scholarly journals Cytochemical localization of hydrogen peroxide production in the rat uterus.

1984 ◽  
Vol 32 (6) ◽  
pp. 674-676 ◽  
Author(s):  
Y Ishikawa ◽  
K Hirai ◽  
K Ogawa
Keyword(s):  
Plasma Membrane ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Surface Epithelium ◽  
Apical Plasma Membrane ◽  
Rat Uterus ◽  
Cytochemical Localization ◽  
Dense Deposits ◽  
Nadph Oxidation ◽  
Endometrial Epithelium ◽  
Reduced Nicotinamide Adenine Dinucleotide

A reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H)-dependent H2O2-generating activity of the rat uterus was investigated both electron cytochemically and biochemically. We tried to cytochemically demonstrate H2O2 generation from the oxidation of reduced NADH or NADPH using the cerium method. NADPH oxidation resulted in electron-dense deposits on the apical plasma membrane covering the microvilli of the surface epithelium of the lightly fixed endometrium. In control specimens incubated in a medium from which substrate was omitted, no such deposits were observed. The reduction of ferricytochrome c due to NADH oxidation was spectrophotometrically detected in the lightly fixed uterus. Absorption at 550 nm increased with the addition of NADH, but not with that of NAD. The reaction was weakened by preheating and adversely affected by the addition of superoxide dismutase, but it was not inhibited by adding 50 mM sodium azide. These results suggest that a kind of NAD(P)H oxidase, generating H2O2 via superoxide formation, may possibly be present on the apical plasma membrane of the rat endometrial epithelium.

Cytoskeleton of intestinal goblet cells: role of actin filaments in baseline secretion

1990 ◽  
Vol 259 (6) ◽  
pp. G991-G997 ◽  
Author(s):  
M. G. Oliver ◽  
R. D. Specian
Keyword(s):  
Plasma Membrane ◽  
Goblet Cell ◽  
Actin Filaments ◽  
Cellular Response ◽  
Apical Cell ◽  
Goblet Cells ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Cytochemical Localization

Although microtubules appear necessary to maintain mucin granule transport in intestinal goblet cells, the role of microfilaments in mucus secretion is unknown. To determine the functional significance of microfilaments in goblet cell secretion, fluorescent cytochemistry of microfilaments and autoradiographic studies on granule movement were performed on rabbit intestinal goblet cells, with and without the actin depolymerizing agents, cytochalasin D (cyto D), and dihydro-cytochalasin B (dihydro B). In normal goblet cells, cytochemical localization of F-actin with NBD-phallacidin demonstrated their restriction to the apical surface of the goblet cell. Visualization of the goblet cell apical surface by electron microscopy revealed the presence of a thin layer of cytoplasm overlying the granule mass. Treatment with cyto D and dihydro B eliminated NBD-phallacidin staining of the apical cell surface. Quantitative analysis of baseline granule translocation demonstrated that treatment with cyto D and dihydro B resulted in dramatic acceleration of granule movement through goblet cells. This cellular response results from an increase in baseline secretion and facilitation of secretion of newly synthesized mucins, not stimulation of an accelerated secretory event. These data imply that actin filaments fulfill a barrier function in baseline secretion by hindering granule access to the plasma membrane; once the granule contacts the plasma membrane, exocytosis occurs. Secretion is balanced by the translocation of subjacent granules. In contrast, an accelerated secretory event is not triggered by plasma membrane access alone; this event requires a regulatory signal. We hypothesize that, unlike accelerated secretion, baseline secretion is constitutive, with exocytosis limited solely by the physical constraint of secretory granule access to the apical plasma membrane.

scholarly journals Coupling of uridine-5'-diphosphate (UDP) formation and nicotinamide adenine dinucleotide (NAD+) reduction for cytochemical localization of glycosyltransferases.

1983 ◽  
Vol 31 (10) ◽  
pp. 1175-1182 ◽  
Author(s):  
G R Matyas ◽  
D J Morré
Keyword(s):  
Golgi Apparatus ◽  
Rat Liver ◽  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine ◽  
Cytochemical Localization ◽  
Permeabilized Cells ◽  
Exogenous Glucose ◽  
Dense Deposits ◽  
Donor And Acceptor ◽  
Reduced Nicotinamide Adenine Dinucleotide

A technique applicable to the cytochemical localization of glycosyltransferases through a series of coupled enzyme reactions is described. Uridine-5'-diphosphate (UDP) formed by glycosyltransferases is first phosphorylated to uridine-5'-triphosphate (UTP) by nucleoside 5'-diphosphate kinase. The UTP plus exogenous glucose-1-phosphate is converted into UDP-glucose by uridine-5'-diphosphoglucose pyrophosphorylase. UDP-glucose is then oxidized by uridine-5'-diphosphoglucose dehydrogenase to form UDP-glucuronic acid and reduced nicotinamide adenine dinucleotide (NADH). The NADH is utilized by membrane-located NADH-ferricyanide oxidoreductases in the presence of a copper salt to form electron-dense deposits of cupric ferrocyanide (Hatchett's brown). Using this technique, galactosyltransferase has been localized in cisternae (including the central midregions of the cisternae) of Golgi apparatus isolated from rat liver. Reactivity is absent from the cis-most cisternae and membrane elements. The reaction is dependent on UDP-galactose and inhibited by ethylene diaminetetraacetic acid and puromycin. the latter is a known inhibitor of galactosyltransferase of rat liver Golgi apparatus. The reaction is adaptable by varying the sugar nucleotide donor and acceptor to any glycosyltransferase utilizing UDP-sugars (except UDP-glucose). Presently it is restricted to isolated membrane fractions and permeabilized cells due to the need for accessibility of reagents and coupling enzymes.

MECHANISM OF METOPIRONE INHIBITION OF A SOLUBLE ADRENAL STEROID 11β-HYDROXYLASE

1967 ◽  
Vol 45 (2) ◽  
pp. 153-163 ◽  
Author(s):  
Denis G. Williamson ◽  
Vincent J. O'Donnell
Keyword(s):  
Enzyme System ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Acetone Powder ◽  
Experimental Conditions ◽  
Adrenal Steroid ◽  
Hydroxylation Reaction ◽  
Nadph Oxidation ◽  
Low Levels ◽  
Reduced Nicotinamide Adenine Dinucleotide ◽  
Similar Decrease

The effect of Metopirone (2-methyl-1,2-bis(3-pyridyl)-1-propanone) (Su-4885) on the 11β-hydroxylation of 11-deoxycorticosterone (DOC) and NADPH oxidation has been examined in a soluble enzyme system extracted from an acetone powder of bovine adrenal mitochondria. Addition of Metopirone in the absence of DOC stimulated the oxidation of reduced nicotinamide–adenine dinucleotide phosphate (NADPH) by the enzyme system. Under the experimental conditions employed, 11β-hydroxylation was inhibited 50% by Metopirone at a final concentration of 4.2 μmoles/l, a quantity 27 times less than that required to produce a twofold increase in NADPH oxidation in the absence of DOC. Low levels of Metopirone that effectively inhibited corticosterone formation produced a similar decrease in NADPH oxidation associated with the hydroxylation reaction, indicating the interrelationship of 11β-hydroxylation and NADPH oxidation. Evaluation of kinetic data showed that the inhibition of 11β-hydroxylation produced by Metopirone was competitive. The Km for the 11β-hydroxylation of DOC was 1.8 × 10−5 mole/l and the Ki for Metopirone was 3.2 × 10−7 mole/l. The ratio Km:Ki indicated that the affinity of Metopirone for the 11β-hydroxylase was 56 times greater than that of DOC.

scholarly journals Redistribution of a rab3-like GTP-binding protein from secretory granules to the Golgi complex in pancreatic acinar cells during regulated exocytosis

1994 ◽  
Vol 124 (1) ◽  
pp. 43-53 ◽  
Author(s):  
BP Jena ◽  
FD Gumkowski ◽  
EM Konieczko ◽  
GF von Mollard ◽  
R Jahn ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Binding Protein ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Apical Plasma Membrane ◽  
Gtp Binding Protein ◽  
Regulated Exocytosis ◽  
Gtp Binding

Regulated secretion from pancreatic acinar cells occurs by exocytosis of zymogen granules (ZG) at the apical plasmalemma. ZGs originate from the TGN and undergo prolonged maturation and condensation. After exocytosis, the zymogen granule membrane (ZGM) is retrieved from the plasma membrane and ultimately reaches the TGN. In this study, we analyzed the fate of a low M(r) GTP-binding protein during induced exocytosis and membrane retrieval using immunoblots as well as light and electron microscopic immunocytochemistry. This 27-kD protein, identified by a monoclonal antibody that recognizes rab3A and B, may be a novel rab3 isoform. In resting acinar cells, the rab3-like protein was detected primarily on the cytoplasmic face of ZGs, with little labeling of the Golgi complex and no significant labeling of the apical plasmalemma or any other intracellular membranes. Stimulation of pancreatic lobules in vitro by carbamylcholine for 15 min, resulted in massive exocytosis that led to a near doubling of the area of the apical plasma membrane. However, no relocation of the rab3-like protein to the apical plasmalemma was seen. After 3 h of induced exocytosis, during which time approximately 90% of the ZGs is released, the rab3-like protein appeared to translocate to small vesicles and newly forming secretory granules in the TGN. No significant increase of the rab3-like protein was found in the cytosolic fraction at any time during stimulation. Since the protein is not detected on the apical plasmalemma after stimulation, we conclude that recycling may involve a membrane dissociation-association cycle that accompanies regulated exocytosis.

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