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ABSTRACT: Organotin compounds such as tributyltin (TBT) are known to cause various forms of cytotoxicity, including developmental toxicity and neurotoxicity. However, the molecular target of the toxicity induced by nanomolar levels of TBT has not been identified. In the present study, we found that exposure to 100 nM TBT induced growth arrest in human pluripotent embryonic carcinoma cell line NT2/D1. Since glucose provides metabolic energy, we focused on the glycolytic system. We found that exposure to TBT reduced the levels of both glucose-6-phosphate and fructose-6-phosphate. To investigate the effect of TBT exposure on glycolysis, we examined glucose transporter (GLUT) activity. TBT exposure inhibited glucose uptake via a decrease in the level of cell surface-bound GLUT1. Furthermore, we examined the effect of AMP-activated protein kinase (AMPK), which is known to regulate glucose transport by facilitating GLUT translocation. Treatment with the potent AMPK activator, AICAR, restored the TBT-induced reduction in cell surface-bound GLUT1 and glucose uptake. In conclusion, these results suggest that exposure to nanomolar levels of TBT causes growth arrest by targeting glycolytic systems in human embryonic carcinoma cells. Thus, understanding the energy metabolism may provide new insights into the mechanisms of metal-induced cytotoxicity.
Metallomics 03/2013; · 3.90 Impact Factor
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ABSTRACT: The α7-nicotinic acetylcholine receptor (α7-nAChR) is widely known as a neurotransmitter receptor in nervous systems. α7-nAChR is also present in a variety of non-neuronal tissues, where it has been implicated in the regulation of essential cellular functions including proliferation, survival, differentiation and communication. We have recently found in breast cancer that α7-nAChR is involved in the proliferation of cancer stem cells, which constitute a minor subpopulation responsible for tumor development and metastasis. Since growing evidence suggests that α7-nAChR is present not only in mature tissues and organs but also in undifferentiated stem cells and progenitor cells, α7-nAChR emerges as a key mediator in the regulation of self-renewal and differentiation. We provide here an overview of the recent works on the expression and function of α7-nAChR in normal and cancer stem cells, and their relevance to disease-related cellular dysfunction. Understanding the role of α7-nAChR in stem cells would be of great interest for its application potential in drug discovery and in regenerative medicine.
Current drug targets 02/2012; 13(5):656-65. · 3.93 Impact Factor
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ABSTRACT: Mesenchymal stem cells (MSC) have the potential to differentiate into various cell lineages, including adipocytes and osteoblasts. The formation of adipose tissue involves the commitment of MSC to the preadipocyte lineage and the differentiation of preadipocytes into mature adipocytes. In the present study, we investigated the involvement of reactive oxygen species (ROS) in adipocyte differentiation from MSC.
ROS signaling was evaluated by the effects of antioxidant N-acetyl-l-cysteine (NAC) or shRNA against NAD(P)H oxidase in the multipotent mesenchymal stem cell line 10T1/2 cells. Intracellular ROS was measured using an H(2)DCF dye.
We found that NAC blocked adipocyte differentiation in MSC. An H(2)DCF assay revealed that differentiation-inducing agents induced ROS generation. These data suggest that ROS is involved in adipocyte differentiation in MSC. Next, we examined the source of ROS. Knockdown of NAD(P)H oxidase 4 (Nox4) by RNA interference inhibited ROS production and adipocyte differentiation by differentiation-inducing agents. Furthermore, treatment with NAC blocked the transcriptional activation of CREB, and the expression of dominant-negative mutants of CREB inhibited adipocyte differentiation.
The findings suggest that the increase in the intracellular ROS level via Nox4 mediates adipocyte differentiation through CREB in MSC. This data will provide new insight into the drug development for obesity.
Life sciences 06/2011; 89(7-8):250-8. · 2.56 Impact Factor
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ABSTRACT: Epidemiological studies have suggested that cigarette smoking is related to increased breast cancer risk. Nicotine is most likely related to the risk in cigarette smoking. However, the mechanisms by which nicotine promotes cancer development are not fully understood. It has recently been suggested that development of breast cancer are originated from cancer stem cells, which are a minor population of breast cancer. In the present study, we investigated the effects of nicotine on the population of cancer stem cells in MCF-7 human breast cancer cells, using flow cytometry with a cancer stem cell marker aldehyde dehydrogenase (ALDH). We found that nicotine increased ALDH-positive cell population in a dose-dependent manner. We further demonstrated that a PKC-Notch pathway is involved in the effect of nicotine. In addition, the effect of nicotine was blocked by treatment with the α7 subunit-selective antagonist of nicotinic acetylcholine receptors (nAChR) α-Bungarotoxin. These data suggest that nicotine increases the stem cell population via α7-nAChR and the PKC-Notch dependent pathway in MCF-7 cells. These findings reveal a relationship between nicotine and the cancer stem cells in human breast cancer.
Biochemical and Biophysical Research Communications 11/2010; 403(1):138-43. · 2.48 Impact Factor
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ABSTRACT: Inescapable shocks (IS) have been reported to reduce the number of 5-bromo-2'-deoxyuridine (BrdU)-positive cells in hippocampus. Antidepressants prevent this reduction, and the role of neurogenesis in depression is now suggested. It has been reported, however, that the number of BrdU-positive cells was not different between the rats that developed learned helplessness and those that did not. This suggests that reduction of neurogenesis does not constitute a primary etiology of depression. It has been previously shown that IS can cause various post-traumatic stress disorder (PTSD)-like behavioral changes in rats. The aim of the present was therefore to examined whether the reduction of BrdU-positive cells relates to any PTSD-like behavioral changes in this paradigm.
Rats were given either inescapable foot-shocks (IS) or not shocked (non-S) treatment in a shuttle box on day 1 and received BrdU injections once daily during the first week after IS/non-S treatment. On day 14, rats treated with IS and non-S were given an avoidance/escape test in the shuttle box and dorsal hippocampal SGZ were analyzed by BrdU immunohistochemistry.
In accordance with previously reported results, IS loading resulted in fewer BrdU-positive cells in the hippocampal subgranular zone (SGZ). Furthermore, in the IS-treated group, the number of BrdU-positive cells in the hippocampal SGZ was negatively correlated at a significant level with several hyperactive behavioral parameters but not with hypoactive behavioral parameters. Earlier findings had indicated that chronic selective serotonin re-uptake inhibitor administration, which is known to increase hippocampal neurogenesis, restored the increase in hypervigilant/hyperarousal behavior but did not attenuate the increase in numbing/avoidance behavior.
The regulatory mechanism responsible for the decreased proliferation and survival of cells in the hippocampus may be related to the pathogenic processes of hypervigilance/hyperarousal behaviors.
Psychiatry and Clinical Neurosciences 01/2009; 62(6):713-20. · 2.13 Impact Factor
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ABSTRACT: GPR56, a member of the G-protein-coupled receptor family, plays a role in the formation of the frontal and parietal brain lobes and cortical lamination in the embryonic stage. A recent report indicated the existence of GPR56 transcripts in the subventricular zone (SVZ) and hippocampal subgranular zone (SGZ) of the adult mouse brain. Both these regions are known to continually produce neural progenitor cells in the adult brain. Here, we demonstrate abundant GPR56 protein expression in the ependymal cell layer and SVZ as well as its reciprocal translational regulation by a 12-day behavioral stress paradigm and 10-day electroconvulsive seizure (ECS) treatment. Our study revealed that GPR56 transcript expression in the hippocampus was regulated by stress and seizure in a manner identical to that in the SVZ. GPR56 expression was downregulated by stress and upregulated by the ECS treatment in both regions, whereas nestin expression showed no changes. Western blot analysis revealed a robust ECS-induced increase in brain-derived neurotrophic factor expression in the wall of the lateral ventricle including the ependymal cell layer and the SVZ, which may provide a possible regulatory mechanism for GPR56 expression. We consider that GPR56 is expressed in the ependymal cell layer and in immature progenitor cells and that its expression is regulated by functional stimulation.
Brain Research 01/2008; 1183:21-31. · 2.73 Impact Factor
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ABSTRACT: We initially examined the effects of apomorphine in vitro using mouse embryonic and adult neural progenitor cells. The effects of apomorphine treatment led to dose-dependent increases in the number of embryonic and adult neural progenitor cells, and dopamine D2 receptor antagonist treatment significantly reduced the increases induced by apomorphine. Next, we investigated the effects of apomorphine in vivo in the adult mouse hippocampus. The effects of single-dose apomorphine administration led to an increase of approximately 30% in the number of bromodeoxyuridine-positive cells in the dentate gyrus. Moreover, the chronic apomorphine administration induced an increase in the number of bromodeoxyuridine-positive cells by about 30%. Thus, we suggest that the stimulation of dopamine D2 receptors increases the proliferation of neural progenitor cells both in vivo and in vitro.
Neuroreport 05/2007; 18(7):659-64. · 1.66 Impact Factor
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ABSTRACT: Cigarette smoke has been firmly established as an independent risk factor for atherosclerosis and other vascular diseases. The proliferation and migration of vascular smooth muscle cells (VSMC) induced by growth factors have been proposed to play an important role in the progression of atherosclerosis. In the present study, we investigated the effects of nicotine, which is one of the important constituents of cigarette smoke, on vascular endothelial growth factor (VEGF) release, in rat VSMC. The stimulation of cells with nicotine resulted in a time- and concentration-dependent release of VEGF. Hexamethonium, an antagonist of nicotinic acetylcholine receptor (nAChR), inhibited nicotine-induced VEGF release. We next investigated the mechanisms by which nicotine induces VEGF release in the cells. The nicotine-induced VEGF release was inhibited by treatment with U0126, a selective inhibitor of MEK, which attenuated the nicotine-induced ERK phosphorylation. Nicotine induced a transient phosphorylation of ERK. Furthermore, AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) kinase, inhibited nicotine-induced ERK phosphorylation and VEGF release. These data suggest that nicotine releases VEGF through nAChR in VSMC. Moreover, VEGF release induced by nicotine is mediated by an EGFR-ERK pathway in VSMC. VEGF may contribute to the risk of cardiovascular diseases in cigarette smokers.
Life Sciences 04/2007; 80(15):1409-14. · 2.53 Impact Factor
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ABSTRACT: The splicing of 26 nucleotides in the coding region of the X-box binding protein-1 (XBP-1) transcript to generate a mature active transcription factor is a part of the unfolded protein response to intracellular endoplasmic reticulum stress. In this study, we demonstrated that XBP-1 splicing is promptly induced in the rat brain including the hippocampus by both inescapable electric foot shock (IS) and pharmacologically manipulated activation of 5-hydroxytryptamine release in a dose-dependent manner. By administering ketanserin, a 5-hydroxytryptamine 2A antagonist, however, we could only partially block the increased splicing by IS and observed that the splicing was not influenced by lithium carbonate pretreatment. Although it is still unclear whether the enhanced unfolded protein response functions neuroprotectively by modulating the rate of general translation and increasing chaperone proteins or whether it eventually induces cellular damage by triggering apoptosis, the present results indicate the possible existence of a new adaptive intracellular signaling pathway in the brain that responds to environmentally challenged behavioral stress loading.
Brain Research 10/2006; 1112(1):26-32. · 2.73 Impact Factor
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ABSTRACT: Thrombin is a mitogen for vascular smooth muscle cells (VSMC) and has been implicated in the development in atherosclerosis. However, little is known about the role of thrombin in glucose transport in VSMC. In this study, we examined the effect of thrombin on glucose uptake in rat A10 VSMC. We found that thrombin induced glucose uptake in a dose-dependent manner while hirudin, a potent thrombin inhibitor, prevented glucose uptake in the cells. PP2, a selective inhibitor of Src, prevented the thrombin-induced glucose uptake, but did not affect insulin-induced uptake. We also examined whether mitogen-activated protein kinase (MAPK) inhibitors influenced thrombin-induced glucose uptake. The p38 MAPK inhibitor (SB203580) inhibited thrombin-induced glucose uptake, but the MEK inhibitor (PD98059) did not. In contrast to thrombin, SB203580 did not affect insulin-induced glucose uptake. Furthermore, thrombin failed to translocate the insulin-sensitive glucose transporter GLUT4. These findings suggest that thrombin stimulates glucose transport via Src and subsequent p38 MAPK activation in VSMC.
British Journal of Pharmacology 10/2005; 146(1):60-7. · 4.41 Impact Factor
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ABSTRACT: This study investigates the involvement of alpha(2)-adrenergic receptors (AR) in mouse brain induced by a low dose of methamphetamine (METH, 2 mg/kg). Immunohistochemical studies show that alpha(2A)-AR increased in the dentate gyrus area of the hippocampus 24 h after five repeated administrations of METH. The hippocampal alpha(2A)-AR proteins rose 3.2-fold when compared to the saline-administered mice. The other adrenergic receptor, alpha(1D)-AR, were not changed by the treatment. Moreover, alpha(o)-subunits of GTP-binding proteins (Galpha(o)), one of the downstream molecules of alpha(2A)-AR, was also increased by the treatment. These suggest that the repeated administration of low-doses of METH causes quantitative changes of the signaling of alpha(2A)-AR in the mouse hippocampus.
Neuroscience Letters 01/2003; 334(3):145-8. · 2.11 Impact Factor
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ABSTRACT: 1. This study deals with phosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) via beta(3)-adrenoceptor (AR) and the signal transduction pathway in 3T3-L1 adipocytes. 2. beta(3)-AR agonist BRL37344A (10 nM) caused phosphorylation and activation of p38 MAPK in 3T3-L1 adipocytes but not in fibroblasts. BRL37344A and also the other beta(3)-AR agonists, CGP12177A and SR58611A, caused p38 MAPK phosphorylation in dose-dependent manners. 3. The p38 MAPK phosphorylations by BRL37344A (10 nM), CGP12177A (100 nM), and SR58611A (10 nM) were not antagonized by beta(1)- and beta(2)-ARs antagonist 1-propranolol (100 nM) but blocked by beta(3)-AR antagonist SR59230A (10 microM), suggesting the phosphorylation was caused via beta(3)-AR. 4. The phosphorylations of p38 MAPK were completely abolished by treatment with cholera toxin (CTX) but not pertussis toxin (100 ng ml(-1), 24 h). Activation of Gs by CTX (100 ng ml(-1)) and adenylyl cyclase by forskolin mimicked p38 MAPK phosphorylation. 5. p38 MAPK phosphorylation by BRL37344A was reduced to almost 50% by cyclic AMP-dependent protein kinase (PKA) inhibitors such as H89 (10 microM) and PKI (10 microM). A src-family tyrosine kinases inhibitor PP2 (1 microM) also halved the p38 MAPK phosphorylation. Combined use of H89 (10 microM) and PP2 (10 microM) did not bring about further inhibition. 6. These results suggest that beta(3)-AR caused phosphorylation of p38 MAPK via Gs protein and partly through a pathway involving PKA and src-family kinase(s), although the contribution of the unidentified pathway remains to be clarified.
British Journal of Pharmacology 03/2002; 135(4):951-60. · 4.41 Impact Factor
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ABSTRACT: The treatment of 3T3-L1 adipocytes with three β3-adrenoceptor agonists, (±)-(R*,R*)-(4-[2-([2-(3-chlorophenyl)-2-hydroxyethyl]amino)propyl]phenoxy)acetic acid (BRL37344), 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1, 3-dihydro-2H-benzimidazol-2-one (CGP12177) and [(7S)7-{(2R)2-(3-chlorophenyl)-2-hydroxyethyl-amino}-5,6,7,8-tetrahydronapht-2-yl]ethyl oxyacetate, hydrochloride (SR58611) induces phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). The phosphorylations were not affected by pretreatment of the adipocytes with pertussis toxin, whereas the same treatment completely abolished lisophosphatidic acid-induced phosphorylation of ERK1/2, suggesting the role of pertussis toxin-insensitive G protein in the ERK1/2 phosphorylation by stimulation with the β3-adrenoceptor agonists. The phosphorylation of ERK1/2 was mimicked by treating the adipocytes with cholera toxin, a direct activator of stimulatory G (Gs) protein. In addition, the ERK1/2 phosphorylations by the β3-adrenoceptor agonists were completely diminished by long-term treatment of the adipocytes with cholera toxin (100 ng/ml, 24 h), whereas that obtained with lisophosphatidic acid stimulation was not. Our findings strongly suggest that the three β3-adrenoceptor agonists induce ERK1/2 phosphorylation in 3T3-L1 adipocytes through a Gs protein-dependent cascade.
European Journal of Pharmacology.
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Yasushi Satoh, Yasunari Kanda,
Mitsuhiro Terakawa,
Minoru Obara,
Katsushige Mizuno,
Yasuhiro Watanabe,
Shogo Endo,
Hidetoshi Ooigawa,
Hiroshi Nawashiro,
Shunichi Sato,
Kunio Takishima
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ABSTRACT: We investigated the feasibility of gene transfer into the mouse central nervous system (CNS) by applying nanosecond pulsed laser-induced stress waves (LISWs). Intraventricular or hippocampal injection of a reporter gene [enhanced green fluorescent protein (EGFP)] followed by application of LISWs showed this method to be efficient in the CNS of newborn and adult mice. Cells expressing EGFP reside at least 3.5 mm from the surface of the tissue, while no apparent damage was detected. Additionally, expression of EGFP was limited to the area that was exposed to LISWs. Using this method, the formulation of plasmid DNA by cationic transfer reagent polyethylenimine proved to be effective for improving transfer efficiency into the CNS.
Journal of Biomedical Optics 10(6):060501. · 3.16 Impact Factor
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ABSTRACT: Recent studies have revealed that activated extracellular signal-regulated kinases (ERKs) 1 and 2 by the stimulation of β3-adrenoceptors played a critical role in cell survival in brown adipocytes. On the other hand, phosphorylation of ERK1/2 via β3-adrenoceptors and its physiological and pathological significance in white adipocyte has remained uncertain despite the increasing significance of functioning white adipocytes. Accordingly, we here studied phosphorylation of ERK1/2 caused by the stimulation of β3-adrenoceptors in 3T3-L1 adipocytes, and the roles of phosphorylated ERK1/2 in lipolysis. Phosphorylation of ERK1/2 was induced by a selective β3-adrenoceptor agonist, dl-4-[2′-{2-hydroxy-2-(3-chlorophenyl)ethylamino}propyl] phenoxyacetic acid sodium salt sesquihydrate (BRL37344), in 3T3-L1 adipocytes in a time- and dose-dependent manner. The phosphorylation of ERK1/2 by BRL37344 was sensitive to the cyclic AMP (cAMP)-dependent protein kinase inhibitor, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide (H89). To elucidate the roles of phosphorylated ERK1/2 in lipolysis, the effect of a selective inhibitor of ERK1/2 phosphorylation, 2′-amino-3′-methoxyflavone (PD98059), was examined. This inhibitor did not alter the lipolytic action caused by BRL37344, even at concentrations sufficient to block phosphorylation of ERK1/2, suggesting that ERK1/2 play no role in the lipolysis caused by BRL37344 in 3T3-L1 adipocytes.
European Journal of Pharmacology.
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ABSTRACT: Na+/H+ exchange has been proposed to be involved in the regulation of cell growth. However, little is known about the regulatory pathway and relationship between Na+/H+ exchange and DNA synthesis. In vascular smooth muscle cells, platelet-derived growth factor (a tyrosine kinase-coupled receptor agonist) and thrombin (a G protein-coupled receptor agonist) stimulate both activation of Na+/H+ exchange and DNA synthesis. In this study, we compared the effect of platelet-derived growth factor (PDGF) and thrombin on the signal transduction pathway leading to the activation of these responses in A10 cells, clonal rat thoracic aortic smooth muscle cells. To investigate the role of mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase as potential mediators, we examined the effect of pharmacological kinase inhibitors on these responses. The Na+/H+ exchange activity induced by thrombin was inhibited by a specific inhibitor of MAPK kinase, 2′-amino-3′-methoxyflavone (PD98059), but was not affected by a specific phosphatidylinositol-3-kinase inhibitor, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). Thrombin-induced DNA synthesis was inhibited by LY294002, but not by PD98059. In contrast, the Na+/H+ exchange activity induced by PDGF was inhibited by neither LY294002 nor PD98059, but PDGF-induced DNA synthesis was inhibited by both LY294002 and PD98059. These data suggest that, in A10 cells, Na+/H+ exchange activation and DNA synthesis are differently regulated by the two extracellular stimuli.
European Journal of Pharmacology.
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ABSTRACT: We investigated whether 17β-estradiol affects the activity of extracellular signal-regulated kinase (ERK) in the central nervous system in vivo. 17β-Estradiol was administered intracerebroventricularly, and then ERK activity in the rat hippocampus was measured. We found that ERK activity in the rat hippocampus had increased to approximately threefold its basal level at 5 min. This rapid effect was mimicked by the membrane-impermeable estradiol, bovine serum albumin-conjugated 17β-estradiol, and was not inhibited by tamoxifen and 7α,9-(4,4,5,5,5-pentafluoropentylsulphinyl)nonylestra-1,3,5(10)-triene-3,17β-diol (ICI 182780), classical nuclear estrogen receptor antagonists. These data suggest that the rapid activation of ERK by estradiol in vivo is mediated through a putative membrane estrogen receptor in the rat hippocampus.
European Journal of Pharmacology.
