Immunocytochemical localization of lung surfactant proteins by cryoultramicrotomy and freeze-substitution embedding

1990 ◽  
Vol 48 (3) ◽  
pp. 38-39
Author(s):  
W.F. Voorhout ◽  
T. Veenendaal ◽  
H.P. Haagsman ◽  
J.W. Slot
Keyword(s):  
Lung Surfactant ◽  
Protein A ◽  
Freeze Substitution ◽  
Transpulmonary Pressure ◽  
Surfactant Protein ◽  
Apical Plasma Membrane ◽  
Type Ii Cells ◽  
Main Function ◽  
Type Ii ◽  
Lamellar Bodies

Lung surfactant is composed primarily of phospholipids but contains also about 10% proteins. Its main function is to decrease alveolar surface tension at low transpulmonary pressure to prevent alveolar collaps. Surfactant is stored in lamellar bodies in alveo1lar type II cells and is transformed after secretion in tubular myelin, a lattice-like structure.We investigated the biogenesis of surfactant and the pathways that the large hydrophilic surfactant protein A (SP-A) and the small hydrophobic surfactant protein B (SP-B) follow in human lung by using two different immunocytochemical techniques, the cryo-ultramicrotomy method and a new post-embedding method.In the non-embedded, ultrathin cryosections of the lung, prepared and immunolabeled for SP-A and SP-B as described before, it was impossible to achieve a satisfying preservation of lipid-rich structures like lamellar bodies (Fig. 1 and 2). Nevertheless SP-A and SP-B are detected in remnants of lamellar bodies (Fig. 1 and 2) and SP-A is further found to be present throughout the biosynthetic route, in some multivesicular bodies and over the apical plasma membrane of type II cells (Fig. 1).

scholarly journals Immunocytochemical localization of lysozyme and surfactant protein A in rat type II cells and extracellular surfactant forms.

1992 ◽  
Vol 40 (10) ◽  
pp. 1491-1500 ◽  
Author(s):  
E M Haller ◽  
S A Shelley ◽  
M R Montgomery ◽  
J U Balis
Keyword(s):  
Lung Surfactant ◽  
Protein A ◽  
Lamellar Body ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Peripheral Compartment ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lamellar Bodies ◽  
Body Protein

Using immunogold labeling of fixed, cryosubstituted tissue sections, we compared the distribution of lysozyme, an oxidant-sensitive lamellar body protein, with that of surfactant protein A (SP-A) in rat Type II cells, extracellular surfactant forms, and alveolar macrophages. Morphometric analysis of gold particle distribution revealed that lysozyme and SP-A were present throughout the secretory and endosomal pathways of Type II cells, with prominent localization of lysozyme in the peripheral compartment of lamellar bodies. All extracellular surfactant forms were labeled for both proteins with preferential labeling of tubular myelin and unilamellar vesicles. Labeling of tubular myelin for SP-A was striking when compared with that of lamellar bodies and other extracellular surfactant forms. Lamellar body-like forms and multilamellar structures were uniformly labeled for lysozyme, suggesting that this protein is rapidly redistributed within these forms after secretion of lysozyme-laden lamellar bodies. By contrast, increased labeling for SP-A was observed over peripheral membranes of lamellar body-like forms and multilamellar structures, apparently reflecting progressive SP-A enrichment of these membranes during tubular myelin formation. The results indicate that lysozyme is an integral component of the lamellar body peripheral compartment and secreted surfactant membranes, and support the concept that lysozyme may participate in the structural organization of lung surfactant.

Pulmonary surfactant protein A-mediated uptake of phosphatidylcholine by alveolar type II cells

1993 ◽  
Vol 265 (2) ◽  
pp. L193-L199 ◽  
Author(s):  
A. Tsuzuki ◽  
Y. Kuroki ◽  
T. Akino
Keyword(s):  
Pulmonary Surfactant ◽  
Protein A ◽  
Lamellar Body ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lamellar Bodies ◽  
Pulmonary Surfactant Protein

Pulmonary surfactant protein A (SP-A)-mediated uptake of phosphatidylcholine (PC) by alveolar type II cells was investigated. SP-A enhanced the uptake of liposomes containing dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl-2-linoleoyl phosphatidylcholine (PLPC), or 1,2-dihexadecyl-sn-glycero-3-phosphocholine (DPPC-ether), a diether analogue of DPPC, but about twice as much DPPC was taken up by type II cells as PLPC or DPPC-ether. When subcellular distribution was analyzed, 51.3 +/- 2.9% (mean +/- SD, n = 3) of cell-associated radiolabeled DPPC was recovered in the lamellar body-rich fraction in the presence of SP-A, whereas only 19.3 +/- 1.9% (mean +/- SD, n = 3) was found to this fraction in the absence of SP-A. When type II cells were incubated either with DPPC at 0 degree C or with DPPC-ether at 37 degrees C, or no cells were included, low proportions of the cell-associated lipids were present in the fractions corresponding to lamellar bodies even in the presence of SP-A. Anti-SP-A antibody significantly reduced the radioactivity incorporated into the lamellar body fraction. Phosphatidylcholine that had been incorporated into lamellar bodies remained largely intact when SP-A was present. Subcellular fractionations of type II cells with radiolabeled SP-A and DPPC revealed that the sedimentation characteristics of cell-associated SP-A are different from those of DPPC, although a small broad peak of radiolabeled SP-A was found in the lamellar body fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Endocytosed SP-A and surfactant lipids are sorted to different organelles in rat type II pneumocytes

2001 ◽  
Vol 281 (2) ◽  
pp. L345-L360 ◽  
Author(s):  
Heide Wissel ◽  
Andrea Lehfeldt ◽  
Petra Klein ◽  
Torsten Müller ◽  
Paul A. Stevens
Keyword(s):  
Cell Surface ◽  
Intracellular Transport ◽  
Protein A ◽  
Lamellar Body ◽  
Surfactant Protein ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lamellar Bodies ◽  
Type Ii Pneumocytes ◽  
Early Endosomes

Intracellular transport of endocytosed surfactant protein A (SP-A) and lipid was investigated in isolated rat type II cells. After internalization, SP-A and lipid are taken up via the coated-pit pathway and reside in a common compartment, positive for the early endosomal marker EEA1 but negative for the lamellar body marker 3C9. SP-A then recycles rapidly to the cell surface via Rab4-associated recycling vesicles. Internalized lipid is transported toward a Rab7-, CD63-, 3C9-positive compartment, i.e., lamellar bodies. Inhibition of calmodulin led to inhibition of uptake and transport out of the EEA1-positive endosome and thus of resecretion of both components. Inhibition of intravesicular acidification (bafilomycin A1) led to decreased uptake of both surfactant components. It inhibited transport out of early endosomes for lipid only, not for SP-A. We conclude that in type II cells, endocytosed SP-A and lipid are transported toward a common early endosomal compartment. Thereafter, both components dissociate. SP-A is rapidly recycled to the cell surface and does not enter classic lamellar bodies. Lipid is transported toward lamellar bodies.

Type II pneumocytes secrete vitamin E together with surfactant lipids

1993 ◽  
Vol 265 (2) ◽  
pp. L133-L139 ◽  
Author(s):  
B. Rustow ◽  
R. Haupt ◽  
P. A. Stevens ◽  
D. Kunze
Keyword(s):  
Vitamin E ◽  
Palmitic Acid ◽  
Lung Surfactant ◽  
Protein A ◽  
Specific Radioactivity ◽  
Surfactant Protein ◽  
Lung Lavage ◽  
Type Ii Cells ◽  
Type Ii ◽  
Alveolar Surfactant

Lung surfactant is exposed to strongly oxidizing conditions. We examined the hypothesis that in lung, lipophilic antioxidants are secreted together with surfactant to counteract the peroxidation of surfactant constituents. Lung lavage and the subfractions of the alveolar surfactant contain the lipophilic antioxidants vitamin E, vitamin A, and plasmalogens. The specific radioactivity of vitamin E isolated from serum, lung homogenate, lamellar bodies, and lung lavage increased linearly up to 3 h after intraperitoneal application of [3H]tocopherol. [3H]tocopherol was secreted in situ together with [14C]palmitic acid-labeled phospholipid in response to isoproterenol. Type II cells cultured in presence of [3H]tocopherol or of [3H]cholecalciferol and [14C]palmitic acid responded to isoproterenol by a time-dependent increase in secretion of [3H]tocopherol and of 14C-labeled phospholipids but not of [3H]cholecalciferol. The isoproterenol-stimulated secretion of [3H]tocopherol and of 14C-labeled phospholipids by type II cells is inhibited by surfactant protein A. We conclude that the alveolar surfactant contains lipophilic antioxidants as integral constituents. [3H]tocopherol seems to be secreted together with surfactant.

scholarly journals Ca2+-dependent binding of annexin IV to surfactant protein A and lamellar bodies in alveolar type II cells

1995 ◽  
Vol 312 (1) ◽  
pp. 175-181 ◽  
Author(s):  
H Sohma ◽  
N Matsushima ◽  
T Watanabe ◽  
A Hattori ◽  
Y Kuroki ◽  
...  
Keyword(s):  
Protein A ◽  
Immunoblot Analysis ◽  
Surfactant Protein ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lamellar Bodies ◽  
Alveolar Type Ii Cells ◽  
Sepharose 4B ◽  
Annexin Iv

Surfactant protein A (SP-A), a lung-specific glycoprotein in pulmonary surfactant, is synthesized and secreted from the alveolar type II cells. It has been shown that SP-A is a Ca(2+)-binding protein with several binding sites and that the high-affinity site(s) is located in the C-terminal region of SP-A. In the present study we isolated the proteins from bovine lung soluble fraction that bind to SP-A in a Ca(2+)-dependent manner using DEAE-Sephacel and SP-A-conjugated Sepharose 4B. At least three different protein bands with molecular masses of 24.5, 32, and 33 kDa were observed on SDS/PAGE. The main protein, with molecular mass of 32 kDa, was identified as annexin IV by the partial-amino-acid-sequence analyses and an immunoblot analysis with anti-(annexin IV) antiserum. We also found from the immunoblot analysis that the cytosolic fraction of isolated rat alveolar type II cells contains annexin IV. In addition, when rat lung cytosol was loaded on to the lung lamellar body-conjugated Sepharose 4B in the presence of Ca2+, two proteins, with molecular masses of 32 and 60 kDa on SDS/PAGE respectively, were eluted with EGTA. The 32 kDa protein was shown to be annexin IV by an immunoblot analysis with the antiserum against annexin IV. The lung annexin IV augmented the Ca(2+)-induced aggregation of the lung lamellar bodies from rats. However, the augmentation of aggregation of the lung lamellar bodies by annexin IV was attenuated when the lamellar bodies were preincubated with polyclonal anti-SP-A antibodies. SP-A bound to annexin IV under conditions where contaminated lipid was removed. These results suggest that SP-A bound to annexin IV based on protein-protein interaction, though both proteins are phospholipid-binding proteins. All these findings suggest that the interaction between SP-A and annexin IV may have some role in alveolar type II cells.

scholarly journals Internalization of surfactant protein A (SP-A) into lamellar bodies of rat alveolar type II cells in vitro.

1993 ◽  
Vol 41 (1) ◽  
pp. 57-70 ◽  
Author(s):  
M Kalina ◽  
F X McCormack ◽  
H Crowley ◽  
D R Voelker ◽  
R J Mason
Keyword(s):  
Protein A ◽  
Fluid Phase ◽  
Surfactant Protein ◽  
Biologically Active ◽  
Surfactant Protein A ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lamellar Bodies ◽  
Alveolar Type Ii Cells

Pulmonary surfactant is thought to be internalized and processed for reuse by alveolar Type II cells. In the present study we followed the internalization and intracellular trafficking of purified surfactant protein A (SP-A) by primary cultures of alveolar Type II cells. Internalization of native rat SP-A was compared with that of recombinant rat and human SP-A isolated from a patient with alveolar proteinosis. All SP-A species were conjugated with colloidal gold for visualization by electron microscopy. The gold conjugates were biologically active, as demonstrated by inhibition of phospholipid secretion from alveolar Type II cells. The SP-A-gold conjugates were internalized to lamellar bodies (LB) via the endosomal system, which included both electron-lucent and -dense multivesicular bodies. Labeling of LB was time dependent, and after 7 hr 30-40% of these organelles were labeled. Alkylation of SP-A greatly reduced internalization, as did an excess of non-conjugated SP-A. No qualitative differences in uptake were observed with the three forms of SP-A. The percent of labeled LB was similar (30-40%) after 7 hr of internalization with the three species of SP-A. The recombinant SP-A produced using a baculovirus vector lacked hydroxyproline and had an altered oligosaccharide, but these features did not affect its internalization or the rate of LB labeling. Internalization of the gold-conjugated SP-A and endocytosis of the fluid-phase marker Lucifer Yellow were related to the shape of Type II cells. Both uptake of SP-A, which is receptor mediated, and fluid-phase endocytosis were found to be less active in the flattened than in the rounded cells. Therefore, cell shape and hence cytoskeletal organization may play an important role in SP-A recycling. However, it is possible that both morphology and decreased endocytosis are independent manifestations related to the loss of differentiated function of cultured Type II cells.

scholarly journals Role of Phosphocholine Cytidylyltransferase α in Lung Development

2006 ◽  
Vol 27 (3) ◽  
pp. 975-982 ◽  
Author(s):  
Yong Tian ◽  
Ruobing Zhou ◽  
Jerold E. Rehg ◽  
Suzanne Jackowski
Keyword(s):  
Epithelial Cell ◽  
Lung Development ◽  
Cultured Cells ◽  
Lung Surfactant ◽  
Surfactant Protein ◽  
Alveolar Type ◽  
Type I ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lamellar Bodies

ABSTRACT Lung development depends upon the differentiation and expansion of a variety of specialized epithelial cell types, including distal type I and type II pneumocytes in the late term. Previous studies have shown a strict dependence on the choline cytidylyltransferase α isoform (CCTα) to mediate membrane phospholipid formation in cultured cells and during preimplantation embryogenesis. CCTα expression is highest in lung, and there has long been speculation about its precise role, due to the dual requirement for phospholipid in proliferating cell membranes and for lung surfactant production from alveolar type II cells. We investigated the function of CCTα in lung development, using an inducible, epithelial cell-specific CCTα knockout mouse line. Deletion of CCTα beginning at embryonic day 7.5 did not restrict lung development but resulted in severe respiratory failure at birth. Alveolar lavage and lung lipid analyses showed significant decreases in the major surfactant phospholipid, dipalmitoyl-phosphatidylcholine. The fatty acids destined for the surfactant phospholipid were redirected to an expanded triglyceride pool. Transcripts encoding type II cell-specific markers were expressed in the knockout mice, indicating the expected progression of differentiation in lung epithelia. However, surfactant protein levels were reduced, with the exception of that for surfactant protein B, which was elevated. Ultrastructural analysis of the type II cells showed Golgi complex abnormalities and aberrant lamellar bodies, which deliver surfactant lipid and protein to the alveolar lumen. Thus, CCTα was not required for the proliferation or differentiation of lung epithelia but was essential for the secretory component of phospholipid synthesis and critical for the proper formation of lamellar bodies and surfactant protein homeostasis.

Regulation of surfactant secretion in alveolar type II cells

2007 ◽  
Vol 293 (2) ◽  
pp. L259-L271 ◽  
Author(s):  
Alexandra V. Andreeva ◽  
Mikhail A. Kutuzov ◽  
Tatyana A. Voyno-Yasenetskaya
Keyword(s):  
Molecular Mechanisms ◽  
Lung Surfactant ◽  
Apical Plasma Membrane ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lamellar Bodies ◽  
Alveolar Type Ii Cells ◽  
Air Liquid Interface ◽  
Recycled Material

Molecular mechanisms of surfactant delivery to the air/liquid interface in the lung, which is crucial to lower the surface tension, have been studied for more than two decades. Lung surfactant is synthesized in the alveolar type II cells. Its delivery to the cell surface is preceded by surfactant component synthesis, packaging into specialized organelles termed lamellar bodies, delivery to the apical plasma membrane and fusion. Secreted surfactant undergoes reuptake, intracellular processing, and finally resecretion of recycled material. This review focuses on the mechanisms of delivery of surfactant components to and their secretion from lamellar bodies. Lamellar bodies–independent secretion is also considered. Signal transduction pathways involved in regulation of these processes are discussed as well as disorders associated with their malfunction.

Confocal imaging of time-dependent internalization and localization of NBD-PC in intact rat lungs

1994 ◽  
Vol 266 (6) ◽  
pp. L713-L721 ◽  
Author(s):  
M. R. Chinoy ◽  
A. B. Fisher ◽  
H. Shuman
Keyword(s):  
Protein A ◽  
Surfactant Protein ◽  
Airway Epithelial Cells ◽  
Confocal Imaging ◽  
Time Dependent ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lamellar Bodies ◽  
Adult Rat

Morphological localization of a fluorescent analogue of phosphatidylcholine (PC), C12-NBD-PC ([1-palmitoyl-2-[12-(7-nitro-2-1,3-benzoxadiazol-4-yl) amino] dodecanoyl]PC) was evaluated in adult rat lung. Fluorescence was observed at various times after intratracheal liposome instillation. Fluorometric analysis of C12-NBD-PC in lung homogenate showed an initial rapid uptake followed by a more gradual uptake phase. Microscopic observation of fixed lung slices showed C12-NBD-PC uptake in alveolar type II cells (identified by surfactant protein A and Maclura pomifera agglutinin colocalization), nonciliated distal airway epithelial cells, but not in subepithelial tissue. Macrophages showed an initial uptake that did not increase with time. Fluorescence measured in type II cells was diffuse at 5 min, became punctate, and intensified gradually by 2 h. Isolation of type II cells and lamellar bodies from instilled lung confirmed localization of fluorophore in them. This study shows time-dependent uptake of C12-NBD-PC by type II and bronchiolar cells and preferential internalization to lung lamellar bodies as previously described for surfactant PC.

Surfactant protein-A plays an important role in lung surfactant clearance: evidence using the surfactant protein-A gene-targeted mouse

2008 ◽  
Vol 294 (2) ◽  
pp. L325-L333 ◽  
Author(s):  
Sandra R. Bates ◽  
Chandra Dodia ◽  
Jian-Qin Tao ◽  
Aron B. Fisher
Keyword(s):  
Lung Surfactant ◽  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Type Ii Cells ◽  
Type Ii ◽  
Rat Lung ◽  
Coated Pits ◽  
Receptor Mediated Endocytosis ◽  
Mouse Lungs

Previous studies with the isolated perfused rat lung showed that both clathrin- and actin-mediated pathways are responsible for endocytosis of dipalmitoylphosphatidylcholine (DPPC)-labeled liposomes by granular pneumocytes in the intact lung. Using surfactant protein-A (SP-A) gene-targeted mice, we examined the uptake of [3H]DPPC liposomes by isolated mouse lungs under basal and secretagogue-stimulated conditions. Unilamellar liposomes composed of [3H]DPPC: phosphatidylcholine:cholesterol:egg phosphatidylglycerol (10:5:3:2 mol fraction) were instilled into the trachea of anesthetized mice, and the lungs were perfused (2 h). Uptake was calculated as percentage of instilled disintegrations per minute in the postlavaged lung. Amantadine, an inhibitor of clathrin and, thus, receptor-mediated endocytosis via clathrin-coated pits, decreased basal [3H]DPPC uptake by 70% in SP-A +/+ but only by 20% in SP-A −/− lung, data compatible with an SP-A/receptor-regulated lipid clearance pathway in the SP-A +/+ mice. The nonclathrin, actin-dependent process was low in the SP-A +/+ lung but accounted for 55% of liposome endocytosis in the SP-A −/− mouse. With secretagogue (8-bromoadenosine 3′,5′-cyclic monophosphate) treatment, both clathrin- and actin-dependent lipid clearance were elevated in the SP-A +/+ lungs while neither pathway responded in the SP-A −/− lungs. Binding of iodinated SP-A to type II cells isolated from both genotypes of mice was similar indicating a normal SP-A receptor status in the SP-A −/− lung. Inclusion of SP-A with instilled liposomes served to “rescue” the SP-A −/− lungs by reestablishing secretagogue-dependent enhancement of liposome uptake. These data are compatible with a major role for receptor-mediated endocytosis of DPPC by granular pneumocytes, a process critically dependent on SP-A.

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