Ultrastructural Localization of NA+-K+ -Atpase in Rabbit Cornea Using 5-Nitroindoxyl Phosphate as Substrate

1977 ◽  
Vol 35 ◽  
pp. 444-445
Author(s):  
K. C. Tsou ◽  
Mahin Khatami
Keyword(s):  
Endothelial Cells ◽  
Comparative Study ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Endothelial Damage ◽  
Previous Method ◽  
New Method ◽  
Rabbit Cornea ◽  
Fixed Tissue ◽  
Nitrophenyl Phosphate

Recently, Leunberger and Novikoff (1) have shown that the endothelial cells are the primary sites of Na+-K+-ATPase, a key mechanism in the control of hydration of cornea. Since this enzyme would be a useful marker for our study of endothelial damage by CO2 laser (2), we have undertaken a comparative study for the localization of this enzyme at ultrastructural level with different methods. In the course of this work we developed a new method with the use of 5-nitroindoxyl phosphate as a synthetic substrate. This new substrate, like p-nitrophenyl phosphate (3), meets the requirements for the demonstration of the K+ activated step of dephosphorylation of ATPase. In addition, it can be used with glutaraldehyde fixed tissue, thereby offering an important advantage over the previous method (3).Rabbit corneas were freshly excised under pentobarbitol anesthesia.

scholarly journals TRANSPORT ADENOSINE TRIPHOSPHATASE CYTOCHEMISTRY I. BIOCHEMICAL CHARACTERIZATION OF A CYTOCHEMICAL MEDIUM FOR THE ULTRASTRUCTURAL LOCALIZATION OF OUABAIN-SENSITIVE, POTASSIUM-DEPENDENT PHOSPHATASE ACTIVITY IN THE AVIAN SALT GLAND

1972 ◽  
Vol 20 (1) ◽  
pp. 13-22 ◽  
Author(s):  
STEPHEN A. ERNST
Keyword(s):  
Enzymatic Activity ◽  
Adenosine Triphosphatase ◽  
Biochemical Characterization ◽  
Metal Salt ◽  
Salt Gland ◽  
Ultrastructural Localization ◽  
Defined Medium ◽  
Fixed Tissue ◽  
Fold Reduction ◽  
Nitrophenyl Phosphate

The optimal kinetic parameters for the K-dependent, ouabain-sensitive hydrolysis of p-nitrophenyl phosphate by K-nitrophenyl phosphatase, under conditions closely approximating those employed for cytochemistry, were determined in the avian salt gland as a necessary prerequisite for the ultrastructural localization of the enzyme. The enzyme was characterized in 50-µ cryostat sections of paraformaldehyde-fixed tissue, incubated at room temperature in a medium containing 5 mM nitrophenyl phosphate, 10 mM MgCl2, 20 mM SrCl2 and 100 mM Tris-HCl buffer (pH 9.0), either with or without 10 mM KCl. For comparison, parallel assays were conducted in the absence of Sr, the heavy metal salt used to precipitate phosphate for cytochemistry. Enzymatic activity was determined by measuring spectrophotometrically the amount of nitrophenol hydrolyzed. In this system, Sr acts as a pure noncompetitive inhibitor of the enzyme, causing 50% inhibition at 3 mM and 87% at 20 mM. The Km for the enzyme is 4.5 mM. Sr (20 mM) causes an 8-fold reduction in the apparent affinity of the enzyme for Mg but has little effect on K affinity. The sensitivity of the enzyme to ouabain is decreased 50-fold in the presence of 20 mM Sr. The relationship of this enzymatic activity to Na-K-activated adenosine triphosphatase and the application of this defined medium to transport adenosine triphosphatase cytochemistry are discussed.

TRANSPORT ADENOSINE TRIPHOSPHATASE CYTOCHEMISTRY II. CYTOCHEMICAL LOCALIZATION OF OUABAIN-SENSITIVE, POTASSIUM-DEPENDENT PHOSPHATASE ACTIVITY IN THE SECRETORY EPITHELIUM OF THE AVIAN SALT GLAND

1972 ◽  
Vol 20 (1) ◽  
pp. 23-38 ◽  
Author(s):  
STEPHEN A. ERNST
Keyword(s):  
Phosphatase Activity ◽  
Adenosine Triphosphatase ◽  
Plasma Membranes ◽  
Salt Gland ◽  
Ultrastructural Localization ◽  
Defined Medium ◽  
Secretory Cells ◽  
Cytochemical Localization ◽  
Fixed Tissue ◽  
Nitrophenyl Phosphate

A cytochemical procedure is described for the ultrastructural localization of K-dependent, ouabain-sensitive nitrophenyl phosphatase activity in avian salt gland. Cryostat sections (50 µ) of paraformaldehyde-fixed tissue were incubated in a kinetically defined medium containing: 5 mM p-nitrophenyl phosphate, 10 mM MgCl2, 10 mM KCl, 100 mM Tris-HCl buffer (pH 8.5 or 9.0) and 20 mM SrCl2 to precipitate hydrolyzed phosphate. After incubation at room temperature, the sections were treated with Pb(NO3)2 to convert SrPi to PbPi precipitates for visualization in the electron microscope. Reaction product was localized on the cytoplasmic side of the secretory cell lateral and basal plasma membranes. Little, if any, reaction product was associated with the apical surfaces of the secretory cells or with endothelial surfaces of capillaries. Appropriate control experiments indicated that deposition of reaction product was dependent on Mg and K and was sensitive to ouabain. Furthermore, nonenzymatic hydrolysis of nitrophenyl phosphate did not occur in the medium, and deposition of artifactually produced precipitates did not resemble deposition of enzymatically produced precipitates. The relationship of this localization to transport adenosine triphosphatase cytochemistry is discussed, and the physiologic implications of the localization for tracing the route of active Na transport in the salt gland are considered.

Ultrastructural Localization of Antigens by Capillary Action Immunocytochemistry

1996 ◽  
Vol 54 ◽  
pp. 40-41
Author(s):  
László G. Kömüves
Keyword(s):  
San Francisco ◽  
Colloidal Gold ◽  
Specific Binding ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Uranyl Acetate ◽  
Adhesive Tape ◽  
Distilled Water ◽  
Capillary Action ◽  
Rabbit Igg

Light microscopic immunohistochemistry based on the principle of capillary action staining is a widely used method to localize antigens. Capillary action immunostaining, however, has not been tested or applied to detect antigens at the ultrastructural level. The aim of this work was to establish a capillary action staining method for localization of intracellular antigens, using colloidal gold probes.Post-embedding capillary action immunocytochemistry was used to detect maternal IgG in the small intestine of newborn suckling piglets. Pieces of the jejunum of newborn piglets suckled for 12 h were fixed and embedded into LR White resin. Sections on nickel grids were secured on a capillary action glass slide (100 μm wide capillary gap, Bio-Tek Solutions, Santa Barbara CA, distributed by CMS, Houston, TX) by double sided adhesive tape. Immunolabeling was performed by applying reagents over the grids using capillary action and removing reagents by blotting on filter paper. Reagents for capillary action staining were from Biomeda (Foster City, CA). The following steps were performed: 1) wet the surface of the sections with automation buffer twice, 5 min each; 2) block non-specific binding sites with tissue conditioner, 10 min; 3) apply first antibody (affinity-purified rabbit anti-porcine IgG, Sigma Chem. Co., St. Louis, MO), diluted in probe diluent, 1 hour; 4) wash with automation buffer three times, 5 min each; 5) apply gold probe (goat anti-rabbit IgG conjugated to 10 nm colloidal gold, Zymed Laboratories, South San Francisco, CA) diluted in probe diluent, 30 min; 6) wash with automation buffer three times, 5 min each; 7) post-fix with 5% glutaraldehyde in PBS for 10 min; 8) wash with PBS twice, 5 min each; 9) contrast with 1% OSO4 in PBS for 15 min; 10) wash with PBS followed by distilled water for5 min each; 11) stain with 2% uranyl acetate for 10 min; 12) stain with lead citrate for 2 min; 13) wash with distilled water three times, 1 min each. The glass slides were separated, and the grids were air-dried, then removed from the adhesive tape. The following controls were used to ensure the specificity of labeling: i) omission of the first antibody; ii) normal rabbit IgG in lieu of first antibody; iii) rabbit anti-porcine IgG absorbed with porcine IgG.

An Electron Microscopic Study of Platelet Thrombus Formation in the Rabbit with Particular Regard to 5-Hydroxytryptamine Release

1967 ◽  
Vol 18 (03/04) ◽  
pp. 592-604 ◽  
Author(s):  
H. R Baumgartner ◽  
J. P Tranzer ◽  
A Studer
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Vessel Wall ◽  
Thrombus Formation ◽  
Endothelial Damage ◽  
Electron Microscopic ◽  
Rabbit Ear ◽  
Basement Lamina ◽  
Platelet Thrombus

SummaryElectron microscopic and histologic examination of rabbit ear vein segments 4 and 30 min after slight endothelial damage have yielded the following findings :1. Platelets do not adhere to damaged endothelial cells.2. If the vessel wall is denuded of the whole endothelial cell, platelets adhere to the intimai basement lamina as do endothelial cells.3. The distance between adherent platelets as well as endothelial cells and intimai basement lamina measures 10 to 20 mµ, whereas the distance between aggregated platelets is 30 to 60 mµ.4. 5-hydroxytryptamine (5-HT) is released from platelets during viscous metamorphosis at least in part as 5-HT organelles.It should be noted that the presence of collagen fibers is not necessary for platelet thrombus formation in vivo.

Biofabrication of Layered Membrane Systems by Using Human Hepatocytes and Endothelial Cells: A Comparative Study

2013 ◽  
Vol 2 (2) ◽  
pp. 109-118 ◽  
Author(s):  
Simona Salerno ◽  
Sabrina Morelli ◽  
Enrico Drioli ◽  
Augustinus Bader ◽  
Loredana Bartolo
Keyword(s):  
Endothelial Cells ◽  
Comparative Study ◽  
Human Hepatocytes ◽  
Membrane Systems

Morphilogy and Ultrastructure of in Vitro Cultures of Aortic Endothelial Cells Interacting with Antibodies

1979 ◽  
Author(s):  
S. Korach ◽  
D. Ngo
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Vascular Endothelial Cells ◽  
Intercellular Junctions ◽  
Cell Types ◽  
Endothelial Damage ◽  
Antibody Concentration ◽  
High Yields ◽  
Scanning Em

Adult pig aortas, sectioned longitudinally, were incubated in 0.1% collagenase-PBS (15 mn, 37°C). Gentle scraping of the lumenal surface resulted in high yields (3-4 x 106 cell/aorta) of viable endothelial cells, essentially devoid of other cell types by morphological and immunochemical (F VIII-antigen) criteria. Confluent monolayers were incubated for various times (5 mn to 1 wk) with decomplemented rabbit antisera raised against pig endothelial cells. Changes in cell morphology appeared to depend on antibody concentration rather than on duration of contact with antiserum. High concentrations of antiserum (5 to 20%) led to cytoplasmic shredding, bulging of cells and extensive vacuolization, whereas at lower concentrations, cells appeared almost normal. Transmission EM studies by the indirect immunoperoxydase method showed antibodies reacting with unfixed cells to be distributed all over the upper cell surface, in the outer parts of intercellular junctions, and within numerous pinocytotic vesicles. Much weaker reactions could also be seen at the lower cell surface. When viewed under the Scanning EM, antiserum-treated endothelial cells also disclosed antibody concentration-dependent bulging and release of cells from their substrate. In vitro studies of gradual modifications of vascular endothelial cells acted upon by antibodies should provide a better understanding of the structural and biochemical processes underlying endothelial damage and detachment.

Maternal Anti-HPA-1a Antibodies Increase Endothelial Cell Apoptosis and Permeability

2021 ◽  
pp. 1-9
Author(s):  
Rima Dardik ◽  
Ophira Salomon
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Intracranial Hemorrhage ◽  
Cell Apoptosis ◽  
Endothelial Damage ◽  
Αvβ3 Integrin ◽  
Endothelial Cell Apoptosis ◽  
Vascular Beds ◽  
Alloimmune Thrombocytopenia

Intracranial hemorrhage (ICH) associated with fetal/neonatal alloimmune thrombocytopenia (FNAIT) is attributed mainly to endothelial damage caused by binding of maternal anti-HPA-1a antibodies to the αvβ3 integrin on endothelial cells (ECs). We examined the effect of anti-HPA-1a antibodies on EC function using 2 EC lines from different vascular beds, HMVEC of dermal origin and hCMEC/D3 of cerebral origin. Anti-HPA-1a sera significantly increased apoptosis in both HMVEC and hCMEC/D3 cells and permeability in hCMEC/D3 cells only. This increase in both apoptosis and permeability was significantly inhibited by a monoclonal anti-β3 antibody (SZ21) binding to the HPA-1a epitope. Our results indicate that (1) maternal anti-HPA-1a antibodies impair EC function by increasing apoptosis and permeability and (2) ECs from different vascular beds vary in their susceptibility to pathological effects elicited by maternal anti-HPA-1a antibodies on EC permeability. Examination of maternal anti-HPA-1a antibodies for their effect on EC permeability may predict potential ICH associated with FNAIT.

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