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.     

Reutilization of phosphatidylglycerol and phosphatidylethanolamine by the pulmonary surfactant system in 3-day-old rabbits

Developing rabbits reutilize the phosphatidylcholine of surfactant with an efficiency of about 95%. The efficiency of reutilization of other components of surfactant have not been determined. 3-day-old rabbits were injected intratracheally with [3H]dipalmitoylphosphatidylcholine (DPPC) mixed with unlabeled natural surfactant and either disaturated [32P]phosphatidylglycerol (DSPG) or [14C]dipalmitoylphosphatidyl-ethanolamine (DPPE). The recovery of [3H]DPPC, [14C]DPPE, and [32P]DSPG in the alveolar wash was measured at different times after injection. By plotting the ratio of [32P]DSPG to [3H]DPPC or [14C]DPPE to [3H]DPPC counts/min in the alveolar wash vs. time after injection we showed that these two phospholipids are reutilized less efficiently than phosphatidylcholine. Based on other studies, several assumptions were made about the kinetics of surfactant phosphatidylethanolamine and phosphatidylglycerol. From the slopes of the semilog plots of total [14C]DPPE and total [32P]DSPG counts/min in the alveolar wash vs. time and these assumptions, we determined that these two phospholipids were reutilized at an efficiency of only 79%.     

Experimental neonatal respiratory failure induced by lysophosphatidylcholine: effect of surfactant treatment

The purpose of this study was to characterize the toxic effectsof lysophosphatidylcholine (lyso-PC) on neonatal lung function. Variousdoses of lyso-PC (from 0 to 40 mg/kg) were administered to near-termnewborn rabbits. Lung-thorax compliance during mechanical ventilationwas significantly decreased by doses 10 mg/kg, and static lungvolumes during deflation were decreased by doses 20 mg/kg. Using thesame experimental model, we investigated the effects of modifiedporcine surfactant (Curosurf, 200 mg/kg). Animals exposed to lyso-PC atbirth and treated simultaneously with surfactant showed a satisfactorytherapeutic response, whereas those treated after 30 min failed torespond. These animals also had a much larger leak of albumin into theair spaces and an elevated minimum surface tension of the lavage fluidin a pulsating bubble surfactometer, suggesting inactivation of theexogenous surfactant. Timing of surfactant administration may thus beessential for the therapeutic effect in this experimental model ofacute lung injury.     

Physical, chemical, and physiological characteristics of isolates of pulmonary surfactant from adult rabbits

Physical and chemical characteristics of two types of preparations of surface active material from adult rabbits were determined. A procedure using multiple centrifugations produced a surface active material (type A) which had 6.6% by weight protein and a phosphorus/protein ratio of 13.1 nmol P/microgram protein. A simpler protocol involving two centrifugations yielded a surface active material (type B) with more protein (10.8%) and a lower phosphorus/protein ratio (8.4 nmol/micrograms). Lipid compositions of both types were similar with phosphatidylcholine being the major phospholipid (80%) and palmitate the major fatty acid in the total lipid (65-71%) and in phosphatidylcholine (80%). Both types exhibited broad thermotropic phase transitions encompassing 37 degrees C. Measurements of aqueous dispersions of surface active material on the surface of a Langmuir-Whilhelmy balance or in a pulsating bubble apparatus indicated that there was variability both between types and between batches of the same type in the capacity to reach low surface tension on the surface balance and in the rates at which low surface tension was achieved on the bubble apparatus. Type A preparations were somewhat more reliable in meeting these ends than were type B. Both types of isolates were effective in normalizing pressure-volume characteristics when instilled into the lungs of immature rabbit fetuses.     

Phosphatidylinositol and not phosphatidylglycerol is the important minor phospholipid in rhesus-monkey surfactant

Pulmonary surfactant was isolated from lung tissue and alveolar washes of lungs of adult rhesus monkeys (Macaca mulatta). The phospholipid composition was determined and compared to the composition of human surfactant fractions. Contrary to human surfactant, phosphatidylinositol is the major acidic phospholipid, whereas phosphatidylglycerol is only a minor component in rhesus-monkey surfactant. These differences are not caused by a difference in plasma myo-inositol concentrations between the two species.     

Convertase activity in alveolar surfactant and lamellar bodies in fetal, newborn, and adult rabbits

Conversion ofheavy-aggregate alveolar surfactant (H) to a light-aggregate,nonsurface active form (L) is believed to involve the activity of anenzyme, namely, convertase. This conversion can bereproduced in vitro by the surface-area cycling technique. The purposeof the present study was to use this technique to investigate thedevelopmental aspects of convertase activity in fetal, newborn, andadult rabbits. H was isolated from alveolar lavage from term[31-day gestation (31d)] fetal rabbit pups, 1-, 4-, and7-day-old newborns, and adults, and the percent conversion to L wasdetermined. To assess lamellar bodies (LB) as a potential source ofactivity in this species, these structures were isolated from lungtissue of 27-day-gestation (27d) and 31d fetuses, 1-, 4-, and 7-day-oldnewborns, and adults and were cycled the same as for H. LB containedconsiderable activity at each developmental stage i.e., ~82% of a27d LB preparation converted to L after 3 h of cycling. In the adult,this value was 78%. Very little conversion of H was obtained fromfetal lung (i.e., <20% of the 31d fetal preparation converted to L),but, by postnatal day 4, this valuewas greatly increased (i.e., >80% conversion) and stayed elevated toadulthood. The activity for each H and LB fraction was temperature andconcentration dependent and diminished with storage at 4°C. Thesedata suggest the LB as the source of convertase activity in the rabbitand demonstrate dramatic developmental changes in this activity afterrelease of the LB contents to the alveoli.

     

Pulmonary surfactant phosphatidylglycerol inhibits Mycoplasma pneumoniae-stimulated eicosanoid production from human and mouse macrophages

Mycoplasma pneumoniae is a human pathogen causing respiratory infections that are also associated with serious exacerbations of chronic lung diseases. Membranes and lipoproteins from M. pneumoniae induced a 4-fold increase in arachidonic acid (AA) release from RAW264.7 and a 2-fold increase in AA release from primary human alveolar macrophages. The bacterial lipoprotein mimic and TLR2/1 agonist Pam3Cys and the TLR2/6 agonist MALP-2 produced effects similar to those elicited by M. pneumoniae in macrophages by inducing the phosphorylation of p38(MAPK) and p44/42(ERK1/2) MAP kinases and cyclooxygenase-2 (COX-2) expression. M. pneumoniae induced the generation of prostaglandins PGD(2) and PGE(2) from RAW264.7 cells and thromboxane B(2) (TXB(2)) from human alveolar macrophages. Anti-TLR2 antibody completely abolished M. pneumoniae-induced AA release and TNFα secretion from RAW264.7 cells and human alveolar macrophages. Disruption of the phosphorylation of p44/42(ERK1/2) or inactivation of cytosolic phospholipase A(2)α (cPLA(2)α) completely inhibited M. pneumoniae-induced AA release from macrophages. The minor pulmonary surfactant phospholipid, palmitoyl-oleoyl-phosphatidylglycerol (POPG), antagonized the proinflammatory actions of M. pneumoniae, Pam3Cys, and MALP-2 by reducing the production of AA metabolites from macrophages. The effect of POPG was specific, insofar as saturated PG, and saturated and unsaturated phosphatidylcholines did not have significant effect on M. pneumoniae-induced AA release. Collectively, these data demonstrate that M. pneumoniae stimulates the production of eicosanoids from macrophages through TLR2, and POPG suppresses this pathogen-induced response.     

Interaction of the lipid and protein components of pulmonary surfactant Role of phosphatidylglycerol and calcium

We investigated the interaction of a major apolipoprotein of pulmonary surfactant with mixtures of lipids analogous to those found in natural surfactant. The apolipoprotein was extracted from canine surfactant and was purified to about 90% homogeneity. The apolipoprotein was mixed with liposomes of lipids in buffers containing 0.1 M sodium chloride and 3 mM calcium chloride at 22°C for 2 h or 37°C for 30 min. Two fractions were separated by centrifugation in sucrose density gradients at 15 000 rev./min. One was comprised of an aggregated, relatively high density recombinant lipoprotein which sedimented to a position toward the bottom of the centrifuge tube; the other remained at the top of the centrifuge tube and was mainly comprised of unbound lipid. The amount of lipid recovered as a sedimenting lipoprotein was dependent upon its composition. Those mixtures of lipids which contained dipalmitoyl phosphatidylglycerol formed sedimenting complexes which comprised 14% to 53% of the recovered lipid; those without phosphatidylglycerol formed such aggregates with less than 13% of the available lipid. Moreover, the lipid-to-protein stoichiometry of the recombinant was also dependent upon phosphatidylglycerol, and lipids containing this phospholipid displayed enhanced binding at a critical concentration of lipid which varied with temperature and composition. Calcium was required to form the sedimenting complex at 37°C. These results suggest that phosphatidylglycerol may be involved in the formation of a micelle-like complex, the stoichiometry of which is regulated over a narrow range of lipid concentration, and the structure of which involves calcium. The physiological advantage of forming this complex has not been determined. We found, however, that lipids containing phosphatidylglycerol absorbed more rapidly to an air/liquid interface than did those without. This rate of adsorption was further increased after interaction with the apolipoprotein.     

Phosphatidylinositol, phosphatidylglycerol and cardiolipin synthesis in adult Dirofilaria immitis females

Srivastava A. K. and Jaffe J. J. 1987. Phosphatidylinositol, phosphatidylglycerol, and cardiolipin synthesis in adult Dirofilaria immitis females. International Journal for Parasitology17:917–920. The pathways leading to the formation of phosphatidylinositol (PI), phosphatidylglycerol (PG) and cardiolipin (CL) in adult Dirofilaria immitis females were investigated. PI was synthesized by both de novo as well as via base exchange pathway in the worms. Under specified assay conditions, the respective rates of PI formation by way of these pathways in crude homogenates of the worms in the order given were around 3.0 and 0.75 nmol min⁻¹ mg⁻¹ protein. PG synthesizing activity in the worms was mainly associated with the particulate fractions and the rate of formation by these fractions was around 1.5 nmol min⁻¹mg⁻¹ protein. The worms were unable to synthesize CL by the pathway found in mammals.     

Lysophospholipid generation and phosphatidylglycerol depletion in phospholipase A(2)-mediated surfactant dysfunction

Pulmonary surfactant's complex mixture of phospholipids and proteins reduces the work of breathing by lowering alveolar surface tension during respiration. One mechanism of surfactant damage appears to be the hydrolysis of phospholipid by phospholipases activated in the inflamed lung. Humans have several candidate secretory phospholipase A(2) (sPLA(2)) enzymes in lung cells and infiltrating leukocytes that could damage extracellular surfactant. We considered two mechanisms of surfactant disruption by five human sPLA(2)s, including generation of lysophospholipids and the depletion of specific phospholipids. All five sPLA(2)s studied ultimately caused surfactant dysfunction. Each enzyme exhibited a different pattern of hydrolysis of surfactant phospholipids. Phosphatidylcholine, the major phospholipid in surfactant and the greatest potential source for generation of lysophospholipids, was susceptible to hydrolysis by group IB, group V, and group X sPLA(2)s, but not group IIA or IID. Group IIA hydrolyzed both phosphatidylethanolamine and phosphatidylglycerol, whereas group IID was active against only phosphatidylglycerol. Thus, with groups IB and X, the generation of lysophospholipids corresponded with surfactant dysfunction. However, hydrolysis of and depletion of phosphatidylglycerol had a greater correlation with surfactant dysfunction for groups IIA and IID. Surfactant dysfunction caused by group V sPLA(2) is less clear and may be the combined result of both mechanisms.     

Adrenalectomy and surfactant in adult rats

We examined the effect of adrenalectomy (ADX) on aspects of the surfactant system of adult rats. Five days after bilateral ADX, ADX rats had about 20% less disaturated phosphatidylcholine (DSPC) in lung lavage returns (airway DSPC) than sham-operated rats, but the amount of tissue DSPC was not different between the groups; airway DSPC formed 12.8 +/- 0.5% of total DSPC (airway + tissue) in ADX and 15.9 +/- 0.7% in sham-ADX rats. An ultrastructural morphometric analysis of alveolar type 2 cells did not reveal an effect of ADX on lamellar body volume density or surface-to-volume ratio. ADX rats had heavier lungs (not as a result of edema) than sham-ADX rats. Treatment of ADX rats with hydrocortisone returned the amount of DSPC toward normal and eliminated the increase of lung weight. ADX did not alter the recoil of saline-filled lungs but did slightly increase the recoil of air-filled lungs. We conclude that corticosteroid hormones influence the in vivo functioning of the surfactant system of adult rats, but this effect seems to be slight.     

The phosphohydrolase activity in lamellar bodies and its relationship to phosphatidylglycerol and lung surfactant formation.

Phosphohydrolase activity of a lamellar body-enriched preparation from pig lung was examined to ascertain if two separate enzymes catalyze the hydrolysis of phosphatidic acid and phosphatidylglycerol 3-phosphate. From sulfhydryl inhibition, heat inactivation and substrate specificity studies, we suggest that one phosphohydrolase may account for the hydrolysis of both substrates. The relationship of the reported experiments to the biosynthesis of the glycerophospholipids present in surfactant is discussed.     

Possible implication of lysophosphatidylcholine in cell fusion accompanying implantation in rabbits.

Implantation in rabbits involves the cellular fusion of trophoblastic and uterine epithelial cells resulting in embryo penetration of the uterine endometrium. Since lysophospholipids, known to have fusigenic properties, could be responsible for this cell fusion, the metabolism of lysophospholipids was studied throughout gestation in blastocyst/yolk sac and extracoelic amnioallantoic fluids. Analysis of phospholipid composition revealed that lysophospholipids are present in blastocyst/yolk sac fluid. Their concentrations and haemolytic activity change during pregnancy. They increase and reach their highest values during days 7 to 9, the implantation days in rabbits. A clear correlation was observed between lysophosphatidylcholine concentrations in blastocyst/yolk sac fluid and haemolysis induced by this fluid. Phosphatidylcholine concentrations, phospholipase A2 activity, which generates lysophospholipids, and lysophospholipase A activity which hydrolyses lysophosphatidylcholine into fatty acid, were at their highest value at day 12. These data suggest that a transient accumulation of lysophospholipids could ensure local cell fusion. Moreover, we propose that the lysophospholipid concentrations in blastocyst/yolk sac fluid are dependent upon activities of phospholipase A2 and lysophospholipase.     

Effect of CO2 concentration on phosphatidylcholine and phosphatidylglycerol metabolism in surfactant and residual lung fractions.

An investigation of the effect of change of total CO(2) concentration from 7 to 43 mM at pH 7.35 in the medium perfusing isolated rat lungs on [U-(14)C]glucose incorporation into lung phospholipids has been carried out. The incorporation of [U-(14)C]glucose into phosphatidylcholine and phosphatidylglycerol of the surfactant fraction and of the remaining lung tissue (residual fraction) was observed. Increased CO(2) concentration increased [U-(14)C]glucose incorporation into phosphatidylcholine of the surfactant fraction and residual fraction by 43 and 50%, respectively, during a 2 hr perfusion. Likewise, incorporation of [U-(14)C]glucose into phosphatidylglycerol was increased 22 and 34% into the surfactant and residual fractions, respectively. The percentage of [U-(14)C]glucose incorporated into the fatty acid moieties of phosphatidylcholine of both fractions increased as a result of increased CO(2) concentration. The increase in the incorporation of [U-(14)C]glucose into the fatty acid moieties of phosphatidylcholine was confirmed by an average increase of 56 and 77% in the specific activity of palmitic acid isolated from phosphatidylcholine of the surfactant and residual fraction, respectively, as a result of increased CO(2) concentration. The results suggest that alteration in extracellular CO(2) concentration affects the de novo synthesis from glucose of phosphatidylcholine and phosphatidylglycerol of the surfactant-lipoprotein fraction of lung.     

Molecular species of phosphatidylcholine and phosphatidylglycerol in rat lung surfactant and different pools of pneumocytes type II.

It is not yet completely understood how a cell is able to export specific phospholipids, like dipalmitoylphosphatidylcholine (dipalmitoyl-PC), which is secreted by pneumocytes type II, into pulmonary surfactant. The acyl species composition of [3H]PC which was synthesized in type II cells in the presence of [2-3H]glycerol resembled the species composition of PC localized in intracellular pneumocyte membranes. This species pattern was different from the pattern of PC of lamellar bodies, i.e., intracellularly stored surfactant, by a higher proportion of dipalmitoyl-PC mainly at expense of 1-palmitoyl-2-oleoyl-PC. Lamellar body PC in turn showed the same species distribution as surfactant PC. The data suggest that subcellular compartmentation and/or intracellular transfer of PC destined to storage in lamellar bodies, but not secretion of lamellar bodies, involves an enrichment of dipalmitoyl-PC and a depletion of 1-palmitoyl-2-oleoyl-PC. In contrast, the acyl species pattern of phosphatidylglycerol does not seem to undergo gross changes on the path from synthesis to secretion.