scholarly journals Quantitative immunocytochemical localization of Na+,K+-ATPase in rat ciliary epithelial cells.

1989 ◽  
Vol 37 (9) ◽  
pp. 1353-1361 ◽  
Author(s):  
T Okami ◽  
A Yamamoto ◽  
K Omori ◽  
M Akayama ◽  
M Uyama ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Aqueous Humor ◽  
Ciliary Body ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Immunoblot Analysis ◽  
Alpha Subunit ◽  
Ciliary Epithelium ◽  
Immunocytochemical Localization ◽  
Gold Particles

Ultrastructural localization of Na+,K+-ATPase in rat ciliary epithelium was investigated quantitatively by the protein A-gold technique, using an affinity-purified antibody against the alpha-subunit of Na+,K+-ATPase. Immunoblot analysis showed that the antibody bound specifically to the alpha-subunit of Na+,K+-ATPase in the ciliary body. Gold particles were found mainly on the basolateral surfaces of both the pigmented epithelial (PE) and nonpigmented epithelial (NPE) cells with an approximately twofold higher labeling density in the PE cells. A few gold particles were also found on the apical and ciliary channel surfaces of the PE cells, whereas no significant binding was found on the apical surfaces of the NPE cells. The basolateral surfaces of PE and NPE cells are markedly infolded and are much greater in area than the apical surfaces. This means that Na+,K+-ATPase is almost exclusively located on the basolateral surfaces of both the NPE and PE cells. We suggest that the Na+,K+-ATPase of both the NPE and PE cells play an important role in the formation of aqueous humor.

scholarly journals Quantitative immunogold localization of Na+,K(+)-ATPase alpha-subunit in the tympanic wall of rat cochlear duct.

1990 ◽  
Vol 38 (2) ◽  
pp. 225-232 ◽  
Author(s):  
T Iwano ◽  
A Yamamoto ◽  
K Omori ◽  
K Kawasaki ◽  
T Kumazawa ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
General Pattern ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Alpha Subunit ◽  
Cochlear Duct ◽  
Gold Particles ◽  
Sensory Hair Cells

Ultrastructural localization of Na+,K(+)-ATPase was quantitatively investigated in the tympanic wall of rat cochlear duct by use of the protein A-gold method, using an affinity-purified antibody against the alpha-subunit of rat kidney Na+,K(+)-ATPase. A moderate number of gold particles were found on the basolateral membrane of the interdental cells of the spiral limbus. A small number of gold particles were found on the basolateral surfaces of the border cells and Hensen's cells. On the inner and outer sensory hair cells, however, the plasma membranes were rarely labeled by gold particles. The general pattern of labeling densities in cochlear structures determined here and in a previous communication from our laboratory shows good correlation with the distribution of Na+,K(+)-ATPase activity as previously estimated biochemically, cytochemically, and autoradiographically.

The Ciliary Body - The Third Organ Found to Synthesize Indoleamines in Humans

1992 ◽  
Vol 2 (2) ◽  
pp. 67-72 ◽  
Author(s):  
X. D. Martin ◽  
H. Z. Malina ◽  
M. C. Brennan ◽  
Ph. H. Hendrickson ◽  
P. R. Lichter
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Pineal Gland ◽  
Electrochemical Detection ◽  
Aqueous Humor ◽  
Ciliary Body ◽  
High Performance ◽  
Ciliary Epithelium ◽  
The Third ◽  
Hydroxyindoleacetic Acid

Indoleamines are associated with circadian rhythms in pineal gland and retina. Because the ciliary epithelium has an embryonic origin similar to that of pineal gland and retina, and intraocular pressure shows circadian variations, indoleamines were searched for in aqueous humor and ciliary body in humans. In aqueous humor, serotonin, 6-hydroxymelatonin, and melatonin were simultaneously detected and measured using high-performance liquid chromatography with electrochemical detection. The concentration was 48.7±10.9 ng/ml for serotonin, 0.47±0.8 ng/ml for melatonin, and 13.9±7.7 ng/ml for 6 hydroxy melatonin. In ciliary bodies from freshly enucleated human eyes, tryptophan, 5-hydroxytryptophan, serotonin, and 5-hydroxyindoleacetic acid were detected using high-performance liquid chromatography with simultaneous fluorescence- and electrochemical detection. Finally, the enzymatic activities of arylalkylamine N-acetyltransferase (NAT) and hydroxyindole-O-methyltrans-ferase (HIOMT), enzymes indispensable in the synthesis of melatonin, were measured. The NAT activity was 273±25 pmol/mg protein/hour and that of HIOMT, 13520±50 pmol/mg protein/hour in ciliary body. Comparison of these activities (NAT versus HIOMT) permits the suggestion that NAT is a limiting enzyme in serotonin metabolism in this tissue. These findings indicate that a circadian rhythm of indoleamines exists in human aqueous humor and that the human ciliary body is the third organ, after the pineal gland and the retina, found to synthesize indoleamines in humans.

scholarly journals Quantitative immunocytochemical analysis of the induction of cytochrome P450IIB in rat hepatocytes.

1992 ◽  
Vol 40 (1) ◽  
pp. 73-82 ◽  
Author(s):  
Y Fukui ◽  
A Yamamoto ◽  
R Masaki ◽  
K Miyauchi ◽  
Y Tashiro
Keyword(s):  
Electron Microscopy ◽  
Particle Density ◽  
Protein A ◽  
Liver Microsomes ◽  
Rat Hepatocytes ◽  
Immunoblot Analysis ◽  
Immunogold Electron Microscopy ◽  
Thin Sections ◽  
Gold Particles ◽  
Rough Er

We examined whether induction of the phenobarbital (PB)-inducible form of cytochrome P450 (P450IIB) in rat hepatocytes could be analyzed quantitatively by immunogold electron microscopy. Rats received intraperitoneal injections of PB every 24 hr and livers at the various stages of PB induction were fixed by perfusion with a mixture of paraformaldehyde (4%) and glutaraldehyde (0.1%) and embedded in LR White. Ultra-thin sections were cut and labeled by the protein A-gold procedure using affinity-purified anti-P450IIB antibody which was previously immunoabsorbed with liver microsomes from a control rat (not treated with PB). We counted the number of gold particles per micron of the rough ER membranes (particle density). Before PB treatment, the particle density of the rough ER in rat hepatocytes was practically zero and increased markedly at 48 and 72 hr after PB treatment. The rough microsomes were prepared from these PB-treated rat livers. The amount of P450IIB was estimated by immunoblot analysis and the number of gold particles bound to the rough microsomal membrane was determined by the same post-embedding immunogold procedure. The particle density of the rough microsomes increased in parallel with the increase in the amount of P450IIB, indicating good correlation of the two variables. Thus, the induction of cytochrome P450IIB can be quantitatively and reliably investigated by immunogold electron microscopy.

scholarly journals Immunocytochemical localization of D-amino acid oxidase in the central clear matrix of rat kidney peroxisomes.

1986 ◽  
Vol 34 (12) ◽  
pp. 1709-1718 ◽  
Author(s):  
N Usuda ◽  
S Yokota ◽  
T Hashimoto ◽  
T Nagata
Keyword(s):  
Amino Acid ◽  
Proximal Tubule ◽  
Rat Kidney ◽  
Protein A ◽  
Immunoblot Analysis ◽  
Acid Oxidase ◽  
Electron Microscopic ◽  
Amino Acid Oxidase ◽  
Thin Sections ◽  
Gold Particles

Light and electron microscopic localizations of D-amino acid oxidase (DAO) in rat kidney was investigated using immunoenzyme and protein A-gold techniques. The enzyme was purified from rat kidney homogenate and its antibody was raised in rabbits. By Ouchterlony double-diffusion analysis and immunoblot analysis with anti-(rat kidney DAO) immunoglobulin, the antibody was confirmed to be monospecific. The tissue sections (200 micron thick) of fixed rat kidney were embedded in Epon or Lowicryl K4M. Semi-thin sections were stained for DAO by the immunoenzyme technique after removal of epoxy resin for LM, and ultra-thin sections of Lowicryl-embedded material were labeled for DAO by the protein A-gold technique for EM. By LM, fine cytoplasmic granules of proximal tubule were stained exclusively. Among three segments of proximal tubules, and S2 and S3 segments were heavily stained but the S1 segment only weakly so. By EM, gold particles indicating the antigenic sites for DAO were exclusively confined to peroxisomes. Within peroxisomes, the gold particles were localized in the central clear matrix but not in the peripheral tubular substructures. The results indicate that D-amino acid oxidase in rat kidney is present exclusively in peroxisomes in the proximal tubule and that within peroxisomes it is found only in central clear matrix and not in the peripheral tubular substructures.

scholarly journals Immunoelectron microscopic demonstration of S-100b protein-like in centriole, cilia, and basal body.

1988 ◽  
Vol 36 (6) ◽  
pp. 693-696 ◽  
Author(s):  
T Uchida ◽  
T Endo
Keyword(s):  
Basal Body ◽  
Colloidal Gold ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Basal Bodies ◽  
Cell Organelles ◽  
Gold Particles ◽  
Microscopic Demonstration ◽  
S 100 ◽  
And Function

We report here the ultrastructural localization of S-100b protein-like immunoreactivity in the centriole, cilia, and basal body. Duodenum and trachea of guinea pigs and rats were fixed and immunostained by the protein A-gold method. All centrioles, cilia, and basal bodies observed showed clear S-100b protein-like immunoreactivity. Specific colloidal gold particles were located over the microtubules in these cell organelles. However, other microtubules scattered throughout the cytoplasm were devoid of immunoreactivity. Although the functional significance of S-100b protein-like immunoreactivity in the centriole, cilia, and basal bodies remains to be elucidated, the present results introduce new perspectives into the investigation of localization and function of S-100 proteins.

Electrogenic Na+-ascorbate cotransport in cultured bovine pigmented ciliary epithelial cells

1989 ◽  
Vol 256 (1) ◽  
pp. C44-C49 ◽  
Author(s):  
H. Helbig ◽  
C. Korbmacher ◽  
J. Wohlfarth ◽  
S. Berweck ◽  
D. Kuhner ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Aqueous Humor ◽  
Time Course ◽  
Transport Systems ◽  
Ciliary Epithelium ◽  
Intracellular Accumulation ◽  
Saturation Kinetics ◽  
Sigmoidal Curve ◽  
High Level ◽  
The Hill

The high level of ascorbic acid (AA) in the aqueous humor of many mammals suggests an active transport of AA across the double-layered ciliary epithelium from blood to aqueous humor. We used [14C]AA to study AA uptake in bovine pigmented ciliary epithelial cells in tissue culture. We observed a 40-fold intracellular accumulation of AA, which was dependent on extracellular Na+. With labeled dehydroascorbate (DHA, the oxidized form of the vitamin) in the medium, there was a 20-fold intracellular accumulation of the label. However, the time course of DHA uptake was different compared with AA uptake and was not Na+ dependent, suggesting different transport systems for AA and DHA. AA uptake was inhibited by 1 mM phloretin and in the presence of isoascorbate. Furthermore, AA uptake was markedly reduced when intracellular Na+ was elevated by preincubation with ouabain or amphotericin B. With increasing AA concentration, Na+-dependent AA uptake exhibited first-order saturation kinetics with half-maximal uptake at 76 microM AA. Na+ dependence of AA uptake revealed a sigmoidal curve of Na+-dependent AA uptake vs. Na+ concentration with a half-maximal AA uptake at 45.4 mM Na+. The slope of the Hill plot from these data was 1.94, suggesting a transport system translocating two or more Na+ for one AA. This stoichiometry implies electrogenicity of the transporter. We, therefore, measured membrane potentials using conventional microelectrodes. Addition of 200 microM AA resulted in a depolarization of the membrane voltage by 4.9 +/- 0.5 mV (n = 22), which was absent in Na+ free medium and was markedly reduced by phloretin.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Immunoelectron microscopic evidence for organ differences in the composition of peroxisome-specific membrane polypeptides among three rat organs: liver, kidney, and small intestine.

1991 ◽  
Vol 39 (10) ◽  
pp. 1357-1366 ◽  
Author(s):  
N Usuda ◽  
T Kuwabara ◽  
R Ichikawa ◽  
T Hashimoto ◽  
T Nagata
Keyword(s):  
Renal Cortex ◽  
Protein A ◽  
Microscopic Analysis ◽  
Immunoblot Analysis ◽  
Jejunal Mucosa ◽  
Peroxisomal Membrane ◽  
Mucosal Epithelium ◽  
Gold Particles ◽  
Ethylhexyl Phthalate ◽  
Specific Membrane

We examined the distribution of peroxisome-specific membrane polypeptides (PMPs) among peroxisomes of the liver, renal cortex, and jejunal mucosa, using antibodies for 70 KD, 26 KD and 22 KD PMPs. Immunoblot analysis showed signals for 70 KD polypeptide in all three kinds of tissue, but for the other two only in the liver and renal cortex, with neither being detected in jejunal mucosa. The total amounts of PMPs increased in all three organs with DEHP (di-(2-ethylhexyl)phthalate) administration. By immunoelectron microscopic analysis using protein A-gold, the three PMPs were localized along the peroxisomal membrane. Quantitation of the gold particles associated with the peroxisomal membrane showed an increase in the density of 70 KD and 26 KD PMPs but a decrease in 22 KD PMP with the administration of DEHP. The presence of tissue-specific localizations of PMPs suggest the 70 KD PMP is a common constituent of peroxisomes of these three tissues, whereas 26 KD and 22 KD PMPs are absent in microperoxisomes of jejunal mucosal epithelium.

scholarly journals In cow anterior pituitary, growth hormone and prolactin can be packed in separate granules of the same cell.

1985 ◽  
Vol 100 (6) ◽  
pp. 2019-2024 ◽  
Author(s):  
G Fumagalli ◽  
A Zanini
Keyword(s):  
Growth Hormone ◽  
Golgi Complex ◽  
Anterior Pituitary ◽  
Relative Proportion ◽  
Protein A ◽  
Central Area ◽  
Ultrastructural Localization ◽  
Specific Antibodies ◽  
Gold Particles ◽  
Pituitary Growth Hormone

The ultrastructural localization of growth hormone and prolactin in cow anterior pituitary was studied by double immunocytochemical labeling using specific antibodies and protein A-gold particles of different sizes. The two hormones were found in specific somatotrophs and mammotrophs as well as in somatomammotropic cells which were multinucleated and predominantly arranged in clusters in the central area of the lobules. In these mixed cells the two hormones were packaged (a) in different granules of the same cell, (b) in the same granules where they were segregated in different portions of the granule content, or (c) in the same granules but evenly intermixed. The relative proportion of these three types of granules varied in somatomammotrophs of different animals. A single large Golgi complex was generally present in somatomammotrophs. Small, immature granules containing either growth hormone or prolactin or both hormones were found randomly distributed along Golgi stacks. This suggests that in these cells the two hormones are processed in the same Golgi cisternae and that mechanism(s) exist(s) to sort out the two hormones from each other.

scholarly journals Immunocytochemical localization of Na+,K(+)-ATPase in rat retinal pigment epithelial cells.

1990 ◽  
Vol 38 (9) ◽  
pp. 1267-1275 ◽  
Author(s):  
T Okami ◽  
A Yamamoto ◽  
K Omori ◽  
T Takada ◽  
M Uyama ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Retinal Pigment Epithelial ◽  
Rat Kidney ◽  
Retinal Pigment ◽  
Protein A ◽  
Retinal Pigment Epithelial Cells ◽  
Alpha Subunit ◽  
Apical Surface ◽  
Pigment Epithelial ◽  
Pigment Epithelial Cells

We investigated quantitatively the ultrastructural localization of the alpha-subunit of Na+,K(+)-ATPase in rat retinal pigment epithelial cells by the protein A-gold technique, using an affinity-purified antibody against the alpha-subunit of rat kidney Na+,K(+)-ATPase. Immunoblot analysis showed that the antibody bound specifically to the alpha- and alpha(+)-subunits of Na+,K(+)-ATPase in the whole retina [the sensory retina plus retinal pigment epithelium (RPE)]. Rat eyes were fixed by perfusion with 4% paraformaldehyde containing 1% glutaraldehyde and embedded in Lowicryl K4M. Ultra-thin sections were incubated with affinity-purified antibody against the alpha-subunit of rat kidney Na+,K(+)-ATPase and subsequently with protein A-gold complex. Light microscopy with a silver enhancement procedure revealed Na+,K(+)-ATPase localized to both the apical and the basal plasma membrane domains of the RPE. Quantitative immunocytochemical analysis by electron microscopy showed a higher density of gold particles on the apical surface than on the basolateral one. Microvilli are so well developed on the apical surface of the RPE that the apical surface profile is much longer than the basolateral one. This means that Na+,K(+)-ATPase is mainly located on the apical surface of the RPE cells.

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