Biography:

In the past Kazutoshi Kiuchi has collaborated on articles with Toshiharu Nagatsu and Akira Ishii. One of their most recent publications is Effects of myosin light-chain kinase inhibitor on catecholamine secretion from rat pheochromocytoma PC12h cells. Which was published in journal Biochemical and Biophysical Research Communications.

More information about Kazutoshi Kiuchi research including statistics on their citations can be found on their Copernicus Academic profile page.

Kazutoshi Kiuchi's Articles: (17)

Effects of myosin light-chain kinase inhibitor on catecholamine secretion from rat pheochromocytoma PC12h cells

AbstractRelease of dopamine from rat pheochromocytoma PC12h cells by high K+ (50 mM) was inhibited by a specific inhibitor of myosin light-chain kinase (ML-9) dose-dependently. The myosin light-chain kinase inhibitor also specifically inhibited the phosphorylation of a 20 KDa protein by myosin light-chain kinase. Myosin light chain kinase may play a stimulatory role in the release reaction of catecholamines from the rat pheochromocytoma cells.

A selective Ca2+calmodulin-dependent protein kinase II inhibitor, KN-62, inhibits the enhanced phosphorylation and the activation of tyrosine hydroxylase by 56 mM K+ in rat pheochromocytoma PC12h cells

AbstractInvolvement of Ca2+calmodulin-dependent protein kinase II (Ca2+CaM-kinase II) on the phosphorylation of tyrosine hydroxylase (TH, EC.1.14.16.2) in rat pheochromocytoma, PC12h cells was examined using KN-62, 1-[N,O-Bis(5-isoquinolinsulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine, a selective inhibitor of Ca2+CaM-kinase II. Both the enhanced phosphorylation of TH and the activated l-3,4-dihydroxyphenylalanine (DOPA) formation in the high K+ depolarization were inhibited by 10 μM KN-62. After incubation of PC12h cells with 10 μM KN-62 for 1 hr, the activation of TH with 3 min incubation of 56 mM K+ was reduced to the basal activity. However, KN-62 did not directly affect the activity of purified rat TH at pH 6.0 or 7.0. These results indicate that Ca2+CaM-kinase II phosphorylates and activates TH of PC12h cells in the high K+ depolarization.

Effects of myosin light-chain kinase inhibitor on catecholamine secretion from rat pheochromocytoma PC12h cells

AbstractRelease of dopamine from rat pheochromocytoma PC12h cells by high K+ (50 mM) was inhibited by a specific inhibitor of myosin light-chain kinase (ML-9) dose-dependently. The myosin light-chain kinase inhibitor also specifically inhibited the phosphorylation of a 20 KDa protein by myosin light-chain kinase. Myosin light chain kinase may play a stimulatory role in the release reaction of catecholamines from the rat pheochromocytoma cells.

A selective Ca2+calmodulin-dependent protein kinase II inhibitor, KN-62, inhibits the enhanced phosphorylation and the activation of tyrosine hydroxylase by 56 mM K+ in rat pheochromocytoma PC12h cells

AbstractInvolvement of Ca2+calmodulin-dependent protein kinase II (Ca2+CaM-kinase II) on the phosphorylation of tyrosine hydroxylase (TH, EC.1.14.16.2) in rat pheochromocytoma, PC12h cells was examined using KN-62, 1-[N,O-Bis(5-isoquinolinsulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine, a selective inhibitor of Ca2+CaM-kinase II. Both the enhanced phosphorylation of TH and the activated l-3,4-dihydroxyphenylalanine (DOPA) formation in the high K+ depolarization were inhibited by 10 μM KN-62. After incubation of PC12h cells with 10 μM KN-62 for 1 hr, the activation of TH with 3 min incubation of 56 mM K+ was reduced to the basal activity. However, KN-62 did not directly affect the activity of purified rat TH at pH 6.0 or 7.0. These results indicate that Ca2+CaM-kinase II phosphorylates and activates TH of PC12h cells in the high K+ depolarization.

A microassay for acid β-galactosidase activity toward asialofetuin

AbstractTo study the enzymatic properties of β-galactosidase from the patients with a β-galactosidase deficiency such as GM1 gangliosidosis, determination of enzymatic activity with naturally occurring substrates, asialofetuin in addition to another natural substrate, GM1 ganglioside, is essentially required. With a previously reported, simple and sensitive fluorometric assay for GM1 ganglioside β-galactosidase using high performance liquid chromatography (HPLC), optimal reaction conditions were determined for the assay of acid β-galactosidase activity toward asialofetuin in skin fibroblast homogenates. Under these conditions, reduced enzymatic activities could be detected in cultured skin fibroblasts from patients with type 1 and 3 GM1 gangliosidoses and mucopolysaccharidosis IV-B (Morquio B syndrome). This method was applicable to study of the enzymatic properties of the mutant β-galactosidase and provided an alternative to assays employing radioactive or artificial substrates.

Effect of (6R)- and (6S)-tetrahydrobiopterin on L-3,4-dihydroxyphenylalanine (DOPA) formation in NRK fibroblasts transfected with human tyrosine hydroxylase type 2 cDNA

Abstractl-erythro-5,6,7,8-Tetrahydrobiopterin (BH4), which is the cofactor of aromatic amino acid hydroxylases, plays an important role in the biosyntheses of monoamine neurotransmitters. BH4 exists as natural (6R)- and unnatural (6S)-isomers. In our previous reports, only (6R)-isomer significantly stimulated cofactor activity for tyrosine, tryptophan and phenylalanine hydroxylases (TH, TPH, PAH) in whole animals or in tissue slices. In this study we have compared the in situ cofactor activity on TH between natural (6R)- and unnatural (6S)-isomers in clonal cells. We have transfected human TH type 2 cDNA into the normal rat kidney (NRK) fibroblasts. These cells expressed TH protein, but had neither DOPA decarboxylase (DDC) nor BH4. Thus, TH activity was observed only in the presence of exogenous BH4. We compared the difference in in situ DOPA formation by TH activity in the presence of (6R)- or (6S)-BH4 in the human TH-transfected cells. The effect of exogenous BH4 was also compared between (6R)- and (6S)-isomers in rat pheochromocytoma PC12h cells, which contained approximately 100 μM endogenous (6R)-BH4. The rate of uptake of both BH4 isomers into these cells increased in proportion to the pterin cofactor concentrations in the incubation medium up to 400 μM but was nearly saturated at 1 mM BH4. TH-transfected NRK fibroblasts formed DOPA only in the presence of exogenously added (6R)- or (6S)-BH4 dose-dependently and released DOPA into the medium. At a saturating concentration of 1 mM, (6R)-BH4 was approximately three times as active as (6S)-BH4. In contrast, in PC12h cells which contained endogenous (6R)-BH4 (approximately 100 μM), exogenous (6R)-BH4 activated DOPA formation maximally at 500 μM about 10-fold, while (6S)-BH4 activated it only slightly, about 2.5-fold. These results suggest that (6S)-isomer has lower cofactor activity with TH in the cells than (6R)-isomer. This TH transfected fibroblasts should be useful to assess cofactor activities of tetrahydropteridines in the cell.

Ferrous ion activates the less active form of human adrenal tyrosine hydroxylase☆

AbstractThe less active form of human tyrosine hydroxylase has been previously reported but its physiological role is unknown. We partially purified the less active form of tyrosine hydroxylase from human adrenals, and examined differences in the properties of the active and less active forms. We succeeded in activation of the less active form of human tyrosine hydroxylase by addition of 100 μM Fe2+. Fe2+ decreased the Kmax for pteridine cofactor in both the active and less active forms, but increased the Vmax only in the less active form. Fe2+ changed the Vmax but not the Km of the less active form for tyrosine. These results suggest that Fe2+ may regulate tyrosine hydroxylase activity in vivo as a result of activation of its less active form.

Effect of the 1-methyl-4-phenylpyridinium ion on phosphorylation of tyrosine hydroxylase in rat pheochromocytoma PC12h cells

AbstractEffects of the 1-methyl-4-phenylpyridinium ion (MPP+) on DOPA formation and phosphorylation of tyrosine hydroxylase (TH) of rat pheochromocytoma PC12h cells were examined after the cells were cultured with MPP+. DOPA formed from endogenous tyrosine in PC12h cells after a 3-day culture with 100 μM MPP+ was decreased to less than 50% as compared to that in the control cells cultured without MPP+. Kinetical study showed that two apparent forms of TH with different Km existed in the cells cultured with 100 μM MPP+ but one form in that of control. Incorporation of radioactive phosphate into TH molecule was also reduced to 50% of its control value following a 3-day exposure to 100 μM MPP+. These results suggest that MPP+ acutely inhibits the phosphorylation of TH to decrease cellular DOPA formation.

Regulatory mechanism of dopamine biosynthesis in the striatum of transgenic mice carrying human tyrosine hydroxylase gene

AbstractWe investigated the regulatory mechanism of dopamine biosynthesis in the striatum of transgenic mice carrying multiple copies of human tyrosine hydroxylase (TH). The in vitro TH activity of transgenic striatum at pH 7.0 was approximately 2.8-fold higher than that of non-transgenic striatum. This augmentation is similar to that of the in vitro TH activity at pH 6.0, indicating that the expression of human TH in transgenic striatum induced little change in the phosphorylation level of TH. l-3,4-Dihydroxyphenylalanine (DOPA) formation in striatal slices of transgenic mice was approximately 2.7-fold higher than that of non-transgenic mice. The addition of 0.5 mM (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (6R-BH4) to the incubation medium brought a negligible increase in DOPA formation in both cases. These results suggest that 6R-BH4 is not the limiting factor of TH in situ both in the transgenic and non-transgenic mice.

Effect of spermine on tyrosine hydroxylase activity before and after phosphorylation by cyclic AMP-dependent protein kinase

The effect of spermine on tyrosine hydroxylase (TH) activity purified from bovine adrenal medulla was examined before and after phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase (A-kinase). Before phosphorylation, spermine (<1 mM) inhibited the enzymatic activity, and negative cooperative effect of spermine on TH (Hill coefficient = 0.7) was observed from the kinetic analysis concerning 6-methyl-5,6,7,8-tetrahydropterin (6MPH4). Spermine interacted noncompetitively toward tyrosine and the Ki for spermine was calculated to be 68 μM. Phosphorylation abolished the ability of spermine to inhibit TH activity in a negative cooperative manner against the pterin cofactor, and also increased four-fold the Ki value against the substrate. These results suggest that spermine may inhibit TH activity by interacting with the pterin binding site of the enzyme molecule in a manner of negative cooperativity, and that this inhibition is reversed by the conformational change of regulatory domain of TH after phosphorylation by A-kinase.

Regular ArticleMolecular Cloning and Expression Analysis ofGFRα-3,a Novel cDNA Related toGDNFRαandNTNRα

AbstractGlial-cell-line-derived neurotrophic factor (GDNF) and neurturin (NTN) are structurally related to TGF-β and are survival factors for sympathetic, sensory, and central nervous system neurons. GDNF transmits its signal primarily through a receptor complex containing the receptor tyrosine kinase Ret and a glycosyl-phosphatidylinositol (GPI)-linked receptor, GDNFRα. NTN utilizes a receptor complex system that consists of Ret and another GPI-linked receptor, NTNRα. We have identified a mouse cDNA, termedGFRα-3,that encodes a putative GPI-linked receptor. At the protein level, mouse GFRα-3 is 35% identical to mouse GDNFRα and 36% identical to mouse NTNRα. Northern blot analysis showed thatGFRα-3is expressed in fetal mouse heart, brain, lung, and kidney and adult heart. These results indicate that the tissue distribution ofGFRα-3mRNA is different from that ofGDNFRαorNTNRαmRNA, and suggest that GFRα-3 may function in differentiation of embryonic cells expressing its mRNA.

Research reportMitogenic effect of glial cell line-derived neurotrophic factor is dependent on the activation of p70S6 kinase, but independent of the activation of ERK and up-regulation of Ret in SH-SY5Y cells

AbstractGlial cell line-derived neurotrophic factor (GDNF) activates c-Ret tyrosine kinase and several downstream intracellular pathways; the biological effects caused by the activation of each of these pathways, however, remain to be elucidated. Here we report the ability of GDNF to induce proliferation, rather than differentiation, of neuroblastoma cells (SH-SY5Y) by targeting the signaling pathway responsible for mediating this proliferative effect. GDNF induces the phosphorylation of Akt and p70S6 kinase (p70S6K) in SH-SY5Y cells in which Ret protein expression is relatively low. Interestingly, treating SH-SY5Y cells with retinoic acid greatly increases Ret protein levels and GDNF-induced Ret tyrosine phosphorylation, but does not affect the mitogenic action of GDNF and the activation of the Akt/p70S6K pathway. In contrast, the activation of the ERK pathway and the resulting induction of immediate-early genes parallel the increases in Ret protein levels. Rapamycin, a specific inhibitor of p70S6K activation by the mammalian target of rapamycin, completely prevents GDNF-induced proliferation and activation of p70S6K. These results suggest that GDNF promotes cell proliferation via the activation of p70S6K, independent of the ERK signaling pathway, and that GDNF activates the Akt/p70S6K pathway more efficiently than the ERK pathway in the cells in which Ret expression is low.

Recruitment of mRNA-destabilizing protein TIS11 to stress granules is mediated by its zinc finger domain

AbstractTIS11, a member of the CCCH zinc finger protein family, was found to be distributed throughout cells with a preferential cytoplasmic localization when transiently expressed in COS-7 cells. Upon treatment with heat shock, TIS11 became localized in discrete particles in the cytoplasm of the transfectants. We showed the TIS11-positive particles to be stress granules (SGs), which are known to be formed in the cytoplasm of eukaryotic cells in response to environmental stresses. By deletion studies using the green fluorescent protein fusion system, we mapped a functional stress granule (SG) localization signal to a region containing two tandem repeats of the zinc finger motif of TIS11. Site-directed mutations of Tyr105/Tyr113, Gly109/Gly 114, and Phe119 in the first zinc finger motif diminished the ability of this TIS11 domain to direct SG localization. Importantly, when the zinc-chelating Cys residues in either the first or second zinc finger were mutated to Ala residues, the recruitment of the TIS11 zinc finger region to SG was significantly inhibited by the mutation and was completely abolished by the mutation in both zinc fingers. These results suggest that recruitment of TIS11 to heat shock-induced SG is governed by the tandem zinc finger domains of this protein.

The role of dopamine transporter in selective toxicity of manganese and rotenone

AbstractThe dopamine transporter has been shown to be the most relevant target site for the specificity of 1-methyl-4-phenylpyridinium ion (MPP+), a neurotoxin for dopaminergic neurons. In contrast, the mechanisms underlying the selective toxicity of manganese and rotenone, potentially toxic agents implicated in dopaminergic neuronal cell death, remain unknown. The aim of this study was to determine the cellular mechanisms of manganese or rotenone uptake in dopaminergic cells via the dopamine transporter. PC12 cells overexpressing the dopamine transporter, which were exposed to 10 μM MPP+, showed extensive DNA fragmentation, a biochemical hallmark of apoptosis, whereas wild-type PC12 cells or vector-transfected PC12 cells, which were exposed to 5 mM MPP+, did not show DNA fragmentation. In contrast, manganese and rotenone induced DNA fragmentation at slightly lower concentrations in PC12 cells overexpressing the dopamine transporter compared to control cells. Dopamine transporter inhibitors, such as mazindol, nomifensine, or GBR12909, inhibited MPP+-induced DNA fragmentation but did not affect manganese- and rotenone-induced DNA fragmentation in PC12 cells overexpressing the dopamine transporter. Finally, manganese accumulated to similar levels in PC12 cells overexpressing the dopamine transporter and control PC12 cells following incubation with manganese chloride. These results suggested that the dopamine transporter dose not confer cytotoxicity to manganese and rotenone.

Prevention of oxytosis-induced c-Raf down-regulation by (arylthio)cyclopentenone prostaglandins is neuroprotective

Highlights•Down-regulation of c-Raf contributed to glutamate-induced oxytosis.•Neuroprotective (arylthio)cyclopentenone prostaglandins preferentially bound to c-Raf.•(Arylthio)cyclopentenone prostaglandins prevented down-regulation of c-Raf, resulting in neuroprotection against oxidative stress.

NF-κB independent signaling pathway is responsible for LPS-induced GDNF gene expression in primary rat glial cultures

AbstractGlial cell line-derived neurotrophic factor (GDNF), a distant member of the transforming growth factor-β superfamily, was originally purified and cloned as a potent survival factor for midbrain dopaminergic neurons. Some studies have characterized the transcriptional regulation of the GDNF gene, but its regulatory mechanisms have yet to be well defined, especially under pathophysiological conditions. In this study, we used a pharmacological approach to study the expression of the rat GDNF gene induced by lipopolysaccharide (LPS) in primary cultures of glial cells. MG132, a blocker of nuclear factor κB (NF-κB) activation, did not apparently affect LPS-induced GDNF gene expression, whereas it attenuated the up-regulation of iNOS genes via Toll-like receptor (TLR) 4. In primary glial cultures, LPS increased the phosphorylation levels of c-Jun amino-terminal kinase 1 (JNK1) and p38 mitogen-activated protein kinase (MAPK); in primary microglial cultures, it enhanced phosphorylation of extracellular signal-regulated kinase (Erk). Of the several MAP kinase inhibitors tested, a JNK-specific inhibitor blocked LPS-induced GDNF transcription in primary cultures of microglia, but not of astrocytes. These results suggest that LPS up-regulates GDNF transcription through an NF-κB independent pathway, and that JNK is responsible for LPS-stimulated GDNF transcription in primary cultures of microglia.

Prevention of dopaminergic neuron death by adeno-associated virus vector-mediated GDNF gene transfer in rat mesencephalic cells in vitro

AbstractGlial cell line-derived neurotrophic factor (GDNF) is known as a potent neurotrophic factor for dopaminergic neurons. Since adeno-associated virus (AAV) vector is a suitable vehicle for gene transfer into neurons, rat E14 mesencephalic cells were transduced with an AAV vector expressing GDNF. When compared with mock transduction, a larger number of dopaminergic neurons survived in AAV-GDNF-transduced cultures (234% and 325% of controls at 1 and 2 weeks, respectively; P<0.01). Furthermore, the dopaminergic neurons in the latter cultures grew more prominent neurites than those in the former. These findings suggest that AAV vector-mediated GDNF gene transfer may prevent dopaminergic neuron death, and is therefore a logical approach for the treatment of Parkinson's disease.

Advertisement
Join Copernicus Academic and get access to over 12 million papers authored by 7+ million academics.
Join for free!

Contact us