Internalized SP-A and lipid are differentially resecreted by type II pneumocytes

2000 ◽  
Vol 278 (3) ◽  
pp. L580-L590 ◽  
Author(s):  
Heide Wissel ◽  
Stefan Zastrow ◽  
Ekkehard Richter ◽  
Paul A. Stevens
Keyword(s):  
Laser Scanning ◽  
Protein A ◽  
Fluorescein Isothiocyanate ◽  
Surfactant Protein ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Type Ii Cells ◽  
Type Ii ◽  
Type Ii Pneumocytes

Biochemical and morphological assays were developed to study surfactant protein A (SP-A) and lipid resecretion kinetics by isolated type II cells in vitro. After a 10-min uptake period with SP-A (3 μg/106 cells) in combination with liposomes (60 μg/106 cells), the cells were allowed to resecrete. After 5 min of resecretion, only 21.7 ± 4.6% of the internalized SP-A remained intracellularly compared with 54 ± 2.9% of the lipids. Extracellular SP-A present during the resecretion period partially inhibited resecretion (SP-A, 36% at 5 min; lipid, ∼16% at 5 min). Lipid resecretion was also dependent on the SP-A concentration present during the uptake period. Although, as shown by confocal laser scanning microscopy, after a 10-min uptake period at 37°C, most of the fluorescein isothiocyanate-labeled SP-A and rhodamine-phosphatidylethanolamine-labeled lipids colocalized within the cells, after an additional 10 min of resecretion, both the strength of the fluorescence signals and the extent of colocalization had markedly decreased. These data indicate that internalized lipid and SP-A can be resecreted rapidly by type II cells, likely via different pathways.

Surfactant protein A and lipid are internalized via the coated-pit pathway by type II pneumocytes

2001 ◽  
Vol 280 (1) ◽  
pp. L141-L151 ◽  
Author(s):  
Paul A. Stevens ◽  
Heide Wissel ◽  
Stefan Zastrow ◽  
Daniela Sieger ◽  
Klaus-Peter Zimmer
Keyword(s):  
Laser Scanning ◽  
Protein A ◽  
Early Time ◽  
Surfactant Protein ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lipid Uptake

Surfactant protein (SP) A and SP-A-mediated lipid uptake by isolated type II cells were investigated with biochemical and morphological methods. Inhibition of coated-pit function by potassium depletion severely reduced both SP-A and SP-A-mediated lipid internalization. Lipid uptake in the absence of SP-A was not affected. With confocal laser scanning microscopy and immunoelectron microscopy, SP-A and lipid predominantly (60%) colocalized in intracellular vesicles carrying early endosomal markers (EEA1) 5 min after endocytosis but were negative for the late endosomal or lysosomal marker LAMP-1. As estimated by subcellular fractionation, at this time point, 23% of the internalized SP-A and 45% of internalized lipid were localized within light (<0.38 M sucrose) fractions, which contain lamellar bodies and are positive for EEA1. The remaining label was predominantly found within EEA1-positive and plasma membrane-containing subfractions (≥1 M sucrose). We suggest that in isolated type II cells in vitro, SP-A and lipid are taken up together via the coated-pit pathway and that at early time points, both components reside in the same early endosomal compartment.

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.

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.

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 The Amelogenin-Derived Peptide TVH-19 Promotes Dentinal Tubule Occlusion and Mineralization

Polymers ◽  
2021 ◽  
Vol 13 (15) ◽  
pp. 2473
Author(s):  
Xiu Peng ◽  
Sili Han ◽  
Kun Wang ◽  
Longjiang Ding ◽  
Zhenqi Liu ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Fluorescein Isothiocyanate ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Dentinal Tubule ◽  
X Ray ◽  
Electron Microscopy Analysis ◽  
Tubule Occlusion ◽  
Demineralized Dentin

In this study, the amelogenin-derived peptide, TVH-19, which has been confirmed to promote mineralization, was evaluated to derive its potential to induce dentinal tubule occlusion. The binding capability of fluorescein isothiocyanate (FITC)-labeled TVH-19 to the demineralized dentin surface was analyzed by confocal laser scanning microscopy (CLSM). Additionally, the sealing function of the peptide was studied through the remineralization of demineralized dentin in vitro. The adsorption results showed that TVH-19 could bind to the hydroxyapatite and demineralized dentin surfaces, especially to periodontal dentin. Scanning electron microscopy analysis further revealed that TVH-19 created mineral precipitates. The plugging rate in the TVH-19 group was higher than that in the PBS group. Moreover, energy-dispersive X-ray spectroscopy (EDX) results indicated that the calcium/phosphorus (Ca/P) ratio of the new minerals induced by TVH-19 was close to that of the hydroxyapatite. Attenuated total internal reflection-Fourier transform infrared (ATR-FTIR) spectrometry and X-ray diffraction (XRD) results indicated that the hydroxyapatite crystals formed via remineralization elongated the axial growth and closely resembled the natural dentin components. These findings indicate that TVH-19 can effectively promote dentin sealing by binding to the periodontal dentin, promoting mineral deposition, and reducing the space between the dentin tubules.

scholarly journals Growth-Inhibitory Effect of Heparin on Babesia Parasites

2004 ◽  
Vol 48 (1) ◽  
pp. 236-241 ◽  
Author(s):  
Sabine Bork ◽  
Naoaki Yokoyama ◽  
Yuzuru Ikehara ◽  
Sanjay Kumar ◽  
Chihiro Sugimoto ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Fluorescein Isothiocyanate ◽  
Inhibitory Effect ◽  
Laser Scanning Microscopy ◽  
Babesia Bovis ◽  
Confocal Laser ◽  
Intracellular Parasites ◽  
Low Concentrations

ABSTRACT We examined the inhibitory effects of three heparins on the growth of Babesia parasites. The multiplication of Babesia bovis, B. bigemina, B. equi, and B. caballi in in vitro cultures and that of B. microti in vivo were significantly inhibited in the presence of heparins, as determined by light microscopy. Treatment with various concentrations of heparin showed complete clearance of the intracellular parasites. Interestingly, a higher percentage of abnormally multidividing B. bovis parasites was observed in the presence of low concentrations of heparin. Furthermore, fluorescein isothiocyanate-labeled heparin was preferably found on the surfaces of extracellular merozoites, as detected by confocal laser scanning microscopy. These findings indicate that the heparin covers the surfaces of babesial merozoites and inhibits their subsequent invasion of erythrocytes.

scholarly journals Lectin activity of the major surfactant protein (SP-A) may participate in, but is not required for, binding to rat type II cells.

1992 ◽  
Vol 40 (5) ◽  
pp. 643-649 ◽  
Author(s):  
T Thorkelsson ◽  
G M Ciraolo ◽  
G F Ross ◽  
J A Whitsett ◽  
R E Morris
Keyword(s):  
Protein A ◽  
Complex Mixture ◽  
Surfactant Protein ◽  
Structural Features ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lectin Activity ◽  
Type Ii Cell

Pulmonary surfactant is a complex mixture of lipids and proteins, of which surfactant protein A (SP-A) is the most abundant glycoprotein. The SP-A molecule has several distinct structural features that include a lectin-like domain, sharing structural features with other mammalian lectins. We have tested the hypothesis that lectin activity of the SP-A molecule is required for the binding to its receptor on the surface of alveolar Type II cells. By using colloidal gold immunocytochemistry in conjunction with electron microscopy, we evaluated the ability of mannosylated proteins to inhibit canine SP-A binding to rat Type II cells in vitro. After preincubation of SP-A with the mannosylated protein horse-radish peroxidase (HRP), SP-A was incubated with isolated filter-grown Type II cells. HRP did not alter the binding of SP-A to the Type II cell surface. Evidence that SP-A did bind to HRP was shown by coincident observation of gold-labeled SP-A and HRP precipitates. These results provide visual evidence that the lectin activity associated with SP-A is not required for its binding to receptor on rat alveolar Type II epithelial cells.

Effect of surfactant protein A on granular pneumocyte surfactant secretion in vitro

2003 ◽  
Vol 285 (5) ◽  
pp. L1055-L1065 ◽  
Author(s):  
Sandra R. Bates ◽  
Jian-Qin Tao ◽  
Kathleen Notarfrancesco ◽  
Kristine DeBolt ◽  
Henry Shuman ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Type Ii Cells ◽  
Type Ii ◽  
Cell Association ◽  
Surfactant Secretion

Surfactant secretion by lung type II cells occurs when lamellar bodies (LBs) fuse with the plasma membrane and surfactant is released into the alveolar lumen. Surfactant protein A (SP-A) blocks secretagogue-stimulated phospholipid (PL) release, even in the presence of surfactant-like lipid. The mechanism of action is not clear. We have shown previously that an antibody to LB membranes (MAb 3C9) can be used to measure LB membrane trafficking. Although the ATP-stimulated secretion of PL was blocked by SP-A, the cell association of iodinated MAb 3C9 was not altered, indicating no effect on LB movement. FM1-43 is a hydrophobic dye used to monitor the formation of fusion pores. After secretagogue exposure, the threefold enhancement of the number of FM1-43 fluorescent LBs (per 100 cells) was not altered by the presence of SP-A. Finally, there was no evidence of a large PL pool retained on the cell surface through interaction with SP-A. Thus SP-A exposure does not affect these stages in the surfactant secretory pathway of type II cells.

scholarly journals Identification of a new surfactant protein A binding protein at the cell membrane of rat type II pneumocytes

1995 ◽  
Vol 308 (1) ◽  
pp. 77-81 ◽  
Author(s):  
P A Stevens ◽  
H Wissel ◽  
D Sieger ◽  
V Meienreis-Sudau ◽  
B Rüstow
Keyword(s):  
Cell Membrane ◽  
Binding Protein ◽  
Protein A ◽  
Surfactant Protein ◽  
Type Ii ◽  
Type Ii Pneumocyte ◽  
Reducing Conditions ◽  
Type Ii Pneumocytes ◽  
Cell Membrane Protein

Antibodies against a type II pneumocyte component were developed by an auto-anti-idiotypic approach using surfactant-associated protein A (SP-A) as the immunogen. The antibodies recognize an SP-A-binding protein (approximately 170-200 kDa under non-reducing conditions, 55 kDa under reducing conditions) on the cell membrane of rat type II pneumocytes. One of the antibodies competes with SP-A for binding to type II cells. In immunization assays in vitro, with this antibody as the antigen, anti-SP-A antibodies have been generated. The SP-A-binding cell membrane protein recognized by this auto-anti-idiotypic antibody may be involved in the interaction of extracellular SP-A with the type II pneumocyte and may play a role in the regulation of alveolar surfactant metabolism.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

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