Lamellar Body Fraction Research Articles

Degradation and reutilization of alveolar phosphatidylcholine by rat lungs.

We have shown previously that phospholipids instilled through the trachea are removed from the air spaces in isolated rat lungs by a process that is stimulated by beta-adrenergic agonists. In this study, we evaluated the fate of radiolabeled lipid vesicles [50% [3H]dipalmitoyl phosphatidylcholine (DPPC), 25% phosphatidylcholine (PC), 15% cholesterol, and 10% phosphatidylglycerol (PG)]. Vesicles were instilled through the trachea of anesthetized rats, and the lungs removed for perfusion. The percent of instilled 3H that could not be removed from lungs by extensive lung lavage increased progressively; at 3 h this fraction was 25.8 +/- 0.63% (mean +/- SE; n = 8). The percent of dpm in the lung homogenate accounted for by PC decreased progressively while dpm in lyso-PC, unsaturated PC, and aqueous soluble metabolites [choline, choline phosphate, glycerophosphorycholine, and cytidine 5'-diphosphate (CDP) choline (CDP-choline) increased. The dpm in microsomal and lamellar body fractions isolated from lung homogenates also increased progressively with time of perfusion. The presence of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) significantly stimulated both uptake of DPPC and the appearance of radioactivity in metabolites and subcellular organelles. This effect of 8-BrcAMP was not due to stimulation of phospholipase A activity. These results indicate that exogenous phospholipids instilled into the air spaces of rat lungs are internalized and degraded by a process that is stimulated by cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization and distribution of a phospholipase A 2 activity from adult rabbit lung

A phospholipase A 2 activity was characterized in adult rabbit lung. This activity was calcium- and deoxycholate-dependent and displayed an alkaline pH optimum. K m and V max were 0.176mM and 256.8 pmoles/min./mg protein respectively. The microsomal fraction displayed the highest enzymatic specific activity; the lowest activity was present in the cytosol. Yet this latter fraction accounted for the majority of the total activity. Although the specific activity was high within the lamellar body fraction this compartment contained only ∼2% of the total activity. Phospholipase A 2 activity was inhibited by bromophenacyl bromide, chlorpromazine and mepacrine in decreasing order of effectiveness. Treatment of the microsomes with increasing concentrations of NaCl indicated that the lung phospholipase A 2 activity was related loosely bound to the microsomal membranes and was maximally removed with salt at a concentration only slightly higher than physiological. Addition of calmodulin to the enzyme assay did not significantly alter hydrolysis of labelled phosphatidylcholine.

Insulin inhibits the accumulation of the major lung surfactant apoprotein in human fetal lung explants maintained in vitro.

In the present study, we characterized the proteins associated with a purified lamellar body fraction isolated from human fetal lung explants. We then raised antibodies directed against the major human surfactant apoprotein, a 35-K glycoprotein, and used the technique of immunoblot analysis to evaluate the content of the surfactant apoprotein in human fetal lung explants maintained in serum-free medium in vitro. We found that the 35K surfactant apoprotein was undetectable in homogenates of fetal lung tissue before culture; the surfactant apoprotein was induced in the cultured explants coincident with the appearance of differentiated type II cells. Insulin, at concentrations as low as 2.5 ng/ml, caused marked inhibition of the accumulation of the 35K protein in the cultured fetal lung tissue. The inhibitory effect of insulin was dose dependent and was apparent as early as day 2 of incubation. When explants were cultured in medium containing insulin plus cortisol, the amount of immunoreactive surfactant apoprotein was reduced compared to that of explants cultured in control medium or explants cultured with cortisol alone. On the other hand, as reported previously, insulin and cortisol, in combination, stimulated phosphatidylcholine synthesis. These findings are indicative that the phospholipid and apoprotein components of surfactant are regulated independently. The results of our studies suggest that fetal hyperinsulinemia may cause the production of a surfactant deficient in the 35K apoprotein, and this may provide an explanation for the increased incidence of respiratory distress syndrome in infants of diabetic mothers.

Isolation, characterization and phospholipid composition of lamellar bodies and subcellular fractions from dog lung

1. 1. Lamellar body fractions from dog lung can be separated by a procedure based on differential centrifugation before ultracentrifugation onto a discontinuous sucrose gradient. This fraction yields about 1 % of total protein from the homogenate. 2. 2. The different fractions obtained in the isolation were assayed for the measurement of four subcellular marker enzymes: β- N-acetylglucosaminidase, acid phosphatase, 5'-nucleotidase and succinate dehydrogenase. 3. 3. Lamellar bodies were not contaminated by mitochondria (0.7 succinate dehydrogenase relative specific activity), whereas high specific hydrolase activities were found ( β- N-acetylglucosaminidase and 5'-nucleotidase were enriched 1.8- and 2.8-fold, respectively). 4. 4. The chemical criterion was established by measuring the specific components of lamellar bodies. The lamellar bodies have the highest phospholipid/protein ratio (0.35); cholesterol/protein ratio (0.15) and the highest phosphatidylglycerol percentages (7.9%). 5. 5. The phospholipid composition of lamellar bodies is distributed among phosphatidylcholine (64.5%), phosphatidylethanolamine (11%), phosphatidylglycerol (7.9%), sphingomyelin (4%), phosphatidylserine and phosphatidylinositol (3%), respectively. The remainder were considered as trace amounts ( < 1%).

Cytochemical and Biochemical Localization of Lipase and Sphingomyelinase Activity in Mammalian Epidermis

Despite a wealth of new information on epidermal lipids and their role in permeability barrier function and desquamation, little is known about the location of the enzymes that regulate their catabolism. In this study we have localized lipase (triacylglycerol hydrolase) and sphingomyelinase in the outer epidermis simultaneously by cytochemical and cell fractionation techniques. Aldehyde-fixed tissues (100-microns slices) incubated in either Tween 85 or triolein plus taurocholate/calcium chloride-containing buffer, pH 7.2 or 4.5, were then exposed to lead to form insoluble soaps, and processed for electron microscopy. Simultaneously, cell homogenates and isolated lamellar body fractions were incubated with methylumbelliferyl oleate under similar conditions, with released, free methylumbelliferone serving as an index of lipase activity. On electron microscopy and cell fractionation, both lipase and sphingomyelinase were localized primarily to intercellular domains in the stratum corneum. In the stratum granulosum lipases were found, both ultrastructurally and biochemically, in lamellar bodies and ultrastructurally in both the perinuclear cistern and mitochondria. In summary, these studies: by demonstrating lipid-catabolic enzymes in the intercellular domains of the stratum corneum, lend further support to the 2-compartment model of the stratum corneum; provide new information about the location of lipid-catabolic enzymes in differentiating epidermis; and provide insights about how lipids are processed during permeability barrier formation and desquamation.

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.

Analysis of pulmonary phospholipid compartments in the unanesthetized rat during prolonged periods of hyperpnea.

We exposed rats to 4% CO2-10% O2-86% N2 for 24 h before infusing with 20 microCi kg-1 (methyl-3H)choline chloride. They were then exposed for periods up to 48 h, at which times their lungs were degassed and lavaged. The lavage fluid was divided into a tubular myelin-rich (PLalv-1) and a tubular myelin-poor (PLalv-2) fraction. Lamellar body (lb) and microsomal (m) fractions were prepared from the lung tissue and the amount of phospholipid (PL) was determined in each of the 4 fractions. Specific activity (sp.act.) curves were constructed for both control and hyperpneic groups. Exposure to the gas doubled both tidal volume and frequency of breathing. Total PLalv, PLlb and PLm were all markedly elevated, which, when taken in conjunction with the changes in sp.act. in these fractions, suggests that the rate of surfactant PL synthesis was increased within 24 h. The shapes of the sp.act. curves suggest precursor-product relationships between PLlb and PLalv-1 and between PLalv-1 and PLalv-2. However, when we applied analysis based on the Zilversmit steady-state equation, instead of the expected straight line, we found a marked clockwise hysteresis that did not return to the origin. Whereas this may reflect PLalv being supplied from 2 tissue pools, we argue that, in fact, we are not dealing with classic compartmental precursor-product relationships.

A specific acid α-glucosidase in lamellar bodies of the human lung

In the present investigation, we have demonstrated that three lysosomal-type hydrolases, α-glucosidase, α-mannosidase and a phosphatase, are present in lamellar bodies isolated from adult human lung. The hydrolase activities that were studied, all showed an acidic pH optimum, which is characteristic for lysosomal enzymes. The properties of acid α-glucosidase in the lamellar body fraction and that in the lysosome-enriched fraction were compared. Using specific antibodies against lysosomal α-glucosidase from human placenta, two α-glucosidases could be distinguished in the lamellar body fraction: one with a high affinity to the antibodies as found in the lysosome-enriched fraction and another with a much lower affinity. Both forms showed an acidic pH optimum. The same heterogeneity of α-glucosidase in the lamellar body fraction could be observed using immobilized concanavalin A. The lectin was able to precipitate nearly all α-glucosidase activity of the lysosome-enriched fraction. In contrast, 30% of the α-glucosidase activity in the lamellar body fraction was not precipitable. Furthermore, the lamellar body α-glucosidase with the low antibody affinity could not be bound to concanavalin A. The results suggest that lamellar bodies contain at least two acid α-glucosidases: one similar to the lung lysosomal α-glucosidase, and another lamellar body-specific isoenzyme with a different immunoreactivity and lectin affinity. The lamellar body-specific α-glucosidase should prove useful as a lamellar body-specific marker enzyme.

Inositol affects the intracellular turnover of pulmonary surfactant phospholipids in the rat

Rats, given a diet supplemented with 20% inositol, had threefold increased plasma inositol concentrations. The pool size of their alveolar surfactant fraction and their lamellar body fraction were the same as in the control rats and differences in phospholipid composition of the surfactant fractions were mainly restricted to changes in the percentages phosphatidylinositol (PI) and phosphatidylglycerol (PG). The change in phospholipid composition did not affect the pressure-volume relationship of the lungs. The labeling of phosphatidylcholine (PC), saturated phosphatidylcholine (SPC) or PI in the alveolar lavage fraction was the same for both groups, whereas labeling of alveolar lavage PG was delayed in the inositol-fed rats. The specific activity-time relationships of the lamellar body phospholipids differed significantly between the control and inositol-fed rats and the differences in disappearance rate of the label from these fractions suggest that approximately 25–30% of the lamellar body material in inositol-fed rats is directed to a third, intracellular pool. We conclude that an increase in PI and a concomitant decrease in PG content of surfactant do not affect the clearance of alveolar surfactant, but enlarge the turnover of the lamellar body fraction because of intracellular degradation.

Lamellar Body-Enriched Fractions from Neonatal Mice: Preparative Techniques and Partial Characterization

Several problems have frustrated the isolation of lamellar bodies (LB) from mammalian epidermis. We obtained pellets enriched in intact LB by utilizing the staphylococcal epidermolytic toxin to provide intact, outer epidermal sheets, by controlled homogenization in a cell disrupter, and by passage of homogenates through a graded series of nuclepore filters (Science 221:962, 1983). Such preparations contained more intact LB than did fractions prepared by a variety of differential or sucrose/metrizamide discontinuous centrifugation methods. Initial characterization of the enzymatic content of this fraction revealed it to be enriched in certain hydrolytic enzymes (acid phosphatase, carboxypeptidase, cathepsin B, acid lipase, sphingomyelinase, and phospholipase A), but strikingly depleted in all sulfatases, beta-glucuronidase, and the non-lysosomal protease, plasminogen activator. Thus, LB show some properties of lysosomes, although certain characteristic lysosomal enzymes are strikingly absent. Lamellar body fractions contained 2-3 times more lipid per unit weight than did homogenates, and were enriched in phospholipids, free sterols, and glycosphingolipids, but not in other neutral lipids or ceramides. In summary, whereas some of the enzymes in LB could participate in the metabolism of LB lipid precursors to hydrophobic barrier constituents, others may attack intercellular constituents, ultimately resulting in desquamation. The lipid profile of these organelles suggests that they deliver precursors of permeability barrier lipids to intercellular domains.

“SURFACTANT REPLACEMENT THERAPY” IN SURFACTANT DEPLETED RABBITS: LUNGFUNCTION, SURVIVAL AND BIOCHEMICAL ASPECTS

To study the effects of surfactant replacement therapy in Respiratory Distress Syndrome we induced surfactant deficiency in adult artificially ventilated (FiO2 1.0) rabbits by lung lavage. 19 Animals received natural sheep surfactant intratracheal, 6 served as controls.Results: In all surfactant treated animals PaO2 rose instantaneously from 11.31±5.00 to 37.78±13.40 kPa (mean±S.D.) (p<0.01). PaCO2 did not change significantly neither did dynamic lungcompliance. 9 Of the 19 surfactant treated animals were succesfully weaned off the ventilator. Static lung compliance was 1.07±0.30 in surviving, 0.71±0.08 in non-surviving (p<0.05) and 0.33±0.09 ml/cm H2O/kg bodyweight in controls (p<0.01). Phospholipid content of the lamellar body fraction, isolated from lung tissue increased from 0.97±0.33 to 2.50±0.10 μmol/g wet weight in the surfactant treated animals.Conclusion: Surfactant replacement results in an instantaneous rise in PaO2. This rise is not associated with a change in dynamic lung compliance, however static lung compliance is improved after surfactant replacement. Phospholipid analysis of the lamellar body fraction indicates that the administered surfactant phospholipids seem to be incorporated by the lamellar bodies of the type II alveolar cells.

316 FETAL RAT SURFACTANT AT THE ONSET OF AIR BREATHING

Morphometric and biochemical measures of type II cell surfactant stores were combined to define late gestational maturation of the alveolar epithelium in its preparation for transition to air breathing. Fetal (G20, G22) and newborn (+10min) lung tissue was obtained from pathogen-free timed pregnant rats. Morphometric analyses of type II cells & their organelles was done using a stratified sample. Lamellar body (LB) fractions were obtained by sucrose density gradients & disaturated phosphatldylcholine (DSPC) was quantitated. The table gives the results of the volume fraction (Vv,%) of type II cell organelle content and the LB fraction and whole lung (WL) DSPC content. There was an increase in LB Vv from day 20 of gestation, with a parallel increase in LB DSPC. Within 10 min after birth, we found a significant increase in intracellular LB by morphologic and biochemical measures. This postnatal accumulation of intracellular surfactant could represent recycling of previously secreted phospholipid from the airspaces. Such an occurrence might supplement de novo pathways of surfactant production by newborn rat type II cells providing an additional source of surfactant flux. Supported by NHLBI R0132188 and by the VA research fund.

244 HYPOXIA, LUNG GROWTH AMD THE SURFACTANT SYSTEM DURING ALVEOLARIZATION

Alveolarization in the rat occurs during the first 21 days and provides surface area proportional to oxygen needs. Lung size is known to increase in distance runners and persons living at altitude. Rat dams with 7–1 day old pups (HR), were placed 0.12 Ox in a recirculating chamber using Nx to reduce O2 and soda lime to absorb CO2. Control dams and comparable litters of pups (CR) were raised in 0.21 O2 under similar conditions. At 20–21 days HR and CR pups were weighed, injected with [14C] methylcholine, and the lungs removed, weighed, and homogenized. A lamellar body (LB) fraction was derived by sucrose density centrifugation and LB disaturated phosphatidylcholine (LBPC) was extracted, quantitated, and the specific activity determined. HR pups were hyperpneic and runted significantly as compared to CR (36.1g vs 51.4g, p<0.001), although HR and CR lung weights were not different (1.41g vs 1.38g) and the % dry weights were comparable (HR 21.0 vs CR 20.6). LW/BW however, was significantly increased in HR (0.039 vs 0.028 p<.005). LBPC/g lung was comparable in HR and CR (209.1 vs 194.2). The accumulations of label in LBPC (dpm/μmol /min) as an index of synthesis was higher in HR (183.7 vs 291.9, p<.001). These data show decreased body growth but not lung growth in response to hypoxia. The increase in LBPC labelling suggest more rapid synthesis of surfactant lipid perhaps in response to hyperpnea.

Effect of cortisol on the synthesis of lamellar body glycerophospholipids in fetal rabbit lung tissue in vitro

The effect of cortisol on the rate of choline incorporation into tissue phosphatidylcholine was investigated in lung explants from fetal rabbits of 19–28 days gestational age. The explants were incubated in medium with or without fetal calf serum for up to 7 days. When lung tissues were incubated in serum-free medium, a stimulatory effect of cortisol on tissue phosphatidylcholine synthesis was found in explants from 21-, 24-, 26- and 28-day fetal rabbits; a stimulatory effect of cortisol was observed in 19-day fetal lung explants only if fetal calf serum was present in the culture medium. To assess directly the effect of cortisol on the synthesis of lamellar body phosphatidylcholine, choline incorporation into phosphatidylcholine associated with a purified lamellar body fraction isolated from lung explants of 21- and 28-day fetal rabbits was also investigated. Cortisol caused a marked stimulation of synthesis and accumulation of lamellar body phosphatidylcholine in lung explants from both 21- and 28-day fetal rabbits. The magnitude of the stimulatory effect of cortisol on the rate of synthesis of lamellar body phosphatidylcholine was always greater than the effect of cortisol on the rate of choline incorporation into lipids of tissue homogenates. The relative rates of synthesis of lamellar body phosphatidylinositol and phosphatidylglycerol were also significantly altered in lung explants from 21- and 28-day fetal rabbits by cortisol treatment. Lamellar bodies that were formed initially in the fetal lung explants were enriched in phosphatidylcholine and phosphatidylinositol and had a relatively low phosphatidylglycerol content. With cortisol treatment there was a decrease in the relative rate of synthesis of lamellar body phosphatidylinositol and an increase in the relative rate of synthesis of phosphatidylglycerol. The stimulatory effect of cortisol on the synthesis of lamellar body phosphatidylcholine was observed at an earlier time-point of incubation than was the effect of cortisol on the relative rates of synthesis of lamellar body phosphatidylinositol and phosphatidylglycerol. The temporal sequence of the cortisol-induced changes in the synthesis of lamellar body glycerophospholipids, therefore, reflects that which occurs with maturation in vivo.

High-frequency oscillation affects surfactant phospholipid metabolism in rabbits.

Surfactant phospholipid metabolism was studied in anesthetized rabbits ventilated with high-frequency oscillation at a frequency of 5 Hz and a mean airway pressure of 5 cm H2O. Blood gases were normal although some atelectasis was evident after 1 h. The static compliance of the lungs and amount and composition of surfactant phospholipids of the lamellar body and alveolar lavage fraction were comparable to values found for spontaneously breathing rabbits. The data obtained for the incorporation of radioactively labeled palmitate into phospholipids are compatible with intracellular degradation of newly synthesized surfactant phospholipids. This hypothesis is supported by two observations. First, the rapid initial increase in specific activity of SPC and PC of the lamellar body fraction is not accompanied by a similar rapid increase in specific activity of SPC and PC of the lamellar body fraction is not accompanied by a similar rapid increase in specific activity of the alveolar lavage fraction. Second, a dissociation occurs between the metabolism of PC and SPC for the lamellar body fraction but not for the alveolar lavage fraction. The change in metabolism might be caused by the absence of large pressure swings during this pattern of ventilation.

Effects of artificial ventilation on surfactant phospholipid metabolism in rabbits.

Surfactant phospholipid metabolism and lung stability were studied in mechanically ventilated and in spontaneously breathing rabbits (control group). During ventilation the dynamic lung-thorax compliance decreased to 79% after 6 h. Static compliance and amount and composition of surfactant phospholipids remained unaltered. These data indicate inactivation of alveolar surfactant during ventilation, which is reversible by a single large inflation. Incorporation of injected radioactively labeled palmitate into saturated phosphatidylcholine (SPC) of the lamellar body fraction increased significantly in the ventilated group; maximal specific activity increased from 20 dpm/nmol SPC at 6 h in the control group to 30 dpm/nmol SPC at 2 h in the ventilated group. The clearance of radioactivity from the lamellar bodies into the alveolar lumen was greatly enhanced in the ventilated group. The results are explained by assuming that as a result of the inactivation of alveolar surfactant, endocytosed surfactant is degraded in the type II cells instead of being recycled and that the degradation products are subsequently reutilized in surfactant synthesis. This interpretation is supported by computer simulations.

Reutilization of phosphatidylcholine analogues by the pulmonary surfactant system: The lack of specificity

Five specific 14C-labelled analogues of l,2-dipalmitoyl- sn-glycero-3-phosphocholine, ( l-α-DPPC) including 1-palmitoyl- sn-glycero-3-phosphocholine, ( l-α-lysoPC) and 2,3-dipalmitoyl- sn-glycero-1-phosphocholine (the d isomer of DPPC) were individually mixed with l-α-[ 3H]DPPC and unlabelled natural surfactant isolated from 3-day-old rabbits. The mixtures were injected intratracheally into 3-day-old rabbits which were then killed at preset times up to 72 h after injection. Phosphatidylcholine was isolated from the alveolar wash and from lamellar body fraction from each rabbit and was analyzed for the ratio of 3H-to- 14C counts/min. These ratios were plotted against the time the rabbits were killed to determine whether a difference existed in the rates of reutilization of the analogues relative to l-α-DPPC. By 60 h l-α-lysoPC was reutilized at 42% of the efficiency of the l-α-[ 3H]DPPC with which it was injected. That part of the l-α-lyso which was reutilized had been converted to [ 14C]phosphatidylcholine. Each of the other four analogues was reutilized by the surfactant system as efficiently as l-α-DPPC. These results are most consistent with a process of bulk uptake of surfactant from the alveolar space by the Type II cell with subsequent processing for resecretion which involves minimal specificity for molecular structure.

REUTILIZATION (RU) OF SURFACTANT (NS) PHOSPHATIDYLCHOLINE (PC) IN ADULT RABBITS (R)

Several studies have demonstrated RU of NS PC in developing and adult R. The magnitude of RU in adult R has not been determined. We injected 87 1 kg R with two solutions simultaneously. [14C]palmitate was given IV and a solution of [3H]choline labeled NS plus [32P]dipalmitoylPC (DPC) was injected intratracheally (IT). R were sacrificed from 10 min to 72 hrs after injection. From each R we collected a complete alveolar wash (AW) and isolated a lamellar body (LB) fraction. We measured total [3H]PC and [32P]PC recovered in the AW and [14C]PC specific activity (SA) in the AW and in LB. From curves of [14C]PC SA in the LB and the AW vs time, we calculated the flux of PC from the LB into the AW (PC flux) and the turnover times (Tt) for alveolar PC (Tt =total AW PC/PC flux). A computer generated equation describing the total AW [3H]PC vs time was obtained and used to independently calculate the PC flux, the Tt, the % RU of NS PC and the LB PC pool size. The results are shown in the table.Also, the ratio of [3H]PC to [32P]PC in the AW did not change with time. We concluded that 1) IT and IV labeling gave comparable results. 2) IT NS and the added DPC behaved metabolically like endogenous NS. 3) That RU of PC in adult R (31%) is less than that of developing R (>90%).

Isolation of lamellar bodies from rat granular pneumocytes in primary culture

A lamellar body fraction was isolated from rat alveolar granular pneumocytes in primary culture by upward flotation on a discontinuous sucrose gradient and compared with a similar fraction isolated from lung homogenates. Lamellar bodies from granular pneumocytes were free of detectable contamination with either succinate dehydrogenase or NADPH-cytochrome c reductase. There was an enrichment of acid phosphatase activity, which, based on distribution of enzyme activity on the gradient, did not appear to be a contamination from other fractions. The lamellar body fraction of granular pneumocytes yielded approx. 1 μ protein/10 6 cells with a phospholipid-to-protein ratio (mg/mg) of 9.6 ± 0.4 ( n = 7). Composition with respect to total phospholipids was 71.0% phosphatidylcholine (disaturated phosphatidylcholine, 45.2%), 8.4% phosphatidylglycerol and 12.8% phosphatidylethanolamine. Palmitic acid comprised 66% of the fatty acids in phosphatidylcholine and 34% of those in phoshatidylglycerol. The lamellar body fraction from granular pneumocytes was similar to that from whole lung with respect to phospholipid-to-protein ratio and phospholipid composition and showed only minor differences in fatty acid composition. Ultrastructurally, lamellar bodies showed generally intact limiting membranes and lamellated structure. Lamellar bodies from granular pneumocytes showed occasional multinucleated whorls which were not seen in those isolated from lung homogenates. This study describes a method for preparing a homogenous fraction of intact lamellar bodies from small amounts of material (6·10 7 granular pneumocytes). The yield on a per cell basis was higher when compared with a similar preparation from whole lung, although overall yield is small, due to loss of cells during the cell isolation procedure. This preparation may be useful to evaluate the role of lamellar bodies in the synthesis and secretion of lung surfactant by isolated granular pneumocytes.

Route of incorporation of alveolar palmitate and choline into surfactant phosphatidylcholine in rabbits

Intratracheal injection of 3-day-old rabbits with radioactively labeled palmitic acid and choline results in an 8–10-fold increase in the efficiency of their incorporation into surfactant phosphatidylcholine when compared to the intravenous injection of these precursors. Based on labeling patterns in microsomal, lamellar body and alveolar wash fractions, the incorporation appears to be via normal surfactant synthetic pathways. Intratracheal injection of phospholipid precursors is useful for producing relatively high specific activity natural surfactant.