RESUMEN
BACKGROUND: Limited research has examined how the functions of self-injurious behaviour (SIB) relate to the production of injuries and the location, type or severity of those injuries. METHODS: Clinical and medical records were coded for 64 individuals hospitalised for SIB. When injuries were present, the physical properties of SIB and injuries were assessed across groups of individuals with automatically and socially maintained SIB. RESULTS: Injuries were observed for 35 of the individuals who engaged in SIB. Individuals who engaged in a single form of SIB were more likely to have injuries (P < .05). Individuals with SIB maintained by automatic reinforcement had significantly more severe injuries to the head than those in the social group (q < .05, P = .0132, H = 12.54). CONCLUSION: Although results are preliminary, the results provide evidence that the function of SIB may influence the severity and location of injuries produced.
Asunto(s)
Discapacidad Intelectual/complicaciones , Conducta Autodestructiva/complicaciones , Conducta Autodestructiva/fisiopatología , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Masculino , Conducta Autodestructiva/clasificación , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
Metastasis is a frequent complication of cancer, yet the process through which circulating tumor cells form distant colonies is poorly understood. We have been able to observe the steps in early hematogenous metastasis by epifluorescence microscopy of tumor cells expressing green fluorescent protein in subpleural microvessels in intact, perfused mouse and rat lungs. Metastatic tumor cells attached to the endothelia of pulmonary pre-capillary arterioles and capillaries. Extravasation of tumor cells was rare, and it seemed that the transmigrated cells were cleared quickly by the lung, leaving only the endothelium-attached cells as the seeds of secondary tumors. Early colonies were entirely within the blood vessels. Although most models of metastasis include an extravasation step early in the process, here we show that in the lung, metastasis is initiated by attachment of tumor cells to the vascular endothelium and that hematogenous metastasis originates from the proliferation of attached intravascular tumor cells rather than from extravasated ones. Intravascular metastasis formation would make early colonies especially vulnerable to intravascular drugs, and this possibility has potential for the prevention of tumor cell attachment to the endothelium.
Asunto(s)
Endotelio Vascular/patología , Fibrosarcoma/patología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Metástasis de la Neoplasia , Animales , Adhesión Celular , Femenino , Fibrosarcoma/irrigación sanguínea , Fibrosarcoma/fisiopatología , Genes Reporteros , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/fisiopatología , Ratones , Ratones Desnudos , Microcirculación/patología , Circulación Pulmonar , Ratas , Ratas Sprague-Dawley , Transfección , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
We investigated metabolic utilization of exogenous (modelled after lung surfactant) phospholipids by granular pneumocytes in primary culture. Cells were incubated for 21, 65, and 140 min with [3H-methyl]dipalmitoylphosphatidylcholine (DPPC) containing liposomes prepared from synthetic lipids. Radioactivity in cellular phosphatidylcholine (PC) declined steadily to 50% of the total trypsin-resistant cell-associated radioactivity. The proportion of radioactivity increased with time in cytidine-5'-diphosphate-choline and phosphorylcholine, which suggested reutilization of choline for PC synthesis. Cells incubated with liposomes for 2 h revealed that of the total cell-associated radioactivity, 7% was in lamellar bodies and 10% in the microsomal fraction. The lipid-associated radioactivity was 24% in "soluble," 96% in lamellar bodies, and 92% in the microsomal fraction. Percent of total PC label recovered in disaturated PC of microsomal fractions decreased (slope = -5.27%/h) with time of incubation (r = 0.67). Incubation of cells with liposomes containing ([3H-methyl]choline-[14C]palmitoyl) DPPC led to altered isotope ratios in both lamellar bodies and microsomes. These observations indicate that granular pneumocytes degrade exogenous PC and resynthesize PC from degradation products.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/metabolismo , Pulmón/citología , Surfactantes Pulmonares/biosíntesis , Cloruro de Amonio/farmacología , Animales , Cloroquina/farmacología , Colina/metabolismo , Liposomas , Pulmón/metabolismo , Metilaminas/farmacología , Organoides/metabolismo , Quinacrina/farmacología , RatasRESUMEN
Total respiratory resistance (R(T)) was measured by the application of a sine wave of airflow to the mouth at the resonant frequency of the respiratory system. The mean respiratory resistance of 42 normal subjects, measured at a mean functional residual capacity of 3.3 liters, was 2.3, SD +/- 0.5, cm H(2)O/liter per sec, and the resonant frequency was between 5 and 8 cycle/sec. The airway resistance measured in these same subjects with the body plethysmograph at a mean panting thoracic gas volume of 3.5 liters was 1.3, SD +/- 0.3, cm H(2)O/liter per sec. Total respiratory resistance was found to vary inversely with lung volume (V) measured plethysmographically; prediction formulae for normal subjects based on this relationship are: R(T) (mean) = 7.1/V, R(T) (range) = 4.0/V to 11.6/V where V is in liters and R(T) is in cm H(2)O/liter per sec. When these criteria were applied to subjects with thoracic disease the following results were obtained: 17 subjects with obstructive lung disease all had elevated total respiratory resistance; 9 subjects with diffuse lung disease without airway obstruction all had normal respiratory resistance; all but 1 of 5 obese subjects and all but 2 of a heterogeneous group of 9 subjects without airway obstruction had normal respiratory resistance. Failure to take lung volume into account resulted in a considerable decrease in the ability to discriminate between obstructive and nonobstructive lung disease on the basis of the forced oscillation test. The resonant frequency of the respiratory system of patients with obesity or nonobstructive lung disease was similar to that obtained in the normal group; accurate evaluation of resonant frequency in subjects with obstructive lung disease was frequently not possible. The combined resistances of lung, thoracic wall and abdominal tissues were found to account for less than 43% of the total respiratory resistance in normal subjects and were only slightly increased by the presence of obesity, restrictive diseases of the thoracic wall, and hyperinflation of the thorax. The forced oscillation method is potentially of value in the study of resistance to breathing of patients who cannot undergo body plethysmography, such as acutely ill, anesthetized, or unconscious subjects. Accurate evaluation of R(T) requires an independent measure of lung volume as well as careful attention during measurements to the airflow rate, phase of respiration, and the adequacy of cheek compression and laryngeal relaxation.
Asunto(s)
Enfermedades Pulmonares/diagnóstico , Pruebas de Función Respiratoria , Fenómenos Fisiológicos Respiratorios , Adolescente , Adulto , Anciano , Femenino , Humanos , Enfermedades Pulmonares/fisiopatología , Masculino , Métodos , Persona de Mediana Edad , Obesidad/fisiopatología , Pletismografía , EspirometríaRESUMEN
Bacterial infection may complicate pulmonary oxygen (O2) toxicity, and animals exposed to high O2 concentrations show depressed in vivo pulmonary bacterial inactivation. Therefore, in vitro studies were undertaken to define the mechanism by which O2 alters pulmonary antibacterial activity. Normal and BCG pretreated rabbits were exposed to 100% O2 for 24, 48, and 72-h periods. Pulmonary alveolar macrophages (PAM) were obtained from the experimental animals and from nonoxygen exposed controls by bronchopulmonary lavage. O2 exposure did not alter cell yield or morphology. PAMs were suspended in 10% serum-buffer, and phagocytosis of (14C)Staphylococcus aureus 502A and (14C)Pseudomonas aeruginosa was measured. Comparison of the precent uptake of the 14C-labeled S. aureus after a 60-min incubation period demonstrated that normal PAMs exposed to O2 for 48 h showed a statistically significant increase in phagocytosis when compared to their controls (43.5 vs. 29.2%). A similar, but smaller increase was seen after 24-h O2 exposures. 48 and 72-h O2 exposures produced no significant changes in phagocytosis in PAMs from BCG-stimulated rabbits. Normal PAMs also showed an increased phagocytosis of Ps. aeruginosa after 48-h oxygen exposure. No impairment of in vitro bactericidal activity against either S. aureus 502A or Ps. aeruginosa could be demonstrated in PAMs from normal rabbits exposed to O2 for 48 h. These results indicate that the in vitrophagocytic and bactericidal capacity of the rabbit PAM is relatively resistant to the toxic effects of oxygen, and that imparied in vivo activity may possibly be mediated by effects other than irreversible metabolic damage to these cells. The mechanism for the observed stimulation of phagocytosis remains to be determined.
Asunto(s)
Macrófagos/efectos de los fármacos , Oxígeno/farmacología , Fagocitosis/efectos de los fármacos , Alveolos Pulmonares/efectos de los fármacos , Animales , Radioisótopos de Carbono , Técnicas In Vitro , Cinética , Macrófagos/ultraestructura , Masculino , Microscopía Electrónica , Pseudomonas aeruginosa/efectos de los fármacos , Conejos , Staphylococcus/efectos de los fármacosRESUMEN
The relationship between alveolar PO2 and the rate of O-demethylation of p-nitroanisole, a model substrate for cytochrome P-450 -linked mixed-function oxidation, was evaluated in the isolated rabbit lung perfused with Krebs-Ringer bicarbonate buffer. The appearance of the product, p-nitrophenol, in the pulmonary perfusate was measured spectrophotometrically, The PO2 of the ventilating gas was varied with an accurate gas mixing pump and measured with an electrochemical O2 analyzer. In control lungs ventilated with 5% CO2 in air, the rate of p-nitrophenol production was approximately equal to 3.1 +/- 0.04 (mean +/- SE; n = 9) mumol/h per g dry wt. p-Nitrophenol production was unaltered when O2 in the ventilating gas was decreased to 1%, but it was depressed reversibly when alveolar O2 WAS 0.1% OR LESS AND WAS ABOLISHED DURING VENTILATION WITH 0.005% O2. The rate of the reaction was inhibited by 50% when alveolar PO2 was 0.3 mm Hg representing and intracellular [O2] OF approximately equal to muM. In the presence of metyrapone (0.1--1 mM), an inhibitor of cytochrome P-450-dependent reactions, p-nitrophenol production was 0.07--0.17 mumol/h per g dry wt. Ventilation of lungs with varying CO concentration in 20% O2 resulted in 50% inhibition of p-nitrophenol production when CO concentration was 10% (CO/O2 = 0.5). These results indicated that O-demethylation of p-nitroanisole by the lung is a cytochrome P-450-dependent reaction and that its rate is not affected until alveolar PO2 is less than 1 mm Hg.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Pulmón/metabolismo , Nitroanisol O-Demetilasa/metabolismo , Oxidorreductasas/metabolismo , Oxígeno , Alveolos Pulmonares/metabolismo , Animales , Monóxido de Carbono/farmacología , Técnicas In Vitro , Metirapona/farmacología , Microsomas/metabolismo , Oxigenasas de Función Mixta/metabolismo , Perfusión , ConejosRESUMEN
The effect of alveolar oxygen tension on lung lipid peroxidation during lung ischemia was evaluated by using isolated rat lungs perfused with synthetic medium. After a 5-min equilibration period, global ischemia was produced by discontinuing perfusion while ventilation continued with gas mixtures containing 5% CO2 and a fixed oxygen concentration between 0 and 95%. Lipid peroxidation was assessed by measurement of tissue thiobarbituric acid-reactive products and conjugated dienes. Control studies (no ischemia) showed no change in parameters of lipid peroxidation during 1 h of perfusion and ventilation with 20% or 95% O2. With 60 min of ischemia, there was increased lipid peroxidation which varied with oxygen content of the ventilating gas and was markedly inhibited by ventilation with N2. Perfusion with 5-, 8-, 11-, 14-eicosatetraynoic acid indicated that generation of eicosanoids during ischemia accounted for approximately 40-50% of lung lipid peroxide production. Changes of CO2 content of the ventilating gas (to alter tissue pH) or of perfusate glucose concentration had no effect on lipid peroxidation during ischemia, but perfusion at 8% of the normal flow rate prevented lipid peroxidation. Lung dry/wet weight measured after 3 min of reperfusion showed good correlation between lung fluid accumulation and lipid peroxidation. These results indicate that reperfusion is not necessary for lipid peroxidation with ischemic insult of the lung and provide evidence that elevated PO2 during ischemia accelerates the rate of tissue injury.
Asunto(s)
Isquemia/metabolismo , Peroxidación de Lípido , Pulmón/irrigación sanguínea , Oxígeno/metabolismo , Ácido 5,8,11,14-Eicosatetrainoico/farmacología , Animales , Eicosanoides/metabolismo , Concentración de Iones de Hidrógeno , Pulmón/metabolismo , Masculino , Ratas , Ratas Endogámicas , Daño por Reperfusión/metabolismoRESUMEN
Lung size was evaluated with pulmonary function tests in 10 patients with acromegaly, 1 pituitary giant, and 1 patient who had acromegaly but now has hypopituitarism. In the six acromegalic men all lung volumes were increased. The average values and per cent of predicted were total lung capacity 9.1 liters. 139%; functional residual capacity 5.2 liters, 145%; vital capacity 6.0 liters, 134%; and tissue volume 1.1 liters. There was no evidence of airflow obstruction or air trapping. Anatomic dead space was increased in proportion to the large lung volumes. Lung compliance was increased, averaging 0.43 liters/cm H(2)O, but lung elastic recoil was normal. These studies show that the lung is involved in the general visceromegaly of acromegaly and that lung size increases in acromegalic men as a result of actual lung growth. Despite the large lung volumes, diffusing capacity was normal suggesting that lung growth resulted from an increase in the size rather than from an increase in the number of alveoli. In contrast to the acromegalic men, lung volumes, anatomic dead space and tissue volume were normal in four acromegalic women, suggesting that sex hormones may modify the effect of growth hormone on the lung. Lung size was large in the pituitary giant but lung volumes were normal according to predicted values based on the patient's great height. Lung volumes were normal in the one male who had been acromegalic but who has been hypopituitary for 21 yr. The role of growth hormone in normal postnatal lung growth and in the maintainance of normal lung size remains to be defined.
Asunto(s)
Acromegalia/fisiopatología , Pulmón/fisiopatología , Acromegalia/complicaciones , Acromegalia/patología , Adulto , Anciano , Femenino , Humanos , Hipertrofia , Pulmón/crecimiento & desarrollo , Pulmón/patología , Rendimiento Pulmonar , Masculino , Persona de Mediana Edad , Espacio Muerto Respiratorio , Pruebas de Función RespiratoriaRESUMEN
Ethanol/ether soluble apoproteins, comprising 17% of the total recovered surfactant-associated proteins, were isolated from rat lung surfactant and purified by silicic acid chromatography. The protein that eluted in 4:1 chloroform/methanol accounted for greater than 85% of protein in the ethanol/ether soluble fraction and was termed surfactant apoprotein Et (Apo Et). By sodium dodecyl sulfate polyacrylamide gel electrophoresis, this protein had an apparent molecular weight of approximately 10,500. Apo Et was evaluated for its effect on uptake of synthetic phospholipids in liposomal form by isolated granular pneumocytes (Type II alveolar epithelial cells) in primary culture. Liposomes were prepared to approximate the phospholipid composition of the alveolar surfactant, and uptake was measured by the accumulation of the radioactively labeled dipalmitoyl phosphatidyl choline fraction. The uptake of liposomal phosphatidylcholine by cells incubated for 2 h with Apo Et was increased by 61% over control. Most of the cell-associated phospholipid uptake was resistant to treatment with trypsin, suggesting an increased internalization of liposomal material in the presence of Apo Et. The effect of Apo Et on uptake was concentration and time dependent and was not associated with cell damage, phospholipase activity, or detergent properties of the protein. Apo Et had no significant effect on phosphatidylcholine uptake by granular pneumocytes maintained for 7 d in primary culture. Apo Et augmented the uptake of phospholipids by alveolar macrophages although total uptake by these cells was less than that observed with granular pneumocytes. Because Apo Et increases the rate of uptake of surfactant phospholipids by alveolar cells (granular pneumocytes and alveolar macrophages), this protein may represent a physiologically important regulator for clearance of lung surfactant phospholipids.
Asunto(s)
Apoproteínas/aislamiento & purificación , Liposomas , Pulmón/fisiología , Animales , Apoproteínas/fisiología , Etanol , Éter , Cinética , Pulmón/citología , Fosfatidilcolinas/metabolismo , Surfactantes Pulmonares/aislamiento & purificación , Ratas , Ratas Endogámicas , SolubilidadRESUMEN
We have previously shown that increased reactive oxygen species (ROS) generation occurs with ischemia in the oxygenated lung and have hypothesized that mechanotransduction is the initiating event. In the present study, we developed an in vitro model of oxygenated ischemia by interrupting medium flow to flow-adapted bovine pulmonary artery endothelial cells in an artificial capillary system. Cellular oxygenation during the "ischemic" period was maintained by perfusing medium over the abluminal surface of porous capillaries. Cells were assessed for ROS generation, nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) binding activities, and DNA synthesis using dichlorofluorescein fluorescence by flow cytometry and spectrofluorometry, electrophoretic mobility shift assay of nuclear extracts with NF-kappaB-specific or AP-1-specific (32)P-labeled oligonucleotides, and (3)H-thymidine incorporation into DNA. Cells that were flow adapted for 2 to 7 days with 1 to 2 dyne/cm(2) shear stress exhibited a 1.6- to 1.9-fold increase in ROS generation during 1 hour of simulated ischemia compared with continuously perfused cells. This effect was abolished by diphenyleneiodonium chloride (DPI), indicating a role for a flavoprotein such as NADPH oxidase. The increase in ROS generation with ischemia was similar for cells from low and high passages. With ischemia, flow-adapted cells exhibited increases of 1.7-fold in nuclear NF-kappaB and 1.5-fold in nuclear AP-1; these changes were abolished by pretreatment with N-acetylcysteine or DPI. Ischemia for 24 hours resulted in a 1.8-fold increase of (3)H-thymidine incorporation into DNA and a significant increase of cells entering the cell cycle, as indicated by flow cytometry with propidium iodide. We conclude that flow-adapted endothelial cells generate ROS with ischemia that results in activation of NF-kappaB and AP-1 and an increase of DNA synthesis. This effect is not mediated by hypoxia, implicating a role for mechanotransduction in ischemia-mediated cell signaling.
Asunto(s)
Endotelio Vascular/metabolismo , Isquemia/fisiopatología , Arteria Pulmonar/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Animales , Bovinos , División Celular , Células Cultivadas , ADN/biosíntesis , Endotelio Vascular/patología , Endotelio Vascular/fisiopatología , Isquemia/metabolismo , Isquemia/patología , FN-kappa B/metabolismo , Perfusión , Arteria Pulmonar/patología , Arteria Pulmonar/fisiopatología , Estrés Mecánico , Timidina/metabolismo , Factor de Transcripción AP-1/metabolismoRESUMEN
In this report we have studied the mechanism by which Transforming Growth Factor beta (TGF beta) inhibits growth of human myeloid leukemia cell lines. TGF beta 1 arrested cells in G1 phase and significantly downregulated the expression of cyclin D2, cyclin D3, cdk4, cyclin A, and cdk2. The downregulation of the molecules resulted in approximately 50-90% decrease of the molecule-dependent kinase activity, varying with each molecule. Although treatment of cells with TGF beta 1 up-regulated accumulation of p27(kip1) in both nucleus and cytoplasm, the association of the p27(kip1) with cdk2, cyclin A, cyclin D2, cyclin D3, and cdk4 was markedly down-regulated, suggesting that p27(kip1) is not responsible for the downregulation of the kinase activity. In contrast, TGF beta 1 upregulated cyclin E-associated p27(kip1) with no effect on the expression of cyclin E. p27(kip1)-immunodepletion upregulated cyclin E-dependent kinase activity by more than 10-fold in TGF beta 1-treated cells but not in proliferating cells; whereas immunodepletion of p27(kip1) from cdk2-immunoprecipitates markedly downregulated cdk2 kinase activity in the lysates extracted from both proliferating and TGF beta-treated cells. Consistent with this observation, TGF beta 1 and p27(kip1) antisense cDNA had a synergistic or additive inhibitory effect on cdk2 but not cyclin E-dependent kinase activity. Our data suggest that (1) TGF beta 1-mediated growth inhibition is accomplished through multiple pathways and (2) p27(kip1) has opposing effects on cdk2 and cyclin E activity in response to TGF beta 1.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas de Ciclo Celular/metabolismo , Leucemia Mieloide/metabolismo , Proteínas Proto-Oncogénicas , Factor de Crecimiento Transformador beta/farmacología , Proteínas de Ciclo Celular/fisiología , División Celular , Ciclina D , Ciclina E/antagonistas & inhibidores , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/biosíntesis , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Regulación hacia Abajo , Fase G1 , Humanos , Leucemia Mieloide/patología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteína de Retinoblastoma/metabolismo , Factor de Crecimiento Transformador beta1 , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
We investigated the effect of exogenous fatty acids on phosphatidylcholine (PC) and disaturated phosphatidylcholine (DSPC) synthesis by rat granular pneumocytes in primary culture. Synthesis of PC and DSPC from [3H-methyl]choline, as evaluated by increasing specific activity (pmol choline incorporated/microgram phosphorus), was linear for 3 h. Exogenous palmitic, oleic, linoleic, or linolenic acid (100 microM each) increased the synthesis of PC by approx. 50% during incubation for 3 h. In contrast, synthesis of DSPC was increased only by palmitic acid. The increase in DSPC synthesis was approx. 150% after 3 h. Conversion of choline phosphate to PC was increased in the presence of palmitic or oleic acid as indicated by pulse-chase studies with [3H-methyl]choline in the intact cells. Cells incubated for 3 h with either oleic or palmitic acid showed increased choline-phosphate cytidyltransferase activity in the cells and the microsomal fraction. In addition, oleic acid increased the activity of this enzyme in the cytosolic fraction. The distribution of this enzyme in cytosolic and microsomal fraction was 24 and 76% in the cells incubated with palmitic acid and 32 and 68% in control cells. These results suggest that exogenous fatty acids stimulate the de novo pathway of PC synthesis in granular pneumocytes by increasing the microsomal choline-phosphate cytidyltransferase activity.
Asunto(s)
Ácidos Grasos/farmacología , Pulmón/metabolismo , Nucleotidiltransferasas/metabolismo , Fosfatidilcolinas/biosíntesis , Animales , Células Cultivadas , Colina/análogos & derivados , Colina/metabolismo , Citidililtransferasa de Colina-Fosfato , Citosol/enzimología , Cinética , Ácido Linoleico , Ácidos Linoleicos/farmacología , Ácidos Linolénicos/farmacología , Pulmón/efectos de los fármacos , Masculino , Microsomas/enzimología , Ácido Oléico , Ácidos Oléicos/farmacología , Ácido Palmítico , Ácidos Palmíticos/farmacología , Ratas , Ratas Endogámicas , Ácido alfa-LinolénicoRESUMEN
Glycerol kinase activity and glycerol utilization by rat granular pneumocytes were determined in order to investigate the rate-limiting step for glycerol incorporation into lung lipids. Granular pneumocytes were isolated in primary culture following trypsinization of rat lungs. Glycerol kinase activity was 8.2 nmol/h per 10(6) cells. Incorporation of [1,3-14C]glycerol into total cell lipids was 0.29 nmol/h per 10(6) cells. In the presence of saturating glycerol concentration, production of 3H2O from [2-3H]glycerol was 13 times greater than incorporation of [14C]glycerol into lipids. Glycerol phosphate dehydrogenase activity in isolated cells was approximately 10 times glycerol kinase activity. In the presence of 5.6 mM glucose, glycerol incorporation into lipids was decreased 79% and detritiation of glycerol was decreased 34%. This effect of glucose was due to a 25% increase in cell glycerol 3-phosphate content, resulting in dilution of the precursor pool and possible inhibition of glycerol phosphorylation. These results indicate that the relatively limited incorporation of glycerol into surfactant phospholipids by lung epithelial cells reflects the relatively high rate of glycerol 3-phosphate oxidation.
Asunto(s)
Glicerol Quinasa/metabolismo , Glicerol/metabolismo , Pulmón/metabolismo , Fosfotransferasas/metabolismo , Animales , Células Cultivadas , Glucosa/metabolismo , Glicerolfosfato Deshidrogenasa/metabolismo , Concentración de Iones de Hidrógeno , Metabolismo de los Lípidos , Fosforilación , RatasRESUMEN
The role of annexin II in the secretion of lung surfactant was investigated using isolated lamellar bodies and/or liposomes as the model system for aggregation and fusion. We first compared membrane aggregation mediated by two forms of annexin II, annexin II monomer (Anx IIm) and annexin II tetramer (Anx IIt). Anx IIt required 20-fold less Ca2+ to mediate phosphatidylserine (PS) liposome aggregation compared to Anx IIm. Aggregation of lamellar bodies mediated by Anx IIt was 4-fold greater than that by Anx IIm at 1 mM Ca2+. These results suggest that Anx IIt may be the more active form in vivo. Fusion of lamellar bodies with PS liposomes was promoted by Anx IIt in a dose-dependent manner, with maximal fusion occurring at 10-15 micrograms/ml of Anx IIt. Fusion was dependent on Ca2+ and the phospholipid composition of liposomes. While the fusion of lamellar bodies with PS liposomes required 100 microM Ca2+, the fusion with PS/phosphatidylethanolamine (PE) (1:3) liposomes required only 10 microM Ca2+. Anx IIt-mediated lamellar body-liposome fusion was enhanced by arachidonic acid, a lung surfactant secretagogue and inhibited by 4.4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of lung surfactant secretion. The data suggest that Anx IIt may play a role in the fusion of lamellar bodies with plasma membranes during lung surfactant secretion.
Asunto(s)
Anexina A2/farmacología , Liposomas , Pulmón/química , Fusión de Membrana/efectos de los fármacos , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Animales , Anexina A2/química , Ácido Araquidónico/farmacología , Calcio/farmacología , Agregación Celular/efectos de los fármacos , Sustancias Macromoleculares , Masculino , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Alveolos Pulmonares/química , Ratas , Ratas Sprague-DawleyRESUMEN
The methylation of phosphatidylethanolamine (PE) to form phosphatidylcholine (PC) was investigated using the isolated rat lung perfused with radiolabeled ethanolamine. Lungs from choline-deficient rats showed increased incorporation of radiolabel into PC at 2 h of perfusion. Increased PC synthesis from PE was also observed with lungs from rats fed a lipotrophic (choline plus methionine deficient) diet when methionine was added to the lung perfusate. These results indicate increased activity of the methylation pathway for lung PC synthesis during choline deficiency.
Asunto(s)
Deficiencia de Colina/metabolismo , Pulmón/metabolismo , Fosfatidilcolinas/biosíntesis , Animales , Radioisótopos de Carbono , Etanolamina , Etanolaminas/metabolismo , Cinética , Masculino , Metionina/metabolismo , Metilación , Fosfatidiletanolaminas/metabolismo , Técnica de Dilución de Radioisótopos , Ratas , Ratas EndogámicasRESUMEN
Incubation of isolated rat alveolar epithelial type II cells with secretagogues (calcium ionophore, ATP or terbutaline) resulted in rapid proteolysis of lung spectrin and appearance of multiple proteolytic products which showed immunoreactivity with an antibody against human erythrocyte spectrin. These proteolytic products were similar to those generated from erythrocyte spectrin or cultured lung tumor cells (A549 cells) incubated with purified calpain. Furthermore, incubation of alveolar type II cells with a calpain-specific inhibitor modulated the secretagogue-induced proteolysis of lung spectrin. Thus, stimulation of secretion appeared to activate endogenous calpain in type II cells, suggesting that calpain-mediated proteolysis of a submembranous cytoskeletal protein could play an important role in the secretory process.
Asunto(s)
Calpaína/fisiología , Pulmón/metabolismo , Alveolos Pulmonares/metabolismo , Espectrina/metabolismo , Adenosina Trifosfato/fisiología , Animales , Western Blotting , Calcimicina/farmacología , Calpaína/antagonistas & inhibidores , Células Cultivadas , Activación Enzimática , Células Epiteliales , Epitelio/metabolismo , Humanos , Fusión de Membrana , Alveolos Pulmonares/citología , Surfactantes Pulmonares/metabolismo , Ratas , Terbutalina/farmacología , Células Tumorales CultivadasRESUMEN
A lamellar body fraction was isolated from rat alveolar granular pneumocytes in primary culture by upward flotation on a discontinuous sucrose gradient and compared with a similar fraction isolated from lung homogenates. Lamellar bodies from granular pneumocytes were free of detectable contamination with either succinate dehydrogenase or NADPH-cytochrome c reductase. There was an enrichment of acid phosphatase activity, which, based on distribution of enzyme activity on the gradient, did not appear to be a contamination from other fractions. The lamellar body fraction of granular pneumocytes yielded approx. 1 microgram protein/10(6) cells with a phospholipid-to-protein ratio (mg/mg) of 9.6 +/- 0.4 (n = 7). Composition with respect to total phospholipids was 71.0% phosphatidylcholine (disaturated phosphatidylcholine, 45.2%), 8.4% phosphatidylglycerol and 12.8% phosphatidylethanolamine. Palmitic acid comprised 66% of the fatty acids in phosphatidylcholine and 34% of those in phosphatidylglycerol. The lamellar body fraction from granular pneumocytes was similar to that from whole lung with respect to phospholipid-to-protein ratio and phospholipid composition and showed only minor differences in fatty acid composition. Ultrastructurally, lamellar bodies showed generally intact limiting membranes and lamellated structure. Lamellar bodies from granular pneumocytes showed occasional multinucleated whorls which were not seen in those isolated from lung homogenates. This study describes a method for preparing a homogeneous fraction of intact lamellar bodies from small amounts of material (6 X 10(7) granular pneumocytes). The yield on a per cell basis was higher when compared with a similar preparation from whole lung, although overall yield is small, due to loss of cells during the cell isolation procedure. This preparation may be useful to evaluate the role of lamellar bodies in the synthesis and secretion of lung surfactant by isolated granular pneumocytes.
Asunto(s)
Alveolos Pulmonares/ultraestructura , Fosfatasa Ácida/aislamiento & purificación , Animales , Fraccionamiento Celular/métodos , Células Cultivadas , Fenómenos Químicos , Química , Ácidos Grasos/aislamiento & purificación , Masculino , Microscopía Electrónica , NADPH-Ferrihemoproteína Reductasa/aislamiento & purificación , Fosfolípidos/aislamiento & purificación , Alveolos Pulmonares/enzimología , Alveolos Pulmonares/metabolismo , Surfactantes Pulmonares/aislamiento & purificación , Ratas , Ratas Endogámicas , Succinato Deshidrogenasa/aislamiento & purificaciónRESUMEN
A novel active-site directed specific inhibitor of phospholipase A2 (PLA2), 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33), administered endotracheally co-dispersed in liposomes, significantly reduced the formation of thiobarbituric acid reactive substances (TBARS) in isolated rat lungs subjected to ischemia-reperfusion. Elevated conjugated dienes were unaffected. This contrasts with the effects of the cyclo-/lipoxygenase inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA), which decreased formation of both TBARS and conjugated dienes (CD). The effects of MJ33 plus ETYA were additive for TBARS but results for CD were similar to ETYA alone. A similar dissociation of inhibition of TBARS and CD formation by MJ33 was observed with t-butyl hydroperoxide induced lipid peroxidation of isolated lung microsomes. Assay of lung homogenate with phosphatidylcholine as substrate showed that MJ33 selectively inhibited the Ca(2+)-independent acidic PLA2. MJ33 had no effect on thromboxane B2 release by the isolated lung, indicating the effects of acidic PLA2 inhibition do not involve the arachidonate cascade. MJ33 also partially prevented lung edema and lactate dehydrogenase release associated with ischemia-reperfusion. The observations show that this PLA2 inhibitor can be delivered to oxidant-sensitive lung sites by its co-dispersal in liposomes, and that oxidant-induced lipid peroxidation in this model of lung injury occurs in a complex lipid prior to PLA2 activity.
Asunto(s)
Glicerofosfatos/farmacología , Isquemia/enzimología , Pulmón/irrigación sanguínea , Fosfolipasas A/antagonistas & inhibidores , Sustancias Reactivas al Ácido Tiobarbitúrico/análisis , Ácido 5,8,11,14-Eicosatetrainoico/farmacología , Animales , Peroxidación de Lípido , Pulmón/enzimología , Masculino , Tamaño de los Órganos , Perfusión , Fosfolipasas A2 , Ratas , Ratas Sprague-DawleyRESUMEN
A marked sequence homology has been noted between lung surfactant protein A (SP-A) and an inhibitor of phospholipase A2 (PLA2) isolated from the serum of Trimeresurus flavoviridis (Habu snake). This study evaluated the effect of SP-A on PLA2 activity from several sources. SP-A was isolated from bovine or rat lung surfactant by extraction with 1-butanol and octyl beta-D-glucopyranoside. The addition of SP-A produced a concentration-dependent inhibition of T. flavoviridis PLA2 that indicated non-competitive kinetics with Ki 5 micrograms/ml. Inhibition was reversed by heat inactivation, disulfide bond reduction or alkylation of SP-A, or by the presence of anti-SP-A antibody. Treatment of SP-A with endoglycosidase F or the presence of variation monosaccharides or lectins did not alter SP-A inhibition. Binding of PLA2 to SP-A was shown by ultrafiltration and was abolished by SP-A alkylation or the presence of SDS. The SP-A/PLA2 complex recovered from the ultrafilter had essentially no enzymatic activity, but activity was restored by treatment with mercaptoethanol. SP-A had no effect on activity of PLA2 from Naja naja, Crotalus atrox, or bovine pancreas. These results indicate that surfactant protein A selectively inhibits Trimeresurus phospholipase A2 activity and suggest that binding to the enzyme is the mechanism for inhibition.
Asunto(s)
Fosfolipasas A/antagonistas & inhibidores , Proteolípidos/farmacología , Surfactantes Pulmonares/farmacología , Animales , Bovinos , Venenos de Crotálidos/antagonistas & inhibidores , Masculino , Peso Molecular , Fosfolipasas A2 , Proteolípidos/química , Proteína A Asociada a Surfactante Pulmonar , Proteínas Asociadas a Surfactante Pulmonar , Surfactantes Pulmonares/química , Ratas , Ratas Sprague-Dawley , TrimeresurusRESUMEN
This study evaluated whether cell membrane depolarization can induce oxidant generation in the isolated perfused rat lung as has been demonstrated with bovine pulmonary artery endothelial cells. Depolarization was produced by perfusing the lungs with high [K+] or with glyburide and was evaluated with bis-oxonol lung surface fluorometry. Lung surface bis-oxonol fluorescence increased above baseline (at 5.9 mM K+) by 18.5% with 24 mM K+, 35% with 48 mM K+, and 67% with 96 mM K+, indicating graded membrane depolarization, and by 75% during perfusion with 10 microM glyburide. Oxidant generation was evaluated with hydroethidine lung surface fluorometry, and with assay of tissue thiobarbituric acid reactive substance (TBARS), conjugated dienes, and perfusate H2O2. Depolarization by high K+ or glyburide led to significant increases in generation of tissue oxidants and lipid peroxidation. Bodipy-FL-glyburide microfluorography showed localization of glyburide binding primarily to vascular endothelial cells vascular and airway smooth muscle cells, alveolar type II cells, and to nonciliated cells of the airway epithelium. These results indicate that cellular depolarization is associated with oxidant generation by the lung and suggests a role for K(+)-channels in these events.