Reutilization of surfactant phosphatidylglycerol and lysophosphatidylcholine by adult rabbits

Adult rabbits reutilize the phosphatidylcholine (PC) of surfactant much less efficiently than developing rabbits (22% vs. 95%). Comparisons of reutilization efficiency of other components of surfactant in adult rabbits have not been determined. We injected adult rabbits intratracheally with [3H]dipalmitoylphosphatidylcholine (DPPG) mixed with [14C]lysophosphatidylcholine (lysoPC) and natural surfactant or [14C]DPPC mixed with [3H]dipalmitoylphosphatidylglycerol (DPPG) and natural surfactant. Recovery in the alveolar wash and lamellar bodies of labelled DPPC, lysoPC and DPPG was determined at different times after injection. By plotting the ratio of [3H]DPPG to [14C]DPPC in the alveolar wash versus time after injection we found that phosphatidylglycerol was reutilized with an efficiency of only 0-7% which was much less than the reutilization of PC in these animals. At early times after injection, adult rabbits injected with [14C]lysoPC had a ratio of [14C]PC in their alveolar wash to lamellar bodies that was larger than 1.0. By comparison, 3-day old rabbits injected intratracheally with [14C]lysoPC had a ratio of [14C]PC in alveolar wash to lamellar bodies less than 1.0 at the earliest times measurable. Thus adult rabbits demonstrate a pathway for accumulation of PC in their alveolar space prior to its appearance in lamellar bodies. This was not detected in developing rabbits. As in developing rabbits, adult rabbits reutilize the phosphatidylglycerol of surfactant less efficiently than the PC of surfactant.     

Metabolism of intratracheally administered unsaturated phosphatidylcholines in adult rabbits

Twenty-five adult rabbits were each injected intratracheally with a solution containing 1-palmitoyl-2-[3H]palmitoyl phosphatidylcholine (DPPC) and 1-palmitoyl-2-[14C]oleoyl-PC that had been associated with with 32P-labeled natural rabbit surfactant. The animals were killed in groups of 5 at 1, 4, 8, 15 and 24 h after isotope injection. Isotope recovery and PC specific activities were measured in alveolar washes, lung homogenates, lamellar bodies and microsomes. The percent clearance per h of PC was very similar for the three labels and were; 3.56, 3.44 and 3.00%, respectively, for the 3H-, 14C- and 32P-labeled PC in the total lung (alveolar wash plus lung homogenate) and 3.84, 3.79 and 3.70%, respectively, for alveolar wash alone. The intracellular pathways of the three labels were assessed by comparing the specific activities in the lamellar bodies over 24 h as well as comparing the ratios of lamellar body to microsome specific activities over this period. These ratios were very similar for the monoenoic and saturated PC labels over time, indicating comparable recycling. In a separate experiment, three other unsaturated species; 1,2-[14C]dioleoyl-PC, 1-palmitoyl-2-[14C]linoleoyl-PC, and 1-palmitoyl-2-[14C]arachidonyl-PC were compared to 1-palmitoyl-2-[14C]oleoyl-PC. Recovery in the alveolar wash and total lung were similar at 16 h for all four labeled phospholipids. The intracellular pathways were also similar, except for the arachidonyl compound. More relative to the lamellar bodies as compared to the other. Thus, the catabolic pathways were similar for the saturated and unsaturated PC species initially present in the airspaces. The only metabolic difference between the compounds appears to be in the intracellular handling of the arachidonic species.     

Altered lung phospholipid metabolism in mice with targeted deletion of lysosomal-type phospholipase A2

Lung surfactant dipalmitoylphosphatidylcholine (DPPC) is endocytosed by alveolar epithelial cells and degraded by lysosomal-type phospholipase A2 (aiPLA2). This enzyme is identical to peroxiredoxin 6 (Prdx6), a bifunctional protein with PLA2 and GSH peroxidase activities. Lung phospholipid was studied in Prdx6 knockout (Prdx6-/-) mice. The normalized content of total phospholipid, phosphatidylcholine (PC), and disaturated phosphatidylcholine (DSPC) in bronchoalveolar lavage fluid, lung lamellar bodies, and lung homogenate was unchanged with age in wild-type mice but increased progressively in Prdx6-/- animals. Degradation of internalized [3H]DPPC in isolated mouse lungs after endotracheal instillation of unilamellar liposomes labeled with [3H]DPPC was significantly decreased at 2 h in Prdx6-/- mice (13.6 +/- 0.3% vs. 26.8 +/- 0.8% in the wild type), reflected by decreased dpm in the lysophosphatidylcholine and the unsaturated PC fractions. Incorporation of [14C]palmitate into DSPC at 24 h after intravenous injection was decreased by 73% in lamellar bodies and by 54% in alveolar lavage surfactant in Prdx6-/- mice, whereas incorporation of [3H]choline was decreased only slightly. Phospholipid metabolism in Prdx6-/- lungs was similar to that in wild-type lungs treated with MJ33, an inhibitor of aiPLA2 activity. These results confirm an important role for Prdx6 in lung surfactant DPPC degradation and synthesis by the reacylation pathway.     

The incorporation of choline into disaturated phosphatidylcholine in the microsomal, lamellar body, and surfactant fractions of hamster lung tissue

The specific activity of disaturated phosphatidylcholine in microsomes and lamellar bodies prepared from hamster lung tissue and in surfactant obtained by lung lavage was determined at various times following the intraperitoneal administration of [Me-3H]choline. The highest specific activity of disaturated phosphatidylcholine in the lung microsomes was attained 1 h after the administration of [3H]choline; thereafter, the specific activity declined. The specific activity of disaturated phosphatidylcholine in lamellar bodies increased steadily for 12 h after [3H]choline administration. The specific activity in lamellar bodies ater 12 h exceeded the maximum specific activity achieved in the microsomal fraction (p less than 0.005). The specific activity of the disaturated phosphatidylcholine in the alveolar lavage increased after an initial lag period of approximately 3 h, attaining the same specific activity as that of the lamellar bodies at the 12-h time point. The reported results are discussed in relation to the biosynthesis, storage, and secretion of the disaturated phosphatidylcholine associated with the lipoprotein, surfactant.     

Role of lamellar inclusions in surfactant production: studies on phospholipid composition and biosynthesis in rat and rabbit lung subcellular fractions.

Lamellar inclusion bodies in the type II alveolar epithelial cell are believed to be involved in pulmonary surfactant production. However, it is not clear whether their role is that of synthesis, storage, or secretion. We have examined the phospholipid composition and fatty acid content of rabbit lung wash, lamellar bodies, mitochondria, and microsomes. Phosphatidylcholine and phosphatidylglycerol, the surface-active components of pulmonary surfactant, accounted for over 80% of the total phospholipid in lung wash and lamellar bodies but for only about 50% in mitochondria and microsomes. Phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin accounted for over 40% of the total in mitochondria and microsomes but for only 6% in lung wash and 15% in lamellar bodies. The fatty acid composition of lamellar body phosphatidylcholine was similar to that of lung wash, but different from that of mitochondria and microsomes, in containing palmitic acid as a major component with little stearic acid and few fatty acids of chain length greater than 18 carbon atoms. The biosynthesis of phosphatidylcholine and phosphatidylglycerol was examined in the mitochondrial, microsomal, and lamellar body fractions from rat lung. Cholinephosphotransferase was largely microsomal. The activity in the lamellar body fraction could be attributed to microsomal contamination. The activity of glycerolphosphate phosphatidyltransferase, however, was high in the lamellar body fraction, although it was highest in the mitochondria and was also active in the microsomes. These data suggest that the lamellar bodies are involved both in the storage of the lipid components of surfactant and in the synthesis of at least one of those components, phosphatidylglycerol.     

Lung phospholipid metabolism in transgenic mice overexpressing peroxiredoxin 6

Previous studies with peroxiredoxin 6 (Prdx6) null mice demonstrated that the phospholipase A(2) activity of this enzyme plays a major role in lung phospholipid metabolism. This study evaluated lung phospholipid metabolism in transgenic mice that over-express Prdx6. Lung lysosomal type PLA(2) activity in transgenic mice was 222% of wild type in lung homogenate and 280% in isolated lamellar bodies. Total phospholipid, phosphatidylcholine (PC) and disaturated PC were decreased approximately 20-35% in bronchoalveolar lung fluid, lung homogenate, and lung lamellar bodies in transgenic mice although lung compliance and type 2 cell ultrastructure were unaltered. To study metabolism, unilamellar liposomes ((3)H-DPPC: PC: cholesterol: PG, 10: 5: 3: 2 mol fraction) were instilled endotracheally in anesthetized mice and lungs were removed for perfusion. Compared to wild type, transgenic mice showed similar net uptake of liposomes in 2 h, but significantly increased (3)H-DPPC degradation (38.9+/-1.1 vs. 29.0+/-1.3% of recovered dpm). The PLA(2) competitive inhibitor MJ33 decreased degradation to 15% of recovered dpm in both transgenic and wild type lungs. Incorporation of [(14)C] palmitate into DSPC at 24 h after its intravenous injection was markedly increased in both the lung surfactant (+100%) and lamellar bodies (+188%) while incorporation of [(3)H] choline was increased by only 10-20%. These results indicate increased DPPC degradation and synthesis by the reacylation pathway with Prdx6 overexpression and provide additional evidence that the PLA(2) activity of Prdx6 has an important role in lung surfactant turnover.     

Altered surfactant function and metabolism in rabbits with acute lung injury

Lung injury was induced in rabbits with N-nitroso-N-methylurethane (NNNMU), and saturated phosphatidylcholine (Sat PC) pool sizes and phospholipid compositions were measured in alveolar wash subfractions isolated by differential centrifugation (large and small surfactant aggregates). Surfactant metabolism also was studied using intravascular and intratracheal radiolabels. Protein permeability, gas exchange, and compliance were significantly abnormal as lung injury progressed. At peak injury, there was a decrease in the large aggregate Sat PC pool size in alveolar wash accompanied by increased uptake of Sat PC from the air space and increased specific activity of both intravascular and intratracheal radiolabels in lamellar bodies. This was followed by a marked rise in the small aggregate pool size in the alveolar wash and increased secretion of Sat PC into the air spaces. Phospholipid compositions, total phospholipid-to-protein ratios, and in vivo functional studies using a preterm ventilated rabbit model were abnormal for both large and small aggregate surfactant fractions from the lung-injured rabbits. These studies characterize quantitative, qualitative, and functional changes of alveolar wash surfactant subfractions in NNNMU-injured lungs.     

Reutilization of surfactant phosphatidylcholine in adult rabbits

32P-saturated phosphatidylcholine was added to [3H]choline-labeled natural surfactant and the mixture was injected intratracheally into 87 adult rabbits. The rabbits were also given [14C]palmitate intravenously at the same time. Rabbits were killed in groups from 10 min to 72 h after injection. In each rabbit we measured the total recovered [3H]phosphatidylcholine (PC) in the alveolar wash, the ratio of [3H]PC to [32P]PC in the alveolar wash, and the specific activity of [14C]PC in the alveolar wash and lamellar bodies. Values were averaged for all rabbits killed at the same times and smooth curves were fit to the data by computer. From the intravenous [14C]palmitate data we calculated a turnover time for alveolar PC of 6.0 h. From the intratracheal labeling data, we calculated a turnover time for alveolar PC of 5.7 h and determined that alveolar PC was reutilized at an efficiency of only 23%. We also concluded that this reutilization occurred as intact molecules.     

Phosphatidylglycerol in lung surfactant. II. Subcellular distribution and mechanism of biosynthesis in vitro.

Lamellar inclusion bodies, apparent precursors for alveolar surfactant lining, have remarkably similar phospholipid composition to surfactant from alveolar lavage, but distinctly different from other fractions studied: mitochondria, microsomal fraction containing endoplasmic reticulum membranes, plasma membranes and nuclei. Surfactant contained (as % of total phospholipid phosphate): 75.5-77.0% lecithin, 11.0-11.2% phosphatidylglycerol, 4.2-4.6% phosphatidylethanolamine, 3.0-3.2% phosphatidylinositol, 1.5-1.7% bis-(monoacylglycerol) phosphate, 1.2-1.9% phosphatidylserine, and 0.7-1.5% sphingomyelin. Fatty acids of phosphatidylglycerol from lamellar bodies were similar to those from microsomes but different from those in mitochondria. Lung homogenate in continuous sucrose density gradient displayed two major activity peaks of phosphatidylglycerol synthesis: the heavier from mitochondria; the lighter from endoplasmic reticulum. Studies on mechanism of phosphatidylglycerol synthesis in vitro revealed (in these two fractions) CDP-diglyceride and sn-glycerol phosphate precursors to phosphatidylglycerol phosphate, that hydrolysed to phosphatidylglycerol. In microsomes disaturated CDP-diglycerides were 1.6-1.9 times more active substrates than in mitochondria, whereas CDP-diglycerides from egg lecithin were almost equally active. In contrast to lung mitochondria no cardiolipin synthesis was detected in microsomes. The highest specific activities for phosphatidate cytidyltransferase, CDP-diglyceride-inositol phosphatidyltransferase, choline phosphotransferase, and phosphatidylethanolamine methyltransferase were all found in microsomes. The present in vitro studies and additional evidence (M. Hallman and L. Gluck, (1975) Fed. Proc. 34, 274) support the hypothesis that de novo synthesis of surfactant lecithin phosphatidylinositol and phosphatidylglycerol takes place in the endoplasmic reticulum of alveolar cells.     

The significance of reutilization of surfactant phosphatidylcholine

To assess the magnitude of reutilization of surfactant phosphatidylcholine, 68 3-day-old rabbits were injected intratracheally with a trace dose of [3H]choline-labeled surfactant mixed with [14C]palmitate-labeled synthetic dipalmitoylphosphatidylcholine. After timed kills we measured the total phosphatidylcholine associated counts/min in whole lung and alveolar wash and the specific activities of phosphatidylcholine in the alveolar wash, lamellar bodies, and microsomes isolated from the lung of each rabbit. Using a modification of the compartment analysis of Skinner et al. (Skinner, S. M., Clark, R. E., Baker, N., and Shipley, R. A. (1959) Am. J. Physiol. 196, 238-244), we found that surfactant phosphatidylcholine was reutilized with greater than 90% efficiency. The turnover time of the alveolar wash phosphatidylcholine was estimated to be 10.1 h and 9.3 h as measured by the 3H and 14C labels, respectively. From the ratios of alveolar wash-associated natural to synthetic phosphatidylcholine specific activities and from similar ratios obtained in 30 additional rabbits using [14C]choline-labeled natural surfactant and [3H]choline-labeled dipalmitoylphosphatidylcholine, we showed that phosphatidylcholine was reutilized intact rather than as component parts. Within 6 h of injection, the synthetic dipalmitoylphosphatidylcholine functioned metabolically as that administered in the form of natural surfactant.     

An apolipoprotein preferentially enriched in cholesteryl ester-rich very low density lipoproteins

Phosphatidyl glycerol is present in lamellar bodies and in the material obtained by alveolar wash representing 12.3 and 11.5%, respectively, of the total phospholipid phosphorus. Lung microsomes catalyze the formation of phosphatidyl glycerol from the known precursors, L-glycerol 3-phosphate and CDP-diglyceride. The rate of [¹⁴C]L-glycerol 3-phosphate incorporation into phosphatidyl glycerol was 30% higher in microsomes as compared to mitochondria. The addition of mercuric chloride inhibited the synthesis of phosphatidyl glycerol, and stimulated the incorporation into another as yet incompletely identified lipid. After pulse labeling of microsomal phosphatidyl glycerol $\text{in vitro}$, further incubation of microsomes with lamellar bodies or alveolar wash resulted in nearly quantitative appearance of label in surfactant.     

Clearance of surfactant phosphatidylcholine from adult rabbit lungs

Rabbits were given various doses of rabbit surfactant and treatment doses of approximately 100 mg/kg body wt of calf surfactant and Surfactant TA by tracheal injection. The linear loss of radiolabeled phosphatidylcholine from the total lung (alveolar wash and lung tissue) was 3.1, 1.5, and 1.8%/h for rabbit surfactant, calf surfactant, and Surfactant TA, respectively. After 24 h only 6% rabbit, 19% calf, and 9.7% Surfactant TA phosphatidylcholine were recovered by alveolar wash, and alveolar macrophage fractions contained less than 1% of the injected labeled phosphatidylcholine. The loss of rabbit surfactant phosphatidylcholine 24 h after tracheal injection did not change for doses in the range of 0.5-70 mumol phosphatidylcholine per kilogram, indicating nonsaturable clearance pathways. Very little of the labeled rabbit surfactant phosphatidylcholine lost from the lungs could be recovered in other organs, and 90% of the recovered labeled phosphatidylcholine in the liver was unsaturated, implying de novo synthesis using precursors from degraded phosphatidylcholine. The surfactant did not change endogenous lung phosphatidylcholine synthesis or its secretion to the alveolus. There were no adverse effects of the surfactant treatments noted in healthy rabbits.     

Glucose effects on lung surfactant kinetics in conscious pigs

The primary goal of this study was to investigate the effects of glucose infusion on surfactant phosphatidylcholine (PC) metabolic kinetics in the lungs. A new stable isotope tracer model was used in which [1,2-(13)C(2)]acetate and uniformly labeled [U-(13)C(16)]palmitate were infused in 12 normal overnight-fasted pigs to quantify lung surfactant kinetics with or without glucose infusion (24 mg. kg(-1). min(-1)). With glucose infusion, the rate of surfactant PC incorporation from de novo synthesized palmitate increased from the control value of 2.1 +/- 0.2 to 15.5 +/- 1.9 nmol PC-bound palmitate. h(-1). g wet lung(-1) (P < 0.05), whereas the incorporation rate from plasma preformed palmitate decreased from the control value of 20.9 +/- 1.9 to 11.6 +/- 1.1 nmol palmitate. h(-1). g wet lung(-1) (P < 0.05). The palmitate composition in lamellar body surfactant PC increased from the control value of 61.7 +/- 2.1% to 75.9 +/- 0.6% (P < 0.05). The surfactant PC secretion rate decreased from the control value of 239.0 +/- 26.1 to 81.9 +/- 5.3 nmol PC-bound palmitate. h(-1). g wet lung(-1) (P < 0.05). We conclude that, whereas surfactant secretion was inhibited by glucose infusion, neither total surfactant PC synthesis nor the surfactant PC pool size was significantly affected due to an increased reliance on de novo synthesized fatty acids.     

Evidence that the rate of phosphatidylcholine catabolism is regulated in cultured rat hepatocytes

The regulation of phosphatidylcholine (PC) catabolism has been studied in choline-deficient rat hepatocytes. Supplementation of choline-deficient hepatocytes, prelabeled with [3H]choline, with 100 microM choline increased the rate of PC catabolism by approx. 2-fold. The major product of PC degradation was glycerophosphocholine in both choline-deficient and choline-supplemented cells. Choline supplementation decreased the radioactivity recovered in lysoPC by 50%. This effect was accompanied by a 2-fold increase of labeled glycerophosphocholine. Comparable results were obtained when PC of the cells was prelabeled with [3H]methionine or [3H]glycerol. The activity of phospholipase A in cytosol, mitochondria and microsomes isolated from choline-deficient rat liver was similar to the activity in control liver, when determined with [3H]PC vesicles as the substrate. Measurement of the activity of phospholipase A with endogenously [3H]choline-labeled PC showed that the formation of lysoPC in mitochondria isolated form choline-supplemented cells was 40% lower than in choline-deficient cells. Alternatively, the formation of [3H]glycerophosphocholine and [3H]choline in microsomes from choline-supplemented cells was significantly higher (1.4-fold) than in microsomes from choline-deficient cells. These results suggest that the rate of PC catabolism is regulated in rat hepatocytes and that the concentration of PC might be an important regulatory factor.     

Association and metabolism of exogenously-derived lysophosphatidylcholine by cultured mammalian cells: kinetics and mechanisms.

The association and metabolism of exogenously-derived lysophosphatidylcholine (lysoPC) with cultured mammalian cells from a variety of sources was studied, and a mechanism was defined by computer modeling for Vero cells. Cell monolayers were incubated with radiolabeled lysoPC, and the kinetics of disappearance from the medium, association with the cells, and metabolism by the cells of lysoPC were monitored both in the absence and in the presence of fetal bovine serum. Exogenously-supplied lysoPC first associated with cell membranes, followed by an almost complete conversion to phosphatidylcholine (PC). The kinetics of partitioning and metabolism were identical regardless of whether the exogenously-supplied lysoPC was labeled with [methyl-3H]choline or with [1-14C]palmitate. A two-step mechanism, consisting of a reversible partitioning of exogenous lysoPC into the cell membrane followed by enzymatic reacylation of PC, was found to adequately describe the observed kinetics in the presence of 0 or 0.5% fetal bovine serum. The effect of temperature on the individual rate constants and on the overall process was examined. An Arrhenius plot indicated an acute temperature sensitivity between 15 and 23 degrees C, consistent with a dependence on the lipid phase of the membrane and a regional phase transition temperature characteristic of mammalian cells. The acute temperature sensitivity was almost entirely due to the temperature dependence of reacylation. A multistep mechanism was established by combining the kinetic constants determined under conditions of low exogenous protein with the binding constant between lysoPC and serum protein. This mechanism accurately predicts the rates of uptake of exogenously-derived lysoPC with cultured cells in the presence of serum concentrations between 0 and 10%. A survey of a variety of cultured cells indicated that the kinetics of association and metabolism of exogenously-derived lysoPC is cell-type specific.     

Regulation of fetal lung disaturated phosphatidylcholine synthesis by de novo palmitate supply

Lung surfactant disaturated phosphatidylcholine (PC) is highly dependent on the supply of palmitate as a source of fatty acid. The purpose of this study was to investigate the importance of de novo fatty acid synthesis in the regulation of disaturated PC production during late prenatal lung development. Choline incorporation into disaturated PC and the rate of de novo fatty acid synthesis was determined by the relative incorporation of [14C]choline and 3H2O, respectively, in 20-day-old fetal rat lung explants and in 18-day-old explants which were cultured 2 days. Addition of exogenous palmitate (0.15 mM) increased (26%) choline incorporation into disaturated PC but did not inhibit de novo fatty acid synthesis, as classically seen in other lipogenic tissue. Even in the presence of exogenous palmitate, de novo synthesis accounted for 87% of the acyl groups for disaturated PC. Inhibition of fatty acid synthesis by agaric acid or levo-hydroxycitrate decreased the rate of choline incorporation into disaturated PC. When explants were subjected to both exogenous palmitate and 60% inhibition of de novo synthesis, disaturated PC synthesis was below control values and 75% of disaturated PC acyl moieties were still provided by de novo synthesis. These data show that surfactant disaturated PC synthesis is highly dependent on the supply of palmitate from de novo fatty acid synthesis.     

Surfactant metabolism in SP-D gene-targeted mice

Mice with surfactant protein (SP)-D deficiency have three to four times more surfactant lipids in air spaces and lung tissue than control mice. We measured multiple aspects of surfactant metabolism and function to identify abnormalities resulting from SP-D deficiency. Relative to saturated phosphatidylcholine (Sat PC), SP-A and SP-C were decreased in the alveolar surfactant and the large-aggregate surfactant fraction. Although large-aggregate surfactant from SP-D gene-targeted [(-/-)] mice converted to small-aggregate surfactant more rapidly, surface tension values were comparable to values for surfactant from SP-D wild-type [(+/+)] mice. (125)I-SP-D was cleared with a half-life of 7 h from SP-D(-/-) mice vs. 13 h in SP-D(+/+) mice. Although initial incorporation and secretion rates for [(3)H]palmitic acid and [(14)C]choline into Sat PC were similar, the labeled Sat PC was lost from the lungs of SP-D(+/+) mice more rapidly than from SP-D(-/-) mice. Clearance rates of intratracheal [(3)H]dipalmitoylphosphatidylcholine were used to estimate net clearances of Sat PC, which were approximately threefold higher for alveolar and total lung Sat PC in SP-D(-/-) mice than in SP-D(+/+) mice. SP-D deficiency results in multiple abnormalities in surfactant forms and metabolism that cannot be attributed to a single mechanism.     

Subcellular site and mechanism of synthesis of disaturated phosphatidylcholine in alveolar type II cell adenomas.

The site of synthesis of 1,2-disaturated-(diacyl)-sn-glycero-3-phosphocholine (Sat2PC) in mouse alveolar type II cell adenomas has been studied by conducting pulse-chase experiments. Isolation of microsomal and lamellar body fractions from adenomas after a 20-min pulse with [methyl-3H]choline demonstrates that Sat2PC first appears in the microsomal fraction, and after a short lag subsequently appears in the lamellar body fraction. The kinetics of labeling of Sat2PC are consistent with the microsomal membranes functioning as the subcellular site of synthesis for this pulmonary surfactant phospholipid. Short term labeling experiments with [9,10-3H]palmitate demonstrate that this fatty acid is incorporated into the sn-2 position of Sat2PC at a faster rate than its incorporation into the sn-1 position. This finding indicates that the synthesis of Sat2PC occurs by a deacylation-reacylation mechanism.     

Cellular cholesterol stimulates acute uptake of palmitate by redistribution of fatty acid translocase in type II pneumocytes

Cholesterol is an abundant lipid of lung surfactant, where its concentration changes relative to phospholipids in response to certain physiological conditions. We investigated the effect of the cellular cholesterol content on uptake and esterification of palmitic acid, and on cellular distribution of fatty acid translocase (FAT/CD36) in alveolar type II cells. Incubation of type II cells with methyl-beta-cyclodextrin-cholesterol complexes increased the cholesterol content of lamellar bodies. The palmitate uptake of type II cells increased in parallel with the cellular cholesterol content. The content of FAT/CD36 increased in membranes and decreased in cytosol in type II cells. The detergent-insoluble fraction (DIGs), isolated from type II cells, was enriched in FAT/CD36 and caveolin-1 after increasing the cellular cholesterol. The total incorporation of labeled palmitic acid into glycerolipids and cholesterol ester (CE) increased by a factor of about 10 when the amount of unbound (14)C-palmitic acid added to type II cells was increased by a factor of about 1000. Under these conditions, a small but significant increase of the palmitate incorporation into PL occurred. Independent from the amount of added palmitate, palmitate incorporation into triacylglycerol decreased and palmitate incorporation into cholesterol ester increased about 40-65-fold. The beta-oxidation of palmitate significantly decreased. We conclude that alveolar type II cells respond to an increase of the cholesterol level with (i) cellular redistribution of FAT/CD36 into DIGs causing enhanced palmitate uptake and increased cholesterol ester-formation, (ii) storage of cholesterol in lamellar bodies, and (iii) induction of the formation of caveolae-like microdomains in the surface membrane, a structure possibly involved in a lamellar body-independent efflux of free cholesterol via the high-density lipoprotein-specific pathway.     

Alterations in rat alveolar surfactant phospholipids and proteins induced by administration of chlorphentermine

Chlorphentermine is a cationic amphiphilic drug which produces a phospholipid storage disorder in rat lungs. Experiments were carried out to characterize changes in the composition of acellular alveolar lavage materials and to study possible mechanisms by which pulmonary surfactant phospholipidosis is produced by administration of the drug. Following ten daily injections of chlorphentermine (25 mg/kg body weight), there are 12.2- and 13.6-fold increases of pulmonary lavage total phospholipids and disaturated phosphatidylcholines (disaturated PC), respectively. In addition, there is a 2.8-fold increase in total protein and a 12.7-fold increase in the surfactant apoprotein group with molecular weights from 28,000 to 32,000. We measured incorporation of labeled palmitate, choline and glycerol into disaturated PC in type II cells and alveolar macrophages isolated from control and chlorphentermine-treated animals. The drug does not affect the incorporation of labeled substrates into disaturated PC in either cell type. However, in alveolar macrophages there is a decrease in the rate of intracellular degradation of recently synthesized disaturated PC in chlorphentermine-treated animals. The drug also inhibits the phospholipase-induced catabolism of rat surfactant disaturated PC which occurs during incubation of alveolar lavage fluid in vitro at 37 degrees C. When the lavage fluid is divided into subfractions by differential centrifugation, a larger percentage of the phospholipid is distributed in the less sedimentable subfractions in chlorphentermine-treated animals relative to controls, suggesting the accumulation of older surfactant materials. These results suggest that chlorphentermine-induced phospholipidosis of pulmonary surfactant materials is due to decreased rates of phospholipid degradation.