Lamellar Body Volume Density Research Articles

Human Umbilical Cord Blood Mononuclear Cells in a Double-Hit Model of Bronchopulmonary Dysplasia in Neonatal Mice

BackgroundBronchopulmonary dysplasia (BPD) presents a major threat of very preterm birth and treatment options are still limited. Stem cells from different sources have been used successfully in experimental BPD, induced by postnatal hyperoxia.ObjectivesWe investigated the effect of umbilical cord blood mononuclear cells (MNCs) in a new double-hit mouse model of BPD.MethodsFor the double-hit, date mated mice were subjected to hypoxia and thereafter the offspring was exposed to hyperoxia. Human umbilical cord blood MNCs were given intraperitoneally by day P7. As outcome variables were defined: physical development (auxology), lung structure (histomorphometry), expression of markers for lung maturation and inflammation on mRNA and protein level. Pre- and postnatal normoxic pups and sham treated double-hit pups served as control groups.ResultsCompared to normoxic controls, sham treated double-hit animals showed impaired physical and lung development with reduced alveolarization and increased thickness of septa. Electron microscopy revealed reduced volume density of lamellar bodies. Pulmonary expression of mRNA for surfactant proteins B and C, Mtor and Crabp1 was reduced. Expression of Igf1 was increased. Treatment with umbilical cord blood MNCs normalized thickness of septa and mRNA expression of Mtor to levels of normoxic controls. Tgfb3 mRNA expression and pro-inflammatory IL-1β protein concentration were decreased.ConclusionThe results of our study demonstrate the therapeutic potential of umbilical cord blood MNCs in a new double-hit model of BPD in newborn mice. We found improved lung structure and effects on molecular level. Further studies are needed to address the role of systemic administration of MNCs in experimental BPD.

Restoration of alveolar type II cell function contributes to simvastatin-induced attenuation of lung ischemia-reperfusion injury

Alveolar type (AT) II cells transdifferentiate into ATI cells and as such represent a promising source for regenerating lung epithelium following lung injury. ATII cells are characterized by the presence of lamellar bodies (LBs), which store and secrete the surfactant protein-C (SP-C). Lung ischemia-reperfusion injury (LIRI) causes a distinct impairment of the ATII cell function, subsequently hindering lung repair by loss of ATI transdifferentiation. In this study, we provide new evidence that the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor simvastatin may restore the function of impaired ATII cells in vitro and in vivo. ATII cell lines, A549 (human) and MLE-12 (mouse), were subjected to hypoxia-reoxygenation (H/R) injury. Simvastatin pretreatment at low (5-20 µM), but not high (50-100 µM) doses markedly reduced apoptosis and increased proliferation and SP-C expression. In a rat lung ischemia-reperfusion (I/R) model, simvastatin treatment also increased ATII cell proliferation in vivo, as demonstrated by proliferating cell nuclear antigen/SP-C double staining. Transmission electron microscopy revealed that the number and volume density of LBs were significantly increased in the simvastatin-treated rat lungs. The protective effects of simvastatin were reversed in vitro by PI3-kinase (PI3K) inhibitors wortmannin and L-mevalonate, indicating that the PI3K/Akt and mevalonate pathways may be involved in simvastatin-induced ATII cell function restoration. These data demonstrate that an appropriate dose of simvastatin has a protective effect on LIRI in vitro and in vivo, due at least partially to restored ATII cell function via the HMG-CoA reductase pathway-dependent activation of PI3K/Akt signaling in a mevalonate pathway-dependent manner.

A REEVALUATION OF THE THRESHOLD EXPOSURE LEVEL OF INHALED JP-8 IN MICE

C57BL/6 mice were nose-only exposed to JP-8 jet fuel at average concentrations of 45, 267, and 406 mg JP-8/m(3) for 1 hr/d for 7 days to further test the hypothesis that exposure to JP-8 concentrations below the current permissible exposure level (PEL) of 350 mg/m(3) will induce lung injury, and to validate a new "in-line, real-time" total hydrocarbon analysis system capable of measuring both JP-8 vapor and aerosol concentrations. Pulmonary function and respiratory permeability tests were performed 24 to 30 hr after the final exposures. No significant effects were observed at 45 or 267 mg/m(3). The only significant effect observed at 406 mg/m(3) was a decrease in inspiratory dynamic lung compliance. Morphological examination and morphometric analysis of distal lung tissue demonstrated that alveolar type II epithelial cells showed limited cellular damage with the notable exception of a significant increase in the volume density of lamellar bodies (vacuoles), which is indicative of increased surfactant production, at 45 and 406 mg/m(3). The terminal bronchial epithelium showed initial signs of cellular damage, but the morphometric analysis did not quantify these changes as significant. The morphometric analysis techniques appear to provide an increased sensitivity for detecting the deleterious effects of JP-8 as compared to the physiological evidence offered by pulmonary function or respiratory permeability tests. These observations suggest that the current 350 mg/m(3) PEL for both JP-8 jet fuel and for other more volatile petroleum distillates should be reevaluated and a lower, more accurate PEL should be established with regard human occupational exposure limits.

Microanatomical changes in alveolar type II cells in juvenile mice intratracheally exposed to Stachybotrys chartarum spores and toxin.

Stachybotrys chartarum is an important environmental fungus. We have shown recently that alveolar type II cells are sensitive to exposure to Stachybotrys chartarum spores and to the trichothecene, isosatratoxin-F, both in vitro and in vivo, in a juvenile mouse model. This sensitivity is manifest as significant changes in the composition and normal metabolic processing of pulmonary surfactant. This study evaluated the effects of a single intratracheal exposure of S. chartarum spores and toxin on ultrastructure and dimensions of alveolar type II cells from juvenile mice. This was to determine whether there are concurrent morphological and dimensional changes in the alveolar type II cell that reflect the metabolic alterations in pulmonary surfactant that we observed in the treated mice. Marked ultrastructural changes were associated with alveolar type II cells in both S. chartarum and isosatratoxin-F treated animals compared to untreated, saline, and Cladosporium cladosporioides spore treated animals. These ultrastructural changes included condensed mitochondria with separated cristae, scattered chromatin and poorly defined nucleolus, cytoplasmic rarefaction, and distended lamellar bodies with irregularly arranged lamellae. Point count stereological analysis revealed a significant increase (p < 0.05) in lamellar body volume density in S. chartarum and isosatratoxin-treated animals after 48 h exposure. Mitochondria volume density was significantly lower in the isosatratoxin-F (48 h exposure) and S. chartarum treated (24 and 48 h exposure) animals compared to those in the other treatment groups. These results reveal that exposure to S. chartarum spores and toxin elicit cellular responses in vivo differently from those associated with exposure to spores of a nontoxigenic mold species. They also indicate that accumulation of newly secreted pulmonary surfactant in the alveolar space of S. chartarum and isosatratoxin-F treated animals might be a consequence of cellular trauma resulting in lamellar body volume density changes leading to increased release of pulmonary surfactant into the alveolar space.

Lung parenchyma and type II cell morphometrics: effect of surfactant treatment on preterm ventilated lamb lungs

The effect of exogenous surfactant treatment on lung and type II cell structure of ventilated lambs of 137-138 days gestational age was studied. Thirty-four lambs were delivered and randomized to control or 100 mg/kg of natural sheep surfactant treatment groups. Lungs from one group of lambs not treated with surfactant were fixed before ventilation, and the other animals were ventilated to maintain normal blood gas values for 3, 24, or 48 h. Morphometric assessment of the inflation-fixed lung parenchyma of ventilated lungs was compared with the architectural appearance of alveoli and alveolar ducts in the unventilated lungs. Mechanical ventilation resulted in distension of alveolar ducts accompanied by the shallowing and loss of well-defined alveoli and areas of atelectasis at 3 h. These abnormalities increased in severity after 24 and 48 h of ventilation. Surfactant treatment before ventilation significantly reduced the extent and degree of dilatation and concomitant atelectasis. The fraction of normal parenchyma was 38 +/- 7% in untreated lambs vs. 64 +/- 6% in treated lambs after 24 h of ventilation. After 48 h of ventilation, significant differences between control (39 +/- 6%) and surfactant-treated (55 +/- 6%) lambs were still evident. Alveolar type II cells contained approximately 15% lamellar bodies by volume. Neither surfactant treatment nor time of ventilation altered the volume density of lamellar bodies or other organelles, except for a decrease in glycogen from 8% in nonventilated lungs to 2.5% in lungs ventilated for 24 h. These findings indicate that a surfactant treatment at birth results in the maintenance of more normal parenchyma with less atelectasis during prolonged ventilation of the immature lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Natural surfactant and hyperoxic lung injury in primates. II. Morphometric analyses

Diffuse lung injury is accompanied by low compliance and hypoxemia with histological evidence of endothelial and alveolar epithelial cell disruption. The histological effects of treatment of an acute diffuse lung injury with a natural surfactant product were evaluated in a primate model because surfactant function and content have been shown to be abnormal in diffuse lung injury in both animals and humans. Ten baboons were ventilated with 100% O2 for 96 h, and 5 were given an aerosol of natural porcine surfactant. Physiological and biochemical measurements of the effects of hyperoxia and surfactant treatment are presented in a companion paper. After O2 exposure, lungs were fixed and processed for quantitative electron microscopy. The responses to O2 included epithelial and endothelial cell injuries, interstitial edema, and inflammation. The hyperoxic animals treated with surfactant were compared with the untreated animals; the treatments altered neutrophil distribution, fibroblast proliferation, and changes in the volumes of type I epithelial cells and endothelial cells. Surfactant-treated animals also had decreased lamellar body volume density in type II epithelial cells and preservation of endothelial cell integrity. These changes suggest complex effects of natural surfactant on the pulmonary response to hyperoxia, including protection against epithelial and endothelial cell destruction as well as significant interstitial inflammation and fibroblast proliferation. We conclude that natural surfactant treatment of hyperoxic lung injury in primates resulted in partial protection of epithelial and endothelial cells but also increased the accumulation of fibroblasts in the lung.

Sympathetic nerve influence on alveolar type II cell ultrastructure

Pulmonary surfactant release from lamellar bodies in alveolar type II cells in response to exogenous and circulating catecholamines is well documented but sympathetic influences from direct nerve stimulation are not as extensively studied. In order to study sympathetic nerve influence on release, ultrastructure of type II cells was examined following one min stimulation of the stellate ganglion (SGS) which sends sympathetic fibers to lung parenchyma. Type II cell secretion was assessed by measuring volume density of lamellar bodies, interpreting decreased volume density to indicate increased secretion according to accepted stereologic principles. The present study showed lamellar body volume density was reduced 20.8% in type II cells of SGS animals. Multivesicular body volume density was increased 150% by SGS, but Golgi bodies were unchanged. These data are consistent with type II cell origin of at least some of the increased saturated phospholipid recovered from airspaces in previous SGS studies, and support the view that sympathetic nerve activity may contribute to surfactant homeostasis under some conditions.

Prostaglandins Regulate Surfactant Protein A (SP-A) Gene Expression in Human Fetal Lungin Vitro*

We previously have observed that dexamethasone has a biphasic effect on surfactant protein A (SP-A) mRNA levels in human fetal lung in vitro. At concentrations of 10(-10)-10(-9) M, dexamethasone increases the levels of SP-A mRNA, whereas, at concentrations greater than 10(-8) M, the steroid is markedly inhibitory. In studies to define the molecular mechanisms for these effects, we observed that dexamethasone causes a dose-dependent stimulation of SP-A gene transcription, but paradoxically causes a dose-dependent inhibition of SP-A mRNA stability. In light of the well-characterized inhibitory effect of glucocorticoids on prostaglandin (PG) synthesis in a number of tissues, it was our objective in the present study to investigate the role of PGs on SP-A gene expression in human fetal lung in vitro and to determine whether the action of dexamethasone (greater than 10(-8) M) to reduce SP-A mRNA levels could be mediated by its effect to inhibit PG synthesis. We found that dexamethasone 10(-7) M) caused a marked decrease in the secreted levels of the PGE2 and PGF2 alpha, the prostacyclin metabolite, 6-keto-PGF1 alpha, and the thromboxane A2 metabolite, thromboxane B2. Indomethacin, which also caused a pronounced reduction in the levels of these secreted prostanoids, had a marked effect to reduce SP-A mRNA levels in human fetal lung in vitro. The inhibitory effects of indomethacin were associated with an 73% reduction in cAMP formation by the fetal lung in culture, and were prevented by simultaneous incubation with dibutyryl cAMP or with PGE2. PGE2 markedly increased cAMP formation by the human fetal lung tissue incubated in the absence or presence of indomethacin. Inhibitory effects of dexamethasone and indomethacin also were observed on two morphological indices of lung differentiation, alveolar lumenal volume density, and lamellar body volume density. PGE2 significantly increased lumenal volume density of the human fetal lung explants. The finding that the inhibitory action of dexamethasone (10(-7) M) on SP-A mRNA levels could not be prevented by simultaneous incubation with either PGE2 or dibutyryl cAMP and that dexamethasone had no apparent effect on cAMP formation by the fetal lung in vitro is suggestive that the action of dexamethasone (greater than or equal to 10(-8) M) to reduce SP-A mRNA levels is mediated at least in part by actions alternative to its inhibitory effects on PG synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Vitamin E Alters Alveolar Type II Cell Phospholipid Synthesis in Oxygen and Air

Newborn rats were injected with vitamin E or placebo daily until 6 days after birth. The effect of vitamin E pretreatment on in vitro surfactant phospholipid synthesis was examined in isolated type II cells exposed to oxygen or air form 24 h in vitro. Type II cells were also isolated from untreated 6-day-old rats and cultured for 24 h in oxygen or air with control medium or vitamin E supplemented medium. These cells were used to examine the effect of vitamin E exposure in vitro on type II cell phospholipid synthesis and ultrastructure. Phosphatidylcholine (PC) synthesis was reduced in cells cultured in oxygen as compared with air. This decrease was not prevented by in vivo pretreatment or in vitro supplementation with vitamin E. Vitamin E pretreatment increased the ratio of disaturated PC to total PC and increased phosphatidylglycerol synthesis. The volume density of lamellar bodies in type II cells was increased in cells maintained in oxygen. Vitamin E did not affect the volume density of lamellar bodies. We conclude that in vitro hyperoxia inhibits alveolar type II cell phosphatidylcholine synthesis without decreasing lamellar body volume density and that supplemental vitamin E does not prevent hyperoxia-induced decrease in phosphatidylcholine synthesis.

Effects of ageing on the alveolar pores of Kohn and on the cytoplasmic components of alveolar type II cells in monkey lungs.

The effects of ageing on the numbers of alveolar pores of Kohn and on the cytoplasmic components of alveolar type II cells were studied in monkey lungs by scanning and transmission electron microscopy. Lung tissue from 26 female and three male pigtail macaques whose ages ranged from 1 month to 31 years (life span is 35 years) was analysed. From the age of 1 month to 10 years there was a significant increase in the number of alveolar pores (r = 0.85, p less than 0. 001); however, between the ages of 14 years to 31 years there was no significant change. In seven animals ranging in age from 1 month to 4 years (mean 2.4 years) the number of pores was 5.8 +/- 3.9 (mean +/- S.D.), whereas in 10 animals aged 16 to 31 years (mean 20.3 years) the number of pores was 32.7 +/- 17.5 (mean +/- S.D.) per alveolar profile, a significant difference (p less than 0.002). In older animals (15-20 years) there was a significant decrease, both in the number of lamellar bodies per alveolar type II cell (p less than 0.01) and in the volume density of lamellar bodies to cytoplasmic volume (p less than 0.05) compared with young animals (1 month to 4.8 years). In older animals, there was also a significant increase in the volume density of a vacuole-like dilatation of the endoplasmic reticulum in alveolar type II cells (p less than 0.05) compared with young animals. These findings suggest impaired pulmonary surfactant production with aging. Both the increased number of alveolar pores and the postulated decrease of surfactant production could play a role in the pathogenesis of pulmonary emphysema.

Influence of maternal diabetes on basement membranes, type 2 cells, and capillaries in the developing rat lung

To determine the effect of maternal diabetes on rat lung development, we studied the ultrastructure of the alveolar wall from the ninteenth day of gestation (term = 22 days) through the eighth postnatal day in fetal and neonatal rats of mothers with streptozotocin-induced diabetes. In normal fetal lung development, epithelial basement membranes develop large discontinuities beneath type 2 cells, through which cytoplasmic foot processes extend into the interstitium. Maternal diabetes delays the appearances of these epithelial basement membrane discontinuities and reduces the number of type 2 cell processes that penetrate it. These alterations in epithelial basement membrane are reversed after birth. There is no ultrastructural evidence of a delay in type 2 cell maturation as assessed by lamellar body volume density morphometry. Endothelial basement membranes, which are not present around the growing pulmonary capillary bed in the pseudoglandular lung, are seen late in normal gestation, primarily around capillaries forming the mature air-blood barrier. This development of endothelial basement membrane may be delayed in the fetuses of diabetic mothers and reflects a significant delay in the expansion of the pulmonary capillary network in these animals as assessed by morphometric volume density measurements. This effect on capillary growth is not reversed in the newborn animals through 8 days after birth. The summation of these effects indicates a generalized slowing of fetal lung development by maternal diabetes, some of which effects persist after birth and may continue to influence lung development during the period of postnatal alveolar septal growth.

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.

Giant lamellar bodies in alveolar type II cells of rats exposed to a low concentration of ozone.

The effect of a low concentration of ozone on pulmonary surfactant release was examined by the ultrastructural morphometry of lamellar bodies in alveolar type II cells. Rats were exposed to approximately 0.3 ppm of ozone for 3 h daily for 16 consecutive days. The volume density of lamellar bodies, relative to the cell, increased significantly following ozone exposure. After 11 days giant lamellar bodies were found in ozone-exposed rats. These findings suggest that ozone exposure causes the impairment of pulmonary surfactant release.

Transpleural CO 2 diffusion in excised dog lungs

Previous studies of transpleural gas diffusion have reported both linear and nonlinear relationships between total gas transfer and the transpleural driving gradient estimated as the difference between pleural and end-tidal gas tensions. The present studies were undertaken to explain these conflicting results by examining the role of convective mixing as well as the validity of using measurements of end-tidal gas to estimate subpleural gas tensions and calculate the transpleural partial pressure gradient. Nine excised nonperfused dog lungs were ventilated with room air under varying conditions of frequency (f), tidal volume ( Vt) and pleural CO 2 concentration (Fpl CO 2 ). The relationship between CO 2 elimination (V CO 2 ) and the transpleural CO 2 concentration gradient estimated by (Fpl CO 2  Fet CO 2 ) was found to be linear for every level of ventilation (constant convective mixing) studied. The slope of this relationship, equal to the pleural CO 2 diffusing capacity (Dpl CO 2 ) was found to decrease as ventilation was decreased whether achieved by reductions in f or Vt. Because changes in membrane geometry were insufficient to explain the differences in Dpl CO 2 , seven additional excised dog lungs were studied ultrastructurally following 4 h of ventilation with room air at a pleura CO 2 concentration of 6%. Previous studies under similar conditions have shown that changes in type II pneumocyte lamellar body volume density Vv(LB/cyto) are related to the alveolar concentration of CO 2. The results indicated that the concentration of CO 2 in alveoli located within 1 mm of the pleural surface was indicated that the concentration of CO 2 in alveoli located within 1 mm of the pleural surface was substantlially greater than the value estimated by Fet CO 2 . Consequently, (Fpl CO 2  Fet CO 2 ) overstimates the transpleural concentration gradient and produces erroneously low estimates of the diffusing capacity of the pleural membrane.

Influence of methylprednisolone on mouse alveolar type 2 cell response to acute lung injury.

To determine whether corticosteroids influence alveolar type 2 cell events after acute lung injury, we administered 30 mg/kg/day of methylprednisolone (MP) to mice at various times after a single injection of butylated hydroxytoluene (BHT). Tritiated thymidine (3H-T) autoradiographs were used to study type 2 cell proliferation and differentiation. Type 2 cell maturation was evaluated by ultrastructural analysis of lamellar body volume density and area in cells labeled with 3H-T. The BHT damaged type 1 cells and induced proliferation of type 2 cells. Sixty per cent of type 2 cells labeled with 3H-T on Day 3 differentiated into type 1 cells over a 4-day period. Lamellar body area per cell was unchanged. Early administration of MP (Days 1 and 2) inhibited type 2 cell proliferation when lung injury was mild; rebound proliferation occurred afer its discontinuation. The MP failed to inhibit proliferation when lung injury was severe. Prolonged MP administration (Days 1 to 5) resulted in greater type 2 cell proliferation, lung injury, and mortality. Late MP administration (Days 3 and 4) impaired differentiation of type 2 to type 1 cells and more than doubled lamellar body area per cell of new type 2 cells. Important interactions appear to exist between proliferation, differentiation, and maturation of alveolar type 2 cells suggesting that agents that influence one of these processes may influence all aspects of type 2 cell function.

Response of normal and beige mouse alveolar type 2 cells to lung injury.

To study the relation between normal and abnormal alveolar type 2 cells and lung repair after injury, and to gain insight into the defect responsible for the giant lamellar bodies in type 2 cells of the beige mouse (an animal model of the Chediak-Higashi syndrome), we produced lung injury with a single injection of butylated hydroxytoluene (BHT). Type 2 cell proliferation and differentiation were analyzed in light microscopic autoradiographs of mice that had received tritiated thymidine (3H-T) at intervals after the injection of BHT. The approximate age of 3H-T labeled type 2 cells and lamellar body volume density and area were correlated by combining light microscopic autoradiography with ultrastructural morphometry. Type 2 cell proliferation and differentiation were identical in normal and in beige mice. In normal mice, the lamellar body area per cell was unchanged at the time periods studied. In beige mice, the lamellar body area per cell, which was initially 9 times that of the normal mice, decreased to nearly normal values after mitosis, and then increased over the subsequent 7 days of study. Thus, the abnormal type 2 cell of the beige mouse was able to proliferate and differentiate in a normal fashion after lung injury. The beige mouse defect, which results in impaired lamellar body release, was transiently corrected in the perimitotic period. The postmitotic enlargement of lamellar bodies provided a model for studying the basic defect in the Chediak-Higashi syndrome and the process of type 2 cell maturation.

Effects of nickel dust on rabbit alveolar epithelium

Eight male rabbits were exposed to metallic nickel dust, during 1 month (5 days/week, 6 hr/day) at a concentration of 1.7 mg/m 3 (SD 0.5), about 40% of which was respirable. Morphometric measurements on the lungs of exposed rabbits showed about a twofold increase in volume density of type II alveolar epithelial cells compared to eight controls. This increment was about the same as the average increase in phospholipids in the lung parenchyma of the rabbits described elsewhere. The volume density of lamellar bodies within the cytoplasm of the type II cell was not significantly increased. Large amounts of laminated structures, resembling those in the alveolar type II cells, were also found free in the alveoli in the exposed rabbits.

Influence of fasting on the lung

We examined the following in fed rats and in rats fasted for 72 h: 1) the dipalmitoyl lecithin (DPL) content of lung lavage fluid and of the remaining lung tissue, 2) descending air and saline pressure-volume curves of excised lungs, and 3) the volume density of granular pneumocyte lamellar bodies. Lung tissue DPL was decreased by 27% and lavage DPL was decreased by 40% in lungs of fasted rats. The decreased lung DPL content was associated with a 20% decrease in the volume density of lamellar bodies of granular pneumocytes. In spite of the decrease in lavage DPL content, air pressure-volume curves of excised lungs were the same as curves of lungs of fed rats. Saline pressure-volume curves of excised lungs were also the same in fed and fasted rats. The amount of lavage DPL obtained from both fed (1.1 +/- 0.1 mg, n=6) and fasted (0.7 +/- 0.1 mg, n=7) rats exceeded the theoretical minimum amount of DPL (0.5 mg) required for a monomolecular film to cover the alveolar surface of the rat at functional residual capacity. If we assume that lavage DPL represents mainly DPL lining the alveolus (surface film and hypophase) the data suggest that there is an alveolar reserve of DPL above that amount needed to maintain normal alveolar stability.