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E Miyamoto

Publications and source records attributed to E Miyamoto.

At least 91 records · Page 5Linked to original sources
PubMed1997

DY-9760e, a novel calmodulin antagonist with cytoprotective action.

We report the pharmacological characterization and cytoprotective effect of DY-9760e, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-( 4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate, a novel antagonist of calmodulin. DY-9760e inhibited calmodulin-dependent enzymes, including calmodulin-dependent protein kinase II and IV, calcineurin, [corrected] calmodulin-dependent phosphodiesterase and myosin light chain kinase with Ki values of 1.4, 12, 2.0, 3.8 and 133 microM, respectively. These antagonistic effects of DY-9760e were more potent than those of W-7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, another calmodulin antagonist. This compound showed little or no effect on calmodulin-independent enzymes, such as protein kinase A and C and calpain I and II. Analysis of the hydrophobic interaction of DY-9760e with calmodulin by using 2-p-toluidinylnaphthalene-6-sulfonate and 9-anthroylcholine revealed that, like W-7, DY-9760e bound to the hydrophobic regions of calmodulin. The [14C]DY-9760e binding assay indicated that DY-9760e bound to calmodulin at one class of binding site. Finally, DY-9760e substantially protected N1E-115 neuroblastoma cells from cytotoxicity induced by the Ca2+ ionophore, A23187. These results indicate that DY-9760e, a novel calmodulin antagonist, possesses a cytoprotective action and suggest that calmodulin plays a critical role in mediating some of the biochemical events leading to cell death following Ca2+ overload.

Calcimycin↗
PubMed1997

Calcineurin in the adult rat hippocampus: different distribution in CA1 and CA3 subfields.

We examined the immunohistochemical regional distribution of calcineurin (Ca2+/calmodulin-dependent protein phosphatase) in the adult rat hippocampus, following various regional destruction. In the normal adult rat hippocampus, the calcineurin immunoreactivity showed a characteristic pattern. This protein phosphatase was detected in all layers of the CA1 subfield, including the cytoplasm of the pyramidal cells, whereas it was strongly evident in the stratum lucidum and moderately so in the cytoplasm of pyramidal cells in the CA3 subfield. Seven days after transient forebrain ischemia, which induced destruction of CA1 pyramidal cells, the calcineurin immunoreactivity decreased in all layers of the CA1 subfield, while the immunoreactivity for synapsin I, a marker of the presynaptic site, was preserved. Seven days after the intraventricular injection of kainate, which induced destruction of CA3 pyramidal cells, the calcineurin immunoreactivity in the stratum lucidum was preserved, although the immunostaining pattern of the stratum lucidum changed when CA3 pyramidal cells were destroyed. Seven days after mechanical destruction of the dentate gyrus and CA4 subfield, which induced destruction of mossy fibers, the calcineurin immunoreactivity in the stratum lucidum was lost, except in the far site of the stratum lucidum. In the CA1 subfield, calcineurin was mainly located in postsynaptic sites, while it was mainly located in the presynaptic sites in the mossy fibers of the CA3 subfield. The immunohistochemistry of adjacent sections with antibodies of microtubule-associated protein 2 and synapsin I, which are markers of postsynaptic and presynaptic sites respectively, supports these results. Thus, calcineurin has a different synaptical distribution in the rat hippocampus.

Animals↗
PubMed1997

Glycation and inactivation of aspartate aminotransferase in diabetic rat tissues.

Glycated cytosolic aspartate aminotransferase was detected in the liver and kidney of streptozotocin diabetic rats using a boronate affinity column for adjacent cis-hydroxyl groups and an immunoblotting technique. The enzymatic activity and amount of immunoreactive substance were determined in the liver, kidney, and erythrocytes of diabetic and control rats. The ratio of enzymatic activity to the amount of enzyme was lower in diabetic rat tissue than in that in the control rats. It has been suggested that there is an inactive aspartate aminotransferase molecule in the tissues of diabetic rats. We therefore suggest that the cytosolic aspartate aminotransferase was inactivated in the diabetic rat tissues by a glycation reaction, accompanied by an impairment in glucose utilization.

Alkaline Phosphatase↗
PubMed1996

Expression of angiotensin-converting enzyme in remaining viable myocytes of human ventricles after myocardial infarction.

BACKGROUND: Local ACE in the heart may be important in the pathophysiological state after myocardial infarction (MI). It is unknown, however, whether ACE is expressed in myocytes of the human heart. METHODS AND RESULTS: Using a newly generated polyclonal antibody to a synthetic peptide corresponding to part of the human endothelial ACE sequence, we examined the localization of ACE in left ventricles of patients (n = 10) with MI obtained at left ventricular aneurysmectomy or autopsy and in the hearts of control subjects at autopsy (n = 10). The avidinbiotinylated peroxidase complex method was used for the immunohistochemical staining for ACE. In the left ventricles, positively stained myocytes for ACE were found in 8 of the 10 patients with MI. ACE immunoreactivity was seen in the remaining viable myocytes located near the infarct scar of the aneurysmal left ventricle and in nonmyocytes such as fibroblasts, macrophages, vascular smooth muscle cells, and endothelial cells within the scarred tissue. On the other hand, no immunoreactivity for ACE was detected in the ventricular myocytes of all control hearts obtained at autopsy. CONCLUSIONS: We observe immunohistochemical staining for ACE in the left ventricular myocytes of the region adjacent to the infarct scar and in nonmyocytes. These results indicate that ACE is markedly increased on the edge of the infarct scar and suggest that local ACE may be important in the ventricular remodeling after MI.

Aged↗
PubMed1996

Regulation of CCAAT/enhancer-binding protein family members by stimulation of glutamate receptors in cultured rat cortical astrocytes.

Regulation of mRNA levels, DNA binding activities, and phosphorylation of CCAAT/enhancer-binding protein (C/EBP) family members by stimulation of glutamate receptors were studied in cultured rat cortical astrocytes. Indirect immunofluorescence and immunoblot analyses with specific antibodies to C/EBP family members revealed that both C/EBPbeta and C/EBPdelta but not C/EBPalpha are expressed in the nuclei of astrocytes. After exposure to glutamate, C/EBPbeta mRNA levels increased within 10 min, reached the maximal level at about 1 h, and returned to the basal level within 6 h. In contrast, C/EBPdelta mRNA levels decreased by 6 h and were recovered within 12 h. These changes in mRNA levels were accompanied by an increase and a decrease in proteins for C/EBPbeta and C/EBPdelta, respectively. Elevation of C/EBPbeta mRNA levels by glutamate treatment required an increase in intracellular Ca2+ concentration and depended on activations of protein kinase C and calmodulin-dependent protein kinases. Gel mobility shift analysis using nuclear extracts from the glutamate-treated cells showed increases in C/EBP site binding activities 2 h after the exposure to glutamate. Moreover, glutamate stimulated phosphorylation of C/EBPbeta in 32P-labeled astrocytes in a Ca2+-dependent manner. These results suggest that glutamate regulates functions of C/EBP family members in brain astrocytes through changes in mRNA levels of C/EBPbeta and C/EBPdelta as well as through phosphorylation of C/EBPbeta.

Animals↗
PubMed1996

Phosphorylation of chromogranin A and catecholamine secretion stimulated by elevation of intracellular Ca2+ in cultured bovine adrenal medullary cells.

We have recently isolated a new endogenous substrate of 70 kDa for Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) from bovine adrenal medullary cells (Yanagihara, N., Toyohira, Y., Yamamoto, H., Ohta, Y., Tsutsui, M., Miyamoto, E., and Izumi, F. (1994) Mol. Pharmacol. 46, 423-430). Here we report the sequence analysis of the 70-kDa protein and examine its phosphorylation by various protein kinases in vitro and by depolarization of the cultured cells. Protein sequencing and immunoblotting revealed that the 70-kDa protein is chromogranin A (CgA) or a closely related protein. Partially purified CgA was phosphorylated by cyclic AMP-dependent protein kinase and protein kinase C as well as CaM kinase II. Tryptic phosphopeptide mapping patterns of CgA differed among these protein kinases. In 32P-labeled bovine adrenal medullary cells, 56 mM K+ increased the phosphorylation of CgA and catecholamine secretion in similar time- and concentration-dependent manners, both of which were inhibited by 20 mM MgSO4, an inhibitor of voltage-dependent Ca2+ channels. These findings suggest that CgA serves as a substrate for several multifunctional protein kinases and that the elevation of the intracellular Ca2+ stimulates the phosphorylation of CgA associated with catecholamine secretion in cultured adrenal medullary cells.

Adrenal Medulla↗
PubMed1996

The effects of 2,5-hexanedione and acrylamide on myosin heavy chain isoforms of slow and fast skeletal muscles of the rat.

We examined the effects of 2,5-hexanedione (2,5-HD) and acrylamide (ACR) on the muscle fiber types and myosin heavy chain (MHC) isoform composition of the slow-twitch soleus and fast-twitch plantaris muscles of rats. We employed two differently designed experiments with respect to the dosage levels and treatment periods for developing clinical neuropathies. When male Wistar rats were subcutaneously injected with 4.5 mmol of 2,5-HD/kg or 0.4 mmol of ACR/kg, 5 days a week, they developed paralysis of the hindlimbs in 4 weeks (high-dosage experiment). When they were subcutaneously injected with 3.5 mmol of 2,5-HD/kg or 0.35 mmol of ACR/kg, 5 days a week, paralysis of the hindlimbs did not develop until 6 weeks (low-dosage experiment). We examined mainly the rats treated with the neurotoxicants for 4 and 8 weeks in the high-and low-dosage experiments, respectively. Significant decreases in the maximum motor conduction velocity of the sciatic nerves were observed in the hindlimbs of the rats in both experiments. The weights of the soleus and plantaris muscles were significantly reduced in the 2,5-HD-treated rats in both experiments, while in the ACR-treated rats, the weights of both muscles decreased only in the low-dosage experiment. We could not detect any changes in the fiber type composition of the muscles by any of the treatments. However, biochemical analysis revealed decreases in the values (percentage) of the relative amounts of fast-type MHC IIa and IIb isoforms to total MHC isoforms in the 2,5-HD-treated rats, but not in the ACR-treated rats in the high-dosage experiment. In contrast, significant differences in the relative amounts of MHC isoforms were not observed after administration of the low dosage of 2,5-HD. These results suggest that 2,5-HD preferentially disorders the muscle fibers which contain the MHC II isoform. These effects may occur only after the relatively acute intoxication of 2,5-HD at a high dosage.

Acrylamide↗
PubMed1996

A role of Ca2+/calmodulin-dependent protein kinase II in the induction of long-term potentiation in hippocampal CA1 area.

Long-term potentiation (LTP) in the CA1 area of the hippocampus is considered to be a synaptic model for learning and memory. The induction of LTP is initiated by activation of the NMDA glutamate receptor in the postsynaptic membrane and a subsequent increase in Ca2+ -influx into the neurons following glutamate release. The action of Ca2+ has been proposed to be mediated by Ca2+ -dependent protein kinases. Recent studies indicate that, among the protein kinases, Ca2+/calmodulin-dependent protein kinase II is implicated in the induction of LTP in the hippocampus.

Animals↗
PubMed1996

CaM kinase II in long-term potentiation.

The observation that autophosphorylation converts CaM kinase II from the Ca(2+)-dependent form to the Ca(2+)-independent form has led to speculation that the formation of the Ca(2+)-independent form of the enzyme could encode frequency of synaptic usage and serve as a molecular explanation of "memory". In cultured rat hippocampal neurons, glutamate elevated the Ca(2+)-independent activity of CaM kinase II through autophosphorylation, and this response was blocked by an NMDA receptor antagonist, D-2-amino-5-phosphonopentanoate (AP5). In addition, we confirmed that high, but not low frequency stimulation, applied to two groups of CA1 afferents in the rat hippocampus, resulted in LTP induction with concomitant long-lasting increases in Ca(2+)-independent and total activities of CaM kinase II. In experiments with 32P-labeled hippocampal slices, the LTP induction in the CA1 region was associated with increases in autophosphorylation of both alpha and beta subunits of CaM kinase II 1 h after LTP induction. Significant increases in phosphorylation of endogenous CaM kinase II substrates, synapsin I and microtubule-associated protein 2 (MAP2), which are originally located in presynaptic and postsynaptic regions, respectively, were also observed in the same slice. All these changes were prevented when high frequency stimulation was applied in the presence of AP5 or a calmodulin antagonist, calmidazolium. Furthermore, in vitro phosphorylation of the AMPA receptor by CaM kinase II was reported in the postsynaptic density and infusion of the constitutively active CaM kinase II into the hippocampal neurons enhanced kainate-induced response. These results support the idea that CaM kinase II contributes to the induction of hippocampal LTP in both postsynaptic and presynaptic regions through phosphorylation of target proteins such as the AMPA receptor, MAP2 and synapsin I.

2-Amino-5-phosphonovalerate↗
PubMed1996

Overexpression of Ca2+/calmodulin-dependent protein kinase II inhibits neurite outgrowth of PC12 cells.

To investigate the physiological role of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in neuronal differentiation, we transfected the cDNA of the alpha subunit of mouse CaM kinase II (CaM kinase II alpha) into PC12 cells and established clonal cell lines that constitutively express the transfected CaM kinase II alpha gene. The expression of CaM kinase II alpha was confirmed by northern blot and immunoblot analyses. Northern blot analysis showed that the gamma and delta subunits of CaM kinase II are mainly expressed in PC12 cells. Treatment of the cells with ionomycin activated CaM kinase II alpha through autophosphorylation and generation of the Ca2+/calmodulin-dependent form. It is interesting that the neurite outgrowth induced by dibutyryl cyclic AMP was inhibited in these cell lines in accordance with the activities of overexpressed CaM kinase II alpha. The activity of cyclic AMP-dependent protein kinase showed similar levels among these cell lines. These results suggest that CaM kinase II is involved in the modulation of the neurite outgrowth induced by activation of the cyclic AMP system.

Animals↗
PubMed1996

Ca(2+)/calmodulin-dependent protein kinase II inhibitor KN-62 inhibits adrenal medullary chromaffin cell functions independent of its action on the kinase.

KN-62, an inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), inhibited significantly catecholamine secretion and tyrosine hydroxylase activity stimulated by acetylcholine in cultured bovine adrenal medullary cells. KN-62, however, showed an additional inhibitory effect on acetylcholine-induced 45Ca2+ influx, which is essential for functional responses. Carbachol-stimulated 22Na+ influx, veratridine-induced 22Na+ influx, and 56 mM K(+)-evoked 45Ca2+ influx were also attenuated by KN-62. Inhibitions by KN-62 of these ion influxes were correlated closely with those of catecholamine secretion. KN-04, which is a structural analogue of KN-62 but does not inhibit CaM kinase II activity, elicited inhibitory effects on the three kinds of stimulant-evoked ion influxes with an inhibitory potency similar to KN-62. These results suggest that KN-62 inhibits catecholamine secretion and tyrosine hydroxylase activation due to mainly its ion channel blockade on the plasma membrane rather than the inhibition of CaM kinase II activity in the cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1996

Cyclic AMP inhibits activation of mitogen-activated protein kinase and cell proliferation in response to growth factors in cultured rat cortical astrocytes.

The cyclic AMP (cAMP)-induced inhibitory effect on cell proliferation was examined through inhibition of mitogen-activated protein kinase (MAP kinase) activation in cultured rat cortical astrocytes. Basic fibroblast growth factor (bFGF) at 10 ng/ml maximally stimulated MAP kinase activity, which peaks during 10 min and prolonged for 24 h. Likewise, DNA synthesis was maximally potentiated with 10 ng/ml bFGF and correlated with MAP kinase activity in a dose-dependent manner. Dibutyryl cAMP (dbcAMP) at 1 mM and isoproterenol at 10 microM inhibited MAP kinase activation and DNA synthesis potentiation with bFGF and platelet-derived growth factor to the control level in cultured astrocytes and C6 glioma cells. The stimulation with bFGF caused a prominent translocation of MAP kinase from the cytosol to the nucleus after 1 h in astrocytes. Treatment of the cells with dbcAMP and isoproterenol completely prevented the translocation of MAP kinase. In experiments with 32P-labeled cultured astrocytes, phosphorylation of Raf-1 was apparently stimulated with bFGF. Treatment with dbcAMP or isoproterenol had a greatly inhibitory effect on the stimulation of Raf-1 phosphorylation with bFGF. Consistent with the effect on Raf-1 phosphorylation, dbcAMP and isoproterenol completely prevented bFGF-induced phosphorylation of MAP kinase kinases, target proteins of Raf-1. Our observations suggest that cAMP-induced suppression of cell growth in astrocytes is due to the inhibitory effect on activation of MAP kinase and its translocation to the nucleus and that the site of the cAMP action is located at Raf-1 or the upstream site of Raf-1.

Adrenergic beta-Agonists↗
PubMed1996

Immunohistochemical examination of phosphorylated tau in granulovacuolar degeneration granules.

Granulovacuolar degeneration (GVD) and neurofibrillary tangles (NFT) are neuropathological features in Alzheimer's disease (AD). The molecular mechanism of GVD formation remains unknown. Recent immunohistochemical investigations suggested a potential link of NFT to GVD formation. Enzyme histochemical studies and electronmicroscopic findings suggested that GVD is formed through lysosomal autophagy of intraneuronal substances. We recently demonstrated that in non-demented cases NFT was phosphorylated at serines 199, 202 and 422 in paired helical filament (PHF)-tau more than in serine 396, while NFT in AD cases was similarly phosphorylated at these four sites in tau. In this study, we demonstrated immunohistochemically a similar phosphorylation state of tau in GVD granules to that in NFT in both non-demented cases and AD patients by using a mouse monoclonal anti-tau antibody and three phosphorylation site-specific antibodies for PHF-tau, indicating that GVD granules and NFT are composed of similar phosphorylated-tau. However, we could not detect PHF structures within any GVD using electronmicroscopy, indicating that PHF itself is not phagocytized by lysosomes during GVD formation. Therefore, the source of GVD granules might be phosphorylated pre-PHF-tau.

Aged↗