Biomedical subjects

S Ohkuma

Publications and source records attributed to S Ohkuma.

At least 73 records · Page 4Linked to original sources

PubMed1996

Expression of 16 kDa proteolipid of vacuolar-type H(+)-ATPase in human pancreatic cancer.

Recent studies have shown that bafilomycin A1-sensitive vacuolar-type H(+)-ATPase (V-ATPase) plays important roles in cell growth and differentiation. However, there is no published study that has focused on the expression of V-ATPase in human tumour tissues. This study was designed to examine the mRNA and protein levels for the 16 kilodalton (kDa) proteolipid of V-ATPase in human pancreatic carcinoma tissues. We first investigated the mRNA level for V-ATPase in six cases of invasive pancreatic cancers and two normal pancreases, using reverse transcription-polymerase chain reaction technique. Then, we examined immunohistochemically the level of V-ATPase protein in 49 pancreatic cancers and ten benign cystic neoplasms of the pancreas, using antisera raised against the 16 kDa proteolipid. There was a notable difference in the level of V-ATPase mRNA between normal and pancreatic carcinoma tissues, with no evident difference in the expression of the beta-actin gene. Immunohistochemically, 42 out of 46 invasive ductal cancers (92%) displayed a mild to marked immunoreactivity for V-ATPase in the cytoplasm, whereas neither non-invasive ductal cancers nor benign cystic neoplasms expressed detectable immunoreactive proteins. These findings suggest that the overexpression of V-ATPase protein is characteristic of invasive pancreatic tumours. V-ATPase may play some crucial roles in tumour progression.

Adenocarcinoma↗

PubMed1996

Properties of H(+)-ATPase from rat liver lysosomes as revealed by reconstitution into proteoliposomes.

The properties of H(+)-ATPase from rat liver lysosomes were analyzed by reconstituting proton pump activity from solubilized enzyme and Escherichia coli phospholipids in proteoliposomes devoid of anion-channels. The reconstitution procedure involved solubilization of the ATPase with n-octyl-beta-D-thioglucoside in the presence of asolectin, and incorporation of the solubilized enzyme into E. coli phospholipid liposomes by dilution, freeze-thawing, and sonication. Proton pump activity of reconstituted H(+)-ATPase as detected by the ATP-dependent quenching of acridine orange fluorescence indicated that ATP can be replaced with dATP and to a lesser extent with GTP, but not with any other nucleotide, that Mg2+ can be replaced with Mn2+, but not with Ca2+, Sr2+, or Ba2+, that Zn2+, Pd2+, Cd2+, and Hg2+ were inhibitory, and that the enzyme was sensitive to inhibitors of v-type H(+)-ATPase, including bafilomycin A1, N-ethylmaleimide, DCCD, DIDS, and tri-n-butyltin. The enzyme showed unique sensitivity to anions and was activated by chloride, fluoride, and bromide from inside, but not from outside the vesicles. It was inhibited by sulfate, sulfite, and thiocyanate from outside the vesicles, and by nitrate from both inside and outside the vesicles.

Animals↗

PubMed1996

A comparative study of (Ca(2+)-Mg2+)-ATPase on the lysosomal membrane and ecto-ATPase on the plasma membrane from rat liver.

We showed that rat liver lysosomes possess at least two types of ATPase besides H(+)-translocating ATPase (H(+)-ATPase); namely, N-ethylmaleimide (NEM)-sensitive and bafilomycin A1-insensitive Mg(2+)-ATPase [ATPase I] and NEM- and bafilomycin A1-insensitive (Ca(2+)-Mg2+)-ATPase [ATPase II] [Hayashi H., et al. Chem. Pharm. Bull., 37, 2783-2786 (1992)]. Subcellular fractionation showed the presence of similar activity of (Ca(2+)-Mg2+)-ATPase not only in the Golgi but also in the plasma membrane fractions, of which plasma membrane had the highest activity, most probably due to the ecto-ATPase. In this study, we partially purified the (Ca(2+)-Mg2+)-ATPases from both lysosomal and plasma membranes and compared their properties. Both enzymes had quite similar characteristics including: (1) broad pH-activity profiles with two pH optima at 4.5 and 7.0, (2) K(m) values for ATP being 21-27 microM (at pH 4.5) and 18-14 microM (at pH 7.0) (in the presence of CaCl2), (3) hydrolysis of both nucleoside triphosphates and diphosphate (ADP), (4) inhibition only by vanadate and 4,4'-diisothiocyanatostilbene 2,2'-disulfonic acid (DIDS) at pH 4.0 (but not at pH 7.0), (5) acidic pI values that were shifted by neuraminidase digestion, and (6) a positive reaction against an antibody to the N-terminal peptide of ecto-ATPase.

Adenosine Triphosphatases↗

PubMed1996

[A new strategy for the therapy of pancreatic cancer by proton pump inhibitor].

Bafilomycin A1 is a specific inhibitor of vacuolar type proton pump (V-ATPase). This study was designed to examine the effect of bafilomycin A1 on the growth of Capan-1 human pancreatic cells which overexpress V-ATPase. Nude mice bearing a xenografted tumor of Capan-1 cell line were treated for 4 weeks with bafilomycin A1 (1.0 mg/kg/day). This treatment inhibited tumor growth, which was significantly reduced as compared with controls after 21 days (p < 0.05). However, there were no significant differences in body weights between groups. Microscopically, a large number of tumor cells in the treated group showed signs of apoptosis. These findings suggest that apoptosis induced by bafilomycin A1 was the event involved in suppression of tumor growth in vivo.

Animals↗

PubMed1996

The role of vacuolar H(+)-ATPase in the control of intragranular pH and exocytosis in eosinophils.

The presence of vacuolar H(+)-ATPase (V-ATPase) on exocytotic granules in eosinophils and the role of this enzyme in exocytosis were explored in this study. Antibody against 116-kd subunit of V-ATPase positively stained eosinophil granules in immunofluorescence analysis. When eosinophil lysate was extracted immunomagnetically with the same antibody, the extracted fraction contained a considerable amount of eosinophil peroxidase, a marker of eosinophil-specific granules, which indicates that V-ATPase was present on the membranes of eosinophil exsosomal granules. The pH of the eosinophil granules, measured fluorometrically with acridine orange as a delta pH-sensitive dye, was estimated to be 5.1. The acidity of the eosinophil granules was perturbed by bafilomycin A1, a potent selective inhibitor of V-ATPase, which indicates that the low pH of these granules is maintained by V-ATPase activity. BafilomycinA1 and NH4Cl, both of which raise the intragranular pH to neutral, inhibited the eosinophil peroxidase exocytosis induced by platelet-activating factor. These agents did not, however, affect the changes in cytosolic free calcium concentration [Ca2+]i induced by platelet-activating factor. These observations suggest that bafilomycin A1 inhibited a delta pH-requiring step in eosinophil exocytosis that was preceded by the [Ca2+]i transient in the signal transduction pathway, and, hence, the findings suggest the pivotal role of V-ATPase in maintaining intragranular pH and its function of eosinophil exosomal granules.

Ammonium Chloride↗

PubMed1995

Ethanol stimulates diazepam binding inhibitor (DBI) mRNA expression in primary cultured neurons.

Changes in expression of diazepam binding inhibitor (DBI) mRNA in cerebral cortical neurons following long-term ethanol (EtOH) exposure were examined. A significant increase in DBI mRNA expression was observed by the exposure of neurons to 50 mM EtOH for up to 5 days and to EtOH (1-100 mM) for 3 days. These EtOH-induced increases in DBI mRNA expression were further elevated after the additional cultivation of neurons under EtOH-free condition. beta-Actin mRNA expression was not altered by similar EtOH treatments. These results indicate that EtOH possesses the activity to increase the expression of DBI mRNA in cerebral cortical neurons.

Animals↗

PubMed1995

Hydroxyl radical scavengers enhance nitric oxide-evoked acetylcholine release from mouse cerebral cortical neurons.

The effect of hydroxyl radical scavengers on acetylcholine (ACh) release evoked by nitric oxide (NO) generators and N-methyl-D-aspartate (NMDA) was investigated. Dimethylthiourea enhanced dose-dependently NO generators-evoked ACh release. Similarly, uric acid and mannitol significantly facilitated ACh release evoked by NO generators. The NMDA-induced ACh release was also significantly facilitated by hydroxyl radical scavengers. These scavengers themselves showed no effects on ACh release. These results suggest that hydroxyl radicals may modify the mechanism for NO-evoked ACh release.

Acetylcholine↗

PubMed1995

Facilitation of N-methyl-D-aspartate-evoked acetylcholine release by hydroxyl radical scavengers.

Effects of hydroxyl radical scavengers such as dimethylthiourea (DMTU), uric acid and mannitol on acetylcholine (ACh) release evoked by N-methyl-D-aspartate (NMDA) were examined in cerebral cortical neurones. NMDA increased dose-dependently ACh release. This increase in the release was significantly suppressed by MK-801, N omega-nitro-L-arginine and haemoglobin, indicating that NMDA evokes ACh release via the formation of NO subsequent to NMDA receptor activation. DMTU dose-dependently enhanced the NMDA-induced ACh release. Uric acid (100 microM) and mannitol (1 mM) facilitated significantly the NMDA-induced ACh release, although these scavengers themselves showed no effects on ACh release in the absence of NMDA. These results indicate that the removal of hydroxyl radical facilitates the NO-induced ACh release.

Acetylcholine↗

PubMed1995

Removal of hydroxyl radical increases nitric oxide generators-induced [3H]GABA release from mouse cerebral cortical neurons.

We investigated the effect of removal of hydroxyl radical on nitric oxide (NO)-induced [3H]GABA release from cerebral cortical neurons. NO generators, S-nitroso-N-acetylpenicillamine and sodium nitroprusside, significantly increased [3H]GABA release, which was completely abolished by hemoglobin. These results indicate that the release of [3H]GABA evoked by these NO generators is mediated by NO formation. N,N'-Dimethylthiourea (DMTU), a hydroxyl radical scavenger, dose-dependently stimulated the increase of NO-induced [3H]GABA release. Similarly, other hydroxyl radical scavengers such as uric acid and mannitol, significantly enhanced the NO-induced [3H]GABA release. On the other hand, each hydroxyl radical scavenger alone showed no effect on [3H]GABA release in the absence of NO generators. These results indicate that the removal of hydroxyl radical enhances NO-evoked [3H]GABA release from cerebral cortical neurons.

Animals↗

PubMed1995

Prodigiosin 25-C uncouples vacuolar type H(+)-ATPase, inhibits vacuolar acidification and affects glycoprotein processing.

Prodigiosin 25-C inhibited the accumulation of 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine and acridine orange in the acidic compartments of baby hamster kidney cells with little perturbation of cellular ATP levels. In rat liver lysosomes, prodigiosin 25-C inhibited the proton pump activity with an IC50 of approximately 30 nM, but did not affect ATPase activity up to 1 microM. It also delayed the transport of vesicular stomatitis virus G protein and induced a drastic swelling of Golgi apparatus and mitochondria. These results indicate that prodigiosin 25-C raises the pH of acidic compartments through inhibition of the proton pump activity of vacuolar type H(+)-ATPase, thereby causing the functional and morphological changes to the Golgi apparatus.

Adenosine Triphosphate↗

PubMed1995

Participation of peroxynitrite in acetylcholine release induced by nitric oxide generators.

Peroxynitrite is a product produced by spontaneous reaction of nitric oxide (NO) with superoxide. Functional roles of peroxynitrite in the release of endogenous acetylcholine evoked by NO generators has been examined using primary-cultured cerebral cortical neurons. NO generators, such as sodium nitroprusside and S-nitroso-N-acetylpenicillamine, dose-dependently increased the release of acetylcholine (ACh); such increase of the release was significantly suppressed by hemoglobin which has the capacity to abolish biological effects of NO. Two types of superoxide scavengers, Cu2+, Zn2+ superoxide dismutase and ceruloplasmin, significantly reduced the NO-evoked ACh release. These results indicate that NO requires superoxide to evoke the release of ACh. Synthesized peroxynitrite evoked the release of ACh from cerebral cortical neurons in a dose-dependent manner. Thus, it is indicated that the NO-evoked ACh release is mediated, at least in part, by peroxynitrite produced by the reaction of NO with superoxide.

Acetylcholine↗

PubMed1995

Expression and localization of mRNA encoding 16 kD subunit of vacuolar H(+)-ATPase in rat brain.

The 16 kD subunit of vacuolar H(+)-ATPase (V-ATPase) is essential for pumping protons into intracellular organella. Northern blot analysis revealed that the 16 kD subunit of V-ATPase is abundantly expressed in rat brain, and that the amount of transcripts is higher in the postnatal 15 days than in the adult age. On in situ hybridization, the mRNA signal for 16 kD subunit of V-ATPase was detected widely over the brain tissues of both adult and postnatal rats, with higher intensity in most of the gray matters located in the cerebellar cortex, hippocampus, thalamic nuclei, pontin nuclei, and olfactory bulb. In contrast, the signal in the cerebral cortex was preferentially enriched in the surface layers in the postnatal 10 days but was distributed diffusely and weakly over the entire layers thereafter. In all regions of brain, the mRNA signal was located predominantly in the neurons rather than in the glial components.

Animals↗

PubMed1995

Involvement of peroxynitrite in N-methyl-D-aspartate- and sodium nitroprusside-induced release of acetylcholine from mouse cerebral cortical neurons.

Functional roles of peroxynitrite in N-methyl-D-aspartate (NMDA)- and sodium nitroprusside (SNP)-evoked releases of acetylcholine (ACh) from cerebral cortical neurons in primary culture have been investigated. NMDA increased the release of ACh in a dose-dependent manner, which was significantly suppressed by (+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cycloheptan-5,10-imine (MK-801), a non-competitive antagonist specific for the NMDA receptor complex, and NO synthase inhibitors. SNP also showed a concentration-dependent increase in ACh release. Hemoglobin significantly abolished the stimulatory effects of both NMDA and SNP on ACh release. In addition, superoxide anion scavengers such as superoxide dismutase and ceruloplasmin significantly reduced the increased ACh release evoked by NMDA and SNP. Synthesized peroxynitrite dose-dependently elevated the release of ACh. These results indicate that the increased release of ACh by NMDA and SNP is mediated through peroxynitrite formed in the reaction of superoxide anion with nitric oxide produced by NMDA receptor activation and liberated from SNP rather than nitric oxide itself.

Acetylcholine↗

PubMed1995

Presence of Vicia graminea lectin- or Vicia unijuga lectin-binding (Vgu) glycoproteins, Vgu glycoproteins with Thomsen-Friedenreich (T) activity and T-active glycoproteins in human meconium.

Perchloric acid-soluble fraction prepared from a mixture of meconiums of 22 newborn babies was subjected to a systematic affinity chromatography using Vicia unijuga lectin-Cellulofine column and Arachis hypogaea lectin-Agarose column and separated into four fractions, Vgu glycoprotein, Vgu glycoprotein with T activity, T-active glycoprotein and another glycoprotein fractions. HPLC analysis showed that the first fraction contained three glycoproteins with molecular weight (Mw) of about 19,400 kDa, 5500 kDa and 1900 kDa, the second fraction consisted of two glycoproteins with Mw of about 460 kDa and 150 kDa and the third fraction composed of several glycoproteins including the main glycoprotein with Mw of about 11,700 kDa.

Amino Acid Sequence↗

PubMed1995

Nitric oxide-induced [3H] GABA release from cerebral cortical neurons is mediated by peroxynitrite.

The functional significance of peroxynitrite in the release of [3H]GABA induced by nitric oxide (NO) liberated from NO generators was investigated using cerebral cortical neurons in primary culture. NO generators such as sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) increased [3H] GABA release in a dose-dependent manner. These increases in [3H]-GABA release were significantly inhibited by hemoglobin, indicating that those NO generators evoke the release of [3H] GABA by the formation of NO. Two types of superoxide scavengers, Cu2+/Zn2+ superoxide dismutase and ceruloplasmin, significantly reduced the increase in [3H]-GABA release induced by both SNP and SNAP, which assumes that NO requires superoxide to induce [3H]-GABA release from the neurons. In addition, synthesized peroxynitrite induced a dose-dependent increase in [3H]-GABA release from the neurons. These results indicate that NO-induced [3H] GABA release is mediated by peroxynitrite formed by the reaction of NO with superoxide.

Animals↗

PubMed1995

Expression and localization of mRNA for the 16 KD subunit of V-ATPase in the rat embryo.

Vacuolar H(+)-ATPase (V-ATPase), an enzyme composed of multisubunits, is located in the membrane of intracellular organelles (e.g., lysosomes, and endosomes) and maintains the intraorganellar acidic pH by pumping protons across the membrane. Although there is growing evidence for some important role of V-ATPase in cell proliferation and differentiation, the functional significance of V-ATPase in vivo during mammalian development remains obscure. In the present study we investigated the expression and localization of mRNA for the 16 KD subunit of V-ATPase, an essential sector for enzymatic activity, in prenatal rat by Northern blot analysis and in situ hybridization with a specific oligonucleotide probe. With Northern blot analysis, consistent expression of the mRNA was observed in the embryos throughout the period examined (E14-E20). On in situ hybridization, mRNA signal was distributed with various intensities in both the epithelial and mesenchymal tissues at embryonic day 14 (E14). In E17 and E20 embryos, localization of strong signal became more restricted to distinct mesenchymal cells such as fibroblasts adjacent to the epithelia of skin, lung, and intestine, the cells of perichondrium, and myoblasts in the process of fusion. These results suggest that V-ATPase performs specific functions during the later stages of embryogenesis, especially at sites of mesenchymal differentiation and epithelium-mesenchyme interaction.

Adenosine Triphosphatases↗

PubMed1995

[Recent advances in studies of GABA receptors: neurochemical aspects].

gamma-Aminobutyric acid (GABA) has been established as a major inhibitory neurotransmitter in the brain. The GABA-induced inhibitory transmission is mediated by two distinct types of GABA receptors which are termed as GABAA and GABAA receptors. The GABAA receptor forms a Cl- channel that consists of several subunits. The recent development of molecular cloning clarified the presence of multiple and heterogeneous molecules in the GABAA receptor subunits. Therefore, it is suggested that multiple GABAB receptors generate various functions in the brain. In contrast, the GABAB receptor exhibits various metabotropic actions for the inhibitory neurotransmission, since it is coupled with GTP-binding proteins. The GABAB receptor of approximately 80 kDa protein in its molecular weight was purified from the bovine cerebral cortex and the presence of another molecular species of the GABAB receptor was also suspected. These results indicate that studies on the molecular diversity of the GABA receptors is important for elucidating the functional roles of GABAergic neurons in the brain.

Amino Acid Sequence↗

PubMed1995

[Functional significance of diazepam binding inhibitor (DBI) in establishment of alcohol dependence].

This review summarizes the data showing the possible involvement of an endogenous ligand for benzodiazepine (BDZ) receptors, diazepam binding inhibitor (DBI), in the development of alcohol dependence. The expression of cerebral DBI mRNA significantly increased in EtOH-inhaled and EtOH-withdrawn mice in comparison with that in EtOH-untreated mice, whereas the DBI mRNA level was not altered after a single administration of EtOH. After EtOH-withdrawal, this increase in the DBI mRNA expression in the mouse cerebral cortex diminished over 14 days despite the disappearance of withdrawal signs within 2 days after the withdrawal of EtOH. Simultaneous administration of flunitrazepam, a BDZ receptor agonist, with EtOH completely abolished the EtOH-induced increase in DBI mRNA expression. These results lead to the assumption that the changes in the expression of cerebral DBI mRNA induced by continuous EtOH treatment may be involved in the establishment of alcohol dependence, and such changes may be also regulated by BDZ receptors.

Alcoholism↗