RESUMO
Type II alveolar epithelial cells were isolated from fetal rat lung by differential adherence in monolayer culture. The preparation had a high degree of purity, as assessed by phase contrast microscopy and immunocytochemistry. Purity, based on reactivity with specific anti-adult lung serum (SAALS), which recognizes only type II cells, was 91% for cells isolated from 19-day fetal lungs and 79% for cells isolated from 21-day fetal lungs. The lower purity of type II cells in cultures derived from 1-day postnatal rat lungs (51% cells reactive with SAALS) is probably due to a lower tendency of the type II cells from neonatal rats to adhere to culture dishes than of type II cells from fetal rats. Type II cells isolated from 21-day fetal lungs contained a higher percentage phosphatidylglycerol and incorporated [Me-3H]choline faster into phosphatidylcholine (PC) than type II cells isolated from 19-day fetal lungs. Moreover, in cell preparations derived from lungs at fetal day 21, a higher percentage of epithelial cells contained lamellar bodies than in preparations derived from lungs at fetal day 19. The observation of these differences in the stage of maturation indicates that these differences, which are typical features of the original material, are not obliterated by differentiation during the culture. Type II cells isolated according to the present procedure were capable of synthesizing PC with a high percentage of the disaturated species. This method for the isolation of fetal type II cells may be a useful tool in studies concerning surfactant synthesis and its regulation in the fetal lung.
Assuntos
Alvéolos Pulmonares/citologia , Animais , Adesão Celular , Células Cultivadas , Colina/metabolismo , Fluoresceína , Fluoresceínas , Imunofluorescência , Soros Imunes/análise , Microscopia de Contraste de Fase , Fosfatidilcolinas/metabolismo , Fosfatidilgliceróis/metabolismo , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/ultraestrutura , Surfactantes Pulmonares/biossíntese , RatosRESUMO
myo-Inositol decreases the synthesis of phosphatidylglycerol by type II cells isolated from fetal rat lung. Inositol addition also increases the synthesized amount of surfactant phosphatidylinositol. These observations indicate that at least part of the decreasing effect of inositol on phosphatidylglycerol formation is the result of competition between phosphatidylglycerol and phosphatidylinositol synthesis for a common pool of CDP diacylglycerol. Studies on the subcellular localization of enzymes measured under optimal conditions suggested that the enzymic activity required for the formation of phosphatidylglycerol is located mainly in the mitochondria, but most likely also for a small part in the endoplasmic reticulum, while the enzymic activity required for phosphatidylinositol formation is located in the endoplasmic reticulum. Inositol was found to inhibit glycerolphosphate phosphatidyltransferase in the microsomal fraction but not in the mitochondrial fraction derived from the type II cells, indicating that the competition between phosphatidylglycerol and phosphatidylinositol synthesis for CDP diacylglycerol takes place in the endoplasmic reticulum. This latter observation together with the observation of a switch-over from surfactant phosphatidylinositol to phosphatidylglycerol production around term indicate that the endoplasmic reticulum is the intracellular site of surfactant phosphatidylglycerol production.
Assuntos
Pulmão/metabolismo , Fosfatidilgliceróis/biossíntese , Fosfatidilinositóis/biossíntese , Surfactantes Pulmonares/biossíntese , Transferases (Outros Grupos de Fosfato Substituídos) , Animais , Sítios de Ligação , Células Cultivadas , Retículo Endoplasmático/enzimologia , Feminino , Inositol/farmacologia , Pulmão/embriologia , Fosfotransferases/metabolismo , Gravidez , Surfactantes Pulmonares/fisiologia , Ratos , Ratos EndogâmicosRESUMO
When organotypic cultures of fetal rat lung epithelial cells are initiated with undifferentiated cells, the cells differentiate into type II cells (Douglas, W.H.J., McAteer, J. A., Smith, J.R. and Braunschweiger, W.R. (1979) Int. Rev. Cytol., Suppl. 10, 45-65). This conclusion was based only on morphologic studies. The present study was undertaken to investigate whether such maturation in culture could also be demonstrated biochemically. In organotypic cultures initiated with epithelial cells from fetal rat lungs at 17-days gestation, the amount of phospholipids increased for at least 10 days. However, no change took place in the percentage of phosphatidylglycerol nor in the ratio of disaturated to total phosphatidylcholine. In cultures initiated with cells obtained at day 17 of gestation the specific activity of cholinephosphate cytidylyltransferase reached a maximum after approximately 3 days, followed by a decrease. A similar profile was obtained, however, if the culture was started at day 20 of gestation. This indicates that the activity profiles obtained in the organotypic cultures reflect changes caused by the culture conditions rather than changes caused by maturation. From these investigations it is concluded that biochemical studies on type II cell development using organotypic cultures as model should be interpreted with caution.
Assuntos
Pulmão/embriologia , Fosfolipídeos/metabolismo , Animais , Colina-Fosfato Citidililtransferase , Epitélio/metabolismo , Feminino , Idade Gestacional , Cinética , Pulmão/enzimologia , Pulmão/ultraestrutura , Microscopia Eletrônica , Nucleotidiltransferases/metabolismo , Técnicas de Cultura de Órgãos , Gravidez , Ratos , Ratos EndogâmicosRESUMO
1. The specific activity of lysolecithin acyltransferase (EC 2.3.1.23) in sonicated adult rat lung alveolar type II epithelial cells, measured either alone or in combination with acyl-CoA synthetase (EC 6.2.1.3), was found to be an order of magnitude greater than that of lysolecithin:lysolecithin acyltransferase. 2. Lysolecithin acyltransferase in type II cells was found to prefer palmitoyl-CoA over oleoyl-CoA as substrate. The combination of lysolecithin acyltransferase and acyl-CoA synthetase was found to prefer palmitate over oleate for incorporation into phosphatidylcholine. 3. Compared to whole lung homogenate, sonicated adult rat type II cells are highly enriched in lysolecithin acyltransferase but not in lysolecithin:lysolecithin acyltransferase. 4. These observations indicate that in normal adult rat type II cells the deacylation-reacylation cycle is more important for the formation of dipalmitoyl phosphatidylcholine than the deacylation-transacylation process.
Assuntos
1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Aciltransferases/metabolismo , Alvéolos Pulmonares/enzimologia , Acil Coenzima A , Animais , Coenzima A Ligases/metabolismo , Pulmão/enzimologia , Lisofosfatidilcolinas , Ácidos Oleicos , Palmitoil Coenzima A , Alvéolos Pulmonares/citologia , Surfactantes Pulmonares/metabolismo , Ratos , Especificidade por SubstratoRESUMO
Rat-liver mitochondria were incubated with [14C]palmitate in the presence of L-malate, fluorocitrate, and L-carnitine. The specific activities of acetyl groups incorporated into citrate, ketone bodies and acetyl-L-carnitine were measured. During state-4 oxidation of [1--14C]palmitate the specific activity of the acetyl-CoA pool was 1.3-times higher than that of the average acetyl group of palmitate, indicating an incomplete breakdown of the palmitate molecule. Accumulation of carnitine esters was observed in this condition. The acyl moieties of carnitine esters formed during the state-4 oxidation of [U-14C]palmitate or [16(-14)C]palmitate were analysed by radioactive gas-chromatography. Substantial amounts of beta-oxidation intermediates were found. The accumulation of carnitine esters of C6-C14 intermediates can quantitatively explain the high specific activity of the acetyl-CoA pool during the state-4 oxidation of [1(-14)C] palmitate. The localization and control of beta-oxidation are discussed.
Assuntos
Carnitina/metabolismo , Mitocôndrias Hepáticas/metabolismo , Ácidos Palmíticos/metabolismo , Animais , Cromatografia Gasosa , Feminino , Cinética , RatosRESUMO
Rat-liver mitochondria oxidized [1-14C]palmitate or [U-14C]palmitate and unlabelled pyruvate in a medium containing fluorocitrate and L-carnitine. The oxidation products (acetyl-L-carnitine, ketone bodies and citrate) were separated by anion-exchange chromatography and their specific activities were determined. The distribution of radioactivity over the two halves of the ketone bodies was essayed. Significant differences between the specific activities of citrate, acetyl-L-carnitine and the carboxylhalf of the ketone bodies were not observed; this was consistently the case, even when pyruvate contributed for more than 80% to the acetyl-CoA pool. Our results argue against compartition of mitochondrial acetyl-CoA. Instead, they strongly suggest that the acetyl-CoA originating from the simultaneous oxidation of pyruvate and palmitate equilibrates before being distributed over the various pathways of further metabolism.
Assuntos
Acetilcoenzima A/metabolismo , Mitocôndrias Hepáticas/metabolismo , Animais , Carnitina/farmacologia , Citratos/farmacologia , Feminino , Corpos Cetônicos/metabolismo , Cinética , Ácidos Palmíticos/metabolismo , Piruvatos/metabolismo , RatosRESUMO
The synthesis of ketone bodies by intact isolated rat-liver mitochondria has been studied at varying rates of acetyl-CoA production and of acetyl-CoA utilization in the Krebs cycle. Factors which enhanced the rate of acetyl-CoA production caused an increase in the fraction of acetyl-CoA which was incorporated into ketone bodies. On the other hand, it was found that factors which stimulated the formation of citrate lowered the relative rate of ketogenesis. It is concluded that acetyl-CoA is preferentially used for citrate synthesis, if the level of oxaloacetate in the mitochondrial matrix space is adequate. The intramitochondrial level of oxaloacetate, which is determined by the malate concentration and the ratio of NADH over NAD+, is the main factor controlling the rate of citrate synthesis. The ATP/ADP ratio per se does not affect the activity of citrate synthase in this in vitro system. Ketogenesis can be described as an overflow of acetyl-groups: Ketone-body formation is stimulated only when the rate of acetyl-CoA production increases beyond the capacity for citrate synthesis. The interaction between fatty acid oxidation and pyruvate metabolism and the effects of long-chain acyl-CoA on mitochondrial metabolism are discussed. Ketone bodies which were generated during the oxidation of [1-14C] fatty acids were preferentially labelled in their carboxyl group. This carboxyl group had the same specific activity as the acetyl-CoA pool, whereas the specific activity of the acetone moiety of acetoacetate was much lower, especially at low rates of ketone-body formation. The activities of acetoacetyl-CoA deacylase and the hydroxymethylglutaryl-CoA (HMG-CoA) pathway were compared in soluble and mitochondrial fractions of rat- and cow-liver in different ketotic states. In rat-liver mitochondria, both pathways of acetoacetate synthesis were stimulated upon starvation or in alloxan diabetes. In cow liver, only the HMG-CoA pathway was increased during ketosis in the mitochondrial as well as in the soluble fraction.