Effect of hydrophobic surfactant peptides SP-B and SP-C on binary phospholipid monolayers. I. Fluorescence and dark-field microscopy. (49/1770)

The influence of the hydrophobic proteins SP-B and SP-C, isolated from pulmonary surfactant, on the morphology of binary monomolecular lipid films containing phosphocholine and phosphoglycerol (DPPC and DPPG) at the air-water interface has been studied using epifluorescence and dark-field microscopy. In contrast to previously published studies, the monolayer experiments used the entire hydrophobic surfactant protein fraction (containing both the SP-B and SP-C peptides) at physiologically relevant concentrations (approximately 1 wt %). Even at such low levels, the SP-B/C peptides induce the formation of a new phase in the surface monolayer that is of lower intrinsic order than the liquid condensed (LC) phase that forms in the pure lipid mixture. This presumably leads to a higher structural flexibility of the surface monolayer at high lateral pressure. Variation of the subphase pH indicates that electrostatic interaction dominates the association of the SP-B/C peptides with the lipid monolayer. As evidenced from dark-field microscopy, monolayer material is excluded from the DPPC/DPPG surface film on compression and forms three-dimensional, surface-associated structures of micron dimensions. Such exclusion bodies formed only with SP-B/C peptides. This observation provides the first direct optical evidence for the squeeze-out of pulmonary surfactant material in situ at the air-water interface upon increasing monolayer surface pressures.  (+info)

Surfactant secretagogue activation of protein kinase C isoforms in cultured rat type II cells. (50/1770)

Several lung surfactant secretagogues are known to activate protein kinase C (PKC) in type II cells. Such agents include 12-O-tetradecanoylphorbol 13-acetate (TPA) and cell-permeable diacylglycerols that directly activate PKC. Other agents include ATP and UTP, which act at P2Y(2) receptors coupled to phosphoinositide-specific phospholipase C, activation of which leads to formation of diacylglycerols and consequent activation of PKC. Activation of PKC is associated with redistribution of enzyme from a cytosolic to a membrane fraction of the cell. We examined the PKC isomers that are translocated by ATP, UTP, TPA, and dioctanoylglycerol in cultured type II cells isolated from adult rats. PKC isoforms were identified by Western blotting using isoform-specific antibodies. Treatment of type II cells with ATP, UTP, TPA, and dioctanoylglycerol resulted in a significant redistribution of PKC-mu from cytosol to membrane. TPA and dioctanoylglycerol also activated PKC-alpha, -betaI, -betaII, -delta, and -eta, but those isoforms were not activated by ATP or UTP. The effects of TPA and dioctanoylglycerol on PKC-mu were more pronounced than those of the P2Y(2) agonists, and the effect of TPA was also more rapid than that of ATP. The data show that direct activators and agents that generate endogenous diacylglycerols have different PKC activation patterns. Because it is activated by different types of secretagogues, PKC-mu may have an important role in the physiological regulation of surfactant secretion.  (+info)

Mechanical ventilation of rat lung: effect on surfactant forms. (51/1770)

Mechanical ventilation of the lung could affect surfactant turnover by alteration of its secretion, recycling, and degradation. In vitro studies of surfactant subfractions recoverable from lavage fluid have led to predictions about surfactant physiology in vivo that include morphological transformations. We used electron microscopy to study in situ lipid forms in alveoli of rat lungs after two ventilation strategies [15 min at pressures (cmH(2)O) of 20/0 or 20/10]. In control animals, 4% of the lipid profile area in the surface lining layer was myelin figures (MF), 14% was tubular myelin, 37% was vesicular forms (VF), and the remainder (45%) was hypophase. Compared with controls, the length-normalized sum of the lipid forms and the hypophase was two times as great in the lungs of the 20/0 group. MF were threefold higher in the 20/0 group and fivefold higher in the 20/10 group. VF doubled after ventilation at 20/0, but VF were the same as control after ventilation at 20/10. The results showed that a ventilation pattern of 20/0 compared with that of 20/10 group was associated with a significantly larger VF, suggesting an increased net production of these surfactant forms during a large tidal volume breathing pattern. These morphological results are consistent with published results using physical methods of fractionating lung lavage.  (+info)

Analysis of genomic regions involved in regulation of the rabbit surfactant protein A gene in transgenic mice. (52/1770)

The gene encoding surfactant protein (SP) A, a developmentally regulated pulmonary surfactant-associated protein, is expressed in a lung-specific manner, primarily in pulmonary type II cells. SP-A gene transcription in the rabbit fetal lung is increased by cAMP. To delineate the genomic regions involved in regulation of SP-A gene expression, lines of transgenic mice carrying fusion genes composed of various amounts of 5'-flanking DNA from the rabbit SP-A gene linked to the human growth hormone structural gene as a reporter were established. We found that as little as 378 bp of 5'-flanking DNA was sufficient to direct appropriate lung cell-selective and developmental regulation of transgene expression. The same region was also sufficient to mediate cAMP induction of transgene expression. Mutagenesis or deletion of either of two DNA elements, proximal binding element and a cAMP response element-like sequence, previously found to be crucial for cAMP induction of SP-A promoter activity in transfected type II cells, did not affect lung-selective or temporal regulation of expression of the transgene; however, overall levels of fusion gene expression were reduced compared with those of wild-type transgenes.  (+info)

Adenovirus-mediated decorin gene transfer prevents TGF-beta-induced inhibition of lung morphogenesis. (53/1770)

Excessive transforming growth factor (TGF)-beta signaling has been implicated in pulmonary hypoplasia associated with bronchopulmonary dysplasia, a chronic lung disease of human prematurity featuring pulmonary fibrosis. This implies that inhibitors of TGF-beta could be useful therapeutic agents. Because exogenous TGF-beta ligands are known to inhibit lung branching morphogenesis and cytodifferentiation in mouse embryonic lungs in ex vivo culture, we examined the capacity of a naturally occurring inhibitor of TGF-beta activity, the proteoglycan decorin, to overcome the inhibitory effects of exogenous TGF-beta. Intratracheal microinjection of a recombinant adenovirus containing decorin cDNA resulted in overexpression of the exogenous decorin gene in airway epithelium. Although exogenous TGF-beta efficiently decreased epithelial lung branching morphogenesis in control cultures, TGF-beta-induced inhibition of lung growth was abolished after epithelial transfer of the decorin gene. Additionally, exogenous TGF-beta-induced antiproliferative effects as well as the downregulation of surfactant protein C were abrogated by decorin in cultured embryonic lungs. Moreover, lung branching inhibition by TGF-beta could be restored by the addition of decorin antisense oligodeoxynucleotides in culture, indicating that decorin is both specifically and directly involved in suppressing TGF-beta-mediated negative regulation of lung morphogenesis. Our findings suggest that decorin can antagonize bioactive TGF-beta during lung growth and differentiation, establishing the rationale for decorin as a candidate therapeutic approach to ameliorate excessive levels of TGF-beta signaling in the developing lung.  (+info)

Pulmonary surfactant in health and human lung diseases: state of the art. (54/1770)

Pulmonary surfactant is a complex and highly surface active material composed of lipids and proteins which is found in the fluid lining the alveolar surface of the lungs. Surfactant prevents alveolar collapse at low lung volume, and preserves bronchiolar patency during normal and forced respiration (biophysical functions). In addition, it is involved in the protection of the lungs from injuries and infections caused by inhaled particles and micro-organisms (immunological, non-biophysical functions). Pulmonary surfactant can only be harvested by lavage procedures, which may disrupt its pre-existing biophysical and biochemical micro-organization. These limitations must always be considered when interpreting ex vivo studies of pulmonary surfactant. A pathophysiological role for surfactant was first appreciated in premature infants with respiratory distress syndrome and hyaline membrane disease, a condition which is nowadays routinely treated with exogenous surfactant replacement. Biochemical surfactant abnormalities of varying degrees have been described in obstructive lung diseases (asthma, bronchiolitis, chronic obstructive pulmonary disease, and following lung transplantation), infectious and suppurative lung diseases (cystic fibrosis, pneumonia, and human immunodeficiency virus), adult respiratory distress syndrome, pulmonary oedema, other diseases specific to infants (chronic lung disease of prematurity, and surfactant protein-B deficiency), interstitial lung diseases (sarcoidosis, idiopathic pulmonary fibrosis, and hypersensitivity pneumonitis), pulmonary alveolar proteinosis, following cardiopulmonary bypass, and in smokers. For some pulmonary conditions surfactant replacement therapy is on the horizon, but for the majority much more needs to be learnt about the pathophysiological role the observed surfactant abnormalities may have.  (+info)

99mTc-ENS: a new radiopharmaceutical for aerosol lung scintigraphy. Comparison between different freeze-dried formulations. (55/1770)

Exogenous natural surfactant (ENS) labeled with 99mTc(99mTc-ENS) is a new radiopharmaceutical for pulmonary aerosol scintigraphy. In this study, different freeze-dried formulations were evaluated to develop a suitable and long-storage method for the ENS, the nonradioactive precursor of this radiopharmaceutical. METHODS: Two freeze-dried formulations were evaluated: the sterile ENS suspension-stannous chloride altogether lyophilized (chlorlioENS) and the lyophilized sterile ENS suspension with the addition of stannous chloride as a solid drug (lioENS). These precursors were stored at room temperature for 3 mo and then labeled with 99mTc. For comparative purposes, the sterile ENS suspension with the addition of stannous chloride labeled with 99mTc(99mTc-chlorENS) was also studied. The quality controls for each radiopharmaceutical were performed by an ascending paper chromatography to determine the labeling yield percentages. The study was performed in 30 female Sprague Dawley rats, which inhaled each radiopharmaceutical by nebulization. Twenty-five minutes after the aerosol inhalation, the animals were killed to extract their organs and measure their activity in a gamma spectrometer. The data are given as the percentage of activity concentration (C%) for each organ. RESULTS: The physicochemical properties of lioENS were adequate for a freeze-dried product. The labeling yields for 99mTc-lioENS and for 99mTc-chlorENS were always greater than 95% even after nebulization. The results of the biologic distribution studies showed that the activity concentration found in lungs for these radiopharmaceuticals were 95.7% +/- 2.6% and 96.7% +/- 2.6% respectively, results that do not differ statistically. On the other hand, the activity concentration found in lungs for the 99mTc-chlorlioENS (31.3% +/- 11.1%) and its labeling yield percentages (<10%) are statistically different (P < 0.05) from the results obtained with the two radiopharmaceuticals mentioned above. CONCLUSION: Taking into account the lioENS physicochemical properties, its long shelf life and that 99mTc-lioENS shows the same radiochemical and radiopharmacological behavior of the 99mTc-chlorENS, it can be concluded that the 99mTc-lioENS can be used for aerosol lung scintigraphy.  (+info)

Aerosolized endotoxin is immediately bound by pulmonary surfactant protein D in vivo. (56/1770)

Collectins are carbohydrate binding proteins that are implicated in innate host defense. The lung collectins, surfactant proteins A and D (SP-A and SP-D), bind a variety of pathogens in vitro and influence phagocytosis by alveolar macrophages. In this report we show that SP-D binds endotoxin (lipopolysaccharide, LPS) in vivo in a rat model of acute respiratory distress syndrome (ARDS). Intratracheal aerosolization of LPS in rats resulted in the typical features of human ARDS. Total amounts of SP-D, as well as the carbohydrate binding properties of SP-D were measured in lung lavage as a function of time. The amount of SP-D did not change during 24 h. Interestingly, SP-D in lung lavage isolated from rats during the first 2 h after LPS treatment, was not able to bind to carbohydrate. Further analysis revealed that the carbohydrate binding sites of SP-D were occupied by LPS, suggesting that SP-D is an LPS scavenging molecule in vivo. Electron microscopic analysis indicated that, 1 h after LPS aerosolization, aggregates of SP-D with LPS were found in lysosomal structures in alveolar macrophages. We conclude that the lung collectin SP-D binds inhaled endotoxin in vivo, which may help to protect the lung from endotoxin-induced disease.  (+info)