RESUMEN
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.
Asunto(s)
Pulmón/microbiología , Micotoxinas/toxicidad , Surfactantes Pulmonares/química , Espectroscopía Infrarroja por Transformada de Fourier , Stachybotrys/patogenicidad , Animales , Pulmón/citología , Pulmón/efectos de los fármacos , Masculino , Ratones , Fosfolípidos/química , Esporas Fúngicas/patogenicidadRESUMEN
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.
Asunto(s)
Butiratos/administración & dosificación , Glucosa/administración & dosificación , Surfactantes Pulmonares/metabolismo , Animales , Glucemia/análisis , Femenino , Sangre Fetal/química , Madurez de los Órganos Fetales , Peso Fetal , Insulina/sangre , Pulmón/embriología , Fosfatidilcolinas/análisis , Fosfatidilinositoles/análisis , Fosfolípidos/análisis , Placebos , Embarazo , Embarazo en Diabéticas/complicaciones , Surfactantes Pulmonares/análisis , ConejosRESUMEN
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.
Asunto(s)
Feto/fisiología , Proteína Quinasa C/fisiología , Proteolípidos/metabolismo , Alveolos Pulmonares/citología , Alveolos Pulmonares/metabolismo , Surfactantes Pulmonares/metabolismo , Animales , Femenino , Glicoproteínas , L-Lactato Deshidrogenasa/metabolismo , Embarazo , Proteína A Asociada a Surfactante Pulmonar , Proteínas Asociadas a Surfactante Pulmonar , ConejosRESUMEN
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.
Asunto(s)
Pulmón/efectos de los fármacos , Compuestos de Metilmercurio/toxicidad , Surfactantes Pulmonares/efectos de los fármacos , Animales , Líquido del Lavado Bronquioalveolar , Pulmón/ultraestructura , Masculino , Ratones , Microscopía Electrónica de Rastreo , Surfactantes Pulmonares/fisiología , Espectroscopía Infrarroja por Transformada de Fourier , Tensión SuperficialRESUMEN
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)
Asunto(s)
Líquido del Lavado Bronquioalveolar/química , Fosfolípidos/análisis , Proteínas/análisis , Surfactantes Pulmonares/química , Espectroscopía Infrarroja por Transformada de Fourier , Amidas/química , Animales , Pulmón/química , Fosfolípidos/química , Conejos , Análisis de RegresiónRESUMEN
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.
Asunto(s)
Alveolos Pulmonares/metabolismo , Surfactantes Pulmonares/metabolismo , Lesión por Inhalación de Humo/metabolismo , Animales , Líquido del Lavado Bronquioalveolar/química , Pulmón/patología , Pulmón/ultraestructura , Masculino , Ratones , Ratones Endogámicos , Tamaño de los Órganos , Fosfatidilcolinas/metabolismo , Fosfolípidos/metabolismo , Surfactantes Pulmonares/clasificación , Lesión por Inhalación de Humo/patologíaRESUMEN
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.
Asunto(s)
Animales Recién Nacidos , Desarrollo Embrionario y Fetal/efectos de los fármacos , Pulmón/química , Palmitatos , Surfactantes Pulmonares , Animales , Autorradiografía , Femenino , Pulmón/efectos de los fármacos , Fosfolípidos/análisis , Embarazo , Alveolos Pulmonares/efectos de los fármacos , Surfactantes Pulmonares/farmacología , Conejos , TritioRESUMEN
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.
Asunto(s)
Agonistas Adrenérgicos beta/farmacología , Isoxsuprina/farmacología , Pulmón/embriología , Alveolos Pulmonares/citología , Surfactantes Pulmonares/biosíntesis , Animales , Diferenciación Celular , Femenino , Pulmón/efectos de los fármacos , Embarazo , ConejosRESUMEN
The role of beta-adrenergic stimulation in surfactant synthesis and secretion was investigated in the fetal lung. Fetuses were treated with isoxsuprine or saline on gestational day 24 by ip injection. Three days later the fetal lungs were lavaged and intracellular surfactant was isolated on a sucrose gradient. Concurrently undifferentiated type II alveolar cells were isolated from 24-day fetal rabbit lung and grown in vitro. In the in vivo portion of the study, examination of surfactant pool sizes revealed that only saline treatment produced a significant elevation in tissue-stored or secreted surfactant compared to untreated controls. Isoxsuprine appeared to inhibit the saline-induced increase. In the case of the intracellular surfactant, the phosphatidylcholine content per gram of lung was significantly increased after saline treatment. In vitro response of isolated type II alveolar cells to isoxsuprine was dependent on prior incubation of the cells for 24 h with conditioned medium. Isoxsuprine stimulated a dose-dependent decrease in the intracellular stores of radioactively labeled DSPC after 24 h of exposure to the drug. A corresponding increase in labeled DSPC in the culture medium was observed. Forth-eight hours after exposure to the drug, those cells that had secreted the highest level of DSPC displayed the highest levels of renewed synthesis of DSPC. This study indicates that the immature fetal lung can be induced to synthesize surfactant-related phospholipid by the stress of laparotomy and/or drug administration. Short-term exposure to beta-agonists is insufficient to stimulate secretion of surfactant stores. In contrast, isolated type II alveolar cells exposed to isoxsuprine respond by secreting DSPC.
Asunto(s)
Agonistas Adrenérgicos beta/farmacología , Isoxsuprina/farmacología , Pulmón/metabolismo , Alveolos Pulmonares/metabolismo , Surfactantes Pulmonares/fisiología , Animales , Separación Celular , Células Cultivadas , Feto/metabolismo , Fibroblastos/metabolismo , Fosfolípidos/biosíntesis , Fosfolípidos/metabolismo , Alveolos Pulmonares/citología , ConejosRESUMEN
Myelin was isolated from the sciatic nerves of chicks of ages 18-day embryonic, 1-day, 4-day, 7-day post-hatch, and adult to study developmental changes in lipid composition of this structure. The yield of myelin increased throughout the early stages of development and the preparations were of high purity. Although the lipid content of the myelin did not change, significant changes took place in lipid composition during development. The most significant changes were a relative increase in cerebrosides, phosphatidalethanolamine and long-chain fatty acids of cerebrosides, and a relative decrease in the content of phosphatidylserine and phosphatidylethanolamine. A second fraction ("lower band") was obtained during the isolation procedure. This "lower band" was present at all developmental stages and layered consistently at the interface of 1.2 and 0.8 M sucrose on a discontinuous gradient. The quantity of this fraction did not change during development and it differed from myelin in electron microscopic appearance. Its lipid composition, which did not change, resembled that of 18-day embryonic myelin in its high phospholipid:cholesterol ratio and low galactolipid content. The enzyme, 2':3'-cyclic-nucleosidemonophosphate phosphodiesterase was found to be present in both the myelin and "lower band" fractions; however there was no enrichment of this enzyme in purified myelin.