Release Of Pulmonary Surfactant Research Articles

Pulmonary surfactant protein SP‐B promotes exocytosis of lamellar bodies in alveolar type II cells

The release of pulmonary surfactant by alveolar type II (ATII) cells is essential for lowering surface tension at the respiratory air-liquid interface, stabilizing the lungs against physical forces tending to alveolar collapse. Hydrophobic surfactant protein (SP)-B ensures the proper packing of newly synthesized surfactant particles, promotes the formation of the surface active film at the alveolar air-liquid interface and maintains its proper structure along the respiratory dynamics. We report that membrane-associated SP-B efficiently induces secretion of pulmonary surfactant by ATII cells, at the same level as potent secretagogues such as ATP. The presence in the extracellular medium of lipid-protein complexes containing SP-B activates the P2Y2 purinergic signaling pathway that ultimately triggers exocytosis of lamellar bodies by ATII cells. Our data suggest that SP-B prompts Ca2+-dependent surfactant secretion via ATP release from ATII cells. This result implies that SP-B is not only an essential component for the biophysical function of surfactant but is also a central element in the alveolar homeostasis by eliciting autocrine and paracrine cell stimulation.-Martínez-Calle, M., Olmeda, B., Dietl, P., Frick, M., Pérez-Gil, J. Pulmonary surfactant protein SP-B promotes exocytosis of lamellar bodies in alveolar type II cells.

Surfactant in Airway Disease

Beta(2)-adrenergic agonists cause a release of pulmonary surfactant into lung airways. The surfactant phospholipids maintain the patency of the conducting airways, but this function is inhibited by plasma proteins entering an inflamed airway. The physical behavior of the surfactant can be studied with a pulsating bubble surfactometer and a capillary surfactometer. Calf lung surfactant extract was found to be inhibited by plasma proteins and by a lowering of temperature. Severe breathing difficulties and malfunctioning surfactant developed in BALB/c mice inhaling ozone or infected with respiratory syncytial virus, mainly as a result of proteins invading the airways. Patients with asthma were challenged with allergens in an area of one lung. BAL fluid (BALF) from such an area contained a surfactant that functioned poorly (ie, an inability to maintain airway openness) compared with BALF from the other lung or from the lungs of healthy volunteers. When proteins in the BALF were removed, surfactant performance clearly improved. Eosinophils, so prominent in asthmatic patients, synthesize the enzyme lysophospholipase, which, together with the enzyme phospholipase A(2), catalyzes the hydrolysis of the main component of the surfactant, phosphatidylcholine. Such hydrolysis incapacitates the ability of the surfactant to maintain airway patency. The treatment of asthma with beta(2)-adrenergic agonists and steroids will have a valuable effect on the surfactant system. It will cause a release of fresh surfactant into terminal airways. Surfactant can also be nebulized and inhaled, which has been shown to be an effective treatment.

Microanatomical changes in alveolar type II cells in juvenile mice intratracheally exposed to Stachybotrys chartarum spores and toxin.

Stachybotrys chartarum is an important environmental fungus. We have shown recently that alveolar type II cells are sensitive to exposure to Stachybotrys chartarum spores and to the trichothecene, isosatratoxin-F, both in vitro and in vivo, in a juvenile mouse model. This sensitivity is manifest as significant changes in the composition and normal metabolic processing of pulmonary surfactant. This study evaluated the effects of a single intratracheal exposure of S. chartarum spores and toxin on ultrastructure and dimensions of alveolar type II cells from juvenile mice. This was to determine whether there are concurrent morphological and dimensional changes in the alveolar type II cell that reflect the metabolic alterations in pulmonary surfactant that we observed in the treated mice. Marked ultrastructural changes were associated with alveolar type II cells in both S. chartarum and isosatratoxin-F treated animals compared to untreated, saline, and Cladosporium cladosporioides spore treated animals. These ultrastructural changes included condensed mitochondria with separated cristae, scattered chromatin and poorly defined nucleolus, cytoplasmic rarefaction, and distended lamellar bodies with irregularly arranged lamellae. Point count stereological analysis revealed a significant increase (p < 0.05) in lamellar body volume density in S. chartarum and isosatratoxin-treated animals after 48 h exposure. Mitochondria volume density was significantly lower in the isosatratoxin-F (48 h exposure) and S. chartarum treated (24 and 48 h exposure) animals compared to those in the other treatment groups. These results reveal that exposure to S. chartarum spores and toxin elicit cellular responses in vivo differently from those associated with exposure to spores of a nontoxigenic mold species. They also indicate that accumulation of newly secreted pulmonary surfactant in the alveolar space of S. chartarum and isosatratoxin-F treated animals might be a consequence of cellular trauma resulting in lamellar body volume density changes leading to increased release of pulmonary surfactant into the alveolar space.

Functional significance and control of release of pulmonary surfactant in the lizard lung.

The amount of pulmonary surfactant in the lungs of the bearded dragon (Pogona vitticeps) increases with increasing body temperature. This increase coincides with a decrease in lung compliance. The relationship between surfactant and lung compliance and the principal stimuli for surfactant release and composition (temperature, ventilatory pattern, and autonomic neurotransmitters) were investigated. We chose to investigate ventilatory pattern (which causes mechanical deformation of the type II cells) and adrenergic agents, because they are the major stimuli for surfactant release in mammals. To examine the effects of body temperature and ventilatory pattern, isolated lungs were ventilated at either 18 or 37 degrees C at different ventilatory regimens. An isolated perfused lung preparation at 27 degrees C was used to analyze the effects of autonomic neurotransmitters. Ventilatory pattern did not affect surfactant release, composition, or lung compliance at either 18 or 37 degrees C. An increase in temperature increased phospholipid reuptake and disproportionately increased cholesterol degradation/uptake. Epinephrine and acetylcholine stimulated phospholipid but not cholesterol release. Removal of surfactant caused a decrease in compliance, regardless of the experimental temperature. Temperature appears to be the principal determinant of lung compliance in the bearded dragon, acting directly to increase the tone of the smooth muscle. Increasing the ambient temperature may result in greater surfactant turnover by increasing cholesterol reuptake/degradation directly and by increasing circulating epinephrine, thereby indirectly increasing phospholipid secretion. We suggest that changing ventilatory pattern may be inadequate as a mechanism for maintaining surfactant homeostasis, given the discontinuous, highly variable reptilian breathing pattern.

Sympathetic nerve influence on alveolar type II cell ultrastructure

Pulmonary surfactant release from lamellar bodies in alveolar type II cells in response to exogenous and circulating catecholamines is well documented but sympathetic influences from direct nerve stimulation are not as extensively studied. In order to study sympathetic nerve influence on release, ultrastructure of type II cells was examined following one min stimulation of the stellate ganglion (SGS) which sends sympathetic fibers to lung parenchyma. Type II cell secretion was assessed by measuring volume density of lamellar bodies, interpreting decreased volume density to indicate increased secretion according to accepted stereologic principles. The present study showed lamellar body volume density was reduced 20.8% in type II cells of SGS animals. Multivesicular body volume density was increased 150% by SGS, but Golgi bodies were unchanged. These data are consistent with type II cell origin of at least some of the increased saturated phospholipid recovered from airspaces in previous SGS studies, and support the view that sympathetic nerve activity may contribute to surfactant homeostasis under some conditions.

Pulmonary surfactant release in fetal rabbits as affected by enprofylline.

At a gestational age of 27.9 days, pregnant rabbits were infused with enprofylline, a new xanthine with potent antiasthmatic effects. Following a priming dose of 5 mg/kg, 20 mg/kg were infused over a 2-h period. Controls received Ringer's solution. On completion of the infusion, the fetuses were delivered and the effect on their surfactant system was evaluated. Surface tension in airway fluid, measured with the pulsating bubble surfactometer, at minimal bubble size in the third cycle, was 21.8 +/- 2.2 (mean +/- SE) versus 28.7 +/- 1.7 mN m-1 (p less than 0.01). Pressure-volume loops of the lungs of the fetuses showed that at a deflation pressure 10 cm H2O the volume was 64 +/- 3 versus 49 +/- 3% (p less than 0.0005). The lung lavage fluid had a phospholipid phosphorus content of 41.9 +/- 3.8 versus 18.5 +/- 1.3 micrograms/g dry lung (p less than 0.0005). The values indicate that enprofylline caused a release of surfactant into the fetal airways.

Radiation induced secretion of surfactant from cell cultures of type II pneumocytes: An in vitro model of radiation toxicity

The pathogenesis of pneumonitis and fibrosis secondary to lung irradiation is incompletly understood. The role of the type II alveolar epithelial pneumocyte in these processes has been under investigation. The type II pneumocyte has been shown in vivo to respond to radiation induced injury with release of pulmonary surfactant. The effect of irradiation on cell cultures of type II pneumocytes was studied to determine if this could be reproduced in vitro. Type II pneumocytes were found to release surfactant material with a threshold of radiation dose between 1000 and 1500 rad. This is similar to the dosage range over which the same effect has been demonstrated in vivo. Experimental results support the concept that the release of surfactant is not due to either cell disruption or non-specific release of phospholipid from cell membranes. Irradiation appears to trigger membrane receptor mediated surfactant release. In addition, irradiation abolishes the ability of cells to subsequently respond to a physiologic agonist, suggesting radiation induced damage to the secretory mechanism. These studies establish that surfactant release in response to irradiation in vivo is a direct effect on type II pneumocytes. Cell cultures of type II pneumocytes can serve as a laboratory model of lung cell radiation toxicity.

Giant lamellar bodies in alveolar type II cells of rats exposed to a low concentration of ozone.

The effect of a low concentration of ozone on pulmonary surfactant release was examined by the ultrastructural morphometry of lamellar bodies in alveolar type II cells. Rats were exposed to approximately 0.3 ppm of ozone for 3 h daily for 16 consecutive days. The volume density of lamellar bodies, relative to the cell, increased significantly following ozone exposure. After 11 days giant lamellar bodies were found in ozone-exposed rats. These findings suggest that ozone exposure causes the impairment of pulmonary surfactant release.

Adrenal epinephrine and the regulation of pulmonary surfactant release in neonatal rabbits

Adrenergic mechanisms influence surfactant metabolism; however the nature and importance of catechol-mediated regulation is unclear. We designed experiments to assess the role of endogenous adrenal epinephrine (E) synthesis in neonatal alveolar surfactant release. We administered the experimental adrenal epinephrine synthesis inhibitor SKF 29661 to pregnant rabbits to reduce fetal adrenal E content. Surfactant release was measured as total alveolar phosphatidylcholine (PC) content recovered by a thorough alveolar wash performed on animals sacrificed at birth and at 1 h of age. At a maternal dose of 100 mg/kg/day, SKF 29661 caused a 57% reduction in fetal adrenal E content (p less than .01), which was associated with a 40% reduction in alveolar PC at birth and a 23% reduction in alveolar PC at 1 h of age (p less than .01). Moreover, adrenal E correlated positively with total alveolar PC content (p less than .001). These results suggest that in the neonatal period, the ability of the fetal adrenal gland to synthesize E for secretion plays a role in the regulation of pulmonary surfactant release.

Pulmonary surfactant release in fetal rabbits: Immediate and delayed response to terbutaline

The fetal lung responds to beta-receptor agonists with a release of surfactant from type II alveolar cells, if such stimulation were intense or long-lasting, the supply might become depleted and, while the supply is being restored, there could be a surfactant deficiency. With experiments on pregnant rabbits, this hypothesis was put to the test and found to be true. At a gestational age of 27.75 days, fetuses received the beta 2-receptor agonist terbutaline, 0.1 or 0.01 mg intramuscularly, or saline only. At the same stage of pregnancy, other does were infused with 7.2 to 9.6 mg of terbutaline or given Ringer's solution. The fetuses were put to death at intervals of 1 to 48 hours, and the lungs were examined. At 1 hour and also at 2 and 3 hours after injection of 0.1 mg of terbutaline, pressure-volume loops demonstrated improved compliance and stability compared to those of controls, but at 24 hours the situation was reversed. At 48 hours there was no difference. At 12 hours from the start of the infusion, pressure-volume loops clearly showed that fetuses of does receiving terbutaline were at a disadvantage. We conclude that terbutaline depleted surfactant stores and, while these stores are being replenished, there is a surfactant deficiency in the alveolar space. Phospholipid analysis supported our conclusion.

The pulmonary consequences of a deep breath

We used the isolated rat lung perfused with Krebs bicarbonate and 4.5% albumin, to examine the effect of a transient increase in peak inspired pressure (PIP). The lung was ventilated with 5% CO 2 in O 2 at a V t of 2.5 ml, an f of 60 min −1 and an end expired pressure of 2 cm H 2O. After 30 min we increased the PIP from 9 to 18 cm H 2O for one breath; following a further 30 sec of normal ventilation we lavaged the lung. The large breath increased the amount of alveolar surfactant phospholipids (PLalv) (control: 7.0 ± 0.73 (11); large breath: 8.3 ± 1.33 (14), mean ± SD in mg·g dry lung −1), and decreased the percentage of PLalv associated with tubular myelin (control: 30.2 ± 3.49% (9); deep breath: 25.4 ± 2.99% (9)). In rats that had received 20μCi · kg −1 of [ methyl 3-H]choline chloride 3 h previously, there was also an increase in the tritium in PLalv expressed as a percent of that in tissue (control: 4.4 ± 0.77% (5); deep breath: 5.7 ± 1.0% (7)). The deep breath also resulted in an increase in oxygen diffusing capacity. We conclude, that a single deep breath results in the opening of atelectatic alveoli, the release of pulmonary surfactant and possibly also the transfer of PLalv from the tubular myelin to the monomolecular phase.

Terbutaline and pulmonary surfactant release in the rabbit fetus.

The influence of terbutaline on content of phospholipids and their fatty acid composition in fetal lung wash and lung homogenates was studied in 28-day-old rabbit fetuses. To obtain both the fatty acid composition and the quantity of lecithin and sphingomyelin gas-liquid chromatography (GLC) was used, which gave a linear relationship when compared to standard solutions of the phospholipids. The amount of lecithin was significantly increased in the lung wash in terbutaline-treated fetuses compared to controls, while the sphingomyelin content was unchanged. In the lung homogenates no quantitative differences were noted between the two groups. The fatty acid composition of lecithin and sphingomyelin did not alter neither in lung wash nor in lung homogenate after terbutaline administration. The present results indicate that beta-mimetics mainly act by favouring the release of preformed surfactant and not by stimulating the synthetic capacity of type II cells in the alveolar lining layer.

1389 SEX DIFFERENCES IN LUNG AND ADRENAL NEUROSYMPATHETIC DEVELOPMENT

Adrenergic mechanisms are known to be important in the synthesis and release of pulmonary surfactant and in the prevention of neonatal respiratory distress syndrome (RDS). We studied neuro-sympathetic development in the lung and adrenal glands in male (M) and female (F) fetal and newborn rabbits. Tissue levels of catecholamines, norepinephrine (NE), epinephrine (E), and dopamine (DA) were measured using a sensitive radioenzymatic assay. Beta adrenergic receptor development in the lung was studied using the tritiated radioligand dihydroalprenolol. Neurosympathetic innervation of the lung was assessed by measuring tissue NE levels which increased gradually from 27 day fetuses to 8 day old newborns. Sex differences were not significant. Lung β-receptor number was significantly elevated in F over M at each gestational age and throughout the neonatal time period investigated (p<0.005) Adrenal gland content of E, NE and DA increased exponentially with advancing developmental age (p<.005). Adrenal E was significantly (p<.01) elevated in F as compared to M fetuses as was the proportion of E. Adrenal NE and DA were similar in M and F fetuses. Sex differences were not significant in the newborn animals. Conclusions: 1) Lung β receptor development but not innervation is accelerated in F fetuses; 2) Adrenal maturation is accelerated in F fetuses; 3) Delayed neurosympathetic and neurohumoral development may explain the male susceptibility to RDS.

Pulmonary surfactant release in fetal rabbits as affected by terbutaline and aminophyllin.

Terbutaline; a beta2-adrenergic agonist, and aminophyllin, a phosphodiesterase inhibitor, were given separately, or in combination, to rabbit fetuses on the 28th day of gestation. The possibility of these drugs causing a release of pulmonary surfactant was evaluated 3 h after i.m. injection directly to the fetus. Lung compliance was studied and lung lavage fluid assessed as regards content to total phospholipid phosphorus, lecithin/sphingomyelin (L/S) ratio, and surface activity. It was found that terbutaline, in a dose of 0.1 mg, had a significant (p less then 0.05) effect on each of the four parameters studied and, when given in a dose of 0.05 mg, significantly affected all except L/S ratio. Aminophyllin, in a dose of 10 mg, resulted in a 40% mortality, but in the surviving fetuses the drug had no significant (p greater than 0.05) effect on any of the four parameters. When 5 mg aminophyllin was given together with 0.05 mg terbutaline, the effect could be attributed to terbutaline.

Isoxsuprine infusion to the pregnant rabbit and its effect on fetal lung surfactant.

Rabbit does, pregnant on the 28th day, were infused with isoxsuprine 2.5 mg/kg body weight/h in 50 ml 5% glucose, or with glucose only. The isoxsuprine caused maternal heart rate to increase and blood pressure, mean and diastolic, to decrease. The volume of fetal pulmonary fluid (FPF) and the wet lung weight/body weight ratio were significantly lowered by isoxsuprine. Lecithin/sphingomyelin ratio of FPF in isoxsuprine-infused animals was higher, compared with controls, not infused. Minimal surface tension of FPF, evaluated with pulsating bubble, was significantly lower in treated fetuses. Histologic examination of the fetal lungs after fixation with potassium dichromate and mercuric chloride showed that in isoxsuprine-treated litters FPF contained more granular and sudanophilic material. The results offer further evidence that isoxsuprine causes dehydration of fetal lungs and a release of pulmonary surfactant.

Isoxsuprine-induced release of pulmonary surfactant in the rabbit fetus

Rabbit fetuses were injected intramuscularly with 0.5 mg. of isoxsuprine on the twenty-eighth day of gestation. They were killed in utero four hours after the injection, and fetal pulmonary fluid (FPF) was collected through a tracheal catheter. The quantity of FPF and the lung weight/body weight ratio were both significantly less in the isoxsuprine-treated fetuses than in control fetuses. Surface activity, evaluated with pulsating bubble, and the lecithin/sphingomyelin (L/S) ratio were greater in FPF from isoxsuprine-treated animals than in control samples. We concluded that isoxsuprine is able to dehydrate the fetal lung and cause a release of surfactant stored in type II pneumocytes. This latter conclusion was supported by a significant decrease in the number of lamellar inclusions observed in these cells.

Effect of postganglionic parasympathetic blockade on pulmonary surfactant.

The existence of a highly surface-active lining at the air-tissue interface in the lungs of a wide variety of species is well established, even though the exact chemical nature of the substance in situ is the subject of controversy (3). Tooley et al. (5) and Bolande and Klaus (1) reported that extracts obtained from the lungs of guinea pigs 2-4 hr after bilateral cervical vagotomy exhibited higher minimum surface tensions than similar extracts obtained from control animals, although the latter investigators felt that the altered surface properties of lung extracts obtained postvagotomy were secondary to the development of pulmonary edema.Pulmonary edema is the inevitable consequence of bilateral cervical vagotomy, and edemagenesis could easily mask any effect which interruption of the parasympathetic nerve supply to the lungs might have on the production and/or release of pulmonary surfactant. It was therefore felt that postganglionic parasympathetic blockade with atropine would aid in revealing whether a...