Phosphatidate phosphohydrolase activity as a marker for surfactant synthesis in organotypic cultures of type II alveolar pneumonocytes

1983 ◽  
Vol 60 (1) ◽  
pp. 199-207
Author(s):  
W.H. Douglas ◽  
S.K. Sommers-Smith ◽  
J.M. Johnston
Keyword(s):  
Pulmonary Surfactant ◽  
Specific Activity ◽  
Type Ii Cells ◽  
Type Ii ◽  
Rat Lung ◽  
Organotypic Cultures ◽  
Foetal Rat ◽  
Phosphatidate Phosphohydrolase ◽  
Type Ii Cell ◽  
Specific Activities

The specific activity of phosphatidate phosphohydrolase (PAPase) (EC 3.1.3.4) has been assayed in organotypic cultures of foetal rat lung type II alveolar pneumonocytes and in L2 cells derived from type II cells of the adult rat lung. This enzyme catalyses a critical step in the synthesis of phosphatidylcholine, the major lipid component of pulmonary surfactant. Surfactant is produced by the mature type II cell in culture as well as in vivo. The specific activity of PAPase in organotypic cultures prepared from foetal rat lung starting at 16 days of gestation increased four- to fivefold during the first 7 days in culture. The specific activity of this enzyme was further increased through the 21 days of culture. In parallel with the increase in PAPase specific activity in the cultures were morphological changes in the type II cells such as the appearance of increased numbers of lamellar bodies. The specific activities of PAPase samples derived from non-type II cell cultures maintained under identical conditions were compared. Organotypic cultures and L2 cells, a culture system that also exhibits type II cell characteristics, show PAPase specific activities five to six times greater than cultures that do not contain type II cells. PAPase activity in the type II cell cultures parallels the development of mature patterns of pulmonary surfactant synthesis storage and secretion.

Localization of a candidate surfactant convertase to type II cells, macrophages, and surfactant subfractions

1999 ◽  
Vol 276 (3) ◽  
pp. L452-L458 ◽  
Author(s):  
Howard Clark ◽  
Lennell Allen ◽  
Erin Collins ◽  
Frederick Barr ◽  
Leland Dobbs ◽  
...  
Keyword(s):  
Bronchoalveolar Lavage ◽  
Alveolar Macrophages ◽  
Pulmonary Surfactant ◽  
Type Ii Cells ◽  
Type Ii ◽  
Clara Cells ◽  
Rat Lung ◽  
Protein Distribution ◽  
Serine Hydrolase ◽  
Surfactant Metabolism

Pulmonary surfactant exists in the alveolus in several distinct subtypes that differ in their morphology, composition, and surface activity. Experiments by others have implicated a serine hydrolase in the production of the inactive small vesicular subtype of surfactant (N. J. Gross and R. M. Schultz. Biochim. Biophys. Acta 1044: 222–230, 1990). Our laboratory recently identified this enzyme in the rat as the serine carboxylesterase ES-2 [F. Barr, H. Clark, and S. Hawgood. Am. J. Physiol. 274 ( Lung Cell. Mol. Physiol. 18): L404–L410, 1998]. In the present study, we determined the cellular sites of expression of ES-2 in rat lung using a digoxygenin-labeled ES-2 riboprobe. ES-2 mRNA was localized to type II cells and alveolar macrophages but not to Clara cells. Using a specific ES-2 antibody, we determined the protein distribution of ES-2 in the lung by immunohistochemistry, and it was found to be consistent with the sites of mRNA expression. Most of the ES-2 in rat bronchoalveolar lavage is in the surfactant-depleted supernatant, but ES-2 was also consistently localized to the small vesicular surfactant subfraction presumed to form as a consequence of conversion activity. These results are consistent with a role for endogenous lung ES-2 in surfactant metabolism.

scholarly journals Maternal administration of dexamethasone stimulates choline-phosphate cytidylyltransferase in fetal type II cells

1987 ◽  
Vol 241 (1) ◽  
pp. 291-296 ◽  
Author(s):  
M Post
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Active Form ◽  
Type Ii Cells ◽  
Type Ii ◽  
Rat Lung ◽  
Dexamethasone Treatment ◽  
Fetal Rat ◽  
Fetal Rat Lung ◽  
Choline Phosphate

Administration of dexamethasone to pregnant rats at 19 days gestation increased phosphatidylcholine synthesis (45%) from radioactive choline in type II cells. This enhanced synthesis of phosphatidylcholine was accompanied by an increased conversion of choline phosphate into CDP-choline. Similar results were obtained by incubating organotypic cultures of 19-day-fetal rat lung with cortisol. The increased conversion of choline phosphate into CDP-choline correlated with an enhanced choline-phosphate cytidylyltransferase activity (31% after dexamethasone treatment; 47% after cortisol exposure) in the cell homogenates. A similar increase (26% after dexamethasone treatment; 39% after cortisol exposure) was found in the microsomal-associated enzyme. No differences in cytosolic enzyme activity were observed. The specific activity of the microsomal enzyme was 3-4 times that of the cytosolic enzyme. Most of the enzyme activity was located in the microsomal fraction (58-65%). The treatments had no effect on the total amount of enzyme recovered from the cell homogenates. These results, taken collectively, are interpreted to indicate that the active form of cytidylyltransferase in type II cells is the membrane-bound enzyme and that cytidylyltransferase activation in type II cells from fetal rat lung after maternal glucocorticoid administration occurs by binding of inactive cytosolic enzyme to endoplasmic reticulum.

Pulmonary lipid phosphate phosphohydrolase in plasma membrane signalling platforms

2001 ◽  
Vol 358 (3) ◽  
pp. 637-646 ◽  
Author(s):  
Meera NANJUNDAN ◽  
Fred POSSMAYER
Keyword(s):  
Cell Lines ◽  
Lung Tissue ◽  
Type Ii Cells ◽  
Type Ii ◽  
Rat Lung ◽  
Caveolin 1 ◽  
Type Ii Cell ◽  
Lipid Phosphate ◽  
Lipid Phosphate Phosphohydrolase ◽  
Isolated Rat

Lipid phosphate phosphohydrolase (LPP) has recently been proposed to have roles in signal transduction, acting sequentially to phospholipase D (PLD) and in attenuating the effects of phospholipid growth factors on cellular proliferation. In this study, LPP activity is reported to be enriched in lipid-rich signalling platforms isolated from rat lung tissue, isolated rat type II cells and type II cell-mouse lung epithelial cell lines (MLE12 and MLE15). Lung and cell line caveolin-enriched domains (CEDs), prepared on the basis of their detergent-insolubility in Triton X-100, contain caveolin-1 and protein kinase C isoforms. The LPP3 isoform was predominantly localized to rat lung CEDs. These lipid-rich domains, including those from isolated rat type II cells, were enriched both in phosphatidylcholine plus sphingomyelin (PC+SM) and cholesterol. Saponin treatment of MLE15 cells shifted the LPP activity, cholesterol, PC+SM and caveolin-1 from lipid microdomains to detergent-soluble fractions. Elevated LPP activity and LPP1/1a protein are present in caveolae from MLE15 cells prepared using the cationic-colloidal-silica method. In contrast, total plasma membranes had a higher abundance of LPP1/1a protein with low LPP activity. Phorbol ester treatment caused a 3.8-fold increase in LPP specific activity in MLE12 CEDs. Thus the activated form of LPP1/1a may be recruited into caveolae/rafts. Transdifferentiation of type II cells into a type I-like cell demonstrated enrichment in caveolin-1 levels and LPP activity. These results indicate that LPP is localized in caveolae and/or rafts in lung tissue, isolated type II cells and type II cell lines and is consistent with a role for LPP in both caveolae/raft signalling and caveolar dynamics.

Generation and characterization of monoclonal antibodies to alveolar type II cell lamellar body membrane

1998 ◽  
Vol 275 (1) ◽  
pp. L172-L183 ◽  
Author(s):  
K. Zen ◽  
K. Notarfrancesco ◽  
V. Oorschot ◽  
J. W. Slot ◽  
A. B. Fisher ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Membrane Protein ◽  
Lamellar Body ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Rat Lung ◽  
Lamellar Bodies ◽  
Alveolar Type Ii Cell ◽  
Type Ii Cell

Monoclonal antibodies against the limiting membrane of alveolar type II cell lamellar bodies were obtained after immunization of mice with a membrane fraction prepared from lamellar bodies isolated from rat lungs. The specificity of the antibodies was investigated with Western blot analysis, indirect immunofluorescence, and electron-microscopic immunogold studies of freshly isolated or cultured alveolar type II cells, alveolar macrophages, and rat lung tissue. One of the monoclonal antibodies identified, MAb 3C9, recognized a 180-kDa lamellar body membrane (lbm180) protein. Immunogold labeling of rat lung tissue with MAb 3C9 demonstrated that lbm180 protein is primarily localized at the lamellar body limiting membrane and is not found in the lamellar body contents. Most multivesicular bodies of type II cells were also labeled, as were some small cytoplasmic vesicles. Golgi complex labeling and plasma membrane labeling were weak. The appearance of lbm180 protein by immunofluorescence in fetal rat lung cryosections correlated with the biogenesis of lamellar bodies. The lbm180 protein decreased with time in type II cells cultured on plastic. The lbm180 protein is an integral membrane protein of lamellar bodies and was also found in the pancreas and the pancreatic βHC9 cell line but not in the rat brain, liver, kidney, stomach, or intestine. The present study provides evidence that the lbm180 protein is a lung lamellar body and/or multivesicular body membrane protein and that its antibody, MAb 3C9, will be a valuable reagent in further investigations of the biogenesis and trafficking of type II cell organelles.

Control of pulmonary surfactant secretion: an evolutionary perspective

2000 ◽  
Vol 278 (3) ◽  
pp. R611-R619 ◽  
Author(s):  
Philip G. Wood ◽  
Olga V. Lopatko ◽  
Sandra Orgeig ◽  
Jean M. P. Joss ◽  
Allan W. Smits ◽  
...  
Keyword(s):  
Nervous System ◽  
Cell Cultures ◽  
Pulmonary Surfactant ◽  
Parasympathetic Nervous System ◽  
Type Ii Cells ◽  
Type Ii ◽  
Surfactant System ◽  
Surfactant Secretion ◽  
Type Ii Cell ◽  
Australian Lungfish

Pulmonary surfactant, a mixture consisting of phospholipids (PL) and proteins, is secreted by type II cells in the lungs of all air-breathing vertebrates. Virtually nothing is known about the factors that control the secretion of pulmonary surfactant in nonmammalian vertebrates. With the use of type II cell cultures from Australian lungfish, North American bullfrogs, and fat-tailed dunnarts, we describe the autonomic regulation of surfactant secretion among the vertebrates. ACh, but not epinephrine (Epi), stimulated total PL and disaturated PL (DSP) secretion from type II cells isolated from Australian lungfish. Both Epi and ACh stimulated PL and DSP secretion from type II cells of bullfrogs and fat-tailed dunnarts. Neither Epi nor ACh affected the secretion of cholesterol from type II cell cultures of bullfrogs or dunnarts. Pulmonary surfactant secretion may be predominantly controlled by the autonomic nervous system in nonmammalian vertebrates. The parasympathetic nervous system may predominate at lower body temperatures, stimulating surfactant secretion without elevating metabolic rate. Adrenergic influences on the surfactant system may have developed subsequent to the radiation of the tetrapods. Furthermore, ventilatory influences on the surfactant system may have arisen at the time of the evolution of the mammalian bronchoalveolar lung. Further studies using other carefully chosen species from each of the vertebrate groups are required to confirm this hypothesis.

Glutathione transport by type II cells in perfused rat lung

1994 ◽  
Vol 267 (4) ◽  
pp. L447-L455 ◽  
Author(s):  
C. Bai ◽  
L. A. Brown ◽  
D. P. Jones
Keyword(s):  
Fluorescence Microscopy ◽  
High Performance ◽  
Cell Types ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Uptake System ◽  
Type Ii ◽  
Rat Lung ◽  
Perfused Lung ◽  
Type Ii Cell

Glutathione (GSH) is an antioxidant that protects the lung against oxidative-injury. Most cells rely on synthesis of GSH to maintain intracellular supply and only a few cell types take up intact GSH. Although isolated type II cells from rat have a Na(+)-dependent uptake system that transports GSH into the cells against a concentration gradient, it is not known whether this occurs from the vasculature in the intact lung or whether other cell types in the lung also transport GSH. Based on the knowledge that gamma-glutamyl analogues of GSH are also transported by the Na(+)-GSH transporter, a method was developed and used to study the cell specificity of GSH uptake in perfused lung. A stable, fluorescent GSH S-conjugate (GSH-I14) was synthesized and separated from the original dye as analyzed by high-performance liquid chromatography. Studies with isolated alveolar type II cells showed that uptake of GSH-I14 was Na+ dependent and inhibited by GSH. In addition, uptake of GSH by the type II cells was inhibited by GSH-I14. After perfusion of the isolated rat lung with GSH-I14, the conjugate accumulated primarily in the alveolar type II cell as observed by fluorescence microscopy. This was confirmed by isolation of type II cells and measurement of GSH-I14 content. Thus these results show that specificity of GSH transport can be studied with the fluorescent derivative, GSH-I14, and that in the isolated perfused lung type II cells can transport and concentrate GSH-I14 from the perfusate. Quantitative fluorescence microscopy will be required to further determine relative transport activities by other cell types.

Intracellular metabolism of the apoproteins of pulmonary surfactant in rat lung

1980 ◽  
Vol 48 (5) ◽  
pp. 812-820 ◽  
Author(s):  
R. J. King ◽  
H. Martin
Keyword(s):  
Pulmonary Surfactant ◽  
Time Course ◽  
Type Ii Cells ◽  
Type Ii ◽  
Rat Lung ◽  
Alveolar Cells ◽  
Dipalmitoyl Phosphatidylcholine ◽  
Surfactant Complex ◽  
Apoprotein B ◽  
Intracellular Metabolism

We studied the metabolic turnover of the major apoproteins of pulmonary surfactant from rat lung and compared it with similar studies on the metabolism of dipalmitoyl phosphatidylcholine (DPPC). At varying times after the injection of tritiated leucine or tritiated palmitate we isolated the alveolar cells and alveolar fluid and measured the incorporation of the radioactive leucine into the 35,000-dalton (apoprotein A) and 10,000-dalton (apoprotein B) apoproteins of surfactant and of the radioactive palmitate into DPPC. Maximum labeling of apoprotein A in type II cells occurred within 1 h after injection of the precursor and declined in the next 19 h. The labeling of intracellular DPPC followed the same time course. The labeling of apoprotein B in type II cells was low, and the change of its labeling with time was not consistent with its being secreted by these cells. Both proteins were labeled in alveolar fluid and in alveolar macrophages. The results indicate that apoprotein A is synthesized by type II cells and is probably secreted as part of the surfactant complex. Apoprotein B is not secreted by type II cells with the same time course as apoprotein A or the lipids of surfactant, but our results do not define its origin or physiological purpose.

P2u purinoceptor stimulation of surfactant secretion coupled to phosphatidylcholine hydrolysis in type II cells

1994 ◽  
Vol 267 (5) ◽  
pp. L625-L633 ◽  
Author(s):  
L. I. Gobran ◽  
Z. X. Xu ◽  
Z. Lu ◽  
S. A. Rooney
Keyword(s):  
Phospholipase D ◽  
Receptor Gene ◽  
Primary Cultures ◽  
Cell Receptor ◽  
Northern Analysis ◽  
Type Ii Cells ◽  
Type Ii ◽  
Type Ii Cell ◽  
Subsequent Activation ◽  
Phosphatidylcholine Secretion

ATP is known to stimulate surfactant phospholipid secretion in type II cells, and there is evidence that this effect is mediated by a P2 purinoceptor. At least five subtypes of the P2 receptor have been reported, but it is not clear which one exists on the type II cell. To determine whether it is the P2u subtype, at which UTP is equipotent with ATP, we have compared the effects of ATP and UTP on phosphatidylcholine secretion and second messenger formation in primary cultures of rat type II cells. ATP and UTP were equally potent in stimulating phosphatidylcholine secretion and phospholipase D activation. The potency order, UTP = ATP > ADP > 2-methylthio-ATP, was the same as that reported for the P2u receptor. UTP stimulated diacylglycerol and phosphatidic acid formation to the same extent as ATP. ATP also increased choline formation. Formation of diacylglycerol was biphasic, and the first peak in response to ATP was previously shown to be associated with inositol trisphosphate formation. Northern analysis showed that the P2u receptor gene was expressed to a greater extent in type II cells than in whole lung. These data suggest that ATP and UTP act via a P2u receptor that is coupled to phosphoinositide-specific phospholipase C with subsequent activation of phospholipase D acting on phosphatidylcholine. ATP has also been reported to act at an additional type II cell receptor coupled to adenylate cyclase. In contrast, UTP did not promote adenosine 3',5'-cyclic monophosphate formation and therefore does not act at that receptor.

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