Biomedical subjects

N Nakahata

Publications and source records attributed to N Nakahata.

At least 19 recordsLinked to original sources

PubMed2001

Preconditioning of 3T3 cells by fresh medium together with genistein enhances prostaglandin E(2) release.

Bradykinin induced prostaglandin E(2) release from the Swiss 3T3 fibroblasts, preconditioned with fresh culture medium. Although treatment with genistein for the entire period of preconditioning and incubation with bradykinin attenuated prostaglandin E(2) release, treatment with fresh culture medium and genistein for only the preconditioning period further augmented the prostaglandin E(2) release. In the cells preconditioned with fresh culture medium and genistein, bradykinin caused the phosphorylation of protein tyrosine and mitogen-activated protein kinase/extracellular-regulated kinase (MAPK/ERK), followed by arachidonic acid release. Interestingly, preconditioning with genistein alone also caused phosphorylation and arachidonic acid release, probably reflecting rebound activation after the washout of genistein. However, preconditioning with genistein alone induced neither the augmentation of prostaglandin E(2) release nor the expression of cyclooxygenase-2. The further potentiation of bradykinin-induced prostaglandin E(2) release by combined preconditioning with fresh culture medium and genistein may be due to the activation of the MAPK/ERK-c phospholipase A(2) pathway by preconditioning with genistein.

3T3 Cells↗

PubMed2001

Furanodictine A and B: amino sugar analogues produced by cellular slime mold Dictyostelium discoideum showing neuronal differentiation activity.

We investigated the constituents of Dictyostelium discoideum to clarify the diversity of secondary metabolites of Dictyostelium cellular slime molds and to explore biologically active substances that could be useful in the development of novel drugs. From a methanol extract of the multicellular fruit body of D. discoideum, we isolated two novel amino sugar analogues, furanodictine A (1) and B (2). They are the first 3,6-anhydrosugars to be isolated from natural sources. Their relative structures were elucidated by spectral means, and the absolute configurations were confirmed by asymmetric syntheses of 1 and 2. These furanodictines potently induce neuronal differentiation of rat pheochromocytoma (PC-12) cells.

Amino Sugars↗

PubMed2001

Scabronine G-methylester enhances secretion of neurotrophic factors mediated by an activation of protein kinase C-zeta.

Glial cells release neurotrophic factors that maintain neurons functionally. Previously, we have shown that the scabronines isolated from Sarcodon scabrosus enhanced the secretion of neurotrophic factors from 1321N1 human astrocytoma cells. In the present study, we examined the mechanism of newly synthesized scabronine G-methylester (ME)-induced secretion of neurotrophic factors from 1321N1 cells. The dramatic neuronal differentiation of rat pheochromocytoma cells (PC-12) was observed by scabronine G-ME-conditioned medium of 1321N1 cells. Scabronine G-ME increased the secretion of nerve growth factor (NGF) and interleukin-6 (IL-6) from 1321N1 cells with the enhancement of their mRNA expressions. Scabronine G-ME concentration-dependently inhibited the carbachol-induced inositol phosphate accumulation in 1321N1 cells, which was reversed by GF109203X, an inhibitor of protein kinase C (PKC) isoforms. Furthermore, GF109203X inhibited the scabronine G-ME-induced mRNA expressions of both NGF and IL-6 and the differentiation of PC-12 cells, showing that scabronine G-ME activated PKC. Although scabronine G-ME enhanced activities of neither conventional nor novel types of PKCs, it translocated PKC-zeta to membranes in intact cells and cell-free condition. Furthermore, recombinant PKC-zeta activity was also increased by scabronine G-ME, suggesting the involvement of PKC-zeta in the effect of scabronine G-ME. Concerning the downstream effectors of the PKC-zeta, scabronine G-ME translocated nuclear factor-kappaB to nucleus, and enhanced its transcriptional activity. In addition, scabronine G-ME caused the degradation of inhibitor of nuclear factor-kappaB concentration-dependently, which was inhibited by GF109203X. These results suggest that scabronine G-ME potentially enhances the secretion of neurotrophic factors from 1321N1 cells mediated via the activation of PKC-zeta.

Animals↗

PubMed2000

Differentiation of rat pheochromocytoma cells by fomitellic acids, specific DNA polymerase inhibitors.

Fomitellic acid (FA) A and B are specific inhibitors of DNA polymerase alpha and beta. They showed cytotoxicity against rat pheochromocytoma cells (PC-12 cells) in a concentration-dependent manner. However, after PC-12 cells were cultivated with low concentrations of FAs, the cells extended neurites in greater degree similar to the cells cultivated with nerve growth factor. Another DNA polymerase alpha inhibitor, aphidicolin, also induced neurite outgrowth. Furthermore, PC-12 cells were strongly immunostained with anti-alpha-tubulin or anti-tau antibody after the treatment with FAs. These results suggest that weak inhibition of DNA polymerase activity induces the neurite outgrowth in PC-12 cells.

Animals↗

PubMed2000

Thromboxane A2 receptor-mediated tonic contraction is attributed to an activation of phosphatidylcholine-specific phospholipase C in rabbit aortic smooth muscles.

Thromboxane A2 (TXA2) analogue STA2 produced a tonic contraction in rabbit aortic smooth muscles. In the present study, we examined phosphatidylcholine (PC) hydrolysis as a signaling pathway for the tonic contraction in rabbit aortic smooth muscles. In the primary cultured cells labeled with [3H]choline, STA2 caused an accumulation of [3H]phosphorylcholine, a metabolite of PC by PC-specific PLC, in a concentration-dependent manner. The accumulation of [3H]phosphorylcholine was inhibited by SQ29548, a TXA2 receptor antagonist. In the muscle strips, STA2-induced tonic contraction was potently inhibited by D609, an inhibitor of PC-specific phospholipase C in a concentration-dependent manner with the IC50 of about 10 microM. Norepinephrine-induced tonic contraction was also inhibited by D609 with a weaker potency. These results strongly suggest that stimulation of TXA2 receptor results in the activation of PC-specific phospholipase C to yield diacylglycerol that contributes to the tonic contraction.

Animals↗

PubMed2000

Discrimination of histamine H1 and muscarinic receptor-mediated signalling pathways by phorbol ester in human astrocytoma cells.

1. Histamine H1 receptor-mediated signalling was compared with muscarinic receptor-mediated signalling in 1321N1 human astrocytoma cells. 2. Short-term (2 min) treatment of cells with phorbol 12-myristate 13-acetate (PMA) resulted in a reduction of increases in intracellular Ca2+ ([Ca2+]i) induced by carbachol or histamine. 3. Carbachol-induced increases in [Ca2+]i were 10-fold more sensitive to PMA than the histamine-induced increases. 4. When cells were treated with PMA for 48 or 72 h (long-term treatment), protein kinase C (PKC) was down-regulated and PMA did not inhibit carbachol-induced increases in [Ca2+]i. 5. Histamine-induced increases in [Ca2+]i were significantly reduced by long-term treatment with PMA. 6. These findings suggest that the signalling pathways mediated by histamine H1 and muscarinic receptors can be distinguished by using PKC in 1321N1 human astrocytoma cells.

Astrocytoma↗

PubMed2000

Involvement of phosphatidylcholine-specific phospholipase C in thromboxane A2-induced activation of mitogen-activated protein kinase in astrocytoma cells.

Thromboxane A2 (TXA2) receptor-mediated signal transduction was investigated in 1321N1 human astrocytoma cells. 9,11-Epithio-11,12-methano-TXA2 (STA2), a TXA2 receptor agonist, induced Ca2+ mobilization and phosphoinositide hydrolysis in a concentration-dependent manner. These responses were inhibited by treatment with U73122, an inhibitor of phosphatidylinositol-specific phospholipase C, or by culturing in 0.5% fetal calf serum containing 0.5 mM dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP) for 2 days. However, the dbcAMP treatment augmented the TXA2 receptor-mediated phosphorylation of mitogen-activated protein kinase (MAPK). These results were confirmed by a functional MAPK assay measuring the incorporation of 32P into the MAPK substrate peptide. The TXA2 receptor-mediated MAPK activation was inhibited by SQ29548, a TXA2 receptor antagonist, and GF109203X, an inhibitor of protein kinase C. Although U73122 did not inhibit or only slightly inhibited the activation of MAPK, D-609, an inhibitor of phosphatidylcholine-specific phospholipase C, potently attenuated the activation in a concentration-dependent manner. Furthermore, STA2 accelerated the release of [3H]choline metabolites from the cells prelabeled with [3H]choline chloride. This release was inhibited by treatment with D-609. These results suggest that phosphatidylcholine-specific phospholipase C and protein kinase C, but not phosphatidylinositol-specific phospholipase C, are involved in TXA2 receptor-mediated MAPK activation in 1321N1 human astrocytoma cells.

Calcium↗

PubMed2000

Inhibition of microtubule polymerization by SK&F 96365, a blocker of receptor-linked Ca2+ entry.

SK&F 96365 (1-[beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl]-1H-imid azole hydrochloride) is widely used as an effective inhibitor of receptor-linked and capacitative Ca2+ entry. Since this inhibitor has additional effects such as inhibition of voltage-dependent Ca2+ channels, sarco- and endoplasmic reticula Ca2+ pumps and cell proliferation, its molecular mechanism of action remains to be solved. In the present study, we have investigated the effect of SK&F 96365 on microtubule protein isolated from bovine brain in vitro. SK&F 96365 depolymerized the polymerized microtubules in a concentration-dependent manner. This result suggests that SK&F 96365 directly depolymerizes microtubules, an effect that may contribute to the various actions of this compound.

Animals↗

PubMed2000

Identification of a 97-kDa mastoparan-binding protein involving in Ca(2+) release from skeletal muscle sarcoplasmic reticulum.

Mastoparan (MP) and radiolabeled [Tyr(3)]MP caused a transient Ca(2+) release from the heavy fraction of sarcoplasmic reticulum, which was inhibited by ryanodine. MP enhanced [(3)H]ryanodine binding in a concentration-dependent manner with an EC(50) value of approximately 0.3 microM. The (45)Ca(2+) release was accelerated by MP, [Tyr(3)]MP, or caffeine in a concentration-dependent manner. The EC(50) values for MP, [Tyr(3)]MP, and caffeine were approximately 2. 0 microM, 7.7 microM, and 1.8 mM, respectively. MP, like caffeine, shifted the stimulatory limb of a bell-shaped curve of Ca(2+) dependence to the left. (45)Ca(2+) release induced by caffeine was completely inhibited by typical blockers of Ca(2+)-induced Ca(2+) release, such as Mg(2+), ruthenium red, or procaine. However, (45)Ca(2+) release induced by MP was completely inhibited by Mg(2+), but it was only partially inhibited by ruthenium red or procaine. The rate of (45)Ca(2+) release induced by MP was further increased in the presence of caffeine, showing that the MP binding site is different from that of caffeine on Ca(2+) release channels. We succeeded in the synthesis of (125)I-[Tyr(3)]MP with a high specific activity. (125)I-[Tyr(3)]MP bound specifically to heavy fraction of sarcoplasmic reticulum with a K(d) value of 4.0 microM and a B(max) value of 3.0 nmol/mg. Furthermore, (125)I-[Tyr(3)]MP specifically cross-linked to the 97-kDa protein without direct binding to ryanodine receptor. The protein was not triadin or Ca(2+)-pump, because antitriadin antibody and anti-Ca(2+)-pump antibody did not immunoprecipitate the protein. These results suggest that the 97-kDa MP-binding protein may have an important role in the excitation-contraction coupling of skeletal muscle.

Animals↗

PubMed1999

Structure-activity relationship of gramine derivatives in Ca(2+) release from sarcoplasmic reticulum.

5,6-Dibromo-1,2-dimethylgramine evoked Ca(2+) release from skeletal muscle sarcoplasmic reticulum through ryanodine receptors in a concentration-dependent manner with an EC(50) of 22.2 microM. Since the EC(50) of caffeine was 0.885 mM, 5,6-dibromo-1,2-dimethylgramine was 40 times more sensitive than caffeine. Among 14 gramine derivatives having different substituents at N-1, C-2, C-5 or C-6 of the indole skeleton, we found that five derivatives were effective. Study of the structure-activity relationship for Ca(2+) release indicated that 1-methylation and/or both 5- and 6-bromination are important for Ca(2+) release. Thus, gramine derivatives are useful tools for the investigation of Ca(2+) release from sarcoplasmic reticulum.

Alkaloids↗

PubMed1999

Identification of 30 kDa protein for Ca(2+) releasing action of myotoxin a with a mechanism common to DIDS in skeletal muscle sarcoplasmic reticulum.

The molecular mechanism of Ca(2+) release by myotoxin a (MTYX), a polypeptide toxin isolated from the venom of prairie rattlesnakes (Crotalus viridis viridis), was investigated in the heavy fraction of sarcoplasmic reticulum (HSR) of rabbit skeletal muscles. [(125)I]MYTX bound to four HSR proteins (106, 74, 53 and 30 kDa) on polyvinylidene difluoride (PVDF) membrane. DIDS, 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, bound predominantly to 30 kDa protein on the PVDF membrane, the molecular weight of which was similar to one of the MYTX binding proteins. The maximum (45)Ca(2+) release induced by caffeine (30 mM) was further increased in the presence of MYTX (10 microM) or DIDS (30 microM), whereas that induced by DIDS (30 microM) was not affected by MYTX (10 microM). MYTX inhibited [(3)H]DIDS binding to HSR in a concentration-dependent manner. Furthermore, [(125)I]MYTX binding to 30 kDa protein was inhibited by DIDS in a concentration-dependent manner. These results suggest that MYTX and DIDS release Ca(2+) from HSR in a common mechanism. The 30 kDa protein may be a target protein for the Ca(2+) releasing action of MYTX and DIDS.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1999

Thromboxane A2-induced phosphatidylcholine hydrolysis in porcine vascular smooth muscle cells.

The effect of 9,11-epithio-11,12-methanothromboxane A2 (STA2), a thromboxane A2 receptor agonist, on phosphatidylcholine hydrolysis was examined in porcine vascular smooth muscle cells. Although STA2 stimulated diacylglycerol production in a concentration-dependent manner, it only caused a slight accumulation of [3H]phosphatidylethanol in the presence of 0.5% ethanol, reflecting its weak stimulation of phosphatidylcholine-specific phospholipase D. STA2-induced diacylglycerol production was potently and concentration dependently inhibited by potassium tricyclo-[5.2.1.0(2.6)]-decyl-(9[8])-xanthogenate (D609), an inhibitor of phosphatidylcholine-specific phospholipase C. These results suggest that the thromboxane A2 receptor in vascular smooth muscles is functionally coupled to phosphatidylcholine-specific phospholipase C to yield diacylglycerol.

Animals↗

PubMed1999

Stimulation of neurotrophic factor secretion from 1321N1 human astrocytoma cells by novel diterpenoids, scabronines A and G.

Glial cells release neurotrophic factors that maintain neurons functionally. When rat pheochromocytoma cells (PC-12) were cultivated with the conditioned medium of human astrocytoma cells (1321N1) incubated with the new diterpenoids, scabronines A and G, isolated from Sarcodon scabrosus, they changed their morphology and there was neurite outgrowth. The scabronines increased the expression of mRNA for nerve growth factor (NGF), and the secretion of NGF from 1321N1 cells in a concentration-dependent manner. However, the enhanced neurite outgrowth produced by the conditioned media was slightly inhibited by NGF neutralizing antibody, and the concentration of NGF released in response to the scabronines was insufficient to cause differentiation. These results suggest that scabronines cause the secretion of other factors together with NGF from 1321N1 cells. The diterpenoids are useful drugs to clarify the mechanism of synthesis and secretion of neurotrophic factors.

Animals↗

PubMed1999

Comparison of maitotoxin with thromboxane A2 in rabbit platelet activation.

Maitotoxin (MTX), a Ca2+ channel-activating marine toxin, caused shape change followed by aggregation in rabbit platelets, like U46619, a thromboxane A2 analogue. Although both drugs failed to cause aggregation in the absence of external Ca2+, U46619, but not maitotoxin, elicited shape change in the absence of external Ca2+. The observations of platelets with a scanning electron microscope showed that both drugs caused contraction of platelets and extension of pseudopodia (shape change) followed by aggregation with a clot in the presence of Ca2+. It is noteworthy that long term exposure to MTX caused the lysis of platelets in the presence of Ca2+. While U46619 transiently increased the internal Ca2+ concentration ([Ca2+]i), maitotoxin slowly but irreversibly increased [Ca2+]i in an external Ca2(+)-dependent manner. MTX-induced phosphoinositide hydrolysis was totally dependent on the presence of external Ca2+, but U46619-induced phosphoinositide hydrolysis was still observed in the absence of external Ca2+. MTX-induced phosphoinositide hydrolysis was partly inhibited by SK&F96365, a voltage-independent Ca2+ channel antagonist, or by genistein, a tyrosine kinase inhibitor. MTX caused phosphorylation of tyrosine residues of several proteins, like U46619. Thus, MTX is similar to U46619 in functions of Ca2+ mobilization, phosphoinositide hydrolysis and tyrosine phosphorylation, but MTX-induced actions are strictly dependent on the presence of external Ca2+.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1999

Decrease in thromboxane A2 receptor expression by differentiation with dibutyryl cyclic AMP in 1321N1 human astrocytoma cells.

Thromboxane A2 (TXA2) receptor expression with its signaling was investigated in 1321N1 human astrocytoma cells differentiated with dibutyryl cyclic AMP (dbcAMP). The cells cultured in 0.5% fetal calf serum containing 0.5 mM dbcAMP for 3 days showed the star-shaped morphology, accompanied with the reduction of a TXA2 mimetic U46619-induced phosphoinositide hydrolysis and Ca2+ mobilization. Immunoblotting analysis revealed that human astrocytoma cells expressed phospholipase C (PLC)-beta1 and -beta3, but not PLC-beta2. The contents of PLC-beta1 and beta3 were not changed by the differentiation. The alpha subunit of Gq/ll bound to TXA2-receptor was reduced by the differentiation, determined by immunoblotting after immunoprecipitation with an anti-TXA2-receptor antibody. Scatchard analysis of the binding of [3H]SQ29548, a TXA2 receptor antagonist, to the membranes revealed that the maximum binding site was reduced by the differentiation. The expression of TXA2 receptor mRNA also was reduced by the differentiation, determined by reverse-transcribed-polymerase chain reaction. Although placental type of TXA2 receptor mRNA expression increased after the differentiation, endothelial type of TXA2 receptor mRNA expression slightly decreased. The results suggest that 1321N1 human astrocytoma cells differentiated with dbcAMP show impaired TXA2 receptor-mediated phosphoinositide hydrolysis and Ca2+ mobilization, due to the decrease in TXA2 receptor number.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1999

Maitotoxin-induced nerve growth factor production accompanied by the activation of a voltage-insensitive Ca2+ channel in C6-BU-1 glioma cells.

1. The aim of the present study was to determine the effects of maitotoxin on nerve growth factor production and the Ca2+ influx in clonal rat glioma cells (C6-BU-1). 2. Maitotoxin (1 - 10 ng ml-1) induced a profound increase in 45Ca2+ influx in an extracellular Ca2+-dependent manner. However, high KCl had no effect at all. These effects were supported by the results from the analysis of intracellular Ca2+ concentration using fura 2. 3. The maitotoxin-induced 45Ca2+ influx was inhibited by inorganic Ca2+ antagonists, such as Mg2+, Mn2+ and Co2+. The inhibitory effect of Co2+ was antagonized by increasing the extracellular Ca2+ concentrations. 4. Maitotoxin (3 ng ml-1) as well as A-23187 (1microM) and dibutyryl cyclic AMP (0.5 mM) caused an acceleration of nerve growth factor (NGF) production in C6-BU-1 cells, as determined by NGF enzyme immunoassay. 5. Reverse transcription polymerase chain reaction (RT - PCR) analysis showed that maitotoxin (10 ng ml-1) enhanced the expression of NGF mRNA, which was abolished by the removal of extracellular Ca2+. A-23187 also accelerated its expression. 6. These results suggest that maitotoxin activates a voltage-insensitive Ca2+ channel and accelerates NGF production mediated through a Ca2+ signalling pathway in C6-BU-1 glioma cells.

Animals↗

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↗