T Kobayashi

Kochi Medical School, Kôti, Kōchi, Japan

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Publications (30)60.66 Total impact

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    ABSTRACT: The effectiveness of granulocyte colony-stimulating factor (G-CSF) for granulocytopenia has been widely recognized. However, although granulocyte counts rapidly increase after a few injection of G-CSF, a large proportion of the increased granulocytes disappear from peripheral blood within a few days following G-CSF withdrawal. Where do they go? In this report, the answer can be seen at a glance. Using MitoCapture and a CCD camera-equipped fluorescence microscope, we succeeded in demonstrating that G-CSF withdrawal induced loss of mitochondrial membrane potential, i.e., an early stage of apoptosis, in human peripheral granulocytes increased by G-CSF.
    Oncology Reports 09/2001; 8(5):1027-9. · 2.30 Impact Factor
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    ABSTRACT: We examined sequential changes in post-irradiated peripheral blood T cells taken from normal volunteers, by using targeted Atlas cDNA Expression Arrays and mitochondrial membrane potential assay. At 1 and 3 hours after 5 Gy irradiation, changes of gene expression were examined by targeted Atlas cDNA Expression Arrays using Apoptosis Array. The Atlas Human Apoptosis Array includes 205 key genes that are known to control apoptosis, including extracellular and cytoplasmic effectors. Concerning Fas, no significant changes of spot intensities were identified between irradiated T cells and non-irradiated ones at both 1 h and 3 h after 5 Gy irradiation. Caspase families, including caspases 9 and 3 also showed no changes between these two groups. An apoptosis regulator bclw showed a remarkable decrease in irradiated T cells. These results suggested that irradiation induced direct apoptosis of T cells by changing the membrane potential of mitochondria. Using a CCD camera-equipped fluorescence microscope and MitoCapture, a mitochondrial membrane potential indicator, we demonstrated 5 Gy radiation induced loss of membrane potential, i.e., an early stage of apoptosis, in human peripheral blood T cells at 10 hours after irradiation.
    International Journal of Molecular Medicine 07/2001; 7(6):603-7. · 1.96 Impact Factor
  • T Kobayashi, S Tsunawaki, H Seguchi
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    ABSTRACT: We present an up-to-date insight into the function of NADPH oxidase in human neutrophils, the signalling pathways involved in activation of this enzyme and the process of association of its components with the cytoskeleton. We also discuss the functional implications of morphological studies revealing localization of the sites of NADPH oxidase activity. An original model of the process of superoxide (O2*-) production in human neutrophils is shown. Organization of NADPH oxidase is associated with several components. Upon stimulation, tri-phox cytosolic components of NADPH oxidase (p40-phox, p47-phox and p67-phox) bind to actin filaments. This process involves other actin-binding proteins, such as cofilin and coronin. Activated protein kinase C, translocated from the plasma membrane, phosphorylates cytosolic components at a scaffold of cytoskeleton. Subsequently, p40-phox, responsible for maintaining the resting state of NADPH oxidase, is separated from other two cytosolic phox proteins following an attachment of the active form of small GTP-binding protein Rac to p67-phox. Cytosolic duo-phox proteins (p47-phox and p67-phox) conjugate with membrane components (gp91-phox, p22-phox and Rapla) of NADPH oxidase residing within membranes of intracellular compartments. This chain of events triggers production of O2*-. Then, oxidant-producing intracellular compartments associate with the plasma membrane. Eventually, intracellularly produced O2*- is released to the extracellular environment through the orifice formed by fusion of oxidant-producing compartments with the plasma membrane. Intracellular movement of the oxidant-producing compartments may be regulated by myosin light chain kinase. The review emphasizes that functional assembly of NADPH oxidase and, therefore, generation of O2*- is accomplished essentially within the intracellular compartments. Upon neutrophil stimulation, intracellularly generated O2*- is transported to the plasma membrane to be released and to ensure host defense against infection.
    Redox Report 02/2001; 6(1):27-36. · 1.66 Impact Factor
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    ABSTRACT: Rebamipide, an antiulcer agent, has been shown to be able to prevent gastric mucosal injury resulting in part from activation of neutrophils. The mechanism of its suppressive action, however, remains to be established. The present study aimed to determine the effect of rebamipide on activation of isolated human neutrophils and to identify the signal transduction pathway involved in its regulation. In unstimulated cells, alkaline phosphatase activity was found residing in short rod-shaped intracellular granules. Upon stimulation with a chemotactic peptide formyl-methionyl-leucyl-phenylalanine, the granules fused to form elongated tubular structures and spherical vacuoles. Rebamipide inhibited reorganization of alkaline phosphatase-containing granules along with upregulation of alkaline phosphatase activity and CD16, a marker of the granules. It also suppressed chemotaxis, an increase in intracellular calcium ion concentration, and NADPH oxidase activation in cells stimulated with formyl-methionyl-leucyl-phenylalanine. In contrast, the drug showed no inhibitory action toward upregulation of alkaline phosphatase activity and CD16, and activation of NADPH oxidase in cells stimulated with phorbol myristate acetate, an activator of protein kinase C. These findings demonstrate that rebamipide exerts a broad spectrum of suppressive actions toward biological functions of human neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine, but not with phorbol myristate acetate, and suggest that the upstream point of protein kinase C is the signal transduction pathway involved in its regulation.
    Histology and histopathology 11/2000; 15(4):1067-76. · 2.28 Impact Factor
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    ABSTRACT: In human neutrophils, superoxide is generated primarily within specialized oxidant-producing intracellular compartments. The present study employs a simple methodological approach to evaluate the intracellular movement of these structures in living human neutrophils. Using a CCD camera system, we monitored fluorescence in cells loaded with the succinimidyl ester of dichlorodihydrofluorescein diacetate, which is nonfluorescent until oxidized by reactive oxygen species. Fluorescence-positive intracellular compartments became detectable after neutrophils were stimulated with phorbol myristate acetate for 1 min. Further stimulation increased the intracellular compartments in both number and size in a time-dependent manner. Upon stimulation with phorbol myristate acetate, no fluorescence was seen in intracellular compartments of neutrophils isolated from patients with X-linked chronic granulomatous disease lacking gp91-phox, a membrane component of NADPH oxidase. The method enables tracking of the movement of a single oxidant-producing intracellular compartment following cell stimulation and visualization of the intracellular structures formed by fusion of oxidant-producing intracellular compartments with endocytotic vesicles and phagosomes. Therefore, it is considered to be an informative tool for evaluation of the intracellular dynamics of oxidant-producing intracellular compartments in living human neutrophils and may have a diagnostic value.
    Histochemie 05/2000; 113(4):251-7. · 2.61 Impact Factor
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    ABSTRACT: Superoxide anion production in neutrophils plays an important role in the microbicidal defense system in the body. In this study, isolated rat neutrophils were stimulated experimentally and examined by electron microscopy to determine the site of superoxide production and its subsequent translocation during different cell stimulation time periods. Blood and peritoneal neutrophils were incubated for periods of 5, 10, and 15 min with phorbol 12-myristate 13-acetate (PMA), N-formyl-Met-Leu-Phe (fMLP), and combinations of PMA and cytochalasin B (CB) and fMLP and CB. Ultracytochemical detection of O(2)(-) was performed with the 3, 3'-diaminobenzidine-manganese (DAB/Mn) cytochemical method and cationized ferritin (CF) particles were added to stimulation media to monitor endocytotic events that occurred during neutrophil stimulation. Unstimulated neutrophils were devoid of O(2)(-) activity in cytoplasmic granules and at the plasma membrane surface. After 5 min stimulations with PMA, PMA + CB, or fMLP + CB, electron-dense DAB/Mn reaction product was detected in small, centrally located tubular compartments within the neutrophils. CF particles which were added to the stimulation media became internalized in endocytotic vesicles after 5 min stimulation; these vesicles were devoid of O(2)(-) activity. At 10 min stimulation with PMA, O(2)(-)-positive granules subsequently fused with each other and translocated to sub-plasma membrane regions where they either contacted the plasma membrane or fused with CF-containing endocytotic vesicles. Little reaction product was observed on the surface of the neutrophils. Spectrophotometric comparison of the stimulatory effects of PMA, fMLP, and fMLP + CB revealed different rates and yields of O(2)(-) production. Results from this study suggest that the O(2)(-)-producing sites of rat neutrophils originate intracellularly and translocate to the plasma membrane surface following stimulation with PMA, PMA + CB, and fMLP + CB, but not with fMLP or CB alone. Furthermore, these compartments appear to possess the ability to fuse with endocytotic vesicles, a process that may be linked to intracellular microbicidal activity in circulating and tissue neutrophils.
    The Anatomical Record 03/2000; 258(2):156-65.
  • V S Zinchuk, T Okada, T Kobayashi
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    ABSTRACT: We used a combined biochemical and histocytochemical approach to study ecto-ATPase in the rat cardiac muscle. The reaction medium employed for histocytochemical detection was optimized in biochemical assays to achieve the highest enzyme activity and lowest inhibition by the capture agent used for visualization of the reaction product. Approximately 70% of the enzyme activity was retained in samples after the fixation procedure. Divalent cations stimulated ecto-ATPase. High activity was detectable within a wide pH range. Histocytochemical reaction was observed at sites at which extracellular ATP can potentially exert its actions on the cardiac muscle: nerve endings, plasma membranes of cardiac myocytes and capillary endothelial cells, and T-tubules. Product of the reaction was found exclusively at the outer surface of the cells. In controls, enzyme activity was abolished by diethyl pyrocarbonate and slightly stimulated by digitonin and concanavalin A, whereas sodium orthovanadate, N-ethylmaleimide, and sodium azide yielded no effect. Our results support the view that cardiac ecto-ATPase is involved in important physiological functions and suggest that its activity may be regulated by the release of ATP from nerve endings.
    Cell and Tissue Research 01/2000; 298(3):499-509. · 3.68 Impact Factor
  • T Okada, V S Zinchuk, T Kobayashi, H Seguchi
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    ABSTRACT: We studied cytochemical localization of ectoadenosine triphosphatase in the rat liver during development from 15-day-old fetus to 4-week-old and adult animal. First signs of the enzyme activity were found in some of the primitive bile canaliculi of 15-day-old fetuses. The majority of canaliculi, however, did not reveal any reaction product. Although intensity of the cytochemical reaction increased at 20 days of gestation, it still remained relatively low. Intensity of the reaction increased significantly and its product became readily detectable in the liver of newborn rats. Liver of 1-, 2-, and 4-week-old animals showed strong reaction for ecto-ATPase at the luminal surface of the plasma membrane of the bile canaliculi. Liver of adult rats contained a prominent reaction product similar to that seen in newborns, 1-, 2-, and 4-week-old animals. At all stages of fetal development, as well as in postnatal and adult rats, reaction was found only within the hepatic bile canalicular system and exclusively at the luminal surface of the canalicular plasma membrane. Using diethyl pyrocarbonate (DEPC), a specific inhibitor of ecto-ATPase activity, cytochemical reaction was blocked in all examined samples. Results of the present study, taken together with established biochemical and immunological data, provide conclusive morphological evidence in support of the view that canalicular ecto-ATPase is involved in bile acid efflux.
    Cell and Tissue Research 01/2000; 298(3):511-8. · 3.68 Impact Factor
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    ABSTRACT: Methods for studying human neutrophils at the ultrastructural level by enzyme cytochemistry and immunocytochemistry are presented. The focus of these methods is on the analysis of the alkaline phosphatase-positive secretory organelle of these cells. These methods provide unique information which, when coupled with biochemical studies, provide for the most complete analysis of neutrophil structure and function.
    Journal of Immunological Methods 01/2000; 232(1-2):169-78. · 2.23 Impact Factor
  • V S Zinchuk, T Okada, T Kobayashi
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    ABSTRACT: This paper describes protocol for the correlated light and electron microscopical histochemical visualization of the reaction product of ecto-ATPase activity in brain. The protocol employs a biochemically optimized incubation medium to adjust the appropriate kinetical parameters for detection of the histochemical reaction product by means of confocal laser scanning and electron microscopy. The reaction product is formed when the liberated inorganic phosphate is captured in histochemical reaction by the cerium ions. Confocal microscopy is performed in reflectance mode due to sufficient reflectance properties of the cerium-containing reaction product. Using this procedure, the prominent reaction of ecto-ATPase activity is readily detectable in hippocampus and cerebellum at sites where ATP is supposed to act as a synaptic neurotransmitter: on synapses of neurons in the pyramidal cell layer of hippocampus, in the granule cell layer of the dentate gyrus, and around synapse-containing areas in the granule cell layer of cerebellum. Reaction product is seen in close association with both pre- and postsynaptic membranes and exclusively extracellularly. Specificity of the visualization is justified in control experiments with diethyl pyrocarbonate, specific inhibitor of ecto-ATPase. The procedure is easy to perform, sensitive, and reproducible. It is recommended as a valuable tool in the arsenal of biochemical, immunochemical, and physiological techniques in studying signal transduction induced by extracellular ATP.
    Brain Research Protocols 01/2000; 4(3):258-65. · 1.82 Impact Factor
  • T Kobayashi, H Seguchi
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    ABSTRACT: We review herein the definition of the NADPH oxidase-activating site in human neutrophils and eosinophils, together with the new biochemical findings of the assembly of NADPH oxidase components and the signal transduction for the activation of NADPH oxidase. The activation of this enzyme is associated with multiple interrelated signaling pathways. Upon cell stimulation, the second messengers act on the assembly of NADPH oxidase components. The cytosolic components are first phosphorylated, and then associated with the membrane components. Small GTP-binding proteins and cytoskeletal components also participate in the activation of the NADPH oxidase. The cytochemical findings demonstrate that the superoxide generated by NADPH oxidase activity is initially localized in distinct types of intracellular granules, and not at the plasma membrane as previously believed. Thus, the assembly of NADPH oxidase components possibly occurs at the limiting membrane of the intracellular compartments. The oxidant-producing compartments mobilize and become associated with the plasma membrane upon cell stimulation with soluble stimulants, or fuse to phagosomes upon stimulation with particulate stimulants. Accordingly, superoxide is released to the extracellular space and into phagosomes in proportion to the oxidant-producing intracellular granule association with the plasma membrane and with the phagosomal membrane, respectively.
    Histology and histopathology 11/1999; 14(4):1295-308. · 2.28 Impact Factor
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    ABSTRACT: The aim of the present study was to detect oxidant-producing sites, and to elucidate their dynamic reorganization in human polymorphonuclear leukocytes (PMNs) fixed with glutaraldehyde which preserves cell structure. In biochemical analyses, the detectable O2- generation in unfixed PMNs upon stimulation with phorbol 12-myristate 13-acetate (PMA) in the presence of cytochalasin B was characterized by a lag period of approximately 10 sec followed by O2- production. The maximal rate reached was 3.18+/-0.07 nmol/min/l x 10(6) cells (mean+/-S.D.; n = 4) after 30 sec of stimulation. PMNs exposed to PMA and cytochalasin B followed by fixation with glutaraldehyde generated O2- without a lag period at a rate of 0.35+/-0.05 n mol/min/l x 10(6) cells (mean+/-S.D.) by the addition of NADPH as substrate to the cell suspension. In the cytochemical assays, we employed both cells exposed to PMA and cytochalasin B, and then fixed with glutaraldehyde followed by incubation in the cytochemical reaction medium (pre-fixed cells) and cells incubated in the medium containing PMA and cytochalasin B followed by fixation with glutaraldehyde (post-fixed cells). Oxidant reaction in the pre-fixed cells was detected by the addition of NADPH and FAD to the reaction medium. No oxidant-reaction product was seen in pre-fixed cells stimulated for 10 sec whereas the oxidant reaction was visualized in intracellular compartments of pre-fixed PMNs stimulated for 20 sec. The fact that the pre-fixed PMNs stimulated for 30 sec showed increased numbers of oxidant-producing structures compared to those seen in the pre-fixed cells stimulated for 20 sec, demonstrates that the amount of the reaction product and the number of oxidant-producing intracellular compartments increases between 20 and 30 sec after start of stimulation with PMA. These cytochemical results using the pre-fixed cells coincided with the findings obtained from the biochemical assays in the pre-fixed cells exposed to PMA and cytochalasin B. The oxidant reaction was observed in elongated tubular structures that were arranged in a radial fashion, and were associated with the plasma membrane in the pre-fixed PMNs, whereas post-fixed PMNs exhibited slender spherical or rod-shaped structures of various lengths. The present results indicate that the pre-fixed PMNs can be employed for elucidating the dynamic reorganization of oxidant-producing sites in human PMNs.
    The Histochemical Journal 04/1999; 31(3):181-94.
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    ABSTRACT: The involvement of ATP in synaptic transmission was examined in synapses on granule cells of the rat cerebellum using ecto-ATPase activity. Reaction product was found in a majority but not all synapses between axodendritic, axoaxonic, and dendrodendritic appositions of granule cells and was associated with extracellular surface of both pre- and postsynaptic membranes. Specificity of the detection was justified by using diethyl pyrocarbonate, specific inhibitor of ecto-ATPase activity. These observations provide direct morphological evidence in support of the view that ATP participates in synaptic transmission and indicate functional heterogeneity of synapses in cerebellum.
    Brain Research 02/1999; 815(1):111-5. · 2.88 Impact Factor
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    ABSTRACT: Human neutrophils possess alkaline phosphatase-containing intracellular granules which are upregulated to the cell surface upon stimulation. The mechanism that governs the intracellular dynamics of these granules is, however, poorly understood. The aim of the present study was to investigate the possible participation of GTP-binding proteins in the reorganization and exocytosis of the alkaline phosphatase-containing granules using electropermeabilized cells. Biochemical assays using intact neutrophils showed that the alkaline phosphatase activity was upregulated and exocytosed into the extracellular space upon stimulation with AIF4 and N-formyl peptide. This upregulation was inhibited by treatment of cells with pertussis toxin and botulinum toxin. Alkaline phosphatase activity was also upregulated in electropermeabilized cells stimulated with guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), but not with guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS). Cytochemically, alkaline phosphatase-containing granules were dispersed throughout the cytoplasm in unstimulated electropermeabilized neutrophils. Upon stimulation with GTPgammaS, but not with GDPbetaS, these granules fused to form elongated tubular structures which eventually became associated with the plasma membrane. Nocodazole disturbed the reorganization of the alkaline phosphatase-containing granules in cells stimulated with GTPgammaS. The results from this study indicate that GTP-binding proteins participate in the reorganization and exocytosis of alkaline phosphatase-containing granules associated with the microtubules in electropermeabilized human neutrophils.
    Histochemie 11/1998; 110(4):395-406. · 2.61 Impact Factor
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    ABSTRACT: Alkaline phosphatase (ALPase) activity was examined by cerium-based ultracytochemistry in isolated rat neutrophils following stimulation with phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP). In control neutrophils, low levels of ALPase activity were detected in small tubular and spherical compartments distributed throughout the cytoplasm. Neutrophils stimulated for 2.5, 5, 15, and 30 min with 50 ng/ml PMA or 10(-7) M fMLP displayed a time-dependent increase in ALPase activity. At 2.5 min, an increase in activity was first identified in compartments that were aggregated in the central regions of the cell. By 15 min, a dense precipitate was seen in tubular or elongated bead-like structures that extended to and made contact with the plasma membrane. Large enzyme-positive vacuoles were also observed in regions near the plasma membrane. At the longer stimulation times, a fine precipitate was present on the cell surface of the neutrophil in regions where subplasmalemmal ALPase activity was present. The results of this study indicate that an increase in activity and a redistribution of ALPase-positive structures occurs in neutrophils in response to stimulation with PMA and fMLP. It is likely that these compartments are latent pools of ALPase which, upon stimulation, fuse and mobilize the enzyme activity to the cell surface.
    Histology and histopathology 02/1998; 13(1):57-65. · 2.28 Impact Factor
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    T Kobayashi, J M Robinson, H Seguchi
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    ABSTRACT: In this study, we show that superoxide production is carried out within intracellular compartments of human neutrophils and not at the plasma membrane following stimulation with phorbol myristate acetate. Oxidant production was not observed in unstimulated cells. Stimulated cells exhibited superoxide production in two distinct types of intracellular organelles. Initially, activity was detected in slender rod-shaped granules and in spherical or elliptical granules. The oxidant-producing granules fused directly with the plasma membrane or fused to form larger intracellular vesicles which then became associated with the plasma membrane. Longer periods of stimulation with PMA resulted in a decrease in the number of vesicles containing oxidant reaction product only, and an increase in structures containing both the oxidant-reaction product and ferritin particles; the latter was used herein as a marker for endocytosis. Thus a complex pattern of intracellular vesicular trafficking occurs in stimulated neutrophils. Alkaline phosphatase activity, a marker enzyme for a type of intracellular neutrophil granule was co-localized in the oxidant reaction-positive intracellular compartments. The time course of up-regulation of alkaline phosphatase activity to the cell surface parallelled the release of superoxide from stimulated cells. Results from this study demonstrate for the first time cytochemical and morphological evidence that superoxide is released from stimulated neutrophils through exocytosis of an oxidant-producing intracellular granule.
    Journal of Cell Science 02/1998; 111 ( Pt 1):81-91. · 5.88 Impact Factor
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    ABSTRACT: We have demonstrated the alteration of the localization of ecto-ATPase activity in human neutrophils after stimulation with phorbol myristate acetate or N-formylmethionyl-leucyl-phenylalanine using a cerium-based cytochemical method. In unstimulated cells, the enzyme activity was observed on the plasma membrane. Both the diazonium salt of sulfanilic acid and diethylpyrocarbonate inhibited the production of the reaction precipitates. Within 2-3 min of stimulation, cells developed cytoplasmic projections (ruffles). The ecto-ATPase activity on the plasma membrane of these ruffles was, however, weaker than that at the non-ruffle-forming side. The ruffle-forming side (RFS) was also the site where elongated tubular structures positive for the enzyme reaction tended to concentrate and associated with the plasma membrane. The enzyme activity was also detected in intracellular compartments, which appeared predominantly in the RFS concomitantly with the disappearance of the enzyme activity from the plasma membrane. Using a series of thick sections and computer-assisted three-dimensional reconstruction, the enzyme reaction-positive internalized membranes were visualized as a complicated mass formed by enzyme reaction-positive vesicles which gathered together and were, at least in part, interconnected. The present results indicate that the detected enzyme reaction is a product of the ecto-ATPase activity, and that RFS possibly serves the membrane flow with respect to endocytosis.
    Histochemie 06/1997; 107(5):353-63. · 2.61 Impact Factor
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    ABSTRACT: Enzyme activity that represents ouabain-insensitive, potassium-dependent p-nitrophenylphosphatase (p-NPPase) was assessed in rat atrial myocytes by biochemical and cytochemical procedures. No activity was detected in parallel experiments with ventricular myocytes. Fixed tissues were incubated in a reaction medium containing Tricine buffer, p-nitrophenylphosphate (p-NPP), KCl, MgCl2, CaCl2, CeCl3. Triton X-100, levamisole, and ouabain. Final pH was adjusted to 7.5. Biochemical studies showed that accumulation of p-nitrophenol in the medium was increased proportionally in accordance with the amount of incubated tissue. This activity was optimal with incubation at pH 7.5 and in the presence of KCl. Approximately 70% of the enzyme was inhibited by 2 mM CeCl3. Electron microscopic observations revealed reaction product (RP) at sites of ouabain-insensitive, potassium-dependent p-NPPase activity as electron-dense precipitate localized at the inner surface of the plasma membrane and at the T-tubules of atrial myocytes. Control experiments indicated that the activity was strongly inhibited by sodium orthovanadate and was repressed by omeprazole and 1,3-dicyclohexylcarbodiimide. X-ray microanalysis confirmed the presence of cerium within the cytochemical RP. The ouabain-insensitive, K-dependent p-NPPase activity detected in the present study is considered to be an isoform of a P-type, H-transporting, K-dependent adenosine triphosphatase (H,K-ATPase).
    Journal of Histochemistry and Cytochemistry 03/1997; 45(2):177-87. · 2.26 Impact Factor
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    ABSTRACT: Alkaline phosphatase (ALPase) activity in neutrophils was examined by enzyme ultracytochemistry at sites of experimentally induced acute inflammation in the rat lung and compared with those in non-inflammatory regions. Neutrophil accumulations in the lung were stimulated by intraperitoneal (IP) injection and intratracheal (IT) instillation of lipopolysaccharide (LPS), an endotoxin from Escherichia coli. Microsliced sections of fixed lung were incubated in a cerium-based reaction medium for the detection of ALPase activity. As cytochemical controls, sections were incubated in a substrate-free medium or in a medium containing 2 mM levamisole for inhibition of ALPase activity. Both IP and IT injections resulted in a significant accumulation of neutrophils in the lung, however, their histological distributions differed, the former stimulating high accumulations within the capillaries and interalveolar spaces, and the latter within the confines of the alveolar spaces. In neutrophils from controls and non-inflamed regions of the lung, ALPase activity was detected as an electron-dense reaction product localized predominantly to small spherical and tubular membrane-bounded cytoplasmic compartments. In the IP-injected rats, prominent ALPase activity was observed at their plasma membrane surfaces, most notably at sites of endothelial cell contact. Following IT instillation of LPS, neutrophils suspended in the alveolar spaces showed a reduced plasma membrane reactivity, however, at type I pneumocyte contact regions, enzyme activity was significantly increased. In all cases, ALPase activity was not detected at the endothelial plasma membranes. Some reaction, however, was seen on the microvilli of the type II surfactant-producing cells. These results indicate that ALPase activity of rat neutrophils in the lung can be increased by LPS injection and that its activity may also be related to the sites of cell-cell interaction and cell surroundings.
    Kaibogaku Zasshi 07/1996; 71(3):183-94.
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    ABSTRACT: Parenchymal and stromal components of the rat parotid and submandibular glands were examined by conventional and high resolution scanning electron microscopy (HRSEM). Freeze-fractured specimens were subjected to HCl and NaOH extraction procedures to better differentiate connective tissue and cellular components. In addition, the internal three-dimensional morphology of the secretory acinar cells and duct cells was revealed by maceration with a dilute osmium tetroxide solution to selectively remove some of the cytoplasmic components. SEM and HRSEM examination of the HCl-treated samples of both glands revealed a fine filamentous network immediately surrounding each acinus. Coarser bundles of collagen that linked neighboring acini were also observed. NaOH-extracted samples selectively removed the cellular components and showed more clearly the three-dimensional structure of the connective-tissue stroma. A dense-collagenous network surrounded each lobule while more internal regions consisted of a honeycomb-like pattern of evacuated spaces previously occupied by secretory acini. These spaces were smoothened in appearance and often interconnected. Apically-located secretory granules and profiles of the rough endoplasmic reticulum and Golgi apparatus in perinuclear regions were encountered in the acinar and duct cells of macerated samples by HRSEM. In addition, a phenylephrine-induced experimental condition performed in some rats resulted in a significant increase in secretory granule size and density of the serous cells.
    Histology and histopathology 02/1996; 11(1):103-10. · 2.28 Impact Factor