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S Ohkuma

Publications and source records attributed to S Ohkuma.

At least 55 records · Page 3Linked to original sources
PubMed1998

ARF-induced lysosomal lysis in vitro.

Cytosol treated with guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) disintegrated lysosomes in a dose-dependent manner, as detected as the release of preloaded fluorescein isothiocyanate-dextran. The effect of GTPgammaS was suppressed by GTP or GDP, indicating a role of a GTP binding protein (G-protein) in the lysis [Sai, Y. et al. (1994) Biochem. Biophys. Res. Commun. 198, 869-877]. Gel filtration of cytosol and GTP-ligand blotting showed that a small GTP-binding protein participated in the lysosomal lysis. We partially purified the G-protein from rat liver cytosol and identified it as ARF1. GTPgammaS-stimulated lysis was reconstituted with ARF1 purified from bovine brain cytosol or recombinant ARF1. ARF bound to lysosomal membranes depending upon GTPgammaS in a dose-dependent manner. These results suggest that the transfer of ARF from the cytosol to the lysosomal membrane is necessary for GTPgammaS-stimulated lysis of lysosomes.

ADP-Ribosylation Factor 1↗
PubMed1998

Prodigiosins uncouple mitochondrial and bacterial F-ATPases: evidence for their H+/Cl- symport activity.

Prodigiosin, metacycloprodigiosin, and prodigiosin 25-C all inhibited the acidification activity of submitochondrial and bacterial (Escherichia coli) F-ATPases (FoF1-ATPases) strongly (IC50 = 20-30 and 24-30 pmol/mg protein, respectively), without affecting significantly the ATP hydrolysis activity. Their effect on the acidification activity was rapid and reversible, showing non-competitive apparent Ki values of the order of nM to sub-nM. However, unlike FCCP (an ordinary uncoupler of oxidative phosphorylation), they showed no protonophoric activity, as demonstrated by the absence of acceleration of ATP hydrolysis. Prodigiosins also inhibited the acidification of proteoliposomes reconstituted from phospholipids and purified F-ATPase of E. coli, suggesting that their acidification-inhibitory effect is not due to the inhibition of anion channels. They did not, however, inhibit the ATP-dependent formation of membrane potential of F-ATPase vesicles. Furthermore, they inhibited and quickly reversed acidification by F-ATPase only in the presence of chloride, and not in the presence of gluconate anion. Finally, they induced swelling of liposomes and submitochondrial particles in isotonic solution of ammonium chloride but not ammonium gluconate, suggesting that intravesicular entry of Cl- is promoted by prodigiosins. These results suggest that prodigiosins uncouple F-ATPases through promotion of H+/Cl- symport (or OH-/Cl- exchange) across vesicular membranes.

Animals↗
PubMed1998

Influence of ammonium chloride on the tissue distribution of anticholinergic drugs in rats.

Ammonium chloride (NH4Cl) increases lysosomal pH and thereby abolishes intralysosomal accumulation of drugs. Its effect on the tissue distribution of biperiden and trihexyphenidyl in rats has been investigated. The tissue-plasma concentration ratios (Kp) of these drugs in various tissues were determined by infusion studies at steady-state in the presence or absence of NH4Cl. Treatment with NH4Cl reduced the Kp values for both drugs, causing the largest reduction in Kp in the lung (52.1 to 11.8 for biperiden and 59.5 to 18.9 for trihexyphenidyl; ratios of decrease 0.77 and 0.68, respectively), followed by the heart and kidneys, with relatively small reductions in the brain, gut, muscle and fat. Subcellular fractionation studies in the lung indicated that the subcellular fraction-plasma concentration ratio of each drug at the steady state (K(p,sf)) was reduced by treatment with NH4Cl, with the largest decrease in the post-nuclear fraction (ratio of decrease 0.82 for biperiden and 0.74 for trihexyphenidyl), followed by the nucleus, microsomes and supernatant, in that order. A strong correlation was found between the ratio of decrease in K(p,sf) after NH4Cl treatment and the specific activity of acid phosphatases, a marker of lysosomes, in each fraction (biperiden, r = 0.948; trihexyphenidyl, r = 0.945). These results suggest that acidic organelles contribute significantly to the distribution kinetics of anticholinergic drugs.

Adipose Tissue↗
PubMed1998

Characteristic subcellular distribution, in brain, heart and lung, of biperiden, trihexyphenidyl, and (-)-quinuclidinyl benzylate in rats.

The subcellular distribution of biperiden (BP), trihexyphenidyl (TP) and (-)-quinuclidinyl benzylate (QNB) in brain, heart and lung following high dose (3.2 mg/kg) i.v. administration was investigated in rats. The subcellular distribution of BP or TP used clinically conformed with that of QNB, a typical potent central muscarinic antagonist. The concentration-time courses of the brain subcellular fractions for these drugs were of two types which decreased slowly and in parallel to the plasma concentration. The subcellular distribution in the brain and heart was dependent on the protein amount of each fraction. The percent post-nuclear fraction (P2) of the total concentration in the lung was characteristically about 3-5 times larger than that in the heart. It was elucidated that the distribution in the lung differs from that in the brain and heart, with high affinity which is not dependent on the protein amount in the P2 fraction containing lysosomes. On the other hand, at a low dose (650 ng/kg) of 3H-QNB, each fraction as a percentage of the total concentration in the brain increased in synaptic membrane and synaptic vesicles and decreased in nuclei and cytosol as compared with the high dose. These results show that although the tissue concentration-time courses of anticholinergic drugs appear to decrease simply in parallel to plasma concentration, the subcellular distribution exhibits a variety of patterns among various tissues.

Animals↗
PubMed1998

Uptake of basic drugs into rat lung granule fraction in vitro.

Although basic drugs are distributed widely in various tissues, they are characteristically concentrated in the lung granule fraction. We examined the uptake of seven lipophilic basic drugs into rat lung granule fraction (P2) in vitro and investigated the contributions of drug lipophilicity and lysosomal trapping to the characteristic lung P2 distribution. The uptake of each drug into P2 was examined at various pH values. The drug concentration in P2 was determined by gas chromatography. Biperiden (BP) was rapidly taken up into P2, reaching a maximal concentration within 1 min at pH 7.4 at both 4 degrees C and 37 degrees C. Both BP and chlorpromazine uptake into P2 was biphasic. Though the uptake rates of the seven drugs into P2 increased with rising pH, the rate of increase varied for each drug. There was a good correlation between the octanol-water partition coefficient of the non-ionized form (P(oct)) of each drug and the uptake into P2 in the presence or absence of NH4Cl, which inhibits lysosomal trapping. However, uptake into P2 in the presence of NH4Cl showed a stronger P(oct)-dependency. We conclude that the distribution of basic drugs into lung P2 is dependent on both drug lipophilicity and lysosomal uptake.

Ammonium Chloride↗
PubMed1998

[Functional involvement of diazepam binding inhibitor (DBI) in the establishment of drug dependence].

In this paper, we have reviewed the possibility of the functional involvement of an endogeneous neuropeptide for benzodiazepine receptors, diazepam binding inhibitor (DBI), in the development of drug dependence and the establishment of its withdrawal symptoms. The cerebral levels of DBI protein and its mRNA significantly increased in alcohol-dependent mice, and alcohol-withdrawal caused further increases of both DBI protein and its mRNA. These increases in the mouse cerebral cortex diminished over 14 days after alcohol-withdrawal. In the cerebrocortical neurons, similar patterns of DBI protein and its mRNA were observed during long-term exposure to alcohol and after its withdrawal. Simultaneous exposures of benzodiazepine receptor agonist, antagonist and inverse agonist with alcohol abolished the alcohol-induced increase in DBI mRNA expression in the neurons. Furthermore, the DBI contents and its mRNA expressions were also increased in the cerebral cortex obtained from nicotine-dependent mice and the neurons after long-term treatment with nicotine. The nicotine-induced increases in DBI content and its mRNA expression in the mouse cerebral cortex and the neurons were completely abolished in the presence of an antagonist for nicotinic acetylcholine receptor. These results suggest that the changes in the levels of cerebral DBI induced by continuous treatments with alcohol and nicotine may be involved in the establishment of dependence by alcohol and nicotine, and such changes may be regulated by the benzodiazepine and nicotinic acetylcholine receptors, respectively.

Alcoholism↗
PubMed1997

Role of cerebral spermidine in the development of sensitization to convulsant activity of cocaine and lidocaine.

We have previously shown that daily treatment with subconvulsant dose of cocaine resulted in the elevation of brain levels of polyamines such as putrescine and spermidine and the development of increased susceptibility to cocaine-induced seizures. The present study examined whether exogenously administered polyamines affect seizure activity caused by various doses of cocaine and lidocaine in mice. Thirty minutes after intracerebroventricular treatments with either saline, putrescine or spermidine (1-4 mumol), animals were injected intraperitoneally with cocaine or lidocaine (60-90 mg/kg); then the occurrence of clonic seizures was observed. Spermidine enhanced cocaine-induced seizure activity, while putrescine had no effect on it. Lidocaine-induced convulsions were also dose-dependently potentiated by spermidine. In addition, spermidine significantly enhanced seizure activity following an injection of N-methyl-DL-aspartate. The results suggest that spermidine plays an important role in the development of sensitization to convulsant activity by cocaine and lidocaine via modulation of N-methyl-D-aspartate receptors.

Animals↗
PubMed1997

Vinconate, a cognitive enhancer, and PI turnover-phospholipase C systems in the brain.

The molecular mechanisms for the stimulation of inositol 1-phosphate (IP1) formation by vinconate were investigated using preparations of rat brain. Vinconate (10(-8)-10(-3) M) dose-dependently inhibited the binding of [3H]quinuclidinyl benzilate ([3H]QNB) to muscarinic acetylcholine receptors and its IC50 value for [3H]QNB binding was 1.7 x 10(-5) M. The rightward shift of carbachol displacement curve of [3H]QNB binding by GTP (10(-4) M) was completely abolished by vinconate (10(-5) M). Carbachol (10(-8)-10(-2) M) increased [3H]IP1 formation in a dose-dependent manner and the carbachol-induced [3H]IP1 formation was significantly accentuated by vinconate (10(-5) M). The enhancement of [3H]IP1 accumulation by vinconate was inhibited by approximately 50% in the presence of atropine (10(-5) M), although phentolamine and ketanserin had no effects on the stimulatory effect of vinconate on [3H]IP1 formation. Vinconate showed no alteration in the binding of [3H]guanosine 5'-(beta, gamma-imino) triphosphate ([3H]Gpp(NH)p) to the crude synaptic membranes. The enhancement of phosphatidylinositol 4,5-biphosphate (PIP2)-specific phospholipase C (PLC) activity by GTP was unaffected in the presence of 10(-3) M vinconate, whereas vinconate alone dose-dependently enhanced the activities of both PIP2-specific and cytosolic PLC. These results suggest that vinconate may induce the facilitation of phosphatidylinositide (PI) turnover via the stimulation of muscarinic acetylcholine receptors, the enhancement of coupling between muscarinic acetylcholine receptors and GTP-binding protein, and the direct activations of PIP2-specific and cytosolic PLC.

Animals↗
PubMed1997

ATP-dependent lysis of isolated lysosomes by basic substances and acidic ionophores.

We established an in vitro cell-free system with which to evaluate the effects of basic substances and acidic ionophores on the internal pH and integrity of FITC-dextran (FD)-loaded lysosomes isolated from the rat liver. In this system, basic substances and acidic ionophores not only increased the internal pH dose-dependently, but also disrupted the lysosomes in the presence of Mg-ATP, which was detected as the release of FD from lysosomes. All of the vacuoligenic bases and acidic ionophores, but none of the non-vacuoligenic bases or neutral ionophores disrupted the lysosomes, suggesting that this phenomenon is an vitro manifestation of vacuole formation induced in vivo by basic substances and acidic ionophores. Lysosome disruption required a functional proton pump as well as permeant anions. It was inhibited by inhibitors of the lysosomal proton pump, including bafilomycin A1, N-ethylmaleimide (NEM), and N, N'-dicyclohexylcarbodiimide (DCCD), or when permeant anions were replaced with impermeant anions. It was also suppressed by increasing the osmotic pressure of the surrounding medium, suggesting that it was caused by osmotic swelling of lysosomes induced by protonated bases or cations characteristic of particular ionophores that accumulated within lysosomes driven by the proton pump. Furthermore, this lysosomal disruption was inhibited by cytosolic factors. This phenomenon will provide an in vitro system for studies on osmoregulation and the intracellular dynamics of the lysosomal system, including membrane fusion.

Adenosine Triphosphate↗
PubMed1997

Investigation of Vicia graminea lectin- and Vicia unijuga lectin-binding glycoproteins as novel oncofetal antigens in cyst and ascitic fluids of human ovarian tumors in benign, dysplastic, and malignant stages.

Perchloric acid-soluble fractions (PASFs) were obtained from cyst fluids of human benign ovarian mucinous cystadenoma, benign dermoid cyst, "borderline" mucinous cystadenoma, and malignant ovarian clear cell carcinoma, and from fluids of malignant embryonal carcinoma and malignant serous cystadenocarcinoma. PASFs from benign lesions did not react with Arachis hypogaea lectin (PNA), Vicia graminea lectin (VGA), or Vicia unijuga lectin (VUA). PASFs from "borderline" lesions reacted with PNA but did not react with VGA and VUA. PASFs from malignant tumors reacted with PNA, VGA, and VUA. These PASFs did not react positively against anti-M or -N sera. These results show that VGA- and VUA-binding glycoprotein (Vgu glycoprotein) exist in PASFs from malignant ovarian tumors, and that PNA-binding glycoprotein was present in PASFs from malignant and "borderline" ovarian tumors. Furthermore, we found the existence of Vgu glycoprotein in PASFs from various human cancer cell lines; however, the antigens were not found in PASFs from benign human cell lines.

Antigens, Neoplasm↗
PubMed1996

Bafilomycin A1 induces apoptosis in PC12 cells independently of intracellular pH.

PC12 cells growth-arrested with bafilomycin A1 died showing apoptotic chromatin condensation in the nuclei. The bafilomycin A1-induced chromatin condensation was preceded by neurite outgrowth (NOG), required higher concentrations of bafilomycin A1 than NOG, and was suppressed by cycloheximide and aurintricarboxylic acid. NH4Cl (10 mM), another acidotropic pH perturbing agent, neither induced apoptotic chromatin condensation by itself nor suppressed that induced by bafilomycin A1, suggesting that bafilomycin A1-induced apoptosis occurs independently of intracellular pH in PC12 cells.

Ammonium Chloride↗
PubMed1996

Role of peroxynitrite in [3H] gamma-aminobutyric acid release evoked by nitric oxide and its mechanism.

Role of peroxynitrite in [3H] gamma-aminobutyric acid (GABA) release evoked by N-methyl-D-aspartate (NMDA) and S-nitroso-N-acetyl-penicillamine (SNAP) and mechanisms of [3H]GABA release induced by peroxynitrite in comparison with those induced by NMDA and SNAP were investigated using cerebrocortical neurons. NMDA dose dependently increased [3H]GABA release, which was significantly inhibited by hemoglobin and superoxide scavengers, Cu2+, Zn(2+)-superoxide dismutase and ceruloplasmin. The NMDA-evoked [3H]GABA release was significantly suppressed by GABA transport inhibitors and inhibitors of voltage-dependent L-typed Ca2+ channel. The SNAP-evoked [3H]GABA release was significantly reduced by Ca2+ withdrawal and by GABA transport inhibitors either in the presence or absence of Ca2+. Similar patterns of [3H]GABA release induced by peroxynitrite were observed. These results indicate that peroxynitrite formed by the reaction of NO with superoxide participates, in part, in the release of [3H]GABA induced by NMDA and SNAP.

Animals↗
PubMed1996

Down-regulation of benzodiazepine receptors by ethyl beta-carboline-3-carboxylate in cerebrocortical neurons.

Effect of exposure of primary cultured cerebral cortical neurons to ethyl beta-carboline-3-carboxylate (beta-CCE) on the function of benzodiazepine receptors was studied. Exposure of neurons to beta-CCE (0.1-10 microM) decreased the binding of [3H]flunitrazepam to extensively washed membrane fractions in a dose- and time-dependent manner, whereas the binding of [3H]flunitrazepam to the cytosolic fractions increased (180%) under the same conditions as described above. Ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a] [1,4] benzodiazepine-3-carboxylate (Ro15-1788), an antagonist of the central type of benzodiazepine receptors, completely abolished the beta-CCE-induced decrease in [3H]flunitrazepam binding and the IC50 value for [3H]flunitrazepam binding to the extensively washed membrane fractions prepared from beta-CCE-treated neurons was similar to that from non-treated neurons. Scatchard analysis revealed that only the Bmax value for [3H]flunitrazepam binding decreased after the exposure to beta-CCE (1 microM) for 12 h, although the Kd value was not altered. These results indicate that beta-CCE induces the down-regulation of benzodiazepine receptors by an increase in benzodiazepine receptor internalization.

Animals↗
PubMed1996

Nitric oxide-evoked [3H]taurine release is mediated by reversal of the Na(+)-dependent carrier-mediated taurine transport system.

The pharmacological characteristics of [3H]taurine release evoked by nitric oxide (NO) were investigated using mouse cerebral cortical neurons in primary culture. N-Methyl-D-aspartate (NMDA) and S-nitroso-N-acetylpenicillamine (SNAP) dose-dependently increased [3H]taurine release from neurons. The NMDA-evoked release of [3H]taurine was reduced to the basal level by N omega-nitro-L-arginine, a NO synthase inhibitor, and MK-801, a noncompetitive antagonist for NMDA receptors. The NMDA- and SNAP-evoked releases of [3H]taurine were completely abolished by hemoglobin, indicating that these [3H]taurine releases were evoked by NO produced by NMDA receptor activation and liberated from SNAP. Withdrawal of Na+ from incubation buffer significantly inhibited the NMDA- and SNAP-induced [3H]taurine releases, whereas removal of Ca2+ produced no alteration in the SNAP-evoked [3H]taurine release. In addition, beta-alanine and guanidinoethane sulfonate, antitransporters of the carrier-mediated taurine transport system, reduced the NMDA- and SNAP-evoked releases of [3H]taurine in a dose-dependent manner. These results indicate that the NO-evoked [3H]taurine release from cerebral cortical neurons is mediated by a reversal of the Na(+)-dependent carrier-mediated taurine transport system.

Animals↗
PubMed1996

Nitric oxide-evoked [3H] gamma-aminobutyric acid release is mediated by two distinct release mechanisms.

Mechanisms underlying the release of [3H] gamma-aminobutyric acid (GABA) evoked by nitric oxide (NO) were investigated by use of primary cultured neurons prepared from the mouse cerebral cortex. NO generators such as sodium nitroprusside (SNP) and S-nitroso-N-a etylpenicillamine (SNAP) increased both [3H]GABA release from the neurons and [45Ca2+] influx into the neurons in a dose-dependent manner, which was significantly diminished by hemoglobin. The removal of Ca2+ significantly reduced the NO-induced [3H]GABA release by about 50%. Nipecotic acid and 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1, 2, 5, 6-tetrahydro-3- pyridinecarboxylic acid (NO-711), GABA uptake inhibitors dose-dependently inhibited the NO-evoked [3H]GABA release in either the presence or absence of Ca2+. The concentration of these GABA uptake inhibitors to suppress the NO-induced release of [3H]GABA was sufficiently lower than that to exhibit the inhibition of [3H]GABA transport into the neurons. In addition, the NO-evoked [3H]GABA release was reduced by approximately 50% when total Na+ in incubation buffer was replaced with equimolar choline, and was also completely abolished by the removal of both Ca2+ and Na+. These results indicate that the release of [3H]GABA evoked by NO is mediated by two release mechanisms, a Ca2+ -dependent release system and the reverse process of the Ca2+ -independent and Na+ -dependent carrier-mediated GABA uptake system.

Animals↗
PubMed1996

Contribution of lysosomes to the subcellular distribution of basic drugs in the rat liver.

PURPOSE: We examined the subcellular distribution of the basic drugs, chlorpromazine (CPZ), imipramine (IMP) and biperiden (BP), in rat liver, and evaluated the contribution of lysosome (Lys) to their intracellular distribution in comparison with that of mitochondria (Mit). METHODS: In an in vivo distribution, the concentrations of CPZ, IMP and BP in the liver subcellular fractions were determined. In an in vitro study, uptake of [3H]IMP into Lys and Mit fractions was determined in the presence or absence of several agents. RESULTS: The distribution of these drugs 10 min after administration was quite similar. However, the relative specific contents (the drug concentration per protein of each fraction divided by that of the total homogenate) in Lys were 7.3, 9.6 and 4.2, respectively for CPZ, IMP and BP, whereas those in the other organella were only 0.4 approximately 1.7. In an in vitro uptake study, the dose response of IMP uptake into Lys was biphasic, while that into Mit fractions was monophasic. The binding of IMP to the high affinity sites of Lys was pH dependent and disappeared in 50 mM NH4Cl or 50 microM CPZ, both of which increased the intralysosomal pH, the low affinity sites were not affected by these drugs. CONCLUSIONS: The results indicated that Lys has the highest affinity for the basic drugs in the liver and that its contribution to their subcellular distribution depends on the intralysosomal pH, which is also affected by these drugs. The importance of these effects may become significant in combination therapy using various basic drugs.

Animals↗