Effect of maternal malnutrition on surface activity of fetal lungs in rats.

The effects of maternal malnutrition on fetal lung growth and surface forces were studied in albino rats. Pregnant albino rats were subjected to one of the following diets: rat chow ad lib. (controls), partial food deprivation (intake one-half that of the controls), complete food deprivation for 4 days (on gestation day 3-7, 9-13, or 17-21), low protein (8%), and fat free. The fetal lungs were studied on the 21st day of gestation (delivered by cesarean section) or at birth (gestation day 22). Fetuses and neonates after maternal food deprivation (FD) on the 17-21 day of pregnancy, and after a low-protein (LP) diet during pregnancy, had significantly smaller body weight and lung wet or dry weight/body weight ratio (hypocellular lungs). The minimum surface tension (gamma min) of fetal lung extracts was significantly increased with FD and LP. This was associated with a reduction of about 35% in lung lecithin content, expressed per lung DNA. The earlier in pregnancy the rat was subjected to 4-day food deprivation the less the effect on the fetus. At birth the gamma min and the lung lecithin content reached control values. This recovery occurred after birth (at age 4-10 h) and prior to first feeding. However, the lungs remained small and hypocellular. The results indicate that the nutritional status of the pregnant rat influences the surface activity and the growth of the fetal lung.     

THE PREPARATION AND USE OF TOBACCO MOSAIC VIRUS CONTAINING RADIOACTIVE PHOSPHORUS

Normal and tobacco mosaic-diseased Turkish tobacco plants were grown in sand for a period of several weeks, during which they were fed daily a complete nutrient solution to which had been added disodium phosphate containing radioactive phosphorus. Determinations were made of the distribution of radioactive phosphorus in different fractions such as the wash from the sand and roots, the press cake obtained on pressing the juice from the plants, the protein and protein-free portions of the supernatant liquids obtained on ultracentrifugation of the juices, and the purified tobacco mosaic virus isolated from the diseased plants. Chemical analyses as well as radiographs of the normal and diseased leaves indicated that they contained the same amount of phosphorus. Approximately 30 per cent of the radioactive phosphorus absorbed by the diseased plants was found to be combined with the purified tobacco mosaic virus that was isolated from these plants. Following the inoculation of purified tobacco mosaic virus possessing high radioactivity to normal Turkish tobacco plants, most of the radioactivity was found to be associated with non-virus components of which about 40 per cent was in the inoculated and 60 per cent in the uninoculated portions of the plants. Although a small amount of radioactive virus was isolated from the uninoculated portions of the plants, it was impossible, because of a number of complicating factors which have been discussed, to draw from the results any reliable conclusions regarding the mode of reproduction of tobacco mosaic virus.     

Phospholipid content, composition and biosynthesis during fetal lung development in the rabbit.

The phospholipid content and composition of lung wash and lung tissue as well as the activities of the enzymes involved in the synthesis of phosphatidylcholine and phosphatidylglycerol (the major surface active components of pulmonary surfactant) were studied in the rabbit during fetal lung development. In lung wash the amount of phospholipid increased four-fold during the period 27-31 day's gestation. There was a further ten-fold increase following the onset breathing. During the same period the amount of phosphatidylcholine in lung wash increased from 29% of the total phospholipid to 80% while the amount of sphingomyelin decreased from 38% to 2%. The amount of phosphatidylcholine in lung tissue also increased during development but to a much lesser extent. During fetal lung development the activities of choline kinase and cholinephosphate cytidyltransferase changed little, cholinephosphotranserase decreased while lysophosphatidic acid acyltransferase and lysolecithin acyltransferase increased. There was a postnatal increase in the activities of cholinephosphate cytidyltransferase, cholinephosphotransferase and both acyltransferases. The amount of phosphatidylglycerol, as a percentage of the total phospholipid, in lung wash and lung tissue as well as the activity of pulmonary glycerolphosphate phosphatidyltransferase did not change appreciably during development.     

Incorporation of acetate into fatty acids and lecithin by lung slices from fetal and newborn lambs

Incorporation of acetate-1-(14)C into phospholipids and fatty acids by lung slices from fetal and newborn lambs and from ewes was studied in vitro. The distribution of radioactivity in the fatty acids of neutral lipids, phospholipids, and lecithin was determined. Acetate-1-(14)C was incorporated into myristic, palmitic, and C(18) fatty acids. Of the lecithin fatty acids, myristic and palmitic were the major radioactive fatty acids. The results indicate that the lung of fetal lambs is able to synthesize lecithin containing saturated fatty acids, a major constituent of pulmonary surfactant. A marked increase in the rate of acetate incorporation into lecithin was observed during maturation, and these rates were higher than those obtained in the ewes. A possible relationship between developmental changes in lecithin biosynthesis and pulmonary surfactant is discussed.     

Characterization of cell-free synthesis of collagen by lung polysomes in a heterologous system.

In normal lung growth, post-pneumonectomy lung growth, and in possibly several lung disorders, there are marked alterations in the density of collagen and changes in the rate of synthesis of collagen relative to the synthesis of other lung proteins. To provide a technology to begin to understand these changes at the molecular level, polysomes were prepared from rabbit lung and translated in a heterologous cell-free system including rabbit reticulocyte 0.5 M KCl ribosomal wash fraction and liver tRNA. Collagen was shown in the cell-free product by collagenase sensitivity, hydroxylation of incorporated proline by peptidyl prolyl hydroxylase, agarose gel chromatography, and sodium dodecyl sulfate acrylamide gel electrophoresis. The cell-free system was optimized with respect to K+, Mg2+, amino acids, and ribosomal wash fraction and used under conditions where total protein synthesis and collagen synthesis are linear with respect to time and amount of polysomes. Under these conditions, collagen synthesis was directed almost entirely by polysomes derived from the endoplasmic reticulum. Polysomes isolated from late fetal lung directed collagen synthesis at twice the rate (per polysome) as those polysomes isolated from adult lung. Similar changes were seen if lung tRNA replaced liver tRNA and if lung ribosomal wash fraction replaced reticulocyte wash fraction. Although these changes in cell-free lung collagen synthesis with tissue explants, further studies will have to be carried out to determine whether, in fact, age-related alterations in control of lung collagen synthesis are truly explained by these findings.     

Oxygen-induced changes in pulmonary phospholipids in the rat.

The composition and synthesis of alveolar and lung tissue phospholipids were investigated in normal and oxygen-poisoned rat lungs. Sixty-hour exposure to oxygen increased the total amount of phospholipids in the endobronchial extracts and lung tissue. Phosphatidyl glycerol was identified in both endobronchial extracts and lung tissue. The amount of unsaturated fatty acids in surfactant lecithin and phosphatidyl glycerol was slightly increased in oxygen-poisoned lungs whereas the composition of phospholipids in the endobronchial extracts was not affected by oxygen. After intraperitoneal administration of [32P]phosphate the specific activities of surfactant lecithin and phosphatidyl glycerol were clearly lower in oxygen-treated animals whereas the specific activities of lung tissue lecithin and phosphatidyl glycerol remained unaffected. The synthesis of lecithin from [14C]methionine through N-methyltransferase pathway was markedly depressed in lung slices but increased in liver tissue taken from oxygen-poisoned rats and incubated under oxygen indicating a difference between lung and liver methyltransferase enzymes. In conclusion, the present work suggests impaired synthesis and removal of alveolar phospholipids in oxygen-poisoned rats.     

Clara cells in the llama.

A study was made by light and electron microscopy of the Clara cells of two llamas born and bred at an altitude of 4,720 m in the Peruvian Andes. The Clara cells were numerous and prominent with big apical caps, many of which had been extruded into the terminal bronchioles. On electron microscopy the caps were found to contain vesicular endoplasmic reticulum. Previous studies have shown this to contain dipalmitoyl lecithin, a known pulmonary surfactant. Acute exposure to a simulated altitude of 4,270 m has been reported to increase surface tension in lung extracts of mice. Hence it may be that an animal, such as the llama, chronically exposed to high altitude requires a persistent secretion of pulmonary surfactant.     

A spin probe study of the influence of cholesterol on motion and orientation of phospholipids in oriented multibilayers and vesicles

Spin probes have been used to study at the molecular level the influence of cholesterol on bilayers of egg lecithin and dipalmitoyl lecithin. Distinct differences between the two lecithin systems were revealed. Increasing amounts of cholesterol result in extension of the fatty acid chains and decreased amplitude of motion of the long axes of the fatty acids in egg lecithin. In dipalmitoyl lecithin cholesterol causes an increase in the mobility and amplitude of motion of the fatty acid side chains, presumably due to alteration of the molecular interactions between phospholipids by relaxing the close packing of these molecules. These data provide an explanation for the condensing and fluidizing effects of cholesterol in water-containing phases and monolayers of egg lecithin and dipalmitoyl lecithin, respectively, and for the permeability behavior of egg lecithin and dipalmitoyl lecithin liposomes in the presence and absence of cholesterol. Differences are revealed between the spin bilayer environments in hydrated phospholipid films and vesicles.     

Phase transition in lipid multilayers induced by benzene. A Raman spectroscopic study

The interaction of increasing amount of benzene with dipalmitoyl lecithin multilayers has been investigated by Raman spectroscopy. It is shown that benzene is able to promote a gel→liquid crystalline phase transition.     

The interaction of 2,4-dinitrophenol with phospholipids at phospholipid-water interfaces

The effect of 2, 4-dinitrophenol, DNP, on monolayers of egg lecithin, hydrogenated egg lecithin, dipalmitoyl lecithin and mitochondrial lipids has been examined. Both the undissociated and dissociated forms of DNP bind to the phospholipid polar groups. Binding of the acid form leads to a decrease in monolayer surface potential and an expansion of the monolayer. The amount of penetration of the acid form into lecithin monolayers appears to depend on the London-Van der Waals attractions between the lecithin hydrocarbon chains. Binding of the 2,4-dinitro-phenolate anion is reflected in a decrease in surface potential for lecithin monolayers, and an increase in surface potential for mitochondrial lipid monolayers. The adsorption of dinitro-phenolate to egg lecithin has been further investigated by micro-electrophoresis of lecithin liposomes. It is suggested that binding of DNP to phospholipid-water interfaces is important in determining its action as an uncoupler of oxidative phosphorylation, and as a compound that increases the electrical conductance of artificial lipid membranes.     

Interaction of phospholipid vesicles with cells. Endocytosis and fusion as alternate mechanisms for the uptake of lipid-soluble and water-soluble molecules

Depending on their phospholipid composition, liposomes are endocytosed by, or fuse with, the plasma membrane, of Acanthamoeba castellanii. Unilamellar egg lecithin vesicles are endocytosed by amoeba at 28 degrees C with equal uptake of the phospholipid bilayer and the contents of the internal aqueous space of the vesicles. Uptake is inhibited almost completely by incubation at 4 degrees C or in the presence of dinitrophenol. After uptake at 28 degrees C, the vesicle phospholipid can be visualized by electron microscope autoradiography within cytoplasmic vacuoles. In contrast, uptake of unilamellar dipalmitoyl lecithin vesicles and multilamellar dipalmitoyl lecithin liposomes is only partially inhibited at 4 degrees C, by dinitrophenol and by prior fixation of the amoebae with glutaraldehyde, each of which inhibits pinocytosis. Vesicle contents are taken up only about 40% as well as the phospholipid bilayer. Electron micrographs are compatible with the interpretation that dipalmitoyl lecithin vesicles fuse with the amoeba plasma membrane, adding their phospholipid to the cell surface, while their contents enter the cell cytoplasm. Dimyristoyl lecithin vesicles behave like egg lecithin vesicles while distearoyl lecithin vesicles behave like dipalmitoyl lecithin vesicles.     

In vitro cytotoxicity of chrysotile asbestos to human pulmonary alveolar macrophages is decreased by organosilane coating and surfactant

Human pulmonary alveolar macrophages were used to quantitate the cytotoxic effect of surface-altered chrysotile asbestos. Little difference was observed in mortality between chrysotile asbestos that was surface-treated to a 42% extent by a hydrophobic organosilane or untreated chrysotile. Little or no effect on mortality was observed when human pulmonary alveolar macrophages were cultured with untreated chrysotile or acid-leached asbestos in the presence of 10 mM dipalmitoyl lecithin. However, when human pulmonary alveolar macrophages were cultured with a hydrophobically-treated (to a 42% or 95% extent) chrysotile asbestos in the presence of 10 mM dipalmitoyl lecithin, a statistically significant decrease in mortality was observed compared to untreated chrysotile. No mutagenic activity was observed when V79 cells were cultured with acid-leached, or 42% hydrophobically-treated chrysotile asbestos, even when human pulmonary alveolar macrophages were included as an activation source. The 95% hydrophobically-treated and acid-leached chrysotile also exhibited decreased binding of benzo[a]pyrene compared to untreated chrysotile asbestos.Abbreviations AHH aryl hydrocarbon hydroxylase - B(a)P benzo[a]pyrene - CA chrysotile asbestos - CHO Chinese hamster ovary - DL dipalmitoyl lecithin - DMEM Dulbecco's Modified Eagle's Medium - FBS fetal bovine serum - O^r resistance to ouabain - PAH polycyclic aromatic hydrocarbon - PAM pulmonary alveolar macrophage - SCE sister chromatid exchange Deceased.     

Lecithins enhance leukotriene production from white cells

36 x 10(7) WBC were isolated from 120 ml heparinized venous blood by 5% dextran T-500 sedimentation. 20 mg egg lecithin and 20 mg dipalmitoyl lecithin were respectively pretreated in 2 ml 0.15 M Tris buffer by vibration and sonication. WBC were incubated with the pretreated lecithins for 20 min. Leukotrienes (LTs) were identified by HPLC and bioassay, and quantified with an RIA Kit. Crude incubation medium of both lecithin groups caused guinea pig ileum contractions which were antagonized with FPL55712. Incubation media were partially purified with Bond elut C18. Purified samples of both lecithin groups showed LTC4 and LTD4 peaks on HPLC. LTC4 production (pg/10(7) WBC, M +/- SD) was 194.5 +/- 61.7 (n = 5) in control group, 348.9 +/- 95.4 (n = 6) in dipalmitoyl lecithin group, 543.8 +/- 105.6 (n = 6) in egg lecithin group and 105.62 +/- 63.2 (n = 6) in AA-861 + dipalmitoyl lecithin group. LTC4 production of both lecithin groups was significantly higher than that of control group (P less than 0.01 in dipalmitoyl lecithin group and P less than 0.001 in egg lecithin group). Both egg lecithin and dipalmitoyl lecithin enhanced LT production from WBC. LT production was suppressed in the presence of AA-861. The mechanism of the enhancement in LT production is unclear, but these lecithins are apparently not substrates because dipalmitoyl lecithin contains no arachidonic acid.     

Polyene antibiotic action on lecithin liposomes: effect of cholesterol and fatty acyl chains

The effect of cholesterol incorporation upon amphotericin B and nystatin susceptibility of lecithin liposome systems containing various fatty acids has been studied. Cholesterol was shown to: 1) confer sensitivity to low concentrations of amphotericin B in liposomes derived from egg lecithin, and 2) suppress the amphotericin B and nystatin-induced response in liposomes derived from dipalmitoyl or distearoyl lecithins. This clear cut difference cannot be explained by mechanisms of drug action so far presented. They are discussed in connection with the possibility that susceptibility to these polyene antibiotics is related to the over-all state of the membrane organization, in particular to the over-all conformation of membrane components.     

Proton NMR bandshape studies of lamellar liquid crystals and gel phases containing lecithins and cholesterol.

Proton NMR spectra for gel and liquid crystalline samples, composed of dimyristoyl and/or dipalmitoyl lecithin, cholesterol and water, can be consistently interpreted in terms of mesophase symmetry and molecular diffusion according to a model proposed by Wennerstrom (Wennerstrom, H. (1973) Chem. Phys. Lett. 18, 41-44). It is shown by computer simulation that the characteristic "super-lorentzian" bandshape of the lamellar mesophase can be described by the superposition of three gaussian curves. The NMR signal of the gel phase can be simulated by the superposition of two gaussian curves with widths at half height of 2.5 kHz and 19 kHz. An upper limit of the lateral diffusion coefficient of the lecithin molecules in the gel phase is calculated to be about 5-10(-15) m-2/s. It is therefore concluded that the static intermolecular dipolar couplings average to zero in the lamellar mesophase. An estimation of the order parameter of the liquid crystalline phase is made from experimental data and a calculated "rigid lattice" linewidth. A two phase system is shown to exist in the temperature range 28-34 degrees C for a mesophase of a mixture of dimyristoyl and dipalmitoyl lecithin. The presence of cholesterol results in enhanced lateral diffusion of the lecithin molecules at temperatures below the Chapman transition point.     

Influence of dexamethasone on the lipid distribution of newly synthesized fatty acids in fetal rat lung

There is a developmental increase in fatty acid biosynthesis and surfactant production in late-gestation fetal lung and both are accelerated by glucocorticoids. We have examined the distribution of the newly synthesized fatty acids to determine whether they are preferentially incorporated into surfactant. Explants of 18 day fetal rat lung were cultured with and without dexamethasone for 48 h and then with [3H]acetate for 4 h after which labeled fatty acids were measured. Incorporation of radioactivity from acetate was considered a measure of newly synthesized fatty acids. Phospholipids contained 86% of the newly synthesized fatty acids of which approx. 80% were in phosphatidylcholine. Phosphatidylcholine and disaturated phosphatidylcholine contained a much greater percentage of the labeled fatty acids than of the phospholipid mass determined by phosphorus assay while phosphatidylethanolamine, phosphatidylserine and sphingomyelin contained less. Dexamethasone increased the rate of acetate incorporation into total lipid fatty acids but it had little effect on fatty acid distribution, except that it increased the percentages in phosphatidylglycerol and disaturated phosphatidylcholine. The hormone also increased the mass of these two phospholipids to a greater extent than that of the total. These data suggested that the newly synthesized fatty acids are preferentially incorporated into surfactant phospholipids and that this process is accelerated by dexamethasone. However, since phosphatidylcholine and phosphatidylglycerol are not exclusive to surfactant, we compared isolated lamellar bodies with a residual fraction not enriched in surfactant. The rate of acetate incorporation into fatty acids in lamellar body phosphatidylcholine as well as its specific activity (radioactivity per unit phosphorus) were both increased by dexamethasone. Specific activity, however, was no greater in the lamellar bodies than in the residual fraction in both control and dexamethasone-treated cultures. Therefore, there is no preferential incorporation of newly synthesized fatty acids into phospholipids in surfactant as opposed to those in other components of the lung.     

Use of liposomes in probing the uptake of liposomal phosphatidylcholine by rabbit lung in vitro.

The purpose of this study was to characterize the uptake of liposomal phosphatidylcholine by lung tissue and its subcellular organelles. Multilamellar liposomes were prepared from egg yolk phosphatidylcholine, dicetyl phosphate, and cholesterol (molar ratio 7 : 2 : 1). Liposomal phosphatidylcholine labeled with [1-14C]dipalmitoyl phosphatidylcholine was taken up by lung slices and incorporated into subcellular organelles including lamellar bodies, mitochondria, and microsomes. In addition, when liposomes were incubated with lamellar bodies, mitochondria, or microsomes, the transfer of liposomal phosphatidylcholine to these subcellular fractions was facilitated by the cytosolic fraction. In tissue slice experiments after 1 h of incubation, about 86% of the total radioactivity absorbed by lung slices and subcellular organelles was recovered in phosphatidylcholine. The ratio of the radioactivity of fatty acids at 1- and 2-positions of dipalmitoyl phosphatidylcholine recovered from all fractions was nearly 1 : 1. This suggests that most phosphatidylcholine molecules were taken up intact. In conclusion, this study provides a method using liposomes as a tool for probing the phosphatidylcholine transfer mechanism in lung.     

Amniotic fluid embolism and leukotrienes--the role of amniotic fluid surfactant in leukotriene production.

Surfactant rich lipid (lipid) was extracted from cell free 10,000 x g pellets of amniotic fluid. White blood cells (WBC) were isolated from human donors. 36 x 10(7) WBC and 5 g rabbit lung were incubated with pretreated lipid or dipalmitoyl lecithin (lecithin). Leukotrienes (LTs) were identified by high performance liquid chromatography (HPLC) and bioassay, and quantified by radioimmunoassay. Peaks of LTC4 and LTD4 on HPLC and guinea-pig ileum contraction could be identified in lipid and lecithin groups, but not in the control group. LTC4 production by lipid and lecithin groups was significantly higher than that by the control group. An involvement of amniotic fluid surfactant in leukotriene production is suggested.     

Hormonal control of pulmonary surfactant synthesis

The specific activities of palmitoyl-CoA synthetase, phospholipase A2, and lysophosphatidylcholine acyltransferase enzymes were low in the lungs of diabetic and hypophysectomized rats as compared to those found in the normal controls. Administration of triiodothyronine (T3), to the diabetic and hypophysectomized rats restored the normal activities of these enzymes. Stimulation of the enzyme activities were also observed when normal rats were injected with the above hormone. The enhancement of the enzyme activities was also found to be dependent on the dose and duration of the hormonal treatment. Optimum levels were achieved at a dose of about 100 micrograms/100 g body weight of T3, 3-4 days after the administration of this hormone. Actinomycin D or cycloheximide abolished the hormone-mediated stimulation of these enzymes in diabetic and hypophysectomized rats. Reduced rate of in vivo palmitoyl-CoA synthetase synthesis was observed in the lungs of diabetic and hypophysectomized animals. Administration of T3 stimulated the rate of synthesis of this enzyme indicating increasing synthesis of this enzyme and not of activation of the pre-existing inactive species. Reduced phospholipid contents, specially decreased amount of lecithin and dipalmitoyl lecithin (DPL) were observed in the lungs of the diabetic and hypophysectomized animals as compared to those in the normal animals. T3 also increased the lecithin and DPL content of the normal rat lungs. These results provide evidence for the involvement of the thyroid hormones in the control of the pulmonary surfactant. The results further suggest that T3 was capable of inducing the enzymes of the "deacylation-reacylation" pathway involved in palmitate incorporation into phosphatidylcholine thereby contributing to the stimulation of dipalmitoyl phosphatidylcholine biosynthesis.     

The ionic structure of lecithin monolayers

Surface potentials of mixed monolayers of dicetyl phosphate and eicosanyl trimethylammonium bromide (1:1) were the same on subsolutions of 0.02 M NaCl or 0.01 M CaCl(2), which indicated that ionic phosphate does not interact with Ca(++) in the presence of a neighboring trimethylammonium group. Surface potential-pH plots of dicetyl phosphate, and of dipalmitoyl, egg, and dioleoyl lecithins showed that as the pH of the subsolution is decreased the phosphate groups in the monolayer are neutralized in the order: dicetyl phosphate > dipalmitoyl lecithin > egg lecithin > dioleoyl lecithin. The binding of cations (Na(+), Ca(++)) to the phosphate group of lecithin also showed the same order. The binding of Ca(++)) to egg phosphatidic acid monolayers, as measured by the increase in surface potential, is considerably greater than that to egg lecithin. These results suggest that there is an internal salt linkage between the phosphate and trimethylammonium groups on the same lecithin molecule. An increase in unsaturation of fatty acyl chains increases the intermolecular spacing, which reduces the ionic repulsion between polar groups, and hence strengthens the internal salt linkage. The results support the concept of a vertical rather than coplanar orientation of the phosphoryl choline group with respect to the interface. A position has been proposed for Ca(++) in the dipole lattice of lecithin from a consideration of the surface potential measurements.