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.

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)

Surface-expressed lamellar body membrane is recycled to lamellar bodies

2000 ◽  
Vol 279 (4) ◽  
pp. L631-L640 ◽  
Author(s):  
S. Schaller-Bals ◽  
S. R. Bates ◽  
K. Notarfrancesco ◽  
J. Q. Tao ◽  
A. B. Fisher ◽  
...  
Keyword(s):  
Cell Surface ◽  
Lamellar Body ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lamellar Bodies ◽  
Double Labeling ◽  
Rat Lungs ◽  
Type Ii Pneumocytes ◽  
Membrane Components

Monoclonal antibody (MAb) 3C9, an antibody generated to the lamellar body of rat lung type II pneumocytes, specifically labels the luminal face of the lamellar body membrane. To follow the retrieval of lamellar body membrane from the cell surface in these cells, MAb 3C9 was instilled into rat lungs. In vivo, it was endocytosed by type II cells but not by other lung cells. In type II cells that were isolated from rat lungs by elastase digestion and cultured on plastic for 24 h, MAb 3C9 first bound to the cell surface, then was found in endosomes, vesicular structures, and multivesicular bodies and, finally, clustered on the luminal face of lamellar body membranes. The amount internalized reached a plateau after 1.5 h of incubation and was stimulated with the secretagogue ATP. In double-labeling experiments, internalized MAb 3C9 did not completely colocalize with NBD-PC liposomes or the nonspecific endocytic marker TMA-DPH, suggesting that lamellar body membrane is retrieved back to existing lamellar bodies by a pathway different from that of bulk membrane and may be one pathway for surfactant endocytosis. The lamellar body membrane components are retrieved as subunits that are redistributed among the preexisting lamellar bodies in the cell.

Lamellar body membrane turnover is stimulated by secretagogues

2000 ◽  
Vol 278 (3) ◽  
pp. L443-L452 ◽  
Author(s):  
Sandra R. Bates ◽  
Jian-Qin Tao ◽  
Susanne Schaller ◽  
Aron B. Fisher ◽  
Henry Shuman
Keyword(s):  
Protein A ◽  
Lamellar Body ◽  
Surfactant Protein ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Dependent Manner ◽  
Type Ii ◽  
Lamellar Bodies ◽  
Membrane Turnover ◽  
Concentration Dependent Manner

Lamellar bodies are specialized cellular organelles used for storage of surfactant by alveolar type II cells of the lung. We utilized monoclonal antibody (MAb) 3C9, which recognizes an integral lamellar body-limiting membrane protein of 180 kDa, to follow lamellar body trafficking. 125I-labeled MAb 3C9 bound to the surface of type II cells and was internalized by the cells in a time- and concentration-dependent manner that was inhibitable by excess unlabeled antibody. The internalized antibody remained undegraded over a 4-h time period. The L2 rat lung cell line that does not have lamellar bodies did not bind iodinated 3C9. Exposure of type II cells to the secretagogues ATP, phorbol 12-myristate 13-acetate, and cAMP resulted in a 1.5- to 2-fold enhancement of binding and uptake of MAb 3C9. Calphostin C inhibited phorbol 12-myristate 13-acetate-stimulated phospholipid secretion and also reduced binding and uptake of MAb 3C9 by type II cells. Treatment of type II cells with phenylarsine oxide to obstruct clathrin-mediated endocytosis had no effect on the internalization of MAb 3C9 while markedly blocking the uptake of surfactant protein A and transferrin. An actin-mediated process was important for lamellar body membrane uptake because incubation with cytochalasin D partially inhibited MAb 3C9 incorporation by type II cells. These studies are compatible with enhanced lamellar body membrane turnover associated with surfactant secretion and indicate that this process can be monitored by the trafficking of the antigen reporter MAb 3C9.

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.

scholarly journals Role of the PI3-kinase signaling pathway in trafficking of the surfactant protein A receptor P63 (CKAP4) on type II pneumocytes

2010 ◽  
Vol 299 (6) ◽  
pp. L794-L807 ◽  
Author(s):  
Altaf S. Kazi ◽  
Jian-Qin Tao ◽  
Sheldon I. Feinstein ◽  
Li Zhang ◽  
Aron B. Fisher ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Protein A ◽  
Surfactant Protein ◽  
Pi3 Kinase ◽  
Surfactant Protein A ◽  
Type Ii Cells ◽  
Type Ii ◽  
Type Ii Pneumocytes ◽  
Kinase Signaling Pathway ◽  
Stimulation Of

Surfactant protein A (SP-A) plays an important role in the maintenance of lung lipid homeostasis. Previously, an SP-A receptor, P63 (CKAP4), on type II pneumocyte plasma membranes (PM) was identified by chemical cross-linking techniques. An antibody to P63 blocked the specific binding of SP-A to pneumocytes and the ability of SP-A to regulate surfactant secretion. The current report shows that another biological activity of SP-A, the stimulation of surfactant uptake by pneumocytes, is inhibited by P63 antibody. cAMP exposure resulted in enrichment of P63 on the cell surface as shown by stimulation of SP-A binding, enhanced association of labeled P63 antibody with type II cells, and promotion of SP-A-mediated liposome uptake, all of which were inhibited by competing P63 antibody. Incubation of A549 and type II cells with SP-A also increased P63 localization on the PM. The phosphatidylinositol 3-kinase (PI3-kinase) signaling pathway was explored as a mechanism for the transport of this endoplasmic reticulum (ER)-resident protein to the PM. Treatment with LY-294002, an inhibitor of the PI3-kinase pathway, prevented the SP-A-induced PM enrichment of P63. Exposure of pneumocytes to SP-A or cAMP activated Akt (PKB). Blocking either PI3-kinase or Akt altered SP-A-mediated lipid turnover. The data demonstrate an important role for the PI3-kinase-Akt pathway in intracellular transport of P63. The results add to the growing body of evidence that P63 is critical for SP-A receptor-mediated interactions with type II pneumocytes and the resultant regulation of surfactant turnover.

Uptake of pulmonary surfactant protein C into adult rat lung lamellar bodies

1993 ◽  
Vol 74 (3) ◽  
pp. 1005-1011 ◽  
Author(s):  
R. A. Pinto ◽  
J. R. Wright ◽  
D. Lesikar ◽  
B. J. Benson ◽  
J. A. Clements
Keyword(s):  
Protein C ◽  
Lamellar Body ◽  
Surfactant Protein ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lamellar Bodies ◽  
Adult Rats ◽  
Alveolar Air ◽  
Surfactant Protein C ◽  
Air Space

Previous studies have provided evidence that a large proportion of secreted surfactant lipids is taken up from the alveolar air space by type II cells, incorporated into lamellar bodies, and resecreted. Our goal was to characterize the clearance of exogenously administered recombinant surfactant protein C (SP-C) and to determine if SP-C is taken up by type II cells and incorporated into lamellar bodies. SP-C was radiolabeled by alkylation with [3H]iodoacetic acid and retained its ability to enhance phospholipid adsorption to an air-liquid interface. A mixture of 100 micrograms phospholipid radiolabeled with [14C]dipalmitoylphosphatidylcholine and 10 micrograms SP-C was instilled into the lungs of spontaneously breathing anesthetized adult rats. At later times, the lungs were lavaged and subcellular organelles were isolated. The radioactivity of both phospholipids and SP-C (expressed as disintegrations per minute per microgram phospholipid) in lamellar body fractions increased up to 4 h postinstillation and began to decline after approximately 4 h. The results of this study suggest that SP-C and dipalmitoylphosphatidylcholine are taken up promptly from the alveolar air space and are incorporated into lamellar bodies with time courses that do not differ greatly.

scholarly journals Role of P63 (CKAP4) in binding of surfactant protein-A to type II pneumocytes

2008 ◽  
Vol 295 (4) ◽  
pp. L658-L669 ◽  
Author(s):  
Sandra R. Bates ◽  
Altaf S. Kazi ◽  
Jian-Qin Tao ◽  
Kevin J. Yu ◽  
Daniel S. Gonder ◽  
...  
Keyword(s):  
Specific Binding ◽  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Type Ii Cells ◽  
Type Ii ◽  
Calcium Dependent ◽  
Type Ii Pneumocytes ◽  
Surfactant Secretion

We have recently described a putative receptor for lung surfactant protein-A (SP-A) on rat type II pneumocytes. The receptor, P63, is a 63-kDa type II transmembrane protein. Coincubation of type II cells with P63 antibody (Ab) reversed the inhibitory effect of SP-A on secretagogue-stimulated surfactant secretion from type II cells. To further characterize SP-A interactions with P63, we expressed recombinant P63 protein in Escherichia coli and generated antibodies to P63. Immunogold electron microscopy confirmed endoplasmic reticulum and plasma membrane localization of P63 in type II cells with prominent labeling of microvilli. Binding characteristics of iodinated SP-A to type II cells in the presence of P63 Ab were determined. Binding (4°C, 1 h) of 125I-SP-A to type II cells demonstrated both specific (calcium-dependent) and nonspecific (calcium-independent) components. Ab to P63 protein blocked the specific binding of 125I-SP-A to type II cells and did not change the nonspecific SP-A association. A549 cells, a pneumocyte model cell line, expressed substantial levels of P63 and demonstrated specific binding of 125I-SP-A that was inhibited by the P63 Ab. The secretagogue (cAMP)-stimulated increase in calcium-dependent binding of SP-A to type II cells was blocked by the presence of P63 Ab. Transfection of type II cells with small interfering RNA to P63 reduced P63 protein expression, attenuated P63-specific SP-A binding, and reversed the ability of SP-A to prevent surfactant secretion from the cells. Our results further substantiate the role of P63 as an SP-A receptor protein localized on the surface of lung type II cells.

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.

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).

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