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Volume 114 (1999)
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Volume 114, Issue supplement
Displaying 1-41 of 41 articles from this issue
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Shuh Narumiya1999 Volume 114 Issue supplement Pages 1-5
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSRho is a member of the Ras-related family of small molecular weight GTP-binding proteins, and works as a molecular switch by shuttling between the GDP-bound inactive form and the GTP-bound active form. Cellular functions of Rho have been studied by two ways; one is to express or microinject constitutively active Rho mutants in cells to identify the active phenotype of Rho, and the other is to use botulinum C3 exoenzyme that specifically ADP-ribosylates and inactivates Rho in cells to find out phenotypes of Rho inactivation. These analyses have revealed that Rho is involved in cell to substratum adhesion and motility, cell contraction and cytokinesis through the reorganization of the actincytoskeleton and modulation of its activity. These actions of Rho are mediated by downstream Rho effectors. Several putative Rho effectors have been isolated on the basis of their selective binding to the GTP-bound form of Rho. Among them, the ROCK family of Rho-associated serine/threonine protein kinases inactivates cofilin and myosin phosphatase to induce stabilization of filamentous actin and increase in the actomyosin-based contractility. mDia binds profilin likely to promote actin polymerization. Thus, these effectors in combination are supposed to work in organization of various forms of the actin cytoskeleton. Furthermore, analyses using a ROCK specific inhibitor Y-27632 have suggested that the Rho-ROCK pathway works in contractions of vascular and bronchial smooth muscles under various pathological conditions and is involved in malignant cell transformation and tumor invasion and metastasis.View full abstractDownload PDF (4972K) -
Yoshiharu Yonekura1999 Volume 114 Issue supplement Pages 6-10
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSPositron emission tomography (PET) and functional magnetic resonance imaging (MRI) have been introduced into the field of brain science as an noninvasive approach to visualize brain function, focusing on the regional distribution of neuronal activity and its connectivity. Measurement of regional cerebral blood flow with PET has been established for mapping human brain function covering the whole brain with various task conditions. Recently introduced functional MRI technique can also detect the signal changes due to the local increase of blood flow by brain activation. Both PET and MRI provide similar activation patterns in cerebral cortical areas, and the study should be designed by considering the characteristics of each modality. Although PET continues to play a major role in imaging of the neurotransmission process, the efforts are now being made to apply this exciting technique to clinical diagnosis by means of single photon emission computed tomography (SPECT). Neuroreceptor imaging is now applied not only for differential diagnosis of neurological diseases but also for determination of optimal dose of appropriate therapeutic drugs in psychiatric patients. Combined use of activation studies with neurotransmission imaging will provide a new insight in understanding the brain mechanism of emotion and behavior.View full abstractDownload PDF (4383K) -
Hitoshi Endou, Yoshikatsu Kanai1999 Volume 114 Issue supplement Pages 11-16
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSAmino acids, especially essential amino acids are required for protein synthesis and as energy sources in all living cells. Since most amino acids are hydrophilic, special membrane proteins are necessary for their transmembrane transport particularly in transformed cells. We cloned a cDNA encoding L-type amino acid transporter, and named it LAT1. LAT1 is highly expressed in most tumor cells and in a few normal tissues such as the bone marrow, spleen, placenta and brain. As a homolog of LAT1, we also cloned LAT2 which is expressed in various normal tissues. In order to clarify the role of LAT1 in tumor cells, we tested a relatively specific inhibitor to LAT1, BCH(2-aminobicyclo-2(2, 2, 1)-heptane-2-caboxylic acid), and found that BCH inhibited mortality of mice treated with sarcoma cells. In addition, BCH also suppressed strongly T24 carcinoma cell growth. Moreover, LAT1 transports amino acid mimetic drugs. Thus, these results indicate that amino acid transporter molecule such as LAT1 can be a drug target,View full abstractDownload PDF (5074K) -
Yukihito Tsukumo, Tsugunobu Andoh, Tomomi Yamaguchi, Hiroshi Nojima, Y ...1999 Volume 114 Issue supplement Pages 17-21
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSNC mice, a model for atopic dermatitis. showed scratching behavior when kept under conventional environment. The scratching behavior of NC mice was suppressed by distraction or by the administration of naltrexone (1 mg/kg, s.c.), an opioid antagonist. These results suggest that such scratching behavior is itchassociated response. The itch-associated response of the NC mice was significantly suppressed by an intravenous injection of nitric oxide (NO) svnthase inhibitor NG-vitro-L-arginine methyl ester (L-NAME, 10 mg/kg), but not D-NAME (10 mg/kg) and saline. Intracutaneous NO production in the rostral back. a region which the NC mice mainly scratched. was markedly increased as compared with the caudal back. a nonscratched region. The increased NO production in the rostral back of NC mice was decreased by the intravenous injection of L-NAME (10 mg/kg). These results suggest that NO and NO sythase are new target in the treatment of atopic pruritus.View full abstractDownload PDF (3061K) -
R.M. Wallis1999 Volume 114 Issue supplement Pages 22-26
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSSildenafil (1-[4-ethoxy-3-(6, 7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4, 3-d]pyrimidin-5-yl) phenylsulphonyl]-4-methylpiperazine) has been shown to be an effective oral treatment for male erectile dysfunction. Sildenafil is a potent competitive inhibitor of PDE5 (IC50 3.5 nM) and is selective over PDEI to 4 (80 to 19, 000-fold) and retinal PDE6 (10-fold). Sildenafil enhanced cGMP accumulation driven with sodium nitroprusside in the corpus cavernosum of rabbits without affecting cAMP formulation. In the absence of nitric oxide drive, sildenafil had no functional effect on the human and rabbit isolated corpus cavernosum, but potently potentiated the relaxant effects of nitric oxide on these tissues. In the anaesthetised dog, sildenafil (ED50: 12 to 16 μg/kg i.v.) enhanced the increase in intracavernosal pressure induced by electrical stimulation of the pelvic nerve or intracavernosal injection of sodium nitroprusside in the absence of meaningful effects on blood pressure. Consistent with its mode of action, sildenafil potentiated the vasorelaxant effects of glyceryl trinitrate on rabbit isolated aortic rings. However, unlike milrinone, sildenafil had no inotropic effects on the dog isolated trabeculae carneae. Thus it is unlikely to have the deleterious effects on cardiac function associated with PDE3 inhibitors. As a consequence of inhibition of PDE6 in the retina, sildenafil (1 to 100 μM) altered the kinetics of the light response of the dog isolated retina. In the anaesthetised dog, sildenafil modified the a and b-wave of the electroretinogram induced by a flash of blue light. These effects were proportional to plasma concentrations, were fully reversible and only occurred following plasma concentrations higher (approximately 30-fold) than those active on intracavernosal pressure. These studies have shown that sildenafil is a potent and selective inhibitor of PDE5. It enhances the effect of nitric oxide on the corpus cavernosum and has been shown to be an effective oral treatment of erectile dysfunction.View full abstractDownload PDF (3888K) -
Yukisato Ishida, Akio Tomoda, Kazutaka Momose1999 Volume 114 Issue supplement Pages 27-32
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSCatalase, myoglobin and NO-synthase are heme proteins. Catalase is capable of producing NO from azide and hydroxylamine (Ignarro LJ, FASEB J 1989; 3:31-36). Heme is the center of catalyzing the production of NO. Thus, we investigated the mode of vasorelaxation induced by azide and nitrite in the endothelium-denuded aorta of guinea pig or rat. Both agents elicited a rapid relaxation of the aorta in a concentration dependent manner: EC50 values for azide and nitrite were 0.1 μM and 0.1 mM, respectively. These relaxation responses were inhibited by the presence of methylene blue, but not by NO-arginine or L-NMMA. Azide rapidly raised the cGMP content of the muscle, which seemed to precede the relaxation response. The catalase activity of the aorta was inhibited by azide and hydroxylamine with the similar IC50 values to EC50 values for relaxation. Myoglobin was found in the vessel tissue by the immunohistological method. Using a NO-sensitive electrode, the NO production from aortas was detected after addition of azide and nitrite. The NO production from nitrite was shown to precede the oxidation of heme moiety of oxymyoglobin. These results suggest that catalase as well as myoglobin, heme proteins, can be the cellular target for pharmacological agents to produce NO leading to vasorelaxation. (Supported in part by a Grant-in-Aid from the Ministry of Education, Sports and Culture of Japan)View full abstractDownload PDF (6025K) -
Hiroshi Azuma1999 Volume 114 Issue supplement Pages 33-40
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSMechanisms of vascular remodelling process are complex and poorly understood. We describe herein the role of L-arginine-endogenous NOS inhibitors-endothelin-1 pathway for the vascular remodelling after endothelial denudation of the rabbit carotid artery. It is reportedly known that NO is a vasodilating substance, an inhibitor of platelet aggregation and adhesion, and an inhibitor of vascular smooth muscle cell proliferation and that endothelin-1, of which production is inhibited by NO, is a potent vasoconstrictor and a potent mitogen. An accumulation of endogenous inhibitors (L-NMMA and ADMA) in regenerated endothelial cells after the endothelial denudation was accompanied by the decreased NO generation, the increased endothelin-1 content within the vessel wall and the occurrence of intimal hyperplasia. Endothelin-1 content within the vessel wall was significantly increased after the exogenous L-NMMA administration for 2 weeks, suggesting that accumulated L-NMMA results in the decreased NO generation and, in turn, increases endothelin-1 content. Endothelin-1 facilitated the [3H]-L-NMMA uptake by endothelial cell and brought about the potentiation of L-NMMA-mediated inhihition of NO generation. These results strongly suggest that the L-arginine - endogenous NOS inhibitors - endothelin-1 pathway plays an active role for vascular remodelling.View full abstractDownload PDF (8694K) -
Shinji Takai, Denan Jin, Mizuo Miyazaki1999 Volume 114 Issue supplement Pages 41-47
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSHuman chymase forms angiotenin (ANG) I to ANG II, whereas the roles of ANG II generated by chymase and the effects of chymase inhibitors have been unclear. On the other hand, rat chymase could not convert ANG I to ANG II. In isolated rat arteries, the ANG I- induced vascular contraction was completely suppressed by angiotensin-converting enzyme (ACE) inhibitor only. However, 30% of ANG I-induced vascular contraction in isolated human arteries was suppressed by an ACE inhibitor, but the remainder was blocked by chymostatin. In hamster hypertensive models, ANG II formation by ACE, but not by chymase, in vascular tissues plays an important role in maintaining hypertension. ANG II formation also induces vascular remodeling such as neointima formation. After balloon injury of vessels in dog, chymase and ACE activities were significantly increased in the injured vessels. In this model, an ANG II receptor antagonist was effective in preventing neointimal formation after balloon injury of vessels in dog, but an ACE inhibitor was ineffective. In dog grafted veins, the activities of chymase and ACEmin the grafted vein were significantly increased 15- and 2-fold, respectively, compared with those in the symmetrical veins. The intimal area of the grafted vein was reduced by a chymase inhibitor. Therefore, chymase-dependent ANG II formation plays an important role in the proliferative response, and chymase inhibitors may appear useful for preventing vascular proliferation.View full abstractDownload PDF (6055K) -
Michiko Muramatsu, Jun Katada, Izumi Hayashi, Masataka Majima1999 Volume 114 Issue supplement Pages 48-54
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSWe investigated the angiogenic effect of chymase, an alternative angiotensin II-generating enzyme, on angiogenesis using hamster sponge implant model. Exogenous administration of angiotensin II (Ang II) or angiotensin I (Ang I) directly into the sponges enhanced angiogenesis, as determined from the hemoglobin contents in the sponge granuloma tissues. Chymostatin, an inhibitor of chymase, inhibited angiogenesis induced by Ang I but not by Ang II, suggesting the presence of a chymase-like Ang II-generating activity in the sponge granuloma. TCV-116 (5 mg/kg p.o.), an antagonist of Ang II type 1 receptor, and chymostatin suppressed bFGF-induced angiogenesis, suggesting the significance of the endogenous angiotensin system. Chymase activity in the sponge granuloma increased in parallel with the rise in hemoglobin contents induced by bFGF. We also examined the effects of direct administration of human pro-chymase gene or purified hamster chymase, and demonstrated that in vivo human pro-chymase gene transfection and direct injection of purified chymase enhanced angiogenesis, which was 50% inhibited by TCV-116. Sponge granulomas treated with Ang II was supressed by vascular endothelial growth factor (VEGF) antisense. Our results suggest that chymase enhanced angiogenesis partly through the local production of Ang II, followed by up-regulation of VEGF.View full abstractDownload PDF (5183K) -
Hiroshi Tsunoda, Hidenori Suzuki, Kenji Kanamaru, Toshio Tanaka1999 Volume 114 Issue supplement Pages 55-59
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSUsing fluorescent differential display and quantitative reverse-PCR, we found in the rat vasospasm model that heme oxygenase-1 messenger RNA was induced in basilar artery. Intracistemal injection of antisense OH-1 oligodeoxynucleotide significantly reduced HO-1 mRNA and HO-1 protein levels and enhanced angiographic vasospasm. Thus, we demonstrate that HO-1 induction may play a important role in the resolution of delayed vasospasm after subarachnoid hemorrhage.View full abstractDownload PDF (3955K) -
Yuzuru Matsuda, Kazuhide Hasegawa1999 Volume 114 Issue supplement Pages 60-65
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSWe have established a novel smooth muscle cell line (SVS) which retains the expression of specific markers for smooth muscle cells, such as smooth muscle myosin-1, calponin and SM22α, from temperature-sensitive SV40 large T-antigen transgenic mice. SVS cells showed temperature-dependent growth and the expression of smooth muscle markers, showing that the differentiation stage can be controlled in this cell line by culture temperature. We have constructed luciferase-reporter cell lines for calponin and SM22α genes by using SVS cells. We detected some compounds including ascorbic acid, which stimulated the expression of smooth muscle markers by the reporter assay. Ascorbic acid and its long lasting derivatives stimulated the expression of markers continuously. Moreover, ascorbic acid activated the expression of markers in synthetic SMCs seen in the intima of a rat balloon injury model. Thus, this reporter system should be useful for discovery of new antiatherosclerotic drugs.View full abstractDownload PDF (6938K) -
Minoru Seto, Yoh Takuwa, Yasuharu Sasaki1999 Volume 114 Issue supplement Pages 66-70
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSIncrease in cytosolic Ca2+ concentration and phosphorylation of myosin light chain (MLC) are key steps of normal and abnormal contractility in smooth muscle. In pathological condition like vasospasm, artery exhibits a hyperreactive contraction associated with an enhanced and sustained MLC phosphorylation, particularly diphosphorylation (Thr18/Ser19). Phosphorylation level is regulated by a balance between phosphorylation and dephosphorylation. Recent studies propose that myosin phosphatase activity is regulated through the phosphorylation of 130K-myosin binding subunit (MBS130K) of the myosin phosphatase in smooth muscle. It is known that in normar artery, receptor agonists like PGF2α induce certain contraction of smooth muscle associated with MLC diphosphorylation. We recently confirm that this PGF2α stimulation also evokes the phosphorylation of MBS130K and fasudil, Rho-kinase inhibitor, inhibits this phosphorylation in parallel with MLC phosphorylation. Furthermore, we reveal that in vasospastic artery, Rho-kinase activity is augmented to induce high level phosphorylation of MBS130K. In this study, we also confirm that phosphorylation of MBS130K is Rho-kinase-dependent and its site is Thr654 residue. Now we speculate that fasudil may shift the phosphorylation-dephosphorylation cycle to dephosphorylation side through the inhibition of Thr654phosphorylation in MBS130K. These results also suggest that augmented activation of Rho-kinase may be involved in the pathophysiology of vasospasm associated with MLC diphosphorylation.View full abstractDownload PDF (3889K) -
Kazuhiro Kohama1999 Volume 114 Issue supplement Pages 71-74
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSWe constructed a plasmid vector to have a 1.4kb insert of myosin light chain kinase (MLCK) cDNA in an antisense direction to express antisense mRNA. The construct was then transfected to SM3, a cell line from vascular smooth muscle cells (VSMCs), producing a few stable transfectants. The down-regulation of MLCK expression in the transfectants was confirmed by both Northern and Western blots. The control SM3 showed chemotaxic motility to the platelet derived growth factor (PDGF), which was supported by the membrane ruffling. However, the transfectants showed neither chemotaxic motility nor developed membrane ruffling, indicating the essential role of MLCK in the motility. The specificity for the targeting was assessed by demonstrating that Rho-kinase activity, which also phosphorylates the myosin light chain (MLC), was well preserved in both SM3 and the transfectants. In spite of this importance of MLCK, PDGF failed to induce MLC phosphorylation in not only the transfectants but also in SM3. The mode in which MLCK was involved in the development of membrane ruffling is discussed with special reference to myosin-binding property of MLCK (Ye et al. Proc Natl. Acad. Sci. USA 96 6666-6671, 1999)View full abstractDownload PDF (3686K) -
Hitoshi Kurose, Motohiro Nishida, Taku Nagao1999 Volume 114 Issue supplement Pages 75-80
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSAlthough ischemia-reperfusion produces reactive oxygen species and induces injury of the heart, the mechanism leading to injury is largely unknown. Hydrogen peroxide (H2O2) is widely used for a reagent to mimic the action of reactive oxygen species produced by ischemia-reperfusion. Treatment of the rat neonatal ryocytes with H2O2 resulted in activation of mitogen-activated protein kinases (MAPKs) such as extracellular signal regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38. To study the involvement of βγ subunit of heterotrimeric G protein in H2O2-induced activation of MAPKs, we expressed the carboxyl terminus of G protein-coupled receptor kinase 2 (GRK2-ct) which can bind βγ subunit and inhibit the interaction with various effector proteins. Expression of GRK2-ct inhibited the H2O2-induced activation of ERK by 70% and also inhibited the activation of Akt by 30%. In contrast with H2O2-induced activation of ERK, the activation of ERK induced by phorbol ester PMA and the activation of JNK and p38 induced by H2O2 were not affected by expression of GRK2-ct, indicating that the activation of ERK but not JNK and p38 is dependent on βγ subunit. Among several inhibitors for analyzing intracellular signaling pathways, wortmannin inhibited the activation of ERK by H2O2 treatment. These data suggest that treatment of the rat neonatal myocytes with H2O2 releases βγ subunit from heterotrimeric G protein, and leads to activation of ERK in part by phosphatidylinositol-3 kinase dependent pathway. Thus βγ subunit may be a novel target molecule to selectively modulate the intracellular signaling cascade.View full abstractDownload PDF (5113K) -
Akinori Hisatsune, Ayako Uto, Takashi Koyanagi, Takahiro Chihara, Take ...1999 Volume 114 Issue supplement Pages 81-85
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSLysozyme is an important component of innate immunity against common pathogens at mucosal surfaces. We previously cloned and characterized the bovine lysozyme 5A (lys5A) promoter with the purpose of determining cis and trans-acting elements controlling airway epithelial cell- specific expression. We found that such expression is controlled by protein binding to an ETS consensus sequence located approximately at - 46 to -40 by from the transcription start site. The identity of the ETS-related protein responsible for gene transactivation was unknown. In this study, we screened six ETS-related proteins by transient transfection into epithelial cells and fibroblasts. Results showed that among these factors, the myeloid Elf-1-like factor (MEF) was the most potent. Gel shift analysis of epithelial cell nuclear extracts using a lys5A probe including the ETS-binding site (-50/-31) yielded a single band with retarded mobility. This band was supershifted by an antibody directed against MEF. Supporting the possibility that MEF is responsible for functional transactivation of lysozyme in epithelial cells, we found that antisense MEF mRNA decreased lys5A promoter activity and that MEF overexpression in stably transfected cells increased lysozyme mRNA and protein expression. We conclude that MEF is required for epithelial cell transactivation of lysozyme.View full abstractDownload PDF (4661K) -
Tetsuhiro Hisayama, Mika Inomoto, Yukari Hioki, Hiroyuki Fukui1999 Volume 114 Issue supplement Pages 86-91
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSProtein kinase C (PKC) family, is now classified into three groups; conventional (cPKC), novel (nPKC) and atypical (aPKC), and to date, 10 members of isozymes have been identified. We have suggested that PKC is essential to interleukin-1 (IL-1)-triggered expression of inducible NO synthase (iNOS), and that by pharmacological analysis, cPKC is not involved in iNOS induction in rat vascular smooth muscle cells (VSMC). In the present study, we identified some PKC isozymes and investigated the effect of PKCα knockdown by antisense oligodeoxynucleotide (AS-ODN) strategy on iNOS expression and nuclear translocation of NF-κB in RASMC. Western blot analysis revealed the presence of cPKC (α), nPKCs (δ and ε) and aPKCs (ι and λ). Short-time (10-20 min) treatment with phorbol 12-myristate 13-acetate (PMA) induced translocation of PKCα from cytosolic to particulate fraction. PKCα was completely downregulated by treatment with 100 nM PMA for 24 hours. Treatment with AS-ODN against PKCα mRNA depleted PKCα specifically, and had no detectable effect on the other PKCs. The production of iNOS mRNA, but not nuclear translocation of NF-κB, stimulated by IL-1β was decreased by PKCα knockdown. These results suggest that there are 5 PKC isozymes in RASMC, and that PKCα is involved in iNOS expression triggered by IL-1β, supporting our previous pharmacological conclusion.View full abstractDownload PDF (5802K) -
Yasuyuki Nomura, Takashi Uehara, Tadashi Nishiya1999 Volume 114 Issue supplement Pages 92-95
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSTo elucidate the intracellular mechanism of NF-κB activation, we performed the involvement of IκBα of NF-κB in the expression of inducible NO synthase (iNOS) and chemokine (CINC) following pretreatment with bacterial endotoxin (LPS) or IL-1β, respectively, using rat C6 glioma cells. We found that herbimycin A, a tyrosine protein kinase inhibitor, blocked: 1) LPS/IFNγ-induced iNOS expression, 2) LPS-induced intranuclear translocation of activated NF-κB (p50·p65) and 3) IFNγ-induced autophosphorylation and activation of Jak 2 and Stat 1 as well as intranuclear translocation of phosphorylated Stat 1. Furthermore, transfection of a dominant negative form of IκBα(SS→AA) suppressed LPS/IFNγ-induced iNOS expression, suggesting that NF-κB, in particular, IκBα molecules could play important roles in the iNOS expression. We also found in IL-1β-induced CINC expression using cultured C6 glioma cells, the transient translocation of NF-κB in response to IL-1β is partly dependent on transient proteasome activation. Thus we suggest that the formation of heterodimer p50·p65 from inactive trimer p50·p65·IκBα particularly, proteolytic degradation and dissociation of IκBα from p50·p65 are a critical phase in NF-κB activation during LPS-induced iNOS and IL-1β-induced CINC expression in astroglial cells.View full abstractDownload PDF (3937K) -
Yasushi Iwamuro, Xiao-Feng Zhang, Yasuo Okamoto, Soichi Miwa, Tomoh Ma ...1999 Volume 114 Issue supplement Pages 96-102
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSTo clarify Ca2+ entry channels involved in the endothelin-1 (ET-1)-induced increase in the intracellular concentration ([Ca2+]i), we performed whole-cell recordings of patch-clamp techniques and monitoring of [Ca2+]i with Ca2+ indicators fura-2 and fluo-3 in A7r5 cells (a cell line derived from rat thoracic aortic smooth muscle cells). With whole-cell recordings, lower concentrations (≤ 1 nM) of ET-1 activated a Ca2+-permeable nonselective cation channel (designated NSCC-1). In contrast, higher concentrations (≤ 1 nM) of ET-1 activated two types of Ca2+-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) and store-operated Ca2+ channel (SOCC). Importantly, we found that these Ca2+ channels can be pharmacologically discriminated using blockers of the so-called receptor operated Ca2+ influx such as SK&F 96365 and LOE 908. That is, NSCC-1 is resistant to SK&F 96365 but sensitive to LOE 908; NSCC-2 is sensitive to both SK&F 96365 and LOE 908; SOCC is sensitive to SK&F 96365 but resistant to LOE 908. Using these blockers, we analyzed the ET-1-induced increase in [Ca2+]i. The increase in [Ca2+]i induced by lower concentrations of ET-1 was resistant to SK&F 96365 but sensitive to LOE 908. In contrast, the increase in [Ca2+]i induced by higher concentrations of ET-1 was partially suppressed to sim;30% of controls by either SK&F 96365 or LOE 908 alone, and it was abolished by their combination. These results show that the increase in [Ca2+]i induced by lower concentrations (≤ 1 nM) of ET-1 results from Ca2+ influx through NSCC-1, whereas the increase in [Ca2+] i induced by higher concentrations (≤ 10 nM) of ET-1 results from Ca2+ influx through NSCC-1, NSCC-2 and SOCC.View full abstractDownload PDF (5523K) -
Xiao-Feng Zhang, Yasushi Iwamuro, Yasuo Okamoto, Soichi Miwa, Tomoh Ma ...1999 Volume 114 Issue supplement Pages 103-109
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESS1) We have recently shown that endothelin-1 (ET-1) activates two types of Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and store-operated Ca2+ channel (SOCC). These channels can be pharmacologically discriminated using Ca2+ channel blockers such as SK&F 96365 and LOE 908. Here we characterized Ca2+ entry channels involved in ET-1-induced contractions of rat thoracic aortic rings and increases in the intracellular free Ca2+ concentration ([Ca2+]i) of single smooth muscle cells using these blockers. 2) LOE 908 or a blocker of voltage-operated Ca2+ channel nifedipine had no effect on the contractions and increases in [Ca2+]i induced by thapsigargin, whereas SK&F 96365 abolished them. 3) The contractions and increases in [Ca2+]i induced by ET-1 depended on extracellular Ca2+ but were resistant to nifedipine. The responses to lower concentrations (≤ 0.1 nM) of ET-1 were abolished by either SK&F 96365 or LOE 908. The responses to higher concentrations (≥ 1 nM) were abolished by SK&F 96365, but were partially resistant to LOE 908. 4) These results show that the contractions and increases in [Ca2+]i of rat aortic smooth muscles at lower concentrations of ET-1 involve only one Ca2+ entry channel which is sensitive to SK&F 96365 and LOE 908 (NSCC-2), whereas those at higher concentrations of ET-1 involve another Ca2+ entry channel which is sensitive to SK&F 96365 but resistant to LOE 908 (SOCC) in addition to the former channel.View full abstractDownload PDF (5420K) -
Takanobu Taniguchi, Rika Inagaki, Rumiko Takauji, Fumiko Suzuki, Ikuno ...1999 Volume 114 Issue supplement Pages 110-112
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSUsing microphysiometer, we have investigated acid expulsion from CHO cells expressing human alpha-1a adrenoceptor. Time course of extracellular acidification rate after noradrenaline stimulation had two phases; one with a peak within 10 s reached several folds of base rate, and another increased gradually to two folds of base rate and reached plateau around two min. Both phases showed concentration-dependent increase of acidification rates in response to noradrenaline but had distinct pEC50 values; 6.0 for rapid phase and 6.6 for late phase. Amiloride and its analogs inhibited both phases entirely, suggesting that Na/H exchanger mainly mediated these acid expulsion responses. Elimination of Ca by BAPTA/EGTA treatment resulted in extensive reductions of the rapid phase response but small decrease of the late phase response. Several Ca channel blockers, Ni and LOE908 also suppressed the rapid phase while nifedipine, verapamil, SKF96365 and ω-conotoxin GIVA did not. Repeated stimulation with noradrenaline enhanced inhibitory effect of blockers. These results indicate that Ca is one of the elements in the rapid phase but not in the late phase of acid expulsion from CHO cells in response to alpha-1 adrenoceptor stimulation and suggest that Ca from both intracellular storage and some type of Ca channel dominantly participates in the rapid phase.View full abstractDownload PDF (2764K) -
Takeyuki Yatsu, Yuichi Tomura, Atsuo Tahara, Koh-ichi Wada, Toshiyuki ...1999 Volume 114 Issue supplement Pages 113-117
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSPharmacology of conivaptan hydrochloride (YM087) was investigated in in vitro and in vivo studies. In radioligand binding study, YM087 showed high affinity for both V1A and V2 receptors in animal and human species. Affinity of YM087 for V1A and V2 receptors was comparable to that of vasopressin (AVP). In functional antagonistic activity study, YM087 concentration-dependently inhibited AVP-induced intracellular Ca2+ elevation via human V1A receptors and AVP-stimulated cAMP accumulation via human V2 receptors. Intravenous administration of YM087 dose-dependently inhibited AVP-induced pressor responses and produced a dose-dependent aquaresis in rats and dogs. Oral administration of YM087 showed a potent and long-lasting antagonistic activity on V1A and V2 receptors. YM087 was effective in dogs with heart failure and in heart failure rats with hyponatremia and edema. These results reveal that YM087 is the first orally active V1A/V2 receptor antagonist and suggest that YM087 may be useful in the treatment of congestive heart failure and hyponatremia.View full abstractDownload PDF (5006K) -
Takahiro Horinouchi, Michihiko Tsujitani, Katsuo Koike1999 Volume 114 Issue supplement Pages 118-122
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSThe purpose of the present study was to characterize the atypical β-adrenoceptors involved in relaxant responses in guinea pig gastric fundus, duodenum and ileum in functional experiments with catecholamines (isoprenaline, noradrenaline and adrenaline), β3-adrenoceptor agonists (BRL37344 and CGP12177A) and a non-selective β1-, β2- and β3-adrenoceptor antagonist bupranolol, and to obtain further evidence to clarify whether there is a tissue difference in atypical β-adrenoceptors in the guinea pig gastrointestinal tissue systems. The atypical β-adrenoceptors are present in gastric fundus, duodenum and ileum of guinea pig. In the presence of propranolol (1 μM) or atenolol (100 μM) plus butoxamine (100 μM), bupranolol caused a concentration-dependent rightward shift of the concentration-response curves for catecholamines and β3-adrenoceptor agonists. There was not a significant difference of pA2 values for bupranolol against these agonists between gastric fundus, duodenum and ileum of guinea pig. These results suggest that guinea pig gastric fundus, duodenum and ileum relaxation are mediated predominantly by an atypical β-adrenoceptor population whereas the classical β1- or/and β2-adrenoceptors play a subordinate function role and that the receptors of three tissues are pharmacological identified by functional approaches. There is not a tissue difference in atypical β-adrenoceptors in the guinea pig gastrointestinal tissue systems between stomach and ileum.View full abstractDownload PDF (4598K) -
Toshio Ohnuki, Takafumi Nagatomo, Masaji Ishiguro1999 Volume 114 Issue supplement Pages 123-126
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSWe deduced the three dimensional structures of transmembrane regions in human β adrenoceptors by computer simulating using the 2D electron density maps of bacteriorhodopsin and frog rhodopsin as templates. The deduced membrane-spanning helical regions, their arrangements and axes to the membrane plain were difrent between two models. Typical β adrenergic antagonist, propranolol, and long-lasting β antagonist, bopindolol, were docked in these models. The receptor-antagonist interactions were difrent between both models, in which the 3, 4, 5 and 6th transmembrane domains might participate in the antagonists-binding in both models, however, the amino acid residues in the domains contributing to the binding might be different between models. Propranolol and bopindolol have high affinity to β1 and β2 subtypes, but low affinity to β3 subtype. The simulated receptor-antagonist relationships could account, in part, for the selectivity of the antagonists to the β subtypes.View full abstractDownload PDF (4167K) -
Takuya Kobayashi, Fumitaka Ushikubi, Shuh Narumiya1999 Volume 114 Issue supplement Pages 127-129
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSProstanoid receptors are the G-protein-coupled, rhodopsin-type receptors with seven transmembrane domains and consist of eight types and subtypes. Although the overall homology is not high, there are several regions specifically conserved among them. These regions are considered to form the ligand binding pocket for the structures common to prostanoid molecules, and the other regions to confer specificity for ligand binding. The PGI and PGD receptors have relatively high homology (40%) at the amino acid level and share the same signalling pathway. To determine which structural domains of these receptors confer ligand binding specificity, we constructed a series of chimeric receptors from the mouse PGI and PGD receptors. These chimeric receptors were expressed in COS-7 cells, and their abilities to bind prostaglandins and their analogues were examined. The region from the sixth transmembrane domain to the carboxyl terminus of the PGI receptor was first replaced by the corresponding region of the PGD receptor. This chimeric receptor binds both PGD2 and PGE2, though the ability to bind iloprost, a PGI receptor agonist, and PGE1 does not change. This result indicates that the sixth and seventh transmembrane domains of the PGI receptor play an important role in distinction of structural difference between PGE1 and PGE2 in the α-side chain. These binding characteristics did not change when the region up to the third transmembrane domain of the PGI receptor was replaced with the corresponding region of the PGD receptor. However, when the first extracellular loop including a portion of the second transmembrane domain was further replaced, the abilities to bind PGE1, PGE2 and iloprost were eliminated. This result indicates that this domain of the PGD receptor is responsible for distinction of structural differences between PGD2 and PGE2 on the cyclopentane ring.View full abstractDownload PDF (2735K) -
Katsuro Tachibana, Shunro Tachibana1999 Volume 114 Issue supplement Pages 138-141
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSUltrasound is frequently used in medicine for diagnostic purposes. Recently, there have been numerous reports on application of ultrasound energy for controlling drug release or targeting. This new concept of therapeutic ultrasound combined with drugs has induced excitement in various areas. Ultrasound energy can enhance effects of thrombolytic agents as urokinase. Ultrasound emitting catheters are currently being developed for cardiovascular diseases. Device with ultrasound transducers implanted in transdermal drug patches are also being evaluated for possible delivery of insulin through the skin. Chemical activation of drugs by ultrasound energy for treatment of cancers is another new field recently termed as “Sonodynamic Therapy”. Various examples of application of ultrasound for drug delivery systems are discussed.View full abstractDownload PDF (3420K) -
Kazuo Ichihara, Kumi Satoh, Atsuko Yamamoto, Katsuji Hoshi1999 Volume 114 Issue supplement Pages 142-149
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSEffects of pravastatin, simvastatin, atorvastatin, fluvastatin and cerivastatin on myocardial contractile dysfunction during reperfusion after brief ischemia were examined in dogs. Pretreatment of the dog with lipophilic HMG-CoA reductase inhibitors for 3 weeks, simvastatin (2 mg/kg/day), atorvastatin (2 mg/kg/day), fluvastatin (4 mg/kg/day), and cerivastatin (40 μg/kg/day) worsened recovery of myocardial contraction during reperfusion after brief ischemia in association with reduced myocardial ATP level. A hydrophilic HMG-CoA reductase inhibitor, pravastatin (2 and 4 mg/kg/day), did not affect the recovery of myocardial contractile function and ATP level during reperfusion following ischemia. The lipophilic inhibitors may enter the myocardial cell, inhibit ubiquinone biosynthesis, and depress ATP generation in mitochondria, leading to worsening of the myocardial stunning after reperfusion subsequent to ischemia.View full abstractDownload PDF (6528K) -
Hitoshi Nakayama, Nobutaka Ohgami, Akihiko Kuniyasu, Akira Miyazaki, H ...1999 Volume 114 Issue supplement Pages 150-153
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSSulfonylureas are generally used in the therapeutic treatment of non-insulin-dependent diabetes mellitus. Little is known, however, whether they also affect the lipid metabolism. Using glibenclamide (GB), a typical sulfonylurea, we have investigated its effects on the lipid metabolism in macrophages, J774 and phorbol ester-treated THP-1 cells. In the whole-cell assay system for cholesteryl ester (CE) accumulation that is inducedby addition of chemically modified low-density lipoprotein (LDL), such as Ac-LDL and ox-LDL, GB effectively inhibited the CE accumulation of J774 cells in dose-dependent manners. The inhibition was resulted from increase in free cholesterol but not from change in total cholesterol amount. The results suggest that GB acts on acyl-CoA: cholestrol acytransferase (ACAT) and inhibits its activity. To confirm the possibility, we then tested GB by another assay system using ACAT that was solubilized from the cells and reconstituted into the liposome composed of phosphatidyl choline- cholesterol. GB inhibition was not so much effective as those by CI-976 and NTE-122, known ACAT inhibitors, but the inhibition was complete in the presence of 100 μM GB. Using cell homogenates of PMA-stimulated THP-1 cells, GB also inhibited the ACAT activity to the level of undifferentiated THP-1 cells. These results indicate that GB acts as ACAT inhibitor but the chemical structure is quite different from the conventional ACAT inhibitors, suggesting it can be a seed to generate potential ACAT inhibitors which do not exhibit toxicity in adrenal gland.View full abstractDownload PDF (3285K) -
Yuta Kobayashi, Hiroaki Shirahase, Hui-fang Zhao, Eiichi Kakizoe, Hide ...1999 Volume 114 Issue supplement Pages 154-158
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSCromoglicate lisetil (CL) is an orally deliverable prodrug of cromoglycic acid, having diethyl promoieties and a lysyl promoiety fir its optimum drug-delivery. We examined the efcts of CL on bovine type II collagen (CII)-induced arthritis (CIA) of male DBA/1J mice, an experimental model fir human rheumatoid arthritis, and its action mechanism. CL (100 mg/kg/day) was given by gavage to CII-immunized mice once daily for 6 weeks, starting when arthritic symptoms became evident. Symptomatic scores of arthritis obviously elevated in non-treated CIA mice at week 6.5 affer initial immunization and continued elevated thereafter throughout the experiment, the elevation which was reduced by CL. CL also improved radiographic score of phalangeal destruction and pathohistological indexes at the end of treatment period. Serum anti-CII antibody titer was increased in non-treated CIA mice and the elevation was reduced by CL treatment. Mast cells (MCs) number in arthritic region was increased in non-treated CIA mice but not by CL treatment. In conclusion, oral CL treatment proved beneficial in CIA mice. Observed correlation between the CL effect on CIA and that on MCs number suggests the potential contribution of MCs to accelerate chronic arthritic processes and may further implicate potential action mechanism of CL, which may act by regulating MC functions for chronic inflammation.View full abstractDownload PDF (4453K) -
Naoaki Harada, Kenji Okajima, Takeshi Katsuragi1999 Volume 114 Issue supplement Pages 159-167
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSTo examine whether adenosine reduces ischemia/reperfusion (I/R)-induced liver injury by inhibiting leukocyte activation via A2 receptor (A2R), we investigated the effects of adenosine and YT-146, selective A2A receptor (A2AR) agonist, on I/R-induced liver injury in rats. Adenosine and YT-146, in the range of concentrations of 10-7sim;10-5 M, significanlty inhibited the formyl-methionyl-leucyl-phenylalanine (fMLP)-induced neutrophil elastase release from isolated neutrophils by about 35% in vitro. Adenosine and YT-146, in the range of concentrations of 10-7 ?? 10-5 M, significantly inhibited the endotoxin-stimulated TNF-α production by monocytes to less than 50% of the control. Although ZM241385, a selective A2AR antagonist, significantly enhanced the fMLP-induced neutrophil elastase release in isolated neutrophils in vitro, this agent did not affect the endotoxin-stimulated TNF-α production by monocytes. Male Wistar rats were subjected to complete ischemia of median and left lobes of liver for 60 min and the subsequent reperfusion. Serum levels of transaminases increased over time after hepatic I/R, peaking at 12 hrs after reperfusion. Intravenous infusion of Adenosine (1 and 10 mg/kg/hr) and YT-146 (0.1 and 1 mg/kg/hr) significantly inhibited the I/R-induced increases in serum transaminase levels 12 hrs after reperfusion. The I/R-induced decrease in hepatic tissue blood flow was significantly inhibited by adenosine and YT-146. Hepatic levels of TNF-α, cytokine-induced neutrophil chemoattractant (a member of interleukin-8), and myeloperoxidase were significantly increased after I/R. These increases were significantly inhibited by administration of adenosine and YT-146. However, ZM241385 did not reduce the I/R-induced liver injury and it inhibited neither the decrease in hepatic tissue blood flow, nor the indicators of leukocyte activation. These findings suggest that adenosine may reduce I/R-induced liver injury mainly by inhibiting hepatic TNF-α production via A2AR, thereby reducing neutrophil activation.View full abstractDownload PDF (7717K) -
Naohisa Ishikawa, Ossama Hamdy, Hiroshi Maekawa, Hidetsugu O. Murakami ...1999 Volume 114 Issue supplement Pages 168-172
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSIn order to find some treatments for respiratory failure caused by pulmonary edema, we investigated the mechanism of neurogenic pulmonary edema. Previously, stimulation of sympathetic nerves caused an increase in pulmonary vascular permeability, possibly due to neuropeptide Y. Neuropeptide Y injected into the trachea increased lung vascular permeability dose-dependently, the ED50 of which was 0.3 - 1 nM. Such an effect remained even after treatment with reserpine, as well as in the presence of α- and β-blockers. And norepinephrine enhanced the effect of neuropeptide Y on lung vascular permeability. These responses were almost similar to those obtained by stimulation of sympathetic nerves. Furthermore, neuropeptide Y, in fibrin-induced pulmonary edema, was localized in alveolar macrophages and alveolar spaces, amounting to approximately 200 nM in edema fluid. The value was significantly greater than that obtained in hydrostatic pulmonary edema by 10 - 30 times. Peptide YY, an analogue of neuropeptide Y, had no action on lung vascular permeability, whereas the effect of neuropeptide Y was inhibited by pretreatment with neuropeptide Y- 13-36, an antagonist for Y3-recetor subtype. These results suggested that neuropeptide Y enhances the lung vascular permeability via Y3-recetor subtype. Neuropeptide Y- 13-36, in fibrin-induced pulmonary edema, decreased a ratio of protein concentration in edema fluid to that in serum, indicating that neuropeptide Y actually acts a role in the development of neurogenic pulmonary edema, via an increase in lung vascular permeability.View full abstractDownload PDF (4124K) -
Atsufumi Kawabata, Ryotaro Kuroda, Morley D. Hollenberg1999 Volume 114 Issue supplement Pages 173-179
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSThe protease-activated receptor (PAR), a G protein-coupled receptor present on cell surface, mediates cellular actions of extracellular proteases. Proteases cleave the extracellular N-terminal of PAR molecules at a specific site, unmasking and exposing a novel N-terminal, a tethered ligand, that binds to the body of receptor molecules resulting in receptor activation. Amongst four distinct PARs that have been cloned, PARs 1, 3 and 4 are activated by thrombin, but PAR-2 is activated by trypsin or mast cell tryptase. Human platelets express two distinct thrombin receptors, PAR-1 and PAR-4, while murine platelets express PAR-3 and PAR-4. Apart from roles of PARs in platelet activation, PARs are distributed to a number of organs in various species, predicting their physiological importance. We have been evaluating agonists specific for each PAR, using multiple procedures including a HEK cell calcium signal receptor desensitization assay. Using specific agonists that we developed, we found the following: 1) the salivary glands express PAR-2 mRNA and secret saliva in response to PAR-2 activation; 2) pancreatic juice secretion occurs following in vivo PAR-2 activation; 3) PAR-1 and PAR-2 modulate duodenal motility. Collectively, PAR plays various physiological and/or pathophysiological roles, especially in the digestive systems, and could be a novel target for drug development.View full abstractDownload PDF (6407K) -
Yoshio Goshima, Nobuya Furukawa, Nobutaka Arai, Takeaki Miyamae, Yukio ...1999 Volume 114 Issue supplement Pages 180-185
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSWe have previously obtained evidence that DOPA is probably involved in an upstream process of mechanisms for in vivo neuronal cell death in striatum. We attempted to clarify whether or not this is also the case in hippocampal region of conscious Wistar rats. Four vessels were occluded for 5 min during microdialysis of hippocampus. DOPA, dopamine and glutamate (Glu) in perfusates collected every 10 min were measured by HPLC-ECD and spectrophotometer. Delayed neuronal cell death in hippocampus was evaluated 96 hr after ischemia. Five-min transient brain ischemia induced Glu release, with the peak being 2.5-fold of a basal release at the fraction immediately after ischemia. The release of DOPA and dopamine was not consistently detectable, but an increase was sometimes observed during and after ischemia. Delayed neuronal cell death was slight to moderate with 5-min ischemia. Intrastriatal perfusion of DOPA cyclohexyl ester (DOPA CHE) at 100 nM, a novel stable potent competitive DOPA antagonist, almost completely inhibited the ischemia-induced glutamate release, and protected hippocampal neurons from delayed cell death. Endogenously released DOPA itself seems to act on its recognition site and to behave as a causal and/or deteriorating factor on glutamate release and resultant delayed neuronal cell death by transient ischemia in rats.View full abstractDownload PDF (5494K) -
Masaru MINAMI, Naoya HAMAUE, Toru ENDO, Masahiko HIRAFUJI, Mutsuko TER ...1999 Volume 114 Issue supplement Pages 186-191
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSIsatin (indole-2, 3-dione), an endogenous inhibitor of monoamine oxidase (MAO), has several physiological properties for stress and anxiety. We previously identified isatin in the brain of stroke-prone spontaneously hypertensive rats (SHRSP) using gas-chromatography mass spectrometry. This study elucidated the effects of isatin on the ACh and DA levels of brain tissues in rats. Furthermore, we evaluated the effect of isatin on DA levels in a rat model of Parkinson's disease induced by Japanese encephalitis virus.
Striatal ACh and DA levels significantly increased at 2 hours after isatin (50-200 mg/kg, i.p.) administration. Perfused through a microdialysis probe, isatin (10-6-10-4 M) also produced a significant and concentration-dependent increase in the ACh and DA concentrations in the perfusate from the rat striatum. Furthermore, urinary isatin concentrations in patients with Parkinson's disease tend to increase according to the severity of disease. Isatin (100 mg/kg, i.p.) significantly increased striatal DA levels in a rat model of Parkinson's disease. These results suggest that urinary isatin may become a diagnostic marker for the clinical severity of Parkinson's disease and that endogenous isatin, a new biological modulator, may play a role in the regulation of the brain levels of ACh by increasing the level of DA under stress.View full abstractDownload PDF (4781K) -
Hiroshi Katsuki, Toshiaki Kume, Shuji Kaneko, Akinori Akaike1999 Volume 114 Issue supplement Pages 192-197
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSWe utilized organotypic midbrain slice cultures for the assessment of survival and degeneration of dopaminergic neurons in the substantia nigra. Application of N-methyl-D-aspartate (NMDA) to midbrain slice cultures for 24 h caused a concentration-dependent decrease in the number of surviving dopaminergic neurons visualized by tyrosine hydroxylase immunohistochemistry. Simultaneous application of (-)-deprenyl significantly attenuated the cytotoxic effect of NMDA. Because pretreatment with (-)-deprenyl failed to reduce NMDA toxicity, it is suggested that the neuroprotective effect of (-)-deprenyl is not mediated by its irreversible inhibitory action on monoamine oxidase B. We also prepared co-cultures of midbrain and striatal slices to investigate whether the presence of target tissue influences toxic actions of several drugs on dopaminergic neurons. Co-cultured dopaminergic neurons formed dense innervation to the striatal tissue. Dopaminergic neurons in midbrain—striatum co-cultures were more resistant to the cytotoxic actions of NMDA and a nitric oxide donor NOC-18, than the same neuronal population in single midbrain cultures. On the other hand, the toxicity of 1-methyl-4 phenylpyridinium ion or buthionine-[S, R]-sulfoximine was more prominent in midbrain—striatum co-cultures than that in single midbrain cultures. Organotypic slice cultures appeared to be a useful system for evaluation of dopaminergic neuronal death under experimental conditons relevant to physiological/pathophysiological situations.View full abstractDownload PDF (5511K) -
Tomoaki Sato, Koh-ichi Tanaka, Yoshiko Ohnishi, Toyonori Teramoto, Ken ...1999 Volume 114 Issue supplement Pages 198-203
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSThe effects of a novel vasopressin fragment analog NC-1900 (pGlu-Asn-Ser-Pro-Arg-Gly-NH2 acetate) were studied on learning and/or memory impairment in passive avoidance task and on cell damage of cultured cerebro-cortical neurocytes induced by glutamic acid. A small dose of NC-1900 (1 ng/kg, s.c.) ameliorated impairments of learning and/or memory induced by intracistemal injection of 467.6 μg of 10 μl glutamic acid. NC-1900 also ameliorated the impairments induced by intracisternal NMDA, AMPA-antagonist CNQX and by metabotropic receptor (mGluR1) agonist 3, 5-dihydroxyphenylglycine but not by kainate agonist domoic acid nor MK-801 in mice. NC-1900 (100 pM, 1nM) ameliorated the cell damage of cultured rat cerebro-cortical neurocytes induced by 100 and 1000 μM of glutamic acid. These results suggest that NC 1900 may serve as a remedies in various patients with certain brain disorders induced by excess glutamic acid.View full abstractDownload PDF (6239K) -
Kumatoshi ISHIHARA, Muhammad AKBAR, Masashi SASA1999 Volume 114 Issue supplement Pages 204-208
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSSigma receptors in the central nervous system have been considered to play an important role in the modulation of mental diseases and memory/learning. However, the physiological function of sigma receptors still remains unknown. To elucidate physiological functions of the sigma receptors in modulation of neuronal activities, the effects of OPC-24439 (a sigma receptor ligand) on neuronal activities in hippocampal slices were studied with electrophysiological methods. Hippocampal slices (thickness ca. 450 μm) were prepared from male Wistar rats (4-7 weeks of age). In extracellular recording, population spikes in the CA1 region evoked by stimulation applied to the Schaffer collateral/commissural fibers were suppressed by OPC-24439 (1-100 μM) in a dose-dependent manner. This inhibition was antagonized by simultaneously applied haloperidol at 1 μM. In intracellular recording experiments. OPC-24439 (100 μM) did not affect the resting membrane potentials of neurons recorded. In addition. OPC-24439 had no effects on depolarization and firing induced by glutamate. These results indicate that sigma receptor activation caused suppression of neuronal activities in the hippocampus via the sigma receptors. This inhibition probably mediated via the suppression of ion channels that are not related to membrane potentials on post-synaptic neurons and/or sigma receptors on pre-synaptic neurons or interneurons.View full abstractDownload PDF (3886K) -
Ryo Saito, Hideto Ariumi, Hisahiko Kubota, Sachiko Nago, Kenji Honda, ...1999 Volume 114 Issue supplement Pages 209-214
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSSubstance P (SP) is a member of the tachykinin family of bioactive peptides and has highest affinity for the NK-1 receptor. We have developed the non-peptide compound HSP-117 as a selective antagonist of the NK-1 receptor. Binding of 3H-SP to the membranes of IM-9 cells was inhibited by the antagonists HSP-117 and CP-99, 994, the inhibitory activity of HSP-117 being 50-fold that of CP-99, 994. The SP-induced firing responses of single neuron activity in slices of the nucleus tractus solitarius of ferrets were inhibited by 10 μM HSP-117. Intracerebroventricular injection of HSP-117 significantly inhibited retching and vomiting induced by copper sulphate and morphine and the inhibitory effect of HSP-117 on emesis was greater than that of CP-99, 994. Moreover, emesis induced by copper sulphate and morphine were inhibited by the microinjection of HSP-117 and CP-99, 994 into the area postrema and by lesion of the area postrema. These results indicate that HSP-117 is a potent anti-emetic agent, blocking NK-1 receptors in the area postrema and that NK-1 receptors in the area postrema play an important role in emesis induced by broad-spectum emetic stimuli.View full abstractDownload PDF (5557K) -
Norimichi NAKAHATA, Reiki KYO, Mizuyo KUTSUWA, Yasushi OHIZUMI1999 Volume 114 Issue supplement Pages 215-219
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSMitogen-activated protein kinase (MAPK) is a family of serine/threonine kinase that appears to be a component common to signalling pathway initiated by a wide range of factors including hormones, differentiation factors and mitogens. Baicalein is a flavonoid derived from the root of Scutellaria baicaleins. From searching the inhibitors of prostaglandin synthesis in C6 rat glioma cells, we found that baicalein had a potent inhibitory activity of prostaglandin synthesis induced by either histamine or A23187, a Ca2+ ionophore. Baicalein inhibited histamine- and A23187-induced phosphorylation of MAPK in the cells, which was known to cause the phosphorylation of cytosolic phospholipase A2. Baicalein also inhibited the phosphorylation of MAPK kinase (MEK) induced by histamine or A23187 in the cells. In vitro kinase assay, baicalein inhibited the activities of raf but not MEK. These results imply that baicalein is a specific inhibitor of MAPK cascade, acting on phosphorylation of MEK by raf.View full abstractDownload PDF (5002K) -
Kimihiro Matsunaga, Keigo Nakatani, Masahiro Murakami, Norimichi Nakah ...1999 Volume 114 Issue supplement Pages 220-224
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSGoniodomin A induced a potent stimulation of the actomyosin ATPase activities of actin-myosin reconstituted system and natural actomyosin. Interestingly, these stimulatory effects of goniodomin A were significantly inhibited by troponin-tropomyosin complex. The ATPase activity of skeletal muscle myofibrils and the contractile response of skinned fibers to Ca2+ were never activated and were decreased by this compound, suggesting inhibition by troponin-tropomyosin complex. In the analysis of the far-ultraviolet circular dichroism spectra, goniodomin A caused a concentration-dependent increase in α-helical content, of actin. Goniodomin A inhibited the ATPase activities of atrial myofibrils, naural actimyosin and reconstituted actomyosin. Interestingly, these ATPase activities of ventricular muscle were enhanced by goniodomin A. The stimulatory effect of goniodomin A was significantly inhibited by troponin-tropomyosin complex. Goniodomin A increased the intracellular content of F-actin and caused the change in its distribution in 1321N1 cells. Goniodomin A-induced inositol phosphates accumulation in 1321N1 cells was reduced by C3 exoenzyme but was not affected by pertussis toxin. These results suggest that goniodomin A affects actin directly to modify the actin-myosin interaction and that goniodomin A-induced activation of the actomyosin ATPase activity may be physiologically significant because its activation is sensitive to the regulatory protein system, troponin-tropomyosin complex.View full abstractDownload PDF (4175K) -
Masatoshi Hori, Yasuhiro Matsuura, Ryo Yoshimoto, Hiroshi Ozaki, Takes ...1999 Volume 114 Issue supplement Pages 225-229
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSPectenotoxin-2 (PCTX-2), which is one of Diarrhetic Shellfish Poisoning (DSP), is a family of cyclic polyether macrolide toxin isolated from scallop Patinopecten yessoensis. Although PCTX-2 has a potent cytotoxic activities against several cancer cell lines, the biochemical activity of PCTX-2 has not been determined yet. To clarify the biochemical activity of PCTX-2 is the aime in this study. PCTX-2 inhibited the contractions elicited by 72.7 mM KCl or 1 μM phenyrephrine in a concentration dependent manner in the isolated rat aorta. In A10 cells, actin stressfiber in center but not in periphery of the cell was disrupted by PCTX-2 without any visible shape change. By monitoring fluorescent intensity of pyrenyl-actin, PCTX-2 was found to inhibit the velocity and the degree of actin polymerization in a concentration dependent manner. In addition, PCTX-2 decreased viscosity of F-actin measured with falling ball viscometry. Stoichiometric analysis indicated that PCTX-2 forms 1:4 complex with G-actin. These results suggest that PCTX-2 is a potent natural actin depolymerizing compound with unique mode of action.View full abstractDownload PDF (4105K) -
Motohiro KOBAYASHI, Tsugitaka NATSUME, Jun-ichi WATANABE, Nami FUJIO, ...1999 Volume 114 Issue supplement Pages 230-235
Published: 1999
Released on J-STAGE: February 27, 2007
JOURNAL FREE ACCESSWe studied the antitumor activity of newly synthesized dolastatin 10 analogs. TZT-1027 showed remarkable activity and was selected for further development. TZT-1027 was found to inhibit the assembly of porcine brain microtubule proteins and to depolymerize the polymerized microtubule proteins. Therefore, its mechanism of antitumor activity seems to be at least partially ascribed to the inhibition of microtubule assembly. We further studied the binding site of TZT-1027 on tubulin. Scatchard analysis of 3H-TZT-1027 binding data suggested two binding sites including a high affinity site and a low affinity site. TZT-1027 affected the binding of vinblastine (VBL) on tubulin but its binding site isn't identical to the VBL binding site. TZT-1027 induced apoptosis within 24 h, not only in human leukemia cells such as HL-60, but also in solid tumors such as human prostate carcinoma cells DU 145 and human mammary carcinoma cells MCF-7. TZT-1027 showed good antitumor activity against human xenografts (MX-1 and LX-1) without causing serious body weight reduction, which resulted in tumor regression. We examined the effect of TZT-1027 on the established tumor vasculature using the dye perfusion into tumor. TZT-1027 exhibited considerable antivascular activity in tumor sections in addition to excellent cytotoxic effect.View full abstractDownload PDF (4486K)
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