The Golgi apparatus of goblet cells in the mouse descending colon: three-dimensional visualization using a confocal laser scanning microscope.

The three-dimensional structure of the Golgi apparatus was studied in goblet cells in lectin-stained sections of the mouse descending colon by using a confocal laser scanning microscope. In the lower part of the crypt, the Golgi apparatus formed a dome- or globe-like structure in the supranuclear region. The wall of the dome had some holes, one of which usually faced toward the nucleus and others toward the apical cytoplasm. Mucous granules seemed to be initially released into the interior of the dome and transported toward the apical cytoplasm through the holes. In the upper part of the crypt, on the other hand, the Golgi apparatus formed a cup- or funnel-like structure with a larger opening toward the cell apex and a smaller opening toward the nucleus. A large mass of mucous granules occupied the inside of the cup to the apical cytoplasm. It is thought that the accumulation of mucous granules enlarges holes at the ceiling of the dome to form a large opening, which makes the configuration of the Golgi apparatus cup-shaped.

Three-dimensional visualization of the Golgi apparatus: observation of Brunner's gland cells by a confocal laser scanning microscope.

The three-dimensional structure of the Golgi apparatus in cells of the Brunner's gland in the mouse was observed by using a confocal laser scanning microscope. Two lectins, FITC-labeled soybean agglutinin and Texas red-labeled Griffonia simplicifolia agglutinin II, were used to visualize the whole Golgi apparatus. Staining with the former lectin, which has been known to label the cis-stacks, showed a lacy dome-like structure situated in the supranuclear region. Staining with the latter lectin, known to label the intermediate-to-trans-stacks and the secretory granules, showed a dome-like structure consisting of network and cobblestone-like patterns in the same region and also granular stainings near the surface of the cobblestone-like patterns and the apical region of a cell. Double-staining demonstrated that the soybean agglutinin-labeled network always surrounded the G. simplicifolia agglutinin II-stained structure. Based on these observations, we propose a new three-dimensional model of the Golgi apparatus: it forms a dome-like structure over a nucleus, a network of cis-stacks forms its outer boundary, and this outer boundary is lined and paved with successive intermediate and trans-stacks. It is thought that secretory granules are released toward the internal space of the Golgi apparatus and transported to the apical cytoplasm through the holes of the network.

Video-rate dynamics of exocytotic events associated with phagocytosis in neutrophils.

Exocytotic responses associated with phagocytosis were investigated in a single neutrophil with a special reference to their dynamic properties and their spatiotemporal relationships with ionic and chemical responses during phagocytosis. The real-time sequence of phagocytosis-exocytosis was directly visualized by video-enhanced contrast differential interference contrast (VEC-DIC) microscopy. The actual release of contents from such a granule was proven by examining a cell loaded with quinacrine with a dual imaging system that allowed us to observe DIC and fluorescence images simultaneously at a high magnification. During the process of phagosome formation in a neutrophil engulfing an opsonized zymosan, the exocytotic response was observed first in a granule located near the cell surface initially attached to the zymosan, and then in other granules sequentially along pseudopodia surrounding the zymosan. When the phagocytosis was induced in a medium containing luminol, a chemiluminescence due to active oxidants was detected exclusively in the region of phagosome, suggesting that exocytosis took place on the phagosomal membrane and not on the plasma membrane. Changes in cytosolic free calcium concentration ([Ca2+]i) were further measured using fura-2 under the dual imaging system. [Ca2+]i transients were more closely related to the extension of pseudopodia for engulfing zymosan and not directly to the exocytosis. These findings lead to a conclusion that exocytosis associated with phagocytosis is initiated by attachment of the cell membrane to the invading organism and mediated by local activation of the phagosomal membrane.

Quantitative analysis of exocytosis visualized by a video-enhanced light/fluorescence microscope reveals two distinct components of exocytosis in RBL-2H3 cells.

Rat basophilic leukemia (RBL-2H3) cells, which exhibit Ca2+-dependent secretion of granules when stimulated with antigen or the Ca2+-ionophore A23187, were observed under a video-enhanced light/fluorescence microscope. Exocytotic events of individual granules were visualized in individual cells stimulated with antigen or A23187. Exocytosis of granules stimulated with A23187 showed two peaks in the time course. The earlier one was inhibited by selective inhibitors of protein kinase C (Ro31-8425, Ro31-8220, and chelerythrine) and the other was inhibited by an inhibitor of phosphatidate hydrolase, propranolol. Exocytosis by antigen stimulation, however, showed only one peak, which was inhibited by the selective inhibitors of protein kinase C, but not by propranolol. These results indicate that at least two distinct components of exocytosis exist in RBL-2H3 cells.

Quantitative analysis of superoxide anion generation in living cells by using chemiluminescence video microscopy.

Superoxide anions (O2-) generated by rabbit neutrophils were detected and quantified by a video microscope equipped with a photon-counting camera. One count obtained by this system was equivalent to 59 amol of O2-. Maximum O2- production was observed at 6-8 min after stimulation and was estimated as 1.9 fmol/min per cell on the average.

A histochemical study on glycoconjugates in epithelial cells in the distal colonic mucosa of adult and developing mice.

Glycoconjugates were histochemically studied in the distal colon of developing ICR mice in view of the presence of goblet and vacuolated cells. Alcian blue, high iron diamine and periodic acid-Schiff stainings were performed to characterize glycoconjugates. In addition, two lectins, Ulex europeus agglutinin I and Limax flavus agglutinin, were applied to detect fucosyl and sialyl residues, respectively. The reactivities to these stainings, noted from day 18 of gestation, did not seem to undergo any major change throughout their development. The present results suggest that: 1) goblet cells secrete sulfated glycoconjugates containing fucosyl and sialyl residues as terminal sugars; 2) vacuolated cells have glycoconjugates containing sialyl residues but no or few fucosyl residues nor sulfonic groups, and certain sialyl residues in the glycoconjugates are probably O-acetylated or O-acylated at least in part; and 3) the brush border of absorptive cells contains glycoconjugates sialylated and fucosylated to same extent but rarely sulfated. Since glycoconjugates elaborated by goblet and vacuolated cells differ from each other, one should be fully aware of the presence of these two types of mucin-producing cells in the distal colon.

[Development of multicomponent immunoassay and micro-localization analysis by laser-photoacoustic and microscopic image analyzer].

Laser photoacoustic spectroscopy (PAS) has a potential to be developed as a sensitive and solid-phase analytical method and was applied to enzyme immunoassay. The test sample for immunoassay was prepared by adsorbing multi-component immunoglobulins on a nitrocellulose membrane filter. Human lambda- and kappa-chains, which are used as a principal indication of malignant lymphoreticular disease, and immunoglobulin G were used as model proteins, and PAS immunoassay was applied to the individual detection of these three proteins in the urine. Furthermore, in order to develop a sensitive analysis for particular biological components in tissues or cells, laser photoacoustic microscopy (PAM) and video intensified microscopy (VIM) were developed. PAM was shown to be applicable to the detection and quantification of human lambda-chain in a micro-region of the tissue sections of the human fetal spleen and pancreas. VIM was applied to the detection of stimulation-response processes in a cell. By using neutrophils which are stimulated by many substances and produce active oxidants as the results, dynamic changes in the stimulation-response process in a living cell were visualized as fluorescence or chemiluminescence images by the VIM system.

Lectin cytochemistry in the gastrointestinal tract with special reference to glycosylation in the Golgi apparatus of Brunner's gland cells.

Two hydrophilic, low temperature-embedding resins, Lowicryl K4M and LR White, were compared in lectin cytochemistry. Post-embedding staining of colloidal gold-labeled Griffonia symplicifolia agglutinin II (GSA-II) resulted in staining of the Golgi apparatus and mucous granules of mucous neck cells in the gastric fundic gland, pylorocytes, and Brunner's gland cells embedded in either resin, although it was much easier to make ultra-thin sections with LR White-embedded material than with the other. Post-fixation with uranyl acetate followed by LR White embedding improved general ultrastructure so that lectin binding sites were identified precisely. All examined lectins, soybean agglutinin (SBA), Maclura pomifera agglutinin (MPA), GSA-II, and Ulex europaeus agglutinin I (UEA-I), stained mucous granules and the Golgi apparatus, in which the staining pattern was characteristic of each lectin: cis cisternae were labeled with SBA and MPA, intermediate cisternae with GSA-II, and trans cisternae and mucous granules with SBA, GSA-II, UEA-I, and lightly with MPA. No labeling was observed in the rough endoplasmic reticulum with any lectin. These findings suggest that the Golgi apparatus is the site of O-linked glycosylation and can be divided into at least three distinct compartments with regard to the glycosylation.

Stimulative effect of chlordane on the various functions of the guinea pig leukocytes.

The effects of the chlordane-related compounds, cis-chlordane, trans-chlordane, heptachlor, and heptachlor epoxide, on stimulation responses of guinea pig polymorphonuclear leukocytes (PMNs) were examined. Treatment of PMN with these compounds stimulated superoxide (O2-) generation, altered membrane potential, and increased intracellular Ca2+ concentration ([Ca2+]i). However, there was no definite tendency among the stimulating effects of chlordane-related compounds, therefore the relationship between the effect and molecular structure of these substances remains unknown. Of these response reactions of PMN stimulated by chlordane-related compounds, stimulation of O2- generation lagged behind others. Increase in [Ca2+]i was due both to acceleration of extracellular Ca2+ penetration and to Ca2+ release from the intracellular pool. These results indicate that chlordane-related compounds stimulate PMN and suggest a causal relationship between the stimulation of O2- generation by these substances and their toxicity.

Inhibitory effect of cholesterol on interaction between cytoplasmic actin and liposomes, and restorative effect of high osmotic pressure.

Although cholesterol is one of the major components of plasma membranes in eukaryotic cells, very little is known about its role in biological membranes. We reported previously (Okimasu et al., Cell Struct. Funct. 11, 273-283, 1986) that introduction of cholesterol into the liposomal membrane caused a decrease in membrane permeability, especially by the binding of cytoplasmic proteins to the liposomal membrane. The present study was carried out to further clarify the biochemical function of cholesterol in the membrane-protein interactions, especially under high osmotic pressure. The association of membranes with cytoplasmic proteins and their permeability were decreased by the introduction of cholesterol, but its effects were diminished in a hypertonic medium. The protein species associated with cholesterol-containing liposomes vary depending on the sort of hypertonic condition. It was suggested that since the degree of lipid packing by the cholesterol was reduced by the locally increased curvature in the lipid bilayer under high osmotic pressure, some cytoplasmic proteins can penetrate into the liposomal membrane.

Influences of isoproterenol pretreatment on cerebral cortical bindings of [3H]clonidine and [3H]dihydroalprenolol in infant and adult rats.

Specific bindings of [3H]clonidine and [3H]dihydroalprenolol [( 3H]DHA) to crude synaptic membranes were measured after cerebral cortical slices or synaptic membranes were pre-incubated with isoproterenol, 200 microM at 37 degrees C for 40 min, in 7- and 70-day-old rats. Isoproterenol caused a significant increase of the Bmax value of [3H]clonidine binding sites at both days 7 and 70, without changing the Kd. Scatchard analysis of [3H]clonidine binding to adult synaptic membranes which was examined by using a wide range of [3H]clonidine concentration (0.05-15 nM), showed that the Bmax in only high-affinity binding sites was 4-fold increased by pretreatment of synaptic membranes with isoproterenol 200 microM. In contrast, the Bmax of [3H]DHA binding sites was significantly reduced by isoproterenol at 7 and 70 days. These results suggest that the possible interrelationship between beta- and alpha 2-adrenoceptors which exists in synaptic membranes, matures by day 7 in the cerebral cortex of rats.