Treatment of mucociliary dysfunction.
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Animals↗
Biomedical subjects
A Wanner
Publications and source records attributed to A Wanner.
Measurement of airway mucosal blood flow with dimethylether: validation with microspheres.
We have recently developed a noninvasive dimethylether (DME) uptake technique to estimate airway mucosal blood flow (Qaw) in humans (12). Because it was not feasible to validate the technique directly, we undertook the present study to compare Qaw as measured by DME (QDME) and by color-coded microspheres (QM) as a standard in seven anesthetized sheep prepared with a carotid and a left atrial catheter. QDME was determined by measuring DME uptake with multiple breath holds after passive inflation with a DME-helium gas mixture, simulating the technique used in humans. After the microspheres were injected into the left atrium, the sheep were killed and the tracheal segment corresponding to the dead space from which DME uptake was determined was removed, and its mucosa was stripped and processed for microsphere counts. Mean QDME was 35.6 ml.min-1.100 g-1 wet tissue (range 9.6-98.0 ml.min-1.100 g-1) and mean QM was 29.1 ml.min-1.100 g-1 (range 7.7-91.5 ml.min-1.100 g-1). There was a strong correlation between QDME and QM (r = 0.89; P = 0.01). Intravenous nitroglycerin and vasopressin caused comparable increases and/or decreases in QDME and QM (r = 0.87; P = 0.02). This suggests that the noninvasive DME uptake method measures Qaw accurately and supports its validity in human studies.
Animals↗
Hydrogen peroxide-scavenging properties of sheep airway mucus.
Reactive oxygen species released from luminal phagocytes in the airway can potentially injure the airway epithelium. Naturally occurring oxygen radical scavengers must therefore exist to protect the epithelium. This study was designed to determine whether the high-molecular-weight fraction of normal sheep tracheal mucus has hydrogen peroxide (H2O2)-scavenging activity. Lyophilized mucus from 10 sheep was reconstituted in phosphate-buffered saline (PBS) or Krebs-Henseleit buffer. H2O2 was added to these mucus samples to a final concentration of 15 microM, and the level of H2O2 remaining was measured over a 10 min period. From a zero-time level of 17 +/- 1.8 microM (mean +/- SD), the H2O2 concentration fell within 10 min to 8 +/- 1.7 microM in 0.05%; to 3.9 +/- 2.2 microM in 0.1%; to 2.6 +/- 2.4 microM in 0.2%; and to 1.2 +/- 1.5 microM in 0.4% mucus reconstituted in PBS. The results obtained in Krebs-Henseleit buffer were similar. The disappearance of H2O2 was not due to the transformation into hydroxyl radicals. Heat and acid denaturation and cleavage of carbohydrate-free peptides from glycoproteins by pronase E treatment abolished the scavenging potential. Fractionation of 0.4% mucus samples according to molecular weight by gel filtration revealed that only one fraction with proteins of M(r) > 110 kD contained the active scavenger. Polyacrylamide gel electrophoresis and lectin blotting with Ulex europaeus I (UEAI) showed that both the whole mucus and the actively scavenging gel filtration fraction contained a glycoprotein that comigrated with a 205 kD molecular weight marker.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Routine use of beta-adrenergic agonists in asthma.
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Adrenergic beta-Agonists↗
Is the routine use of inhaled beta-adrenergic agonists appropriate in asthma treatment? Yes.
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Administration, Inhalation↗
Actual pentamidine dose delivered by Respigard II nebulizer.
The Respigard II nebulizer system is the approved method to deliver pentamidine aerosols in the USA. Although continuous operation of the nebulizer until dryness is a designated regimen, the actual pentamidine dose delivered under the operating condition has not been thoroughly studied. Pentamidine solutions (300 mg in 6 mL water) were nebulized continuously with the Respigard II nebulizer system until dryness (40 min operation). Aerosols were delivered to the lower airways via an oropharyngeal model and sampled on a filter with a standard breathing mode of 20 breaths.min-1 frequency and 750 mL tidal volume. Intermediate samples were also obtained for the initial 20 min delivery. The pentamidine dose delivered to the mouth was 1.6% of the dose placed in the nebulizer. Of the dose delivered to the mouth, 92% was delivered during the initial 20 min period. Aerosol loss in the oropharyngeal model was 15% of the dose delivered to the mouth or 0.24% of the dose initially placed in the nebulizer. Pentamidine dose delivered to the lower airways was a very small fraction of the initial dose in the nebulizer. A partial delivery for the initial 20 min was nearly comparable to complete delivery.
Humans↗
Effect of lung volume and intrathoracic pressure on airway mucosal blood flow in man.
We have recently described an inert soluble gas uptake technique (using dimethyl-ether, DME) for the non-invasive measurement of airway mucosal blood flow (Qaw) in humans. In the present study, we assessed the effects of lung volume and intrathoracic pressure on Qaw, in healthy non-smokers (age range 19-52 years). Qaw was calculated from the steady-state uptake of DME from a 50 ml segment of the anatomic dead space. The mean (+/- SD) Qaw of three consecutive measurements at a lung volume of FRC + 300 ml was 8.3 +/- 2.3, 8.6 +/- 2.6 and 8.3 +/- 2.7 ml.min-1 (n = 13; coefficient of variation 14 +/- 7%). At zero airway pressure, there was an inverse relationship between apparent Qaw on the one hand and lung volume and anatomic dead space (DS) on the other: mean Qaw was 12.2 +/- 5.3, 8.2 +/- 2.5 and 5.3 +/- 2.2 ml.min-1 at RV + 300 ml (DS = 131 +/- 11 ml), FRC + 300 ml (DS = 153 +/- 12 ml) and TLC (DS = 206 +/- 22 ml) positions, respectively (n = 11; P < 0.05 among all three). At a lung volume of FRC + 300 ml, an increase in intrathoracic pressure to +25 cmH2O (modified Valsalva maneuver) decreased mean Qaw to 3.3 +/- 2.8 ml.min-1 while a decrease in intrathoracic pressure to -35 cmH2O (modified Müller maneuver) increased mean Qaw to 17.1 +/- 7.4 ml.min-1 from a control value of 7.2 +/- 2.2 ml.min-1 (n = 7; P < 0.05 among all three). These results indicate that lung volume has an effect on apparent Qaw, presumably by influencing the depth to which the analyzed anatomical dead space segment extends into the bronchial tree. The results also show that changes in intrathoracic pressure alter Qaw, possibly reflecting concomitant changes in left ventricular output and its distribution to intrathoracic and extrathoracic vascular beds.
Adult↗
Effect of antigen on the glycoconjugate profile of tracheal secretions and the epithelial glycocalyx in allergic sheep.
To characterize the glycoconjugate composition of tracheal secretions and the apical glycocalyx of the tracheal epithelium under baseline conditions and after antigen challenge, sheep allergic to Ascaris suum were intubated with a double-balloon nasotracheal tube to create a tracheal chamber. After an initial tracheal lavage, the animals were either exposed to intratracheally nebulized phosphate-buffered saline (PBS) (3 ml, n = 6) or A. suum antigen (251,000 protein nitrogen units in 3 ml of PBS, n = 6). Tracheal lavage was repeated 2 hours later, and the animals were killed. An enzyme-linked lectin assay and lectin histochemical analysis were used to characterize carbohydrate residues in lyophilized, resuspended tracheal secretions and the apical glycocalyx of the tracheal epithelium, respectively. Eight lectins were used to detect GalNAc, alpha-Gal, beta-Gal, alpha-Fuc (1-3)Man, alpha-Man/Glu, alpha-Man, and alpha-(2-3)sialyl residues. The amounts of total nondialyzable solids, proteins, and lipids in tracheal secretions were approximately twice as high after exposure to A. suum than after exposure to PBS. All carbohydrate residues were present in tracheal secretions after exposure to PBS and A. suum, but the reactivity was higher after exposure to A. suum for beta-Gal (+125%), alpha-Man/Glu (+150%), alpha-(1-3)Man (+287%), alpha-(2-3)sialyl (+353%), and alpha-Man (+448%) (p < 0.05). Likewise, the apical glycocalyx contained all carbohydrate residues after exposure to PBS and A. suum; afer exposure to A. suum, the reactivity was greater for alpha-GalNAc (+18%), alpha-(2-3)sialyl (+90%), beta-Gal(1-3)GalNAc (+433%), and alpha-(1-3)Man (+482%) (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Tracheal mucosal edema in hydrostatic pulmonary edema.
Airway edema has been described in heart failure, and, in animal experiments, airway narrowing was observed with elevated left atrial pressure (Pla). On the basis of double-indicator-dilution principles using helium and dimethylether, we were able to measure a water compartment of the tracheal mucosa (VH2O) in dogs. Hypervolemia with an attendant increase in Pla caused by infusion of 2 liters of dextran increased VH2O from 368 +/- 71 (SE) to 794 +/- 177 microliters (P < 0.01). Pulmonary arterial wedge and central venous pressures (Pcv) rose concomitantly. Increases in pulmonary arterial wedge and Pcv by a left atrial balloon catheter produced similar increases in VH2O, whereas increases in Pcv alone by a right atrial balloon did not increase VH2O. Increasing VH2O by dextran infusion was associated with an increase in pulmonary resistance from 1.16 +/- 0.19 to 2.15 +/- 0.24 cmH2O.l-1.s (P < 0.01). These observations show that fluid accumulation in the lung during pulmonary congestion also involves extraparenchymal airways and is related to Pla rather than right atrial pressure. This indicates that sufficient collateral drainage exists during right-sided but not left-sided pressure elevations.
Anesthesia↗
Airway mucosal blood flow in humans. Response to adrenergic agonists.
We measured the uptake of the soluble inert gas dimethyl ether (DME) from a segment of the conducting airways to estimate mucosal blood flow (Qaw) noninvasively. The subjects inhaled, from the functional residual capacity position, a 300-ml gas mixture containing 35% DME, 8% helium, 35% oxygen, and the balance nitrogen; they held their breath for 5 s and then exhaled into a spirometer. During exhalation, the instantaneous concentrations of DME and helium were recorded together with expired gas volume. The maneuver was repeated with breathhold times of 5, 10, 15, and 20 s. We calculated Qaw using the time-dependent decrease in DME concentration in relation to the helium concentration in an expired volume fraction between 80 and 130 ml (representing an anatomic dead-space segment distal to the glottis) and the mean DME concentration. In 10 healthy nonsmokers, mean (+/- SE) Qaw was 8.0 +/- 1.3 ml/min, or 8 +/- 2 microliters/min/cm2 mucosal surface. We obtained a value of 12 +/- 3 microliters/min/cm2 in a validation experiment in sheep. Inhaled methoxamine (nebulized dose 10 mg) caused a 65 +/- 19% decrease (p < 0.05), and albuterol (nebulized dose 2.5 mg) a 92 +/- 17% increase (p < 0.05), in mean Qaw in seven subjects, with the maximum changes occurring immediately or 15 min postinhalation. We conclude that the DME uptake method is an acceptable noninvasive means of estimating airway mucosal blood flow in humans and its modification by vasoactive substances.
Adult↗
Endothelin-1 promotes mitogenesis in airway smooth muscle cells.
Endothelin exists as three isoforms (ET-1, ET-2, and ET-3) and exhibits vasoconstricting, bronchoconstricting, and growth-promoting properties in vascular smooth muscle. In the airways, ET-1 immunoreactivity and mRNA have been detected and localized to the epithelium, smooth muscle, and endothelium in different species, including humans. It has been suggested that ET-1 may have a role in the airway smooth muscle hyperplasia and hypertrophy seen in patients with bronchial asthma. We studied ovine airway smooth muscle cells (SMC) in vitro and showed saturable binding of [125I]ET-1 with a dissociation constant (Kd) of 0.4 nM and high affinity binding sites (Bmax) for ET-1 (104 fmol/10(6) cells). This binding was functional as ET-1 promoted mitogenesis of these muscle cells as measured by increased cell number in the absence of serum. Twenty-four hours after exposing the cells to graded doses of ET-1 from 1 pM to 1 microM, cell number increased significantly over control in a dose-dependent manner. ET-1 also enhanced the transient expression of c-fos mRNA by 2.5-fold over control, with maximal expression occurring at 30 min. These observations provide evidence that: (1) airway SMC possess high affinity binding sites for ET-1, and (2) ET-1 is mitogenic for airway SMC as determined by increased cell number and amplification of c-fos mRNA expression. ET-1 may have a fundamental role in influencing the growth of smooth muscle in the airways.
Animals↗
Oxygen radicals contribute to antigen-induced airway hyperresponsiveness in conscious sheep.
We previously showed that oxygen radicals can induce airway hyperresponsiveness (AHR) in allergic sheep. The purpose of this study was to determine whether antigen challenge results in the generation of free oxygen radicals and if these radicals contribute to antigen-induced AHR. We first determined baseline airway responsiveness in seven Ascaris suum-sensitive sheep by calculating the cumulative provocative concentration of carbachol in breath units (BU; one BU defined as one breath of a 1% wt/vol carbachol solution) that increased specific lung resistance (SRL) 400% over baseline (PC400). On a different day, the sheep underwent inhalation challenge with A. suum antigen, SRL was measured before and immediately after challenge and then hourly for 2 h, at which time SRL had returned to baseline. The postchallenge PC400 was then measured. This procedure was repeated on separate occasions, each at least 14 days apart, except that the sheep were treated with an aerosol of catalase (CAT; 38 mg in 3 ml deionized water), the enzyme that catalyzes the decomposition of hydrogen peroxide (H2O2), at three different times: Trial 1, before antigen and then every 30 min after antigen challenge for 2 h; Trial II, 1 and 2 h after antigen challenge; and Trial III, only at 2 h after antigen challenge. In the control trial, antigen challenge caused a transient (mean +/- SEM) 303 +/- 48% increase in SRL over baseline (p < 0.05), and 2 h later, PC400 was reduced to 11.0 +/- 1.7 BU from a prechallenge value of 24.8 +/- 1.9 BU (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Aerosols↗
Lectin-detectable glycoconjugate profile of the tracheal secretions and epithelial glycocalyx in sheep. Effect of muscarinic stimulation.
Reflex mucus secretion in the airways serves a defense function that includes the binding of bacteria to mucus glycoconjugates thereby preventing bacterial adherence to the epithelium. We therefore compared the lectin-detectable glycoconjugate profile of the epithelial glycocalyx and luminal secretions under baseline conditions and after muscarinic receptor stimulation in the sheep trachea. The sheep were intubated with a double-balloon nasotracheal tube to create a tracheal chamber for collection of secretions. After an initial lavage of the chamber to clear it of secretions, the sheep received an intravenous injection of normal saline, 0.5 mg/kg pilocarpine, or 0.5 mg/kg pilocarpine after pretreatment with 0.2 mg/kg atropine. Tracheal lavage was repeated 2 h later, and the sheep were then killed. An enzyme-linked lectin assay and lectin histochemistry were used to characterize glycoconjugate residues in tracheal secretions and in the apical epithelial glycocalyx, respectively. Eight different lectins were used to detect N-acetyl galactosamine, alpha-galactose, alpha-galactose-N-acetyl galactosamine, beta-galactose, beta-galactose-N-acetyl galactosamine, alpha-fucose, alpha-glucose, alpha-mannose and alpha-(2-3)sialyl residues. After normal saline, reactivity was present for all glycoconjugates in secretions and in the glycocalyx.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Cyclic AMP-dependent phosphorylation of a 26 kD axonemal protein in ovine cilia isolated from small tissue pieces.
To study cyclic adenosine monophosphate (cAMP)-dependent phosphorylation events in ovine cilia in vitro, we adapted published axonemal isolation methods to obtain pure mammalian axonemal proteins from small ovine tracheal mucosa pieces with a surface area of only 1 cm2. The isolated axonemes could be reactivated in vitro upon ATP addition, thereby attesting to their functional integrity. The axonemal protein yield from these small mucosa pieces was high enough to allow protein concentration measurements of each sample and axonemal polypeptide analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). cAMP is known to increase ciliary beat frequency, possibly through a phosphorylation event in the axoneme. To study cAMP-dependent phosphorylation events in ovine tracheal cilia, these axonemal preparations were exposed to [gamma-32P]ATP under conditions that stimulated or inhibited kinase activity. Analysis of axonemal polypeptides by SDS-PAGE and subsequent autoradiography showed that an axonemal protein with a M(r) of 26 kD is the only polypeptide consistently phosphorylated in a cAMP-dependent manner. The phosphorylation of this protein could be diminished by a highly specific inhibitor of cAMP-dependent protein kinase, KT-5720. The addition of calcium did not affect label incorporation into this protein during cAMP treatment. In the presence of cAMP and calcium, inhibitors of protein kinase C and calcium/calmodulin-dependent kinase did not change the level of phosphorylation of the 26 kD protein. We conclude that cAMP treatment of isolated mammalian cilia results in the phosphorylation of a single protein with a M(r) of 26 kD (p26).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Protein kinase C-dependent phosphorylation of a ciliary membrane protein and inhibition of ciliary beating.
The present study examined whether protein kinase C phosphorylated a ciliary protein and whether this phosphorylation event was temporally correlated with a decrease in ciliary beat frequency. Activation of protein kinase C decreased ciliary beat frequency of sheep tracheal epithelium, an effect fully blockable by pretreatment of the tissue pieces with H-7, a protein kinase inhibitor. Using cilia removed from these epithelial surfaces and incubated in solutions containing stimulators of protein kinase C along with [gamma-32P]ATP or [gamma-35S]ATP, a single protein target of ciliary protein kinase C activity was identified. The protein is a polypeptide of molecular mass 37 kDa (p37) as estimated by SDS-polyacrylamide gel electrophoresis. Protein kinase C dependency of p37 phosphorylation was proven by showing that Calphostin C, a specific protein kinase C inhibitor, blocked label incorporation into p37 completely, and by demonstrating that purified protein kinase C phosphorylated p37. Inhibitors of cAMP-dependent kinase and calcium/calmodulin-dependent kinase did not change the phosphorylation of p37 in the presence of protein kinase C activators. p37 was recovered in a Triton X-100-extractable fraction of this ciliary preparation, suggesting that p37 is membrane associated. This hypothesis was further supported by the fact that p37 was present in a pellet representing reconstituted membranes. Thin-layer electrophoresis revealed that p37 was phosphorylated on serine and tyrosine residues, suggesting that the activation of protein kinase C also stimulated tyrosine kinase activity. p37 did not precipitate with annexin I or II antibodies. These results show that sheep tracheal cilia contain protein kinase C activity and that activated protein kinase C phosphorylates a membrane-associated ovine ciliary target, an effect temporally related to a protein kinase C-mediated decrease in ciliary beat frequency.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Microvascular and macrovascular endothelial cells produce different constrictor substances.
The media from cultured microvascular and macrovascular endothelial cells (conditioned media, CM) were collected and tested for constrictor activity in sheep coronary artery rings and tracheal smooth muscle strips in vitro (isometric force), expressed as percentage of contraction produced by 80 mM KCl. Both microvascular (micro) and macrovascular (macro) CM caused a sustained slow-onset contraction (P less than 0.05) of the coronary artery rings by 71 +/- 10% (micro; n = 7) and 67 +/- 8% (macro; n = 6) and tracheal smooth muscle strips by 33 +/- 14% (micro; n = 6) and 34 +/- 6% (macro; n = 11); the calcium antagonist gallopamil (10(-7) M) attenuated these effects by 25-55%. Unconditioned medium and medium conditioned by cultured tracheal smooth muscle cells had no constrictor activity on coronary artery rings or tracheal smooth muscle strips. Synthetic endothelin (ET-1) also produced contraction of coronary artery rings and tracheal smooth muscle strips. The mean levels of ET-1 measured by radioimmunoassay were 1,200 pg/ml in the macro CM and 33 pg/ml in the micro CM. Depleting macro CM of ET-1 by affinity columns constructed with protein A agarose and anti-ET-1 antibody removed the contractile activity for coronary artery rings and tracheal smooth muscle strips. Thus ET-1 did not appear to be the contractile substance in the micro CM. Preliminary characterization of the contractile substance in micro CM revealed that it was heat stable, had a molecular weight of less than 10,000, was inactivated by trypsin, and retained its activity after two cycles of freeze-thawing.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Systemic pilocarpine increases deposition of and decreases responsiveness to inhaled carbachol in sheep.
The purpose of this study was to determine whether excessive airway secretions could serve as a barrier function against inhaled particulate matter. To increase airway secretions, six conscious sheep were treated with pilocarpine (0.8 mg/kg i.v.). Pilocarpine increased pulmonary resistance (RL) and total aerosol deposition within five breaths (AD5) as determined by the rebreathing of an inert monodisperse aerosol. When RL had returned to baseline, AD5 remained elevated [21 +/- 2% (SE), P < 0.05] and tracheal secretions were increased (237 +/- 77%, P < 0.05) above the values before pilocarpine administration. A carbachol aerosol dose-response curve was carried out at this time and compared with a control carbachol dose-response curve by calculating the dose of carbachol required to increase RL by 400% (PD400). Mean PD400 was increased postpilocarpine by 53 +/- 18 (P < 0.05) and 85 +/- 25% (P < 0.05) when normalized for increased aerosol deposition. Thus, pilocarpine decreased airway responsiveness to inhaled carbachol despite increasing aerosol deposition. The pilocarpine-induced airway hyporesponsiveness to inhaled carbachol is consistent with the hypothesis that excessive secretions have a protective role in the airways.
Administration, Inhalation↗
Impairment of airway mucociliary transport by Pseudomonas aeruginosa products. Role of oxygen radicals.
We previously showed that the supernatant of a Pseudomonas aeruginosa (PA) culture and its constituents pyocyanin and 1-hydroxyphenazine inhibit ciliary activity of dispersed tracheal epithelial cells in vitro via the generation of oxygen radicals by phagocytes. In the present study, we wished to determine if tracheal mucus velocity (TMV) is also impaired by PA supernatant and if oxygen radicals have a mediating role. In conscious sheep, TMV (measured with a radiographic method) was determined before and serially following aerosol challenge with the cell-free supernatant of a PA culture or unconditioned culture medium (control). TMV decreased from a mean (+/- SEM) baseline of 6.7 +/- 1.1 mm/min (n = 6) by 29, 35, and 25% at 0.5, 3, and 24 h after challenge, respectively (p < 0.05), and returned to baseline 1 wk later (-6%, p = NS). Control medium had no effect on TMV (maximum decrease by 15% at 0.5 h). Aerosolized catalase blunted the effect of PA supernatant on TMV. To determine if the impairment of TMV involved ciliary inhibition, tissues were mounted in a chamber and ciliary beat frequency (CBF) and surface liquid velocity (SLV) were measured with a microscopic method. PA supernatant decreased both CBF (maximum mean decrease 12%; n = 5, p < 0.05) and SLV (maximum mean decrease 78%; n = 5, p < 0.05) in a dose-dependent fashion, with a correlation between the two parameters; these effects were blocked by catalase.(ABSTRACT TRUNCATED AT 250 WORDS)
Administration, Inhalation↗