A mode of formation of tubular myelin from lamellar bodies in the lung

A mechanism is suggested by which the membranes of lamellar bodies are converted to tubular myelin (TM) in the lung. It is argued that a simple corrugation of the membranous sheets can produce the TM formation. Such corrugation would occur in response to simple stresses acting on the lamellar body membranes. The intersections of the tubular figures are formed by fusion of adjacent corners in the corrugations. This results in a more stable hydrophobic bonding of phospholipid molecules. Strong supportive evidence for the mechanism is given by electron micrographs of TM formations.     

Proteomic analysis of lamellar bodies isolated from rat lungs

Background  

Lamellar bodies are lysosome-related secretory granules and store lung surfactant in alveolar type II cells. To better understand the mechanisms of surfactant secretion, we carried out proteomic analyses of lamellar bodies isolated from rat lungs.     

A method for microinjection into subpleural alveoli of rat lung in situ

A new technique is described for the micropuncture of rat lung in the intact thorax. Under pentobarbital sodium anesthesia a glass micropipette is passed through a small area of the parietal pleura, which has been cleared of overlying intercostal muscle. The micropipette is passed into the lung parenchyma and withdrawn again without collapsing the lung. The current application of this technique is in the microinjection of fluid into subpleural alveoli, which is illustrated using a suspension of colloidal gold. The gold particles are immediately dispersed over the surface of many alveoli, a small proportion spreading laterally as far as 4-6 mm. There is no evidence of alveolar flooding.     

Uptake of lung surfactant subfractions into lamellar bodies of adult rabbit lungs

The goals of this investigation were to determine whether subfractions of alveolar surfactant that have different physical and biochemical properties are preferentially taken up from the alveolar air space into lamellar bodies and to correlate the magnitude of the uptake with the properties of the fractions. Radiolabeled subfractions were obtained by differential centrifugation of lavage fluid from rabbits that had been intravenously injected with radioactive palmitate. The subfractions were P (pellet) 3 (1,000 g, 20 min), P4 (60,000 g, 60 min), P5 (100,000 g, 16 h). Subfractions were instilled into the lungs of anesthetized spontaneously breathing adult rabbits, and lavage and lamellar body fractions were isolated at later times. P3 and P4 were taken up to a larger extent than was P5 or liposomes prepared from a P4 lipid extract. The fractions that were preferentially taken up (P3 and P4) contained surfactant apoprotein (APO) 36, tubular myelin, multilamellar vesicles, and were rapidly adsorbed to an air-water interface. P3 also contained APO 10. These results demonstrate that different forms of surfactant are recycled at different rates and suggest that there is specificity in the recycling process.     

Experimental oligohydramnios decreases collagen in hypoplastic fetal rat lungs

Neonates with premature rupture of the membrane and oligohydramnios have an increased risk of acute respiratory morbidity. The aims of this study are to investigate the effects of experimental oligohydramnios on transforming growth factor (TGF)-beta1 and connective tissue growth factor (CTGF) expressions and collagen level in fetal rat lungs. On day 16 of gestation, we anesthetized timed pregnant Sprague-Dawley dams, punctured the uterine wall and fetal membranes of each amniotic sac which resulted in oligohydramnios. Fetuses in the opposite uterine horn served as controls. On days 19 and 21 of gestation, fetuses were delivered by cesarean section. Rats exposed to oligohydramnios exhibited significantly lower lung weight/body weight ratios on days 19 and 21 of gestation than did the control rats. Lung type I collagen and TGF-beta1 mRNA expressions and lung collagen levels were significantly decreased in rats exposed to oligohydramnios on days 19 and 21 of gestation. Type I collagen and inhibitors of metalloproteinase-1 (TIMP-1) proteins were decreased and matrix metalloproteinase-1 (MMP-1) was increased in oligohydramnios-exposed rats on days 19 and 21 of gestation. CTGF mRNA expressions were comparable between control and oligohydramnios-exposed rats on days 19 and 21 of gestation. These data suggest that downregulation of collagen might be involved in the pathogenesis of oligohydramnios-induced respiratory morbidity.     

Calcitonin-sensitive adenylate cyclase in rat renal tubular membranes.

1. Renal tubular membranes from rat kidneys were prepared, and adenylate cyclase activity was measured under basal conditions, after stimulation by NaF or salmon calcitonin. Apparent Km value of the enzyme for hormone-linked receptor was close to 1 x 10(-8) M. 2. The system was sensitive to temperature and pH. pH was found to act both on affinity for salmon calcitonin-linked receptor and maximum stimulation, suggesting an effect of pH on hormone-receptor binding and on a subsequent step. 3. KCl was without effect areas whereas CoCl and CaCl2 above 100 muM and MnCl2 above 1 muM inhibited F- -and salmon calcitonin-sensitive adenylate cyclase activities. The Ca2+ inhibition of the response reflected a fall in maximum stimulation and not a loss of affinity of salmon calcitonin-linked receptor for the enzyme. 4. The measurement of salmon calcitonin-sensitive adenylate cyclase activity as a function of ATP concentration showed that the hormone increases the maximum velocity of the adenylate cyclase. GTP, ITP and XTP at 200 muM did not modify basal, salmon calcitonin- and parathyroid hormone-sensitive adenylate cyclase activities. 5. Basal, salmon calcitonin- and F- -sensitive adenylate cyclase activities decreased at Mg2+ concentrations below 10 mM. High concentrations of Mg2+ (100 mM) led to an inhibition of the F- -stimulated enzyme. 6. Salmon calcitonin-linked receptor had a greater affinity for adenylate cyclase than human or porcine calcitonin-linked receptors. There was no additive effect of these three calcitonin peptides whereas parathyroid hormone added to salmon calcitonin increased adenylate cyclase activity, thus showing that both hormones bound to different membrane receptors. Human calcitonin fragments had no effect on adenylate cyclase activity. 7. Salmon calcitonin-stimulated adenylate cyclase activity decreased with the preincubation time. This was due to progressive degradation of the hormone and not to the rate of binding to membrane receptors.     

Differential effect of colchicine upon the entry of proteins into myelin and myelin related membranes

Brain slices from 20 day old rats were incubated with radioactive aminoacids in the presence and absence of 500 M colchicine and the appearance of labeled proteins in myelin and in a myelin-like fraction (SN₄ fraction) was measured. In the presence of the inhibitor, the entry of proteolipid proteins was decreased to 55% in myelin and to 45% in SN₄ fraction with reference to control values while the entry of basic proteins and other minor protein components was unaffected in both fractions. The synthesis of proteolipid proteins was not affected by the presence of colchicine; moreover, a slight accumulation of these proteins was observed in microsomes. The results suggest that the microtubular system is involved in the transport of proteolipid proteins from their site of synthesis to their site of deposition and that the various types of myelin proteins follow different transport routes to enter into this special type of membrane.     

Escherichia coli administered into pig amniotic cavity appear in fetal airways and attract macrophages into fetal lungs

Escherichia coli (2 x 10(4) bacteria) of the non-pathogenic O86 strain or enteropathogenic O55 strain were administered into the pig amniotic cavity at 79 to 86 days of gestation for six or ten hours. Translocation of bacteria into fetal lungs was confirmed by cultivation as well as by light and electron microscopy. Infection caused an influx of macrophages that were immunostained in cryostat sections by monoclonal antibody recognizing calprotectin.     

Antisense inhibition of surfactant protein A decreases tubular myelin formation in human fetal lung in vitro

Surfactant protein A (SP-A) is the most abundant of the surfactant-associated proteins. SP-A is involved in the formation of tubular myelin, the modulation of the surface tension-reducing properties of surfactant phospholipids, the metabolism of surfactant phospholipids, and local pulmonary host defense. We hypothesized that elimination of SP-A would alter the regulation of SP-B gene expression and the formation of tubular myelin. Midtrimester human fetal lung explants were cultured for 3-5 days in the presence or absence of an antisense 18-mer phosphorothioate oligonucleotide (ON) complementary to SP-A mRNA. After 3 days in culture, SP-A mRNA was undetectable in antisense ON-treated explants. After 5 days in culture, levels of SP-A protein were also decreased by antisense treatment. SP-B mRNA levels were not affected by the antisense SP-A ON treatment. However, there was decreased tubular myelin formation in the antisense SP-A ON-treated tissue. We conclude that selective elimination of SP-A mRNA and protein results in a decrease in tubular myelin formation in human fetal lung without affecting SP-B mRNA. We speculate that SP-A is critical to the formation of tubular myelin during human lung development and that the regulation of SP-B gene expression is independent of SP-A gene expression.     

Extramembraneous particles and structural variations of tubular myelin figures in rat lung surfactant

Tubular myelin figures of pulmonary surfactant were examined by electron microscopy after fixation in glutaraldehyde and postfixation in an osmium tetroxide-ferrocyanide mixture. Bilayered membranes were seen as parallel arrays or as lattices with spacings varying from about 36 to 50 nm. This method also produced good visualization of drumstick-like particles, 5 nm in diameter and about 15 nm in length. The particles were regularly spaced at intervals of 16 nm in rows along the rectangular angles of myelin membranes. Depending on the size of the tubules the particles contacted each other in the center of the tubules at low diameters (tubular diameter less than 40 nm) and formed a continuous filamentous central core, or they were separated from one another (tubular diameter greater than 40 nm). In the latter case the central core had a hollow appearance. Based on further findings employing tannic acid, lipid extraction with 2,2-dimethoxypropane, and a ruthenium red-osmium tetroxide technique for the demonstration of polyanionic proteins it is suggested that these particles are protein in nature and that they are involved in the formation and maintenance of the structure of tubular myelin. A new concept of the ultrastructure of tubular myelin figures is proposed.     

Angiotensin II mediates Tyr-dephosphorylation in rat fetal kidney membranes

Angiotensin II (Ang II) elicits a variety of physiological effects through specific Ang II receptors in numerous tissues. In addition, Ang II is a modulator of cellular growth and exerts a positive or negative effect on cell growth depending on which receptor subtype is activated. Expression of the intrarenal AT₂ receptors occurs at its highest levels in the fetal kidney, with a rapid decline after birth. In the present paper, we performed a study on the signaling mechanism of Ang II receptors in rat fetal (E20) kidney, a rich source of AT₂ receptors, where both Ang II receptor subtypes are present. Ang II induces Tyr-dephosphorylation of proteins in rat fetal kidney membranes. The response is dose-dependent, with a reduction of 20% with respect to the control (100%), signal that is completely reversed by Ang II AT₂ competitor PD123319. Orthovanadate, the inhibitor of phospho-Tyr-phosphatases (PTPase), reverts Ang II effect, suggesting the involvement of a protein tyrosine phosphatase. The peptide analog of Ang II, CGP42112, exhibits an agonist effect, which is dose-dependent. Thus, in rat fetal (E20) kidney, the Ang-induced protein Tyr-dephosphorylation of several proteins is mediated by AT₂ receptors, mechanism that involves an orthovanadate sensitive PTPase.     

The cellular mechanism for the formation of lamellar bodies in type-II pneumocytes in rat lungs]

Pneumocyte type II cells from lungs of native rats and of rats that inspired a hypoxic mixture of gases were investigated by transmission electron microscopy. In cells of experimental rats, membrane structures were found that well compare with lamellar bodies. Experimental results and analysis of literature allowed to put forward a hypothesis about the cell mechanism of formation of lamellar bodies from the spiral twisted membranes of the endoplasmic reticulum.     

In vitro insertion of the myelin proteolipid apoprotein into oligodendrocyte plasma membranes

Uncoated vesicles (UCV) loaded with the myelin proteolipid apoprotein covalently tagged with fluorescein (PLP_F) were found to interact with isolated oligodendrocytes from bovine brain at 4°C as well as at 37°C. After 1.5 hours of incubation, the labeled protein was localized in the cell membranes. After 2.5 hours the fluorescence intensity associated with the oligodendrocytes decreased and completely disappeared at t=3.5 hours. Addition of KCl or EDTA in the incubation medium significantly hindered the interaction with cells. In contrast, the elimination of membrane proteins from UCV did not perturb cell labeling. A specific role of PLP was suggested since UCV loaded with a soluble protein (BSA_F) led to a weak cell labeling.Abbreviations IAF 5-iodacetamidofluorescein - BSA bovine serum albumin - BSA BSA labelled with IAF - PLP proteolipid apoprotein - PLP_F aqueous form of PLP tagged with IAF - CV coated vesicles - UCV uncoated vesicles - UCV^*PLP_F UCV loaded with PLP_F - MV model vesicles This work was suported by Cnrs and INSERM.     

Exchange of sterols between myelin and other membranes of developing rat brain

1. The effect of inhibition of cholesterol synthesis by a hypocholesterolaemic drug (AY-9944) was studied in rat brain during development. 2. At 2 weeks after administration of AY-9944 to young rats 7-dehydrocholesterol accounted for half the total sterol of myelin and other subcellular components. 3. At 4 weeks after injection of the drug 7-dehydrocholesterol had disappeared whereas the cholesterol content of myelin had increased by an equivalent amount. Our studies show that purified myelin has low 7-dehydrocholesterol reductase activity and suggest that 7-dehydrocholesterol is largely converted into cholesterol outside the myelin sheath. 4. Resultant cholesterol may be re-incorporated into myelin by an exchange process. 5. The metabolism of sterols in developing brain is discussed.     

Absorption by rat fetal tissues of 14C-labeled phospholipids injected into the rat body in the late stages of pregnancy

The authors studied the possibility of 14C-phospholipid transplacental penetration after 15C-phospholipid injection into rats at the 20th day of pregnancy. The preparation of 14C-phospholipids (total phospholipids) was isolated by thin-layer chromatography from the liver of rats injected with 2-14C-sodium acetate. One hour after its injection into the rat, 14C-phospholipids were detectable in total phospholipids of the pulmonary and cerebral fetal tissues. It was discovered that specific radioactivity of phospholipids contained by these tissues was 2--5 times higher when 14C-phospholipids were injected subcutaneously as compared with intramuscular injection. It is concluded that exogenous phospholipids entrapped in the mother's circulation penetrate the placental barrier of the fetus and the blood-brain barrier of the mature fetus, being consumed by different fetal tissues for forming membrane structures of the fetal tissues.     

Early biological effects of whole body irradiation on rat lungs

Summary The influence of whole-body irradiation (WBI) with 4, 8 and 15 Gyionizing radiation upon some biochemical indices in the brochoalveolar lavage fluid (BALF) of rat lungs was studied. It was established that lactate dehydrogenase (LDH), alkaline phosphatase, (APH) and acid phosphatase (AcPH) activities show a dose-dependent decrease on the day 1 and day 5 after the irradiation. A similar trend was observed in the total protein content on the day 1. Angiotensin converting enzyme (ACE) activity was increased on the day 1 in the groups irradiated with 8 Gy and 15 Gy in comparison with the controls (190,2% and 187,5%, respectively). It was concluded that WBI decreass LDH, APH and AcPH levels in the lung cells, which secrete them into bronchoalveolar spaces. An irradiation with 8 Gy and 15 Gy WBI provokes an early damage on cytoplasmic membranes of the endothelial cells in lung capillars. It was considered that the bronchoalveolar lavage can find a more wide application for evaluation of the biological effect of ionizing radiation in lungs.