RESUMEN
Dipalmitoylphosphatidylcholine, (DP-PtdCho), the major phospholipid component of lung surfactant is biosynthesized via a de novo pathway, the last step of which is catalyzed by CDP-choline:cholinephosphotransferase (CPT) and two remodeling steps: a deacylation and a reacylation one, catalyzed by an acidic, Ca²âº-independent phospholipase A2 (aiPLA2) and a lyso-phosphatidylcholine acyltransferase (LPCAT), respectively. The aim of our study was to investigate whether a low magnitude, non-injurious static mode of mechanical stretch can induce phosphatidylcholine (PtdCho) biosynthesis and its remodeling to DP-PtdCho in the A549 cell-line, a model of alveolar type II cells. The deformation of A549 cells did not cause any release of lactate dehydrogenase, or phospholipids into the cell culture supernatants. An increase in PtdCho levels was observed after 1 h of static stretching, especially among the DP-PtdCho molecular species, as indicated by targeted lipidomics approach and site-directed fatty acyl-chain analysis. Moreover, although sphingomyelin (CerPCho) levels were unaffected, the DP-PtdCho/CerPCho ratio increased. Induction was observed in CPT, LPCAT and aiPLA2 enzymatic activities and gene expression. Finally, incubation of the cells with MJ33 suppressed aiPLA2 activity and DP-PtdCho production. Our data suggest that mild static mechanical stretch can promote the biosynthesis of PtdCho and its remodeling to DP-PtdCho in lung epithelial cells. Thus, low magnitude stretch could contribute to protective mechanisms rather than to injurious ones.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/biosíntesis , Alveolos Pulmonares/citología , 1-Acilglicerofosfocolina O-Aciltransferasa/genética , 1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Línea Celular/efectos de los fármacos , Supervivencia Celular , Diacilglicerol Colinafosfotransferasa/metabolismo , Regulación de la Expresión Génica , Glicerofosfatos/farmacología , Humanos , L-Lactato Deshidrogenasa/metabolismo , Peroxiredoxina VI/genética , Fosfolipasas A2/metabolismo , Fosfolípidos/metabolismo , Alveolos Pulmonares/metabolismo , Esfingomielinas/metabolismo , Estrés MecánicoRESUMEN
Acyl-CoA:lysophosphatidylcholine acyltransferase 1 (LPCAT1) is a relatively newly described and yet indispensable enzyme needed for generation of the bioactive surfactant phospholipid, dipalmitoylphosphatidylcholine (DPPtdCho). Here, we show that lipopolysaccharide (LPS) causes LPCAT1 degradation using the Skp1-Cullin-F-box ubiquitin E3 ligase component, ß-transducin repeat-containing protein (ß-TrCP), that polyubiquitinates LPCAT1, thereby targeting the enzyme for proteasomal degradation. LPCAT1 was identified as a phosphoenzyme as Ser(178) within a phosphodegron was identified as a putative molecular recognition site for glycogen synthase kinase-3ß (GSK-3ß) phosphorylation that recruits ß-TrCP docking within the enzyme. ß-TrCP ubiquitinates LPCAT1 at an acceptor site (Lys(221)), as substitution of Lys(221) with Arg abrogated LPCAT1 polyubiquitination. LPS profoundly reduced immunoreactive LPCAT1 levels and impaired lung surfactant mechanics, effects that were overcome by siRNA to ß-TrCP and GSK-3ß or LPCAT1 gene transfer, respectively. Thus, LPS appears to destabilize the LPCAT1 protein by GSK-3ß-mediated phosphorylation within a canonical phosphodegron for ß-TrCP docking and site-specific ubiquitination. LPCAT1 is the first lipogenic substrate for ß-TrCP, and the results suggest that modulation of the GSK-3ß-SCFß(TrCP) E3 ligase effector pathway might be a unique strategy to optimize dipalmitoylphosphatidylcholine levels in sepsis.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/biosíntesis , 1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Lipopolisacáridos/farmacología , Surfactantes Pulmonares/metabolismo , Mucosa Respiratoria/metabolismo , Ubiquitinación/efectos de los fármacos , Proteínas con Repetición de beta-Transducina/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/genética , 1-Acilglicerofosfocolina O-Aciltransferasa/genética , Animales , Células Cultivadas , Estabilidad de Enzimas/efectos de los fármacos , Estabilidad de Enzimas/genética , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Ratones , Fosforilación/efectos de los fármacos , Fosforilación/genética , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Ligasas SKP Cullina F-box/genética , Proteínas Ligasas SKP Cullina F-box/metabolismo , Sepsis/genética , Sepsis/metabolismo , Ubiquitinación/genética , Proteínas con Repetición de beta-Transducina/genéticaRESUMEN
We have investigated the role of carnitine palmitoyltransferase (EC 2.3.1.21) in pulmonar type II pneumocyte, a lung cell responsible for the synthesis of surface active lipids. Adult type II pneumocytes were isolated from rat lung and purified by differential adherence. When these lung cells were incubated with radioactive palmitate, the percentage of radioactivity recovered into dipalmitoylphosphatidylcholine (DPPC), a major surface active lipid, was almost 60% with respect to total phosphatidylcholine (PC) molecular species. Cellular lysates from type II pneumocytes contained detectable amount of carnitine palmitoyltransferase (CPT) activity (1 nmol/min/mg). Most of the CPT activity found in these cells could be inhibited by incubating them for 60 min with 5 microM tetradecylglycidic acid (TDGA), a specific and irreversible CPT inhibitor of the malonyl-CoA sensitive CPT isoform (CPT I). TDGA treatment of adult type II pneumocytes caused a significant reduction in the incorporation of radioactive palmitate into PC, though this effect did not seem to be specific for DPPC. TDGA affected the incorporation of radioactive palmitate at the sn2 rather than the sn1 position of the glycerol backbone of PC. The incorporation of radioactive palmitate into DPPC was also observed when these lung cells were incubated with palmitate-labeled palmitoyl-L-carnitine. Our data suggest that type II pneumocyte CPT may play an important role in remodelling PC fatty acid composition and hence DPPC synthesis.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/biosíntesis , Carnitina O-Palmitoiltransferasa/metabolismo , Pulmón/citología , Alveolos Pulmonares/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/aislamiento & purificación , Animales , Células Cultivadas , Cromatografía en Capa Delgada , Compuestos Epoxi/antagonistas & inhibidores , Compuestos Epoxi/farmacología , Ácidos Grasos/antagonistas & inhibidores , Ácidos Grasos/farmacología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
This study evaluated the role of a lysosomal-type phospholipase A2 (aiPLA(2)) in the degradation of internalized dipalmitoylphosphatidylcholine (DPPC) and in phospholipid synthesis by the rat lung. Uptake and degradation of DPPC were measured in isolated perfused rat lungs over 3 h following endotracheal instillation of [(3)H]DPPC in mixed unilamellar liposomes plus or minus MJ33, a specific inhibitor of lung aiPLA(2). Uptake of DPPC was calculated from total tissue-associated radiolabel, and degradation was calculated from the sum of radiolabel in degradation products. Both uptake and degradation were markedly stimulated by addition of 8-bromo-cAMP to the perfusate. MJ33 had no effect on DPPC uptake but decreased DPPC degradation at 3 h by approximately 40-50%. The effect of MJ33 on lung synthesis of DPPC was evaluated with intact rats over a 12- to 24-h period following intravenous injection of radiolabeled palmitate and choline. MJ33 treatment decreased palmitate incorporation into disaturated phosphatidylcholine of lamellar bodies and surfactant by approximately 65% at 24 h but had no effect on choline incorporation. This result is compatible with inhibition of the deacylation/reacylation pathway for DPPC synthesis. These results obtained with intact rat lungs indicate that aiPLA(2) is a major enzyme for degradation of internalized DPPC and also has an important role in DPPC synthesis.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/metabolismo , Lisosomas/enzimología , Fosfolipasas A/fisiología , Alveolos Pulmonares/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/biosíntesis , 1,2-Dipalmitoilfosfatidilcolina/farmacocinética , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Glicerofosfatos/farmacología , Masculino , Inhibidores de Fosfodiesterasa/farmacología , Fosfolipasas A2 , Ratas , Ratas Sprague-DawleyRESUMEN
Dipalmitoyl phosphatidylcholine (deltaPC) synthesis by lung epithelium occurs in part by a deacylation/reacylation pathway utilizing phospholipase A2 (PLA2) and an acyl transferase. The role of acidic Ca2+-independent PLA2 (aiPLA2) in this pathway was investigated using a transition-state analog enzyme inhibitor [1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33)]. Granular pneumocytes were isolated from rat lung with elastase and were maintained in primary culture for 24 h on microporous membranes in the presence of radiolabeled choline or free fatty acids (palmitate plus oleate). Disaturated phosphatidylcholine (DSPC) was determined by osmication chromatography. Incorporation (nmol/mg protein) into DSPC at 24 h incubation was 11.9 +/- 0.2 for [3H]choline and 12.1 +/- 0.04 for [3H]palmitate. In the presence of 3 mol% MJ33, incorporation of [3H] choline and [3H]palmitate was decreased by 37 and 69%, respectively, and DSPC pool size (microg/mg cell protein) decreased by 9% (P < 0.05). A similar decrease in radiolabel incorporation was observed with 2 h of incubation. The presence of p-bromophenacyl bromide (20 microm) had a significantly smaller effect that was additive with that of MJ33. After 24 h of labeling and 4 h of chase with unlabeled substrate, there was a significant decrease of radiolabel in DSPC that was inhibited by MJ33. Under all experimental conditions, MJ33 resulted in either no change or a modest increase of radiolabel in the cellular unsaturated PC fraction. These results indicate that aiPLA2 has a major role in DSPC synthesis by granular pneumocytes.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/biosíntesis , Ácidos/metabolismo , Calcio/fisiología , Pulmón/metabolismo , Fosfolipasas A/fisiología , Animales , Inhibidores Enzimáticos/farmacología , Ácidos Grasos/farmacología , Glicerofosfatos/farmacología , Pulmón/citología , Pulmón/efectos de los fármacos , Masculino , Metilaminas/farmacología , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A2 , Ratas , Ratas Sprague-Dawley , Especificidad por SustratoRESUMEN
La ausencia de surfactantes pulmonares trae como consecuencia el incremento de la tensión superficial a lo largo del epitelio alveolar, provocando un colapso alveolar y la lisis de las células epiteliales. Este proceso culmina con la aparición de un síndrome de insuficiencia respiratoria, que es la causa principal de morbimortalidad en niños prematuros. Recientemente, la aplicación de mezclas de agentes surfactantes con fines terapéuticos ha constituido un gran apoyo para la terapia respiratoria, ya que permite una evolución más rápida de los niños que padecen este síndrome. Por todo esto, resulta de gran importancia el conocimiento más detallado de la función, el metabolismo y la regulación de la expresión genética de las proteíinas surfactantes, para el diseño de nuevas y mejores estrategias terapéuticas para combatir este síndrome
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/biosíntesis , 1,2-Dipalmitoilfosfatidilcolina/química , Fosfolípidos/biosíntesis , Fosfolípidos/química , Lectinas/química , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/fisiología , Surfactantes Pulmonares/química , Surfactantes Pulmonares/genética , Surfactantes Pulmonares/ultraestructuraRESUMEN
We examined the effects of parathyroid hormone (PTH) and PTH-related protein (PTHrP) on rat fetal lung fibroblast and pneumocyte cell signalling. We also studied the effects of PTH and PTHrP on surfactant phospholipid synthesis to determine whether these peptides can modulate pulmonary maturation. Exposure of fibroblasts (gestational days 18-21) to PTH(1-34) or PTHrP(1-34) produced time- and dose-dependent stimulations of cAMP and inositol phosphate accumulation. Maximal stimulation of cAMP accumulation occurred with 1 x 10(-8) M of either peptide. These effects upon cAMP accumulation were competitively inhibited by the PTH antagonist, [Nle8, Nle18, Tyr34]bPTH(3-34)amide. Maximal stimulation of fibroblast inositol phosphates was reached at 1 x 10(-7) M of either peptide. In contrast, PTH and PTHrP at these concentrations produced no changes in cAMP or inositol phosphate metabolism in isolated type II pneumocytes. When pneumocytes were exposed to PTH or PTHrP and pulse-labelled with [methyl-3H]choline chloride, no hormone-stimulated changes in saturated phosphatidylcholine (PC) synthesis were detected. However, PTH and PTHrP stimulated saturated PC synthesis in rat fetal lung explants (gestational day 19-20) by 46% and 106%, respectively. When fibroblasts and pneumocytes were co-cultured, PTH and PTHrP again stimulated saturated PC synthesis by 45% and 73%, respectively. Taken together, these findings suggest that PTH and PTHrP may be endocrine and/or paracrine regulators of fetal lung development.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/biosíntesis , Pulmón/metabolismo , Hormona Paratiroidea/farmacología , Proteínas/farmacología , Animales , Células Cultivadas/efectos de los fármacos , AMP Cíclico/metabolismo , Desarrollo Embrionario y Fetal/efectos de los fármacos , Epitelio/metabolismo , Edad Gestacional , Fosfatos de Inositol/metabolismo , Pulmón/efectos de los fármacos , Pulmón/embriología , Proteína Relacionada con la Hormona Paratiroidea , Ratas , Ratas Sprague-DawleyRESUMEN
Endogenous content of and incorporation of labelled glycerol into alkenylacyl-, alkylacyl- and diacyl-glycerol, -glycerol-3-phosphocholine and -glycero-3-phosphoethanolamine of pulmonary type II cells were measured. On prolonged incubation of type II cells with labelled glycerol, the proportion of label incorporated into the diacyl subclass of these glycerolipids increased and the proportion of label incorporated into the ether lipids declined. Endogenous phosphatidylcholine (PtdCho) of type II cells contained 38.4% of the dipalmitoyl species, but endogenous phosphatidylethanolamine (PtdEtn) only 2.5%. In contrast, similar proportions of labelled glycerol were incorporated into dipalmitoyl-PtdCho and -PtdEtn after short-time incubation but, with prolonged incubation time the proportion of labelled dipalmitoyl-PtdCho increased from 11.3 to 18.8%, whereas that of dipalmitoyl-PtdEtn did not change significantly. Type II cell membranes were found to exhibit cofactor-independent and CoA-mediated transacylations of [1-14C]palmitoyl-lyso-PtdCho and -lyso-PtdEtn. The distribution of label among the palmitic acid-containing species of PtdCho and PtdEtn formed by both transacylation activities was determined. Cofactor-independent and CoA-mediated transacylation showed a strong selectivity for palmitate and arachidonate and a strong discrimination against oleate. The amount (nmol) of dipalmitoyl-PtdEtn formed by both transacylation activities after short-time incubation (2 min) decreased with prolonged incubation time (60 min). In contrast, the nmol of dipalmitoyl-PtdCho formed by cofactor-independent transacylation remains nearly the same after short-time and longer incubation. The nmol of dipalmitoyl-PtdCho formed by CoA-mediated transacylation increased strongly in the same time interval. Beside synthesis de novo via the CDP-choline pathway and reacylation of lyso-PtdCho with palmitoyl-CoA, the CoA-mediated transacylation of lyso-PtdCho may be an effective pathway for the formation of dipalmitoyl-PtdCho in pulmonary type II cells.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/biosíntesis , Pulmón/metabolismo , Fosfatidilcolinas/biosíntesis , Fosfatidiletanolaminas/biosíntesis , Acilación , Animales , Células Cultivadas , Pulmón/citología , Lisofosfatidilcolinas/metabolismo , Masculino , Metilación , Palmitoil Coenzima A/metabolismo , Fosfatidiletanolaminas/metabolismo , Ratas , Ratas EndogámicasRESUMEN
Lipids, particularly surface-active phospholipids, have been proposed to provide an important protective barrier in the gastric mucosa. The predominant surface-active phospholipid in the pulmonary surfactant complex is dipalmitoylphosphatidylcholine. To determine whether the gastric epithelium synthesizes and secretes this phospholipid, primary cultures of canine gastric mucous cells isolated by counterflow elutriation were studied. During the 24-hour period of culture, the gastric mucous cells incorporated 3H-choline into phosphatidylcholine, with dipalmitoylphosphatidylcholine representing 13.8% +/- 0.6% of the phosphatidylcholine synthesized. When mucous cell preparations with greater chief cell contamination were studied, they incorporated significantly less precursor into dipalmitoylphosphatidylcholine. Administration of prostaglandin E2, a cytoprotective agent, to the cultured mucous cells for 1 hour led to a significant increase in phosphatidylcholine release, reaching a maximum of 120.4% +/- 4.2% (P less than 0.001) at 10(-6) mol/L. No significant stimulation of phospholipid release by prostaglandin E2 was seen in the fractions containing a greater proportion of chief cells. To further establish the relationship between mucin and phospholipid secretion, two gastric cancer cell lines, Hs746T and KATO III, were studied. Using immunocytochemical and biochemical techniques, mucin synthesis and secretion were confirmed by these cell lines. The Hs746T cells were significantly more active in the secretion of both mucin and phospholipid than the KATO III cells. The Hs746T line secreted 5.7-fold more mucin and 7.3-fold more phospholipid than KATO III cells during a 24-hour period of culture. The association between mucin and phospholipids in an aqueous solution was also studied. Purified mucin in the concentration of 0.5-2 mg/mL of glycoprotein led to a significant dose-dependent increase in phospholipid solubility, suggesting the formation of a glycoprotein-phospholipid complex. The current studies indicate that the gastric mucous cell is the source of surfactant phospholipids as well as mucin. The synthesis and release of mucin and phospholipid are functions of the mucous cell that play a critical role in the primary defense of gastric epithelium.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/biosíntesis , Dinoprostona/farmacología , Mucinas Gástricas/metabolismo , Mucosa Gástrica/metabolismo , Fosfolípidos/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Animales , Células Cultivadas , Perros , Epitelio/metabolismo , Mucinas Gástricas/biosíntesis , Mucinas Gástricas/química , Mucosa Gástrica/efectos de los fármacos , Fosfatidilcolinas/metabolismo , Células Tumorales CultivadasRESUMEN
Fly ash contains many polycyclic aromatic hydrocarbons and genotoxic trace elements. In rats, fly ash exposure profoundly affects lung and liver histology. In the present study, the effect of fly ash inhalation on lung and liver lipids of rats was examined. Male Wistar strain rats were exposed daily to fly ash (0.27 +/- 0.01 mg/L air) in an inhalation chamber, 6 hr daily over a period of 15 days, and were killed on various days, i.e. 16, 30, 60, and 120. Fly ash inhalation significantly (P less than 0.05) increased total phospholipids (PL), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in lungs. PC and dipalmitoylphosphatidylcholine (DPPC) contents in microsomes and lung surfactant also were significantly (P less than 0.05) higher in rats exposed to fly ash compared to control group animals. Radiolabeled precursor incorporation studies indicated that fly ash induced the synthesis of PC and DPPC by both CDP-choline pathway and N-methylation of PE in lung microsomes and enhanced their secretion into lung surfactant. In liver, PC and PE contents were elevated significantly (P less than 0.05) by fly ash exposure on days 16 and 30 respectively. A similar elevation of PC was observed in hepatic microsomes; this increase was due to its increased synthesis. However, the increased synthesis of PC in liver occurred to a greater extent by the N-methylation pathway than by the CDP-choline pathway.
Asunto(s)
Carbono/farmacología , Hígado/efectos de los fármacos , Pulmón/efectos de los fármacos , Fosfolípidos/biosíntesis , Surfactantes Pulmonares/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/biosíntesis , Administración por Inhalación , Animales , Carbono/administración & dosificación , Carbón Mineral , Ceniza del Carbón , Hígado/metabolismo , Pulmón/metabolismo , Masculino , Material Particulado , Fosfatidilcolinas/biosíntesis , Fosfatidiletanolaminas/biosíntesis , Ratas , Ratas EndogámicasRESUMEN
Alveolar type II cells require a source of palmitic acid for synthesis of dipalmitoyl phosphatidylcholine (DPPC), a major constituent of pulmonary surfactant. Previous studies indicated that maximal rates of DPPC synthesis are achieved only if exogenous palmitate is available to the type II cell. Little is known of the mechanisms by which fatty acids enter type II cells. To determine if uptake is mediated by a membrane carrier system, as described in other cell types, we examined the kinetics of palmitate uptake. Using freshly isolated rabbit type II cells, we demonstrated that radiolabeled palmitate uptake was maximal and linear for 45 s; after 1 min the apparent rate of uptake declined. The initial uptake phase was taken as a measure of cellular fatty acid influx because intracellular radiolabeled palmitate remained 80% nonesterified at this time but was 55% esterified by 2 min. Cellular influx of palmitate showed saturation kinetics with increasing concentration of nonalbumin bound palmitate. Michaelis constant was 52.6 nM, and maximum velocity was 152 pmol.10(6) cells-1.min-1. The hypothesis that saturable cellular influx of palmitate is likely linked to the previously identified membrane fatty acid binding protein (MFABP) was supported by Western-blot analysis of rat lung tissue with an antibody to MFABP that demonstrated the presence of this carrier protein in lung tissue. These data suggest that palmitate uptake by type II cells is saturable and may be mediated by a membrane-associated carrier as described in other cell types.
Asunto(s)
Proteínas de Neoplasias , Proteínas del Tejido Nervioso , Ácidos Palmíticos/metabolismo , Alveolos Pulmonares/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/biosíntesis , Animales , Transporte Biológico , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Técnicas In Vitro , Cinética , Masculino , Ácido Palmítico , Alveolos Pulmonares/citología , Conejos , Técnica de Dilución de Radioisótopos , Albúmina Sérica Bovina/metabolismo , TritioRESUMEN
Presentamos un paciente de 3 años de edad con intoxicación por Paraquat, a quien se le practicaron las medidas de prevención de absorción del tóxico, tres y media horas después de su ingestión accidental. A las 20 horas posteriores se consideró otra alternativa terapéutica, la hemoperfusión con carbón, debido a sus condiciones clínicas estables, sin alteraciones sistémicas aparentes, pero con persistencia de la prueba cualitativa del Paraquat en orina fuertemente positiva, lo que indicaba mal pronóstico. A las 22 horas se inicia la técnica en el Servicio de Nefrología del Hospital "J. M. de los Ríos", utilizando un Hemo-Adsorda 150 C (GAMBRO), durante un período de 3 horas. No se presentaron complicaciones de trombocitopenia, hipotensión, hipoglicemia, o trastornos electrolíticos. Al finalizar, se repitió la prueba cualitativa de orina, apreciándose disminución significativa de la reacción. La evolución fue satisfactoria, sin observarse complicaciones hepáticas, renales, ni respiratorias. Su seguimiento ambulatorio 5 meses después es adecuado. Realizamos revisión de la literatura especializada, y se recomiendan pautas del manejo
Asunto(s)
Preescolar , Humanos , Femenino , Carbón Orgánico , Hemoperfusión , Paraquat/toxicidad , Orina/análisis , 1,2-Dipalmitoilfosfatidilcolina/biosíntesis , Albúminas/uso terapéutico , Ditionita , Furosemida/uso terapéutico , Sulfato de Magnesio/uso terapéuticoRESUMEN
Sepsis is characterized by Adult Respiratory Distress Syndrome (ARDS)-like pulmonary dysfunction largely attributed to alveolar capillary endothelial cell injury which causes increased microvascular permeability and interstitial edema formation. In addition, quantitative and qualitative abnormalities of the pulmonary surfactant system may be important features of some clinical and experimental lung injuries. This study was designed to investigate the relationship of bacteremia and endotoxemia to pulmonary surfactant production in vivo. A technique for estimation of pulmonary surfactant phospholipid synthesis measuring incorporation of a stable isotope precursor [( 2-13C]acetate) into dipalmitoylphosphatidylcholine (DPPC) in alveolar lavage fluid was developed. Male 350 g Sprague-Dawley rats had placement of central venous catheters. After overnight recovery, sublethal bacteremia (Escherichia coli, 1 x 10(8) organisms, iv) and sublethal endotoxemia (Difco; 10 mg/kg, iv) were induced. Both were associated with lung microvascular permeability increases consistent with capillary endothelial injury. Eight-hour infusions of [2-13C]acetate were given. After sacrifice, bronchoalveolar washings and lung tissue were obtained and [2-13C] incorporation into lavage and lung DPPC was measured by gas chromatography mass spectroscopy. Endotoxin-treated animals had a 21.5% reduction in label incorporation into DPPC [1.215 +/- 0.145 APE (%) sham versus 0.954 +/- 0.144 APE (%) experimental, P greater than 0.05] and bacteremic animals had a 56.9% diminution of [2-13C]acetate incorporation [1.215 +/- 0.145 APE (%) sham versus 0.524 +/- 0.56 APE (%) experimental, P less than 0.05]. Bacteremia-induced dysfunction of alveolar type II epithelial cells manifested as diminished alveolar surfactant phospholipid production may be a contributing factor to sepsis-induced respiratory failure.
Asunto(s)
Infecciones por Escherichia coli/metabolismo , Alveolos Pulmonares/metabolismo , Surfactantes Pulmonares/biosíntesis , Sepsis/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/biosíntesis , Acetatos/metabolismo , Animales , Líquido del Lavado Bronquioalveolar/metabolismo , Permeabilidad Capilar , Endotelio Vascular/patología , Infecciones por Escherichia coli/patología , Cromatografía de Gases y Espectrometría de Masas , Pulmón/irrigación sanguínea , Pulmón/patología , Masculino , Ratas , Ratas Endogámicas , Sepsis/patologíaRESUMEN
The most important component of pulmonary surfactant is dipalmitoyl phosphatidylcholine, or DPPC. At the end of an expiration, when the surfactant film is maximally compressed, DPPC is probably almost the sole component in the monomolecular film, but, during inspiration, as the film expands, other elements move in. They may be phospholipids with different fatty acids, saturated or unsaturated, or with the alcohol choline replaced with ethanolamine, inositol, glycerol or serine. The elements moving into the expanding film may also be free fatty acids, di- or triglycerides, etc. The synthesis of DPPC, similar to that of other phospholipids, has been thoroughly studied and is reviewed. The building blocks, glycerol, palmitic acid, choline and phosphate, are generally produced in the lung itself, but choline is generously supplied with the bloodstream. The slowest and, therefore, rate-limiting reaction is conversion of choline phosphate to cytidine diphosphate choline (CDP choline). The enzyme required, choline phosphate cytidyltransferase, is, therefore, crucial. It exists in two forms and can be stimulated to be converted to the more active H-form by the presence of phosphatidylglycerol. This would give PG a specific role; it has no unique ability to affect the surface tension, but it stimulates the synthesis of DPPC.