017

he purpose of this investigation was to evaluate the effects of bacterial products derived from Pseudomonas aeruginosa on the function of airway cilia and to assess the role of phagocytes and oxygen radicals in the observed responses. Ciliary beat frequency (CBF) was measured in a perfusion chamber with a microscopic technique using tracheal epithelial cells obtained from normal sheep by brush biopsy (70% epithelial cells, 18% macrophages, 11% neutrophils). Baseline CBF ranged between 678 and 1,126 min-1. After 20 min of perfusion with the cell free supernatant of P. aeruginosa culture (mucoid strain), a concentration-dependent depression of CBF was observed with a 58% inhibition at a 1:1 dilution (P less than 0.05). The P. aeruginosa-derived products pyocyanin and 1-hydroxyphenazine also decreased CBF in a dose-related fashion. The cilion-inhibitory effects of the supernatant and bacterial products were markedly attenuated after centrifugation of the brush preparation (80% epithelial cells, 16.5% macrophages, 3.5% neutrophils). Glucose/glucose oxidase also caused a rapid, concentration-dependent cilioinhibition or ciliostasis. Catalase blocked or attenuated the ciliary effects of the supernatant, bacterial products and glucose/glucose oxidase. Thus bacterial products released from P. aeruginosa impaired ciliary activity by a pathway which involved neutrophils and was mediated by toxic oxygen radicals.
Interactions of oxidant stress and vascular reactivity
Authors: Gurtner G. H. , Burke-Wolin T.
Published: December 2017

Oxidants have complex effects on pulmonary vascular reactivity. They can stimulate production of vasoconstrictor arachidonate mediators and can also cause vasodilation through activation of guanylate cyclase. Oxidants can also inactivate vasomotor phenomenon by interfering with mechanisms of signal transduction or smooth muscle contraction. The final physiological response depends on the balance of these complex actions.
Roles of neutral endopeptidase in airways
Authors: Borson D. B.
Published: December 2017

In recent years, studies of the regulation of the airways have focused to an increasing degree on the roles of neuropeptides. Several peptides have been shown to be present in airways and mediate such diverse responses as ion transport, mucus secretion, bronchospasm or relaxation, edema, cough, changes in vascular permeability, and neutrophil chemotaxis. More recently, studies have described the roles of peptidases, most notably neutral endopeptidase (NEP, also known as enkephalinase, or E.C. 3.4.24.11) and kininase II (also known as angiotensin-converting enzyme, or E.C. 3.4.15.1) in modulating peptide-induced responses. The enzymes cleave a wide variety of peptides, generating metabolites that are inactive in the systems studied to date. Thus inhibitors of NEP potentiate responses to peptides that are cleaved by it. Therefore, NEP plays roles in modulating peptide-induced effects analogous to the role of acetylcholinesterase in modulating cholinergic neurotransmission. In several experimental respiratory diseases, the activity of neutral endopeptidase is decreased, resulting in increased responses to peptides. The therapeutic application of recombinant NEP protects the airways from the adverse actions of stimuli that release inflammatory peptides, and induction of the NEP gene expression by glucocorticoids suggest a possible mechanism for the action of these steroids in treating airway diseases such as asthma, chronic bronchitis, or cystic fibrosis.
Characterization of L-glutamine transport by pulmonary artery endothelial cells
Authors: Herskowitz K. , Bode B. P. , Block E. R. , Souba W. W.
Published: December 2017

This study characterized the transport of L-glutamine by porcine pulmonary artery endothelial cells (PAECs). Uptake of 50 microM glutamine was determined and found to be linear for at least 45 min. The sodium-dependent velocity represented greater than 95% of the total uptake at all time points. Kinetic studies of glutamine uptake at concentrations between 0.005 and 10 mM showed a single saturable high-affinity carrier with a Michaelis constant of 100 +/- 6 microM and a maximal transport velocity of 1.0 +/- 0.08 nmol.mg protein-1.30s-1. Glutamine uptake by PAECs was markedly inhibited in the presence of L-cysteine, L-threonine, or L-alanine; lesser degrees of inhibition occurred when histidine and arginine were added. 2-Methylaminoisobutyric acid and 2-aminobicyclo [2,2,1]heptanedicarboxylic acid had little effect on glutamine uptake. Lithium did not substitute for sodium, strongly suggesting that L-glutamine was not transported by system N. Furthermore, transport of glutamine was not affected by hormones or by changes in external pH. Based on the intolerance of this high-affinity carrier to N-methylated substrate, its insensitivity to pH and hormonal regulation, and the failure of lithium to substitute for sodium, as well as its inhibition by alanine and cysteine, we conclude that in porcine pulmonary artery endothelial cells L-glutamine is predominantly taken up through system ASC.
Primary translation products of pulmonary surfactant protein D
Authors: Crouch E. , Rust K. , Persson A. , Mariencheck W. , Moxley M. , Longmore W.
Published: December 2017

Surfactant protein D (SP-D) is a collagenous, surfactant-associated, carbohydrate-binding protein that is synthesized by alveolar type II epithelial cells. To further characterize SP-D, we isolated RNA from adult rat lungs and rat type II cells and translated mRNAs in vitro. [35S]methionine-labeled translation products were precipitated with antibodies to rat SP-D, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by fluorography. Immune precipitates of translation reactions for rat lung or rat type II cells demonstrated a single collagenous polypeptide (39.3 kDa) that was smaller than surfactant-associated SP-D (43 kDa, reduced) but larger than the mature secreted form of rat SP-A. This component was not identified in translation reactions of rat liver, gut, brain, mammary gland, or rat L2 cell RNA. There was a fivefold enrichment of SP-D mRNA in freshly isolated type II cells relative to lung; however, the levels of translatable SP-D mRNA decreased rapidly during the first 24 h of cell culture. The SP-D translation product migrated faster than the major cellular form of SP-D but approximately 1 kDa slower than cellular SP-D synthesized in the presence of 2,2'-dipyridyl plus tunicamycin. Translation in the presence of canine pancreatic microsomes gave a single glycosylated, endoglycosidase F-sensitive form (40.6 kDa) and demonstrated cleavage of a small signal peptide. These results indicate that SP-D is a secretory product of differentiated type II epithelial cells and that SP-D is secreted in a mature form that does not undergo further proteolytic processing in vivo.
Effects of PAF on isolated rat airways perfused with blood-free solution
Authors: Munoz N. M. , Kirchhoff C. F. , Strek M. E. , Blumenthal R. N. , Leff A. R.
Published: December 2017

We examined the mechanism of constriction and muscarinic augmentation of contraction of airway smooth muscle caused by platelet-activating factor (PAF) in airways from 55 Sprague-Dawley rats perfused through the isolated bronchial circulation (BC) and pulmonary circulation (PC) and isometrically in tissue perfusion chambers. Dose-response curves were generated cumulatively by infusing 10(-10) to 10(-7) mol PAF dissolved in Krebs-Henseleit solution buffer containing 4% bovine serum albumin into the BC or PC. The efficacy of PAF in central airways (BC) was approximately twofold greater in increasing lung resistance (RL) than for more peripheral airways perfused by the PC (P less than 0.05). Tachyphylaxis was demonstrated in both BC and PC for preparations in which a second PAF dose-response curve was generated. Bolus injection of 10(-6) mol of the PAF antagonist, CV-6209, plus 10(-7) mol PAF caused 81% reversal of the maximal BC response. The same dose of CV-6209 reversed the response to PAF in the PC by 99.2%. Initial administration of 10(-6) mol CV-6209 with PAF prevented completely contraction elicited by PAF in the BC and PC. Concentration-response studies also were generated isometrically in tissue perfusion chambers from 64 tracheal smooth muscle strips. Maximal contraction elicited by 10(-6) M PAF was blocked completely with 10(-6) M CV-6209. In separate studies, addition of 10(-6) mol CV-6209 to the BC perfusate caused 93% blockade of the RL response to PAF and 100% inhibition when administered in the PC. Prior administration of PAF caused two- to fourfold augmentation of the contractile response to acetylcholine (ACh) within the same preparation; in the presence of CV-6209, the response to ACh was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)
Uptake and reutilization of surfactant phospholipids by type II cells of isolated perfused lung
Authors: Moxley M. A. , Jacoby J. , Longmore W. J.
Published: December 2017

To investigate the role of type II alveolar cells in surfactant uptake and reutilization while cells are in situ, the combination of isolated perfused rat lung and type II cell isolation has been used. After an equilibration period, isolated perfused rat lungs received an intratracheal injection of labeled rat surfactant or a labeled bovine-derived preparation, and the perfusion continued. At time intervals less than or equal to 3 h, lungs were lavaged and type II cells isolated. Freshly isolated cells from perfused lungs were highly viable as judged by trypan blue exclusion and by linear incorporation of labeled leucine into trichloroacetic acid precipitable protein during perfusion. After cell isolation, total phospholipid or phosphatidylcholine was extracted from cells. Incorporation of surfactant phosphatidylcholine label into cellular phosphatidylcholine was shown to be linear with time whether lungs received natural rat surfactant or bovine-derived surfactant preparation. Uptake of natural surfactant from alveoli into type II cells was approximately three times greater than uptake of bovine-derived material. Results of administration of 0.5 mumol surfactant phospholipid or 2.0 mumol were similar except that administration of 2.0 mumol of natural rat surfactant caused uptake to become saturated at 1 h of perfusion. An increase in cell-associated phosphatidylcholine occurred whether results were expressed on basis of cellular DNA or specific activity of cellular phosphatidylcholine. Majority of administered surfactant label remained lavage fluid associated at 3 h, whereas remainder was associated with isolated cells or debris from cell preparation. Little label was detected in perfusion medium.(ABSTRACT TRUNCATED AT 250 WORDS)
Maturational changes in endothelium-derived relaxing factor activity of ovine pulmonary arteries in vitro
Authors: Abman S. H. , Chatfield B. A. , Rodman D. M. , Hall S. L. , McMurtry I. F.
Published: December 2017

To examine mechanisms underlying fetal pulmonary vascular vasodilator responses and maturational changes in endothelial function, we studied effects of endothelium-dependent (acetylcholine, ACh: adenosine diphosphate, ADP; and A23187) and -independent (sodium nitroprusside, SNP) vasodilators on tone of small (third generation) pulmonary artery rings isolated from late-gestation fetal, newborn, and adult sheep. Changes in isometric force of phenylephrine-contracted rings were measured after the cumulative addition of vasodilators in baths aerated with 21% O2 and 5% CO2. Pulmonary artery rings from fetal lambs demonstrated minimal relaxation to ACh, ADP, and A23187, achieving only 17 +/- 3, 14 +/- 5, and 23 +/- 8% relaxation, respectively. In contrast, fetal rings relaxed completely (100%) to SNP. Rings from newborn and adult animals had significantly greater maximal relaxation in response to ACh. ADP, and A23187 than fetal rings (at least P less than 0.05 for each comparison with fetal rings), but responses to SNP were not different. Hemoglobin (10(-5) M), an inhibitor of endothelium-derived relaxing factor, caused less augmentation of phenylephrine contraction in fetal than adult pulmonary artery rings (11 +/- 4% vs. 49 +/- 8%; P less than 0.01). We conclude that in comparison with pulmonary artery rings from postnatal animals, fetal pulmonary artery rings have diminished endothelium-derived relaxation factor activity. We speculate that maturational changes in endothelial cell function contribute to ontogenetic differences in pulmonary vasoreactivity.
Surfactant subtypes in experimental lung damage: radiation pneumonitis
Authors: Gross N. J.
Published: December 2017

Radiation pneumonitis, a chronic form of adult respiratory distress syndrome (ARDS), is known to be associated with physiological and biophysical abnormalities of the surface element of the lungs suggesting an impairment of the surfactant system. The alveolar surfactant of mice with radiation pneumonitis was fractionated into subtypes on continuous sucrose density gradients to examine their relative amounts, composition, ultrastructure, surface activity, and turnover kinetics. The total phospholipid and protein contents of the alveolar lavage were increased. The proportions of high buoyant density subtypes (normally surface active) were increased about twofold and that of the low buoyant density subtype (not surface active) was decreased or absent. The buoyant densities, ultrastructure, and phospholipid compositions of the major surfactant subtypes were not significantly altered. The surface activity of the normally surface-active subtypes, when purified free of extraneous material, was close to those of normal controls. Turnover studies of the kinetics of surfactant subtype phospholipids suggested increased secretion of surfactant but a delay in the conversion of the heavier subtypes into their low-density product. Most of the heavier material appeared not to enter the lighter pool, in contrast to findings in control mice. It is concluded that in this form of ARDS the surfactant subtypes are qualitatively normal but that their surface activity is impaired, presumably by extraneous material in the alveoli, and that proportions of surfactant subtypes are radically altered by a combination of increased synthesis and decreased metabolism of the heavier subtypes.
Limited division of low-density adult rat type II pneumocytes in serum-free culture
Authors: Tanswell A. K. , Byrne P. J. , Han R. N. , Edelson J. D. , Han V. K.
Published: December 2017

A previously reported serum-free medium for the culture of adult rat type II pneumocytes has been further defined by the addition of 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer and 700 U/ml catalase. Both additions significantly enhanced [3H]thymidine incorporation into pneumocyte DNA between days 5 and 6 of culture. In the presence of 100 micrograms/ml bovine pituitary extract, both insulin-like growth factor (IGF) I (20 ng/ml) and II (25 ng/ml) further increased [3H]thymidine incorporation into pneumocyte DNA over that observed with the basal medium. When type II pneumocytes were plated at 2.75 X 10(5) cells/cm2 there was a plating efficiency of 50 +/- 5%, and the increased DNA synthesis in response to IGFs was not associated with an increase in cell number. When the plating number was reduced to 1.6 X 10(5) cells/cm2 the plating efficiency was only 23 +/- 2%. At this low cell density a doubling of cell number was observed in response to IGF-I between days 4 and 9 of culture.
Regulation of manganese superoxide dismutase in porcine pulmonary artery endothelial cells
Authors: Visner G. A. , Block E. R. , Burr I. M. , Nick H. S.
Published: December 2017

Bacterial lipopolysaccharide (LPS) was shown to produce an induction of manganese superoxide dismutase (Mn SOD) mRNA levels in porcine pulmonary artery endothelial cells (PAEC). Additional studies in porcine PAEC also demonstrated induction of Mn SOD mRNA in response to the inflammatory mediators interleukin 1 and tumor necrosis factor. On the other hand, we observed no change in Mn SOD mRNA within 24 h of a hyperoxic exposure. The induction of Mn SOD by LPS was blocked by both a RNA synthesis inhibitor, actinomycin D, and a protein synthesis inhibitor, cycloheximide. The data implicate the involvement of Mn SOD in the acute phase response of pulmonary endothelial cells.
Developing fetal alveolar epithelial cells secrete fluid in primary culture
Authors: McCray P. B. , Welsh M. J.
Published: December 2017

The developing pulmonary epithelium secretes a Cl(-)-rich fluid during fetal life by a process involving active transport. To determine if alveolar cells contribute to this fluid production, we studied developing fetal rat alveolar epithelial cells (18-day gestation) in primary culture. Fetal alveolar epithelial cells aggregated to form cystic, alveolar-like structures with a fluid-filled lumen. On light and transmission electron microscopy, the cells were polarized with microvilli facing the lumen. Dexamethasone and triiodothyronine stimulated lamellar body production in many of the epithelial cells of the cyst wall. The transepithelial voltage in the cyst was -2.4 +/- 0.4 mV (lumen negative), suggesting the presence of active electrolyte transport. Bumetanide, an inhibitor of Cl- secretion in other systems, decreased the size and number of cysts. A membrane-permeant analogue of adenosine 3',5'-cyclic monophosphate (cAMP) and 3-isobutyl-1-methylxanthine increased the size of cysts, an effect that was blocked by coincubation with bumetanide. The increased size of the cysts did not result from stimulation of cell growth; in fact, [3H]thymidine incorporation was inhibited to 40% control values by cAMP, suggesting that growth was inhibited rather than stimulated. These results suggest that fetal alveolar epithelial cells secrete fluid via a cAMP-mediated Cl(-)-secretory process. Secretion of fluid by the developing alveolar epithelium may play an important role in lung development.
Isolation of an inhibitor of neutrophil function from bronchial lavage of normal volunteers
Authors: Cooper J. A. , Sibille Y. , Zitnik R. J. , Bayles G. , Buck M. G. , Merrill W. W.
Published: December 2017

Bronchial inflammation is associated with a reduction in airway caliber. Factors that may dampen this inflammation are ill defined. We have developed a model of airway inflammation using an extract of cotton bracts (CBE). In the current study we characterize inhibitor(s) of polymorphonuclear neutrophil (PMN) function in bronchial lavage (BL) fluid and examine the relationship between inhibitor concentrations and induction of bronchial inflammation or bronchoconstriction by CBE. We report that BL obtained contains factors that inhibit PMN hydrogen peroxide production and chemotaxis to formylmethionylleucyl-phenylalanine (FMLP). In two different subject populations the relative degree of a one molecular mass inhibitor was greatest in BL from subjects that manifested little bronchoconstriction to CBE compared with other subjects. In one subject population the relative amount of this inhibitor correlated inversely with the bronchoconstricting response to CBE and a number of parameters of airway inflammation including PMN noted on bronchial biopsy after CBE instillation. Partial chemical characterization of one molecular mass BL-derived inhibitor reveals it is of low molecular mass (less than 1,000 Da), nonpolar, and sensitive to aminopeptidase digestion. The finding that the bronchial environment contains variable concentrations of an oligopeptide that inhibits PMN function has important implications for treatment of inflammatory airway diseases
Components of electrogenic transport in unstimulated equine tracheal epithelium
Authors: Joris L. , Quinton P. M.
Published: December 2017

Basic components of unstimulated electrolyte transport across equine tracheal mucosa were characterized. After the tissue was mounted in Ussing chambers, both current and tissue resistance gradually increased for approximately 60 min before reaching stable values. Thereafter, under open-circuit conditions, the tissue had a resistance of 250 +/- 14 omega.cm2, generated a transepithelial potential difference of -34 +/- 1.7 (SE) mV (referenced to the serosal side) and an equivalent short-circuit current (Ieqsc) of -149 +/- 10.2 microA/cm2. Even though 10(-5) M amiloride reduced the current by approximately 65%, mucosal Na+ substitution with choline decreased the current significantly more (approximately 80%), indicating that part of the Na(+)-dependent current was amiloride insensitive. No current decrease occurred after serosal application of 10(-4) M bumetanide, which was expected to inhibit Na(+)-K(+)-2Cl(-)-mediated Cl- secretion, even though bilateral Cl- substitution with gluconate reduced Ieqsc by approximately 30 microA/cm2. Continuous short-circuit conditions caused a reversible fall in the short-circuit current that was inhibited by amiloride but not by Cl- depletion, suggesting that sustained short circuiting leads to a significant underestimation of the amiloride-sensitive Na+ transport. In the absence of Cl-, the response to amiloride was significantly smaller, which suggests that Cl- depletion also has an inhibitory effect on electrogenic, amiloride-sensitive Na+ absorption.
Protein phosphorylation and dephosphorylation in type II pneumocytes
Authors: Warburton D. , Tayag A. , Buckley S. , Cosico L. , Seth R.
Published: December 2017

Protein phosphorylation and dephosphorylation are a major mechanism for regulating cellular activity. Substantial evidence exists for ascribing a key role of protein phosphorylation and dephosphorylation in the regulation of surfactant secretion from type II pneumocytes, yet understanding of the specific molecular mechanisms is generally lacking. Herein, we report two-dimensional electrophoretic mapping of proteins phosphorylated in type II pneumocytes in primary culture, the response to protein kinase C simulation with phorbol ester, and the response to protein phosphatase inhibition with okadaic acid. Exposure of cells for 15 min to phorbol ester at a concentration (10(-4) M) which maximally stimulated both translocation of protein kinase C from cytosol to membranes and surfactant secretion increased phosphorylation (50-80% compared with control) of three specific proteins (50 kDa, pI 5.8 and 5.7; 25 kDa, pI 5.7). Exposure of cells for 2.5 h to okadaic acid (10(-6) M), a concentration that inhibited 90% of protein phosphatase activity, resulted in greatly increased phosphorylation (200-1,500% compared with control) of five specific proteins (50 kDa, pI 5.7 and 5.6; 45 kDa, pI 5.5; 40 kDa, pI 5.5; 25 kDa, pI 5.5). Combined treatment with okadaic acid and phorbol ester resulted in further increases (145-3,080% compared with control) in phosphorylation of four specific proteins (50 kDa, pI 5.6; 45 kDa, pI 5.5; 40 kDa, pI 5.5; 25 kDa, pI 5.5). We conclude that these respective proteins comprise major substrates for protein kinase C-dependent phosphorylation and for protein phosphatases in type II pneumocytes in primary culture. Furthermore, we speculate that these proteins will prove to play key roles in the regulation of type II pneumocyte function.
Myosin phosphorylation/dephosphorylation and regulation of airway smooth muscle contractility
Authors: de Lanerolle P. , Paul R. J.
Published: December 2017

Airway smooth muscles contract due to the activation of a highly sophisticated signal transduction mechanism. Signal transduction in muscle must include 1) a mechanism for converting chemical energy (i.e., ATP) into mechanical work (energy transduction) and 2) a mechanism for integrating the response to multiple stimuli (signal integration). In smooth and striated muscles, ATP hydrolysis due to the cyclic interaction of actin and myosin is the final site for both energy transduction and signal integration. There is growing consensus that this interaction in smooth muscles is regulated by the phosphorylation/dephosphorylation of the 20-kDa light chain of smooth muscle myosin. By phosphorylation/dephosphorylation we mean the enzyme-catalyzed transfer of the terminal phosphate of ATP to a serine or threonine residue on a protein, by a class of enzymes known as protein kinases, with the formation of a covalent phosphoester linkage and the enzyme-catalyzed removal of the phosphate group by phosphoprotein phosphatases. Smooth muscles contain many protein kinases and phosphatases, and the research emphasis on the regulation of smooth muscle contraction has focused on how these enzymes act individually and in concert to regulate the actin-myosin interaction. This review will describe the biochemical and physiological experiments that have been performed to understand the role of myosin phosphorylation/dephosphorylation in regulating smooth muscle contraction. Although data from studies on vascular and other smooth muscles will be summarized, this review will focus on studies performed on airway smooth muscle. More detailed reviews of studies on nonairway smooth muscles can be found in Refs. 47 and 79.
Rapid procedure for obtaining tracheal apical membranes
Authors: Shen B. Q. , Yang C. M. , Widdicombe J. H.
Published: December 2017

Apical membranes from cow tracheal epithelium were prepared in a two-step process. First, most of the unwanted membranes in the crude homogenate were aggregated with Mg and removed by a low-speed spin. The membranes remaining in the supernatant were pelleted by a high-speed spin, resuspended, and exposed to ouabain-affinity chromatography. This step removed approximately 50% of the protein, all the Na-K-adenosinetriphosphatase, but had no effect on total levels of alkaline phosphatase (a marker for apical membranes). The specific activity of the apical membrane marker, alkaline phosphatase, was 21 +/- 7-fold (mean +/- SD) greater in the apical membranes than in the homogenate. Markers for nuclei, mitochondria, and basolateral membranes were excluded compared with the homogenate. Similar results were obtained with primary cultures of cow tracheal epithelium. The vesicular nature of the membranes was demonstrated in isotope uptake studies that revealed an osmotically active space.
Dibutyryl cAMP, prostaglandin E2, and antioxidants protect cultured bovine bronchial epithelial cells from endotoxin
Authors: Koyama S. , Rennard S. I. , Claassen L. , Robbins R. A.
Published: December 2017

Acute bronchitis secondary to bacterial infection in the airway is accompanied by an acute inflammatory response composed predominantly of neutrophils. Mucosal injury with denudation of the airway epithelium to basement membrane frequently occurs. We postulated that endotoxin might explain this cytotoxicity and neutrophil influx. To test this hypothesis, bovine bronchial epithelial cells were cultured, and the culture supernatant fluids were evaluated for neutrophil chemotactic activity (NCA) and lactate dehydrogenase (LDH) after exposure to endotoxin. Escherichia coli endotoxin stimulated the release of NCA and LDH in a dose-dependent manner. Because intracellular augmentation of adenosine 3',5'-cyclic monophosphate (cAMP) has anti-inflammatory effects, we postulated that dibutyryl cAMP (DBcAMP) and prostaglandin E2 (PGE2) might modulate the effect of endotoxin. DBcAMP and PGE2 decreased the release of NCA and LDH. Because cAMP might exert its effect by decreasing intracellular release of oxidants, we investigated the capacity of the antioxidants dimethyl sulfoxide (DMSO) and allopurinol to attenuate the effects of endotoxin. DMSO and allopurinol alone or in combination attenuated the effects of endotoxin-induced NCA and LDH release. These data suggest that endotoxin may account for the pathophysiological changes seen with bronchial bacterial infection or endotoxin inhalation and that the inflammatory responses may be attenuated by DBcAMP, PGE2, and antioxidants.
Ontogeny of ion transport across fetal pulmonary epithelial cells in monolayer culture
Authors: Rao A. K. , Cott G. R.
Published: December 2017

The transition of the fetal lung from a fluid-secreting to a fluid-absorbing organ is dependent on ion transport across the pulmonary epithelium. This study examined the ion transport characteristics of distal pulmonary epithelial cells isolated from rat fetuses in late gestation and maintained in differentiation-arrested monolayer cultures. The response to inhibitors of active ion transport suggested the presence of apical to basolateral Na+ transport in monolayers derived from each gestational age. However, amiloride inhibition of short-circuit current (Isc) varied with gestational age, decreasing Isc by 30% in monolayers derived from day 18 fetuses and by 55% in monolayers from day 21 fetuses. A portion (10%) of the residual Isc remaining after amiloride addition to monolayers from day 18 fetuses could be inhibited by bumetanide, suggesting the induction of net Cl- transport. Ion-substitution experiments confirmed the presence of Na+ and inducible Cl- transport mechanisms in monolayers from day 18 fetuses and only Na+ transport mechanisms in monolayers from day 21 fetuses. beta-Adrenergic stimulation increased Isc but maintained the age-dependent characteristics of Na(+)- and Cl(-)-dependent ion transport. In summary, monolayer cultures of fetal pulmonary epithelial cells exhibit age-dependent differences in ion transport properties that are consistent with a transition or maturation of the distal pulmonary epithelium from an epithelium capable of Na+ absorption and Cl- secretion preterm to one capable of only Na+ absorption at term.
Method for rapid evaluation of topically applied agents to cystic fibrosis airways
Authors: Wine J. J. , King V. V. , Lewiston N. J.
Published: December 2017

The proposal is to target a single maxillary sinus for treatment with agents designed to reverse or ameliorate the cystic fibrosis (CF) defect in airway mucosa, with the opposite sinus serving as a control. Selected CF patients have undergone maxillary antrostomy and antibiotic lavage to help relieve severe pulmonary disease and chronically impacted and infected sinuses. After treatment, the mucosa in the maxillary sinuses of these patients are accessible and can be bathed with fluids introduced via the stomas with procedures that restrict the fluid to a single sinus. The ability of an agent to reverse mucosal pathology can therefore be determined easily with the mucosa of the contralateral sinus serving as a control. Electrophysiological properties, amounts and composition of fluid and mucus, immune functions, and bacterial colonization can be measured accurately and repeatedly. The consistent observation that sinus involvement in CF is near universal and bilaterally symmetric offers a unique opportunity for a simultaneous within-subject, double-blinded control paradigm. This approach should speed evaluation of any agent designed to improve airway mucosal function.
Control of type I collagen formation in the lung
Authors: Goldstein R. H.
Published: December 2017

Type I collagen is a major structural protein in the lung, the accumulation of which is stimulated during certain inflammatory reactions in the lung. Accumulating evidence suggests that type I collagen formation parallels changes in steady-state mRNA levels. Specific inflammatory substances modulate transcription of collagen genes and stabilization of collagen mRNA in vitro. However, the precise role for any particular mediator during fibrotic processes is difficult to identify because of the complex nature of the inflammatory reaction and potential interaction among mediators. The signal transduction mechanisms that regulate collagen accumulation remain to be defined. This review focuses on the regulation of collagen accumulation in the lung by specific inflammatory substances.
Structural and functional changes of surfactant protein A induced by ozone
Authors: Oosting R. S. , van Greevenbroek M. M. , Verhoef J. , van Golde L. M. , Haagsman H. P.
Published: December 2017

The major surfactant protein SP-A is thought to play an important role in the regulation of the structure and the metabolism of pulmonary surfactant. We investigated whether this protein may be a target in ozone toxicity. Several properties were studied that are probably relevant to the physiological functions of SP-A. In vitro exposure of human and canine SP-A to ozone led to decreases in 1) self-association of SP-A, 2) SP-A-mediated lipid aggregation, and 3) binding of SP-A to immobilized mannose. SP-A was neither degraded nor cross-linked by ozone exposure. Exposure of canine SP-A to ozone led to an increase in the apparent molecular weight of monomeric SP-A. Human SP-A did not show this change. Oxidation of methionine and tryptophan residues in canine SP-A was detected following ozone exposure. Reactions of ozone with other amino acid residues were not observed. The impairments of structure and properties of SP-A may contribute to the toxic action of ozone in the lungs.
Type II pneumocyte proliferation in vitro: problems and future directions
Authors: Uhal Bruce D. , Flowers Kevin M. , Rannels D. Eugene
Published: December 2017

In adult animals, the type II pneumocyte is progenitor of both the type I and type II alveolar epithelium. In primary culture, however, the fate of this cell is uncertain. Type II cells in culture lose their differentiated properties and eventually resemble type I cells, but a lack of specific markers has complicated the characterization of the phenotype acquired in vitro. Furthermore, limited proliferation of these cells in vitro has precluded definition of the relationship between type II cell proliferation and differentiation. Recent work in this laboratory has involved the correlation of flow cytometric cell cycle analysis with phenotype markers. Initial results indicate that isolation of type II cells induces cell cycle block similar to that sustained by other cell types in response to stress. In addition, preliminary evaluation of phenotype suggests that traditional markers become ambiguous beyond the 1st day of primary culture. These results raise concern related to the interpretation of experiments conducted in vitro. This report discusses the implications of these findings and directions for future work.

alveolar epithelium; bromodeoxyuridine; cell cycle block; flow cytometry; lung injury
Time course of mechanical efficiency during afterloaded contractions in isolated cardiac muscle
Authors: Peterson Jon N. , Alpert Norman R.
Published: December 2017

The time course of mechanical efficiency during working contractions in rabbit papillary muscle is presented. Efficiency is found to remain relatively constant during the working portion of the twitch, when the muscle is contracting against a constant load. As afterload was decreased, efficiency increased to 65 ± 11% (mean ± SE, n = 3) at 10% developed force at maximum length. This is in contrast to muscle work, which reached a peak of 3.0 ± 0.3 (n = 6) mJ/g at 50% developed force at maximum length.

heart; initial heat; rabbit; working contractions