Biomedical subjects

S Ohkuma

Publications and source records attributed to S Ohkuma.

At least 37 records · Page 2Linked to original sources

PubMed2000

N-ethylmaleimide-enhanced phosphatidylserine externalization of human pancreatic cancer cells and immediate phosphatidylserine-mediated phagocytosis by macrophages.

The purpose of this study was to examine whether N-ethylmaleimide (NEM) can enhance phosphatidylserine (PS) externalization on the outer surface of cancer cells, and PS-externalized cancer cells can be phagocytosed immediately by macrophages. NEM could externalize PS of cancer cells in concentration- and time-dependent manners. When treated with 5 mM NEM for 1 h, 80% of the carcinoma cells externalized their PS. In phagocytosis assay, these cells were engulfed immediately by macrophages before undergoing apoptosis. These results suggest that NEM can immediately externalize PS of cancer cells, leading to their recognition and phagocytosis by macrophages before undergoing apoptosis morphologically.

Animals↗

PubMed2000

Uptake of imipramine in rat liver lysosomes in vitro and its inhibition by basic drugs.

We investigated the uptake of imipramine (IMP) in highly purified lysosomes from rat liver and its inhibition by a variety of basic drugs in vitro. The uptake of [(3)H]IMP into lysosomes peaked in less than 20 s, showing little temperature dependency or countertransport phenomena. It was accelerated by increase of extralysosomal pH, stimulated by Mg(2+)-ATP in KCl buffer, and suppressed by acidic ionophores. However, the uptake of [(3)H]IMP in lysosomes was approximately 140-fold higher than the value expected from the pH-partition theory. IMP and other weak lipophilic bases like chlorpromazine and propranolol raised the intralysosomal pH, and their potency was stronger than that of NH(4)Cl, a typical pH-perturbing weak base. A variety of basic drugs inhibited the uptakes of [(3)H]IMP and [(14)C]methylamine into lysosomes, their 50% inhibitory concentrations (IC(50)) being almost the same for [(3)H]IMP and [(14)C]methylamine uptake (r = 0.842). A high correlation (r = 0.946) was observed between the IC(50) values (for the inhibition of [(3)H]IMP uptake) and the lipophilicity (P(oct) values). These results suggest that the accumulation of lipophilic basic drugs is driven primarily by the transmembrane pH difference (pH-partition theory) but with the involvement of some additional mechanism(s) related to drug lipophilicity, possibly binding (partition or adsorption) to lipophilic substance(s) and/or aggregation within lysosomes. Based on this idea, we have established a model that described and successfully simulated the weak base-induced pH increase, the accumulation of a lipophilic weak base (IMP), and the inhibition of accumulation of IMP by lipophilic basic drugs.

Animals↗

PubMed1999

Choice of vincristine or 6-mercaptopurine on the basis of polyamine level in tumor-bearing regions of the brain.

The effects of vincristine (VC) and 6-mercaptopurine (6-MP) on body weight, regional weights, and the contents of putrescine, spermidine, and spermine in six regions of the brain were examined in rats that had been given these drugs for 5 consecutive days. VC is recommended for management of tumors in the corpus striatum and/or hippocampus, and cortex although its efficacy is dependent on the doubling time of the tumor cells, whereas 6-MP is recommended for the management of tumors in the cortex, thalamus and/or hypothalamus, and diencephalon. VC and 6-MI are chosen for treatment of the brain tumors because they reduce polyamines which are associated with the reduction of drug-sensitive cells and an inhibition of tumor growth.

Animals↗

PubMed1999

Effects of methotrexate and cyclophosphamide on polyamine levels in various tissues of rats.

The effects of methotrexate (MTX) and cyclophosphamide (CYP) on body weight, organ weight, and the concentration of putrescine, spermidine, and spermine in 14 different tissues were measured in rats that had been given these compounds for 5 consecutive days. These three polyamines in both the thymus and spleen of rats treated with MTX and CYP showed a statistically significant decrease. Further, putrescine in the seminal vesicles, kidney, liver, and small intestine of MTX-treated rats, and in the prostate, seminal vesicles, kidney, heart, liver, small intestine, and lung of CYP-treated rats, spermidine in the prostate, seminal vesicles, testis, thymus, spleen, kidney, heart, small intestine, and skeletal muscle of CYP-treated rats, and spermine in the prostate, seminal vesicles, kidney, heart, small intestine, and stomach of CYP-treated rats showed statistically significant decreases. Recognition of the significance of polyamine levels and attention to their response in anti-cancer drug therapy may have clinical implications.

Animals↗

PubMed1999

Activated lansoprazole inhibits cancer cell adhesion to extracellular matrix components.

Integrins play an important role in tumor dissemination. The purpose of this study was to examine whether a SH-reactive reagent inhibits the adhesion of the human pancreatic cancer cell line AsPC-1 to extracellular matrix components. Activated lansoprazole (AG-2000) was used as the SH-reactive reagent because this compound is known to react with SH groups but does not permeate the cell membrane. The effect of AG-2000 on the adhesion of AsPC-1 cells to matrix was examined, using both an in vitro adhesion assay and an in vivo nude mouse xenograft model of peritoneal implantation. In the in vitro adhesion assay, a 60-min exposure of AsPC-1 cells to AG-2000 resulted in a dose-dependent inhibition of AsPC-1 cell adhesion to laminin, fibronectin and type IV collagen, although AG-2000 did not affect the viability of AsPC-1 cells by MTT assay. In the in vivo assessment of AsPC-1 cell implantation, the AsPC-1 cells were initially preincubated with AG-2000 for 60 min to ensure adequate exposure of the AsPC-1 cells to AG-2000 before intraperitoneal injection. AG-2000 significantly inhibited the peritoneal implantation of the AsPC-1 cells in nude mice. These findings suggest that a short exposure of cancer cells to AG-2000 can inhibit cancer cell adhesion to extracellular matrix components.

Animals↗

PubMed1998

Removal of hydroxyl radical facilitates Ca2+-dependent [3H]GABA release by peroxynitrite.

We investigated mechanisms for enhancement of peroxynitrite (OONO-; 5 microM)-evoked [3H] gamma-aminobutyric acid (GABA) release. Hydroxyl radical scavengers such as N,N'-dimethylthiourea (DMTU), mannitol, and uric acid, significantly increased OONO--evoked [3H]GABA release, whereas urea showed no effects on the release. Removal of Ca2+ from incubation buffer abolished the enhancement of the release by DMTU, although DMTU showed no effects on the basal release with and without Ca2+ in extracellular space. These results indicate that hydroxyl radical scavengers facilitate OONO--evoked [3H]GABA release dependent on Ca2+.

Animals↗

PubMed1998

Prodigiosins uncouple lysosomal vacuolar-type ATPase through promotion of H+/Cl- symport.

We reported previously [Kataoka, Muroi, Ohkuma, Waritani, Magae, Takatsuki, Kondo, Yamasaki and Nagai (1995) FEBS Lett. 359, 53-59] that prodigiosin 25-C (one of the red pigments of the prodigiosin group produced by micro-organisms like Streptomyces and Serratia) uncoupled vacuolar H+-ATPase, inhibited vacuolar acidification and affected glycoprotein processing. In the present study we show that prodigiosin, metacycloprodigiosin and prodigiosin 25-C, all raise intralysosomal pH through inhibition of lysosomal acidification driven by vacuolar-type (V-)ATPase without inhibiting ATP hydrolysis in a dose-dependent manner with IC50 values of 30-120 pmol/mg of protein. The inhibition against lysosomal acidification was quick and reversible, showing kinetics of simple non-competitive (for ATP) inhibition. However, the prodigiosins neither raised the internal pH of isolated lysosomes nor showed ionophoric activity against H+ or K+ at concentrations where they strongly inhibited lysosomal acidification. They required Cl- for their acidification inhibitory activity even when driven in the presence of K+ and valinomycin, suggesting that their target is not anion (chloride) channel(s). In fact, the prodigiosins inhibited acidification of proteoliposomes devoid of anion channels that were reconstituted from lysosomal vacuolar-type (V-)ATPase and Escherichia coli phospholipids. However, they did not inhibit the formation of an inside-positive membrane potential driven by lysosomal V-ATPase. Instead, they caused quick reversal of acidified pH driven by lysosomal V-ATPase and, in acidic buffer, produced quick acidification of lysosomal pH, both only in the presence of Cl-. In addition, they induced swelling of liposomes and erythrocytes in iso-osmotic ammonium salt of chloride but not of gluconate, suggesting the promotion of Cl- entry by prodigiosins. These results suggest that prodigiosins facilitate the symport of H+ with Cl- (or exchange of OH- with Cl-) through lysosomal membranes, resulting in uncoupling of vacuolar H+-ATPase.

Adenosine Triphosphate↗

PubMed1998

Prodigiosins as a new group of H+/Cl- symporters that uncouple proton translocators.

We reported previously (Kataoka, T., Muroi, M., Ohkuma, S., Waritani, T., Magae, J., Takatsuki, A., Kondo, S., Yamasaki, M., and Nagai, K. (1995) FEBS Lett. 359, 53-59) that prodigiosin 25-C uncoupled vacuolar H+-ATPase, inhibited vacuolar acidification, and affected glycoprotein processing. In the present study we show that prodigiosins (prodigiosin, metacycloprodigiosin, and prodigiosin 25-C) inhibit the acidification activity of H+-ATPase chloride dependently, but not membrane potential formation or ATP hydrolysis activity, and suggest that they promote H+/Cl- symport (or OH-/Cl- exchange, in its equivalence) across vesicular membranes. In fact, prodigiosins displayed H+/Cl- symport activity on liposomal membranes. First of all, they decreased the internal pH of liposomes depending on the external chloride, and raised it depending on the internal chloride when external buffer was free from chloride. Second, their effect was electroneutral and not seriously affected by the application of an inside positive membrane potential generated by K+ and valinomycin. Finally, they promoted the uptake of [36Cl] from external buffers with concomitant intraliposomal acidification when external pH was acidic relative to liposome interior. As prodigiosins hardly inhibit the catalytic activity (ATP hydrolysis) unlike well known OH-/Cl- exchangers (for example, tributyltin chloride), they should provide powerful tools for the study of molecular machinery and cellular activities involving transport of protons and/or chloride.

Animals↗

PubMed1998

V-ATPase of Thermus thermophilus is inactivated during ATP hydrolysis but can synthesize ATP.

The ATP hydrolysis of the V1-ATPase of Thermus thermophilus have been investigated with an ATP-regenerating system at 25 degreesC. The ratio of ATPase activity to ATP concentration ranged from 40 to 4000 microM; from this, an apparent Km of 240 +/- 24 microM and a Vmax of 5.2 +/- 0.5 units/mg were deduced. An apparent negative cooperativity, which is frequently observed in case of F1-ATPases, was not observed for the V1-ATPase. Interestingly, the rate of hydrolysis decayed rapidly during ATP hydrolysis, and the ATP hydrolysis finally stopped. Furthermore, the inactivation of the V1-ATPase was attained by a prior incubation with ADP-Mg. The inactivated V1-ATPase contained 1.5 mol of ADP/mol of enzyme. Difference absorption spectra generated from addition of ATP-Mg to the isolated subunits revealed that the A subunit can bind ATP-Mg, whereas the B subunit cannot. The inability to bind ATP-Mg is consistent with the absence of Walker motifs in the B subunit. These results indicate that the inactivation of the V1-ATPase during ATP hydrolysis is caused by entrapping inhibitory ADP-Mg in a catalytic site. Light-driven ATP synthesis by bacteriorhodopsin-VoV1-ATPase proteoliposomes was observed, and the rate of ATP synthesis was approximately constant. ATP synthesis occurred in the presence of an ADP-Mg of which concentration was high enough to induce complete inactivation of ATP hydrolysis of VoV1-ATPase. This result indicates that the ADP-Mg-inhibited form is not produced in ATP synthesis reaction.

Adenosine Diphosphate↗

PubMed1998

Bafilomycin A1 induces apoptosis in the human pancreatic cancer cell line Capan-1.

Bafilomycin A1, a specific inhibitor of vacuolar type H(+)-ATPase, can inhibit the growth of a variety of cultured cells in a dose-dependent manner, but its mechanism is unclear. The aim of this study was to examine whether bafilomycin A1 inhibits the growth of Capan-1 human pancreatic cancer cells through apoptosis. The effect of bafilomycin A1 on tumour growth in vitro and in vivo was examined using an MTT assay and an in vivo tumour model. The presence or absence of apoptosis was determined by morphology and DNA analysis of tumour cells. The concentration of bafilomycin A1 for 50 per cent inhibition of cell viability during 72 h by the MTT assay was 5 nm. In DNA analysis, a ladder of fragmented DNA was detected in Capan-1 cells treated with bafilomycin A1 at concentrations greater than 10 nm for 24 h. Nude mice bearing a xenografted Capan-1 cell line tumour received 4 weeks of bafilomycin A1 (1.0 mg/kg per day). This treatment significantly inhibited tumour growth compared with controls after 21 days (P < 0.05). Histopathological examination of tumour cells in the treated group demonstrated signs of apoptosis with chromatin condensation and cell shrinkage. These observations suggest that bafilomycin A1 inhibits the growth of Capan-1 human pancreatic cancer cells through apoptosis.

Adenocarcinoma↗

PubMed1998

Enhancement of peroxynitrite-evoked acetylcholine release by hydroxyl radical scavengers from mouse cerebral cortical neurons.

We investigated the effects of hydroxyl radical scavengers on peroxynitrite (OONO-)-evoked acetylcholine (ACh) release from mouse cerebral cortical neurons. N,N'-dimethylthiourea, a hydroxyl radical scavenger, dose-dependently increased OONO(-)-evoked ACh release. Other hydroxyl radical scavengers such as uric acid and mannitol, also enhanced OONO(-)-evoked ACh release, although these enhancing effects were not found in the absence of OONO-. In addition, OONO(-)-induced [45Ca2+]influx was significantly facilitated by the scavengers, whereas no effects of the scavengers on [45Ca2+]influx was observed in the absence of OONO-. These results indicate that hydroxyl radical scavengers enhance OONO(-)-evoked ACh release via the facilitation of OONO(-)-induced [45Ca2+]influx.

Acetylcholine↗

PubMed1998

Functional involvement of benzodiazepine receptors in ethanol-induced increases of diazepam binding inhibitor (DBI) and its mRNA in the mouse brain.

We have attempted to clarify the mechanisms for alcohol (EtOH)-induced elevation of diazepam binding inhibitor (DBI) mRNA and to investigate whether the increase in DBI mRNA is paralleled with that in DBI using EtOH-treated mice and primary cultured neurons. Both the DBI content and the expression of DBI mRNA were elevated in the cerebral cortex of EtOH-inhaled and -withdrawn mice. Simultaneous administration of flunitrazepam (FLN) and Ro15-1788 with EtOH vapor completely abolished the EtOH-induced elevation of DBI mRNA. In addition, the exposure of the neurons for 3 days significantly elevated the expression of DBI mRNA, which was completely inhibited by concomitant exposure of FLN, Ro15-4513 and Ro-15-1788 with EtOH, while muscimol and bicuculline showed no effects on the EtOH-induced increase of DBI mRNA expression. These results indicate that functional interaction between EtOH and benzodiazepine (BDZ) receptors is a critical role in the increased expression of DBI mRNA.

Administration, Inhalation↗

PubMed1998

Multiple actions of nitric oxide on voltage-dependent Ca2+ channels in mouse cerebral cortical neurons.

We investigated the effects of nitric oxide (NO) on voltage-dependent Ca2+ channels (VDCCs) by examining [45Ca2+]influx into mouse cerebral cortical neurons. S-nitroso-N-acetylpenicillamine (SNAP) induced a dose-dependent increase in [45Ca2+]influx, which was completely abolished by hemoglobin, tetrodotoxin and dibucaine. The NO-induced [45Ca2+influx was significantly inhibited by verapamil and omega-agatoxin VIA (omega-AGX), whereas omega-conotoxin GVIA (omega-CTX) had no effects on the NO-induced [45Ca2+]influx. KCl (30 mM) stimulated [45Ca2+]influx, and verapamil, omega-CTX and omega-AGX reduced the KCl-induced [45Ca2+]influx by about 40, 26 and 34%, respectively, indicating that the neurons used here possess L-, N- and P-typed VDCCs. SNAP itself reduced KCl-induced [45Ca2+]influx by about 28.5%. In the presence of both KCl and SNAP, omega-CTX showed no effects on the influx, while verapamil and omega-AGX significantly inhibited the influx and the concomitant presence of verapamil and omega-AGX completely abolished the influx. These results indicate that NO induces [45Ca2+] influx via the opening of L- and P-typed VDCCs subsequent to neuronal membrane depolarization and that NO itself inhibited the function of N-typed VDCC in the cerebral cortical neurons.

Animals↗

PubMed1998

Continuous treatment with nicotine increases diazepam binding inhibitor (DBI) and its mRNA in the mouse brain.

Effect of chronic treatment with nicotine on DBI and its mRNA in mouse cerebral cortex were examined. Continuous treatment of mice with nicotine significantly increased DBI content and its mRNA expression, which was completely abolished by simultaneous administration of mecamylamine (1 mg/kg, i.p.). These results indicate that chronic functional interaction between nicotine and nicotinic acetylcholine receptors has a critical role in increases in DBI content and its mRNA expression.

Animals↗

PubMed1998

Enhanced acetylcholine release from cells that have more 15-kDa proteolipid in their membrane, a constituent V-ATPase, and mediatophore.

Mediatophore is a protein that translocates acetylcholine (ACh) on calcium action. It is a homopolymer of a 15-kDa proteolipid that is also a constituent of the membrane sector of vacuolar H+-adenosine trisphosphatase (V-ATPase; vacuolar proton pump). Experiments on neuroblastoma cell lines (N18TG-2) that are deficient for ACh release and on cells that are competent for release, such as the glioma C6BU-1 or the N18TG-2/C6BU-1 fusion product NG108-15, show that there is a correlation between ACh release and the 15-kDa proteolipid content of the cell membrane. In another cell line, L-M(TK-), it has been possible to up-regulate ACh release and the membrane proteolipid content after treating the cells with dibutyryl-cyclic AMP or dexamethasone. As mediatophore translocates ACh and as V-ATPase may help vesicular ACh storage, it was interesting to determine the respective role of the two proteins in the observed correlation between release and proteolipid content. After blocking vesicular loading with vesamicol, we did not affect release from these cells, suggesting that the observed correlation may be attributed to mediatophore. The acquisition of an ACh release mechanism would then depend on the process that guides the proteolipid to the plasma membrane of the cell.

Acetylcholine↗

PubMed1998

Mechanisms for facilitation of nitric oxide-evoked [3H]GABA release by removal of hydroxyl radical.

We have investigated the mechanisms for enhancement of nitric oxide (NO)-evoked gamma-[3H]aminobutyric acid ([3H]GABA) release from mouse cerebrocortical neurons by hydroxyl radical (.OH) scavengers. .OH scavengers, such as N,N'-dimethylthiourea (DMTU), uric acid, and mannitol, dose-dependently facilitated NO-evoked [3H]GABA release evoked by NO liberated from S-nitroso-N-acetylpenicillamine. Ionomycin-evoked [3H]GABA release, which was significantly inhibited by hemoglobin and an NO synthase, N(G)-methyl-L-arginine, was also enhanced by DMTU. These results indicate that GABA release evoked by both endogenous and exogenous NO is facilitated by .OH scavengers. These enhancing actions of .OH scavengers were completely abolished by Ca2+ removal from incubation buffer and by an L-type voltage-dependent Ca2+ channel (VDCC) inhibitor, nifedipine, whereas each .OH scavenger showed no effects on [3H]GABA release in the absence of NO. Inhibitors for P/Q- and N-type VDCCs had no effects on the enhancement. NO-induced 45Ca2+ influx was also dose-dependently enhanced by .OH scavengers, although 45Ca2+ influx was not altered by .OH scavengers in the absence of NO. Nifedipine abolished this enhancement of the NO-induced 45Ca2+ influx by .OH scavengers. These results indicate that the removal of .OH by its scavengers facilitates the NO-evoked [3H]GABA release dependent on Ca2+ and that this enhancement is due to the increase in Ca2+ influx via L-type VDCCs.

Animals↗

PubMed1998

Continuous treatment with morphine increases diazepam binding inhibitor mRNA in mouse brain.

Effects of acute and chronic morphine treatment on the expression of diazepam binding inhibitor (DBI) mRNA in the mouse brain were examined. Cerebral DBI mRNA expression significantly increased in morphine-dependent mice, and this increase is more remarkable in morphine-withdrawn mice, whereas a single administration of morphine (50 mg/kg) produced no changes in the expression. Simultaneous administration of naloxone (3 mg/kg) with morphine completely abolished the increase in cerebral DBI mRNA expression observed in morphine-dependent and -withdrawn mice. These results indicate that a chronic functional interaction between morphine and opioid receptors has a critical role in increases in DBI mRNA expression.

Animals↗

PubMed1998

Disintegration of lysosomes mediated by GTPgammaS-treated cytosol: possible involvement of phospholipases.

We showed previously that cytosol treated with guanosine 5'-O-(3-thiotriphosphate) (GTP-gammaS) disintegrated lysosomes in vitro [Sai, Y. et al. (1994) Biochem. Biophys. Res. Commun. 198, 869-877] in time-, temperature-, and dose-dependent manners. This also requires ATP, however, the latter can be substituted with deoxy-ATP, ADP, or ATPgammaS, suggesting no requirement of ATP hydrolysis. The lysis was inhibited by several chemical modifiers, including N-ethylmaleimide, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, and by various phospholipase inhibitors (trifluoperazine, p-bromophenacyl bromide, nordihydroguaiaretic acid, W-7, primaquine, compound 48/80, neomycin, and gentamicin), but not by ONO-RS-082, an inhibitor of phospholipase A2. The reaction was also inhibited by phospholipids (phosphatidylinositol, phosphatidylserine, phosphatidic acid, and phosphatidylcholine) and diacylglycerol. Among the phospholipase A2 hydrolysis products of phospholipids, unsaturated fatty acids (oleate, linoleate, and arachidonate) and lysophospholipid (lysophosphatidylcholine) by themselves broke lysosomes down directly, whereas saturated fatty acids (palmitate and stearate) had little effect. We found that GTPgammaS-stimulated cytosolic phospholipase A2 activity was highly sensitive to ONO-RS-082. These results suggest the participation of phospholipase(s), though not cytosolic phospholipase A2, in the GTPgammaS-dependent lysis of lysosomes.

Animals↗