Biomedical subjects

N Nakahata

Publications and source records attributed to N Nakahata.

At least 37 records · Page 2Linked to original sources

PubMed1999

Antagonism of saikosaponin-induced prostaglandin E2 release by baicalein in C6 rat glioma cells.

There are several Kampo medicines (Chinese herbal medicines) containing both Bupleuri Radix and Scutellariae Radix, which are used for the treatment of inflammation. Saikosaponins are derived from Bupleuri Radix, and baicalein is from Scutellariae Radix. The present study was undertaken to investigate the pharmacological interaction of saikosaponin b1 and baicalein in prostaglandin E2 (PGE2) release from C6 rat glioma cells in vitro. Saikosaponin a, b1 and d potently stimulated PGE2 release, while saikosaponin b2 and c moderately stimulated PGE2 release. Saikosaponin b1 caused an irreversible elevation of intracellular Ca2+ concentration, which was eliminated by removing extracellular Ca2+. On the other hand, baicalein inhibited saikosaponin b1-induced PGE2 release in a concentration-dependent manner. These results suggest that saikosaponins are activators of PGE2 release, and baicalein is one of the functional inhibitors of PGE2 release by saikosaponins.

Animals↗

PubMed1999

[Inhibition of mitogen-activated protein kinase cascade by baicalein, a flavonoid of natural origin].

Mitogen-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.

Animals↗

PubMed1999

[Goniodomin A induces modulation of actomyosin ATPase activity mediated through conformational change of actin].

Goniodomin 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 alpha-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.

Actins↗

PubMed1999

Maitotoxin-induced phosphoinositide hydrolysis is dependent on extracellular but not intracellular Ca2+ in human astrocytoma cells.

Since maitotoxin, a potent marine toxin, is known to cause not only Ca2+ influx but also phosphoinositide hydrolysis, we investigated the Ca2+ dependency of maitotoxin-induced phosphoinositide hydrolysis in 1321N1 human astrocytoma cells. Maitotoxin elicited inositol 1,4,5-trisphosphate accumulation in a time-dependent manner. In [3H]inositol-labeled cells, maitotoxin stimulated phosphoinositide hydrolysis in an extracellular Ca2+-dependent manner. Maitotoxin also caused an intracellular Ca2+ elevation, which was abolished by an intracellular Ca2+ chelater BAPTA-AM. Interestingly, maitotoxin still caused phosphoinositide hydrolysis in the BAPTA-AM-treated cells. These results indicate that maitotoxin-induced phosphoinositide hydrolysis is dependent on extracellular but not intracellular Ca2+ in 1321N1 human astrocytoma cells.

Astrocytoma↗

PubMed1998

A new factor derived from 1321N1 human astrocytoma cells causes differentiation of PC-12 cells mediated through mitogen-activated protein kinase cascade.

Glial cells play an important role in maintaining neural function. In the present study, we examined the effects of a factor derived from human astrocytoma cells (1321N1) on differentiation of rat pheochromocytoma cells (PC-12). The conditioned medium which had been used for culture of 1321N1 cells caused the differentiation of PC-12 cells, suggesting that 1321N1 cells release a neurotrophic factor. The factor was apparently distinct from well-known neurotrophic factors, such as nerve growth factor (NGF), since it was resistant to boiling and trypsin treatment. The molecular size of the factor was assumed to be below 1000 through dialysis and ultrafiltration experiments. Furthermore, PC-12 cells were differentiated synergistically by the combined addition of NGF and the conditioned medium of 1321N1 cells. Partially purified fraction of the factor by Sephadex G-15 gel filtration column caused the prolonged activation of mitogen-activated protein kinase (MAPK). The differentiation of PC-12 cells induced by the fraction or NGF disappeared after the treatment with PD98059, a specific inhibitor of MAPK kinase (MEK), suggesting the involvement of MAPK in the differentiation. These results suggest that the new low-molecular factor derived from glial cells causes differentiation of PC-12 cells mediated through an activation of MAPK.

Animals↗

PubMed1998

Medium change amplifies mitogen-activated protein kinase-mediated prostaglandin E2 synthesis in Swiss 3T3 fibroblasts.

In Swiss 3T3 fibroblasts, changing the culture medium prior to stimulation resulted in an augmentation of bradykinin-induced prostaglandin E2 synthesis. The augmentation depended on the duration of the exposure to the fresh medium, with a maximum effect at 1 h. Fetal calf serum in the fresh medium was essential for augmented prostaglandin E2 synthesis. The medium change slightly augmented the bradykinin-induced increase in intracellular free Ca2+ concentration and phosphoinositide hydrolysis with a different time course from that for prostaglandin E2 synthesis. 4',5,7-Trihydroxyisoflavone (genistein) and 3,4-dihydroxybenzylidene-malononitrile (tyrphostin 23), inhibitors of tyrosine kinases, and 2'-amino-3'-methoxyflavone (PD98059), an inhibitor of mitogen-activated protein kinase (MAPK) kinase, attenuated the increase in prostaglandin E2 synthesis. Bradykinin caused phosphorylation of cytosolic phospholipase A2 and p42/p44 MAPK, which was augmented by the medium change. From the results, it is concluded that activation of MAPK and cytosolic phospholipase A2 is involved in the augmentation of prostaglandin E2 synthesis produced by the medium change.

3T3 Cells↗

PubMed1998

Characterization of mastoparan-induced histamine release from RBL-2H3 cells.

Mastoparan (5-30 microM), a tetradecapeptide isolated from wasp venom, caused histamine release from RBL-2H3 cells in a concentration- and time-dependent manner. Mastoparan-induced histamine release remained after removing the extracellular Ca2+, whereas the antigen-induced one disappeared. Pertussis toxin did not inhibit mastoparan-induced histamine release from the cells, and mastoparan did not stimulate phosphoinositide hydrolysis. In agreement with the results, RBL-2H3 cells had a small amount of ADP-ribosylation substrates for pertussis toxin. Neomycin (1-5 mM) suppressed mastoparan-induced histamine release and phospholipase D activation. However, butanol slightly inhibited mastoparan-induced histamine release. Moreover, 2,3-diphosphoglycerate inhibited mastoparan-induced phospholipase D activation, but not it's histamine release. On the other hand, mastoparan caused the leakage of lactate dehydrogenase from the cells in a similar concentration range to the histamine release. This leakage was also suppressed by neomycin. These results suggest that mastoparan enhances the membrane permeability, resulting in histamine release in a pertussis toxin-insensitive manner, and that mastoparan-induced phospholipase D activation may not relate to histamine release.

1-Butanol↗

PubMed1998

Baicalin and baicalein, constituents of an important medicinal plant, inhibit intracellular Ca2+ elevation by reducing phospholipase C activity in C6 rat glioma cells.

Glial cells have a role in maintaining the function of neural cells. This study was undertaken to clarify the effects of baicalin and baicalein, flavonoids isolated from an important medicinal plant Scutellariae Radix (the root of Scutellaria baicalensis Georgi), on glial cell function using C6 rat glioma cells. Baicalin and baicalein caused concentration-dependent inhibition of a histamine-induced increase in intracellular Ca2+ concentrations ([Ca2+]i). The potency of baicalein was significantly greater than that of baicalin. The noradrenaline- and carbachol-induced increase in [Ca2+]i was also inhibited by baicalein and both drugs inhibited histamine-induced accumulation of total [3H]inositol phosphates, consistent with their inhibition of the increase in [Ca2+]i. These results suggest that baicalin and baicalein inhibit [Ca2+]i elevation by reducing phospholipase C activity. The inhibitory effects of baicalin and baicalein on [Ca2+]i elevation might be important in the interpretation of their pharmacological action on glial cells, such as inhibition of Ca2(+)-required enzyme phospholipase A2.

Animals↗

PubMed1998

Goniodomin A, an antifungal polyether macrolide, increases the filamentous actin content of 1321N1 human astrocytoma cells.

Goniodomin A, a polyether macrolide isolated from the dinoflagellate Goniodoma pseudogoniaulax, caused morphological change in 1321N1 human astrocytoma cells. Observation by scanning electron microscopy revealed that the shape of the cell became refractile from flat in response to goniodomin A. This morphological change was accompanied by the outgrowth of a needle-like structure from the cell surface. Goniodomin A increased the intracellular content of filamentous actin in a concentration- and time-dependent manner. It also caused the change in filamentous actin distribution in the cells. The elongation of filamentous actin was observed in goniodomin A-treated cells. These results suggest that goniodomin A induces morphological change by increasing the content of filamentous actin in non-muscle cells.

Actins↗

PubMed1998

Analysis of inhibitory effects of scutellariae radix and baicalein on prostaglandin E2 production in rat C6 glioma cells.

Inhibitory mechanism of the water extract of Scutellariae Radix on prostaglandin E2 (PGE2) release was examined in C6 rat glioma cells. Scutellariae Radix reduced a Ca2+ ionophore A23187-induced PGE2 release by inhibition of arachidonic acid (AA) liberation. Sho-saiko-to and San'o-shashin-to, which contain Scutellariae Radix, also inhibited PGE2 release. A23187 caused phosphorylation of mitrogen-activated protein kinase (MAPK), resulting in activation of cytosolic phospholipase A2 (cPLA2). Scutellariae Radix and baicalein inhibited the phosphorylation of MAPK. Baicalein, but not baicalin, inhibited A23187-induced PGE2 release. These results suggest that baicalein in Scutellariae Radix reduces AA liberation through the inhibition of the MAPK-cPLA2 pathway.

Amino Acid Sequence↗

PubMed1998

Effects of Sho-saiko-to, San'o-shashin-to and Scutellariae Radix on intracellular Ca2+ mobilization in C6 rat glioma cells.

Glial cells are able to support neurons physically and functionally. The present study was undertaken to determine the effects of Kampo medicines on glial cell function, especially Ca2+ mobilization. C6 rat glioma cells expressed H1-histamine, muscarinic cholinergic and adrenergic alpha1-receptors, stimulation of which resulted in phosphoinositide hydrolysis and increase in intracellular Ca2+ concentrations ([Ca2+]i). The water extracts of Sho-saiko-to and San'o-shashin-to, Kampo medicines which contain Scutellariae Radix (Ogon, the root of Scutellaria baicalensis GFORGI), inhibited histamine (100 microM)-induced increase in [Ca2+]i in a concentration-dependent manner. The water extract of Scutellariae Radix potently decreased [Ca2+]i in a concentration-dependent manner. Sho-saiko-to, San'o-shashin-to and Scutellariae Radix significantly inhibited histamine-induced accumulation of total [3H]inositol phosphates, consistent with their inhibition of the increase in [Ca2+1]i. These results suggest that Sho-saiko-to, San'o-shashin-to and Scutellariae Radix inhibit Ca2+ mobilization mediated via an inhibition of phospholipase C. The inhibitory effect may be important in interpreting the pharmacological actions of above Kampo medicines.

Animals↗

PubMed1998

[Thromboxane A2 receptor-mediated signal transduction in glial cells].

Thromboxane A2 (TXA2) receptor subtypes and their signal transduction were examined in 1321N1 human astrocytoma cells. Placental and endothelial types of TXA2 receptor mRNA were found in astrocytoma cells by RT-PCR procedure. Using immunoaffinity column conjugated with anti-TXA2 receptor antibody, two TXA2 receptors (58 and 55 kDa) were partially purified. The partially purified TXA2 receptor fraction contained Gq/11 and G12. The incubation of the cells with dibutyryl cyclic AMP (dbcAMP) caused a differentiation of human astrocytoma cells. DbcAMP treatment resulted in the reduction of Ca2+ elevation and phosphoinositide hydrolysis induced by TXA2 receptor agonist. Whereas the responsiveness of Ca2+ signaling was weakened by dbcAMP treatment, phosphorylation of mitogen-activated protein kinase was increased in dbcAMP-treated cells. These results suggested that human astrocytoma cells express placental and endothelial TXA2 receptors. The TXA2 receptors were coupled with Gq/11 and G12. DbcAMP treatment discriminates TXA2 receptor-mediated MAPK activation from the Ca2+ signaling pathway.

Bucladesine↗

PubMed1998

Two distinct effects of 12S-hydroxyeicosatetraenoic acid on platelet aggregation by zooxanthellatoxin-A and ionomycin.

The marine toxin zooxanthellatoxin-A (ZT-A) and the Ca2+ ionophore ionomycin caused aggregation in rabbit platelets. While ZT-A-induced platelet aggregation was inhibited by indomethacin, ionomycin-induced aggregation was potentiated by the drug. In contrast, both ZT-A- and ionomycin-induced accumulations of thromboxane B2 (TXB2), a stable metabolite of thromboxane A2 (TXA2), were completely inhibited by indomethacin. 12S-Hydroxyeicosatetraenoic acid (12-HETE), which could be accumulated in the presence of indomethacin, inhibited ZT-A-induced aggregation, but it potentiated the ionomycin-induced one. These results suggest that the different effects of indomethacin may be attributed to the distinct effects of 12-HETE on ZT-A- and ionomycin-induced platelet aggregations.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1998

Contribution of endothelium in contractile response of isolated rabbit aortic preparation to electrical field stimulation.

An isolated rabbit aortic preparation, on which drugs selectively act from the intimal surface was made. The contractile response of the aortic preparation to norepinephrine (NE) (1 nM-10 microM) administered from the intimal surface was considerably reduced by removal of the endothelium, whereas the response to KCl was not changed. The contractile response of the aortic preparation to electrical field stimulation (EFS) was abolished by the treatment with tetrodotoxin (1 microM), guanethidine (1 microM) or prazosin (10 microM), indicating that the response was elicited by the release of NE from the adrenergic nerve terminals. The contractile response of the aortic preparation to EFS was also considerably reduced by the removal of the endothelium. A nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine (100 microM) had almost no effect on the contractile response to EFS, indicating that the involvement of NO in the contractile response was negligible. In prostaglandin F2 alpha (PGF 2 alpha)-contracted aortic preparation, the contractile response to EFS was never converted into relaxing response by the treatment with prazosin (10 microM). The finding confirmed that adrenergic beta action of NE released from nerve terminals on the aorta was minor. These findings indicate that endogenously released NE from adrenergic nerve terminals as well as exogenously administered NE acts on the endothelium and may release endothelium derived contracting factor(s) which act together with NE on the vascular smooth muscle.

Animals↗

PubMed1997

Selective inhibition of collagen-induced arachidonic acid liberation by 1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7), a myosin light chain kinase inhibitor, in washed rabbit platelets.

Effects of myosin light chain (MLC) kinase inhibitor ML-7 [1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine hydrochloride] and protein kinase C inhibitor H-7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydro-chloride] on collagen-induced platelet activation were investigated in washed rabbit platelets. Upon stimulation with collagen (1 microg/mL), H-7 decreased protein kinase C-mediated pleckstrin phosphorylation, but had no inhibitory effect on thromboxane (TX) A2 formation or platelet aggregation. In contrast, ML-7 produced a concentration-dependent inhibition of the collagen-induced platelet aggregation and TXA2 formation by preventing arachidonic acid (AA) liberation from membrane phospholipids. However, ML-7 had little effect on AA liberation induced by thrombin, Ca2+ ionophore A-23187 or melittin, suggesting that ML-7 may affect the signal transduction pathway specific for collagen-induced AA liberation, without direct inhibition of phospholipase A2 activity. In indomethacin-treated platelets, collagen caused MLC phosphorylation and AA liberation in the absence of a significant increase in intracellular Ca2+ concentration ([Ca2+]i) or protein tyrosine phosphorylation. ML-7 inhibited both MLC phosphorylation and AA liberation induced by collagen in indomethacin-treated platelets. These results demonstrate that MLC phosphorylation and AA liberation are early events detectable in collagen-stimulated platelets, and suggest that ML-7 inhibits these early steps of collagen-induced signal transduction pathway in rabbit platelets.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1997

Tyrphostin 23 blocks phosphorylation of p42 but not p38 mitogen-activated protein kinase by zooxanthellatoxin-A.

Zooxanthellatoxin-A isolated from a symbiotic dinoflagellate, caused aggregation in rabbit platelets that was inhibited by genistein (50 microM) and tyrphostin 23 (500 microM). Zooxanthellatoxin-A increased tyrosine phosphorylation of 42-kDa proteins which were identified as p42 and p38 mitogen-activated protein kinase (MAPK) by immunoprecipitation. Tyrphostin 23 inhibited the tyrosine phosphorylation of p42 MAPK but not p38 MAPK. In contrast, genistein abolished zooxanthellatoxin-A-induced tyrosine phosphorylation of both p42 and p38 MAPK. The results suggest that tyrphostin 23 selectively inhibits tyrosine phosphorylation of p42 MAPK. The p38 MAPK tyrosine phosphorylation is not involved in zooxanthellatoxin-A-induced platelet aggregation.

Animals↗

PubMed1997

Effects of ATP on phosphoinositide hydrolysis and prostaglandin E2 generation in rabbit astrocytes.

Extracellular ATP secreted from stimulated nerves plays a role in neurotransmission. This study examined the effects of extracellular ATP on phospholipase A2 and C signalling pathways in rabbit astrocytes. ATP caused prostaglandin E2 (PGE2) generation and phosphoinositide hydrolysis in a time- and concentration-dependent manner. A P2y purinoceptor-selective agonist, 2-methylthio-ATP also caused phosphoinositide hydrolysis, but not PGE2 generation. A P2x purinoceptor-selective agonist, alpha, beta-methylene-ATP did not cause either phosphoinositide hydrolysis or PGE2 generation. Although pertussis toxin had no effect on 2-methylthio-ATP-induced phosphoinositide hydrolysis, it markedly decreased ATP-induced PGE2 generation, with significant inhibition of phosphoinositide hydrolysis. Dexamethasone and indomethacin which potently inhibited ATP-induced PGE2 generation, caused partial inhibition of phosphoinositide hydrolysis, suggesting that pertussis toxin-sensitive component of ATP-induced phospholipase C activation is mediated by cyclo-oxygenase metabolites of arachidonic acid. These results suggest that a stimulation of P2y receptor results in phospholipase C activation in a pertussis toxin-insensitive manner, and that a P2 receptor other than the P2y or P2x subtypes is involved in ATP-induced phospholipase A2 activation via a pertussis toxin-sensitive G protein.

Adenosine Triphosphate↗

PubMed1997

[Effect of zooxanthellatoxin-A, an unique marine product, on arachidonic acid cascade in rabbit platelets].

Zooxanthellatoxin-A (ZT-A), a bioactive substance isolated from a symbiotic marine alga Symbiodinium sp., caused rabbit platelet aggregation. ZT-A-induced aggregation was dependent on the presence of external Ca2+, and was inhibited by several Ca2+ channel antagonists except L-type one. Furthermore, ZT-A-induced aggregation was attenuated by genistein, indomethacin and SQ29548, indicating that tyrosine phosphorylation and thromboxane A2 (TXA2) are involved in the aggregation. In fact, ZT-A released arachidonic acid and accumulated TXB2, a stable metabolite of TXA2, which was inhibited by genistein. ZT-A caused phosphorylation and activation of mitogenactivated protein kinase (MAPK), which was known to activate cytosolic phospholipase A2 (cPLA2). ZT-A caused the activation of phospholipase C (PLC)-gamma 2, resulting in an accumulation of diacylglycerol that activates protein kinase C (PKC). The MAPK activation was inhibited by genistein and staurousporine. ZT-A is not a Ca(2+)-lonophore, since its different responsibility from ionomycin to external Ca2+, indomethacin and 12-HETE, a platelet lipoxygenase product. These results suggest that ZT-A stimulates PKC a tyrosine kinase with influxed Ca2+, resulting in the activation PLC-gamma 2 that stimulates via diacylglycerol. Then, MAPK is activated by a PKC pathway, then cPLA2 is activated by MAPK. The released arachidonic acid is rapidly converted to TXA2 which causes platelet aggregation.

Animals↗