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.

Analysis of pulmonary surfactant by Fourier-transform infrared spectroscopy following exposure to Stachybotrys chartarum (atra) spores.

Lung cells are among the first tissues of the body to be exposed to air-borne environmental contaminants. Consequently the function of these cells may be altered before other cells are affected. As gas exchange takes place in the lungs, changes in cellular function may have serious implications for the processes of oxygen uptake and carbon dioxide elimination. In order for these processes to occur, the lung must maintain a high degree of expandability. This latter function is accomplished in part by the pulmonary surfactant which is synthesized and released by alveolar type II cells. Earlier studies have shown that exposure to gas phase materials such as smoke or organic solvents can alter the composition and function of the surfactant. The present study examines the ability of highly toxigenic mold spores to alter surfactant composition. Stachybotrys chartarum spores suspended in saline were instilled into mouse trachea as described earlier. After 24 h, the lungs were lavaged and the different processing stages of surfactant isolated by repeated centrifugation. Intracellular surfactant was isolated from the homogenized lung tissue by centrifugation on a discontinuous sucrose gradient. Samples were extracted into chloroform-methanol, dried and analyzed by Fourier-Transform infrared spectroscopy (FTIR). Exposure to S. chartarum induced an overall reduction of phospholipid among the three surfactant subfractions. The intermediate and spent surfactant fractions in particular were reduced to about half of the values observed in the saline-treated group. The relative distribution of phospholipid was also altered by spore exposure. Within the intracellular surfactant pool, higher levels of phospholipid were detected after spore exposure. In addition, changes were observed in the nature of the phospholipids. In particular strong intramolecular hydrogen bonding, together with other changes, suggested that spore exposure was associated with absence of an acyl chain esterified on the glycerol backbone, resulting in elevated levels of lysophospholipid in the samples. This study shows that mold spores and their products induce changes in regulation of both secretion and synthesis of surfactant, as well as alterations in the pattern of phospholipid targeting to the pulmonary surfactant pools.

Effects of Stachybotrys chartarum on surfactant convertase activity in juvenile mice.

We have shown recently that alveolar type II cells are sensitive to exposure to Stachybotrys chartarum spores, both in vitro and in an in vivo juvenile mouse model. In mice, this sensitivity is manifest in part as a significant increase in the newly secreted, biologically active, heavy aggregate form of alveolar surfactant (H) and the accumulation of the lighter, "metabolically used", biologically inactive alveolar surfactant forms (L(vivo)) in the interalveolar space. Conversion of the heavy, surface-active alveolar surfactant to the light metabolically used, nonsurface active forms is believed to involve the activity of an enzyme, namely convertase, which is thought to be derived from lamellar bodies (LB) in alveolar type II cells. The purpose of this study was to evaluate the effects of S. chartarum spores on mouse H and LB convertase activity by measuring their rates of conversion to L(vivo) using the in vitro surface area cycling technique. It was determined whether there were concurrent changes in the protein and phospholipid concentrations of the raw bronchoalveolar lavage fluid (RL) and LB fractions that could be correlated with changes in convertase activity. Conversions of H to L(vivo) in untreated control mice and saline-, isosatratoxin F-, and Cladosporium cladosporioides-exposed mice were not significantly different (p > 0.05). However, conversion from H to L(vivo) in the mice exposed to S. chartarum spores was significantly higher than all other treatment groups (p < 0.001). LB to L(vivo) conversions in untreated and saline-exposed mice were not significantly different, although they were significantly higher than the H to L(vivo) conversions in these two animal treatment groups (p < 0.005), which supports the position that LB is a source of convertase activity in animals. LB to L(vivo) conversion from C. cladosporioides-, isosatrotoxin F-, and S. chartarum-exposed mice were all significantly depressed (p < 0.003) compared to the LB to L(vivo) conversion values obtained from untreated and saline-exposed mice. Protein concentrations in RL, H, L(vivo), and LB from mice exposed to S. chartarum spores were significantly elevated compared to those from the other treatment groups (p < 0.001). Protein concentration in H isolated from C. cladosporioides-exposed mice was also significantly elevated above untreated and saline control animal levels. Phospholipid concentrations in H isolated from S. chartarum-exposed mice were significantly elevated compared to those from other treatment groups, while LB phospholipid concentrations were significantly increased compared to saline and untreated control animal groups. These results show that S. chartarum spores significantly alter convertase activity in both the H and LB surfactant fractions in juvenile mice and that these changes can be related to changes in protein and phospholipid concentrations in alveolar lavage fractions. As surfactant promotes lung stability by reducing the surface tension of the air-alveolar interface, these results further support our position that inhalation exposure to S. chartarum spores in exposed individuals may lead to altered surfactant metabolism, and possibly to lung dysfunction through diminished alveolar surfactant surface tension attributes, and lung stability.

Alveolar metabolism of natural vs. synthetic surfactants in preterm newborn rabbits.

We compared the recoveries of four surfactant preparations: two natural [term fetal rabbit surfactant (FRS) and adult rabbit surfactant (ARS)] and two commercially available preparations [apoprotein-based Survanta (S) and synthetic Exosurf (E)] from 27-day gestation rabbit pups treated at birth and ventilated up to 120 min. At 5, 60, and 120 min, we measured the recovery of the heavy-aggregate, metabolically active form (H) and the light-aggregate, nonsurface active metabolic breakdown form (L) of alveolar surfactant and determined the phospholipid content and composition of the intracellularly stored lamellar body (LB) pool. Pups treated with FRS had <15% loss of H by 2 h. ARS-treated pups had a >50% loss of H by 1 h, and E- and S-treated pups had approximately 50% loss by 5 min, with a slower rate of continuing loss of up to 80% by 2 h. The major losses of H phospholipid were not explained by the L-form recovery. LB phospholipid significantly increased only in the E-treated pups and only at 2 h. FRS provides a biologically active form (H) of surfactant that appeared to remain in the airway for a significantly longer time than the other surfactant preparations. The unique properties of FRS merit further study.

Comparative effects of chronic exposure to glucose or sodium butyrate on surfactant development in fetal rabbits.

BACKGROUND: Infants of diabetic mothers (IDM) often have delayed lung development and are thus at an increased risk of Respiratory Distress Syndrome (RDS). Both hyperglycemia and/or hyperinsulinemia have been implicated in this delay but the precise mechanism has not been clarified. Another metabolite, sodium butyrate, which is increased in IDM has been shown to decrease surfactant production in vitro but its effects on the development of the fetal lung surfactant system in vivo have not been studied. AIM: To investigate the in vivo effects of high glucose and sodium butyrate treatment on maternal and fetal glucose and insulin levels and on fetal lung surfactant maturation using timed-pregnant New Zealand White rabbits. METHODS: On the 24th day of gestation the doe was implanted s.c. with time release pellets containing either glucose (300 mg), sodium butyrate (200 mg) or matching placebo. On the 27th or 30th day maternal (ear vein) and fetal (cardiac puncture) blood samples were drawn for glucose and insulin determinations. Fetal surfactant pools (both intra- and extracellular) were quantitatively harvested using differential and density gradient centrifugation and their phospholipid profiles determined. Data were statistically compared with ANOVA and Duncan's Multiple Range Test. RESULTS: Neither glucose nor sodium butyrate affected maternal plasma glucose or insulin. Both metabolites significantly increased fetal plasma insulin, decreased fetal plasma glucose but did not delay any of the parameters of surfactant maturation examined. CONCLUSIONS: Fetal hyperinsulinemia, whether attained by prolonged exposure to elevated glucose or sodium butyrate in vivo does not appear to be the causative agent for delayed lung maturity which frequently occurs in infants of diabetic mothers.

Calcium-PS-dependent protein kinase C and surfactant protein A in isolated fetal rabbit type II alveolar cells and surfactant-related material.

The fetal lung secretes significant quantities of surfactant during late gestation to prepare for initiation of respiration at birth. However, the mechanism by which this occurs has not been determined. Since Ca2+-phosphatidylserine (PS)-dependent protein kinase C has been implicated in surfactant secretion in adult lung, the present study was done to determine whether this enzyme is also involved in the initiation of surfactant release from fetal type II cells. Type II cells isolated from gestational day-24 fetal rabbits were used. Cells were prelabelled with [32P] and [3H]choline and exposed to 4beta phorbol ester (10(-5) M) for 2 h. Secretion product and subcellular fractions were isolated by removing the culture medium, mixing with homogenate from adult rabbit lung, and subfractionating by centrifugation on a sucrose gradient. Samples of secretion product were also prepared for electron microscopy. Ca2+-PS-dependent protein kinase C was also assayed in some samples, and an add-back technique was used to determine whether enzyme activity in the intracellularly stored surfactant fraction was due to contamination. The results showed that material released by fetal type II cells after exposure to phorbol ester coprecipitated with adult rabbit lung lamellar bodies and microsomes. Morphologically, a range of forms, including lamellar-body-like structures, was detected. The released material originated largely from the lamellar body compartment of the fetal type II cells and displayed immunoreactivity with antibody to surfactant protein A (SP-A) at 35 and 70 kDa apparent molecular mass. Assay of protein kinase C in fetal type II cells showed that exposure to conditioned medium, which induces differentiation, increased activity. Incubation with phorbol ester induced translocation of activity to the microsomal fraction. Add-back assays suggested that protein kinase C activation by treatment with phorbol ester induced translocation of enzyme activity to the lamellar body fraction; none was detected prior to treatment. These results support a role for Ca2+-PS-dependent protein kinase C in initiation of surfactant release by interaction with the developing lamellar body compartment in fetal type II cells.

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

Conversion of heavy-aggregate alveolar surfactant (H) to a light-aggregate, nonsurface active form (L) is believed to involve the activity of an enzyme, namely, convertase. This conversion can be reproduced in vitro by the surface-area cycling technique. The purpose of the present study was to use this technique to investigate the developmental aspects of convertase activity in fetal, newborn, and adult rabbits. H was isolated from alveolar lavage from term [31-day gestation (31d)] fetal rabbit pups, 1-, 4-, and 7-day-old newborns, and adults, and the percent conversion to L was determined. To assess lamellar bodies (LB) as a potential source of activity in this species, these structures were isolated from lung tissue of 27-day-gestation (27d) and 31d fetuses, 1-, 4-, and 7-day-old newborns, and adults and were cycled the same as for H. LB contained considerable activity at each developmental stage i.e., approximately 82% of a 27d LB preparation converted to L after 3 h of cycling. In the adult, this value was 78%. Very little conversion of H was obtained from fetal lung (i.e., <20% of the 31d fetal preparation converted to L), but, by postnatal day 4, this value was greatly increased (i.e., >80% conversion) and stayed elevated to adulthood. The activity for each H and LB fraction was temperature and concentration dependent and diminished with storage at 4 degreesC. These data suggest the LB as the source of convertase activity in the rabbit and demonstrate dramatic developmental changes in this activity after release of the LB contents to the alveoli.

Effects of Stachybotrys chartarum (atra) conidia and isolated toxin on lung surfactant production and homeostasis.

This study evaluated the effects of Stachybotrys chartarum conidia and a trichothecene, isosatratoxin-F, on choline incorporation into DSPC by fetal rabbit alveolar type II cells and on alveolar surfactant subtypes in mice. Exposure of fetal rabbit type II cells to S. chartarum conidia at concentrations of 10(3) to 10(6) conidia ml(-1) significantly depressed [3H] choline incorporation after 24 h of exposure. Exposure of the rabbit cells to 10(5) to 10(6) conidia ml(-1) also resulted in significantly depressed [3H] choline uptake after 48 h. Additionally, fetal rabbit alveolar type II cells exposed to isosatratoxin-F in concentrations ranging from 10(-9) to 10(-4) M showed a significant reduction in [3H] choline incorporation into DSPC. Alveolar surfactant phospholipid concentrations in the different metabolic subfractions of lung lavage fluid of mice intratracheally exposed to either 50 microl of 10(7) ml(-1) S. chartarum conidia or 50 microl 10(-7) M isosatratoxin-F showed some significant changes at 12, 24, 48, and 72 h post-exposure, compared to the surfactant subfractions of control mice which were either untreated, exposed to saline or to 50 microl of 10(-7) ml(-1) Cladosporium cladosporioides conidia. In both the S. chartarum- and the isosatratoxin-F-treated mice, exposure significantly increased P10, P100, and S100 phospholipid concentrations, while the P60 phospholipid concentrations were depressed. In contrast, C. cladosporioides-treated mice showed only one significant change in subfraction phospholipid concentration: P60 was depressed at 48 h post-exposure. These results reveal that alveolar type II cells are sensitive to exposure to S. chartarum conidia and to isosatratoxin F. Sensitivity is manifest by alterations in the normal metabolic processing of alveolar surfactant. In exposed mice, this effect appears to involve a significant increase in newly secreted surfactant and an accumulation of the used surfactant forms.

Methylmercury-induced alterations in lung and pulmonary surfactant properties of adult mice.

Exposure to methylmercuric chloride (MMC) has been shown to significantly affect development of the lung and pulmonary surfactant system of the fetus. Preliminary results suggest it may also affect adult lung and associated bronchoalveolar lavage (BAL), which represents the extracellular surfactant pool. To determine if mercury exposure has the potential to alter surfactant function, adult mice were treated with MMC, 15 mg/kg by intragastric intubation on 4 successive days. BAL was collected by repeated intratracheal lavage 24 h after the last treatment. Nucleated cell numbers in lavage were determined. Tissue was prepared for scanning electron microscopy (SEM). Lavage fluid was extracted into chloroform:methanol and phospholipid concentration determined. A sample of the extract was used at a constant phospholipid concentration to measure surface activity on a bubble surfactometer. Lung weight to body weight ratio increased whereas total numbers of nucleated cells in BAL were not altered by MMC. SEM of samples from lungs of animals exposed to MMC showed normal architecture. Surface tension measurements suggest that the mean time to minimum surface tension and the minimum surface tension were greater in BAL from mice exposed to MMC for 4 days. In addition samples of BAL were prepared for Fourier-transform infrared spectrophotometry (FT-IR). Spectra showed changes in both lipid and protein components of BAL. Morphometric analyses of micrographs showed that mean alveolar diameter was reduced and wall thickness increased after mercury exposure. These results suggest that methylmercury exposure may significantly affect surface tension characteristics and composition of BAL, possibly through leakage of edematous interstitial tissue.

Fourier-transform infrared spectroscopic analysis of rabbit lung surfactant: subfraction-associated phospholipid and protein profiles.

Surfactant obtained from bronchoalveolar lavage (BAL) can be separated into subfractions based on sedimentation characteristics. It has been suggested that the 10,000 x g, 60,000 x g and 100,000 x g subfractions isolated by this approach represent stages of surfactant extracellular processing. These three subfractions have been reported to differ in their morphology, composition and ability to lower surface tension. We wished to determine if infrared spectroscopy, which may be applied as a non-invasive technique could potentially prove useful for characterization and quantification of bronchoalveolar lavage (BAL) protein and phospholipid, and if this approach could detect differences in intermediate surfactant processing stages. Subfractions were collected from adult rabbit lungs by BAL and differential centrifugation and analyzed by Fourier transform infrared (FT-IR) spectroscopy. Biochemical assay of phospholipid and protein showed differences between subfractions that correlated well with the phospholipid/protein ratios obtained from FT-IR spectra (r = 0.939; r2 = 0.882). The subfraction sedimenting at 100,000 x g (P100) exhibited spectral shifts in the Amide I band, suggesting that the protein secondary structure was different compared to other fractions. Spectra obtained after separation of lipids and protein components showed an apparent disordering of protein secondary structure but little or no effect on the structure or mobility of phospholipids. These results support the idea that subfractions represent various processing stages of surfactant. In addition, they show that results from FT-IR analyses correlate significantly with traditional biochemical assay methods which may prove of clinical use.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of smoke inhalation on alveolar surfactant subtypes in mice.

The effects of smoke inhalation on alveolar surfactant subtypes were examined in mice exposed for 30 minutes to smoke generated from the burning of a flexible polyurethane foam. At 4 or 12 hours after the exposure, three surfactant pellets, P10, P60, and P100, and a supernatant, S100, were prepared by sequential centrifugation of lavage fluids at 10,000 g for 30 minutes (P10), 60,000 g for 60 minutes (P60), and 100,000 g for 15 hours (P100 and S100). Phospholipid analysis and electron microscopy were performed on each fraction. Smoke exposure dramatically altered the normal distributions of these fractions: it significantly increased the phospholipid content of the heavier subtype, P10, which is thought to represent newly secreted surfactant; had no effect on the intermediate form, P60; and dramatically increased the phospholipid content (approximately fivefold) of the lighter subtypes, P100 and S100, which are believed to represent catabolic end-products of alveolar surfactant. Only P100 was structurally altered by the smoke. These results represent alterations of the normal metabolic processing of alveolar surfactant. Whereas the mechanism is yet to be defined, it seems to involve a small but significant increase in the newly secreted surfactant, as well as an excessively high accumulation of the structurally altered catabolic forms of the secreted surfactant.

Mouse alveolar surfactant: characterization of subtypes prepared by differential centrifugation.

To characterize the properties of alveolar surfactant subfractions obtained from mouse lung by differential centrifugation, lavage fluid, following a preliminary centrifugation at 140 x g for 5 min to yield a cellular pellet (Pc), was sequentially centrifuged at 10,000 x g for 30 min, 60,000 x g for 60 min and 100,000 x g for 15 h; and the resultant pellets, respectively referred to as P10, P60 and P100, were harvested for electron microscopy, phospholipid analysis and surface tension measurements. Ultrastructural differences were observed, in that P10 contained large multilamellated structures which were typical of newly secreted surfactant, P100 contained small unilamellar vesicular structures, typical of catabolic end products of alveolar surfactant and P60 appeared to contain a mixture of structures present in P10 and P100 in addition to numerous, large unilamellar vesicles which were not present in either P10 or P100. Slight but significant differences were found in the phospholipid compositions of the three subfractions but not in the fatty acid composition of their phosphatidylcholine (PC) component. There were no significant differences in their disaturated PC/total PC ratios, but significant differences in their phospholipid/protein ratios. P60 had the highest proportion of phospholipid to protein. P10 and P60 demonstrated surface activity but P100 did not. Total alveolar surfactant phospholipid was evenly distributed among the three fractions. This pattern of distribution was significantly different from that observed in rabbit subfractions prepared by the same procedure. These data indicate that mouse alveolar surfactant consists of three distinct subfractions or subtypes which can be separately and quantitatively isolated by differential centrifugation.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased inorganic sulfate concentrations in amniotic fluid.

We assayed inorganic sulfate by ion chromatography in 49 amniotic fluid samples from pregnancies of 14 to 38 weeks gestation. In second trimester samples (14 to 26 weeks gestation), amniotic fluid sulfate concentrations (317 +/- 22 mumol/L, mean +/- SE; n = 32) were not different from previously reported maternal serum values but were significantly lower (p < 0.001) than in the third trimester (693 +/- 42 mumol/L; n = 16). In third trimester samples, sulfate concentrations were significantly correlated with creatinine and uric acid but not chloride, suggesting that renal excretion may be the major source of the amniotic fluid sulfate in the late stages of gestation.

Autoradiographic localization and effects of exogenous radioactively labelled surfactant in the lung of the prematurely delivered fetal rabbit.

Adult rabbit lung surfactant was radioactively labelled with [3H]palmitate and isolated by centrifugation. This material was instilled into the trachea of fetal rabbits prematurely delivered on the 27th gestational day. A similar preparation of unlabelled surfactant was used to measure the effects on pressure-volume characteristics in lungs of 27th day fetuses. Tissue sections were prepared from the lungs of all animals and morphometric and autoradiographic determinations made. Surfactant instillation improved pressure-volume relationships in fetal rabbit lungs. Histologically, although only the middle right lobe seemed to show significant qualitative improvement in expansion after surfactant treatment, quantitative assessment indicated that the surfactant preparation had significantly increased the mean alveolar cross-sectional areas in all three lobes of right lungs. In addition, distribution of autoradiographic grains indicated that 8-25% were located over the alveolar spaces while approximately half this percentage was present over tissue at the level of the alveolus. These results indicate that intratracheal instillation of surfactant supplements the endogenous surfactant at the level of the alveolus.

Effects of smoke inhalation on surfactant phospholipids and phospholipase A2 activity in the mouse lung.

The effects of smoke inhalation on the pulmonary surfactant system were examined in mice exposed for 30 minutes to smoke generated from the burning of polyurethane foam. At 8 or 12 hours after exposure, surfactants were isolated separately from lung lavage (extracellular surfactant) and residual lung tissue (intracellular surfactant) for phospholipid analysis. Calcium-dependent phospholipase A2 (PLA2) was measured on a microsomal fraction prepared from the tissue homogenate. Smoke inhalation produced a twofold increase in extracellular surfactant total phospholipid. While there was no change in the total phospholipid or phosphatidylcholine (PC) content of the intracellular surfactant, smoke inhalation significantly decreased the disaturated species of PC (DSPC). The specific activity of PLA2 was reduced by more than 50% in both groups of exposed mice. Smoke inhalation appears to result in selective depletion of the DSPC of intracellular surfactant and PLA2 involved in its synthesis. This depletion may be compensated for by increased secretion or slower breakdown of the material present in the extracellular compartment.

Correlation of absorbance at 650 nm with the presence of phosphatidylglycerol in amniotic fluid.

Amniotic fluid absorbance at 650 nm was correlated with the presence of phosphatidylglycerol (PG) in the isolated surfactant fraction (10,000-g pellet). Shake test results were included. Two hundred ninety-seven samples were analyzed. PG was present in 222 of 226 samples in which the absorbance was greater than or equal to 0.250 and absent from 48 of 71 with an absorbance less than 0.250. PG was present in all 166 samples with a positive shake test and absent in 52 of 131 samples with a negative one. In 65 samples in which the shake test was negative and the absorbance greater than or equal to 0.250, PG was present in all but 4. The false-positive rate for the prediction of respiratory distress syndrome was 0.8% for the Shake test and 0.6% for the absorbance measurement. The results support the usefulness of the absorbance measurement as a simple and reliable procedure for assessing fetal lung maturity.

Gestation-dependent effects of the combined treatment of glucocorticoids and thyrotropin-releasing hormone on surfactant production by fetal rabbit lung.

The effects of cortisol (0.1 mg per dose, administered intraperitoneally to fetal rabbits at 24 to 27 days' gestation), thyrotropin-releasing hormone (40 micrograms/kg per dose administered intravenously to the doe at 24 to 26 days' gestation), or a combination of the two on surfactant pool size (both intracellular and extracellular) at 27 or 28 days' gestation was investigated. Cortisol increased both surfactant pools only when administered on the twenty-fourth or twenty-fifth gestational day. Thyrotropin-releasing hormone, whether administered in single or multiple doses, had no effect on the extracellular pool but increased the intracellular pool; the magnitude of the response (approximately twofold) was similar to that observed with the cortisol response. All combinations of cortisol and thyrotropin-releasing hormone resulted in an increased response over either drug given alone. The greatest response (almost tenfold) resulted from cortisol administration at 24 days' gestation plus thyrotropin-releasing hormone administration at 24+ 25+ 26 days. These data demonstrate differential effects of glucocorticoids and thyrotropin-releasing hormone on developing lung and furthermore show that the timing of their combined treatment may be crucial to achieving maximal response.

Pulmonary toxicity of trichloroethylene: induction of changes in surfactant phospholipids and phospholipase A2 activity in the mouse lung.

Trichloroethylene (TCE) is a common organic solvent in use as a dry cleaning agent as well as an inhalant anesthetic. Nevertheless the effects of this material on the pulmonary surfactant which prevents alveolar collapse at maximal expiration is not known. Therefore, we have examined the effect of TCE on the intra- and extracellular surfactant pools and the activity of phospholipase A2, an enzyme which controls the remodeling of phosphatidylcholine to dipalmitoylphosphatidylcholine, the primary constituent of the pulmonary surfactant. Male CD-1 mice were treated ip with 2500 or 3000 mg/kg TCE. Twenty-four hours later mice were anesthetized and the lungs lavaged. Mice were then killed, the lungs perfused and excised, and subcellular fractions including lamellar bodies prepared. Some lungs were prepared for ultrastructural examination. Phospholipase A2 was assayed in all subcellular fractions. Phospholipid was assayed in the lavage (extracellular surfactant) and the lamellar bodies (intracellular surfactant). TCE (2500 mg/kg) caused selective exfoliation of Clara cells. However, only the dose of 3000 mg/kg TCE produced a significant decrease in the intracellular surfactant phospholipid. Minimal changes occurred in the phospholipid profiles. Phospholipase A2 specific activity was significantly decreased at both dosages within the lung microsomal fraction. In addition after treatment with 3000 mg/kg TCE the enzyme activity in the lamellar body fraction was significantly increased. These data suggest that inhalation of TCE may damage the enzymes which are responsible for synthesizing the pulmonary surfactant resulting in lower amounts of surfactant being stored and available for secretion into the alveolus.

Beta-adrenergic-induced surfactant synthesis, secretion, and reutilization in fetal rabbit lung and isolated differentiating type II alveolar cells.

In vivo and in vitro approaches were used to examine the role of beta-adrenergic agonists in the regulation of surfactant synthesis and secretion in the lung. Rabbit fetuses of either 28 or 30 gestational days were treated with isoxsuprine. Fetuses from half of the does in each group were removed and allowed to breathe for 30 minutes. The others were left in utero. Intracellular and extracellular surfactant pools were isolated. Breathing increased secreted surfactant. On the twenty-eighth day without breathing, isoxsuprine treatment increased secretion of surfactant. The reverse effect was noted in the group that received the drug and also breathed. In contrast, on the thirtieth day, the drug inhibited surfactant release in those fetuses that did not breathe. In in vitro studies, undifferentiated type II alveolar cells were isolated and stimulated to differentiate. Subsequent exposure to isoxsuprine (5 or 10 mumol/L) stimulated both the synthesis and secretion of radiolabeled disaturated phosphatidylcholine. Concurrent incubation of those cells exposed to 10 mumol/L isoxsuprine with either unsaturated or disaturated phosphatidylcholine that was carbon 14 labeled showed a strong preference for incorporation of the latter phospholipid into total cellular phosphatidylcholine. These results suggest that beta-adrenergics may inhibit as well as stimulate secretion of surfactant by type II alveolar cells and that these cells may reincorporate secreted disaturated phospholipid.

Subcellular distribution of disaturated phosphatidylcholine in developing rabbit lung.

To determine the subcellular distribution of disaturated phosphatidylcholine (DSPC) in lung tissue during perinatal development, fetal rabbits at 24, 26, 28 and 31 (term) days gestation and newborns were studied. Following alveolar lavage, fractions enriched in nuclei-cellular debris, mitochondria, microsomes, surfactant (lamellar bodies) and cytosol were prepared from the residual tissue homogenate, and their DSPC content was determined. The DSPC content of the unfractionated residual lung tissue homogenate progressively and significantly increased during fetal development, rising from 9.09 +/- 0.91 to 17.45 +/- 2.88 mg/g dry lung between 24 days gestation, and term. Between 24 and 26 days gestation the overall increase in tissue DSPC was due to a two-fold increase in the mitochondrial, microsomal and cytosolic pools. Lamellar bodies were first isolable at 26 days gestation. The DSPC content of this fraction increased six-fold (from 0.10 +/- 0.02 to 0.67 +/- 0.15 mg/g dry lung) between 26 and 28 days gestation and a further seven-fold (to 4.63 +/- 1.06 mg/g dry lung) by term, accounting for the overall increase in the tissue homogenate value during this time period. By the first postnatal day, microsomal and cytosolic DSPC increased another two-fold, but no significant change occurred in the other subcellular fractions. Alveolar lavage DSPC progressively increased over the time period studied. While there was no change in the lamellar body DSPC/total PC ratio during fetal development, each of the mitochondrial, microsomal and cytosolic ratios decreased between days 26 and 28 of gestation and then increased at term.(ABSTRACT TRUNCATED AT 250 WORDS)